Use of micronucleus and comet assay to evaluate evaluate the genotoxicity of oregano essential oil (Origanum vulgare l. Virens) in rats orally exposed for 90 days

1 Use of micronucleus and comet assay to evaluate the genotoxicity of oregano essential oil (Origanum vulgare l. Virens) in rats administered orally for 90 days. María Llana-Ruiz-Cabello1, María Puerto1, Sara Maisanaba1, Remedios Guzmán- Guillén1, Silvia Pichardo1, Ana M. Cameán1 1Area of Toxicology, Faculty of Pharmacy, Universidad de Sevilla, Profesor García González n°2, 41012 Seville. Spain. Running title: Genotoxicity study of oregano essential oil in rats *Corresponding author: Silvia Pichardo Sánchez Area of Toxicology, Faculty of Pharmacy, Universidad de Sevilla, Profesor García González n°2, 41012 Seville. Spain. E-mail address: [email protected] 2 Abstract Essential oils from Origanum spp. exhibit antioxidant and antimicrobial activities making them suitable for use as food additives. The incorporation of oregano essential oil in active food packaging is under study; however, it has been not authorized for this purpose thus far. In order to fulfill the requirements of the European Food Safety Authority (EFSA), the aim of the present study was to determine the genotoxic potential of oregano essential oil using both micronucleus (MN) test and comet (standard and enzyme-modified) assays in Wistar rats treated with 50, 100 or 200 mg/kg body weight administered daily for 90 days. MN was performed in cells from the bone marrow and standard and enzyme-modified comet assays were conducted in stomach, liver and blood cells. The major compound detected in the analytical study of oregano essential oil from Origanum vulgare L. virens, was carvacrol (55.82%) followed by thymol (5.14%), as well as their precursors, γ-terpinene (16.39%) and ρ-cimne (4.71%). The results obtained in the genotoxicity assays indicated lack of effect in MN and standard comet assay under the conditions tested. Further, no apparent oxidative damage was observed in the enzyme-modified comet assay in any of the tissues examined of rats exposed to oregano essential oil for 90 days. Therefore, this oregano essential oil appears to be safe in Wistar rats and might be considered as a potential active material in food packaging industry. Keywords: genotoxicity, micronucleus, comet assay, oregano essential oil, in vivo 3 Introduction Herbs and spices containing essential oils (EO) are widely used as flavoring substances in food, soft drinks and beverages (Liju et al. 2013) and might be considered to be used as food additives as an attractive alternative to the synthetic ones in order to improve food shelf life (Hylgaard et al. 2012). Therefore, the safety of these EO needs to be assessed prior their utilization in the food industry either as additive in foods or as active substances in food packaging. In this regard, although antioxidant and antimicrobial studies regarding botanical preparation and/or its respective components are known (Bakkali et al. 2008; Burt 2004; Lang and Buchbauer 2012), toxicological data for EO are still scarce (Hwang and Kim 2012; Escobar et al. 2012, 2015), especially in vitro and in vivo data on genotoxic potential. Further, the disparity in these results needs to be investigated due to the lack of awareness if some of the compounds detected in the EO exhibit genotoxic, carcinogenic profile (Mellado-Garcia et al. 2016a). Among the predominant EO employed due to their extraordinary antimicrobial and antioxidants properties, the oregano essential oil (OEO) requires critical attention (Burt 2004; Olmedo et al. 2014; Viuda-Martos et al. 2009). This OEO is usually obtained from the aerial part of Origanum spp. by steam distillation, hydrodistillation or simultaneous distillation-extraction methods (Bayramoglu et al. 2008). OEO is included as well in the GRAS list by the FDA, although in Europe more studies are required to be approved for this purpose. Regarding OEO safety, varying findings were noted regarding in vitro toxicity potential of parent compound and main components as previously reported (Llana-Ruiz-Cabello et al. 2015). In general, the OEO tested did not display mutagenic activity with or without metabolic activation in the Ames test at the conditions tested (Ipek et al. 2005; De Martino et al. 2009; Gulluce et al. 2012); however, a reduction in mitotic index, chromosomal and nuclear irregularities were 4 found (Hamedo and Abdelmigid 2009). The nature of OEO makes it problematic for evaluation due to (1) its mixture in aqueous solutions, such as culture media of cell models, (2) results in turbid suspension due to hydrophobicity; and consequently, endpoints of in vitro assays are difficult to determine (Hanlon et al. 2011). Hence, the assessment of their main compounds is an alternative when these are well defined (EFSA, 2009). Similarly to OEO, contradictory results were found regarding its main components, such as carvacrol and thymol (Llana-Ruíz-Cabello et al. 2015). Absence of mutagenic activity for carvacrol in the Salmonella/microsome reversion test (Stammatti et al. 1999) and in the sister chromatid exchange assay (Ipek et al. 2003) was reported. However, Ipek et al. (2005) and Llana-Ruíz-Cabello et al. (2014) demonstrated that carvacrol is a potent, direct-acting mutagen in the Ames test without and with external metabolic activation. Similar to mutagenic assays, in vitro studies of genotoxic activity of carvacrol provided contradictory observations because this substance induced both positive (Aydin et al. 2005a; 2005b; Llana-Ruíz-Cabello et al. 2014) and negative (Horváthová et al. 2006) results. In case of positive in vitro results EFSA urged to conduct in vivo tests. In addition, the Scientific Committee notes small number of substances that are negative in vitro might exert positive in vivo results as in vitro metabolic activation system does not encompass the full spectrum of potential genotoxic metabolites generated in vivo (EFSA, 2011). Consequently, in vivo data as well as an appropriate OEO profile characterization are necessary (EFSA 2009). In this regard, no apparent in vivo genotoxic effect for carvacrol was reported in rats using a combined micronucleus (MN) and comet assays (Llana-Ruiz-Cabello et al. 2016a). In the present investigation, the chemical composition of OEO obtained from Origanum vulgare L. virens grown in Almería (Spain) was examined taking into account that botanical preparations need to be characterized due to variability. Further, 5 both MN and comet assays were conducted to evaluate in vivo potential genotoxic effects of OEO in bone marrow, as well as in stomach, liver, and blood cells of Wistar rats administered OEO for 90 days. Materials and Methods Supplies and chemicals Chemicals, including Endo III and FPG, were purchased from Sigma-Aldrich (Madrid, Spain), VWR Eurolab (Madrid, Spain), C-viral S.L. (Seville, Spain) and BioWhittaker (Madrid, Spain). Isolation of essential oil and gas chromatography analysis The isolation of the OEO was performed in El Jarpil S.L. (Almería, Spain). Leaves and aerial parts of oregano (Origanum vulgare L. virens) were collected from Bédar (Almeria, Spain) in June 2012 and stored for 24-48 hr at 23ºC before distillation. A continuous water steam distillation extraction was performed for approximately 3.5 hr and the oil collected and stored at 4°C. The analysis was carried out according to Llana- Ruíz-Cabello et al. (2017), using a Hewlett Packard 5890 chromatograph interfaced to a Hewlett Packard 5970 mass selective detector (Hewlett Packard, Palo Alto, USA). Components found over 0.01% are presented in Llana-Ruíz-Cabello et al. (2017). Animal housing and feeding conditions The Ethical Animal Experimentation Committee of the University of Córdoba and the Junta de Andalucía approved the procedures developed in this study (project no. 05/10/2015-339). In addition, all animals received humane care in compliance with the guidelines for the protection of animals used for scientific purposes (Directive, 2010/63/EU, Decision, 2012/707/UE and RD 53/2013). 6 Forty-six Wistar rats (23 males and 23 females) strain Crl.WI (Han) were randomly distributed into 5 groups per gender (5 rats/group and 3 rats/positive control group), with similar mean body weight (297.5 ± 2 g for males and 188.7 ± 3.6 g for females) for each group, and individually housed in cages. Rats were fed with standard lab feed (Harlan 2014, Harlan Laboratories, Barcelona, Spain) and water ad libitum. Experimental design and treatment In order to reduce animal usage in genotoxicity testing, the European Food Safety Authority (EFSA) recommends to integrate both MN and comet assay into repeateddose toxicity study (EFSA, 2011). Hence, in this investigation, the MN and comet assay, described in the Organisation for Economic Co-operation and Development (OECD) protocols (OECD 474 and 489, respectively) were developed associated into a repeated-dose toxicity study (OECD 408). After acclimatization, animals were treated daily for 90 days with 50, 100 or 200 mg/kg/day of OEO incorporated to neutral gelatin according to Mellado-García et al. (2016a). These doses were selected according to an acute oral toxicity study “Up and Down Procedure” OECD 425 (OECD 2008) and after a palatability experiment which evidence that 200 mg/kg/day was the highest dose tolerated by animals. Finally, two additional spaced dose levels separated by a factor of 2 were employed. Neutral gelatin without OEO was used as negative control. Acute oral exposure was selected for the positive control group (three male and three female rats) which was administered 200 mg/kg/day ethylmethanesulfonate (EMS) using a stomach tube (Vygon, Ecouen, France). In order to obtain the positive controls for both assays three dosages were used as described by Bowen et al. (2011) and Mellado-García et al. (2016b). Sample collection 7 Samples of bone marrow, stomach and liver were collected according to Mellado-García et al. (2016b). Blood samples were obtained by intracardiac injection (lightly anesthetized with isoflurane) and maintained in Vacutainer® sodium heparin tubes (Becton Dickinson, Rutherford, NJ). Micronucleus (MN) assay The recommendations of Corcuera et al. (2015) and the principles of OECD guideline 474 were followed to perform the mammalian erythrocyte MN test in bone marrow of Wistar rats exposed to OEO (OECD 2014a). Briefly, bone marrow extensions were performed on slides, fixed in methanol and stained with Giemsa (10 %). Then, the polychromatic erythrocytes (PCE) among total erythrocytes (normochromatic erythrocytes (NCE) + PCE) ratio and PCE among NCE ratio were determined by counting 500 erythrocytes per animal. The incidence of micronucleated immature erythrocytes (MNPCE) expressed as % MN was analyzed by counting a total of 5000 cells per animal. Isolation of single-cell suspensions for the comet assay Single cell suspensions from stomach and liver were isolated based upon the method of Corcuera et al. (2015) and Mellado-García et al. (2016b). Briefly, tissues were washed with Merchant´s buffer (MB) (0.14 M NaCl, 1.47 nM KH2PO4, 2.7 mM KCl, 8.1 mM Na2HPO4, 10 mM Na2EDTA, pH 7.4) and medium discarded. After that a portion of each tissue were homogenized in cold at 40C. Finally, homogenates were centrifuged, filtered and mixed with 5ml buffer until slide preparation. Heparinized blood samples were mixed v/v (1/1) with phosphate buffered saline (PBS) solution and lymphocytes isolated using histopaque® (Sigma-Aldrich, Madrid, Spain) and centrifuged at 1200g for 30 min. Finally, cells were washed twice with PBS and resuspended in PBS at a concentration of 2 x 105 cells/ml. 8 Standard and enzyme-modified comet assay For blood samples, 30 µl cells suspension was mixed with 140 µl 0.5% low-melting point agarose and 12 drops of 5 µl were placed on a microscope slide. For stomach and liver cell suspensions were mixed with 1% low-melting point agarose and mixtures placed on a microscope slide in the same manner as blood samples. Then, the standard comet assay was performed as previously described by Mellado-García et al. (2016b) and according to the recommendations of OECD guideline 489 (OECD, 2014b). Combining the comet assay with restriction enzymes such as Endo III, which responds to oxidized pyrimidines, and formamidopyrimidine (DNA) glycosylase (FPG) , which recognizes altered purine bases including 8-oxo-guanine, enabling detection of lesions that are not severe breaks (Azqueta et al., 2009). Briefly, 30 µl cell suspension were mixed with 140 µl pre-warmed low-melting-point agarose in PBS, and immediately 12 aliquots of each cell suspension were placed on agarose precoated glass slides. After lysis, slides were washed three times for 5 min with enzyme buffer (40 mM HEPES, 0.1M KCl, 0.5 mM EDTA, 0.2 mg/ml bovine serum albumin, pH 8). Subsequently, two gels in each slide were exposed to 30 µl lysis solution, enzyme buffer alone (buffer F), buffer F containing FPG (10 U/ml) or buffer F containing Endo III (14 U/ml) for up to 30 min in a metal box at 37 ºC. Then, nuclei were denatured and electrophoresis performed. Finally, DNA was neutralized in PBS, washed with water and fixed with 70% and absolute ethanol before staining. Images of at least 100 randomly selected nuclei per animal were analyzed with the image analysis software Comet Assay IV (Perceptive Instruments, UK). The % DNA in tail represents DNA strand breaks and oxidized damage in DNA bases. Results obtained for control groups exhibited 10 % DNA in tail indicating that cells are in satisfactory conditions for comet assay (Collins 2004). 9 Statistical analysis Results from the MN assay are presented as mean ± SD for each group of animals and statistical analysis was performed using the analysis of variance (ANOVA) followed by Dunnett’s multiple comparison tests. For the standard and enzymemodified comet assay, means ± SD were calculated and total scores of different groups compared using the non-parametric Kruskal–Wallis test followed by the Mann–Whitney U-test when the first test showed differences. Analysis was performed using Graph-Pad InStat software (GraphPad Software Inc., La Jolla, USA). The criterion for significance was set at p<0.05. Results No mortality occurred during the study. Body weight gain or food/water consumption were not significantly altered through the experiment. Additional data is available in Llana-Ruiz-Cabello et al. (2017). Chemical composition of OEO Gas chromatography-mass spectrometry (GC-MS) analysis of Origanum vulgare L. virens essential oil resulted in identification of 108 compounds, representing 99.9% of the essential oil. 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Bone marrow cytotoxicity expressed as micronucleated polychromatic erythrocytes (PCE) among total erythrocytes; PCE among total erythrocytes (normochromatic erythrocytes (NCE) + PCE) ratio; and the micronuclei induction expressed as % MN. The values are expressed as mean±SD. * Significant from control *p < 0.05 23 Groups N Doses Sex PCE/Total PCE/NCE % MN Negative Control 5 - Male 0.49±0.02 0.98±0.07 0.34±0.10 5 Female 0.50±0.02 1.00±0.10 0.23±0.06 Positive Control (EMS) 3 200 mg/Kg Male 0.31±0.03* 0.45 ±0.07* 8.17±1.33* 3 Female 0.31±0.03* 0.46±0.06* 7.55±0.20* Oregano essential oil 5 50 mg/Kg Male 0.50±0.01 0.99±0.04 0.33±0.13 5 Female 0.49±0.03 0.98±0.13 0.21±0.11 5 100 mg/kg Male 0.45±0.03* 0.83±0.09* 0.27±0.20 5 Female 0.47±0.04 0.88±0.15 0.28±0.18 5 200 mg/kg Male 0.41±0.02* 0.75±0.12* 0.35±0.21 5 Female 0.40±0.02* 0.68±0.06* 0.24±0.07 0 10 20 30 40 50 60 Control - 81mg/Kg 256mg/Kg 810mg/Kg Control + Control - 81mg/Kg 256mg/Kg 810mg/Kg Control + Control - 81mg/Kg 256mg/Kg 810mg/Kg Control + % DNA in tail Comet Assay (SBs) Endo III-sensitive sites FPG-sensitive sites Stomach male female *** *** *** *** *** *** 0 10 20 30 40 50 60 Control - 81mg/Kg 256mg/Kg 810mg/Kg Control + Control - 81mg/Kg 256mg/Kg 810mg/Kg Control + Control - 81mg/Kg 256mg/Kg 810mg/Kg Control + % DNA in tail Comet Assay (SBs) Endo III-sensitive sites FPG-sensitive sites Liver male female *** *** *** *** *** *** 0 10 20 30 40 50 60 Control - 81mg/Kg 256mg/Kg 810mg/Kg Control + Control - 81mg/Kg 256mg/Kg 810mg/Kg Control + Control - 81mg/Kg 256mg/Kg 810mg/Kg Control + % DNA in tail Comet Assay (SBs) Endo III-sensitive sites FPG-sensitive sites Blood male female *** *** *** *** ****** A B C