N-acetyltransferase (NAT) 1 and NAT2 enzyme activities drive interindividual variability in sulfamethoxazole...

Sulfamethoxazole (SMX) is associated with idiosyncratic drug-induced liver injury, which remains difficult to predict. SMX is metabolized by N-acetyltransferases (NAT1/NAT2) to form N₄-acetyl sulfamethoxazole (NA-SMX), and by cytochrome P450-mediated oxidation to form SMX-hydroxylamine. This study aimed to characterize SMX metabolism in vitro and investigate how NAT1 and NAT2 variation influences NA-SMX formation, including the relationship between NAT2 protein levels and metabolite formation. Human liver microsomes, S9 fractions, and primary human hepatocytes were used to generate SMX metabolites. NA-SMX was the most abundant metabolite in primary human hepatocytes, showing 4.2-fold variability across n = 26 donors. Interestingly, NAT2 genotype-inferred acetylator phenotype did not reliably predict NA-SMX formation in 6 of 9 slow acetylators, whose formation exceeded the mean of intermediate acetylators. However, N-acetyl sulfamethazine (NA-SMZ) formation was accurately predicted using the NAT2 probe substrate, SMZ, revealing significant differences between NAT2 phenotype groups (P < .05). Activities of NAT1 and NAT2, as measured by p-aminobenzoic acid and SMZ N-acetylation, respectively, significantly correlated with NA-SMX formation (r = 0.576, P = .006; r = 0.459, P = .036). The stronger correlation with NAT1 activity supports the relationship of NAT1 to SMX metabolism. Novel targeted proteomic quantification of NAT2 showed significant correlations between NAT2 protein concentration and NAT2 activity (r = 0.823; P < .0001 and r = 0.734, P = .0002; for 2 peptides). This work demonstrates interindividual variability in SMX metabolism and highlights the importance of considering genetic and nongenetic factors in SMX-induced drug-induced liver injury risk. SIGNIFICANCE STATEMENT: This study provides new insights into sulfamethoxazole (SMX) metabolism using in vitro hepatic systems and quantifies interindividual variability in N₄-acetyl SMX formation. Although NAT2 genotype did not predict SMX slow acetylator metabolism in all individuals, N₄-acetyl SMX formation was significantly correlated with NAT1 and NAT2 enzyme activity. These findings show the importance of considering both genetic and phenotypic data to better understand SMX metabolism and individual risk for drug-induced liver injury.