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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 &(&&&&)&–&
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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).
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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
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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.
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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).
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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.
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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 &(&&&&)&–&
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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. In addi-
ion, me al-based d ugs may offe he possibili y o
o e -coming he eme ging p oblem o esis ance o
con en ional an i- ungal d ugs (Van den Bossche e al.,
1998; Kon oyiannis & Lewis, 2002).
Unci ed e e ences
B and , 1954; McNaugh and Owen, 1949 and Tu -
ian, 1951 a e no ci ed in he ex .
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