Mutation Research, 283 (1992) 7-11
7
© 1992 Elsevier Science Publishers B.V. All rights reserved 0165-7992/92/$05.00
MUTLET 0694
Evaluation of amitrole mutagenicity in Salmonella typhimurium using prostaglandin synthase activation P. Croker, A.M. Bonin and N.H. Stacey Toxicology Unit, National Institute of Occupational Health and Safety (Worksafe Australia), The University of Sydney, Sydney, N.S. Wales 2001, Australia (Received 6 January 1992) (Revision received 30 April 1992) (Accepted 4 May 1992)
Keywords: Amitrole; Metabolic activation; Prostaglandin synthase
Summary Amitrole is a herbicide which has been found to induce thyroid and liver tumours in rodents, yet demonstrates limited genotoxic activity. The lack of mutagenicity of this compound in Salmonella typhimurium when employing a standard liver microsomal fraction, combined with evidence of activation of amitrole by peroxidases, warranted an investigation employing this other pathway of metabolic activation. Using prostaglandin H synthase as the activating system, the aromatic amine 2-aminofluorene provided a con~cenient positive control for optimisation of the metabolising system. Under such conditions, amitrole did not induce elevated numbers of revertant colonies in Salmonella typhimurium TA98, neither did it display evidence of interference with histidine biosynthesis as had been reported. Amitrole also remained nonmutagenic when preincubated at varying pHs. Thus, it has been shown that the alternative activation system, prostaglandin H synthase, does not produce metabolites which are mulagenic in the Ames test.
The herbicide amitrole (3-amino-l,2,4-triazole) is a known goitrogen and thyroid carcinogen in rats and mice when administered for prolonged periods (Steinhoff et al., 1983; Innes et al., 1969). Its mechanism of carcinogenicity has been considered to be due to hormone imbalance and pro-
The views expressed in this article are those of the authors and do not necessarily reflect those of the National Occupational Health and Safety Commission. Correspondence: Dr. Neill Stacey, Toxicology Unit, NIOHS, G.P.O. Box 58, Sydney, NSW 2001, Australia.
longed stimulation of the thyroid gland by thyrotropin, by inhibiting the enzyme thyroid peroxidase (TPO) and hence preventing iodination of tyrosine residues and coupling of the iodinated tyrosines to form thyroid hormone (Alexander, 1959; Strum and Karnovsky, 1971). Consistent with this proposed epigenetic mechanism of carcinogenesis is the observation that amitrole has been negative in a variety of genotoxicity tests with and without hepatic microsomal enzyme activation. In contrast, however, there is also evidence which supports a mode of action based on geno-
toxicity. Krauss and Eling (1987), for example, have shown that amitrole (50 /~M) may be metabolised in vitro by the enzyme prostaglandin H synthase (PHS) to intermediates that bind to nucleic acids and proteins. The implication of PHS's ability to generate genotoxic metabolites of amitrole is further enhanced by the reported induction of cell transformation and gene mutations in cultured Syrian hamster embryo (SHE) cells at concentrations up to 0.1 m g/ m l (Inoue et al., 1981; Tsutsui et al., 1984). These cells have been shown to carry out PHS-mediated metabolism of other compounds including the "hormonal carcinogen" diethylstilboestrol (Degen et al., 1983). PHS, a two-part enzyme system containing cyclooxygenase and peroxidase, has been demonstrated in almost every mammalian tissue, in membrane proteins and in the microsomal fraction of tissue homogenates. The dissimilarity between cytochrome P-450 monooxygenases and PHS with respect to tissue distribution, reactions catalysed and response to inhibitors suggests that PHS could serve as an alternative enzyme for biotransformation or activation of chemicals, particularly in extrahepatic tissues that are low in monooxygenase activity (Eling et al., 1990). PHS has recently been used to activate chemicals to metabolites that are mutagenic in the Ames test (Boyd et al., 1985; Petry et al., 1988, 1989; Josephy, 1989) and it has been suggested by Degen (1989) that PHS should be used as an additional activating system for Salmonella mutagenicity testing, particularly for compounds with extrahepatic carcinogenicity. Thus, it was of interest to investigate the effect of PHS metabolic activation of amitrole in a Salmonella mutagenicity assay because the sensitivity of the thyroid to the effects of amitrole suggests that extrahepatic metabolism may be important in the carcinogenicity of this compound. Materials and methods
Chemicals. Amitrole was obtained from Aldrich Chemical Company, Milwaukee, USA. Hematin was obtained from Sigma Chemical
Company, St. Louis, USA. 2-Aminofluorene (AF) was obtained from ICN Pharmaceuticals, Plainview, NY, USA. PHS (10000 units per ml) was obtained from Cayman Chemical Company, Michigan, USA and stored at -70°C.
Mutagenesis assays with bacteria. TA98 strain of Salmonella typhimurium, used in all experiments, was obtained from Professor Bruce Ames, University of California, Berkeley, USA, and cultured as described (Bonin et al., 1989). The preincubation technique, based on Josephy et al. (1989), was conducted as follows: a 100/zl volume of amitrole at various concentrations (dissolved in water) was added to 500 units of PHS in phosphate buffer (0.5 ml, pH 7.4 unless otherwise specified), hematin (1/zM) and 0.1 ml of bacteria (approximately 2 × 108/ml), and incubated at 37°C. After 30 min, 2 ml of standard histidine/ biotin-supplemented top agar was added to the preincubation mixture, and plated. Duplicate plates at each dose level were incubated at 37°C for 72 h before counting revertant colonies with an Artek model 880 counter. Results
The effect of amitrole on growth of his + and his- cultures o f TA98 were compared (Table 1). His + bacteria are able to grow on minimal agar plates whereas his- strains require exogenous histidine. Top agar, supplemented with biotin but not histidine, was used for the his + strain, while biotin and an excess of histidine were added for
TABLE 1 E F F E C T O F A M I T R O L E O N G R O W T H O F his + A N D h i s - S. typhimurium TA98 Amitrole
TA98 colonies a
(mg/plate)
his-
his +
0 0.1 0.316 1.0 3.16 10
102+ 8 96+ 6 103+ 9 110+ 7 9 4 + 10 102_+ 8
97+ 6 104-1-11 108+ 9 96+ 6 9 9 + 10 1075: 8
a Values are mean + SD, n = 3.
9 TABLE 2
TABLE 4
PHS-ACTIVATED MUTAGENICITY F L U O R E N E IN TA98
OF
PHS (units/plate)
TA98 revertants a
0 100 250 500 750
16_+ 2 107_+4 226_+ 7 311 _+8 423 _+9
a
2-AMINO-
Values are mean-+SD, n = 3.
E F F E C T OF V A R Y I N G pH O N P H S - M E D I A T E D METABOLISM OF A M I T R O L E IN TA98 Amitrole
TA98 revertants a
(rag/plate)
pH 6.4
pH 7.4
pH 8.0
0 1 3.16 10
24 28 30 29
34 36 31 24
26 34 23 17
2-Aminofluorene (20 ~ g / p l a t e )
75
76
84
a Mean of 2 plates.
the his- strain. The bacteria were diluted to an extent where they were countable with accuracy so that growth characteristics could be more readily discerned. Both the his + and h i s - strains grew equally well even in the presence of 10 m g / p l a t e amitrole, compared with untreated control plates. Thus under these conditions, there was no evidence of interference with histidine biosynthesis or cell growth in either strain of bacteria. The PHS-mediated activity of the positive control mutagen, 2AF, is illustrated in Table 2. At a fixed substrate concentration of 10 / x g / p l a t e of 2AF, there was a linear dose-related increase in revertant colonies in response to increasing enzyme concentration. Similar test conditions were then applied for the investigation of the genotoxic potential of amitrole. Enzyme concentration of 500 u n i t s / p l a t e was chosen for metabolic activation as this lay in the upper portion of the d o s e response curve and resulted in about a 20-fold induction of revertants with respect to 2AF. The
TABLE 3 E F F E C T OF PHS A C T I V A T I O N OF A M I T R O L E IN TA98 Amitrole
TA98 revertants a
(mg/plate)
Expt. I
0 1 3.16 10 2-Aminofluorene (10/xg/plate) a
43_+ 40_+ 40_+ 40_+
Expt. II 3 3 2 2
42_+3 43_+4 40_+3 43_+3
358 ± 10
336 -+8
Values are mean + SD, n = 3.
results are shown in Table 3 and even at a concentration of 10 m g / p l a t e no genotoxic or cytotoxic effects were observed, as monitored by examination of lawn colonies under low power light microscopy. Further experiments were conducted to investigate the influence of p H of the preincubation conditions on mutagenicity (Table 4). No adverse effects were noted on bacterial survival but, again, amitrole was nonmutagenic under these conditions. Discussion
Our results on cytotoxic effects of amitrole towards Salmonella typhimurium TA98 are at variance with Bamford et al. (1976) who described an inhibition of bacterial growth with 0.1% amitrole (2 m g / p l a t e ) in Escherichia coli K 1 2 / A and Salmonella typhimurium LT 2 trp A8 by this compound. Indeed, it had also been reported that amitrole interfered with the biosynthesis of histidine (Hilton et al., 1965), which is of greater relevance to this study. We were not able to demonstrate any difference in the growth of his + and his- S. typhimurium TA98 with amitrole even at 10 mg per plate, compared with untreated controls. Thus, the differences in the various studies could be due to the genotypically different strain of S. typhimurium used. The activity of the PHS metabolising system was established using 2AF as an appropriate substrate and produced comparable numbers of revertant colonies as reported by Boyd et al. (1985).
10
The findings of Boyd et al. (1985) and Josephy et al. (1989) using 2AF and PHS were confirmed, thereby indicating that the metabolising system was effective. Genotoxicity may be demonstrated by varying the experimental conditions, for example addition of arachidonic acid or peroxide to the incubation mix. Other variables such as ionic strength, temperature and pH also have a strong influence on enzymatic activity. Although short-term tests for genotoxic activity are usually performed at pH 7.4, the majority of the microsomal cytochrome P-450-dependent monooxygenases and FAD-containing oxygenases have pH optima at higher pH values (Paolini et al., 1988). Varing the pH of the PHS-mediated preincubation mixture to pH 6.4 and 8.0 did not reveal any latent mutagenic potential of amitrole. The reports of cell transformation and gene mutation in cultured SHE ceils induced by amitrole (Tsutsui et al., 1984) and PHS-activated binding of amitrole to protein and nucleic acid (Krauss and Eling, 1987) cannot be ignored. It is possible that in this experimental system, amitrole inactivates the peroxidase enzyme before sufficient reactive metabolite is generated to result in a biologically detectable genotoxic event in the Ames test. Results of a study by Krauss and Eling (1987), using amitrole and peroxidase enzymes were consistent with a mechanism-based (suicide) inactivation of the enzyme by amitrole. In their study PHS was an order of magnitude more active than lactoperoxidase, and two orders of magnitude more active than thyroid peroxidase in mediating binding of amitrole to protein and nucleic acid. Thus, under these conditions amitrole is not mutagenic in the Ames test with PHS activation, consistent with the proposed non-genotoxic mechanism of carcinogenicity. References Alexander, N.M. (1959) Antithyroid action of 3-amino-l,2,4triazole, J. Biol. Chem., 234, 148-150. Bamford D., M. Sorsa, U. Gripenberg, I. Laamanen and T. Meretoja (1976) Mutagenicity and toxicity of amitrole, III. Microbial tests, Mutation Res., 40, 197-202.
Bonin, A.M., C.A. Rosario, C.C. Duke, R.S.U. Baker, A.J. Ryan and G.M. Holder (1989) The mutagenicity of dibenz[a,j]acridine, some metabolites and other derivatives in bacteria and mammalian cells, Carcinogenesis, 10, 1079-1084. Boyd J.A., E. Zeiger and T.E. Eling (1985) The prostaglandin H synthase-dependent activation of 2-aminofluorene to products mutagenic to S. typhimurium strains TA98 and TA98NR, Mutation Res., 143, 187-190. Degen, G.H., A. Wong, T.E. Eling, J.C. Barrett and J.A. McLachlan (1983) Involvement of prostaglandin synthase in peroxidative metabolism of diethylstilbestrol in Syrian hamster embryo fibroblast cell cultures, Cancer Res., 43, 992-996. Eling, T.E., D.C. Thompson, G.L. Foureman, J.F. Curtis and M.F. Hughes (1990) Prostaglandin H synthase and xenobiotic oxidation, Annu. Rev. Pharmacol. Toxicol., 30, 1-45. Hilton, J.L., P.C. Kearney and B.N. Ames (1965) Mode of action of the herbicide, 3-amino-l,2,4-triazole (amitrole): inhibition of an enzyme of histidine biosynthesis, Arch. Biochem. Biophys., 112, 544-547. Innes, J.R.M., B.M. Ulland, M.G. Valerio, L. Petrucelli, L. Fishbein, E.R. Hart, A.J. Pallotta, R.R. Bates, H.L. Falk, J.J. Gart, M. Klein, I. Mitchell and J. Peters (1969) Bioassay of pesticides and industrial chemicals for tumorigenicity in mice: a preliminary note, J. Natl. Cancer Inst., 42, 1101-1114. Inoue, K., Y. Katoh and S. Takayama (1981) In vitro transformation of hamster embryo cells by 3-(N-salicyloyl)amino1,2,4-triazole, Toxicol. Lett., 7, 211-215. Josephy, P.D. (1989) Activation of aromatic amines by prostaglandin H synthase, Free Radical Biology and Medicine, 6, 533-540. Josephy, P.D., A.L.H. Chiu and T.E. Eling (1989) Prostaglandin H synthase-dependent mutagenic activation of benzidine in a Salmonella typhimurium Ames tester strain possessing elevated N-acetyltransferase levels, Cancer Res., 49, 853-856. Krauss, R.S., and T.E. Eling (1987) Macromolecular binding of the thyroid carcinogen 3-amino-l,2,4-triazole (amitrole) catalyzed by prostaglandin H synthase, lactoperoxidase and thyroid peroxidase, Carcinogenesis, 8, 659-664. Paolini, M., E, Sapigni, P. Hrelia, S. Grilli, G. Lattanzi and G. Cantelli-Forti (1988) Improvement of short-term tests for mutagenicity: on the optimal pH for the liver microsomal assay, in press. Perry, T.W., T.E. Eling, A.L.H. Chiu and P.D. Josephy (1988) Ram seminal vesicle microsome-catalyzed activation of benzidine and related compounds: dissociation of mutagenesis from peroxidase-catalyzed formation of DNA-reactive material, Carcinogenesis, 9, 51-57. Petry, T.W., P.D. Josephy, D.A. Pagano, E. Zeiger, K.T. Knecht and T.E. Eling (1989) Prostaglandin hydroperoxidase-dependent activation of heterocyclic aromatic amines, Carcinogenesis, 10, 2201-2207. Steinhoff, D., H. Weber, U. Mohr and K. Boehme (1983) Evaluation of amitrole for potential carcinogenicity in
11 orally dosed rats, mice and golden hamsters, Toxicol. Appl. Pharmacol., 69, 161-169. Strum, J.M., and M.J. Karnovsky (1971) Aminotriazole goitre. Fine structure and localization of thyroid peroxidase activity, Lab. Invest., 24, 1-12. Tsutsui, T., H. Maizumi and J.C. Barrett (1984) Amitrole-in-
duced cell transformation and gene mutations in Syrian hamster embryo cells in culture, Mutation Res., 140, 205207.
Communicated by J.M. Gentile
7
© 1992 Elsevier Science Publishers B.V. All rights reserved 0165-7992/92/$05.00
MUTLET 0694
Evaluation of amitrole mutagenicity in Salmonella typhimurium using prostaglandin synthase activation P. Croker, A.M. Bonin and N.H. Stacey Toxicology Unit, National Institute of Occupational Health and Safety (Worksafe Australia), The University of Sydney, Sydney, N.S. Wales 2001, Australia (Received 6 January 1992) (Revision received 30 April 1992) (Accepted 4 May 1992)
Keywords: Amitrole; Metabolic activation; Prostaglandin synthase
Summary Amitrole is a herbicide which has been found to induce thyroid and liver tumours in rodents, yet demonstrates limited genotoxic activity. The lack of mutagenicity of this compound in Salmonella typhimurium when employing a standard liver microsomal fraction, combined with evidence of activation of amitrole by peroxidases, warranted an investigation employing this other pathway of metabolic activation. Using prostaglandin H synthase as the activating system, the aromatic amine 2-aminofluorene provided a con~cenient positive control for optimisation of the metabolising system. Under such conditions, amitrole did not induce elevated numbers of revertant colonies in Salmonella typhimurium TA98, neither did it display evidence of interference with histidine biosynthesis as had been reported. Amitrole also remained nonmutagenic when preincubated at varying pHs. Thus, it has been shown that the alternative activation system, prostaglandin H synthase, does not produce metabolites which are mulagenic in the Ames test.
The herbicide amitrole (3-amino-l,2,4-triazole) is a known goitrogen and thyroid carcinogen in rats and mice when administered for prolonged periods (Steinhoff et al., 1983; Innes et al., 1969). Its mechanism of carcinogenicity has been considered to be due to hormone imbalance and pro-
The views expressed in this article are those of the authors and do not necessarily reflect those of the National Occupational Health and Safety Commission. Correspondence: Dr. Neill Stacey, Toxicology Unit, NIOHS, G.P.O. Box 58, Sydney, NSW 2001, Australia.
longed stimulation of the thyroid gland by thyrotropin, by inhibiting the enzyme thyroid peroxidase (TPO) and hence preventing iodination of tyrosine residues and coupling of the iodinated tyrosines to form thyroid hormone (Alexander, 1959; Strum and Karnovsky, 1971). Consistent with this proposed epigenetic mechanism of carcinogenesis is the observation that amitrole has been negative in a variety of genotoxicity tests with and without hepatic microsomal enzyme activation. In contrast, however, there is also evidence which supports a mode of action based on geno-
toxicity. Krauss and Eling (1987), for example, have shown that amitrole (50 /~M) may be metabolised in vitro by the enzyme prostaglandin H synthase (PHS) to intermediates that bind to nucleic acids and proteins. The implication of PHS's ability to generate genotoxic metabolites of amitrole is further enhanced by the reported induction of cell transformation and gene mutations in cultured Syrian hamster embryo (SHE) cells at concentrations up to 0.1 m g/ m l (Inoue et al., 1981; Tsutsui et al., 1984). These cells have been shown to carry out PHS-mediated metabolism of other compounds including the "hormonal carcinogen" diethylstilboestrol (Degen et al., 1983). PHS, a two-part enzyme system containing cyclooxygenase and peroxidase, has been demonstrated in almost every mammalian tissue, in membrane proteins and in the microsomal fraction of tissue homogenates. The dissimilarity between cytochrome P-450 monooxygenases and PHS with respect to tissue distribution, reactions catalysed and response to inhibitors suggests that PHS could serve as an alternative enzyme for biotransformation or activation of chemicals, particularly in extrahepatic tissues that are low in monooxygenase activity (Eling et al., 1990). PHS has recently been used to activate chemicals to metabolites that are mutagenic in the Ames test (Boyd et al., 1985; Petry et al., 1988, 1989; Josephy, 1989) and it has been suggested by Degen (1989) that PHS should be used as an additional activating system for Salmonella mutagenicity testing, particularly for compounds with extrahepatic carcinogenicity. Thus, it was of interest to investigate the effect of PHS metabolic activation of amitrole in a Salmonella mutagenicity assay because the sensitivity of the thyroid to the effects of amitrole suggests that extrahepatic metabolism may be important in the carcinogenicity of this compound. Materials and methods
Chemicals. Amitrole was obtained from Aldrich Chemical Company, Milwaukee, USA. Hematin was obtained from Sigma Chemical
Company, St. Louis, USA. 2-Aminofluorene (AF) was obtained from ICN Pharmaceuticals, Plainview, NY, USA. PHS (10000 units per ml) was obtained from Cayman Chemical Company, Michigan, USA and stored at -70°C.
Mutagenesis assays with bacteria. TA98 strain of Salmonella typhimurium, used in all experiments, was obtained from Professor Bruce Ames, University of California, Berkeley, USA, and cultured as described (Bonin et al., 1989). The preincubation technique, based on Josephy et al. (1989), was conducted as follows: a 100/zl volume of amitrole at various concentrations (dissolved in water) was added to 500 units of PHS in phosphate buffer (0.5 ml, pH 7.4 unless otherwise specified), hematin (1/zM) and 0.1 ml of bacteria (approximately 2 × 108/ml), and incubated at 37°C. After 30 min, 2 ml of standard histidine/ biotin-supplemented top agar was added to the preincubation mixture, and plated. Duplicate plates at each dose level were incubated at 37°C for 72 h before counting revertant colonies with an Artek model 880 counter. Results
The effect of amitrole on growth of his + and his- cultures o f TA98 were compared (Table 1). His + bacteria are able to grow on minimal agar plates whereas his- strains require exogenous histidine. Top agar, supplemented with biotin but not histidine, was used for the his + strain, while biotin and an excess of histidine were added for
TABLE 1 E F F E C T O F A M I T R O L E O N G R O W T H O F his + A N D h i s - S. typhimurium TA98 Amitrole
TA98 colonies a
(mg/plate)
his-
his +
0 0.1 0.316 1.0 3.16 10
102+ 8 96+ 6 103+ 9 110+ 7 9 4 + 10 102_+ 8
97+ 6 104-1-11 108+ 9 96+ 6 9 9 + 10 1075: 8
a Values are mean + SD, n = 3.
9 TABLE 2
TABLE 4
PHS-ACTIVATED MUTAGENICITY F L U O R E N E IN TA98
OF
PHS (units/plate)
TA98 revertants a
0 100 250 500 750
16_+ 2 107_+4 226_+ 7 311 _+8 423 _+9
a
2-AMINO-
Values are mean-+SD, n = 3.
E F F E C T OF V A R Y I N G pH O N P H S - M E D I A T E D METABOLISM OF A M I T R O L E IN TA98 Amitrole
TA98 revertants a
(rag/plate)
pH 6.4
pH 7.4
pH 8.0
0 1 3.16 10
24 28 30 29
34 36 31 24
26 34 23 17
2-Aminofluorene (20 ~ g / p l a t e )
75
76
84
a Mean of 2 plates.
the his- strain. The bacteria were diluted to an extent where they were countable with accuracy so that growth characteristics could be more readily discerned. Both the his + and h i s - strains grew equally well even in the presence of 10 m g / p l a t e amitrole, compared with untreated control plates. Thus under these conditions, there was no evidence of interference with histidine biosynthesis or cell growth in either strain of bacteria. The PHS-mediated activity of the positive control mutagen, 2AF, is illustrated in Table 2. At a fixed substrate concentration of 10 / x g / p l a t e of 2AF, there was a linear dose-related increase in revertant colonies in response to increasing enzyme concentration. Similar test conditions were then applied for the investigation of the genotoxic potential of amitrole. Enzyme concentration of 500 u n i t s / p l a t e was chosen for metabolic activation as this lay in the upper portion of the d o s e response curve and resulted in about a 20-fold induction of revertants with respect to 2AF. The
TABLE 3 E F F E C T OF PHS A C T I V A T I O N OF A M I T R O L E IN TA98 Amitrole
TA98 revertants a
(mg/plate)
Expt. I
0 1 3.16 10 2-Aminofluorene (10/xg/plate) a
43_+ 40_+ 40_+ 40_+
Expt. II 3 3 2 2
42_+3 43_+4 40_+3 43_+3
358 ± 10
336 -+8
Values are mean + SD, n = 3.
results are shown in Table 3 and even at a concentration of 10 m g / p l a t e no genotoxic or cytotoxic effects were observed, as monitored by examination of lawn colonies under low power light microscopy. Further experiments were conducted to investigate the influence of p H of the preincubation conditions on mutagenicity (Table 4). No adverse effects were noted on bacterial survival but, again, amitrole was nonmutagenic under these conditions. Discussion
Our results on cytotoxic effects of amitrole towards Salmonella typhimurium TA98 are at variance with Bamford et al. (1976) who described an inhibition of bacterial growth with 0.1% amitrole (2 m g / p l a t e ) in Escherichia coli K 1 2 / A and Salmonella typhimurium LT 2 trp A8 by this compound. Indeed, it had also been reported that amitrole interfered with the biosynthesis of histidine (Hilton et al., 1965), which is of greater relevance to this study. We were not able to demonstrate any difference in the growth of his + and his- S. typhimurium TA98 with amitrole even at 10 mg per plate, compared with untreated controls. Thus, the differences in the various studies could be due to the genotypically different strain of S. typhimurium used. The activity of the PHS metabolising system was established using 2AF as an appropriate substrate and produced comparable numbers of revertant colonies as reported by Boyd et al. (1985).
10
The findings of Boyd et al. (1985) and Josephy et al. (1989) using 2AF and PHS were confirmed, thereby indicating that the metabolising system was effective. Genotoxicity may be demonstrated by varying the experimental conditions, for example addition of arachidonic acid or peroxide to the incubation mix. Other variables such as ionic strength, temperature and pH also have a strong influence on enzymatic activity. Although short-term tests for genotoxic activity are usually performed at pH 7.4, the majority of the microsomal cytochrome P-450-dependent monooxygenases and FAD-containing oxygenases have pH optima at higher pH values (Paolini et al., 1988). Varing the pH of the PHS-mediated preincubation mixture to pH 6.4 and 8.0 did not reveal any latent mutagenic potential of amitrole. The reports of cell transformation and gene mutation in cultured SHE ceils induced by amitrole (Tsutsui et al., 1984) and PHS-activated binding of amitrole to protein and nucleic acid (Krauss and Eling, 1987) cannot be ignored. It is possible that in this experimental system, amitrole inactivates the peroxidase enzyme before sufficient reactive metabolite is generated to result in a biologically detectable genotoxic event in the Ames test. Results of a study by Krauss and Eling (1987), using amitrole and peroxidase enzymes were consistent with a mechanism-based (suicide) inactivation of the enzyme by amitrole. In their study PHS was an order of magnitude more active than lactoperoxidase, and two orders of magnitude more active than thyroid peroxidase in mediating binding of amitrole to protein and nucleic acid. Thus, under these conditions amitrole is not mutagenic in the Ames test with PHS activation, consistent with the proposed non-genotoxic mechanism of carcinogenicity. References Alexander, N.M. (1959) Antithyroid action of 3-amino-l,2,4triazole, J. Biol. Chem., 234, 148-150. Bamford D., M. Sorsa, U. Gripenberg, I. Laamanen and T. Meretoja (1976) Mutagenicity and toxicity of amitrole, III. Microbial tests, Mutation Res., 40, 197-202.
Bonin, A.M., C.A. Rosario, C.C. Duke, R.S.U. Baker, A.J. Ryan and G.M. Holder (1989) The mutagenicity of dibenz[a,j]acridine, some metabolites and other derivatives in bacteria and mammalian cells, Carcinogenesis, 10, 1079-1084. Boyd J.A., E. Zeiger and T.E. Eling (1985) The prostaglandin H synthase-dependent activation of 2-aminofluorene to products mutagenic to S. typhimurium strains TA98 and TA98NR, Mutation Res., 143, 187-190. Degen, G.H., A. Wong, T.E. Eling, J.C. Barrett and J.A. McLachlan (1983) Involvement of prostaglandin synthase in peroxidative metabolism of diethylstilbestrol in Syrian hamster embryo fibroblast cell cultures, Cancer Res., 43, 992-996. Eling, T.E., D.C. Thompson, G.L. Foureman, J.F. Curtis and M.F. Hughes (1990) Prostaglandin H synthase and xenobiotic oxidation, Annu. Rev. Pharmacol. Toxicol., 30, 1-45. Hilton, J.L., P.C. Kearney and B.N. Ames (1965) Mode of action of the herbicide, 3-amino-l,2,4-triazole (amitrole): inhibition of an enzyme of histidine biosynthesis, Arch. Biochem. Biophys., 112, 544-547. Innes, J.R.M., B.M. Ulland, M.G. Valerio, L. Petrucelli, L. Fishbein, E.R. Hart, A.J. Pallotta, R.R. Bates, H.L. Falk, J.J. Gart, M. Klein, I. Mitchell and J. Peters (1969) Bioassay of pesticides and industrial chemicals for tumorigenicity in mice: a preliminary note, J. Natl. Cancer Inst., 42, 1101-1114. Inoue, K., Y. Katoh and S. Takayama (1981) In vitro transformation of hamster embryo cells by 3-(N-salicyloyl)amino1,2,4-triazole, Toxicol. Lett., 7, 211-215. Josephy, P.D. (1989) Activation of aromatic amines by prostaglandin H synthase, Free Radical Biology and Medicine, 6, 533-540. Josephy, P.D., A.L.H. Chiu and T.E. Eling (1989) Prostaglandin H synthase-dependent mutagenic activation of benzidine in a Salmonella typhimurium Ames tester strain possessing elevated N-acetyltransferase levels, Cancer Res., 49, 853-856. Krauss, R.S., and T.E. Eling (1987) Macromolecular binding of the thyroid carcinogen 3-amino-l,2,4-triazole (amitrole) catalyzed by prostaglandin H synthase, lactoperoxidase and thyroid peroxidase, Carcinogenesis, 8, 659-664. Paolini, M., E, Sapigni, P. Hrelia, S. Grilli, G. Lattanzi and G. Cantelli-Forti (1988) Improvement of short-term tests for mutagenicity: on the optimal pH for the liver microsomal assay, in press. Perry, T.W., T.E. Eling, A.L.H. Chiu and P.D. Josephy (1988) Ram seminal vesicle microsome-catalyzed activation of benzidine and related compounds: dissociation of mutagenesis from peroxidase-catalyzed formation of DNA-reactive material, Carcinogenesis, 9, 51-57. Petry, T.W., P.D. Josephy, D.A. Pagano, E. Zeiger, K.T. Knecht and T.E. Eling (1989) Prostaglandin hydroperoxidase-dependent activation of heterocyclic aromatic amines, Carcinogenesis, 10, 2201-2207. Steinhoff, D., H. Weber, U. Mohr and K. Boehme (1983) Evaluation of amitrole for potential carcinogenicity in
11 orally dosed rats, mice and golden hamsters, Toxicol. Appl. Pharmacol., 69, 161-169. Strum, J.M., and M.J. Karnovsky (1971) Aminotriazole goitre. Fine structure and localization of thyroid peroxidase activity, Lab. Invest., 24, 1-12. Tsutsui, T., H. Maizumi and J.C. Barrett (1984) Amitrole-in-
duced cell transformation and gene mutations in Syrian hamster embryo cells in culture, Mutation Res., 140, 205207.
Communicated by J.M. Gentile
