Yasushi Yamazoe

INTRODUCTION OF AN N-GLYCOSYLATION SITE INTO UDP-GLUCURONOSYLTRANSFERASE 2B3 ALTERS ITS SENSITIVITY TO CYTOCHROME P450 3A1-DEPENDENT MODULATION

Our previous studies have demonstrated functional protein–protein interactions between cytochrome P450 (CYP) 3A and UDP-glucuronosyltransferase (UGT). However, the role of carbohydrate chains of UGTs in the interaction with CYP is not well understood. To address this issue, we examined whether CYP3A1 modulates the function of UGT2B3 which lacks potential glycosylation sites. We also examined whether the introduction of N-glycosylation to UGT2B3 affects CYP3A-dependent modulation of UGT function. To introduce a potential glycosylation site into UGT2B3, Ser 316 of UGT2B3 was substituted with Asn by site-directed mutagenesis. A baculovirus-Sf-9 cell system for expressing CYP3A1 and UGT2B3/UGT2B3(S316N) was established using a Bac-to-Bac system. Glycosylation of UGT2B3(S316N) was demonstrated in this expression system. The microsomal activity of recombinant UGT was determined using 4-methylumbelliferone as a substrate. The effect of CYP3A1 co-expression on UGT function was examined by comparing the kinetic profiles between single (UGT alone) and double expression (UGT plus CYP) systems. The kinetics of the two expression systems fitted a Michaelis-Menten equation. When the 4-MU concentration was varied, co-expression of CYP3A1 lowered the Vmax of UGT2B3-mediated conjugation. Conversely, for UGT2B3(S316N), the Vmax in the dual expression system was higher than that in the single expression system. The data obtained demonstrate that the introduction of N-glycosylation to UGT2B3 alters its sensitivity to CYP3A1-dependent modulation while CYP3A1 enhanced UGT2B3(S316N) activity, and wild-type UGT2B3 was suppressed by CYP3A1. These data suggest that N-glycosylation of UGT is one of the determinants regulating the interaction between CYP3A and UGT.

Prediction of regioselectivity and preferred order of metabolisms on CYP1A2-mediated reactions. Part 2: Solving substrate interactions of CYP1A2 with non-PAH substrates on the template system

In our previous paper (Drug Metabolism Parmacokinet31: 363, 2016), a simulation system for ligand interactions of human CYP1A2 was developed using Template composed of hexagonal grids focusing on polyaromatic hydrocarbons (PAHs). The system has been expanded for the application of non-PAH chemicals including medical drugs, industrial chemicals and natural products in the present study. Additions of two Templates C and D around Ring C/E and Ring B, respectively, perpendicular each to Template A, offered the accommodation of non-PAH substrates. The size and shape of Templates C and D were defined from the reciprocal comparison of experimental data of ligands with simulation on Templates. The requirements of occupancies at Trigger region (Ring B) and at region of Facial-side Movement (Ring C) as well as Site of Oxidation were found to be mutual throughout CYP1A2 good substrates tested for over the 450 reactions, irrespective of their shapes and flexibilities. The CYP1A2 Template system was also verified with distinct types of poor substrates (47 chemicals) and inhibitors (37 inhibitors) and found to offer the information on probable structural causes. Present CYP1A2 Template system offers a unified evaluation of human CYP1A2 ligands, which aids for toxicological assessments as well as drug metabolism studies.

Assessment of Nongenotoxic Mechanisms in Carcinogenicity Test of Chemicals; Quinone, Quinone Imine, and Quinone Methide as Examples

Abstract Chemicals show carcinogenicity in rodent assays through genotoxic and nongenotoxic mechanisms. No threshold levels are defined for genotoxic carcinogens, and thus genotoxic carcinogens are tightly restricted. Nongenotoxic carcinogens often evoke tumorigenicity after chronic administration of high doses and show a threshold when testing multiple doses. However, verification of genotoxic and nongenotoxic carcinogens is not readily achievable in most cases. This is mainly because of overlapping of biological effects. Therefore, mechanistic support is of importance in the assessment of chemicals with tumorigenic potentials. From pathological and pharmaco (toxico) kinetic points of view, quinoids and precursors may be a category of genotoxicants different from those acting through a direct electrophilic reaction with DNA. Many food additives are capable of producing free radicals, and many food additives are able to act as free-radical scavengers. Free- and oxygen-radical scavenging mechanisms also function in the body, whereas such mechanisms are generally not present in tests of genotoxicity in vitro. Detailed investigations of in vivo pathological examinations and pharmaco (toxico) kinetic profiles on quinoid-related chemicals offer us clear assessments with regard to human safety.