83
Mutanon Research, 260 (1991) 83-87 © 1991 Elsewer Scmnce Pubhshers B.V. 0165-1218/91/$03.50 ADONIS 016512189100082J MUTGEN 01644
Induction of mutation in V79 Chinese hamster cells by tetrachlorohydroquinone, a metabolite of pentachlorophenol Kristian Jansson and Vuokko Jansson Department of Cell Btology, Untverstty of Jyv~iskyld, SF-40100 Jyoaskyli~ (Finland) (Received 21 June 1990) (Revision received 5 October 1990) (Accepted 10 October 1990)
Keywords" Chlorophenols; Pentachlorophenol; Carcinogens, enxaronmental, Mutagemclty tests; Cells, cultured
Summary Tetrachlorohydroquinone (TCHQ) and tetrachlorocatechol (TCC), two metabolites of the environmental mutagen and carcinogen pentachlorophenol, were tested without exogenous activation in V79 Chinese hamster cells for the induction of mutation at the hypoxanthine phosphoribosyl transferase (HPRT) locus to 6-thioguanine resistance (TG r) and at the N a / K - A T P a s e locus to ouabain resistance ( O u a R ) . T r e a t m e n t was for 24 h at 37 o C. T C H Q produced statistically significant increases in the frequency of T G r mutants. The lowest observed effective dose (LOED) was 20 /tM, where the relative cloning efficiency was 63%. The relationship between the dose of T C H Q and the frequency of T G r mutants was approximately linear over the range of 0-60 /~M with an estimated slope ( + 95% confidence limits) of 1.1 _ 0.3 mutants per 10 6 clonable cells per ktM. At the highest tested dose of TCHQ, 60 /tM, the relative cloning efficiency was reduced to 7%. In contrast to TCHQ, TCC was unable to induce T G r mutants at doses up to 120/tM. The relative cloning efficiency at this dose was 5%. Both T C H Q and TCC were unable to induce Oua R mutants. The results suggest that T C H Q is at least partly responsible for the genotoxic activity of pentachlorophenol. T C H Q can produce reactive oxygen species, which may cause large genetic damage such as deletions, resulting in mutation to T G ' but not to Oua R.
Tetrachlorohydroquinone (TCHQ), a major metabolite of the environmental contaminant pentacb_lorophenol (PCP) in mice and rats (Jacobson and Yllner, 1971; Ahlborg et al., 1974, 1978),
Correspondence: Knstmn Jansson, Department of Cell Btology, Umvers~ty of Jyv~iskyl~i, Vapaudenkatu 4, SF-40100 Jyvaskylh (Finland).
binds covalently to calf thymus D N A and causes single-strand breaks in bacteriophage PM2 D N A in contrast to PCP (Witte et al., 1985). In addition to TCHQ, tetrachlorocatechol (TCC) has been identified as a metabolite of PCP in rats (Edgerton et al., 1979). Metabolism of PCP by rat liver microsomes results in formation of T C H Q and TCC, and in covalent binding to microsomal protein and to added D N A (van Ommen et al., 1986).
84 Evidently, both T C H Q and TCC are metabolites of PCP in humans (Ahlborg et al., 1974; Edgerton et al., 1979, 1981; Juhl et al., 1985). Previous studies have shown that PCP is a mouse somatic mutagen (Fahrig et al., 1978; reviewed by Styles and Penman, 1985) but not a direct gene mutagen in V79 Chinese hamster cells (Jansson and Jansson, 1986). More recently, PCP has been shown to cause cancer in mice (National Toxacology Program, 1989), to induce chromosome aberrations in Chinese hamster ovary (CHO) cells in the presence but not the absence of $9 metabolic activation (Galloway et al., 1987), and to induce prophage lambda in Eschenchia coh only in the presence of $9 (DeMarini et al., 1990). In the present study, we tested T C H Q and TCC without exogenous activation in V79 cells for the reduction of mutation at the hypoxanthine phosphoribosyl transferase (HPRT) locus to 6thloguanme resistance (TG r) and at the N a / K ATPase locus to ouabain resistance (OuaR). Materials and methods Chemtcals
T C H Q (CAS No. 87-87-6, purity > 99% by titration) was purchased from Eastman Kodak Co., Rochester, NY. Ethyl methanesulfonate (EMS, CAS No. 62-50-0) was obtained from Sigma Chemical Co., St. Louis, MO. TCC (CAS No. 1198-55-6, purity > 99.9% by gas chromatography) was kindly supplied by Dr. Juha Knuutinen (for synthesis see Knuutinen, 1981). Stock solutions of the chemicals were prepared in dimethyl sulfoxide (E. Merck, Darmstadt, F.R.G.) immediately before use. Mutatton assay
V79 Chinese hamster cells, originally obtained from Dr. Eliezer Huberman (Oak Ridge National Laboratory, Oak Ridge, TN), were cloned to reduce the spontaneous background frequency of T G r and Oua R mutants, checked for the presence of mycoplasma by the method of Chen (1977), and stored in ampoules frozen in liquid nitrogen. No HAT (hypoxanthine, aminopterin, thymidine) treatments were performed. Before each experiment, an ampoule of cells was thawed and maintained in exponential growth for 3 days. The cells
were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, penicillin (100 I U / m l ) and streptomycin (100 # g / m l ) (Gibco Ltd., Paisley, Scotland) at 3 7 ° C with a humidified atmosphere of 10% CO 2 in air. Cell-culture dishes and flasks were purchased from Nunc (Roskilde, Denmark). The mutation assay was essentmlly as described previously (Jansson and Jansson, 1986), but modified to detect resistance to Oua in addition to TG. In an experiment, 1 x 106 cells were plated in 10 ml of medium per 100-mm dish (2 dishes/dose) and allowed to grow for 24 h. The medium was then replaced with 10 ml of fresh medium containing the test chemical. Each experiment included a solvent control and a positive control of EMS at 1.6 mM. The final solvent concentration was 0.5% for all samples. After 24 h of treatment, the cells were rinsed with Dulbecco's phosphate-buffered saline (PBS) and dissociated with 0.05% trypsin and 0.02% EDTA (Gibco Ltd.) in PBS. To determine cloning efficiency, 200 cells were plated in 4 ml of medium per 60-mm dish (4 dishes/dose), and after 6 days, the colonies were fixed in ethanol and stained with Giemsa. For phenotyplc expression, 2 x 10 6 cells were plated into a 80-cm2 flask in 20 ml of medium. The cells were subcultured every 2 days, maintaining 2 x 106 cells at each subculture. After a 6-day expression period, the cells were plated at 1 x 105 cells per 100-mm dish (20 dishes/dose) in 10 ml of medium containing 30 /~M T G (Sigma Chemical Co.) and at 2 × 105 cells per 100-mm dish (10 dishes/dose) in 10 ml of medium containing 1 mM Oua (Sigma Chemical Co.). The mutant colonies were fixed and stained 10 days later. To determine clomng efficiency at the time of mutant selection, 200 cells were plated m 4 ml of medium per 60-mm dish (4 dishes/dose), and after 6 days, the colonies were fixed and stained. The results were calculated as follows: Cloning efficiency (CE) number of colonies formed number of cells plated
Relative CE =
CE (treatment) CE (solvent control)
85
Mutant frequency (MF)
of M F is proportional to the expectation of MF. The relative CE is presented as the mean of the 2 experiments.
number of mutant colomes number of cells plated × CE M F is expressed as mutants per 1 0 6 clonable cells. When no mutant colonies were observed, the M F was calculated on the basis of an observation of a single mutant colony and thus represents the lower limit of detection. Each experiment was conducted twice. Combined analyses of the 2 experiments were made. The M F data were evaluated by analysis of variance and Dunnett's test after a variance-stabilizing log transformation. Where a statistically significant ( P < 0.05) response occurred, the lowest observed effective dose (LOED) was noted. The slope ( _ 95% confidence limits) for the linear regression of M F on dose was calculated by the method of weighted least squares, with the weights estimated to be inversely proportional to the square of the expectation of MF, E [ M F Idose] = a + fl(dose). This weighting scheme and the above log transformation are based on the assumption that for a fixed dose, the true standard deviation
Results and discussion
Table 1 shows that treatment of V79 cells with both T C H Q and T C C resulted in dose-related decreases in CE. At a dose of 60 #M, T C H Q and TCC reduced the relative CE to 7 and 31%, respectively. For comparison, PCP reduced the relative CE of V79 cells to about 66% at the same dose (Jansson and Jansson, 1986). T C H Q produced statistically significant increases in the frequency of T G r mutants. The L O E D was 20 #M, where the relative CE was 63%. The relationship between the dose of T C H Q and the frequency of T G r mutants was approximately linear over the range of 0 - 6 0 / ~ M with an estimated slope ( + 95% confidence limits) of 1.1 + 0.3 mutants per 1 0 6 clonable cells per #M. In contrast to T C H Q , T C C was unable to induce T G r mutants at doses up to 120 #M. The relative CE at this dose was 5%. Both T C H Q and T C C were unable to induce
TABLE 1 M U T A T I O N T O O U A B A I N RESISTANCE (Oua R) A N D 6 - T H I O G U A N I N E R E S I S T A N C E ( T G r) IN V79 C H I N E S E H A M STER CELLS A F T E R T R E A T M E N T W I T H T E T R A C H L O R O H Y D R O Q U I N O N E (TCHQ) A N D T E T R A C H L O R O C A T E C H O L (TCC) F O R 24 h Treatment
Cloning efficiency (%) a
Oua g
Mutant frequency b TG r
Expt. 1
Expt. 2
Expt. 1
Expt. 2
G.M.
Expt. 1
Expt. 2
G.M
Solvent control TCHQ 4 #M 20#M 40 # M 60 # M PosRlve control c
86 75 48 17 6 51
61 57 43 23 4 46
3 (4) 1 (1) 1 (1) 2 (3) 2 (3) 191 (228)
2 (3) 2 (3)
Mutanon Research, 260 (1991) 83-87 © 1991 Elsewer Scmnce Pubhshers B.V. 0165-1218/91/$03.50 ADONIS 016512189100082J MUTGEN 01644
Induction of mutation in V79 Chinese hamster cells by tetrachlorohydroquinone, a metabolite of pentachlorophenol Kristian Jansson and Vuokko Jansson Department of Cell Btology, Untverstty of Jyv~iskyld, SF-40100 Jyoaskyli~ (Finland) (Received 21 June 1990) (Revision received 5 October 1990) (Accepted 10 October 1990)
Keywords" Chlorophenols; Pentachlorophenol; Carcinogens, enxaronmental, Mutagemclty tests; Cells, cultured
Summary Tetrachlorohydroquinone (TCHQ) and tetrachlorocatechol (TCC), two metabolites of the environmental mutagen and carcinogen pentachlorophenol, were tested without exogenous activation in V79 Chinese hamster cells for the induction of mutation at the hypoxanthine phosphoribosyl transferase (HPRT) locus to 6-thioguanine resistance (TG r) and at the N a / K - A T P a s e locus to ouabain resistance ( O u a R ) . T r e a t m e n t was for 24 h at 37 o C. T C H Q produced statistically significant increases in the frequency of T G r mutants. The lowest observed effective dose (LOED) was 20 /tM, where the relative cloning efficiency was 63%. The relationship between the dose of T C H Q and the frequency of T G r mutants was approximately linear over the range of 0-60 /~M with an estimated slope ( + 95% confidence limits) of 1.1 _ 0.3 mutants per 10 6 clonable cells per ktM. At the highest tested dose of TCHQ, 60 /tM, the relative cloning efficiency was reduced to 7%. In contrast to TCHQ, TCC was unable to induce T G r mutants at doses up to 120/tM. The relative cloning efficiency at this dose was 5%. Both T C H Q and TCC were unable to induce Oua R mutants. The results suggest that T C H Q is at least partly responsible for the genotoxic activity of pentachlorophenol. T C H Q can produce reactive oxygen species, which may cause large genetic damage such as deletions, resulting in mutation to T G ' but not to Oua R.
Tetrachlorohydroquinone (TCHQ), a major metabolite of the environmental contaminant pentacb_lorophenol (PCP) in mice and rats (Jacobson and Yllner, 1971; Ahlborg et al., 1974, 1978),
Correspondence: Knstmn Jansson, Department of Cell Btology, Umvers~ty of Jyv~iskyl~i, Vapaudenkatu 4, SF-40100 Jyvaskylh (Finland).
binds covalently to calf thymus D N A and causes single-strand breaks in bacteriophage PM2 D N A in contrast to PCP (Witte et al., 1985). In addition to TCHQ, tetrachlorocatechol (TCC) has been identified as a metabolite of PCP in rats (Edgerton et al., 1979). Metabolism of PCP by rat liver microsomes results in formation of T C H Q and TCC, and in covalent binding to microsomal protein and to added D N A (van Ommen et al., 1986).
84 Evidently, both T C H Q and TCC are metabolites of PCP in humans (Ahlborg et al., 1974; Edgerton et al., 1979, 1981; Juhl et al., 1985). Previous studies have shown that PCP is a mouse somatic mutagen (Fahrig et al., 1978; reviewed by Styles and Penman, 1985) but not a direct gene mutagen in V79 Chinese hamster cells (Jansson and Jansson, 1986). More recently, PCP has been shown to cause cancer in mice (National Toxacology Program, 1989), to induce chromosome aberrations in Chinese hamster ovary (CHO) cells in the presence but not the absence of $9 metabolic activation (Galloway et al., 1987), and to induce prophage lambda in Eschenchia coh only in the presence of $9 (DeMarini et al., 1990). In the present study, we tested T C H Q and TCC without exogenous activation in V79 cells for the reduction of mutation at the hypoxanthine phosphoribosyl transferase (HPRT) locus to 6thloguanme resistance (TG r) and at the N a / K ATPase locus to ouabain resistance (OuaR). Materials and methods Chemtcals
T C H Q (CAS No. 87-87-6, purity > 99% by titration) was purchased from Eastman Kodak Co., Rochester, NY. Ethyl methanesulfonate (EMS, CAS No. 62-50-0) was obtained from Sigma Chemical Co., St. Louis, MO. TCC (CAS No. 1198-55-6, purity > 99.9% by gas chromatography) was kindly supplied by Dr. Juha Knuutinen (for synthesis see Knuutinen, 1981). Stock solutions of the chemicals were prepared in dimethyl sulfoxide (E. Merck, Darmstadt, F.R.G.) immediately before use. Mutatton assay
V79 Chinese hamster cells, originally obtained from Dr. Eliezer Huberman (Oak Ridge National Laboratory, Oak Ridge, TN), were cloned to reduce the spontaneous background frequency of T G r and Oua R mutants, checked for the presence of mycoplasma by the method of Chen (1977), and stored in ampoules frozen in liquid nitrogen. No HAT (hypoxanthine, aminopterin, thymidine) treatments were performed. Before each experiment, an ampoule of cells was thawed and maintained in exponential growth for 3 days. The cells
were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, penicillin (100 I U / m l ) and streptomycin (100 # g / m l ) (Gibco Ltd., Paisley, Scotland) at 3 7 ° C with a humidified atmosphere of 10% CO 2 in air. Cell-culture dishes and flasks were purchased from Nunc (Roskilde, Denmark). The mutation assay was essentmlly as described previously (Jansson and Jansson, 1986), but modified to detect resistance to Oua in addition to TG. In an experiment, 1 x 106 cells were plated in 10 ml of medium per 100-mm dish (2 dishes/dose) and allowed to grow for 24 h. The medium was then replaced with 10 ml of fresh medium containing the test chemical. Each experiment included a solvent control and a positive control of EMS at 1.6 mM. The final solvent concentration was 0.5% for all samples. After 24 h of treatment, the cells were rinsed with Dulbecco's phosphate-buffered saline (PBS) and dissociated with 0.05% trypsin and 0.02% EDTA (Gibco Ltd.) in PBS. To determine cloning efficiency, 200 cells were plated in 4 ml of medium per 60-mm dish (4 dishes/dose), and after 6 days, the colonies were fixed in ethanol and stained with Giemsa. For phenotyplc expression, 2 x 10 6 cells were plated into a 80-cm2 flask in 20 ml of medium. The cells were subcultured every 2 days, maintaining 2 x 106 cells at each subculture. After a 6-day expression period, the cells were plated at 1 x 105 cells per 100-mm dish (20 dishes/dose) in 10 ml of medium containing 30 /~M T G (Sigma Chemical Co.) and at 2 × 105 cells per 100-mm dish (10 dishes/dose) in 10 ml of medium containing 1 mM Oua (Sigma Chemical Co.). The mutant colonies were fixed and stained 10 days later. To determine clomng efficiency at the time of mutant selection, 200 cells were plated m 4 ml of medium per 60-mm dish (4 dishes/dose), and after 6 days, the colonies were fixed and stained. The results were calculated as follows: Cloning efficiency (CE) number of colonies formed number of cells plated
Relative CE =
CE (treatment) CE (solvent control)
85
Mutant frequency (MF)
of M F is proportional to the expectation of MF. The relative CE is presented as the mean of the 2 experiments.
number of mutant colomes number of cells plated × CE M F is expressed as mutants per 1 0 6 clonable cells. When no mutant colonies were observed, the M F was calculated on the basis of an observation of a single mutant colony and thus represents the lower limit of detection. Each experiment was conducted twice. Combined analyses of the 2 experiments were made. The M F data were evaluated by analysis of variance and Dunnett's test after a variance-stabilizing log transformation. Where a statistically significant ( P < 0.05) response occurred, the lowest observed effective dose (LOED) was noted. The slope ( _ 95% confidence limits) for the linear regression of M F on dose was calculated by the method of weighted least squares, with the weights estimated to be inversely proportional to the square of the expectation of MF, E [ M F Idose] = a + fl(dose). This weighting scheme and the above log transformation are based on the assumption that for a fixed dose, the true standard deviation
Results and discussion
Table 1 shows that treatment of V79 cells with both T C H Q and T C C resulted in dose-related decreases in CE. At a dose of 60 #M, T C H Q and TCC reduced the relative CE to 7 and 31%, respectively. For comparison, PCP reduced the relative CE of V79 cells to about 66% at the same dose (Jansson and Jansson, 1986). T C H Q produced statistically significant increases in the frequency of T G r mutants. The L O E D was 20 #M, where the relative CE was 63%. The relationship between the dose of T C H Q and the frequency of T G r mutants was approximately linear over the range of 0 - 6 0 / ~ M with an estimated slope ( + 95% confidence limits) of 1.1 + 0.3 mutants per 1 0 6 clonable cells per #M. In contrast to T C H Q , T C C was unable to induce T G r mutants at doses up to 120 #M. The relative CE at this dose was 5%. Both T C H Q and T C C were unable to induce
TABLE 1 M U T A T I O N T O O U A B A I N RESISTANCE (Oua R) A N D 6 - T H I O G U A N I N E R E S I S T A N C E ( T G r) IN V79 C H I N E S E H A M STER CELLS A F T E R T R E A T M E N T W I T H T E T R A C H L O R O H Y D R O Q U I N O N E (TCHQ) A N D T E T R A C H L O R O C A T E C H O L (TCC) F O R 24 h Treatment
Cloning efficiency (%) a
Oua g
Mutant frequency b TG r
Expt. 1
Expt. 2
Expt. 1
Expt. 2
G.M.
Expt. 1
Expt. 2
G.M
Solvent control TCHQ 4 #M 20#M 40 # M 60 # M PosRlve control c
86 75 48 17 6 51
61 57 43 23 4 46
3 (4) 1 (1) 1 (1) 2 (3) 2 (3) 191 (228)
2 (3) 2 (3)
