Modification of serine 360 by a reactive intermediate of 17-alpha-ethynylestradiol results in mechanism-based inactivation of cytochrome P450s 2B1 and 2B6

17-alpha-Ethynylestradiol (17EE) is a mechanism-based inactivator of P450 2B1 and P450 2B6 in the reconstituted monooxygenase system. The loss in enzymatic activity was due to the binding of a reactive intermediate of 17EE to the apoprotein. P450 2B1 and P450 2B6 were inactivated by 17EE and digested with trypsin. The peptides obtained following digestion with trypsin of 17EE-inactivated P450 2B1 and P450 2B6 were separated by liquid chromatography and analyzed by ESI-MS. Adducted peptides exhibiting an increase in mass consistent with the addition of the mass of the reactive intermediate of 17EE were identified for each enzyme. Analysis of these modified peptides by ESI-MS/MS and precursor ion scanning facilitated the identification of the Ser360 in both enzymes as a site that had been adducted by a reactive intermediate of 17EE. A P450 2B1 mutant where Ser360 was replaced by alanine was constructed, expressed, and purified. Activity and inactivation studies indicated that mutation of the Ser360 residue to alanine did not prevent inactivation of the mutant enzyme by 17EE. These observations suggest that Ser360 is not critical for the catalytic function of these P450s. Spectral binding studies of the 17EE-inactivated P450 2B1 and P450 2B6 indicated that modification of the enzymes by the reactive intermediate of 17EE resulted in an enzyme that was no longer capable of binding substrates. These results suggest that the inactivation by 17EE may be due to modification of an amino acid residue in the substrate access channel near the point of entry into the active site.     

Bioactivation of phencyclidine in rat and human liver microsomes and recombinant P450 2B enzymes: evidence for the formation of a novel quinone methide intermediate

The hypothesis that the psychological side effects associated with the anesthetic phencyclidine (PCP) may be caused by irreversible binding of PCP or its reactive metabolite(s) to critical macromolecules in the brain has resulted in numerous in vitro studies aimed at characterizing pathways of PCP bioactivation. The studies described herein extend the current knowledge of PCP metabolism and provide details on a previously unknown metabolic activation pathway of PCP. Following incubations with NADPH- and GSH-supplemented human and rat liver microsomes and recombinant P450 2B enzymes, two sulfhydryl conjugates with MH+ ions at 547 and 482 Da, respectively, were detected by LC/MS/MS. Shebley et al. [(2006) Drug Metab. Dispos. 34, 375-383] have also observed the GSH conjugate 1 with MH+ at 547 Da in PCP incubations with rat P450 2B1 and rabbit P450 2B4 isoforms fortified with NADPH and GSH. The molecular weight of 1 is consistent with a bioactivation pathway involving Michael addition of the sulfhydryl nucleophile to the putative 2,3-dihydropyridinium metabolite of PCP obtained via a four-electron oxidation of the piperidine ring in the parent compound. The mass spectrum of the novel GSH adduct 2 with an MH+ ion at 482 Da was suggestive of a unique PCP bioactivation pathway involving initial ortho- or para-hydroxylation of the phenyl ring in PCP followed by spontaneous decomposition to piperidine and an electrophilic quinone methide intermediate, which upon reaction with GSH yielded adduct 2. The LC retention times and mass spectral properties of enzymatically generated 2 were identical to those of a reference standard obtained via reaction of GSH with synthetic p-hydroxyPCP in phosphate buffer (pH 7.4, 37 degrees C). 1H NMR and 13C-distortionless enhancement by polarization transfer (DEPT) NMR spectral studies on synthetically generated 2 suggested that the structural integrity of the p-hydroxyphenyl and cyclohexyl rings likely was preserved and that the site of GSH addition was the benzylic carbon joining the two scaffolds. The formation of 2 in human microsomes was reduced upon addition of the dual P450 2C19/P450 2B6 inhibitor (+)- N-3-benzylnirvanol. Consistent with this finding, both recombinant P450 2B6 and P450 2C19 catalyzed PCP bioactivation to 2. In the absence of GSH, synthetic p-hydroxyPCP underwent rapid decomposition (t1/2 approximately 5.2 min) to afford p-hydroxyphenylcyclohexanol and p-hydroxyphenylcyclohexene, presumably via the quinone methide intermediate. Overall, our findings on the facile degradation of synthetic p-hydroxyPCP to yield an electrophilic quinone methide intermediate capable of reacting with nucleophiles, including GSH and water, suggest an inherent instability of the putative phenolic PCP metabolite. Thus, if formed enzymatically in vivo, p-hydroxyPCP may not require further metabolism to liberate the quinone methide, which can then react with macromolecules. To our knowledge, this is the first report of a quinone methide reactive intermediate obtained in human-liver microsomal metabolism of PCP.     

Investigation of the mechanisms underlying the differential effects of the K262R mutation of P450 2B6 on catalytic activity

Human P450 2B6 is a polymorphic enzyme involved in the oxidative metabolism of a number of clinically relevant substrates. The lysine 262-to-arginine mutant of cytochrome P450 2B6 (P450 2B6.4) has been shown to have differential effects on P450 2B6 catalytic activity. We reported previously that the mutant enzyme was unable to metabolize 17-alpha-ethynylestradiol (17EE) or become inactivated by 17EE or efavirenz, which are inactivators of the wild-type enzyme. Studies were performed to elucidate the mechanism by which this mutation affects P450 2B6 catalytic activity. Studies using phenyldiazene to investigate differences between the active site topologies of the wild-type and mutant enzymes revealed only minor differences. Likewise, Ks values for the binding of both benzphetamine and efavirenz were comparable between the two enzymes. Using the alternate oxidant tert-butyl hydroperoxide, the mutant enzyme was inactivated by both 17EE and efavirenz. The stoichiometry of 17EE and efavirenz metabolism by P450s 2B6 and 2B6.4 revealed that the mutant enzyme was more uncoupled, producing hydrogen peroxide as the primary product. Interestingly, the addition of cytochrome b5 improved the coupling of the mutant, resulting in increased catalytic activity. In the presence of cytochrome b5 the variant readily metabolized 17EE and was inactivated by both 17EE and efavirenz. It is therefore proposed that the oxyferrous or iron-peroxo intermediate formed by the mutant enzyme in the presence of 17EE and efavirenz may be less stable than the same intermediates formed by the wild-type enzyme.     

Mechanism of inactivation of human cytochrome P450 2B6 by phencyclidine.

The mechanism behind the observed inactivation of human P450 2B6 by phencyclidine (PCP) has been evaluated over the past 2 decades. The scope of the current investigation was to contribute to the fundamental knowledge of PCP oxidation and perhaps the mechanism behind P450 inactivation. To study the chemistry of PCP oxidation, we subjected PCP to the Fenton reagent. Under Fenton chemistry conditions, oxidation on all three PCP rings was observed by liquid chromatography/tandem mass spectrometry (LC-MS/MS). When PCP was incubated with the Fenton system in the presence of glutathione (GSH), three GSH-PCP conjugates were identified. Subsequent LC-MS/MS analysis of these conjugates revealed two species that had GSH attached to the cyclohexane ring of PCP and a third conjugate in which GSH was adducted to the piperidine ring. When PCP was incubated across a panel of P450 enzymes, several enzymes, including P450s 2D6 and 3A4, were able to catalyze the formation of the PCP iminium ion, whereas P450s 2B6 and 2C19 were exclusively able to hydroxylate secondary carbons on the cyclohexane ring of PCP. Subsequent mechanistic experiments revealed that only P450s 2B6 and 2C19 demonstrated loss of catalytic activity after preincubation with 10 microM PCP. Finally, investigation of P450 2B6 inactivation using structural analogs of PCP revealed that blocking the para-carbon atom on the cyclohexane ring of PCP from oxidation protected the P450 2B6 from inactivation, which suggests that a reactive intermediate generated during the hydroxylation of the cyclohexane ring may be linked to the mechanism of inactivation of P450 2B6 by PCP.     

Mechanism-based inactivation of cytochromes P450 2B1 and P450 2B6 by 2-phenyl-2-(1-piperidinyl)propane.

2-Phenyl-2-(1-piperidinyl)propane (PPP), an analog of phencyclidine, was tested for its ability to inactivate cytochrome P450s (P450s) 2B1 and 2B6. PPP inactivated the 7-(benzyloxy)resorufin O-dealkylation activity of liver microsomes obtained from phenobarbital-induced rats with a K(I) of 11 microM. The 7-ethoxy-4-(trifluoromethyl)coumarin O-deethylation activity of purified rat liver P450 2B1 and expressed human P450 2B6 was inactivated by PPP in a reconstituted system containing NADPH-cytochrome P450 reductase and lipid. In the presence of NADPH, the loss of activity was time- and concentration-dependent, and followed pseudo first order kinetics. The rate of inactivation for P450 2B1 was 0.3 min(-1), and the concentration of PPP required to achieve half-maximal inactivation was 12 microM. The time for 50% of the P450 2B1 to become inactivated at saturating concentrations of PPP was 2.5 min. P450 2B6 was inactivated with a k(inact) of 0.07 min(-1), a K(I) of 1.2 microM, and a t(1/2) of 9.5 min. The inactivated P450s 2B1 and 2B6 lost about 25 and 15%, respectively, of their ability to form a CO-reduced complex, suggesting that the loss of activity was caused by a PPP modification of the apoprotein rather than the heme. The estimated partition ratio for P450s 2B1 and 2B6 with PPP was 31 and 15, respectively. The inactivation was not reversible and reductase activity was not affected. Coincubation of P450 2B1 and 2B6 with PPP and NADPH in the presence of an alternate substrate protected both enzymes from inactivation. The exogenous nucleophile GSH did not affect the rate of inactivation. PPP-inactivated P450s 2B1 and 2B6 were recognized on Western blots by an antibody generated to phencyclidine that had been conjugated to BSA. Stoichiometries of 1.4:1 and 0.7:1 were determined for the binding of a [3H]PPP metabolite to P450 2B1 and 2B6, respectively.     

Mutation of a single residue (K262R) in P450 2B6 leads to loss of mechanism-based inactivation by phencyclidine.

Human cytochrome P450 (P450) 2B6 plays an important role in the metabolism of many drugs used in the clinic, and it has been shown to be highly polymorphic and inducible by a variety of substrates. The metabolism of phencyclidine (PCP) by P450 2B6 results in mechanism-based inactivation of the enzyme. We investigated the effects of a naturally occurring mutation of P450 2B6 where a lysine 262 is changed to an arginine (K262R) on PCP metabolism and mechanism-based inactivation of 2B6 by PCP. The K262R mutant retained the 7-ethoxy-4-trifluoromethylcoumarin O-deethylation activity when it was incubated with PCP and NADPH in the reconstituted system, whereas the wild-type enzyme was readily inactivated by PCP. Spectral binding studies showed that PCP was reversibly bound in the active site of the K262R mutant with slightly higher affinity (156 muM) compared with the wild-type 2B6 (397 muM). In addition, all the metabolites of PCP (M1-M8) that were formed by the wild-type enzyme were also formed by the K262R mutant. Although the K262R mutant metabolized PCP to give similar metabolite profiles, the overall rate of metabolite formation was lower than the wild-type enzyme. A reactive intermediate of PCP was formed by wild-type P450 2B6 and trapped with glutathione (GSH). However, no GSH conjugates were detected from incubations with the K262R mutant. These data suggest that the lysine 262 residue plays an important role in the formation of a reactive intermediate of PCP that leads to the mechanism-based inactivation of P450 2B6.     

Selective pathways for the metabolism of phencyclidine by cytochrome p450 2b enzymes: identification of electrophilic metabolites, glutathione, and N-acetyl cysteine adducts.

The metabolism of phencyclidine (PCP) has been studied previously in cytochrome P450 (P450)-containing microsomal systems. However, the reactive intermediate(s) that covalently binds to the P450 and leads to inactivation or leaves the active site to modify other proteins has not been identified. In this study two electrophilic intermediates of PCP were identified by mass spectrometry and by trapping with reduced glutathione (GSH) or N-acetyl cysteine (NAC). The tentative structures of these electrophilic intermediates were determined using mass spectrometry. P450s 2B1 and 2B4 formed a metabolite that exhibited an m/z of 240 corresponding to the mass of the 2,3-dihydropyridinium species of PCP or its conjugate base, the 1,2-dihydropyridine. Chemical reduction of the incubation mixture using NaBH4 resulted in the disappearance of the signal at m/z 240, consistent with reduction of a 2,3-dihydropyridinium species. Furthermore, the reactive metabolite trapped by GSH resulted in an adduct exhibiting an m/z of 547, consistent with the mass of the 2,3-dihydropyridinium species of PCP (m/z 240), that has reacted with a molecule of GSH (m/z 308). However, P450 2B6 formed a different reactive intermediate of PCP that was isolated as a GSH adduct exhibiting an m/z of 581 and an NAC adduct with an m/z of 437. Liquid chromatography-tandem mass spectrometry analysis of these adducts suggested that a di-oxygenated iminium metabolite of PCP could be the reactive intermediate formed by P450 2B6 but not by the other 2B isoforms. These data suggest that P450 2B6 favors oxidation pathways for PCP metabolism that are different from those of P450s 2B1 and 2B4.     

Time-dependent inactivation of P450 3A4 by raloxifene: identification of Cys239 as the site of apoprotein alkylation

Time-dependent inactivation of cytochrome P450s is typically a result of substrate bioactivation to form reactive species that subsequently alkylate the heme group, apoprotein, or both. The chemical identity of many reactive intermediates is generally proposed based on the products of trapping reactions with nucleophilic agents as only a few P450-drug adducts have been directly characterized. We describe the use of mass spectrometry to show that a single equivalent of raloxifene is bound to the intact P450 apoprotein. Furthermore, mass analysis of peptides following digestion with proteinase K revealed that the covalently bound drug is localized to residue Cys239. A mass shift of 471 Da to the intact protein and peptide, relative to control samples, indicated that time-dependent inactivation of P450 3A4 occurred through the raloxifene diquinone methide intermediately prior to nucleophilic attack of the sulfur of Cys239. Association between raloxifene adduction to P450 3A4 apoprotein and the observed time-dependent inactivation was further investigated with the use of cysteine-specific modifying reagents. When P450 3A4 was treated with iodoacetamide or N-(1-pyrene)iodoacetamide, which alkylated residue Cys239 exclusively, time-dependent inactivation of P450 3A4 by raloxifene was prevented. The change in protein mass of 471 Da combined with the protection from inactivation that occurred through pre-alkylation of Cys239 provided conclusive evidence that raloxifene-mediated P450 3A4 inactivation occurred through the bioactivation of raloxifene to the diquinone methide and subsequent alkylation of Cys239.     

Thr302 is the site for the covalent modification of human cytochrome P450 2B6 leading to mechanism-based inactivation by tert-butylphenylacetylene

The mechanism-based inactivation of human CYP2B6 by tert-butylphenylacetylene (BPA) in the reconstituted system was investigated. The inactivation of CYP2B6 by BPA is time-, concentration-, and NADPH-dependent and exhibits a K(I) of 2.8 μM, a k(inact) of 0.7 min(-1), and a t(1/2) of 1 min. The partition ratio is ～5. Unlike CYP2B1 and CYP2B4, in addition to the formation of an apoprotein adduct and a glutathione conjugate, a small heme adduct was observed when CYP2B6 was incubated with BPA. The mass increase of the adducted apoprotein and GSH conjugate is 174 Da, equivalent to the mass of one molecule of BPA plus one oxygen atom. To identify the adducted residue, BPA-inactivated CYP2B6 was digested with trypsin, and the digest was then analyzed by liquid chromatography-tandem mass spectrometry. A mass shift of 174 Da was used for the SEQUEST database search, and the identity of the modified residue was confirmed by MS/MS fragmentation of the modified peptide. Two residues, Lys274 and Thr302, were identified as having been modified. Further mutagenesis studies have demonstrated that the residue that is modified to result in inactivation is Thr302, not Lys274. Docking studies show that in the enzyme-substrate complex, Thr302 is in close contact with the triple bond of BPA with a distance of 3.8 ? between the terminal carbon of BPA and the oxygen in the hydroxyl group of Thr302. In conclusion, Thr302 of CYP2B6 is covalently modified by a reactive metabolite of BPA, and this modification is responsible for the mechanism-based inactivation.     

Mechanism-based inactivation of cytochromes P450 2B1 and P450 2B6 by n-propylxanthate

n-Propylxanthate (nPX) inactivated the 7-ethoxy-4-(trifluoromethyl)coumarin (7-EFC) O-deethylation activity of purified, reconstituted rat hepatic P450 2B1 or human P450 2B6 in a mechanism-based manner. The inactivation followed pseudo-first-order kinetics and was entirely dependent on both NADPH and nPX. The maximal rate constant for inactivation of P450 2B1 at 30 degrees C was 0.2 min-1. The apparent KI was 44 microM, and the half-time for inactivation was 4.1 min. Purified, reconstituted human P450 2B6 was also inactivated by nPX with a KI of 12 microM. The kinactivation for P450 2B6 was 0.06 min-1, and the t1/2 was 11 min. Incubations of P450 2B1 with nPX and NADPH for 20 min resulted in a 75% loss in enzymatic activity and a concurrent 25% loss of the enzyme's ability to form a reduced CO complex. Little loss in the absolute spectrum of nPX-inactivated P450 2B1 was observed. With P450 2B6, an 83% loss in enzymatic activity and a 12% loss in the CO-reduced spectra were observed. The extrapolated partition ratio for nPX with P450 2B1 was 32. P450 2B1 could be protected from inactivation by nPX by adding an alternate substrate to the reaction mixture. Removal of unbound nPX by dialysis did not reverse the inactivation. The alternate oxidant iodosobenzene was able to partially restore enzymatic activity to nPX-inactivated P450 2B1 samples. A stoichiometry for labeling of 1.2:1 for binding of radiolabeled nPX metabolite to P450 2B1 was seen. These results indicated that nPX inactivated P450 2B1 and P450 2B6 in a mechanism-based manner. P450 2B1 was inactivated primarily by a nPX reactive intermediate that bound to the apoprotein.     

NADPH-dependent covalent binding of [3H]paroxetine to human liver microsomes and S-9 fractions: identification of an electrophilic quinone metabolite of paroxetine

The primary pathway of clearance of the methylenedioxyphenyl-containing compound and selective serotonin reuptake inhibitor paroxetine in humans involves P450 2D6-mediated demethylenation to a catechol intermediate. The process of demethylenation also results in the mechanism-based inactivation of the P450 isozyme. While the link between P450 2D6 inactivation and pharmacokinetic interactions of paroxetine with P450 2D6 substrates has been firmly established, there is a disconnect in terms of paroxetine's excellent safety record despite the potential for bioactivation. In the present study, we have systematically assessed the NADPH-dependent covalent binding of [(3)H]paroxetine to human liver microsomes and S-9 preparations in the absence and presence of cofactors of the various phase II drug-metabolizing enzymes involved in the downstream metabolism/detoxification of the putative paroxetine-catechol intermediate. Incubation of [(3)H]paroxetine with human liver microsomes and S-9 preparations resulted in irreversible binding of radioactive material to macromolecules by a process that was NADPH-dependent. The addition of reduced glutathione (GSH) to the microsomal and S-9 incubations resulted in a dramatic reduction of covalent binding. Following incubations with NADPH- and GSH-supplemented human liver microsomes and S-9, three sulfydryl conjugates with MH(+) ions at 623 Da (GS1), 779 Da (GS2), and 928 Da (GS3), respectively, were detected by LC-MS/MS. The collision-induced dissociation spectra allowed an insight into the structure of the GSH conjugates, based on which, bioactivation pathways were proposed. The formation of GS 1 was consistent with Michael addition of GSH to the quinone derived from two-electron oxidation of paroxetine-catechol. GS 3 was formed by the addition of a second molecule of GSH to the quinone species obtained via the two-electron oxidation of GS 1. The mechanism of formation of GS 2 can be rationalized via (i) further two-electron oxidation of the catechol motif in GS 3 to the ortho-quinone, (ii) loss of a glutamic acid residue from one of the adducted GSH molecules, and (iii) condensation of a cysteine-NH 2 with an adjacent carbonyl of the ortho-quinone to yield an ortho-benzoquinoneimine structure. Inclusion of the catechol-O-methyltransferase cofactor S-adenosylmethionine (SAM) in S-9 incubations also dramatically reduced the covalent binding of [(3)H]paroxetine, a finding that was consistent with O-methylation of the paroxetine-catechol metabolite to the corresponding guaiacol regioisomers in S-9 incubations. While the NADPH-dependent covalent binding was attenuated by GSH and SAM, these reagents did not alter paroxetine's ability to inactivate P450 2D6, suggesting that the reactive intermediate responsible for P450 inactivation did not leave the active site to react with other proteins. The results of our studies indicate that in addition to the low once-a-day dosing regimen (20 mg) of paroxetine, efficient scavenging of the catechol and quinone metabolites by SAM and GSH, respectively, serves as an explanation for the excellent safety record of paroxetine despite the fact that it undergoes bioactivation.     

Bioactivation of Fluorinated 2-Aryl-benzothiazole Antitumor Molecules by Human Cytochrome P450s 1A1 and 2W1 and Deactivation by Cytochrome P450 2S1

Both 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole (5F 203) and 5-fluoro-2-(3,4-dimethoxyphenyl)-benzothiazole (GW 610) contain the benzothiazole pharmacophore and possess potent and selective in vitro antitumor properties. Prior studies suggested the involvement of cytochrome P450 (P450) 1A1 and 2W1-mediated bioactivation in the antitumor activities and P450 2S1-mediated deactivation of 5F 203 and GW 610. In the present study, the biotransformation pathways of 5F 203 and GW 610 by P450s 1A1, 2W1, and 2S1 were investigated, and the catalytic parameters of P450 1A1- and 2W1-catalyzed oxidation were determined in steady-state kinetic studies. The oxidations of 5F 203 catalyzed by P450s 1A1 and 2W1 yielded different products, and the formation of a hydroxylamine was observed for the first time in the latter process. Liquid chromatography-mass spectrometry (LC-MS) analysis with the synthetic hydroxylamine and also a P450 2W1/5F 203 incubation mixture indicated the formation of dGuo adduct via a putative nitrenium intermediate. P450 2W1-catalyzed oxidation of GW 610 was 5-fold more efficient than the P450 1A1-catalyzed reaction. GW 610 underwent a two-step oxidation process catalyzed by P450 1A1 or 2W1: a regiospecific O-demethylation and a further hydroxylation. Glutathione (GSH) conjugates of 5F 203 and GW 610, presumably through a quninoneimine and a 1,2-quinone intermediate, respectively, were detected. These results demonstrate that human P450s 1A1 and 2W1 mediate 5F 203 and GW 610 bioactivation to reactive intermediates and lead to GSH conjugates and a dGuo adduct, which may account for the antitumor activities of 5F 203 and GW 610 and also be involved in cell toxicity. P450 2S1 can catalyze the reduction of the hydroxylamine to the amine 5F 203 under anaerobic conditions and, to a lesser extent, under aerobic conditions, thus attenuating the anticancer activity.     

Bioactivation of 2,3-diaminopyridine-containing bradykinin B1 receptor antagonists: irreversible binding to liver microsomal proteins and formation of glutathione conjugates

The 2,3-diaminopyridine (DAP) moiety was found to represent a core structure essential for the potency of a new series of human bradykinin B(1) receptor antagonists. However, incubation of (14)C-labeled 2,3-DAP derivatives with rat and human liver microsomes resulted in substantial irreversible binding of radioactive material to macromolecules by a process that was NADPH-dependent. Trapping the reactive species with GSH led to significant reduction of the irreversible binding of radioactivity, with concomitant formation of abundant GSH adducts. One type of thiol adducts (detected in both human and rat liver microsomes), resulting from addition of 305 Da to the parent compound, was observed with all 2,3-DAP compounds. These adducts also were detected in rat hepatocyte incubates, as well as in rat bile, following intravenous administration of 2,3-DAPs. Formation of the conjugates appeared to involve modification of the DAP ring, based upon mass spectral analysis of a number of representative GSH adducts; this was corroborated by detailed LC NMR analysis of one compound. Formation of this type of GSH conjugate was markedly reduced when the 2-amino methyl group linking the 2,3-DAP and the biphenyl moiety was replaced with an ether oxygen atom. It is postulated, therefore, that oxidation of the 2-amino group serves as a key step leading to the formation of reactive species associated with the DAP core. In addition, this step appears to be mediated primarily by P450 3A, as evidenced by the marked decrease in both the irreversible binding of radioactivity and the formation of the GSH adducts in human liver microsomes following treatment with ketoconazole and monoclonal antibodies against P450 3A. A mechanism for the bioactivation of 2,3-DAP is proposed wherein oxidation (dehydrogenation or N-hydroxylation followed by dehydration) of the 2-amino group, catalyzed by P450 3A, results in the formation of a highly electrophilic species, pyridine-2,3-diimine.     

Metabolism of pigment yellow 74 by rat and human microsomal proteins.

Pigment Yellow 74 (PY74) is a monoazo pigment that is used in yellow tattoo inks. The metabolism of PY74 was investigated using rat liver and human liver microsomes and expressed human cytochromes P450 (P450s). Two phase I metabolites were isolated and characterized by mass spectrometry and NMR techniques. One metabolite (PY74-M1) was a ring hydroxylation product of PY74, 2-((2-methoxy-4-nitrophenyl)azo)-N-(2-methoxy-4-hydroxyphenyl)-3-oxobutanamide. The second metabolite (PY74-M2) was identified as 2-((2-hydroxy-4-nitrophenyl)azo)-N-(2-methoxy-4-hydroxyphenyl)-3-oxobutanamide, which is the O-demethylation product of PY74-M1. These metabolites were formed by in vitro incubations of PY74 with 3-methylcholanthrene-induced rat liver microsomes and to a much lesser extent by liver microsomes from untreated or phenobarbital-induced rats. The role for CYP1A in the metabolism of PY74 was confirmed using expressed human P450s. The catalytic ability of the P450s for metabolism of PY74 was CYP 1A2 > CYP 1A1 > CYP 3A4 approximately CYP 1B1 (no activity with CYP 2B6, 2C9, 2D6 or 2E1). The metabolism of PY74-M1 to PY74-M2 was catalyzed only by CYP 1A2 and CYP 1A1 (no activity from CYP 1B1, 2B6, 2C9, 2D6, 2E1, or 3A4). These results demonstrate that the tattoo pigment PY74 is metabolized in vitro by P450 to metabolites that should be available for phase II metabolism and excretion.     

Organochlorine insecticides: impacts on human HepG2 cytochrome P4501A, 2B activities and glutathione levels.

This study examined the effects of the organochlorine (OC) insecticides chlordane, o,p'-DDT, dieldrin, endosulfan, kepone, methoxychlor, and toxaphene on human HepG2 cytochrome P450 (1A-EROD and 2B-PROD) activities and glutathione (GSH) levels. Cells were exposed for 24 h at high concentrations (1, 5 or 10 mM) and for 48 h at lower concentrations ranging from 0.01 to 1 mM to evaluate dose responses. Our results show that after 48 h all but dieldrin significantly induced both P4501A and 2B. P4502B responses were greater at all exposure concentrations and times. Mixed responses in GSH levels were observed. All OCs except dieldrin and MXC significantly depleted GSH after 24 h. At 48 h, chlordane, endosulfan and toxaphene significantly increased GSH at low levels and decreased GSH at high levels, while kepone and methoxychlor produced significant declines in GSH at all concentrations. These results support findings of OC insecticides inducing CYP1A, 2B in rats, with CYP2B responses more important. GSH levels declined when P4502B activity was significantly elevated and were significantly increased in the absence of significant P450 activity, suggesting that GSH levels influence the catalytic activity of the cytochrome P450s and the cytochrome P450s influence the cell's ability to regulate GSH.     

Mechanism-based inactivation of cytochrome P450 2D6 by 1-[(2-ethyl-4-methyl-1H-imidazol-5-yl)methyl]- 4-[4-(trifluoromethyl)-2-pyridinyl]piperazine: kinetic characterization and evidence for apoprotein adduction

The kinetics for inactivation of cytochrome P450 2D6 by (1-[(2-ethyl-4-methyl-1H-imidazol-5-yl)methyl]-4-[4-(trifluoromethyl)-2-pyridinyl]piperazine (EMTPP) were characterized, and the mechanism was determined in an effort to understand the observed time-based inactivation. Loss of dextromethorphan O-demethylase activity following coincubation with EMTPP followed pseudo-first-order kinetics and was both NADPH- and EMTPP-dependent. Inactivation was characterized by an apparent Ki of 5.5 microM with a maximal rate constant for inactivation (kinact) of 0.09 min(-1), a t1/2 of 7.7 min, and a partition ratio of approximately 99. P450 2D6 inactivation was unaffected by coincubation with exogenous nucleophiles or reactive oxygen scavengers and was protected by the competing inhibitors N-4-(trifluoromethyl)benzyl quinidinium bromide and quinidine. After a 30 min incubation with 100 microM EMTPP, dextromethorphan O-demethylase activity was decreased approximately 76%, with a disproportionate loss ( approximately 35%) in carbon monoxide binding. Additional mechanistic studies showed no evidence of either metabolite inhibitory complex formation or heme adduction. However, a P450 2D6 apoprotein adduct was characterized that had a mass shift relative to unadducted P450 2D6 apoprotein consistent with the molecular mass of EMTPP (353 Da). In vitro metabolism studies revealed that EMTPP is susceptible to P450 2D6-mediated hydroxylation and dehydrogenation, postulated to both form via initial hydrogen atom abstraction from the alpha-carbon of the imidazole ethyl substituent. Additional studies demonstrated that while a dehydrogenated EMTPP metabolite was apparently stable and observable, we propose that a thermodynamic partitioning may exist, which results in formation of a second dehydrogenated imidazo-methide-like metabolite that may serve as the reactive species causing mechanism-based inactivation of P450 2D6. Last, trapping studies with EMTPP yielded an N-acetyl cysteine conjugate, which upon tandem MS and NMR analysis revealed adduction to the alpha-carbon of the imidazole ethyl substituent. Overall, evidence suggests that nucleophilic attack of an imidazo-methide-like intermediate by a P450 2D6 active site residue leads to apoprotein adduction and consequent inactivation.     

Decomposition rates of isothiocyanate conjugates determine their activity as inhibitors of cytochrome p450 enzymes

Thiol conjugates of isothiocyanates (thiol-ITCs) are metabolites of ITCs formed in the mercapturic acid pathway in mammals. They are effective chemopreventive agents in mouse lung tumor bioassays and in other models. Thiol-ITCs are inhibitors of P450s, but it has not been determined if P450 inhibition is due to conjugates themselves or to parent ITCs released by deconjugation reactions. In studies of mechanism of chemopreventive action of thiol-ITCs, rates of deconjugation of Cys, GSH, and N-acetyl-L-cysteine (NAC) conjugates of benzyl isothiocyanate (BITC), phenethyl isothiocyanate (PEITC), 6-phenylhexyl isothiocyanate (PHITC), and sulforaphane (SFN), expressed as the first-order rate constant k(1) and the half-life of decomposition Dt(1/2), were measured in aqueous solutions at pH 7.4 and 37 degrees. The Dt(1/2)s for the Cys conjugates were severalfold shorter than the Dt(1/2)s for respective GSH conjugates, while the Dt(1/2)s for the NAC conjugates were the longest. Cleavage of thiol conjugates was pH dependent, much slower under acidic conditions than at pH 7.4. Inhibition of P450 enzymes by thiol-ITCs was followed using PROD (pentoxyresorufin O-dealkylation) for P450 2B1 and EROD (ethoxyresorufin O-dealkylation) for P450 1A1. The inhibition of PROD and EROD by aqueous thiol-ITCs increased with preincubation time and was roughly parallel to the extent of decomposition of the conjugate that had occurred, indicating that both potency of the respective parent ITC and the rate of reductive cleavage of the conjugate influenced enzyme inhibition. In the presence of 250-1000 microM GSH, comparable to physiological levels, rates of deconjugation of thiol-ITCs were markedly reduced; inhibition of PROD was also proportionately reduced. Slow rates of decomposition of thiol-ITCs anticipated in plasma and tissues suggests that inhibition of P450 enzymes involved in carcinogen activation by ITCs released from thiol-ITCs may not be a principal mechanism for their tumor inhibitory activity; other mechanisms probably contribute to their chemopreventive activity.     

Structure-activity relationship and elucidation of the determinant factor(s) responsible for the mechanism-based inactivation of cytochrome P450 2B6 by substituted phenyl diaziridines.

It has been demonstrated previously that several 3-trifluoromethyl-3-(4-alkoxyphenyl)diaziridines inhibit the 7-ethoxy-4-(trifluoroethyl)coumarin (7-EFC) O-deethylation activity of P450 2B6 in a mechanism-based manner. In contrast, 3-trifluoromethyl-3-(4-methylthio)phenyl)diaziridine did not have any effect on the activity of P450 2B6. It is interesting that both the alkoxy and the thiophenyl compounds were metabolized by P450 2B6. In this report, the structure-activity relationships for the mechanism-based inactivation of cytochrome P450 2B6 by a series of aryl diaziridines were investigated. Three diaziridines that did not contain a 4-alkoxy-substituent on their phenyl ring, namely, 3-trifluoromethyl-3-(3-methoxyphenyl)diaziridine, 3-trifluoromethyl-3-phenyl diaziridine, and 3-trifluoromethyl-3-(4-chlorophenyl)diaziridine had no effect on the P450 2B6 7-EFC activity. Another analog that did not contain a diaziridine substructure, 3-trifluoromethyl-3-(4-methoxyphenyl)ethanone, also had no effect on the activity of P450 2B6. Glutathione ethyl ester adducts of the phenyldiaziridine reactive intermediates were isolated from reaction mixtures of the inactivated samples and analyzed by liquid chromatography-tandem mass spectrometry. The structures of the conjugates suggested that the electrophilic reactive intermediate in each case was a quinone methide (quinomethane), 4-ethylidene-cyclohexa-2,5-dienone, generated from the 4-alkoxyphenyldiaziridines by removal of both of the diaziridine and the 4-alkyl groups. In conclusion, the determinant factor for the mechanism-based inactivator activity of the aryl diaziridines seems to be the formation of the reactive quinomethane intermediate, which is generated from the 4-alkoxyphenyl diaziridines by a cytochrome P450-catalyzed metabolic reaction.     

Mechanism-based inactivation of cytochromes P450 2E1 and 2E1 T303A by tert-butyl acetylenes: characterization of reactive intermediate adducts to the heme and apoprotein

The kinetics for the inactivation of cytochrome P450 2E1 and the mutant P450 2E1 T303A by tert-butyl acetylene (tBA) and tert-butyl 1-methyl-2-propynyl ether (tBMP) were investigated. The two acetylenes inactivated the 7-ethoxy-4-(trifluoromethyl)coumarin (7-EFC) O-deethylation activity of purified rabbit P450s 2E1 and 2E1 T303A in a reconstituted system in a time-, concentration-, and NADPH-dependent manner. The K(I) values for the inactivation of P450s 2E1 and 2E1 T303A by tBA were 1.0 and 2.0 mM, the k(inact) values were 0.20 and 0.38 min(-)(1), and the t(1/2) values were 3.5 and 1.8 min, respectively. The K(I) values for the tBMP-inactivated P450s were 0.1 and 1.0 mM, the k(inact) values were 0.12 and 0.07 min(-)(1), and the t(1/)(2) values were 5.9 and 10.2 min, respectively. Losses in enzyme activity occurred with concurrent losses in the P450 CO spectrum and P450 heme, which were accompanied by the appearance of two different tBA- or tBMP-modified heme products in each inactivated sample. LC-MS analysis of the adducts showed masses of 661 or 705 Da, consistent with the mass of an iron-depleted heme plus the masses of a tBA or tBMP reactive intermediate and one oxygen atom, respectively. Only the tBA-inactivated P450 2E1 revealed a tBA-adducted apoprotein with an increase in mass of 99 Da, corresponding to the mass of tBA plus one oxygen atom. Surprisingly, the inactivation, CO spectral and heme loss, and heme adduct formation of the tBA-inactivated T303A mutant were completely reversible after dialysis. In addition, metabolism of para-nitrophenol was not compromised by the tBA-inactivated T303A mutant. Therefore, our studies on the inactivation of P450s 2E1 and 2E1 T303A by tBA and tBMP suggest the existence of three distinct mechanisms for inactivation, among which includes a novel, reversible heme alkylation that has not been previously described with P450 enzymes.     

Metabolic activation of a pentafluorophenylethylamine derivative: Formation of glutathione conjugates in vitro in the rat

The aim was to investigate the metabolic activation potential of a pentafluorophenylethylamine derivative (compound I) in vitro in the rat and to identify the cytochrome P450 (CYP) enzymes that catalyse these metabolic activation processes. Reduced glutathione (GSH) was fortified in rat hepatocytes and liver microsomes to trap possible reactive intermediates. Four glutathione conjugates (M1–4) were identified by LC-MSⁿ following incubation of compound I in GSH-enriched rat hepatocytes and liver microsomes. Three of these conjugates (M2–4) have not been reported previously for pentafluorophenyl derivatives. Elemental composition analysis of these conjugates was obtained using high-resolution quadrupole time-of-flight mass spectrometry. The formation of GSH conjugate M1 was rationalized as a direct nucleophilic replacement of fluoride by glutathione, whereas the formation of the GSH conjugates M2–4 was proposed to occur by NADPH-dependent metabolic activation of the pentafluorophenyl ring via arene oxide, quinone and/or quinoneimine reactive intermediates. Formation of these conjugates was enhanced three- to five-fold in liver microsomes obtained from phenobarbital- and dexamethasone-treated rats. In incubations with pooled rat liver microsomes and recombinant rat CYP3A1 and CYP3A2, troleandomycin (TAO) reduced the formation of GSH conjugates M2–4 by 80–90%, but it had no effect on the formation of M1. Incubation of compound I with rat supersomes indicated that only CYP3A1 and CYP3A2 were capable of mediating these metabolic activation processes.