Quantitative tissue and gene-specific differences and developmental changes in Nat1, Nat2, and Nat3 mRNA expression in the rat.

Human N-acetyltransferase 1 (NAT1) and 2 (NAT2) are important phase II enzymes involved in the biotransformation of xenobiotics. In toxicity and carcinogenicity studies, functional polymorphism of rat N-acetyltransferase is considered a model for similar human variability. To accurately quantitate expression of the three rat N-acetyltransferases, we developed sensitive, specific assays for Nat1, Nat2, and Nat3 mRNAs. In male F344 rats, tissue-specific expression varied over a limited range for both Nat1 (approximately 19-fold) and Nat2 (approximately 30-fold), with the highest expression of both genes in colon. Expression of Nat3 mRNA was at least 2 to 3 orders of magnitude less than that of Nat1 or Nat2. Comparison of Nat1 and Nat2 mRNA expression in bladder, colon, liver, and lung of male and female F344 rats detected no significant gender-specific difference. In Sprague-Dawley and F344 rats ranging in age from neonate to mature adult, colon showed a >10-fold increase in Nat2 during the first postnatal month that did not correlate with changes in Nat1. In contrast, Nat2 showed no developmental change in Sprague-Dawley or F344 liver as Nat1 increased modestly. These measures of rat Nat expression confirm that Nat3 expression is negligible and that Nat1 and Nat2 are the primary determinants of arylamine acetylation activity in all tissues tested. The findings demonstrate differential tissue-specific and developmental regulation of the rat Nat1 and Nat2 genes and contribute to more complete understanding of tissue-, gender-, and development-specific expression patterns of the cognate N-acetyltransferase genes of humans and other species.

Structure/function evaluations of single nucleotide polymorphisms in human N-acetyltransferase 2.

Arylamine N-acetyltransferase 2 (NAT2) modifies drug efficacy/toxicity and cancer risk due to its role in bioactivation and detoxification of arylamine and hydrazine drugs and carcinogens. Human NAT2 alleles possess a combination of single nucleotide polymorphisms (SNPs) associated with slow acetylation phenotypes. Clinical and molecular epidemiology studies investigating associations of NAT2 genotype with drug efficacy/toxicity and/or cancer risk are compromised by incomplete and sometimes conflicting information regarding genotype/phenotype relationships. Studies in our laboratory and others have characterized the functional effects of SNPs alone, and in combinations present in alleles or haplotypes. We extrapolate this data generated following recombinant expression in yeast and COS-1 cells to assist in the interpretation of NAT2 structure. Whereas previous structural studies used homology models based on templates of N-acetyltransferase enzyme crystal structures from various prokaryotic species, alignment scores between bacterial and mammalian N-acetyltransferase protein sequences are low (approximately 30%) with important differences between the bacterial and mammalian protein structures. Recently, the crystal structure of human NAT2 was released from the Protein Data Bank under accession number 2PFR. We utilized the NAT2 crystal structure to evaluate the functional effects of SNPs resulting in the protein substitutions R64Q (G191A), R64W (C190T), I114T (T341C), D122N (G364A), L137F (A411T), Q145P (A434C), E167K (G499A), R197Q (C590A), K268R (A803G), K282T (A845C), and G286E (G857A) of NAT2. This analysis advances understanding of NAT2 structure-function relationships, important for interpreting the role of NAT2 genetic polymorphisms in bioactivation and detoxification of arylamine and hydrazine drugs and carcinogens.

Structure-function analyses of single nucleotide polymorphisms in human N-acetyltransferase 1.

Human N-acetyltransferase 1 (NAT1) alleles are characterized by one or more single nucleotide polymorphisms (SNPs) associated with rapid and slow acetylation phenotypes. NAT1 both activates and deactivates arylamine drugs and carcinogens, and NAT1 polymorphisms are associated with increased frequencies of many cancers and birth defects. The recently resolved human NAT1 crystal structure was used to evaluate SNPs resulting in the protein substitutions R64W, V149I, R187Q, M205V, S214A, D251V, E261K, and I263V. The analysis enhances knowledge of NAT1 structure-function relationships, important for understanding associations of NAT1 SNPs with genetic predisposition to cancer, birth defects, and other diseases.

Tissue expression and genomic sequences of rat N-acetyltransferases rNat1, rNat2, rNat3, and Functional characterization of a novel rNat3*2 genetic variant.

Human arylamine N-acetyltransferases NAT1 and NAT2 are highly polymorphic genes that modify individual susceptibility to cancers caused by exposure to arylamine procarcinogens. Strong similarities exist between rat Nats and human NATs, and rat Nat2 polymorphisms result in slow acetylator phenotype. Recently, a third rat Nat, rNat3*1, was reported. Although in vivo toxicological and carcinogenic studies are often conducted in rats, relatively little is known about Nat sequences among available inbred rat strains. We report here that rNat1 and rNat2 open reading frames (ORFs) in 12 inbred rat strains (ACI, BN, BUF, CDF, COP, DA, LEW, LOU/M, MW, PVG, SHR, WF) corresponded to reference rNat1*13 and rNat2*20. While 10 of the 12 strains had reference rNat3*1 ORFs, strains ACI and COP had a variant rNat3*2 ORF characterized by a G619>T transversion (A207S). The rNat3*2 single nucleotide polymorphism reduced Nat3 protein levels and N- and O-acetyltransferase activity when recombinantly expressed in bacteria. Recombinant expression of rNat3 1 and rNat3 2 in COS-1 cells yielded equivalent protein levels but undetectable catalytic activities. Relative tissue expressions of rNat1, rNat2, and rNat3 mRNAs were assessed in liver and 12 extrahepatic tissues (lung, spleen, kidney, heart, esophagus, stomach, urinary bladder, prostate, colon, duodenum, jejunum, ileum) from male F344 rats exsanguinated prior to sacrifice. Semiquantitative RT-PCR experiments demonstrated that the relative expression of the rNat transcripts in liver and 12 extrahepatic tissues was rNat1 > rNat2, while rNat3 transcripts were not detected. This study concludes that rNat1 and rNat2 are primarily responsible for acetylation phenotype in rats.

Computational and experimental analyses of mammalian arylamine N-acetyltransferase structure and function.

Arylamine N-acetyltransferases (NATs) play an important role in the metabolism of arylamine and hydrazine drugs and many arylamine procarcinogens. The two human N-acetyltransferases, NAT1 and NAT2, are widely distributed in human tissues and are highly polymorphic. Although many xenobiotic procarcinogens and drugs are known mammalian NAT substrates, it is unclear what physiological roles these enzymes might play, what endogenous substrates they primarily act upon, or the mechanisms underlying the functional effects of specific NAT gene coding region single-nucleotide polymorphisms. Analyses of mammalian NAT protein structures can greatly help to answer these questions. Homology modeling techniques can be used to approximate mammalian NAT structures using known bacterial NAT crystal structures as templates. In comparison to the bacterial template NATs used for homology modeling, mammalian NATs have a 17-residue insert of unknown structure and function. Homology modeling analyses yielded two different alignments (Modeler 8v1 or 3DCoffee algorithms) that placed this insert in two likely alternative locations. Secondary structure prediction techniques and experimental analyses of a series of human NAT2 mutants with artificial deletions/replacements of the insert region distinguished one of these alternatives as the most likely insert location and provided a better understanding of its structure and function. This study demonstrates both the utility and limitations of computational structural modeling with proteins that differ as much as the mammalian and bacterial NATs.

Identification and characterization of functional rat arylamine N-acetyltransferase 3: comparisons with rat arylamine N-acetyltransferases 1 and 2.

Arylamine N-acetyltransferases (NATs; EC 2.3.1.5) catalyze both the N-acetylation and O-acetylation of arylamines and N-hydroxyarylamines. Humans possess two functional N-acetyltransferase genes, NAT1 and NAT2, as well as a nonfunctional pseudogene, NATP. Previous studies have identified Nat1 and Nat2 genes in the rat. In this study, we identified and characterized a third rat N-acetyltransferase gene (Nat3) consisting of a single open reading frame of 870 base pairs encoding a 290-amino acid protein, analogous to the previously identified human and rat N-acetyltransferase genes. Rat Nat3 nucleotide sequence was 77.2 and 75.9% identical to human NAT1 and NAT2, respectively. Rat Nat3 amino acid sequence was 68.6 and 67.2% identical to human NAT1 and NAT2, respectively. Rat Nat1, Nat2, and Nat3 were each cloned and recombinantly expressed in Escherichia coli. Recombinant rat Nat3 exhibited thermostability intermediate between recombinant rat Nat1 and Nat2. Recombinant rat Nat3 was functional and catalyzed the N-acetylation of several arylamine substrates, including 3-ethylaniline, 3,5-dimethylaniline, 5-aminosalicylic acid, 4-aminobiphenyl, 4,4'-methylenedianiline, 4,4'-methylenebis(2-chloroaniline), and 2-aminofluorene, and the O-acetylation of N-hydroxy-4-aminobiphenyl. The relative affinities of arylamine carcinogens such as 4-aminobiphenyl, N-hydroxy-4-aminobiphenyl, and 2-aminofluorene for N- and O-acetylation via recombinant rat Nat3 were comparable with recombinant rat Nat1 and higher than for recombinant rat Nat2. This study is the first to report a third arylamine N-acetyltransferase isozyme with significant functional capacity.

4,4'-methylenedianiline-induced hepatotoxicity is modified by N-acetyltransferase 2 (NAT2) acetylator polymorphism in the rat.

4,4'-Methylenedianiline (MDA) is widely used in the manufacturing of polyurethane foam, epoxy resins, and polymers. Exposure to MDA induces liver damage in humans and rats. MDA undergoes N-acetylation catalyzed by N-acetyltransferase 1 (NAT1) and 2 (NAT2) in the liver. Both human and rat NAT2 are polymorphic, and human NAT2 genetic polymorphism modifies the frequency and/or severity of drug and xenobiotic toxicity in human populations. Recombinant expression of rat Nats in Escherichia coli showed that MDA was acetylated by both recombinant rat Nat1 and Nat2 and was catalyzed at substantially higher rates by rapid acetylator Nat2 compared with slow acetylator Nat2. Rapid acetylator F344 rat liver cytosols catalyzed the N-acetylation of MDA at significantly higher rates than those from slow acetylator Wistar-Kyoto (WKY) inbred rats. To test the effect of NAT2 genetic polymorphism on hepatotoxicity from acute MDA exposure, we compared hepatotoxicity in rapid (F344) and slow (WKY) Nat2 acetylator inbred rats that were administered MDA. Based on the results of dose-response studies ranging up to 150 mg/kg MDA administered by intragastric gavage, the effect of a moderately hepatotoxic dose (37.5 mg/kg) was compared in rapid versus slow acetylator rats. Plasma alanine transaminase enzyme activities were approximately 5-fold higher (p < 0.05) in rapid versus slow acetylator rats after MDA treatment, and necrotizing hepatitis with portal damage consisting of bile ductular necrosis, portal expansion, and inflammation was clearly more prominent. These results suggest that acetylator phenotype is an important factor for susceptibility toward MDA hepatotoxicity.