Treatment of Rats with Apocynin Has Considerable Inhibitory Effects on Arylamine N-Acetyltransferase Activity in the Liver

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.

Figure 1. Impact of a diet containing apocynin on the activity of rat liver NATs.

The NAT activity of the liver S9 fractions (3–8 μg/μl protein) obtained from rats fed a diet free of apocynin (untreated rats) or varying amounts of apocynin (treated rats) was determined ex vivo using pANS and AcCoA, as described in methods. The average value of NAT activity for the untreated group was compared with each NAT activity value for the treated groups, and statistical significance at p < 0.001 is indicated by an asterisk (*). Assays were conducted in triplicate and all values are expressed as mean ± standard deviation values.

Figure 2. Impact of varying preincubation times of liver homogenates with apocynin and AcCoA on the inhibition of rat liver NAT activity.

NAT activity in the S9 fractions (9.60 μg protein) from untreated rats was determined in vitro as described in methods (A). The inhibitory potency of apocynin (0.49 mM) was tested at different assay conditions: in assays (B–D) apocynin was preincubated with S9 fractions for 0, 5′ or 15′ respectively; in assays E and F, S9 fractions were preincubated with AcCoA for 5′ or 15′ respectively. Appropriate enzymatic control assays were performed for each condition. Assays (B–F) were all statistically different from assay A (p < 0.05); ** denotes statistically significant difference (p < 0.001) between assays E and F.

Figure 3. In vitro inhibition of NAT activity from rat liver samples by apocynin.

The impact of varying concentrations of apocynin (0 and 10 mM) on the activity of rat liver NAT from S9 fractions (9.60 μg protein) of untreated rat livers was investigated in-vitro, as described in methods. Assays were conducted in triplicate and all values are expressed as mean ± standard deviation values.

Figure 4. Hypothetical mechanisms of NAT inhibition by apocynin.

(a) Tautomers of apocynin (1). (b) Possible Michael addition between Cys68 thiolate and apocynin quinonemethide tautomer (2) within NAT active site. Cys68, His107 and Asp122 constitute the catalytic triad in mammalian NAT enzymes. (c) Possible interaction between exposed thiols groups of NATs and radical apocynin metabolites (3).

Figure 5. Substrate binding pockets of (HUMAN)NAT1 and (HUMAN)NAT2 with apocynin tautomers docked.

(a,b) The active site of (HUMAN)NAT1 (a) and (HUMAN)NAT2 (b) with docked apocynin quinonemethide tautomer (2) is shown in surface and stick representation respectively. The residues involved in ligand binding, substrate catalysis and substrate selectivity are shown in stick representation and labelled with carbon atoms in dark blue (a) or dark pink (b), nitrogen in blue, oxygen in red, and sulphur in orange. 2 is labelled with carbon atoms in green, oxygen in red, and hydrogen in white. (c,d) The active site of (HUMAN)NAT1 (c) and (HUMAN)NAT2 (d) with docked 1 is shown in surface and stick representation respectively. The residues involved in ligand binding, substrate catalysis and substrate selectivity are shown in stick representation and labelled with carbon atoms in dark blue (a) or dark pink (b), nitrogen in blue, oxygen in red, and sulphur in orange. 1 is labelled with carbon atoms in green, oxygen in red, and hydrogen in white.

Figure 6. Reversible inhibition of liver NAT activity by apocynin.

Apocynin inhibition of liver NAT activity appears to be reversible upon dilution. Liver lysates were incubated with varying amounts of apocynin (0–1 mM) in the assay for 30 min. The samples were subsequently diluted 1000 times, and evaluation of residue liver NAT activity was conducted using AcCoA and pANS as substrates, as described in methods. Assays were conducted in triplicates.

Figure 7. Position of cysteine residues in human NAT crystal structures.

Overall surface structures of (HUMAN)NAT1 (a) and (HUMAN)NAT2 (b) are shown with cysteine side chains highlighted in stick representation as modelled in the original PDB files. Sections of protein surface corresponding to outward cysteine thiols are coloured in dark red.