Environmentally persistent free radicals lead to selective inhibition of CYP1 monooxygenase activities, and increased production of reactive oxygen species by reaction uncoupling

Abstract

This study focuses on the effect of Environmentally Persistent Free Radicals (EPFRs) on the P450 enzymes of the CYP1 family. EPFRs are a component of particulate pollutants, that are stable in the environment, but can generate free radicals, leading to oxidative stress and subsequent toxicity of the respiratory, cardiovascular, and immune systems once they enter an organism. The results show differences in the ability of EPFRs to inhibit CYP1-dependent substrate metabolism, with CYP1B1 being inhibited to the greatest extent. There also were differences in the ability of EPFRs to disrupt the POR•CYP1 complex, with CYP1B1 being the only form where EPFRs disrupted POR•CYP1B1 complex formation. Despite the inhibition of substrate metabolism, each CYP1 enzyme, when reconstituted with NADPH-cytochrome P450 reductase (POR) was able to synergistically stimulate the generation of reactive oxygen (ROS) in the presence of particulate matter. Interestingly, both POR and the CYP1 enzymes were able to stimulate ROS generation, even when in partial reconstituted systems where only one of the proteins was present. However, when both POR and CYP1 were combined in a complete reconstituted system, ROS generation was synergistically stimulated.

Keywords: CYP1A1; CYP1A2; CYP1B1; cytochrome P450; environmentally persistent free radicals; enzyme inhibition; protein-protein interactions; reactive oxygen species.

Figure 1

Effect of EPFR and non-EPFR particles on CYP1-mediated 7-benzyloxyresorufin metabolism as a function of substrate concentration. 7BR metabolism was examined in reconstituted systems containing 0.02 μM CYP1 and 0.2 μM POR in a reconstituted system in DLPC (160:1 DLPC:CYP1) as a function of substrate concentration in the presence and absence of 0.15 mg/ml non-EPFR (CuO-Si) and EPFR (MCP230) particles. The data points represent the mean ± SD for 3 determinations. (A) CYP1A1; (B) CYP1A2; and (C) CYP1B1.

Figure 2

Effect of EPFR and non-EPFR particles on the POR•CYP1 redox complexes. HEK 293T/17 cells were transfected with plasmids coding for POR-Rluc and each of the CYP1-GFP constructs. After 24 h, complex formation between POR and the CYP1 enzymes was examined using BRET at a range of particle concentrations. Data points represent the mean ± the SD for triplicate determinations from a single group of cells. Previous experiments were conducted with adjustments to transfection conditions for optimization of protein expression, generating similar results (not shown). (A) CYP1A1, (B) CYP1A2, and (C) CYP1B1. ^*p < 0.05.

Figure 3

Effect of EPFRs on CYP1-mediated ROS formation. ROS generation was monitored using the formation of the fluorescent product DCF in the presence of 0.1 mg/ml of particles as a function of the POR/CYP1 reconstituted system. The POR:CYP1 ratio was 2:1. The results are the mean ± SD for at least 3 determinations. (A) CYP1A1: The slopes of the lines were 3.9, 56.6, and 51.1 (min)⁻¹(μM CYP1A1)⁻¹. Both treatment groups were determined to be significantly different from the no particle group (p < 0.01). (B) CYP1A2: The slopes of the lines, taken over the linear response range were 11.1, 67.2, and 79.6 (min)⁻¹(μM CYP1A2)⁻¹. Both treatment groups were determined to be significantly different from the no particle group (p < 0.01). (C) CYP1B1. The slopes of the lines were 6.1, 55.1, and 52.9 (min)⁻¹(μM CYP1B1)⁻¹. Both treatment groups were determined to be significantly different from the no particle group ^**(p < 0.01).

Figure 4

Identification of the components of the CYP1-containing reconstituted systems responsible for synergistic ROS generation. ROS formation was measured in the absence and presence of particulate matter when combined with components of a CYP1-containing reconstituted system. The Figure is divided into three sections, no particles (left), the non-EPFR, CuO-Si (center), and the EPFR, MCP230. In each case, ROS generation was measured in the presence of POR (black), CYP1A1 (red), CYP1A2 (green), CYP1B1 (blue). The hatched bars represent partial reconstituted systems using only the proteins indicated. The solid bars represent complete reconstituted systems that contain both POR and the indicated CYP1 enzyme. Data are shown as the mean ± SD. Because of lack of protein availability, there is only a single determination for CYP1A1 in the absence of particles, and duplicate determinations for CYP1A1 in the CuO-Si and MCP230 groups. N = 4 for all other conditions. Because of the low N for the CYP1A1 data, it was not included in the statistical analysis. Statistics were performed using a one-way analysis of variance and Bonferroni's multiple comparison test (significantly different from the corresponding group in the absence of particles – **, p < 0.01; and ***, p < 0.001). ‡ – significantly different from the corresponding CuO-Si group (p < 0.01). In all cases, a synergistic stimulation of ROS production was observed when comparing the complete reconstituted system (POR•CYP1) vs. the sum of POR and CYP1 alone (p < 0.01, not shown for clarity).