235

Mutation Research, 259 (1991) 235-250 © 1991 Elsevier Science Publishers B.V. 0165-1218/91/$03.50 ADONIS 016512189100061M MUTGEN 00034

Mammalian cell mutagenicity and metabolism of heterocyclic aromatic amines Hans-Ulrich Aeschbacher and Robert J. Turesky Nestl$ Research Centre, CH-IO00 Lausanne 26 (Switzerland) (Received 15 March 1990) (Accepted 17 April 1990)

Keywords: Heterocyclic aromatic amines; Genotoxicity; Metabolism; N-Oxidation; Adduct formation; Bacterial transformation

Contents Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . DNA damage in mammalian ceils in vitro and in vivo . . . . . . . . . . . . ....................................... Gene mutations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ................... Chromosome aberrations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Nuclear aberrations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sister-chromatid exchanges . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . DNA-repair synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . DNA-strand breaks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Oncogene activation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Monooxygenase and peroxidase metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . DNA adduct formation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Protein adduct formation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . In vivo metabolism and biodisposition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Biotransformation in the digestive tract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Human studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

235 236 236 236 237 237 237 238 238 239 239 239 240 242 242 243 244 244

Summary Heterocyclic aromatic amines are bacterial mutagens which also induce D N A damage in mammalian cells. Damage has been demonstrated using a number of endpoints, including gene mutation, chromosome aberrations, sister-chromatid exchange, DNA-strand breaks, DNA repair and oncogene activation.

Correspondence: Dr. H.-U. Aeschbacher, Nestl6 Research Center, P.O. Box 44, Vers-chez-les-Blanc, CH-1000 Lausanne 26 (Switzerland).

Abbreviations: Heterocyclic amine nomenclature: Trp-P-1, 3amino-l,4-dimethyl-5H-pyrido[4,3-b]indole; Trp-P-2, 3-amino1-methyl-5H-pyrido[4,3-b]indole; Glu-P-1, 2-amlno-6-methyldipyrido[1,2-a : 3',2'-d]imidazole; GIu-P-2, 2-aminodipyrido[1,2-a : 3',2'-d]imidazole; AaC, 2-amino-9H-pyrido[2,3-b]indole; MeAaC, 2-amino-3-methyl-gH-pyrido[2,3-b]indole; IQ,

2-amino-3-methylimidazo[4,5-f]quinoline; MelQ, 2-amino-3,4dimethylimidazo[4,5-f]quinoline; MelQx, 2-amino-3,g-dimethylimidazo[4,5-f]quinoxaline; PhlP, 2-amino-l-methyl-6-phen. ylimidazo[4,5-b]pyridine; other compounds: DMABP, 3,2'dimethyl-4-aminobiphenyl; 4-ABP, 4-aminobiphenyl; BNF, B-naphthoflavone; HPRT, hypoxanthine-guanine phosphoribosyl transferase; APRT, adenosine phosphoribosyl transferase; DCNP, 2,6-dichloro-4-nitrophenol; PCP, pentachlorophenoL

236

Although the responses in mammalian cells are weak when compared to bacterial mutagenicity, heterocyclic aromatic amines are rodent carcinogens. Metabolic N-oxidation by cytochrome P450 is an initial activation step with subsequent transformation of the N-hydroxy metabolites to the ultimate mutagenic species by O-acetyltransferase or sulfotransferase. Major routes of detoxification include cytochrome P450-mediated ring oxidation followed by conjugation to glucuronic or sulfuric acid. Direct conjugation to the exocyclic amine group also occurs. Major reactions include N-glucuronidation and sulfamate formation.

Introduction

In the past 10 years a number of heterocyclic aromatic amines have been isolated from heated meat and fish products and been found to be potent bacterial mutagens (Sugimura, 1982; Felton et al., 1986). A number of these compounds have been shown to be multisite rodent carcinogens (Sugimura, 1988; Esumi et al., 1989; Sugimura et al., 1990; Sugimura, present issue) and recently one of these chemicals, 2-amino-3methyfimidazo[4,5-f]quinoline was also found to induce hepatocellular carcinoma in monkeys (Adamson et al., 1990). A number of mammalian cell mutagenicity tests and whole-animal assays have been used to evaluate the genotoxicity of these chemicals but have given conflicting results partly due to the different metabolic activation systems and endpoints used for evaluation. Most of these amines are multipotential carcinogens in rodent bioassays (Sugimura et al., 1990). However the daily doses administered were extraordinarily high, at least a million times greater than that to which humans are chronically exposed, which makes an extrapolation of the actual risk to humans difficult. As is the case with many carcinogens, heterocyclic aromatic amines must be rrietabolically activated in order to exert genotoxicity. The initial activation step is N-oxidation by cytochrome P450. The liver is the most active tissue in transforming these compounds to mutagenic agents as demonstrated in a number of species including rat, mouse, rabbit, guinea pig, hamster, monkey and man (Kato et al., 1983; Kato, 1986; Yamazoe et al., 1988; Shimada et al., 1988, 1989; McManus et al., 1988, 1989; Butler et al., 1989). The goal of this review is to present the current knowledge on mutagenic damage in mammalian cells and the metabolic activation and detoxification processes which occur both in vivo and in

vitro for these food-related mutagens and potential human carcinogens. DNA damage in mammalian cells in vitro and in vivo

Two groups of food-borne carcinogens, the protein pyrolysates (Trp-P-2, Trp-P-1, Glu-P-1, Glu-P-2, AaC and MeAaC) and aminoimidazoazaarenes (IQ, MelQ, MelQx and PhlP), have been studied using mammalian cell culture systems. In these experiments primary cultures of rodent hepatocytes, human lymphocytes or CHO cells served as target cells. In many instances $9 was used as an exogenous metabolic activation system. The genotoxic effects of the compounds described by different groups remain difficult to evaluate due to variable test conditions employed. Mammalian tissues from different origins were used for metabolic activation and animal pretreatments with enzyme inducers were not necessarily the same (Takayama et al., 1983; Nakayasu et al., 1983; Thompson et al., 1983, 1987; Barnes et al., 1985; Loury et al., 1985; Holme et al., 1987; Schmuck et al., 1988; Aeschbacher et al., 1989). The differences in these enzyme preparations and their ability to generate the ultimate mutagenic/ carcinogenic species most likely lead to profound differences in the outcome of the experiments. In addition, hepatocytes isolated from different rodent species led to different results implying that the species had different metabolizing capacity (Loury et al., 1985; Yoshimi et al., 1988). Other factors, such as media, incubation conditions and dose ranges of test compounds were not standardized and may have contributed to the variables of these studies (see IARC, 1986). Gene mutations

Heterocyclic amines showed a considerably lower capacity to induce gene mutations in roam-

237 malian cells than in bacteria (Thompson et al., 1983; Felton et al., 1988; Aeschbacher et al., 1989). Trp-P-2 and PhlP were significantly mutagenic in CHO cells (HPRT and APRT loci) following activation with hamster liver $9 whereas IQ, MelQ and MelQx had unexpectedly weak activity and produced definite responses only in repair-deficient cells (Thompson et al., 1983, 1987). When mutations at the HPRT and APRT loci were compared for IQ and Trp-P-2, the estimated levels of adducts and mutagenic efficiency of the adducts for both compounds were found to be the same (Brookman et al., 1985). From these results it was concluded that IQ was a weak mutagen in this system because the reactive metabolite(s) produced extracellularly either did not reach or did not react efficiently with the DNA of CHO cells. Despite the high sensitivity of CHO cells deficient in DNA repair to detect DNA damage, heterocyclic amines did not induce strong responses when co-cultured with hepatocytes obtained from PCB-treated rats (Holme et al., 1987) or with Syrian hamster embryo cells (Takayama et al., 1983). There was no significant induction of gene mutations in Chinese hamster V79 cells by heterocyclic amines with the exception of Trp-P-2 (Takayama et al., 1983). Trp-P-2 also strongly induced mutations in a forward-mutation test system (diphtheria toxin resistance) using hamster lung ceils, whereas Trp-P-1, MelQx, Glu-P-1 and Glu-P-2 only caused a relatively weak response (Nakayasu et al., 1983). These in vitro test results demonstrate that protein pyrolysis mutagens, in particular Trp-P-2, can readily induce point mutations in different cell systems with different endpoints whereas the 'IQ' compounds consistently produced negative or weak effects in such systems. These observations were confirmed in vivo by Wild et al. (1985) who showed that IQ did not induce point mutations (coat pigmentation in mice) whereas Trp-P-2 and Glu-P-1 significantly induced point mutations (Jensen, 1983). It could be argued that the negative or weak effect of heterocyclic amines in mammalian cells could be attributed to the inability of the cells to detect 'frameshift' mutations as might be the case for ouabain resistance (Takayama et al., 1983). However, thioguanine and diphtheria toxin resistance (Nakayasu et al., 1983; Holme et al., 1987) are likely to detect 'frameshift' mutations since they are considered appropriate markers (Gupta et al., 1980).

Chromosome aberrations The results reported in the literature demonstrate that IQ, MeIQ and MelQx do not induce chromosomal aberrations in hamster ovary cells (Thompson et al., 1983, 1987) or in human lymphocyte s (Aeschbacher et al., 1989) despite the very high doses used in the presence of exogenous $9 activation preparations from PCB-pretreated rodents. However, IQ did have a weak effect on micronucleus induction in cultured Chinese hamster cells (Schmuck et al., 1988), while Trp-P-2 systematically induced chromosome aberrations in these cells (Sasaki et al., 1980; Ishidate et al., 1981; Thompson et al., 1983). These results are generally confirmed by in vivo findings where IQ did not significantly induce chromosomal aberrations (micronuclei) in mouse bone marrow even at very high doses (Minkler et al., 1984; Wild et al., 1985). Trp-P-2 only showed a weak effect (Minkler et al., 1984), while PhlP induced chromosomal aberrations in mouse blood lymphocytes but not in the bone marrow test (Tucker et al., 1989). In conclusion, the in vivo results are in agreement with the in vitro findings showing chromosomal aberrations for protein pyrolysates but not for aminoimidazoazaarenes (Table 1). Nuclear aberrations The induction of nuclear aberrations in mouse colon epithelial cells has been proposed as a predictor of colon carcinogens (Wargovich et al., 1983). In this test system heterocyclic amines, in particular MelQ and IQ (Bird et al., 1984; Dolora et al., 1986), gave a much greater effect than protein pyrolysates (see Table 1). Although nuclear aberrations have been associated with cancer initiation, none of these heterocyclic amines was found to be a colon carcinogen in mouse bioassays. Several compounds including IQ, MelQ, Glu-P-1 and Glu-P-2 do cause cancer in the small and large intestines of rats (Sugimura et al., 1990). Sister-chromatid exchange All the heterocyclic amines studied so far have induced sister-chromatid exchanges (SCEs) in various mammalian cell systems (see Table 1). IQ-type compounds that did not cause point mutations or chromosome aberrations induced low numbers of SCEs which were mostly not dose-dependent (Minkler et al., 1984; Thompson et al., 1987; Couch et al., 1987; Aeschbacher et al., 1989). However, relatively strong and dose-dependent ef-

238 TABLE 1 S U M M A R Y OF STUDIES ON D N A D A M A G E BY HETEROCYCLIC A R O M A T I C A M I N E S IQ Gene mutation (point mutation) in vitro - (17) + (5, 8, 13, 19) in vivo - (22) Chromosome aberration in vitro - (1, 16, 19)

MelQ - (17) + (8, 13, 20)

- (20)

MelQx

PhlP

Trp-P-2

-

(17)

+ (13, 20) + (20) + (5, 13, 17, 19) + (13) + (11) + (11)

Glu-P-2 Glu-P-1 A a C -

(17)

-

(17)

-

(17)-

MeAaC (17)

+ (13) + (13) + (13) + (13)

- (1, 20) + (20) + (10, 15, 19)

+ (10,15) + (10) + (10) + ( 1 0 ) + (10)

+ (21) + (14)

in vivo - (14, 22) Micronucleated cells in the colonic epithelium in vivo + (3, 4, 6) + (3, 4) Sister-chromatid exchange in vitro + (1, 8, 19, 24) in vivo + (14)

Trp-P-1

+ (8, 20, 24) + (7)

+ (3) + (1, 20) + (20) + (18, 19) - (21) + (21) - (7) + (14)

lnduced DNA-repair synthesis in vitro - (9, 12) - (9, 23) + (2, 8, 9, 12, 16, 23, 24) + (8, 9, 12, 23, 24) + (9, 23)

-

(9, 23)

+ (3)

+ (3)

+ (18)

+ (18) + (18)

-

(9)

-

(23)

+ (9, 23) + (23) + (23) + (23) + (23)

Neoplastic transformation was observed for Trp-P-1, Trp-P-2, Glu-P-1 (Takayama et al., 1979) and for IQ (Cortesi et al., 1983). - represents a negative or borderline effect; + represents a significant and reproducible positive effect. References: 1, Aeschbacher et al., 1989; 2, Barnes et al., 1985; 3, Bird et al., 1984; 4, Bird, 1986; 5, Brookman et al., 1985; 6, Dolora et al., 1986; 7, Couch et al., 1987; 8, Holme et al., 1987; 9, Howes et al., 1986; 10, Ishidate et al., 1981; 11, Jensen, 1983; 12, Loury et al., 1985; 13, Nakayasu et al., 1983; 14, Minkler et al., 1984; 15, Sasaki et al., 1980; 16, Schmuck et al., 1988; 17, Takayama et al., 1983; 18, Thoda et al., 1980; 19, Thompson et al., 1983; 20, Thompson et al., 1987; 21, Tucker et al., 1989; 22, Wild et al., 1985; 23, Yoshimi et al., 1988; 24, Brunborg et al., 1988.

fects were observed for Trp-P-2 (Thompson et al., 1983) and PhlP (Thompson et al., 1987). The response obtained for SCEs compared to point mutations and chromosomal aberrations was generally shifted upwards due to the extreme sensitivity of SCEs as an endpoint (Latt et al., 1981). The mechanisms and biological implications of SCEs are still under debate. Some experiments suggest that SCEs are not involved in mutational events (Bradley et al., 1979) but more recent work reports a linkage between mutations and malignant transformations (Morales Ramirez et al., 1988).

DNA-repair synthesis There are several reports that heterocyclic amines induce DNA-repair synthesis in rodent hepatocytes indicating that uninduced hepatocytes are able to activate heterocyclic amines to metabolites capable of causing D N A damage (Barnes et al., 1985; Loury et al., 1985; Howes et al., 1986; Holme et al., 1987; Yoshimi et al., 1988). Some species differences have been observed which differ between laboratories. Howes et al. (1986) reported that D N A repair of rat hepatocytes was

not readily induced by heterocyclic amines whereas D N A repair synthesis in rat hepatocytes was observed for the same compounds by another group (Yoshimi et al., 1988). Remarkably, Trp-P-2 failed to induce DNA-repair synthesis (Howes et al., 1986; Yoshimi et al., 1988) although it was the strongest inducer of other types of D N A damage such as point mutations, SCEs and chromosome aberrations (see above).

DNA-strand breaks Some of the heterocyclic amines were investigated for induction of DNA-strand breaks which served as an endpoint for D N A damage in mammalian cells. In these investigations alkaline elution procedures were used to detect single-strand breaks in mouse leukemia cells or in Chinese hamster V79 cells. However, it is not clear if and how such breaks are linked to malignancy. MelQ, IQ and PhlP readily induced DNA-strand breaks (Caderni et al., 1983; Dolora et al., 1985; Brunborg et al., 1988; Holme et al., 1989) whereas MelQx only caused a borderline effect (Dolora et al., 1985). These positive results were only ob-

239 tained when an activation system was present in the incubation medium such as rat hepatocytes or rat liver homogenate.

Oncogene activation Rat c-raf was found to be activated in a transformant obtained with DNA of a hepatocellular carcinoma induced by IQ in the NIH 3T3 cell assay. It was suggested that the DNA recombination was responsible for the transforming activity of the activated c-raf (Ishikawa et al., 1985, 1986). Furthermore it was observed that activated N-ras oncogene also occurred in a transformant derived from a rat small intestinal adenocarcinoma induced by Glu-P-2 (Ishizaka et al., 1987). From these data it was concluded that either the activation of these oncogenes had occurred during the transfection or that these oncogenes had been present in a minor population of cells in the original tumors. These in vitro findings have not yet been substantiated by in vivo results. Metabolism

Monooxygenase and peroxidase metabolism Studies using reconstituted cytochrome P450 enzymes from rat, rabbit or human liver have shown that the P450IA2 isozyme has the highest activity for N-oxidation of heterocyclic aromatic amines (Kato et al., 1983; Yamazoe et al., 1983, 1988; Kato, 1986; Shimada et al., 1988, 1989; McManus et al., 1988, 1989) although P450IA1 also possesses notable activity, particularly with PhlP (McManus et al., 1989; Wallin et al., 1990). These chemicals may induce their own metabolism and activation. Single i.p. injections of Trp-P1,2, Glu-P-1,2, AaC, MeAaC, IQ or MelQx in rats led to an induction of cytochrome P450IA2 in liver but not in extrahepatic tissue (Degawa et al., 1989). Similar findings were reported in a second study on IQ (Rodrigues et al., 1989). The doses given were rather high and the effect of induction by chronic low-level exposure has not been reported. Rats fed BNF as part of the diet were reported to have higher levels of O-deethylase activity of ethoxyresorufin in the small intestine but not in the liver (Knize et al., 1989). Consumption of fried beef also leads to similar enzyme induction in rats (Lindeskog et al., 1988). O-Deethylase activity is principally catalyzed by cytochrome P450IA1 and P450IA2 (Guengerich et al., 1982) and might indicate an increase in metabolic

activation of heterocyclic amines in rat intestine, a target organ of tumorigenesis for several of these compounds. Most metabolism studies have been conducted with rodents following a single oral acute dose. The effect of chronic exposure, such as occurs during carcinogen bioassays, may provide valuable information about changes in metabolism. Humans are exposed to numerous cytochrome P450 inducers in the environment (Conney, 1982) and animals pretreated with known cytochrome P450 inducers may more closely simulate human metabolism. In general, cytochrome P450 is the most efficient enzyme system to activate these amines, but other enzyme systems are capable of activation. The flavin-containing monooxygenase (FMO), which is distinct from cytochrome P450, has been shown to be a major contributor to the metabolic activation of N-methylaminoazobenzene and a minor contributor to the activation of 2-aminofluorene (Frederick et al., 1982). However, FMO does not appear to be involved in metabolic activation of heterocyclic amines. This is suggested by the significant decrease in mutagenic activity when the specific inhibitor of cytochrome P450IA2, a-naphthoflavone, or the antibody, anti-NADPH-cytochrome P450 reductase (Yamazoe et al., 1988; McManus et al., 1988; Shimada et al., 1989), are incorporated into bacterial mutation assays. Methimazole, an inhibitor of FMO with a very low gm, has also been shown to have no effect on the activation and binding of IQ to macromolecules (Loretz and Pariza, 1984). Aromatic and heterocyclic aromatic amines may also be activated by peroxidases where the amines serve as reducing co-factors for prostaglandin H synthase (PHS), an arachidonic acid-dependent peroxidase, and thereby undergo peroxidative metabolism to form reactive species. IQ, MelQ and Glu-P-1 were reported to be activated by PHS from ram seminal vesicles to reactive species which bound to protein and DNA (Nemoto and Takayama, 1984; Wild and Degen, 1987; Petry et al., 1989). PHS-activated IQ and MelQ were found to be mutagenic in TA98 while Glu-P-1 was mutagenic only in TA1538/1,8-DNP 6 (pYG121), a tester strain containing elevated levels of Nacetyltransferase (Watanabe et al., 1987). Human tissues also support the arachidonic acid-dependent peroxidative activation of several aromatic and heterocyclic aromatic amines to species capable of binding to DNA (Flammang et al., 1989).

240

Peroxidase activity was detected in human colon and bladder. Benzidine was the most active substrate for both colon and bladder microsomal preparations. Lower, but notable activity was detected for 2-naphthylamine, Glu-P-1 and 4-ABP followed by IQ and Trp-P-2. Peroxidation of aromatic amines by PHS may be an important route of activation in vivo, particularly in extrahepatic tissues, where the cytochrome P450 content is low. DNA adduct formation Mechanisms of DNA adduct formation and enzymes involved in activation of N-hydroxy metabolites have been studied extensively in vitro. The covalent binding of carcinogens to DNA is regarded as a critical event in cancer initiation. The elucidation of DNA carcinogen adduct structures and knowledge about their frequencies of formation and relative stabilities provide important information on metabolism, chemical reactivity and carcinogenic potential (Miller, 1970). The N-hydroxy metabolites of several of these amines including IQ, MelQx, Glu-P-1 and Trp-P-2 have been found to react directly with DNA but

~

their chemical reactivity differs from the aromatic amine bladder carcinogens 4-aminobiphenyl or 2naphthylamine (Kadlubar et al., 1977). DNA binding of heterocyclic amines is not significantly increased by acidic pH and suggests that acid does not greatly enhance nitrenium ion formation (Mita et al., 1982; Snyderwine et al., 1988a; Negishi et al., 1989). In contrast, a pronounced increase in DNA binding is observed for the N-hydroxy derivatives of 4-aminobiphenyl and 2-naphthylamine by lowering the pH from 7 to 5 (Kadlubar et al., 1977). However, DNA binding of heterocyclic amines is greatly increased by in situ generation of the N-acetoxy esters using acetic anhydride or ketene, indicating more facile nitrenium ion formation (Hashimoto et al., 1980a,b; Hashimoto et al., 1982a; Snyderwine et al., 1988a; Negishi et al., 1989). The metabolic pathways of activation of carcinogenic primary aromatic amines are well documented. A number of aromatic amines serve as good substrates for N-acetyltransferase (King and Glowinsld, 1983). The N-acetylarylamines then may undergo further transformation through N-oxidation to form the corresponding arylhyOH

NH2 - -

~,

~

NHAc

NA¢

OH

> 1

NH

~

OSO3H

I

NAc OAc NH

NH

I

DNA N--H

DNA

NA¢

Fig. 1. Metabolic pathways leading to activation and D N A adduct f o r m a d o n from aromatic and heterocyclic aromatic amines. The bold-faced arrows represent pathways leading to activation through O-acetylation of the arylhydroxylamine.

241

droxamic acids which serve as substrates for N,O-acetyltransferase, and sulfotransferase (Beland and Kadlubar, 1985). The resulting N-acetoxy or sulfonyloxy esters are believed to be the ultimate carcinogenic species which react spontaneously with DNA to form covalently bound carcinogen DNA adducts. Heterocyclic aromatic amines appear to be very poor substrates for mammalian N-acetyltransferase and metabolic activation does not follow the pathway of primary aromatic amines (Shinohara et al., 1984; Brunborg et al., 1988). The major metabolic activation pathway of heterocyclic amines is believed to occur through direct O-acetylation of the N-hydroxylamines rather than direct acetylation and subsequent N-hydroxylation or N-acetylation of the hydroxylamine followed by subsequent N,Oacetyltransfer (see Fig. 1) (Shinohara et al., 1985, 1986b; Kato, 1986; Flammang et al., 1988). Two other mammalian enzyme systems which may be involved in activation are sulfotransferase and prolyl tRNA synthetase (Kato et al., 1983; Yamazoe et al., 1985; Kato, 1986 and references within). The cytos01s of both rat and human liver and colon have been found to activate N-hydroxy metabolites of aromatic amines and liver and colon cytosols of rat activate N-hydroxy metabolites of heterocyclic aromatic amines through acetyl CoA-dependent O-acetyltransferase (Shinohara et al., 1985, 1986; Kato, 1986; Flammang et al., 1988, 1989; Yamazoe et al., 1989). In preliminary studies, acetyl CoA-dependent DNA binding has also been observed for both liver and colon cytosols of humans for the N-hydroxy derivatives of IQ, MelQx, PhlP and Glu-P-1 (Turesky et al., 1990a). The importance of O-acetyltransferase in bacterial mutagenesis of heterocyclic amines has been demonstrated through a number of studies using mutagenicity and DNA binding as endpoints. The incorporation of DCNP, a specific inhibitor of sulfotransferase (Mulder and Scholtens, 1977), in bacterial reversion assays results only in a slight diminution of mutagenicity of IQ and MelQx in TA98 cells, but incorporation of PCP, an inhibitor of both acetyltransferase and sulfotransferase (Saito et al., 1985), dramatically decreases mutagenicity (Nagao et al., 1983; Paterson and Chipman, 1987; Negishi, 1989). A dramatic decrease in mutagenicity is also observed in the Salmonella typhimurium acetyltransferasedeficient strain TA98/1,8-DNP 6 when compared

to TA98 for IQ, MelQ, MelQx, Glu-P-1, Glu-P-2 and MeAaC but the decrease in activity is less pronounced for Trp-P-1, Trp-P-2 and AaC (Nagao et al., 1983; Saito et al., 1983b). Similar observations have been made with PhlP (Holme et al., 1989). Other esterifying enzymes may be involved in activation of these compounds. Alternatively, the N-hydroxy derivatives may be sufficiently reactive to bind directly with DNA and cause mutagenicity (Kato, 1986 and references within). It is important to note that the activity of bacterial and mammalian enzymes may be very different. For example, the K m value for acetyl CoA using HNOH-Glu-P-1 as a substrate was reported to be about 100-fold higher in hamster liver acetyltransferase than in S. typhimurium (Saito et al., 1986). In addition, O-acetyltransferase from S. typhimurium did not show N,O-acetyltransfer activity which is in contrast to some mammalian enzymes (Saito et al., 1985, 1986). Recently it was discovered that neither TA98 nor TA98/1,8-DNP 6 possess sulfotransferase activity (Yamazoe et al., 1989). These findings demonstrate that there are important differences in metabolic capacity and substrate specificity for enzymes in bacterial and mammalian systems which may explain some of the genotoxic discrepancies found between bacterial and mammalian cells. DNA adducts formed in vitro through the reaction of the N-hydroxy metabolites of Trp-P-2, Glu-P-1 and IQ (Hashimoto et al., 1980a,b; Snyderwine et al., 1988a) have been identified as C8-deoxyguanosine-substituted products with adduction occurring through the exocyclic amine group. C8-deoxyguanosine adducts have also been isolated from the livers of rats treated with Glu-P-1 and Trp-P-2 (Hashimoto et al., 1982a,b). At least 8 adducts of IQ were detected in liver of male monkeys fed IQ (Snyderwine et al., 1988b) using the 32p-postlabeling method developed by Randerath and co-workers (1985). The C8-guanine IQ adduct accounted for approximately 50% of these adducts. A similar adduct profile was observed in kidney, colon, stomach and bladder tissue. An identical adduct profile was obtained from DNA modified in vitro with N-hydroxy-IQ, verifying that the N-hydroxy metabolite was responsible for adduct formation in vivo. In another study, 5 DNA adducts were detected by 32P-postlabeling of liver, small and large intestines of rats following i.p. administration of IQ (Schut et al., 1988). Similar DNA adduct patterns have been identified

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in vitro by incubation of nitro-IQ or IQ activated by $9 with Salmonella suggesting that the same ultimate electrophile is formed (Assan et al., 1987). Studies conducted with rats treated with MeIQx, Glu-P-1 and Trp-P-1 revealed that DNA adduct profiles from liver tissue were very similar to those generated in vitro following reactions with the N-hydroxy derivatives of MeIQx and Glu-P-1 but not for Trp-P-1 with DNA (Yamashita et al., 1988). Other metabolic pathways may be involved in the activation of Trp-P-1 in vivo. Using the DNA covalent binding index as an indication of carcinogenic potency in mice, MeIQx was found to be of similar magnitude to that of 2-aminofluorene, a strong carcinogen (Alldrick and Lutz, 1989), and animal pretreatment with cytochrome P450 inducers led to higher levels of DNA adduct formation of MeIQx in the liver (Turteltaub et al., 1990). Protein adduct formation Many of the carcinogenic metabolites which react with DNA may also react with proteins. Protein carcinogen adduct formation provides complementary information to DNA adduct studies on metabolism of carcinogens and chemical reactivity. In the past few years highly sensitive methods have been developed to measure protein carcinogen adducts for the purpose of monitoring human exposure (Osterman-Golkar et al., 1976; Neumann et al., 1977; Skipper and Tannenbaum, 1990). Such measurements also provide valuable information about the capacities of individuals to metabolically activate chemicals. Several studies have been reported on the interaction of heterocyclic amines with proteins. IQ was found to bind to blood proteins in the rat in a dose-dependent fashion and it was distributed amongst a number of proteins including hemoglobin and albumin (Turesky et al., 1987). When expressed on a permole basis, binding was 3-4-fold higher for albumin than hemoglobin. One of the adducts formed with albumin was identified as a sulfinamide linkage occurring at the cysteine34 residue. In a study conducted in vitro, synthetic N-hydroxyGlu-P-1 was found to bind to hemoglobin thiol groups through its oxidized nitroso derivative (Umemoto et al., 1988) and Glu-P-1 formed adducts with hemoglobin in both rats and rabbits (Umemoto et al., 1986; Yin et al., 1989). A major proportion of Glu-P-1 bound to hemoglobin in rabbits could be hydrolyzed with acid to regener-

ate the parent amine. Such findings suggest formation of a sulfinic acid amide linkage. Formation of a similar protein adduct has been reported for 4-ABP in rodents and humans (Green et al., 1984; Bryant et al., 1987). The structure of this adduct was shown by X-ray crystallography to be formed at the 93/3 cysteine of human hemoglobin (Ringe et al., 1988). A quantitative method using gas chromatography-mass spectrometry for the analysis of 4-ABP bound as this linkage in humans revealed that blood samples from cigarette smokers had consistently higher adduct levels than nonsmokers (Bryant et al., 1987). Monitoring longterm human exposure to and metabolic activation of Glu-P-1 through this adduct may also be possible. Interactions of other heterocyclic amines with proteins have not been reported and merit further investigations for application in human dosimetry. In vivo metabolism and biodisposition Most metabolism studies conducted in vivo with heterocyclic amines have been performed with rodents. These chemicals are rapidly absorbed from the gastrointestinal tract of rodents following oral administration and are transformed into a number of detoxified products by the liver (Sjrdin and J~igerstad, 1984; Turesky et al., 1986, 1988a,b; Rafter and Gustafsson, 1986; Gooderham et al., 1987; Alldrick and Rowland, 1988; Sj~Sdin et al., 1988; Luks et al., 1989; Isamasu et al., 1989). Once absorbed, the compounds and metabolites are distributed by the blood stream to most parts of the body except the central nervous system (Brandt et al., 1983; Bergman, 1985). Elimination through urine and feces is rapid and recovery of material is nearly quantitative within 72 h with residual activity remaining principally in liver and kidney. The routes of in vivo metabolism best understood are those for IQ and MeIQx, followed by PhIP. In most of the studies a single acute oral dose was given. Routes of detoxification for IQ and MeIQx include cytochrome P450-mediated ring hydroxylation at the C-5 position followed by conjugation to sulfuric or glucuronic acid (Gooderham et al., 1987; Turesky et al., 1988a,b; Luks et al., 1989; Wallin et al., 1989; Sj/Sdin et al., 1989). Direct conjugation to the exocyclic amine group also occurs. Major routes include Nglucuronidation and the uncommon pathway of sulfamate formation. N-Acetylation has been found to be a minor pathway (Stormer et al.,

243 1987; Hayatsu et al., 1987). The N-glucuronide and sulfamate conjugates have been found to be very resistant towards hydrolases but they are hydrolyzed in acid with quantitative recovery of the parent amine (Turesky et al., 1986, 1988a,b). These conjugates are considerably more stable than N-glucuronides or sulfamates of primary aromatic amines and arylhydroxylamines (Kadlubar et al., 1977) which may explain their large contribution to metabolism. The ring-hydroxylated glucuronide and sulfate conjugates of IQ and MelQx as well as the sulfamates and N-glucuronides have been identified in bile. However, the sulfamate is the principal metabolite to survive passage through the intestinal tract aad is recovered in feces. All of these metabolites have been identified in varying degrees hl urine, Metabolites of MelQ produced in vitro with freshly isolated hepatocytes have been characterized and metabolic pathways are similar to those reported for IQ and MelQx (Alexander et al., 1989b). All of the above metabolites are detoxified products with the exception of the N-acetyl derivatives which require further metabolic activation in order to exert genotoxicity. The lack of reactive metabolites may be attributed to the fact that many of these studies were performed with uninduced animals where N-oxidation is low. Recently, the N-hydroxy metabolite of PhlP and the metastable N-glucuronide conjugate of N-hydroxy-PhlP have been identified, respectively, in isolated hepatocytes and in bile of rats pretreated with PCB (Alexander et al., 1989c). Ring oxidation at the 4 position of the phenyl ring followed by sulfation is a major route of detoxification of PhlP (Alexander et al., 1989a). In contrast to IQ, MelQ and MelQx, sulfamate formation does not appear to be an important route of detoxification of PhlP in the rat. As many as 11 metabolites of PhlP were detected in urine while only 2 major metabolites were found in feces of mice pretreated with Aroclor 1254 (Turteltaub et al., 1989). The structures have not yet been elucidated. Relatively less is known about the in vivo metabolism of the amino acid pyrolysate mutagens. The N-acetylated derivatives of Trp-P-1 and Glu-P-1 have been identified as minor components in bile and urine of rats (Rafter and Gustafsson, 1986; Negishi et al., 1986). Several monohydroxylated metabolites of Trp-P-1 have also been reported to be present in urine (Rafter

and Gustafsson, 1986). The role of glutatllione in the detoxification of a wide variety of carcinogens including aromatic amines is well documented (Ketterer et al., 1983), but its role in the detoxification of heterocyclic aromatic amines is less clear. Conjugation to glutathione has not been found to be a significant route of in vivo metabolism and detoxification of heterocyclic amines. The studies performed in vitro are few and inconclusive. In one study conducted in vitro glutathione reacted enzymatically with N-hydroxy-Trp-P-2 to form 3 conjugates (Saito et al., 1983a). Two of the conjugates were detoxification products, but the third conjugate was found to be a more potent mutagen than the N-hydroxy metabolite. Another study demonstrated that glutathione did have a protective role against TrpP-2 in hepatocytes as depletion of cellular glutathione resulted in elevated levels of Trp-P-2 metabolites bound to DNA (Mita et al., 1983). Cellular depletion of glutathione also led to increased levels of IQ bound to protein and DNA (Loretz and Pariza, 1984). A recent study has suggested that a small portion of MelQx is conjugated with glutathione in vitro using $9 mix or with freshly isolated hepatocytes but the structure of the metabolite is not known (Wallin et al., 1989). The nitroso derivative of Glu-P-1 was found to react non-enzymatically with glutathione to form both sulfinamide and sulfenamide linkages (Saito and Kato, 1984). The adducts were so unstable that they could be detected only in situ inside a fast atom bombardment mass spectrometer. The formation of such labile GSH conjugate intermediates is a possible means of detoxification of the reactive N-hydroxy metabolites and could be involved in the detoxification process of heterocyclic amines. However, due to their instability and rearrangements, such metabolites may remain undetected. Recently, the glutathione sulfinic acid amide of PhlP was identified in hepatocytes of rats pretreated with PCB and serves as a major route of detoxification (Alexander et al., 1990). Biotransformation in the digestive tract The involvement of intestinal bacterial flora in the overall metabolism and biodisposition of heterocyclic amines has not been adequately addressed. Two studies have been reported on the comparison of metabolism of Trp-P-1 (Rafter and Gustafsson, 1986) and 4,8-DiMelQx (Knize et al., 1989) in germ-free and conventional rats. The

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investigators' conclusions in both studies were that the intestinal flora did not appear to have a major role in metabolism. Slower rates of excretion of metabolites via feces were found in germ-free animals due to longer transit times. Several of these heterocyclic amines including IQ, MelQ, Glu-P-1 and Glu-P-2 are strong intestinal carcinogens in rats and the involvement of bacteria in tumor initiation merits further investigations. Several amines including IQ, MelQ, Trp-P-1,2, and Glu-P-1 induce nuclear aberrations in the colonic epithelial cells of mice but none has been found to produce intestinal tumors (Tudek et al., 1989). However, IQ, MelQ, Glu-P-1 and Glu-P-2 are potent intestinal carcinogens in rats (Sugimura, 1988). Production of intestinal tumors suggests the possibility that the critical metabolites are formed in the liver and excreted in bile. This proposal is supported by work conducted on DMABP, a primary aromatic amine which is an intestinal carcinogen. The N-hydroxy-N-glucuronide has been identified as a major metabolite in bile (Nussbaum et al., 1983). Hydrolysis of this metabolite with fl-glucuronidase yields N-hydroxy-DMABP, a metabolite which reacts with DNA and whose binding is catalyzed by acetyl CoA O-acetyltransferase (Flammang et al., 1988). Recently the N-glucuronide conjugates of N-hydroxy-PhlP (Alexander et al., 1989c) and N-hydroxy MelQx (Turesky et al., 1990b) have been identified in bile and hepatocytes of rats indicating that glucuronidation is an important route of metabolism and transport of carcinogenic aromatic and heterocyclic aromatic amines (Radomski et al., 1977; Kadlubar et al., 1977; Nussbaum et al., 1983). Alternatively, the genotoxicity could be attributed to mixed-function oxidase or PHS activity present in the intestinal tract which directly activates these procarcinoger!s. A recent publication has reported that the bacterial flora in the gut may directly contribute to the activation of heterocyclic amines. IQ was found to be oxidized at the 7 position by Eubacterium to form 2-amino3,6-dihydro-3-methyl-7H-imidazo-[4,5-f]quinoline7-one, a direct acting mutagen (Carman et al., 1988). This metabolite was also identified in human feces following consumption of fried meat (Van Tassell et al., 1989). The formation of this metabolite by the colonic flora is significant because it may be genotoxic to the colonic epithelium without undergoing enterohepatic circulation. Further genotoxicity studies are necessary in

order to evaluate the biological activity of this metabolite. The contribution of bacteria 'to the direct activation of heterocyclic amines needs further investigation. Human studies

Preliminary studies with humans have shown that heterocyclic amines present in foods are absorbed through the gastrointestinal tract as evidenced by an increase in mutagenicity in urine and feces following consumption of fried beef (Hayatsu et al., 1985, 1986). It was suggested that some of the mutagenicity may be attributed to MelQx and its metabolites, but the mutagenic principles were not identified. A subsequent study confirmed the presence of a small amount of MelQx in urine following consumption of fried beef (Murray et al., 1989). Several heterocyclic amines, including the glutamic acid and tryptophan pyrolysates and MelQx, have been reported in plasma of patients with uremia at levels ranging from 10 to several hundred pM (Yanagisawa et al., 1986; Manabe et al., 1987a,b). The Nacetylated metabolites of Glu-P-1 and Glu-P-2 have also been identified at very low levels in bile and urine (Kanai et al., 1988). This is the first demonstration of metabolism of heterocyclic amines by humans in vivo. Human hepatic microsomes activate heterocyclic aromatic amines to bacterial mutagens (Yamazoe et al., 1988; McManus et al., 1988; Shimada et al., 1989) and have been found to N-oxidize heterocyclic amines at rates comparable to 4-ABP, a known human carcinogen (Butler et al., 1989). Several of these heterocyclic amines have also been found to serve as substrates for hepatic and colonic O-acetyltransferase (Turesky et al., 1990). Thus, human tissues can appreciably activate heterocyclic amines to biologically reactive species and these genotoxins may be a factor in the etiology of human cancer. Conclusions

Many data have been gathered on human exposure to heterocyclic amines and mechanisms of genotoxicity have been elucidated. However, risk estimation of cancer development due to the daily ingestion of low amounts of these chemicals is still difficult. Heterocyclic amines caused only weak induction of DNA damage in vitro, usually only with sensitive conditions such as repair-deficient

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cells and with highly active tissue preparations for metabolic activation. The greater capacity of protein pyrolysate compounds to induce DNA damage in several in vitro mammalian cell assays as compared to aminoimidazoazaarenes does not correspond to their carcinogenic potential. Trp-P2, the strongest DNA-damaging agent in mammalian cells, was a potent liver carcinogen in rodents but did not affect other organs. In contrast, the very weakly mutagenic 'IQ-type' compounds caused multiorgan carcinogenicity in both rats and mice. The daily consumption of these compounds is very low compared to the TDs0 values obtained from rodent experiments and it does not appear that development of human cancer can be attributed only to exposure to a single chemical. However, these compounds are consumed simultaneously from a number of sources and the combined effects on carcinogenicity are unknown. A preliminary study in the rat has indicated that tumor induction following simultaneous administration of several amines may be greater than an additive effect (Takayama et al., 1987). There may be differences between man and rodents in the absorption, metabolism and activation versus detoxification of heterocyclic amines. The rates of excretion may also be different. Humans are exposed to many other carcinogens and enzyme inducers and their effects on metabolism and activation are not known. Humans are also exposed to tumor promoters which may enhance the carcinogenic potential of heterocyclic amines (Sugimura, 1982). Fortunately, there are tumor-inhibiting factors in the diet which may weaken the biological activity of heterocyclic amines (Hayatsu et al., 1981; Wattenberg, 1985; Arimoto and Hayatsu, 1989). With the recent advances in the molecular biology of cytochrome P450, cDNAs of P450 can now be expressed in a number of different cell lines (Battula et al., 1987; Snyderwine and Battula, 1989; Gonzalez, 1989). Cells containing expressed cytochrome P450 may be more advantageous than highly purified cytochrome P450s for metabolism studies because the enzymes are in an environment which more closely simulates the normal physiology of the living cell. The use of such genetically engineered cells may provide even greater insight into the genotoxicity of heterocyclic amines. Human metabolism studies following consumption of foods containing heterocyclic amines are

necessary in order to evaluate human health risks. The application of techniques such as 32p-postlabeling of DNA and quantification of protein adducts will provide information about individual capacities to activate these compounds. Recent work has shown that human hepatic cytochrome P450IA2 metabolic activation of primary aromatic and heterocyclic aromatic amines also catalyzes 3-demethylation of caffeine (Butler et al., 1989). The investigators proposed the use of caffeine as a means to characterize arylamine N-oxidation phenotypes of individuals in order to assess the role of P450 in determining individual susceptibilities to arylamine-induced cancers. Such non-invasive metabolism studies as 32p-postlabeling and protein adduct analysis in conjunction with cytochrome P450IA2 phenotyping could provide valuable information on the bioactivation of heterocyclic aromatic amines by humans and their role in carcinogenesis.

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