Cawley George F, Connick J Patrick, Eyer Marilyn K, Backes Wayne L
Department of Pharmacology and Experimental Therapeutics, Louisiana State University Health Science Center, New Orleans, Louisiana; The Stanley S. Scott Cancer Center, Louisiana State University Health Science Center, New Orleans, Louisiana.
Department of Pharmacology and Experimental Therapeutics, Louisiana State University Health Science Center, New Orleans, Louisiana; The Stanley S. Scott Cancer Center, Louisiana State University Health Science Center, New Orleans, Louisiana.
Drug Metab Dispos. 2025 Jan;53(1):100012. doi: 10.1124/dmd.124.001939. Epub 2024 Nov 22.
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 cytochrome P450 (P450) function, inducing the expression of some forms through the Ah receptor. However, another characteristic of EPFRs is their ability to inhibit P450 activities. CYP2E1 is one of the P450s that is inhibited by EPFR (MCP230, the laboratory-generated EPFR made by heating silica 5% copper oxide, and silica [<0.2 μm in diameter] and 2-monochlorophenol at ≥230 °C) exposure. Because 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. Because EPFR-mediated inhibition was more robust at subsaturating NADPH-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 was inhibited by EPFRs. Examination of ROS production showed that MCP230 generated a greater amount of ROS than the non-EPFR control particle (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 the uncoupling of NADPH and oxygen consumption from substrate metabolism to the generation of reactive oxygen species. These results show that environmentally persistent free radicals block the metabolism of foreign compounds and synergistically stimulate the formation of reactive oxygen species that lead to oxidative damage within the organism.
环境持久性自由基（EPFRs）是颗粒物中最近才被认识到的一种成分，会导致呼吸道和心血管毒性。EPFR毒性的机制似乎与其产生活性氧（ROS）的能力有关，从而造成氧化损伤。研究表明，EPFRs会影响细胞色素P450（P450）的功能，通过芳烃受体诱导某些形式的表达。然而，EPFRs的另一个特性是它们抑制P450活性的能力。CYP2E1是受EPFR（MCP230，通过加热含5%氧化铜的二氧化硅、直径<0.2μm的二氧化硅和2-一氯苯酚在≥230℃下制备的实验室生成的EPFR）暴露抑制的P450之一。由于已知CYP2E1也会产生活性氧，因此了解EPFRs影响该酶功能的能力并确定其中涉及的机制很重要。研究表明，CYP2E1受EPFRs抑制，受非EPFR颗粒抑制的程度较小。由于在次饱和烟酰胺腺嘌呤二核苷酸磷酸-细胞色素P450还原酶（POR）浓度下，EPFR介导的抑制作用更强，因此研究了POR•CYP2E1复合物形成和电子转移的破坏情况。令人惊讶的是，EPFRs既不抑制POR与CYP2E1之间的复合物形成，也不抑制电子转移。对活性氧产生的检测表明，MCP230产生的活性氧比非EPFR对照颗粒（CuO-Si）更多。当将含POR/CYP2E1的重组系统添加到污染物颗粒系统中时，会协同刺激活性氧的产生。结果表明，EPFRs会抑制CYP2E1介导的底物代谢，但不会改变电子转移，实际上还会刺激活性氧的产生。综上所述，这些结果与EPFRs通过抑制底物代谢和增加活性氧的产生来影响CYP2E1功能一致。意义声明：环境持久性自由基通过抑制单加氧酶活性来影响CYP2E1功能。这种抑制不是由于POR•CYP2E1复合物的破坏或电子转移的抑制，而是由于烟酰胺腺嘌呤二核苷酸磷酸和氧消耗从底物代谢到活性氧产生的解偶联。这些结果表明，环境持久性自由基会阻碍外来化合物的代谢，并协同刺激活性氧的形成，从而导致生物体内的氧化损伤。
