Functional characterization of the G162R and D216H genetic variants of human CYP17A1.

Cytochrome P450 17A1 (CYP17A1) is a dual-function enzyme catalyzing reactions necessary for cortisol and androgen biosynthesis. CYP17A1 is a validated drug target for prostate cancer as CYP17A1 inhibition significantly reduces circulating androgens and improves survival in castration-resistant prostate cancer. Germline CYP17A1 genetic variants with altered CYP17A1 activity manifesting as various endocrinopathies are extremely rare; however, characterizing these variants provides critical insights into CYP17A1 protein structure and function. By querying the dbSNP online database and publically available data from the 1000 genomes project (http://browser.1000genomes.org), we identified two CYP17A1 nonsynonymous genetic variants with unknown consequences for enzymatic activity and stability. We hypothesized that the resultant amino acid changes would alter CYP17A1 stability or activity. To test this hypothesis, we utilized a HEK-293T cell-based expression system to characterize the functional consequences of two CYP17A1 variants, D216H (rs200063521) and G162R (rs141821705). Cells transiently expressing the D216H variant demonstrate a selective impairment of 16α-hydroxyprogesterone synthesis by 2.1-fold compared to wild-type (WT) CYP17A1, while no effect on 17α-hydroxyprogesterone synthesis was observed. These data suggest that substrate orientations in the active site might be altered with this amino acid substitution. In contrast, the G162R substitution exhibits decreased CYP17A1 protein stability compared to WT with a near 70% reduction in protein levels as determined by immunoblot analysis. This variant is preferentially ubiquitinated and degraded prematurely, with an enzyme half-life calculated to be ∼2.5 h, and proteasome inhibitor treatment recovers G162R protein expression to WT levels. Together, these data provide new insights into CYP17A1 structure-function and stability mechanisms.

CYP-independent inhibition of platelet aggregation in rabbits by a mixed disulfide conjugate of clopidogrel.

Dual antiplatelet therapy with clopidogrel and aspirin has been the standard of care in the United States for patients with acute coronary syndromes (ACS) and/or undergoing percutaneous coronary interventions (PCI). However, the effectiveness of clopidogrel varies significantly among different sub-populations due to inter-individual variability. In this study we examined the antiplatelet potential of a novel mixed disulfide conjugate of clopidogrel with the aim to overcome the inter-individual variability. In the metabolic studies using human liver microsomes and cDNA-expressed P450s, we confirmed that multiple P450s are involved in the bioactivation of 2-oxoclopidogrel to H4, one of the diastereomers of the pharmacologically active metabolite (AM) possessing antiplatelet activity. Results from kinetic studies demonstrated that 2C19 is the most active in converting 2-oxoclopidogrel to H4 with a catalytic efficiency of 0.027 µM⁻¹min⁻¹ in the reconstituted system. On the basis of this finding, we were able to biosynthesise the conjugate of clopidogrel with 3-nitropyridine-2-thiol, referred to as clopNPT, and examined its antiplatelet activity in male New Zealand white rabbits. After administration as intravenous bolus at 2 mg/kg, the clopNPT conjugate was rapidly converted to the AM leading to the inhibition of platelet aggregation (IPA). Analyses of the blood samples drawn at various time points showed that intravenous administration of clopNPT led to ~70% IPA within 1 hour and the IPA persisted for more than 3 hours. Since the antiplatelet activity of clopNPT does not require bioactivation by P450s, the mixed disulfide conjugate of clopidogrel has the potential to overcome the inter-individual variability in clopidogrel therapy.

Mechanism-based inactivators as probes of cytochrome P450 structure and function.

The cytochromes P450 superfamily of enzymes is a group of hemeproteins that catalyze the metabolism of an extensive series of compounds including drugs, chemical carcinogens, fatty acids, and steroids. They oxidize substrates ranging in size from ethylene to cyclosporin. Although significant efforts have been made to obtain structural information on the active sites of the microbial P450s, relatively little is currently known regarding the identities of the critical amino acid residues in the P450 active sites that are involved in substrate binding and catalysis. Since information on the crystal structures of the eukaryotic P450s has been relatively limited, investigators have used a variety of other techniques in attempts to elucide the structural features that play a role in the catalytic properties and substrate specificity at the enzyme active site. These include site-directed mutagenesis, natural mutations, homology modeling, mapping with aryl-iron complexes, affinity and photoaffinity labeling, and mechanism-based inactivators. A variety of different mechanism-based inactivators have proven to be useful in identifiying active site amino acid residues involved in substrate binding and catalysis. In this review we present a sampling of the types of studies that can be conducted using mechanism-based inactivators and highlight studies with several classes of compounds including acetylenes, isothiocyanates, xanthates, aminobenzotriazoles, phencyclidine, and furanocoumarins. Labeled peptides isolated from the inactivated proteins have been analyzed by N-terminal amino acid sequencing in conjunction with mass spectrometry to determine the sites of covalent modification. Mechanistic studies aimed at identifying the basis for the inactivation following adduct formation are also presented.

Spectral studies of tert-butyl isothiocyanate-inactivated P450 2E1.

Inactivation of cytochrome P450 2E1 by tert-butyl isothiocyanate (tBITC) resulted in a loss in the spectrally detectable P450-reduced CO complex. The heme prosthetic group does not appear to become modified, since little loss of the heme was observed in the absolute spectra or the pyridine hemochrome spectra, or in the amount of heme recovered from HPLC analysis of the tBITC-inactivated samples. Prolonged incubations of the inactivated P450 2E1 with dithionite and CO resulted in a recovery of both the CO complex and the enzymatic activity. Inactivated samples that were first reduced with dithionite for 1 h prior to CO exposure recovered their CO spectrum to the same extent as samples not pretreated with dithionite, suggesting that the major defect was an inability of the inactivated sample to bind CO. Spectral binding studies with 4-methylpyrazole indicated that the inactivated P450 2E1 had an impaired ability to bind the substrate. Enzymatic activity could not be restored with iodosobenzene as the alternate oxidant. EPR analysis indicated that approximately 24% of the tBITC-inactivated P450 2E1 was EPR-silent. Of the remaining tBITC-inactivated P450 2E1, approximately 45% exhibited an unusual low-spin EPR signal that was attributed to the displacement of a water molecule at the sixth position of the heme by a tBITC modification to the apoprotein. ESI-LC-MS analysis of the inactivated P450 2E1 showed an increase in the mass of the apoprotein of 115 Da. In combination, the data suggest that tBITC inactivated P450 2E1 by binding to a critical active site amino acid residue(s). This modified amino acid(s) presumably acts as the sixth ligand to the heme, thereby interfering with oxygen binding and substrate binding.

Differential effects of naturally occurring isothiocyanates on the activities of cytochrome P450 2E1 and the mutant P450 2E1 T303A.

The effects of benzyl (BITC) and phenethyl isothiocyanate (PEITC) on the activity of a P450 2E1 mutant where the conserved threonine at position 303 was replaced with an alanine residue (P450 2E1 T303A) were examined. PEITC inactivated the mutant enzyme with a K(I) of 1.6 microM. PEITC also inactivated the wild-type P450 2E1 as efficiently with a K(I) of 2.7 microM. The inactivation was entirely dependent on NADPH and followed pseudo-first-order kinetics. Previously we reported the mechanism-based inactivation of wild-type P450 2E1 by BITC with a K(I) of 13 microM. In contrast to the wild-type enzyme, the P450 2E1 T303A mutant was not inactivated by BITC but it was inhibited in a competitive manner with a K(i) of 3 microM. The binding constants determined by spectral binding studies were similar for both enzymes. The binding of BITC produced characteristic Type I spectral changes in the wild-type and mutant enzyme. A radiolabeled BITC metabolite bound to P450 2E1 and to P450 2E1 T303A when both enzymes were incubated with [(14)C]BITC and NADPH. Whole protein electrospray ion trap mass spectrometry indicated that a mass consistent with one molecule of benzylisocyanate and oxygen was adducted to the wild-type enzyme. The mass adducted to the T303A mutant was consistent with the addition of one hydroxylated BITC or of one benzylisocyanate moiety and one sulfur molecule. Analysis of the metabolites of BITC indicated that each enzyme produced similar metabolites but that the mutant enzyme generated significantly higher amounts of benzaldehyde and benzoic acid when compared to the wild-type enzyme.

N-nitrosodimethylamine-mediated formation of oxidized and methylated dna bases in a cytochrome P450 2E1 expressing cell line.

The metabolic activation of N-nitrosodimethylamine (NDMA) to reactive metabolites is a critical step for the expression of its toxic and carcinogenic potential. We have previously reported that a P450 2E1 expressing cell line, GM2E1, can metabolize NDMA to toxic reactive metabolites and cause apoptotic cell death. To investigate whether DNA is a critical target for the reactive metabolites of NDMA, we measured the levels of DNA adducts in untreated and NDMA-treated GM2E1 cells. 8-Hydroxydeoxyguanosine (8-OHdG), a biomarker for oxidative DNA damage, was analyzed following enzymatic hydrolysis of DNA. 7-Methylguanine (7-mGua), the most suitable marker for the DNA adducts formed by methylating agents, was released by thermal depurination of DNA. The modified guanine adducts were separated by HPLC and quantified using electrochemical detection. The levels of 8-OHdG and 7-mGua in GM2E1 cells treated with NDMA increased up to approximately 4- and 100-fold over those in the untreated cells, respectively. The addition of ascorbic acid, an antioxidant, to the NDMA-treated cells resulted in a significant decrease in the cytotoxicity with a concomitant decrease in the levels of 8-OHdG, but not the levels of 7-mGua. Our results demonstrate that the metabolism of NDMA in GM2E1 cells causes both DNA methylation and oxidation and support the hypothesis that NDMA-mediated DNA damage may play an important role in its toxic effects.

Metabolism of the beta-oxidized intermediates of N-nitrosodi-n-propylamine: N-nitroso-beta-hydroxypropylpropylamine and N-nitroso-beta-oxopropylpropylamine.

The rat liver carcinogen N-nitrosodi-n-propylamine (NDPA) is metabolized to a propylating and methylating species in vivo. Metabolism to a methylating species is believed to require an initial hydroxylation by cytochrome P450s (P450s) to N-nitroso-beta-hydroxypropylpropylamine (NHPPA), which is oxidized to N-nitroso-beta-oxopropylpropylamine (NOPPA), followed by a P450-mediated depropylation to beta-oxopropyldiazotate, which non-enzymatically breaks down to the methylating agent. Purified rat liver P450 2B1 and rabbit liver 2E1 in the reconstituted system and liver microsomes from phenobarbital (PB) and pyridine (Pyr) treated rats readily metabolized NOPPA to a methylating species as determined by the in vitro formation of 7-methylguanine (m7Gua) in DNA. Exposure of cells derived from the human liver epithelium transfected with human 2E1 (T5-2E1) to NOPPA resulted in the formation of m7Gua DNA adducts and a dose dependent toxicity. In vitro incubation of NHPPA with microsomes from PB, Pyr and non-treated (NT) rats and a human microsomal sample also resulted in m7Gua formation. P450s 2B1 and 2E1 oxidized NHPPA to NOPPA, forming 16.5 +/- 3.1 and 20.0 +/- 4.4 pmol NOPPA/pmol P450 in 1 h, respectively. Rat liver cytosol, in the presence of NAD+, oxidized NHPPA to NOPPA at a rate of 13.7 +/- 3.0 pmol/min/mg protein while microsomes from NT rats catalyzed this reaction at 95.6 +/- 16.5 pmol/min/mg protein. Cells derived from hamster lung tissue (V79 control) and T5-neo cells oxidized NHPPA to NOPPA. This oxidation was about 15 fold higher in T5-2E1 or V79 cells transfected with human 2E1 or rat 2B1, respectively. The results are consistent with the putative sequential oxidation pathway and suggest that, at the concentrations tested, oxidation of NHPPA to NOPPA may be predominantly mediated by cytochrome P450s. In addition, it appears that rabbit, rat and human P450 2E1 can catalyze both oxidations.

Differential induction of rat hepatic cytochromes P450 3A1, 3A2, 2B1, 2B2, and 2E1 in response to pyridine treatment.

Pyridine (PY) effects on rat hepatic cytochromes P450 (CYP) 3A1 and 3A2 expression were examined at the levels of metabolic activity, protein, and mRNA and were compared with those of CYP2B1/2 and CYP2E1. CYP3A metabolic activity as well as CYP3A protein and mRNA levels increased following treatment of rats with PY. CYP3A1 and CYP3A2 were differentially affected by PY treatment in terms of induction levels, dose dependence, and stability of mRNA. CYP3A1 mRNA levels maximally increased ~42-fold after PY treatment, whereas CYP3A2 mRNA level increased ~4-fold. Moreover, CYP3A1 mRNA levels decreased more rapidly than those of CYP3A2 as determined following inhibition of transcription with actinomycin D or cordycepin. Treatment of rats with PY resulted in a dose-dependent increase in CYP3A1, CYP3A2, and CYP2B1/2B2 protein levels. In contrast to the effects of PY treatment on CYP3A1 and 2B, CYP2E1 protein levels increased in the absence of a concomitant increase in CYP2E1 mRNA levels. Treatment of rats with PY at 200 mg/kg/day for 3 days increased both protein and mRNA levels of CYP3A2, whereas treatment with higher than 200 mg/kg/day for 3 days increased CYP3A2 protein levels without an increase in CYP3A2 mRNA levels. These data demonstrated that PY regulates the various CYPs examined in this study at different levels of expression and that PY regulates CYP3A1 expression through transcriptional activation and CYP3A2 expression through transcriptional and post-transcriptional activation at a low- and high-dose PY treatment, respectively.

Effects of benzyl isothiocyanate on rat and human cytochromes P450: identification of metabolites formed by P450 2B1.

Naturally occurring isothiocyanates, such as benzyl isothiocyanate (BITC), are potent and selective inhibitors of carcinogenesis induced by a variety of chemical carcinogens. These effects appear to be mediated through favorable modification of both phase I and II enzymes involved in carcinogen metabolism. The inactivation of rat and human cytochromes P450 (P450s) in microsomes and the reconstituted system by BITC was investigated. BITC is a mechanism-based inactivator of rat P450s 1A1, 1A2, 2B1, and 2E1, as well as human P450s 2B6 and 2D6. BITC was most effective in inactivating P450s 2B1, 2B6, 1A1, and 2E1, whereas the activities of human P450 2C9 and rat P450 3A2 were not altered. The concentrations required for half-maximal inactivation (K(I)) of P450s 1A1, 1A2, 2B1, and 2E1 were 35, 28, 16, and 18 microM, respectively. The corresponding values for k(inact) were 0.26, 0.09, 0.18, and 0.05 min(-1), respectively. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of P450 2B1 inactivated by [(14)C]BITC indicated specific and covalent modification of the P450 apoprotein by a metabolite of BITC. High-performance liquid chromatography analysis of the BITC metabolites revealed that benzylamine was the major metabolite and there were lesser amounts of benzoic acid, benzaldehyde, N,N'-di-benzylurea, and N,N'-di-benzylthiourea. Presumably, BITC was metabolized to the reactive benzyl isocyanate intermediate that covalently modified the P450 apoprotein or hydrolyzed to form benzylamine. BITC was an efficient inactivator of P450 2B1 with a partition ratio of approximately 11:1. This irreversible inactivation of P450s by BITC could contribute significantly to its chemopreventative action.

Inactivation of cytochrome P450 2B1 by benzyl isothiocyanate, a chemopreventative agent from cruciferous vegetables.

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.

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.

Identification of the human liver microsomal cytochrome P450s involved in the metabolism of N-nitrosodi-n-propylamine.

The ability of human liver cytochrome P450s to metabolize the environmental carcinogen N-nitrosodi-n-propylamine (NDPA) was investigated. The maximum rate of NDPA depropylation in seven human liver microsomal samples was 1.15 nmol/min/mg (range 0.53-2.60). Troleandomycin, a P450 3A4/5 inhibitor, inhibited depropylation modestly (10-60%) in three of seven samples. Diethyldithiocarbamic acid, a potent 2E1 inhibitor, and a 2E1 inhibitory monoclonal antibody (mAb) inhibited the reaction in all samples (23 to almost 100%). No significant inhibition was observed with the 2C9 inhibitor sulfaphenazole or with mAbs to 3A4, 2A6 and 2D6. The 2C8/9/18/19 mAb inhibited depropylation in one sample by approximately 25% and approximately 25% of the activity in another sample could not be accounted for by the inhibitors. Denitrosation of NDPA by three of the microsomal samples exhibited low K(m) values (51-86 microM) while two of these also had high K(m) values (2.6 and 4.6 mM). Purified human P450 2B6 and 3A4 and human P450 2A6, 2C8, 2C9 and 2D6 membranes had high K(m) values relative to their maximum turnover rates and are unlikely to participate in NDPA metabolism at micromolar concentrations. Conversely, purified rabbit 2E1 exhibited K(m) and V(max) values for depropylation of 52 microM and 13.4 nmol propionaldehyde/min/nmol P450, respectively. Values for denitrosation were 66 microM and 1.44 nmol nitrite/min/nmol P450, respectively. The toxicity of NDPA in transfected human liver epithelial cells expressing 2E1 was dose dependent down to 50 microM. No toxicity was observed in control cells or those expressing 2A6. These results indicate that 2E1 is the major human liver microsomal isoform responsible for NDPA metabolism at low micromolar concentrations. We also show that purified P450s catalyze the denitrosation of NDPA at approximately 10-20% of the rate of depropylation and K(m) values for both reactions are the same for each isozyme. This is consistent with the formation of an initial intermediate common to both pathways, presumably an alpha-nitrosamino radical.

Mechanistic studies of cytochrome P450 2B1 inactivation by xanthates.

Xanthates have previously been shown to inactivate the phenobarbital-inducible rat cytochrome P450 2B1 as well as its human homologue P450 2B6. The inactivation was mechanism-based and the loss in enzymatic activity was due to covalent binding of a reactive xanthate intermediate to the P450 2B1 apoprotein. In this report, we investigated various mechanistic events to elucidate the individual step(s) in the P450 catalytic cycle that are compromised due to the inactivation by xanthates. Different xanthates displayed typical type I binding spectra and the spectral binding constants were in the low-millimolar range. A dramatic loss in 7-ethoxy-4-(trifluoromethyl)coumarin activity was observed when P450 2B1 was incubated with five different xanthates in the presence of NADPH. With the exception of the C14 xanthate, virtually no loss of absorbance at 418 or 450 nm in the reduced-CO complex was observed. Long-chain xanthates were able to affect the rate of the first electron transfer in the P450 catalytic cycle by stabilizing the heme in its low-spin state. n-Octyl xanthate (C8) metabolism led to very little observable oxy-ferro intermediate complex formation. The alternate oxidant tert-butyl hydroperoxide was able to support the inactivation reaction of C8 in the absence of reductase or NADPH. The rates of reduction of native, C8-exposed, and C8-inactivated P450 2B1 were measured. The C8-inactivated P450 had a 62% lower rate of reduction in the absence or presence of benzphetamine compared to the native enzyme. Product formation of the three enzyme preparations was quantified with benzphetamine as the substrate. The C8-inactivated P450 2B1 exhibited a much lower rate of NADPH consumption and formation of formaldehyde. However, the ratio of H2O2 to formaldehyde production increased from 1:1 for the native enzyme to 2.8:1 for the inactivated P450. Together these observations indicate that the covalent modification of P450 2B1 by a reactive intermediate of xanthates reduces the rate of the first electron transfer by the reductase and also leads to uncoupling of electron transfer from product formation by diverting a greater proportion of the electrons to H2O2 formation.

Mechanism-based inactivation of cytochrome P450 2B1 by 7-ethynylcoumarin: verification of apo-P450 adduction by electrospray ion trap mass spectrometry.

7-Ethynylcoumarin was synthesized as a potential mechanism-based inhibitor, and it was found to be an effective inactivator of 7-ethoxy-4-(trifluoromethyl)coumarin (7EFC) O-deethylation catalyzed by purified, reconstituted P450 2B1. In contrast, 7-ethynylcoumarin demonstrated minimal inactivation of P450 2A6-mediated 7-hydroxycoumarin formation. The inactivation of P450 2B1 demonstrated pseudo-first-order kinetics and was NADPH- and inhibitor-dependent. The maximal rate constant for the inactivation of 2B1 was 0.39 min(-)(1) at 30 degrees C, and thus, the time required to inactivate 50% of the P450 2B1 that was present (t(1/2)) was 1.8 min. The estimated concentration which led to half-maximal inactivation (K(I)) was 25 microM. No protection from inactivation was seen in the presence of nucleophiles (glutathione and sodium cyanide), an iron chelator (deferroxamine), or superoxide dismutase and catalase. Addition of the substrate (7EFC) protected P450 2B1 from inactivation, in a concentration-dependent manner. The partition ratio for P450 2B1 was 25; i.e., the number of metabolic events was 25-fold higher than the number of inactivating events. Incubations of 7-ethynylcoumarin with P450 2B1 for 10 min resulted in an 80% loss in enzymatic activity, while 90% of the ability to form a reduced-CO complex remained. This activity loss was not recovered following dialysis, indicative of irreversible inactivation. Covalent attachment of the entire inhibitor and oxygen to apo-P450 2B1, in a 1:1 ratio, was shown via electrospray ion trap mass spectrometry. This method also verified the absence of modification to the heme or the cytochrome P450 reductase. Taken together, the characterization of the inhibition seen with P450 2B1 and 7-ethynylcoumarin was consistent with all of the criteria required to distinguish a mechanism-based inactivator. In addition, electrospray ion trap mass spectrometry has the potential to be applied to protein adducts above and beyond those associated with the mechanism-based inactivation of cytochrome P450s.

Expression of human cytochrome P450 2B6 in Escherichia coli: characterization of catalytic activity and expression levels in human liver.

Expression of human cytochrome P450 (P450) 2B6 in Escherichia coli was achieved following supplementation of the expression medium with chloramphenicol. The recombinant protein was purified using Ni(2+)-nitrilotriacetate chromatography and was characterized with regard to its spectral properties and catalytic activities toward typical P450 substrates. The purified recombinant protein was also used to raise polyclonal antibodies in rabbits. Examination of a panel of human liver microsomal preparations revealed expression of P450 2B6 in most samples, with levels of <1 to 30 pmol 2B6/mg microsomal protein. Examination of purified P450 2B6 preparations revealed the presence of a protease-sensitive site located 126 residues away from the N-terminus. The identity of the cleavage boundary was verified by protein sequence analysis. Cleavage of P450 2B6 at that site results in the presence of a lower molecular weight fragment of approximately 35 kDa in purified preparations. An immunoreactive peptide of a similar molecular weight was consistently observed in some but not all human liver microsomal preparations suggesting cleavage at the same site. Examination of catalytic activities of the purified reconstituted protein indicated the potential utility of (S)-mephenytoin N-demethylation and testosterone 16beta-hydroxylation as markers for P450 2B6.

Rat liver cytosol catalyzes a reaction involving activated N-nitrosodimethylamine and a carbohydrate from the pentose phosphate pathway.

N-Nitrosodimethylamine is a liver toxin and mutagen following activation by cytochrome P450. The role of the cytosol in N-nitrosodimethylamine metabolism is not well understood. The effect of cytosol on N-nitrosodimethylamine metabolism was investigated using microsomes and cytosol from rat liver in in vitro reactions with N-nitrosodimethylamine and an NADPH generating system. Studies in which [(14)C]-N-nitrosodimethylamine and calf thymus DNA were used indicated that the addition of cytosol to the microsomal reaction mixture resulted in >200% enhancement of the radioactivity associated with DNA after the DNA was isolated from the reaction mixture by phenol extraction followed by ethanol precipitation. This stimulatory effect was associated with a cytosolic protein and was found to be dependent on both the microsomes and the carbohydrate used in the glucose-6-phosphate dehydrogenase system for the generation of NADPH. The carbohydrate requirement was found to be specific for intermediates of the pentose phosphate pathway, and maximum stimulation occurred with ribulose 5-phosphate. Most of the counts from [(14)C]-N-nitrosodimethylamine which were isolated with DNA after the addition of cytosol to reaction mixtures were not covalently bound to the DNA. HPLC analysis identified four radiolabeled metabolites derived from [(14)C]-N-nitrosodimethylamine following the in vitro incubations. One of the four products was formed only when both cytosol and ribulose 5-phosphate were added to the enzymatic incubations. This product also formed from [(14)C]-alpha-acetoxy nitrosodimethylamine in the absence of microsomes, only when cytosol and ribulose 5-phosphate were added to the reaction mixtures. Thus, these data demonstrate that an enzyme in the cytosol catalyzes a reaction involving a metabolite of N-nitrosodimethylamine (which is formed following cytochrome P450-mediated activation) and a carbohydrate related to the pentose phosphate pathway. A similar reaction also occurs with N-diethylnitrosamine but not with N-dipropylnitrosamine or N-dibutylnitrosamine.

Inactivation of cytochrome P450 2E1 by benzyl isothiocyanate.

The cytochrome P450 enzymes constitute a family of phase I enzymes that play a prominent role in the metabolism of a great variety of endogenous and xenobiotic compounds. In this study, the kinetics for the inactivation of cytochrome P450 2E1 by benzyl isothiocyanate (BITC) were elucidated. BITC is a naturally occurring compound found in cruciferous vegetables such as broccoli. BITC inhibited the 7-ethoxy-4-(trifluoromethyl)coumarin (7-EFC) O-deethylation activity of purified and reconstituted P450 2E1 in a time- and concentration-dependent manner. The concentration of inactivator needed for half-maximal inactivation (K(I)) was 13 microM, and the maximum rate of inactivation at saturation (k(inact)) was 0.09 min-1. The partition ratio for the inactivation of P450 2E1 by BITC was found to have an approximate value of 27. Inactivation of P450 2E1 by BITC was dependent on the presence of NADPH. Following incubation for 5 min with BITC, a 65% loss in enzymatic activity was observed, while approximately 74% of the spectrally detectable enzyme remained. 7-Ethoxycoumarin (7-EC), a substrate of P450 2E1, protected P450 2E1 from BITC inactivation, reducing the loss in 7-EFC O-deethylation activity from 50 to 18% when a 1:20 molar ratio of BITC:7-EC was used. Inactivation of P450 2E1 by BITC was irreversible, and no activity was regained after extensive washes to remove BITC. Addition of cytochrome b(5) to the reconstituted system did not affect the rate of inactivation. Reductase activity was unaffected by BITC. The results reported here indicate that BITC is a mechanism-based inactivator of cytochrome P450 2E1 and that the inactivation was primarily due to a modification of the apoprotein by BITC.

Metabolism of genistein by rat and human cytochrome P450s.

The metabolism of genistein (4',5,7-trihydroxyisoflavone), a phytoestrogen derived from soy products, was investigated using rat and human liver microsomes and recombinant human cytochrome P450 enzymes. Metabolism of genistein by microsomes obtained from rats treated with pyridine, phenobarbital, beta-naphthoflavone, isosafrole, pregnenolone-16alpha-carbonitrile, or 3-methylcholanthrene resulted in very different product profiles consisting of five different NADPH- and time-dependent metabolites as observed by HPLC reverse-phase analysis at 260 nm. The metabolism of genistein was also investigated with recombinant human cytochrome P450 1A1, 1A2, 1B1, 2B6, 2C8, 2E1, or 3A4. P450s 1A1, 1A2, 1B1, and 2E1 metabolized genistein to form predominantly one product (peak 3) with smaller amounts of peaks 1 and 2. P450 3A4 produced two different products (peaks 4 and 5). Product peaks 1-3 eluted off the HPLC column prior to the parent compound genistein, and the UV/vis spectra, GC/MS, and ESI/MS/MS analyses support the conclusion that these products result from hydroxylation of genistein. The product peak 3 has been identified by tandem mass spectrometry as 3',4',5, 7-tetrahydroxyisoflavone, also known as orobol, and peaks 1 and 2 appear to be hydroxylated at position 6 or 8.

Identification of selective mechanism-based inactivators of cytochromes P-450 2B4 and 2B5, and determination of the molecular basis for differential susceptibility.

Rabbit cytochromes P-450 (P-450) 2B4 and 2B5 differ by only 12 amino acid residues yet they exhibit unique steroid hydroxylation profiles. Previous studies have led to the identification of active site residues that are determinants of these specificities. In this study, mechanism-based inactivators were identified that discriminate between the closely related 2B4 and 2B5 enzymes. A previously characterized inhibitor, 2-ethynylnaphthalene (2EN), was found to be selective for 2B4 inactivation. As inhibitor metabolism and the partition ratio affect susceptibility, molecular dynamics simulations were performed to assess the stability of the productive binding orientation of 2EN within 2B4 and 2B5 three-dimensional models. Although 2EN was stable within the 2B4 model, it exhibited substantial movement away from the heme moiety in the 2B5 model. However, heterologously expressed 2B5 was found to catalyze the oxidation of 2EN to the stable product 2-naphthylacetic acid. Thus, the increased mobility of 2EN may result in reduced susceptibility of 2B5 by increasing the probability that the reactive ketene intermediate hydrolyzes with water instead of reacting with active site residues. Another compound, 1-adamantyl propargyl ether (1APE), selectively inactivated 2B5. The structural basis for 2EN and 1APE susceptibility was assessed using active site mutants. Interconversion of 2EN susceptibility was observed for 2B4 or 2B5 mutants containing a single alteration at residue 363. Single substitutions in 2B4 also conferred susceptibility to 1APE; however, multiple alterations were required to reduce the susceptibility of 2B5. These alterations may influence inhibitor susceptibility by affecting the stability of the productive binding orientation.

N-Nitrosodimethylamine-mediated cytotoxicity in a cell line expressing P450 2E1: evidence for apoptotic cell death.

N-Nitrosodimethylamine (NDMA) is an acute hepatotoxin and potent carcinogen. The metabolic activation of NDMA to reactive metabolites is a critical step for the expression of its toxic and carcinogenic potential. We have previously demonstrated a strong correlation between methylation of cellular macromolecules and NDMA-mediated cytotoxicity, and we have demonstrated that reactive oxygen species may partially contribute to the toxic effects in P450 2E1-expressing cells. The mode of cell death in NDMA-treated monolayer cultures exhibited the following characteristics: (i) condensation of nuclear chromatin as demonstrated by using Hoechst 33258 staining, (ii) DNA fragmentation as detected by combining pulsed field and conventional agarose gel electrophoresis, and (iii) DNA double strand breaks determined by using the in situ terminal deoxynucleotidyl transferase assay and flow cytometric analysis. These results indicate that reactive metabolites of NDMA trigger activation of the signal pathway for apoptotic cell death in these P450-expressing cells. The NDMA-mediated cell death was partially prevented by the endonuclease inhibitor, aurintricarboxylic acid, as well as the caspase inhibitors, acetyl-Asp-Glu-Val-Asp-CHO and acetyl-Tyr-Val-Ala-Asp-CHO. The cell cycle distribution was altered in NDMA-treated cells resulting in an increase in the G2/M phase and a decrease in the G1 phase. Our results suggest that DNA degradation, the inability to complete DNA repair, the biochemical events associated with G2/M arrest, and the process of apoptotic death all result from P450 2E1-catalyzed metabolism of NDMA.