Generation and analysis of mice with a targeted disruption of the arylamine N-acetyltransferase type 2 gene

Cornish, Valerie; Pintér, Katalin; Boukouvala, Sotiria; Johnson, Nichola; Labrousse, Claire; Payton, Mark; Priddle, Helen; Smith, Andrew J.H.; E, Sim

doi:10.1038/sj.tpj.6500170

Cited by 51 publications

(59 citation statements)

References 34 publications

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“…23 Since these models have limitations, efforts have been underway in several laboratories to develop strains over-or underexpressing NAT genes. [19][20][21][22] The current study supports and expands previous work showing that it is very difficult to significantly raise NAT levels using transgenes. Viable animals generally had few copies of the transgene, regardless of whether it was human NAT1 or NAT2 (Tables 1 and 2).…”

Section: Discussionsupporting

confidence: 84%

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Only low levels of exogenous N-acetyltransferase can be achieved in transgenic mice

et al. 2005

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Therapeutic and environmental aromatic amines and hydrazines are substrates for the arylamine N-acetyltransferases (NAT). In all, 10 transgenic lines containing either the human NAT1 or NAT2 transgene were developed using multiple promoters. The presence of the transgene was confirmed by determining copy number, mRNA and enzyme activity. Despite some lines having high copy numbers of the transgene, only modest or no increases in enzymatic activity could be found in a variety of tissues. The NAT1 transgene could not be bred to homozygosity. The cytomegalovirus (CMV)-promoted NAT1 transgene increased endogenous Nat1 mRNA levels in liver and had little effect on endogenous Nat2 mRNA levels. The presence of the CMVpromoted NAT2 transgene appeared to suppress endogenous hepatic Nat2 mRNA, but did not alter Nat1 mRNA levels. The failure to achieve high expression of any of the transgenes suggests that overexpression of NAT genes may have harmful effects during development.

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Section: Discussionsupporting

confidence: 84%

“…18 The Nat2 and the Nat1,2 knockouts have no grossly observable phenotype. 19,20 Efforts to increase activity of NATs by the addition of transgenes have not been readily achieved. There was a 15-fold increase in sulfamethazine NAT activity in mouse prostate, using the prostate-specific probasin promoter to drive human NAT2.…”

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confidence: 99%

Only low levels of exogenous N-acetyltransferase can be achieved in transgenic mice

et al. 2005

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“…A: representative Western blot showing mNAT2 protein (red band) and ␤-actin (green band) detected on proximal (P), central (C), and distal (D) colon mucosa homogenates from a mouse with DSS colitis. mNAT2 protein was detected with antisera 195, previously validated in mouse tissues (12). L, liver homogenate.…”

Section: Discussionmentioning

confidence: 99%

“…Colon mucosal homogenates were separated using 10% SDS-PAGE (Bio-Rad, Hercules, CA) and transferred to a polyvinylidene fluoride membrane (Millipore, Billerica, MA). Membranes were incubated with a blocking buffer (Li-Cor Biosciences, Lincoln, NE) and then probed with rabbit antisera specific for mNAT2 protein (antiserum 195; Dr. E. Sim, University of Oxford, Oxford, UK) at 1:10,000 dilution (12) and Alexa Fluor 680 goat anti-rabbit (1:5,000; Invitrogen, Carlsbad, CA). Loading control was ␤-actin [primary antibody 1:20,000 mouse monoclonal (Cell Signaling Technology, Danvers, MA), secondary antisera 1:10,000 Alexa Fluor 800 goat anti-mouse (Invitrogen)].…”

Section: Methodsmentioning

confidence: 99%

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

Ramírez-Alcántara

Montrose

2014

American Journal of Physiology-Gastrointestinal and Liver Physi

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Ramírez-Alcántara V, Montrose MH. Acute murine colitis reduces colonic 5-aminosalicylic acid metabolism by regulation of N-acetyltransferase-2. Am J Physiol Gastrointest Liver Physiol 306: G1002-G1010, 2014. First published April 17, 2014 doi:10.1152/ajpgi.00389.2013.-Pharmacotherapy based on 5-aminosalicylic acid (5-ASA) is a preferred treatment for ulcerative colitis, but variable patient response to this therapy is observed. Inflammation can affect therapeutic outcomes by regulating the expression and activity of drug-metabolizing enzymes; its effect on 5-ASA metabolism by the colonic arylamine N-acetyltransferase (NAT) enzyme isoforms is not firmly established. We examined if inflammation affects the capacity for colonic 5-ASA metabolism and NAT enzyme expression. 5-ASA metabolism by colonic mucosal homogenates was directly measured with a novel fluorimetric rate assay. 5-ASA metabolism reported by the assay was dependent on Ac-CoA, inhibited by alternative NAT substrates (isoniazid, p-aminobenzoylglutamate), and saturable with Km (5-ASA) ϭ 5.8 M. A mouse model of acute dextran sulfate sodium (DSS) colitis caused pronounced inflammation in central and distal colon, and modest inflammation of proximal colon, defined by myeloperoxidase activity and histology. DSS colitis reduced capacity for 5-ASA metabolism in central and distal colon segments by 52 and 51%, respectively. Use of selective substrates of NAT isoforms to inhibit 5-ASA metabolism suggested that mNAT2 mediated 5-ASA metabolism in normal and colitis conditions. Western blot and real-time RT-PCR identified that proximal and distal mucosa had a decreased mNAT2 protein-to-mRNA ratio after DSS. In conclusion, an acute colonic inflammation impairs the expression and function of mNAT2 enzyme, thereby diminishing the capacity for 5-ASA metabolism by colonic mucosa. drug metabolism; fluorescence; dextran sulfate sodium; myeloperoxidase; kinetics; enzymatic assay; inflammatory bowel disease; enzyme isoform THE TWO MAJOR INFLAMMATORY bowel diseases (IBD), ulcerative colitis (UC) and Crohn's disease, afflict 1.4 million individuals in the United States (USA) and 2.2 million in Europe (42). To induce and maintain mucosal healing of the colonic mucosa, the major goal of UC therapy is to reduce or eliminate colonic inflammation. For mild to moderate inflammation, therapy based on the nonsteroidal anti-inflammatory compound 5-aminosalicylic acid (5-ASA) is frequently prescribed. In information compiled by the National Institutes of Health from the 2004 Verispan database of prescriptions filled at retail pharmacies in the USA, 5-ASA or its prodrugs accounted for 44% of Crohn's disease prescriptions and 81% of UC prescriptions (23). It is not well understood how 5-ASA induces its antiinflammatory effect on colonic mucosa, but multiple mechanisms have been proposed, including inhibition of intestinal macrophage chemotaxis (47), inhibition of proinflammatory cytokine release (25), inhibition of cyclooxygenase and prostaglandin pathways (28), inhibition of nuclear fa...

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“…These animal models offer the advantage of investigating genetic variation under controlled exposures and environmental and secondary genetic influences. We and others have generated knockout mouse models lacking both Nat1 and Nat2 (Sugamori et al, 2003) or Nat2 alone (Cornish et al, 2003). However, mice possess a third Nat gene, Nat3 (Kelly and Sim, 1994;Fretland et al, 1997).…”

mentioning

confidence: 99%

Effect of Arylamine AcetyltransferaseNat3Gene Knockout onN-Acetylation in the Mouse

Sugamori¹,

Brenneman²,

Wong³

et al. 2007

Drug Metab Dispos

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ABSTRACT:Arylamine N-acetyltransferases (NAT) catalyze the biotransformation of many important arylamine drugs and procarcinogens. NAT can either detoxify or activate procarcinogens, complicating the manner in which these enzymes may participate in enhancing or preventing toxic responses to particular agents. Mice possess three NAT isoenzymes: Nat1, Nat2, and Nat3. Whereas Nat1 and Nat2 can efficiently acetylate many arylamines, few substrates appear to be appreciably metabolized by Nat3. We generated a Nat3 knockout mouse strain and used it along with our double Nat1/2(؊/؊) knockout strain to further investigate the functional role of Nat3. Nat3(؊/؊) mice showed normal viability and reproductive capacity. Nat3 expression was very low in wild-type animals and completely undetectable in Nat3(؊/؊) mice. In contrast, greatly elevated expression of Nat3 transcript was observed in Nat1/2(؊/؊) mice. We used a transcribed marker polymorphism approach to establish that the increased expression of Nat3 in Nat1/2(؊/؊) mice is a positional artifact of insertion of the phosphoglycerate kinase-neomycin resistance cassette in place of the Nat1/Nat2 gene region and upstream of the intact Nat3 gene, rather than a biological compensatory mechanism. Despite the increase in Nat3 transcript, the N-acetylation of p-aminosalicylate, sulfamethazine, 2-aminofluorene, and 4-aminobiphenyl was undetectable either in vivo or in vitro in Nat1/2(؊/؊) animals. In parallel, no difference was observed in the in vivo clearance or in vitro metabolism of any of these substrates between wild-type and Nat3(؊/؊) mice. Thus, Nat3 is unlikely to play a significant role in the N-acetylation of arylamines either in wild-type mice or in mice lacking Nat1 and Nat2 activities.

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Generation and analysis of mice with a targeted disruption of the arylamine N-acetyltransferase type 2 gene

Cited by 51 publications

References 34 publications

Only low levels of exogenous N-acetyltransferase can be achieved in transgenic mice

Only low levels of exogenous N-acetyltransferase can be achieved in transgenic mice

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

Effect of Arylamine AcetyltransferaseNat3Gene Knockout onN-Acetylation in the Mouse

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