1,8-DNP6

Mutation Research, 144 (1985) 159-163

159

Elsevier

MRLett 0763

M e t a b o l i s m and mutagenesis o f benzidine in Salmonella strains T A 9 8 and T A 9 8 / 1 , 8 - D N P 6

typhimurium

Bryon F. De France, Margaret H. Carter, P. David Josephy, Douglas W. Bryant I and Dennis R. McCalla 1 Guelph- Waterloo Center for Graduate Work in Chemistry, Department of Chemistry and Biochemistry, University of Guelph, Guelph, Ont. N1G 2W1, and IDepartment of Biochemistry, McMaster University, Hamilton, Ont. L8S 4J9 (Canada) (Accepted 5 July 1985)

Benzidine has been identified as a bladder carcinogen in humans and a liver carcinogen in rats, hamsters, guinea pigs and mice (Garner et al., 1984). In the rat, benzidine undergoes extensive biotransformation (Lynn et al., 1984). The identified urinary and biliary metabolites include Nacetylbenzidine (ABZ), and N,N'-diacetylbenzidine (DABZ), hydroxylated derivatives, and a glutathione conjugate of DABZ (ibid.). Benzidine is mutagenic in the Ames test using Salmonella typhimurium strain TA98 and rat liver $9 activation (Ames et al., 1973). Other mammalian activation systems have also been used, such as hamster liver $9 (Phillipson and Ioannides, 1983) and ram seminal vesicle microsomal preparation (Robertson et al., 1983). ABZ and DABZ are more mutagenic than benzidine (Reid et al., 1984; Kennelly et al., 1984). Benzidine mutagenicity is enhanced several-fold by the addition of acetylcoenzyme A to the Ames assay incubation mix (Kennelly et al., 1984). Furthermore, benzidine is metabolized to ABZ and DABZ by rat liver $9 supplemented with acetyl-coenzyme A (ibid.). McCoy et al. have isolated a derivative of Salmonella typhimurium which is resistant to the mutagenicity of 1,8-dinitropyrene, a compound which does not require $9 activation. This strain, TA98/1,8-DNP6 is also resistant to 2-aminofluorene mutagenicity (McCoy et al., 1981, 1983).

The yield of revertant colonies per microgram of 2-aminofluorene was reduced 10-fold in TA98/1,8-DNP6 compared to TA98. By comparison, the response to 1,8-dinitropyrene itself was reduced about 100-fold in the mutant. McCoy et al. ( 1 9 8 1 , 1 9 8 3 ) also showed that TA98/1,8-DNP6 is probably deficient in an enzyme capable of catalyzing the acetylation of aromatic amines and, presumably, arylhydroxylamines. Saito et al. (1983) showed that this enzyme, isolated from TA98, catalyzes the acetylcoenzyme A-dependent binding of the heterocyclic hydroxylamines N-OH-Trp-2 and N-OH-GIu-P-1 to DNA. TA98/I,8-DNP6 strain is deficient in this activity. Recently, these workers have shown that TA98/1,8-DNP6 is less sensitive to mutagenesis by these compounds, and have partially purified the acetylase enzyme (Saito et al., 1985). In this paper, we show that intact cells of Salmonella typhimurium strain TA98 metabolize benzidine to ABZ and DABZ, in the absence of exogenous mammalian enzymes or acetyl-coenzyme A. TA98/I,8-DNP6 is both deficient in this acetylation activity and resistant to benzidine mutagenesis. This suggests that endogenous bacterial metabolism plays a previously unsuspected role in benzidine mutagenesis.

0165-7992/85/$ 03.30 (c') 1985 Elsevier Science Publishers B.V. (Biomedical Division)

160

Methods

Benzidine and DABZ were obtained from Pfaltz and Bauer, Stamford, CN. ABZ was obtained from ICN Pharmaceuticals, Inc., Plainview, NY, ABZ was found to contain about 15070 DABZ, and DABZ was found to contain about 2007o ABZ, as impurities, by H P L C analysis. [14C]Benzidine (18.8 mCi/mmole) was obtained from New England Nuclear, Boston, MA; the radiolabeled benzidine was purified by H P L C using the system described below to a radiochemical purity of >99.9°70. Solutions of mutagens were prepared fresh in DMSO for each mutagenicity experiment. Conditions for the Ames test were as described previously (Josephy et al., 1985), with the following exceptions. Glucose 6-phosphate and N A D P ÷ were purchased from Boehringer-Mannheim Canada, Dorval, P.Q. Male Syrian golden hamsters (100-120 g) were obtained from High Oak Ranches, Goodwood, Ontario. $9 protein concentrations were food to be approximately 40 mg/ml. "In each experiment, the protein concentration was adjusted so that 0.5 ml o f a 10°70 $9 mix contained 1.5 mg protein. The H P L C equipment configuration used was as described previously (Josephy and Iwaniw, 1985), with the addition of a Gilson Model 202 fraction collector. H P L C conditions were as follows: solvent A: 90070 phosphate buffer, 10 mM, pH 7.4/10°70 methanol; solvent B: 10°70 buffer/90070 methanol. The gradient program was as follows: initial conditions, 100070 A, 2 ml/min; linear gradient of flow rate to 6 ml/min from 0 to 2 rain; solvent composition gradient using Waters program No. 5 to 50070 A/50070 B from 2 to 6 min; hold to 8.5 rain; linear gradient of solvent composition to 100070 B at 9.5 rain; hold to 11.5 min; return to initial conditions. T L C was performed using silica gel plates (Type GF, Analtech), in solvent CHCl~/acetone (90/10), developed twice, and zones were scraped for scintillation counting. Fractions from H P L C analysis were counted in Anderson's scintillation cocktail (xylene, PPO,

Triton X-100) using a Packard Minaxi Tri-Carb model 4430 scintillation counter. Salmonella cultures were grown overnight in Oxoid nutrient broth No. 2 (37°C, with shaking) to a density of A65o = 1.0. The cultures were concentrated 10-fold by centrifugation and resuspension in nutrient broth. The incubation mixture (12 ml total volume) contained benzidine (20 #M) made up o f radiolabeled and cold carrier benzidine, total activity 2.5 × 106 cpm, added as a stock solution in DMSO (0.3 ml). The bacteria were incubated at 37°C with shaking. At appropriate times, samples (2 ml) were withdrawn. The incubations were terminated by adding an equal volume of methanol containing unlabeled benzidine (200 #g), ABZ (20 #g), and DABZ (20 #g). (This cold carrier was required to prevent non-enzymatic decomposition of the metabolites during extraction and work-up). The samples were centrifuged (13 750 × g for 15 min), and the supernatants were extracted twice with ether (8 ml). An aliquot of the aqueous layer was taken for scintillation counting. The two ether extracts were pooled, evaporated, and resuspended in H P L C solvent B (0.6 ml). An aliquot (50/zl) was counted to determine total recovered activity, and 200/zl was injected into the H P L C . The UV trace (254 nm) was used to mark the elution of the benzidine, ABZ, and DABZ standards, and the eluate was collected in 5.1 l-sec fractions for scintillation counting. Results

Benzidine was metabolized by Salmonella typhimurium strain TA98 to two major products (Fig. 1A). These metabolites were identified as ABZ and DABZ, based on co-chromatography with authentic standards in the H P L C system and in the TLC system (data not shown). 1°70 of the initial radioactivity was found to remain in the aqueous phase at zero time, increasing to approximately 807o by 24 h incubation (Fig. 2A), presumably due to formation of unidentified polar metabolites. The levels of ABZ and DABZ increased with time of incubation (Fig. 3A). TA98/1,8-DNP6, in contrast, yielded no detec-

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ed with T A 9 8 (Fig. 2B). We studied the relative sensitivities of TA98 a n d T A 9 8 / I , 8 - D N P 6 to mutagenesis by benzidine, A B Z , a n d D A B Z (Figs. 4 a n d 5). T h e h a m s t e r liver $9 a c t i v a t i o n system was used in these studies.

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Fig. 4. Mutagenicity of benzidine. Ames test was performed as described in text, using Salmonella typhimuriurn strains TA98 (open bar) and TA98/I,8-DNP6 (hatched bar).

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Dose (nmol per plate) Fig. 5. Mutagenicityof ABZ and DABZ. At both doses, data for the two strains are shown superimposed. Left bar: ABZ - TA98 (open bar) and TA98/1,8-DNP6 (cross-hatched). Right bar: DABZ- TA98 (open bar) and TA98/1,8-DNP6 (solid bar).

that this is a more general phenomenon than has been appreciated. One might expect the acetylation capacity of the $9 fraction to exceed that of the bacteria themselves; however, the studies of Kennelly et al. (1984) suggest that the availability of acetyl-coenzyme A limits the rate of acetylation by the $9 fraction; indeed, no detectable acetylation by the $9 occurred in the absence of exogenous acetyl-coenzyme A. Furthermore, in the presence o f acetyl-coenzyme A there was no detectable level o f DABZ. Thus, under the standard conditions of the Ames assay, the bacterial enzyme probably predominates. The role of this bacterial enzyme in mutagenesis must be not only the conversion of benzidine to ABZ and DABZ, but also the subsequent steps in activation, since the response of the mutant strain to all 3 compounds is similarly reduced.

Discussion Acknowledgments The possible role of bacterial metabolism in benzidine mutagenesis has been given little attention, because benzidine mutagenesis in the standard Ames assay is dependent upon the addition of $9 fraction or other mammalian activation system. However, in a modified Ames test involving incubation at low pH and addition of H202, benzidine mutagenesis is detected in the absence o f exogenous activation systems (Josephy and Subden, 1984). This phenomenon may reflect the role of an endogenous bacterial peroxidase, supporting the peroxidase-dependent oxidative activation o f ben° zidine (Josephy et al., 1983). Recent work by Ames and colleagues has demonstrated the importance of endogenous peroxidases as defenses against oxidative stress in Salmonella typhimurium (Christman et al., 1985). We undertook the present investigation in an attempt to detect the metabolism of benzidine by a peroxidase activity in Salmonella typhimurium; however, the major metabolites in strain TA98 proved to be acetylated derivatives of benzidine. The relative resistance of Salmonella typhimurium strain TA98/I,8-DNP6 to arylamine mutagenicity has been observed previously (McCoy et al., 1983). The present work suggests

This work was supported by the Natural Sciences and Engineering Research Council of Canada and the National Cancer Institute of Canada.

Note added in proof Further studies have shown that at least part of the non-organic-extractable radioactivity (Fig. 2) is due to non-enzymatic decomposition of benzidine at 37°C. The heat lability and possible nonenzymatic decomposition of benzidine during extraction and work-up have been noted elsewhere (Riggin, R.M., and C.C. Howard (1979) Determination of benzidine, dichlorobenzidine, and diphenylhydrazine in aqueous media by high performance liquid chromatography, Anal. Chem., 51, 210-214).

References Ames, B.N., W.E. Durston, E. Yamasaki and F.D. Lee (1973) Carcinogens are mutagens: A simple test system combining liver homogenates for activation and bacteria for detection, Proc. Natl. Acad. Sci. (U.S.A.), 70, 2281-2285. Christman, M.F., R.W. Morgan, F.S. Jacobson and

163 B.N. Ames (1985) Positive control of a regulon for defences against oxidative stress and some heat shock proteins in Salmonella typhimurium, Cell, in press. Garner, R.C., C.N. Martin and D.B. Clayson (1984) Carcinogenic aromatic amines and related compounds, in: C.E. Searle (Ed.), Chemical Carcinogens, 2nd Edn., Vol. 1, American Chemical Society, Washington, DC, pp. 175-277. Josephy, P.D., and D.C. lwaniw (1985) Identification of the Nacetylcysteine conjugate of benzidine formed in the peroxidase activation system, Carcinogenesis, 6, 155-158. Josephy, P.D., and R.E. Subden (1984) Hydrogen peroxidedependent activation of benzidine to mutagenic species, Mutation Res., 141, 23-28. Josephy, P.D., T.E. Eling and R.P. Mason (1983) Cooxidation of benzidine by prostaglandin synthase and comparison with the action of horseradish peroxidase, J. Biol. Chem., 258, 5561-5569. Josephy, P.D., M.H. Carter and M.T. Goldberg (1985) Inhibition of benzidine mutagenesis by nucleophiles: a study using the Ames test with hamster hepatic $9 activation, Mutation Res., 143, 5-10. Kennelly, J.C., C.A. Stanton and C.N. Martin (1984) The effect of acetyl-CoA supplementation on the mutagenicity of benzidines in the Ames assay, Mutation Res., 137, 39-45. Lynn, R.K., C.T. Garvie-Gould, D. Milam, K.F. Scott, C.L. Eastman, A.M. Ilias and R.M. Rodgers (1984) Disposition of the aromatic amine, benzidine, in the rat: Characterization of mutagenic urinary and biliary metabolites, Toxicol. Appl. Pharmacol. 72, 1-14. Maron, D.M., and B.N. Ames (1983) Revised methods for the Salmonella mutagenicity test, Mutation Res., 113, 173-215.

McCoy, E.C., H.S. Rosenkranz and R. Mermelstein (1981) Evidence for the existence of a family of bacterial nitroreductases capable of activating nitrated polycyclics to mutagens, Environ. Mutagen., 3, 421-427. McCoy, C., M. Anders and H.S. Rosenkranz (1983) The basis of the insensitivity of Salmonella typhimurium strain TA98/1,8-DNP6 to the mutagenic action of nitroarenes, Mutation Res., 121, 17-23. Phillipson, C.E., and C. loannides (1983) Activation of aromatic amines to mutagens by various animals species including man, Mutation Res., 124, 325-336. Reid, T.M., C.Y. Wang, C.M. King and K.C. Morton (1984) Mutagenicity of some benzidine congeners and their Nacetylated and N,N'-diacetylated derivatives in different strains of Salmonella typhimurium, Environ. Mutagen., 6, 145-151. Robertson, 1.G.C., K. Sivarajah, T.E. Eling and E. Zeiger (1983) Activation of some aromatic amines to mutagenic products by prostaglandin endoperoxide synthetase, Cancer Res., 43, 476-480. Saito, K.,~ Y. Yamazoe, T. Kamataki and R. Kato (1983) Mechanism of activation of proximate mutagens in Ames' tester strains: The acetyl-CoA dependent enzyme in Salmonella typhimurium TA98 deficient in TA98/I,8-DNP6 catalyzes DNA-binding as the cause of mutagenicity, Biochem. Biophys. Res. Commun., 116, 141-147. Saito, K., A. Shinohara, T. Kamataki and R. Kato (1985) Metabolic activation of mutagenic N-hydroxyarylamines by O-acetyltransferase in Salmonella typhimurium TA98, Arch. Biochem. Biophys., 239, 286-295.