Role of Human N‐Acetyltransferase 2 Genetic Polymorphism on Aromatic Amine Carcinogen‐Induced DNA Damage and Mutagenicity in a Chinese Hamster Ovary Cell Mutation Assay

Abstract

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 NAT2*4 (rapid acetylator) or NAT2*5B (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/NAT2*4 acetylator cell line compared to the UV5, UV5/CYP1A2, and UV5/CYP1A2/NAT2*5B 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.

Human N-Acetyltransferase 1 and 2 Differ in Affinity Towards Acetyl-Coenzyme A Cofactor and N-Hydroxy-Arylamine Carcinogens

“…Our findings are consistent with the NAT2-genotype-dependent O-acetylation of N-OH-ABP observed in cryopreserved human hepatocytes (Doll et al, 2010) in which both NAT1 and NAT2 are expressed. The higher affinity of N-OH-AF and N-OH-ABP for human NAT2 also is consistent with recent findings in which CHO cells expressing CYP1A2 and rapid acetylator NAT2*4 experienced greater DNA adducts and mutations than CHO cells expressing CYP1A2 and slow acetylator NAT2*5B following incubations with low concentrations of AF and ABP (Baldauf et al, 2020).…”

“…N-OH-ABP metabolic activation via O-acetylation also has been reported in cryopreserved human hepatocytes (Doll et al, 2010). CHO cells expressing CYP1A2 and rapid acetylator NAT2*4 exhibit greater DNA adducts and mutations than CHO cells expressing CYP1A2 and slow acetylator NAT2*5B following incubations with low concentrations of 2aminofluorene (AF) and 4-aminobiphenyl (ABP) (Baldauf et al, 2020) suggesting an important role of NAT2-catalyzed O-acetylation in the metabolic activation of arylamine carcinogens in tissues expressing NAT2.…”

“…Metabolic activation of N-OH-AF and N-OH-ABP via O-acetylation is catalyzed by recombinant human NAT1 and NAT2 expressed in bacteria (Hein et al, 1993, Hein et al, 1995Doll and Hein, 2017), yeast (Fretland et al, 2002), and both COS-1 (Zang et al, 2007;Zhu et al, 2011) and Chinese hamster ovary (CHO) (Millner et al, 2012;Baldauf et al, 2020) cells. N-OH-ABP metabolic activation via O-acetylation also has been reported in cryopreserved human hepatocytes (Doll et al, 2010).…”

Combined in vitro and cell‐based selection display method producing specific binders against IL‐9 receptor in high yields

“…To increase binding specificity, we utilized protein-free blocking reagents in the ribosome display process. In the standard protocols, the target protein is immobilized on the surface of microplates and their surface is blocked by protein-based blockers, BSA protein [19,20], non-fat-skimmed milk [21] or gelatin [22,23]. These proteins can and apparently do serve as unwanted selection targets, lowering the specificity of the selected variants.…”