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Effects of iron chelators and glutathione depletion on the induction and repair of chromosomal aberrations by tert-butyl hydroperoxide in cultured Chinese hamster cells
Mutation Research, 213 (1989) 243-248
243
Elsevier MUT 04775
Effects of iron chelators and glutathione depletion on the induction and repair of chromosomal aberrations by tert-butyl hydroperoxide in cultured Chinese hamster cells Takafumi Ochi Department of Environmental Toxicology, Faculty of Pharmaceutical Sciences, Teikyo University, Sagamiko, Kanagawa 199-01 (Japan) (Received 2 January 1989) (Revision received 7 March 1989) (Accepted 8 March 1989)
Keywords: Hydroperoxide; Chromosomal aberrations; Iron chelators; Glutathione depletion
Summary The effects of iron chelators and glutathione (GSH) depletion on the induction of chromosomal aberrations by tert-butyl hydroperoxide (t-BuOOH) were investigated in cultured Chinese hamster V79 cells. t-BuOOH in a concentration range of 0.1-1.0 mM induced chromosomal structural aberrations, consisting mainly of chromatid gaps and breaks, in a dose-dependent fashion. The divalent iron chelator o-phenanthroline almost completely suppressed the formation of chromosomal aberrations while the trivalent chelator desferrioxamine was less effective. GSH depletion did not affect the formation of chromosomal aberrations and DNA single-strand breaks (ssb) by t-BuOOH. DNA ssb by 0.5 mM t-BuOOH were repaired within 60 min of treatment in both GSH-depleted ( G S H - ) and non-depleted (GSH ÷) cells. In contrast, chromosomal aberrations increased a tittle during the 60 min after treatment in both G S H - and GSH ÷ cells. The aberrations were then repaired in GSH + cells but those in G S H - cells were maintained to a great extent during 20 h of post-treatment incubation. These results indicate that divalent iron mediates the induction of chromosomal aberrations by t-BuOOH. That t-BuOOH-induced chromosomal aberrations remain even after DNA ssb were repaired suggests involvement of other lesions than DNA ssb in the formation of chromosomal aberrations by the hydroperoxide.
Organic hydroperoxides generate reactive oxygen free radicals and cause a variety of deleterious effects on biological systems (Logani and Davies, 1980; Trotta et al., 1982; Halliwell and Gut-
Correspondence: Dr. T. Ochi, Department of Environmental Toxicology, Faculty of Pharmaceutical Sciences, Teikyo University, Sagamiko, Kanagawa 199-01 (Japan).
teridge, 1984; Thornally et al., 1984; Inouye, 1984; Aust et al., 1985; Ochi and Cerutti, 1987). In our earlier studies, tert-butyl hydroperoxide (tBuOOH) used as a model of alkyl hydroperoxides induced cytotoxicity in cultured Chinese hamster V79 cells (Ochi, 1988; Ochi and Miyaura, 1989) and DNA single-strand breaks (ssb) in the alkaline elution assay (Ochi and Cerutti, unpublished). Both cytotoxicity and DNA ssb required
0027-5107/89/$03.50 © 1989 Elsevier Science Pubfishers B.V. (Biomedical Divsion)
244 free iron, in particular divalent, for their induction by t-BuOOH. Further, depletion of cell glutathione (GSH) with L-buthionine-SR-sulfoximine, a selective inhibitor of "t-glutamylcysteine synthetase, markedly enhanced t-BuOOH-induced cytotoxicity. However, GSH depletion did not influence the efficiency of induction of D N A ssb by the hydroperoxide. Thus, there was no correlation between t-BuOOH-induced cytotoxicity and D N A ssb with regard to their sensitivity to GSH depletion while iron was required for both. Here, our study is extended to the cytogenetic level in order to examine the relation between t-BuOOH-induced cytotoxicity, DNA ssb and chromosomal aberrations. The effects of iron chelators and GSH depletion on the formation and repair of chromosomal aberrations by t-BuOOH were investigated and compared with D N A ssb. Materials and methods Chemicals L-Buthionine-SR-sulfoximine (BSO), reducedform glutathione (GSH), glutathione reductase (type IV, 200 U / m g protein) and t-BuOOH were obtained from Sigma Chemical Co., St. Louis, MO (U.S.A.). o-Phenanthroline was purchased from Wako Pure Chemical Co., Osaka (Japan), tetra-npropylammoniumhydroxide from Tokyo Kasei Kogyo Co., Tokyo (Japan), desferrioxamine (desferal mesylate) from Ciba Geigy, Basel (Switzerland), and Colcemid from Grand Island Biological Co., Grand Island, NY (U.S.A.). [2-14C]Thymidine (58 mCi/mmole) was obtained from ICN Radiochemicals, Irvine, CA (U.S.A.). Cell cultures and media V79 cells from lung fibroblasts of male Chinese hamster were grown in a monolayer in minimum essential medium (MEM) supplemented with 10% fetal bovine serum (FBS), penicillin (100 U / m l ) and streptomycin (100 #g/ml). The cells were cultured in a CO 2 incubator with 5% CO 2 in humidified air. 10 mM Hepes-buffered (pH 7.4) MEM medium without FBS was used as a medium for treatment with t-BuOOH. Cell treatment and chromosomal preparation Cells were seeded at a density of 5 × 105 cells in 6-cm Petri dishes. After a 20-h incubation, cells
were incubated in the medium with or without 0.2 mM BSO for 6 h and were placed in Hepesbuffered MEM including BSO for challenge with t-BuOOH. In some experiments, cells were preincubated with or without iron chelators for 15 min before the addition of t-BuOOH, followed by incubation with the hydroperoxide in the presence or absence of the chelators for 1 h. After a 1-h incubation with t-BuOOH, cells were washed twice with Hanks' balanced salt solution (BSS) and placed in prewarmed control medium for further incubation. 0.1/~g/ml Colcemid was added to the cultures 1 h before harvesting the cells. At 0 h of the post-treatment incubation, cells were incubated with t-BuOOH in the presence of Colcemid for 1 h and chromosomes were prepared immediately after the treatment. Chromosomal preparations were made using the flame-drying method. 100 metaphases were analyzed for chromosomal aberrations. DNA-strand breaks and their repair Cells were seeded at a density of I × 105 cells in 3.5-cm Petri dishes. After a 20-h incubation, [laC]thymidine at 0.05 # C i / m l was added to the cultures and D N A was labelled by a 24-h incubation. After removal of the radioactive medium the cells were grown in fresh medium with or without 0'.2 mM BSO for 6 h. Prior to treatment with t-BuOOH, medium was placed in Hepes-buffered MEM without FBS. Following treatment with tBuOOH for 1 h, the cells were immediately chilled on ice or placed in prewarmed control medium for repair incubation. D N A single-strand breaks were measured by the alkaline elution method of Kohn et al. (1976). Results Effects of iron chelators on the induction of chromosomal aberrations by t-BuOOH After treatment of V79 cells with t-BuOOH for 1 h followed by a 1-h incubation in control medium, chromosomal structural aberrations consisting mainly of chromatid gaps and breaks appeared in a concentration-dependent fashion. Fig. 1 shows the suppression by iron chelators of tBuOOH-induced chromosomal aberrations. The
245 diffusible divalent iron chelator o - p h e n a n t h r o l i n e almost completely suppressed the f o r m a t i o n of c h r o m o s o m a l aberrations. I n contrast, the trivalent chelator desferrioxamine was less effective.
m e n t i n c u b a t i o n were n o t different for the 2 types of cells.
Effect of GSH depletion on the induction of chromosomal aberrations by t-BuOOH
The efficiency of i n d u c t i o n of D N A ssb b y t - B u O O H a n d the rate of resealing of the ssb were c o m p a r e d b e t w e e n G S H ÷ a n d G S H - cells. Fig. 2 shows the alkaline e l u t i o n profiles of D N A d u r i n g 60 m i n of p o s t - t r e a t m e n t i n c u b a t i o n after exposure to 0.5 m M t - B u O O H . T h e efficiency of f o r m a t i o n of D N A ssb b y t - B u O O H was somewhat lower in G S H - cells t h a n G S H ÷ cells. T h e rate of resealing of D N A ssb b y 0.5 m M t - B u O O H was very fast d u r i n g the first 10 min. Most of the D N A ssb were repaired within 60 m i n i n b o t h
Cell G S H was depleted as described before (Ochi et al., 1988). T r e a t m e n t of V79 cells with 0.2 m M BSO for 6 - 7 h depleted G S H to 5 - 6 % of the c o n t r o l value. T h e efficiency of f o r m a t i o n of c h r o m o s o m a l a b e r r a t i o n s b y t - B u O O H was c o m p a r e d b e t w e e n c o n t r o l ( G S H ÷) a n d G S H - d e p l e t e d ( G S H - ) cells. As shown i n T a b l e 1, incidences a n d types of a b e r r a t i o n s a p p e a r i n g after 1 h of the post-treat-
Effect of GSH depletion on the repair of t-BuOOHinduced DNA ssb
TABLE 1 INDUCTION OF CHROMOSOMAL ABERRATIONS BY t-BuOOH IN GSH + AND GSH- CELLS Treatment 0.5 mM t-BuOOH
Cells GSH +
GSH-
0.2 mM t-BuOOH
GSH ÷
GSH-
0.15 mM t-BuOOH
GSH ÷
GSH-
Control
GSH +
GSH -
Culture
Type of aberration (%) CG CB E
1)(2
F
Aberrant metaphases (%) + SD
1 2 3 1 2 3
47 40 40 46 40 46
2 6 3 3 6 1
1 0 2 0 0 0
0 0 1 0 0 0
0 4 0 0 0 0
49] 45 1 45.3 + 3.5 42 48] 44~ 46.3+2.1 47J
1 2 3 1 2 3
26 28 32 25 27 28
5 5 4 3 4 1
1 0 1 0 1 0
1 4 1 2 1 0
0 0 0 0 1 0
32~ 35~ 34.0+ 1.7 35 28 1 34I 30.3+ 3.2 29
1 2 3 1 2 3
25 25 26 20 28 20
3 1 3 1 3 8
1 0 0 0 0 0
1 1 0 1 2 1
0 0 0 0 1 0
297 27~ 28.3+ 1.2 29 22 ] 32f 27.3+ 5.0 28
1 2 3 1 2 3
8 4 8 9 4 8
0 0 0 0 0 0
0 0 0 0 0 0
0 0 0 0 0 0
0 0 0 0 0 0
8] ~I
6.7+2.3
9] ~
7.0+2.6
GSH-, glutathione-depleted cells; GSH +, cells not depleted of glutathione. CG, chromatid gaps; CB, chromatid breaks; E, exchanges; DC, dicentrics; F, fra~nentations.
246 60-
50.
50--
g U 40 "T: 30
c~
20
o 0
0.5
l.O
~ 0
I0
Concentration of t-BuOOH (mM)
20
Time of post-treatment incubation (h)
Fig. 1. Effects of iron chelators on the induction of chromosomal aberrations by t-BuOOH. V79 cells were preincubated with or without iron chelators f o r l 5 rain before the addition of t-BuOOH. The cultures were then incubated with the hydroperoxide in the presence or absence of the chelators for 1 h. • t-BuOOH alone; o t-BuOOH+100 ttM ophenanthroline; ,x t-BuOOH + 1 mM desferrioxamine.
Fig. 3. Repair of chromosomal aberrations by t-BuOOH in GSH-depleted and control cultures. Cells preteated with (closed symbols) or without (open symbols) 0.2 mM BSO for 6 h were incubated with or without t-BuOOH for 1 h. The cells were then washed and incubated in control medium for the indicated time prior to chromosomal preparation, zxA controls; [] • 0.2 mM t-BuOOH; ©O 0.5 mM t-BuOOH.
Fraction 0
l.O
l I
2
3
4
5
6
7
I
I
I
I
I
I
0
1
2
3
4
5
6
7
0.5 L%
o.~.
U
O.O5-
i
GSH+
GSH-
LL
Fig. 2. Induction and repair of D N A single-strand breaks by t-BuOOH in GSH-depleted and control cultures. Cells preincubated with ( G S H - ) or without (GSH + ) 0.2 mM BSO for 6 h were treated with 0.5 mM t-BuOOH for 1 h. After a 1-h incubation, cells were washed and incubated for 0 (O), 10 (zx), 30 (n) or 60 min ( o ) in control medium. Broken line indicates untreated control.
247
G S H ÷ and G S H - cells. Little difference in the rate of resealing of the D N A ssb was observed between the 2 cell types.
Effect of GSH depletion on the formation and repair of chromosomal aberrations by t-BuOOH To relate t-BuOOH-induced D N A ssb to the chromosomal aberrations, formation and repair of chromosomal aberrations by the hydroperoxide were investigated in both GSH ÷ and G S H - cells. As shown in Fig. 3, the incidence of chromosomal aberrations induced by 0.2 and 0.5 mM t-BuOOH was somewhat increased after 1 h of the posttreatment incubation compared to that at just the end of the treatment. Thereafter, the incidence of aberrations in GSH ÷ cells decreased rapidly to 18% for 0.5 mM t-BuOOH and 12% for 0.2 mM at 6 h of the post-treatment incubation and to control levels after 20 h. In contrast, the aberrations in G S H - cells remained high during 20 h of the post-treatment incubation. Discussion The effects of iron chelators and GSH depletion on the formation of chromosomal aberrations by t-BuOOH in V79 cells were investigated and compared with the effects on t-BuOOH-induced D N A ssb. The divalent iron chelator o-phenanthroline almost completely suppressed the formation of chromosomal aberrations while the trivalent chelator desferrioxamine was less effective. This may be due to the fact that only o-phenanthroline readily penetrates the cellular and nuclear membranes. Suppression by iron chelators of t-BuOOH-induced D N A ssb has also been observed (Ochi and Cerutti, unpublished), suggesting the presence of some common steps in the pathway leading to the formation of D N A ssb and chromosomal aberrations by the hydroperoxide. On the other hand, there seems to be a difference between t-BuOOH-induced D N A ssb and chromosomal aberrations with regard to their formation and repair. While the D N A ssb induced by 0.5 m M t-BuOOH were repaired in 60 min after treatment in both G S H + and G S H - ceils, chromosomal aberrations rather increased a little during this time in both cells and then were repaired
only in G S H + cells. Thus, chromosomal aberrations remain even after D N A ssb are repaired to control level, suggesting the involvement of other lesions than D N A ssb in the formation of chromosomal aberrations by t-BuOOH. In an earlier study, G S H depletion did not influence the formation of D N A ssb by t-BuOOH, although cytotoxicity was increased. Likewise, as shown in Table 1, G S H depletion did not affect the formation of chromosomal aberrations by tBuOOH. However, GSH depletion affected subsequent recovery of the aberrations. As shown in Fig. 3, the incidence of chromosomal aberrations induced by t-BuOOH in G S H + cells increased somewhat during the first 60 min after t-BuOOH treatment and then decreased markedly at 6 h of the post-treatment incubation and after 20 h the incidence was as low as in controls. In contrast, the aberrations in G S H - cells remained high even after 20 h of the post-treatment incubation. The failure of G S H - cells to repair t-BuOOH-induced chromosomal aberrations may be a cause of the enhanced cytotoxicity after G S H depletion or may conversely be a consequence of it. Depletion of cell GSH with BSO had no cytotoxic or clastogenic effect for at least the period of 24 h examined by Ochi et al. (1988). Therefore, the depleted cells provide a useful system for understanding the role of GSH in the detoxification or activation of mutagens inside cells, as well as the use of bacterial GSH-deficient strains (Kerklaan et al., 1983, 1985).
References Aust, S.D., L.A. Morhouse and C.E. Thomas (1985) Role of metals in oxygen radical reactions, J. Free Radical Biol. Med., 1, 3-25. Halllwell, B., and J.M.C. Gutteridge (1984) Oxygen toxicity, oxygen radicals, transition metals and disease, Biochem. J., 219, 1-14. Inouye, S. (1984) Site-specific cleavage of double-strand DNA by hydroperoxide of linoleic acid, FEBS Lett., 172, 231-234. Kerklaan, P., S. Bouter and G. Mohn (1983) Isolation of a mutant of Salmonella typhimurium strain TA1535 with decreased level of glutathione ( G S H - ) . Primary characterization and chemical mutagenesis studies, Mutation Res., 122, 257-266. Kerklaan, P.R.M., C.E.M. Zoetemelk and G.R. Mohn (1985) Mutagenic activity of various chemicals in Salmonella strain
248 TA100 and glutathione-deficient derivatives: on the role of glutathione in the detoxification or activation of mutagens inside bacterial cells, Biochem. Pharmacol., 34, 2151-2156. 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, 4628-4637. Logani, M.K., and R.E. Devies (1980) Lipid oxidation: biological effect and antioxidant, a review, Lipid, 15, 485-495. Ochi, T. (1988) Effects of glutathione depletion and induction of metallothioneins on the cytotoxicity of an organic hydroperoxide in cultured mammalian cells, Toxicology, 50, 257-268. Ochi, T., and P.A. Cerutti (1987) Clastogenic action of hydroperoxy, 5,8,11,13-icosatetraenoic acids on mouse embryo fibroblasts C3H 10T1/2, Proc. Natl. Acad. Sci. (U.S.A.), 84, 990-994. Ochi, T., and S. Miyaura (1989) Cytotoxicity of an organic
hydroperoxide and cellular antioxidant defense system against hydroperoxides in cultured mammalian cells, Toxicology, 55, 69-82. Ochi, T., F. Otsuka, K. Takahashi and M. Ohsawa (1988) Giutathione and metallothioneins as cellular defense against cadmium toxicity in cultured Chinese hamster cells, Chem.Biol. Interact., 65, 1-14. Thomally, P.J., R.J. Trotta and A. Stem (1984) Free radical production from the reaction of t-butylhydroperoxide with iron complex, in: B. Wolf, S. Manfred and T. David (Eds.), Oxygen Radicals in Chemistry and Biology, Walter de Gruyter and Co., Berlin, New York, pp. 215-218. Trotta, R.J., S.G. Sullivan and A. Stem (1982) Lipid peroxidation and haemoglobin degradation in red blood cells exposed to t-butyl hydroperoxide: effects of the hexose monophosphate shunt as mediated by glutathione and ascorbate, Biochem. J., 204, 405-415.
243
Elsevier MUT 04775
Effects of iron chelators and glutathione depletion on the induction and repair of chromosomal aberrations by tert-butyl hydroperoxide in cultured Chinese hamster cells Takafumi Ochi Department of Environmental Toxicology, Faculty of Pharmaceutical Sciences, Teikyo University, Sagamiko, Kanagawa 199-01 (Japan) (Received 2 January 1989) (Revision received 7 March 1989) (Accepted 8 March 1989)
Keywords: Hydroperoxide; Chromosomal aberrations; Iron chelators; Glutathione depletion
Summary The effects of iron chelators and glutathione (GSH) depletion on the induction of chromosomal aberrations by tert-butyl hydroperoxide (t-BuOOH) were investigated in cultured Chinese hamster V79 cells. t-BuOOH in a concentration range of 0.1-1.0 mM induced chromosomal structural aberrations, consisting mainly of chromatid gaps and breaks, in a dose-dependent fashion. The divalent iron chelator o-phenanthroline almost completely suppressed the formation of chromosomal aberrations while the trivalent chelator desferrioxamine was less effective. GSH depletion did not affect the formation of chromosomal aberrations and DNA single-strand breaks (ssb) by t-BuOOH. DNA ssb by 0.5 mM t-BuOOH were repaired within 60 min of treatment in both GSH-depleted ( G S H - ) and non-depleted (GSH ÷) cells. In contrast, chromosomal aberrations increased a tittle during the 60 min after treatment in both G S H - and GSH ÷ cells. The aberrations were then repaired in GSH + cells but those in G S H - cells were maintained to a great extent during 20 h of post-treatment incubation. These results indicate that divalent iron mediates the induction of chromosomal aberrations by t-BuOOH. That t-BuOOH-induced chromosomal aberrations remain even after DNA ssb were repaired suggests involvement of other lesions than DNA ssb in the formation of chromosomal aberrations by the hydroperoxide.
Organic hydroperoxides generate reactive oxygen free radicals and cause a variety of deleterious effects on biological systems (Logani and Davies, 1980; Trotta et al., 1982; Halliwell and Gut-
Correspondence: Dr. T. Ochi, Department of Environmental Toxicology, Faculty of Pharmaceutical Sciences, Teikyo University, Sagamiko, Kanagawa 199-01 (Japan).
teridge, 1984; Thornally et al., 1984; Inouye, 1984; Aust et al., 1985; Ochi and Cerutti, 1987). In our earlier studies, tert-butyl hydroperoxide (tBuOOH) used as a model of alkyl hydroperoxides induced cytotoxicity in cultured Chinese hamster V79 cells (Ochi, 1988; Ochi and Miyaura, 1989) and DNA single-strand breaks (ssb) in the alkaline elution assay (Ochi and Cerutti, unpublished). Both cytotoxicity and DNA ssb required
0027-5107/89/$03.50 © 1989 Elsevier Science Pubfishers B.V. (Biomedical Divsion)
244 free iron, in particular divalent, for their induction by t-BuOOH. Further, depletion of cell glutathione (GSH) with L-buthionine-SR-sulfoximine, a selective inhibitor of "t-glutamylcysteine synthetase, markedly enhanced t-BuOOH-induced cytotoxicity. However, GSH depletion did not influence the efficiency of induction of D N A ssb by the hydroperoxide. Thus, there was no correlation between t-BuOOH-induced cytotoxicity and D N A ssb with regard to their sensitivity to GSH depletion while iron was required for both. Here, our study is extended to the cytogenetic level in order to examine the relation between t-BuOOH-induced cytotoxicity, DNA ssb and chromosomal aberrations. The effects of iron chelators and GSH depletion on the formation and repair of chromosomal aberrations by t-BuOOH were investigated and compared with D N A ssb. Materials and methods Chemicals L-Buthionine-SR-sulfoximine (BSO), reducedform glutathione (GSH), glutathione reductase (type IV, 200 U / m g protein) and t-BuOOH were obtained from Sigma Chemical Co., St. Louis, MO (U.S.A.). o-Phenanthroline was purchased from Wako Pure Chemical Co., Osaka (Japan), tetra-npropylammoniumhydroxide from Tokyo Kasei Kogyo Co., Tokyo (Japan), desferrioxamine (desferal mesylate) from Ciba Geigy, Basel (Switzerland), and Colcemid from Grand Island Biological Co., Grand Island, NY (U.S.A.). [2-14C]Thymidine (58 mCi/mmole) was obtained from ICN Radiochemicals, Irvine, CA (U.S.A.). Cell cultures and media V79 cells from lung fibroblasts of male Chinese hamster were grown in a monolayer in minimum essential medium (MEM) supplemented with 10% fetal bovine serum (FBS), penicillin (100 U / m l ) and streptomycin (100 #g/ml). The cells were cultured in a CO 2 incubator with 5% CO 2 in humidified air. 10 mM Hepes-buffered (pH 7.4) MEM medium without FBS was used as a medium for treatment with t-BuOOH. Cell treatment and chromosomal preparation Cells were seeded at a density of 5 × 105 cells in 6-cm Petri dishes. After a 20-h incubation, cells
were incubated in the medium with or without 0.2 mM BSO for 6 h and were placed in Hepesbuffered MEM including BSO for challenge with t-BuOOH. In some experiments, cells were preincubated with or without iron chelators for 15 min before the addition of t-BuOOH, followed by incubation with the hydroperoxide in the presence or absence of the chelators for 1 h. After a 1-h incubation with t-BuOOH, cells were washed twice with Hanks' balanced salt solution (BSS) and placed in prewarmed control medium for further incubation. 0.1/~g/ml Colcemid was added to the cultures 1 h before harvesting the cells. At 0 h of the post-treatment incubation, cells were incubated with t-BuOOH in the presence of Colcemid for 1 h and chromosomes were prepared immediately after the treatment. Chromosomal preparations were made using the flame-drying method. 100 metaphases were analyzed for chromosomal aberrations. DNA-strand breaks and their repair Cells were seeded at a density of I × 105 cells in 3.5-cm Petri dishes. After a 20-h incubation, [laC]thymidine at 0.05 # C i / m l was added to the cultures and D N A was labelled by a 24-h incubation. After removal of the radioactive medium the cells were grown in fresh medium with or without 0'.2 mM BSO for 6 h. Prior to treatment with t-BuOOH, medium was placed in Hepes-buffered MEM without FBS. Following treatment with tBuOOH for 1 h, the cells were immediately chilled on ice or placed in prewarmed control medium for repair incubation. D N A single-strand breaks were measured by the alkaline elution method of Kohn et al. (1976). Results Effects of iron chelators on the induction of chromosomal aberrations by t-BuOOH After treatment of V79 cells with t-BuOOH for 1 h followed by a 1-h incubation in control medium, chromosomal structural aberrations consisting mainly of chromatid gaps and breaks appeared in a concentration-dependent fashion. Fig. 1 shows the suppression by iron chelators of tBuOOH-induced chromosomal aberrations. The
245 diffusible divalent iron chelator o - p h e n a n t h r o l i n e almost completely suppressed the f o r m a t i o n of c h r o m o s o m a l aberrations. I n contrast, the trivalent chelator desferrioxamine was less effective.
m e n t i n c u b a t i o n were n o t different for the 2 types of cells.
Effect of GSH depletion on the induction of chromosomal aberrations by t-BuOOH
The efficiency of i n d u c t i o n of D N A ssb b y t - B u O O H a n d the rate of resealing of the ssb were c o m p a r e d b e t w e e n G S H ÷ a n d G S H - cells. Fig. 2 shows the alkaline e l u t i o n profiles of D N A d u r i n g 60 m i n of p o s t - t r e a t m e n t i n c u b a t i o n after exposure to 0.5 m M t - B u O O H . T h e efficiency of f o r m a t i o n of D N A ssb b y t - B u O O H was somewhat lower in G S H - cells t h a n G S H ÷ cells. T h e rate of resealing of D N A ssb b y 0.5 m M t - B u O O H was very fast d u r i n g the first 10 min. Most of the D N A ssb were repaired within 60 m i n i n b o t h
Cell G S H was depleted as described before (Ochi et al., 1988). T r e a t m e n t of V79 cells with 0.2 m M BSO for 6 - 7 h depleted G S H to 5 - 6 % of the c o n t r o l value. T h e efficiency of f o r m a t i o n of c h r o m o s o m a l a b e r r a t i o n s b y t - B u O O H was c o m p a r e d b e t w e e n c o n t r o l ( G S H ÷) a n d G S H - d e p l e t e d ( G S H - ) cells. As shown i n T a b l e 1, incidences a n d types of a b e r r a t i o n s a p p e a r i n g after 1 h of the post-treat-
Effect of GSH depletion on the repair of t-BuOOHinduced DNA ssb
TABLE 1 INDUCTION OF CHROMOSOMAL ABERRATIONS BY t-BuOOH IN GSH + AND GSH- CELLS Treatment 0.5 mM t-BuOOH
Cells GSH +
GSH-
0.2 mM t-BuOOH
GSH ÷
GSH-
0.15 mM t-BuOOH
GSH ÷
GSH-
Control
GSH +
GSH -
Culture
Type of aberration (%) CG CB E
1)(2
F
Aberrant metaphases (%) + SD
1 2 3 1 2 3
47 40 40 46 40 46
2 6 3 3 6 1
1 0 2 0 0 0
0 0 1 0 0 0
0 4 0 0 0 0
49] 45 1 45.3 + 3.5 42 48] 44~ 46.3+2.1 47J
1 2 3 1 2 3
26 28 32 25 27 28
5 5 4 3 4 1
1 0 1 0 1 0
1 4 1 2 1 0
0 0 0 0 1 0
32~ 35~ 34.0+ 1.7 35 28 1 34I 30.3+ 3.2 29
1 2 3 1 2 3
25 25 26 20 28 20
3 1 3 1 3 8
1 0 0 0 0 0
1 1 0 1 2 1
0 0 0 0 1 0
297 27~ 28.3+ 1.2 29 22 ] 32f 27.3+ 5.0 28
1 2 3 1 2 3
8 4 8 9 4 8
0 0 0 0 0 0
0 0 0 0 0 0
0 0 0 0 0 0
0 0 0 0 0 0
8] ~I
6.7+2.3
9] ~
7.0+2.6
GSH-, glutathione-depleted cells; GSH +, cells not depleted of glutathione. CG, chromatid gaps; CB, chromatid breaks; E, exchanges; DC, dicentrics; F, fra~nentations.
246 60-
50.
50--
g U 40 "T: 30
c~
20
o 0
0.5
l.O
~ 0
I0
Concentration of t-BuOOH (mM)
20
Time of post-treatment incubation (h)
Fig. 1. Effects of iron chelators on the induction of chromosomal aberrations by t-BuOOH. V79 cells were preincubated with or without iron chelators f o r l 5 rain before the addition of t-BuOOH. The cultures were then incubated with the hydroperoxide in the presence or absence of the chelators for 1 h. • t-BuOOH alone; o t-BuOOH+100 ttM ophenanthroline; ,x t-BuOOH + 1 mM desferrioxamine.
Fig. 3. Repair of chromosomal aberrations by t-BuOOH in GSH-depleted and control cultures. Cells preteated with (closed symbols) or without (open symbols) 0.2 mM BSO for 6 h were incubated with or without t-BuOOH for 1 h. The cells were then washed and incubated in control medium for the indicated time prior to chromosomal preparation, zxA controls; [] • 0.2 mM t-BuOOH; ©O 0.5 mM t-BuOOH.
Fraction 0
l.O
l I
2
3
4
5
6
7
I
I
I
I
I
I
0
1
2
3
4
5
6
7
0.5 L%
o.~.
U
O.O5-
i
GSH+
GSH-
LL
Fig. 2. Induction and repair of D N A single-strand breaks by t-BuOOH in GSH-depleted and control cultures. Cells preincubated with ( G S H - ) or without (GSH + ) 0.2 mM BSO for 6 h were treated with 0.5 mM t-BuOOH for 1 h. After a 1-h incubation, cells were washed and incubated for 0 (O), 10 (zx), 30 (n) or 60 min ( o ) in control medium. Broken line indicates untreated control.
247
G S H ÷ and G S H - cells. Little difference in the rate of resealing of the D N A ssb was observed between the 2 cell types.
Effect of GSH depletion on the formation and repair of chromosomal aberrations by t-BuOOH To relate t-BuOOH-induced D N A ssb to the chromosomal aberrations, formation and repair of chromosomal aberrations by the hydroperoxide were investigated in both GSH ÷ and G S H - cells. As shown in Fig. 3, the incidence of chromosomal aberrations induced by 0.2 and 0.5 mM t-BuOOH was somewhat increased after 1 h of the posttreatment incubation compared to that at just the end of the treatment. Thereafter, the incidence of aberrations in GSH ÷ cells decreased rapidly to 18% for 0.5 mM t-BuOOH and 12% for 0.2 mM at 6 h of the post-treatment incubation and to control levels after 20 h. In contrast, the aberrations in G S H - cells remained high during 20 h of the post-treatment incubation. Discussion The effects of iron chelators and GSH depletion on the formation of chromosomal aberrations by t-BuOOH in V79 cells were investigated and compared with the effects on t-BuOOH-induced D N A ssb. The divalent iron chelator o-phenanthroline almost completely suppressed the formation of chromosomal aberrations while the trivalent chelator desferrioxamine was less effective. This may be due to the fact that only o-phenanthroline readily penetrates the cellular and nuclear membranes. Suppression by iron chelators of t-BuOOH-induced D N A ssb has also been observed (Ochi and Cerutti, unpublished), suggesting the presence of some common steps in the pathway leading to the formation of D N A ssb and chromosomal aberrations by the hydroperoxide. On the other hand, there seems to be a difference between t-BuOOH-induced D N A ssb and chromosomal aberrations with regard to their formation and repair. While the D N A ssb induced by 0.5 m M t-BuOOH were repaired in 60 min after treatment in both G S H + and G S H - ceils, chromosomal aberrations rather increased a little during this time in both cells and then were repaired
only in G S H + cells. Thus, chromosomal aberrations remain even after D N A ssb are repaired to control level, suggesting the involvement of other lesions than D N A ssb in the formation of chromosomal aberrations by t-BuOOH. In an earlier study, G S H depletion did not influence the formation of D N A ssb by t-BuOOH, although cytotoxicity was increased. Likewise, as shown in Table 1, G S H depletion did not affect the formation of chromosomal aberrations by tBuOOH. However, GSH depletion affected subsequent recovery of the aberrations. As shown in Fig. 3, the incidence of chromosomal aberrations induced by t-BuOOH in G S H + cells increased somewhat during the first 60 min after t-BuOOH treatment and then decreased markedly at 6 h of the post-treatment incubation and after 20 h the incidence was as low as in controls. In contrast, the aberrations in G S H - cells remained high even after 20 h of the post-treatment incubation. The failure of G S H - cells to repair t-BuOOH-induced chromosomal aberrations may be a cause of the enhanced cytotoxicity after G S H depletion or may conversely be a consequence of it. Depletion of cell GSH with BSO had no cytotoxic or clastogenic effect for at least the period of 24 h examined by Ochi et al. (1988). Therefore, the depleted cells provide a useful system for understanding the role of GSH in the detoxification or activation of mutagens inside cells, as well as the use of bacterial GSH-deficient strains (Kerklaan et al., 1983, 1985).
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