Inhibition of human cytochrome P450-catalyzed oxidations of xenobiotics and procarcinogens by synthetic organoselenium compounds

The effects of synthetic chemopreventive organoselenium compounds 1,2-, 1,3-, and 1,4-phenylenebis(methylene)selenocyanate (o-, m-, and p-XSC, respectively), benzyl selenocyanate (BSC), and dibenzyl diselenide (DDS) and inorganic sodium selenite on the oxidation of xenobiotics and procarcinogens by human cytochrome P450 (P450 or CYP) enzymes were determined in vitro. Spectral studies showed that BSC and three XSC compounds (but not sodium selenite or DDS) induced type II difference spectrum when added to the suspension of liver microsomes isolated from beta-naphthoflavone-treated rats, with m-XSC being the most potent in inducing spectral interactions with P450 enzymes; m-XSC also produced a type II spectral change with human liver microsomes. o-, m-, and p-XSC inhibited 7-ethoxyresorufin O-deethylation catalyzed by human liver microsomes when added at concentrations below 1 microM levels, but BSC and DDS were less effective. All of these compounds inhibited the oxidation of model substrates for human P450s to varying extents. We studied the effects of these compounds on the activation of procarcinogens by recombinant human CYP1A1, 1A2, and 1B1 enzymes using Salmonella typhimurium NM2009 tester strain for the detection of DNA damage. The three XSCs were found to be very potent inhibitors of metabolic activation of 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole, 2-amino-3,5-dimethylimidazo[4,5-f]quinoline, and 2-aminoanthracene, catalyzed by CYP1A1, 1A2, and 1B1, respectively. The potency of inhibition of m-XSC on CYP1B1-dependent activation of 2-aminoanthracene was compatible to those of alpha-naphthoflavone. These inhibitory actions may, in part, account for the mechanisms responsible for cancer prevention by organoselenium compounds in laboratory animals.     

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.     

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.     

Childhood hepatoblastomas frequently carry a mutated degradation targeting box of the beta-catenin gene

The enzyme system responsible for the N-deacetylation of eprinomectin in rats was characterized. Tissue and subcellular studies showed that the hydrolysis activity was localized mainly in liver microsomes. Apparent KM and Vmax values calculated from Lineweaver-Burk plots were 53 microM and 0.81 nmol/mg/min for male rats and 70 microM and 4.99 nmol/mg/min for female rats, respectively. Pretreatment of male rats with dexamethasone, phenobarbital, and pregnenolone 16alpha-carbonitrile increased the activity by more than 3-fold. Paraoxon and bis-4-nitrophenylphosphate strongly inhibited the deacetylase activity at concentrations as low as 1 microM. The hydrolysis activity also was inhibited by SKF525, but less effectively. Eserine strongly inhibited the activity at 1 x 10(-4) M. HgCl2 decreased the activity to about 40% at a concentration of 1 x 10(-4) M. FeCl3, CaCl2, MgCl2, and EDTA had little effect on the hydrolysis of eprinomectin, whereas NaF slightly increased the activity to 118%. Thus, the inhibition study suggested that eprinomectin deacetylase resembled "B" type carboxylesterase/amidases. The hydrolysis activity of eprinomectin and isocarboxazid, a specific substrate of RL2 [Hosokawa, M, Maki T and Satoh T (1987) Mol Pharmacol 31:579-584], by liver microsomes from rats treated with various cytochrome P-450 inducers correlated well (r = 0.92). Also, elusion profiles of esterase by gel filtration and ion exchange chromatography demonstrated that the active protein(s) for eprinomectin and isocarboxazid hydrolysis coeluted. Thus, RL2 or an enzyme system similar to RL2 is responsible for the N-deacetylation of eprinomectin.     

Studies of postnatal diabetes mellitus in women who had gestational diabetes. Part 2. Prevalence and predictors of diabetes mellitus after delivery

Diclofenac antiserum was previously developed and used to detect protein adducts of metabolites of dichlofenac in livers of mice and rats. In this study, the antibody has been used to facilitate the purification of a major 51 kDa microsomal adduct of diclofenac from the liver microsomes of male rats that were treated with diclofenac. The adduct was identified as male-specific cytochrome P4502C11 based on its N-terminal amino acid sequence, reaction with a cytochrome P4502C11 antibody, and by its absence from liver microsomes of diclofenac-treated female rats. When diclofenac was incubated with liver microsomes of control rats in the presence of NADPH, only the 51 kDa adduct was produced. The formation of the adduct was inhibited by a cytochrome P4502C11 monoclonal antibody, but not by reduced glutathione or N-alpha-acetyl-L-lysine. No adduct was detected when diclofenac was incubated with liver microsomes from female rats. Moreover, adduct formation in vivo appeared to lead to a 72% decrease in the activity of cytochrome P4502C11. The results indicate that cytochrome P4502C11 metabolizes diclofenac into a highly reactive product that covalently binds to this enzyme before it can diffuse away and react with other proteins.     

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.     

Lithium exerts a time-dependent and tissue-selective attenuation of the dexamethasone-induced polyamine response in rat brain and liver

Recently El-Bayoumy and coworkers have reported that 1,4-phenylene-bis(methylene)selenocyanate (p-XSC) was very effective in inhibiting 7,12-dimethylbenz(a)anthracene (DMBA)-induced mammary carcinogenesis and adduct formation during the initiation phase (Cancer Res., 52, 2402-2407, 1992). Furthermore, this compound was found to be well tolerated by rats at high doses. The present study was designed to extend these earlier observations by investigating the response to lower levels of p-XSC given either before or after DMBA administration. At a level of 15 p.p.m. Se, p-XSC suppressed total mammary tumor yield by 80% and 52% in the initiation phase and post-initiation phase, respectively. A dose-response effect was evident in the range 5-15 p.p.m. Se. When p-XSC was given at a level of 5 p.p.m. Se during the entire course of the experimental period, total tumor yield was reduced by half. This dose is about 4 x less than the maximum tolerable dose (MTD). Other selenocyanate analogs were also examined in an attempt to obtain information on their respective chemopreventive index, which is calculated as the ratio of MTD to the effective dose which produces approximately a 50% inhibition in total tumor yield (ED50). The reagents studied included potassium selenocyanate, methyl selenocyanate and benzyl selenocyanate, as well as sodium selenite (reference compound). Compared to p-XSC, which has a chemopreventive index of 4.0, the other four compounds have a lower index ranging from 1.3 for sodium selenite and potassium selenocyanate to 2.0 for methyl selenocyanate and 2.5 for benzyl selenocyanate. A high chemopreventive index signifies that a compound is well tolerated at doses required for cancer suppression. The last component of the present study involved the repletion assay of liver glutathione peroxidase in selenium-deficient rats as a biomarker to estimate the metabolizability of the above selenium compounds. The bioavailability data suggest that the selenium from p-XSC is not as efficiently incorporated into glutathione peroxidase as the selenium from selenite or the other selenocyanate analogs. Currently, we are working under the hypothesis that the chemical structure of the RSeCN compound could affect activity per se and also influence the rate of release of selenium from the parent compound, thereby impacting on the anticarcinogenic efficacy, tolerance and bioavailability of the compound.     

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.     

Amination of tyrosine in liver cytosol protein of male F344 rats treated with 2-nitropropane, 2-nitrobutane, 3-nitropentane, or acetoxime

Previously, the secondary nitroalkane 2-nitropropane, a strong hepatocarcinogen in rats, had been shown to induce the formation of 8-aminoguanine in both DNA and RNA of rat liver through a sulfotransferase-mediated pathway. This pathway was postulated to convert the carcinogen into an aminating species [Sodum, R. S., et al. (1994) Chem. Res. Toxicol. 7, 344-351]. To submit this postulate to further test, we examined liver proteins of rats treated with 2-nitropropane, other carcinogenic secondary nitroalkanes, or the related rat liver tumorigen acetoxime for the presence of 3-aminotyrosine, the expected product of tyrosine amination. Using ion-pair and/or cation-exchange high-performance liquid chromatography with electrochemical detection, we found that the liver cytosolic proteins of these animals contained 0.1-1.5 mol of 3-aminotyrosine/10(3) mol of tyrosine. Treatment with the noncarcinogenic primary nitroalkane 1-nitropropane or with other primary nitroalkanes did not produce an analogous increase in the aminated amino acid (level of detection estimated at approximately 0.01 mol/10(3) mol of tyrosine). To our knowledge, this is the first report of the modification of protein tyrosine in vivo by a carcinogen. In vitro studies with acetoxime-O-sulfonate and hydroxylamine-O-sulfonate showed that these proposed intermediates in the activation pathway of 2-nitropropane react with guanosine to give 8-aminoguanosine, N1-aminoguanosine, and 8-oxoguanosine and also react with tyrosine to give 3-aminotyrosine and 3-hydroxytyrosine. The in vitro amination and oxidation of guanosine at C8 were also produced by acetophenoxime-O-sulfonate and 2-heptanoxime-O-sulfonate. These results provide additional evidence for the production of a reactive species capable of aminating nucleic acids and proteins from 2-nitropropane and other carcinogenic secondary nitroalkanes by a pathway involving oxime- and hydroxylamine-O-sulfonates as intermediates.     

Ring hydroxylation of [o-3H]methoxychlor as a probe for liver microsomal CYP2B activity: potential for in vivo CYP2B assay

An in vitro radiometric assay selective for inducible CYP2B activity is described. The assay is based on the quantification of 3H2O release that occurs during o-ring hydroxylation of [o-3H]methoxychlor by liver microsomes in the presence of NADPH. 3H2O is isolated by removing > 99.9% of the parent compound and organic metabolites by facile charcoal extraction and filtration. There was no evidence for an NIH shift during ring hydroxylation, and there was little or no isotope effect. Selectivity for CYP2B was demonstrated using liver microsomes prepared from rats and mice treated with inducers of different CYP isoforms. Ring hydroxylation of [o-3H]methoxychlor was elevated 11.4-fold over control values in liver microsomes from male rats treated with phenobarbital. With mice, phenobarbital treatment elevated liver microsomal ring hydroxylation 7.1-fold. Clofibrate, 3-methylcholanthrene, or beta-naphthoflavone treatment of male rats or pyridine treatment of female rats did not elevate liver microsomal ring-hydroxylase activity, indicating that CYP4A, 1A, and 2E1 do not support this reaction. In female rats, dexamethasone and pregnenolone-16 alpha-carbonitrile treatment elevated ring hydroxylation up to 5.5- and 3.2-fold, respectively, an activity that may be attributed to CYP2B induction in those animals. Incubation of liver microsomes from phenobarbital-treated males with monospecific anti-CYP2B monoclonal antibodies (Mab) inhibited ring-hydroxylase activity up to 86%, demonstrating predominantly CYP2B-mediated catalysis. An 86% inhibition by these Mabs was also observed using liver microsomes from male mice treated with phenobarbital, indicating the assay is not limited to rats. The CYP2B mechanism-based inhibitor orphenadrine caused a 76% decline in activity, providing further evidence for CYP2B involvement. Unlike other CYP2B-selective assays, this method may be readily adapted to in vivo studies, by measuring urinary excretion of 3H2O as an indication of total body CYP2B activity.     

Enzymes involved in the bioactivation of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in patas monkey lung and liver microsomes

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a potent tobacco-specific carcinogen in animals. Our previous studies indicated that there are differences between rodents and humans for the enzymes involved in the activation of NNK. To determine if the patas monkey is a better animal model for the activation of NNK in humans, we investigated the metabolism of NNK in patas monkey lung and liver microsomes and characterized the enzymes involved in the activation. In lung microsomes, the formation of 4-oxo-1-(3-pyridyl)-1-butanone (keto aldehyde), 4-(methylnitrosamino)-1-(3-pyridyl-N-oxide)-1-butanone (NNK-N-oxide), 4-hydroxy-1-(3-pyridyl)-1-butanone (keto alcohol), and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) was observed, displaying apparent Km values of 10.3, 5.4, 4.9, and 902 microM, respectively. NNK metabolism in liver microsomes resulted in the formation of keto aldehyde, keto alcohol, and NNAL, displaying apparent Km values of 8.1, 8.2, and 474 microM, respectively. The low Km values for NNK oxidation in the patas monkey lung and liver microsomes are different from those in human lung and liver microsomes showing Km values of 400-653 microM, although loss of low Km forms from human tissue as a result of disease, surgery or anesthesia cannot be ruled out. Carbon monoxide (90%) significantly inhibited NNK metabolism in the patas monkey lung and liver microsomes by 38-66% and 82-91%, respectively. Nordihydroguaiaretic acid (a lipoxygenase inhibitor) and aspirin (a cyclooxygenase inhibitor) decreased the rate of formation of keto aldehyde and keto alcohol by 10-20 % in the monkey lung microsomes. Alpha-Napthoflavone and coumarin markedly decreased the oxidation of NNK in monkey lung and liver microsomes, suggesting the involvement of P450s 1A and 2A6. An antibody against human P450 2A6 decreased the oxidation of NNK by 12-16% and 22-24% in the patas monkey lung and liver microsomes, respectively. These results are comparable to that obtained with human lung and liver microsomes. Coumarin hydroxylation was observed in the patas monkey lung and liver microsomes at a rate of 16 and 4000 pmol/min/mg protein, respectively, which was 5-fold higher than human lung and liver microsomes, respectively. Immunoblot analysis demonstrated that the P450 2A level in the individual patas monkey liver microsomal sample was 6-fold greater than in an individual human liver microsomal sample. Phenethyl isothiocyanate, an inhibitor of NNK activation in rodents and humans, decreased NNK oxidation in the monkey lung and liver microsomes displaying inhibitor concentration resulting in 50% inhibition of the activity (IC50) values of 0.28-0.8 microM and 4.2-6.8 microM, respectively. The results demonstrate the similarities and differences between species in the metabolic activation of NNK. The patas monkey microsomes appear to more closely resemble human microsomes than mouse or rat enzymes and may better reflect the activation of NNK in humans.     

beta-Naphthoflavone- and self-induced metabolism of 3,3',4,4'-tetrachlorobiphenyl in hepatic microsomes of the male, pregnant female and foetal rat

1. The in vitro metabolism of 3,3',4,4'-tetrachloro-[14C]-biphenyl ([14C]-TCB) by hepatic microsomes from the Wistar rat was investigated with liver microsomes from the male, pregnant female and foetus. 2. Three hydroxylated metabolites (4-OH-3,3',4,5'-tetrachlorobiphenyl, 5-OH-3,3',4,4'-tetrachlorobiphenyl, and 6-OH-3,3',4,4'-tetrachlorobiphenyl) were identified by hplc and gc-ms after incubations of liver microsomes from the beta-naphthoflavone-pretreated male rat and TCB-treated pregnant rat. No metabolites of [14C]-TCB were found after incubation with foetal liver microsomes from dams pretreated with [14C]-TCB. The results indicate that the in vivo accumulation of 4-OH-tetraCB in the foetal compartment is probably due to transplacental transport rather than the formation of this metabolite in the foetus. 3. Pretreatment of the male rat with beta-naphthoflavone substantially induced the formation of hydroxylated metabolites, but pretreatment with phenobarbital and dexamethasone was without effect. Based on in vitro incubations of liver microsomes from the beta-naphthoflavone pretreated male rat, an apparent Km and Vmax of 4.5 microM and 240 pmol/mg protein/min respectively was determined for the metabolism of [14C]-TCB. The formation of phenolic metabolites of [14C]-TCB was most likely dependent on P4501A induction.     

Inhibition of DNA cytosine methyltransferase by chemopreventive selenium compounds, determined by an improved assay for DNA cytosine methyltransferase and DNA cytosine methylation

The organoselenium compounds benzyl selenocyanate (BSC) and 1,4-phenylenebis(methylene)selenocyanate (p-XSC), as well as sodium selenite, are effective chemopreventive agents for various chemically induced tumors in animal models at both the initiation and postinitiation stages. The mechanisms involved at the postinitiation stage are not clear. Because several lines of evidence indicate that inhibition of excess DNA (cytosine-5)-methyltransferase (Mtase) may be a sufficient factor for the suppression or reversion of carcinogenesis, we examined the effects of sodium selenite, BSC, p-XSC and benzyl thiocyanate (BTC), the sulfur analog of BSC, on Mtase activity in nuclear extracts of human colon carcinomas, and of p-XSC on the Mtase activity of HCT116 human colon carcinoma cells in culture. For this purpose, we developed an improved Mtase assay, in which the incorporation of the methyl-[3H] group from S-adenosyl[methyl-3H]methionine into deoxycytidine of poly(dI-dC)-poly(dI-dC), is specifically determined by HPLC with radioflow detection after enzymatic hydrolysis, enhancing specificity and reliability. In a variation, using SssI methyltransferase and labeled S-adenosylmethionine, the overall methylation status of DNA in various tissues can also be compared. Selenite, BSC and p-XSC inhibited Mtase extracted from a human colon carcinoma with IC50s of 3.8, 8.1 and 5.2 microM, respectively; BTC had no effect. p-XSC also inhibited the Mtase activity and growth of human colon carcinoma HCT116 cells, with an IC50 of approximately 20 microM. The improved Mtase assay should prove to be a reliable method for screening potential Mtase inhibitors, especially using cells in culture. We suggest that inhibition of Mtase may be a major mechanism of chemoprevention by selenium compounds at the postinitiation stage of carcinogenesis.     

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.     

Development of a highly efficient purification process for recombinant adenoviral vectors for oral gene delivery

This study compared catalytic and immunochemical properties of drug metabolizing phase I and II enzyme systems in houbara bustard (Chlamydotis undulata) liver and kidney and rat liver. P450 content in bustard liver (0.34 +/- 0.03 nmol mg-1 protein) was 50% lower than that of rat liver (0.70 +/- 0.02 nmol mg-1 protein). With the exception of aniline hydroxylase activity, monooxygenase activities using aminopyrine, ethoxyresorufin and ethoxycoumarin as substrates were all significantly lower than corresponding rat liver enzymes. As found in mammalian systems the P450 activities in the bird liver were higher than in the kidney. Immunohistochemical analysis of microsomes using antibodies to rat hepatic P450 demonstrated that bustard liver and kidney express P4502C11 homologous protein; no appreciable cross-reactivity was observed in bustards using antibodies to P4502E1, 1A1 or 1A2 isoenzymes. Glutathione content and glutathione S-transferase (GST) activity in bustard liver were comparable with those of rat liver. GST activity in the kidney was 65% lower than the liver. Western blotting of liver and kidney cytosol with human GST isoenzyme-specific antibodies revealed that the expression of alpha-class of antibodies exceeds mu in the bustard. In contrast, the pi-class of GST was not detected in the bustard liver. This data demonstrates that hepatic and renal microsomes from the bustard have multiple forms of phase I and phase II enzymes. The multiplicity and tissue specific expression of xenobiotic metabolizing enzymes in bustards may play a significant role in determining the pharmacokinetics of drugs and susceptibility of the birds to various environmental pollutants and toxic insults.     

Hepatic metabolism of skatole in pigs by cytochrome P4502E1

High concentrations of skatole in fat are a major cause of boar taint in intact male pigs. Skatole is metabolized in the liver, and this metabolism could affect concentrations of skatole in fat. In this study, we evaluated the involvement of cytochrome P450, in particular cytochrome P4502E1, in skatole metabolism in pig liver. Liver microsomes from F4 European Wild Pig x Swedish Yorkshire intact male pigs were incubated in a buffer containing NADPH, NADH, and skatole. Several skatole metabolites were detected by HPLC, including 6-hydroxyskatole (pro-MII), 3-hydroxy-3-methyloxyindole (MIII), and five others not identified in this study. Inhibitors of P450 were added to microsomal incubations, and their effect on the formation of skatole metabolites and skatole disappearance was evaluated. The general cytochrome P450 inhibitors SKF 525A, at a concentration of .2 mM and metyrapone, at a concentration of .1 mM decreased the formation of pro-MII (P = .001) to 38.2 and 11.6%, respectively, of that of controls. The SKF 525A also reduced the synthesis of MIII and three other metabolites, whereas metyrapone only reduced the disappearance of skatole and synthesis of pro-MII. Inhibitors specific for cytochrome P4502E1 were more effective in reducing the formation of skatole metabolites than SKF 525A and metyrapone. Chlorzoxazone and diallyl sulfide reduced (P = .001) the synthesis of pro-MII to 9.7 and 30.9% of the control rate. The formation of most of the other skatole metabolites and disappearance of skatole were also reduced with these inhibitors. These results indicate that skatole is metabolized in pig liver to pro-MII and other metabolites by cytochrome P4502E1.     

Phylogenetic analysis of tnpR genes in mercury resistant soil bacteria: the relationship between DNA sequencing and RFLP typing approaches

The purpose of this study was to characterize hepatic cytochrome P450 induction in the dog by phenobarbital, beta-naphthoflavone, dexamethasone, and isoniazid using catalytic activities and Western blots with antibodies prepared against rat cytochrome P450 isozymes. Male beagle dogs were treated with phenobarbital (10 mg/kg for 2 days and 30 mg/kg for the following 5 days), beta-naphthoflavone (50 mg/kg for 5 days), or isoniazid (10 mg/kg for 2 days and 30 mg/kg for the following 5 days). Female beagle dogs were treated with dexamethasone (50 mg/kg for 5 days). Increases in the liver/body weight ratio were observed after treatment of dogs with phenobarbital (133% of control) and dexamethasone (153%). Total cytochrome P450 content was increased as a percentage of control after treatment with phenobarbital (264%) and (3-naphthoflavone (186%), while it slightly decreased after treatment with isoniazid (54%) and dexamethasone (71%). Dog liver microsomes hydroxylated testosterone mainly at the 6-beta and 16-alpha positions but also at the 6-alpha-, 15-beta-, 15-alpha-, 16-beta-, 18-, 2-beta-, and 17-positions. There were no sex differences in terms of regio-selectivity of testosterone metabolism between control male and female dogs. Treatment of dogs with phenobarbital produced increases in 6-beta- (184%), 16-alpha- (379%), 16-beta- (210%), 18- (195%), and 2-beta-testosterone (203%) hydroxylase and pentoxyresorufin 0-dealkylase (651%) activities. On Western blots, phenobarbital treatment produced induction of P450 3A- and 2B1-related proteins. Although treatment with dexamethasone resulted in a large increase in liver weight, no significant increase in P450 3A-related protein or 6-beta-hydroxylase activity was detected. However, dexamethasone and isoniazid treatment produced slight increases in chlorzoxazone hydroxylase activity. Treatment with isoniazid induced a P450 2E1-related protein. Treatment with (beta-naphthoflavone produced increases that were 689 and 357% of control in ethoxyresorufin 0-deethylase and chlorzoxazone hydroxylase activities, respectively. Beta-Naphthoflavone treatment increased the amount of two proteins immunochemically related to the cytochrome P450 1A subfamily. Thus, although generally similar to other species, the response of the dog to cytochrome P450 inducers differs significantly from the rat and human in some cases.     

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.     

Participation of cytochrome P450-2B and -2D isozymes in the demethylenation of methylenedioxymethamphetamine enantiomers by rats

The cytochrome P450 isozymes in rat liver microsomes that catalyze the demethylenation of methylenedioxymethamphetamine enantiomers to the corresponding dihydroxymethamphetamine were characterized. Dihydroxymethamphetamine formation in liver microsomes from male Sprague-Dawley rats exhibited multienzyme kinetics, with Km values in the micromolar/millimolar range. The stereoselectivity [(+)-isomer versus (-)-isomer] varied from 0.78 to 1.94 after pretreatment of the rats with phenobarbital, 3-methylcholanthrene, pregnenolone-16 alpha-carbonitrile, or pyrazole, suggesting that different isozymes participate in the reaction. The low-Km demethylenation was not induced by these compounds and was not inhibited by antibodies raised against CYP2C11. Liver microsomes from female Dark-Agouti rats, a strain genetically deficient in CYP2D1, exhibited demethylenation activities that were 9% of those in microsomes from male Sprague-Dawley rats. The low-Km demethylenation was also inhibited by CYP2D substrates such as sparteine, bufuralol, or desipramine and was almost completely inhibited by antibodies against P450 BTL, which belongs to the CYP2D family. The higg-Km demethylation activity was induced by phenobarbital and pregnenolone-16 alpha-carbonitrile and the activity in both untreated and phenobarbital-induced microsomes was suppressed by anti-CYP2B1 IgG. Experiments with IgG raised against cytochrome b5 suggested that the hemoprotein contributed to the low-Km activity but not the high-Km activity. These results indicate that cytochrome P450 isozymes belonging to the CYP2D subfamily catalyze demethylenation with low Km values and that the reaction occurring with high Km values is likely to be mediated by members of the CYP2B family, but with the possible participation of other phenobarbital-inducible isoforms.     

Lectin-binding patterns in the olfactory epithelium and vomeronasal organ of the common marmoset

The role of different cytochrome P450 isozymes (CYP) in the N-demethylation of chlorimipramine and chlorpromazine has been investigated in liver microsomes from rats by studying the effects of multiple subchronic doses of chlorimipramine, chlorpromazine, phenobarbital and beta-naphthoflavone on the N-demethylation of ethylmorphine, mono-N-demethyl-chlorimipramine and chlorpromazine and on the hydroxylation of aniline. With control microsomes, CYP-dependent metabolism of chlorimipramine and chlorpromazine (100 nmol; 30 min incubation) resulted in the formation of predominantly chlorimipramine (46.5 +/- 4.9 nmol) whereas chlorpromazine (14.1 +/- 0.9 nmol) accounted for only part of the overall metabolism of chlorpromazine. Multiple doses of chlorimipramine increased the capacity of microsomes to N-demethylate ethylmorphine (9.8 +/- 0.73 and 6.08 +/- 0.06 nmol min(-1) (mg protein)(-1) for chlorimipramine-treated and control rats, respectively) as well as itself (4.65 +/- 0.25 and 3.10 +/- 0.33 nmol min(-1) (mg protein)(-1), respectively). Multiple doses of chlorpromazine induced aniline-hydroxylase activity (1.11 +/- 0.16 and 0.94 +/- 0.06 nmol min(-1) (mg protein)(-1) for chlorimipramine and control microsomes, respectively) but the capacity to N-demethylate itself was unchanged. Phenobarbital treatment induced ethylmorphine N-demethylation activity, but did not affect N-demethylation activity, towards chlorimipramine and chlorpromazine. In control microsomes the N-demethylation capacity of chlorimipramine or chlorpromazine (0.160 +/- 0.025 and 0.015 +/- 0.003 nmol min(-1) (mg protein)(-1), respectively) was one order of magnitude lower than that of chlorimipramine or chlorpromazine. The capacity to N-demethylate either chlorimipramine or chlorpromazine was increased by treatment with either phenobarbital or beta-naphthoflavone. In control microsomes, sulphaphenazole markedly inhibited both chlorimipramine-N-mono- and di-N-demethylation, whereas quinidine markedly inhibited the rate of formation of chlorpromazine. The CYP2C and CYP2D subfamilies seem to be involved in the mono N-demethylation of chlorimipramine and chlorpromazine, respectively. Moreover the CYP1A and CYP2B subfamilies might participate in the N-demethylation of either chlorimipramine or chlorpromazine. This could have important implications in the clinical use of chlorimipramine and chlorpromazine in view of the genetic polymorphism of CYP2C and CYP2D isozymes in man.