Induction of apoptosis in yeast and mammalian cells by exposure to 1,10-phenanthroline metal complexes

TIV 1118 Disk used No. pages 8, DTD=4.3.1 ARTICLE IN PRESS UNCORRECTED PROOF Induc ion o apop osis in yeas and mammalian cells by exposu e o 1,10-phenan h oline me al complexes Ba y Coyle a , Paula Kinsella b , Malachy McCann a , Michael De e eux c , Robe O’Conno b , Ma in Clynes b , Ke in Ka anagh d, * a Depa men o Chemis y, NUI Maynoo h, Co. Kilda e, I eland b Na ional Ins i u e o Cellula Bio echnology, Dublin Ci y Uni e si y, Glasne in, Dublin 9, I eland c Dublin Ins i u e o Technology, Ca hal B ugha S ., Dublin 1, I eland d Depa men o Biology, NICB, NUI Maynoo h, Co. Kilda e, I eland Accep ed 31 Augus 2003 Abs ac 1,10-Phenan h oline (phen) and me al–phen complexes display ungicidal and ungis s ic ac i i y, dis up mi ochond ial unc ion and induce oxida i e s ess. We ha e examined he effec o hese d ugs on he s uc u e o yeas and mammalian cell o ganelles and he in eg i y o cellula DNA. Exposu e o Candida albicans o [Mn(phen) 2 (mal)].2H 2 O o [Ag 2 (phen) 3 (mal)].2H 2 O (mal H 2 =malonic acid) esul ed in DNA deg ada ion whe eas exposu e o phen o [Cu(phen) 2 (mal)].2H 2 O did no . All d ugs induced ex ensi e changes o he in e nal s uc u e o yeas cells including e ac ion o he cy oplasm, nuclea agmen a ion and dis up ion o he mi ochond ion. In he case o cul u ed mammalian cells [Cu(phen) 2 (mal)].2H 2 O induced apop osis as e idenced by he ladde pa e n o DNA agmen s ollowing gel elec opho esis and also he blebbing o he cell memb ane. The o he d ugs p oduced non-specific DNA deg ada ion in mammalian cells. In conclusion, phen and me al–phen complexes ha e he po en ial o induce apop osis in ungal and mammalian cells. Gi en hei dis inc mode o ac ion compa ed o con en ional an i- ungal d ugs, phen and me al–phen complexes may ep esen a no el g oup o an i- ungal agen s o use ei he in combina ion wi h exis ing d ugs o in cases whe e esis ance o con en ional d ugs has eme ged. #2003 Published by Else ie L d. Keywo ds: Apop osis; Candida; Me al-based d ug; Fungicidal; Fungis a ic 1. In oduc ion Fungal pa hogens a e a se ious cause o in ec ion and dea h in pa ien s immuno-comp omised as a esul o disease (e.g. leukaemia) o he apeu ic p ocedu es (e.g. b oad spec um an ibio ics, immuno-supp ession p io o o gan ansplan a ion) (De Pauw, 1997). The yeas Candida albicans is an oppo unis ic ungal pa hogen which causes a ange o diseases in suscep ible indi i- duals (P alle e al., 1998). These can ange om supe - ficial in ec ions in ol ing he o al ca i y, agina o skin o se e e li e- h ea ening in ec ions in ol ing many essen ial o gans. The e has been a conside able inc ease in he incidence o disease a ibu able o his yeas in ecen yea s wi h he sp ead o AIDS, he widesp ead use o immuno-supp essi e he apy and he p olonged su i al o pa ien s wi h c i ical illnesses (Lunel e al., 1999). Con en ional he apy o he con ol o ungal in ec ions elies upon he use o polyene o azole d ugs. The mos widely used polyene an i- ungal d ug is ampho e icin B which unc ions by binding o e gos- e ol in he ungal cell memb ane c ea ing po es h ough which in acellula cons i uen s leak (Abu-Salah, 1996). Azoles a ge he e gos e ol biosyn he ic pa hway lead- ing o cells deple ed in e gos e ol and wi h ele a ed le els o oxic in e media es which p o e a al o he cell. The eme gence o C.albicans isola es esis an o an i- ungal d ugs has se ious implica ions o he con- inued success o con en ional an i- ungal he apy (Van den Bossche e al., 1998; Kon oyiannis & Lewis, 2002). 0887-2333/$ - see on ma e #2003 Published by Else ie L d. doi:10.1016/j. i .2003.08.011 Toxicology in Vi o &(&&&&)&–& www.else ie .com/loca e/ oxin i 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 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 Abb e ia ions: mal, malonic acid; MIC, minimum inhibi o y concen a ion; MM, Minimal medium; Phen, 1,10-phenan h oline. * Co esponding au ho . Tel.: +353-1-708-3859; ax: +353-1-708- 3845. E-mail add ess: ke in.ka [email protected] (K. Ka anagh). TIV 1118 Disk used No. pages 8, DTD=4.3.1 ARTICLE IN PRESS UNCORRECTED PROOF Me al-based d ugs ep esen a no el g oup o an i- ungal agen s wi h po en ial applica ions o he con ol o ungal in ec ions. 1,10-Phenan h oline (phen) and subs i u ed de i a i es, bo h in he me al- ee s a e and as ligands co-o dina ed o ansi ion me als, dis u b he unc ioning o a wide a ie y o biological sys ems (Bu le e al., 1969). Fu he mo e, when he me al- ee N,N-chela ing bases a e ound o be bioac i e i is usually assumed ha he seques e ing o ace me als is in ol ed, and ha he esul ing me al complexes a e he ac i e species (MacLeod, 1952; Dwye e al., 1969)P e- ious wo k has demons a ed ha in RPMI medium a 37 C he me al-based d ugs [Cu(phen) 2 (mal)].2H 2 O, [Mn(phen) 2 (mal)].2H 2 O and [Ag 2 (phen) 3 (mal)].2H 2 O (phen=1,10-phenan h oline; malH 2 =malonic acid) inhibi he g ow h o C. albicans by a ound 95% a a concen a ion o 5 mg/ml (Coyle e al., 2003). I was es ablished ha bo h me al- ee phen and he me al– phen complexes affec mi ochond ial unc ion, e a d he syn hesis o cy och omes b and c and uncouple espi a ion. T ea men o ungal cells wi h he Cu(II) and Ag(I) complexes esul ed in a educed amoun o e gos e ol in he cell memb ane and subsequen inc ease in i s pe meabili y. Cells exposed o me al- ee phen and he Cu(II) and Mn(II) complexes [bu no he Ag(I) complex] demons a ed an ele a ion in oxygen up ake. Indeed, pa o he mode o ac ion o his g oup o d ugs seems o lie in hei abili y o induce oxida i e s ess wi hin he cell as e idenced by he dec eased educed:oxidized glu a hione a ios (GSH:GSSG) and inc eased le els o lipid pe oxides in Candida cells ea- ed wi h [Cu(phen) 2 (mal)].2H 2 O(McCann e al., 2000). The aim o he wo k p esen ed he e was o u he cha ac e ise he mode o ac ion o hese d ugs in e ms o hei effec s on he mo phology o ungal and mam- malian cells. Due o hei diffe en mode o ac ion compa ed o he polyene and azole an i- ungal d ugs (Coyle e al., 2003), me al based d ugs may ep esen a no el g oup o an i- ungal agen s wi h po en ial appli- ca ions ei he alone o in combina ion wi h con en- ional an i- ungals. In addi ion, hey may be applicable in si ua ions whe e esis ance o con en ional an i- ungal d ugs has eme ged. 2. Ma e ials and me hods 2.1. Fungal s ain and cul u e condi ions C. albicans ATCC 10231 was ob ained o m he Ame ican Type Cul u e Collec ion, (VA, USA). Cul- u es we e g own on Sabou aud dex ose aga (SDA) pla es a 37 C and main ained a 4 C o sho - e m s o age. Cul u es we e ou inely sub-cul u ed e e y 4–6 weeks. Cul u es we e g own o he s a iona y phase (app oxima ely 110 8 /ml) o e nigh a 30 C and 200 pm in minimal medium (MM) [2% w/ glucose, 0.5% w/ yeas ni ogen base (wi hou amino acids o ammonium sulpha e), 0.5% w/ ammonium sulpha e]. 2.2. Human cell cul u e The HEp-2 cell line (ATCC CCL23) was ob ained om he Ame ican Type Cul u e Collec ion (VA, USA) and cells we e g own in MEM (Sigma Ald ich Chemical Co., Do se , UK) supplemen ed wi h 5% / oe al cal se um (Gibco, Paisley, UK), 4 mM l-glu amine and 1% / Penn-S ep (Sigma Ad ich). The DLKP cell line was ob ained om he Na ional Cell and Tissue Cul u e Cen e (Dublin, I eland) and is de i ed om a poo ly diffe en ia ed cell ca cinoma om a lymph node me a- s asis o a p ima y lung umou . DLKP cells we e cul- u ed unde he same condi ions as HEp-2 cells. Adhe en cells we e g own in 80 cm 2 cul u e flasks a 37 C and 5% CO 2 in a humidified a mosphe e and sub- cul u ed by ypsinisa ion e e y 3–4 days. 2.3. D ugs Chemicals we e ob ained om comme cial sou ces and used wi hou u he pu ifica ion. [Cu(phen) 2 (mal)].2H 2 O, [Mn(phen) 2 (mal)].2H 2 O and [Ag 2 (phen) 3 (mal)].2H 2 O we e p epa ed as p e iously desc ibed (McCann e al., 2000). 2.4. In i o oxici y es ing Sub-confluen DLKP cells we e ha es ed by ypsi- nisa ion washed and esuspended in PBS. Cells we e enume a ed mic oscopically and dilu ed wi h MEM o gi e a final cell densi y o 210 4 /ml. Nine y-six-well pla es (NUNC) we e seeded wi h 100 ml o his suspen- sion pe well and incuba ed a 37 C and 5% CO 2 in a humidified a mosphe e o 24 h o allow cell a ach- men . Subsequen ly, a ange o concen a ions o me al based d ug was added o he ows o wells and he pla es we e e-incuba ed un il con ols eached 80–90% confluency ( ypically 5–6 days). Cell g ow h in oxici y assays was quan ified as desc ibed p e iously (Ma in & Clynes, 1993). 2.5. Ex ac ion o DNA om C. albicans Yeas cells we e g own in he p esence o d ug (10 mg/ ml) o he la e exponen ial phase (18–24 h) in MM a 30 C in an o bi al incuba o . Cells we e ha es ed by cen i uga ion and washed wi h 1 mmEDTA. Cells we e esuspended in sphe oplas ing buffe (1 M so bi ol, 0.1 M EDTA, 6 mg/ml ly icase and 0.05 M di hio he i ol, pH 7.5) and incuba ed a 37 C o 2 h. Sphe oplas s we e ha es ed by cen i uga ion, esuspended in lysing buffe (50 mmEDTA, 50 mmT is (pH 8), 1% w/ SDS 2B. Coyle e al. / Toxicology in Vi o &(&&&&)&–& 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 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 TIV 1118 Disk used No. pages 8, DTD=4.3.1 ARTICLE IN PRESS UNCORRECTED PROOF and 8 mg/ml p o einase K) and incuba ed a 65 C o 0.5 h. DNA was p ecipi a ed wi h wo olumes o chil- led e hanol (95% / ) and incuba ed a 20 C o e - nigh . DNA was ha es ed by cen i uga ion, washed wi h e hanol (20% / ), ha es ed by cen i uga ion a 4000g o 25 min and allowed ai d y. The DNA was esuspended in TE buffe , 1 mg/ml RNase and incu- ba ed a 37 C o 0.5 h. E hanol (95% / ) and 3 m sodium ace a e solu ion (pH 5.2) was added and he sample s o ed a 20 C o e nigh . 2.6. Ex ac ion o DNA om cul u ed human cells Sub-confluen HEp-2 cells ha had been cul u ed in he p esence o 3 mmo me al-based d ug o 24 h we e ha es ed by ypsinisa ion, washed and esuspended in PBS. Cells (110 6 ) we e esuspended in lysis buffe [20 mmEDTA, 0.8% (w/ ) sodium lau yl sa cosia e, 100 mmT is (pH 8.0)] and 10 mg/ml RNase (Boeh inge Manneheim, Sussex, UK) and incuba ed a 37 C o 18 h. P o einase K (1 mg/ml) was subsequen ly added and he samples we e incuba ed o a u he 2 h a 50 C. 2.7. DNA gel elec opho esis Pu i y and concen a ion o DNA was de e mined by UV spec oscopy (260–280 nm). DNA om yeas and mammalian cells was un a a concen a ion o 40 mg/ml on a 0.8% (w/ ) aga ose gel a 40 V o 18 h. Following s aining wi h e hidium b omide DNA was isualised using a UV ansillumina o . 2.8. Elec on mic oscopy P ima y fixa ion o s a iona y phase yeas cells was in a 3% solu ion o glu e aldehyde in 0.1 mphospha e buffe o 2 h. Seconda y fixa ion was in a 2% solu ion o osmium e oxide in 0.1 mphospha e buffe o 1 h. Dehyd a ion o samples was in an alcohol se ies o 10, 30, 50, 75, 95 and 100%, each o 15 min. Samples we e embedded in Aga 100 esin (Aga Scien ific L d., UK) and iewed using a Hi achi H-7000 T ansmission Elec on Mic oscope ope a ing a 100 k accele a ing ol age. 3. Resul s 3.1. De e mina ion o effec o me al-based d ugs on yeas cell DNA The aim o he wo k p esen ed he e was o es ablish whe he d ug-induced oxida i e s ess al e ed he s uc- u e o he cellula o ganelles and affec ed he in eg i y o yeas DNA. The minimum inhibi o y concen a ion o each d ug was used as de e mined p e iously (Coyle e al., 2003). Cells o C. albicans we e g own o he s a- iona y phase in MM con aining 10 mg/ml o each d ug o 24 h. Cells we e ha es ed by cen i uga ion, he DNA was ex ac ed as desc ibed and isualised by e hi- dium b omide s aining ollowing aga ose gel elec o- pho esis. The DNA ex ac ed om yeas cells exposed o [Ag 2 (phen) 3 (mal)].2H 2 O shows ex ensi e deg ada ion (Fig. 1). Smalle amoun s o deg ada ion, as demon- s a ed by smea ing, a e also isible in cells ea ed wi h [Cu(phen) 2 (mal)].2H 2 O and [Mn(phen) 2 (mal)].2H 2 O, whe eas incuba ion o cells wi h me al- ee phen causes li le o no DNA b eakdown unde he condi ions employed he e. 3.2. Elec on mic oscopic examina ion o C. albicans ol- lowing g ow h in he p esence o me al-based d ugs Cul u es o C. albicans we e g own o he s a iona y phase o e nigh in MM medium a 30 C and 200 pm in he p esence o each d ug a a final concen a ion o 10 mg/ml. Cells we e ha es ed by cen i uga ion, washed wi h PBS (pH 7.2) and placed on ice p io o p epa a ion o TEM examina ion (as desc ibed). Cells g own in he absence o d ug showed no mal cellula mo phology wi h a dis inc cell wall, an in ac nucleus and nume ous memb anous o ganelles (Fig. 2a). In con as , cells g own in he p esence o me al- ee phen demons a ed a dis ended cell wall, up u ed in e nal o ganelles and he wi hd awal o he cy oplasmic mem- b ane om wi hin he cell wall (Fig. 2b). Cells ea ed wi h [Ag 2 (phen) 3 (mal)].2H 2 O possessed a dis ended cell wall, up u ed o ganelles and, in some cases, a ag- men ed nucleus (Fig. 2c). The mos ob ious ea u e o [Cu(phen) 2 (mal)].2H 2 O- ea ed cells was he occu ence Fig. 1. DNA banding pa e n o C. albicans cells ea ed wi h phen and me al-phen complexes o 24 h. Lane 1: DNA om con ol cells, Lane 2: phen, Lane 3: [Cu(phen) 2 (mal)].2H 2 O, lane 4: [Mn(phen) 2 (- mal)].2H 2 O, Lane 5: [Ag 2 (phen) 3 (mal)].2H 2 O. B. Coyle e al. / Toxicology in Vi o &(&&&&)&–&3 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 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 TIV 1118 Disk used No. pages 8, DTD=4.3.1 ARTICLE IN PRESS UNCORRECTED PROOF o an enla ged nucleus which, in some cells, was c escen shaped (Fig. 2d). Wi h his d ug he in e nal o ganelles appea ed in ac bu some sh inkage o he cy oplasm wi hin he cells was appa en . Cells exposed o [Mn(phen) 2 (mal)].2H 2 O had, in mos cases, comple ely dis up ed o ganelles. Some o hese cells possessed enla ged nuclei while o he s appea ed o con ain dis inc nuclea agmen s (Fig. 2e). 3.3. Effec o me al-based d ugs on in eg i y o mamma- lian DNA P e ious s udies in o he effec o me al-phen com- plexes on ungal cell iabili y indica ed ha he d ugs dis up mi ochond ial unc ion (Coyle e al., 2003). In i o oxici y assays we e pe o med o es ablish he concen a ion o 1,10-phen and [Cu(phen) 2 (mal)].2H 2 O Fig. 2. Elec on-mic og aphs o C. albicans cells exposed o phen and me al-phen complexes o 24 h. (a) Con ol, (b) Phen, (c) [Ag 2 (phen) 3 (mal)].2H 2 O, (d) [Cu(phen) 2 (mal)].2H 2 O, (e) [Mn(phen) 2 (mal)].2H 2 O. (Ba =1 mM). 4B. Coyle e al. / Toxicology in Vi o &(&&&&)&–& 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 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 TIV 1118 Disk used No. pages 8, DTD=4.3.1 ARTICLE IN PRESS UNCORRECTED PROOF capable o killing cul u ed human cells. The esul s indica ed IC 50 alues o 0.002 mg/ml and 0.001 mg/ml o each d ug, espec i ely agains DLKP cells. HEp-2 cells we e chosen o de e mine whe he he me al–phen complexes induced apop osis since he appea ance o he DNA agmen a ion pa e n asso- cia ed wi h his mode o cell dea h (Ve haegen, 1998)is easie o isualise in his cell line han in he DLKP line. HEp-2 cells we e cul u ed o 24 h in he p esence o 3 mmo me al- ee phen o he Cu(II), Mn(II) and Ag(I) phen complexes, and he DNA was subsequen ly ex ac ed and sepa a ed by aga ose gel elec opho esis. Deg ada ion o high molecula weigh DNA was e i- den in hose cul u es ea ed wi h Cu(II), Mn(II) and Ag(I) phen. Cells ea ed wi h me al- ee phen also showed ex ensi e deg ada ion o DNA. T ea men o cells wi h he coppe –phen complex p oduced a DNA pa e n which was di ided in o dis inc agmen s. The appea ance o his DNA agmen a ion ‘ladde ’ is indi- ca i e o apop osis (o p og ammed cell dea h) occu - ing in he cells in esponse o he me al based d ug. Cul u es ea ed wi h Mn o Ag complexes demon- s a ed DNA deg ada ion bu did no p oduce a specific ‘ladde ’ agmen a ion pa e n a his concen a ion. 3.4. Mic oscopic examina ion o cul u ed human cells g own in he p esence o [Cu(phen) 2 (mal)].2H 2 O P e ious wo k has es ablished ha he me al-phen complexes affec he oxygen up ake a e o C. albicans and induced oxida i e s ess wi hin he ungal cell (McCann e al. 2000; Coyle e al., 2003). We sough o de e mine whe he hese complexes affec ed he g ow h and cellula mo phology o cul u ed mammalian cells in o de o assess hei oxici y in i o owa ds cells de i ed om human issue. In o de o examine he mo phological changes aking place in cul u ed cells ollowing exposu e o he mos oxic o he me al–phen complexes sub-confluen DLKP cells we e exposed o [Cu(phen) 2 (mal)].2H 2 O(3mM) and incuba ed a 37 C o 48 h. Un ea ed cells show he ypical mo phology o adhe en epi helial cells (Fig. 4A). Exposu e o [Cu(phen) 2 (mal)].2H 2 O o 24 h p oduced cells showing e idence o memb ane ‘blebbing’ (Fig. 4b)— a ea u e o cell dea h by apop osis (Ve - haegen, 1998). A e 48 h incuba ion a numbe o cells had agmen ed o gi e clus e s o small sub-cellula packe s (Fig. 4c)— en a i ely iden ified as apop o ic bodies, a ea u e o he la e s ages o apop osis (Ve - haegen, 1998). 4. Discussion The in i o an ibac e ial ac ion o phen has been demons a ed on se e al species o bac e ia (Dwye e al., 1969; Feeney e al., 1957). Whe eas me al–phen complexes can be bac e is a ic (Dwye e al., 1969) and bac e icidal (Bu le e al., 1969) owa ds many G am- posi i e bac e ia hey a e ela i ely ineffec i e agains G am-nega i e o ganisms. In addi ion, dilu e aqueous solu ions o phen and i s Cu(II) and Mn(II) complexes we e highly oxic o clinical isola es o Candida species (Ge agh y e al., 2000; Ge agh y e al., 1998). Ea lie in i o expe imen s in ou labo a o ies ha e shown ha phen and a numbe o ansi ion me al complexes inco po a ing his chela ing ligand a e ex e- mely ac i e an i- ungal d ugs (Ge agh y e al., 1999a,b,c; De e eux e al., 2000a,b; McCann e al., 2000; Ge agh y e al., 2000). The compounds ha e minimum inhibi o y concen a ions in he ange 1.25– 5.0 mg/ml and, a a concen a ion o 10 mg/ml, display some ungicidal ac i i y. T ea ing exponen ial and s a- iona y phase yeas cells wi h phen and he Cu(II) and Mn(II) complexes induces a d ama ic inc ease in oxygen consump ion. All o he d ugs cause educ ions in he le els o cy och omes b and c in he cells, while he Ag(I) complex also lowe s he amoun o cy och ome aa 3 . Cells ea ed wi h phen and he Cu(II) and Ag(I) species show educed le els o e gos e ol while he Mn(II) complex induces an inc ease in he s e ol con- en . Ex ensi e s udies wi h [Cu(phen) 2 (mal)].2H 2 O indica ed ha his d ug induces significan cellula oxi- da i e s ess (dec eased educed:oxidized glu a hione a ios (GSH:GSSG) and inc eased le els o lipid pe - oxides). Fu he mo e, as he d ugs we e no uni o mly ac i e his sugges ed ha hei bioac i i y has a deg ee o me al-ion dependency. The d ugs dis up mi ochon- d ial unc ion, uncouple espi a ion and p omo e oxi- da i e s ess in he o ganism (Coyle e al., 2003). As such, phen and he me al–phen complexes may ep e- sen a no el se o highly ac i e an i- ungal agen s whose mode o ac ion is significan ly diffe en o ha o he polyene and azole p esc ip ion d ugs. Fig. 3. DNA banding pa e n om HEp-2 cells exposed o phen and me al–phen complexes o 24 h. Lane 1: molecula weigh s anda ds, Lane 2: con ol, Lane 3: phen, Lane 4: [Cu(phen) 2 (mal)].2H 2 O, lane 5: [Mn(phen) 2 (mal)].2H 2 O, Lane 6: [Ag 2 (phen) 3 (mal)].2H 2 O. B. Coyle e al. / Toxicology in Vi o &(&&&&)&–&5 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 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 TIV 1118 Disk used No. pages 8, DTD=4.3.1 ARTICLE IN PRESS UNCORRECTED PROOF The wo k p esen ed he e is a p og ession o he abo e mechanis ic s udies and examines he effec s o he me al–phen complexes on he in eg i y o DNA and cellula mo phology o C. albicans and cul u ed mam- malian cells. Exposing C. albicans o [Ag 2 (phen) 3 (mal)].2H 2 O o [Mn(phen) 2 (mal)].2H 2 O leads o non- specific DNA clea age. In he case o he Mn(II) com- plex his may be as a esul o oxida i e damage o he cells caused by ele a ed le els o oxygen up ake (Coyle e al., 2003). The Cu(II) complex and me al- ee phen seem o ha e li le effec on ungal DNA. This la e finding is su p ising since phen and coppe (I)–phen complexes a e known o clea e DNA wi h he same p e e ences as mic ococcal nuclease (Jessee e al., 1982; Chen & Sigman, 1986). I is possible ha ei he he concen a ion used was no op imal o induce ungal cell DNA deg ada ion o ha he cells we e capable o epai ing he damage o e he ime ame o he expe i- men . In addi ion, i is possible ha he oxida i e s ess induced by hese compounds can inhibi Caspase ac i - i y which is sensi i e o he edox balance wi hin he cell (G een & K oeme , 1998). Elec on-mic og aphic examina ion o ungal cells exposed o phen and he me al–phen complexes e eals se e e dis up ion o in e nal cellula s uc u es (Fig. 2a–e). In pa icula , nuclea dis up ion is e iden ollowing exposu e o [Ag 2 (phen) 3 (mal)].2H 2 O [Mn(phen) 2 (mal)].2H 2 O and [Cu(phen) 2 (mal)].2H 2 O. This is con- sis en wi h he clea age o ungal DNA e iden in Fig. 1. Nuclea agmen a ion is cha ac e is ics o apop osis as is nuclea ‘c escen ’ o ma ion e iden in many cells (Cohen, 1993). Apop osis in ungal cells ol- lows many o he same s eps e iden in animal cells including agmen a ion o he nucleus, deg ada ion o DNA and dis up ion o in e nal o ganelles (Roze & Linz, 1998). Disc e e apop o ic bodies a e no o med. Exposu e o mammalian cells o [Cu(phen) 2 (mal)].2H 2 O a a concen a ion o 0.003 mg/ml esul s in a DNA agmen a ion pa e n which is cha ac e is ic o cells dying by apop osis (Cohen, 1993; Ve haegen, 1998). In his mode o cell dea h, he inju ed cell plays an ac i e ole in i s own demise and one pa o he p ocess is he clea age o nuclea DNA in o specific sized agmen s by an endonuclease gi ing ise o a ‘ladde ’ pa e n o ag- men s upon gel elec opho esis (Cohen, 1993; Co e and Al-Rubeai, 1995). Coppe –phen complexes ha e p e- iously been shown o induce apop osis in a ange o cell lines (Zhou e al., 2002 a,b; De Vizcaya-Ruiz e al., 2002). Exposu e o cul u ed mammalian cells o me al- ee phen, [Ag 2 (phen) 3 (mal)].2H 2 O o [Mn(phen) 2 (mal)].2H 2 O did Fig. 4. Mic og aphs o DLKP cells cul u ed in he p esence o [Cu(phen) 2 (mal)].2H 2 O. (a) Cells showing memb ane ‘blebbing’ a e 24 h exposu e, (b) Apop o ic bodies a e 48 h exposu e, (c) Dead and de ached cells a e 96h exposu e. (MB: memb ane blebbing; AB: Apop o ic bodies). (O i- ginal magnifica ion: (a) and (b) 400, (c) 100). 6B. Coyle e al. / Toxicology in Vi o &(&&&&)&–& 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 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 TIV 1118 Disk used No. pages 8, DTD=4.3.1 ARTICLE IN PRESS UNCORRECTED PROOF no gi e ise o a specific DNA agmen a ion ladde pa e n bu ex ensi e non-specific DNA agmen a ion is isible. F om his i is possible o conclude ha [Cu(phen) 2 (mal)].2H 2 O ac i a es mammalian cell dea h by apop osis and ha he o he d ugs induce non-spe- cific clea age o DNA possibly due o he nuclease-like ac i i y o he phen ligand (Jessee e al., 1982; Chen and Sigman, 1986). Examina ion o cul u ed mammalian cells exposed o [Cu(phen) 2 (mal)].2H 2 O o 24 h show cells unde going memb ane ‘blebbing’. A e 48 h a numbe o cells we e obse ed o ha e unde gone agmen a ion o yield s uc u es en a i ely iden ified as ‘apop o ic bodies’ (Fig. 4). These findings and he DNA deg ada ion pa - e n obse ed p e iously wi h his d ug (Fig. 3) a e consis en wi h he induc ion o cell dea h by apop osis (Ve haegen, 1998). A simila finding was made by Viz- caya-Ruiz e al. (2000) who demons a ed he abili y o coppe based an i-cance d ugs o induce apop osis in human o a ian ca cinoma cells. In ha case he induc- ion o apop osis was moni o ed by changes in cell mo phology, ac i a ion o caspases and he deg ada ion o DNA o gi e a ladde pa e n o agmen s. Coppe – 1,10-phenan h oline complexes ha e been shown o induce G1-phase specific apop osis in a li e ca cinoma cell line (Zhou e al., 2002a,b), u he suppo ing he iew ha he [Cu(phen) 2 (mal)].2H 2 O employed in his wo k is capable o ac i a ing cell dea h by apop osis. Coppe chela o s ha e been shown o accumula e cop- pe wi hin hymocy es which can igge oxida i e-s ess induced apop osis (Nobel e al., 1995). In conclusion, he me al-phen complexes examined he e ha e a po en an i- ungal effec being capable o inhibi ing g ow h o C. albicans by 95% a a concen- a ion o 5 mg/ml (Coyle e al., 2003). Yeas and mam- malian cells exposed o hese complexes a a concen a ion o 10 mg/ml show DNA clea age. In he case o mammalian cells [Cu(phen) 2 (mal)].2H 2 O induces a DNA agmen a ion pa e n indica i e o apop osis. TEM examina ion o yeas cells e eals g oss dis o ion o cellula s uc u es and nuclea agmen a ion. This wo k indica es ha , in addi ion o i s effec on mi o- chond ial unc ion and oxygen up ake (Coyle e al., 2003), [Cu(phen) 2 (mal)].2H 2 O also plays a ole in indu- cing cell dea h by apop osis in yeas and mammalian cells. The mi ochond ion plays a cen al ole in go e n- ing he induc ion o apop osis (G een & K oeme , 1998) and he d ugs examined he e, pa icula ly [Cu(phen) 2 (mal)].2H 2 O, in e e e wi h mi ochond ial unc ion (Coyle e al., 2003) and in eg i y which may be sufficien o push he cell owa ds apop o ic cell dea h. Whe he o no apop osis is a di ec effec o exposu e o he me al based d ugs o is ela ed o an effec on he mi o- chond ion induced by hem (inc eased oxygen up ake, dis up ion o cy och ome syn hesis (Coyle e al., 2003) is cu en ly being in es iga ed. The con en ional polyene and azole an i- ungal d ugs a ge e gos e ol in he ungal cell memb ane o inhibi e gos e ol biosyn hesis, espec i ely. We ha e demon- s a ed ha he me al–phen complexes examined he e ha e a dis inc mode o ac ion and may ep esen a no el g oup o an i- ungal agen s o be used alone o in combina ion wi h exis ing an i- ungal d ugs. 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