Participation of active oxygen species in the induction of chromosomal aberrations by cadmium chloride in cultured Chinese hamster cells

137

Mu tation Research, 143 (1985) 137-142 Elsevier

MTR 0696

Participation of active oxygen species in the induction of chromosomal aberrations by cadmium chloride in cultured Chinese hamster cells Takafumi Ochi* and Motoyasu Ohsawa Department of Environmental Toxicology, Faculty of Pharmaceutical Sciences, Teikyo University, Sagamiko, Kanagawa 199-01 (Japan) (Accepted 18 February 1985)

Summary The effect of various scavengers of active oxygen species on the induction of chromosomal aberrations by cadmium chloride (CdCIz) was investigated in cultured Chinese ham ster V79 cells. Incid ences of chromosomal aberrations by CdCIz were partially or fully reduced by the presence of catalase, mannitol (a scavenger of hydroxyl radicals) and but ylated hydroxytoluene (BHT , an antioxidant). These findings ma y indicate participation o f the acti ve oxygen species such as hydrogen peroxide (H 20 2) or hydrox yl radicals in the c1astogenicity of cadmium. In cont rast, superoxide dismutase (SOD) and dimethylfuran (a scavenger of singlet oxygen) did not influence incidences of chromosomal aberrations by CdCh. The se results suggest that superoxide anion and singlet oxygen are not directly involved in the c1astogenicity of the metal. The presence of am inotriazole (an inhibitor of catalase) increased incidences of chromosomal aberrations by CdCh, Thi s emphasizes participation of H 202 in the clastogenicity of cadmium.

The carcinogenic activity of cadmium compounds is well documented (Heath et aI., 1962; Kazantzis, 1963; Gunn et aI., 1963, 1964; Guthrie, 1964). Mutagenic activity of cadmium has been sho wn in the assa y system for mutation at the hypoxanthine-guanine pho sphoribosyltransferase (Och i and Ohsawa, 1983) or th ymidine kinase (Amacher and Paillet, 1980; Oberly et aI., 1982) locus in cultured mammalian cells. In contrast, cadmium was not mutagenic in most microbial assa ys (Venitt and Levy, 1974; Nishioka, 1975; Heddle and Bruce, 1977; Putrament et aI., 1977), although the rec assa y in Bacillus subtilis gave "To whom all cor respondence should be addressed .

positive results (Nishioka, 1975; Kanematsu et aI., 1980). Cadmium has also been shown to be c1astogenic in human lymphocytes when given as CdS (Shiraishi et aI. , 1972) or CdCl , (Cea et aI. , 1983) or cultured hamster cells when given as CdS0 4 (Rohr and Bauchinger, 1976). We have also shown that structur al and numerical changes in chromosomes were induced with a concen tration dependency by the short-term treatment of cultured V79 cells with CdCl, follo wed by recovery culture (Ochi et aI., 1984). Confirmation of a do se-effect relationship is a prerequisite for investigating the mechanism for the induction of chro mosomal aberrations by cadmium .

0165-7992/ 85/ $ 03.30 © 1985 Elsevier Science Publishers B.Y. (Biomedical Division)

138

On the other hand, repairable DNA singlestrand scissions were induced in the cells treated with CdCb for 2 h by use of the technique of alkaline elution (Ochi and Ohsawa, 1983; Ochi et al ., 1983a). Moreover, participation of acti ve oxygen species in the induction of DNA single-strand scissions by Cd'Cl, has been shown in the experiment in which cadmium failed to induce DNA single- strand scissions in anaerobic culture conditions or in the medium containing superoxide dismutase (SOD) (Ochi er al ., 1983b) . These results sug gest that cadmium induced DNA single- strand scissions via indirect action, i.e. the mediation of active oxygen species. DNA single-strand scissions by cadmium ma y be an important DNA lesion which is responsible for ' the biological effects such as chromosomal aberrations or mutations by the metal. Accordingly , chromosomal aberrations or mutations by cadmium may also be attributable to the indirect action of the metal. In the present st udy, we invest iga ted th e effect of vario us sca vengers o f active oxygen species on the induction of chromosomal aberrations by CdCIz and found that so me active oxygen species participated in the induction of chromosomal aberrations by the metal.

cernid from Grand Island Biological Co., Grand Island , NY (U.S.A.); dimethylfuran from Aldrich Chemical Co ., Wisconsin (O .S.A .); D-mannitol from Wako Pure Chemical Co ., Osaka (Japan); superoxide dismutase (SO D, 3000 U/ mg solid) from Toyobo Co ., Ltd, Osaka (Japan) .

Chromosomal preparation Detection of chro moso mal aberrations was performed by a protocol described in our pre viou s experiment (Ochi et al., 1984). Briefl y, V79 cells (triplicate cultures) were plated into 6-cm petri dishes at a cell density of I x 106 cells per dish (5-ml culture). After a 24-h culture, the scavengers of active oxygen species or the inhibitor of catalase were added to the cultures 30 min before addition of CdC I2. The cells were incubated with CdCb for 2 h in the presence of the scavengers of the inhibitor. The cells were then washed twice with Hanks' balanced salt solution , and 1 x 106 cells were replated into 6-cm dishe s containing the control med ium. Chromosomal preparations were made 24 h after addition of cadmium according to the 50

til

.;:" ...

Materials and methods

" '"

III .0

Cell line and medium V79 cells from lun g fibroblasts of Chinese hamster were grown in a monolayer in Eagle's minimal essential medium (MEM) supplemented with 100/0 fetal bovine serum (FBS), penicillin (100 U /ml) and strepto mycin (100 jLg/ml) . The cells were cultured in a CO 2 incubator with 5% C02 in air.

Chemicals Cadmium chloride (CdCIz) wa s obtained from Mer ck , Darmstadt (Germany). But ylated hydroxytoluene (BHT) was purchased from Tokyo Kasei Kogyo Co., Tokyo (Japan); 3-amino-l ,2,4-triazole (aminotriazole) and catalase (20000 U lmg protein) from Sigma Chemical Co. , St. Louis (U.S.A.); col-

40

"" 30

....o

zo

til III

u

c:

III

-e

.... U ....e

10

o

Iii i

o

1

5 (x 10 - SM )

Concent r at i on of CdC1

z

Fig. I. Incidence s of chromosomal aber rations in V79 cells induced by treatment with CdCh. Bars indicate the sta ndard deviati on fro m 5 diffe rent experiments. Symbols without bar indicate the mean of 2 different experiments.

139

flame-d rying method . Colcemid at a final con centr at ion of 0.1 iLg /ml was added to the cultures for 2 h before the preparation of chro mosomes. 100 metaphases per culture were a na lysed for chromosomal ab errations.

TAB LE I EFFE CT OF SCAV E NG ERS O F AC TIV E O XYGE N SP EC IES ON TH E INDUCTION OF C H RO MOSOMA L ABERRA T IO NS BY caci, IN C ULT U RED C HI NESE HAM ST ER CEL LS Tr eatmen t

fJ70 cells with aberrations ± S.E.

Control 1000 U/m l SO D 2 x 10- 5 M CdCh 2 x 10- 5 M caci,

2.7 ±O.6 (3) 2 21.7 ± 3.2 (3)

+ lOoo U/m l SO D Co nt ro l 100 I'gl nl catalase 2 x 10- 5 M caci, 2 x 10- 5 M CdCIz

20.3 ± 2.3 (3)

4.0±2.0 (3)

2 28.3 ± 1.5 (3)

+ 50 I'g/m l catalase 2 x 10- 5 M cacr, + 100 I'g/ ml cata lase

21.3 ± 2.9 (3)*

Contro l

3.0 ± 1.0 (3) 2 20. 7± 4.6 (3)

5 x 10 - z M mannitol 2 x 10- 5 M CdCh 2 x 10- 5 M CdC Iz + I x 10- z M mannitol 2 x 10- 5 M caci, + 5 x 10- z M mannitol

14.7± 2.1 (3)**

10.3 ± 2.9 (3)*

10.0 ± 0.0 (3)**

Co ntrol I mM dime thylf ura n 2 x 10- 5 M CdCIz 2 x 10- 5 M CdClz + 1 mM dimethylfuran

2.0 (2) 3 19.0 ± 2.6 (3)

Co ntro l 50 I'M BHT 2 x 10- 5 M CdCIz

2.0 ±0.0 (3) 3 17.3 ± 2.5 (3) 9.7 ±0.6 (3)* 4.01: 2.0 (3)*

2 x 10- 5 M CdCIz + 10 I'M BHT 2 x 10 - 5 M CdCIz +50 I'M BHT

18.3 ± 3.5 (3)

T he number of cu ltures is ind icated in parentheses. T he aberratio n ra tes co mp rise all types of aberratio ns. *P < 0.01, ** P
Results and discussion Inducibilit y of chromosomal aberra tions by Cd CIz was examined according to the protocol of

our previous study with a 2-h tr eatment with the metal and subsequent reco ver y culture for 22 h (Oc hi et al., 1984). Chromosome struc tur al aberration s were observed in the cells tre ated with CdCIz with a concentration dep end ency (Fig. 1). A ma ximum incidence of chromosomal aberrations was ob served at 5 x 10- 5 M of CdCIz, but the metal at the co ncentration was very to xic to the cells and the number of metaphase cells was markedly decreased when compared to that of the control. Therefore, 2 x 10 - 5 M of CdCIz , which was subtoxic and increased chromosom al aberra tions , was used for the subsequent chromosom e experiments. Effects of va rious scave ngers o f active oxygen species on the induction of chro mosoma l aberrations by CdC Iz are show n in Table I . No increase in inciden ce of chromosomal aberra tion was ob served by tre atment with the scavengers alone. Our previou s studies ha ve show n that DNA single-strand scissions induced by Cd Ch were partly suppressed in the presence o f 1000 U /ml of SOD. There for e, we at first examined the effect of SOD on the induction o f chromosoma l aberratio ns by Cd CIz. Ho wever, SOD showed no effe ct on the inciden ce of chro mosomal ab erration s by 2 x 10- 5 M o f Cd Ch. In contrast to SOD , catalase signi ficantly suppressed the indu cibility of chromosom al aberrations by CdCh with a concentrat ion-dependency. The presence of cat alase at 100 iLg/ml reduced th e incid ences to appr oxima tely 50% of those in the cells treated with ca dmium alone . n-Mannitol , a sca venger of hydr oxyl rad icals (Misra a nd Frido vich, 1976), also signif icantly suppressed the incidences . A ppro ximatel y 50010 suppression in the incidence of a berr ations was ob served in th e presence of the chemical in a concentration exceeding 10- 2 M. Dimethylfuran , a scavenger o f singlet oxygen (Emerit et aI. , 1983), was not prot ective at a concentration of 1 mM. BHT , a well-know n antiox idant , was markedly protective in the indu ction of chromosoma l aberrations by CdCIz and at a co n-

140 TA BLE 2 EFFECT OF CATALASE ON T HE IND UCT ION OF CHROMOSOMAL ABERRAT IONS BY HAM STER CE LLS Tr eatm ent

C ulture number

caci,

Type of aberration (070)

IN CU LTURE D CH IN ESE

CG

ICG

ICB

CB

E

DC

F

Aberra nt meta phases (%) (mean ± S.D.)

I 2 3

16 17

I 0 0

0 0 0

10 11 12

3 I 0

3 I 3

I 0 0

30 27 (28.3 ± 1.5) 28

5O l'g / ml catalase + CdC Iz

I 2 3

13 12

0 0 I

9 8 9

0 4

II

0 0 0

I

2 0 I

0 0 0

23 23 (21.3 ± 2.9)* 18

100 I'g/ml cat a lase + Cd C h

I 2 3

6 5 6

I 0 I

0 0 0

10 6 6

I I 2

0 I 1

I I 0

14 (I4.7 ± 2.1)** 13

2

0

0

0

0

0

0

2

2

0 0 0

0 0 0

I 2 I

0 0 0

I

0 0

0 0 0

4 6 (4.0 ±2 .0)

2 x 10 - 5 M CdC h

100 I'g/ml ca ta lase Control

2 3

17

4 1

17

2

CG , chr omatid gaps; ICG , iso-chro ma tid gap s; ICB ; iso-chro matid br eak s; CB, chro mat id breaks; E , excha nges; DC , dicentri cs; F, fragmenta tio ns. As terisk s den ote chan ges in the cata lase-treated gro up significantly different (* P< O.OI , ** P< O.OO I) from tho se in the group treate d with CdCIz alone .

centration of 50 J.tM red uced the incidence o f abe rrations nearly to co nt ro l value s. T abl e 2 shows t he types of aberrations in duc ed by th e treatment wit h CdCIz in the presence o r absence of ca tala se . The presence o f catalas e as well as ma nnitol or BHT did not alter the distributio n amo ng the different typ es of ab errations. Types of aberratio ns induced by CdCIz alone mainly co nsisted of chr omatid gaps an d chromatid b reaks and a few exchanges and d icentrics were also obser ved. Aminotriazole, an inhibitor of catalase (Heim et aI. , 1955), significa nt ly enha nced inducibility of chromo somal aberrations by cad mi um. As shown in Table 3, no increase in the incidence of chromosomal a be rrations was obser ved by t he t reatment wit h 20 m M arni notriazole alo ne. T he co- existence of aminotriazole increa sed the incidence of chro moso mal aberrations to 26.70/0 for 2 mM of aminotriazole or 29.0% fo r 20 mM in

contrast to 19.3% in the cells treated with cadmium alone. T he p resent study shows that the induction o f chro m osomal aberr ations by CdCIz can be partially or fully suppressed by some of the scavengers of ac tive oxygen species or by an antioxidant , suggesting that ca dmium ind uced the cla stogenicit y via indire ct action , i. e., the mediation of active ox ygen species. T he suppressive effect o f catalase suggests participation of HzOz in the cla stogeni cit y o f cadmium . Moreover, the enhancement by aminotriazole of t he clastogenicity of cadmium may be d ue to t he acc umu lat ion of HzOz by the inhib ition of catalase . T his empha sizes the pa rt icipatio n of HzOz in the cla sto genicity of cad mium . Indeed , HzOz has been shown to induce chromosomal aberratio ns in mouse ascite s tumor cells (Schoneich , 1967) and in cult ur ed Chinese hamster cells (Tsuda , 1981). The suppressive effect o f mannitol implicates the participation o f hydrox-

141 TABLE 3 EF FECT OF AMINOT RIAZOLE ON THE INDU CT ION OF CHROMOSOMAL ABERRATIONS BY CdCh IN CULTU RED CHIN ESE HAMSTER CELLS Tre atm ent

Culture number

Type of ab erration (070 ) CG

zx 10- 5 M

I

CdCh

2 3

2 mM aminotria zole + CdC h

2 3

20 mM aminotriazole + CdCh

2 3

20 mM aminotria zole

Control

I

I

I

2 3 I

2 3

7 6

lCG I

13

0 0

12 8 10

0 0

I

Aberrant metaphases (070) (mean ± S.D.)

CB

E

DC

F

9 9 5

2 3

I 0

2

I

I

20 16 (I9 .3±3.l) 22

12 16 15

4 5 5

1 0 0

2 2 6

28 25 (26.7 ± 1.5)* 27

I

28 27 (29.0 ± 2.6)** 32

18 18 15

0 I I

13 10 12

2 2 3

2

2 2

2 4 2

0 0 0

0 0 0

0 0 0

0 0 0

0 0 0

2 4 (2.7 ± 1.2) 2

2 2 4

0 0 0

0 0 0

0 0 0

0 0 0

0 0 0

2 2 (2.7 ± 1.2) 4

CG , chromatid gaps; ICG , iso-chr om atid gap s; CB , chromatid breaks; E, exchan ges; DC, dicentrics; F, fragmentation s. Asterisk s denote chan ges in the aminotriazole-treat ed gro up significantly dif ferent (*P < 0.05 , **P < 0.02 ) from those in the group treated with CdC h alone.

yl radicals, but this conclusion may be tentative because of the relatively high concentrations of the compound that were needed to demonstrate anticlastogenicity. Singlet oxygen does not ap pear to be involved in the c1asto genicity of cadmium because dimethylfur an, a scavenger of singlet oxygen, was not protective of the effect of cadmium . SOD was not protective of the c1astogenicity of CdCh. It suggests that superoxide anions did not directly participate in the c1astogenicity of the metal. That SOD had no effect on the clastogenicity of cadmium is not in accordance with our previous observation on the partial suppressio n by SO D of Cd -induced DNA single -strand scissio ns in cu ltured mammalian cells (Ochi et al ., 1983). DNA sing le-strand scissions and chromosomal aberrations induced by CdC\z may be due to different mechanisms. Meanwhile, BHT markedly suppressed the c1astogenicity of cadmium, but the specific

intermediates of active oxygen metabolism to be scavenged were unclear because of the limited specificity of action of the compound. Cadmium at a co ncentration exceeding 5 x IO ~ 5 M has been sho wn to induce lipid peroxidation (formation of thiobarbituric acid-reacting substances) in isolated hepatocytes (Stacey et aI., 1980). Malondialdehyde, a secondary product of lip id hydroperoxide , has been sho wn to react with DNA (Brooks and Klamerth , 1968) and to be mutagenic (Mukai and Goldstein , 1976) and carcinogenic (Schamberger et aI., 1974). NA DP Hdependent microsomal lipid peroxid ation in vitro was reduced by SO D (Kameda et aI., 1979), the spin-trap for hydroxyl radicals (Ching-San Lai and Pette, 1978) or BHT (Shire s, 1975), suggesting partic ipation of some acti ve oxygen species in lipid peroxidation . These findings a llow us to speculate on the possible involvement of lipid peroxidation in the c1astogenicity of cadmium .

142

Acknowledgement

We wish to thank Miss Eiko Ik egaya for her technical assistance.

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