Isolation and reconstitution of cytochrome P450ox and in vitro reconstitution of the entire biosynthetic pathway of the cyanogenic glucoside dhurrin from sorghum

A cytochrome P450, designated P450ox, that catalyzes the conversion of (Z)-p-hydroxyphenylacetaldoxime (oxime) to p-hydroxymandelonitrile in the biosynthesis of the cyanogenic glucoside beta-D-glucopyranosyloxy-(S)-p-hydroxymandelonitrile (dhurrin), has been isolated from microsomes prepared from etiolated seedlings of sorghum (Sorghum bicolor L. Moench). P450ox was solubilized using nonionic detergents, and isolated by ion-exchange chromatography, Triton X-114 phase partitioning, and dye-column chromatography. P450ox has an apparent molecular mass of 55 kD, its N-terminal amino acid sequence is -ATTATPQLLGGSVP, and it contains the internal sequence MDRLVADLDRAAA. Reconstitution of P450ox with NADPH-P450 oxidoreductase in micelles of L-alpha-dilauroyl phosphatidylcholine identified P450ox as a multifunctional P450 catalyzing dehydration of (Z)-oxime to p-hydroxyphenylaceto-nitrile (nitrile) and C-hydroxylation of p-hydroxyphenylacetonitrile to nitrile. P450ox is extremely labile compared with the P450s previously isolated from sorghum. When P450ox is reconstituted in the presence of a soluble uridine diphosphate glucose glucosyltransferase, oxime is converted to dhurrin. In vitro reconstitution of the entire dhurrin biosynthetic pathway from tyrosine was accomplished by the insertion of CYP79 (tyrosine N-hydroxylase), P450ox, and NADPH-P450 oxidoreductase in lipid micelles in the presence of uridine diphosphate glucose glucosyltransferase. The catalysis of the conversion of Tyr into nitrile by two multifunctional P450s explains why all intermediates in this pathway except (Z)-oxime are channeled.     

Spectral intermediates during the reduction of hepatic microsomal cytochrome P-450

The slow reduction of microsomal cytochrome P-450 by dithionite consists of an initial fast and then a slow phase. During reduction of aniline and azide complexes with cytochrome P-450, an intermediate spectrum developed in the fast phase and changed to that of the reduced form in the slow phase. Only the spectra in the slow phase had an isosbestic point. No intermediate spectrum detectable during reduction of the cyanide complex and native-cytochrome P-450. Carbon monoxide accelerated the reaction, causing complete reduction in the initial phase. The electron spin resonance spectrum of cytochrome P450 was greatly reduced in the initial phase of reduction with dithionite. These results indicate that reduction of the aniline and azide complexes of cytochrome P-450 involves two steps: first reduction of cytochrome P-450 and then some changes in reduced state. The aniline and cyanide difference spectra of reduced cytochrome P-450 showed peaks at 423 nm and 429 nm, respectively, while that of azide had a peak at 445 nm and a trough at 404 nm. An essay method to obtain the difference spectrum of reduced minus oxidized cytochrome P-450 using a spectral data processor is reported. The effects of other NADH-nonreducible pigments on the spectrum is eliminated by this procedure, provided these pigments are rapidly reduced by dithionite. Therefore, the spectrum obtained was slightly differed from that measured by the usual method, especially in the region of 425 nm.     

Cloning of three A-type cytochromes P450, CYP71E1, CYP98, and CYP99 from Sorghum bicolor (L.) Moench by a PCR approach and identification by expression in Escherichia coli of CYP71E1 as a multifunctional cytochrome P450 in the biosynthesis of the cyanogenic glucoside dhurrin

A cDNA encoding the multifunctional cytochrome P450, CYP71E1, involved in the biosynthesis of the cyanogenic glucoside dhurrin from Sorghum bicolor (L.) Moench was isolated. A PCR approach based on three consensus sequences of A-type cytochromes P450- (V/I)KEX(L/F)R, FXPERF, and PFGXGRRXCXG-was applied. Three novel cytochromes P450 (CYP71E1, CYP98, and CYP99) in addition to a PCR fragment encoding sorghum cinnamic acid 4-hydroxylase were obtained. Reconstitution experiments with recombinant CYP71E1 heterologously expressed in Escherichia coli and sorghum NADPH-cytochrome P450-reductase in L-alpha-dilaurylphosphatidyl choline micelles identified CYP71E1 as the cytochrome P450 that catalyses the conversion of p-hydroxyphenylacetaldoxime to p-hydroxymandelonitrile in dhurrin biosynthesis. In accordance to the proposed pathway for dhurrin biosynthesis CYP71E1 catalyses the dehydration of the oxime to the corresponding nitrile, followed by a C-hydroxylation of the nitrile to produce p-hydroxymandelonitrile. In vivo administration of oxime to E. coli cells results in the accumulation of the nitrile, which indicates that the flavodoxin/flavodoxin reductase system in E. coli is only able to support CYP71E1 in the dehydration reaction, and not in the subsequent C-hydroxylation reaction. CYP79 catalyses the conversion of tyrosine to p-hydroxyphenylacetaldoxime, the first committed step in the biosynthesis of the cyanogenic glucoside dhurrin. Reconstitution of both CYP79 and CYP71E1 in combination with sorghum NADPH-cytochrome P450-reductase resulted in the conversion of tyrosine to p-hydroxymandelonitrile, i.e. the membranous part of the biosynthetic pathway of the cyanogenic glucoside dhurrin. Isolation of the cDNA for CYP71E1 together with the previously isolated cDNA for CYP79 provide important tools necessary for tissue-specific regulation of cyanogenic glucoside levels in plants to optimize food safety and pest resistance.     

Liver microsomal drug-metabolizing enzyme system: functional components and their properties

The liver microsomal drug-metabolizing enzyme system consists of two protein components, cytochrome P-450 and NADPH-cytochrome c reductase, and a lipid, phosphatidylcholine. Cytochrome P-450 serves as the binding site for oxygen and substrate while the reductase acts as an electron carrier shuttling electrons from NADPH to cytochrome P-450. The phospholipid facilitates the transfer of electrons from NADPH-cytochrome c reductase to cytochrome P-450 but itself is not an electron carrier. Different cytochromes P-450 and P-448 have been purified; the spectral, catalytic, and immunological properties as well as the molecular weight (determined by SDS-gel electrophoresis) of all these hemeproteins differ from one another. The presence of multiple cytochrome P-450s may explain the species, strain, age, tissue, and sex differences as well as the effect of inducers and nutritional status in mammlian drug metabolism.     

Methodology of the survey]

To evaluate the effects of various barbiturates on lidocaine metabolism by cytochrome P-450 (P-450), enzyme kinetics were analyzed in an in vitro study using rat hepatic microsomes. Phenobarbital, amobarbital, hexobarbital, pentobarbital, and thiamylal showed the mixed type inhibition of lidocaine metabolism with inhibition constants being 4.89, 1.08, 2.76, 0.77 and 0.65 mM, respectively. Same as lidocaine, all barbiturates used in the present study, corresponding to binding with P-450, induced the I type of spectral change of P-450. Since these did not affect cytochrome C reductase activity, it was suggested that this inhibition of lidocaine metabolism in hepatic microsomes may have been caused by the reduction of activity on P-450 by the barbiturates.     

Regio- and stereoselective propranolol metabolism by 15 forms of purified cytochromes P-450 from rat liver

Regio- and stereoselectivity of cytochrome P-450-mediated propranolol metabolism (4-, 5- and 7-hydroxylations and N-desisopropylation) was studied using 15 purified cytochrome P-450 species. With each purified cytochrome P-450 species, the regioselectivity was distinct and different between the two optical isomers used as substrates. The stereoselectivity was different depending on the position of propranolol to be metabolized. The regio- and stereoselectivity was altered when substrate concentration was altered, suggesting that the kinetics of the reactions are different depending on the positions of propranolol to be metabolized. Furthermore, the selectivity and its manner of alterations with substrate concentrations were different among all cytochrome P-450 species used. Propranolol, with its multiple metabolic pathways and optical isomers, is an extremely interesting substrate for characterization of cytochrome P-450 species.     

The changing sex ratio of the navajo tribe

Two approaches may be used to study the function of cytochrome P-450 in insects: (a) an evaluation of the spectral and catalytic properties of the hemoprotein while associated with microsomal membranes; (b) the solubilization, resolution and purification of the microsomal mixed-function oxidase system. The first approach has provided some understanding of the biochemical factors involved in the metabolism of a variety of compounds, including pesticides, drugs, hormones and many other xenobiotics. However, solubilization of the monooxygenase system allows the study of each of its components individually, providing a better insight on the sequence of events leading to the hydroxylation of a substrate, the type of intermediates formed, and the rate-limiting step(s). This report discusses studies carried out with the monooxygenase system associated with microsomal membranes, as well as procedures to solubilize and partially purify its components from housefly microsomes. The latter involves solubilization with either Triton X-100 or sodium cholate, followed by either ammonium sulfate fractionation, Sephadex G-200, DEAE-Sephadex A-50 column chromatography or by omega-amino-n-octyl-Sepharose 4B affinity chromatography. These procedures have shown that two cytochrome P-450 species (P-450 and P-450I) are present in microsomes isolated from a resistant housefly strain. Induction with either naphthalene or phenobarbital appears to increase cytochrome P-450I preferentially.     

Preparation and properties of partially purified pulmonary cytochrome P-450 from rabbits

Cytochrome P-450 from rabbit pulmonary microsomes was purified approximately 32-fold. The purification method involved solubilization of microsomes using sodium cholate, and recovery of cytochrome P-450 in the precipitate formed between 25 to 42% saturation of the digested microsomes with ammonium sulfate in the absence of glycerol. Further purification was achieved by chromatography on DEAE-cellulose and hydroxylapatite using Emulgen 913 as an eluent. Partially purified preparations containing up to 7.4 nmol of cytochrome P-450 per mg of protein were essentially free of NADPH-cytochrome c reductase activity and cytochromes b5 and P-420. However, epoxide hydrase was found to co-purify with cytochrome P-450. The CO-difference spectrum of dithionite-reduced purified cytochrome showed the expected peak at 450 nm. However, the magnitude of the peak was dependent on added microsomal lipid fraction in the assay medium. Purified pulmonary cytochrome P-450 formed typical types I and II substrate difference spectra with benzphetamine and pyridine, respectively. Sodium dodecyl sulfate-gel electrophoresis of partially purified cytochrome P-450 gave two major bands when stained with Coomassie blue. The faster moving band which contained peroxidase activity had an estimated molecular weight of 49,000 +/- 1,200. The cytochrome P-450 fraction, when combined with solubilized pulmonary microsomal NADPH-cytochrome c reductase and lipid fractions, was active in the O-deethylation of 7-ethoxycoumarin and the N-demethylation of benzphetamine.     

Genomic cloning and protein expression of a novel rat brain cytochrome P-450 CYP2D18* catalyzing imipramine N-demethylation

We have previously reported the isolation of two cDNA clones, designated 2d-29 and 2d-35, which have identical open reading frames and code for a novel brain cytochrome P-450 (P-450) belonging to the CYP2D subfamily, and noted that the mRNA of clone 2d-35 seems to be expressed in the brain but not in the liver (1). Although the deduced amino acid sequence of these clones differs from that of the liver CYP2D4 by only 5 amino acids distributed in the C-terminal region, this new P-450 cDNA clone contained a unique 5'-extension, and we posit in this report by analysis of a genomic clone that this 5'-untranslated sequence is derived from a gene distinct from that of CYP2D4. Thus, this novel P-450 was named P-450 2D18 according to the recommended nomenclature (2). The expressibility of this cDNA was confirmed by in vitro translation using a reticulocyte system, and protein expression was performed using COS-M6 cells. Immunoblot analysis showed a cross-reacting band of the predicted size range with anti-P-450 2D6 antiserum, which was not seen in control cells. Furthermore, the CYP2D18-expressed COS cell lysate showed N-demethylation activity toward imipramine, whereas another brain P-450 CYP4F6-expressed COS cell lysate showed 10-hydroxylation activity. This is the first report that associates an individual P-450 isozyme in brain with a particular metabolic alteration of the antidepressant imipramine.     

Purification and comparative characterization of cytochrome P-450scc (CYP XIA1) from sheep adrenocortical mitochondria

Cytochrome P-450scc (CYP XI A1) was purified from sheep adrenocortical mitochondria. The purified cytochrome was found to be homogeneous on SDS-polyacrylamide gel electrophoresis and to have a heme content of 20.8 nmol/mg of protein. Its amino acid composition and NH2-terminal amino acid sequence were determined, and compared with those of other known mammalian and fish cytochromes P-450scc. EPR spectra of the cytochrome P-450scc were measured for oxidized and NO-reduced forms in the presence or absence of cholesterol and/or adreno-ferredoxin. Spectral properties of these various forms were very similar to those of the bovine enzyme. Circular dichroism spectra of the purified sheep cytochrome P-450scc in the oxidized and dithionite-reduced forms, and of their complexed forms with cholesterol or adreno-ferredoxin were analyzed in the region from 200 to 700 nm. The difference CD spectrum of the oxidized cytochrome P-450scc complexed with adreno-ferredoxin minus the oxidized form suggests an increase in the high-spin form upon the addition of adreno-ferredoxin. This may suggest a direct influence of the adreno-ferredoxin binding to the heme moiety of the oxidized cytochrome P-450scc.     

Electron transfer to cytochrome P-450scc limits cholesterol-side-chain-cleavage activity in the human placenta

The aim of this study was to determine whether electron transfer from adrenodoxin reductase and adrenodoxin limits the activity of cytochrome P-450scc in mitochondria from the human placenta. Mitochondria were disrupted by sonication to enable exogenous adrenodoxin and adrenodoxin reductase to deliver electrons to cytochrome P-450scc. After sonication, the rate of pregnenolone synthesis was greatly decreased relative to that by intact mitochondria, due to dilution of endogenous adrenodoxin and adrenodoxin reductase into the incubation medium. The addition of saturating concentrations of bovine or human adrenodoxin and bovine adrenodoxin reductase to the disrupted mitochondria gave an initial rate of pregnenolone synthesis that was 6.3-fold higher than that for intact mitochondria. Similar results were observed when 20alpha-hydroxycholesterol was used as substrate rather than endogenous cholesterol. The turnover number of cytochrome P-450scc in sonicated placental mitochondria supplemented with adrenodoxin and adrenodoxin reductase was comparable to that for the purified enzyme assayed under conditions where electron transfer was not limiting. Addition of exogenous adrenodoxin and adrenodoxin reductase to sonicated mitochondria from the pig corpus luteum and rat adrenal had a much smaller effect on pregnenolone synthesis compared with intact mitochondria, than observed for the placenta. We conclude that in the human placenta, electron transfer to cytochrome P-450scc is limiting, permitting pregnenolone synthesis to proceed at only 16% maximum velocity.     

Steroidogenic electron transport in adrenal cortex mitochondria

The flavoprotein NADPH-adrenodoxin reductase and the iron sulfur protein adrenodoxin function as a short electron transport chain which donates electrons one-at-a-time to adrenal cortex mitochondrial cytochromes P-450. The soluble adrenodoxin acts as a mobile one-electron shuttle, forming a complex first with NADPH-reduced adrenodoxin reductase from which it accepts an electron, then dissociating, and finally reassociating with and donating an electron to the membrane-bound cytochrome P-450 (Fig. 9). Dissociation and reassociation with flavoprotein then allows a second cycle of electron transfers. A complex set of factors govern the sequential protein-protein interactions which comprise this adrenodoxin shuttle mechanism; among these factors, reduction of the iron sulfur center by the flavin weakens the adrenodoxin-adrenodoxin reductase interaction, thus promoting dissociation of this complex to yield free reduced adrenodoxin. Substrate (cholesterol) binding to cytochrome P-450scc both promotes the binding of the free adrenodoxin to the cytochrome, and alters the oxidation-reduction potential of the heme so as to favor reduction by adrenodoxin. The cholesterol binding site on cytochrome P-450scc appears to be in direct communication with the hydrophobic phospholipid milieu in which this substrate is dissolved. Specific effects of both phospholipid headgroups and fatty acyl side-chains regulate the interaction of cholesterol with its binding side. Cardiolipin is an extremely potent positive effector for cholesterol binding, and evidence supports the existence of a specific effector lipid binding site on cytochrome P.450scc to which this phospholipid binds.     

Chemical modification of Tyr34 and Tyr129 in rabbit liver microsomal cytochrome b5 affects interaction with cytochrome P-450 2B4

Rabbit liver microsomal cytochrome b5 was allowed to react with tetranitromethane. Up to three tyrosine residues in each cytochrome b5 molecule were found to be accessible to the nitrating agent. Co-modification of tryptophan and histidine residues could be disregarded. CD-spectral measurements disproved gross changes in cytochrome b5 structure as a consequence of derivatization. Introduction of 1.6 nitro groups/polypeptide chain resulted in a fivefold increase in binding affinity for cytochrome P-450 2B4 (P-450 2B4), whereas spectral interaction with cytochrome c remained unaffected. Furthermore, the capacity of nitrated cytochrome b5 to shift the spin equilibrium to the high-spin conformer of P-4502B4 was diminished by 44% compared with the control. This corresponded with the partial disruption of NADH-dependent electron flow to ferric P-450 2B4. Changes in the redox potential of cytochrome b5 could be discounted as being responsible for this effect. The overall oxidative turnover of 4-nitroanisole did not respond to cytochrome b5 modification. MS analysis and sequencing of peptide fragments produced by tryptic digestion of modified cytochrome b5 permitted the detection of three nitrated tyrosine residues located at positions 11, 34 and 129. Derivatization of cytochrome b5 in the presence of a protective amount of P-450 2B4 provided evidence of the involvement of Tyr34 and Tyr129 in complexation of the two hemoproteins. It is proposed that Tyr129 might control docking of cytochrome b5 to P-450 2B4, whereas Tyr34 could be of functional importance in electron transfer.     

Multigenerational study of the effects of consumption of PCB-contaminated carp from Saginaw Bay, Lake Huron, on mink. 2. Liver PCB concentration and induction of hepatic cytochrome P-450 activity as a potential biomarker for PCB exposure

This study examined the effect of polychlorinated biphenyls (PCBs) from Saginaw Bay (Lake Huron) carp on the hepatic cytochrome P-450 activity in mink (Mustela vison). Hepatic cytochrome P-450 activities are of interest for their possible use as biomarkers to indicate consumption and biological effects of PCBs in the environment. Adult mink were fed diets containing ocean fish (control diet, 0.0 ppm) or Saginaw Bay carp toprovide 0.25, 0.5, or 1.0 ppm PCBs. Mink were bred after 3 mo of exposure, and half of the parental mink (P1) and kits (F1-1) previously consuming diets containing Saginaw Bay carp were switched to control diet at weaning of the F1-1 kits. P1 and F1-1 mink were then bred within their age and dietary groups after 15 mo of exposure, to produce the second-year F1 (F1-2) and F2 kits. Mink were killed when the new kits were weaned. Transfer of half the animals to the control diet examined whether the effects of the PCB-containing diet on hepatic cytochrome P-450 activity were permanent. Continual exposure to diets containing PCBs from Saginaw Bay carp induced cytochrome P-450 activity in a generally dose-dependent manner. Cytochrome P-450 activity was not different from untreated controls in animals switched to the control diet from the PCB-containing diet. The response of cytochrome P-4501A1 (EROD) activity in a dose-dependent manner and the lack of induction after transfer to noncontaminated diets suggest that this hepatic enzyme activity is a potential biomarker for current exposure to PCBs and other similar cytochrome P-450 inducers.     

Flavonoid-induced alterations in cytochrome P450-dependent biotransformation of the organophosphorus insecticide parathion in the mouse

The majority of insecticides currently in use throughout the world belong to the class of the organophosphorus insecticides. Many of these compounds, such as the phosphorothioate insecticides, exert their mammalian toxicity only after undergoing metabolic activation by a variety of cytochrome P450 isoforms to produce their corresponding oxygen analogs (or oxons), which are potent inhibitors of the critical enzyme acetylcholinesterase. Of the many chemicals identified that can modulate cytochrome P450-dependent activities, the flavonoids represent some of the most unusual compounds in that they have been reported to both inhibit and stimulate certain activities. The present study was undertaken to determine if representative flavonoids (at in vitro concentrations of 1-100 microM) can alter the mammalian cytochrome P450-dependent biotransformation and acute toxicity of the phosphorothioate insecticide parathion. The flavonoids 5,6-benzoflavone, flavone, and quercetin had the biphasic effect of stimulating mouse hepatic microsomal parathion oxidation at a concentration of 1 microM, and inhibiting this same activity when increased to 100 microM. In contrast, 7,8-benzoflavone was only inhibitory at all concentrations examined. All the flavonoids examined except quercetin altered the ratio of activation/detoxification of parathion by mouse hepatic microsomes, but had no effect on this same ratio with human CYP1A2. These data suggest that the changes in the activation/detoxification ratio observed with mouse hepatic microsomes resulted from selective inhibition or stimulation of various cytochrome P450 isoforms rather than a flavonoid-induced alteration in the nonenzymatic rearrangement of the putative phosphooxythirane intermediate generated by cytochromes P450 from parathion. Surprisingly, however, none of the four flavonoids in the current study affected the lethality of parathion in vivo, suggesting that the flavonoid-induced alterations in cytochrome P-450-dependent metabolism of parathion documented in vitro were simply not great enough to be of any significance in vivo.     

Expression of functionally active hyman cytochrome P-450c21 (CYPXXIA2) in Escherichia coli and single-stage purification of it using metal-affinity chromatography

Active human cytochrome P-450c21 was expressed in Escherichia coli and purified to homogeneity. To increase expression, cDNA encoding for the N-terminal fragment of cytochrome P-450c21 was modified. Four histidine codons were added to cDNA encoding for the C-terminus of the protein; thus, recombinant protein could have been rapidly and effectively purified by metal-affinity chromatography. Modified human cytochrome P-450c21 was expressed (40-50 nmoles/l of culture according to spectrophotometry) which was able to bind to bacterial membrane. Modifications of N- and C-terminal regions of cytochrome P-450c21 did not change Km and Vmax for hydroxylation of progesterone and 17 alpha-hydroxyprogesterone in reconstituted system. Recombinant cytochrome P-450c21 was purified to apparent homogeneity from Escherichia coli membrane extract by metal-affinity chromatography. Purified cytochrome P-450c21 migrates as a single 54 kD band on polyacrylamide gel and exhibits type I spectral changes during interaction with progesterone and 17 alpha-hydroxyprogesterone. Activity of purified cytochrome P-450c21 was reconstituted with mouse liver microsomal NADPH-cytochrome P-450-reductase and NADPH-regenerating system. Purified enzyme had Km 12.2 and 3.21 microM and Vmax 192.9 and 198 nmoles/min/nmole of P-450c21 for 17 alpha-hydroxyprogesterone and progesterone, respectively. According to titration spectra, dissociation constants for progesterone and 17 alpha-hydroxy-progesterone were 14.7 and 31.1 microM, respectively.     

A detailed molecular model for human aromatase

Using a variety of techniques, including sequence alignment, secondary structure prediction, molecular mechanics and molecular dynamics, we have constructed a model for the three-dimensional structure of P-450arom (human aromatase) based on that of P-450cam, the only cytochrome P-450 enzyme for which the crystal structure is known. The predicted structure is found to be in good agreement with current experimental data; both direct, from site-directed mutagenesis studies, and indirect, from the consideration of the structures and activities of known substrates and inhibitors.     

Influence of mutation of the amino-terminal signal anchor sequence of cytochrome P450 2B4 on the enzyme structure and electron transfer processes

The role of the NH2-terminal hydrophobic patch of cytochrome P4502B4 (CYP2B4) in interactions with NADPH-cytochrome P450 reductase (P450R) and cytochrome b5 (b5) was assessed using a variant lacking the signal anchor sequence (Delta2-27). CD, second-derivative, and fluorescence emission spectra indicated that the structure of the deletion mutant slightly differed from that of the native CYP2B4. Fitting of the initial-velocity patterns for P450R- and b5-directed electron transfer to the ferric CYP2B4 forms to Michaelis-Menten kinetics revealed an approximately 2.3-fold decrease in the affinity of the two electron donors for the engineered enzyme, while the reductive efficiency remained unaffected. Circumstantial analysis suggested that impaired association of the redox proteins with P4502B4(Delta2-27) accounted for this phenomenon. Interestingly, spectral docking of P450R to the truncated pigment was not hampered, while the binding of b5 was blocked. The rates of substrate-triggered aerobic NADPH consumption in systems containing CYP2B4(Delta2-27) and P450R were 16 to 56% those obtained with the unchanged hemoprotein. Decelerated cofactor oxidation did not arise on defective substrate binding or perturbed utilization of the substrate-bound oxy complex. Experiments with b5 as the ultimate electron donor hinted at some damage to second-electron transfer to the truncated enzyme. The results are consistent with the proposal that the NH2-terminal hydrophobic region of CYP2B4 might be of importance in preservation of the catalytic competence of the enzyme.     

Chemoprotective effects of capsaicin and diallyl sulfide against mutagenesis or tumorigenesis by vinyl carbamate and N-nitrosodimethylamine

Capsaicin (trans-8-methyl-N-vanillyl-6-nonenamide) is a major pungent and irritating ingredient of hot chilli peppers, which are frequently consumed as spices. This dietary phytochemical has been found to interact with microsomal xenobiotic metabolizing enzymes in rodents. Capsaicin and its saturated analog dihydrocapsaicin (trans-8-methyl-N-vanillyl-6-nonanamide) have been proposed to inactivate cytochrome P-450 HE1 by irreversibly binding to the active sites of the enzyme. Besides cytochrome P-450 HE1, other isoforms of the P-450 superfamily were also reported to be inhibited by capsaicin. The inhibition by capsaicin of microsomal monooxygenases involved in carcinogen activation implies its chemopreventive potential. As part of a program to investigate chemoprotective properties of capsaicin we initially determined the effect of capsaicin on vinyl carbamate (VC)- and N-nitrosodimethylamine (NDMA)-induced mutagenesis in Salmonella typhimurium TA100. Capsaicin (0.42 mM) attenuated the bacterial mutagenicity of VC and NDMA by 50% and 42% respectively. Diallyl sulfide, a thioether found in garlic with selective P-450 HE1 inhibitory activity, also lessened the mutagenicity of the above carcinogens in a concentration-dependent manner. The suppression of VC- and NDMA-induced mutagenesis by capsaicin and diallyl sulfide correlated with their inhibition of P-450 IIE1-mediated p-nitrophenol hydroxylation and NDMA N-demethylation. Pretreatment of female ICR mice with a topical dose of capsaicin lowered the average number of VC-induced skin tumors by 62% at 22 weeks after promotion. A similar degree of protection was attained with oral administration of diallyl sulfide before carcinogen treatment. The results of this study suggest that capsaicin and diallyl sulfide suppress VC- and NDMA-induced mutagenesis or tumorigenesis in part through inhibition of the cytochrome P-450 IIE1 isoform responsible for activation of these carcinogens.