Department of Pharmacology and Toxicology and James Graham Brown Cancer Center, Louisville, Kentucky.
Environ Mol Mutagen. 2020 Feb;61(2):235-245. doi: 10.1002/em.22331. Epub 2019 Sep 30.
Carcinogenic aromatic amines such as 4-aminobiphenyl (ABP) and 2-aminofluorene (AF) require metabolic activation to form electrophilic intermediates that mutate DNA leading to carcinogenesis. Bioactivation of these carcinogens includes N-hydroxylation catalyzed by CYP1A2 followed by O-acetylation catalyzed by arylamine N-acetyltransferase 2 (NAT2). To better understand the role of NAT2 genetic polymorphism in ABP- and AF-induced mutagenesis and DNA damage, nucleotide excision repair-deficient (UV5) Chinese hamster ovary (CHO) cells were stably transfected with human CYP1A2 and either NAT24 (rapid acetylator) or NAT25B (slow acetylator) alleles. ABP and AF both caused significantly (P < 0.001) greater mutagenesis measured at the hypoxanthine phosphoribosyl transferase (hprt) locus in the UV5/CYP1A2/NAT24 acetylator cell line compared to the UV5, UV5/CYP1A2, and UV5/CYP1A2/NAT25B cell lines. ABP- and AF-induced hprt mutant cDNAs were sequenced and over 80% of the single-base substitutions were at G:C base pairs. DNA damage also was quantified by γH2AX in-cell western assays and by identification and quantification of the two predominant DNA adducts, N-(deoxyguanosin-8-yl)-4-aminobiphenyl (dG-C8-ABP) and N-(deoxyguanosin-8-yl)-2-aminofluorene (dG-C8-AF) by liquid chromatography-mass spectrometry. DNA damage and adduct levels were dose-dependent, correlated highly with levels of hprt mutants, and were significantly (P < 0.0001) greater in the UV5/CYP1A2/NAT2*4 rapid acetylator cell line following treatment with ABP or AF as compared to all other cell lines. Our findings provide further clarity on the importance of O-acetylation in CHO mutagenesis assays for aromatic amines. They provide evidence that NAT2 genetic polymorphism modifies aromatic amine-induced DNA damage and mutagenesis that should be considered in human risk assessments following aromatic amine exposures. Environ. Mol. Mutagen. 61:235-245, 2020. © 2019 Wiley Periodicals, Inc.
致癌芳香胺如 4-氨基联苯 (ABP) 和 2-氨基芴 (AF) 需要代谢激活才能形成导致致癌的亲电中间体。这些致癌物质的生物活化包括 CYP1A2 催化的 N-羟化，然后是芳香胺 N-乙酰转移酶 2 (NAT2) 催化的 O-乙酰化。为了更好地了解 NAT2 遗传多态性在 ABP 和 AF 诱导的突变和 DNA 损伤中的作用，核苷酸切除修复缺陷 (UV5) 中国仓鼠卵巢 (CHO) 细胞被稳定转染了人 CYP1A2 和 NAT24(快速乙酰化酶)或 NAT25B(慢乙酰化酶)等位基因。与 UV5、UV5/CYP1A2 和 UV5/CYP1A2/NAT25B 细胞系相比，ABP 和 AF 均在 UV5/CYP1A2/NAT24 乙酰化酶细胞系中导致明显 (P<0.001) 更高的诱变，这是在次黄嘌呤磷酸核糖转移酶 (hprt) 基因座上测量的。测序 ABP 和 AF 诱导的 hprt 突变 cDNA，超过 80%的单碱基替换发生在 G:C 碱基对。通过 γH2AX 细胞内 Western 测定和通过液相色谱-质谱法鉴定和定量两种主要的 DNA 加合物 N-(脱氧鸟嘌呤-8-基)-4-氨基联苯 (dG-C8-ABP) 和 N-(脱氧鸟嘌呤-8-基)-2-氨基芴 (dG-C8-AF)，也定量了 DNA 损伤。DNA 损伤和加合物水平呈剂量依赖性，与 hprt 突变体水平高度相关，并且在用 ABP 或 AF 处理后，在 UV5/CYP1A2/NAT2*4 快速乙酰化酶细胞系中，与所有其他细胞系相比，显著更高 (P<0.0001)。我们的研究结果进一步阐明了 O-乙酰化在芳香胺 CHO 致突变试验中的重要性。它们提供了证据表明，NAT2 遗传多态性修饰了芳香胺诱导的 DNA 损伤和突变，这在芳香胺暴露后的人类风险评估中应予以考虑。环境。分子突变。61:235-245,2020. Wiley Periodicals, Inc.
