Mouse arylamine N-acetyltransferase 2 (Nat2) expression during embryogenesis: a potential marker for the developing neuroendocrine system.

Arylamine N-acetyltransferase (NAT) genes in humans and in rodents encode polymorphic drug metabolizing enzymes. Human NAT1 (and the murine equivalent mouse Nat2) is found early in embryonic development and is likely to have an endogenous role. We report the detailed expression of the murine gene (Nat2) and encoded protein in mouse embryos, using a transgenic mouse model bearing a lacZ transgene inserted into the coding region of mouse Nat2. In mouse embryos, the transgene was expressed in sensory epithelia, epithelial placodes giving rise to visceral sensory neurons, the developing pituitary gland, sympathetic chain and urogenital ridge. In Nat2+/+ mice, the presence and activity of Nat2 protein was detected in these tissues and their adult counterparts. Altered expression of the human orthologue in breast tumours, in which there is endocrine signalling, suggests that human NAT1 should be considered as a potential biomarker for neuroendocrine tissues and tumours.

Deletion of a xenobiotic metabolizing gene in mice affects folate metabolism.

The mouse arylamine N-acetyltransferase 2 (Nat2) and its homologue (NAT1) in humans are known to detoxify xenobiotic arylamines and are also thought to play a role in endogenous metabolism. Human NAT1 is highly over-expressed in estrogen receptor positive breast tumours and is implicated in susceptibility to neural tube defects. In vitro assays have suggested an endogenous role for human NAT1 in folate metabolism, but in vivo evidence to support this hypothesis has been lacking. Mouse Nat2 provides a good model to study human NAT1 as it shows similar expression profiles and substrate specificities. We have generated transgenic mice lacking a functional Nat2 gene and compared the urinary levels of acetylated folate metabolite para-aminobenzoylglutamate in Nat2 knockout and Nat2 wild-type mice. These results support an in vivo role for mouse Nat2/human NAT1 in folate metabolism. In addition, effects of the Nat2 deletion on sex ratios and neural tube development are described.

N-acetyltransferase (Nat) 1 and 2 expression in Nat2 knockout mice.

Arylamine N-acetyltransferases (Nat) 1 and 2 catalyze the N-acetylation 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 > 0.05) between Nat2 KO and WT mice. Nat1 catalytic activity was present in all tissues examined and did not differ significantly (p > 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.

Arylamine N-acetyltransferase 2 expression in the developing heart.

Murine arylamine N-acetyltransferase 2 (NAT2) is expressed in the developing heart and in the neural tube at the time of closure. Classically described as a xenobiotic metabolizing enzyme, there is increasing evidence for a distinct biological role for murine NAT2. We have characterized the expression of arylamine N-acetyltransferase 2 during cardiogenesis, mapping its expression in vivo, using a lacZ insertion deletion, and also in vitro, by measuring NAT2 enzyme activity. These findings show that cardiac Nat2 expression is both temporally and spatially regulated during development. In neonatal mice, cardiac Nat2 expression is most extensive in the central fibrous body and is evident in the atrioventricular valves and the valves of the great vessels. Whereas Nat2 expression is not detected in ventricular myocardial cells, Nat2 is strongly expressed in scattered cells in the region of the sinus node, the epicardium of the right atrial appendage, and in the pulmonary artery. Expression of active NAT2 protein is maximal when the developing heart attains the adult circulation pattern and moves from metabolizing glucose to fatty acids. NAT2 acetylating activity in cardiac tissue from Nat2(-/-) and Nat2(+/-) mice indicates a lack of compensating acetylating activity either from other acetylating enzymes or by NAT2 encoded by the wild-type Nat2 allele in Nat2(+/-) heterozygotes. The temporal and spatial control of murine Nat2 expression points to an endogenous role distinct from xenobiotic metabolism and indicates that Nat2 expression may be useful as a marker in cardiac development.