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PubMedDate not supplied

A comparative study of benzo[a]pyrene hydroxylase and epoxide hydrolase derived from isolated rat hepatic nuclei and microsomes.

Several properties of benzo[a]pyrene hydroxylase (AHH) and epoxide hydrolase (EH) in isolated rat hepatic nuclei were investigated. Comparisons were made with microsomal AHH and EH in order to determine whether the nuclear enzymes were distinct from those of endoplasmic reticulum origin. The ratio of EH to AHH in the nuclei of adult male rats was 4.5 compared to 1.6 in the microsomes. Phenobarbital increased AHH in nuclei from immature males and females. However, the increase were markedly lower than those observed in the microsomes. In contrast, 3-methylcholanthrene increased nuclear AHH to a greater extent than the microsomal enzyme. The age and sex dependence, the inhibition by SKF 525-A, the stimulatory and inhibitory effects of 7,8-benzoflavone and the decrease produced by trans-stilbene oxide administration were the same for both nuclear and microsomal AHH. Phenobarbital, l-alpha-acetyulmethadol, and trans-stilbene oxide increased nuclear EH. The increases were approximately 3-fold lower than observed in the microsomes. EH activity in both nuclei and microsomes was inhibited by 1,2-epoxy-3,3,3-trichloropropane. The data indicate that both the constitutive and induced form(s) of AHH in isolated nuclei and endoplasmic reticulum have a number of common properties. The constitutive form(s) of microsomal and nuclear EH also appear to be similar. However, AHH and EH in isolated nuclei differ from their microsomal counterparts in quantitative inducibility. These differences in inducibility support the suggestion that AHH and EH are located in isolated nuclei rather than resulting from contamination by endoplasmic reticulum.

Animals↗
PubMed1981

Cytosolic and microsomal epoxide hydrolases are immunologically distinguishable from each other in the rat and mouse.

Antibodies raised to homogeneous rat liver microsomal epoxide hydrolase were used to distinguish microsomal epoxide hydrolase from epoxide hydrolase of cytosolic origin in mice and rats. Using double diffusion analysis in agarose gels, we show that anti-rat liver microsomal epoxide hydrolase forms a single precipitin line with solubilized microsomes from rat and mouse liver, but no reaction is seen with the corresponding cytosolic fractions. Rat or mouse microsomal epoxide hydrolase activity (using benzo[a]pyrene 4,5-oxide as substrate) can be completely precipitated out of solubilized preparations by the antibody, which is equipotent against rat and mouse microsomal epoxide hydrolase. No precipitation of cytosolic hydrolase activity (using trans-beta-ethyl styrene oxide as substrate) is seen with any concentration of the antibody tested. Thus, in the case of microsomal epoxide hydrolase, extensive immunological cross-reactivity exists between the two species, rat and mouse. In contrast, no cross-reactivity is detectable between cytosolic and microsomal epoxide hydrolase, even when enzymes from the same species are compared. We conclude that microsomal and cytosolic epoxide hydrolase activities represent distinct and immunologically non-cross-reactive protein species.

Animals↗
PubMed1978

Covalent binding of the carcinogen trichloroethylene to hepatic microsomal proteins and to exogenous DNA in vitro.

Studies were carried out on the in vitro covalent binding of the carcinogen trichloroethylene (TCE) to liver microsomal preparations and to exogenous DNA. The binding of TCE to liver microsomal proteins of male C57BL/6 X C3H/He F1 (hereafter called B6C3F1) hybrid mice, a species and strain susceptible to TCE-induced liver tumorigenesis, was 46% higher than that of [14C]TCE to microsomal proteins from male Osborne-Mendel rats, a species and strain resistant to TCE-induced hepatocellular carcinoma. The in vitro binding of [14C]TCE to liver microsomal proteins was 37% higher for male B6C3F1 mice; female B6C3F1 mice that have been reported to show a lower incidence of TCE-induced hepatocellular carcinoma than do males. Microsomal proteins from the lung, stomach, and kidney of B6C3F1 hybrid mice also metabolized TCE, as indicated by the covalent binding of [14C]TCE to microsomal proteins from these organs. For rats the binding of TCE to liver microsomal proteins of Sprague-Dawley animals was higher than that of Osborne-Mendel and Fischer 344 rats. Incubation of [14C]TCE with salmon sperm DNA in the presence of microsomal preparations from B6C3F1 hybrid mice resulted in covalent binding of [14C]TCE to DNA. This binding was much higher in the presence of microsomal proteins from male rather than female mice. The binding to DNA and protein was enhanced by in vivo phenobarbital administration. The effects of 1,2-epoxy-3,3,3-trichloropropane on the covalent binding of [14C]TCE to protein and DNA were also examined.

Animals↗
PubMed1983

Stimulation of mouse heart and liver microsomal lipid peroxidation by anthracycline anticancer drugs: characterization and effects of reactive oxygen scavengers.

The interaction of adriamycin or several other quinone-containing anthracycline anticancer drugs with mouse heart or liver microsomes resulted in greatly enhanced NADPH oxidation and 5- to 10-fold stimulation of NADPH-dependent, reactive oxygen-mediated microsomal lipid peroxidation. Adriamycin, daunorubicin, desacetyladriamycin and aclacinomycin A all stimulated lipid peroxidation maximally when included at concentrations of 100 to 200 microM, whereas carminomycin and 4-demethoxydaunorubicin produced equivalent enhancement of peroxidation at lower concentrations of 30 to 50 microM. Mitomycin C markedly increased heart microsomal lipid peroxidation, but, interestingly, had little effect when liver microsomes were used. In contrast, 5-imino-daunorubicin and alkylaminoanthracenedione inhibited both heart and liver microsomal lipid peroxidation. NADPH supported anthracycline-stimulated lipid peroxidation, however, an NADPH-generating system provided greater activity. NADH was only about 50% as effective as NADPH and served as cofactor with liver microsomes but not with heart microsomes. Scavengers of reactive oxygen such as superoxide dismutase, reduced glutathione and 1,3-dimethylurea diminished the anthracycline-stimulated heart and liver microsomal lipid peroxidation, indicating that superoxide radical and hydroxyl radical participated in the peroxidation reactions. Ethylenediaminetetraacetic acid was also inhibitory, which suggests trace amounts of iron were also required. Adriamycin, 4-demethoxydaunorubicin, carminomycin, aclacinomycin A and mitomycin C strikingly enhanced peroxidation of unsaturated lipids in mouse lung and kidney microsomes, indicating that anthracycline-mediated enhanced reactive oxyradical generation may have toxic consequences in those organs as well as in the heart. These observations support the proposal that anthracycline-accentuated membrane lipid peroxidation may be relevant to the pathogenesis of anthracycline cardiotoxicity.

Animals↗
PubMed1984

Esophageal microsomal metabolism of N-nitrosomethylbenzylamine in the zinc-deficient rat.

Epidemiological studies in China suggest that dietary zinc deficiency and environmental exposure to N-nitrosamine carcinogens, such as N-nitrosomethylbenzylamine (NMBA), are among the factors associated with an increased incidence of esophageal carcinoma in humans. NMBA belongs to a class of nitrosamines which require activation by the cytochrome P-450-dependent mixed-function oxidases in order to be mutagenic. Rats maintained on a zinc-deficient diet exhibited an increased incidence of NMBA-induced esophageal carcinoma when compared to rats on a control diet. The increased tumor formation was associated with an alteration of the microsomal metabolism of NMBA. Weanling male Sprague-Dawley rats were raised on egg protein diets containing 2.3 ppm zinc (low zinc) or 50 ppm zinc (control zinc). Analysis of tissues revealed a rapid decline in the levels of zinc in serum and esophagi of the animals fed the low-zinc diet. Gastric and hepatic zinc content did not differ significantly between the animals fed the low-zinc diet and the animals fed the control zinc diet, even after 6 weeks. Microsomes were prepared from esophageal mucosa, livers, and forestomachs from weanling animals fed these diets for 3 weeks. The rate of formation of benzaldehyde from NMBA by esophageal mucosal microsomes prepared from the rats fed the low-zinc diet was nearly 10-fold higher than that of the rats fed the control zinc diet [0.230 +/- 0.047 (S.E.) versus 0.024 +/- 0.008 nmol/min/mg microsomal protein; p less than 0.001]. The rate of benzaldehyde formation by hepatic microsomes was 0.062 +/- 0.005 nmol/min/mg microsomal protein in the rats fed the low-zinc diet and 0.042 +/- 0.002 nmol/min/mg microsomal protein in the rats fed the control zinc diet (p less than 0.01). The rate of benzaldehyde formation by forestomach microsomes was not detectable in tissue from rats on either diet. This increased rate of NMBA metabolism by esophageal mucosal microsomes from the zinc-deficient rats may explain the increased incidence of esophageal carcinoma in these animals.

Animals↗
PubMed1978

Red cell damage induced by peroxidized microsomes: the relationship between hemolytic activity and peroxide content.

Rat red blood cells will hemolyze if they are present in vitro in mixtures of rat liver microsomes in which lipid peroxidation has been initiated by NADPH. Recent work from this laboratory indicated that a toxic factor not having radical properties could be generated from the lipids of the peroxidizing microsomes. This toxic factor produced prelytic damage in rat red blood cells. In this communication we show that if Ca(++)-aggregated microsomes are first peroxidized and then sedimented by centrifugation, the resuspended peroxidized microsomes are capable of hemolyzing red cells in the absence of any further microsomal lipid peroxidation. This result shows conclusively that the microsomal lipid peroxidation step can be separated from the attack on red cells leading to frank hemolysis. Furthermore, lipids extracted from the peroxidized microsomes with chloroform-methanol account quantitatively for the degree of hemolysis produced. The active hemolytic material could not be detected in resuspended microsomal centrifugates obtained during the first 10 minutes of NADPH-stimulated microsomal lipid peroxidation. It appeared rapidly after 10 minutes. It was maximal at 20 minutes, and fell to a low level of activity by 60 minutes. Peak hemolytic activity correlated with peak generation of lipid soluble peroxides. High, but less than maximal levels of peroxides appearing at 10 minutes did not cause hemolysis, and high, but less than maximal levels remaining at 60 minutes were only weakly hemolytic. The extracted lipoidal material with hemolytic potency is more reactive than hydrogen peroxide in a peroxide assay.

Animals↗
PubMed1981

Practolol metabolism. III. Irreversible binding of [14C]practolol metabolite(s) to mammalian liver microsomes.

The irreversible binding of [14C]practolol metabolites to mammalian liver microsomes was studied in vitro. Binding occurred by covalent linkage to amino acids of microsomal proteins. The binding reaction was dependent on oxygen, NADPH, reaction time, microsomal protein content and substrate concentration and was inhibited by carbon monoxide, glutathione and albumin. Albumin inhibition resulted in the binding of reactive metabolite(s) to this serum protein. Hamster (Syrian) liver microsomes had the highest rate of binding and 3-hydroxypractolol formation and marmoset the lowest. The reduced binding of [acetyl-14C]practolol compared to [phenyl-14C]practolol to hamster liver microsomes, but not rat, indicated that metabolites with and without the acetyl grouping were bound in this species. The binding of [14C]desacetylpractolol to hamster liver microsomes was greater than the binding of practolol. Sodium fluoride caused both an increase and a decrease of practolol binding to microsomes from pooled hamster livers, whereas there was a consistent increases in binding to microsomes from individual hamster livers. The collective data indicate that electrophilic metabolites of practolol, possibly N-hydroxylation of the acetyl-amino grouping are formed by the microsomal cytochrome P-450-dependent drug metabolizing enzyme system. These metabolites can react irreversibly with tissue and serum proteins which could be sufficiently altered to be recognized as antigens.

Animals↗
PubMed1983

Biotransformation of phenol to hydroquinone and catechol by rat liver microsomes.

Hepatic microsomal biotransformation of phenol to hydroquinone and catechol has been investigated with special reference to the covalent binding to microsomal protein of reactive metabolites formed during microsomal metabolism of phenol. Incubation of [14C]phenol with microsomes from phenobarbital-treated rat liver in the presence of an NADPH-generating system resulted in the formation of hydroquinone and catechol in the ratio of 20:1. No significant formation of 1,2,4-benzenetriol was observed. The biotransformation of phenol to both hydroquinone and catechol required NADPH and molecular oxygen. NADH was much less effective than NADPH as an electron donor and exhibited no significant synergistic effect when used together with NADPH. The biotransformation was inhibited by typical cytochrome P-450 inhibitors such as carbon monoxide, SKF 525-A, and metyrapone. These results indicated the involvement of cytochrome P-450 in the microsomal hydroxylation of phenol at both the ortho- and para-positions. Covalent binding of radioactivity to microsomal protein was observed when [14C]phenol was incubated with rat liver microsomes in the presence of an NADPH-generating system. The covalent binding was also found to require NADPH and molecular oxygen. Inclusion of cytochrome P-450 inhibitors in the incubation mixture resulted in a decrease in the covalent binding. These results indicated that at least one step in the metabolic activation of phenol to the metabolites responsible for covalent binding to microsomal protein was mediated by cytochrome P-450. Inclusion of N-acetylcysteine in the incubation mixture resulted in the complete inhibition of the covalent binding of radioactivity derived from [14C]phenol to microsomal protein, and there was a concomitant formation of N-acetylcysteine adducts of hydroquinone and catechol. These results indicated that hydroquinone and catechol were both precursors to reactive metabolites responsible for the covalent binding.

Animals↗
PubMed1981

Dietary fat and 3-MC induction of hepatic nuclear and microsomal cytochrome P-450.

Hepatic microsomes from male Holtzman albino rats fed a synthetic fat-free diet for 21 days had significantly less cytochrome P-450 and exhibited less binding capacity (delta Amax/mg protein) for aniline and octylamine than microsomes from similar rats fed a diet containing 10% corn oil. Treatment with 3-methylcholanthrene (3-MC) increased the concentrations of cytochrome P-450 (as measured by CO binding spectra) to nearly equal levels in both dietary groups, but the binding of aniline and octylamine to microsomes of rats fed the fat diet exceeded the increase in cytochrome P-450 concentration. Nuclear envelope concentrations of cytochrome P-450 were unaffected by diet. The administration of 3-MC to rats fed a fat-free diet failed to induce nuclear envelope P-450; however, in rats fed the corn oil diet, 3-MC increased this CO binding pigment over twofold. The affinity of nuclear envelope P-450 towards type II substrates was at least equal to that of microsomes, except in control rats fed the fat-free diet. In general, 3-MC pretreatment increased the binding affinity of nuclear envelop and microsomes toward aniline, while increasing affinity for SKF 525-A binding only to nuclear envelope. Molecular weight species in the region known to contain the cytochrome P-450 were quantified by fluorescence gel electrophoresis. Molecular weight species of 48,000 and 53,000 in the nuclear envelope had their counterparts in the microsomal preparation, but a 50,000 dalton component of nuclear envelope was not detected in microsomes. 3-Methylcholanthrene increased only a species with molecular weight 45,500 in the microsomal and nuclear envelope preparations. Rats fed the diet containing corn oil had microsomes with increased capacity for binding CO, but this was not accompanied by increased cytochrome P-450 protein concentration, as measured by quantitative fluorescence gel electrophoresis.

Animals↗
PubMed1982

Chronic ethanol ingestion increases calcium uptake and resistance to molecular disordering by ethanol in liver microsomes.

Calcium uptake and molecular ordering were studied in hepatic microsomes from rats treated chronically with ethanol and were compared with the effects of ethanol in vitro. Calcium uptake was increased by 30% in microsomes from ethanol-fed animals, the Vmax being increased and the Km remaining unchanged. In Arrhenius plots of calcium uptake, the transition temperature in the 25-30 degrees C range was higher by 1.5 degrees C in microsomes from ethanol-fed rats. Chronic ethanol administration resulted in more rigid microsomal membranes, as evidenced by EPR spectra of the spin probe 5-doxylstearic acid. In a plot of the order parameter, S, as a function of temperature, microsomes from ethanol-fed rats showed a discontinuity at a higher temperature than those from controls (29.9 versus 24.2 degrees C). Ethanol, in vitro, inhibited microsomal calcium uptake in both preparations. However, the degree of inhibition was greater in controls. The rate of calcium uptake by microsomes from ethanol-treated animals in the presence of 100 mM ethanol was the same as those from controls in the absence of ethanol. In vitro, increasing concentrations of ethanol progressively decreased molecular ordering of control microsomes, whereas little change was noted in microsomes after chronic ethanol treatment.

Adenosine Triphosphate↗
PubMed1995

N4-hydroxylation of sulfamethoxazole by cytochrome P450 of the cytochrome P4502C subfamily and reduction of sulfamethoxazole hydroxylamine in human and rat hepatic microsomes.

The N4-hydroxylation of sulfamethoxazole (SMX) to its hydroxylamine (SMX-HA) metabolite is the first step in the formation of reactive metabolites responsible for mediating hypersensitivity reactions associated with this compound. In rat hepatic microsomes, the NADPH-dependent oxidation of SMX to SMX-HA was increased 3-fold by pretreatment of rats with phenobarbital. Other cytochrome P450 (CYP) inducers were ineffective. The constitutive and induced SMX N-hydroxylation activities were inhibited by tolbutamide, and induction of SMX-HA activity paralleled the induction of progesterone 21-hydroxylase activity, a marker for CYP2C6. SMX N-hydroxylation in phenobarbital-treated rat hepatic microsomes was inhibited 70% by anti-CYP2C6 antisera. Thus, the N4-hydroxylation of SMX by rat hepatic microsomes was mediated by members of the CYP2C subfamily, probably CYP2C6. In a panel of human microsomes, SMX-HA formation correlated with tolbutamide hydroxylase activity (r = 0.75; p = 0.01); CYP2C9 content (r = 0.79; p < 0.01) and was inhibited 70% by 500 microM tolbutamide and 90% by 100 microM sulfaphenazole. Recombinant CYP2C9 catalyzed the N-hydroxylation of SMX. SMX-HA formation in human hepatic microsomes was therefore mediated predominantly by CYP2C9. CYP-mediated reduction of SMX-HA to SMX was markedly induced in dexamethasone and phenobarbital-treated rat hepatic microsomes, and was attributed to CYP3A and CYP2B forms. In uninduced rat and human hepatic microsomes, SMX-HA reduction was mediated predominantly by an NADH-dependent microsomal hydroxylamine reductase under aerobic conditions. Under anaerobic conditions, troleandomycin at > or = 1 microM inhibited the reduction of SMX-HA in human hepatic microsomes by 45%, whereas sulfaphenazole had no effect.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1975

Multiplicity of cytochrome P-450 hemoproteins in rat liver microsomes. Preparation and specificity of an antibody to the hemoprotein induced by phenobarbital.

Rat liver microsomes have previously been shown to contain hemoproteins having molecular weights of 53,000, 50,000, and 45,000. The 45,000-dalton hemoprotein, which is induced in rat liver microsomes by pretreatment of animals with phenobarbital, is resistant to proteolysis by trypsin. This characteristic was used to purify it from the other microsomal hemoproteins. In the procedure used, a sodium cholate-solubilized microsomal fraction from phenobarbital-pretreated rats was treated with trypsin and chromatographed on Sephadex G-100 to separate the hemoprotein from preolytic degradation products. The hemoprotein thus isolated was homogenous on the basis of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and was identified spectrally as a cytochrome P-420 hemoprotein. This hemoprotein was free of cytochrome b5 and NADPH-cytochrome c reductase activity. Antibody prepared against the protease-treated cytochrome P-420 hemoprotein will not cross-react with the 53,000- and 50,000-dalton hemoproteins. This was assessed by three criteria. First, immunoprecipitation studies were conducted with detergent-solubilized partially purified cytochrome P-450 preparations isolated from the liver microsomes of control and phenobarbital- and 3-methylcholathrene-retreated rats. The antibody immunoprecipitated only the 45,000-dalton hemoprotein from these partially purified cytochrome P-450 preparations, each of which contains all three hemoproteins. Second, the antibody demonstrated specificity with regard to the microsomal hydroxylation reactions it would inhibit in a reconstituted hydroxylation system containing partially purified cytochrome P-450 (448) fractions isolated from the liver microsomes from phenobarbital- or 3-methylcholathrene-pretreated rats. The antibody would inhibit benzphetamine-N-demethylation catalyzed by both cytochrome P-450 fractions but would not inhibit benzpyrene hydroxylation catalyzed by either. Third, agglutination and complement fixation assays were performed to assess the binding of the antibody to liver microsomes isolated from control and phenobarbital- or 3- methylcholanthrene-pretreated rats. These studies demonstrated that the antibody binds preferentially to the liver microsomes isolated from phenobarbital-pretreated rats, in which the 45,000-dalton hemoprotein has been shown to be induced. It is hypothesized that there are very significant structural and catalytic differences among the cytochrome P-450 hemoproteins.

Agglutination Tests↗
PubMed1975

Influence of cholesterol feeding on liver microsomal metabolism of steroids and bile acids in conventional and germ-free rats.

The present investigation has aimed at defining the factor responsible for the differences in microsomal metabolism of steroids between germ-free and conventional rats. Cholesterol, cholic acid, taurocholic acid, and chenodeoxycholic acid were fed to conventional and germ-free male rats and the effects on liver microsomal metabolism of 4-[4-14C]androstene-3,17-dione, 5alpha-[4-14C]androstane-3alpha,17beta-diol, [4-14C]-cholesterol, 7alpha-hydroxy-4-[6beta-3H]cholesten-3-one, and [24-14C]lithocholic acid were studied. The most consistent effects were found with dietary cholesterol that stimulated the activities of several of the hydroxylases active on 4-androstene-3,17-dione and 5alpha-androstane-3alpha,17beta-diol and that decreased the 5alpha reduction of 4-androstene-3,17-dione, increased the 7alpha hydroxylation of cholesterol, decreased the 12alpha hydroxylation of 7alpha-hydroxy-4-cholesten-3-one, and increased by 6beta hydroxylation of lithocholic acid. These effects of cholesterol feeding on the microsomal metabolism of steroids in conventional rats made the pattern of microsomal enzyme activities resemble that characteristic of germ-free rats. Cholesterol feeding led to a pronounced increase in the intestinal concentration of beta-muricholic acid in conventional rats. Furthermore, cholesterol feeding to conventional animals led to an intestinal ratio of chenodeoxycholic acid (including its metabolites alpha- and beta-muricholic acid and hyodeoxycholic acid) to cholic acid (including deoxycholic acid) that was almost identical to that in germ-free rats. Conventionalization of germ-free rats for a period of up to 56 days led only to a partial normalization of the liver microsomal metabolism of 5alpha-[4-14C]androstane-3alpha, 17beta-diol and 7alpha-hydroxy-4-[6beta-3H]cholesten-3-one and of the liver microsomal concentration of cytochrome P-450. The concentration of cholesterol was higher in both total liver homogenate and liver microsomal fraction of germ-free rats than in corresponding preparations from conventional rats. In conclusion, it is suggested that cholesterol is one of the factors responsible for the different microsomal metabolism of steroids in germ-free and conventional rats. It is also suggested that cholesterol may play a role as regulator of microsomal enzyme activities.

Androstane-3,17-diol↗
PubMed1993

Differential activation of cyclophosphamide and ifosphamide by cytochromes P-450 2B and 3A in human liver microsomes.

The present study identifies the specific human cytochrome P-450 (CYP) enzymes involved in hydroxylation leading to activation of the anticancer drug cyclophosphamide and its isomeric analogue, ifosphamide. Substantial interindividual variation (4-9-fold) was observed in the hydroxylation of these oxazaphosphorines by a panel of 12 human liver microsomes, and a significant correlation was obtained between these 2 activities (r = 0.85, P < 0.001). Enzyme kinetic analyses revealed that human liver microsomal cyclophosphamide 4-hydroxylation and ifosphamide 4-hydroxylation are best described by a 2-component Michaelis-Menten model composed of both low Km and high Km P-450 4-hydroxylases. To ascertain whether one or more human P-450 enzymes are catalytically competent in activating these oxazaphosphorines, microsomal fractions prepared from a panel of human B-lymphoblastoid cell lines stably transformed with individual P-450 complementary DNAs were assayed in vitro for oxazaphosphorine activation. Expressed CYP2A6, -2B6, -2C8, -2C9, and -3A4 were catalytically competent in hydroxylating cyclophosphamide and ifosphamide. Whereas CYP2C8 and CYP2C9 have the characteristics of low Km oxazaphosphorine 4-hydroxylases, CYP2A6, -2B6, and -3A4 are high Km forms. In contrast, CYP1A1, -1A2, -2D6, and -2E1 did not produce detectable activities. Furthermore, growth of cultured CYP2A6- and CYP2B6-expressing B-lymphoblastoid cells, but not of CYP-negative control cells, was inhibited by cyclophosphamide and ifosphamide as a consequence of prodrug activation to cytotoxic metabolites. Experiments with P-450 form-selective chemical inhibitors and inhibitory anti-P-450 antibodies were then performed to determine the contributions of individual P-450s to the activation of these drugs in human liver microsomes. Orphenadrine (a CYP2B6 inhibitor) and anti-CYP2B IgG inhibited microsomal cyclophosphamide hydroxylation to a greater extent than ifosphamide hydroxylation, consistent with the 8-fold higher activity of complementary DNA-expressed CYP2B6 with cyclophosphamide. In contrast, troleandomycin, a selective inhibitor of CYP3A3 and -3A4, and anti-CYP3A IgG substantially inhibited microsomal ifosphamide hydroxylation but had little or no effect on microsomal cyclophosphamide hydroxylation. By contrast, the CYP2D6-selective inhibitor quinidine did not affect either microsomal activity, while anti-CYP2A antibodies had only a modest inhibitory effect. Overall, the present study establishes that liver microsomal CYP2B and CYP3A preferentially catalyze cyclophosphamide and ifosphamide 4-hydroxylation, respectively, suggesting that liver P-450-inducing agents targeted at these enzymes might be used in cancer patients to enhance drug activation and therapeutic efficacy.

Antibodies↗
PubMed1994

Generation of reactive oxygen intermediates by human liver microsomes in the presence of NADPH or NADH.

Studies were carried out to evaluate the ability of human liver microsomes to generate superoxide radical and hydrogen peroxide, and to interact with ferric chelates to produce more potent oxidizing species such as the hydroxyl radical (.OH). In the presence of either NADPH or NADH, human liver microsomes produced superoxide and H2O2 at rates about 20 to 30% of that found with rat liver microsomes. These lower rates are caused, in part, by the 3-fold lower content of total cytochrome P450 in the human liver microsomes. NADH-dependent rates were about 25 to 30% of the NADPH-dependent rates. In the presence of appropriate ferric complexes, human liver microsomes generated .OH, promoted cleavage of vicinal diols, and underwent lipid peroxidation. In contrast to results with rat liver microsomes, NADH-dependent rates of .OH production or lipid peroxidation by human liver microsomes were similar to the NADPH-dependent rates. Human liver microsomes reduced ferric ATP or ferric EDTA at nearly comparable rates with NADPH and NADH. Sensitivity of the various iron-dependent reactions to antioxidants was found to be characteristic of the particular system. These results suggest the possibility that human liver microsomes are an important source of reactive oxygen intermediates, especially under conditions of increased NADH or NADPH availability and elevated iron concentration.

Animals↗
PubMed1995

Microsomal metabolism of the 5-lipoxygenase inhibitors L-746,530 and L-739,010 to reactive intermediates that covalently bind to protein: the role of the 6,8-dioxabicyclo[3.2.1]octanyl moiety.

Hepatic microsomes from different species were used to study the oxidative metabolism of L-746,530 and L-739,010, two potent and specific 5-lipoxygenase inhibitors. HPLC analysis of the incubates obtained from the microsomal incubations of L-739,010 and L-746,530 showed only traces of metabolites. However, recovery of the starting material in the supernatant was less than quantitative in all of the species studied (approximately 90% in rat, approximately 70% in the dexamethasone-induced rat, approximately 70-90% in humans, and approximately 60% in the rhesus monkey for both compounds). The recovery of the starting material was found to be time- and NADPH-dependent, suggesting that metabolite(s) were formed and reacting with the microsomal proteins. Evidence that the cytochrome P4503A (CYP3A) contributed to the formation of the reactive metabolite(s) was shown by the low recovery of material that was observed upon incubation with microsomes obtained from dexamethasone-treated rats (a CYP3A inducer), compared with microsomes obtained from untreated rats. Also, the recovery of material was improved when troleandomycin, a CYP3A inhibitor, was added to rhesus monkey microsomal incubations (25% more parent compound detected in the supernatant with 100 microM of troleandomycin). Using radiolabeled L-746,530 and gel electrophoresis analysis, it was confirmed that radiolabeled material was covalently bound to the microsomal protein. Incubations of L-739,010 and L-746,530 in the presence of semicarbazide resulted, in both cases, in the formation of two adducts. Using a combination of NMR, liquid secondary-ion MS, and UV techniques, these adducts were identified as isomers of an oxidized metabolite that had been trapped by semicarbazide. The site of oxidation was determined to be on the dioxabicyclo moiety. The importance of this moiety in the formation of reactive metabolite(s) was verified by incubating analogs of the 5-lipoxygenase inhibitors that contained blocking methyl groups at the proposed site of oxidation on the bicyclo moiety. Incubations of these gemdimethyl analogs of L-746,530 and L-739,010 with microsomes from different species resulted in significantly improved recovery of the starting material (approximately 94% in the rat, 85% in the dexamethasone-induced rat, 95% in humans, and 85% in the rhesus monkey for both compounds) and significantly less radioactive binding to the microsomal protein.

Animals↗
PubMed1996

Effect of 3-methylcholanthrene on bunitrolol metabolism. Kinetics and immunological studies on 4-hydroxylation of bunitrolol catalyzed by two species of cytochromes P450 in rat liver microsomes.

Effect of the induction of cytochrome P4501A1 with 3-methylcholanthrene (3-MC) treatment on kinetics of bunitrolol (BTL) 4-hydroxylase activity of rat liver microsomes was investigated. The relationship between the rate and BTL concentration showed monophasic kinetics (KM = 0.74 +/- 0.13 microM) when microsomes from untreated rats were used, whereas microsomes from 3-MC-treated rats showed biphasic kinetics (KM1 = 0.76 +/- 0.13, KM2 = 646 +/- 16 microM). Anti-cytochrome P450 (P450) BTL (P4502D subfamily) antiserum inhibited the reaction > 90% when low concentrations (approximately 10 microM) of BTL were used in both microsomes. However, at high BTL concentrations (approximately 2,000 microM), the inhibition was only up to a half of control in microsomes from 3-MC-treated rats, whereas in microsomes from untreated rats, the rates were suppressed > 90%. Kinetics observed in microsomes from 3-MC-treated rats changed to nearly monophasic, with a KM value corresponding to KM2. Anti-P4501A1 IgG, on the other hand, hardly inhibited the reaction conducted by liver microsomes from 3-MC-treated rats when substrate concentrations were low, whereas at higher concentrations, it inhibited up to 50%, resulting in a monophasic kinetics with a KM value corresponding to KM1. These results clearly indicate that the biphasicity of kinetics in BTL metabolism in liver microsomes from 3-MC-treated rats is caused by the involvement of two P450 species: P450 BTL and P4501A1 in the reaction. This is the first direct evidence to the theoretical hypothesis that the biphasic kinetics of the enzyme reaction is caused by the involvement of at least two enzymes with different kinetic parameters.

Animals↗
PubMed1996

Inhibition and kinetics of cytochrome P4503A activity in microsomes from rat, human, and cdna-expressed human cytochrome P450.

Midazolam (MDZ) is metabolized in human liver microsomes by the cytochrome P450 (CYP) 3A subfamily to 1'-hydroxy (1'-OH) and 4-hydroxy (4-OH) metabolites. MDZ is metabolized in the rat primarily to 4-OH MDZ, 1'-OH MDZ, and 1',4-dihydroxy (1',4-diOH) MDZ. The kinetics of 4-OH and 1'-OH metabolite formation were determined using hepatic microsomes from control, Ro 23-7637 and dexamethasone-treated male rats. KM values for the major metabolite, 4-OH MDZ, were 24.5, 43.1, and 32.8 microM, and the corresponding Vmax values were 5.9, 28.9, and 13 nmol/mg/min for the control, DEX, and Ro 23-7637-treated animals, respectively KM values for 1'-hydroxylation of MDZ (the major metabolite) after incubation with human liver microsomes from three individuals were 5.57, 2.50, and 3.56 microM, and the corresponding Vmax values were 4.38, 0.49, and 0.19 nmol/mg/min, respectively. In parallel studies using cDNA-expressed human CYP3A4 microsomes, the KM for 1'-OH formation was 1.56 microM, and the corresponding Vmax was 0.16 nmol/mg/min. MDZ was not metabolized by cDNA-expressed human CYP2D6, CYP2E1, or CYP1A2, thus confirming that these isoforms were not responsible for its biotransformation. The formation of 1',4-diOH metabolite in rat and 1'-OH formation in cDNA-expressed human CYP3A4 microsomes showed a decrease in velocity at high substrate concentrations. Inhibition studies showed that MDZ hydroxylation was strongly inhibited by ketoconazole and Ro 23-7637 in rat, human, and cDNA-expressed human CYP3A4 microsomes. alpha-Naphthoflavone stimulated 1'-OH metabolite formation in human and cDNA-expressed human CYP3A4 microsomes at low concentration (10 microM). Naringenin, a flavonoid present in grapefruit juice, also inhibited MDZ metabolism in human liver microsomes. Immunoinhibition studies revealed that polyclonal anti-rat CYP3A2 antibody inhibited MDZ metabolism 80-90% in rat, human, and cDNA-expressed human CYP3A4 microsomes, thus suggesting that members of the CYP3A4 subfamily were involved in the metabolism.

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