Environmentally Persistent Free Radicals stimulate CYP2E1-mediated generation of reactive oxygen species at the expense of substrate metabolism.

Environmentally persistent free radicals (EPFRs) are a recently recognized component of particulate matter that cause respiratory and cardiovascular toxicity. The mechanism of EPFR toxicity appears to be related to their ability to generate reactive oxygen species (ROS), causing oxidative damage. EPFRs were shown to affect P450 function, inducing the expression of some forms through the Ah receptor. However, another characteristic of EPFRs lies in their ability to inhibit P450 activities. CYP2E1 is one of the P450s that is inhibited by EPFR (MCP230) exposure. As CYP2E1 is also known to generate ROS, it is important to understand the ability of EPFRs to influence the function of this enzyme and to identify the mechanisms involved. CYP2E1 was shown to be inhibited by EPFRs, and to a lesser extent by non-EPFR particles. As EPFR-mediated inhibition was more robust at subsaturating NADPH-cytochrome P450 reductase (POR) concentrations, disruption of POR·CYP2E1 complex formation and electron transfer were examined. Surprisingly, neither complex formation nor electron transfer between POR and CYP2E1 were inhibited by EPFRs. Examination of ROS production showed that MCP230 generated a greater amount of ROS than the non-EPFR CuO-Si. When a POR/CYP2E1-containing reconstituted system was added to the pollutant-particle systems there was a synergistic stimulation of ROS production. The results indicate that EPFRs cause inhibition of CYP2E1-mediated substrate metabolism, yet do not alter electron transfer and actually stimulate ROS generation. Taken together, the results are consistent with EPFRs affecting CYP2E1 function by inhibiting substrate metabolism and increasing the generation of ROS. Significance Statement Environmentally persistent free radicals affect CYP2E1 function by inhibition of monooxygenase activity. This inhibition is not due to disruption of the POR·CYP2E1 complex or inhibition of electron transfer, but due to uncoupling of NADPH and oxygen consumption from substrate metabolism to the generation of ROS. These results show that EPFRs block the metabolism of foreign compounds, and also synergistically stimulate the formation of reactive oxygen species that lead to oxidative damage within the organism.