N-Acetyltransferase (Nat) 1 and 2 Expression in Nat2 Knockout Mice

Abstract

Arylamine N-acetyltransferases (Nat) 1 and 2 catalyze the Nacetylation of aromatic amine and hydrazine drugs and carcinogens. After N-hydroxylation, they also catalyze the metabolic activation of N-hydroxy-arylamines via O-acetylation. Functional characterization of mouse Nat1 and Nat2 was investigated in an Nat2 knockout (KO) model and compared with the wild-type (WT) strain. Nat1-and Nat2-specific mRNA, determined by quantitative real-time polymerase chain reaction, was detected in all tissues examined and did not differ significantly (p Ͼ 0.05) between Nat2 KO and WT mice. Nat1 catalytic activity was present in all tissues examined and did not differ significantly (p Ͼ 0.05) between the Nat2 KO and WT mice. In contrast, Nat2 catalytic activity was present in all tissues examined from male WT mice but was below the limit of detection in all tissues of Nat2 KO mice. N-acetyltransferase activity toward the aromatic amine carcinogen 4-aminobiphenyl and O-acetyltransferase activity toward its proximate metabolite N-hydroxy-4-aminobiphenyl were both present in tissue cytosols of WT mice but were undetectable in Nat2 KO mice. Nat2 protein was readily detectable in liver cytosols of WT mice but not in liver cytosols from Nat2 KO mice. Since the reductions in Nat2 activity correlated with reductions in Nat2-specific protein but not mRNA, these results strongly suggest that insertion of the LacZ ablation cassette eliminated Nat2 protein and catalytic activity via disruption of the Nat2 protein, without significantly affecting transcription rates or transcript stability. The Nat2 KO model will be useful in future studies to assess the role of Nat2 in arylamine carcinogenesis.

Acute murine colitis reduces colonic 5-aminosalicylic acid metabolism by regulation of N-acetyltransferase-2

“…Unfortunately, this has only been examined in small studies using IBD patients with diverse clinical status, and results are conflicting. It has been reported that IBD patients with active disease have a lowered plasma Ac-5-ASA-to-5-ASA ratio vs. healthy controls (19), but this conflicts with observations of others who saw no change in 5-ASA acetylation rates in colonic biopsy homogenates or isolated colonocytes from IBD patients (2,36,41). At present, we cannot firmly predict if decreased 5-ASA metabolism is a reproducible finding in inflamed tissue or if such a change in metabolism would improve or reduce the clinical benefit of 5-ASA therapy.…”

“…Total RNA was extracted (TRI Reagent; Molecular Research Center, Cincinnati, OH) and then reverse transcribed (iScript cDNA Synthesis Kit; Bio-Rad). cDNA was amplified by real-time PCR using primers for mnat2 (41) and gapdh using a Fast SYBR Green Master Mix kit (AB Applied Biosystems, Foster City, CA) in a thermal cycler (StepOne Real-Time PCR System, with StepOne Software version 2.1; AB Applied BioSystems). Raw cycle threshold values (C t values) obtained from mnat target samples were deducted from the Ct value obtained from internal control (gapdh) amplification and calculated relative to a positive control (liver), using the ⌬⌬Ct method as follows: ⌬⌬Ct ϭ (Ct target Ϫ Ct target GAPDH) Ϫ (Ct liver Ϫ Ct liver GAPDH).…”

Functional Analysis of the Human N-Acetyltransferase 1 Major Promoter: Quantitation of Tissue Expression and Identification of Critical Sequence Elements

“…4) and matches well with the M00196 Sp1 consensus matrix. Mouse Nat1 and Nat2 are both expressed in a wide range of tissues (Sugamori et al, 2003;Loehle et al, 2006), but little is known concerning tissue-specific expression of the bovine or rat genes. The human NAT2 promoter is notably divergent at the Sp1 site location, however, and shows multiple sequence discordances with highly conserved positions in the M00196 matrix.…”

“…In each gene, this segment includes approximately 800 bp of genomic sequence upstream of the known or suspected transcription start site region. Although no spliced mouse Nat1 cDNA has been reported, expression of the Nat1 isoenzyme and Nat1 mRNA in mouse tissues is well documented (Hein et al, 1988;McQueen and Chau, 2003;Loehle et al, 2006). A candidate promoter region for mouse Nat1 was detected by alignment of the 1200-bp rat promoter segment within a 12.5-kb mouse genomic segment upstream of the mouse Nat1 coding exon.…”

Identification ofN-Acetyltransferase 2 (NAT2) Transcription Start Sites and Quantitation ofNAT2-Specific mRNA in Human Tissues

“…A large number of studies have also examined the possible involvement of NAT2 in the etiology of cancers of various other organs (reviewed in Hein et al, 2000a,b). A critical question is whether vulnerability to neoplastic transformation is related to specific expression of NAT2 in the target organ (Williams, 2001), and studies have investigated NAT2 expression in various human (reviewed in Boukouvala and Fakis, 2005) and recently in Syrian hamster and mouse (Loehle et al, 2006) tissues. We hypothesized that human NAT2 expression is highest in liver and gut but is also present in other human tissues.…”