ABSTRACT
The effect of apocynin on the activity of arylamine N-acetyltransferases (NATs) in excised liver samples was examined using eighteen Sprague-Dawley rats. Three groups of six animals each were fed a normal diet alone or a treatment of 50 or 100 mg/kg/day of apocynin via gavages for eight (8) weeks. Chronic in vivo administration of apocynin led to significant (p < 0.001) reduction of in vitro liver NAT activity up to 93% as compared with untreated rats (18.80 ± 2.10 µmols p-anisidine/min/µg liver protein). In vitro exposure of untreated liver homogenates to apocynin led to a dose-dependent inhibition of NAT activity with IC50 = 0.69 ± 0.02 mM. In silico modelling of apocynin tautomers and radical species into human NAT crystal structures supported the hypothesis that thiol functionalities in NAT enzymes may be crucial in apocynin binding. The involvement of human NAT enzymes in different pathological conditions, such as cancer, has encouraged the research for selective NAT inhibitors in both humans and animal models with possible chemopreventive properties.
Subject(s)
Acetophenones/chemistry , Antineoplastic Agents/chemistry , Arylamine N-Acetyltransferase/antagonists & inhibitors , Enzyme Inhibitors/chemistry , Acetophenones/metabolism , Administration, Oral , Amino Acid Sequence , Animals , Antineoplastic Agents/metabolism , Arylamine N-Acetyltransferase/chemistry , Arylamine N-Acetyltransferase/metabolism , Catalytic Domain , Complex Mixtures/chemistry , Enzyme Inhibitors/metabolism , Humans , Isoenzymes/antagonists & inhibitors , Isoenzymes/chemistry , Isoenzymes/metabolism , Kinetics , Liver/chemistry , Liver/drug effects , Liver/enzymology , Male , Mice , Molecular Docking Simulation , Protein Binding , Protein Interaction Domains and Motifs , Protein Structure, Secondary , Rats , Rats, Sprague-Dawley , Sequence Alignment , Sequence Homology, Amino AcidABSTRACT
N-Acetyltransferase 2 (NAT2) metabolizes a variety of xenobiotics that includes many drugs, chemicals and carcinogens. This enzyme is genetically variable in human populations and polymorphisms in the NAT2 gene have been associated with drug toxicity and efficacy as well as cancer susceptibility. Here, we have focused on the identification of NAT2 variants in Brazilian individuals from two different regions, Rio de Janeiro and Goiás, by direct sequencing, and on the characterization of new haplotypes after cloning and re-sequencing. Upon analysis of DNA samples from 404 individuals, six new SNPs (c.29T>C, c.152G>T, c.203G>A, c.228C>T, c.458C>T and c.600A>G) and seven new NAT2 alleles were identified with different frequencies in Rio de Janeiro and Goiás. All new SNPs were found as singletons (observed only once in 808 genes) and were confirmed by three independent technical replicates. Molecular modeling and structural analysis suggested that p.Gly51Val variant may have an important effect on substrate recognition by NAT2. We also observed that amino acid change p.Cys68Tyr would affect acetylating activity due to the resulting geometric restrictions and incompatibility of the functional group in the Tyr side chain with the admitted chemical mechanism for catalysis by NATs. Moreover, other variants, such like p.Thr153Ile, p.Thr193Met, p.Pro228Leu and p.Val280Met, may lead to the presence of hydrophobic residues on NAT2 surface involved in protein aggregation and/or targeted degradation. Finally, the new alleles NAT2*6H and NAT2*5N, which showed the highest frequency in the Brazilian populations considered in this study, may code for a slow activity. Functional studies are needed to clarify the mechanisms by which new SNPs interfere with acetylation.