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Participation of active oxygen species in the induction of DNA single-strand scissions by cadmium chloride in cultured Chinese hamster cells
Mutation Research, 122 (1983) 169-175
169
Elsevier
MRLett 0462
Participation of active oxygen species in the induction of DNA single-strand scissions by cadmium chloride in cultured Chinese hamster cells T a k a f u m i Ochi*, T o h r u Ishiguro and M o t o y a s u Ohsawa Division of Environmental Toxicology and Environmental Health, Faculty of Pharmaceutical Sciences, Teikyo University, Sagamiko, Kanagawa 199-01 (Japan) (Accepted 13 July 1983)
Summary A mechanism for the induction of DNA single-strand scissions in cultured Chinese hamster cells by cadmium chloride (CdCI2) was investigated by use of the technique of alkaline elution. Inducibility of DNA single-strand scissions by cadmium was examined under an aerobic or anaerobic culture condition. About 62°70 of the total cellular DNA was eluted throughout the filter within 10 h of elution time by treatment with 4 x 10- 5 M CdCI2 for 2 h in our usual aerobic medium. In contrast, no difference in elution profiles of DNA was observed between untreated control cells and the cells treated with CdCI2 in the anaerobic medium which was prepared by N2 gas bubbling of aerobic medium for 60 min. Furthermore, elution of DNA from cells treated with cadmium decreased markedly in the presence of superoxide dismutase (SOD) when compared with that in the absence of SOD. Inhibition of the cell growth by cadmium was significantly protected by the presence of SOD in the medium although the cell growth was not restored to the control level. These results indicate that active oxygen species participate in Cd-induced DNA single-strand scissions and also in the growth inhibition of the cells by the metal.
Ochi and Ohsawa (1983) found that exposure of cultured Chinese hamster cells to CdCI2 (2-5 x 10-5 M) for 2 h results in DNA single-strand scissions that are detectable by the technique of alkaline elution combined with digestion of cell lysates by proteinase-K. Moreover, DNA single-strand scissions induced by cadmium are rejoined after 4-20 h of repair incubation, and DNA single-strand scis* To whom correspondence should be addressed. 0165-7992/83/$ 03.00 © 1983 Elsevier Science Publishers B.V.
170
sions accumulate on simultaneous treatment with inhibitors of DNA repair replication, hydroxyurea and 1-/3-D-arabinofuranosylcytosine (Ochi et al., 1983). Thus, cadmium-induced DNA lesions are apparently repairable in cultured mammalian cells. It remains unclear what kind of repair system participates in the repair of DNA lesions induced by cadmium, because the mechanism for the induction of DNA single-strand scissions by the metal is not clear. In the present study, we found that cadmium failed to induce DNA single-strand scissions in anaerobic culture conditions in the medium containing superoxide dismutase, indicating participation of active oxygen species on the induction of DNA single-strand scissions by cadmium.
Materials and methods
Cell line and culture medium V79 cells derived from Chinese hamster lung were grown in a monolayer in Ham's F12 medium containing 2°70 foetal bovine serum (FBS). Eagle's MEM medium containing 10°70 FBS was used as the medium for prelabelling cellular DNA with [4C]thymidine because F12 medium contains 2.9 x 10-6 M thymidine. Cells were cultured in a CO2 incubator with 5070 CO2 in air. Chemicals Cadmium chloride (CdC12) and proteinase K were obtained from E. Merck AG, Darmstadt (Germany). Bovine superoxide dismutase (SOD) was kindly donated by Dr. M. Nishimura and Toyobo Co. Ltd, Osaka (Japan). Sarkosyl (N-lauroylsarcosine) was purchased from Nakarai Chemical Co., Kyoto (Japan); Na3EDTA, Na2EDTA and H4EDTA from Wako Pure Chemical Co., Osaka (Japan); tetra-npropylammoniumhydroxide from Tokyo Kasei Ind., Tokyo (Japan). [2-14C]Thymidine (58 mCi/mmole) was obtained from Amersham International Ltd. (Great Britain). Treatment o f V79 cells with cadmium [14C]Thymidine (0.02/zCi/ml) was added to V79 cells (1 x 105 cells per ml) grown in 25-cm 2 flasks (3012, Tissue Culture Flask, Falcon, U.S.A.) containing MEM medium supplemented with 10% FBS, and mature DNA was labelled by a 48-h incubation. After removal of the radioactive medium, the cells were incubated in F12 plus 2% FBS for 1.5 h before cadmium treatment. Cells labelled with [a4C]thymidine were then treated with CdC12 for 2 h in the anaerobic medium which was prepared by bubbling with N2 gas for 30 or 60 rain or in the medium containing SOD at 1000 U / m l . The flasks for the anaerobic culture were sealed after N2 gassing for 30 sec.
171
Measurement of cell growth Growth curves of cells were obtained by the replicate culture method. V79 cells (duplicate cultures) were plated into the multi-well plate (Linbro ® , 12 flat bottom wells, 2.4 x 1.7, Connecticut, U.S.A.) at a cell density of 1 x 105 cells/ml/well. After a 24-h culture, cadmium chloride was added to the cultures. SOD was added 4 h before the addition o f cadmium. The cells were counted with a haemocytometer 24 and 48 h after the addition of cadmium.
Alkaline elution of DNA from V79 cells Alkaline elution of DNA from cells treated with cadmium was performed by a modification of the procedures o f Kohn et al. (1976) and Ewig and Kohn (1977). Briefly, 106 cells were loaded onto the surface of 25-mm diameter and 2-t~m pore size polyvinyl chloride filter (Millipore Corp., Bedford, MA) and lysed on the filter with 5 mi of 0.2% Sarkosyl, 2 M NaCI and 0.04 M Na3EDTA (pH 10). The lysates on the filter were then treated with 5 ml of 0.5% SDS, 0.01 M NaC1, 0.01 M NazEDTA and 0.01 M Tris (pH 8.2) containing 0.5 mg of proteinase K per ml for 60 min at 30°C. After each filter had been washed with 5 ml of 0.02 M NaEEDTA (pH 10), the filter was washed with the eluting solution (2% SDS, 0.02 M H4EDTAtetra-n-propylammoniumhydroxide added to give a pH of 12.2) at 30°C. DNA was eluted in the dark at a flow rate of 0.04 ml/min. Eluted fractions were collected at 60-min intervals. Radioactivities for each fraction and filter were determined as described previously (Ewig and Kohn, 1977).
Results and discussion
Hakkinen et al. (1983) found that lung damage in mice by methylcyclopentadienyl manganese tricarbonyl, cadmium chloride, oleic acid, cyclophosphamide and bleomycin is potentiated by hyperoxia. Among these compounds, bleomycin induces DNA strand scissions that are increased by the combined action of the chemical and superoxide radical (Ishida and Takahashi, 1975). Because of these findings, we first investigated the effect of oxygen on the induction of DNA singlestrand scissions by CdC12. Fig. 1 shows the inducibility of DNA single-strand scissions by cadmium in aerobic or anaerobic culture conditions, by use o f the technique of alkaline elution. About 62°7o of the total cellular DNA was eluted throughout the filter within l0 h of elution time by treatment with 4 x 10- 5 M CdCI2 for 2 h in an aerobic medium, in contrast to 8% in DNA from the control cells. A marked decrease in elution of DNA from the cells treated with 4 x 10-5 M CdC12 was observed in an anaerobic culture medium prepared by N2 gas bubbling for 30 or 60 min. No difference in elution profiles o f DNA was observed between untreated control cells and the cells treated with cadmium in the medium bubbled with N2 gas for 60 min.
172
Elution 2 ~
1.0
~
0.5
4
time (h) 6
8
i0
4..1
z o o 4-1
0.I Fig. 1. Alkaline elution profiles of D N A from cells treated with CdC12 in an aerobic or anaerobic culture condition. V79 cells prelabelled with [14C]thymidine were treated with 4 x 10- 5 M CdCI2 for 2 h in the usual aerobic medium (©) and in anaerobic media prepared by N2 gas bubbling for 30 min (o) or 60 min (zx). o, control. Alkaline elution of D N A was performed after proteinase-K digestion of the cell lysates on the filter.
This oxygen requirement for the induction of DNA single-strand scissions by cadmium suggests that an active oxygen species but not cadmium itself may play a crucial role for the induction of DNA lesions after exposure to cadmium. A second experiment with superoxide dismutase (SOD), which catalyses the dismutation of superoxide anion O z- to the less toxic H202 and molecular oxygen (McCord and Fridovich, 1969), was performed to clarify the possible participation of active oxygen species in the induction of DNA lesions by cadmium. Cells were treated with 4 × 10 -5 M CdC12 for 2 h in the presence or absence of SOD, 1000 U/ml, and then analysed by the technique of alkaline elution. Fig. 2 shows that the elution of DNA from cells treated with cadmium decreased markedly in the presence of SOD when compared with that in its absence, apparently indicating participation of active oxygen species in the induction of DNA single-strand scissions by the metal. Details of the mechanism by which cadmium produces DNA lesions via active oxygen species are now under investigation. Amoruso et al. (1982) showed that the production of 0 2 - is significantly enhanced in human granulocytes or in rat alveolar macrophages that have been activated by digitonin in the presence of 3.6 × 10 -5 M cadmium. They suggested that a stimulation of membrane-bound pyridine nucleotide oxidase by cadmium may be responsible for production of superoxide radical.
173
Elution time 2
1
.
4
6
0
(h) 8
I0
~
~3 4.3
< z o © 43
° 0.i Fig. 2. Alkaline elution profiles of D N A from cells treated with CdCl2 in the presence or absence of superoxfide dismutase (SOD). V79 cellsprelabelled with ['4C]thymidine were treated with 4 × 10-5 M CdCl2 for 2 h in the presence or absence of SOD, I000 U/ml. Alkaline elution of D N A was performed after proteinase-K digestion of the cell ;ysates on the filter.O, control; ©, 4 x I0-5 M CdCl2; m, 4 >( 10 - 5 M CdCI2 + 1 0 0 0 U S O D p e r ml.
Cadmium interacts not only with DNA but also with other cellular macromolecules, thereby giving rise to cellular toxicity (Webb, 1979; Verma et al., 1982). Therefore, the contribution of the DNA single-strand scissions on the growth inhibition of the cells by cadmium was investigated to clarify the biological significance of DNA lesions induced by the metal. Fig. 3 shows the growth curves of V79 cells in medium containing various concentrations of CdCI2 with or without SOD. The duration of cadmium exposure was prolonged to 24-48 h instead of a 2-h exposure to amplify the effect of the metal on cell growth. Inhibition of cell growth by cadmium was partially protected by the presence of SOD which suppressed cadmium-induced DNA single-strand scissions in the technique of alkaline elution. This result indicates that DNA single-strand scissions by cadmium may be one of the causes of cellular damage by the metal. Mitra and Bernstein (1977) found that DNA ligase but not DNA polymerase I is involved in the repair of cadmium-induced DNA lesions in E. coli. Ochi et al. (1983) also found that cadmium-induced DNA single-strand scissions are rejoined by repair incubation in cultured mammalian cells and that the DNA lesions are accumulated by the inhibition of repair replication. Hence, cadmium-induced DNA lesions are apparently repairable, but details of the mechanism of the repair are not understood, because the mechanism for the induction of DNA single-strand scis-
174
Control 2 x 10-6M Cd + SOD 2 x 10-6M Cd 5 x 10-6M Cd + SOD 5 x 10-6M Cd
r-4
E 0.~ ~8
0 0
8 x 10-6M Cd + SOD
d
Z
8 x 10-6M Cd
1 x 10-5M Cd
i
2
3
Days after inoculation of cells Fig. 3. Effect o f S O D on the g r o w t h o f cells after addition o f CdC12. V 7 9 cells were plated at a density o f l × 105 cells per ml. After a 2 4 - h c u l t u r e , CdC12 w a s a d d e d to the cultures. SOD ( 1 0 0 0 U / m l ) was a d d e d 4 h before the addition of cadmium. Open symbols, without S O D ; c l o s e d s y m b o l s , w i t h S O D . o , e , c o n t r o l ; [], • , 2 x 1 0 - s M CdCI2; A , • , 1 × 1 0 - 5 M CdC12; V , • , I ~ 1 , 5 × 10 - 6 M CdC12; o , o , 2 x
8 × 1 0 - 6 M CdCI2; I>,o,
10 - s M CdC12.
sions by cadmium is unclear. Accordingly, a participation of active oxygen species in the induction of D N A single-strand scissions after exposure to cadmium in the present study would be important to suggest that the repair may be due, not to a UV-type excision-repair system which is processed by enzymatic incision and exci-
175
sion of damaged DNA, but to a repair system similar to that of the DNA damage induced by X-rays which is known to be suppressed by various radical scavengers (Misra and Fridvich, 1976).
References Amoruso, M.A., G. Witz and B.D. Goldstein (1982) Enhancement of rat and human phagocyte superoxide anion radical production by cadmium in vitro, Toxicol. Lett., 10, 133-138. Ewig, R.A.G., and K.W. Kohn (1977) DNA damage and repair in mouse leukemia L1210 cells treated with nitrogen mustard, 1,3-bis-(2-chloroethyl)-l-nitrosourea, and other nitrosourea, Cancer Res., 37, 2114-2122. Hakkinen, P.J., C.C. Morse, F.M. Martin, W.E. Dalbey, W.M. Haschek and H.R. Witschi (1983) Potentiating effects of oxygen in lung damaged by methylcyclopentadienyl manganese tricarbonyl, cadmium chloride, oleic acid and antitumor drugs, Toxicol. Appl. Pharmacol., 67, 55-69. lshida, R., and T. Takahashi (1975) Increased DNA chain breakage by combined action of bleomycin and superoxide radical, Biochem. Biophys. Res. Commun., 66, 1432-1438. Kohn, K.W., L.C. Erickson, R.A.G. Ewig and C.A. Friedman (1976) Fractionation of DNA from mammalian cells by alkaline elution, Biochemistry, 15, 4629-4637. McCord, J.M., and I. Fridovich (1969) Superoxide dismutase, an enzymatic function for erythrocuprein, J. Biol. Chem., 244, 6049-6055. Misra, H.P., and I. Fridovich (1976) Superoxide dismutase and oxygen enhancement of radiation lethality, Arch. Biochem. Biophys., 176, 577-581. Mitra, R.S., and I.A. Bernstein (1977) Nature of the repair process associated with the recovery of Escherichia coli after exposure to Cd 2+, Biochem. Biophys. Res. Commun., 74, 1450-1455. Ochi, T., and M. Ohsawa (1983) Induction of 6-thioguanine-resistant mutants and single-strand scission of DNA by cadmium chloride in cultured Chinese hamster cells, Mutation Res., in press. Ochi, T., M. Takayanagi and M. Ohsawa (1983) Cadmium-induced DNA single-strand scissions and their repair in cultured Chinese hamster cells, Toxicol. Lett., in press. Verma, M.P., R.P. Scharma and D.R. Bourcier (1982) Macromolecular interactions with cadmium and the effect of zinc, copper, lead and mercury ions, Biol. Trace Element Res., 4, 35-43. Webb, M. (1979) Interaction of cadmium with cellular components, in: M. Webb (Ed.), The Chemistry, Biochemistry and Biology of Cadmium, Elsevier Biomedical, Amsterdam, pp. 285-340.
169
Elsevier
MRLett 0462
Participation of active oxygen species in the induction of DNA single-strand scissions by cadmium chloride in cultured Chinese hamster cells T a k a f u m i Ochi*, T o h r u Ishiguro and M o t o y a s u Ohsawa Division of Environmental Toxicology and Environmental Health, Faculty of Pharmaceutical Sciences, Teikyo University, Sagamiko, Kanagawa 199-01 (Japan) (Accepted 13 July 1983)
Summary A mechanism for the induction of DNA single-strand scissions in cultured Chinese hamster cells by cadmium chloride (CdCI2) was investigated by use of the technique of alkaline elution. Inducibility of DNA single-strand scissions by cadmium was examined under an aerobic or anaerobic culture condition. About 62°70 of the total cellular DNA was eluted throughout the filter within 10 h of elution time by treatment with 4 x 10- 5 M CdCI2 for 2 h in our usual aerobic medium. In contrast, no difference in elution profiles of DNA was observed between untreated control cells and the cells treated with CdCI2 in the anaerobic medium which was prepared by N2 gas bubbling of aerobic medium for 60 min. Furthermore, elution of DNA from cells treated with cadmium decreased markedly in the presence of superoxide dismutase (SOD) when compared with that in the absence of SOD. Inhibition of the cell growth by cadmium was significantly protected by the presence of SOD in the medium although the cell growth was not restored to the control level. These results indicate that active oxygen species participate in Cd-induced DNA single-strand scissions and also in the growth inhibition of the cells by the metal.
Ochi and Ohsawa (1983) found that exposure of cultured Chinese hamster cells to CdCI2 (2-5 x 10-5 M) for 2 h results in DNA single-strand scissions that are detectable by the technique of alkaline elution combined with digestion of cell lysates by proteinase-K. Moreover, DNA single-strand scissions induced by cadmium are rejoined after 4-20 h of repair incubation, and DNA single-strand scis* To whom correspondence should be addressed. 0165-7992/83/$ 03.00 © 1983 Elsevier Science Publishers B.V.
170
sions accumulate on simultaneous treatment with inhibitors of DNA repair replication, hydroxyurea and 1-/3-D-arabinofuranosylcytosine (Ochi et al., 1983). Thus, cadmium-induced DNA lesions are apparently repairable in cultured mammalian cells. It remains unclear what kind of repair system participates in the repair of DNA lesions induced by cadmium, because the mechanism for the induction of DNA single-strand scissions by the metal is not clear. In the present study, we found that cadmium failed to induce DNA single-strand scissions in anaerobic culture conditions in the medium containing superoxide dismutase, indicating participation of active oxygen species on the induction of DNA single-strand scissions by cadmium.
Materials and methods
Cell line and culture medium V79 cells derived from Chinese hamster lung were grown in a monolayer in Ham's F12 medium containing 2°70 foetal bovine serum (FBS). Eagle's MEM medium containing 10°70 FBS was used as the medium for prelabelling cellular DNA with [4C]thymidine because F12 medium contains 2.9 x 10-6 M thymidine. Cells were cultured in a CO2 incubator with 5070 CO2 in air. Chemicals Cadmium chloride (CdC12) and proteinase K were obtained from E. Merck AG, Darmstadt (Germany). Bovine superoxide dismutase (SOD) was kindly donated by Dr. M. Nishimura and Toyobo Co. Ltd, Osaka (Japan). Sarkosyl (N-lauroylsarcosine) was purchased from Nakarai Chemical Co., Kyoto (Japan); Na3EDTA, Na2EDTA and H4EDTA from Wako Pure Chemical Co., Osaka (Japan); tetra-npropylammoniumhydroxide from Tokyo Kasei Ind., Tokyo (Japan). [2-14C]Thymidine (58 mCi/mmole) was obtained from Amersham International Ltd. (Great Britain). Treatment o f V79 cells with cadmium [14C]Thymidine (0.02/zCi/ml) was added to V79 cells (1 x 105 cells per ml) grown in 25-cm 2 flasks (3012, Tissue Culture Flask, Falcon, U.S.A.) containing MEM medium supplemented with 10% FBS, and mature DNA was labelled by a 48-h incubation. After removal of the radioactive medium, the cells were incubated in F12 plus 2% FBS for 1.5 h before cadmium treatment. Cells labelled with [a4C]thymidine were then treated with CdC12 for 2 h in the anaerobic medium which was prepared by bubbling with N2 gas for 30 or 60 rain or in the medium containing SOD at 1000 U / m l . The flasks for the anaerobic culture were sealed after N2 gassing for 30 sec.
171
Measurement of cell growth Growth curves of cells were obtained by the replicate culture method. V79 cells (duplicate cultures) were plated into the multi-well plate (Linbro ® , 12 flat bottom wells, 2.4 x 1.7, Connecticut, U.S.A.) at a cell density of 1 x 105 cells/ml/well. After a 24-h culture, cadmium chloride was added to the cultures. SOD was added 4 h before the addition o f cadmium. The cells were counted with a haemocytometer 24 and 48 h after the addition of cadmium.
Alkaline elution of DNA from V79 cells Alkaline elution of DNA from cells treated with cadmium was performed by a modification of the procedures o f Kohn et al. (1976) and Ewig and Kohn (1977). Briefly, 106 cells were loaded onto the surface of 25-mm diameter and 2-t~m pore size polyvinyl chloride filter (Millipore Corp., Bedford, MA) and lysed on the filter with 5 mi of 0.2% Sarkosyl, 2 M NaCI and 0.04 M Na3EDTA (pH 10). The lysates on the filter were then treated with 5 ml of 0.5% SDS, 0.01 M NaC1, 0.01 M NazEDTA and 0.01 M Tris (pH 8.2) containing 0.5 mg of proteinase K per ml for 60 min at 30°C. After each filter had been washed with 5 ml of 0.02 M NaEEDTA (pH 10), the filter was washed with the eluting solution (2% SDS, 0.02 M H4EDTAtetra-n-propylammoniumhydroxide added to give a pH of 12.2) at 30°C. DNA was eluted in the dark at a flow rate of 0.04 ml/min. Eluted fractions were collected at 60-min intervals. Radioactivities for each fraction and filter were determined as described previously (Ewig and Kohn, 1977).
Results and discussion
Hakkinen et al. (1983) found that lung damage in mice by methylcyclopentadienyl manganese tricarbonyl, cadmium chloride, oleic acid, cyclophosphamide and bleomycin is potentiated by hyperoxia. Among these compounds, bleomycin induces DNA strand scissions that are increased by the combined action of the chemical and superoxide radical (Ishida and Takahashi, 1975). Because of these findings, we first investigated the effect of oxygen on the induction of DNA singlestrand scissions by CdC12. Fig. 1 shows the inducibility of DNA single-strand scissions by cadmium in aerobic or anaerobic culture conditions, by use o f the technique of alkaline elution. About 62°7o of the total cellular DNA was eluted throughout the filter within l0 h of elution time by treatment with 4 x 10- 5 M CdCI2 for 2 h in an aerobic medium, in contrast to 8% in DNA from the control cells. A marked decrease in elution of DNA from the cells treated with 4 x 10-5 M CdC12 was observed in an anaerobic culture medium prepared by N2 gas bubbling for 30 or 60 min. No difference in elution profiles o f DNA was observed between untreated control cells and the cells treated with cadmium in the medium bubbled with N2 gas for 60 min.
172
Elution 2 ~
1.0
~
0.5
4
time (h) 6
8
i0
4..1
z o o 4-1
0.I Fig. 1. Alkaline elution profiles of D N A from cells treated with CdC12 in an aerobic or anaerobic culture condition. V79 cells prelabelled with [14C]thymidine were treated with 4 x 10- 5 M CdCI2 for 2 h in the usual aerobic medium (©) and in anaerobic media prepared by N2 gas bubbling for 30 min (o) or 60 min (zx). o, control. Alkaline elution of D N A was performed after proteinase-K digestion of the cell lysates on the filter.
This oxygen requirement for the induction of DNA single-strand scissions by cadmium suggests that an active oxygen species but not cadmium itself may play a crucial role for the induction of DNA lesions after exposure to cadmium. A second experiment with superoxide dismutase (SOD), which catalyses the dismutation of superoxide anion O z- to the less toxic H202 and molecular oxygen (McCord and Fridovich, 1969), was performed to clarify the possible participation of active oxygen species in the induction of DNA lesions by cadmium. Cells were treated with 4 × 10 -5 M CdC12 for 2 h in the presence or absence of SOD, 1000 U/ml, and then analysed by the technique of alkaline elution. Fig. 2 shows that the elution of DNA from cells treated with cadmium decreased markedly in the presence of SOD when compared with that in its absence, apparently indicating participation of active oxygen species in the induction of DNA single-strand scissions by the metal. Details of the mechanism by which cadmium produces DNA lesions via active oxygen species are now under investigation. Amoruso et al. (1982) showed that the production of 0 2 - is significantly enhanced in human granulocytes or in rat alveolar macrophages that have been activated by digitonin in the presence of 3.6 × 10 -5 M cadmium. They suggested that a stimulation of membrane-bound pyridine nucleotide oxidase by cadmium may be responsible for production of superoxide radical.
173
Elution time 2
1
.
4
6
0
(h) 8
I0
~
~3 4.3
< z o © 43
° 0.i Fig. 2. Alkaline elution profiles of D N A from cells treated with CdCl2 in the presence or absence of superoxfide dismutase (SOD). V79 cellsprelabelled with ['4C]thymidine were treated with 4 × 10-5 M CdCl2 for 2 h in the presence or absence of SOD, I000 U/ml. Alkaline elution of D N A was performed after proteinase-K digestion of the cell ;ysates on the filter.O, control; ©, 4 x I0-5 M CdCl2; m, 4 >( 10 - 5 M CdCI2 + 1 0 0 0 U S O D p e r ml.
Cadmium interacts not only with DNA but also with other cellular macromolecules, thereby giving rise to cellular toxicity (Webb, 1979; Verma et al., 1982). Therefore, the contribution of the DNA single-strand scissions on the growth inhibition of the cells by cadmium was investigated to clarify the biological significance of DNA lesions induced by the metal. Fig. 3 shows the growth curves of V79 cells in medium containing various concentrations of CdCI2 with or without SOD. The duration of cadmium exposure was prolonged to 24-48 h instead of a 2-h exposure to amplify the effect of the metal on cell growth. Inhibition of cell growth by cadmium was partially protected by the presence of SOD which suppressed cadmium-induced DNA single-strand scissions in the technique of alkaline elution. This result indicates that DNA single-strand scissions by cadmium may be one of the causes of cellular damage by the metal. Mitra and Bernstein (1977) found that DNA ligase but not DNA polymerase I is involved in the repair of cadmium-induced DNA lesions in E. coli. Ochi et al. (1983) also found that cadmium-induced DNA single-strand scissions are rejoined by repair incubation in cultured mammalian cells and that the DNA lesions are accumulated by the inhibition of repair replication. Hence, cadmium-induced DNA lesions are apparently repairable, but details of the mechanism of the repair are not understood, because the mechanism for the induction of DNA single-strand scis-
174
Control 2 x 10-6M Cd + SOD 2 x 10-6M Cd 5 x 10-6M Cd + SOD 5 x 10-6M Cd
r-4
E 0.~ ~8
0 0
8 x 10-6M Cd + SOD
d
Z
8 x 10-6M Cd
1 x 10-5M Cd
i
2
3
Days after inoculation of cells Fig. 3. Effect o f S O D on the g r o w t h o f cells after addition o f CdC12. V 7 9 cells were plated at a density o f l × 105 cells per ml. After a 2 4 - h c u l t u r e , CdC12 w a s a d d e d to the cultures. SOD ( 1 0 0 0 U / m l ) was a d d e d 4 h before the addition of cadmium. Open symbols, without S O D ; c l o s e d s y m b o l s , w i t h S O D . o , e , c o n t r o l ; [], • , 2 x 1 0 - s M CdCI2; A , • , 1 × 1 0 - 5 M CdC12; V , • , I ~ 1 , 5 × 10 - 6 M CdC12; o , o , 2 x
8 × 1 0 - 6 M CdCI2; I>,o,
10 - s M CdC12.
sions by cadmium is unclear. Accordingly, a participation of active oxygen species in the induction of D N A single-strand scissions after exposure to cadmium in the present study would be important to suggest that the repair may be due, not to a UV-type excision-repair system which is processed by enzymatic incision and exci-
175
sion of damaged DNA, but to a repair system similar to that of the DNA damage induced by X-rays which is known to be suppressed by various radical scavengers (Misra and Fridvich, 1976).
References Amoruso, M.A., G. Witz and B.D. Goldstein (1982) Enhancement of rat and human phagocyte superoxide anion radical production by cadmium in vitro, Toxicol. Lett., 10, 133-138. Ewig, R.A.G., and K.W. Kohn (1977) DNA damage and repair in mouse leukemia L1210 cells treated with nitrogen mustard, 1,3-bis-(2-chloroethyl)-l-nitrosourea, and other nitrosourea, Cancer Res., 37, 2114-2122. Hakkinen, P.J., C.C. Morse, F.M. Martin, W.E. Dalbey, W.M. Haschek and H.R. Witschi (1983) Potentiating effects of oxygen in lung damaged by methylcyclopentadienyl manganese tricarbonyl, cadmium chloride, oleic acid and antitumor drugs, Toxicol. Appl. Pharmacol., 67, 55-69. lshida, R., and T. Takahashi (1975) Increased DNA chain breakage by combined action of bleomycin and superoxide radical, Biochem. Biophys. Res. Commun., 66, 1432-1438. Kohn, K.W., L.C. Erickson, R.A.G. Ewig and C.A. Friedman (1976) Fractionation of DNA from mammalian cells by alkaline elution, Biochemistry, 15, 4629-4637. McCord, J.M., and I. Fridovich (1969) Superoxide dismutase, an enzymatic function for erythrocuprein, J. Biol. Chem., 244, 6049-6055. Misra, H.P., and I. Fridovich (1976) Superoxide dismutase and oxygen enhancement of radiation lethality, Arch. Biochem. Biophys., 176, 577-581. Mitra, R.S., and I.A. Bernstein (1977) Nature of the repair process associated with the recovery of Escherichia coli after exposure to Cd 2+, Biochem. Biophys. Res. Commun., 74, 1450-1455. Ochi, T., and M. Ohsawa (1983) Induction of 6-thioguanine-resistant mutants and single-strand scission of DNA by cadmium chloride in cultured Chinese hamster cells, Mutation Res., in press. Ochi, T., M. Takayanagi and M. Ohsawa (1983) Cadmium-induced DNA single-strand scissions and their repair in cultured Chinese hamster cells, Toxicol. Lett., in press. Verma, M.P., R.P. Scharma and D.R. Bourcier (1982) Macromolecular interactions with cadmium and the effect of zinc, copper, lead and mercury ions, Biol. Trace Element Res., 4, 35-43. Webb, M. (1979) Interaction of cadmium with cellular components, in: M. Webb (Ed.), The Chemistry, Biochemistry and Biology of Cadmium, Elsevier Biomedical, Amsterdam, pp. 285-340.