4Mechanistic and Other Relevant Data

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4.1. Absorption, distribution, metabolism, elimination

4.1.1. Humans

Because of the known carcinogenicity of 2-naphthylamine in humans, few metabolic studies have been carried out. However, N-(2-naphthyl)-hydroxylamine and bis-(2-amino-1-naphthyl) phosphate have been identified in the urine of some hospital patients given small doses of 2-naphthylamine (Troll & Nelson, 1961; Troll et al., 1963).

Grimmer et al. (2000) analysed the amounts of the aromatic amines 1- and 2-naphthylamine and 2- and 4-aminobiphenyl in the urine of 48 German smokers and non-smokers. The results indicate that both groups excrete these four aromatic amines, with smokers excreting approximately twice as much (736 ng/24 hours vs 303 ng/24 hours). Similar amounts of urinary 2-naphthylamine and 4-aminobiphenyl were found in the two groups. The excreted aromatic amines decompose in the urine within a few hours, which explains why aromatic amines are difficult to detect in this matrix (in this study para-toluidine was added to the urine as a stabilizer). The origin of the aromatic amines found in the urine of non-smokers is unknown at present. Based on the cotinine levels in the urine of non-smokers, environmental tobacco smoke can be excluded as a major source of aromatic amines. In addition, neither diesel exhaust-related nitroarenes, nor the corresponding amino-derivatives to which they may be metabolically converted, were detected. The aromatic amines in urine arising from sources other than tobacco smoke or diesel exhaust may play a role in the etiology of bladder cancer in non-smokers.

4.1.2. Animals

Boyland (1958) and Boyland & Manson (1966) identified 24 metabolites of 2-naphthylamine in the urine of rats, rabbits, dogs or monkeys. Metabolism occurs along four main pathways: (i) N-hydroxylation followed by conversion to 2-amino-1-naphthyl-mercapturic acid, 2-nitrosonaphthalene and rearrangement to 2-amino-1-naphthol; (ii) oxidation at positions C5 and C6 to an arene oxide, which rearranges to 5-hydroxy-2-naphthylamine, reacts with water to form a 5,6-dihydroxydihydro derivative and forms a 5-hydroxy-6-mercapturic acid; (iii) conjugation of the amino group with acetic, sulphuric or glucuronic acid; (iv) secondary conjugation of the hydroxyl group with phosphate sulphuric or glucuronic acid. The proportion of these metabolites excreted in the urine of various experimental animals is different (Bonser et al., 1951; Boyland & Manson, 1966; Conzelman et al., 1969), although in most species 2-amino-1-naphthyl sulfate is the predominant metabolite. In early studies it was proposed that the ortho-hydroxylation metabolite 2-amino-1-naphthyl glucuronide was hydrolysed by β-glucuronidase present in urine, yielding 2-amino-1-naphthol (Allen et al., 1957), which may act as the proximate carcinogen. The latter compound may be oxidized to form 2-amino-1-naphthoquinone, which can form DNA adducts (Beland & Kadlubar, 1985; Yamazoe et al., 1985).

In dogs a small percentage of 2-naphthylamine was metabolized to bis-(2-amino-1-naphthyl) hydrogen phosphate (Boyland et al., 1961; Troll et al., 1963). N-Oxidation of 2-naphthylamine leads to more reactive metabolites (Deichmann & Radomski, 1969). Radomski & Brill (1971) showed that after a single oral dose of 70 mg/kg bw of both the carcinogenic 2-isomer and the weakly active or inactive 1-isomer of naphthylamine, the proportion of the dose converted into the corresponding N-hydroxylamine and nitroso compound and excreted in the urine was about the same. When a dose of 5 mg/kg bw was given, the proportion of the 2-isomer converted to these metabolites in the urine remained about the same (0.2%), whereas the 1-isomer gave rise to barely detectable traces of these compounds. The unstable N-hydroxy metabolites are excreted in the urine as glucuronic acid conjugates and hydrolysed by acid or β-glucuronidase. These conjugates have been considered as the carcinogenic urinary metabolites of aromatic amines in the dog (Radomski et al., 1973). Methaemoglobinaemia, which is a measure of N-hydroxy compounds in the blood, was higher in the dog with the 2-isomer at the 70-mg/kg bw dose (Radomski & Brill, 1971).

4.1.3. In-vitro systems

Dermal penetration of 2-naphthylamine and ortho-toluidine through human skin was studied by use of diffusion cells. Both compounds penetrate rapidly (lag time approximately 1.2 and 0.8 hours, respectively) and in high percentages (54 and 50%, respectively, of the applied dose within 24 hours) (Lüersen et al., 2006).

Duverger-van Bogaert et al. (1991) evaluated the ability of the cytosol from human red blood cells to activate aromatic amines using the Ames test with S. typhimurium strain TA98 under liquid preincubation conditions. While negative results were obtained with 1-naphthylamine, a slight response was observed for 2-naphthylamine.

The cytosol from nine fresh autopsy specimens of human bladder tissue was analysed for the presence of N-acetyltransferase (NAT) activity towards para-aminobenzoic acid, 4-aminobiphenyl, 2-aminofluorene, and 2-naphthylamine. Apparent Km values indicated little difference in NAT affinity (100–300 microM) for any of the substrates between the nine individual bladders. However, the apparent Vmax values showed that the bladders could be divided in rapid or slow acetylator phenotypes, based on their NAT activity towards 4-aminobiphenyl, 2-aminofluorene, and 2-naphthylamine. Four of the bladder cytosols had mean activities significantly higher (approximately 10-fold, P < 0.01) than the mean NAT activities of the other five bladder cytosols towards each arylamine carcinogen. However, no significant difference was detected in their NAT activities when para-aminobenzoic acid ws used as a substrate. The human bladder cytosols were also tested for their capacity to activate N-hydroxy-3,2′-dimethyl-4-aminobiphenyl to a DNA-binding electrophile through direct O-acetyltransferase (OAT)-mediated catalysis. The N-hydroxyarylamine OAT activity also discriminated between two levels of activation, being significantly higher (about twofold, P = 0.0002) in the rapidly N-acetylating bladder cytosols, which correlated (r = 0.94) with the measured levels of NAT activity in each cytosol. These results suggest that NAT activity and OAT activity of the human bladder vary concordantly with N-acetylator phenotype. The polymorphic expression of these acetylation activities may be important risk factors in human susceptibility to bladder cancer from arylamine carcinogens (Kirlin et al., 1989).

The metabolic activation of 4-aminobiphenyl, 2-naphthylamine, and several heterocyclic amines has been shown to be catalysed by rat cytochrome P450ISF-G [CYP1a2] and by its human ortholog, cytochrome P450PA [CYP1A2] (Butler et al., 1989a). In humans, hepatic microsomal caffeine 3-demethylation is the initial major step in caffeine biotransformation, which is selectively catalysed by cytochrome P450PA. Caffeine 3-demethylation was highly correlated with 4-aminobiphenyl N-oxidation (r = 0.99; P <0.0005) in hepatic microsomal preparations obtained from 22 human organ donors, and both activities were similarly decreased by the selective inhibitor 7,8-benzoflavone. A rabbit polyclonal antibody raised against human cytochrome P450PA strongly inhibited these activities as well as the N-oxidation of the carcinogen 2-naphthylamine and other amines. Human liver cytochrome P450PA also catalysed caffeine 3-demethylation, 4-aminobiphenyl N-oxidation, and phenacetin O-deethylation. Thus, estimation of caffeine 3-demethylation activity in humans may be useful in the characterization of arylamine N-oxidation phenotypes and to assess whether or not the hepatic levels of cytochrome P450PA, as affected by environmental or genetic factors, contribute to inter-individual differences in susceptibility to arylamine-induced cancers (Butler et al., 1989b).

Moore et al. (1984) investigated the metabolism of benzidine and 2-naphthylamine in organ cultures of human and rat bladder. There was little oxidative metabolism of either carcinogen in either species. In particular, N-hydroxy-2-naphthylamine, a proximate carcinogen of 2-naphthylamine could not be detected. In contrast, large amounts of the acetylated metabolites, viz. N-acetylbenzidine, N,N-diacetylbenzidine and N-acetyl-2-naphthylamine were formed both in rat and human bladder cultures.

Microsomal enzyme preparations from dog liver, kidney, and bladder were used to assess the prostaglandin H synthase-catalysed activation of carcinogenic aromatic amines to bind covalently to proteins and nucleic acids. Bladder transitional epithelial microsomes activated ortho-dianisidine, 4-aminobiphenyl, and 2-naphthylamine to bind to protein and tRNA, and benzidine and ortho-dianisidine to bind to DNA Co-substrate and inhibitor specificities were consistent with activation by prostaglandin H synthase. Binding of benzidine to protein was not observed with either hepatic or renal cortical microsomes upon addition of arachidonic acid or reduced nicotinamide adenine dinucleotide phosphate. Prostaglandin H synthase and mixed-function oxidase-catalysed binding of 2-naphthylamine to protein and to tRNA were compared by use of liver and bladder microsomes. Only mixed-function oxidase-catalysed binding was observed in liver, and only prostaglandin H synthase-catalysed binding was seen in bladder. The rate of binding catalysed by bladder microsomes was considerably greater than that catalysed by hepatic microsomes. In addition, the bladder content of prostaglandin H synthase activity was approximately 10 times that in the inner medulla of the kidney, a tissue reported to have a relatively high content of this enzyme in other species. These results are consistent with involvement of bladder transitional epithelial prostaglandin H synthase in the development of primary aromatic amine-induced bladder cancer (Wise et al., 1984)

Hammons et al. (1985) studied the in-vitro hepatic metabolism of 2-aminofluorene (2-AF), 2-naphthylamine and 1-naphthylamine by use of high-pressure liquid chromatography. Hepatic microsomes from rats, dogs, and humans were shown to catalyse the N-oxidation of 2-AF and of 2-naphthylamine, but not of 1-naphthylamine; and the rates of N-oxidation of 2-AF were 2- to 3-fold greater than the N-oxidation rate of 2-naphthylamine. In each species, rates of 1-hydroxylation of 2-naphthylamine and 2-hydroxylation of 1-naphthylamine were comparable and were 2- to 5-fold greater than 6-hydroxylation of 2-naphthylamine or 5- and 7-hydroxylation of 2-AF. Purified rat hepatic monooxygenases, cytochromes P450UT-A, P450UT-H, P450PB-B, P450PB-D, P450BNF-B, and P450ISF/BNF-G but not P450PB-C or P450PB/PCN-E, catalysed several ring oxidations as well as the N-oxidation of 2-AF. Cytochromes P450PB-B, P450BNF-B, and P450ISF/BNF-G were most active; however, only cytochrome P450ISF/BNF-G, the isosafrole-induced isozyme, catalysed the N-oxidation of 2-naphthylamine. The purified porcine hepatic flavin-containing monooxygenase is known to carry out the N-oxidation of 2-AF, but only ring-oxidation of 1-naphthylamine and 2-naphthylamine was detected. No N-oxidation of 1-naphthylamine was found with any of the purified enzymes, which is in line with the fact that no carcinogenicity is observed with this compound. Furthermore, carcinogenic arylamines appear to be metabolized similarly in humans and experimental animals. Enzyme mechanisms accounting for the observed product distributions were evaluated by Hückel molecular-orbital calculations on neutral, free-radical, and cation intermediates. The authors proposed a reaction pathway that involves two consecutive one-electron oxidations to form a paired substrate cation-enzyme hydroxyl-anion intermediate that collapses to ring- and N-hydroxy products (Hammons et al., 1985; see also Sasaki et al., 2002).

Boyd and Eling (1987) examined the oxidation of the bladder carcinogen 2-naphthylamine by prostaglandin H synthase (PHS) in vitro. Oxygen-uptake studies of 2-naphthylamine oxidation in the presence of glutathione, as well as extensive product analysis yielded data that were consistent with a one-electron mechanism of 2-naphthylamine oxidation by PHS. The formation of 2-nitroso-naphthalene was not observed under any condition. Metabolism studies with a purified PHS preparation confirmed that 2-naphthylamine oxidation is dependent on the peroxidase activity of the enzyme complex, and that a variety of organic hydroperoxides may support the reaction. Horseradish peroxidase oxidized 2-naphthylamine to the same products but, depending on the pH, in very different proportions from those obtained with PHS. Oxidation of 2-naphthylamine by a one-electron chemical oxidant resulted in a product profile similar to that obtained in the enzymatic systems. These results are consistent with a one-electron mechanism of 2-naphthylamine oxidation by PHS. The metabolism data also provide evidence for the formation of two types of potentially reactive electrophile: 2-imino-1-naphthoquinone and a free-radical species.

Poupko et al. (1983) studied microsome-mediated N-hydroxylation of 4-aminobiphenyl in mucosal tissue of bovine and canine bladder relative to the activity in liver. Bovine bladder microsomes mediated the N-hydroxylation of this amine at an exceptionally high rate, whereas no detectable activity was found with bovine liver microsomes. Dog-bladder microsomes were 40–100 times less active than bovine bladder microsomes and contained approximately one third the amount of cytochrome P450 (CYP). Dog liver microsomes were as active as dog bladder microsomes per nanomole CYP, and an order of magnitude more active when normalized to microsomal protein. Rat liver microsomes contained the highest level of CYP of all the preparations studied, and N-hydroxylase activity was approximately twice that in dog liver. Metabolic conversion of 4-ABP, 2-naphthylamine, and 1-naphthylamine into mutagens with S9 from bovine bladder mucosa was investigated in Salmonella typhimurium and found to parallel the carcinogenic potency of these compounds. These results demonstrate considerable tissue-, species-, and compound-specificity for the metabolic activation of aromatic amines, and provide further evidence in support of activation of the amines in the bladder as a mechanism of aromatic amine-induced bladder cancer (Poupko et al., 1983).

4.2. Genetic and related effects

4.2.1. Experimental systems

(a) DNA adducts of 2-naphthylamine

Three DNA adducts are formed by the reaction of N-hydroxy-2-naphthylamine with DNA in vitro at pH 5.0 (Beland et al., 1983; Beland & Kadlubar, 1985). The major adduct has been characterized as an imidazole ring-opened derivative of N-(deoxyguanosin-8-yl)-2-naphthylamine (50% of the total adducts); there were smaller amounts of 1-(deoxyguanosin-N2-yl)-2-naphthylamine (30% of total adducts) and 1-(deoxyadenosin-N6-yl)-2-naphthylamine (15% of total adducts). These same three DNA adducts were formed in target (urothelium) and non-target (liver) tissues of dogs two days after the oral administration of 2-naphthylamine (Beland & Kadlubar, 1985). A four-fold higher binding level of 2-naphthylamine was found in urothelial DNA compared with that in the liver. The major adduct in both tissues was the ring-opened derivative of N-(deoxyguanosin-8-yl)-2-naphthylamine; there were smaller amounts of 1-(deoxy-adenosoin-N2-yl)-2-naphthylamine and 1-(deoxyguanosin-N2-yl)-2-naphthylamine. The N2-deoxyguanosine adduct persisted in the dog liver, and both this adduct and the ring-opened C8-deoxyguanosine adduct persisted in the bladder. The differential loss of adducts indicates that active repair processes are ongoing in both tissues, and the relative persistence of the ring-opened C8-deoxyguanosine adduct in the target but not the non-target tissue suggests that this adduct is a critical lesion for the initiation of urinary bladder tumours.

Peroxidative enzymes, such as prostaglandin H synthase (PHS), catalyse both the N-oxidation and ring-oxidation of 2-naphthylamine, a major ring-oxidation product being 2-amino-1-naphthol (Yamazoe et al., 1985). When PHS was used to catalyse the binding of 2-naphthylamine to DNA, the same three adducts arising from N-hydroxy-2-naphthylamine were detected. In addition there were three other adducts, which appeared to be formed from 2-imino-1-naphthoquinone, the oxidative product of 2-amino-1-naphthol. The major product was characterized as N4-(deoxyguanosin-N2-yl)-2-amino-1,4-naphthoquinone-imine; two minor products were tentatively identified as N4-(deoxyadenosin-N6-yl)-2-amino-1,4-naphthoquinone-imine and a deoxyguanosin-N2-yl adduct of a naphthoquinone-imine dimer (Beland & Kadlubar, 1985; Yamazoe et al., 1985). These DNA adducts, formed via peroxidation, accounted for approximately 60% of the total DNA binding that was observed by incubation of 2-naphthylamine with PHS in vitro. In vivo, in dogs, the DNA adducts derived from 2-imino-1-naphthoquinone accounted for approximately 20% of the 2-naphthylamine bound to urothelial DNA, but they were not detected in liver DNA (Yamazoe et al., 1985). The remaining adduction products were derived from N-hydroxy-2-naphthylamine. Thus, PHS expressed in the bladder could play a significant role in bioactivation of arylamines directly in the bladder and could contribute to carcinogenesis of 2-naphthylamine and other arylamines that serve as substrates of PHS.

(b) Genotoxicity of 2-naphthylamine

Most of the genetic effects of 2-naphthylamine have been reviewed (Mayer 1982) and are summarized in Supplement 7 of the IARC Monographs (IARC, 1987).

2-Naphthyl-amine exhibits weak mutagenicity in the microbial assays and in some mammalian systems in vitro. Results for the mutagenicity of 2-naphthylamine in yeast assays have been inconsistent. In the assays in vivo, 2-naphthylamine was positive in the sex-linked recessive lethal assay with Drosophila melanogaster, but it failed to induce sister chromatid exchange in the mouse. Several micronucleus tests and sperm-abnormality assays with mice yielded inconclusive results. In the bone-marrow micronucleus assay, very high doses of 2-naphthylamine (200–800 mg/kg bw) were required to elicit effects in C57BL6 male mice (Mirkova & Ashby, 1988). 2-Naphthylamine was positive in the mammalian spot test in T stock mice given nine doses of 2-naphthylamine (50 to 100 mg/kg bw), but there was no clear dose-response effect (Chauhan et al., 1983). Few other data exist about the mutagenic effects of 2-naphthylamine in whole organisms, although it is known to induce DNA fragmentation in rodent liver after treatment in vivo (Parodi et al., 1981).

(i) Bacterial mutagenesis

The metabolic conversion of 2-naphthylamine (5–50 µg/plate) to a bacterial mutagen in S. typhimurium TA100 was catalysed much more efficiently by the hamster than by any of the other species tested (human, pig, rat, mouse). Mouse preparations displayed the weakest activity (Phillipson & Ioannides, 1983). In two other studies, the activities of liver-S9 preparations from rats pre-treated with PCBs or 3-methylcholanthrene (3-MC) were comparable to the activities of liver-S9 obtained from guinea-pigs pre-treated with the same CYP450 inducers in bio-activation of 2-naphthylamine (2.5–10 µg/plate) to a bacterial mutagen in strain TA100 (Baker et al., 1980). Liver-S9 preparations from rats pre-treated with PCB or 3-MC also displayed comparable activity in bio-activation of 2-naphthylamine (10–50 µg/plate) in strain TA1535 (Bock-Hennig et al., 1982). Bovine urinary bladder cells, but not hepatocytes, were able to activate 2-naphthylamine to bacterial mutagens in S. typhimurium TA98; the minimal amount of 2-naphthylamine required to increase the level of revertants over background was 20 µg/plate (Hix et al., 1983). Bovine bladder cells were also able to bioactivate 2-naphthylamine (20–80 µg/plate) to a mutagen in S. typhimurium strain TA100, with the activity being about six-to eight-fold higher than in TA98; however, bladder cells did not activate 2-naphthylamine (20 µg/ml) to a mutagen in hamster V79 cells when resistence to 6-thioguanine was used as a genetic marker (Oglesby et al., 1983). The finding of bacterial mutagenicity of 2-naphthylamine (4–5000 µg/ml) was corroborated by bioactivation with rat-liver S9 (PCB-pretreatment) in the Ames II assay (Flückiger-Isler et al., 2004).

Recombinant human CYPs 1A1, 1A2 and 1B1 were unable to activate 2-naphthylamine (5 µM) to a DNA-damaging agent, when the induction of umu was used as an endpoint, in S. typhimurium strain NM2009, which expresses multiple copies of bacterial O-acetyltransferase (Shimada et al., 1996). 2-Naphthylamine (up to 100 µM) also failed to induce the umu response in S. typhimurium tester strains expressing human CYP1A1, 1A2, 1B1, 2C9, 2D6, 2E1 or 3A4 with bacterial O-acetyltransferase (Oda et al., 2001). However, 2-naphthylamine (≥ 1 µM) in the presence of recombinant human CYP1A2 did induce the umu response in S. typhimurium tester strains NM6001 and NM6002, which expressed recombinant human acetyltransferase NAT1 and NAT2 isoforms, respectively, but not in strain NM6000, which is O-acetyltransferase-deficient (Oda 2004). The level of umu induction by 2-naphthylamine was about twofold higher in strain NM6001 than in NM6002 (Oda 2004). Purified rat CYP4B1 was the most efficient haemoprotein among 10 different CYP450s in bioactivating 2-naphthylamine (10 µM) and inducing the umu response in S. typhimurium NM2009 (Imaoka et al., 1997).

In the E. coli K-12 uvrB/recA DNA-repair host-mediated assay in male NMRI mice, 2-naphthylamine (i.p. 200 mg/kg bw) elicited differential killing in E. coli retrieved from the kidney and testicles, but no activity was observed in blood, liver or lung. Surprisingly, 1-naphthylamine (i.p. 33 and 100 mg/kg bw) showed a quite different pattern, inducing differential killing of E. coli retrieved from blood, liver, lung and kidneys (Hellmér & Bolcsfoldi, 1992).

The results of studies on bacterial mutagenesis of 2-naphthylamine with recombinant CYP450 enzymes are inconsistent. This may be due to varying assay conditions and the presence of different phase-II enzymes that modulate the metabolism of 2-naphthylamine.

(ii) Mammalian mutagenesis

2-Naphthylamine (50 µg/mL and 100 (µg/mL) in the presence of liver microsomes from PCB-pretreated rats was shown to increase the level of mutations by up to tenfold over background levels at five independent genetic loci in Chinese hamster ovary (CHO) cells (Gupta & Singh, 1982). Mutation induction was also observed at these loci in the absence of a metabolic activation system, indicating that the CHO cells are capable of bioactivating 2-naphthylamine (Gupta & Singh, 1982). However, 2-naphthylamine (20 µg/ml) was not mutagenic in hamster V79 cells, based on selection of 6-thioguanine-resistant mutants in the presence of bovine urinary bladder cells for bioactivation (Oglesby et al., 1983).

(iii) DNA damage induced by human carcinogens in cell-free assays

2-Naphthylamine (0.2–1 mM) induced DNA damage—measured by 32P-postlabelling—when calf-thymus DNA was co-incubated with 2-naphthylamine and liver microsomes from rats pretreated with phenobarbital or β-naphthoflavone as CYP inducers. However, the bioactivated 2-naphthylamine did not induce DNA fragmentation (Adams et al., 1996).