Inactivation of Cytochrome P450 2B1 by Benzyl Isothiocyanate, a Chemopreventative Agent from Cruciferous Vegetables

Abstract

A series of arylalkyl isothiocyanates were evaluated for their ability to inactivate purified cytochrome P450 2B1 in a reconstituted system. Benzyl isothiocyanate (BITC) and phenethyl isothiocyanate (PEITC) occur naturally in several cruciferous vegetables, and the inhibition of cytochrome P450 (P450) enzymes has been implicated in their chemopreventative abilities. The naturally occurring isothiocyanates BITC and PEITC inactivated P450 2B1 in a time- and concentration-dependent manner, whereas the synthetic isothiocyanates phenylpropyl and phenylhexyl isothiocyanate did not result in inactivation, but were potent competitive inhibitors of P450 2B1 activity. The kinetics of inactivation of P450 2B1 by BITC were characterized. The 7-ethoxy-4-(trifluoromethyl)coumarin O-deethylation activity of P450 2B1 was inactivated in a mechanism-based manner. The loss of O-deethylation activity followed pseudo-first-order kinetics, was saturable, and required NADPH. The BITC concentration required for half-maximal inactivation (K(I)) was 5.8 microM, and the maximal rate constant for inactivation was 0.66 min(-)(1) at 23 degrees C. BITC was a very efficient inactivator of P450 2B1 with a partition ratio of approximately 9. The mechanism of BITC-mediated inactivation of P450 2B1 was also investigated. More than 80% of the catalytic activity was lost within 12 min with a concomitant loss of approximately 45% in the ability of the reduced enzyme to bind CO. The magnitude of the UV/visible absorption spectrum of the inactivated protein did not decrease significantly, and subsequent HPLC analysis indicated no apparent modification of the heme. HPLC and protein precipitation analyses indicated that the P450 apoprotein was covalently modified by a metabolite of BITC. Determination of the binding stoichiometry indicated that 0.90 +/- 0. 16 mol of radiolabeled metabolite was bound per mole of enzyme that was inactivated, suggesting the modification of a single amino acid residue per molecule of enzyme that was inactivated. The results reported here indicate that BITC is a mechanism-based inactivator of P450 2B1 and that inactivation occurs primarily through protein modification.

Effects of kale ingestion on pharmacokinetics of acetaminophen in rats

“…Previous reports (4,10,17,20,26) have shown that CYP3A4, CYP1A2, CYP2D6, and CYP2E1 are involved in the metabolism of AA. In addition, it has been reported that ITCs had an inhibitory effect on CYP activity (6,7,12,14,23). In our experiment, the plasma level and AUC 0-120 of AA and the AUC 0-120 of AA-G were raised by pretreatment with kale, whereas there were no changes in the plasma levels or pharmacokinetics parameters of AA-S.…”

Suppressive effect of 1,4-phenylene diisothiocyanate onN-butyl-N-(4-hydroxybutyl)nitrosamine-induced urinary bladder carcinogenesis in male ICR mice

“…Because the inhibitory action of ITC has been attributed to their ability to modulate xenobiotic-metabolizing enzymes (e.g., cytochrome P-450) that are required for the activation of many carcinogens or to induce Phase II detoxifying enzymes. 44,[63][64][65] . One possibility is that cytochrome P-450 are critical targets in inhibition of the chemical-induced tumorigenesis by ITC, the distinct patterns of P-450 isozymes for carcinogen activation in rats and mice may be partially responsible for the differences observed.…”

Cruciferous Vegetables: Dietary Phytochemicals for Cancer Prevention

“…It is possible that the effect of isothiocyanates on the O-deethylation of ethoxyresorufin is due, to some extent, mechanism-based inhibition associated with these compounds (Yoxall et al, 2005;Hanlon et al, 2008a). However, it is important to point out that it is the phenobarbital-inducible CYP2B enzymes that catalyse the metabolism of phenethyl isothiocyanate to the metabolite(s) liable for mechanismbased inhibition, in contrast to benzo[a]pyrene inducible CYP1 enzymes, suggesting that mechanism-based inhibition is unlikely to be the dominant mechanism (Lee, 1996;Goosen et al, 2000;Nakajima et al, 2001;Konsue and Ioannides, 2010c). Thus, it can be inferred that isothiocyanates are effective antagonist of the Ah receptor in rat and human liver, and this potential may contribute to their established chemopreventive activity.…”