Differences between human slow N-acetyltransferase 2 alleles in levels of 4-aminobiphenyl-induced DNA adducts and mutations.

Aromatic amines such as 4-aminobiphenyl (ABP) require biotransformation to exert their carcinogenic effects. Genetic polymorphisms in biotransformation enzymes such as N-acetyltransferase 2 (NAT2) may modify cancer risk following exposure. Nucleotide excision repair-deficient Chinese hamster ovary (CHO) cells stably transfected with human cytochrome P4501A1 (CYP1A1) and a single copy of either NAT2*4 (rapid acetylator), NAT2*5B (common Caucasian slow acetylator), or NAT2*7B (common Asian slow acetylator) alleles (haplotypes) were treated with ABP to test the effect of NAT2 polymorphisms on DNA adduct formation and mutagenesis. ABP N-acetyltransferase catalytic activities were detectable only in cell lines transfected with NAT2 and were highest in cells transfected with NAT2*4, lower in cells transfected with NAT2*7B, and lowest in cells transfected with NAT2*5B. Following ABP treatment, N-(deoxyguanosin-8-yl)-4-aminobiphenyl (dG-C8-ABP) was the primary adduct formed. Cells transfected with both CYP1A1 and NAT2*4 showed the highest concentration-dependent cytotoxicity, hypoxanthine phosphoribosyl transferase (hprt) mutants, and dG-C8-ABP adducts. Cells transfected with CYP1A1 and NAT2*7B showed lower levels of cytotoxicity, hprt mutagenesis, and dG-C8-ABP adducts. Cells transfected with CYP1A1 only or cells transfected with both CYP1A1 and NAT2*5B did not induce cytotoxicity, hprt mutagenesis or dG-C8-ABP adducts. ABP-DNA adduct levels correlated very highly (r>0.96) with ABP-induced hprt mutant levels following each treatment. The results of the present study suggest that investigations of NAT2 genotype or phenotype associations with disease or toxicity could be more precise and reproducible if heterogeneity within the "slow" NAT2 acetylator phenotype is considered and incorporated into the study design.

Effect of N-acetyltransferase 2 polymorphism on tumor target tissue DNA adduct levels in rapid and slow acetylator congenic rats administered 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine or 2-amino-3,8-dimethylimidazo-[4,5-f]quinoxaline.

2-Amino-3,8-dimethylimidazo-[4,5-f]quinoxaline (MeIQx) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) are suspected human carcinogens generated in well done meats. After N-hydroxylation, they are O-acetylated by N-acetyltransferase 2 (NAT2) to electrophiles that form DNA adducts. dG-C8-MeIQx and dG-C8-PhIP adducts have been identified in human tissues. In the female rat, administration of PhIP leads to mammary and colon tumors, whereas MeIQx induces liver tumors. Both humans and rats exhibit NAT2 genetic polymorphism yielding rapid and slow acetylator phenotypes. Because O-acetylation is an activation pathway, we hypothesized that MeIQx- and PhIP-induced DNA damage would be greater in tumor target tissues and higher in rapid than slow NAT2 acetylators. Adult female rapid and slow acetylator rats congenic at the Nat2 locus received a single dose of 25 mg/kg MeIQx or 50 mg/kg PhIP by gavage, and tissue DNA was isolated after 24 h. Deoxyribonucleoside adducts were identified and quantified by capillary liquid chromatography-tandem mass spectrometry using isotope dilution methods with deuterated internal standards. Major adducts were those bound to the C8 position of deoxyguanosine. dG-C8-PhIP DNA adducts were highest in colon, lowest in liver and did not significantly differ between rapid and slow acetylator congenic rats in any tissue tested. In contrast, dG-C8-MeIQx adducts were highest in liver and significantly (p < 0.001) higher in rapid acetylator liver than in slow acetylator liver. Our results are consistent with the tumor target specificity of PhIP and MeIQx and with increased susceptibility to MeIQx-induced liver tumors in rapid NAT2 acetylators.

Bone selective effect of an estradiol conjugate with a novel tetracycline-derived bone-targeting agent.

In this study a novel bone-targeting agent containing elements of the tricarbonylmethane system of ring A of tetracycline was developed and was shown to bind to the mineral constituent of bone, hydroxyapatite. Conjugation of this bone-targeting agent to estradiol resulted in a bone-targeted estrogen (BTE(2)-A1) with an enhanced ability to bind to hydroxyapatite. In an ovariectomized rat model of osteoporosis a partial separation of the skeletal effects of estradiol from the uterine effects was observed following subcutaneous administration of BTE(2)-A1. This novel bone-targeting estradiol delivery system has the potential to improve the safety profile of estradiol in the treatment of osteoporosis.

METHODS FOR AROMATIC AND HETEROCYCLIC AMINE CARCINOGEN-DNA ADDUCT ANALYSIS BY LIQUID CHROMATOGRAPHY-TANDEM MASS SPECTROMETRY.

Xenobiotic-DNA adducts are used as biomarkers to assess the genotoxic effects of carcinogens. Rats were dosed with 4-aminobiphenyl (4-ABP), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), or 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). DNA was isolated from the colons of vehicle and carcinogen-treated rats and digested using different nucleases and alkaline phosphatase. Deoxyribonucleoside adducts were quantified by capillary liquid chromatography-tandem mass spectrometry (LC-MS/MS) using isotope dilution methods with deuterated internal standards. Major adducts were those bound to the C8 position of deoxyguanosine. 3'- and 5'-Exonucleases were the most efficient nucleases at isolating dG-C8-ABP adducts. However, bulky adducts such as dG-C8-MeIQx and dG-C8-PhIP were better isolated using nuclease P1 rather than a combination of micrococcal nuclease and spleen phosphodiesterase. The use of DNase I enhanced the detection of all three adducts. We describe LC-MS/MS methods for DNA adduct detection and support the testing of different nucleases that increase DNA digestion efficiency and make available more DNA adducts for detection.