Use of Phenoxyaniline Analogues To Generate Biochemical Insights into the Interactio n of Polybrominated Diphenyl Ether with CYP2B Enzymes.

Human hepatic cytochromes P450 (CYP) are integral to xenobiotic metabolism. CYP2B6 is a major catalyst of biotransformation of environmental toxicants, including polybrominated diphenyl ethers (PBDEs). CYP2B substrates tend to contain halogen atoms, but the biochemical basis for this selectivity and for species specific determinants of metabolism has not been identified. Spectral binding titrations and inhibition studies were performed to investigate interactions of rat CYP2B1, rabbit CYP2B4, and CYP2B6 with a series of phenoxyaniline (POA) congeners that are analogues of PBDEs. For most congeners, there was a <3-fold difference between the spectral binding constants (KS) and IC50 values. In contrast, large discrepancies between these values were observed for POA and 3-chloro-4-phenoxyaniline. CYP2B1 was the enzyme most sensitive to POA congeners, so the Val-363 residue from that enzyme was introduced into CYP2B4 or CYP2B6. This substitution partially altered the protein-ligand interaction profiles to make them more similar to that of CYP2B1. Addition of cytochrome P450 oxidoreductase (POR) to titrations of CYP2B6 with POA or 2'4'5'TCPOA decreased the affinity of both ligands for the enzyme. Addition of cytochrome b5 to a recombinant enzyme system containing POR and CYP2B6 increased the POA IC50 value and decreased the 2'4'5'TCPOA IC50 value. Overall, the inconsistency between KS and IC50 values for POA versus 2'4'5'TCPOA is largely due to the effects of redox partner binding. These results provide insight into the biochemical basis of binding of diphenyl ethers to human CYP2B6 and changes in CYP2B6-mediated metabolism that are dependent on POA congener and redox partner identity.

Reduction of thyroid hormones triggers down-regulation of hepatic CYP2B through nuclear receptors CAR and TR in a rat model of acute stroke.

Stroke is a neurological condition and may cause changes in hepatic drug-metabolizing enzymes. Hepatic CYP2B is involved in the metabolism of a variety of centrally active substances. The purpose of this study was to investigate the possible down-regulation mechanism of hepatic CYP2B after acute stroke. Using a rat model of acute stroke induced by middle cerebral artery occlusion, we studied the influence of brain ischemia/reperfusion (I/R) injury on CYP2B expression. Effects of 3,5,3'-triiodo-L-thyronine (T3) treatment on constitutive androstane receptor (CAR) and thyroid hormone receptors (TRs, including TRα and TRß) proteins were detected in Huh7 cells. We found dramatic decreases in the levels of plasma free triiodthyronine, free thyroxine and hepatic CYP2B expression. Both CAR and retinoid X receptor alpha (RXRα) were significantly dissociated from the phenobarbital-responsive enhancer module (PBREM) of the CYP2B1 promoter in the early stages of the acute stroke. The levels of the polymer of TRs, CAR, and RXRα were time-dependently decreased after brain I/R injury. T3 regulated the CAR expression at the transcriptional level, and facilitated the translocation of TRα/ß proteins as well as the binding of TRs, RXRα, and CAR to PBREM region. The reduction of thyroid hormone levels after a brain I/R injury may be the initial trigger for the down-regulation of hepatic CYP2B1 via induction of the dissociation of CAR from the TRs and from the PBREM region. Our data suggest that patients with acute ischemic stroke may have a decreased CYP2B-mediated metabolism of exogenous and endogenous compounds because of the low level of thyroid hormones.

Anticarcinogenic activity of meso-zeaxanthin in rodents and its possible mechanism of action.

Anticarcinogenic activity of meso-zeaxanthin (MZ), a xanthophyll carotenoid with profound antioxidant activity, was evaluated against 3-methylcholanthrene (3-MC)-induced sarcoma in mice. Oral administration of MZ at different doses significantly increased tumor latency period. In 3-MC control group, animals started developing sarcoma on 6th week. However animals treated with 3-MC and MZ (50 and 250 mg/kg b.wt) started developing sarcoma only on 15th and 18th week, respectively. Survival of tumor-bearing mice was significantly increased by MZ treatment. Animals in 3-MC control group started dying due to tumor burden from 8th week. All animals treated with MZ (50 and 250 mg/kg b.wt) along with 3-MC were found to be alive even after 16 and 20 wk, respectively. Oral administration of MZ inhibited different CYP450 isoenzymes like CYP1A1 (PROD), CYP1A2 (MROD), and CYP2B1/2 (EROD), which are involved in carcinogen metabolism in a dose-dependent manner. Moreover, levels of phase II enzymes like UDP-glucuronyl transferase and glutathione-S-transferase, which are involved in detoxification of carcinogens, were significantly increased by MZ treatment. Results indicated that mode of action of MZ may be through inhibition of carcinogen activation coupled with enhancement of detoxification process. MZ may also inhibit promotion phases of carcinogenesis by its antioxidant activity.

Cancer prevention mediated by caffeic acid phenethyl ester involves cyp2b1/2 modulation in hepatocarcinogenesis.

Studies of cancer chemoprevention with caffeic acid phenethyl ester (CAPE) in the resistant hepatocyte model of hepatocarcinogenesis have shown the participation of CYP drug metabolizing enzymes. To prevent neoplastic and preneoplasic lesions, we must specifically identify which CYP activities are modified in the mechanism of action of CAPE. Male Fischer-344 rats were pretreated with CAPE twelve hours before administration of diethylnitrosamine (DEN) and were sacrificed twelve hours after CAPE and twelve hours, twenty-four hours, twenty-four days, and twelve months after DEN. Other rats were treated with the CYP inhibitors α-naphthoflavone or SKF525A and sacrificed twenty-four hours and twenty-four days after DEN. Microsomes were obtained from livers to quantify protein using Western blot. Diethylnitrosamine metabolism was measured based on nitrite formation and liver histology using GGT histochemistry. Caffeic acid phenethyl ester diminished the protein levels of CYP1A2 and CYP2B1/2. The inhibition of CYP2B1/2 prevented the appearance of preneoplastic lesions. Microsomal assays demonstrated that CAPE interfered with DEN activation diminishing nitrites similar to SKF525A and probably mediated by CYP2B1/2 inhibition. A single dose of CAPE before DEN treatment reduced the appearance of tumors by 43%. These results confirmed that CAPE is a promising agent to confer chemoprotection in liver cancer and should be considered for human therapies.

Bupropion hydroxylation as a selective marker of rat CYP2B1 catalytic activity.

Benzyloxyresorufin-O-dealkylation (BROD) is usually used as a marker of cytochrome P450 (P450) 2B1 in rat. However, some reports show that CYP1A2 is also highly implicated. The purpose of the present study was to establish bupropion (BUP) hydroxylation, but not BROD, as a selective in vitro marker of CYP2B1 catalytic activity. IC(50) for BROD and BUP hydroxylation were equivalent (40.8 ± 4.6 and 41.8 ± 3.4 µM, respectively) when using liver microsomes from ß-naphthoflavone-pretreated rats in the presence of metyrapone (CYP2B1 inhibitor). When using the same microsomes in the presence of CYP1A1/2-selective inhibitor α-naphthoflavone, we found an IC(50) of 2.5 × 10(-3) ± 0.8 × 10(-3) µM for BROD and >100 µM for BUP hydroxylation. These results suggest that CYP2B1 is similarly involved in both activities, whereas CYP1A2 is involved in BROD activity but not in BUP hydroxylation. BUP hydroxylation was assessed in microsomes from baculovirus-infected insect cells coexpressing NADPH-P450 oxidoreductase, and 14 rat P450s and kinetic parameters (K(m) and V(max)) were determined. BUP hydroxylation was predominantly catalyzed by CYP2B1 (75% of total hydroxybupropion formation), low activity was detected with CYP2E1 and CYP2C11 (10.9 and 8.7% of total hydroxybupropion, respectively), and activity was almost undetectable with the other P450 isoforms at saturating substrate concentrations (2500 µM), thereby validating the use of BUP as a diagnostic in vitro marker of CYP2B1 catalytic activity in rat.

In vitro metabolism and covalent binding of ethylbenzene to microsomal protein as a possible mechanism of ethylbenzene-induced mouse lung tumorigenesis.

This study was conducted to determine species differences in covalent binding of the reactive metabolites of ethylbenzene (EB) formed in the liver and lung microsomes of mouse, rat and human in the presence of NADPH. These data further the understanding of the mechanism by which EB causes mouse specific lung toxicity and a follow-up to our earlier report of the selective elevation, although minor, of the ring-oxidized reactive metabolites in mouse lung microsomes (Saghir et al., 2009). Binding assays were also conducted with or without 5-phenyl-1-pentyne (5P1P), an inhibitor of CYP 2F2, and diethyldithiocarbamate (DDTC), an inhibitor of CYP 2E1 to evaluate their role in the formation of the related reactive metabolites. Liver and lung microsomes were incubated with (14)C-EB (0.22 mM) in the presence of 1mM NADPH under physiological conditions for 60 min. In lung microsomes, binding activity was in the order of mouse (812.4+/-102.2 pmol/mg protein)>>rat (57.0+/-3.2 pmol/mg protein). Human lung microsomes had little binding activity (15.7+/-1.4 pmol/mg protein), which was comparable to the no-NADPH control (9.9-16.7 pmol/mg protein). In liver microsomes, mouse had the highest activity (469.0+/-38.5 pmol/mg protein) followed by rat (148.3+/-14.7 pmol/mg protein) and human (89.8+/-3.0 pmol/mg protein). Presence of 5P1P or DDTC decreased binding across species and tissues. However, much higher inhibition was observed in mouse (86% [DDTC] and 89% [5P1P]) than rat (56% [DDTC] and 59% [5P1P]) lung microsomes. DDTC showed approximately 2-fold higher inhibition of binding in mouse and human liver microsomes than 5P1P (mouse=85% vs. 40%; human=59% vs. 36%). Inhibition in binding by DDTC was much higher (10-fold) than 5P1P (72% vs. 7%) in rat liver microsomes. These results show species, tissue and enzyme differences in the formation of reactive metabolites of EB. In rat and mouse lung microsomes, both CYP2E1 and CYP2F2 appear to contribute in the formation of reactive metabolites of EB. In contrast, CYP2E1 appears to be the primary CYP isozyme responsible for the reactive metabolites of EB in the liver.

Mechanism-based inactivation of CYP2B1 and its F-helix mutant by two tert-butyl acetylenic compounds: covalent modification of prosthetic heme versus apoprotein.

The mechanism-based inactivation of cytochrome CYP2B1 [wild type (WT)] and its Thr205 to Ala mutant (T205A) by tert-butylphenylacetylene (BPA) and tert-butyl 1-methyl-2-propynyl ether (BMP) in the reconstituted system was investigated. The inactivation of WT by BPA exhibited a k(inact)/K(I) value of 1343 min(-1)mM(-1) and a partition ratio of 1. The inactivation of WT by BMP exhibited a k(inact)/K(I) value of 33 min(-1)mM(-1) and a partition ratio of 10. Liquid chromatography/tandem mass spectrometry analysis (LC/MS/MS) of the WT revealed 1) inactivation by BPA resulted in the formation of a protein adduct with a mass increase equivalent to the mass of BPA plus one oxygen atom, and 2) inactivation by BMP resulted in the formation of multiple heme adducts that all exhibited a mass increase equivalent to BMP plus one oxygen atom. LC/MS/MS analysis indicated the formation of glutathione (GSH) conjugates by the reaction of GSH with the ethynyl moiety of BMP or BPA with the oxygen being added to the internal or terminal carbon. For the inactivation of T205A by BPA and BMP, the k(inact)/K(I) values were suppressed by 100- and 4-fold, respectively, and the partition ratios were increased 9- and 3.5-fold, respectively. Only one major heme adduct was detected following the inactivation of the T205A by BMP. These results show that the Thr205 in the F-helix plays an important role in the efficiency of the mechanism-based inactivation of CYP2B1 by BPA and BMP. Homology modeling and substrate docking studies were presented to facilitate the interpretation of the experimental results.

Docosahexaenoic acid downregulates phenobarbital-induced cytochrome P450 2B1 gene expression in rat primary hepatocytes via the c-Jun NH2-terminal kinase mitogen-activated protein kinase pathway.

Mitogen-activated protein kinase (MAPK) pathways play central roles in the transduction of extracellular stimuli into cells and the regulation of expression of numerous genes. Docosahexaenoic acid (DHA) was shown to be involved in the regulation of expression of drug metabolizing enzymes (DMEs) in rat primary hepatocytes in response to xenobiotics. Cytochrome P450 2B1 (CYP 2B1) is a DME that is dramatically induced by phenobarbital-type inducers. The constitutive androstane receptor (CAR) plays a critical role in regulating the expression of DMEs, and the phosphorylation/dephosphorylation of CAR is an important event in CYP 2B1 expression. In the present study, we determined the effect of DHA on MAPK transactivation and its role in CYP 2B1 expression induced by phenobarbital. c-Jun NH2-terminal kinase (JNK) JNK1/2 and ERK1/2 were activated by phenobarbital in a dose-dependent manner. DHA (100 muM) inhibited JNK1/2 and ERK2 activation induced by phenobarbital in a time-dependent manner. Both SP600125 (a JNK inhibitor) and SB203580 (a p38 MAPK inhibitor) inhibited CYP 2B1 protein and mRNA expression induced by phenobarbital. SB203580 significantly increased the intracellular 3'-5'-cyclic adenosine monophosphate (cAMP) concentration compared with a control group (p < 0.05). Our results suggest that inhibition of JNK activation by DHA is at least part of the mechanisms of DHA's downregulation of CYP 2B1 expression induced by phenobarbital.

Brain drug-metabolizing cytochrome P450 enzymes are active in vivo, demonstrated by mechanism-based enzyme inhibition.

Individuals vary in their response to centrally acting drugs, and this is not always predicted by drug plasma levels. Central metabolism by brain cytochromes P450 (CYPs) may contribute to interindividual variation in response to drugs. Brain CYPs have unique regional and cell-type expression and induction patterns, and they are regulated independently of their hepatic isoforms. In vitro, these enzymes can metabolize endogenous and xenobiotic substrates including centrally acting drugs, but there is no evidence to date of their in vivo function. This has been difficult to demonstrate in the presence of hepatically derived metabolites that may cross the blood-brain barrier. In addition, because of the membrane location of brain CYPs and the rate limiting effect of endogenous heme levels on the activity and appropriate membrane insertion of some induced CYPs, it has been unclear whether sufficient cofactors and coenzymes are present for constitutive and induced CYP forms to be enzymatically active. We have developed a method using a radiolabeled mechanism-based inhibitor of CYP2B1, (3)H-8-methoxypsoralen, to demonstrate for the first time that both the constitutive and induced forms of this enzyme are active in situ in the living rat brain. This methodology provides a novel approach to assess the function of enzymes in extrahepatic tissues, where expression levels are often low. Selective induction of metabolically active drug metabolizing enzymes in the brain may also provide ways to control prodrug activation in specific brain regions as a novel therapeutic avenue.

Inhibitory effects of a dietary phytochemical 3,3'-diindolylmethane on the phenobarbital-induced hepatic CYP mRNA expression and CYP-catalyzed reactions in female rats.

3,3'-diindolylmethane (DIM), derived from indole-3-carbinol (I3C), is used as a dietary supplement for its putative anticancer effects that include suppression of mammary tumor growth in female rats. The mechanism of action DIM may involve its interaction(s) with hepatic cytochromes P450 (CYPs) catalyzing oxidations of 17beta-estradiol (E2). Our study showed that DIM added to hepatic microsomes of female Sprague-Dawley rats was primarily a competitive inhibitor of beta-naphthoflavone (beta-NF)- or I3C-induced CYP1A1 probe activity, and a potent mixed or uncompetitive inhibitor of phenobarbital (PB)-induced CYP2B1 or CYP2B2 probe activity, respectively. Microsomal metabolites of DIM were tentatively identified as two mono-hydroxy isomers of DIM, each formed preferentially by CYP1A1- or CYP2B1/2-catalyzed reaction. Evaluation of the effects of co-treatment of rats with PB and DIM by a full factorial ANOVA showed that DIM decreased the PB-induced CYP2B1 and CYP2B2 mRNA expression levels, and the rates of 2- and 4-hydroxylation of E2, and total E2 metabolite formation. The results suggest that interactions of DIM, and/or its mono-hydroxy metabolites, with CYP2B1 and CYP2B2 found to occur in hepatic microsomes upon addition of DIM or co-treatment of rats with DIM affect the rates of relevant oxidations of E2, and potentially protect against estrogen-dependent tumorigenesis.

Identification of 17-alpha-ethynylestradiol-modified active site peptides and glutathione conjugates formed during metabolism and inactivation of P450s 2B1 and 2B6.

The oral contraceptive 17-alpha-ethynylestradiol (17EE) is a mechanism-based inactivator of cytochrome P450s (P450s) 2B1 and 2B6. Inactivation of P450s 2B1 and 2B6 in the reconstituted system by [3H]17EE resulted in labeling of the P450 apoprotein. Mass spectral analysis of 17EE-inactivated P450 2B1 showed an increase in the mass of the apoprotein by 313 Da, consistent with the mass of 17EE plus one oxygen atom. P450s 2B1 and 2B6 were inactivated with [3H]17EE and digested with CNBr. Separation of these peptides resulted in the identification of one major labeled peptide for each enzyme. N-Terminal sequencing of these peptides yielded the amino acid sequences PYTDAVIHEI (for P450 2B1) and PYTEAV (for P450 2B6) that corresponded to amino acids P347-M376 and P347-M365 in P450s 2B1 and 2B6, respectively. Electrospray ionization (ESI)-liquid chromatography-mass spectrometry (LC-MS) and matrix-assisted laser desorption ionization (MALDI)-MS analysis of the P450 2B1-derived peptide resulted in a mass of 3654 Da consistent with the mass of the P347-M376 peptide (3385 Da) plus a 268 Da 17EE adduct. Chemically reactive intermediates of 17EE that were generated during the metabolism of 17EE by P450s 2B1 and 2B6 were trapped with gluthathione (GSH). ESI-LC-MS/MS analysis of 17EE-GSH conjugates from the incubation mixtures indicated that P450s 2B1 and 2B6 generated different reactive 17EE intermediates that were responsible for the inactivation and protein modification or the formation of GSH conjugates by these two enzymes.

Proteasome-dependent degradation of cytochromes P450 2E1 and 2B1 expressed in tetracycline-regulated HeLa cells.

The degradation of ethanol-inducible cytochrome P450 2E1 (CYP2E1) and phenobarbital-inducible cytochrome P450 2B1 (CYP2B1) expressed in tetracycline (Tc)-inducible HeLa cell lines was characterized. A steady-state pulse-chase analysis was used to determine a half-life of 3.8 h for CYP2E1 while the half-life of CYP2B1 was 2.3-fold greater in the same cell line. In contrast, NADPH cytochrome P450 reductase which is constitutively expressed in Tc-HeLa cells had a half-life of about 30 h. Lactacystin and other selective proteasome inhibitors including N-benzyloxycarbonyl-leucyl-leucyl-leucinal (MG132) and N-benzyloxycarbonyl-L-leucyl-L-leucyl-L-norvalinal (MG115) significantly inhibited both CYP2E1 and CYP2B1 degradation. The turnover of CYP2E1 was slightly inhibited by calpain inhibitors while CYP2B1 turnover was not altered. Inhibitors of lysosomal proteolysis had no effect on the degradation of either protein. Treatment of cells with brefeldin A did not alter the degradation of either P450 which suggested the degradation occurred in the endoplasmic reticulum (ER). Even in the presence of proteasome inhibitors high molecular weight ubiquitin conjugates were not observed. Mutagenesis of two putative ubiquitination sites (Lys 317 and 324) did not alter the degradation of CYP2E1. The role of ubiquitination in the degradation of CYP2E1 was also examined in a Chinese hamster mutant cell line E36ts20 that contains a thermolabile ubiquitin-activating enzyme (E1). The turnover of CYP2E1 was not significantly different at the nonpermissive temperature in the ts20 when compared to the control E36 cells. Furthermore, the addition of the hsp90 inhibitors geldanamycin, herbimycin, and radicicol had no effect on the turnover of CYP2E1, differentiating the degradation of CYP2E1 from other substrates for proteasome-dependent degradation.

Metabolism of N,N',N"-triethylenethiophosphoramide by CYP2B1 and CYP2B6 results in the inactivation of both isoforms by two distinct mechanisms.

The anticancer drug N,N,"N"-triethylenethiophosphoramide (tTEPA) inactivated CYP2B6 and CYP2B1 in the reconstituted system in a time-, concentration-, and NADPH-dependent manner indicative of mechanism-based inactivation. The KI value for the inactivation of CYP2B1 was 38 microM, the kinact was 0.3 min(-1), and the t1/2 value was 2.5 min. Spectral carbon monoxide (CO) binding and high-performance liquid chromatography heme studies of the tTEPA-inactivated CYP2B1 suggest that the loss in the enzymatic activity was primarily due to the binding of a reactive tTEPA intermediate to the 2B1 apoprotein. Inactivation by tTEPA in the presence of 7-ethoxycoumarin, an alternate substrate, reduced the rate of inactivation of CYP2B1. Incubations with tTEPA and NADPH resulted in greater than 90% loss in the 7-ethoxy-4-(trifluoromethyl)coumarin O-deethylation and testosterone hydroxylation activity of CYP2B1. In contrast, benzphetamine metabolism was significantly less inhibited (47%). CYP2B6 was inactivated by tTEPA with a KI value of 50 microM, a k inact value of 0.1 min(-1), and a t1/2 value of 14 min. However, unlike CYP2B1, the tTEPA-inactivated human isoform showed losses in the cytochrome P450 (P450) CO spectrum, the pyridine hemochrome spectrum, and in the amount of native heme that were comparable with the loss in the 7-EFC and benzphetamine activity, suggesting that activity loss was brought about by a tTEPA-reactive intermediate damaging the CYP2B6 heme. CYP2B6 could only be protected from the tTEPA-dependent inactivation by the 2B6-specific substrate bupropion but not by other substrates of CYP2B such as benzphetamine, testosterone, or 7-ethoxycoumarin. The data indicate that tTEPA metabolism by these two 2B isoforms results in inactivation of the P450s by two distinct mechanisms.

Role of cytochrome P450 2B1 in puromycin aminonucleoside-induced cytotoxicity to glomerular epithelial cells.

Puromycin aminonucleoside (PAN)-induced glomerular injury in rats mimics minimal-change nephrotic syndrome (NS) in humans. We have demonstrated an important role of cytochrome P450 (CYP) as a significant source of catalytic iron in this model of NS. The current study was designed to identify CYP isozyme(s) present in the rat glomerular epithelial cells (GEC) and to explore the role of the specific CYP isozyme in PAN-induced cytotoxicity. CYP2B1 was identified in GEC by immunocytochemistry and Western blot. Treatment of GEC with PAN resulted in a marked generation of hydrogen peroxide (H(2)O(2)) and reduction of CYP2B1 content associated with significant increase in catalytic iron and hydroxyl radical formation. Preincubating GEC with CYP2B1 inhibitors (piperine and cimetidine) and H(2)O(2) scavenger (pyruvate) significantly reduced H(2)O(2 )generation, preserved CYP2B1 content, prevented the increase in catalytic iron and hydroxyl radical formation including PAN-induced cytotoxicity. We also observed the induction of heme oxygenase (HO-1) in PAN-treated GEC, and this up-regulation was reduced by pretreatment of the CYP inhibitors and pyruvate. Our data thus indicate an important role of CYP2B1 in PAN-induced cytotoxicity by serving as a site of reactive oxygen metabolite generation and a significant source of catalytic iron.

Inhibition of clastogenicity of benzo[a]pyrene and of its trans-7,8-dihydrodiol in mice in vivo by fruits, vegetables, and flavonoids.

In the in vivo mouse bone marrow micronucleus assay, homogenates of spinach, artichoke, peaches, and blue grapes as well as commercial concentrates of these vegetables and fruits reduced induction of micronuclei by benzo[a]pyrene (BaP) by 43-50%. Concentrates of strawberries (31% reduction) and of cauliflower (20% reduction) were less potent. Inhibition of genotoxicity by spinach and peaches was not caused by any delay in maturation of micronucleated erythrocytes as shown by experiments with sampling times of 24, 48, and 72 h after dosing of BaP. Pre-treatment of the mice with spinach 48, 24, and 12h before application of BaP resulted in a 44% reduction of micronuclei while peaches generated only a marginal effect. A post-treatment procedure administering spinach or peaches 6h after dosing of BaP did not indicate any protective effects. When trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (BaP-7,8-OH) was applied for induction of micronuclei spinach and peaches reduced the number of micronuclei by 55 and 48%, respectively. Pre-treatment of mice with spinach 96, 72, and 60 h before sacrifice caused a decline of hepatic 7-ethoxyresorufin-O-dealkylase (EROD) and of 7-pentoxyresorufin-O-dealkylase (PROD) activities by factors of 2.2 and 1.4, respectively. However, statistical significance was not reached. On the other hand, peaches had no influence on hepatic EROD or PROD activities. The flavonoids quercetin and its glucoside isoquercitrin, administered orally in doses of 0.03 mmol/kg body weight simultaneously with intraperitoneally given BaP, reduced the number of micronuclei in polychromatic erythrocytes of the bone marrow of mice by 73 and 33%. Ten-fold higher concentrations, however, reversed the effects with a particular strong increase observed with isoquercitrin (+109%; quercetin: +16%).

Effect of naturally occurring plant phenolics on the induction of drug metabolizing enzymes by o-toluidine.

Plant phenolics modify the metabolic activation of several carcinogens, including aromatic amines. In this study, we have evaluated the effects of three structurally diversified plant phenolics, protocatechuic acid (PCA), tannic acid (TA) and ellagic acid (EA) on cytochrome p450-dependent enzymes and glutathione S-transferase (GST) activities after oral administration alone or in combination with o-toluidine in rat liver and kidney. Protocatechuic and ellagic acids significantly decreased the activities of ethoxy- (EROD), methoxy- (MROD) and penthoxyresorufin (PROD) dealkylases in liver. In kidney, all phenolics inhibited only the activity of PROD. Enzyme modulation in liver correlated with CA metabolism measured in plasma. Treatment of rats with ellagic acid 1 h before o-toluidine administration diminished the activities of all hepatic alkoxyresorufine dealkylases induced by o-toluidine but increased renal EROD. In contrast to EA, protocatechuic and tannic acids increased the activities of p450-dependent enzymes in liver. All phenolics administered in combination with o-toluidine increased the activity of GST, which was reduced after the treatment with o-toluidine alone. In addition, CA metabolism in plasma resulting from oral treatment with CA was measured. The formation of CA metabolites was reduced by PCA and EA, and the metabolism of CA induced by o-toluidine was depressed by administration of all three phenolics. Our results indicate that plant phenolics, especially EA, may modulate the genotoxic effects of o-toluidine by modifying pathways leading to the formation of its reactive metabolite. Moreover, as the result of CYP1A modification these compounds may affect the metabolism of CA.

Hemin-mediated restoration of allylisopropylacetamide-inactivated CYP2B1: a role for glutathione and GRP94 in the heme-protein assembly.

Administration of the cytochrome P450 (P450) suicide inactivator allylisopropylacetamide (AIA) to phenobarbital (PB)-pretreated rats results in rapid and marked inactivation of several liver endoplasmic reticulum (ER)-bound P450s. A few of these such as CYP2B1, inactivated due to AIA-mediated prosthetic heme N-alkylation, can be structurally and functionally restored nearly completely by exogenous hemin in vivo or in vitro. Such in vitro hemin-mediated reassembly is unsuccessful with purified AIA-inactivated CYP2B1 and, as shown herein, is not very effective even when heme is incubated with just the corresponding liver microsomes that contain the reconstitutable CYP2B1 protein, thereby implicating a requirement for additional factors provided by the intact liver cell homogenates, ER, and/or cytosol. Using various approaches that include high-performance liquid chromatographic fractionation of the liver cytosolic subfraction as well as chemical and immunological probes such as the Hsp90/GRP94-specific inhibitor geldanamycin (GA) and polyclonal anti-GRP94 antibodies, respectively, we now demonstrate that the in vitro hemin-mediated reassembly of heme-stripped microsomal CYP2B1 requires GSH as well as the ER chaperone GRP94, but not the cytosolic chaperone heat shock protein 90. It remains to be determined whether GSH acts directly or indirectly, via a putative ER thiol reductase, to maintain the conserved active site cysteine-thiol (Cys436 in CYP2B1) in a reduced state, competent for heme binding and repair.

Effects of plant-derived phenols on rat liver cytochrome P450 2B1 activity.

Dietary constituents contain a variety of compounds that are known to modulate liver enzyme activity. In this report, the plant-derived phenols catechin, chlorogenic acid, diosmin, epigallo-catechin gallate (EGCG), naringenin, quercetin and resveratrol were studied for their effects on the activity of cytochrome P450 2B1 in liver microsomes from 6- and 20-month male Fisher F344 rats. The compounds at two concentrations (0.1 and 0.25 mM) were incubated with 0.2 mg liver microsomal protein and 50 microM 7-ethoxy-4-trifluoromethyl coumarin (EFC). O-deethylation of EFC to the fluorescence product 7-hydroxy-4-trifluoromethyl coumarin (HFC) is catalyzed by CYP450 2B1. EGCG, naringenin, quercetin and resveratrol inhibited the in vitro O-deethylation of EFC in liver microsomes from both 6- and 20-month rats. Quercetin was the most effective inhibitor. Catechin inhibited the in vitro O-deethylation of EFC only in microsomes from 6-month-old rats whereas diosmin inhibited the reaction only in microsomes from 20-month-old rats. Chlorogenic acid inhibited the in vitro O-deethylation of EFC in microsomes from both age groups at the 0.25 mM concentration only. These results suggest that plant phenols have varied effects on liver microsomal cytochrome P450 2B1 activity that may be influenced by the age of the animal.

Effect of alpha-methyldopa on pentoxyresorufin O-dealkylation in liver microsomes from rats treated with phenobarbital.

Loss of pentoxyresorufin O-dealkylation (PROD) was observed when microsomes from PB-treated rats were preincubated in the presence of NADPH. PROD proved to be quite sensitive towards inactivation. Decrease in cytochrome P450 (CYP) dependent activity was accompanied by simultaneous formation of thiobarbituric acid reactive substances (TBARS) indicating the occurrence of lipid peroxidation. The presence of 50 microM alpha-methyldopa (AMD) during preincubation with NADPH resulted in complete protection against enzyme activity loss and the extent of lipid peroxidation was also diminished. Addition of ascorbate or GSH in combination with AMD reduced the protective effect of the drug on PROD. AMD probably exerts its effect by scavenging reactive oxygen species but chelation of ferric ions can also contribute to the protective effect of the drug on PROD activity.

Inhibition of cytochrome P450 isozymes by curcumins in vitro and in vivo.

To study the mechanism(s) of turmeric-mediated chemoprevention and to compare the chemopreventive efficacy of turmeric/curcumin(s) against benzo[a]pyrene (B(a)P) and 4-methylnitrosamino-1-(3-pyridyl)-1-butanone (NNK, a tobacco-specific carcinogen), the effects of turmeric/curcumin (C), demethoxycurcumin (dmC), bis-demethoxycurcumin (bdmC) and phenyl and phenethyl-isothiocyanates (PITC and PEITC) on the dealkylation of ethoxyresorufin (ER), methoxyresorufin (MR) and pentoxyresorufin (PR) by rat liver microsomes (in vitro) were studied. These reactions are predominantly mediated by cytochrome P450 (CYP450) isozymes 1A1, 1A2 and 2B1, respectively. In vitro incubation of rat liver microsomes with each of the compounds--C, dmC, bdmC, PITC and PEITC--showed a dose-dependent decrease in carbon monoxide binding to microsomes and also showed a dose-dependent inhibition of CYP 1A1, 1A2 and 2B1 activity, as judged by a decrease in formation of resorufin from respective biochemical probes used. Both the isothiocyanates inhibited activity of CYP 2B1 more readily than that of CYP 1A1/1A2. Significantly lower concentrations of curcumin(s) than isothiocyanates achieved 50% inhibition of activity of CYP 1A1 and 1A2, while concentrations of C (4 microM), bdmC (2.5 microM) required to inhibit CYP 2B1 were slightly higher than that of PEITC (1.3 microM), suggesting curcumin(s) to be effective inhibitors of CYP 2B1 as well. Pretreatment of rats with 1% turmeric through the diet resulted in a significant decrease in induction of B(a)P-induced CYP 1A1 and 1A2 and phenobarbitone (PB)-induced CYP 2B1 in liver, lung and stomach, although the extent of the decrease was different. These results suggest that turmeric/curcumin(s) as in the case of isothiocyanate, PEITC, are likely to inhibit activation of carcinogens metabolized by CYP450 isozymes, namely, CYP 1A1, 1A2 and 2B1.