Identification of 2-amino-1,7-dimethylimidazo[4,5-g]quinoxaline: an abundant mutagenic heterocyclic aromatic amine formed in cooked beef.

A previously unknown isomer of the carcinogenic heterocyclic aromatic amine (HAA) 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (8-MeIQx) was recently discovered in the urine of meat eaters and subsequently detected in cooked ground beef (Holland, R.D., et al. (2004) Chem. Res. Toxicol. 17, 1121-1136). In this current investigation, the identity of the analyte was determined through a comparison of its chromatographic tR by HPLC and through UV and mass spectral comparisons to the synthesized isomers of 8-MeIQx. Angular tricyclic isomers of 8-MeIQx were excluded as potential structures of the newly discovered HAA, on the basis of dissimilar tR and product ion mass spectral data. The linear tricyclic isomers 2-amino-1,6-dimethylimidazo[4,5-g]quinoxaline (6-MeIgQx) and 2-amino-1,7-dimethylimidazo[4,5-g]quinoxaline (7-MeIgQx) were postulated as plausible structures. Both compounds were synthesized from 4-fluoro-5-nitro-benzene-1,2-diamine in five steps. The structure of the analyte was proven to be 7-MeIgQx, on the basis of co-injection of the compound with the synthetic isomers, and corroborated by comparisons of the UV and mass spectral data of the analyte and MeIgQx isomers. 7-MeIgQx induced 348 revertants/microg in the S. typhimurium tester strain YG1024, when liver S-9 homogenate of rats pretreated with polychlorinated biphenyls (PCBs) was used for bioactivation. This newly discovered 7-MeIgQx molecule is one of the most abundant HAAs formed in cooked ground beef patties and pan-fried scrapings. The human health risk of 7-MeIgQx requires investigation.

Mutagenic potency of food-derived heterocyclic amines.

The understanding of mutagenic potency has been primarily approached using "quantitative structure-activity relationships" (QSAR). Often this method allows the prediction of mutagenic potency of the compound based on its structure. But it does not give the underlying reason why the mutagenic activities differ. We have taken a set of heterocyclic amine structures and used molecular dynamic calculations to dock these molecules into the active site of a computational model of the cytochrome P4501A2 enzyme. The calculated binding strength using Boltzman distribution constants was then compared to the QSAR value (HF/6-31G* optimized structures) and the Ames/Salmonella mutagenic potency. Further understanding will only come from knowing the complete set of mutagenic determinants. These include the nitrenium ion half-life, DNA adduct half-life, efficiency of repair of the adduct, and ultimately fixation of the mutation through cellular processes. For two isomers, PhIP and 3-Me-PhIP, we showed that for the 100-fold difference in the mutagenic potency a 5-fold difference can be accounted for by differences in the P450 oxidation. The other factor of 20 is not clearly understood but is downstream from the oxidation step. The application of QSAR (chemical characteristics) to biological principles related to mutagenesis is explored in this report.

The effect of UDP-glucuronosyltransferase 1A1 expression on the mutagenicity and metabolism of the cooked-food carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine in CHO cells.

UDP-glucuronosyltransferase proteins (UGT) catalyze the glucuronidation of both endogenous and xenobiotic compounds. In previous studies, UGT1A1 has been implicated in the detoxification of certain food-borne carcinogenic-heterocyclic amines. To determine the importance of UDP-glucuronosyltransferase 1A1 (UGT1A1) in the biotransformation of the cooked-food carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), genetically modified CHO cells that are nucleotide excision repair-deficient, and express cytochrome P4501A2 (UV5P3 cell line) were transfected with a cDNA plasmid of human UGT1A1 to establish the UDP-glucuronosyltransferase 1A1 expressing 5P3hUGT1A1 cell line. Expression of the UGT1A1 gene was verified by screening neo gene expressing clonal isolates (G-418 resistant) for their sensitivity to cell killing from PhIP exposure. Five of 11 clones were chosen for further analysis due to their resistance to cell killing. Western blot analysis was used to confirm the presence of the UGT1A1 and CYP1A2 proteins. All five clones displayed a 52-kDa protein band, which corresponded to a UGT1A1 control protein. Only four of the clones had a protein band that corresponded to the CYP1A2 control protein. Correct fragment size of the cDNAs in the remaining four clones was confirmed by RT-PCR and quantification of the mRNA product was accomplished by real-time RT-PCR. Expression of UGT1A1 in the transfected cells was 10(4)-10(5)-fold higher relative to the UV5P3 parental cells. One clone (#14) had a 10-fold higher increase in expression at 1.47 x 10(5) over the other three clones. This clone was also the most active in converting N-hydroxy-PhIP to N-hydroxy-PhIP glucuronide conjugates in microsomal metabolism assays. Based on the D50 values, the cytotoxic effect of PhIP was decreased approximately 350-fold in the 5P3hUGT1A1 cells compared to the UV5P3 control cells. In addition, no significant increase in mutation frequency was observed in the transfected cells. These results clearly indicate that UGT1A1 plays a critical role in PhIP biotransformation, providing protection against PhIP-mediated cytotoxicity and mutagenicity.

Development and characterization of CHO repair-proficient cell lines for comparative mutagenicity and metabolism of heterocyclic amines from cooked food.

In order to understand the role of repair and metabolism in the mutagenicity of heterocyclic amines from cooked foods, we previously developed the nucleotide excision repair-deficient CHO 5P3NAT2 cell line engineered to coexpress the mouse CYP1A2 and human N-acetyltransferase genes. In the present study, we have made a matched repair-competent cell line by mutagenizing 5P3NAT2 cells with ethyl methanesulfonate and selecting for resistance to cytotoxicity by 2-amino-3-methylimidazo[4,5-f]quinoline (IQ). In the differential cytotoxicity (DC) assay, 4 out of 15 clones showed no cytotoxic effect with IQ at the highest dose (30 microg/ml) tested, in contrast to repair-deficient 5P3NAT2 cells, which showed approximately 100% cytotoxicity at 0.3 microg/ml. Subsequently, these IQ-resistant clones were examined for resistance to killing by UV irradiation. All four IQ-resistant clones, which show resistance to UV similar to that of repair-proficient AA8 cells, still express both the CYP1A2 and N-acetyltransferase genes. Sequence analysis of CXPD cDNA from the 5P3NAT2R9 clone revealed an A:T-->G:C reversion event at the site of the UV5 mutation. This base change results in reversion of the codon 116 tyrosine in UV5 cells back to the original cysteine in AA8 cells, thereby restoring wild-type CXPD activity and repair function. In contrast to 5P3NAT2 cells, the repair-proficient 5P3NAT2R9 revertant cell line shows little IQ-induced cell killing, and dramatically lower levels of induced mutation at the adenine phosphoribosyltransferase (Aprt) gene locus over the range of 2-40 microg/ml IQ. This matched pair of repair-proficient/deficient cell lines can provide insight not only into the genotoxicity of heterocyclic amine dietary carcinogens such as IQ and PhIP, but also into the effects of nucleotide excision repair on the ultimate mutagenicity of these compounds.