Activation of aminoimidazole carcinogens by nitrosation: mutagenicity and nucleotide adducts

Abstract

2-Amino-3-methylimidazo[4,5-f]quinoline (IQ) and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline(MeIQx) are heterocyclic amines (HCAs) derived from high temperature cooking of meat and thought to cause colon cancer in humans. Reactive nitrogen oxygen species, which are mediators of the inflammatory response, can convert these amines to the corresponding N-nitrosamines, N-NO-IQ and N-NO-MeIQx. This study was designed to evaluate whether these N-nitrosamines are genotoxic and could be responsible, in part, for the high incidence of colon cancer in individuals with colitis. Such an association would counsel reduced intake of well-done red meat by colitis patients. Mutagenicity was evaluated by reversion of a lacZ frameshift allele in three different E. coli strains. Strains DJ701 and DJ702 express recombinant(S. typhimurium) aromatic amine N-acetyltransferase (NAT); DJ702 also expresses recombinant human cytochrome P450 1A2 and NADPH-P450 reductase; and DJ2002 served as an N-acetyltransferase negative control. In strain DJ701, N-NO-IQ and N-NO-MeIQx elicited dose-dependent mutagenicity,which was not further increased in DJ702. Neither nitrosamine was mutagenic in strain DJ2002. While both N-nitrosamines are stable for >4 h (pH 7.4, 37 degrees C), they react with DNA or 2'-deoxyguanosine 3'-monophosphate at lower pH (5.5) to form adducts. HOCl, a component of the inflammatory response,increased adduct formation, as measured by 32P-postlabeling. Following treatment with nuclease P1and separation by two-dimensional thin-layer chromatography and then HPLC, N-NO-IQ and N-NOMeIQxwere shown to form the same adducts as those formed by N-OH-MeIQx or N-OH-IQ, namely N-(deoxyguanosin-8-yl) adducts. In summary, these N-nitrosamines are genotoxic and might be alternatives to their hydroxylamine analogues as activated intermediates leading to initiation of colon cancer in individuals with colitis.

Figure 1

N-Nitrosoacetanilide: structure and decomposition pathway.

Figure 2

N-Nitroso-N-2-fluorenylacetamide: structure.

Figure 3

Tautomeric equilibrium of N-nitrosobenzimidazoles.

Figure 4

2-Nitrosoamino-3-methylimidazo[4,5-f]quinoline (N-NO-IQ) and 2-nitrosoamino-3,8-dimethylimidazo[4,5-f]quinoxaline (N-NO-MeIQx): structures.

Figure 5

Mutagenicity of N-NO-IQ and N-NO-MeIQx in the E. coli lacZ reversion assay. Each data point represents the mean ± standard error of at least two independent triplicate experiments (at least six plates per point). The E. coli strains used were DJ2002 (NAT-minus; diamonds), DJ701 (expressing human NAT2; filled circles), and DJ702 (expressing human NAT2 and P450 1A2; open squares).

Figure 6

³²P-Postlabeling of adducts. N-OH and N-NO heterocyclic amines were incubated with 2′ deoxyguanosine 3′ monophosphate (dGp). Adducts were ³²P-postlabeled and initially separated by thin-layer chromatography on PEI-cellulose plates. For analysis, samples were scraped off PEI-cellulose plates, treated with nuclease P1, and, as illustrated here, analyzed by HPLC.

Figure 7

Hypothetical mechanism for NAT2-catalyzed bioactivation of N-NO-MeIQx (shown) and N-NO-IQ. N-Acetylation of the amino N of the N-nitroso group generates a nitrosoacyl heterocyclic amine. Migration of the acetyl group from the amino N to the O atom of the nitroso group generates a reactive intermediate which decomposes by loss of acetate anion to give a diazonium ion.

Figure 8

Model of the postulated relationships among chronic inflammation/infection/injury, well-done red meat in the diet, and development of colon cancer. The inflammatory process leads to expression of inducible nitric oxide synthase (iNOS), which converts arginine (L-arg) to nitric oxide. NO and other factors generated during inflammation, such as peroxidases, hydrogen peroxide, hypochlorite, reactive nitrogen-oxygen species (RNOS), and acid pH (pH 5.5), facilitate this process in a temporal and spatial manner [, –55]. Well-done red meat in the diet is a source of exposure to heterocyclic amines (HCA). The RNOS species nitrosate HCA to produce N-nitroso derivatives (N-NO-HCA). These N-nitroso compounds, which may be further activated by acetylation or oxidation, bind to DNA and cause damage that contributes to somatic mutation and carcinogenesis.