A genetic polymorphism at the NAT2 gene locus, encoding for polymorphic N-acetyltransferase (NAT2), segregates individuals into rapid, intermediate or slow acetylator phenotypes. Both rapid and slow acetylator phenotypes have been associated with increased incidence of cancer in certain target organs related to arylamine exposure, suggesting a role for acetylation in both the activation and deactivation of arylamine carcinogens. A second gene (NAT1) encodes for a different acetyltransferase isozyme (NAT1) that is not subject to the classical acetylation polymorphism. In order to assess the relative ability of NAT1 and NAT2 to activate and deactivate arylamine carcinogens, we tested the capacity of recombinant human NAT1 and NAT2, expressed in Escherichia coli XA90 strains DMG100 and DMG200 respectively, to catalyze the N-acetylation (deactivation) and O-acetylation (activation) of a variety of carbocyclic and heterocyclic arylamine carcinogens. Both NAT1 and NAT2 catalyzed the N-acetylation of each of the 17 arylamines tested. Rates of N-acetylation by NAT1 and NAT2 were considerably lower for heterocyclic arylamines such as 2-amino-3-methyl-imidazo[4,5-f]quinoline (IQ), particularly those (e.g. IQ) with steric hindrance to the exocyclic amino group. For carbocyclic arylamines such as 4-aminobiphenyl and beta-naphthylamine, the apparent affinity was significantly (P < 0.05) higher for NAT2 than NAT1. NAT1/NAT2 activity ratios and clearance calculations suggest a significant role for the polymorphic NAT2 in the N-acetylation of carbocyclic arylamine carcinogens. Both NAT1 and NAT2 catalyzed acetyl coenzyme A-dependent O-acetylation of N-hydroxy-2-aminofluorene and N-hydroxy-4-aminobiphenyl to yield DNA adducts. NAT1 catalyzed paraoxon-resistant, intramolecular N,O-acetyltransferase-mediated activation of N-hydroxy-2-acetylaminofluorene and N-hydroxy-4-acetylaminobiphenyl at low rates; catalysis by NAT2 was not readily detectable in the presence of paraoxon. In summary these studies strongly suggest that the human acetylation polymorphism influences both the metabolic activation (O-acetylation) and deactivation (N-acetylation) of arylamine carcinogens via polymorphic expression of NAT2. These findings lend mechanistic support for human epidemiological studies suggesting associations between both rapid and slow acetylator phenotype and cancers related to arylamine exposure.

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Cutaneous reactions are the most common manifestation of delayed-type hypersensitivity caused by sulfamethoxazole and dapsone. In light of the recognized metabolic and immunologic activity of the skin, we investigated the potential role of normal human epidermal keratinocytes in the development of these reactions. Adult and neonatal normal human epidermal keratinocytes metabolized sulfamethoxazole and dapsone to N-4-hydroxylamine and N-acetyl derivatives in a time-dependent manner. The latter was catalyzed by N-acetyltransferase 1 alone as normal human epidermal keratinocytes did not express mRNA for N-acetyltransferase 2. Investigation of metabolism-dependent toxicity of sulfamethoxazole and dapsone, and subsequent incubation of normal human epidermal keratinocytes with the respective hydroxylamine metabolites, demonstrated that these cells were resistant to the cytotoxic effects of sulfamethoxazole hydroxylamine but not dapsone hydroxylamine. With prior depletion of glutathione, however, normal human epidermal keratinocytes became susceptible to the toxicity of sulfamethoxazole hydroxylamine. Covalent adduct formation by sulfamethoxazole hydroxylamine was detected in normal human epidermal keratinocytes, even in the absence of cell death, and was increased with glutathione depletion. Major protein targets of sulfamethoxazole hydroxylamine were observed in the region of 160, 125, 95, and 57 kDa. Dapsone hydroxylamine also caused covalent adduct formation in normal human epidermal keratinocytes. Together, these observations provide a basis for our hypothesis that normal human epidermal keratinocytes are involved in the initiation and propagation of a cutaneous hypersensitivity response to these drugs.

Cisplatin, a chemotherapeutic used for the treatment of solid cancers, has nephrotoxic side effects leading to acute kidney injury (AKI). Cisplatin cannot be given to patients that have comorbidities that predispose them to an increased risk for AKI. Even without these comorbidities, 30% of patients administered cisplatin will develop kidney injury, requiring the oncologist to withhold or reduce the next dose, leading to a less effective therapeutic regimen. Although recovery can occur after one episode of cisplatin-induced AKI, longitudinal studies have indicated that multiple episodes of AKI lead to the development of chronic kidney disease, an irreversible disease with no current treatment. The standard mouse model of cisplatin-induced AKI consists of one high dose of cisplatin (>20 mg/kg) that is lethal to the animal 3 days later. This model does not accurately reflect the dosing regimen patients receive nor does it allow for the long-term study of kidney function and biology. We have developed a repeated dosing model whereby cisplatin is given once a week for 4 wk. Comparison of the repeated dosing model with the standard dosing model demonstrated that inflammatory cytokines and chemokines were induced in the repeated dosing model, but levels of cell death were lower in the repeated dosing model. The repeated dosing model had increased levels of fibrotic markers (fibronectin, transforming growth factor-β, and α-smooth muscle actin) and interstitial fibrosis. These data indicate that the repeated dosing model can be used to study the AKI to chronic kidney disease progression as well as the mechanisms of this progression.

Aim Humans exhibit genetic polymorphism in NAT2 resulting in rapid, intermediate and slow acetylator phenotypes. Over 65 NAT2 variants possessing one or more SNPs in the 870-bp NAT2 coding region have been reported. The seven most frequent SNPs are rs1801279 (191G>A), rs1041983 (282C>T), rs1801280 (341T>C), rs1799929 (481C>T), rs1799930 (590G>A), rs1208 (803A>G) and rs1799931 (857G>A). The majority of studies investigate the NAT2 genotype assay for three SNPs: 481C>T, 590G>A and 857G>A. A tag-SNP (rs1495741) recently identified in a genome-wide association study has also been proposed as a biomarker for the NAT2 phenotype. Materials & methods Sulfamethazine N-acetyltransferase catalytic activities were measured in cryopreserved human hepatocytes from a convenience sample of individuals in the USA with an ethnic frequency similar to the 2010 US population census. These activities were segregated by the tag-SNP rs1495741 and each of the seven SNPs described above. We assessed the accuracy of the tag-SNP and various two-, three-, four- and seven-SNP genotyping panels for their ability to accurately infer NAT2 phenotype. Results The accuracy of the various NAT2 SNP genotype panels to infer NAT2 phenotype were as follows: seven-SNP: 98.4%; tag-SNP: 77.7%; two-SNP: 96.1%; three-SNP: 92.2%; and four-SNP: 98.4%. Conclusion A NAT2 four-SNP genotype panel of rs1801279 (191G>A), rs1801280 (341T>C), rs1799930 (590G>A) and rs1799931 (857G>A) infers NAT2 acetylator phenotype with high accuracy, and is recommended over the tag-, two-, three- and (for economy of scale) the seven-SNP genotyping panels, particularly in populations of non-European ancestry.