Flavone modulators of rat hepatic aryl hydrocarbon hydroxylase

The cytochrome P-450-dependent aryl hydrocarbon hydroxylase (AHH) metabolizes a wide variety of endogenous and exogenous compounds to nontoxic metabolites and/or toxic products. We have utilized a series of 18 flavone modulators of AHH to distinguish and probe for different cytochrome P-450 isozymes in liver microsomes from control and 3-methylcholanthrene (MC)-injected rats. some flavones (maackiain acetate, flavanone, mollisacacidin, embinin, sciadopitysin) activated, while most of the tested compounds inhibited the MC-induced type of AHH. Although all flavones either inhibited or had little effect on the constitutive AHH in microsomes from control rats, the degree of inhibition varied greatly: some flavones (chrysin, chrysoeriol, baicalein, maackiain acetate, isoliquiritigenin, sciadopitysin) inhibited over 75% of the AHH. The various flavones we screened may prove useful in defining the cytochrome P-450 content of tissues and for probing the active sites of individual isozymes. The modulatory effects of the naturally occurring flavones assume additional importance in that they may be factors in animal and human responsiveness to cytochrome P-450 substrates.     

Monoclonal antibodies to phenobarbital-induced rat liver cytochrome P-450

Somatic cell hybrids were made between mouse myeloma cells and spleen cells derived from BALB/c female mice immunized with purified phenobarbital-induced rat liver cytochrome P-450 (PB-P-450). Hybridomas were selected in HAT medium, and the monoclonal antibodies (MAbs) produced were screened for binding to the PB-P-450 by radioimmunoassay, for immunoprecipitation of the PB-P-450, and for inhibition of PB-P-450-catalyzed enzyme activity. In two experiments, MAbs of the IgM and IgG1 were produced that bound and, in certain cases, precipitated PB-P-450. None of these MAbs, however, inhibited the PB-P-450-dependent aryl hydrocarbon hydroxylase (AHH) activity. In two other experiments, MAbs to PB-P-450 were produced that bound, precipitated and, in several cases, strongly or completely inhibited the AHH and 7-ethoxycoumarin deethylase (ECD) activities of PB-P-450. These MAbs showed no activity toward the purified 3-methylcholanthrene-induced cytochrome P-450 (MC-P-450), β-naphthoflavone-induced cytochrome P-450 (BNF-P-450) or pregnenolone 16-α-carbonitrile-induced cytochrome P-450 (PCN-P-450) in respect to RIA determined binding, immunoprecipitation, or inhibition of AHH activity. One of the monoclonal antibodies, MAb 2-66-3, inhibited the AHH activity of liver microsomes from PB-treated rats by 43% but did not inhibit the AHH activity of liver microsomes from control, BNF-, or MC-treated rats. The MAb 2-66-3 also inhibited ECD in microsomes from PB-treated rats by 22%. The MAb 2-66-3 showed high cross-reactivity for binding, immunoprecipitation and inhibition of enzyme activity of PB-induced cytochrome P-450 from rabbit liver (PB-P-450_LM2). Two other MAbs, 4-7-1 and 4-29-5, completely inhibited the AHH of the purified PB-P-450. MAbs to different cytochromes P-450 will be of extraordinary usefulness for a variety of studies including phenotyping of individuals, species, and tissues and for the genetic analysis of P-450s as well as for the direct assay, purification, and structure determination of various cytochromes P-450.     

Induction of rat-hepatic microsomal cytochrome P-450 and aryl hydrocarbon hydroxylase by 1,3-benzodioxole derivatives

Several 1,3-benzodioxoles (BD) and related compounds were studied in relation to their ability to generate metabolite complexes with hepatic cytochrome P-450 following administration in vivo to rats. BD derivatives that formed stable metabolite complexes with cytochrome P-450 were considerably more effective inducers of cytochrome P-450 and aryl hydrocarbon (benzo[alpha]pyrene) hydroxylase (AHH) activity than derivatives that did not form stable complexes. Linear regression analysis showed that AHH activity was well correlated (r = 0.980) with total (i.e. complexed plus uncomplexed) cytochrome P-450 content and was not correlated with levels of uncomplexed cytochrome P-450. Aminopyrine N-demethylase (APDM) activity in hepatic microsomes from rats treated with 1,3-benzodioxoles was moderately correlated in a linear relationship with uncomplexed levels of cytochrome P-450 and not with total cytochrome P-450.     

Induction of rat-hepatic microsomal cytochrome P-450 and aryl hydrocarbon hydroxylase by 1,3-benzodioxole derivatives

1. Several 1,3-benzodioxoles (BD) and related compounds were studied in relation to their ability to generate metabolite complexes with hepatic cytochrome P-450 following administration in vivo to rats.

2. BD derivatives that formed stable metabolite complexes with cytochrome P-450 were considerably more effective inducers of cytochrome P-450 and aryl hydrocarbon (benzo[α]pyrene) hydroxylase (AHH) activity than derivatives that did not form stable complexes.

3. Linear regression analysis showed that AHH activity was well correlated (r = 0.980) with total (i.e. complexed plus uncomplexed) cytochrome P-450 content and was not correlated with levels of uncomplexed cytochrome P-450.

4. Aminopyrine N-demethylase (APDM) activity in hepatic microsomes from rats treated with 1,3-benzodioxoles was moderately correlated in a linear relationship with uncomplexed levels of cytochrome P-450 and not with total cytochrome P-450.     

Structure-activity relationships in the interactions of alkoxymethylenedioxybenzene derivatives with rat hepatic microsomal mixed-function oxidases in vivo

Following in vivo administration to rats of equimolar amounts of a series of 4-n-alkoxymethylenedioxybenzene (AMDB) derivatives, hepatic microsomal aryl hydrocarbon hydroxylase (AHH) activities, total cytochrome P-450 levels, and AMDB metabolite-cytochrome P-450 spectral complex (455 nm) formation were well correlated in parabolic relationships with pi, the hydrophobic constant of the n-alkoxy substituent. Each of these parameters increased progressively over control values with increasing carbon chain length of the alkoxy substituent, passed through an optimal value in compounds containing five or six carbon atoms, and subsequently decreased with the higher homologues. AHH activity was highly correlated in linear relationships with total (complexed plus uncomplexed) cytochrome P-450 content and intensity of the 455-nm spectral complex. Aminopyrine N-demethylase activities in microsomes from AMDB-treated rats were not well correlated with cytochrome P-450 levels or spectral complex formation. AMDB metabolite-ferricytochrome P-450 complexes varied considerably in their relative ease of displacement following treatment with 2-n-heptylbenzimidazole, those derived from the n-butoxy to n-hexoxy derivatives being particularly stable toward the displacer. The results are discussed in relation to the possible mechanisms involved in the interactions of methylenedioxyphenyl compounds with cytochrome P-450 and drug oxidation.     

Monoclonal antibody-directed determination of cytochrome P-450 types expressed in a human lymphoblastoid cell line

Cytochrome P-450-dependent aryl hydrocarbon hydroxylase (AHH) and 7-ethoxycoumarin O-deethylase activities of a cloned line of human lymphoblastoid AHH-1 cells are inhibited by a monoclonal antibody (MAb 1-7-1) prepared to a 3-methylcholanthrene-induced rat liver cytochrome P-450. The monoclonal antibody inhibition determined that a single MAb 1-7-1-sensitive type of cytochrome P-450 is responsible for all of AHH expression in both the basal and benz[a]anthracene-induced cells. Partial inhibition by the MAb 1-7-1, however, indicates that at least two forms of cytochrome P-450 catalyze 7-ethoxycoumarin O-deethylase in both the basal and the induced cells, one form of which is identical to the MAb-sensitive cytochrome P-450 responsible for all of the AHH. Thus, a single cloned cell line is capable of expressing two classes of cytochromes P-450, and the observed multiplicity of cytochrome P-450 in animal tissues does not necessarily depend on cell heterogeneity. A sensitive MAb 1-7-1-based radioimmunoassay also directly demonstrates the presence in these cells of a MAb 1-7-1-specific type of cytochrome P-450 as well as its elevation in the induced cells. These MAb-based methods thus can determine the contribution of specific MAb-defined types of cytochromes P-450 to the cellular metabolism of specific xenobiotics.     

Spectral and inhibitory interactions of methylenedioxyphenyl and related compounds with purified isozymes of cytochrome P-450

Spectral and inhibitory interactions of two methylenedioxyphenyl (MDP) compounds (dihydrosafrole (DHS) and 4,5-dichloro-1,2-methylenedioxybenzene (DCMB] and 4-n-butyl dioxolane (BD) were studied in vitro in reconstituted systems incorporating cytochromes P-450b and P-450c, purified respectively from hepatic microsomes of phenobarbital (PB)- and beta-naphthoflavone (beta NF)-treated rats. In NADPH-fortified reconstituted systems containing P-450b, DHS yielded a stable type III spectral complex with peaks at 428 and 458 nm; a complex with a single 456 nm peak was formed in systems containing cytochrome P-450c. DCMB formed unstable 456-458 nm spectral complexes with both isozymes, and BD generated an unstable complex with a single Soret peak near 428 nm with cytochrome P-450b; no spectral interaction occurred between BD and cytochrome P-450c. Carbon monoxide was formed in incubations of DCMB with both isozymes but was not observed with either DHS or BD. Marked selectivity was observed in the ability of the test compounds to inhibit selected mono-oxygenase reactions in the reconstituted systems. Thus, while DHS was an effective inhibitor of cytochrome P-450b-mediated ethoxycoumarin O-deethylase (ECD), it failed to inhibit aldrin epoxidase (AE) in the same system; DCMB and BD inhibited both of these reactions. In reconstituted systems incorporating cytochrome P-450c, DHS and DCMB, but not BD, were effective inhibitors of ethoxyresorufin O-deethylase (ERD) activity but none of the compounds showed any inhibitory activity towards aryl hydrocarbon (benzo[alpha]pyrene)hydrolase (AHH) activity. The results indicate that metabolite complex formation with cytochrome P-450 is not the sole criterion for inhibition of mono-oxygenase activity by MDP and related compounds, and that in some cases type I competitive interactions at the substrate binding sites may be the primary contributing factor.     

Influence of liver disease and environmental factors on hepatic monooxygenase activity in vitro

Summary The effects of liver disease and environmental factors on hepatic microsomal cytochrome P-450 content, NADPH-cytochrome c reductase (reductase) activity and aryl hydrocarbon hydroxylase (AHH) activity have been simultaneously investigated in 70 patients undergoing diagnostic liver biopsy. The activity of reductase was not significantly affected by the presence of liver disease or any of the environmental factors studied. Cytochrome P-450 content decreased with increasing severity of liver disease whereas AHH activity was only significantly reduced in biopsies showing hepatocellular destruction. None of the parameters of monooxygenase activity varied significantly with the age or sex of the patients. Alcohol excess was associated with decreased cytochrome P-450 content and AHH activity and this effect was independent of the histological status of the biopsy. Both high caffeine intake and cigarette smoking increased AHH activity in the absence of any change in cytochrome P-450 content. There was a positive correlation between the number of meat meals eaten per week and cytochrome P-450 content. Chronic treatment with enzyme-inducing anticonvulsants appeared to increase both cytochrome P-450 content and AHH activity. Despite differential effects of liver disease and environmental influences on cytochrome P-450 content and AHH activity there was a highly significant correlation between the two parameters. The results of the present study correlate well with the known effects of disease and environment on drug metabolism in vivo.     

The effects of dietary brussels sprouts and Schizandra chinensis on the xenobiotic-metabolizing enzymes of the rat small intestine

After an initial equilibration period of 7 days on a semi-synthetic basal diet, male Sprague-Dawley rats were fed for 2 wk on either the basal diet (controls), the basal diet containing 5% Schizandra chinensis or 25% Brussels sprouts, or on rat chow. One group of chow-fed rats was pretreated with 20 mg 3-methylcholanthrene (3-MC)/kg body weight, 24 hr before they were killed. Microsomal and cytosolic fractions were prepared from small intestine mucosa. Microsomes were assayed for cytochrome P-450, aryl hydrocarbon hydroxylase (AHH), ethoxycoumarin O-deethylase (ECD) and epoxide hydrolase (EH) activities, and for metabolism of benzo[a]pyrene (BaP), Cytosols were assayed for glutathione S-transferase (GST) activity. The largest increase in intestinal mixed-function oxidase activity over levels in the controls was seen in the 3-MC-treated group. However, EH and GST activities in these animals were not significantly increased. Increases in cytochrome P-450 levels and significant increases in AHH, ECD, EH and GST activities occurred in the rats fed Brussels sprouts. Rats in the S. chinensis group showed inhibition of AHH activity relative to controls, but increased activity of ECD, EH and GST. In the rats fed chow there were significant increases in the activities of all the enzymes assayed except GST. The percentage conversion of BaP to metabolites reflected the results of the AHH assay and the groups were ranked in the following order: 3-MC greater than Brussels sprouts greater than rat chow greater than basal diet greater than S. chinensis. The profile of BaP metabolites showed a larger proportion of the BaP diols and 3-hydroxybenzo[a]pyrene, and a smaller proportion of BaP-4,5-epoxide and the BaP quinones, for the Brussels sprouts- and S. chinensis-fed groups. The significance of these results is discussed in regard to the role of the small intestine as a mediator of toxicity induced by ingested chemicals.     

Spectral and inhibitory interactions of methylenedioxyphenyl and related compounds with purified isozymes of cytochrome P-450

1. Spectral and inhibitory interactions of two methylenedioxyphenyl (MDP) compounds (dihydrosafrole (DHS) and 4,5-dichloro-1,2-methylenedioxybenzene (DCMB)) and 4-n-butyl dioxolane (BD) were studied in vitro in reconstituted systems incorporating cytochromes P-450b and P-450c, purified respectively from hepatic microsomes of phenobarbital (PB)- and β-naphthoflavone (βNF)-treated rats.

2. In NADPH-fortified reconstituted systems containing P-450b, DHS yielded a stable type III spectral complex with peaks at 428 and 458 nm; a complex with a single 456?nm peak was formed in systems containing cytochrome P-450c. DCMB formed unstable 456–458?nm spectral complexes with both isozymes, and BD generated an unstable complex with a single Soret peak near 428?nm with cytochrome P-450b; no spectral interaction occurred between BD and cytochrome P-450c. Carbon monoxide was formed in incubations of DCMB with both isozymes but was not observed with either DHS or BD.

3. Marked selectivity was observed in the ability of the test compounds to inhibit selected mono-oxygenase reactions in the reconstituted systems. Thus, while DHS was an effective inhibitor of cytochrome P-450b-mediated ethoxycoumarin O-deethylase (ECD), it failed to inhibit aldrin epoxidase (AE) in the same system; DCMB and BD inhibited both of these reactions. In reconstituted systems incorporating cytochrome P-450c, DHS and DCMB, but not BD, were effective inhibitors of ethoxyresorufin O-deethylase (ERD) activity but none of the compounds showed any inhibitory activity towards aryl hydrocarbon (benzo[a]pyrene)hydrolase (AHH) activity.

4. The results indicate that metabolite complex formation with cytochrome P-450 is not the sole criterion for inhibition of mono-oxygenase activity by MDF and related compounds, and that in some cases type I competitive interactions at the substrate binding sites may be the primary contributing factor.     

Inhibition of coal fly ash polycyclic aromatic hydrocarbons and metals induced mixed-function oxidase activity in rat lung and liver by vitamin A and citrate

Administration of benzene-soluble fraction (FAE) and benzene-insoluble fraction (FAR) of fly ash for 3 consecutive days to rats significantly raised cytochrome P-450 levels, aryl hydrocarbon hydroxylase (AHH) activity, and glutathione S-transferase activity in liver. This treatment also significantly increased pulmonary AHH and glutathione S-transferase activity. Intratracheal administration of FAR (5 mg/100 g body weight) alone for 6 consecutive days also significantly increased hepatic cytochrome P-450 levels and the activity of glutathione S-transferase. Intragastric administration of retinyl palmitate (5000 IU/100 g body weight), along with intratracheal FAE and FAR administration, significantly reduced P-450 levels, activity of glutathione S-transferase in liver, and activity of AHH and glutathione S-transferase in lung of rats. Intraperitoneal administration of citrate (40 mg/100 g body weight) along with FAR significantly reduced FAR-induced increase in hepatic cytochrome P-450 levels and glutathione S-transferase activity. The activity of AHH was not affected by these treatments.     

Monoclonal antibody characterization of hepatic and extrahepatic cytochrome P-450 activities in rats treated with phenobarbital or methylcholanthrene and fed various cholesterol diets

Monoclonal antibodies (MAb) against 3-methylcholanthrene (MC)- and phenobarbital (PB)-inducible forms of cytochrome P-450 isozyme were used to characterize changes in aryl hydrocarbon hydroxylase (AHH) and ethoxycoumarin O-deethylase (ECDE) activities modulated by dietary cholesterol. Rats were induced by MC or PB, and immunochemical inhibition of AHH and ECDE activities was studied as an indication of changes in cytochrome P-450 isozyme patterns. Feeding of a cholesterol-free diet markedly decreased enzyme activities both in liver and in small intestinal mucosa, and the highest activities were observed after feeding rats a high (2%)-cholesterol diet for one month. As a control, a normal pelleted diet (0.1% cholesterol) was used; in rats fed this diet, intermediate levels of monooxygenase activities were present. Although no diet-dependent change in total AHH and ECDE activities was observed in kidneys and lungs, diet apparently modulated isozyme composition in the lungs, as indicated by a change in the immunochemical inhibition pattern with MAb; no such shift was observed in the kidneys. In liver and intestine, in addition to changes in total activity, isozyme composition was also altered, as indicated by inhibition of the catalytic activities of cytochrome P-450 by MAb. Our data infer that dietary cholesterol can: (i) modulate total monooxygenase activities, especially in the intestine; (ii) change the cytochrome P-450 isozyme composition in liver and intestine; (iii) change isozyme composition without changing overall enzyme activity, e.g. in lungs; and (iv) have no effect in a tissue (e.g. kidney) that lacks constitutionally the P-450 isozyme responsive to cholesterol.     

Drug Metabolism Activities of Isolated Rat Hepatocytes in Monolayer Culture

Abstract: The levels of cytochrome P-450 in hepatocytes cultured as monolayers for 22 hrs in Dulbecco's modified Eagle medium supplemented with serum and insulin was reduced to approximately 40% of initial values of freshly isolated hepatocytes. In correspondance with this the activities of the cytochrome P-450 monooxygenases aryl hydrocarbon (benzo(a)pyrene) hydroxylase (AHH) and ethylmorphine (EM) N-demethylase were reduced to 40 and 22% of their initial activities, respectively. Modifying the culture medium through omission of cysteine and cystine, and adding dexamethazone and delta-amino levulinic acid, increased the content of cytochrome P-450 to 59 % and EM N-demethylase to 46 % of initial values, but was without effect on AHH activity. However, further modifications by adding high concentrations of asparagine and leucine increased AHH activity to 62% of initial values, but did not further enhance the total content of cytochrome P-450 or the EM N-demethylase activity. The activities of cytochrome P-450 reductase, flavin containing monooxygenase, epoxide hydrolase and glutathione S-transferase decreased less (to about 70–80% of initial values) than cytochrome P-450 associated monooxygenase activities, whereas UDP-glucuronyl transferase decreased to about 50% of initial values. In contrast to what was observed regarding cytochrome P-450 and associated monooxygenase activities, modification of the incubation conditions did not affect the non-cytochrome P-450 enzymatic activities.     

Effect of o-benzyl-p-chlorophenol on drug-metabolizing enzymes in rats

o-Benzyl-p-chlorophenol (BCP) is widely used as a broad spectrum disinfectant. Treatment of male Fischer 344 rats with BCP resulted in an increase in cytochrome P-450 content and an accompanying decrease in aryl hydrocarbon hydroxylase (AHH) activity in both liver and kidney microsomes. Several other drug-metabolizing enzymes were not affected by BCP treatment. However, in kidney, BCP induced NADPH-cytochrome c reductase and uridine diphosphate glucuronyl transferase activities and caused a small increase in total cytochrome P-450 content and glutathione concentration. The cytochrome P-450 isozymes induced by BCP were fractionated by high pressure liquid chromatography (HPLC). The HPLC profile following BCP treatment most closely resembled that seen after phenobarbital. Using an immunoblotting procedure and a radioimmunoassay, it was shown that the increase in cytochrome P-450 content in the liver after BCP treatment was, in part, due to an increase in the phenobarbital-inducible isozymes, P-450b + e. In the kidney, the increase in total cytochrome P-450 content after BCP exposure was not due to an increase in P-450b + e. The decrease in AHH activity appeared to be caused by noncompetitive inhibition of constitutive AHH activity by BCP. BCP also inhibited benzphetamine demethylation, although to a lesser extent. The failure to observe an increase in benzphetamine demethylase activity in vivo, despite the induction of P-450b, was probably due to the concomitant induction and inhibition of drug-metabolizing enzymes by BCP.     

Differential effects of 12-O-tetradecanoylphorbol 13-acetate on cytochrome P-450-dependent monooxygenase activities in rat hepatoma cells: induction of P-450I and suppression of P-450II

We have studied the effects of the tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA) on cytochrome P-450-dependent monooxygenase activities in several differentiated and dedifferentiated Reuber rat hepatoma cell lines using aryl hydrocarbon (benzo[a]pyrene) hydroxylase (AHH), ethoxyresorufin O-deethylase (EROD), and aldrin epoxidase (AE) as test systems. The following results were obtained: (1) Exposure of cultures to 400 nM TPA for 18-24 h increased AHH activities in the differentiated lines 2sFou, H41IEC3/G- and Fao as well as in the dedifferentiated line 5L, 1.5-2.5-fold. The phorbol ester did not affect AHH activity in the dedifferentiated line H5. (2) EROD, a marker for P-450I, was induced by the phorbol ester to a similar degree as AHH. (3) A monoclonal antibody directed against P-450I strongly inhibited the AHH activity induced by TPA. (4) The onset of AHH or EROD induction by TPA was much later than that elicited by benz[a]anthracene. (6) In contrast to the induction of AHH and EROD, TPA decreased AE activity, a marker for P-450II, by about 50% in all the cell lines containing this monooxygenase activity. (7) The half-maximum-effect concentration of TPA for inducing or suppressing AHH and AE, respectively, was approximately 20 nM. (8) TPA did not interfere with AHH induction by benz[a]anthracene. However, the phorbol ester moderately decreased AHH induction and markedly suppressed AE induction by dexamethasone. The results indicate that TPA simultaneously induces P-450I and suppresses P-450II forms in rat hepatoma cells. P-450I induction by TPA in these cells did not appear to depend on their status of differentiation. Furthermore, the results suggest that the mechanism of P-450I induction by TPA differs from that elicited by polycyclic aromatic hydrocarbons or glucocorticoids.     

6-Methyl-1,3,8-trichlorodibenzofuran as a 2,3,7,8-tetrachlorodibenzo-p-dioxin antagonist: inhibition of the induction of rat cytochrome P-450 isozymes and related monooxygenase activities

In addition to being one of the most toxic chemicals known, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is the most potent inducer of rat liver microsomal cytochrome P-4501A1 (P-450c). Previous studies have demonstrated that a high affinity, low capacity cytosolic receptor (the Ah receptor) mediates the activity of TCDD to induce cytochrome P-4501A1, which catalyzes benzo[a]pyrene hydroxylation [aryl hydrocarbon hydroxylase (AHH]) and 7-ethoxyresorufin O-dealkylation (EROD). The results of the present study indicate that 6-methyl-1,3,8-trichlorodibenzofuran (MCDF) effectively competes with [3H]TCDD for binding to the Ah receptor in rat liver cytosol. The concentration of MCDF effecting 50% displacement of [3H]TCDD was 4.9 X 10(-8) M, which is approximately 50 times greater than the EC50 for unlabeled TCDD (approximately 1 X 10(-9) M). However, in contrast to TCDD, MCDF was only a weak inducer of AHH and EROD activity in rat hepatoma H-4-II cells in culture. When co-incubated, MCDF diminished in a concentration-dependent manner the ability of TCDD to induce AHH and EROD activity in vitro. Treatment of rats with 20-200 mumol/kg MCDF in vivo had little or no effect on liver microsomal AHH and EROD activity, whereas treatment of rats with 16 nmol/kg TCDD caused a 6- and a 70-fold induction of AHH and EROD activity, respectively. When co-administered, MCDF diminished by approximately 50% the ability of TCDD to induce AHH and EROD activity in vivo. The partial antagonism produced by 50 mumol/kg MCDF could be partially overcome by doubling the dosage of TCDD from 16 to 32 nmol/kg. Immunochemical analysis of rat liver microsomes revealed that treatment of rats with 20-200 mumol/kg MCDF caused little or no induction of cytochromes P-4501A1 and P-4501A2 (P-450d), whereas these isozymes were induced 33- and 5-fold, respectively, in rats treated with 16 nmol/kg TCDD. When co-administered, MCDF diminished by approximately 50% the ability of TCDD to induce cytochrome P-4501A1 in vivo, which established that MCDF was not simply acting as an inhibitor of AHH and EROD activity. MCDF also antagonized the ability of TCDD to induce cytochrome P-4501A2, which suggests that the induction of both cytochromes P-4501A1 and P-4501A2 is regulated by the Ah receptor. These results indicate that MCDF binds with high affinity to the Ah receptor in rat liver cytosol and competitively blocks the binding of TCDD.(ABSTRACT TRUNCATED AT 400 WORDS)     

Biochemical toxicology of argemone oil. I. Effect on hepatic cytochrome P-450 and xenobiotic metabolizing enzymes

The in vivo effect of argemone oil on hepatic xenobiotic metabolizing enzymes was investigated in albino rats following either a single (10 ml kg-1 body wt.) or multiple intraparenteral doses (5 ml kg-1 body wt.) for three days. Animals sacrificed 72 h after a single intraparenteral dose of argemone oil exhibited a significant loss of hepatic cytochrome P-450 (35%) and cytochrome b5 (34%) contents and inhibition of aminopyrine-N-demethylase (APD), aryl hydrocarbon hydroxylase (AHH) and ethoxycoumarin-O-deethylase (ECD) activities (21-39%). Three successive 24-hourly intraparenteral injections of argemone oil followed by sacrificing the animals after 24 h of the last injection, showed a greater degree of inhibition of the content of cytochrome P-450 (58%) and its dependent mixed-function oxidases (35-63%). Also, multiple treatment of argemone oil caused a depletion of endogenous hepatic glutathione (GSH) content (72%) with a concomitant increase in lipid peroxidation (177%) and decrease in glutathione-S-transferase (GST) activity (30%). A significant decrease in relative liver weight (39%) was observed in animals treated with multiple treatment of argemone oil. These results suggest that argemone oil can alter both membrane and cytosolic defences and destabilizes the hepatic cytochrome P-450 dependent mixed-function oxidase system, so that it tips in the direction of autooxidative peroxidation of lipids.     

Effects of inhibitors of RNA and protein synthesis on hepatic microsomal cytochrome P-450 concentration in fasted and fed rats

A number of sex-related differences in the metabolism of drugs, steroids and xenobiotics have been reported in studies on rats. Generally, male rats tend to metabolize these compounds more efficiently than females. In the studies presented here, male and female rats were fasted for 24–48 hr, and the effects of fasting on total hepatic microsomal cytochrome P-450 were examined. Hepatic cytochrome P-450, as determined by CO difference spectra, was increased significantly as a percentage of control in microsomes from fasted female rats when compared to fasted male rats. Cytochrome P-450 concentration increased from 0.57 ± 0.07 nmole/mg protein to 0.99 ± 0.08 nmole/mg protein following a 24-hr fast. In male rats, cytochrome P-450 levels were essentially unaffected by the 24-hr fast. Cytochrome b₅ concentration was not altered by fasting. When female rats were fasted for 24 hr and refed, cytochrome P-450 levels were not significantly different from cytochrome P-450 levels in continuously fed animals. Treatment of fasted female rats with the protein synthesis inhibitor ethionine, or the RNA synthesis inhibitor actinomycin D, prevented the induction of cytochrome P-450 in the fasting animal. Cytochrome P-450 concentration in fed animals was not affected significantly by either inhibitor. Induction of cytochrome P-450 by phenobarbital (PB) and 3-methylcholanthrene (MC) under fed and fasted conditions was also investigated in male and female rats. Xenobiotic-induced cytochrome P-450 concentration was significantly higher in fasted female hepatic microsomes when compared to microsomes from fed female rats. Fasting did not significantly affect xenobiotic-induced cytochrome P-450 in male rats. Our results suggest that fasting in female rats results in an increase in cytochrome P-450 which is dependent upon synthesis of RNA and protein.     

The role of heme oxygenase and aryl hydrocarbon hydroxylase in the protection by cysteamine from acetaminophen hepatotoxicity

Administration of cysteamine to rats depressed hepatic aryl hydrocarbon hydroxylase (AHH) activity, cytochrome P-450, and total heme at 24 hr. Total heme remained decreased at 48 hr when all other parameters returned to control values. A significant 5-fold increase in heme oxygenase activity occurred in rat liver 5 hr after treatment, when AHH activity and total heme were unchanged. Histological examination of liver biopsies from rats treated with cysteamine revealed normal hepatic architecture. The observed effects of cysteamine on hepatic drug-metabolizing enzymes in vivo were not due to cysteamine-induced hepatotoxicity. Our results indicate that cysteamine increases heme oxygenase activity in rat liver, with a subsequent decrease in total heme, AHH activity, and cytochrome P-450 content. The depression of P-450 by cysteamine is likely to be an important mechanism for its protection in acetaminophen overdose. The protection studies illustrate this mechanism. Centrilobular hepatic necrosis and elevation in transaminase activity following a toxic dose of acetaminophen were prevented by treatment with cysteamine. The hepatoprotective effect of cysteamine was evident when acetaminophen was administered 24 hr after cysteamine but did not occur when acetaminophen was administered 5 hr after cysteamine or simultaneously. All groups of rats receiving cysteamine showed decreased mortality compared to the group receiving acetaminophen alone.     

Species differences in stimulation of intestinal and hepatic microsomal mixed-function oxidase enzymes

The effect of intraperitoneal (i.p.) administration of phenobarbital (PB) or 3-methylchol-anthrene (3-MC) on some mixed-function oxidase (MFO) enzymes was studied in small intestine and liver of male rats, mice, guinea pigs and rabbits. PB treatment enhanced intestinal and 7-ethoxycoumarin deethylase activities in the mouse and rat, whereas benzo[a]pyrene hydroxylase (AHH) activity was increased only in the mouse. Ethylmorphine demethylase and aniline hydroxylase activities in small intestine were not stimulated by PB in any species. Administration of 3-MC increased the activity of intestinal AHH in rat, mouse and guinea pig, but intestinal 7-ethoxycoumarin deethylase activity was elevated only in the rat. The guinea pig and mouse intestinal ethoxycoumarin deethylase activity was inhibited by 3-MC treatment. None of the enzymes tested in rabbit intestine was induced by PB or 3-MC. The hepatic activities of ethylmorphine demethylase, aniline hydroxylase, 7-ethoxycoumarin deethylase and AHH, and the cytochrome P-450 content were increased by PB in all species. In contrast, 3-MC enhanced hepatic aniline hydroxylase and AHH activities in rats, mice and guinea pigs, and hepatic 7-ethoxycoumarin deethylase activity in mice and rats. In rabbits, these hepatic enzymes were inhibited by 3-MC pretreatment. The hepatic cytochrome P-450 absorption spectra was shifted to 448 nm in all species. These results suggest that there are differences in induction of intestinal and hepatic MFO enzymes which are influenced by the type of inducing agent, substrate and animal species used.