Desarrollo y evaluación toxicológica de nuevos materiales para su aplicación en la conservación de alimentos.

UNIVER SIDAD D E SEVILLA FACUL TAD DE FARMACIA DEPARTAMEN TO DE NUT RICIÓN Y B ROMATOLOG ÍA, TO X ICOLOGÍA Y MEDICINA L EGAL “ DE SARROLLO Y EVA LUAC IÓN TOXICOLÓG ICA DE N UEVOS MATER IALES PA RA SU APLICACIÓN EN LA CONSERVA CIÓN D E ALIMENTOS ” Memor ia que presen ta la Licenciada SARA MAISANABA HERNÁNDEZ para optar al título de Doctor por la Universidad d e Sevilla con la Mención Internacional Sevilla, 2015 UNIVERSIDAD DE SEVIL LA AREA DE T OXICOLOGÍA C/ Profesor García Go nzále z, nº 2 4101 2 Sevilla (España) Teléfono: 954 5 5 67 62 Fax: 954 55 64 22 Este trabajo ha sido realizado en el Área de Toxicología del Departamento de Nutrición y Bromatología, Toxicología y Medicinal L egal de la Facultad de Farmacia de la Universidad de Sevilla, y se ha financiado con los siguientes Pro y ectos de Investig ación:  P10- AGR -5969, Proy ect o de Excelencia de la Junta de Andalucía: Desarrollo y Evaluación Toxicológica de Nanomateriales para su Aplicación en Conservación d e Alimentos (I nvestigadora Principal: Ángeles Mencía Jos Gallego).  AGL2010-21210, Plan Nacional de 2010 (Ministerio de Economía y Comp etitividad ) : Desarrollo y Evaluación Toxicológica de Nanoarcillas de Aplicación en el Envasado de Alimentos (Investigadora Principal: Ángeles Mencía Jos Galleg o ).  AGL2012-38357-C02-01, Plan Nacional de 2012 (Minist erio de Economía y Competitividad): Evaluación de l a Seguridad par a C ontacto Alimenta rio de Envas es Activos Basa dos en Polímeros Biodegradables y Extractos Natura les (Investiga do ra Principal: Ana María Cameán Ferná nd ez).  P11- AGR -7252, Pro y ect o de Excelencia de la Junta de Andalucía: Desarrollo y Evaluación Toxicológica de Env ases Activos Aptos para Contacto Alimentario (Investigadora Principal: Ana Mar ía Cameán Fernández). La Doctor anda Dª Sara Maisanaba Hernández ha disfrutado de un a a yuda P redoctora l de Personal Investigador en Formación (PIF), asociada a un P royec to de Excelencia de la Junta de Andalucía (P10- AGR - 5969), desde Febrero de 2012. As í mismo, para la realización de la est ancia en el extranjero, la Doctoranda Dª S ara Maisanaba Hernández ha disfrutado de una A y uda de Movilidad derivada del V P lan Propio de Investigac ión de la Universidad de Sevilla (2014). ÍNDICE / INDEX Índice de Abreviaturas / Abbreviations Index VII ÍNDICE DE A BREVIATURAS / ABBREVIA TIONS IND EX ADN: ácido desoxirribonucleico Al: aluminio AlO 6 : óxido de alumnio APTES : aminopropiltrietoxisilano ARN : ácido ribonucleico ARN : ácido ribonucleico mensajero Ca : calcio Caco -2: línea celular humana de adenocarcinoma de colon CAT : catalasa CEC : capac idad de intercambio iónico CHO : células de ovario de hámster c hino CITIUS : Centro de I nve stigac ión, T ecnología e Innovación de la Universidad de Sevilla CNa + : Cloisite®Na + C15A : Cloisite®15A C10A : Cloisite®10A C20A : Cloisite®20ª C93A : Cloisite®93A d: espesor de la membrana polimérica d´ : tra y ectoria/lon gitud de la dif usión del gas en prese ncia de las nanoláminas de arcillas EE.UU: Estados Unidos EFSA: European Food Safe t y Authorit y ERO: especies reactivas de oxígeno FDA: F ood and Drug Ad ministration Fe: hierro FTIR: espectroscopía infrarr oja g: gramo GPX: glutatión peroxidasa GR: gluta tión reductasa Índice de Abreviaturas / Abbreviations Index VIII GRAS: Generally R ecogniz ed as Safe GSH: glutatión h: hora s HEK293: línea celular rena l HeLa: línea ce lular epitelial HepG2: línea celular humana de hepatoma HMy2.CIR : línea ce lul ar linfoblástica HUVEC: línea celular humana de ve n a endotelial umbilical IA: industria alimentaria IARC: Agencia I nternacional de Inve sti gación sobre el Cáncer IL -6: interleucina-6 IMR32 : lín ea c elula r neuroblástica IMR90 : línea ce lul ar fibroblástica INT -407: línea celular intestinal kg: kilogramo L : espesor de las nanoláminas de arc illa L5178Y : línea celular linfoide L929 : línea celular f ibroblástica LDH : lactato deshidrogenasa Li: litio LPO: peroxidación lipídica M: molar MCF- 7: línea celular epitelial MDA: malondialde hído mequiv.: miliequivalente mg: miligra mo mL: mililitr o Mg: magne sio MgO 6 : óxido de magnesio Índice de Abreviaturas / Abbreviations Index IX MLA: mouse ly mphom a assa y / ensa y o d e linfoma de ra tón mM: milimolar MN: micronúcleos Mt: montmorillonita MTS: 3-(4,5-dimethy lthi azol -2-y l) -5- (3 -carbox ymethox y phenyl)- 2- (4 -su lfopheny l) -2H- tetrazolium MTT: Bromuro de 3-(4,5-dimetiltiazol-2-ilo)-2,5-difeniltetrazol N1E- 115: línea celula r neuronal N2a: línea celular ne u ronal Na: sodio NIB: National Institute of B iol og y NIH3T3: línea celular fibroblástica nm: nanómetro O 2 : oxíge no OCDE: Organización para la Coope r ación y Desa rrollo Económico OMS: Organización Mundial de la Salud PCR: reacción en cade na de la polimerasa PET: polietileno PLA: ácido poliláctic o PP: polipropileno ppm: partes por millón q-PCR: PCR cuantitativa a tiempo real RN: rojo neutro ROC: línea celular ne u ronal S9: fracción metabólica ex terna SCE: intercambio de cromátidas hermanas Si: silicio SiO4: tetaedro de silicato SOD: superóxido dismutasa TGA: ter mogravimetría Índice de Abreviaturas / Abbreviations Index X TNF- α: factor de necrosis tumoral - α U937: línea celular monocítica UE: Unión Europea V79: línea celular pulmonar VMTS: viniltrimetoxisilano W : grosor de las nanoláminas de a rcilla µg : microg ramo(s) µmol: micromol( es ) µm: micrómetro µM : micromolar I. RESUMEN / SUMMARY Resumen / Summary 1 RESUMEN En la actualidad, l a indus tria alimentaria está apos tando por la incorpo rac ión de sustan cias naturales a envases alimentarios con el fin de incrementar la pe rdurabilidad del alimento en el mercado. D ebido al empleo de estas sustancias e n esta nueva aplicación, la seg u ridad de los consumidores y d el medio ambiente puede v erse comprom etida debido a una ma y o r exposición, desconoc iéndose en gran medida, hasta el momento, las posibles consecuencias. De entre las sust ancias naturales s elecc ionadas por la industria alimentaria pa ra este fin , destacan l as arcillas y minerales de arcillas, y los ace ites esenciales y sus respectivos componentes ma y orita rios , constitu y endo las primeras (arcillas) el p rincipal objeto de estudio de la presente Tesis Doctoral. Por todo ello, decidimos realizar una evaluación tox icológica mediante una batería de ensa yos tanto in vitro como in vivo , para inve stigar los posibles efec tos tóxicos que pueden desencadenarse tras la exposición a estas sustanc ias, tanto puras como ya incorporadas en el envase. La relevancia de la información toxicológica de estas sustancias es tal que constituy e un requisito reglament ario por parte de las autoridades competentes antes de su comercialización. En el c aso de las arcillas y minerales d e arcill a, se ha demostrado un a mejora de las propiedades mecánicas, térmicas y barrera de las matrices poliméricas de bido a su incorporación, lo que est á, íntimamente r elacionado con el increm ento de la vida útil de los alimentos. Con el fin de contextualizar la sit uación ante l a que se encontraban las arcillas y sus deriva dos en el área del envasado de la industria alimentari a y así abordar posteriores estudios, nos pareció important e realizar una exhaustiva revisión bibliográ fica sobre los datos de toxicidad publicados en la literatura científica hasta la actualidad. La disparidad de los resultados disponibles hasta el momento hace nec esaria una evaluación toxicológica caso por caso. Diferentes parámet ros pueden estar involucrados en la respu esta obtenida, inclu yendo: (i) condiciones de exposición tales como con centraciones s eleccionadas o t iempos ensa y ados; (ii) modelos ex perimentales eleg idos; (iii) modificadores o sur factantes incorporados a la estructura de la arcilla, (iv) sensibilidad de los ensay os llevados a cabo , etc. Dicha revisión bibliográfica ha dado lu gar a la siguiente publicación:  TOXICOLOGICAL E V AL UATION OF CL AY MINERALS AND DERIVED NANOCOMPOSITES: A RE V IEW. (Maisanaba y col., 2015; Environmental Research 138, 233-254). Resumen / Summary 2 La vía de exposi ción más importante en el contex to que nos engloba es la vía oral, dado que las a rcillas van a s er incorporad as a un en vase que posteriormente se va a poner en contacto con un alimento, y éste últ imo será inger ido por los consumidores. En este caso lo s órganos que principalmente se podrían v er afectados serán los qu e componen el sistema digestivo, destacando entre ellos el intestino, encargado de l a absorción de nutrientes, y el hígado, c rucial en la biotransformación de xenobióticos. Se llevó a cabo una bater ía de ensayos de los materiales objeto de estudio con el fin de esclarecer sus principales efectos tóxicos, teniendo en c uenta la esc as ez y disparidad de los da tos disponibles . La s lí neas celulare s seleccionadas f ueron una línea celular de hepatoma humano ( HepG2) y una lí nea celular de adenocarcinoma de colon (Caco -2). Éstas fueron expuestas a diversas arcillas no modificadas y modificadas con sales de amonio cuater nario , tanto comerc ializadas (C loisite® Na + (CNa + ), Cloisite®20A (C20A) y Cloisite®30B (C30B)) o desarrolladas por el Instituto Tecnológico de Embalaje, Transporte, y Logística (ITENE) de Valencia (Cla y 1 y Clay2). Los experimentos llevados a cabo inclu ye ron la evaluación de : c itotoxicidad basal, análisis de la morfología celular, genotox icidad (ensa y o cometa y ensay o de micronúcleos (MN)) y estrés oxidativo (producción de esp ecies reactivas de ox ígeno (ERO) y contenido de glutatión (GSH) ). De for ma general se obtuvo ausencia d e toxicidad en el caso d e las arcillas CNa + , C 20A y Cla y1, y sólo en el caso de la primera arcilla mencio nada se obtuvo una inducción de MN si gnificativa y alguna alteración de la morfología celular. Sin embargo, pudimos observar daño en ambas lí neas celulare s tras la ex posición a C30B y Cla y 2 en la mayor ía de los parámetros evaluados. Además, se ha llevado a cabo por primera vez en la línea celular HepG2 el es tudio de la influencia de las arcillas en la modulación de la expresión gé nica de un extenso grupo de genes involucrados en activación/destoxicación y otros mecanismos de acción tóxica. CN a + y Clay2 sí demostraron alterar la regulación de dist intos ge nes, no así C30B y Cla y 1 . L os resultados de estos ex perimentos ha n dado lu gar a las siguientes publicaciones:  IN V ITRO TOXICOLOGIC AL AS SESSMENT OF CLA YS FOR THEIR USE IN FOOD PACKA GING A PPLICATIONS. (Maisanaba y col., 2013, Food and Chemical Toxicology 37, 266-275).  GENETIC POTENTIAL OF MONTMORILLONITE CLAY MINERAL AND ALTERATION IN THE EX PRESSION OF GENES INVOLVED IN TOX ICITY MECHANISMS IN THE HUMAN HE PATOMA CELL LI NE HEPG2. ( Maisanaba y col., 2015 ; Journal of Hazardous Materials (en revision/ under revision) ). Resumen / Summary 3  INDUCTION OF MICRONUCLEI AND ALTERATION OF GENE EXPRESSION BY AN ORGANOMODIFIED CLAY IN HEP G2 CELLS. ( Maisanaba y col., 2015; Archives of Toxicology (en rev isi ón/ under revision)).  EFFECTS OF TWO ORGAN OMODIFIED C LAYS INTENDED TO FOOD CONTACT MA TERIA LS ON THE GENOMIC INSTABILITY AND GENE EXPRESSION OF HEPATOMA CELLS. (M aisanaba y col., 2015 ); Toxicology Letters (en rev ision/ under revision).  TOXIC EFFECTS OF A MODIFIED MONTMORILLONITE CLAY ON THE HUMAN INTESTINAL CELL LINE C ACO-2. (Maisanaba y col., 2014 ; Journal of Applied Toxicology 34, 714-725).  TOXICITY ASSESSM ENT OF ORGANOMODIFIED CLAYS USED IN FOOD CONTACT MATER IALS ON HUMAN TAR GET CELL LI NES. (Houtman y col., 2014; Applied Clay Science 90, 150-158). Aunque los ór ganos y tejidos del sistema digestivo s erían p robablemente los más afec tados por exposición a las arcillas, ha y otras dianas que también podr ían verse d añadas , destacando el tejido endotelial vascular, involuc rado en la dist ribución y absorción. Hasta el momento los datos disponibles acerca de los efe ct os de este modelo experimental expuesto a arcillas son limitados. Por ello , se ha llevado a cabo la evaluac ión de la viabili dad de la línea celular endotelial de vena umbilical humana (HUVEC) expuestas a CNa + , C30B, Clay 1 y Clay2. Los resultados obtenidos indi caron que las células HUVEC seguían un patrón de respuesta mu y similar al de HepG2 y Caco -2 ante la exposición a las arcillas, presentando una sensibilidad parecida a la línea celular hepática . Además, el estudio del pot encial mutagénico mediante el Test de Ames es una prueba exigida antes de la comercialización de cualquier producto que va y a a ent rar en contacto con alimentos (EFSA 2011 b, 20 15), siendo en este caso necesaria la evalu ación de las arcillas que estén destinadas a s er incorp oradas al envasado alimentario. El modelo experimental se leccionado fue Salmonel la typhi mu rium (5 cepas) , el cua l se expuso a las c uatro arcillas menc ionadas , en ausencia y presencia de fracción metabólica externa. Únicamente pudimos observar una respuesta m utagénica positiva en el ca so de C30B y C lay 1 . Los resultados obtenidos se re flejan en la siguiente publicación:  CYTOTOXICITY AND MUTAGENI CITY A SSESSME NT OF ORGANOMODIFIED CLAYS POTENTIALLY USED IN F OOD PACKAGING. (Maisanaba y col., 2015; Toxicology In Vitro 29, 1222-1230). Resumen / Summary 4 La Autoridad Europea de Seguridad Alimentaria (EFSA) no sólo propone la evaluación de los materiales que se inc orporan a l as matrices po liméricas de env asado, sino también, siendo incluso de may or importancia, la evaluación de los envases resultantes (EFSA, 2011a,b). En este sentido, ITENE desa rrolló dos materiales nanocompuestos basados en ác ido poli(láctico) (PLA) y C lay1/Clay2, ambas arcillas diseñ adas por ellos. Con estos materia les se llevaron a cabo ensayos de mi gración, uti lizando como simulante alimentario a gua destilada, aceptado por el Reglame nto UE Nº 10/2011 dado el tipo de alimentos hacia los que van diri gidos los mismos (hidrófilos). Una vez obtenidos los e xtractos de migración de P L A -Clay 1 y PLA-Cla y2, se ev aluaron distintos parámetros con el fin de determinar los posibles efectos tóx icos debido a la migración de las arcillas tra s la e xposición a l os mismos. En este sentido, se evaluó la viabilidad celular de HepG2 y Caco-2 expuestas a ambos extractos, así como el potencial mutagé nico de los mi smos mediante el Test de Ames. En los ensayos ll evados a cabo s e observó la ausencia de citotoxicidad y mut agenicidad en las condi ciones ensa y adas. Por otro lado, teniendo en cuenta que la presencia de los metales ma y orita rios de la estructur a de las arcillas son Al, Ca, Fe, Mg y S i, se evaluó el contenido de los mismos en los extractos de migración mediante Espectrometría d e Masas con P lasma Acoplado Inductivamente (ICP- MS ) y Espectroscopía d e Plasma Induc ti vo acoplado a Espectroscopía de Emisión Óptica (I CP-OES), no obteniéndose diferencias si gnificativas con respecto al grupo control . Estos resultados se recoge n en la siguiente publicación:  CYTOTOXICITY AND MUTAGENICITY S TUDIES ON MIGRATION EXTRACTS FROM NANOCOMPOSI TES W ITH POTENT IA L USE IN F OOD PACKAGING. (Maisanaba y col., 2014 Food and Chemical Toxicology 66, 366 -372). Una vez realizado el estudio toxicológico in vitro de las arc illas y considerando las mejoras tecnológicas presentadas por los polímeros nanocompuestos resultantes, se seleccionó la arcilla de mejor perfil tox icológico y tecnológico, siendo en este caso Clay1 la seleccionada para continuar la investigación. Con el fin de completar los resultados obtenidos in vitro y dada la escasez de información in vivo , se realizó un ensayo de to xicidad subcrónica durante 90 días en ratas Wistar ex puestas a Clay 1 (40 mg/kg/día) en la di eta y su extracto de migración (PLA-Clay 1) como ag u a de bebida ( a d libitum ). En este sentido, transcurrido el periodo de ex posición, los animales fueron sacrifica dos y se extrajeron s us órganos (híga do, Resumen / Summary 11 EXPRESSION OF HEPATOMA CELLS. (M aisanaba y col., 2015); Toxicology Letters (en rev ision/ under revision).  TOXIC EFFECTS OF A MODIFIED MONTMORILLONITE CLAY ON THE HUMAN INTESTINAL CELL LINE C ACO-2. (Maisanaba y col., 2014 ; Journal of Applied Toxicology 34, 714-725).  TOXICITY ASSESSM ENT OF ORGANOMODIFIED CLAYS USED IN FOOD CONTACT MATER IALS ON HUMAN TAR GET CELL LI NES. (Houtman y col., 2014; Applied Clay Science 90, 150-158). Further more, althou gh th e organs and tissues of t he digestive s y stem would probabl y be the most affected b y the ex posure to cla y s, other targets could also be da maged, hi ghlig hti ng the vascular endothelial tissue, invol ved in dist ribution and absorption. In this concern, data on the effec ts of this experimental model after exposure to clay s ar e limited s o far. Therefore, cell viability evaluation of human umbilical vein endothelial cells (HUV EC) exposed to CNa + , C30B, Cla y 1 and C lay 2 was carried out. The results showed that HUVEC re sponse followed a similar pattern than HepG2 and Caco-2 upon exposure to clay s , with a sensitivit y close to the hepatic c ell line. I n addition, the study of the mutagenic potential using the Ames test is required before marketing an y produ ct t hat come into contact with food (EF S A 2011b, 2015), such as clay s, w hich are intended to be used in food packaging . The ex perimental model selec ted was Salmonella typhimurium (5 strains), which were ex posed to the four mentioned cla ys, in the absence and presence of e xternal metabolic activation. We only observed a positi ve mutagenic response in the ca s e of C30B and Cla y 1. The results obtained are c ompiled in the following publication:  CYTOTOXICITY AND MUTAGENI CITY A SSESSME NT OF ORGANOMODIFIED CLAYS POTENTIALLY USED IN F OOD PACKAGING. (Maisanaba y col., 2015; Toxicology In Vitro 29, 1222-1230). The European Food Safety Authorit y (EFSA) proposes , not onl y the evaluation of materials that ar e incorporated into packa g ing polymer matrices, but also the evaluation of the resulting pack ages (EFS A, 2011a, b). Thus, I TE NE developed two nanocomposites based on poly (l actic acid) (PLA) and Cla y1, and PLA-Clay 2, both modi fied cla y s designed by them. With these materials migration tests were c arried out. The food stimul ant used was distilled Resumen / Summary 12 water, accepted b y the EU Regulation No. 10/2011 , due to the type of food products the se nanocomposites will be used for (hy drophilic food ). Once the mi gra ti on ex tracts of P LA-Clay 1 a nd P L A-Cla y2 were o btained, several parameters were evalu ated in order to determine the potential toxi c effect s induced after their exposure. Cell viabili ty of HepG2 and Caco-2 exposed to both extracts and the mut agenic potential by the Ames te st were evalu ated. The results shown an absence of c y totox icit y and mutage nicit y under the condit ions tested. Moreove r, considering that the main metals prese nt in clay s structure are Al , Ca, Fe, Mg and Si, the ir contents in the migration extracts were analy z ed b y Inductive l y Coupled Plasma Mass Spectrometry ( I CP -MS) and I ndu ctively Coupled Plasma Optical Emi ssion Spectroscop y (I CP -OES). N o sig nificant differences with respec t to the control group were found. The se results a re summarized in the following publication:  CYTOTOXICITY AND MUTAGENICITY S TUDIES ON MIGRATION EXTRACTS FROM NANOCOMPOSI TES W ITH POTENTIAL USE IN FOOD PACKAGING. (Maisanaba y col., 2014 Food and Chemical Toxicology 66, 366 -372). Once the in vitro tox icology studies were conducted, the cla y with the be st technological properties and tox icolog ical profile was selected for fu rther experiments, Cla y 1. In order to complete the in vitro result s and taking into account that in vivo data a re scarce, a repeated dose 90-day oral t oxicit y study in W istar rats exposed to Clay1 (40 m g/ kg/day ) with the diet and its migration extract (PLA-Cla y 1) as drinking water ( ad libitum ) w as performed. In this sense, after the exposure period, the animals wer e sacrificed and th eir organs (liver, kidne y, intestine, brain, heart, t esticles, lungs and sple en) and blood b y cardiac puncture were removed. Several parameters were evaluated, including: his topathological analysis, clinical biochemistry of blood serum, interleukin 6 ( I L-6) leackage, biomarkers involved in oxidative stress response in liver and kidne y , su ch as reduced/oxidized glutathione ratio (GSH /GSS G), lipid peroxidation (L P O), and activities of antioxidant enz y mes (superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and glutathione S -Transferase (GST). Moreover, gene ex pression and protein abundance of SOD and CAT in li ve r and kidne y were also evaluated. We c an hig hl ight an increase of CAT activity in kidney , as we ll as an alteration in g ene expression and protein abundance after exposure to Clay 1. All these biomarkers remained unaltere d after the exposure to P L A -Cla y 1 mi gration extract. Resumen / Summary 13 In addition, it was interesting to ev aluate the content of the t y pi cal met als presented in clay s composition in the spleen of exposed rats. Sig nificant differences were not observed compared to control rats. The results obtained in these experiments have led to the following publi catio ns:  EFFECTS OF THE SUBCH RONIC EXPOSURE TO AN ORGANOMODIFIED CLAY MINERAL FOR FOOD PACKAGING APPLICAT IONS ON WISTAR RATS. (Maisanaba y col., 2014; Applied Clay Scienc e 95, 37 -40).  IN V I VO E V ALUA TION O F ACTI V ITIES AND EX PR ES S ION OF ANTIOXIDANT ENZYMES IN WISTAR RATS EXPOSED FOR 90 DAY S TO A MODIFIED CLAY. (Maisanaba y col., 20 14; Journal of T oxicology and Environmental Health, Part A: Current Issues 77,456-466).  IN V I VO TOXICITY EV A LUATION OF THE MIGR ATION EXTRA C T OF AN ORGANOMODIFIED CLAY-POLY(LACTIC) ACI D NANOC OMPOSITE. (Maisanaba y col., 2014 ; Journal of Toxicology and Environmental H ealth, Part A: Current Issues 77,731-446).  EV ALUACIÓN DE LA S EGURIDAD DE UNA ARCILLA MODIFICADA Y S U EXTRACTO DE MIGRACIÓN EN BAZO DE RATAS WISTAR EXPU ESTAS DE FORMA SUBC RÓNICA. (Maisanaba y col., 2 013 Revista de T oxicología 30, 125 - 130). For the fulfillment of this thesis, the PhD stude nt performed an internship in I TE NE, under the direction of Drs. Susana Aucejo and Maria Jordá, where different objectives were addressed. During the internship, new sil anes-modified cla y s based in C Na + , Cla y 3, Clay 4A and Clay4B, were developed intended to food packaging . The characterization of these cla y s was performed b y infrared spectroscopy (FTIR), X -ray diffraction and thermogravimetric analysis (TGA). Preliminar y result s showed good incorporation of modifiers and improved technolog ical profile in comparison to the raw clay. Subsequently, a toxicological evaluation of the three s y nthesiz ed clays was performed. Biomarkers studied included basal c y tot oxicit y , ge notox icit y (by the comet assa y ) and oxidative stress induction (production of ROS a nd GSH content) in H epG2 and Caco -2 cell Resumen / Summary 14 lines. Furthermore, the possible mechanism of cell death of Ca co-2 ce ll s exposed to Cla y 4A and Clay4B was studi ed by flow c ytometry. In ad dition, the mutagenic potential of clays was evaluated b y the Ames test. I n general, the preliminary results led to an absence of tox ic effects b y Cla y 3; however, both Cla y4A and Clay4B show ed tox ic ity in al most all parameters tested. I n this sense, Clay3 showed the best technological and tox icological profile. Therefore, it was selec t ed for food shelf- life testing , giving an increase of approxim ately 24 hours compared with the contr ol. Moreover, the migration of metals (Al, Ca, F e, Mg and Fe) from nan ocomposites of polypropylene (PP) -Clay3 or PP -Clay 4A w as evaluated. In all cases differences from controls in some of the specific metals studied were obtained. The results of thes e experiments will be include d in different publications, pending to b e send:  DEVELOPMENT, CHARA CTERIZATION AND CY TOTOXICITY OF NOV EL SILANES MODIFIED CLAYS I NTENDED TO PACKAGING (provisional title ).  TOXICITY EVALUATION OF A NEW SILANE -MODIFIED CLAY AND ITS MIGRATION EXTRA CT FROM A NANOCOMPOSITE INTENDED TO FOOD PACKAGING (provisional title ).  TOXICOLOGICAL AS SESSME NT OF TWO SILANE -MODIFIED CLAYS IN HUMAN HEPATOMA CELLS AND SALMONELLA TYPHIMURIUM STRAINS (provisional title ).  CYTOTOXICITY, OXIDATIVE STRES S AND GENOT OXICITY ASS AYS OF SILANES-MODIFIED CLAYS IN THE HU MAN INTES TINAL CELL LINE CACO -2 (pr ovisional title ). Regarding to the ess ential oils and their main components, the y are also h aving a boom in the food industr y , b eing used in a new t y p e of packaging known as active packaging . In t his case, the te chnological improvements are du e to the antioxidant and antimicrobial natural properties presented by this kind of substances. However, it should be established a safe range of use to avoid the possible adverse effects due to their abuse. First, we carr ied out a contextualization of the current use of this type of packaging and the antimicrobial/ antioxidant properties th e y show ed, and also, a re view of the ir ma in cy totox ic effe cts in differe nt cell lines of human origin. That review has resulted in this publication: Resumen / Summary 15  NEW ADVANCES IN ACTI VE PACKA GING INCORPO RATED WITH ESSENTIAL OILS OR THEIR MAIN COMPONENTS FOR FOOD PRESERVATION. (Maisanaba y col., 2015. Food Reviews International (en revisión, under revision). Finally , the evaluation of the major components of the essential oils before their incorporation to th e pa ckaging is also mand atory, as established by the EFS A (EFSA, 2011b). In this regard, ore g ano essential oil is one of the most used for its outstan ding antimicrobial properties, bein g th ymol and carvacrol their two main components. In the li teratur e, the re a re severa l data about their genotoxic profiles (Azizan and Blevins, 1995; S tammati et al., 1999; Ipek et al., 2005; B u yukleyla and Recuzogullari, 2009; L lana -Ruiz-Cabello et a l, 2014). These r esults are inconclusive in some aspects, so a comp rehensive assessment of t hy mol and carvacrol, focussed in their ge notox ic profile, is necessar y . Therefor e, it was interesting to conduct the MN test and, for the first time, the mous e l y mphoma assay. The re sult s showed no significant biolo gical relevance fo r an y of the compounds under the conditions tested. These re sults are describ ed in the following publi cation:  IN V ITRO GENOTOXICIT Y TESTING OF CARV AC ROL AND THYMOL USING THE MICRONUCLEU S AND MOUSE LYMPHOMA ASSAY S. ( Ma isanaba y col., 2015; Mutation Research 784-785, 37-44). II. INTROD UCCIÓN / INTRODU CTION Introducción/Introduction 19 1. ORIGEN DE LAS ARCILLAS Y MINERALES DE ARCILLA La s a rcillas y minerales de arcilla (también conocidos como láminas de silic atos) pertenecen al grupo de filosilicatos (del griego “ phyllon ”: hoja , y del latín “ silic ”: piedra) (M eunier, 2005) . Estos materiales se ca r ac terizan por ser mu y pequeños (un tamaño máximo de micrómetros), pudiéndolos encontrar de forma de f orma natural, provenientes de rocas o cenizas volcánicas, bajo superficie (su elos y sedim entos), como es el caso de las arcillas, o bien, como resultado de la meteorización química (diagénesis y alteraciones hidroterm ales) de otros minerales de sil icato, en el caso de los m inerales de a rcilla (Meunier, 200 5; Bergaya y Lagaly, 20 06; C hoy y col, 2007; Floody y col, 2009). A p esar de p resentar características muy similares , ex isten varios aspectos qu e diferencian a las arc illas y minerales de arcilla qu e se e xponen en la Tabla 1. Arcilla Minerales de Arcilla Natural Natural o sintética Grano fino ( <2 -4 µm ) No hay criterio establec i do para el tamaño Filosilicatos como principales constituye ntes Pueden no incluirse filosilicatos en su estructura Plásticas (con excepc ión de arc il las con orige n pedroso) Plásticas Se endurece con el secado o cocción Se endurece c on el secado o cocción Tabla 1. Dife r encia s entre ar cillas y m inerales de arc i llas (tom ada de Bergay a y Lagaly, 2006). 2. CLASIFICACIÓN Y PRINCIPALES CARACTERÍST ICAS DE ARCILLAS Y MINERALES DE ARCILLA La s p ropiedades físicas y químicas de las arcillas y minerales de arcillas dependen d e su estructura y composición, pudiendo clasificar de tal forma un amplio grupo según las caracter ísti cas pre sentad as ( Fig. 1 ). La s arcillas son mate ria les conformados en láminas de tetraedros de silicato (SiO 4 ) y octaedros (cont eniendo Al, Mg y Fe). Estos m inerales arcillosos naturales se construy en basados en u nidades estructurales la minadas con un espesor individual de cada lámina de aproximadamente uno a unos po cos nanómetros y dimensiones laterales Introducción/Introduction 20 que van desde 30 nm a varios micrómetros, obteniendo una relación de longitud - espesor superior a 1000 nm (Z hu y Nju guna, 2014). De acuerdo con su composición química, características de la estructura y la carga net a, podemos clasificar siete grupos: (1) caoli nita-serpe ntina, (2) talco- pirofilita, (3) esmectita, (4) vermiculita, (5) mica, (6) clorita, y (7) arcillas inte restratificadas (Martin y col., 1991). El grupo m ás simple presenta una estructura con una relación 1:1, en el que una capa tetraé drica de sílice se fusiona por compartición de átomos de ox ígeno a un octaedro de aluminio (por ejemplo, caolinita). El s iguiente grupo presenta una estructura con re lación 2:1, conocido ge neralmente como el g rupo de los filosilica tos, el cual consta de un a lámina octaédrica central de alum inio ubicada entre dos láminas tetraédr icas de sílice. Las unidades laminadas del silicato, a menudo conocidas como estructura inte rlaminar o ga lería laminar, se encuentran posicionadas en paralelo y están unidas por fuerzas de Van der W aals y el ectrostáticas. La sustitución isomórfic a dent ro de las l áminas genera cargas n egativas que normalmen te se ven contrarrestadas por sodio o calcio presentes en el e spacio interlaminar. Estos cat iones intercambiables pueden ser r eemplazados por cationes or gá nicos e inorgánicos a través de re acciones de intercambio iónico. Dentro de la familia de los filosilicatos 2:1 se encuentran los silicatos más ampliamente conocidos en el mundo científico y usados por la industria, la s esmectitas (Bignon, 1990; Gilman, 1999; Williams y col., 2004; Goettler y col., 2007; Leszczy nska y col. , 2007a, 2007b; Drumm y y col. ,2010; Garrido-Ramírez y col., 2010; Gatica y Vida l, 2010; Kil iaris y Papa sp y rid es, 2010; Yamada y col., 2011). Figura 1. Cla sifi cac i ón de si licato s y princip al es subg rupos de arcillas ( tomada de OMS, 2005). Introducción/Introduction 27 4.3. ARCILLAS Y MODIFICADORES ORGÁN ICOS Como se h a expuesto en el apartado anterior, debi do al bajo grado de ex foliación que presentan las arcillas en la matriz polimérica por la naturaleza hidrofílica con la que cuentan y el carácter hidrófob ico presentado po r las cadenas del polí mero, el sector industrial lleva a cabo una seri e de modificaciones en la estru ctura de l as m is mas con el fin de mejorar este a specto. Los métodos llevados a cabo para conseguir una arcilla más o rganofílica son varios, destacando principalmente a) l a reac ción de intercambio iónico ( ion exchange reaction ) y b) la reacción de injerto ( grafting reaction ). a. Reacc ión de intercambio iónico La reacción de intercambi o iónico , un proceso de modificación qu ímica ampliamente utilizado para dar lugar a arc illas más organofílicas, se de fine como una reacción r eversible en la que los cationes se intercambian estequiométricamente entre una solución electrolítica y la fase sup erficial del material intercambiador (Bladel y La udelout, 1996; P isticelli y c ol., 2010), en nuestr o caso, la arc illa. Cabe considerar a las arcillas como polielectroli tos multivalentes ideales para este ti po de reacciones. El exceso de carga n egativa d e las capas d e silicatos y su capacidad de intercambio de iones se puede cuantificar mediante una específica propiedad conocida como la capacidad d e int ercambio catiónico (C I C) , expresada en mequiv./g (Alex andre y Dubois, 2000; Manias y col., 2001). En el caso de las esmectitas, g rupo de filo silicatos con mayor CEC (0.7-1.2 moles/kg) (Sherman, 2011), cuentan con una capa de int ermedia de cationes hidratados, pudiendo ser desplazados por otros grupos orgánicos mediante una solución acuosa dando lu gar a la modificación en su composición y por tanto obteniendo una arcilla modificada con características mejoradas de compatibilidad (Sherman, 2011). Más específicamente, la Mt sódica (C I C 110 mequiv./100g (Ra y y Okamoto, 2003)) , formadapor dos capas tetraédrica s constituidas por átomos de Si 4+ y O 2- , y, fusionadas con una capa octaédri ca con átomos Al 3+ y Mg 2+ unidos a grupos oxígeno e hidroxilo , es ampliamente uti lizada en este tipo de modificaciones. Debi do a una sustitución isomórfica, el Al 3+ puede ser sustituido por el Mg 2+ , creando un exceso de carga negativa, que se equilibra por cationes (p. ej. Na + , Ca 2+ ) coloca do en el espacio entre la capa intermedia plaquetas (Jordá-Bene y to y col., Introducción/Introduction 28 2014). Para la mejora de la miscibilidad e int eracción de las láminas de silicato entre esta ar cilla y el polí mero, uno debe convertir la superficie hidrófila del sil icato a una organófila. Generalmente, esto se lleva a cabo gracias a la reacción de interca mbio iónico mediante la incor poración de tensioactivos catiónicos como modificadores. Entre los modifica dores más utilizados pa ra obtener estas orga noarcillas encontramos los cationes cuater narios de alquilamonio, comúnm ente conocidos como sales de amonio cuaternario, siendo los más destacados en la industria del envasado alimentario ( Fig. 5 ), junto con los cationes alquilofosfonio. Estos cationes modificadores, una vez que son incorporados, reducen l a energía superficial de las láminas de arcilla y mejoran las caracter ísticas d e humectaci ón de la m atriz polimérica, d ando como resultado un ma y or espaciamiento de las mi smas. Además, estos modificadores pueden propo rcionar grupos funcional es qu e pueden r eaccionar con la matriz poli mérica, o, en algunos casos, inician la polimerización de monómeros para mejorar l a resistencia ent re la int erfase inor gánica y la matriz polimérica or gá ni ca (Blumstein, 1965; Krishnamoorti y col., 1996). Figura 5. Mod i ficadore s com er ciales de sa les de amonio cuaternario m ás usados en arcill as m odificadas. Introducción/Introduction 29 La mejora por la incorporación de arcillas modi ficada s con sales de amonio cuaternarias a polímeros bien conocidos está más que corroborada. Por ejemplo, Fukushima y col. (2012) describieron como dos tipos de nanocompuestos de ácido poliláctico (P L A ), polí mero orgá nico y biodegradable (Jamshidian y col., 2010), uno con un 5% de una Mt modi ficada or gá nicamente, Cloisit e®20A (C20A), y otro con sepiolita no modificada, presentaron una mejora tras la incorporación de las arcillas, siendo más destacadas en aquel b asado en la Mt modificada. Ambos tipos de nanocompuestos dieron lugar a propiedades térmicas y de viscosida d mejoradas Además, no se afectó la transpare ncia d el po límero tras la incorporación de las partículas. Sin embargo, la perme abilidad del pl ástic o únicamente tuvo una variación significa tiva en el caso d el nanoc ompuesto de PLA -C20A. b. Reac ción de inserción De bido a la degradación de la s sal es de amonio cuaternario a altas temperaturas a través de la rea cción de Hoffman, la inestabilidad térmica de la arcilla org ánica se convierte e n una notable limitación en el proc esamiento de nanocompuestos poli méricos de arcilla modificadas con estos tensioactivos ( Xie y col, 2001, 2002 ; Wang y col , 2012 ). Por esta r azón, la reacción de injerto de grupos hidrófobos en las superficies de la capa ha supuesto una alterna tiva alca nz able. Est a reacción de injerto, se ll eva a ca bo ge neralmente entre los agentes de acoplamiento de silano s y los grupos sil anol es reac tivos qu e s e encuentran en los bordes de las plaquetas de a rcilla y en los defectos estructurales situados en la superficie de la capa intermedia y externa ( Di Gianni y col., 2008; Herrera y col., 2010; Pist icelli y col., 2010 ). En resumen, la reacción de inj erto es una solución irreversible para obtener arcilla modificadas químicamente en las qu e las moléculas del modificador silano ( Fig. 6 ) están unidas a l as capas de la arcilla seleccionada ma y oritariamente, en este caso M t (Wypyc h, 2004). Varios autores h an investigado la modificación de arcillas con silanos incorporados a diferentes polí meros teniendo en cuenta los diferentes m étodos de modificación y la obtención de varios resultados, la mejora o l a disminución de las propiedades en comp aración con la arcilla sin modificar y los polí meros a granel ( Di Gianni y otros, 2008; Shen y col., 2007; Pisticelli y col., 2010; Silva y col., 2011). Introducción/Introduction 30 Figura 6. Silano s utilizados com o modificadores de ar ci llas. En la actualidad, en la industria alimentaria podemos encontrar arcillas organomodificadas con estos tipos de modificadores ya patentados y comercializados , y otras tantas, bajo desarro llo y evaluación. El fin último es dar lugar a una arcilla con el mejor perfil tecnológ ico y tox icológico, con el objetivo de obtener el nanocompuesto polimérico de arcilla con las mejores propiedades para incrementar la vida úti l del alimento y qu e sea se guro para el consumidor. L a s principales arcillas comercializadas y en desarrollo se muestran en la Tabla 2 . Nombre Composición química Modificador orgánico Concentrac ión del modificador Cloisite®Na + (CNa + , comercial) No modificada ( Mt natur al) No presenta Cloisite®30B(C30B, comercial) Mt modificada con sa l de amonio cuaternario (MT2EtOH) 90meq/100g c la y Cloisite®20A(C20A, comercial) Mt modificada con sa l de amonio cuaternario (2M2HT) 95meq/100g c la y Cloisite®93A(C93A, comercial) Mt modificada con sa l de amonio terciar io (M2HT) 90meq/100g c la y Cloisite®15A(C15A, comercial) Mt modificada con sal de amonio cuaternario (2M2HT) 125meq/100g c la y Cloisite®10A(C10A, comercial) Mt modificada con sa l de amonio cuaternario (2MBHT) 125meq/100g c la y Clay1(en desarrollo) Mt modificada con sa l de amonio cuaternario (HDTA) 6 veces del CEC de la Mt, CEC de Mt= 92.6 meq/100g Clay2(en desarrollo) Mt modificada con sa l de amonio cuaternario (HDTA+ACO) HDTA en 5.75 y ACO en 0.25 veces del CEC Introducción/Introduction 31 de Mt , CEC de Mt = 92.6meq/100g Clay3(en desarrollo) Mt modificada con silano (3 -Aminopropiltrietoxisi la no) 4 veces del CEC de la Mt Clay4A(en de sarrollo) Mt modificada con silano (Viniltrimetoxisilano) 4 veces del CEC de la Mt Clay4B(e n desarrollo) Mt modificada con silano (Viniltrimetoxisilano) 8 veces del CEC de la Mt Tabla 2. Prin ci pale s arcilla s c om er ciales y en desarrollo destinadas a la in dustria a limentaria . 5. EVALUACIÓN TOXICOLÓGICA DE ARCILLAS NO MODIF IC ADAS, ORGANOARCILLAS Y MATERIALES NANOCOMPUESTOS. Teniendo en cuenta que l os materiales n anocompuestos poliméricos de arcill a están teniendo una g ran potencialidad de aplicaciones en el sector del envasa do de alimentos como un innovador mat erial de embal aje con pr opiedades funcionales nuevas que dan lugar a la protección y mejor calidad de los ali mentos. Se ha ce nec esario evaluar la seguridad hacia los consumidores, y a que esta mos ante mate ri ales en contacto con alimentos que posteriormente serán ingeridos (Rhim y col., 2013). Aunque los efec tos tecnológicos son conocidos y están generalmente bien descritos desde hace años, los pot enciale s efectos toxicológicos y el impa cto de la s arcillas sin modificar o modificados y d e los nano compuestos poli méricos deriv ados sobre la s alud humana y el medio amb iente, están actualmente siendo investigados ex haustivament e con mayor interés. Hay que tener en cuenta que l a exposición de la población en general a bajas concentraciones de Mt, caolinita y otros mi nerales de arcilla en s u forma natural es ubicua (OMS, 2005). Respecto a la incorporación de las arcillas a los polímeros empleados en la industria alimentaria pued e r esultar como una exposición no intencionada a ellas para el consumidor, por lo que es necesario no sólo evaluar la arcilla microestructurada modificada o no, sino también e l nanocompuesto resultante una vez imbuida ésta , debido a la posible migración al producto alimenticio (EFSA , 2011a; Guillard y col, 2010; Song y col., 2011; EFSA, 2015 ). Introducción/Introduction 32 Es razonable asumir que esta mi grac ión se pr oduce a causa de las pequeñas dimensiones que presentan estos nanocomponentes. También, hay indi cios de que la migración podría est ar relacionada con el desgaste en el tiempo del film /plástico (Rejinders, 2006). En consecuencia, la principal preocupación sobre la aplicación de nanocompuestos poliméricos usados en envas ado en contacto con alimentos está relacionada con la exposición indirecta debido a la potencial migración del envase al alimento. Por ello, para los consumidores, la prime ra preo cupación es verificar la medida de la mi gración de las n anoarcillas del film al alimento, y , posteriormente, si esto ocurre, a qué efectos pueden dar lugar des de que las ingieren h asta el final del tracto gastrointestinal. Actualmente, en relación a las ar cillas, modi ficadas o no, podemos encontrar varios trabajos sobre la evaluación tox icológica de a rcillas y a comerciales utilizadas en envasado y los ensa yos tox icológicos que se han llevado a cabo con las mismas. L os resultados publicados por diversos autores p resentan resultados contradictorios en muchos casos (Sharma y col., 2010; Baek y col., 2012), h aciendo necesaria una evaluac ión tox icológica exhaustiva y consideración indi vidualizada de cada una. En el caso de extractos de mig ración obtenidos a partir de materiales nanocompuestos, los estudios son mucho más escasos (Avella y col., 2005; Simon y col., 2008). Debido al auge que está presentando el uso de arcillas y nano compuestos poliméricos de arcilla, s ería neces aria h acer una revisión bibl iográfica, c aso por c aso, con el fin de ir recopila ndo la ma y or información posi ble y poder deter mi nar el estado del arte de cada material con el fin de encaminar y completar una a d ecuada evaluación toxicológica y ser conscientes de la informac ión sobre seguridad que hay hasta el momento. La Autoridad Europea d e S eguridad Alim entaria (EFSA) es la piedra an gu lar de la Unión Europea (UE) centrada en la evaluación de riesg os en relación con la alimentación y la se g uridad de los piensos. En estrecha colaboración con las autoridades nacionales y en consu lta abierta con grupos de interés, la E FSA proporciona asesoramie nto científico independiente y comunicación cla ra sobr e los ries gos existentes y emerge nt es. La EFSA ha desarrollado un en foque p ráctico para evaluar los riesg os potenc iales derivados desde las aplicaciones de la nano cienc ia y la nanotecnología en la cadena alimentaria human a y animal (EFSA, 2011). En este Introducción/Introduction 33 aspecto, para llevar a cabo una rigurosa evaluación de las arcillas, como nanoarcillas, se tienen en cuenta los pasos propuestos po r esta autoridad, con el fin de s er comercializada s en un futuro cercano en Europa. En su informe, la EFSA proporciona ori entación sobre: (I) los requisi tos de caracter ización físico-quím ica de n anomateriales artificiales utiliz ados por ejemplo como aditivos aliment arios, en zimas, aromat izantes, materiales en contacto con alimentos, alimentos nuevos, aditivos p ara piensos y pl aguicidas y ; (II) las pruebas para identificar y caracterizar los peligros de rivados de las propiedades d e los materiales en evaluac ión que, en general, inclu y e , info rmación sobe genotoxicidad in vitr o , absorc ión, distribución, metabolismo y excreción, ade más de ensa y os de tox icidad oral de dosis repetida durante 90 dí as en roedo res. Esta guía indica todas las incerti dumbres que deben ser consideradas para ll evar a cabo una correcta y completa evaluac ión de riesgo (EFSA, 2011a). Además, la EFSA ha publicado recientemente , “ Acontec imi entos recientes en la evaluación del riesgo de sustancias químicas en alimentos y su potencial impacto en la evaluación de la seguridad de sustancias utilizadas en los materiales en contacto con alimentos ”, un bo ceto-guía con estructura sim ilar a la anteriormente mencionada , destacando que una evaluación genotóxica es requerida en cualquier caso ( EFSA, 2015). Éste último documento completaría en parte a la información proporc ionada por el primero. Muchos de estos aspectos tox icológicos de las nanoarcillas han sido abor dado por numerosos autores. A continuación se expondrán los meca nism os de t oxicidad que hasta el momento se han descrito sobre los mat eriales que nos compete n, esbozando resultados disponibles más relevantes. 5.1. ENSAYOS TOXICOLÓG ICOS IN VITRO Y MECANISMOS DE TOXICIDAD Los métodos in vitro pr oporcionan un medio efectivo y rápido para la selección ( screening ) y clasificac ión de compuestos, además de ser ética y mor almente más correctos que los ensay os in vivo . Entre otras ven tajas destac an que el uso de animales es conside rableme nte menor, e incluso nulo en muchas ocasiones, se ut iliz a material muy homogéneo obtenido con técnicas estandarizadas, posibilitan el uso d e material de Introducción/Introduction 34 orige n humano, lo que se puede simplificar por e xtrapolación, son más fác il mente objetivables y cuantificables que los ensa y os in vivo y los resultados presentan ma y or reproduc ibilidad. A todo ello se le une un menor coste, ma yor rapidez e inst alaciones menos complejas (Eisenbrand y c ol., 2002). La s evaluaciones toxicológicas in vitro evaluac io nes tox icológicas son úti les para la obtención de infor maci ón acerca del meca nismo de tox icidad de los materia les de estudio. L as células resp onden rápidamente al estrés que genera el tóx ico mediante la alterac ión, por ejemplo, de diferentes biomarcadores basales, tales como la generación de especies reactivas de ox ígeno (ERO) y conten ido de glutatión (GSH), o alteraciones en varios orgánulos, como las mitocondrias y lisosomas. Teniendo esto en cuenta, una cuestión importante de resolver es cómo los materiales estudiados, en este caso, organoarcillas y nanocompuesto s que las contengan, son capaces de altera r estos parámetro s y cómo estas altera ciones pueden afect ar a la tasas metabólic as, crecimiento celular y la transcr ipción génica (Eisenbrand et al., 2002). La inte racción, influencia y potencial de tox icidad de lo s materiales con las proteínas y las células son un enfoque esencial para evaluar y entender la compatibilidad de los mater iales frente a la tox icidad (J ones y Grainger, 2009). L as reacciones de células de interés como material de absorción ce lul ar, las pe rturbaciones integrida d de la m embrana, o la alteración en la viabilidad de orgánulos vitales. Por lo tanto, los puntos finales tales como la generación de ERO y otros parámetros relacionados con la respuesta al estrés celular (GSH, la a ctividad de l a supe róx ido dismutasa (SOD), la peroxidación lipídica (L PO ), etc.), daño en el ADN, la respuesta de la inflamación y l a necr osis y apoptosis proporc ionan una valiosa in formación para la comprensión de la respuesta tóxica. Entre los indicadores de tox icidad que se pueden determinar con los métodos in vitro , destacan que a c ontinuación se exponen. a) Citotoxicidad basal Los cultivos de células juegan un papel esencial en la evaluación toxicológica de los minerales de arcilla, y a que proporcionan los sis temas de alto rendimiento para la detecc ión rápida y rentable de los peli gros. Sele ccionadas las lí neas celulares de trabajo, teniendo un gran int erés las líneas de órganos di ana del aparato di gestivo, los ensayos de citotox icidad son los llevados a cabo en primera instancia par a valorar los efectos Introducción/Introduction 35 adversos o int erfe r encias con estructuras y/o propiedades esencia les para la supervivencia, proliferación y /o función celular una ve z que se les ha ex puesto el compuesto objeto de estudio. Diversos grupos han evaluado mediante diferentes biomarcadore s de citotox icidad la alteración funcional p roducida por exposición de diferentes líne as celulares a dich as arcillas orga nomodi ficadas. Son difere ntes los métodos que podemos encontrar para evalua r el d escenso d e viabilidad tras exposición de las arcillas, así com o extractos de migración de materiales nanocompuestos. Entr e ell os destacan, la reducción de Bromur o de 3 -(4,5-dimetilt iazol - 2-ilo)-2,5-difeniltetrazol (MTT) y s al de t etrazolio MTS (MTS) (Baltrop y col., 1991), comprobando la funcion alidad mitocondrial tras la ex posición al compuesto; liberación de la enzima L actato Deshidrogenasa (LDH) p ara eva lua r el daño en la membrana celular; el ensa y o Azul Tr y p an (Binderup y col. , 2002) pa ra evaluar la proliferación celular; el ensa yo de Ro jo Neutro (RN) (Borenfreud y Puerner., 1984) q ue constata el daño lisosomal y la valoración del contenido de proteinas (Bradford, 1976), con el fin de evaluar la prolifera ción celular. La Organización Mundia l de la Salud (OMS) revisó los efectos en sistemas in vitro de bentonita, caolín y ot ros mi nerales de arcilla (OMS, 2005). Sin embargo, las obras mencionadas fechan del período 1969-1996, no resultando ser una información actual y extrapolable a la realidad, haciéndose de tal forma necesa ria una profunda revisión de los datos disponibles hasta día de hoy. Estudios más actuales han demostrado difer entes comportamientos de líneas celulare s tras la exposición de la Mt o Cloi site ®Na + (CNa + ). Por ejemplo, Sharma y col., (2010) observaron u n efecto no citotóxico en la línea de adenocarcino ma de colon Caco-2 ex puesta a la arcill a en las condi ciones en say a das, sin embargo, Li y col. (2010) y Baek y col., (2012), o btuvieron un desce nso de viabilidad tras la exp osición de la misma en la línea celular de ovario de hámster chino (CHO) y en células int estinales humanas, respectivamente. Respecto a otras arcillas modi ficadas disponibles comercialmente, los resultados obtenidos también fueron muy divers os, presentándose de forma general efectos tóxicos tras la exposición a las arc illas. En el c aso de C loisite®30B (C30B) y Cloisite®93A Introducción/Introduction 36 (C93A)) se observaron efectos citotóxicos tras su exposición en Caco-2 y la línea de hepatoma humano HepG2 (Sharma y col., 2010; L o rdan y col., 2011). Por otro lado, Liu y col. (2011) también observaron un des censo de la viabili dad celular y un aumento en la liberación d e la ctato deshidroge nasa (LDH) en fibroblastos embrionarios de ratón (NIH 3T3) y células de riñón embriona rio 293 (293 HEK ) tras la exposición a una Mt modificada c on oli go (estire no- co - acrilonitrilo). En definitiva, cada a rcilla, pued e presentar un perfil toxicológico diferente teniendo en cuenta las líneas celulares expuestas, ti empos ensayados y concen traciones de exposición, obteniendo de tal forma una amplia di versidad en los resultados, haciéndose necesaria una evaluación caso por caso. Respecto los ensa y os de citotocidad llevados a cabo con extractos de migración d e materiales n anocompuestos poliméricos de arcilla so n mu y es casos hast a el momento, siendo necesaria una exhaustiva investigación de los mismos antes de la comercializac ión del envase. Z ia y col. (2011), obtuvieron una mejor adhesión de células en envases con arcillas sin modi ficar que con arcillas modifi cadas, viendo efec tos adversos ante la presencia de modificadores . b) Estré s oxidativo La expresión / generación de respuestas de estr és y las alter aciones bioquímicas posteriores puede se r potenciale s ma rcadores para la tox icidad inducida por el compuesto (Eisenbra nd et al., 2002). Existe una considerable evid encia q ue indica que muchas de las respuestas de estrés se producen antes de qu e cualquier citotox icidad medible; por lo tanto, ta mbién se recomienda la vigilanc ia de las vías de estrés a nivel subtóxicas. La producción de formas reactivas de oxígeno es tóxica para los organismos y un exceso de las mismas da lugar a lo que se conoce como estrés oxidativo ( Fig. 7 ). Introducción/Introduction 43 Respecto a la absorción y acumulación en el organismo de este tipo de compuestos poco se sabe, siendo muy limitados los estudios encontrados en bibliogra fía, requiriéndose una prof unda revisión debido a la a ntigüeda d d e muchos de los artículos disponibles. Destacar algunos resultados publicados que constatan la acumulación progresiva e n or ina y e n de terminados órganos (r iñón>hí gado>cora z ón>cerebro) de restos de arcillas destinadas a diferentes usos en el ámbito de la medicina (Mascolo y col., 1999; 2004). Sin e mbargo, en el informe m encionado de la EF SA, ni bentonita ni sepiolita, comparadas con otras arcillas, presentan un grado de abso rción medible (EFSA, 2013). En cuanto a la evaluación tox icológica in vivo de materiales n anocompuestos de arcillas sólo hemos encontrado en bibl iografía un único trabajo, donde se ve una mayor compatibilidad in vivo de un polímero refor z ado con Mt comparado con el p olí mero puro (Hsu y col., 2012). 5.3.ENSAYOS TOXICOLÓGICOS A NIVEL MOLECULAR La evaluación de efectos a nivel mole cular nos da una info rmación a nivel gé nico y proteico de los efectos desencadenados tras la ex posición a un x enobiótico. En este sentido los datos obtenidos completarían el perfil tox icológ ico de una sustancia, y a que los ens a y os llevados a cabo para determinar estos parámetros pueden realizarse tanto in vitro como in vivo . Los ensayos más destac ados a nivel mole cular son la Reacc ión en Cadena Po limerasa Cuantitativa a Tiempo Rea l (qPCR ), que eva lú a la expresión génica, y, el W estern Blot, destinado a la evaluación de l a abundancia proteica. La invención de la reacción en caden a de la polimerasa (PCR) por K. Mullis y sus colaboradore s en 1985 ha revolucionado la biología y la medicina molecular (Saiki y col., 1985). La reacción en cadena de la polimerasa es una técnica in vitro utilizada para amplificar enzimáticamente una región determinada de ADN situada entre dos regiones de ADN cuya s ecuencia se conoce . Mientras que antes solo podían obtenerse cantidades mínimas d e un gen específico, ahora incluso un único ejemplar de un gen puede amplificarse con la PCR hasta un millón de ejemplares en poco tiempo. La s técnicas de P CR se han hecho indispensables para muchos procedimientos comunes, como la clonac ión de fragmentos específicos de ADN, la detección e identificación de ge nes pa ra di agnóstico y medicina le gal, y en la i nvestigac ión de mod elos de expresión Introducción/Introduction 44 de los genes, siendo este último el aspecto que más nos interesa. Más recientemente, la PCR ha permitido la investigación de nuevo s campos, como el control de la autenticidad de los alimentos, la presenc ia de ADN modificado genéticamente y la contaminación microbiológica (Somma y Querci, 2007) . En el caso de la qPCR, se mide la cantidad de AD N después de cada ciclo por el uso de marcadores fluorescentes que s e incorporan en la r eacción final o bien en la mezcla del gen seleccionado (sonda+gen). El aumento de la señal fluorescente es directamente proporcional al número de moléculas del gen (a mplicones) generados en la fase exponencial de la de reacción. Se usan indicadores fluorescentes que se incluyen en el ADN de doble cadena a colora ntes de unión, moléculas de colorante unidas a los cebadores d e PCR o sondas que son incorporadas en el producto durante la amplificación. Se mide el cambio en la fluorescencia durante el transcurso de la reacción por un instrume nto que combina el ciclo térmico con capacidad d e escaneado. El inst rumento de qPCR g enera una trama de amplificación que represe nt a la acumulación de p roducto durante la duración de t oda la reacción de P CR, finalmente se compara con la expresión de un estándar conocido norm alizándose de tal forma la cantidad expresada del gen seleccionado. L os d atos obtenidos mostrarán una baja o sobreexpresión con respecto al grupo control, determinando el tipo de afectación producido por la sustanc ia objeto de estudio ( I nvit rogen, Life Technologies). Por otro lado, el W estern blot se utiliza a menudo en la investigación para separar e identificar las proteín as. Esta técnica s e centra en la separación de prot eínas en base al peso mol ecular de cada una a través de electroforesis en gel. Estos re sultados son transferidos a un a me mbrana donde aparece una banda pa ra cada proteína. La membrana se incuba a continuación con anticuerpos específicos para l a proteína de interés, uniéndose a ella y d ando lugar a la banda de señal . El espesor de la banda corre sponde a la cantidad de proteína presente; por lo tanto , haciendo un estándar pued e indicar la cantidad de proteína pre sent e (Mahmood y Y ang, 2012). En literatura no existen hasta el mom ento estudi os que evalúen la influe ncia de las arcillas destinadas a la i ndustria alimenta ria en la modulación de la ex presión génica y abundancia proteica tras su ex posición. Introducción/Introduction 45 6. COMERCIALIZAC IÓN Y ASPECTOS REGULATO RIOS Como hemos estado viendo a lo lar go de esta introducción, no sólo es importante la optimización de proceso s y la creación de p roductos nanocompu estos poli méricos de arcilla destinados al me rcado, sino que también ha y otr as dos cuestiones que hasta el momento ha n re cibido poca atención: la mi gración y la toxicidad de estos nuevos materiales. De esta forma, la evaluac ión de organoarcillas debe centrarse no solo en la arcilla modificada incorporada y a al polímero, sino también, en la propia arcilla antes y después de la modificación e incorporación. Hasta la fecha, no existe una evidencia científica rotunda que sugiera mot ivo de preocupación por el uso de arcillas modificadas; sin embargo, es ló gico q ue los nuevos alimentos y materiales de envasado d eben ser minuciosamente evaluado s con el fin de cumplir con los re quisitos de la EFSA y otros organismos regula dores, siendo probable que la investigación so bre propiedades de mi gración y la tox icidad de polímeros nanocompuestos usados en la industria alimentaria como materiales en contacto con alimentos se amplíe en un futuro muy cercano (Pl ackett y Siró, 2012). Si bien es c ie rto, que hasta el momento no se han comercializado en Europa, productos nanocompuestos de arcilla destinados al envasado de alimentos , la EFSA, si propone diferentes guías, y a mencionadas en el apartado 5, para la evaluaci ón de nuevos materiales destinados al contacto de alimentos ( EFSA, 2011a, 2011b, 2015) .Para ello, sería necesaria realizar una exhaustiva evaluación en la actualidad, con el fin de que en un futuro temprano se puedan comercializar es tos envases mejorados, y sobre todo , seguros. Además, es mu y importante destacar el R eg lamen to UE Nº 10/2011 de la C omisión de 14 de ene ro de 2011 sobre materiales y objetos plásticos destinados a entrar en contacto con alimentos y la Directiva del Consejo del 18 de octubre de 1982, por la que se estable cen las normas básicas n ecesarias para evalu ar la mi gración de los constituye ntes de m ateriales y objetos plásticos destinados a entrar en contacto con productos alimenticios. En ambos se recoge n los aspectos r elacionados con los materiales pl ásticos y objetos destinados a entrar en cont acto con los alimentos (en nuestro caso, mat eriales nanocompuestos poliméricos de arcilla) y los productos alimenticios, así como los simuladores utilizados en los ensayos de migración, Introducción/Introduction 46 estableciendo que la evaluación del riesgo deb e cubrir la mi g ración pot encial en l as peores con diciones previsibl es de uso y toxicidad. Por otra parte, el R eglamento UE Nº 10/2011 de la Comisión también establece un límite de migración global de 10 mg / dm 2 , basándose en que los materiales plásticos y artículos derivados no d eberán ceder sus componentes a los alimentos simuladores en cantidades supe riores a 10 mg de los componentes totales liberados por dm 2 de superficie en contacto con el alimento. Como curiosidad, comentar que en otros países ajenos a la UE, como es el caso de EE . UU ., podemos encontrar mate riales y a c omercializados y apro bados por la Admistración de Drogas y Alimentos (U.S. Food and D rug Administration, FDA), Autoridad Americana de Alimentación, homóloga a la EFSA en Europa. P or ejemplo, Bayer Polymers (Pittsbur gh, PA, E E.UU) ha int roducido Durethan®, una poli amida que contiene silicato en capa s como nanorrelleno para proveer mejores propi edades contra el gas y la humedad al p olímero, y así proporcionar una mayor vida útil a los productos alimenticios (Anon, 2009). Na nocor (Arlington Heig hts, IL, EE.UU.) anuncia la oferta de nanocristales qu e pu eden ser usados para mejorar l as propiedades d e barrera de plásticos de botellas de cerveza y mejorar así la vida útil de la bebida. Además, e l ejército de EE . UU . ha llevado a cabo una investigación acerca de la producción de bolsas de comidas preparadas li stas para con sumi r a b ase d e polietil eno de b aja densidad con un refuerzo de un 7,5% de lámina de silicatos, m ás específicamente Mt, como nanore lleno. Por la incor poración de estás láminas de silicato se han conseguido mejoras térmicas, m ecá ni cas y de b arrera mu y signi ficativas comparadas c on el material de partida (National Nanotechnology Initiative, 2 009). Por otro lado, Honeywell ofrece seis resinas con diferentes grados d e rellenos basados en n anoarcillas-ny lon , bajo el nombre comercial Aegis®. Estos productos han sido utilizados por Anchor B rewing Company (San Francisco) para botellas de cervezas y films especiale s. Un ejemplo de estos materiales es Nano cor Nano- PA -6,conocido por ser el primer plásti co comercial nanocompuesto usado co mo empaquetado flexibl e, así como re vestimiento para cartón con fin de aumentar la barr era contra la humedad y el oxígeno, por ejemplo pa ra tetrabricks de l eche y zumos. Además se ha visto que proporc iona un a me jor rigidez y resistencia al calor. Por otro lado, otros nanocompuestos de resinas de Nanocor se han utilizado para botellas multil aminadas de poli etileno (PET) y en botellas que alberguen alimentos sensibles al dióxido de carbono y al oxíg eno. En diciembre de 2009, la FDA Introducción/Introduction 47 aprobó la comercialización de el producto Nanocor-M t, tratado con sale s de amonio cuaternario, destinado a incrementar las propiedades barrera de botellas de PET (Plackett y S iró, 2012). Siempre que sea posible , teniendo en cuenta co mo fin último la seguridad de los consumidores, la produ cción de polímeros nanocompuestos en ár eas e specializadas podría tener v entajas si gnificativas en términos d e manejo y se guridad, especialmente teniendo en cuenta los mater iales para envases de alimentos y bebidas. 7. OTROS USOS Y VÍAS DE EXPOSICIÓN A lo largo de la hist oria las arcillas h an sido utiliz adas por el se r humano con fines terapéutic os. Los minerales de arcilla pueden ser beneficiosos para la salud humana, sirviendo como principios activos o excipientes en forma de preparados f armacéuticos , en los balnearios y e n l a medicina terapéutica de belleza (Carretero y col., 2006). El uso de minerales d e arcilla en las formulaciones farmacéuticas h a sido descrito por muchos autores, des tacando el uso de caolinita, talco, paligorskita, y esme ctitas, como principios activos o excipientes de las mismas (Viseras y López-Galindo, 1999; Lópe z-Galindo y Viser as, 2000; C arr etero, 2002 ). Las propiedades fund amentales p or las que los minerales de arcilla se utilizan en la industria farmacéutica son las comentadas a lo largo de sec ciones anteriores: el alta área específica y capacidad de absorción, contar con características reológicas favorables, la ine rcia quí mica, baja o nula toxicidad para el pa ciente (aspecto que habría que corroborar), y bajo precio (Carre tero y col., 2006). La s arcillas pued en ser administradas a lo s pacientes como pr otectores ga strointestinales, antiácidos y/o antidiarreicos, bi en por vía or al en fo rma de píldoras, polvos, suspensiones, y emulsiones, o bien, por vía tópica usándose como protectores dermatológ icos o por razones de estéticas. Además, la caolinita, el talco, la paligorskita, y l as esmectitas también se utilizan como excipientes en cosmética y preparaciones farmacéuticas, funci onando como: lubricantes para facilit ar la f abricación de pastillas; agentes pa ra a y ud ar a la desintegrac ión a través de su capacidad de hincharse en presencia de agua o por medio Introducción/Introduction 48 de la dispersión de fibras , promoviendo la libe ración de l a droga cuando ll eg a al estómag o; y, como emulsionantes, geles po lares y agentes esp esantes por sus caracter ísticas coloidales , evitando la segregación de los componentes de la formulación farmacéutica y la formación de un sedimento que precipite y no se distribuya (Carretero y col., 2006). Por otro lado, Carretero y col. (2006) también describieron que l as arcillas puede n utilizarse también en spas y tra t amientos de belleza, como es el caso de las caolinitas y esmectitas. La s principales propieda des de los mine rales de arcilla que determinan su utilidad en el spa y medi cina estética, son: a) la suavidad y el pequeño tamaño de la partícula , ideal para la aplicación del lodo o la m ascarilla facial o corporal; b ) las propiedades reológ icas adecuadas pa ra la formación de una viscosa y consistente pa sta, y buenas propiedades plásticas para una fácil aplic ación y adhesión a la pie l durante el tratamiento; c) la similitud en el pH al de la piel con el fin de evitar la irr itación u otra al teración dermatoló gica ; d) gran capacidad de absorción (l as arcillas pueden eliminar el exceso de gra sa y las s ustancias tóxicas de la piel, siendo muy eficaces contra las enfermedade s dermatológicas tales como for únculos, acné, úlc eras, absce sos y seborrea); e) alta capacidad de intercambio iónico, permitiendo un intercambio de nutrientes (Ca + ó Na + ) mientras la arc il la está en contacto con la piel ; f) alta capacidad de retención de calor. C omo el calor es t ambién un a ge nte ter apéutico, l as arcilla s se aplican en caliente para tratar inflamaciones reumáticas, tr aumatismos deportivos crónico s, y problema s d ermatológicos. A part e de las dos vías d e exposi ción ya nombrad as, la oral y la dérmica, la vía de entrada d e minera les de arcillas al organismo más común es la vía inhalatoria (Zhao y Castranova, 2011 ; Silvestre y col., 2011 ). No obstante, en la actualidad existe esca sa información sobre la exposición ocupacional a los minerales de arc illa en las minas, plantas de pro cesamiento, y l as industrias. Sin embargo, se s abe n que el alto s niveles de minerales d e arcilla o la exposición inadecuada a estos en el medio ambien te y los seres humanos pueden dar lugar a efectos indeseables ( Zhu y Njuguna, 2014). Los polvos minerales causan daños por inhalación, y rara vez por inge sti ón o penetración en la piel. En los pulm ones, los mi nerales pueden producir diver sas patologías como c áncer de pulmón o mesotelial y neumoconiosis. Las patologías Introducción/Introduction 49 provocada s po r mi nerales puede ser determinada mediante estudios epid emiológicos, basados en la evaluación de la relac ión que ha y entre la ex posición humana a una sustancia peligrosa y los efe ctos potenc iales para la salud que se puede n dar tras esa exposición; además puede evaluarse mediante estudios in vivo e in vitro , estudiando los efec tos de la exposición a polvos minerales en mo delos animales o bien determinando la actividad biol ógica de un mineral en líneas celulares específicas, respectivamente (Guthrie, 1992). Aunque los datos son antiguos, hasta el momento, el efecto nocivo causado por inhalación de la caolinita, está principalmente relacionado con la presencia de diversos minerales en las rocas, entre los que se encuentra el cuarzo. Trabajadores expuestos al polvo de la caolinita han desarrolla do en muchos casos neumoconiosis ; sin embargo no hay un gran número de d atos relac ionados con el cáncer de pulm ón (Ross y col., 1993). Los estudios epide mioló gicos lleva dos a cabo con trabajadores expuestos a sepiolita no mostraron datos que supusiesen riesg o d e pad ecer enfermedad pulmonar , pudiendo concluir que la exposición a este tipo de minerales no supone ningún riesgo (Mc Connochie y col., 1993; Ross y col., 1993; S antaren y Alvarez, 1994). Respecto al grupo de las esmectitas, en biblio grafía científica h a y pocos estudios epidemiológicos disponi bles de enfermedades respiratorias causadas por la exposición a ellas. Al gunos estudios sugieren que determinadas muestras pu eden provoca r una respuesta fibro génica d ependiente de l a dosis ante altos niveles de ex posición. Generalmente, sin embarg o, ha y una exposición concomitante a otros minerales (por ejemplo, sílice y anfíboles), complicándose la int erpr etación de los datos obtenidos. Así , los depósitos de bentonita ge neralmente contienen otros minerales, incluyendo cuarzo de grano mu y fino y sílice. En el caso de W y oming (EE.UU.) el contenido de bentonitas en sílice y cuarzo oscila entre un 0 a 24% (Ross y col., 1993). En conclusión, para tod o tipo de aplicaciones sería necesaria una evaluación caso por caso, teniendo en cuenta tipo de arcilla, t iempo y vía de ex posición, y dosis empleadas como fac tores más im portantes para una cor recta evaluación de riesgo. Introducción/Introduction 50 8. ACEITES ESENCIALES, OTRA ALTERNATIVA P ARA EL ENVASADO ALIMENTARIO La industria alimentaria, como hemos visto en el caso de materiales nanocompuestos, con idea de suplir carencias presentes en la conservación de alimentos, han hecho n ecesaria la aparición de un nuevo s tipos de envas es que permitan cubrir tanto las n ecesidades de los consumidores , enfocadas a alimentos más sanos y naturales, como las derivadas de la globalización de la alimentación, que necesita alimentos que permanezcan con sus cualidades inalteradas du rante períodos de tiempo más prolongados. Los materiales nanocompuestos polim éricos de arcilla son una alternativa real en diversas zonas del mundo, que pe rsigue el aumento de la vida útil del alimento centrándose en las propiedades tecnológicas del envasado en sí, dando lugar a las mejoras descritas, pero no ejerce ning ún efecto dir ecto sobre el propio alimento. Sin embargo, no es la única opción qu e se baraja dentro del área del envasado, valorándose y d esarrollándose una perspectiva e cológ ica y sostenible, compartida por las empresas de la ind ustria alimentaria, el e nvasado activo (Tharanathan, 2003 ) . Además, la p resión ej ercida por parte de los consumidores, siendo cada vez más conscientes de los problemas derivados del uso de aditivos sintéticos en los alimentos, unida a la necesidad de ge stionar los subproductos de las industrias y buscar fuentes renovables p ara la c reación de polímeros, ha ce que empiecen a desarrollarse envases con compuestos natura le s (Llana-Ruíz Cabello, 2014). El envasado activo permite que los alimentos puedan estar en una atmósfera natural, donde constituy entes del envase, generalmente aceites esenciales, interaccionan durante el tiempo que el alimento está envasado, aprovechando estas propiedades antibacter ianas y antioxidantes que pr esentan esto s ex tractos naturales y au mentando así la durabilidad d e los alimentos . Este nuevo concepto de envase en el que se fuerza la interacc ión del mismo con el alimento plantea una serie de retos, e n relac ión a la evaluac ión de la seguridad, en comparación con el envasado tradicional que buscaba materiales ine rtes que ac tuasen como mera barrera frente al ambiente externo ( Danielli y col., 2008). Introducción/Introduction 51 En la actualidad, este tip o de enva sado está siendo utiliz ado en determinados países como J apón y EE.UU.; sin embargo, la legislación europea, más restrictiva, ha retrasado su incorporación al mercado comunitario por demanda r estudios que aseguren la utilidad e inocuidad de estos nuevos material es. La nueva normativa comunitaria (EFSA, 2009b, 2011b; Reglamento UE 119/20 11 ) intenta agilizar y sistematiz ar los procedimientos de presentación de solicitudes de autorización de sustan cias activas, la evaluac ión de los ri esgos inherentes a estos materiales y las norma s relativas al etiquetado. Sin embargo, estos nuevos materiales son mí nimamente uti lizados por la industria alimentaria, pudiendo ser debido a factores socio ‐ económicos, i nquietudes y elecc iones de los consumidores y , cue sti ones no resueltas e inseguridades sobre la implementación (Chaves Sánchez, 2011 ). Entre los constituyentes principales de estos nuevos envasados se encu entran los y a mencionados ac eites ese nciales, cu yo uso , con el fin de beneficiarse de sus propiedades antibacter ianas, antiox idantes y /o aromáticas una vez que se ponen en co ntacto con los alimentos, se ha demostrado a lo largo d e los años en muchas partes del mundo (Dainielli y col., 2008 ). Estos conocimientos previos sobre los aceites es enciale s h an hecho posible que aumente el interés del uso de los mi smos, respondiendo a las demandas d e la pobl ación y aumentando la vi da úti l de los alimento s, cubriendo debilidades presentes en la industria alimentaria. L os aceites esenciales y extractos de diferentes gé neros de especies vegetales se han convertido, así, en un recurso a estudiar en el desarrollo de envases activos. Muchos de estos ace it es y sus componentes principales están recogidos en la Decisión de la C omisión 2002/113/EC como aromatizantes permitidos en la UE , por ejemplo el ti mol, linalol, carvacrol, limoneno o eugenol, para los que no se considera que exista riesgo para la salud del consumidor ( H y ld gaard y col., 2012). En EE.UU., existen aceites esenciale s (orégano, ti mo, mostaza o albahaca) que y a están clasific ados como Gene rall y Reco gnized as Safe (GRAS) por la F DA (Zhou y col., 2006; Lópe z y col., 2007 ). A parte de sus aplicaciones conocidas como aromatizantes se han desarrolla do aplic aciones como conservantes en alimentación por las propiedades antioxidantes y antibacteriana s qu e presentan ( Dusan y col., 2006). Existen algunos trabajos en bibliografía que combinan la presencia de arcillas y aceites esenciale s en matrices poliméricas, destacando la mejora de la s propieda des barrera ( causado por las arcillas) y el aumento de las propiedades anti microbianas (debido a los ace ites Introducción/Introduction 52 esenciale s de mejor ana o clavo), dando lugar a un producto final con un perfil mu y mejorado (Alboofetileh y col., 2014). Teniendo en cuenta que l a se gur idad alimentaria y la calidad son las dos principales preocupaciones p ara la i ndustria ali mentaria, si el uso de estos a ceites cond uce a una mejora por su potencial anti microbiano en el deterioro producido por microor ga nism os de los productos alimenticios y , consecuente mente, una disminución de diversas enfermedade s en los seres humanos, estamos ante una opción mu y atractiva. Además, la oxidación como proceso de de gra d ación tam bién juega un papel e n numerosos productos alimenticios, siendo el potencial a ntioxidante de estas sustancias bien reconoc ido, pr esentándose así un papel crucial e n este aspecto. Ortega-Ramírez y col. (2014) declararon que las propiedades anti microbianas y antioxidantes de los compuestos bioactivos s e deben principalmente a sus propiedades redox, su capacidad quelante de metales, y la c apacidad d e ex tinguir a especies reactivas de oxígeno singlete. Por esta razón, l os ace ites esenciales o sus componentes individuales son una buena opción para ser utili zados en la industria ali mentaria para la mejora de la vida útil de los productos alimenticios. Sin embargo, ha y a lguna s limitaciones p ara la aplicación de estas sustancias, ya que la variabilidad química en su composición podría desempeñar un pape l im portante e n su actividad ( Prakash y c ol., 2015) . Solórzano-Santos y Miranda-Novales (2012) pu blicaron que los efe ctos deseables (antimicrobiano/antioxidante) de los aceites esenciales o sus componen tes añadidos directamente a los alimentos , se observaban a d eterminadas concentraciones , ya qu e a concentraciones superiores se producían cambios indeseables en el sabor, el olor, etc. Alcanzar concentraciones efectivas sin añadir g r andes cantidades d e estas sustancias ha llevado a la industria alimentaria a desarrollar diversos métodos de envasado activo en los que el aceite no es añ adido como un ingrediente di recto del alimento, sino como un componente del envase, produciéndose una liberación controlada de l os mismos y ejerciéndose los efectos beneficiosos de manera gradual. Con esta nueva propuesta de envasado alimentari o, en los que se produce migración de las sustancias al espacio de ca b eza y se genera inevitablemente contacto con el alimento, es necesario realizar una evaluación del riego ex haustiva para salvaguardar la salud del consumidor y cumplir así con el r equisito de eva luación de la seguridad exigido por el Re glamento 1935/2004 y EFSA (2011b, 2015) sob re los materiales y Justificación y Objetivos/ Significance and Purposes 59 competentes para la comercialización de nuevos productos que van a estar en contacto con alimentos; c) apren dizaje de técnicas de biología molecular como aislamiento de ARN y PCR cuantitativa a tiempo real (q -PCR), investigando la influencia que tienen las arcillas destinadas al envasado de alimentos en la modulación de la expresión génica a nivel de ARNm. Por todo ello, los objetivos específicos establecidos en la pres ente Tesis D octoral han sido: 1. Realizar una exhaustiva valora ción y revisión bibliográ fi ca del estado d el arte actual y los datos de toxicidad disponibles de arcillas no modificadas y modificadas destinadas a la industria alimentaria, así como de los materiales poliméricos resultantes que las contienen. 2. Evaluar el potencial tóxico in vitro de la arcilla no modi ficada , la montmorilloni ta (Cloisite®Na + (CNa + )), y de arcillas modificadas con sales d e amonio cuaternario (Cloisite®30B (C30B), Cloisite20A (C20A), Cla y 1 y Cla y2) en lí neas celulares de órganos diana (HepG2, C aco-2 y HUVEC), determinando los mecanismos de acción tóxica mediant e la realización de ensa y os de genotoxicidad/mutagenicidad , estrés ox idativo, inflamación, etc., y de finiendo el perfil tox icológico de cada una, además de una evaluación mediante microscop ía de posibles alteraciones c elulare s ultraestructurales. 3. Poner a punto e investi gar por primera vez, la i nfluencia que tienen las arcillas (CNa + , C30B, Clay 1 y Clay2) en la modulación de la ex presión génica a nivel de ARNm de células HepG2 mediante la técnica q-PCR. 4. Estudiar el potencial citotóxico y mutagénico de los extractos de mig r ación obtenidos a partir de polímeros (ácido poli láctico) con org anoarcillas incorporadas (Clay1 y Cla y 2) , además del contenido de metales (Al, Ca, Fe, Mg, Si) que ha y an migra do del envase al simulante alimentario. 5. Evaluar el pot encial t óx ico in vivo, mediante un ensayo de tox icidad oral subcrónica de 90 días en roedores (rata Wist ar), de una arcilla modificada (Cla y 1) y su extracto de migración, investigando la indu c ción de estrés ox idativo como mecanismo de acción t óxica, los cambios y alterac iones histopatoló gicas de distintos órg anos tras la exposi ción, marcadores de inflamación y análisis de la bioquímica clínica. Justificación y Objetivos/ Significance and Purposes 60 6. Explorar las alterac iones a nivel de ex presión génica mediante qPC R y Western Blot de enzim as involucradas en la defensa antioxidante (CAT y SOD) en órganos diana (hígado y riñón) tras la exposición subcr ónica de roedores a la ar cilla modificada Clay1 y su extracto de migración. 7. Analizar la presencia de metales comunes de l a estructura de l as arcillas en el bazo de roedores, tr as la exposición durante 90 días a la arcilla modi ficada Clay1 y su extracto de migración. 8. Desarrollo y caracterización de tres nu evas arcillas modificadas con silanos (Clay3, Cla y 4A y Cla y 4 B) destinadas a ser incor porada s a polipropileno con el fin de mejorar sus propiedades para su aplicación en envasado alimentario y evaluac ión del incremento de la vida útil de a limentos . 9. Investigar e l potencial tóx ico in vitro de las n uevas arcillas modificadas c on silanos y sus extractos de migración en dos lí neas celulares d e órganos diana (HepG2 y Caco-2) mediante ensayos de citoto xicidad, ge notoxicidad/mu tagenicidad, estrés oxidativo y citometría de flujo. 10. Evaluar la mi grac ión de metales propios de la estructura d e las arcillas en simulantes alimentarios que ha n estado en contacto con materiales nanocompuestos poliméricos conteniendo Cla y 3 y Clay4B. 11. Re visar la bibliografía disponible sobre los efectos antiox idantes y antimicrobianos de los ac eites es enciales y sus componentes m a y o ritarios incorporados a envases y l as propiedades de lo s envases resultantes, as í como llevar a cabo un e studio de sus principales efec tos citotóx icos. 12. Estudiar la genotoxicidad de los compuestos mayoritarios del aceite ese ncial de orégano, ti mol y carvacrol, mediante el ensa y o de mi cronúcleos y , po r primera vez, el ensayo de linfoma de ra tón. El trabajo experimental s e ha realizado en el Área de Tox icología de la Facultad de Farmacia de la Univ ersidad de S evilla, haciendo uso , así mismo, del Servicio de Biolog ía y de Microscopía del Centro de I nvesti gación, Tecnolo gía e Innovación de la Universidad de Sevilla (CITI US ). El desarr oll o de las arcillas se ha llevado a cabo en colaboración con el Área de M ateriales del I nstituto Tecnológico de l Embalaje, Transporte y Logística ( ITENE) de Valencia , gracias a las Dras. Susana Aucejo y María Jordá. Parte de la investi gación llevada a cabo a nivel molecular, así como el ensayo de Justificación y Objetivos/ Significance and Purposes 61 ge notoxicidad de mi cronúcleos en células HepG2 , fueron llevados a cabo en el Departa mento de Tox icología Genética y Biología del Cáncer, del Instituto Nacional de Biolog ía (N I B ) de Ljubljana (Eslovenia), bajo la dirección y sup ervisión de la Dras. Metka Filipic y Bojana Zeg ura. El análisis morfológico a nivel celular ha s ido realizado en colaboración con el Dr. J avier Moreno Onorato del grupo de C itoquímica Ultraestructural del Departamento de B iología Celul ar d e la Facultad de Biología de la Universidad de Sevilla. As í mismo, los estudios histopatológicos s e han realizado en colaboración con el Área de Tox icología y el Departamento de An atomía y Anatomía Patológica Comparadas, ambos de la Universidad de Córd oba, gracias a la colaboración de la Dra. Rosario Mo yano y el Dr. Alfonso Blanc o . La evaluación de l a bioquímica clínica del su ero san guíneo se ha llevado a cabo en la Unidad d e Bioquímica C línica del Hospital Universitario Virgen Macarena de Sevilla. P ara el aprendizaje de los ensayos de MN y linfoma de ratón se contó con la colabora ción del Grupo de Mut agéne sis del Departa mento de Genética y Mic robiología de la Universidad Autónoma de Barcelon a bajo la dirección del Dr . Ricard Marcos. Siguiendo la no rmativa de la Universidad de S evilla, el resumen, la justificación y obj etivos, y las conclusiones se r edactan tanto e n español como en inglés para optar a la “Mención I nternacional en el Título de Doctor”. Justificación y Objetivos/ Significance and Purposes 62 Taking into account the state of the art previously described, it is important to note that although the new incorporation of natural subs tances, such as clays or essential oils, int o food packa ging has a great interest in or der increase the shelf li fe of food, the y also poses a potential risk for humans and the environment. This risk is closely linked to the possible migration o f the materials incorpor ated into packaging polymers, which would be ingested with food by th e consumer. Therefore, a comprehensive assessment of human ex posure risks to these substan ces, cla y s o r essential oil s, to gether with the new packaging mat erials created with them, is mandator y . This assessme nt requires a detailed characterization of the hazard, ex ploring the toxic effects tha t occur after exposure of several ce ll lines from target organs, as well as, the r esult of the exposure with in vivo ex perimental models, in order to extrapolate the data obtained to real human ex posure scenario. I n this sense, after a t horough literature review in which the disparity of the available results has been verified, the study of the toxic effects of differe nt organomodified cla y s with quaternar y ammonium salts and silan es, both in vitro and in vivo, has been performed. I n addit ion, some of these clays have been rece ntl y develop ed, therefore no available data in the literature have been found, being th e to xicolog ical evaluation even more necessar y . On the other hand, in vitro genotoxic potential of major compounds of the oregano essential oil was eva luated, being one of the most used essential oils in active food pac ka g ing. In this sense, consid ering that the oral rout e is the most im portant to human exposure in thi s kind of substance s, it is of great interest to evaluate the response of target cell lines f rom organs involved in th e digestive process, emph asizing the first organ involved in the absorption, intestine, and the main organ invol ved in x enobiotics biotransformation, liver. On the other h and, it would be also intere sting to check the toxic effect after a possi ble distribution through the bloodstream of the m aterials under study. We decided to investigate in vitro , in huma n cell lines such as Caco- 2 (intestinal), HepG2 (liver), and HUVEC (endothelial), th e c y totox ic potential and the main mechanisms of toxicity at different exposure t imes of several clays i ntended to be incorporated into food pa ckaging. Moreover, the effects produced at the mo lecular level were also evaluated through the gene expression anal y sis at subcytotoxic concentrations. I n addition, the toxicological evaluation of migration ex tra cts obtained from polymer-clay nano composites was necessary , being ev aluated in different in vitro experimental models. Justificación y Objetivos/ Significance and Purposes 63 After the in vitro tox icit y approach, the cla y with the best technolo g ical and toxicological profile was selected to perform a repeated dose 90 -day oral toxicit y study in Wistar rats, based on the wor st scena rio of migration which could be exposed the consumer. Histopathological studies were perf o rmed in a wide varie t y of organs that could be affected and different markers of ox idative stress, inflammation, gene expression and protein abundance w ere evaluated. S imilarly , the presence of the major metal pr esent in c lays struc ture w as de termine d in order to identify other possible alterations. Regarding to the essential oils and their main components the ava ilable d ata on the ir use in food packaging, are contradictor y so far, bein g n ecessary an ex haustive review of the main antimicrobial/antioxidant eff ects of the r esultant active packaging on the food, and the main cytotoxic effects on h uman cell li nes. Moreover, oregano essential oil and its major compon ents, c arvacrol and thymol, a re on e o f the options most used in the food industry . The use o f the se compounds has been described b y severa l authors but the results published do not indi cate a similar b ehavior of them so far. Therefore, we found it interest ing to evaluate the genotoxicit y of carvacrol and thymol b y two tests required b y EFSA, the micronucleus test and mous e l y mphom a assay, the second one ha s not been carried out until now, being completed the available toxicological information of these substances. For the fulfillment of this thesis, the PhD student performed internships, two nationals and one international, in which several objectives w ere addressed: a) development and design of new silane-modified cla ys intended to be used in food packaging as well as an approach to techniques to evaluation their technological properties: mechanical, thermal and barrier; b) learn new genotoxicit y tests according to the rules of the Org anization for Economic C oopera tion and Deve lopment (OECD), such as the micronucleus test and mouse l y mph oma assa y , required b y the competent authorities to commerciali ze new products th at will be in contact with food; c) learn molecular biology techniques a s RNA isol ation a nd real-time quantitative PCR (q- PCR), to investigate th e influence o f cl a y s in the modulation of gene expression at the mRNA level. Therefore, the specific objectives in this PhD thesis were: Justificación y Objetivos/ Significance and Purposes 64 1. To perform a comprehensive assessment and litera ture review of the current state of art and tox icity data of unmodified and modified cla y s int ended to be used in the food indus try, as w ell as, the resulting polymeric materials that contain them. 2. To e valuate th e in vitro toxicit y potential of the unmodified clay, montmorillonite (Cloisite®N a + (CNa + )) and quaternar y ammonium salts - modified cla y s (Cloisite®30B (C30 B), Cloisit e20A (C20A) and Cla y2 clay1 ) in target cell lines (HepG2, Caco-2 and HU VEC), determining the ac tion mechanisms by test ing genotox icity /muta genicity, ox idat ive stress, inflammation, etc., d efining each toxicolog ic al profile, a nd possi ble ultrastructural cellular a lterations b y microscopy . 3. To deve lop and investi g ate the influence of clay s (CNa + , C30B, Cla y1 and Clay2) in the modulation of gene expression at th e mRNA level of HepG2 cells by q-PCR. 4. To stud y th e cytotoxic and mutagenic potential of migration ex tracts ob tained from pol y mers (pol y la ctic acid) w ith organomodified clays (Cla y1 and C lay2) and the migration of metals content (Al, Ca, F e, Mg, Si) presented in th e food simulants. 5. To anal y z e the in vivo to xicit y potential, b y performing a repeated dos e 9 0-da y oral toxicit y stud y in Wistar rats exposed to a modified clay (Cla y 1 ) and its migra tion extract, re sear ching the induction of oxidative stre ss as a toxicit y mechanism, histopathological alterations of different organs, inflammation re sponses and anal y sis of clinical biochemistry . 6. To ex plore the alterations in gene expression b y qPCR and Western blotti ng of enzymes involved in antiox idant defense (SOD and CAT) from target organs (liver and kidne y ) of rat s ex posed subchronically to modified cla y , Cla y1, and its migration extract. 7. To determine the presen ce of major metals of clay s structure in the sple en of rodents after exposure during 90 da y s to a modified cla y , C lay1, and its migra tion extract. 8. To develop and charact erize three new sil ane-modified cla y s (Cla y3, Cla y 4A and Clay4B) intended to be incorpora t ed to polypr op y len e (PP) in order to improve their properties for application in food packaging and evaluation of the increase of food shelf life. Justificación y Objetivos/ Significance and Purposes 65 9. To investigate the in vit ro toxicit y potential of new silane-modified cla y s and their migra ti on ex tracts in two targ et cell lines (HepG2 and Caco-2) b y t esting cy totoxicit y , genotoxicity / mutagenicit y , ox idative stress and f low c ytome try. 10. To evaluate the metals migra tion presented in clays structure in food sim ulants which have been in contact with Clay3 a nd Cl a y 4 A polymer nanocomposites. 11. To review the available literature on antiox idants and antimicrobial effe cts of essential oils and their major components incorporated into packaging and properties of the resulting packa g es , as well as, conducting a stud y o f their main cy totoxic effects. 12. To study the genotoxicity o f the main compounds of oregano essential oil, thymol and carvacrol, using the micronucleus test and , fo r the first ti me, the mouse lymphoma a ssa y . The ex perimental work has been performed in the Area of Toxicolog y , Facult y o f Pharmacy, Universit y o f S evilla, usin g also th e Biology S ervices fro m Centro de Investigación, Tecnología e I nnov ación from the University of Sevilla (C I T I US). The development of the c lays was carried out in c ollaboration with the D epartment of Materials from the T echnological I nstitute of Packaging, Tr ansport and Logistics (ITENE) f rom Valencia, thanks to Drs. Susana Aucejo and Maria J orda. Part of the research conducted at the molecular level, as well as , genotoxicit y testing of micronuclei in HepG2 cells were carried out in t he Department of G enetic Tox icology and Cancer Biology, National Institute of Biology (NIB) to Ljubljana (Slovenia ), under the direction and supervision o f Drs. Metka Filipic and Bojana Zegura. Cellular morphologica l analysis has been conducted in collaboration with Dr. Javier Moreno Onorato, belon ging to Ultrastructural C ytochemistry group from the Department of Cell Biology , Facult y of Biolog y , Univ ersity o f Sevilla. Histopathological studies have been performed in the Area of Toxicolog y and i n the Dep artment of Anatom y and Comparative Pathology and Anatom y , both from the Universit y of Córdoba, thanks to the collaboration of Dr. Rosario Mo y ano and Dr. Alfo nso Blanco, respectively. The clinical biochemistry evaluation of blood serum has been carried out in the Clinical Biochemistry Unit of the University Hospital Virgen Macarena in Seville. MN and mouse l y mphoma assa ys were learn ed thanks to the collaboration with the Group o f Mutage nesis of the Department of Genetics and Microbiolog y at the Universit y Autónoma of Barcelona under the direction of Dr . Ricard Marcos. Justificación y Objetivos/ Significance and Purposes 66 Following the regul ations from the Universit y of Sevilla, the summary , significa nce and purposes, and c onclusions have be en written both in Spanish and English to aim for a P hD with I nternational Menti on. IV. RESULT ADOS Y DISCUSIÓN/ RESULTS AND DISCUSSION T able 3 Cyto to xic effects of unmodi fi ed/modi fi ed clays and derived nanocomposites. Material tested Modi fi er Experimental model Assays performed Concentration range Exposure time Main results Refer ence - Sepiolite Unmodi fi ed Primary rat hepatocytes Lactate deh ydrogenase (LDH) re- lease assay 1-1 0 m g/mL 20 h No signi fi cant differences were observed in compar - ison with the controls. Denizeau et al. (1 985) - Mt, bentonite, kaolinite and erionite Unmodi fi ed HUVE, N1E-1 1 5 and ROC-1 cell lines LDH release assay , Trypan Blue e x- clusion test 0. 1 mg/mL 24 h Only HUVE cells experienced signi fi cant LDH release after exposure t o bentonite, kaoli- nite and Mt. Similarly , all clays changed signi fi cantly cell viability in HUVE cells, but only Mt and erionite did in N1E-1 1 5. No effect was reported in R OC-1 cells Murph y et al. (1 993a) - Quartz – Kaolin (Both untreated and treated) Dipalmitoy l phosphatidylcoline (DPPC) Rat pulmonary al- veolar macro- phages cells Live-Dead assay 0-80 m g/cm 2 0-40 m g/cm 2 1 , 3, 5 days Concentration and time cy- toto xicity was observed in cells exposed for 1 , 3 and 5 days to untreated q uartz and kaolin. DPPC-surfactant pretreatment delay ed the to xic effects Gao et al. (20 0 0) - Nativ e Bentonite - Modi fi ed Bentonites Unmodi fi ed α -quartz þ chemical modi fi cations (alkalin, acid and organic) IMR90 cell line Alarm Blue assay 1-50 m g/cm 2 24 h All bentonite samples in- duced cytoto xic effects, being more evident in bentonites with higher quartz contents than in untreated bentonites or those with low er quartz content Geh et al. (20 06) - Native Bent onite (BPN) - Active Bent onite (BP A) Unmodi fi ed H 2 SO 4 HMy2.CIR cell line Cell Counting, neutral red upt ake, LDH release assay 0-1 0 0 0 m g/mL 4 and 24 h Cell viability decrease with the exposur e concentrations and time. BP A being more cytoto xic than BPN Meibian et al. (20 1 0) - Mt Unmodi fi ed CHO cell line 3-(4,5-dimethy lthiazol-2-yl)-2,5- diphen yltetrazolium bromide (MTT) assay , LDH release 0-1 0 0 0 m g/mL 3, 1 2, 24 h Reduction of 40% cell viabili- ty and increase in 40% in LDH levels after 24 h of exposure to 1 0 0 0 m g/mL Mt Li et al. (20 1 0) - CNa þ - C30B (Un fi lt ered and fi ltered) Unmodi fi ed Quaternary ammonium salt (MT2EtOH) Caco-2 cell line Alarmar Blue assay 0-226 m g/mL 24 h Only the highest concentra- tion used (226 m g/mL) of the un fi ltered e xposure under - went signi fi cant reduction in cell viability (40%) Sharma et al. (20 1 0) - Halloysite Clay Nano tubes (HNT) unmodi fi ed and functionalized Unmodi fi ed Aminoprop yltrietho xysilane HeLa and MCF-7 cell line MTT assay , Trypan Blue assay 1-1 0 0 0 m g/mL 24, 48, 72 h Both HNTs exhibit growth inhibition in a concentration and time dependent manner at both cell lines and assays. The cell viability w as pre- served up to 75 m g/mL. No effect of functionalization was r ecorded Vergaro et al. (20 1 0) - Mt (Na-MMT) - Oligo(styreme-co-acrylo- nitrile)-modi fi ed Mt (PSAN-MMT) Unmodi fi ed Oligo(styrene-co-acrylonitrile) HEK 293 and NIH 3T3 cell lines MTT assay , LDH release 0. 1 – 2 g/L 24 h Signi fi cant cell viability de- crease was observed in both cell lines exposed to both Mt in the LDH release assay and MTT assay . PSAN-MMT was less toxic than MMT Liu et al. (20 1 1) S. Maisanaba et al. / Enviro nmental Research ∎ ( ∎∎∎∎ ) ∎∎∎ – ∎∎∎ 5 Please cite this article as: Maisanaba, S., et al., T oxicological ev aluation of clay minerals and deriv ed nanocomposites: A review . Environ. Res. (20 1 5), ht tp://dx.doi.org/1 0. 1 0 1 6/j.envr es.201 4. 1 2.024 i T able 3 ( continued ) Material tested Modi fi er Experimental model Assays performed Concentration range Exposure time Main results Refer ence - CNa þ - C93A Unmodi fi ed T ernary ammonium salt (M2HT) HepG2 cell line MTT assay , LDH release 0 – 10 0 0 m g/mL 24 h Concentration-dependent ef- fect was observed in cells treated with bo th clays, being signi fi cant from 1 m g/mL in the MTT assay and from 50 m g/mL in the LDH release assay Lordan et al. (20 1 1) - Aminoprop yl magnesium ph yllosilicate (AMP) - Aminoprop yl calcium ph yllosilicate (ACP) Aminoprop yl CCD-986sk, A549, MRC-5, HT -29 cell lines MTT assay , LDH release 1 – 10 0 0 m g/mL 24, 48, 72 h Decrease in cell viability and membrane damage appeared at the highest concentrations of both organoclays assay ed Han et al. (20 1 1) - Nanocomposite of halloy- site clay nanotubes (HNT) incorporated int o chitosan Unmodi fi ed NIH3T3 cell line MTT assay 2, 5, 7 .5 and 1 0% 1 , 3 and 7d a y s Both chitosan and chitosan/ HNT s nanocomposites fi lms are biocompatible since no visible reduction in viability was found Liu et al. (20 1 2) - Mt Unmodi fi ed INT -407 cell line 2-(4-Iodophen yl)-3-(4-ni- trophen yl)-5-(2,4-disulfopheny l)- 2 H-tetrazolium (WST -1) assay, MTT assay , LDH release assay 0-1 0 0 0 m g/mL 24, 48, 72 h and 1 0 day s Cell proliferation was in- hibited in a concentration and time dependent in the short-term WST -1 assay . Si- milarly , after 1 0 days of e x- posure, all concentrations tested signi fi cantly r educed cell viability . Only 10 0 0 m g/mL induced a sig- ni fi cant release of LDH Baek et al. (20 1 2) - Platelet nanoclays (Ben- tone MA, ME-1 0 0, Cloisite Na þ , Nanomer PG V, De- lite L VF) Tubular nano- clays (Halloy site and Hal- loysit e MP1) Not speci fi ed A549 cell line High content screening and r eal- time impedance sensing 1 – 250 m g/mL 24 h No to xic effect was recorded for nanoclays up to 1 0 m g/mL, ex cept for Delite L VF . At higher concentration, cells grow decreased in a con- centration dependent man- ner . Tubular nanoclay s showed lower cyto toxicity compared to platelet type, with Bentone MA and ME- 1 0 0 showing the better re- sults in this group Verma et al. (20 1 2) - Nanocomposites of an un- modi fi ed sepiolite, un- modi fi ed and modi fi ed Mt and fl uorohectorites Poly(butylene adipat e-co-terephthalate) and its nanocomposites based on 1 0 wt.% clay minerals. Modi fi ed Mt contained a q uaternary ammonium salt (MT2EtOH), and modi fi ed fl uorohectorite contained a dih ydroxy organic modi fi er . L929 cell line MTT assay 1 0 wt% clay miner als 1 , 2 and 5d a y s No visible reduction in via- bility was observed at an y experimental condition. Fukushima et al. (20 1 2) - Nanocomposites of an un- modi fi ed sepiolite, un- modi fi ed and modi fi ed Mt and fl uorohectorites Poly(butylene adipat e-co-terephthalate) and its nanocomposites based on 1 0 wt.% clay minerals. Modi fi ed Mt contained a q uaternary ammonium salt (MT2EtOH), and modi fi ed fl uorohectorite contained a dih ydroxy organic modi fi er . Fibroblast and os- teoblast cell lines MTT assay 1 0 wt% clay miner als 1 , 4 and 7d a y s No signi fi cant reduction in viability was observed in both cells in all times of exposure Fukushima et al. (20 1 3) - Halloysite clay nanotubes (HNT) Unmodi fi ed Caco-2/HT29-MTX cells in co-culture 2,3-bis-(2-methoxy-4-nitr o-5-sul- fophen yl)-2 H-tetrazolium-5-car - boxanilide (XXT) assay , LDH release and Lucifer yellow permeability assay 1, 10 a n d 10 0 m g/mL 6 h No cytoto xic effects were observed in an y assay performed Lai et al. (20 1 3) S. Maisanaba et al. / Environmental Resear ch ∎ ( ∎∎∎∎ ) ∎∎∎ – ∎∎∎ 6 Please cite this article as: Maisanaba, S., et al., T oxicological evaluation of clay minerals and derived nanocomposites: A review . Environ. Res. (20 1 5), ht tp://dx.doi.org/1 0. 1 0 1 6/j.envr es.201 4. 1 2.024 i - CNa þ - C30B Unmodi fi ed Quaternary ammonium salt (MT2EtOH) HepG2 cell line Protein content, neutral red upta ke, 3-(4,5-dimethy lthiazol-2-yl)-5-(3- carboxymetho xyphenyl)-2-(4-sul- fophen yl)-2 H-tetrazolium (MTS) reduction assay 0-62.5 m g/mL 0-50 0 m g/mL 24, 48 h Only C30B showed cytoto xic effects (EC 50 ¼ 88 m g/mL) Maisanaba et al. (20 1 3) - 6-MP þ Mt-PLA nanocomposite 6-Mercapt opurine (6-MP) encapsulated with Mt IMR32 cell line Try an blue dye e xclusion test and MTT assay 1 0 ppm/test composite 24 h Cell viability was greater after exposure to the nano- composite containing the drug (6-MP) Kev adiya et al. (20 1 3) -C20A -Clay1 -Clay2 Quaternary ammonium salt (2M2HT) Hexadecy ltrimethylammonium bromide (HDT A) HDT A þ Acetylcoline (ACO ) Caco-2 and HepG2 cell lines Protein conten t, MTS reduction assay 0-62.5 m g/mL 0-8 m g/mL 0-1 25 m g/mL 24, 48 h Only Clay2 induced cyto- to xicity in both cell lines, being more sensitive Caco-2 than HepG2 (EC 50 ¼ 34 m g/mL and EC 50 ¼ 88 m g/mL, respectivel y) Houtman et al. (20 1 4) - CNa þ - C30B Unmodi fi ed Quaternary ammonium salt (MT2EtOH) Caco-2 cell line Protein content, neutral red uptake, MTS reduction assay 0-1 25 m g/mL 0-250 m g/mL 24, 48 h Only C30B showed cytot oxic effects (EC 50 ¼ 40 m g/mL) Maisanaba et al. (20 1 4a) -Clay1 -Clay2 Hexadecy ltrimethylammonium bromide (HDT A) HDT A þ Acetylcoline (ACO ) Caco-2 and HepG2 cell lines Neutral red uptake 0-8 m g/mL 0-1 25 m g/mL 24, 48 h No signi fi cant effects were observed, ex cept for a de- crease in NR uptake in HepG2 cells exposed t o 8 m g/mL Clay1 Jorda-Bene- yto et al. (20 1 4) -PLA-Clay1 e xtract -PLA-Clay2 e xtract HDT A þ Mt þ PLA HDT A þ ACO þ Mt þ PLA Caco-2 and HepG2 cell lines Protein conten t, neutral red uptake, MTS reduction assay 0-1 0 0% of extract 24, 48 h No cytoto xic effects were re- corded in both cell lines at an y concentration and ex- posure time Maisanaba et al. (20 1 4b) - CAP þ Mt-PLA nanocomposite Capecitabine (CAP) encapsulated with Mt IMR32 cell line Try an blue dye e xclusion test and MTT assay 1 0 ppm/test composite 24 h Cell viability was greater when cells were e xposed to the nanocomposite contain- ing the drug (CAP) than when exposing cells only to CAP Kev adiya et al. (20 1 4) - CNa þ - C30B - Clay1 - Clay2 Unmodi fi ed Quaternary ammonium salt (MT2EtOH) HDT A HDT A þ ACO HUVEC cell line Protein content, neutral red uptake, MTS reduction assay 0-1 25 m g/mL 0-250 m g/mL 0-8 m g/mL 0-1 25 m g/mL 24, 48 h CNa þ and Clay1 showed in general no signi fi cant chan- ges in any of the endpoints assayed after 2 4 and 48 h of exposure. C30B and Clay2 induced cytoto xic effects (EC 50 ¼ 21 . 4 7 11 . 4 m g/mL and EC 50 ¼ 55.4 7 6. 1 m g/mL, respectivel y) both at MTS after 24 h of exposure Maisanaba et al. (20 1 4c) - Unmodi fi ed MT (MMTdell and MMTdells) Dimeth yl dihydrog enated tallow ammonium Ramos, A-549, HCT -1 1 6, SK MEL 28 and HepG2 cell lines Alarm Blue assay Serial dilutions from 1 0  2 to 10 2 m g/mL 72 h The nanoclays modi fi ed with dimethy l benzyl hydro- genated tallow ammonium showed higher to xicity than those modi fi ed with the di- meth yl dih ydrogenated tal- low ammonium Janer et al. (20 1 4) - Modi fi ed MT (MMTdell 43B, MMTdell 67G, MMTdell 72T , MMTdell 43Bs, MMTdell 67Gs and MMTdell 72T s) Dimeth yl benzyl hydrog enated tallow ammonium Cell lines: A-549: human alveolar adenocarcinoma cell line; Caco-2: colorectal adenocarcinoma cell line; CCD-986sk: human skin fi broblast ; CHO: Chinese hamster ov ary cell line; HCT -1 1 6: human colorectal car cinoma cell line; HEK 293: human embry onic kidney cell line; HeLa: human epithelial adenocarcinoma cell line; HepG2: liver hepatocellular carcinoma cell line; HMy2.CIR: human B lymphoblast cell line; HT -29: human colon epithelial carcinoma cell line; HT29-MTX: human colon adenocarcinoma cells treated with metho trexate; HUVEC: human endothelial cell line; IMR32: human neur oblastoma cell line; IMR90: human lung fi broblast cell line; INT -407 : human embryonic intestine cell line; L929: murine fi broblast cell line; MRC-5: human lung fi broblast cell line; MCF-7 : human breast cancer cell line; NIH3T3: mouse fi broblast cell line; N1E-1 1 5: neuroblasto ma cell line; SK MEL 28: human melanoma cell line; Ramos: human Burkitt ’ s lymphoma cell line; ROC-1: oligodendr oglial cell line. S. Maisanaba et al. / Enviro nmental Research ∎ ( ∎∎∎∎ ) ∎∎∎ – ∎∎∎ 7 Please cite this article as: Maisanaba, S., et al., T oxicological ev aluation of clay minerals and deriv ed nanocomposites: A review . Environ. Res. (20 1 5), ht tp://dx.doi.org/1 0. 1 0 1 6/j.envr es.201 4. 1 2.024 i ( Meibian et al., 201 0 ). In fact, it has been reported that different modi fi ers have different effects; hence, Mt delite modi fi ed with dimeth yl benzyl h ydrogenated tallow ammonium showed higher to xicity than those modi fi ed with dimethyl dih ydrog enated tallow ammonium in fi ve cell lines ( Janer et al., 20 1 4 ). This fi nding w as corroborated by analysing the cytot oxic effects of the modi fi ers in the same cell lines. S tudies evaluating the to xicity of q uaternary ammonium compounds potentially used as clay modi fi ers are scarce. Thus, Jodynis-Liebert et al. (201 0) obtained a mean in- hibitory concentration for didecyldimeth ylammonium sacchar - inate among 1 .44 and 5.4 7 m M for the MTT assay in six human cell lines. Moreov er , native Mt (CNa þ ) showed low er toxicity than the modi fi ed Mt (C30B) in HepG-2 and Caco-2 cells ( Maisanaba et al., 20 1 3 , 20 1 4a ). Additionall y , the same cell lines showed the different cytot oxic pro fi les of the three organo-modi fi ed clay minerals, evidencing that the type of clay mineral, the concentration range and the origin of the cell line play an important role in the ob- served to xicity ( Houtman et al., 20 1 4 ; Jordá-Beneyto et al., 20 1 4 ). How ever , the modi fi cation of Mt w as also demonstrated to de- crease the to xicity of clay minerals. For instance, the modi fi cation of Mt with oligo (styr ene-co-acrylonitrile) result ed in a less to xic material compared with the unmodi fi ed Mt ( Liu et al., 201 1 ). In addition, the modi fi cation of the organoclay could result in a non- to xic material, which is the main objective of the synthesis of these materials intended for human contact. Han et al. (201 1) re- ported no signi fi cant membrane damage and change in cell via- bility in four cell types exposed to magnesium and calcium organoph yllosilicates. The nature of the clay mineral plays an important role in the observed to xicity . In this regard, Murph y et al. (1 993a) report ed the following to xicity scale in HUVE cells af ter 24 h of incubation with 0. 1 mg/mL clay minerals: Mt 4 bentonit e ¼ kaolinit e b erionite. In addition to the abov e-mentioned work, most studies hav e evaluated the to xicity of Mt and bentonit e. T o the best of our knowledge, the toxicity of kaolinit e has only been studied by Gao et al. (20 0 0) , who compared the results with another silicate, namely quartz. Both compounds induced cytoto xic effects on rat pulmonary alveolar macrophag e cells starting on the fi rst day of exposure, although pretr eatment with surfactant delay ed the to xicity . In addition to the platelet clay s, tubular nanoclays, another ph ysical structure of clay minerals, have been assayed. Verma et al. (20 1 2) compared the to xicity of both structur es in the lung epi- thelial cells A54 9 and showed that the platelet-structur ed nano- clays wer e more cyt oto xic than the tubular types. It is important to note that the structure was not the only difference among them because the nature of the clay mineral also differed. In this sense, platelet clays wer e mainly bentonites, whereas tubular nanoclays were halloy sites. In this regard, the toxicity of halloy site nanotubes (HNT) has also been evaluated in a co-culture of intestinal cells exposed to 0 – 10 0 m g/mL HNT , which result ed in no cytoto xic effect ( Lai et al., 201 3 ). However , unmodi fi ed and functionalized HNT s show ed cytoto xic effects on HeLa and MCF-7 cells at concentra- tions higher than 75 m g/mL ( Vergaro et al., 20 1 0 ). No in fl uence on the functionalization of the HNT was observed. Liu et al. (201 2) assessed the safety of HNT incorporated into chitosan to form bionanocomposite fi lms. The Chitosan/HNT s nanocomposite show ed similar cytocompatibility to the chitosan nanocomposite without the clay nanotube. Despite the latte r work, very few stu- dies have been conducted to date in composites containing clay minerals. Only Kev adiya et al. (20 1 3 , 20 1 4) ha ve reported the in- teresting potential of clay -based composites as reservoirs of cy- to to xic drugs because they hav e been proven to reduce the to xic effects of the drugs and t o also play an important role in the de- liv ery pro fi ciency . Among the most freq uently used endpoints in cellular to xicity testing are those based on changes in cell morphology ( Boren- freund and Borrero, 1 984 ). The most remarkable morphological features altered by clay minerals are listed in T able 4 . The mor - phological changes induced by different clay minerals have been studied in HUVE cells, which were markedly changed af ter 24 h of exposure to 0. 1 mg/mL Mt, bentonite and kaolinite, including cell ly sis ( Murphy et al., 1 993a ). The ly sis was greater in the case of Mt compared with bentonit e and kaolinite, with only limited ly sis observed after exposure to erionite. This fi nding agreed with the cytot oxicity assay results. In the same manner , in shorter exposure times, Mt and bentonite wer e also able to cause complete cell lysis in the neuronal cultures within 60 min ( Murphy et al., 1 993b ). Similarly , HepG2 cells exposed for 24 h to 50 and 1 0 0 0 m g/mL CNa þ and C93A exhibited a dose-dependent cell death induction ( Lordan et al., 20 1 1 ). Moreover , C30B induced morphological changes in HepG2 and Caco-2 cells af ter 24 and 48 h of exposure, undergoing ultrastructur al features characteristic of cell impair - ment, such as damage to the mitochondria, nucleus and en- domembrane systems ( Maisanaba et al., 201 3 , 20 1 4a ). In contrast, no cell organelles were affected in the lung fi broblast cell line IMR90 exposed to 1 0 m g/cm 2 Mt for 24 h ( Geh et al., 20 0 6 ). The degeneration of the mitochondria reported in HepG2 and Caco-2 cells exposed t o C30B is in agreement with the changes observed in the MTT , MTS, WST -1 and XXT (different tetrazolium salts) assays in man y cell types exposed to unmodi fi ed and func- tionalized Mt and HNT ( Baek et al., 201 2 ; Houtman et al., 201 4 ; Lai et al., 20 1 3 ; Li et al., 20 1 0 ; Liu et al., 201 1 ; Lor dan et al., 20 1 1 ; Maisanaba et al., 201 3 , 20 1 4a , b ,c; Vergaro et al., 201 0). Ad- ditionally , the presence of fat droplets and nuclear lipid inclusions in HepG2 and Caco-2 cells exposed to C30B r evealed an alteration in the lipid metabolism, which is consistent with the release of fatty acids reported by Murphy et al. (1 993a) . In addition to the studies on clay minerals, the biocompatibility of bio-nanocomposites containing these materials has also been assessed by analysing the attachment and spr eading of L-929 cells ( Zia et al., 20 1 1 ). The cells showed higher attachment and growth on bio-nanocomposites without Mt than those containing the nanoclay at a concentration of 1%, 2%, 4% and 8%. The staining results also showed that an increased in the concentration of Mt has adverse effects on the biocompatibility of the samples. In contrast, Liu et al. (20 1 2) examined the cytocompatibility of chit- osan/HNT s nanocomposites fi lms, evidencing a positive r esponse in NIH3T3 cells even at the highest content of HNT s (1 0%). When all data above ar e considered, it is evidenced that in vitro to xicological research on clays is of high interest now adays. Howev er , reports dealing with nanocomposites containing clays are scarcer . Different clays have their o wn cytoto xic pro fi le with dependence on the experimental conditions (type of clay , modi- fi er , cell line, concentrations used, etc .). But they have been shown to be able t o induce deleterious effects on cells, and this deserves to be investigated, taking into account their multiple applications. 2.2. T oxicity mechanisms In vitro to xicological assessments are useful for obtaining me- chanism-derived information. Cells respond rapidly to to xic stress by altering, for example, different basal biomarkers, such as the generation of reactiv e oxy gen species (ROS) and glutathione con- tent (GSH), and making alter ations to sever al organelles, such as the mitochondria and lysosomes. T aking this into account, im- portant q uestions are how the studied materials, in this case, clay minerals, and derived nanocomposites are able to alter these paramet ers and how these alterations can affect the metabolic rates, cell gro wth and gene transcrip tion ( Eisenbrand et al., 20 02 ). The interaction, in fl uence and potential to xicity of materials with prot eins and cells are an essential focus in assessing and S. Maisanaba et al. / Environmental Resear ch ∎ ( ∎∎∎∎ ) ∎∎∎ – ∎∎∎ 8 Please cite this article as: Maisanaba, S., et al., T oxicological evaluation of clay minerals and derived nanocomposites: A review . Environ. Res. (20 1 5), ht tp://dx.doi.org/1 0. 1 0 1 6/j.envr es.201 4. 1 2.024 i T able 4 Morphological changes evaluation of unmodi fi ed/modi fi ed clays and deriv ed nanocomposites. Material tested Modi fi er Experimental model Concentration range Exposure time Main results Referen ce - Mt and bentonite Unmodi fi ed HUVE cell line 0. 1 mg/mL 24 h All clays induced morphological changes, including lysis of the cells. The toxicity observed w as: Mt 4 bentonite E kaolinite»erionite Murph y et al. (1 993a) - Mt, bentonite and erionite Unmodi fi ed Culture of primary murine spinal cord neurons and differentiated N1E-1 1 5 cell line 0. 1 mg/mL 5, 1 5, 60 min and 1 8 h Mt and bentonite caused complete cell lysis in the neur onal cultures within 60 min after exposure. None of the clay s appeared to be cytoto xic to the differentiated NlE-1 1 5 cells even at 1 8 h Murph y et al. (1 993b) - Native bent onite Unmodi fi ed IMR90 cell line 1 0 m g/cm 2 24 h No organelles were affected in IMR90 cells exposed to clays; despite Mt crystal was found intracellularl y Geh et al. (20 06) - Modi fi ed bentonites α -Quartz þ chemical mod- i fi cations (alkalin, acid and organic) - CNa þ Unmodi fi ed HepG2 cell line 50 and 1 00 0 m g/mL 24 h Cell death w as observed after exposure to nanoclays Lordan et al. (20 1 1) - C93A T ernary ammonium salt (M2HT) - Polyurethane bio- nanocomposites 4,4 ’ - diphen ylmethane diisocyanate L-929 cell line PUBNC1: without nanoclay , PUBNC2: 1 .0%, PUBNC3: 2.0%, PUBNC4: 4.0% and PUBNC5: 8.0% nanoclay 48 h No cytoto xicy was observed in cells exposed to the nano- composites without nanoclay and that containing only 1% nanoclay . However , those with higher cont ents of bentonite nanoclay showed some toxic and incompatible behaviour Zia et al. (201 1) - Nanocomposite of halloysit e clay nanotubes (HNT) in- corporated into chit osan Unmodi fi ed NIH3T3 cell line Chitosan/HNTs with 5% and 1 0% HNT s 1 , 3, and 7d a y s Both chitosan and chitosan/HNT s nanocomposite fi lms are cytocompatibility even when the loading of HNTs is 1 0% Liu et al. (20 1 2) - C30B Quaternary ammonium salt (MT2EtOH) HepG2 cell line 0 – 88 m g/mL 24 and 48 h Mitochondrial degeneration, dilated endomembrane sys- tems, heterophagosomes formation, fat droplets appearance and presence of nuclear lipid inclusions were oberved Maisanaba et al. (20 1 3) - C30B Quaternary ammonium salt (MT2EtOH) Caco-2 cell line 0 – 40 m g/mL 24 and 48 h Dilated cisternae edge in the Golgi apparatus and nucleolar segregation w as observed Maisanaba et al. (20 1 4a) Caco-2: colorectal adenocarcinoma cell line; HepG2: liver hepatocellular carcinoma cell line; HUVE: human umbilical vein endo thelial cell line; IMR90: human lung fi broblast cell line; L-929: fi broblast cell line; NIH3T3: mouse fi broblast cell line; and N1E-1 1 5: neuroblastoma cell line. S. Maisanaba et al. / Enviro nmental Research ∎ ( ∎∎∎∎ ) ∎∎∎ – ∎∎∎ 9 Please cite this article as: Maisanaba, S., et al., T oxicological ev aluation of clay minerals and deriv ed nanocomposites: A review . Environ. Res. (20 1 5), ht tp://dx.doi.org/1 0. 1 0 1 6/j.envr es.201 4. 1 2.024 i understanding the material compatibility versus to xicity ( Jones and Grainger , 20 09 ). The cell-material reactions of inter est include cellular uptake, membrane integrity perturbations, or alteration in the viability or vital organelles. Thus, endpoints such as the gen- eration of ROS and other paramet ers related with the cellular stress r esponse (GSH, superoxide dismutase activity (SOD), lipid pero xidation (LPO), etc .), DNA damage, in fl ammation response and cell necrosis and apoptosis pr ovide valuable information for un- derstanding the to xic response. Below , a descrip tion of the main studies that focused on the to xicity mechanisms in different cell lines exposed to clay minerals and derived nanocomposites is presented. Mor eover , the simila- rities and differences in the results due to different experimental models and e xposures will hopefully be identi fi ed. 2.2. 1. Oxidative stress generation Increases in the intracellular leve l of ROS represent a pot ential to xic mechanism, which if not counteract ed will lead to other disorders, such as membrane dysfunction, LPO, which means a higher malondialdeh yde level (MD A) in the cell membrane, DN A damage and a drastic inactivation of pro teins. T o prev ent damage to cellular components, there are numerous enzymatic antioxidant defences designed to sca venge ROS in the cell. Examples of these enzymes are the SOD, catalase (CA T), glutathione pero xidase (GPx) and glutathione reductase (GR) ( Puerto et al., 20 09 , 201 0 ). Activity measurements of this group of antioxidant enzymes may provide a marker of oxidativ e stress because they are able to be completed with the study of other parameters, such as the GSH, MD A and carbon yl levels ( Puerto et al., 20 1 4 ). Several resear chers hav e studied the effects related with R OS generation and other disorders in several target cell lines exposed to different unmodi fi ed and modi fi ed clay minerals and derived micro/nanocomposites ( Baek et al., 20 1 2 ; Gov erna et al., 1 995 ; Hansen and Mossman, 1 987 ; Houtman et al., 201 4 ; Kev adiya et al., 20 1 3 , 20 1 4 ; Lordan et al., 201 1 ; Maisanaba et al., 20 1 3 , 20 1 4a ; Meibian et al., 201 0 ; Sharma et al., 201 0 )( T able 5 ). Contradictory results wer e observed when various cell lines from digestiv e origin, such as HepG2, Caco-2 and INT -407 , were exposed to unmodi fi ed Mt, CNa þ . Baek et al. (20 12) show ed that the exposure of the INT -407 cell line for 7 2 h to the low est con- centration of Mt tested result ed in ROS generation. How ever , sig- ni fi cant differences with respect to the control were only observed with the highest concentration assayed, 1 0 0 0 m g/mL, after 24 h. Similarly , Lor dan et al. (201 1) also evidenced ROS generation in HepG2 cells exposed to CNa þ starting with the lower concentra- tions (50 – 1 000 m g/mL) and at all times of e xposure (4, 1 6 and 24 h). Nevertheless, o ther researchers did not observe any effects in ROS g eneration in Caco-2 cells exposed to the unmodi fi ed clay at the concentrations (the highest was 226 m g/mL) and times as- say ed ( Sharma et al., 201 0 ). The stress response generated depends on the cell lines exposed and the concentration rang es assayed. Lordan et al. (20 1 1) also evaluat ed the possible ROS gener ation in the HepG2 cell line e xposed to C93A , a modi fi ed Mt with a ternary ammonium salt. The results obtained were similar to those obtained after exposure to CNa þ , but the effect of C93A on in- tracellular R OS production was less prominent because the in- creased levels observed were not as substantial as those obtained with the unmodi fi ed clay and were not consistent over 24 h of exposure. Sharma et al. (20 1 0) observed that C30B, a quaternary ammonium salt-modi fi ed clay , did not induce ROS production at the conditions assayed in Caco-2 cells. In contrast, our research group ( Maisanaba et al., 20 1 3 ; 20 1 4a ) also ev aluated the stress response by analysing the ROS and GSH content of HepG2 and Caco-2 cells e xposed to C30B. In our case, when HepG2 cells were exposed to the modi fi ed clay , no signi fi cant alteration in ROS production w as observed at all concentrations (22, 4 4 and 88 m g/mL) and times of exposure (2 4 and 48 h). In contrast, a concentration-dependent decrease in the GSH content was ob- tained at the highest concentration tested and at both times of exposure . According to the results obtained by Maisanaba et al. (20 1 3) , Caco-2 cells e xposed to C30B experienced a signi fi cant decrease in the GSH content at 40 m g/mL af ter 48 h. Moreover , an alteration in ROS gener ation was also observed at 40 m g/mL and at both times of e xposure (24 and 48 h). The production of R OS may be related to the cell damage induced by C30B at this concentra- tion, as w as also corroborated by an aforementioned morpholo- gical study ( Maisanaba et al., 20 1 4a ). In contrast, Houtman et al. (201 4) also evaluated the stress response in HepG2 and Caco-2 cell lines e xposed to the other nov el modi fi ed clay , Clay2, in the presence of the other quater nary ammonium salt modi fi er , HDT A þ ACO. These resear chers only observed an alter ation in the GSH content in HepG2 cells e xposed to the modi fi ed clay , and signi fi cantly differences were obtained at all concentrations test ed (22, 4 4 and 88 m g/mL) and both times of exposure (24 and 48 h). In addition to Mt, studies with other clay minera ls with a si- milar structure have been published in the scienti fi c literatur e. For example, Meibian et al. (20 1 0) studied the ROS g eneration, SOD activity and MDA levels of a lymphoblast cell line exposed to two bentonites, an unmodi fi ed or nativ e bentonite (BPN) and a mod- i fi ed or activ e bentonite (BP A) activ ated with H 2 SO 4 . In this study , the authors observed that the highest oxidativ e response w as shown after exposure to BP A at all of the conditions tested. Little is known about in vitro stress assays of nanocomposites obtained from clays and polymeric matrices. However , some stu- dies of microcomposite materials used for different biological ap- plications have been performed. K evadiy a et al. (201 3) evaluat ed the stress effects of 6-mercaptopurine (an antineoplastic drug)- Mt-poly ( L -lactide) acid (PLA) microcomposite (6-MPMtPLA) in the neuroblastoma cell line IMR32. The MD A levels, SOD activity , prot ein carbonyl levels and GSH content were evaluat ed, and the results show ed that all of the endpoints in the assayed cells e x- posed to the test composite (1 0 ppm) did not show signi fi cant differences with respect to the control group. Speci fi cally , an in- crease in the protein carbony l groups was expressed in pristine 6-MP-tr eated cells, whereas 6-MP-Mt and microcomposite spheres (MPs) proved to be somewhat less toxic in this parameter . Intracellular glutathione estimation also revealed similar results as 6-MPMtPLA and MPs, i.e. , it was less damaging than the pristine antineoplastic drug. Similar results were obtained in another study in which the IMR32 cell line was exposed to capecitabine (CAP) (other antineoplastic drug) encapsulated in Na þ -Mt and further compounded with poly ( L -lactide) acid. In all cases, cells treated with CAP-Mt and MPs exhibited beha viour similar to that of the control group, showing low er oxidativ e responses compared with the pristine CAP cell group ( Kev adiya et al., 20 1 4 ). In this sense, the encapsulation of the antineoplastic drugs into the clay s has ame- liorat ed the to xicity . 2.2.2. Genotoxicity assays Due to the v ariety of mechanisms leading to xenobiotic-in- duced DN A damage and the range of mutagenic event s that may occur as a result, a battery of testing systems is req uired for the establishment of the genot oxic po tential of a substance under in- vestigation ( Doak et al., 20 1 2 ). At present, in vitro mutagenicity assays ar e included among the core set of to xicity tests for the safety assessment required by European legislation to authorize different substances. The most freq uently used genoto xicity assays are the Comet assay , Ames test and micronucleus assay ( Maisa- naba et al., 20 1 3 , 20 1 4a , b , c ). In this regard, the genoto xicity evaluation of clays and derived nanocomposites used in or destined for the food industry is of S. Maisanaba et al. / Environmental Resear ch ∎ ( ∎∎∎∎ ) ∎∎∎ – ∎∎∎ 10 Please cite this article as: Maisanaba, S., et al., T oxicological evaluation of clay minerals and derived nanocomposites: A review . Environ. Res. (20 1 5), ht tp://dx.doi.org/1 0. 1 0 1 6/j.envr es.201 4. 1 2.024 i T able 5 Oxidative str ess evaluation of unmodi fi ed/modi fi ed clays and derived micro/nanocomposit es. Material tested Modi fi er Experimental model Assays performed Concentration range Main results Refer ence - Sepiolite U nmodi fi ed Hamster and rat al- veolar macrophagues Reduction of cytocrome C (SOD) 5 m g/cm 2 dish and 25 m g/cm 2 dish in each cell line, respectivel y The clay mineral caused a sig- ni fi cant increase in the release of O 2  in both cell lines Hansen and Mossman (1 987) - Sepiolite U nmodi fi ed (both of them) PMN cell line R OS generation Not speci fi ed Sepiolite w as unreactive in ROS production while a high R OS gen- eration w as observed after kaolinite exposure Governa et al. (1 995) - Kaolinite AM cell line - Native bent onite (BPN) Unmodi fi ed HMy2.CIR cell line ROS g eneration, SOD activity , MDA levels 0 – 240 m g/mL The highest oxidative r esponse was presented by BP A exposure Meibian et al. (20 1 0) - Activ e bentonite(BP A) H 2 SO 4 - CNa þ Unmodi fi ed Caco-2 cell line ROS gener ation 0 – 226 m g/mL R OS production was not observed Sharma et al. (20 1 0) - C30B (un fi ltered and fi ltered) Quaternary ammonium salt (MT2EtOH) - CNa þ Unmodi fi ed HepG2 cell line R OS generation 0 – 10 0 0 m g/mL ROS production was observed with both cla ys Lordan et al. (20 1 1) - C93A T ernary ammonium salt (M2HT) - Mt Unmodi fi ed INT -407 cell line ROS generation 0 – 10 0 0 m g/mL ROS production was observed Baek et al. (201 2) - 6-MP þ Mt-PLA microcomposite 6-Mercapt opurine (6-MP) encapsulated with Mt IMR32 cell line MDA levels, SOD activ - ity , protein carbon yl levels, GSH content 1 0 ppm/test composite In all cases cells exposed to test composites showed lower oxidativ e responses than the other groups assay ed Kev adiya et al. (20 1 3) - C30B Quaternary ammonium salt (MT2EtOH) HepG2 cell line ROS generation, GSH content 0 – 88 m g/mL No signi fi cant altera tion in ROS generation w as observed but a de- crease of GSH content w as showed Maisanaba et al. (20 1 3) - Clay2 Hexadecyltrimeth ylammonium bromide (HDT A) þ Acetyl coline (AC O) Caco-2 and HepG2 cell lines ROS g eneration, GSH content 0 – 34 m g/mL and 0 – 88 m g/mL Both cell lines exposed to Clay2 did not present R OS generation. HepG2 showed a signi fi cant decreased in GSH content Houtman et al. (20 1 4) - CAP þ Mt-PLA microcomposite Capecitabine (CAP) encapsulated with Mt IMR32 cell line MDA lev els, SOD activ- ity , Protein Carbon yl levels, GSH content 1 0 ppm/test composite In all endpoints, cells exposed to test composites show ed lower o xi- dative r esponses than the other groups assay ed Kev adiya et al. (20 1 4) - C30B Quaternary ammonium salt (MT2EtOH) Caco-2 cell line ROS generation, GSH content 0 – 40 m g/mL Increased ROS and decr eased GSH content at the highest concentration Maisanaba et al. (20 1 4a) Cell lines: PMN: human polymorphonuclear leucocytes; AM: bovine al veolar macrophagues; HMy2.CIR: human B lymphoblast cell line; Caco-2: colorectal adenocarcinoma cell line; HepG2: liver hepatocellular carcinoma cell line; INT -407 : human normal intestinal cell line: and IMR32: human neuroblastoma cell line. S. Maisanaba et al. / Enviro nmental Research ∎ ( ∎∎∎∎ ) ∎∎∎ – ∎∎∎ 11 Please cite this article as: Maisanaba, S., et al., T oxicological ev aluation of clay minerals and deriv ed nanocomposites: A review . Environ. Res. (20 1 5), ht tp://dx.doi.org/1 0. 1 0 1 6/j.envr es.201 4. 1 2.024 i great interest. In previous opinions ( EFSA, 201 1b and 20 1 2 ), the EFSA Panel on Additives and Products or Substances used in Ani- mal Feed (FEED AP) has assessed the safety of bentonite and con- cluded that “ montmorillonites are not genoto xic based on a series of tests ” ( EFSA, 20 1 3 ), but different results have been obtained to date. An important number of studies related to the possible carci- nogenic and mutagenic effects of clay minerals in different cell lines are av ailable in the scienti fi c literature ( T able 6 )( Denizeau et al., 1 985 ; Gao et al., 20 0 0 ; Houtman et al., 20 1 4 ; Li et al., 20 1 0 ; Maisanaba et al., 201 3 ; Maisanaba et al., 201 4a , b , c; Meibian et al., 20 1 1 ; Sharma et al., 20 1 0). Several research groups have evaluat ed the DNA damage in- duced by unmodi fi ed Mt and CNa þ through different methods and experimental models, and all of them agree that Mt does not ex- hibit genot oxic or mutagenic potential ( Li et al.,201 0 ; Maisanaba et al., 20 1 4c ; Sharma et al., 201 0 ). However , different results were obtained in the case of modi fi ed clays. Maisanaba et al. (20 1 3) ev aluated the DN A damage using the comet assay in HepG2 cells exposed to C30B, evidencing DNA strand breaks at the highest concentration assay ed (88 m g/mL) af ter 48 h of exposure. How ever , a similar e xperiment in another cell line, Caco-2, resulted in no DN A damage ( Maisanaba et al., 20 1 4a ). In contrast, Sharma et al. (20 1 0) also studied the exposure to C30B in Caco-2 cells, and genoto xic effects in a concentration-related manner were ob- served. In this case, the differences between the results could be directly related to the concentration assay ed because Sharma et al. (20 1 0) used higher concentrations in comparison to Maisanaba et al. (20 1 4a) . Moreov er , the geno to xic potential of two novel modi fi ed clays, Clay1 and Clay2, has also been evaluated by the comet assay and compared with that of another modi fi ed clay , C20A. In this case, only DNA damage w as observed in cells (Caco-2 and HepG2) exposed to Clay2 at the highest concentrations tested (34 and 4 4-88 m g/mL, respecti vely) ( Houtman et al., 20 1 4 ). Other researchers ha ve studied similar structural clay s, such as bentonite and kaolinite. Gao et al. (20 0 0) evaluated the genot oxic effects in rat pulmonary alveolar macrophages exposed to un- modi fi ed quartz and kaolinit e and their modi fi cations with di- palmitoy lphosphatidylcholine (DPPC) by a single-cell gel electro- phoresis assay to detect the DN A damage induced. The results show that quartz and kaolin, regardless of whether it is treated, causes genoto xic effects, but DPPC-treated kaolin show ed no sta- tistically signi fi cant activity until day 5 at the highest concentra- tion (40 m g/mL), whereas the other samples induce DNA damage at lower e xposure times. Apart from the comet assay , other mutagenicity trials are per - formed to evaluate different alterations that could occur in the genetic material. The Ames test, according to the OECD (1 99 7) guideline for the testing of chemicals 4 7 1 , is the bacterial rev erse mutation test used to determine the mutagenicity of ex ogenous substances. The test identi fi es mutagenic compounds as those capable of reverting point mutations in histidine or tryptophan biosynthesis genes in Salmonella typhimurium or Escherichia coli , respectiv ely , restoring the ability of the bacteria to g enerate these essential amino acids. Usually , a combination of 5 S. typhimurium strains or 4 S. typhimurium strains plus 1 or 2 E. coli strains are req uired to detect a range of base substitution or frameshift events. The ease and cost effectiveness of the test system make it widely used in the safety analysis of chemical substances. It is an essential test within the current battery of assays req uired for genoto xicity ev aluation ( Doak et al., 20 1 2 ). According t o the OECD (20 1 0) guideline 487 , the in vitro mi- cronucleus assay (MNvit) is a test that rapidly determines the freq uency of gross chromosomal damage induced by a test agent. It has gained popularity as the test of choice over the chr omosome aberration assay because it is substantially quicker to perform and easier to anal yse and readily detects aneugens and clastogens (which the chromosome aberration assay canno t do unless it is speci fi cally modi fi ed). Thus, the MNvit is now recommended as one of the in vitro test systems to characterize the g enoto xicity of chemical and pharmaceutical agents ( Kirkland et al., 20 1 1 ). Sharma et al. (20 1 0) studied the mutagenic potential of CNa þ and C30B in both fi ltered and un fi ltered suspensions. In an y case, there were no indications of mutage nic activity in the two strains , T A98 and TA1 0 0, assayed either with or without metabolic acti- v ation up to the highest soluble concentration (1 4 1 m g/mL). How- ever , Maisanaba et al. (201 4c) also evaluat ed the mutagenic po- tential of CNa þ and C30B by the Ames test, obtaining positive results only in the case of the modi fi ed clay C30B in the presence of the S9 fract ion at four of the fi ve concentrations assayed (3 1 .25 – 250 m g/mL). In the same wor k , other quaternary ammonium salt- modi fi ed clays, Cla y1 and Clay2, showed a different mutage nic pro fi le, and only Clay1 showed positive results at the four highest concentrations (1 – 8 m g/mL) with S9. The presence of mutagenicity may be related to the modi fi er selected, the range of concentra- tions assay ed or the strains used in the test. In addition to the mutagenic evaluation of CNa þ , T ay lor et al. (20 1 4) also investigat ed the mutagenicity potential of halloy site. The most remarkable results from the Ames mutagenicity test show ed a slight but reproducibly elev ated growth at all con- centrations of CNa þ assay ed. The authors associated this fi nding to the presence of a bene fi cial trace mineral component in CNa þ . Howev er , in the case of halloy site, a diminished growth in the bacterial strains suggested to xicity rather than a mutagenic effect. Meibian et al. (201 1) also evaluated the possible genoto xic al- teration in the MHy2.CIR cell line exposed to BPN and BP A through the comet and micronucleus assays. These results correlate well with those from the afore-mentioned oxidativ e stress studies. Reports on the mutagenicity of nanocomposite materials are even scarc er . Maisanaba et al. (20 1 4b) analysed the mutage nic potential of PLA-Clay1 and PLA-Clay2 extracts by the Ames test. These were obtained from a nanocomposite formed by Clay1 þ PLA and Clay2 þ PLA, respectively , and did not present mutagenic po- tential in an y case. Also, genot oxicty and mutagenicity studies on potential modi fi ers are lacking and the results a vailable shown both positiv e and negative effects ( Dmochowska et al., 20 1 1 ; Grabinska-Sota, 20 1 1 ). Anew , different results dependent on the clays assay ed, the present modi fi er (if an y) and the concentration range test ed are presented in this section. 2.2.3. In fl ammation responses Among the biomarkers that are most freq uently assayed in relation to the in fl ammation process is the cytokine release, mainly inter leukin-6 (IL-6) and tumour necrosis factor - α (TNF- α ) ( Lopresti et al., 20 1 4 ). In fl ammation studies related with modi fi ed clays used in the food industry are scarce. The Caco-2 and HepG2 cell lines did not experiment an increase in IL-6 release af ter e xposure to the modi fi ed clays, Clay2 and C30B ( Houtman et al., 20 1 4 ; Maisanaba et al., 20 1 3 ). However , Elmore (20 03) reported that different clays, including Mt, induced cytoto xicity in several macrophage-type cell lines and have haemolytic activity tow ard the red blood cells of different species. Addi tionally , it has been reported that Mt clay may promote infection by a direct cytoto xic effect on neutrophils, making them unav ailable for bacterial phagocytosis ( Dougherty et al., 1 985 ). 2.2.4. Cell death Different markers are used to determine the cell death me- chanism af ter e xposure to a toxic substance. Among the most used are fl o w cytometry and caspase activities ( Napierska et al., 20 09 ; S. Maisanaba et al. / Environmental Resear ch ∎ ( ∎∎∎∎ ) ∎∎∎ – ∎∎∎ 12 Please cite this article as: Maisanaba, S., et al., T oxicological evaluation of clay minerals and derived nanocomposites: A review . Environ. Res. (20 1 5), ht tp://dx.doi.org/1 0. 1 0 1 6/j.envr es.201 4. 1 2.024 i T able 6 Genoto xicity of unmodi fi ed/modi fi ed clays and derived nanocomposites. Material tested Modi fi er Experimental model Assays performed Concentration range Main results Refer ence - Sepiolite Unmodi fi ed Rat hepatocytes Unscheduled DN A synth- esis (UDS) 1 and 1 0 m g/mL Sepiolite did not lead to signi fi cant UDS-induction Denizeau et al. (1 985) - Quartz Dipalmito yl phosphatidylcoline (DPPC) Rat pulmonary alveolar mac- rophagues cell line Single-cell gel electrophor - esis assay for DN A damage 0 – 40 m g/mL Genoto xic effects were observ ed in all cases Gao et al. (20 0 0) - Kaolin (both un- treated and treated) - NSP U nmodi fi ed Salmonella typhimurium strains (T A98,TA1 00, TA1 535, T A1 537 , TA1 02) Ames test 0 – 10 0 0 m g/plate No signi fi cant genot oxic effects were observed in an y assay Li et al. (201 0) Rat peripheral-blood cell line Micronucleus assay 0 – 50 0 mg/kg bw CHO cell line Comet assay 0 – 10 0 0 m g/mL - CNa þ Unmodi fi ed Salmonella typhimurium strains (T A98,TA1 00) Ames test 0 – 14 .1 m g/plate Genoto xic effects were observed by Comet assay in Caco-2 cells exposed to C30B Sharma et al. (20 1 0) - C30B (un fi ltered and fi ltered) Quaternary ammonium salt (MT2EtOH) Caco-2 cell line Comet assay 0 – 17 0 m g/mL (un- fi ltered)/0 – 226 m g/mL ( fi ltered) - Native bent onite (BPN) Unmodi fi ed H 2 SO 4 HMy2.CIR cell line Comet assay 0 – 240 m g/mL The highest genoto xic effects were ob- served after BP A exposure Meibian et al. (20 1 1) - Active bentonite(BP A) Micronucleus assay - Bentonite Unmodi fi ed Salmonella typhimurium strains (T A97 , TA1 02) Ames test 0 – 1 0 mg/plate No mutagenic activity was observed EFSA (20 1 3) - C30B Quaternary ammonium salt (MT2EtOH) HepG2 cell line Comet assay 0 – 88 m g/mL DNA damage w as observed in HepG2 exposed t o C30B Maisanaba et al. (20 1 3) - C20A Quaternary ammonium salt (2M2HT) Caco-2 and HepG2 cell lines Comet assay 0 – 34 m g/mL and 0 – 88 m g/mL Genoto xic effects were observed only in cells exposed to Clay2 Houtman et al. (20 1 4) - Clay1 Hexadecyltrimeth ylammonium bromide (HDT A) - Clay2 HDTA þ Acetylcoline (AC O) - C30B Quaternary ammonium salt (MT2EtOH) Caco-2 cell line Comet assay 0 – 40 m g/mL No DN A damage was observed Maisanaba et al. (20 1 4a) - PLA-Clay1 extract HDT A þ Mt þ PLA Salmonella typhimurium strains (T A97 , TA98,T A1 0 0, T A1 02, TA1 04) Ames test 0 – 1 00% of extract No mutagenic potential w as observed Maisanaba et al. (20 1 4b) - PLA-Clay2 extract HDT A þ ACO þ Mt þ PLA - CNa þ Unmodi fi ed Salmonella typhimurium strains (T A97 , TA98,T A1 0 0, T A1 02, TA1 04) Ames test 0 – 12 5 m g/mL Mutagenic activity w as observed for C30B and Clay1 e xposure Maisanaba et al. (20 1 4c) - C30B Quaternary ammonium salt (MT2EtOH) 0 – 250 m g/mL - Clay1 HDTA 0 – 8 m g/mL - Clay2 HDTA þ ACO 0 – 12 5 m g/mL - CNa þ Unmodi fi ed Salmonella typhimurium strains (T A1 537 , TA1 538, T A1 02) Ames test 0 – 0.0 1 g A slight elevat ed growth was observed in CNa þ exposure while a disminished growth w as detected in Hallosyte exposure T aylor et al. (20 1 4) - Hallosyte Unmodi fi ed CHO: Chinese hamster ovary cell line; HMy2.CIR: human B lymphoblast cell line; Caco-2: colorectal adenocarcinoma cell line; and HepG2: liver hepatocellular carcinoma cell line. S. Maisanaba et al. / Enviro nmental Research ∎ ( ∎∎∎∎ ) ∎∎∎ – ∎∎∎ 13 Please cite this article as: Maisanaba, S., et al., T oxicological ev aluation of clay minerals and deriv ed nanocomposites: A review . Environ. Res. (20 1 5), ht tp://dx.doi.org/1 0. 1 0 1 6/j.envr es.201 4. 1 2.024 i Lordan et al., 20 1 1 ) Some studies related t o the ev aluation of cell death produced by exposure to clays can be found in the scienti fi c literature ( Ta- ble 7 )( Geh et al., 20 0 6 ; Janer et al., 20 1 4 ; Lordan et al., 20 1 1 ; Liu et al., 20 1 1 ; Maisanaba et al., 20 1 3 ; Meibian et al., 20 1 0 ). Liu et al. (20 1 1) studied the enzymatic activity of caspase 3 and the per - centage of apoptotic/necr otic cells by fl ow cytometry in two dif- ferent cell lines, NIH3T3 and HEK293, exposed to unmodi fi ed Mt and oligo(S tyrene-Co-Acrylonitrile)-modi fi ed Mt (PSAN-Mt) at 1 g/ L. The results of the two assays pr ovided substantial evidence that less cell apoptosis w as induced by PSA-Mt compared with Mt in both treated cells, although signi fi cant differences wer e observed with respect to the control in both clay exposures. Similar results were obtained by Janer et al. (201 4) , who evaluated the same parameters in the HepG2 and SKMEL28 cell lines exposed to Mt and other modi fi ed montmorillonites with quaternary ammonium modi fi ers. An increase in the caspase 3/7 activities were observed after 4.5 h only in the HepG2 cell line and in both cell lines at the highest time of e xposure (48 h). R egarding the fl ow cytometry results, HepG2 exposed t o 1 0 0 m g/mL large pristine nanoclays for 48 h showed a sevenfold incre ase in the percentage of cells stained with Annexin V. Ho wever , these results did not match those ob- tained by Lordan et al. (201 1) because after 24 h, no increase in caspase 3/7 activity was det ected in the cells treated with CNa þ , the unmodi fi ed Mt, and C93A. Similarl y , Maisanaba et al. (201 3) did not detect an y variation in caspase 3/7 acti vity in the HepG2 cell line e xposed to C30B for 24 and 48 h. Nativ e and modi fi ed bentonites ha ve also been evaluated by other resear ch groups. Geh et al. (20 06) used sever al techniq ues to show that the IMR90 cell line e xposed to native or alkali-, acid- or organo-modi fi ed bentonites at a speci fi c concentration (20 m g/mL) suffered necrosis and apoptosis as the cell death response. Meibian et al. (20 1 0) also detected apoptotic cells after exposure to BPN and BP A in the HMy2.CIR cell line. Regar ding to the toxicity mechanisms of clays, cell death, genoto xicity , mutagenicity and oxidative stress have been widely investigated in the recent years, as it has been described above. Again, clays show speci fi c to xicity pro fi les. On the other hand, the research on inmunomodulatory effects of clays is still scarce and it should be promoted as allergic disorders have a high prevalence now adays. 2.3. Antimicrobial activity In this era, when many bacteria ha ve developed antibiotic re- sistance and may produce more harmful toxins as a defence me- chanism ( e.g. , Shiga-to xin), the investigation of natural alternativ e antibacterials is of paramount importance. Na tural clays ha ve been used for healing singe the earliest recor ded history , but their medicinal properties have largely not been scienti fi cally re- cognized ( Williams et al., 20 1 1 ). A v ariety of ph ysical and/or che- mical processes can provide antibacterial properties to clays. Ph ysical bactericide can occur by surface attraction between clay minerals and bacteria, which can hamper the passive and acti ve uptake of essential nutrients, disrupt cell envelopes or impair the ef fl ux of metabolites ( Ferris et al., 1 987 ). The natural antibacterial clays studied do not kill by ph ysical associations between the clay and bacterial cells ( Williams and Haydel, 20 1 0 ). Williams et al. (20 1 1) described various studies verifying the antimicrobial po- tential against E. coli using clay minerals and Fe-rich phases. How ever , two works related to the Mt antibacterial action by na- noh ybrids or derived nanocomposites with polyurethane ar e a vailable in the literature ( Su et al., 20 09 ; W ang et al., 20 12a ). Su et al. (20 09) ev aluated the potential against several bacterial strains ( Staphy lococcus aureus, Pseudomonas aeruginosa, Strept o- coccus pyrogens, E. coli and the methicillin- and oxacillin-resistant T able 7 Cell death evaluation of unmodi fi ed/modi fi ed clays and deriv ed nanocomposites. Material tested Modi fi er Experimental model Assays performed Concentration range Main results Reference - Native bent onite Unmodi fi ed IMR90 cell line Flow cytometry , gel electrophoresis 20 m g/mL The bentonite samples induced ne- crosis as well as apopto tic cell death Geh et al. (20 06) - Modi fi ed bentonites α -Quartz þ chemical modi fi cations (alka- lin, acid and organic) - Native bent onite (BPN) Unmodi fi ed HMy2.CIR cell line Flow cytometry 0 – 24 0 m g/mL Bentonite particles induced apoptosis Meibian et al. (20 1 0) - Active bentonit e (BPA) H 2 SO 4 - Mt Unmodi fi ed NIH3T3 cell line Caspase 3 enzymatic activ - ity , Flow cytometry 1 g/L Caspase 3 activity and apoptosis were observed in both cases, but lower with PSAN-MMT Liu et al. (20 1 1) - Oligo(styrene- Co- acrylonitrile) modi fi ed Mt (PSAN-Mt) Oligo(styrene-Co-acrylonitrile) HEK293 cell line - CNa þ Unmodi fi ed HepG2 cell line Caspase 3/activity 0 – 10 0 0 m g/mL No increase in caspase 3/7 activity w as detected Lordan et al. (20 1 1) - C93A T ernary ammonium salt (M2HT) - C30B Quaternary ammonium salt (MT2EtOH) HepG2 cell line Caspase 3/7 activity 0 – 50 0 m g/mL The caspase 3/7 activity did not show an y signi fi cant variation Maisanaba et al. (20 1 3) - Mt Unmodi fi ed HepG2 cell line Caspase 3/7 activity , fl ow cytometry 0 – 50 0 m g/mL Signi fi cant differences were observed in both biomarkers assay ed Janer et al. 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(Eds.), Health and Environ- mental Safety of Nanomaterials. W oodhead Publishing, Elsevier, The Nether - lands, pp. 1 33 – 14 6 . Ziegler, J.L., 1 997. Geophagy: a vestige of palaeonutrition? Trop. Med. Int. Health 7, 609 – 61 1 . S. Maisanaba et al. / Environmental Resear ch ∎ ( ∎∎∎∎ ) ∎∎∎ – ∎∎∎ 22 Please cite this article as: Maisanaba, S., et al., T oxicological evaluation of clay minerals and derived nanocomposites: A review . Environ. Res. (20 1 5), ht tp://dx.doi.org/1 0. 1 0 1 6/j.envr es.201 4. 1 2.024 i Resultados y Discusión / Results and Discussion 93 CAPÍTULO 2 / CHAPTER 2 Sara Maisanaba, María Puerto, Silvia Pichardo, María Jordá, F . Javier Moreno, Susana Aucejo, Ánge les J os IN VITRO TOXICOLOGICAL ASSESSMENT OF CLAYS FOR THEI R USE IN FOOD PACKAGING APPLICATIONS Food and Chemical Toxicology 57, 266-275, 2013 Our reference: FCT 7238 P-authorquery-v11 AUTHOR QUERY FORM Journal: FCT Article Number: 7238 Please e-mail or fax your responses and any corrections to: E-mail: correctio ns.eseo@else vier.sps.co. in Fax: +31 2048 52799 Dear Author , Please check your proof carefully and mark all corrections at the appropri ate place in the proof (e.g., by using on-scree n annotation in the PDF file) or compile them in a separate list. Note: if you opt to annotate the file with software other than Adobe Reader then please al so highlight the appropriate place in the PDF file. To ensure fast publicati on of your paper please return your correction s within 48 hours. For correction or revision of any artwork, please consult http://www .elsevier.com/ artworkinst ructions . Any queries or remar ks that have arisen during the processing of your manuscri pt are listed below and highlight ed by flags in the proof. Click on the ‘Q ’ link to go to the location in the proof. Location in article Query / Remar k: click on the Q link to go Please insert your reply or correction at the correspond ing line in the proof Q1 Please confirm that given names and surnam es have been identi fied correct ly . Q2 The following refer ences ‘Gutie ´ rrez-Praena (2011b, 2013) and Sharma et al. (2000) ’ were cited in the text but not listed. Kindly check and correct if necessary . Q3 This section compri ses references that oc cur in the reference list but not in the body of the text. Please position each reference in the text or, altern atively, delete it. Any reference not dealt with will be retained in this section . Thank you for your assistance. Please check this box if you ha ve no corrections to make to the PDF f ile Highlights  Clays used in food applications induce cytotoxic effects in HepG2 cell line.  The modifiers of the clays have a role in their toxicity .  Cloisite 30B induces cyto and genotoxici ty, GSH decrease and morphologi cal damage. FCT 7238 No. of Pages 1, Model 5G 13 April 2013 1 1 3 In vitro toxicological assessment of clays for their use in food packaging 4 applications 5 6 7 Sara Maisanaba a , María Puerto a , Silvia Pichardo a , María Jordá b , F. Javier Moreno c , Susana Aucejo b , 8 Ángeles Jos a , ⇑ 9 a Area of Toxicology, Faculty of Pharmacy, University of Seville, Profesor García González n ° 2, 41012 Seville, Spain 10 b Packaging, Transport, & Logistics Research Institute (ITENE), C/Albert Einstein 1, Parque Tecnológico, Paterna, Spain 11 c Area of Cellular Biology, Faculty of Biology, University of Seville, Avda. Reina Mercedes s/n, 41012 Seville, Spain 12 13 14 16 article info 17 Article history: 18 Received 21 January 2013 19 Accepted 29 March 2013 20 Available online xxxx 21 Keywords: 22 Clay 23 Montmorillonite 24 Cytotoxicity 25 HepG2 cell line 26 27 abstract 28 Montmorillonite based clays have a wide range of applications that are going to contribute to increase 29 human exposure to these materials. One of the most promising uses of clays is the development of rein- 30 forced food contact materials that results in nanocomposites with improved barrier properties. Different 31 organoclays have been developed introducing modifiers in the natural clay which is commercially avail- 32 able. However, the toxicological aspects of these materials have been scarcely studied so far. In the pres- 33 ent study, the cytotoxic effects of a non-modified clay (Cloisite Ò Na+) and an organoclay (Cloisite Ò 30B) 34 have been investigated in the hepatic cell line HepG2. Only Cloisite Ò 30B showed cytotoxicity. In order to 35 elucidate the toxic mechanisms underlying these effects, apoptosis, inflammation, oxidative stress and 36 genotoxicity biomarkers were assayed. Moreover, a morphology study with light and electron micros- 37 copy was performed. Results showed genotoxic effe cts and glutathione decrease. The most relevant 38 ultraestructural alterations observed were mitochondrial degeneration, dilated endomembrane systems, 39 heterophagosomes formation, fat droplets appearance and presence of nuclear lipid inclusions. Cloisite Ò 40 30B, therefore, induces toxic effects in HepG2 cells. Further research is needed to assess the risk of this 41 clay on the human health. 42 Ó 2013 Published by Elsevier Ltd. 43 44 45 1. Introduction 46 Clays have an array of commercial applications in different 47 fields: the manufactur e of inks, paints, greases and cosmetics, water 48 treatment processes, the controlled release of therapeutic agents, 49 food packaging, etc. ( Lordan et al., 2011 ). In regard to food packag- 50 ing applications, clays are used to improve the barrier properties of 51 food contact materials. This results in a length of the storage time 52 while keeping the product fresh. Polymers incorporating clay nano- 53 particles are among the first polymer nanocompo sites to emerge on 54 the market as improved materials for food packaging. This is due to 55 the easy availabili ty of the raw clay materials and because their 56 cation exchange chemistry has been intensive ly studied. In addi- 57 tion, these clay nanoparticl es have unique properties such as large 58 surface areas, large aspect ratios and improved mechanical , thermal 59 and optical properties ( Utracki and Kamal, 2002; Pavlidou and 60 Papaspyrides , 2008 ). The most frequently used clay in the prepara- 61 tion of polymer nanocompo sites is montmorill onite, which is the 62 major constituent of bentonite . Montmorillonite is a natural clay 63 which occurs as plate-like particles called platelets. These platelets 64 have an average thickness of only 1 nm, while its dimensions in 65 length and width can be measured up to 1 mm ( Lordan et al., 66 2011 ). One limitation of clays is the incompa tibility between the 67 hydrophi lic clay and a hydrophobi c polymer, which could cause 68 agglomer ation of clay in polymeric matrices ( Elmore and Andersen, 69 2003; Zeng et al., 2005 ). Therefore, surface modification of clay 70 minerals is an important step to achieve polymer nanocom posites. 71 By cation exchange with organic cations, clays become hydrophobic 72 and thereby compatible with polymers. Such modified clays are 73 referred to as organoclay s( Sharma et al., 2010 ). The most widely 74 known theories to explain the improved barrier properties of 75 polymer–clay nanocom posites are based on a theory develope d 76 by Nielsen (1967) , which focuses on a tortuous path around the clay 77 plates, forcing the gas permeant to travel a longer path to diffuse 78 through the film. 79 Successful technical developmen t of nanocom posites for food 80 packagin g has to overcome barriers in safety, technology, regula- 81 tion, standardizati on, etc. ( Silvestre et al., 2011 ). Regarding to 82 safety, since the use of clays and organoclays is increasing, it is 83 important to take into account their toxicity. Clays are natural 84 materials but they are not free of possible side-effects. In this 85 sense, toxicity studies of clays have been scarcely performed . The 86 toxicolog ical evaluation of these products can be faced using both 0278-6915/$ - see front matter Ó 2013 Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.fct.2013.03.043 ⇑ Corresponding author. Tel.: +34 954 556762; fax: +34 954 556422. E-mail address: [email protected] (Á. Jos). Q1 Food and Chemical Toxicology xxx (2013) xxx–xxx Contents lists available at SciVerse ScienceDi rect Food and Chemi cal Toxic ology journal homepage: www. elsevier.com/loc ate/foodchemtox FCT 7238 No. of Pages 11, Model 5G 13 April 2013 Please cite this article in press as: Maisan aba , S. , et al. In vitro toxicolog ical assessmen t of clays for their use in food packagin g applica tions. Food Chem. Toxi col. (2013), htt p://dx.doi.org /10.1016/ j.fct.20 13.03.043 87 in vivo and in vitro methods. The primary aim of in vitro testing is 88 toxicity screening and the understand ing of biological responses 89 and underlying mechanisms ( EFSA, 2011 ). Moreover, in vitro meth- 90 ods are encouraged in toxicologica l research for ethical reasons. 91 Toxic effects of clay minerals have been shown to occur mainly 92 after inhalation ( Carretero et al., 2006; Sharma et al., 2010 ). But 93 also, one of the most likely routes of exposure to these clays for 94 the general population is the oral pathway, since they are present 95 in food contact materials. Tateo and Summa (2007) reported that 96 the ingestion of clays is common at low doses in food preparations , 97 in pharmac euticals for oral administ ration, and as herbal remedies. 98 Moreover, distribution studies performed with different nanoparti- 99 cles showed that they can translocate to several organs such as li- 100 ver, kidney, lungs. ( Kim et al., 2008; Kwon et al., 2008; EFSA, 2009) 101 so for clays this possibility cannot be discarded. 102 In view of the limited toxicolog ical information of clays, in this 103 study we aim to evaluate the toxicity of an unmodified (Cloisite Ò 104 Na+) and an organomodified clay (Cloisite Ò 30B) in the human 105 hapatocellul ar cell line HepG2. For this purpose, basal cytotoxicity 106 biomarkers and mechanistic biomarkers of oxidative stress, 107 inflammation and genotoxicity have been investigated . Moreover, 108 a histopatholo gical study has been also performed. 109 2. Materials and methods 110 2.1. Clay materials and characterization 111 Unmodified montmorillonite (Cloisite Ò Na+) and the organically modified one 112 (Cloisite Ò 30B) were obtained from Southern Clay Products, INC. (modifier: methyl, 113 tallow, bis-2-hydroxyethyl, quaternary ammonium, concentration: 90 meq/100 g 114 clay). Both clays were characterized by thermogravimetric analysis (TGA), X-ray dif- 115 fraction (XRD) and particle size distribution (PSD). 116 TGA analysis of Cloisite Na+ and Cloisite 30B were performed on a Q5000IR 117 thermobalance (TA Instruments) by heating the samples from room temperature 118 up to 900 ° C with heating rate of 10 ° C/min, in nitrogen atmosphere. Approximately 119 7 mg of each finely ground sample was heated in a platinum crucible. Powder XRD 120 analyses were performed using a D8ADVANCE A25 Bruker diffractometer. The clay 121 powders were mounted on a sample holder with a large cavity and a smooth sur- 122 face was obtained by pressing the powders with a glass plate. The X-ray diffraction 123 patterns were measured from 1.5 ° to 25 ° (2 h ) at a scan rate of 0.02 s. Particle size 124 distributions of the samples were estimated with a Mastersizer 3000 (Malvern 125 Instruments Ltd., UK). Dispersion in dry basis was the selected method to perform 126 the different assays, with controllable airflow and feed rate. This method allows 127 measuring particle sizes between the range 0.1–3500 l m. 128 2.2. Clays test solutions 129 The test concentrations for both clays were selected taking into account previ- 130 ous dispersion experiments in order to avoid interferences with the measurement 131 system. The highest concentrations tested were 62.5 and 500 l g/mL for Cloisite Ò 132 Na+ and Cloisite Ò 30B, respectively. Test solutions were prepared in serum-free 133 medium. Three sonication steps of 10 s each one at an amplitude of 40% were per- 134 formed using an ultrasonic tip (Dr. Hielscher, Germany) to disperse the test 135 concentrations. 136 2.3. Cell culture 137 HepG2 (human hepatocellular carcinoma epithelial cell line) (HB-8065) was ob- 138 tained from the American Type Culture Collection (ATCC, Manassas, VA, USA). Cells 139 were cultured in monolayer in Eagle’s Minimum Essential Medium (ATCC) supple- 140 mented with 10% of fetal calf serum (FCS, Gibco, New Zealand), 100 U/mL penicillin 141 and 100 l g/mL streptomycin (Gibco, New Zealand). Cells were grown at 37 ° C and 142 5% CO 2 in a humidified atmosphere. 143 2.4. Cytotoxicity assays 144 For cytotoxicity assays, exposure concentrations for Cloisite Ò Na+ were set at 0, 145 0.49, 0.98, 1.95, 3.91, 7.81, 15.63, 31.25, 62.5 l g/mL and for Cloisite Ò 30B at 0, 3.91, 146 7.81, 15.63, 31.25, 62.5, 125, 250, 500 l g/mL. HepG2 cells were seeded at a density 147 of 7.5  10 5 cell/mL in 96 wells plates and exposed to the clays for 24 and 48 h. 148 Total cellular protein content (PC) was quantified following the method of Brad- 149 ford (1976) . After exposure, cells were washed with phosphate buffer saline (PBS) 150 and incubated with 0.1 N NaOH for 2h at 37 ° C. Later, a 22% Coomassie brilliant 151 blue solution (Biorad, Spain) was added to the wells and within 30 min absorbance 152 was read at 595 nm on a microplate reader (Biotek, USA). 153 Neutral Red (NR) uptake is a suitable endpoint to determine viable cells, be- 154 cause this dye is taken up by viable lysosomes. This assay was performed according 155 to Borenfreund and Puerner (1984) . Briefly, NR in medium is absorbed and concen- 156 trated in lysosomes of cells. NR uptake is proportional to the concentration of the 157 NR solution and the numbers of viable cells. NR can be extracted from lysosomes 158 for quantitative measurement at 540 nm. 159 The MTS tetrazolium reduction assay was performed according to a procedure 160 based on Baltrop et al. (1991) , being MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carb- 161 oxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt bioreduced by 162 cells into a colored formazan product soluble in culture medium. Cells were directly 163 incubated with MTS tetrazolium salt for 2 h at 37 ° C and absorbance was read at 164 492 nm. 165 Only when cytotoxicity was observed, mechanistic biomarkers were deter- 166 mined. In this case, the mean effective concentration (EC 50 ) of the most sensitive 167 cytotoxicity endpoint was chosen as the higher exposure concentration to investi- 168 gate mechanistic biomarkers along with the fractions EC 50 /2 and EC 50 /4. 169 2.5. Caspase-3/7 activity 170 Caspase-3 and -7 activities were used as apoptosis biomarkers in cells exposed 171 for 24 and 48 h to the clays. Manufacturer instructions from the kit (Caspase-Glo Ò 172 3/7 Assay, Promega, USA) were followed. 173 2.6. ROS generation 174 The production of ROS was assessed in 96 wells microplates using the dichloro- 175 fluorescein (DCF) assay. Cells were incubated with 200 l L, 40 l M 2 0 ,7 0 -dichloroflu- 176 orescein diacetate (DCFH-DA) in culture medium at 37 ° C for 30 min, and then 177 washed with PBS and resuspended in 200 l L of PBS. The formation of the fluores- 178 cence oxidized derivative of DCF-DA was monitored at emission wavelength of 179 535 nm and excitation wavelength of 485 nm. ROS production was expressed as 180 fluorescence arbitrary units ( Puerto et al., 2010 ). 181 2.7. GSH content 182 Cellular glutathione (GSH) content was evaluated by reaction with the 183 fluorescent probe monochlorobimane (mBCl) ( Jos et al., 2009 ). This molecule forms 184 a thioether adduct with GSH in a reaction catalyzed by the enzyme glutathione- 185 S-transferase (GST). After the cell exposure the medium was discarded and cells 186 were incubated at 37 ° C for 20 min in the presence of 40 l M mBCl. Later on cells 187 were washed with PBS and the fluorescence was recorded in a spectrofluorometer 188 (Biotek, USA) at the following excitation/emission wavelengths: 355/460. Results 189 were expressed as fluorescence arbitrary units. 190 2.8. Interleukin-6 leakage 191 For this assay the culture medium of the cells after 24 and 48 h exposure to the 192 clays was used. Manufacturer instructions from the kit (EH2IL6, Thermo Scientific, 193 USA) were followed. 194 2.9. Comet assay 195 The comet assay was performed to detect DNA strand breaks. HepG2 cells were 196 seeded into 12-well tissue culture treated plates (Corning Costar Corporation, New 197 York, USA) and left overnight at 37 ° C in 5% CO 2 to attach to the plates. Approxi- 198 mately 3.5  10 5 cells in each well were exposed with different concentrations of 199 Cloisite Ò 30B (0, 22, 44 or 88 mg/mL) after 24 and 48 h to exposure. 200 In order to monitor the ongoing process of the assay, a negative control (cells 201 treated with medium without fetal calf serum) and a positive control (cells treated 202 with a solution of 100 l MH 2 O 2 ) were included. After treatments cells were washed 203 and detached in PBS. The comet assay was applied as previously described by 204 Collins et al. (1997) with modifications ( Corcuera et al., 2011 ). Briefly, cells were 205 resuspended in PBS at a concentration of 2.5  106 cells/mL. This suspension 206 were mixed with 1% low melting point agarose and placed on a microscope slide. 207 Once the gels had become solid, the slides were dipped into lysis solution at 4 ° C. 208 All nucleotides were denatured in a high-pH buffer. Electrophoresis was carried 209 out approximately at 25 V (300 mA) and the DNA was gently reneutralized in PBS 210 and washed in H 2 O. After neutralization, microscope slides are fixed in 96% ethanol 211 and absolute ethanol. Finally, DNA was stained with SYBR Gold nuclei acid gel stain 212 and was visualized with an Olympus BX61 fluorescence microscope (20  objective) 213 coupled via a CCD camera to an image-analysis system (DP controller-DP manager). 214 Images of randomly selected nuclei ( P 100) per experimental point were analyzed 215 with the image analysis software (Comet Assay IV, Perceptive Instruments, UK). 2 S. Maisanaba et al. / Food and Chemical Toxicology xxx (2013) xxx–xxx FCT 7238 No. of Pages 11, Model 5G 13 April 2013 Please cite this article in press as: Maisan aba , S. , et al. In vitro toxicolog ical assessmen t of clays fo r their use in food packagin g applica tions. Food Chem. Toxicol. (2013), http ://dx.doi. org/10.1016/ j.fct.20 13.03.043 216 2.10. Morphology 217 Cells were exposed to different concentrations of Cloisite Ò 30B (0, 22, 44 or 218 88 mg/mL) during 24 and 48 h of exposure. Afterwards, cultured cells were fixed di- 219 rectly in the cell culture dish in 1.6% glutaraldehyde in 0.1 M cacodylate buffer, pH 220 7.2, for 60 min at 4 ° C. They were all postfixed in 1% osmium tetroxide for 60 min at 221 4 ° C. Samples were dehydrated in ethanol at progressively higher concentrations 222 and embedded in Epon (epoxy embedding medium). Toluidine blue-stained semi- 223 thin sections (0.5 l m thick) used as controls were viewed in a Leitz (Aristoplan) 224 light microscope. Thin sections (60–80 nm thick) were cut on a Reichert-Jung Ultra- 225 cut E ultramicrotome, stained with uranyl acetate and lead citrate, and examined in 226 a Philips CM-10 transmission electron microscope. 227 2.11. Calculations and statistical analysis 228 All experiments were performed at least three times and at least in duplicate 229 per concentration. Statistical analysis was carried out using analysis of variance 230 (ANOVA), followed by Dunnett’s multiple comparison tests. Differences were con- 231 sidered significant from p < 0.05. EC 50 values, mean effective concentration, concen- 232 tration of test chemical that modified each biomarker by 50% (positive or negative) 233 in comparison with appropriate untreated controls, were determined by linear 234 interpolation. 235 3. Results 236 3.1. Characterizati on of Cloisite Ò Na+ and Cloisite Ò 30B 237 TGA results of both clays are presented in Fig. 1 . Loss weight 238 percent (%), and derivative weight (%/ ° C) are presented versus tem- 239 perature. It can be observed that only the sample Cloisite Ò 30B has 240 big loss step in the range between 200 and 500 ° C, indicating that 241 an organic compound is being released in this step. The percentage 242 of organic modifier in Cloisite Ò C30B is 35.98%, as calculated in the 243 TGA curves. Cloisite Ò Na+ hardly presents any weight change in 244 this range of temperature. 245 XRD results of both clays are presente d in Fig. 2 . The signal 246 intensity is plotted versus de 2 h . Results show the typical patterns 247 associated with a montmorilloni te material. The main differenc e 248 corresponds to the signal associate d to the interlayer space in the 249 clays (corresponding to the d(0 0 1) diffraction peak); in the case 250 of Cloisite Ò Na+ is 8.8 (2 h ), and in the case of Cloisite Ò 30B is 4.7 251 (2 h ). The distances of silicate layers can be calculated by Bragg’s 252 Law ( n k =2 d sin h , d = layer distance). The measured d 0 0 1-spacing 253 of Cloisite Ò Na+ is 10.0 Å (2 h = 8.8), but after cation exchange with 254 the modifier (Cloisite Ò 30B) d 0 0 1-spacing became 18.7 Å (2 h = 4.7). 255 PSD of the samples has been calculated and results are pre- 256 sented in Table 1 . It can be observed that particle size of Cloisite Ò 257 30B is lower for all the values (Dv10, Dv50, and DV90) than those 258 of Cloisite Ò Na+. 259 3.2. Cytotoxici ty assays 260 Cloisite Ò Na+ did not induce cytotoxic ity after the exposure of 261 the cells for 24 and 48 h at the concentratio ns assayed ( Fig. 3 ). Only 262 the PC assay showed a small significant reduction at the higher 263 concentr ation (62.5 l g/mL). The EC 50 could not be calculated in 264 any of the biomarkers. 265 Protein content was the less sensitive endpoint of Cloisite Ò 30B 266 cytoxicity . After 24 h exposure to Cloisite Ò 30B, this endpoint de- 267 creased from 250 l g/mL, with a calculated EC 50 of 285 ± 9 l g/mL. 268 Similar reductions of protein content were remarkable after 48 h, 269 although after 2 days of exposure, the decrease could be apprecia- 270 ble already from 125 l g/mL in comparison to 24 h ( Fig. 4 a). 271 Regarding to NR uptake, no significant changes were observed in 272 the four lower concentratio ns used, although a concentratio n- 273 depende nt decrease was observed from 62.5 l g/mL with EC 50 val- 274 ues of 88 ± 4 and 55 ± 3 l g/mL at 24 and 48 h, respectively 275 ( Fig. 4 b). MTS metabolizati on decreased substantially , with a 276 reduction of around 90% at the higher concentration tested com- 277 pared to the control. After 48 h, MTS metaboli zation was signifi- 278 cantly reduced between 62.5 and 500 l g/mL. EC 50 values for this 279 endpoint varied between 158 ± 7 and 79 ± 5 l g/mL after 24 h and 280 48 h respectively ( Fig. 4 c). 281 The most sensitive endpoint for Cloisite Ò 30B was the neutral 282 red uptake, therefore the EC 50 value of this biomarker (88 l g/mL) 283 was chosen as the higher exposure concentratio n for the mecha- 284 nistic studies in the human hepatocellular cell line along with 285 the fractions EC 50 /2 and EC 50 /4, being the concentr ations used 286 88, 44 and 22 l g/mL. 287 3.3. Caspase-3/7 activity 288 The caspase activity assay did not show any significant varia- 289 tion with any of the concentrations of Cloisite Ò 30B and at any time 290 of exposure ( Fig. 5 ). 291 3.4. ROS and GSH content 292 When HepG2 cells were exposed to Cloisite Ò 30B during 24 and 293 48 h, no significant alteration on ROS was observed at any of the Fig. 1. TGA results for Cloisite Ò Na+ (square) and Cloisite Ò 30B (round). S. Maisanaba et al. / Food and Chemical Toxicology xxx (2013) xxx–xxx 3 FCT 7238 No. of Pages 11, Model 5G 13 April 2013 Please cite this article in press as: Maisan aba , S. , et al. In vitro toxicolog ical assessmen t of clays for their use in food packagin g applica tions. Food Chem. Toxi col. (2013), htt p://dx.doi.org /10.1016/ j.fct.20 13.03.043 294 exposure concentratio ns in comparison to the control group 295 ( Fig. 6 a). In contrast, a concentratio n-dependent decrease of GSH 296 was observed being the GSH content 10-fold lower at the higher 297 concentratio n assayed. Moreove r, there were not statistical differ- 298 ences with respect to the control in any concentration tested and 299 times of exposure ( Fig. 6 b). 300 3.5. IL-6 leakage 301 The IL-6 assay showed no increase in the IL-6 content in the cell 302 culture medium of the cytotoxicity assays in any of the treatments 303 performed (data not shown). 304 3.6. Genotoxicity : comet assay 305 Cloisite Ò 30B induced time-dependent increases of DNA strand 306 breaks in HepG2 cells ( Fig. 7 ). After 24 h of exposure no changes 307 were observed after exposure to the clay; in contrast a significant 308 increase of DNA damage was observed after 48 h incubation with 309 the highest concentratio n (88 l g/mL). The mean value of the% tail 310 DNA was 23.4, while in the control group was only 2.9%. The per- 311 centage of DNA in the tail for 88 l g/mL is not significantly different 312 from the percentage of DNA found for the positive control. 313 3.7. Morphologic study 314 3.7.1. Light microscope observatio ns 315 Control HepG2 cells observed under light microscope are shown 316 in Fig. 8 a. After 24 h of exposure to 22 l g/mL Cloisite Ò 30B, they 317 are still growing with similar morphological characteri stics to the 318 control group. However, after 48 h of exposure to 88 l g/mL Cloi- 319 site Ò 30B, a decrease in the growing rate is observed, which could 320 be due to a cellular cycle stop and cell death ( Fig. 8 b). Moreover, 321 cells show severe morphological changes such as intense 322 vacuolizati on. Fig. 2. DRX results for Cloisite Ò Na+ (rhombus) and Cloisite Ò 30B (square). Table 1 PSD results for Cloisite Ò Na+ and Cloisite Ò 30B. Sample Dv 10 ( l m) Dv 50 ( l m) Dv 90 ( l m) Cloisite Ò Na+ 5.33 15.71 33.23 Cloisite Ò 30B 2.63 9.20 24.58 (A) (B) (c) MTS 0 20 40 60 80 100 120 % of Control 24 h 48 h Cloisite® Na+ (µg/mL) PC 0 20 40 60 80 100 120 0 0.49 0.98 1.95 3.91 7.81 15.63 31.25 62.50 0 0.49 0.98 1.95 3.91 7.81 15.63 31.25 62.50 0 0.49 0.98 1.95 3.91 7.81 15.63 31.25 62.50 % of Control 24h 48h Cloisite® Na+ (µg/mL) * * NR 0 20 40 60 80 100 120 % of Control 24 h 48 h Cloisite® Na+ (µg/mL) Fig. 3. Protein content, PC (a); neutral red uptake, NR (b); and reduction of tetrazolium salt, MTS (c) of HepG2 cells after 24 h and 48 h of exposure to 0– 62.5 l g/mL Cloisite Ò Na+. All values are expressed as mean ± SD.  Significantly different from control ( p 6 0.05). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) 4 S. Maisanaba et al. / Food and Chemical Toxicology xxx (2013) xxx–xxx FCT 7238 No. of Pages 11, Model 5G 13 April 2013 Please cite this article in press as: Maisan aba , S. , et al. In vitro toxicolog ical assessmen t of clays fo r their use in food packagin g applica tions. Food Chem. Toxicol. (2013), http ://dx.doi. org/10.1016/ j.fct.20 13.03.043 Resultados y Discusión / Results and Discussion 105 CAPÍTULO 3 / CHAPTER 3 Sara Maisanaba , Klara Herc og, Metka Filipic, Ángeles Jos, Bojana Zegura GENOTOXIC POTENTIAL OF MONTMORILLONITE CLAY MINERAL AND ALTERATION IN THE EXPRESSION OF GENES INV OLVED IN TOXICITY MECHANISMS IN THE HUMAN HEPATOMA CELL LINE HE PG2 Enviado a Journal of Hazardous Materials/ Sent to J ournal of Hazardous Materials Elsevier Editori al System (tm) for Jo urnal of H azardous Ma terials Manuscri pt Draf t Manus cript N u mber: HAZ MAT-D- 15 - 03066 Title: Gen o to x ic po tential o f Montm orillonite clay min eral and alteration i n the e x pre ss ion of gene s involved i n toxicity mec hanisms in th e human hepat oma cell lin e HepG 2 Article Typ e: Resear ch Pap er Keyword s: Cloisite®N a+ ; t o xicity; mi cronucle us; g ene expre ssion; qPC R. Corresp onding Aut hor: Mr s. Sara Maisanaba, Corresp onding Aut hor's In st itution: First Au thor: Sara Maisana ba Order of Auth o rs: Sara Maisanaba; Klar a Hercog; Metka Fili pic; Angeles Jos; Bo ja na Zegura Abstract: Montmorill onite, also kn own as C loisite® Na+ (CNa+), is a natural cl ay with a wide rang e of well-doc umente d and n ovel appli cation s, such as p harmace u tic al produ cts or f ood packaging . Although considere d a low toxic pr oduct, the e xpected i ncreased expos ure to CNa +a rise s concern on the potential c o n sequen ces on human and env ironmental hea lth especially as its gen otoxicity has sc arc ely been inv estigated s o far. Thus, w e inves tigated, for t he fir st time, t he influ ence of non-cytotoxi c concentra tions of CN a+ (1 5.65, 31.25 an d 62.5 µg/mL ) on genomic i nstability o f human h epat oma cell line (Hep G2) by determi ning the forma tion of micron uclei (MNi), n ucleoplasmi c bridges (NPBs ) and nuclear bu ds ( NBUDs ) with the Cytoki nesis block micr onucleus cytome as say. Further o n we st udied the influ ence of C Na+ on th e expressi on of severa l genes involved in toxicity mec hanisms usi ng the real-time q uantitative P CR. The res u lts s h owed t hat CNa+ incre ased the n umber of MNi , while the number s of NBU Ds and NP Bs were n ot affected. I n addi tion it dereg ulated gen es i n all the gro u ps studied, m ainly after long er tim e of e xposur e. These findings provi de th e eviden ce that CNa + is potential ly genot oxic. Th erefore fur ther studie s that will elucidate t h e m o lecul ar mech ani sms involved in toxic a ctivity o f CNa+ are needed for haz ard iden tification and h uman safety as se ssmen t. Novelty statemen t The authors decla re that the results included in the present m anuscrip t hav e not been submitted bef ore to any other scientifi c journal. To the extent o f our knowledge, this is th e first study that r eports that Cloisite®Na + induces altera tions in the gene expr ession as well as genotoxic effe cts. *Novelty Statement Highlights - Cloisite®Na + has a w id e ran ge o f well-d o cu mented and novel app lications - Cloisite®Na + ind uces micronu cleus, but not nuclear bridges or nuclear bud s in HepG2 cells - Cloisite®Na + ind uces chang es in the gene expression - Gene alterati o n is pr ese nt ed mainly after 24h of exp osure to Cloisite®Na + Highlights (for review) Sara Maisan aba Hernán dez Area of T oxicolog y. Faculty of Pharm acy. Univ ersit y of Sev i lla C/Profesor G arcía Gonz ál ez 2, 410 12 Sevilla, Sp ai n Tel.: +34- 954556762; fax: +34- 954556422. E-m ail address: saram [email protected] s 24 th July , 2015 Dear E ditor, We would be ver y gr ateful if y ou consider the manuscript entitled “ Genotoxic potential of Montmorillonite clay mineral and alteration in the expres sion of gene s involved in toxicity mechanism s in the human h epatom a cell li ne HepG2 ” for its publication in “ Journal of Hazardous Materials ” . Although cla y mineral s have a wide range of applications, in rega rd to their tox icological profile sc arce data are available in the scientific literature about their effects on the genetic material. Thus, t o the ex tent of our knowled ge , this is the first work that stud ies the influen ce o f Montmorillonite (Cloisite®Na + ) on genomic inst ability b y the C y t okinesis block micronucleus c y tome assay, as well as, the Cloisite®Na + induced modulation of the expression of selected ge nes involved in the main toxicity mechanisms in HepG2 cell li ne. The results provide new data about this clay mineral, as it has shown to be potentially genotoxic. I am looking f orward to receiving a positive answer from you. Sincerely, Sara Maisanaba Hernández P.S. Total number of words: 4994 Cover Letter 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 1 Genotoxic po tential o f Montmo rillonite c l ay mine ral and alteration i n the exp ression o f genes invo lved in toxici ty mechanis ms in the human hepato ma cell line Hep G 2 Sara Maisanaba a* , K lara H ercog b , Metka Filipic b , Ángeles Jos a , Bojana Zegura b a Area of Toxi cology, Facu lty of Phar macy, Universi ty of Seville , Profesor García González n° 2, 41012 Seville. Sp ain. b National Institut e of Biology, Depart ment for Genetic T oxicology and Cancer Biol o gy, Vecna pot 111, 10 0 0 Ljubljan a, Slovenia. *Corresponding author: Sara Maisanaba Area of Toxic o logy, Facult y of Pharmacy, University of Seville, Profesor García G onzález n°2, 41012 Seville. Sp ain. E-mail addre ss: sara [email protected] Tel: +34 95 4 55 6762 *Manuscript Click here to view linked References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 2 Abstract Montmorilloni te, also known as Cloisit e ®Na + (CNa + ), is a natural cla y with a wide rang e of well-docu mented and n ovel applications, such as pharmaceutical pr oducts or food packaging . Although c o nsidered a low t o xic pr oduct, the expe cte d incr eased exposure to CNa + arises concern on the potential cons equences on human and environ mental health especially as i ts gen o toxicit y has scarcel y been investi gated so far. Thus, we inve stigated, for the first ti m e, the influenc e of non-cytotoxi c concentr ations of CNa + (15.65, 31.25 and 62. 5 µg/mL ) on genomic instabil ity of human hepat oma cell lin e (HepG2) by det erminin g the formation of m icronuclei (MNi), nucle oplasmic bridges (NPBs ) and nuclear bud s (NBUDs) with the Cytokinesis bl ock micronucleus cyto m e assa y . Furt her on we stud ied the influence of CNa + on the expr essio n of several g enes in v olved in toxicit y mechanisms using the real -tim e quantitative P CR. The results showed that CNa + incr eased the number o f MNi, while the numbers of NBU Ds and NPBs wer e not aff ected. In add itio n it deregu lated gen es in all the groups studied, m ainly aft er longer time o f exposur e. These find ings pr ov ide the evidenc e that CNa + is potentially genotoxic. Theref o re further studies that will elucid ate the molecul ar mechanisms in v olved in toxic activit y of CNa + are ne eded for hazard iden tification and human safety assess m ent. Keywords: Cl oisite®Na + ; t oxicity; micronucleus; gene expression; qPCR. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 3 1. Introd uction Clays and cla y minerals are recognized as the materials o f the 21st century be cause they are abund ant, inexpensive and envir o nmentall y fri endly [1].There ar e well over 100 documented industrial applications of clay m a terials. Clays ar e utilized in the pr oc ess industries, in agr icultural a pplications, in engineerin g and construction app licatio ns, in environmen tal remedia tion, in geology [2], as ph armaceutical and co sm et ic pr oducts [3], and in many other miscellaneous appli cations. They belon g to the ph yllosilicate gr oup that comprises diff erent structurally rela te d co m pounds [4 ]. Amo ng them, montmorill onite (Mt) is a smectite, 2:1 phyllosilicat e, meaning that it has two tetrahedral sheets sand wic hing a central octahedral shee t, and th e particles ar e plate-shaped [ 5 ]. It has the following molecular formula: (Na, Ca) 0.33 (Al, Mg ) 2 (Si 4 O 10 ) (OH) 2 ∙ nH 2 O [6]. Mt, also com m ercial ly known as Cl o isite®Na + ( CNa + ), h as some of the p ote n tial applications mentioned ab o ve. For exa m ple, it is used as drug delivery sy ste m [7] because i t is generally consid ered as a low toxic material compared to other inorgan ic deliver y carriers [8] . Also, in the f o od industr y , it is chemicall y modified t o be incorporat ed in the fo od packagin g in order to give new ma terials known as nan ocom posite s, which r epresent a new al ternative to conventional te chnologies for i mproving pol y mer pr operties [9]. Taking into acc o unt all thes e well-known and new applications that clay minerals have, the human an d environ m en tal exposure to thes e compounds will pr o bably incr ease in the near future. Therefor e, the potential cons equences on hum an and envir onmental heal th are of concern and a saf ety evaluation of cla y s is requ ired . Recently the toxic effec ts and the underlying me chanisms of different clays, including CN a + , have b een revi ewed by Maisanab a et al. [10] . Toxic eff ect s of cla y minerals in general ha ve been shown to occur m ainl y after inhalation [5, 11], but one of the most likely ro utes o f e xposure to these cla y mine rals fo r the general populati o n is b y or al ingestion, since they are potentially present in f ood and pharmaceuti cal products, etc. Due to the co mmercial availab ility of CNa + reports on its toxicity are more abundant in the scientific li terature c ompared to others clay mineral s . Mor eov er , due to i ts wide ap plication and increasing use the int erest in toxicological a spects of CNa + and its deri v atives has increased in the recent y ears [1 0 ]. CN a + has been r eported t o be n ot toxic in ani mals [12], how ever, i n vitro , CNa + has be en shown to redu ce the pr oliferation o f human nor mal intestinal cells (IN T - 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 4 407) and has b ee n cyt o toxi c at high c oncentrations [6] . Similarly, Lordan e t al., [ 13] reported cytotoxic acti vity of CNa + in human hepa toma c ell line (Hep G2), while Maisanab a et al. [ 14] at lower tested co ncentrati ons did not detect c y tot oxic a ctivity of CNa + in the sa me cell line as well as in a hum an colon ce ll line (Caco-2) [15]. Genotoxicity st udies showed that CNa + is not mut agen ic in Salmonella typhimurium [5, 16] and d oes not induce D NA strand breaks in Caco-2 cells [5] . In vit r o data on the induction o f reactive ox ygen species (R OS) by CNa + are contradic tory. Some literatur e data report that CNa + induces ROS f o rmati o n [13] , while at l o wer c oncentrati o ns no oxid ative stress ha s been detected [5] . Al tho ugh the se scarce data point o ut tha t CNa + is n o t genot o xic there is a n eed for further res earch on its g enotoxic pot ential to obtain inf ormation that will enable the elucidation of the underlying mechanis ms of its ac tion at the mole cular level . Therefore, the aim of our study w as to evaluate wh et her CN a + had in fluence on genomic instability by analyzing the formation o f m icr onuclei (MNi), nucl ear buds (NBUD), and nu cleoplasmic bridg es (NPB) in the hu man hepatoma cell lin e (HepG2 cells) b y the Cytokin esis bl o ck mic ronucleus cytome assay (CBMN) . Moreover, the CNa + induced m o dulation of the expressi o n of selected genes involv ed in the metabolism, imm ediate-early re sponse/signali ng, DNA da mage response, oxidati ve stress and programmed cell deat h was investigat ed by real-time quantitative P CR ( qR T-PCR). 2. Material s and Me thods 2.1. Chemical s Minimum e ssential m edium (MEM), TRIz ol® reag ent and B27 sup plement were from Gibco BRL (Paisl ey , Sco tland) ; foetal bovine s erum (FBS ), non-essential aminoacid s ( NEAA), penicillin/strep tomycin, L-glutamine and ph osphate buffered saline (P BS ) from PAA (Pasching , Austria). Cytochala sin B (Cyt-B), acridine o range (AO), dimeth yl sulfoxide (D MSO [CAS 67 - 68 - 5]), trypsin, e toposide (E T) [CAS 33419- 42 - 0] and benzo( a )pyrene ( B( a )P) [CAS 5 0- 32 - 8], were obtained fro m Sigma – Ald rich (St. Louis, USA ). High Capacity cDN A Archi v e Kit an d Taqman Gene Expressi o n Assa ys were fro m Applied Bios y ste ms (Forest City, CA, USA), T a qMan Universal PCR M aster M ix f rom Applied Biosys tems, (Bran chburg, NJ , USA) and Hu man GAPDH from Applied Bi osy s tems (Warrington, UK ) . Cloisite® Na + was o btained from Southern Cla y Produc ts, INC (Austin, Texas, USA) . The clay was characterized b y th ermogravim et ric analysis (TGA), X-ray dif- 11 5 fraction (XR D ) an d particle size distribution (P S D) as d escribed in Maisanab a et al . [14]. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 11 conjugation with ph ase II e nzymes is considered the d et oxificati on phase of xen obiotic metabolis m, although in c ertain situations, it could res ult in activated metab olites and increase in toxicity [2 4 ]. Some of the im mediate early resp onse genes exhibit a very rapid and significant enhancement o f their mRNA levels when liv er cells are induced to proliferate. These genes include man y proto-onc o genes, such as c-MYC, c-FOS , c-JUN, JUNB, JUND [25] an d their deregulation contributes t o the genesis o f most hum an tumours [26]. In HepG 2 cells all studied genes involved in signaling an d immediate-early response ( FOS, JUN B, MYC, TGFB 2 ) ha ve shown strong up-regu lation after 24 ho urs of exposure t o CNa + . The induction of FOS is known to be indicativ e for DN A-damage and is increas ed by a wide variety of DNA-damag ing agents [27], while MYC proto- oncogene r egulates m any bi ological functi ons, including cell growth, proliferation, ap optosis, differentiati o n, and t ransformation [28]. JUNB is a key c ell cycl e regulator ab le to arrest the G1/S phase transiti o n throug h transcriptional inh ibiti on of Cyclin D1 [29] . TGFB2 , a potent a nti-inflamatory cytokine, b elongs to the tr ansforming g rowth factor Bs (TGF-Bs) that regulate a wide variety of c ellular pro cesses, and were shown t o inhib it epithelial c ell proliferati on by delaying or arr est ing pr ogression through the late portion of G1 [30]. The result s of the present stud y show that CNa + influences c ell signaling processes t hat determine th e faith of the cell t hat can be either cell-cycle arrest, proliferation, apoptosis, or DNA damag e repair. The tumor-suppre sso r gen e, TP53 , plays th e central r ole in the cellu lar resp onse to agents or c onditions that d amage DNA by act i vating the transcripti on of several essential genes contr o lling cell c y cl e arrest /DNA repair, s enesce nce, di fferentiati o n and ap o ptosis [3 1]. In the present stud y we did not detect any changes in the expr ession of TP53 . This is no t unusual, as it is known that DNA damage a ctivates the p53 pr o tein predominantly thr ough its phosphorylation b y DNA damage- r esponsive kinases and, to lesser extent, throu gh up - regulation of gene expressi on [32]. Af ter 24 h exposure TP53 downstream regula ted genes, CDKN1A and GA DD45 A , were up-regulated, which can be associated w ith the c ell cycle arres t due to DNA da m age . Cyclin -dependent kinase inh ibitor 1A ( CDKN 1A ), is an important C D KI that induces cell cycle arre st, inhib its c ell pr oliferation and is directl y involved in D NA repair, includi ng nu cleotide excision repair ( NER) [ 3 3]. The r ole o f GADD45A is to control the cell c ycle G2 -M checkpoin t, the DN A repair p ro cess and apopt o sis [34]. On the contrary, th e gene expression o f checkpoin t ki nase 1 ( CHEK1 ), a Ser /Thr kinas e that i s involved in me diating the cellular resp o nse to DNA-d amage (review ed in Oza et al. [35]), and ERCC4 that is i nvo l v ed in 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 12 NER, as well as MD M2 that enhan ces the tu morigenic po tential o f the cells and p ro motes c ell survival and cell cycle progression [36] , were not aff ected by CNa + . To date, there are n o published reports conce rning po ssible changes in the expression of genes inv olved in oxidati v e stress in cells exposed to CNa + . Theref o re in the pr esent study we measur ed the expr ession of the most impor tant antioxidan t enzy m es at the mRNA lev el . As already menti o ned ab ove CNa + increased the mRNA level of GST1A1 , a m em b er of glutathi o ne S-transferase ( GST ) famil y , which participate in a det oxification pathway that acts via the conjugation of t he subs tance with glutathi one (GSH). GSH plays a central ro l e in i ntracellular antioxidant d efense. It can exist in either a reduced (G SH) or oxidized (GSSG ) for m . Under steady state c o nditions , cells maintain a resting level o f GSH/GSSG know as the redox state [37]. Two enzy m es are involved in maintainin g cellular redox, namely GPX that cat alysis th e reduction of p eroxides and the f ormation of GSSG and GSR that r educes the GSSG back t o GSH [38]. As a resp o nse to oxidative stre ss GSH can be depleted in c ells and is then consequ ently synthesized d e novo in a so call ed  -gluta myl cycle that requires two ATP dependent enzy m es, glutamate-cys te ine ligase (GCLC) and glu tathione synthase (GS) [ 3 9]. The up-regu lation of th e gene coding fo r GCLC in HepG2 cell s exposed to CNa + , indicated possible resp onse of cells t o a depletion of GS H and its bi o synth esis due to the toxic effects of CNa + . The enz ymes inv o lved in maintaining cellu lar redox, GPX1 and G S R, wer e not af fected at the transcrip tional lev el by CNa + at none o f the exp o sed times and concentr ations. Furthermore, we explore d the gene expression o f the primar y antioxidant enzymes in cells, superoxid e dis m utase (SO D ) and catalase (CAT) that are more imp o rtant in the protection of cells than the glutathi one r edox cycle when se vere oxidative stress o ccurs [40]. The mRNA level of SO D1A was not chan ged, while CAT was significantly dose depend ently down-re gulated after 24h exp o sure of HepG2 cell to CNa + . There is onl y limited da ta d escribing the indu ction of oxidative stress by CNa + . In HepG2 cells CNa + a t relatively high c o ncentra tio ns ran ging from 50-1000 µ g/ml in creased the formation of re activ e oxyg en species (ROS), which c oincided with increased cell membrane damage [13]. On the contrary Shar ma et al. [5] did not dete ct inducti on of ROS in Caco-2 cells at concentra tions up to 1 70 µg/ m l. A subchr onic in vivo study on rat s t hat we re orally exposed to Clay1 (40 m g/kg/da y in the diet), a Mt modified wi th a quaternary ammonium salt for 90 days, showed in the liver n o induction of oxidative str ess markers such as GSH /G SSG levels and their ratio [ 4 1], lipid per o xid ation, and the ac tiv iti es and protein c o ntent of antioxidative enzymes such as SOD, CAT, GPX and GST. In additi o n no chang es in expressi on of SOD and CAT 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 13 at the gene and pr otein level determin ed w ith the qRT- PCR and West ern blot, r espectively, were confir med [41]. The most c ommon for m of cell death that pla y s a piv otal role in the m aintenanc e of tissue homeos tasis is apopt o sis, progra mmed cell death. The major apoptoti c pathways include the extrinsic or death recep tor pathwa y and the in trinsic or mitochondri al pathway [42]. In this respe ct we measured the ex pres sion of se veral genes invol ved in the apo ptotic processes . In th e present s tud y we showed that expos ure o f H epG2 cells to CNa + in duced significant transcrip tional chang es of anti-apoptoptic gene BCL2 , while the e xpression o f pr o - apoptotic gen e BAX was not affected. From all caspas e genes included in our stud y only CASP3 was up-regulated by CNa + , while the mRNA le vels of CASP9 and CA SP8 were not c hanged. Caspases are pr oteases that act as es sential initi ators and executioners of t he apoptotic process . Classicall y , the cas pase cascade is initi ated via clea vage of the s o -called in itiator caspases (-2, - 8, -9, and - 10), most likel y by aut o prote o lysis. In itiator caspases, in turn, cleave and activate the executioner caspas es ( -3, - 6, and - 7) [43]. This results in mediation and amplification o f the death signal and eventually c ell death. The results of gene der egulations of pro- and anti-apop totic genes do not allo w for clear i nterpretation whether CNa + induced apoptosis or no t. The up-re gulation of BCL2 sugges t s that apopt o sis is supp resse d; while on the other hand th e up-regulation of CASP3 indicat es potenti al involvement of intrinsic path way of apoptosis in CN a + toxicit y . As th e obtained re sults are contradict ory the mecha nisms involved in c ell death ind uced by CNa + n ee d to b e furt her elucidated. Regarding to program med cell death, other authors s uch as Lordan et al. [13] r eported that after 24 h, n o increase in caspase- 3/7 activi ty was detec te d in HepG2 cells tr eated with CNa + and Cloisit e 93A , an organical ly modified Mt. Simil arly also Maisanab a et al. [14] r epo rted no induction of apoptosis with the commercial CNa + - based clay C lo isi te®30B in t he same cell type. In another st udy on HepG2 cells, CNa + s tatisticall y significantly increased the activit y of caspase 3/7 af te r 4.5h (at 500 µg/mL) and 48 h (3 3 -500 µg/m L) of exposure [4 4]. On the contrary, it ind uced apopto sis in SK -MEL 28 hu man m elanoma cells only after 48 h ours of exposure at 33 µg/ mL and higher [44]. More over, Liu et al. [12] observed an increase in the caspase 3 activit y in hu m an embryonic kidney (HE K) 293 cells treated wi th 1 g/L CNa + , where as with the modified oligo(styrene- co -a crylonitrile)-mont m orill onite (PSAN- MMT) the increas e was lower c o mpar ed to CN a + . In addition PSAN-MM T an d CNa + influ ence on p53 m RNA and protein lev el showed highe r effect of CNa + clay in HE K 293 c ells . 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 14 5. Conclusion s Based on the results o f the present study we c an concl ude that CNa + is po te ntiall y genotoxic as it ind uced the formation of micronuclei at non-cytotoxi c concentr ations and can therefore re present a risk f or human health especially when consid ering l o ng ter m exposure. The changes of the expression of several studied gen es provide new insig hts into the mechanisms of CNa + potential t oxicity. Neverth eless , t here is still a need t o further c o nfirm these results on the protei n level, which will help to clarify the mechan isms involved in CNa + genotoxic acti vity . Acknowledgemen ts The authors wi sh to thank Junta de An dalucía (AGR 5969) and Sloveni an Research Agency (ProgramP1- 0245) f or the f inancial supp o rt and to the Technological Institute of Packaging , Transport, and Logistics (IT ENE) for the do nati o n of th e clay. Sara M aisanaba als o gratefully acknowledges Jun ta de An dalucia for her pred octoral grant associated to the AGR59 69 project. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 15 References [1] F.Bergaya , G. 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Guti érrez -Praena, M. Llana-Ruíz-Cabello , S. Pi chardo, 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 18 A. Mate, M. J ordá-Beneyt o, A.M. Cameán, S. Auc ejo, A. Jos, In vivo evaluati o n of activities and expression o f antioxidan t en zym es in Wis tar rats exposed for 90 days to a modifi ed clay , J. Toxicol. Envir on. Health A. 77 (2014) 4 56 – 466. [42] B. Zhiv o tovsky, G. Kroem er, Apop to sis and geno m ic instab ility , Nature R eviews M o l. Cell Biol. 5 (2004) 75 2-762. [43] S.E. Logue , S.J. Martin, Caspase acti vation cascades in apop to sis, Bioche m. Soc. Trans. 36 (20 08) 1- 9. [44] G. Janer, E. F ernández-Rosas, E. Mas del Molino, D. G onzález-Gálvez, G. Vi lar, C. López-Iglesias , V. Ermini, S. Vázquez-Campos, In v itro toxicit y of functi onalised nanoclays in mainly dri ven by the pres ence of organic modifiers, Nanotoxic o logy, 8 (2014) 279-294. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 19 Figure caption s Figure 1 . Flourescent microg raphs of HepG2 c ells exposed to CNa + (62.5 µ g/ml f o r 24 h) a ) binucleated c ell, b) binucle ated cell with M N, c) binucleated cell with two MNi, d) binucleated cells with NBU D, e) binucleated c ells with N BUD and MN and f) binucl eated cell with NBP. Figure 2 . Number of binucleated cells with a ) micr onucleated cells (M Ned cells) , b ) micronuclei (MNi), c ) nucl ear bridges ( NPBs) and d ) c ells with nuclear bu ds (NBUDs) per 100 0 binucleated HepG2 cells aft er th e exposu re to CNa + (0, 15. 6 5, 3 1.25 and 62.5 µ g/m L ) for 24 h. The number of MN cells shows h ow many cells c ontain ed MN, while MNi sh ows the overall number of MN as some cells c an p o ssess m ore than o ne MN. Signifi cant di ff erenc es between C Na + treated cells and th e control group (0 ) is ind icated by * P < 0.05, ** P < 0.01, ** * P < 0.001, and **** P < 0 .000 1 . Benz o ( a )pyrene (2.5 µM) and Etop o sid e (1 µg/mL) we r e used as t he positive controls. Figure 3 . The influen ce of CNa + (0, 15. 6 5, 3 1.25 and 62.5 µg/mL) on the nuclear division index (NDI) after 24 h expo sure. Sig nificant difference s betw een C Na + -tr eated cells and the control group (0) is ind icated by * P < 0.05 and ** P < 0 . 0 1. Ben zo( a )pyrene ( 2 .5 µM) and E to posid e (1 µg/mL) w ere used as the positi v e contr ols. Table capt ion Table 1. Effect o f CNa + on e xpressio n of mRNAs of se l ected gene s involved in me tabolism , inmediate-early response/sig naling, DNA damage, apo pto sis /survival and o xidati ve stress responses in HepG 2 cells. Mechan isms involved Gene symbol C Na + (µg/mL) 4h 24h Entrez gene name Mean±SD Mean±SD Metabolism (activation /detoxification ) CYP1A1 6.25 0.95±0.07 1. 70±0. 13 * Cytochrome P450 fam ily 1. Subfamily A. p olypeptide 1 62.5 0.95 ±0. 05 1.72 ±0.3 B(a)P 30µM 1.91 ±0. 43 130.32 ±9.22 ** CYP1A2 6.25 0.80±0.15 2.6 0±2.45 Cytochrome P450 fam ily 1. Subfamily A. p olypeptide 2 62.5 1.19±0. 18 1.82 ±0.78 B(a)P 30µM 1.76 ±0. 73 79.78 ±15.01 * CYP3A4 6.25 0.91±0.24 2.9 0±1.16 Cytochrome P45 0 family 3. Su bfamily A. polypeptid e 4 62.5 0.80±0.33 4.06 ±1.84 B(a)P 30µM 1.11±0.17 2.66 ±0. 18 ** GST1A1 6.25 0.80±0.15 1.88 ±0.34 Glutathione S -transferase alp ha 1 62.5 0.85±0.07 1.9 0±0.48 B(a)P 30µM 0.75±0.01 *** 0.6 0±0.25 UGT1A1 6.25 0.92±0.13 0.84±0.26 UDP glucuronosyltra nsferase 1 family. polypeptide A1 62.5 0.85±0.23 0.69 ±0.04 ** B(a)P 30µM 0.97±0.02 5.57 ±0.96 * Immediat e-early response/signaling FOS 6.25 1.05±0.12 10.35 ±2.32 * FBJ murine ost eosarcoma via oncogene homolog B 62.5 0.94±0.68 20.38 ±6.74 B(a)P 30µM 1.23±0.73 12.49 ±5.54 JUNB 6.25 0.94±0.01 ** 1.77±0.35 Jun B proto -oncogen 62.5 0.63 ±0.09 * 2.76 ±0.02 *** B(a)P 30µM 1.13±0.36 2.41±0.46 * MYC 6.25 1.06±0.08 1.29±0.14 V-Myc avian my elocytomatosi s viral oncogene ho molog 62.5 1.00±0.06 1.66 ±0.32 B(a)P 30µM 1.07±0.07 0.78±0.06 * TGFB2 6.25 1.26±0.34 4.04±1.05 Transforming gro wth factor. beta 2 62.5 1.07±0.7 2.94 ±0.62 * B(a)P 30µM 0.10±0.77 0.36 ±0.09 * Table 13 When Caco-2 or HepG2 cells were exposed to Clay 2 during 24 and 48 h, no sign ificant alterati on 295 on ROS was obser ved at any of the exposure concentrations in comparison to the control group 296 (Fig. 6a & 6b). Similarly, GSH content was not affe cted when Caco-2 was exposed to Clay 2 ( Fig. 297 7 a) In contrast, GSH co ntent significantly ( p <0.01) depleted in HepG2 cells, especially from 44 298 µg/mL o nwar ds. The percentage of GSH fo r 44 µg/ml was 8 fold lower in comparison to th e 299 control group (Fig. 7 b) . ** p <0.0 1 significantly different f rom control. 300 3.3. IL -6 leakage 301 The IL -6 assay showed no increase in th e IL -6 content in the cell culture m edium of t he 302 cytotoxicity a ssays in any of t he treatments performed (data not show n). 303 304 3.4. C omet assay 305 Cloisite®20A a nd Clay 1 d id not induce DNA strand-breaks in Cac o -2 and Hep -G2 cells at the 306 concentrations te sted after 24 and 48h o f exposure (Fig. 8 and 9) . In regard to Caco-2 cells 307 exposed to 8.5 and 17 µg/mL of Clay 2 , no si gnificant variations of the DNA strand breaks were 308 detected after 2 4 h and 48 h of exposure. However, the highest exp osu re concentration (34 309 µg/mL) led to a significant ( p <0.05) increase of DNA in the tails, compared to the control (Fi g. 310 10 a) . The percentage of DNA in th e tail with this concentration was not significantly different from 311 the results obtained for the positive control H 2 O 2 ( * p<0.05 significantly different from co ntrol) . In 312 treated HepG2 cells a significant ( p <0.01) increase of DNA damage was observed after 24h 313 exposure at the highest c oncentrat ion only (88 µg/mL). In contrast, afte r 48h, a significant 314 increase of DNA str and br eaks was dete cted in cells exp osed to 44 ( p <0.05 ) and 88 ( p <0.01 ) 315 µg/mL (Fig. 10 b) . T he pe rcentage of DNA in the tail for 44 and 88 µg/mL was 3.5 and 13 fold 316 higher respectively, in co mparison to the control group. * p <0.05 ** p<0.01 significantly different 317 from control. 318 319 14 4. Discussi on 320 The use of m odified clay s is a great option to obtain nanocompo sites with improved properties 321 that have been shown to be very useful in food packaging applications. Massive quantities of 322 nanomaterials would need to be produced , the reby increasing the potential risk of human 323 exposure and raising additional conc ern about their short a nd long term toxicological effects 324 (Hussain et al., 2009). Due to th is lack of information, a spe cific toxicity assessment of these 325 materials and their precursors (non modified a nd chemically organomodified clays) is n ecessary . 326 Therefore, in the present study, biochemical alterations with three different montmorillonite -based 327 clays, all of them chemically modified with quatern a ry ammonium salts; a comm ercial one, 328 Cloisite®20A, and two developed by ITENE, Clay 1 and Clay 2, were studied in th e human cell 329 lines Caco-2 and HepG2. The results obtained sho w ed that, at the concen tratio ns assayed, the 330 main cytotoxic eff ects are induced by Clay 2 with a ti me- de pendent pattern . Nev ertheless, t he 331 toxicity assessment of Cloisite®20A and Clay 1 showed no toxic (cytotoxic and genotoxic) effects 332 at the concentrations assa yed , although they were lower than those used for Clay 2.In this sense, 333 the importance o f the mod ifiers that are used to improve th e c ompatibility with th e polymer matr ix 334 must be highlighted. T he incorporation of ACO in Clay 2 structure could be related with the 335 different toxicity profile of Clay 1 and Clay 2 ob served in both cell lines. ACO pro vides a lower 336 hydrophobicity to the clay that allows testing higher co ncent rations in the aqueous cu lture 337 medium. When the same test concentration (8 µg/m L) of both clays is comp ared in Caco -2 cells, 338 results are q uite similar with a low decrease of viabilit y. In HepG2 on the other hand, 8 µg/m L 339 Clay 1 ind uced a significa nt decrease of protein co nten t, b ut 8 µg/mL Clay 2 did not induce any 340 effect. Unfortunately, higher concentrations of Clay 1 could be not a ssayed due to the presence 341 of interferenc e s with the measurement system. A similar comparison performed with th e same 342 concentration (62.5 µg/ml) of Clay 2 and Cloisite®20A shows the higher toxicity of Clay 2. 343 15 Others au thors have evaluated the toxicity of the commercial non m odified montmorillonite in the 344 same cell lines (Gu tierrez- Praena et al., 2011 ; Lordan et al., 2011; Ma is anaba et al., 2013a ,c ; 345 Sharma et al., 2010). The toxicity assa ys carried o ut by Lordan et al. (2011) with the commercial 346 unmodified clay, designated as Cloisite®Na+ showed a significant cell viability decrease in all the 347 concentrations assa yed, from 1 to 1 000 µg/mL, in the Hep G2 ce ll line. T hese r esults do n ot 348 agree with Maisanaba et al. (2013a ,c), who observed that HepG2 and Caco -2 exposed to 349 Cloisite®Na + did not present higher significant reductions of viab ility with respect to the controls in 350 the range of concentrations assaye d, from 0-62.5 and 0 -125 µg/mL, after 24 and 48h of 351 exposure, re spectively. M oreove r, Sharma et al. (2010), did not obtain any cytotoxic eff ects i n 352 Caco-2 exposed to Cloisite®Na + , in agreement with our results. In this case, the concentration 353 range used of the m odified clays Cloisite ®20A and Clay 1 showed the same b ehaviour compared 354 to the starting material, unmodified montmorillonite, indicating that the modifiers e mployed could 355 not involve changes in the safety profile of the modified clays. 356 There are a limited number of toxicological studies in th e literature a bout commercial mo difie d 357 clays in Caco-2 an d HepG2. Lordan et al. ( 2011) also evaluated a commercial m odified clay, 358 Cloisite®93A, in the hepatic cell line, obtaining cytotoxic effects at all concentrations tested (1- 359 1000µg/mL). Other commercial modified montmorillonite, Cloisite®30B, ha s been evaluated in 360 Caco-2 cells by Sharma e t al. (2010 ). The research group evaluated filtered and u nfilter ed 361 suspensions of Cloisite®30B. T he r esults sh owed a notably cytotoxic e ffect a t the highe st 362 concentrations assayed in both cases, reporting a 4 0% cell viability reduction at 226 µg/mL. The 363 cytotoxic effects of this modified clay in Caco-2 a nd HepG2 have been also s tudied by our 364 research group, obtain ing cyt otoxicity for all the biomarkers assayed, being the highest 365 concentration assayed 250 and 50 0 µg/mL, respectively (Maisanaba et al., 2013a ,c ). 366 367 16 The resu lts obtained showed that the intestinal cell line, Caco -2 was more sen sitive in cytotoxicity 368 par ameters to the m odified clays exp osure. Thu s, in the present study the EC50 (24h) for the PC 369 assay calculated after Clay 2 exposure was 93 µg/mL for HepG2 and 34 µg/mL for Caco - 2. 370 These results agree with th ose o btained by the oth e r authors, t hat also obse rved a higher 371 sensitivity after exposure to Cloisite®30B in Caco -2, with an EC50 = 40µg/mL ( Maisanaba et al., 372 2013c) versus an EC50 = 88 µg/mL for HepG2 (Maisanaba e t al., 2013a). However, biomarkers 373 showed a different sensitivity in both cell lines, the P C was th e m ost sensitive in Caco-2 whereas 374 MTS was the m ost sensitive in HepG2. This cou ld be related with their different origin and 375 therefore to their different capacity to face toxic insults. 376 377 Others authors have studie d the cytotoxic effects of commercial clays in o ther target cell lines. 378 Baek et al. (2012) evaluated the toxicity effects in human normal intestinal c ells (INT -407) in a 379 short and long term exposure, 24, 48, 72h, and, 10 days, to M MT. Ther eby, a decrease in cell 380 proliferation showe d at a ll times assayed. On the one hand, significant differences in the short 381 term assays were found above 100 µg/mL concentrati on levels, on the other hand, a sign ificant 382 inhibition of normal colon y forma tion in the long term was ob served at all conce ntrations tested. 383 Even though, alterations in LDH r elease were only observed at the highest concentrations at 48 384 and 72h. Li et al. (2010) also studied CHO ce ll viability , when exposed to 62.5 – 1000 µg/mL 385 nan osilicate platelets, obta ining a slight decrease in MTT and LDH ass ays. Also, the oligo 386 (styrene- co - acrylonitrile)- modified clay m ontmorillo nite showed an increased LDH release 387 activity and ce ll viability reduction at a concentration of 1g/L in m ouse em bryonic fibroblast (NIH 388 3T3) cells and human embryonic kidney 293 (HE K 293) cells (Liu et al., 2011). 389 390 Taking into account th at Clay 2 showed a clear cytotoxic p rofile a t the concentration range tested 391 in comparison to Closite®20A and Clay 1, the toxic mec hanisms of this organoclay were 392 17 considered to require further study. The implication of oxidative stress, inflammation or DNA 393 damage, among others, could be related to micro and nanoparticles exposure (Bouwmeester et 394 al., 2009). For this reason, levels of ROS and GSH were assayed in this study, obtaining only 395 significant differences with respect to the control group on the GSH leve ls of HepG2. The r esult s 396 showed therefore a higher sensitivity of the HepG2 cell line to o xidative stress biomarkers in 397 contrast to the r esults obtained in the cytotoxicity study. Other authors have previously re ported 398 that He pG2 cells are generally more susceptible to oxidant -induced stress than Caco -2 ce ll s 399 (Martin et al., 1997). Our research group ha s also observed a deep decreas e in GSH conte nt in 400 HepG2 exposed to Cloisite®30B at all timepoints assayed, however no changes were observed 401 in ROS levels (M aisanaba et al., 2013a). T his might be due beca use GSH have many biological 402 functions apart from ROS sca venging such as signal transduction, gene expr ession and 403 apoptosis, etc. ( Sies 1999). Moreover, Sharma e t al. (2010) also reported that Closite®Na + and 404 Closite®30B did n ot induce ROS production in Caco- 2, in agreement with our res u lts for thos e 405 clays (Maisanaba et al., 2013 a) and although there was no change in ROS leve ls they observed 406 genotoxic effects. By con trast, Lordan et al. (2011) reported that Cloisite®Na + induced 407 intracellular ROS formation, wher eas, the e ffect of Cloisite®93A in the generation of ROS was 408 less p rominent. Furthermore, Baek e t al. (2012) evalu a ted the ROS production in INT - 407 cells 409 exposed to MMT, obtaining significant levels of ROS a t the h ighes t concentration (1000 µg/ mL) 410 at all three timepoints assayed (24, 48 and 72h). 411 412 Clay 2 did not induce leaka ge of IL-6, biomar ke r of an inflammatory response, in any of the cell 413 lines. MMT, on the other hand, has been reported to rapidly lyses ne utrophils and erythrocytes in 414 vitro. F urthermore, it can stimulate chemiluminescence, the neutrophil oxidative metabolic b urst 415 (Dougherty et al., 1985). 416 417 18 In relation to genotoxicity, Clay 2 induced DNA str and b reaks in both cell lines Moreover, Clay 2 418 induced time-dependent increases o f DNA s trand breaks in HepG2 cells. Similar results were 419 also found by Maisanaba et al ., (2013a) for Cloisite30®B in HepG2 cells. Other authors evaluate d 420 the genotoxicity of other commercial clays in similar cell lines. S harma et al. ( 2010), showed no 421 DNA dam age in Caco-2 exposed to u nfiltered and filtered Cloisite®Na + samples. Never theless , 422 DNA d amage was observed in the intestinal cell line Caco -2 e xposed to Cloisite®30B fr om 113 423 µg/mL a fter 24h. By contrast, the results obtained by M aisanaba et al ., (2013c) with the intestinal 424 cell line exposed to Cloisite®30B, did not coincide with the aforementioned research group, and 425 this ca n be r elated with the lower concentrations tested in th is case (10, 20 and 40 µg/mL). 426 Maisanaba et al. (2013a), however, showed differen ces with respe ct to the control at the highest 427 concentration assayed (8 8 µ g/mL) in HepG2 after 48 h. In this case, it is difficult to compare 428 because Sharma et al. ( 2010) only presented results after 24h, and at this concentration DNA 429 damage in the intestinal cell line was not observed. 430 Sharma et al. (2010), also indicated that the modifiers used to modify the clay contributed to the 431 genotoxic effects, and these effects were not due to oxidative damage. In this se nse, the absence 432 of ROS obtained in this wo rk after exposure to Clay 2 coincides with the Sharma´s hypothesis. 433 On the other hand, Li et al. (2010) o bserved no genotoxic results in the comet assay with CHO 434 cells exposed up to 1000µg/mL nanosilicate platelets d e rived from MMT. 435 Regarding to in vivo to xicity d ata, there is no information about the three organoclays selected in 436 the present study, but Li e t al. (2010) did not reported acute oral toxicity of MMT in rats exposed 437 to a single dose of 5700 mg/kg. Also Baek et al. (2012) did not f ind any remarkable toxicity i n 438 mice orally exposed to a single dose of 1000 mg/kg MTT. 439 440 In summary, our r esults showed that a t the concentrations assayed Cloisite®20A and Clay 1 did 441 not induce cytotoxicity. Clay 2, however, showed cytot oxic an d genotoxic e ffe cts, as well as an 442 19 alteration in GSH content. Therefore, a case by case toxicological assessment is r equired , as the 443 modifier has a role in the toxicity observed. Moreover, a different sensitivity was observed 444 depending on the cell line employed. 445 More studies are require d to elucidate the r isks of these clays for human health as their use is 446 going to increase widely in the near future. 447 448 449 Acknowledgements 450 The a uthors wish to thank the Ministerio de Ciencia e Innovación (AGL2010 -21210) and Junta de 451 Andalucía (AGR5969) for the financial support for this study, and the Cell Culture Service and 452 Microscopy Service of Centro de Investigación, Tecnología e In novac ión de la Universidad de 453 Sevilla (CITIUS) for providing technical assistance. 454 455 Formatted: Spanish (Internationa l Sort) 20 References 456 Avella, M., De Vlieger, J. J., Errico, M. E ., F ischer, S., Vacca, P., & Volpe, M. G. 2005. 457 Biodegradable starch/clay nanocomposite films for food packaging applications. Food Chem. 93, 458 467 – 474.Azeredo, H. M . C., 2009. Nanoco mposites for food packaging applications. Food. 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UV-sensitive rodent mutant 480 cell lines of complementation g roups 6 and 8 differ ph enotypically from their human counterparts . 481 Environ. Mol. Mutagen . 29, 152 -160. 482 Corcuera, L.A., Arbillaga, L., Vettorazzi, A., Azqueta, A., López de Cerain, A., 2011. Ochratoxin A 483 reduces aflatoxin B1 induced DNA damage detected by the comet assay i n Hep G2 cells. F ood. 484 Chem. Toxicol . 49, 2883-2889. 485 Dougherty, S.H., F iegel, V.D., Nelson, R.D., Rodeheaver, G.T., Simmons, R.L., 1985. Effects of 486 soil infection potentiating factors on neutrophils in vitro. A m. J. Surg . 150, 306 – 311. 487 Duncan, T.V., 2011. Applications of nanotechnology in food packagin g and food safety: ba rrier 488 materials, antimicrobials and sensors. J.Colloid. Interf. S ci . 363, 1- 24. 489 Gutiérrez-Praena, D., Pichardo, S., Jordá, M., Bermúdez, J.M., Aucejo, S., Jos, A., 2011. 490 Citotoxicidad de la nanoarcilla cloisite 30b e n la línea ce lular inte stinal humana Caco -2. 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Mater . 21 , 1549-1559. 499 Jordá-Beneyto, M., Alonso, J., Salas, J., Gallur, M., Aucejo, S., Clegg, F., Breen, C., 2008. 500 Processed biopolymer fil ms filled with modified montmorillonite for food packaging applications. 501 Proceedings of the Polymer Processing Society 24 th Annual Meeting, PPS-24, June 1 5-19, 2008, 502 Salerno (Italy). 503 22 Jorda-Beneyto, M., Ortuño, N., Devis, A., Aucejo, A., Gutierrez -Praena, D., Puerto, M ., Pichardo, 504 S., Houtman, J., Jos, A., 2013. Use of nanoclay platelets in food packagi ng materials. Technical 505 and toxicological aspects. Food. Addit. Contam . (under revision). 506 Jordan, J., Jaco b, K.I., Tanenbaum, R., Sharaf, M., Jasiuk, I., 2005 Experimental trends in 507 polymer nanocomposites: a review. Mater. Sci. Eng. A. 393, 1 – 11. 508 Jo s, A., Camean, A.M ., Pflugmacher, S., Segner, H. 2009. The antioxidant glutathione in the fish 509 cell lines E PC and BCF -2: Response to model p r o-oxidants as measured by three d ifferent 510 fluorescent dyes. Toxicol. In. Vitro. 23, 546-553. 511 Lagaron, J.M. 2006. Higher barriers and better perfor mance. Food Eng Ingredients 31(2) , 50 – 51. 512 Li, P.R., Wei, J.C., Chiu, Y.F., Su, H.L., Peng, F.C., Lin, J.J. , 2010. Evaluation on cytotoxicity and 513 genotoxicity of the exfoliated silicate nanoclay. Appl. Mater. Interf. 2(6), 1608-1613. 514 Liu, Q., Li u, Y., Xiang, S., Mo, X., Su, S., Zhang, J., 2011. Apoptosis and cytotoxicity of 515 oligo(styrene- co -acrylonitrile)-modified montmorillonite. Appl. Clay. Sci . 51, 214-219. 516 Lordan, S., Kennedy, J.E., Higginb othamb, C.L., 2011. Cytotoxic effects induc e d by unmodified 517 and organically modified nanoclays in the human hepatic HepG2 cell line. J. Appl. Toxicol . 31, 518 27 – 35. 519 Madejova, J., 2003. FTIR techniques in clay mineral studies, Vibrational Spectrosc. 31, 1- 10. 520 Maisanaba, S., Puerto, M., Pichardo, S., Jordá, M., Moreno, F.J., Aucejo, S., Jos, A., 2013a. In 521 vitro toxicity evaluation of unmodified commercial clays on the human hepatic cell line HepG2. 522 Food. Chem. Toxicol. 57, 266-275. 523 Maisanaba, S., Pichardo, S., Jordá- Beneyto, M., Aucejo, S., Jos, A., 20 13b. Toxicity of M igration 524 Extracts from Nanocompo site s used i n Fo od Packaging in Caco-2 and HepG2 Cell Lines. 525 Toxicol. In. Vitro. (under revision). 526 Formatted: English (U.S.) 2 Abstract 25 Nowadays, the inco rporati on of organomodified clays based on montmorillonite into po lymers 26 intended for p ackaging ind u stry is a reality. The final resu lt is a polymer nanocomposite with 27 enhanced b arrier properties. Different organomodified clays are already commerciall y ava ilable 28 and others new ones are being developed, however littl e is known about their safety. 29 In the present work, the cytotoxic effects (a tetrazolium salt reduction a nd p r o tein content) of 30 three o rganomodified clays, Cloisite®20A, a co mmercial clay, and Cl ay 1 and Clay 2, two novel 31 modified clays developed by the P a ckaging, Transport, & Logistics Resea rch Institute, were 32 evaluated in Caco-2 and HepG2 cells afte r 24 and 48h of exposure. Our results sh o wed th at only 33 Clay 2 induced toxic effects in both cell l ines. The mean effective concentration was calculated for 34 each case, showing Caco-2 to b e more sensitive than HepG2. Moreover, in order to elucidate the 35 toxicity mechanisms of Clay 2, different mechanistic biomarkers were inve stigat ed. Interleukin 36 leakage and gene ration of intracellular reactive o xygen species was not obser ved , whereas 37 glutathione content decreased in HepG2. DNA d amage (comet a ssay) was induced in both cell 38 lines at th e highest concentration tested. Overall, results sh ow that the type of cl ay, the 39 concentrations range and the type of cell line play an important role in the toxicity o b served. 40 41 Keywords: clay, montmorillonite, cytotoxicity, cell lines, DNA damage 42 43 44 3 1. Introduction 45 46 Over the last decades, the use of polymers as food packaging materials has increased 47 enormously due to their advantages o ver other trad iti onal materials (Jordan e t al., 2005; Silvestre 48 et al., 2011). Latest innovations include the us e of “improved” food contact ma te rials, th is is th e 49 addition of different reinforceme nts, for e x ample nanoparticles, to the polymeric matrix in order to 50 enhance the functional prope rties o f p ackaging materials, and thus improve the s helf life of foo d 51 and beverage p roducts. The resu lting nano composit es are a multi -ph ase material in which t he 52 majority of the dispersed phase components have one or more dimensions of the o rder of 100 nm 53 or less (SCENIHR, 2007). 54 55 Smectite clays, such as montmo rillonite (MMT), b elong to the structural famil y ca lled 2 :1 56 phyllosilicates, which p rese n t a structure composed by two tetrahedral layers formed by Si an d O 57 atoms, fused with an octahedral layer with aluminum and magnesium atoms bonded to oxyge n 58 and h ydroxyl groups (Jordá-Beneyto et al., 2008). They a re one o f the main choices for d esigning 59 polymer nanocomposites due to their low cost and rich intercalation chemistry allowing them to 60 be ch emically mod ified (organoclays) and to improve the co mpatibility with th e polymer matrix 61 (Bitinis et al., 2011). Once the final clay nanocomp osites a re ready, the resulting material 62 presents a specific d isposition of the clays, su ch as p l atelets, g iving a to rtuous path to the gas 63 permeant, forcing it to travel a longer pa th to d iffuse t hrough the film (Nielsen, 1967). Se v eral 64 authors revie w ed the advan tag es, and in few ca ses the limitations, of the layere d 65 nanocomposites. Great improvements in thermal, mechanical and barrier (pe rmeability) 66 properties a re p resent ed , as well as strength, stiffness, dimensional stability, a nd heat resistance 67 (De Azeredo, 2009; Duncan et al., 2011; Hatzigrigoriou et al., 2011; Hetzer et al., 2 008) . 68 Moreover, they also offer good barrier properties under different packing, handling , shipp ing, and 69 4 storage conditions (Avella et al., 2005; Brody, 2006; Ray e t al., 2006; Timoty and Duncan 2011; 70 Volpe, 2005), therefore they co uld improve the qua lity and sa fety of packaged food 71 (Lagaron,2006) . 72 73 Although there is a lot of evidence for the good technological p erformance of nanocomposites, 74 safety issues a re a lso of importance. Available data on clay’s toxicity is still scarce , bu t different 75 authors h ave already d escribed toxic effects induced b y montmorillonite a nd organoclays ( Lordan 76 et a l., 2011; Maisanaba e t al., 2013a ,c; Sha rma et al., 2 010). Moreo ver, the modifier used to 77 synthesize the organoclay has a role in the toxicity observed (Maisanaba et al., 2013a). 78 79 In the case of organoclays, the oral pa thway is the most important entrance rou te for the 80 consumers, as they are exposed to the po ssi b le migrants from packaging nanocomposites to the 81 food products, and they sho uld know the possible e ffects of the ingestion of these 82 nanosubstances to the ga strointestinal tract (Silvestre et al., 2011) . When the nanoparticles reach 83 the blood circulation, the liver is among the main organs fo r distribution (SCENIHR, 2 009), so for 84 clays imbued in the nanocomposite material this possibil ity canno t be discard ed. 85 86 Taking into account that the prod uction of n anocomposi te s is going to increase in the near fu ture, 87 the human exposure will also increase. The refore, potential toxic effects sho u ld be inve stigated in 88 order to guarantee the safety of the food pro ducts. 89 90 The aim of this study is to evaluate the toxicit y of three modified montmorillonite clays used in the 91 production of nanocomposites with applications as food contact materials in two target human cell 92 lines of the dige stive system, Caco -2 a nd HepG2 from intestin al a nd hepatocellular origin, 93 respectively. For this issue, b asal cytotoxicity biomarkers a nd mechanistic biomarkers of 94 5 inflammation, oxidative stress and genotoxicity have be en investigated with Cloisite ® 20A, a 95 commercial clay, and Clay 1 and Clay 2, two novel modified organoclays d eveloped by the 96 Technological Institute of Packag ing, Transport a nd Logistics (ITENE). 97 98 2. Materials and Methods 99 100 2.1. Supplies and Chemicals 101 Culture medium, fetal bo vi n e serum and cell culture r e agents were obta ined from BioWhittaker 102 (Spain). Chemicals fo r the different a ssays were provided by Sigma -Aldrich (Spain) an d VWR 103 International Eurolab (Spain). Protein reagent assay wa s o btained from BioRad (Sp a in). 104 105 2.2. Clays 106 Clay1 and Clay2 are two novel microsized clays that have been developed and characterized by 107 Thermogravimetric Analysis (TGA) a nd Fourier Transform In fraRed (FTIR), as d escribed in 108 Maisanaba et al, (2013b) and Jordá -Beneyto et al., (2008 , 2013) .Clay 1 contains as modifier 109 quaternary ammonium salt hexadecyltrimethyl -ammonium bromide (HDTA) a nd Clay 2 contains 110 HDTA and acetylcholine chloride (ACO). Both clays are obtained by cation exchange rea ctio n 111 from Cloisite®Na + (Southern Clay Products, INC.). This raw clay ha s a typ ical dr y particle size 112 less than 25μm (d50) . Cloisite®20A was also obtained from Southern Clay Prod ucts, INC. 113 (modifier: dimeth yl, dehydrogenated ta llow, quaternary ammonium, concentratio n: 95 meq/100g 114 clay). This modified clay has a typical dry particle size less th an 10 μm (d5 0) and ha s been 115 characterized by Thermogravimetric Analysis (TGA ) an d Fourier Transform InfraRe d (FTIR). 116 TGA analysis of Cloisite®20A and Cloisite ® Na + (the original non -mo dified clay included as 117 control) were p erformed on a Q5000IR t hermobalance (TA Instruments) by heating the s a mples 118 6 from room te mperature up to 900 °C with hea ting rate o f 1 0 °C/min, in n itrogen a tmosphere. 119 Approximately 7 mg of each finely ground sample was heated in a platinum crucible. 120 121 FTIR Spectra were obtained o n an Equinox 55 s pe ctrometer (Bruk e r), coupled to a microscope 122 modulus with ATR ob jetive ( Hiperion, Bruker). This technique was used to characteri ze both clays 123 Cloisite ® Na + and Cloisite ® 20A. For each sample 128 sca ns were recorded with a resolution o f 4 124 cm- 1 . 125 126 2.3. Model systems 127 Caco-2 cell line derives from a hu man colon carcinoma (A TCC® HTB- 37) and HepG2 is a human 128 hepatocellular carcinoma epithelial cell line ( ATCC® HB -8065). Both of th em were obtained from 129 the American Type Culture Collection. Caco-2 cell line was maintained in Eagle’s medium 130 (EMEM) su pplemented with 10 % feta l bovine serum (FBS), 1% non -essential amino acids, 50 131 μg/mL gentamicine, 2 mM L -glut amine, and 1 mM p yruvate. HepG2 cell line was maintained in 132 EMEM supp lemented with 10% FBS, 100 U/mL penic illin, an d 100 μg/mL streptomycin. Cells 133 were grown nea r confluenc e in 75 -cm 2 p lastic flasks at 37ºC in an atmosphere con taining 5% 134 CO 2 at 95% relative humidity (CO 2 incub ator, NuAire ® , Sp ain) and harvested weekly with 0.25% 135 trypsin. They were co unt ed in a n improved Neubauer haemocytometer and viab ility was 136 determined by the Trypan Blue exclusio n test. The cells were u sed at passages between 10 and 137 21 . Both cell lines were plat ed at a density of 7.5 x 10 5 cells/mL to perform all experiments. 138 139 2.4. Clays test solutions 140 Test concentrations of clays were determined individually in p revious experiments in orde r to 141 avoid interference with the method of mea surement. With this purpose the ab so rba nce of clay 142 solutions (1000 µ g/mL and serial ½ dilution s) we re measured at 0, 24 and 48h. The 143 7 concentrations selected were the highest ones that did not show sta tistical d ifferences versus the 144 control. T hu s, the maximu m concentration s were 8 μg/mL for Clay 1, 125 μg/mL fo r Clay 2 , and 145 62.5 μg/mL for Cloisite®20A. T est sol utions were prepared in serum -free medium. An ultr a sonic 146 tip (Dr. Hielscher, Germany) at an amplitude of 40% for a to tal time of 30 s was employed to 147 disperse the test concentrations. 148 149 2.5. Cytotoxicity assays 150 From th e initial solution s, serial d ilutions in medium withou t se rum were prepared. Cultur e 151 medium without clay was used as control group. After replacing the previous medium, the 152 exposure solutions were ad ded to the systems, and incubated a t 37ºC for 24 and 48 hours. The 153 basal cytotoxicity en dpoints assa yed were te trazolium salt reduction (MTS) and protein content 154 (PC) . 155 156 MTS reduction is carried out by dehydroge nases , enz ymes present in mitochondri a , making this 157 endpoint a g ood marker for the damage induc ed in this o rganelle. MTS red uction was measured 158 according to the procedure of Baltrop et al. (1991). The MTS (3 -(4,5-dimethylthiazol-2-yl)-5- (3 - 159 carboxymethoxyphenyl)-2 - (4 -sulfophenyl)-2H tetrazolium salt) added to the medium is reduced 160 by the e nzymes to a colored formazan product soluble in culture me dium and is measured by a 161 spectrophotometer at 490 nm after 2 hours of i n cubation in the dark. 162 163 Protein content (PC) is a very use ful endpoint to assess cytotoxicity, since it g ives da ta about ce l l 164 damage with independenc e o f the toxic mechanism involved (Pichardo e t al. 2007). PC was 165 quantified in situ , according to the procedu re given b y Bradford ( 1976), using Coomassie Brilliant 166 Blue G-250 in the same 96-well tissue culture plates in which e xposure originally to ok place, i n 167 order to determine the total cell number present in the wells. The culture medium was replaced by 168 8 200 μL NaOH and after 2 h of incu bation at 37ºC, 180 μL were replaced by the same volume of a 169 22% Coomassie Brill ian t Blue G-250 solut ion. A fter 30 min incubation at room te mperature, 170 absorbance was read at 59 5 n m in a microplate spectrophotometer (Tecan Infinite M200, 171 Austria). 172 173 2.6 . Oxidative stress assays 174 Considering that only Clay 2 showed remarkable cyt oto xic e ffects, t his clay w as chosen t o 175 perform mechanistic studies. For this purpose low cytotoxic concentrations were selected: 0, 8.5, 176 17 and 34 μg/mL for Caco -2 cells and 22 , 44 and 88 µg/mL for HepG2 cultures. These 177 concentrations correspond to the mean effective concentration (EC 50 ) value obtained for the most 178 sensitive cytotoxicity endpoint at 24 h along with t he fr actions E C 50 /2 and EC 50 /4 . After replacin g 179 the previous me dium, the exposure s olutions were added to the cells, and incubated at 37ºC for 180 24 and 48 hours. Cultu re medium without clay was used as control group. The o xidative stress 181 endpoints measured were reactive oxygen species (ROS) content an d glutathione (GSH) le vels. 182 183 The production of ROS was assessed in 96 well micr oplates using the dichlorofluorescein (DCF) 184 assay. Th e probe 2’,7 ’ -dichlorofluorescein diacetate (DCFH-DA) (Molecular probes, Invitrogen) 185 readily diffuses through th e cell membrane and is hydrolyzed b y intrace llul a r esterases to non - 186 fluorescent compound (DCFH), which is rapidly oxidized in the presence of ROS to the highly 187 fluorescent DC F. Specifically, cells were incubated with 2 00 µl 2 0 µM DCFH -DA in culture 188 medium a t 37ºC for 30 min, a nd then washed with phosphate buffered saline (PBS) and 189 resuspended in 200 µl of PBS. The fo rmation of the fluorescence oxidized derivative o f DCF -DA 190 was monitored a t emission wavelength of 535 nm and excitation wavelength of 485 nm. ROS 191 production was expressed as fluorescence arbitrary units (Puerto et al. 2010). 192 193 9 GSH content in cells was eva luated by reaction with the fluorescent probe monochlorobimane 194 (mBCl, Molecu lar probes, In vitrogen) (Jos et al. 200 9) . This molecule fo rms a thioether adduct 195 with GS H in a reaction catalyzed by the enzyme GST. After the cell exp osure to the clay, medium 196 was d iscarded and cells were incubated at 37°C fo r 20 min in the presence of 40 μM mBCl. Later 197 on, cells were washed with PBS and the flu orescence was recorded in a spectrofluorometer 198 (Biotek, USA) at the e xcitation/emission wave l engths of 380/460 nm . Resu lts were exp ressed as 199 arbitrary units. 200 201 2.7. Interleukin-6 leakage 202 For this assay, the culture medium of the cells after 24 and 48h exp osure to Clay 2 was used. 203 Manufacturer instructions from the kit (EH2IL6, Thermo Scientific, USA) were foll owed. 204 205 2.8 . C omet assay 206 The comet assay was performed to detect DNA strand breaks. The cells lines were seeded into 207 12 -well tissue culture trea ted plates (Corning Costar Corporation, New York, USA) and left 208 overnight at 37ºC in 5 % CO 2 to attach to the plates. Ap proximately 3.5×10 5 Cac o -2 or HepG2 209 cells were expo sed to d ifferent concentra tions of Clay 2: 0, 8.5, 17, 34 µ g/ml an d 0, 22, 44 an 88 210 µg/ml, respectively, for 24 and 48 h. 211 212 In order to monitor the ongoing process of the a ssay, a negative control (cells treated with culture 213 medium) and a positive control (cells treate d with a so l ution of 1 00 µM H 2 O 2 ) were includ ed. After 214 treatments cells were washed and detached in PBS. T he comet assay was applied as previously 215 described b y Collins et al. (19 97) with mod ifications (Corcuera et al., 2011). Briefly, cells wer e 216 resuspended in P BS at a concentration of 2.5x10 6 cells/mL. This suspension was mixed with 1% 217 low melting point agarose and placed on a microscope slide (12 gels per slide). On ce the gels 218 10 bec ame so lid, the slides were dipp ed into lysis solution a t 4ºC. All nucleotides were denatured in 219 a high-pH buffe r. Electrophoresis was ca rried out at approximately 25 V (300 mA , ~ 1 V/cm),and 220 the DNA was gently reneutralized in PBS and washed in H 2 O. After n eutralization, microscope 221 slides are fixed in 96% ethan ol and absolute ethanol. Fi n ally, DNA was stained with S YBR Gold 222 nucleic acid gel stain and was visua lized wit h an Olympus BX61 fl uorescence microscope (20× 223 objective) coupled via a CCD camera to an image -analysis system (DP controller -DP manager). 224 Images of randomly se l ected nuclei (≥100) pe r expe rim en tal p oint were analyzed with the image 225 analysis software (Comet Assay IV, Percep tive Instruments, UK). The results from four 226 independent experiments are expressed as % of tail DNA. 227 228 2.9 . C alculations and statistical analysis 229 All expe riments were pe rform ed at lea st three times and at least in d uplicate per con centratio n 230 (n=3). Data for the conce ntration -dependent cytotoxicity relation ships of all experiments were 231 presented a s the arithmetic mean percentage ± standard deviation (SD) in relation to control. 232 Statistical analysis was carried ou t with GraphPad InSta t3 software ( La Jolla, CA , USA ) using 233 analysis of variance (ANOVA), followed b y Dunnett’s multiple comparison tests. This test 234 compares th e response of the diffe rent exposure co ncentrations versus the controls. Previous to 235 ANOVA, normality was checked with Ko lmogorov – Smirnov te st ( p >0.05) and variance 236 homogeneity among groups assesse d with Bartlett's homocedasticity test ( p >0.05). Differences 237 among groups were considered significant at p <0.05. 238 EC 50 values (mean effective concentration, co ncentration that modified each biom arker by 5 0%, 239 positive or negative, in co mparison with appropriate u ntreated controls) were derived by linea r 240 regression in the concentration-response cu rv e s. 241 242 3. Results 243 11 3.1. Characterization of Cloisite® 20A 244 TGA results o f both clays ( Cloisite ® Na + as control a nd Cloisite ®20A) are presented in Figure 1. 245 Loss weight p ercent (%), a nd de rivative weight (%/°C) are p resented versus t emperature. It can 246 be ob serv ed that on ly the sample Cloisite ® 20A has a big loss ste p in the ran ge between 200 and 247 500 ºC, indicating that an organic co mpound is decomposed. The percentage of organic modifier 248 in Cloisite ® 20A is 24.87 % w/w, as calculated in the TGA curves. Cloisite ® Na + hardly presents 249 any weight change in this range of temperature. 250 The presence of modifiers in the clays was de termined by FTIR. The ATR spectra are shown in 251 Figure 2. Characteristic Si-O stretching v ibration band of the unmodified mont morillonit e (cloisite Na+) 252 occurs at 1030 cm -1 , whereas in the spectra of the corresponding organoclay (cloisite20A) the band 253 appears slightly shifted (1000 cm -1 )(Madejov a, 2003) . Inner hydroxyl g roups, lying be tween the 254 tetrahedral and octahedral sheets, gives the abso rptio n near 3620 cm -1 , as it can be seen in both 255 spectra in Figu re 2. The C-H stretching bands of alkylammnium cation s occur in th e range o f 256 3020-2800 cm -1 . The C- H stretching bands at 2930 cm -1 and at 2850 cm -1 ar e present i n spectra 257 of Cloisite ® 20A, which is a o rgano-modified clay. A ch aracteristic C -H be nding vibration o f the 258 (CH3)4N + cation at 1487 cm -1 is seen in the spectrum of the modified clay, which correspon ds 259 with the a lkylammonium modifier in this clay. The spectrum o f Cloisite ® Na + does not present 260 these peaks, showing that in this clay ther e is no presence of any organic modifier. 261 262 3. 2. Cytotoxicity assays 263 MTS red uction performed by Caco -2 and HepG2 cells exposed to Clay 1 remained unaltered with 264 respect to the control cells after 24 and 48 h (Fig. 3a & 3 b). In respect to PC, Caco-2 cell line 265 showed no alterations a t 24 h , whe reas at 48 h only experienced a sign ificant ( p <0.05) d ecrease 266 respect t o the control at 8 µg/mL (Fig. 3c). In the case of H e pG2 cells, PC did not show 267 18 cell line exposed to Cloisite®30B, did not coincide with the aforementioned research group, and 417 this can be related with the lower concentrations tested in this case (10, 20 and 4 0 µg/mL). 418 Maisanaba et al. (201 3a), however, showed differen ces with respect to the control at the highest 419 concentration assayed (88 µg/mL) in HepG2 after 48 h. In this case, it is d ifficult to compar e 420 because Sharma et a l. (2010) only presented results after 24h, and at this concentration DNA 421 damage in the i ntestinal cell line was not observed. 422 Sharma et al. (2010), also indicated that the modifiers used to modify the clay contributed to the 423 genotoxic effects, and these effects were not due to o xi dative damage. In this sen se, the absence 424 of ROS obtained in this wo rk after exposure to Clay 2 coincides with the Sharma´s hypothesis. 425 On the other han d, Li et al. (2010) o bserved no genot o xic results in t he comet assay with CHO 426 cells exposed up to 1000µg/mL nanosilicate platelets d erived from MMT. 427 Regarding to in vivo t oxicity data , there is no information about the three o rganoclays selected in 428 the p r esent study, but Li et al. (2010) did not reported acute oral to xicity of MMT in rats exposed 429 to a single dose of 5700 mg/kg. Also Baek e t al. (2012) did not f ind any remarkable toxicity in 430 mice orally exposed to a single dose of 1000 mg/kg MTT. 431 432 In summary, o ur results sh owed that at the concentrations assayed Cloisite®20A and Clay 1 did 433 not induce cyto toxicity. Clay 2 , however, showed cytotoxic and genotoxic effe cts, a s well as an 434 alteration in GSH content. Therefore, a case b y case toxicological assessment is required , as th e 435 modifier has a role in the toxicity o bserved. Moreover, a different sensitivity was obse rved 436 depending on the cell line employed. 437 More studies a re required to elucidate the risks of these clays for human he alth as their use is 438 going to increase widely in the near future. 439 440 441 19 Acknowledgements 442 The authors wish to thank the Ministerio d e Ciencia e Inno vación (AGL2010 -21210) a nd Junta de 443 Andalucía (AGR5969) for the financial su pport for this stu dy, and the Cell Culture Service and 444 Microscopy Service of Centro de Investigación, Tecnología e Innovac ión de l a Universidad de 445 Sevilla (CITIUS) for providing technical assistance. 446 447 20 References 448 Avella, M., De Vliege r, J. J., Errico, M. E., Fischer, S., Vacca, P ., & Vo lpe, M. G. 2005. 449 Biodegradable starch/clay nanocomposite films for food packaging applications. Food Chem. 93, 450 467 – 474.Az eredo, H. M. C., 2009. Nanocomposites for food packaging applications. Food. 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FTIR techniques in clay mine ral studies, Vibrational Spectrosc. 31, 1- 10. 512 Maisanaba, S., Puerto, M., Pichardo, S., Jordá, M., Moreno, F.J., Aucejo, S., Jos, A., 2013a. In 513 vitro toxicity evalua tion o f unmodified commercial clays on the human hepatic cell line HepG2. 514 Food. Chem. Toxicol. 57, 266 -275. 515 Maisanaba, S., Pichardo, S., Jordá -Beneyto, M., Aucejo, S., Jos, A., 2013b. Toxicity of Migration 516 Extracts from Nanocomposites used in Food Packaging in Caco -2 and HepG2 Cell Lines. 517 Toxicol. In. Vitro. (under revisi on). 518 23 Maisanaba, S ., Gutierrez- Praena, D., P ichardo, S., Moreno, F.J., Jordá, M., Cameán, A.M., 519 Aucejo, S., Jos, A., 2013 c.Toxic effe cts induced by mon morillonite -based clays on the human 520 intestinal cell line Caco-2. J. Appl . Tox icol. (under revision). 521 Martin, K .R., Failla, M.L., Smith, J.C., 1997. Differential susceptibility of Caco -2 and H epG2 522 human cell lines to oxidative stress. J . Elisha Mitchell S ci . 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European Commission & Directorate - General for 542 Health and Consumers. 1 9 Ja nuary, 2009. 543 24 Sharma, A .K., Schmidt, B, Fran dsen, H., Jacobsen, N.R., Larsen, E.H., Binder up, M.L., 2010. 544 Genotoxicity of unmodified and organo-modified montmorillonite. Mutat. Res . 700, 18 – 25. 545 Silvestre, C., Duraccio, D., Cimmino, S., 20 11. Food packag ing ba sed on polymer nano materials. 546 Prog. Polym. Sci . 36, 1766-1782. 547 Sies, H. 1999. Glutathione and its role in cellularfunctio ns. Free Radical Bio Med. 27 , 916-921. 548 Volpe, M.C. 20 05. Biodegradable clay nanocomposite film s fo r fo od packaging applications.Food 549 Chem. 93; 467 – 474. 550 Zhao, J., Castranova, V., 2011. Toxicology o f nanoma te rials used in nanomedicin e . J. Toxicol. 551 Env. Heal B. 14, 593-632. 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 25 Table captions 569 Table 1. EC50 values (µg/mL) obtained after 24 and 48h exposure to Clay 2 in bot h cell lines . 570 571 Figure captions 572 Figure 1. TGA results for Cloisite ® Na + (round) and Cloi site ® 20A (square). 573 574 Figure 2. FTIR results for Cloisite ® Na + (up) and Cloisit e ® 20A (down). 575 576 Figure 3. MTS te trazolium salt red uction (a, b) a nd total protein content (c,d) of Ca co -2 (a,c) and 577 HepG2 (b,d) cells after 24h and 48h of exposure to 0 - 8 μg/mL Clay 1. Results from 3 independent 578 experiments with 6 rep licates/experiment. All values are expressed as mean ± s.d. Diffe rence s 579 were considered significant from p <0.05 (*). 580 581 Figure 4. MTS te trazolium salt red uction (a, b) a nd total protein content (c,d) of Ca co -2 (a,c) and 582 HepG2 (b,d) cells after 24h and 48 h of e xposure to 0 - 125 μg /mL Clay 2 . Results from 3 583 independent experiments with 6 replicates/experiment. All values are exp ressed as mean ± s.d. 584 Differences were considered significant from p <0 .05 (*) . 585 586 Figure 5. MTS te trazolium salt red uction (a, b) a nd total protein content (c,d) of Ca co -2 (a,c) and 587 HepG2 (b,d) cells after 24h and 48h of exposure to 0 - 62.5 μg/mL Cloisite ® 20A. Results from 3 588 independent experiments with 6 replicates/experiment. All values are exp ressed as mean ± s.d. 589 Differences were considered significant from p <0 .05 (*) . 590 591 Figure 6 . ROS content (a) in Caco-2 cells after 24 and 48h of exposure to 8.5, 17, or 34 µg/mL 592 Clay 2. ROS content (b) in HepG2 c ells after 24 an d 48h of exposure to 2 2, 44, o r 88 µg/mL Clay 593 26 2. Results f r om 3 independent experiments with 3 replicates/experiment. All va lues are expressed 594 as mean ± s.d . 595 596 Figure 7 . GS H content (a) in Caco -2 cells after 24 and 48h of exposure to 8.5, 17, or 34 µg/mL 597 Clay 2. GSH content (b) in HepG2 c ells after 24 an d 48h of exposure to 2 2, 44, o r 88 µg/mL Clay 598 2 . Results from 3 independent experiments with 3 replicates/experiment. All values are expressed 599 as mean ± s.d. ** significantly different from control ( p < 0.01). 600 601 Figure 8. Comet assay results (a) of Caco - 2 and (b) HepG2 ce lls after 24 and 48h of exposure to 602 15.65, 3 1.25, or 6 2.5 µg/ mL Cl o isite ® 20A . R e sults from 3 independent e xperi men ts with 2 603 replicates/experiment. All value s are expressed as mean ± s.d. * significantly d ifferent from 604 control ( p < 0.05). 605 606 Figure 9. Comet assay results (a) of Caco - 2 and (b) HepG2 ce lls after 24 and 48h of exposure to 607 2, 4 or 8 µg /mL Clay 1. Results from 3 independe nt experiments with 2 replicates/experiment. All 608 values are expressed as mean ± s.d. * significantly diff e rent from control ( p < 0.05). 609 610 Figure 10 . Comet assay results (a) of Caco- 2 cells after 2 4 and 48h of exposure to 8.5, 17, or 34 611 µg/mL Cl a y 2. Comet assay results (b) of HepG2 cells after 24 and 48h of exposure to 22, 44 and 612 88µg/mL. Resu lts from 3 independent experiments with 2 rep licates/experiment. A ll va lues are 613 expressed as mean ± s.d. * significantly different from control ( p < 0.0 5). ** sign ificantly different 614 from control ( p ≤ 0.01). 615 Table 1 EC50 (µg/ mL) Cell line Exposure ti m e MTS PC Caco-2 24h 55 ±6 34±3 48h 26 ±3 18 ±2 HepG2 24h 88±4 93±5 48h 51±3 74±3 Table 1 Click here to download Table: Table 1.docx a b 8.5 Figure 7 a b 0 20 40 60 80 100 Control 15.65 31.25 62.5 H2O2 % DNA in t ali Cloisite® 20A (µg/mL ) Comet A ssay Caco-2 24h 48h * * 0 20 40 60 80 100 Control 15.65 31.25 62.5 H2O2 % DNA in tail Cloisite®20A (µg/mL) Comet A ssay HepG2 24h 48h * * Figure 8 a b 0 20 40 60 80 100 Control 2 4 8 H2O2 % DNA in tail Clay 1 (µg/mL) Comet A ssay Caco-2 24h 48h * * 0 20 40 60 80 100 Control 2 4 8 H2O2 % DNA in tail Clay 1 (µg/mL) Comet A ssay HepG2 24h 48h * * Figure 9 a b Figure 10 Resultados y Discusión / Results and Discussion 225 CAPÍTULO 8 / CHAPTER 8 Sara Maisanaba, Ana I. Prieto, Silvia Pichardo, María Jordá -Beneyto, Susana Aucejo, Ángeles Jos CYTOTOXICITY AND MUTAGENICITY ASSESSMENT OF ORGANOMODIFIED CLAYS POTENTIALLY USED IN FOOD PACKAGING Toxicology In Vitro 20, 1222-1230, 2015 Elsevier Editori al System (tm) for To x icology in Vi tro Manuscri pt Draf t Manus cript N u mber: Title: Cyt otoxici ty and m u tag enicity ass essment of o rganomo dified clay s potenti all y us ed in food packaging Article Typ e: SI:The E STIV 20 14 proce eding Keyword s: clay minera ls , c ytotoxi city; mut agenicity; Ames test; HUVE C Corresp onding Aut hor: Dr. S ilvia Pichardo, PhD Corresp onding Aut hor's In st itution: Universidad de Sevilla First Au thor: Sara Maisana ba Order of Auth o rs: Sara Maisanaba; Ana I Prieto; Silvia Pichardo, P hD; Mar ia Jordá-Beneyto; Susana Aucejo; Ang eles Jos Abstract: Modern foo d packaging has made g reat advances as resul t of gl o bal tre nds and cons u mer prefere nces, whi ch are oriented to ob tain improve d food q uality an d safety. In this regard, clay minerals, an d mainly M ontmori llonite ( Mt) are attra cting consider able interest i n food packagi ng because of the impr ovements dev eloped in mechanical and bar rier proper tie s.. H ence, the pr esent work aim to a ssess t he toxici ty of fo u r Mo ntmori llo nit e-based clay miner als, an u nmodified clay , Cloisite ®Na+ (CNa+ ), and three m odified Mt clay s: Cloi site®30 B (C30B ), a comm ercial clay, a nd Clay1 and Clay2, two novel m odified organoclay s developed by the Packagi ng, Tr ansport, & Logi stics Researc h Instit ute (ITEN E). Fi rst, the cyto toxic effects w ere studie d in the h uman endo thelial cell line (HUVEC ). In ad dition, the poten tial mutageni city of the clays w as evaluated by the Ames te st. Clay1 di d not ind uce any cyt otoxic ef fects in H UVE cells, alth ou g h it ex hibited po tential mut agenicity in TA98 S. thyphim urium strain. I n c ontrast, Clay2 prod u ced cy totoxicity in en dothelial cell s but no mutageni city was rec orded. H o w ever, C Na+ was n ot cytot o xic n either mutageni c. And finally, C3 0B showe d positive results in both assays. T herefore, r esults sh owed t h at clay mineral s have a diff erent to xicity profile an d a case by c ase toxicity ev aluati on is requi re d. Silvia Pichardo Area of T oxicolog y. Faculty of Pharm acy. University of Sevil le C/Profesor G arcía Gonz ál ez 2, 41012 Seville, Spain Tel.: +34- 954556762; fax: 34- 954556422. E-m ail address: spicha r do @us.es 23 rd August, 2014 Dear E ditor, We would be very grateful if y ou conside r the manuscript entitled “ Cytotoxicity and mutagenicity assessme nt of organom od ified clays potentially used in food packaging ” for its publication in “Toxicolog y in Vitro”. The work include d in the present manuscript has been presented in the Conference ESTIV 2014 by Ms Sara Maisanaba with the following titles: - “ CYTOTOXI C I TY O F UNMODIFIED AND MODIFIED CLAYS IN AN ENDOTHELI A L CELL LINE ” - “ MUTAGENICITY AND GENOTOXICITY IN A TARGET CELL LINE OF THE MODIFIED CLAYS: CLAY1 AND CL AY2 ” Unfortunately, the ma nuscript was not read y to be sent as proceedings in this Conference. But I hope this t ime it could be considere d to be published in y o ur journal. To the extent of our knowledge this is the first wor k dealing c y tot oxic effects of four montmorillonite-based clay minerals, an unmodified cla y , Cloisite®Na+, a nd three modified Mt clay s: C loisite®30B, a commercial clay , and Clay1 and Clay2, two novel modified organoclay s, on the human cell line HUVE cells; as well as stud y i ng their mutage nic potential in or der to assess their safet y in regard to be used in food packaging. The authors declare that there a re no conflicts of interest. I am looking f orward to receiving a positive answer from you. Sincerely, Silvia Pichardo Cover Letter 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 1 Cytotoxicity and mutagenicity assessme nt of organomodified clays potential ly used in food packaging Sara Maisanaba a , Ana I Prieto a , Silvia Pichardo a* , María Jordá-Beneyto b , Susana Aucejo b , Ángeles Jos a a Area of Toxicology, Faculty of Pharmacy, U niversity of Seville, Profesor G arcía González n°2, 41012 Seville. Spain. b Area of Packaging Materials and Systems, ITE NE, C/ Albert Einstein 1, 469 80 Paterna (Valencia), Spain. * To whom correspondence should be addressed a t : Silvia Pichardo Sánchez Area of Toxicology, Faculty of Pharmacy, Universit y o f Seville, Profesor García González n°2, 41012 Seville. Spain. E-mail address: [email protected] Tel: +34 954 556762 Fax: +34 954 556422 Manuscript Click here to view linked References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 8 viable cells. The NR can be extracted from lysosomes for quantitative measurement of cell viability and cytotoxic ity of xenobiotics . Culture medium was replaced by 100 μL me dium without serum containing 10 mg/mL NR. The 96 -well plate with the NR- containing medium was returned to the incu bator for another 3 h to allow the uptake of NR into the lysosomes of viable, intact cells. Thereafter, the medium was remove d and cells were fixed for 1 min with a formaldehyde-CaCl 2 solution. By adding 0.2 mL of acetic acid-ethanol solution to the wells, the NR a bsorbed by the cells was extracted, brought into solution and quantified at 540 nm (Infinite M200, Tecan, Austria). MTS (3-(4,5-dimethylthiazol-2-yl)-5- (3 -carboxymethoxyphenyl)-2- (4 -sulfophenyl)- 2H - tetrazolium salt) reduction is carried out by dehy d rogenases enzymes present in mitochondria, being this endpoint a good marker of the damage induced i n this organelle. MTS reductio n was me asured according to the procedure o f Baltrop et al. (1991). The MTS tetrazolium compound ad ded to the medium is bioreduced by cells seeded in the 96-well plate into a colored formazan product soluble i n culture medium and is directly measured spectrophotom etrically a t 490 nm (Infinite M200, Tecan, Austria) after 2 hours of incubation in t he dark. Ames Test The incorporation version of the Ames test w as performed according to the recommendations of Maron & Ames (1983) and f ollowing the principles of OCDE guideline 471 (1997). Five Salmonella typhimurium h istidine-auxotrophic strains TA97A, TA98, TA100, TA102 and TA104 wer e used for the assay. Cultures of eac h [Document text truncated for crawler view.]