The Inactivation of Human CYP2E1 by Phenethyl Isothiocyanate, a Naturally Occurring Chemopreventive Agent, and Its Oxidative Bioactivation

Abstract

Phenethylisothiocyanate (PEITC), a naturally occurring isothiocyanate and potent cancer chemopreventive agent, works by multiple mechanisms, including the inhibition of cytochrome P450 (P450) enzymes, such as CYP2E1, that are involved in the bioactivation of carcinogens. PEITC has been reported to be a mechanismbased inactivator of some P450s. We describe here the possible mechanism for the inactivation of human CYP2E1 by PEITC, as well as the putative intermediate that might be involved in the bioactivation of PEITC. PEITC inactivated recombinant CYP2E1 with a partition ratio of 12, and the inactivation was not inhibited in the presence of glutathione (GSH) and not fully recovered by dialysis. The inactivation of CYP2E1 by PEITC is due to both heme destruction and protein modification, with the latter being the major pathway for inactivation. GSH-adducts of phenethyl isocyanate (PIC) and phenethylamine were detected during the metabolism by CYP2E1, indicating formation of PIC as a reactive intermediate following P450-catalyzed desulfurization of PEITC. Surprisingly, PIC bound covalently to CYP2E1 to form protein adducts but did not inactivate the enzyme. Liquid chromatography mass spectroscopy analysis of the inactivated CYP2E1 apo-protein suggests that a reactive sulfur atom generated during desulfurization of PEITC is involved in the inactivation of CYP2E1. Our data suggest that the metabolism of PEITC by CYP2E1 that results in the inactivation of CYP2E1 may occur by a mechanism similar to that observed with other sulfur-containing compounds, such as parathion. Digestion of the inactivated enzyme and analysis by SEQUEST showed that Cys 268 may be the residue modified by PIC.

Translational Implications of the Alcohol-Metabolizing Enzymes, Including Cytochrome P450-2E1, in Alcoholic and Nonalcoholic Liver Disease

“…These antioxidants, contained in many fruits and vegetables, show beneficial effects on AFLD, NAFLD, and DILI, although some of these antioxidants also exhibit significant protection in other tissues such as heart, muscle, and brain (Pallauf, Giller, Huebbe, & Rimbach, 2013;Rodriguez et al, 2015). Some of these agents include inhibitors of CYP2E1 such as diallyl sulfide in garlic, phenethyl isothiocyanate (Stice et al, 2015;Yoshigae, Sridar, Kent, & Hollenberg, 2013) in crucible vegetables, or dioscin in edible plants , although these inhibitors seem less potent than the synthetic CYP2E1 inhibitor CMZ (Hu et al, 1994) or YH439 ( Jeong et al, 1996). In addition, general antioxidants such as SAMe and betaine were shown to preserve mitochondrial function and proteome in the animal models of AFLD Purohit et al, 2007) and NAFLD (Santamaria et al, 2003), partly through blocking the ROS/RNS production and restoring the physiological levels of GSH, SAMe, and S-adenosyl-homocysteine.…”

A principal mechanism for the cancer chemopreventive activity of phenethyl isothiocyanate is modulation of carcinogen metabolism

“…Indeed, PEITC is a potent mechanism-based inhibitor of cytochrome P450 enzymes Modulation of carcinogen metabolism by PEITC 7 both in rat and human microsomes, far more effective than the aliphatic isothiocyanates sulforaphane and erucin (Konsue & Ioannides, 2010a,b;Yoshigae et al, 2013). Using recombinant human cytochrome P450 enzymes, it was established that PEITC was a mechanism-based inhibitor of CYP2E1 (Nakajima et al, 2001).…”

“…Using recombinant human cytochrome P450 enzymes, it was established that PEITC was a mechanism-based inhibitor of CYP2E1 (Nakajima et al, 2001). In the same system, it was demonstrated that PEITC acted as a mechanism-based inhibitor by interacting with both the heme and protein moieties of cytochrome P450, with the latter being the principal inactivation route (Yoshigae et al, 2013). Phenethyl isocyanate, generated by the cytochrome P450-catalyzed oxidative desulfuration of PEITC was initially considered the likely metabolite responsible for the inhibitory effect (Lee, 1996) but this was shown not to be the case in subsequent studies (Konsue & Ioannides, 2010b).…”

Mechanisms of the Anticancer Effects of Isothiocyanates

“…Inhibition of Phase I metabolizing enzymes is one of the prominent mechanisms of chemoprevention by ITCs (Fig. 3) [112][113][114][115]. Morse et al and several other studies have mechanistically shown that ITCs prevent tumorigenesis caused by various chemical carcinogens in vivo [115][116][117].…”

“…3) [112][113][114][115]. Morse et al and several other studies have mechanistically shown that ITCs prevent tumorigenesis caused by various chemical carcinogens in vivo [115][116][117]. PEITC exhibits marked chemopreventive effect against tobacco-induced lung and esophageal cancer in rodent models [118,119].…”