Reduced 4-aminobiphenyl-induced liver tumorigenicity but not DNA damage in arylamine N-acetyltransferase null mice

Abstract

The aromatic amine 4-aminobiphenyl (ABP) is a liver procarcinogen in mice, requiring enzymatic bioactivation to exert its tumorigenic effect. To assess the role of arylamine N-acetyltransferase (NAT)-dependent acetylation capacity in the risk for ABP-induced liver tumors, we compared 1-year liver tumor incidence following the postnatal exposure of wild-type and NAT-deficient Nat1/2(−/−) mice to ABP. At an ABP exposure of 1200 nmol, male Nat1/2(−/−) mice had a liver tumor incidence of 36% compared to 69% in wild-type males, and at 600 nmol there was a complete absence of tumors compared to 60% in wild-type mice. Only one female wild-type mouse had a tumor using this exposure protocol. However, levels of N-deoxyguanosin-8-yl-ABP-DNA adducts did not correlate with either the strain or sex differences in tumor incidence. These results suggest that female sex and NAT deficiency reduce risk for ABP-induced liver tumors, but by mechanisms unrelated to differences in DNA-damaging events.

Introduction

Metabolic bioactivation of chemical carcinogens into DNA damaging agents is a causal factor in a multi-step process leading to the development of tissue-specific tumors [1]. While genotoxic effects are believed to be a necessary prerequisite for chemically-induced carcinogenesis, it has become clear that non-genotoxic effects also contribute to the development of tumors [2], [3] by sustaining or promoting a proliferative environment, which is one of the proposed hallmarks of cancer [4].

The aromatic amine 4-aminobiphenyl (ABP), which is found in cigarette smoke and hair dyes, is a recognized human bladder carcinogen (for review see [3]). Since environmental exposure to ABP may thus contribute to the occurrence of some tissue-specific cancers, many studies have been undertaken to measure and correlate potential biomarkers of ABP exposure and cancer risk. ABP-DNA adducts have been detected in the DNA from bladder cancer patients [5], exfoliated ductal epithelial cells isolated from human breast milk [6] and from hepatocytes obtained from hepatocellular carcinoma cases [7], and a higher urinary excretion of ABP is found in smokers versus non-smokers [8]. In addition, ABP can bind to proteins such as hemoglobin and albumin, and thus protein adducts such as ABP hemoglobin adducts have also been used as a surrogate or biomarker for exposure and potential risk [9].

The first step in one proposed bioactivation model for ABP is its N-oxidation to N-hydroxy-ABP (N-OH-ABP). The hepatic cytochrome P450 (CYP) isoform CYP1A2 has been implicated in this reaction in the liver [10], whereas CYP1A1 [11] or peroxidases [12] may be involved in its localized oxidation within other tissues. O-acetylation (OAT) of N-OH-ABP by arylamine N-acetyltransferases (NATs) can yield an acetoxy ester, which is chemically unstable and decomposes into a highly reactive arylnitrenium ion that can bind to and damage DNA. The major DNA adduct formed as a result of ABP bioactivation is N-deoxyguanosin-8-yl-ABP (dG-C8-ABP) which causes primarily G:C → T:A transversion mutations [13] that have been mapped as a common mutation in codon 61 of the H-ras gene of ABP-exposed mice [14]. Competing with N-oxidation in the liver is the protective NAT-mediated N-acetylation which leads to the production of an innocuous acetamide metabolite. Thus, it is unclear whether NAT enzymes facilitate (by O-acetylation) or prevent (by N-acetylation) ABP-induced tumorigenesis.

Two functional NAT isozymes, NAT1 and NAT2, are present in humans and display distinct tissue distributions and substrate specificities [15]. In human liver, both ABP N-acetylation and N-OH-ABP O-acetylation activities are mediated predominantly by NAT2 [16], whereas NAT1 has a widespread tissue distribution and may be involved in the localized activation of aromatic amines within other tissues such as the bladder [17]. In addition, NAT1 is transcribed by alternatively spliced promoters, NATa and NATb, giving rise to multiple transcripts which differ in tissue specificity and the ability to form ABP-DNA adducts and mutations in vitro[18]. Thus, the role that NAT enzymes may have in either the bioactivation or detoxification of aromatic amines such as ABP may ultimately depend on their tissue expression profiles, the balance of competing enzyme pathways, the inducibility of these pathways, the dose and route of exposure, and organ-specific metabolism that can have a significant effect on the rate of clearance [19], [20]. Furthermore, NAT enzyme activity can be modulated by environmental factors such as oxidative stress arising from inflammation or environmental exposure [21], and by folate levels which have been shown to affect the methylation status of the mouse Nat2 promoter [22]. Thus, genotype is not the only factor that can influence functional NAT enzyme activity.

Since human NAT1 and NAT2 are both highly polymorphic enzymes (for review see [15], [23]), several epidemiological studies have been undertaken to investigate the role of rapid versus slow acetylator phenotype or NAT genotype to tissue-specific cancer risk, particularly in relation to arylamine or heterocyclic amine exposure. NAT1 and/or NAT2 genotypes have been investigated with respect to cancers of the bladder, colon, breast and liver [24], [25], [26], [27]. Although results from epidemiological studies have tended to be contradictory and indicate that there is no clear or consistent correlation between acetylator status and cancer risk, more recent meta-analyses have indicated an increased risk of either breast or bladder cancer in NAT2 slow acetylators that is dependent on smoking intensity [25], [26]. Limitations to epidemiological studies include small sample sizes, genotype misclassification errors and lack of quantitative information on chemical exposures due to the complex mixtures of aromatic amines and other chemicals to which individuals may be exposed.

To investigate the role of NAT-dependent acetylation in ABP-induced tumorigenesis, in the present study we have used the neonatal carcinogen bioassay and our Nat1/2(−/−) mice, which are deficient in both mouse Nat1 and Nat2 enzyme activities [28]. The neonatal mouse carcinogen bioassay is an accelerated carcinogenicity assay that has been validated using ABP as a positive control for liver tumors [29]. Using this assay, chemical carcinogens requiring metabolic bioactivation tend to show a restricted tissue tumor sensitivity that is reflective of the distribution of available drug metabolizing enzymes, whereas direct-acting genotoxic agents such as the alkylating agent N-ethyl-N-nitrosourea are more likely to result in a widespread tissue distribution of tumors. On the other hand, non-genotoxic chemical carcinogens that mediate increased lipid peroxidation or oxidative stress do not produce tumors using this bioassay [30]. The assay has also been used to demonstrate that in contrast to the bioactivation model described above, mouse Cyp1a2 may not be the initial enzyme involved in ABP-induced carcinogenesis [31]. ABP N-oxidation activity was still evident in Cyp1a2(−/−) mice, albeit at reduced levels, and ABP-DNA adduct levels were not reduced in these animals [32]. These findings have all challenged the view that mouse Cyp1a2 is involved in the metabolic bioactivation of aromatic amines such as ABP, and reinforces the necessity for conducting bioactivation and carcinogenicity studies invivo to determine the role that specific biotransformation enzymes such as the NATs may play in determining tissue-specific tumor risk.