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

Area of Science:

• Biochemistry
• Pharmacology
• Toxicology

Background:

• Arylamine N-acetyltransferases (Nat) 1 and 2 are crucial enzymes involved in metabolizing drugs and carcinogens.
• These enzymes catalyze N-acetylation and O-acetylation, impacting the activation or detoxification of various compounds.
• Understanding the specific roles of Nat1 and Nat2 is essential for assessing risks associated with aromatic amine exposure.

Purpose of the Study:

• To functionally characterize mouse Nat1 and Nat2 enzymes.
• To investigate the efficacy of a Nat2 knockout (KO) mouse model.
• To evaluate the impact of Nat2 deficiency on arylamine metabolism and activity.

Main Methods:

• Quantitative real-time polymerase chain reaction (qPCR) to measure Nat1 and Nat2 mRNA levels.
• Assay of N-acetyltransferase and O-acetyltransferase catalytic activities in tissue cytosols.
• Comparison of enzyme activity and protein expression between Nat2 KO and wild-type (WT) mice.
• Detection of Nat2 protein using Western blotting in liver cytosols.

Main Results:

• Nat1 mRNA and catalytic activity were comparable between Nat2 KO and WT mice across all examined tissues.
• Nat2 catalytic activity and N-/O-acetyltransferase activities were absent in all tissues of Nat2 KO mice, but present in WT mice.
• Nat2 protein was detectable in WT mouse liver cytosols but absent in Nat2 KO mice.
• The absence of Nat2 activity correlated with reduced Nat2 protein, not mRNA, suggesting protein-level disruption.

Conclusions:

• The Nat2 knockout mouse model effectively eliminates Nat2 protein and catalytic activity.
• The LacZ insertion in the KO model disrupted Nat2 protein expression without significantly affecting mRNA levels.
• This validated Nat2 KO model is a valuable tool for future research into the role of Nat2 in arylamine-induced carcinogenesis.