Dibenzyl trisulfide binds to and competitively inhibits the cytochrome P450 1A1 active site without impacting the expression of the aryl hydrocarbon receptor

Abstract

The toxicological manifestation of many pollutants relies upon their binding to the aryl hydrocarbon receptor (AHR), and it follows a cascade of reactions culminating in an elevated expression of cytochrome P450 (CYP) 1 enzymes. CYP1A1 and CYP1B1 are associated with enhanced carcinogenesis when chronically exposed to certain polyaromatic hydrocarbons, and their inhibition may lead to chemoprevention. We evaluated dibenzyl trisulfide (DTS), expressed in the ethnomedical plant, Petiveria alliacea, for such potential chemoprevention. Using recombinant human CYP1A1 and CYP1B1 bactosomes on a fluorogenic assay, we first demonstrated that DTS moderately inhibited both enzymes with half maximal inhibitory concentration (IC₅₀) values of 1.3 ± 0.3 and 1.7 ± 0.3 μM, respectively. Against CYP1A1, DTS was a reversible, competitive inhibitor with an apparent inhibitory constant (K_i) of 4.55 ± 0.37 μM. In silico molecular modeling showed that DTS binds with an affinity of -39.8 kJ·mol^-1, situated inside the binding pocket, approximately 4.3 Å away from the heme group, exhibiting interactions with phenylalanine residue 123 (Phe-123), Phe-224, and Phe-258. Lastly, zebrafish (Danio rerio) embryos were exposed to 0.08-0.8 μM DTS from 24 to 96 h post fertilization (hpf) with the in vivo ethoxyresorufin-O-deethylase (EROD) assay, and, at 96 hpf, DTS significantly suppressed EROD CYP1A activity in a dose-dependent manner, with up to 60% suppression in the highest 0.8 μM exposure group. DTS had no impact on gene transcription levels for cyp1a and aryl hydrocarbon receptor 2 (ahr2). In co-exposure experiments, DTS suppressed CYP1A activity induced by both B[a]P and PCB-126, although these reductions were not significant. Taken together, these results demonstrate that DTS is a direct, reversible, competitive inhibitor of the carcinogen-activating CYP1A enzyme, binding in the active site pocket close to the heme site, and shows potential in chemoprevention.

Keywords: AHR pathway; Aryl hydrocarbon receptor; CYP1; Carcinogenesis; Chemoprevention; Cytochrome P450 1; Dibenzyl trisulfide; Ethnomedicine; Petiveria alliacea.