Influence of conjugation reactions on the mutagenicity of aromatic amines

Mutation Research, 107 (1983) 239-247 Elsevier Biomedical Press

239

Influence of conjugation reactions on the mutagenicity of aromatic amines Jan Hongslo a, Lise Timm Haug b, Peter J. Wirth c, Mona Moiler Erik Dybing b,, and Snorri S. Thorgeirsson c

a,

a Central Institute oflndustrial Research, P.O. Box 350, Blindern, Oslo 3 (Norway), b Department of Toxicology, National Institute of Public Health, Postuttak, Oslo I (Norway), "Laboratory of Carcinogen Metabolism, National Cancer Institute, Bethesda, MD 20205 (U.S.A.)

(Received 25 May 1982) (Revision received 23 August 1982) (Accepted 25 August 1982)

Summary 2-Acetylaminofluorene (AAF) and 2-aminofluorene (AF), as well as their N-hydroxylated metabolites, N-OH-AAF and N-OH-AF, were studied for mutagenic effects in Salmonella typhimurium with rat- and mouse-liver $9 and microsomal subfractions in the presence of cofactors for glucuronidation and glutathione (GSH) transfer. Addition of UDPGA did not affect the mutagenicity of AAF, AF or N-OH-AAF under any experimental condition. Addition of GSH, on the other hand, markedly inhibited AAF, AF and N-OH-AAF. This seemed to be due to the direct effect of GSH, and not through an enzyme-catalyzed conjugation. Further, GSH inhibited the direct mutagenicity of N-OH-AF.

The Salmonella typhimurium reverse mutation assay (Ames et al., 1975) has enjoyed wide popularity as a screening system for identifying potential mammalian carcinogens and/or mutagens. Atfempts have also been made to correlate mutagenic potency in this in vitro test with carcinogenic potency in vivo (Meselson and Russell, 1977; Ames and Hooper, 1978). However, it seems unreasonable that such a simplistic correlation should exist when the highly artificial conditions of the Salmonella test are compared with the complex events taking place in a mammalian * Address for correspondence. Abbreviations: AAF, 2-acetylaminofluorene; AF, 2-aminofluorene; N-OH-AAF, N-hydroxy-2acetylaminofluorene; N-OH-AF, N-hydroxy-2-aminofluorene; GSH, glutathione; UDPGA, uridine diphosphoglucuronic acid; MC, 3-methylcholanthrene; $9, 9000 × g supernatant fraction.

0027-5107/83/0000-0000/$03.00 © Elsevier Biomedical Press

240 organism. One of the differences between the test system in vitro and the situation in vivo is the relative role of competing activation versus detoxication pathways. In the mutation assay, only cofactors for the polysubstrate mono-oxygenases are added, whereas endogenous levels of cofactors for e.g. glucuronidation and GSH conjugation are markedly diluted and not re-added. Further, enzyme preparations are usually made from animals maximally induced with substances such as PCBs, which may lead to both quantitative and qualitative differences in metabolic pathways compared with uninduced animals in carcinogen bioassays. There is obviously also a difference in the handling of a carcinogen in vivo if this occurs under conditions of first-order kinetics compared with an experiment in vitro where zero-order kinetics may prevail. To assess the role of conjugation reactions in the activation/detoxication of premutagens in the Salmonella test, cofactors for such reactions may be added. In this investigation, the effect of cofactors for glucuronide and GSH transfer on the mutagenicity of the model aromatic amines A A F and AF, as well as their proximate mutagenic and carcinogenic metabolites, N-OH-AAF and N-OH-AF, were studied. These phase-II metabolic reactions are generally involved in detoxication pathways, but may in certain instances convert xenobiotics to electrophilic products (Irving, 1971; Rannug et al., 1978; van Bladeren et al., 1979).

Materials and methods

Materials. AAF, AF, NADP, glucose 6-phosphate and glucose-6-phosphate dehydrogenase were purchased from Koch-Light Laboratories (Colnbrook, UK), and 4-nitrophenol was obtained from The Norwegian Medicinal Depot. N-OH-AAF and N-OH-AF were synthesized according to published procedures (Lotlikar et al., 1965). UDPGA, UDP N-acetylglucosamine and GSH were obtained from Sigma (St. Louis, MO, USA), and MC from Ferak AG (Berlin, FRG). The Salmonella typhimurium strain TA98 was kindly provided by Dr. Bruce N. Ames, University of California (Berkeley, CA, USA). Animals. Male Wistar rats (150-200 g) and male C57/BL/6 mice (20-25 g) were obtained from Molleghrd Breeding and Research Centre (Ejby, Denmark). They were pretreated with MC (80 mg/kg) in corn oil intraperitoneally 40 h before death. Preparation of liver subfractions, Liver from 2 rats or 20 mice were pooled and homogenized in 2 vol. of sterile, ice-cold 20 mM Tris buffer, pH 7.4, containing 1.15% KC1, in a Teflon-glass homogenizer. Liver $9, microsomes and 105000 × g supernatant fraction were prepared as previously described (Dybing and Thorgeirsson, 1977). Supernatant fractions were frozen as such whereas microsomes were frozen in 30% glycerol-Tris-KC1 buffer, and kept at - 8 0 ° C until use. Assays. The mutagenicity of AAF, AF and N-OH-AAF was assayed in vitro in the Salmonella plate test system of Ames et al. (1975) with various concentrations of protein and test substances in the presence of NADPH-generating cofactors as previously described. In some experiments, the Salmonella pre-incubation test of

241

Matsushima et al. (1980) was used as described (Aune et al., 1980). The mutagenicity of N-OH-AF was tested directly with bacteria, with or without protein and N A D P H cofactors. Protein concentrations were determined by the method of Lowry et al. (1951), whereas GSH was measured by the method of Tietze (1969). U DP glucuronyl transferase activity in native microsomes was determined with 4-nitrophenol as substrate (Winsnes, 1969).

Results

Effects of UDPGA addition on the mutagenicity of aromatic amines Experiments performed to assess the role of glucuronidation in the activation of the aromatic amines AAF, AF and N-OH-AAF to mutagens were carried out under 3 different experimental conditions. The test substances were either plated together with Salmonella typhimurium TA98 in the presence of liver $9 or microsomal subfractions plus N A D P H cofactors (plate test) or they were pre-incubated with bacteria and liver $9 plus cofactors before the plating (pre-incubation test). These systems were then assayed in the presence or absence of U D P G A with liver $9 or microsomes from mice and rats (Figs. 1 and 2). None of the various experimental conditions revealed any marked effect of U D P G A addition. These experiments were carried out with a U D P G A concentration of 4 mM. Raising the U D P G A concentration to 8 mM did not change this picture. Some experiments were performed with

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Fig. 1. Effects of UDPGA and GSH on mutagenicity of AF, AAF and N-OH-AAF with mouse liver subfractions. Mutagenicity was assayed with S. typhimurium TA98 and 1.0 mg $9 protein ( A - F ) or 0.5 mg microsomal protein from MC-pretreated mouse liver and N A D P H cofactors in the plate test (A-C, G - I ) or in the pre-incubation test ( D - F ) without (e), with 4 mM UDPGA (It) or with 10 mM GSH (-). Each point is the mean of duplicate estimations from a typical experiment with pooled fractions from 2 rats.

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Fig. 2. Effect of UDPGA and GSH on mutagenicity of AF, AAF and N-OH-AAF with rat-liver subfractions. Mutagenicity was assayed with S. typhimurium TA98 and 1.0 mg $9 protein ( A - F ) or 0.5 mg microsomal protein ( G - I ) from MC-pretreated rat liver and N A D P H cofactors in the plate test (A-C, G - I ) or in the pre-incubation test ( D - F ) without (e), with 4 mM U D P G A ( I ) or with 10 mM GSH (-). Each point is the mean of duplicate estimations from a typical experiment with pooled fractions from 2 rats.

the UDP-glucuronyl transferase activator UDP-N-acetylglucosamine (Winsnes, 1969), which has been used in similar mutagenicity experiments with benzo[a]pyrene (Bock et al., 1981). However, the activator inhibited the conversion of AAF to mutagens (data not shown). It is readily apparent that mouse-liver preparations were much more active than rat-liver preparations in activating the aromatic amines, especially with respect to AAF and N-OH-AAF. No qualitative differences were seen between the 3 different test conditions. Glucuronidation may not only affect aromatic amine mutagenicity through detoxifying pathways, but mutagenicity might be enhanced through formation of a TABLE 1 E F F E C T OF PARAOXON A N D U D P G A ON N - O H - A A F M U T A G E N I C I T Y SALMONELLA PLATE TEST WITH RAT- A N D MOUSE-LIVER SUBFRACTIONS Species

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Fig. 3. Effect of GSH on mutagenicityof N-OH-AF. Mutagenicitywas assayed with S. typhimuriumTA98 without (A, C), or in the presence of 1.0 mg $9 protein from MC-pretreated mouse liver and NADPH cofactors (B, D) in the plate test with (C, D) or without (A, B) 10 mM GSH. Each point is the mean of duplicate estimations from 2 Expts.

reactive glucuronide of hydroxamic acid (Irving, 1971). N - O H - A A F was therefore tested with or without paraoxon, a potent inhibitor of both N - O H - A A F deacetylase and N - O H - A A F mutagenicity (Schut et al., 1978) in combination with U D P G A (Table 1). There was no increase in N - O H - A A F mutagenicity in the presence of paraoxon plus U D P G A compared with that in the presence of paraoxon alone; in fact a small decrease was found, with both rat- and mouse-liver preparations. Paraoxon had only a minimal effect (9-179~ inhibition) on 4-nitrophenol glucuronidation with rat- and mouse-liver microsomes.

Effects of GSH addition on the mutagenicity of aromatic amines In contrast to the lack of effect by the cofactor for glucuronidation, addition of G S H led to a considerable inhibition of aromatic amine mutagenicity (Figs. 1-3). This occurred under most experimental conditions, i.e. with all substrates, A A F and AF, as well as their N-hydroxylated metabolites, and both with $9 and microsomes from mouse, as well as rat liver; the only exception being that G S H had little or no effect on N - O H - A A F mutagenicity with rat-liver $9 (Fig. 2C, F). Of interest is that G S H affected the mutagenicity of AAF, AF and N-OH-AAF activated by the microsomal fraction to a similar extent as with the $9 (Fig. I G - I versus A - F and Fig. 2 G - I vs. A - F ) . The endogenous levels of G S H in the S9s from mouse and rat liver were in the order of 50-60 n m o l e s / m g protein and 30-40 n m o l e s / m g protein, respectively. In separate experiments, various amounts of G S H gave a clear concentration-de-

244 pendent response (data not shown). N-OH-AF, the presumptive proximate mutagenic species of AAF, N-OH-AAF and AF in the Salmonella test system, which is mutagenic without the addition of an activating system (McCann et al., 1975), was also markedly less mutagenic in the presence of GSH (Fig. 3). Also, the addition of an activating system reduced N-OH-AF mutagenicity.

Discussion

The major pathway for activation of AAF to a mutagen in the Salmonella test involves an initial N-hydroxylation by the microsomal cytochrome P-450 monooxygenase system and a subsequent microsomal deacetylation, thereby forming N-OH-AF (Felton et al., 1976; Schut et al., 1978; Weeks et al., 1978). Mutagenic activation of AF presumably only needs a one-step N-hydroxylation to produce the directly mutagenic hydroxylamine. The final, chemical conversion to the ultimate electrophilic species presumably occurs inside the bacteria (Schut et al., 1978; Weeks et al., 1978). Other pathways for N-OH-AAF mutagenicity in vitro include activation by a cytosolic acyltransferase (Weeks et al., 1978; Wirth and Thorgeirsson, 1981) and by an unknown non-acyltransferase cytosolic enzyme (Kaneda et al., 1981). Glucuronidation might influence the mutagenic activation of the aromatic amines in several ways. Firstly, glucuronides are major urinary excretion products of N- as well as C-hydroxylated metabolites (Weisburger et al., 1956). Secondly, the glucuronide of N-OH-AAF is moderately electrophilic (Irving, 1971). In the present study, however, glucuronidation did not seem to affect the mutagenic activation of AAF, AF or N-OH-AAF (Figs. 1 and 2). Addition of UDPGA to the $9 or microsomal activation systems could increase the detoxication of the proximate mutagenic species or channel more of the parent compounds through non-mutagenic ring hydroxylations, if non-saturating conditions prevail. The K m of N-hydroxylation of AAF and AF in the rat has been reported to be 4.4E-6M and 0.56E-6M, and in the mouse 1.6E-6M and 1,0E-6M, respectively (Razzouk et al., 1980; Razzouk and Roberfroid, 1982). Because of the methodology, the exact mutagen concentration in the plate assay is not known. Paraoxon effectively blocks the major pathway of AAF mutagenic activation after either oxidation to N-OH-AAF or deacetylation to AF (Schut et al., 1978). Under such inhibitory conditions, any small mutagenic effect of an N-OH-AAF glucuronide should presumably have been revealed. No such effect was observed (Table 1). Conjugation with GSH, either directly or indirectly via GSH S-transferases, is an important defense mechanism against many electrophilic and thereby potentially toxic metabolites. GSH conjugates of AAF have recently been identified (Meerman et al., 1982). Further, AAF administration to mice does not deplete liver GSH levels (Thorgeirsson and Nebert, 1977). Of interest is that AAF and AF are not substrates for the GSH S-transferases (Ketterer, 1982). In the present experiments, GSH clearly protects against the mutagenicity of AAF, AF and N-OH-AAF (Figs. 1-3). This seems to be due to the direct, nucleophilic activity of GSH, rather than through an enzyme-catalyzed conjugation, because GSH inhibits N-OH-AF mutagenicity in

245 the absence of an activation system and because G S H inhibits AAF, AF and N - O H - A A F mutagenicity when microsomes are used as source of activating enzymes. Similar inhibitory effects have been reported with respect to trans-4-acetylaminostilbene mutagenicity (Glatt et al., 1980). In this situation, conjugation via G S H S-transferases seemed to be necessary for the inhibitory effect. The present experiments, however, do not answer the question whether cellular G S H is an important modifier of genotoxic effects of the aromatic amines in an intact cellular system. In a mutagenic activation system with intact, isolated hepatocytes, depletion of cellular G S H did not affect the mutagenic activation of N - O H - A A F (unpublished results). In the Salmonella test, only cofactors for the cytochrome P-450 systems are added, since these enzymes are those mostly involved in the conversion of premutagens to mutagens. However, this may create an artificial situation compared with conditions in Vivo, from both a quantitative and a qualitative viewpoint. Further, for some mutagens, other pathways than mono-oxygenation are involved: non-cytochrome P-450-mediated reduction (Sugimura et al., 1979), hydrolysis (Schut et al., 1978) and conjugation reactions (Rannug et al., 1978; van Bladeren et al., 1979) participate in metabolic activation. One way to assess the role of other metabolic pathways for mutagen activation is to add lacking cofactors to the Salmonella test system, as was done in the present study. Another possibility in the study of this problem is to use a whole cell system of isolated hepatocytes for activation, as has been reported in several instances (Dybing et al., 1979; Polley et al., 1980; Glatt et al., 1981). For some classes of chemical carcinogens, there seems to be a better correlation between mutagenic and carcinogenic effects when mutagenicity results from the whole cell system are used rather than those from the subcellular system of the Salmonella test (Polley et al., 1980; Glatt et al., 1981). Further studies o.f aromatic amine mutagenicity in a hepatocyte system may thus prove of value in assessing the role of competing metabolic pathways; such studies have been initiated (Holme and Dybing, 1982).

Acknowledgement This study was supported by grants from the Royal Norwegian Council for Scientific and Industrial Research.

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