Identification of CYP4A11 as the major lauric acid omega-hydroxylase in human liver microsomes

Human liver microsomes are capable of oxidizing lauric acid (laurate), a model medium-chain fatty acid, at both the omega- and omega-1 positions to form 12- and 11-hydroxylaurate, respectively. These laurate hydroxylation reactions are apparently catalyzed by distinct P450 enzymes. While the P450 responsible for microsomal laurate omega-1 hydroxylation in human liver has been identified as CYP2E1, the enzyme catalyzing omega-hydroxylation remains poorly defined. To that end, we employed conventional purification and immunochemical techniques to characterize the major hepatic laurate omega-hydroxylase in humans. Western blotting with rat CYP4A1 antibodies was used to monitor a cross-reactive P450 protein (M(r) = 52 kDa) during its isolation from human liver microsomes. The purified enzyme (7.4 nmol P450/mg protein) had an NH2-terminal amino acid sequence identical to that predicted from the human CYP4A11 cDNA over the first 20 residues found. Upon reconstitution with P450 reductase and cytochrome b5, CYP4A11 proved to be a potent laurate omega-hydroxylase, exhibiting a turnover rate of 45.7 nmol 12-hydroxylaurate formed/min/nmol P450 (12-fold greater than intact microsomes), while catalyzing the omega-1 hydroxylation reaction at much lower rates (5.4 nmol 11-hydroxylaurate formed/min/nmol P450). Analysis of the laurate omega-hydroxylation reaction in human liver microsomes revealed kinetic parameters (a lone Km of 48.9 microM with a VMAX of 3.72 nmol 12-hydroxylaurate formed/min/nmol P450) consistent with catalysis by CYP4A11. In fact, incubation of human liver microsomes with antibodies raised to CYP4A11 resulted in nearly 85% inhibition of laurate omega-hydroxylase activity while omega-1 hydroxylase activity remained unaffected. Furthermore, a strong correlation (r = 0.89; P < 0.001) was found between immunochemically determined CYP4A11 content and laurate omega-hydroxylase activity in liver samples from 11 different subjects. From the foregoing, it appears that CYP4A11 is the principle laurate omega-hydroxylating enzyme expressed in human liver.     

Effects of the agrochemicals butachlor, pretilachlor and isoprothiolane on rat liver xenobiotic-metabolizing enzymes

1. The herbicides butachlor (2-chloro-2',6',diethyl-N-[buthoxymethyl] acetanilide) and pretilachlor (2-chloro-2',6'-diethyl-N-[2-propoxyethyl] acetanilide) are widely used in Asia, South America, Europe and Africa. Isoprothiolane (diisopropyl-1,3-dithiolan-2-ylidenemalonate) is used as a fungicide and an insecticide in rice paddies. We administered these agrochemicals to the male rat and examined their effects on cytochrome P450 (P450), glutathione S-transferase (GST), UDP-glucuronosyltransferase (UDPGT), and NAD(P)H-quinone oxidoreductase 1 (NQO1)-related metabolism in the liver. 2. Administration of isoprothiolane, butachlor or pretilachlor to rat induced hepatic P4502B subfamily-dependent enzyme activities (pentoxyresorufin O-depentylation and testosterone 16 beta-hydroxylation) up to 271-413% of control, which coincided with the increase in expression levels of the P4502B apoprotein. 3. Activities of GST toward 1-chloro-2,4-nitrobenzene and 3,4-dichloronitrobenzene were slightly induced (127-133% of control) in the liver of the rat treated with these pesticides. On the other hand, marked elevations of UDPGT activities toward p-nitrophenol (164-281% of control) were observed. NQO1-related metabolism (menadione reductase activity) was also induced (123-176% of control) in the liver of rat treated with these agrochemicals. 4. These results indicate that some of the agrochemicals currently in use are capable of inducing phase I and II xenobiotic-metabolizing enzyme activities in an isozyme selective manner. The induction of these activities may disrupt normal physiologic functions related to these enzymes in exposed animals.     

Activation of the anti-cancer drug ifosphamide by rat liver microsomal P450 enzymes

The NADPH-dependent metabolism of ifosphamide catalyzed by rat liver microsomes was investigated in order to identify individual P450 enzymes that activate this anti-cancer drug and to ascertain their relationship to the P450 enzymes that activate the isomeric drug cyclophosphamide. Pretreatment of rats with phenobarbital or clofibrate increased by up to 8-fold the activation of both ifosphamide and cyclophosphamide catalyzed by isolated liver microsomes. Studies using P450 form-selective inhibitory antibodies demonstrated that constitutively expressed P450s belonging to subfamily 2C (forms 2C11/2C6) make significant contributions to the activation of both oxazaphosphorines in uninduced male rat liver microsomes, while the phenobarbital-inducible P450 2B1 was shown to be a major catalyst of these activations in phenobarbital-induced microsomes. Pretreatment of rats with dexamethasone increased liver microsomal activation of ifosphamide approximately 6-fold without a corresponding effect on cyclophosphamide activation rates. Ifosphamide activation catalyzed by dexamethasone-induced liver microsomes was minimally inhibited by anti-P450 2B or anti-P450 2C antibodies, but was selectively inhibited by anti-P450 3A antibodies. Selective inhibition of liver microsomal ifosphamide activation was also effected by the macrolide antibiotic triacetyloleandomycin, an inhibitor of several dexamethasone-inducible 3A P450s. These studies establish that a dexamethasone-inducible family 3A P450 can make an important contribution to rat liver microsomal ifosphamide activation, and suggest that dexamethasone pretreatment might provide a useful approach for modulation of ifosphamide metabolism in order to improve its therapeutic efficacy in cancer patients.     

Identification of human liver cytochrome P450 isoforms involved in the in vitro metabolism of cyclobenzaprine

Cyclobenzaprine (Flexeril) is a muscle relaxant, possessing a tricyclic structure. Numerous therapeutic agents containing this structure are known to be metabolized by polymorphic cytochrome P4502D6. The aim of this study was to determine if cytochrome P4502D6 and other isoforms are involved in the metabolism of cyclobenzaprine in human liver microsomes. Selective cytochrome P450 inhibitors for CYP1A1/2 (furafylline and 7,8-benzoflavone) and CYP3A4 (troleandomycin, gestodene, and ketoconazole) inhibited the formation of desmethylcyclobenzaprine, a major metabolite of cyclobenzaprine, in human liver microsomes. Antibodies directed against CYP1A1/2 and CYP3A4 inhibited the demethylation reaction whereas anti-human CYP2C9/10, CYP2C19, and CYP2E1 antibodies did not show any inhibitory effects. When a panel of microsomes prepared from human B-lymphoblastoid cells that expressed specific human cytochrome P450 isoforms were used, only microsomes containing cytochromes P4501A2, 2D6, and 3A4 catalyzed N-demethylation. In addition, demethylation catalyzed by these recombinant cytochromes P450 can be completely inhibited with selective inhibitors at concentrations as low as 1 to 20 microM. Interestingly, cyclobenzaprine N-demethylation was significantly correlated with caffeine 3-demethylation (1A2) and testosterone 6 beta-hydroxylation (3A4) but not with dextromethorphan O-demethylation (2D6) in human liver microsomes. To further determine the involvement of cytochrome P4502D6 in cyclobenzaprine metabolism, liver microsomes from a human that lacked CYP2D6 enzyme activities was included in this study. The data showed that cyclobenzaprine N-demethylation still occurred in the incubation with this microsome. These results suggested that cytochrome P4502D6 plays only a minor role in cyclobenzaprine N-demethylation whereas 3A4 and 1A2 are primarily responsible for cyclobenzaprine metabolism in human liver microsomes. Due to the minimum involvement of CYP2D6 in the vitro metabolism of cyclobenzaprine, the polymorphism of cytochrome P4502D6 in man should not be of muci concern in the clinical use of cyclobenzaprine.     

Involvement of CYP2D1 in the metabolism of carteolol by male rat liver microsomes

1. The metabolism of carteolol, a beta-adrenoceptor blocking drug, was investigated in male Sprague-Dawley rat liver microsomes. 2. The formation of 8-hydroxycarteolol was the principal metabolic pathway of carteolol in vitro and followed Michaelis-Menten kinetics with a K(m) = 11.0 +/- 5.4 microM and a Vmax = 1.58 +/- 0.64 nmol/min/nmol P450 respectively (mean +/- SD, n = 5). Eadie-Hofstee plot analysis of carteolol 8-hydroxylase activity confirmed single-enzyme Michaelis-Menten kinetics. 3. The cytochrome P450 isoforms involved in 8-hydroxylation of carteolol were investigated using selective chemical inhibitors and polyclonal anti-P450 antibodies. Quinine (Ki = 0.06 microM) and quinidine (Ki = 2.0 microM), selective inhibitors of CYP2D1, competitively inhibited 8-hydroxycarteolol formation. Furthermore, only anti-human CYP2D6 antibody inhibited this reaction. 4. These results suggest that carteolol is metabolized to 8-hydroxycarteolol by CYP2D1. The K(m) of carteolol for CYP2D1 in male rat liver microsomes was much greater than those of propranolol or bunitrolol, indicating that carteolol has a lower affinity for CYP2D1 compared with these other beta-adrenoceptor blocking drugs.     

Modulation of cytochrome P-450-dependent monooxygenases, glutathione and glutathione S-transferase in rat liver by geniposide from Gardenia jasminoides

Geniposide is an iridoid glycoside extracted from the fruits of Gardenia jasminoides, which are used as a food colorant and as a traditional Chinese medicine for treatment of hepatic and inflammatory diseases. The effects of geniposide and G. jasminoides fruit crude extract on liver cytochrome P-450 (P-450)-dependent monooxygenases, glutathione and glutathione S-transferase were investigated using rats treated orally with the iridoid glycoside (0.1 g/kg body weight/day) or the fruit crude extract (2 g/kg/day) for 4 days. The treatments decreased serum urea nitrogen level but increased liver to body weight ratio, total hepatic glutathione content and hepatic cytosolic glutathione S-transferase activity. Treatments with geniposide and G. jasminoides decreased P-450 content, benzo[a]pyrene hydroxylation, 7-ethoxycoumarin O-deethylation, and erythromycin N-demethylation activities in liver microsomes without affecting aniline hydroxylation activity. The natural products had no effect on glutathione content and monooxygenase activities in kidney microsomes. Immunoblotting analyses of liver microsomal proteins using mouse monoclonal antibody 2-13-1 to rat P4503A1/2 revealed that geniposide and G. jasminoides crude extract decreased the intensity of a P4503A-immunorelated protein. Protein blots probed with mouse monoclonal antibody 1-12-3 to rat P4501A1 and rabbit polyclonal antibody against human P4502E1 showed that both treatments had little or no effect on P4501A and 2E proteins. The present findings demonstrate that geniposide from G. jasminoides has the ability to inhibit a P4503A monooxygenase and increase glutathione content in rat liver.     

Bioactivation and irreversible binding of the cognition activator tacrine using human and rat liver microsomal preparations. Species difference

Tacrine's [1,2,3,4-tetrahydro-9-acridinamine monohydrochloride monohydrate, (THA)] metabolic fate was examined using human and rat liver microsomal preparations. Following 1-hr incubations with human microsomes, [14C]THA (0.4 microM) was extensively metabolized to 1-hydroxyTHA with trace amounts of 2-, 4-, and 7-hydroxyTHA also produced. Poor recovery of radioactivity in the postreaction incubates suggested association of THA-derived radioactivity with precipitated microsomal protein. After exhaustive extraction, 0.034, 0.145, 0.126, and 0.012 nmol eq bound/mg protein/60 min of THA-derived radioactivity was bound to human liver preparations H109, H111, H116, and H118, respectively. Preparations H109 and H118 were lower in P4501A2 content and catalytic activity as compared with preparations H111 and H116. Incubations of equimolar [14C]1-hydroxyTHA with human liver microsomes also resulted in binding to protein, although to a lesser extent than observed with THA. [14C]THA (0.4 microM) was incubated for 1 hr with rat liver microsomes (1 microM P-450) prepared from noninduced (N), phenobarbital (PB), isoniazid (I), and 3-methylcholanthrene (3-MC)-pretreated animals. In all incubations, 1-hydroxyTHA was the major biotransformation product detected. After exhaustive extraction, 0.048, 0.054, 0.049, and 0.153 nmol eq/mg protein/60 min of THA-derived radioactivity was bound to microsomal protein from N, PB, I, and 3-MC pretreated rats. Increased binding with 3-MC induced rat liver preparations suggests the involvement of the P-450 1A subfamily in THA bioactivation. Glutathione (5 mM) coincubation inhibited the irreversible binding of THA-derived radioactivity in both human and 3-MC-induced rat liver preparations, whereas human epoxide hydrase (100 micrograms/incubate) had a relative minor effect. A mechanism is proposed involving a putative quinone methide(s) intermediate in the bioactivation and irreversible binding of THA. A species difference in THA-derived irreversible binding exists between human and noninduced rat liver microsomes, suggesting that the rat is a poor model for studying the underlying mechanism(s) of THA-induced elevations in liver marker enzymes found in clinical investigations.     

Vitamin E in therapy of rheumatic diseases]

Methyl t-butyl ether (MTBE) and ethyl t-butyl ether (ETBE) are commonly used in unleaded gasoline to increase the oxygen content of fuel and to reduce carbon monoxide emissions from motor vehicles. This study was undertaken to investigate: (1) the effect of administration to rats of ETBE and its metabolite, t-butanol, on the induction and/or inhibition of hepatic P450 isoenzymes; (2) the oxidative metabolism of MTBE and ETBE by liver microsomes from rats pretreated with selected P450 inducers and purified rat P450(s), (2B1, 2E1, 2C11, 1A1). ETBE administration by gavage at a dose of 2 ml/kg for 2 days induced hepatic microsomal P4502E1-linked p-nitrophenol hydroxylase and the P4502B1/2-associated PROD and 16beta-testosterone hydroxylase, verified by immunoblot experiments. t-Butanol treatments at doses of 200 and 400 mg/kg i.p. for 4 days did not alter any liver microsomal monoxygenases. Both MTBE and ETBE were substrates for rat liver microsomes and were oxidatively dealkylated to yield formaldehyde and acetaldehyde, respectively. The dealkylation rates of both MTBE and ETBE were increased c. fourfold in phenobarbital (PB)-treated rats. In rats pretreated with pyrazole, an inducer of 2E1, only the demethylation of MTBE was increased (c. twofold). When the oxidations of MTBE and ETBE were investigated with purified P450(s) in a reconstituted system, it was found that P4502B1 had the highest activities towards both solvents, whereas 1A1 and 2C1 were only slightly active; P4502E1 had an appreciable activity on MTBE but not against ETBE. Metyrapone, a potent inhibitor of P450 2B, consistently inhibited both the MTBE and ETBE dealkylations in microsomes from PB-treated rats. Furthermore, 4-methylpyrazole (a probe inhibitor of 2E1) and anti-P4502E1 IgG showed inhibition, though modest, only on MTBE demethylation, but not on ETBE deethylation. Inhibition experiments have also suggested that rat 2A1 may exert an important role in MTBE and ETBE oxidation. Taken together, these results indicate that 2B1, when expressed, is the major enzyme involved in the oxidation of these two solvents and that 2E1 may have a role, although minor, in MTBE demethylation. The implications of these data for MTBE and ETBE toxicity remain to be established.     

Suppression of constitutive and inducible cytochrome P450 gene expression by alpha-hederin in mice

The effects of alpha-Hederin, a triterpenoid saponin which exists in some oriental herbs, on the expression of liver cytochrome P450s were examined in mice. The administration of alpha-Hederin to mice significantly decreased the hepatic content of P450 and the activities of microsomal ethoxyresorufin O-deethylase, methoxyresorufin O-demethylase, and aniline hydroxylase, representative activities of cytochrome-P4501A1, -P4501A2, and -P4502E1, respectively, in a dose- and time-dependent manner. However, pentoxyresorufin O-dealkylase, a representative activity of cytochrome P4502B1/2, was decreased to a lesser extent. alpha-Hederin also decreased inducible monooxygenase activities in the same manner. Suppressions of P450 isozyme expression occurred in alpha-Hederin treated hepatic microsomes, as determined by immunoblot analysis in a manner consistent with that of the enzyme activity levels. Levels of mRNA of P4501A1/2 and P4502B1/2 were also decreased by alpha-Hederin as shown by Northern blot analysis. In contrast, the level of P4502E1 mRNA in the liver of alpha-Hederin treated mice was unchanged. These results suggest that alpha-Hederin may act as a more specific suppressor for P4501A and P4502E1 than P4502B and that the suppression involves decreases in mRNA levels except in the case of P4502E1.     

Studies on the effect of intratesticular administration of opioid peptides, naloxone or N-acetyl beta-endorphin antiserum on some testicular parameters in rats

Substrate specificity and other properties of a fatty acid monooxygenase system in kidney microsomes of the Japanese house musk shrew (Suncus murinus) were examined. The suncus kidney microsomes catalyzed the hydroxylation of various saturated and unsaturated fatty acids to the omega- and (omega-1)-hydroxy derivatives. Laurate was most effectively hydroxylated among saturated and unsaturated fatty acids. The specific activity (53.79 +/- 5.59 [mean +/- SD, n = 6] nmol/nmol cytochrome P450/min) of laurate in suncus kidney microsomes was very high compared with that in liver and kidney microsomes of other species. C18 unsaturated fatty acids were converted to epoxides by a cytochrome P450-dependent fatty acid monooxygenase system in suncus kidney microsomes, in addition to omega- and (omega-1)-hydroxylation products. The monooxygenase system metabolized arachidonic acid only to omega- and (omega-1)-hydroxylation products, not to epoxidation products.     

Reductive activation of 1,1-dichloro-1-fluoroethane (HCFC-141b) by phenobarbital- and pyridine-induced rat liver microsomal cytochrome P450

1. During anaerobic reductive incubation of liver microsomes, from either the pyridine- or phenobarbital-treated rat, with 1,1-dichloro-1-fluoroethane (HCFC-141b) in the presence of a NADPH-regenerating system, a time- and dose-dependent formation of reactive metabolites was detected as indicated by a depletion of added exogenous glutathione. 2. A statistically significant, dose-dependent loss of both cytochrome P450 and microsomal haem was also observed under these experimental conditions. Furthermore, a statistically significant decrease of p-nitrophenol hydroxylase and pentoxyresorufin O-depentylase activity was measured in microsomes from the pyridine- and phenobarbital-induced rat, respectively indicating that both P4502E1 and P4502B undergo substrate-dependent inactivation. 3. Both reactive metabolite formation and P450 inactivation were almost completely inhibited by previous bubbling of the incubation mixture with carbon monoxide, indicating that interaction of the substrate with a free and reduced P450 haem iron is required for substrate bioactivation and enzyme loss. 4. The presence in the incubation mixture of the spin-trap N-t-butyl-alpha-phenylnitrone (PBN) and the carbene trap 2,3-dimethyl-2-butene (DMB) largely prevented both glutathione depletion and P450 loss. This suggests that free radical and carbene intermediates formed by the metabolic activation of the substrate are involved in the inactivation of P450 and the loss of its prosthetic haem group.     

Comparison of laparoscopic versus open repair of paraesophageal hernia

Of seven cDNA-expressed human cytochrome P450 (P450) enzymes (P450s 1A2, 2B6, 2C9, 2C19, 2D6, 2E1, and 3A4) examined, P450 1A2 was the most active in catalyzing 2- and 4-hydroxylations of estradiol and estrone. P450 3A4 and P450 2C9 also catalyzed these reactions although to lesser extents than P450 1A2. P450 1A2 also efficiently oxidized estradiol at the 16alpha-position but was less active in estrone 16alpha-hydroxylation; the latter reaction and also estradiol 16alpha-hydroxylation were catalyzed by P450 3A4 at significant levels. Anti-P450 1A2 antibodies inhibited 2- and 4-hydroxylations of these two estrogens catalyzed by liver microsomes of some of the human samples examined. Estradiol 16alpha-hydroxylation was inhibited by both anti-P450 1A2 and anti-P450 3A4, while estrone 16alpha-hydroxylation was significantly suppressed by anti-P450 3A4 in human liver microsomes. Fluvoxamine efficiently inhibited the estrogen hydroxylations in human liver samples that contained high levels of P450 1A2, while ketoconazole affected these activities in human samples in which P450 3A4 levels were high. alpha-Naphthoflavone either stimulated or had no effect on estradiol hydroxylation catalyzed by liver microsomes; the intensity of this effect depended on the human samples and their P450s. Interestingly, in the presence of anti-P450 3A4 antibodies, alpha-naphthoflavone was found to be able to inhibit estradiol and estrone 2-hydroxylations catalyzed by human liver microsomes. The results suggest that both P450s 1A2 and 3A4 have major roles in oxidations of estradiol and estrone in human liver and that the contents of these two P450 forms in liver microsomes determine which P450 enzymes are most important in hepatic estrogen hydroxylation by individual humans. P450 3A4 may be expected to play a more important role for some of the estrogen hydroxylation reactions than P450 1A2. Knowledge of roles of individual P450s in these estrogen hydroxylations has relevance to current controversies in hormonal carcinogenesis [Service, R. F. (1998) Science 279, 1631-1633].     

Effects of motorcycle exhaust on cytochrome P-450-dependent monooxygenases and glutathione S-transferase in rat tissues

The effects of motorcycle exhaust (ME) on cytochrome P-450 (P-450)-dependent monooxygenases were determined using rats exposed to the exhaust by either inhalation, intratracheal, or intraperitoneal administration. A 4-wk ME inhalation significantly increased benzo[a]pyrene hydroxylation, 7-ethoxyresorufin O-deethylation, and NADPH-cytochrome c reductase activities in liver, kidney, and lung microsomes. Intratracheal instillation of organic extracts of ME particulate (MEP) caused a dose- and time-dependent significant increase of monooxygenase activity. Intratracheal treatment with 0.1 g MEP extract/kg markedly elevated benzo[a]pyrene hydroxylation and 7-ethoxyresorufin O-deethylation activities in the rat tissues 24 h following treatment. Intraperitoneal treatment with 0.5 g MEP extract/kg/d for 4 d resulted in significant increases of P-450 and cytochrome b5 contents and NADPH-cytochrome c reductase activity in liver microsomes. The intraperitoneal treatment also markedly increased monooxygenases activities toward methoxyresorufin, aniline, benzphetamine, and erythromycin in liver and benzo[a]pyrene and 7-ethoxyresorufin in liver, kidney, and lung. Immunoblotting analyses of microsomal proteins using a mouse monoclonal antibody (Mab) 1-12-3 against rat P-450 1A1 revealed that ME inhalation, MEP intratracheal, or MEP intraperitoneal treatment increased a P-450 1A protein in the hepatic and extrahepatic tissues. Protein blots analyzed using antibodies to P-450 enzymes showed that MEP intraperitoneal treatment caused increases of P-450 2B, 2E, and 3A subfamily proteins in the liver. The ME inhalation, MEP intratracheal, or MEP intraperitoneal treatment resulted in significant increases in glutathione S-transferase activity in liver cytosols. The present study shows that ME and MEP extract contain substances that can induce multiple forms of P-450 and glutathione S-transferase activity in the rat.     

Induction of cytochrome P450 1A in hamster liver and lung by 6-nitrochrysene

The present study has determined the effect of 6-nitrochrysene (6-NC) on hepatic and pulmonary cytochrome P450 (P450)-dependent monooxygenases using hamsters pretreated with the nitrated polycyclic aromatic hydrocarbon (nitro-PAH) at 5 mg/kg per day for 3 days. Pretreatment with 6-NC elevated serum gamma-glutamyltranspeptidase, lactate dehydrogenase, and bilirubin levels. Liver S9 fractions prepared from controls and hamsters pretreated with 6-NC markedly increased mutagenicity of the nitro-PAH in Salmonella typhimurium tester strains TA98, TA100, and TA102. The pretreatment selectively increased 1-nitropyrene reductase activities of lung cytosol and liver and lung microsomes. Pretreatment with 6-NC resulted in increases of microsomal 7-ethoxyresorufin and methoxyresorufin O-dealkylases activities in liver and lung without affecting the monooxygenase activities in kidney. Immunoblot analysis of microsomal proteins using mouse monoclonal antibody 1-12-3 to rat P450 1A1 revealed that 6-NC induced P450 1A-immunorelated proteins in liver and lung. RNA blot analysis using mouse P450 1A1 cDNA showed that 6-NC increased liver and lung P450 1A mRNA. 6-NC had no effect on the kidney P450 protein and mRNA. The present study demonstrates that the hamster enzymes can support 6-NC metabolic activation and the nitro-PAH induces liver and lung P4501A via a pretranslational mechanism.     

Impact of lipid and ACE inhibitor therapy on cardiovascular disease and metabolic abnormalities in the diabetic and hypertensive patient

We examined the effect of 1,1-dichloroethylene (1,1-DCE) on microsomal cytochrome P450 (P450) enzymes in rat liver and kidney. Rats were treated intraperitoneally with 1,1-DCE daily for 4 days, at doses of 200, 400, and 800 mg/kg. Among the P450-dependent monooxygenase activities in liver microsomes, testosterone 2alpha-hydroxylase (T2AH), which is associated with CYP2C11 activity, was remarkably decreased by 800 mg/kg 1,1-DCE. The level relative to control activity was < 10%. Furthermore, immunoblotting showed that 1,1-DCE (> or = 400 mg/kg) significantly decreased CYP2C11/6 protein levels in liver microsomes. In addition, 7-methoxyresorufin O-demethylase (MROD), 7-ethoxycoumarin O-deethylase (ECOD), benzphetamine N-demethylase (BZND), chlorzoxazone 6-hydroxylase (CZ6H), and testosterone 6beta-hydroxylase (T6BH) activities were significantly decreased by the highest dose of 1,1-DCE (by 40-70%). However, the activities of other P450-dependent monooxygenases, namely 7-ethoxyresorufin O-deethylase (EROD), 7-benzyloxyresorufin O-debenzylase (BROD), aminopyrine N-demethylase (APND), erythromycin N-demethylase (EMND), lauric acid omega-hydroxylase (LAOH), and testosterone 7alpha-hydroxylase (T7AH) were not affected by 1,1-DCE at any dose. Immunoblotting showed CYP1A1/2, CYP2B1/2, CYP2E1, and CYP3A2/1 protein levels were significantly decreased by 60-66% by 1,1-DCE (800 mg/kg), whereas that of CYP4A1/2 was not affected by any dose of 1,1-DCE. By contrast, among the P450-dependent monooxygenase activities in kidney microsomes, only CZ6H activity was increased by 1,1-DCE (1.6-fold at 800 mg/kg). Also, it was observed that 1,1-DCE (800 mg/kg) significantly increased CYP2E1 protein levels by immunoblotting (approximately 1.5-fold). These results suggest that 1,1-DCE changes the constitutive P450 isoforms in the rat liver and kidney, and that these changes closely relate to the toxicity of 1,1-DCE.     

Purification and characterization of constituent testosterone 2 alpha-hydroxylase (cytochrome P450(2)alpha) from mouse liver

Hepatic microsomal testosterone/androstenedione 2 alpha-hydroxylase (i.e., cytochrome P450(2)alpha) was purified from female CD-1 mice. Protein purification was monitored in eluates from Fractogel, DEAE-sephacel, and hydroxylapatite columns at heme absorbing 417 nm and by cytochrome P450 content, reactivity to a monoclonal antibody against female-specific rat cytochrome P450 2C12, and testosterone 2 alpha-hydroxylase activity. The catalytic activity of the purified cytochrome P450(2)alpha, exhibiting a high degree of regioselectivity and stereospecificity, was basically restricted to the 2 alpha-hydroxylation of testosterone and androstenedione; representing > 96% and > 92% of these respective metabolites. Polyclonal antibodies against cytochrome P450(2)alpha exhibited a dose-dependent and very selective inhibition of testosterone 2 alpha-hydroxylation. The specific cytochrome P450 content of the purified cytochrome P450(2)alpha fraction was 12.06 nmol/mg protein. The specific testosterone 2 alpha-hydroxylase activity of the purified protein was 14 nmol/min/nmol cytochrome P450, which was about 60-fold higher than the respective microsomes. The apparent subunit molecular weight of cytochrome P450(2)alpha was 51,000 and the protein appeared as a single band on sodium dodecyl sulfate polyacrylamide gels. The amino-terminal sequence analysis indicates that cytochrome P450(2)alpha is a member of the murine cytochrome P450 2d family.     

Respective roles of human cytochrome P-4502E1, 1A2, and 3A4 in the hepatic microsomal ethanol oxidizing system

The microsomal ethanol oxidizing system comprises an ethanol-inducible cytochrome P-4502E1, but the involvement of other P-450s has also been suggested. In our study, human CYP2E1, CYP1A2, and CYP3A4 were heterologously expressed in HepG2 cells, and their ethanol oxidation was assessed using a corresponding selective inhibitor: all three P-450 isoenzymes metabolized ethanol. Selective inhibitors-4-methylpyrazole (CYP2E1), furafylline (CYP1A2), and troleandomycin (CYP3A4)-also decreased microsomal ethanol oxidation in the livers of 18 organ donors. The P-450-dependent ethanol oxidizing activities correlated significantly with those of the specific monooxygenases and the immunochemically determined microsomal content of the respective P-450. The mean CYP2E1-dependent ethanol oxidation in human liver microsomes [1.41+/-0.11 nmol min(-1) (mg protein)(-1)] was twice that of CYP1A2 (0.61+/-0.07) or CYP3A4 (0.73+/-0.11) (p < 0.05). Furthermore, CYP2E1 had the highest (p < 0.05) specific activity [28+/-2 nmol min(-1) (nmol CYP2E1)(-1) versus 17+/-3 nmol min(-1) (nmol CYP1A2)(-1), and 12+/-2 nmol min(-1) (CYP3A4)(-1), respectively]. Thus, in human liver microsomes, CYP2E1 plays the major role. However, CYP1A2 and CYP3A4 contribute significantly to microsomal ethanol oxidation and may, therefore, also be involved in the pathogenesis of alcoholic liver disease.     

Examples of pitfalls in statistical analysis--6. Evaluation of data consisting of the elapsed time between two events

beta-Carotene and other carotenoids are widely regarded as biological antioxidants. However, recent clinical trials indicate that beta-carotene supplements are not effective in disease prevention and raise questions about the biological significance of carotenoid antioxidant actions. To further explore this issue, we have reevaluated the antioxidant actions of beta-carotene in liposomal and biological membrane systems. In dilinoleoylphosphatidylcholine liposomes in which 0.35 mol % beta-carotene was incorporated into the bilayer during liposome preparation, the carotenoid inhibited lipid peroxidation initiated by 10 mm azobis[amidinopropane HCl] (AAPH). In carotenoid-free liposome suspensions to which the same amount of beta-carotene was added, no antioxidant effect was observed. Supplementation of rat liver microsomes with beta-carotene in vitro yielded microsomes containing 1.7 nmol beta-carotene mg-1 and 0.16 nmol alpha-tocopherol mg-1 microsomal protein. In beta-carotene supplemented microsomes incubated with 10 mm AAPH under an air atmosphere, lipid peroxidation did not occur until alpha-tocopherol was depleted by approximately 60%. beta-Carotene exerted no apparent antioxidant effect and was not significantly depleted in the incubations. Similar results were obtained when the incubation was done at 3.8 torr O2. In liver microsomes from Mongolian gerbils fed beta-carotene-supplemented diets, beta-carotene levels were 16-37% of alpha-tocopherol levels. The kinetics of AAPH-induced lipid peroxidation were no different in beta-carotene-supplemented microsomes than in microsomes from unsupplemented animals, although the kinetics of beta-carotene and alpha-tocopherol depletion were similar. The results indicate that beta-carotene is ineffective as an antioxidant when added to preformed lipid bilayer membranes and that alpha-tocopherol is a much more effective membrane antioxidant than beta-carotene, regardless of the method of carotenoid-membrane incorporation. These results support a reevaluation of the proposed antioxidant role for beta-carotene in biological membranes.     

Cytochrome P4502E1 hydroxyethyl radical adducts as the major antigen in autoantibody formation among alcoholics

BACKGROUND & AIMS: We have previously reported that alcoholics have increased titers of immunoglobulins reacting with protein adducts of hydroxyethyl free radicals. Because hydroxyethyl radicals are produced during ethanol metabolism by liver microsomes, the aim of this study was to determine whether such antibodies recognize microsomal proteins complexed with hydroxyethyl radicals. METHODS: Liver microsomal proteins reacting with the anti-hydroxyethyl radical antibodies were characterized by an enzyme-linked immunosorbent assay and Western blotting. RESULTS: Alcoholic cirrhotics, but not patients with nonalcoholic cirrhosis or healthy subjects, had increased serum levels of immunoglobulin G and A directed against antigens produced in microsomes incubated with reduced nicotinamide adenine dinucleotide phosphate (NADPH) and ethanol. Such immunoreactivity was completely blocked when microsomes were incubated with ethanol in the presence of the spin-trapping agent 4-pyridyl-1-oxide-t-butyl nitrone or by preincubating the sera with hydroxyethyl radical-bound human albumin. Immunoblotting of proteins from human liver microsomes incubated with NADPH and ethanol showed that 86% of the sera from alcoholic cirrhotics reacted with a 52-kilodalton protein, whereas variable reactivity was observed with proteins of 78, 60, and 40 kilodaltons, respectively, The 52-kilodalton protein was identified by immunoblotting and immunoprecipitation as ethanol-inducible cytochrome P4502E1. CONCLUSIONS: Antibodies from alcoholic cirrhotics specifically recognized hydroxyethyl radical-cytochrome P4502E1 adducts, suggesting the possible implication of these antigens in the development of autoimmune reactions in alcoholic liver disease.