Mutation Research, 238 (1990) 235-243 Elsevier
235
MUTREV 02808
Endogenous mutagens derived from amino acids Hansruedi Glatt Department of Toxicology, Universityof Mainz, D-6500Mainz (F.R.G.) (Accepted 12 October1989)
Keywords: N-Acetyl-L-cysteine;ChinesehamsterV79 cells; L-Cysteine;Levodopa;Homogentisicacid; Salmonella typhimurium
Summary L-Cysteine, glutathione and the therapeutically used L-cysteine precursor, N-acetyl-L-cysteine, induced strong mutagenic effects in Salmonella typhimurium (reversion of the his- strains TA97, TA92 and TA104), when tested in the presence of subcellular kidney preparations. The tyrosine metabolites, levodopa (an ortho-hydroquinone) and homogentisic acid (a para-hydroquinone) reverted various hisstrains as well. This mutagenicity did not require the presence of mammalian enzymes, and was relatively weak. The induction of gene mutations was also studied in mammalian cells (V79 Chinese hamster cells), using acquisition of resistance toward 6-thioguanine as the marker. L-Cysteine and N-acetyl-L-cysteine were found to be inactive, levodopa was weakly mutagenic, and homogentisic acid was strongly mutagenic (enhancing the mutation frequency 135-fold above background at an exposure concentration of 50/~M). This finding is striking as the urinary concentration of homogentisic acid is about 1000 times higher in patients with a genetic defect in homogentisic acid 1,2-dioxygenase (alkaptonuria). Genotoxic and carcinogenic effects of other amino acids and metabolites, reported in the literature, are discussed as well.
It is a widespread prejudice that physiological compounds are non-genotoxic and non-mutagenic, and therefore only very few of them have been experimentally investigated in this respect. Some years ago, we observed pronounced mutagenic effects in Salmonella typhimurium of glutathione, tested in the presence of subcellular kidney preparations, and of L-cysteine, tested in the presence of kidney or liver preparations (Glatt et al., 1983). We then learned that similar mutagenic effects had also been observed by others in L-cysteine (Yamaguchi and Yamashita, 1981). The mutagenicity of these compounds was further in-
Correspondence: Dr. HansruediGlatt, Departmentof Toxicology, Universityof Mainz,Obere ZahlbacherStrasse67, D-6500 Mainz 1 (F.R.G.).
vestigated with regard to metabolic aspects, stereospecificity, responsiveness of various bacterial strains and mammalian cells (Glatt and Oesch, 1985; Ross et al., 1986; Carter and Josephy, 1986; Stark et al., 1987, 1988; Glatt, 1989; Glatt et al., 1990). Here, some findings are reported on the L-cysteine precursor, N-acetyl-L-cysteine. This compound is used as a mucolytic and as an antidote in acetaminophen poisoning. In the latter situation (Prescott and Critchley, 1983), very high doses are used. A typical treatment consists of a loading dose of 140 mg/kg, followed by administration of 70 mg/kg every 4 h for 17 doses. N-Acetyl-L-cysteine probably acts, in part, by replenishing hepatic stores of glutathione, depleted by acetaminophen. N-Acetyl-t-cysteine is more effective than direct administration of L-cysteine or glutathione.
0165-1110/90/$03.50 © 1990 ElsevierSciencePublishersB.V. (BiomedicalDivision)
236 CO0+ I H3N-C-H
Dopamine, Adrenatine, Noradrenaline
C,H2 +
CO0I
H3N-C-H Metanin
OH Dopa OH Tyrosine
~
OH I~CH2-CO0 L
---~
Acetoacetate, + Fumarate
OH Homogentisic acid
Fig. 1. Structures and metabolic relations of tyrosine, dopa and homogentisic acid.
We recently observed mutagenic effects of the benzene metabolites, catechol and p-benzohydroquinone, in V79 Chinese hamster cells (Glatt et al., 1989). These structures are also contained in the tyrosine metabolites, levodopa (L-dopa) and homogentisic acid (Fig. 1). L-Dopa is the physiological metabolic precursor of the catecholamines dopamine, adrenaline and noradrenaline (which are catechols as well) and of the melanins. It is the major drug used in the treatment of parkinsonism. Homogentisic acid is an intermediate in the catabolism of tyrosine. In normal individuals, its concentration is very low, due to efficient further metabolism by homogentisic acid 1,2-dioxygenase. However, in a heritable disorder, alkaptonuria, this enzyme is defective and the patients excrete 4-8 g of homogentisic acid in their urine per day (McKusick, 1972). For these reasons, L-dopa and homogentisic acid have been investigated for mutagenicity in V79 cells and in bacteria. Materials and methods
Test compounds N-Acetyl-L-cysteine and L-dopa were obtained from Sigma (Deisenhofen, F.R.G.), homogentisic acid was purchased from Aldrich (Steinheim, F.R.G.). The compounds were dissolved in water immediately before use. The solutions were adjusted to pH 6-7 by adding dilute NaOH. Bacteria Bacteria were grown overnight in Nutrient broth No. 2 (Oxoid GmbH, Wesel, F.R.G.), 25 g/1. For
inoculation, stock cultures that were stored at - 7 0 °C were used. The overnight cultures were centrifuged, resuspended in medium A (1.6 g Bacto nutrient broth + 5 g NaC1/1) and adjusted nephelometrically to a titer of about 2 × 10 9 bacteria (colony-forming units)/ml. In each experiment the presence of the R-factor pKM 101 was ascertained by growing diluted cultures on ampicillin-containing and ampicillin-free, histidine-supplemented agar plates. With strains TA92, TA97, TA100, TA102 and TA104 the numbers of colonies were always virtually identical under the 2 culture conditions, whereas strains TA1535 and TA1537 grew only in the absence of ampicillin. Tissue preparations Male Sprague-Dawley rats (200-300 g) were obtained from Wiga, Sulzfeld (F.R.G.). The kidneys and livers of animals that had not been deliberately treated with an inducing agent were homogenized in 3 volumes of sterile, cold KCI (150 mM), buffered with 10 mM sodium phosphate, pH 7.4. The homogenate was centrifuged at 10 000 x g for 10 min. The resulting supernatant, termed $9 fraction, was used on the day of preparation or stored at - 7 0 °C until use. Bacterial mutagenicity assay Test compound (in neutralized aqueous solution, 100-200 #1), buffer (as used for preparation of the tissue fraction, 500 ffl), or $9 fraction (equivalent to 100 mg tissue, 333/~1) and cofactor solution (for details see legends to figures), and bacteria (100 #1 of the resuspension) were mixed in a glass tube, and incubated for 20 min (' preincubation assay') or 0 rain ('standard plate-incorporation assay') at 37 °C. Then, 2 ml of 45°C warm soft agar (0.55% agar, 0.55% NaC1, 50 /~M histidine, 50 #M biotin, 25 mM sodium phosphate buffer, pH 7.4) was added, and the mixture was poured on to a Petri dish containing 24 ml minimal agar (1.5% agar in Vogel-Bonner E medium with 2% glucose). After incubation for 3 days in the dark, colonies (his + revertants) were counted. Mammalian cell mutagenicity assay Chinese hamster V79 cells were obtained in 1984 from Dr. G. Turchi (Consiglio Nazionale delle Ricerche, Pisa, Italy). They were cloned in
237
order to eliminate mutants. The modal number of chromosomes of our strain was 22. The cells were free of mycoplasms. They were maintained in Dulbecco's modified minimal essential medium supplemented with 5% fetal calf serum and penicillin/streptomycin (DME-FCS). On day 1 of the mutagenicity test, 1.5 x 106 cells were seeded with 30 ml of medium in 15-cm Petri dishes. After 18 h the test compound was added. 24 h later, the exposure was terminated by a change of the medium. On day 4, the cells were detached by treating with trypsin, counted, and subcultured at a density of 3 x 106 cells per 15-cm Petri dish. On day 8, they were subcultured again at a density of 1 0 6 cells per 15-cm Petri dish in medium containing 6-thioguanine (7 #g/ml) to determine the number of mutants (6 replicate plates). The cloning efficiency was determined by plating 100 cells with 5 ml of 6-thioguanine-free medium in 6-cm Petri dishes (3 replicate plates). The colonies were counted after incubating for about 8 days (cloning efficiency plates) and 10 days (selection plates). Results
Mutagenicity in Salmonella typhimurium For L-cysteine and glutathione the results have been reported previously (Glatt et al., 1983; Glatt and Oesch, 1985; Glatt, 1989). N-Acetyl-L-cysteine was tested in 3 strains (TA92, TA97, TA104), in which L-cysteine and glutathione show strong effects. In the absence of a mammalian metabolizing system, no increases in the number of mutants were observed (at all doses used, 3.75-120 /zmole/plate, numbers of colonies were less than 1.2-fold the value of the solvent controls). However, in the presence of rat kidney $9 fraction pronounced mutagenic effects were found (Fig. 2). TA97 was the most responsive strain, with impressive increases in the number of colonies per plate from 180 in the negative control to 4700 at the highest dose used. Dose-response curve, maximal effect and strain preference were very similar to those reported for t-cysteine and glutathione (Glatt, 1989). In the direct test, L-dopa enhanced the number of colonies about 2-fold with strain TA104 and 1.5-fold with strain TA100 (Fig. 3). These effects were confirmed in repeat experiments (data not
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235
MUTREV 02808
Endogenous mutagens derived from amino acids Hansruedi Glatt Department of Toxicology, Universityof Mainz, D-6500Mainz (F.R.G.) (Accepted 12 October1989)
Keywords: N-Acetyl-L-cysteine;ChinesehamsterV79 cells; L-Cysteine;Levodopa;Homogentisicacid; Salmonella typhimurium
Summary L-Cysteine, glutathione and the therapeutically used L-cysteine precursor, N-acetyl-L-cysteine, induced strong mutagenic effects in Salmonella typhimurium (reversion of the his- strains TA97, TA92 and TA104), when tested in the presence of subcellular kidney preparations. The tyrosine metabolites, levodopa (an ortho-hydroquinone) and homogentisic acid (a para-hydroquinone) reverted various hisstrains as well. This mutagenicity did not require the presence of mammalian enzymes, and was relatively weak. The induction of gene mutations was also studied in mammalian cells (V79 Chinese hamster cells), using acquisition of resistance toward 6-thioguanine as the marker. L-Cysteine and N-acetyl-L-cysteine were found to be inactive, levodopa was weakly mutagenic, and homogentisic acid was strongly mutagenic (enhancing the mutation frequency 135-fold above background at an exposure concentration of 50/~M). This finding is striking as the urinary concentration of homogentisic acid is about 1000 times higher in patients with a genetic defect in homogentisic acid 1,2-dioxygenase (alkaptonuria). Genotoxic and carcinogenic effects of other amino acids and metabolites, reported in the literature, are discussed as well.
It is a widespread prejudice that physiological compounds are non-genotoxic and non-mutagenic, and therefore only very few of them have been experimentally investigated in this respect. Some years ago, we observed pronounced mutagenic effects in Salmonella typhimurium of glutathione, tested in the presence of subcellular kidney preparations, and of L-cysteine, tested in the presence of kidney or liver preparations (Glatt et al., 1983). We then learned that similar mutagenic effects had also been observed by others in L-cysteine (Yamaguchi and Yamashita, 1981). The mutagenicity of these compounds was further in-
Correspondence: Dr. HansruediGlatt, Departmentof Toxicology, Universityof Mainz,Obere ZahlbacherStrasse67, D-6500 Mainz 1 (F.R.G.).
vestigated with regard to metabolic aspects, stereospecificity, responsiveness of various bacterial strains and mammalian cells (Glatt and Oesch, 1985; Ross et al., 1986; Carter and Josephy, 1986; Stark et al., 1987, 1988; Glatt, 1989; Glatt et al., 1990). Here, some findings are reported on the L-cysteine precursor, N-acetyl-L-cysteine. This compound is used as a mucolytic and as an antidote in acetaminophen poisoning. In the latter situation (Prescott and Critchley, 1983), very high doses are used. A typical treatment consists of a loading dose of 140 mg/kg, followed by administration of 70 mg/kg every 4 h for 17 doses. N-Acetyl-L-cysteine probably acts, in part, by replenishing hepatic stores of glutathione, depleted by acetaminophen. N-Acetyl-t-cysteine is more effective than direct administration of L-cysteine or glutathione.
0165-1110/90/$03.50 © 1990 ElsevierSciencePublishersB.V. (BiomedicalDivision)
236 CO0+ I H3N-C-H
Dopamine, Adrenatine, Noradrenaline
C,H2 +
CO0I
H3N-C-H Metanin
OH Dopa OH Tyrosine
~
OH I~CH2-CO0 L
---~
Acetoacetate, + Fumarate
OH Homogentisic acid
Fig. 1. Structures and metabolic relations of tyrosine, dopa and homogentisic acid.
We recently observed mutagenic effects of the benzene metabolites, catechol and p-benzohydroquinone, in V79 Chinese hamster cells (Glatt et al., 1989). These structures are also contained in the tyrosine metabolites, levodopa (L-dopa) and homogentisic acid (Fig. 1). L-Dopa is the physiological metabolic precursor of the catecholamines dopamine, adrenaline and noradrenaline (which are catechols as well) and of the melanins. It is the major drug used in the treatment of parkinsonism. Homogentisic acid is an intermediate in the catabolism of tyrosine. In normal individuals, its concentration is very low, due to efficient further metabolism by homogentisic acid 1,2-dioxygenase. However, in a heritable disorder, alkaptonuria, this enzyme is defective and the patients excrete 4-8 g of homogentisic acid in their urine per day (McKusick, 1972). For these reasons, L-dopa and homogentisic acid have been investigated for mutagenicity in V79 cells and in bacteria. Materials and methods
Test compounds N-Acetyl-L-cysteine and L-dopa were obtained from Sigma (Deisenhofen, F.R.G.), homogentisic acid was purchased from Aldrich (Steinheim, F.R.G.). The compounds were dissolved in water immediately before use. The solutions were adjusted to pH 6-7 by adding dilute NaOH. Bacteria Bacteria were grown overnight in Nutrient broth No. 2 (Oxoid GmbH, Wesel, F.R.G.), 25 g/1. For
inoculation, stock cultures that were stored at - 7 0 °C were used. The overnight cultures were centrifuged, resuspended in medium A (1.6 g Bacto nutrient broth + 5 g NaC1/1) and adjusted nephelometrically to a titer of about 2 × 10 9 bacteria (colony-forming units)/ml. In each experiment the presence of the R-factor pKM 101 was ascertained by growing diluted cultures on ampicillin-containing and ampicillin-free, histidine-supplemented agar plates. With strains TA92, TA97, TA100, TA102 and TA104 the numbers of colonies were always virtually identical under the 2 culture conditions, whereas strains TA1535 and TA1537 grew only in the absence of ampicillin. Tissue preparations Male Sprague-Dawley rats (200-300 g) were obtained from Wiga, Sulzfeld (F.R.G.). The kidneys and livers of animals that had not been deliberately treated with an inducing agent were homogenized in 3 volumes of sterile, cold KCI (150 mM), buffered with 10 mM sodium phosphate, pH 7.4. The homogenate was centrifuged at 10 000 x g for 10 min. The resulting supernatant, termed $9 fraction, was used on the day of preparation or stored at - 7 0 °C until use. Bacterial mutagenicity assay Test compound (in neutralized aqueous solution, 100-200 #1), buffer (as used for preparation of the tissue fraction, 500 ffl), or $9 fraction (equivalent to 100 mg tissue, 333/~1) and cofactor solution (for details see legends to figures), and bacteria (100 #1 of the resuspension) were mixed in a glass tube, and incubated for 20 min (' preincubation assay') or 0 rain ('standard plate-incorporation assay') at 37 °C. Then, 2 ml of 45°C warm soft agar (0.55% agar, 0.55% NaC1, 50 /~M histidine, 50 #M biotin, 25 mM sodium phosphate buffer, pH 7.4) was added, and the mixture was poured on to a Petri dish containing 24 ml minimal agar (1.5% agar in Vogel-Bonner E medium with 2% glucose). After incubation for 3 days in the dark, colonies (his + revertants) were counted. Mammalian cell mutagenicity assay Chinese hamster V79 cells were obtained in 1984 from Dr. G. Turchi (Consiglio Nazionale delle Ricerche, Pisa, Italy). They were cloned in
237
order to eliminate mutants. The modal number of chromosomes of our strain was 22. The cells were free of mycoplasms. They were maintained in Dulbecco's modified minimal essential medium supplemented with 5% fetal calf serum and penicillin/streptomycin (DME-FCS). On day 1 of the mutagenicity test, 1.5 x 106 cells were seeded with 30 ml of medium in 15-cm Petri dishes. After 18 h the test compound was added. 24 h later, the exposure was terminated by a change of the medium. On day 4, the cells were detached by treating with trypsin, counted, and subcultured at a density of 3 x 106 cells per 15-cm Petri dish. On day 8, they were subcultured again at a density of 1 0 6 cells per 15-cm Petri dish in medium containing 6-thioguanine (7 #g/ml) to determine the number of mutants (6 replicate plates). The cloning efficiency was determined by plating 100 cells with 5 ml of 6-thioguanine-free medium in 6-cm Petri dishes (3 replicate plates). The colonies were counted after incubating for about 8 days (cloning efficiency plates) and 10 days (selection plates). Results
Mutagenicity in Salmonella typhimurium For L-cysteine and glutathione the results have been reported previously (Glatt et al., 1983; Glatt and Oesch, 1985; Glatt, 1989). N-Acetyl-L-cysteine was tested in 3 strains (TA92, TA97, TA104), in which L-cysteine and glutathione show strong effects. In the absence of a mammalian metabolizing system, no increases in the number of mutants were observed (at all doses used, 3.75-120 /zmole/plate, numbers of colonies were less than 1.2-fold the value of the solvent controls). However, in the presence of rat kidney $9 fraction pronounced mutagenic effects were found (Fig. 2). TA97 was the most responsive strain, with impressive increases in the number of colonies per plate from 180 in the negative control to 4700 at the highest dose used. Dose-response curve, maximal effect and strain preference were very similar to those reported for t-cysteine and glutathione (Glatt, 1989). In the direct test, L-dopa enhanced the number of colonies about 2-fold with strain TA104 and 1.5-fold with strain TA100 (Fig. 3). These effects were confirmed in repeat experiments (data not
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