Pentoxyresorufin O-dealkylase Research Articles

Opposite Effects of 2,2′,4,4′,5,5′-Hexachlorobiphenyl and 2,3,7,8-Tetrachlorodibenzo-p-dioxin on the Antibody Response to Sheep Erythrocytes in Mice

The effect that cotreatment with 2,2',4,4',5,5'-hexachlorobiphenyl (PCB153) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) has on the antibody plaque-forming cell (PFC) response to sheep red blood cells (SRBCs) was determined in female B6C3F1 mice. Groups of eight mice per group were given a single oral dose of PCB153 alone (0, 3.58, 35.8, or 358 mg/kg), TCDD alone (0, 0.1, 1, or 10 micrograms/kg), and all possible combinations of these doses in corn oil 7 days prior to immunization with SRBCs. Separate groups of mice were given phenobarbital (PB) parenterally by intraperitoneal injection at a dosage of 160 mg/kg/day for 3 days. Four days after intravenous immunization, body, spleen, thymus, and liver weights and the PFC response to SRBCs were determined. Exposure to TCDD alone resulted in a dose-related suppression of the PFC response, with significant suppression at 1 and 10 micrograms/kg. In contrast, exposure to PCB153 alone resulted in the enhancement of the PFC response at 358 mg/kg. Combined exposure to 358 mg/ kg PCB153 and TCDD resulted in no change (PCB153 + 0.1 microgram/ kg TCDD) or suppression (PCB153 + 1 or 10 micrograms/kg TCDD) of the PFC response relative to PCB153 alone; however, the PFC response was enhanced (PCB153 + 0.1 microgram/kg TCDD), unaffected (PCB153 + 1 microgram/kg TCDD), or suppressed (PCB153 + 10 micrograms/kg TCDD) relative to corn oil controls. PB did not affect the PFC response to SRBCs, despite a 13-fold induction of hepatic pentoxyresorufin O-dealkylase (PROD) activity. These results suggest that PCB153 enhancement of the PFC response is not related to PROD induction and that it acts as a functional antagonist rather than an aryl hydrocarbon receptor or dispositional antagonist. By enhancing the PFC response to SRBCs, PCB153 raises the "setpoint" response level. Consequently, cotreatment with an immunosuppressive dose of TCDD fails to suppress the PFC response relative to corn oil controls, while clearly suppressing it relative to the appropriate control, PCB153 alone.

Induction of rat hepatic cytochrome P4501A and P4502B by the methoxsalen.

The effect of methoxsalen, the inhibitor of the hepatic mixed function oxidase, on the expression of liver cytochrome P450s was examined in rats. Administration of methoxsalen to rats significantly increased the hepatic content of P450 and activities of microsomal ethoxyresorufin O-deethylase (EROD), methoxyresorufin O-demethylase (MROD), pentoxyresorufin O-dealkylase (PROD), a representative activity of P4501A1, P4501A2 and P4502B1/2, respectively, in a dose-dependent manner. In contrast, there was no effect on the P4502E1 catalyzed aniline hydroxylase. In the time-course experiment, methoxsalen exhibited a biphasic effect on EROD, MROD, and PROD activities, an initial inhibitory phase was followed by a phase of induction following a single treatment. Immunoblot analysis using anti-rat liver P4501A and P4502B revealed that increase in the apoprotein levels of P4501A1/2 and P4502B1/2 by methoxsalen was consistent with those in enzyme activity levels. Levels of mRNA of P4501A1/2 and P4502B1/2 were also increased by methoxsalen in Northern blot analysis. These results demonstrated that methoxsalen acts as an inducer of the hepatic microsomal mixed function oxidase; selective induction of P4501A and P4502B families involved increases in mRNA levels.

Differential induction of isozymes of drug-metabolizing enzymes by butylated hydroxytoluene in mice and Chinese hamsters

Induction of isozymes of drug-metabolizing enzymes by butylated hydroxytoluene (BHT) was studied in the male ddY mouse and Chinese hamster. In mice given 0.05% BHT in the diet for 14 days cytochrome P-450 contents and the activities of uridine diphosphate-glucuronyl transferase (UDP-GT) and pentoxyresorufin O-dealkylase were markedly increased, while in those fed 0.15% BHT testoterone 6α-, 16α- and 16β-hydroxylases were greatly increased, while indicated induction of cytochrome P-450 isozymes of the CYP2B family. Western blot analysis also showed and increased level of the isozyme immunorelated to rat CYP2B2 by BHT feeding. The activities of aryl hydrocarbon hydroxylase, ethoxycoumarin O-deethylase (ECOD), erythromycin N-demethylase and glutathione S-transferase (GST) remained unchanged. In Chinese hamster given 0.05 and 0.15% BHT in the diet for 14 days activities if ECOD and GST were induced, but cytochrome P-450 contents and the activities of other enzymes were unaffected. Testosterone 15α-hydroxylase was induced in hamster fed 0.15% BHT. These findings suggested that BHT administration in the hamster induced CYP2A2-type isozyme, which was confirmed by Western blot analysis. BHT treatment enhanced activation of benzo[ a] pyrene (B[ a]P) as determined by the mutagenicity test, especially in Chinese hamster. The results suggest that BHT treatment induces specific isozymes of drug-metabolizing enzymes and might modify the expression of toxicities of other chemicals.

Genetic and non-genetic biomarkers related to carcinogenesis in evaluating toxicological risk from Fenarimol

A multibiomarker approach based on the study of toxicity mechanisms at both genetic and metabolic levels has been applied to Fenarimol. With regard to genotoxicity, particular attention was given to assays for chromosomal aberration and micronuclei; clastogenic potential was assessed in human peripheral blood lymphocytes in vitro, while the induction of micronuclei was studied in male CD1 mouse bone marrow polychromatic erythrocytes (PCE). Fenarimol did not induce any significant increase in chromosomal aberrations in vitro, either in the presence or absence of rat liver S9 fraction. In contrast, a significant dose-related increase in micronucleated PCEs, up to 4-fold above the control level at a single dose of 75 mg/kg b.w., was observed 24 h after treatment. Using selective biochemical markers of effect Fenarimol was found to induce CYP 2B1 isoforms in liver, kidney and lung microsomes of Swiss Albino CD1 male and female mice, as shown by the significant increase in specific 2B1-probe pentoxyresorufin O-dealkylase activity. On the contrary, CYP 3A, probed by N-demethylation of aminopyrine, were only induced in the liver. Results were corroborated by by means of Western immunoblotting using rabbit polyclonal antibodies anti-CYP 2B1 and 3A. Northern blotting analysis with CYP 2B1 and 3A cDNA biotinylated probes showed that the expression of such isoforms is regulated at mRNA level. Taken as a whole, these data indicate the possible (mutagenic) cotoxic/cocarcinogenic and promoting potential of this fungicide.

Distinct responses of mouse hepatic CYP enzymes to corn oil and peroxisome proliferators

We studied the response of male DBA/2N mouse liver monooxygenases to acute (one-day) and subacute (7-day) exposure to clofibrate, gemfibrozil, and com oil. The day following a single treatment with clofibrate (200 mg/kg), coumarin 7-hydroxylase (COH) activity decreased significantly (by 70%) with a concomitant decrease in the CYP2A4/5 protein and mRNA levels. The 7-day treatment schedule also decreased COH activity but only by 30%, though the levels of CYP2A4/5 protein and mRNA were still low. Treatment 1 and 7-day with clofibrate decreased 7-pentoxyresorufin O-dealkylase (PROD) activity by 40%. No changes were seen in testosterone 15α-hydroxylase (TISαOH) activity after 1 day of treatment with clofibrate but, after 7 days, it was decreased by 50%. Clofibrate treatment had no significant effects on testosterone 7α-hydroxylase (T7αOH), 7-ethoxyresorufin O-deethylase (EROD), or benzphetamine N-demethylase (BZDM) activities. Gemfibrozil (200 mg/kg) did not alter COH activity or CYP2A4/5 protein content after a single treatment, but a slight decrease was seen in the mRNA level. Treatment for 7 days significantly increased (2.5-fold) the activity and mRNA content but the amount of protein remained unchanged. Gemfibrozil enhanced (2–2.7-fold) PROD and EROD (2–2.5-fold) activities by both treatments, whereas T15αOH, T7αOH, or BZDM activities were not significantly affected. Treatment with corn oil for 7 days significantly decreased (65%) COH activity and CYP2A4/5 protein and mRNA levels. PROD (55%) and T15αOH (65%) activities were significantly decreased even after a single dose although injection for 7 days had no effect. Neither of the corn oil schedules had any marked effect on T7αOH, EROD, or BZDM activities. These results demonstrate: 1. a decrease in the expression of CYP2A4/5 gene by clofibrate and corn oil; 2. substantial differences within the CYP2A subfamily in their responses to corn oil, clofibrate, and gemfibrozil; and 3. distinct responses of other xenobiotic metabolizing CYP subfamily enzymes to clofibrate and gemfibrozil.

Induction effects of polychlorinated biphenyls, polycyclic aromatic hydrocarbons and other widespread aromatic environmental pollutants on microsomal monooxygenase activities in chick embryo liver.

Cytochrome P450-dependent 7-ethoxyresorufin O-deethylase (EROD), 7-pentoxyresorufin O-dealkylase (PROD) and 7-ethoxycoumarin O-deethylase (ECOD) activities in 14-day-old chick embryo livers were determined 24 h after pretreatment with selected widespread aromatic environmental contaminants, including polychlorinated biphenyls (PCBs), polycyclic aromatic hydrocarbons (PAHs), hexachlorobenzene, and dialkylesters of phthalic acid, and compared with the inducing potencies of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and the coplanar and mono-o-chlorinated PCBs. The effects of other model inducers, i.e. phenobarbital and pyrazole, were also examined. Specificity of EROD induction was estimated with regard to contaminants frequently present in environmental samples and dose-response curves for EROD induction were determined. A strong induction (comparable with that by mono-o-chlorinated biphenyl treatment) by dibenzo[a,h]anthracene, benzo[k]fluoranthene or benzo[b]fluoranthene was found, but the maximal level of EROD activity inducible by TCDD was not achieved, partly due to the high toxicity of the tested PAHs. 3-Methylcholanthrene showed moderate inducing potencies; benz[a]anthracene, benzo[a]pyrene, chrysene and 2,2',3,4,4',5'-hexachlorobiphenyl appeared to be weak inducers. Other PAHs and PCBs tested, as well as hexachlorobenzene, dialkyl phthalates, phenobarbital and pyrazole had no marked effects on the EROD level. ECOD activities were increased non-specifically by TCDD, 3-methylcholanthrene, hexachlorobenzene and phenobarbital. A significant enhancement of PROD activity by TCDD and related inducers was observed, while phenobarbital induced the PROD activity only weakly; SDS-PAGE analysis showed that the chicken phenobarbital-inducible cytochromes P4502H with apparent molecular weights 50 kDa were not markedly induced by the TCDD- or 3-methylcholanthrene treatments. Inhibition of EROD and PROD by 9-hydroxyellipticine, a specific inhibitor of rat hepatic cytochrome P4501A1, revealed that PROD induction by TCDD and other P4501A-inducers was probably a result of a broader substrate specificity of chick embryo P4501A. Measurement of EROD activities in chick embryo liver is highly sensitive, specific and suitable for the determination of TCDD-type toxicity of new drugs, agrochemicals, and industrial pollutants.

Regenerative changes in hepatic morphology and enhanced expression of CYP2B10 and CYP3A during daily administration of cocaine.

The effects of daily cocaine administration for up to 14 days were studied in terms of hepatic morphology and the expression of cytochrome P450 (CYP) enzymes in the mouse liver. Daily intraperitoneal doses of 60 mg/kg of cocaine for 3 days induced severe hepatocellular necrosis in the pericentral zone and decreased activities of CYP1A2, CYP2A4/5, and CYP2Cx. The microsomal CYP2B10 protein content was increased by about 2.5-fold, but 2B10-dependent 7-pentoxyresorufin O-dealkylase (PROD) activity was barely detectable. Five or seven daily cocaine doses caused prominent pericentral inflammation and a significant (up to 14-fold) increase in the microsomal protein content and PROD activity. An increase in microsomal CYP3A was also evident, but CYP2A5 and CYP1A2 still remained at a low level. Immunohistochemical examination showed that the relative induction of CYP2B10 and CYP3A after treatment with cocaine was strongest in perivenous hepatocytes. Immunoinhibition experiments showed that CYP2B10 accounted for catalysis of only 15% to 20% of the enhanced microsomal cocaine N-demethylase (CNDM) activity, which correlated well with immunoreactive 3A protein, and could be blocked 70% to 90% by triacetyloleandomycin. After 10 or 14 daily doses of cocaine, regenerative changes with hepatocyte ballooning were observed, coinciding with increases in CYP1A2, CYP2A4/5, and CYP3A. These results suggest the following: (1) cocaine enhances its own cytochrome P450-dependent metabolism; (2) increased production of norcocaine in microsomes is catalyzed mainly by CYP3A enzyme(s), whereas 2B10, although markedly increased by cocaine treatment, has only a minor role in cocaine hepatotoxicity; and (3) despite increased microsomal CNDM activity, cocaine-induced liver injury is reversible in mice.

Regenerative changes in hepatic morphology and enhanced expression of CYP2B10 and CYP3A during daily administration of cocaine

The effects of daily cocaine administration for up to 14 days were studied in terms of hepatic morphology and the expression of cytochrome P450 (CYP) enzymes in the mouse liver. Daily intraperitoneal doses of 60 mg/kg of cocaine for 3 days induced severe hepatocellular necrosis in the pericentral zone and decreased activities of CYP1A2, CYP2A4/5, and CYP2Cx. The microsomal CYP2B10 protein content was increased by about 2.5-fold, but 2B10-dependent 7-pentoxyresorufin O-dealkylase (PROD) activity was barely detectable. Five or seven daily cocaine doses caused prominent pericentral inflammation and a significant (up to 14-fold) increase in the microsomal protein content and PROD activity. An increase in microsomal CYP3A was also evident, but CYP2A5 and CYP1A2 still remained at a low level. Immunohistochemical examination showed that the relative induction of CYP2B10 and CYP3A after treatment with cocaine was strongest in perivenous hepatocytes. Immunoinhibition experiments showed that CYP2B10 accounted for catalysis of only 15% to 20% of the enhanced microsomal cocaine N-demethylase (CNDM) activity, which correlated well with immunoreactive 3A protein, and could be blocked 70% to 90% by triacetyloleandomycin. After 10 or 14 daily doses of cocaine, regenerative changes with hepatocyte ballooning were observed, coinciding with increases in CYP1A2, CYP2A4/5, and CYP3A. These results suggest the following: (1) cocaine enhances its own cytochrome P450-dependent metabolism; (2) increased production of norcocaine in microsomes is catalyzed mainly by CYP3A enzyme(s), whereas 2B10, although markedly increased by cocaine treatment, has only a minor role in cocaine hepatotoxicity; and (3) despite increased microsomal CNDM activity, cocaine-induced liver injury is reversible in mice. (Hepatology 1996 Mar;23(3):515-23)

Regenerative changes in hepatic morphology and enhanced expression of CYP2B10 and CYP3A during daily administration of cocaine

The effects of daily cocaine administration for up to 14 days were studied in terms of hepatic morphology and the expression of cytochrome P450 (CYP) enzymes in the mouse liver. Daily intraperitoneal doses of 60 mg/kg of cocaine for 3 days induced severe hepatocellular necrosis in the pericentral zone and decreased activities of CYP1A2, CYP2A4/5, and CYP2Cx. The microsomal CYP2B10 protein content was increased by about 2.5-fold, but 2B10-dependent 7-pentoxyresorufin O-dealkylase (PROD) activity was barely detectable. Five or seven daily cocaine doses caused prominent pericentral inflammation and a significant (up to 14-fold) increase in the microsomal protein content and PROD activity. An increase in microsomal CYP3A was also evident, but CYP2A5 and CYP1A2 still remained at a low level. Immunohistochemical examination showed that the relative induction of CYP2B10 and CYP3A after treatment with cocaine was strongest in perivenous hepatocytes. Immunoinhibition experiments showed that CYP2B10 accounted for catalysis of only 15% to 20% of the enhanced microsomal cocaine N-demethylase (CNDM) activity, which correlated well with immunoreactive 3A protein, and could be blocked 70% to 90% by triacetyloleandomycin. After 10 or 14 daily doses of cocaine, regenerative changes with hepatocyte ballooning were observed, coinciding with increases in CYP1A2, CYP2A4/5, and CYP3A. These results suggest the following: (1) cocaine enhances its own cytochrome P450-dependent metabolism; (2) increased production of norcocaine in microsomes is catalyzed mainly by CYP3A enzyme(s), whereas 2B10, although markedly increased by cocaine treatment, has only a minor role in cocaine hepatotoxicity; and (3) despite increased microsomal CNDM activity, cocaine-induced liver injury is reversible in mice. (Hepatology 1996 Mar;23(3):515-23)

Differential Effects of Nonhydroxylated Flavonoids as Inducers of Cytochrome P450 1A and 2B Isozymes in Rat Liver

Flavanone, flavone, and tangeretin differentially affected the activities of cytochrome P540 1A and 2B isozymes in rat liver. Flavone and, to a lesser extent, tangeretin, increased activities of ethoxyresorufinO-deethylase, methoxyresorufinO-demethylase, and pentoxyresorufinO-dealkylase (PROD), whereas flavanone mainly enhanced PROD activity. Immunoblot analysis indicated that flavone and tangeretin increased cytochrome P450 1A1, 1A2, and 2B1,2 forms, whereas flavanone only enhanced the cytochrome P450 2B isozymes. Northern blot study showed that flavone and tangeretin increased the level of the cytochrome P450 1A2 mRNAs. The concentration of the other mRNAs were slightly or not affected by flavonoids. These results suggest that the induction of P450 1A2 by flavone and tangeretin might involve a transcriptional and/or post-transcriptional mechanism.

Modulation of constitutive and inducible hepatic cytochrome(s) P-450 by interferon β in mice

Aims/Methods: Interferon β is used as a therapeutic agent, but its effects on the hepatic cytochrome P-450-dependent drug metabolizing system have not yet been characterized. We investigated the effect of interferon β on cytochrome P-450 in mice. Results: Interferon β (2×10 5 units/mouse) significantly reduced total hepatic cytochrome P-450 (20%) and the activity of NADPH cytochrome C reductase (12%) 24 h after administration; lower doses had no such effect. Various monooxygenase activities were slightly reduced, the one most affected being 7-ethoxycoumarin O-deethylase (29%). In phenobarbital-treated mice, interferon β reduced the induction of total cytochrome P-450 (22%), the activities of pentoxyresorufin O-dealkylase (38%), benzyloxyresorufin O-dealkylase (30%), erythromycin N-demethylase (30%), 7-ethoxycoumarin O-deethylase (16%) and cytochrome P-450 2B1 (33%) and 3A (45%) proteins. In β-naphthoflavone-treated mice, interferon β lowered the induction of total cytochrome P-450 (18%), the activities of ethoxyresorufin O-deethylase (31%) and of 7-ethoxycoumarin O-deethylase (25%) and of cytochrome P-450 1A1 protein (31%). Conclusions: Thus it appears that induced cytochrome(s) P-450 were susceptible to interferon β, this effect not being influenced by the type of inducer. Since various members of the same cytochrome P-450 subfamilies catalyze oxidation of drugs in humans, our findings have potential significance as regards the fate of drugs or exogenous compounds given to patients receiving interferon β.

Organochlorine accumulation by sentinel mallards at the Winston-Thomas sewage treatment plant, Bloomington, Indiana

Farm-raised 12-month-old female mallards (Anas platyrhynchos) were released at the Winston-Thomas sewage treatment plant, Bloomington, Indiana. Five mallards were sacrificed at the start of the study and at approximately 10-day intervals through day 100. Concentrations of polychlorinated biphenyls (PCBs) in carcasses increased linearly with time of exposure and exceeded 16 μg/g wet weight by day 100; PCBs in breast muscle exceeded 3.9 μg/g by day 100. These PCB values are among the highest recorded for wild or sentinel waterfowl. PCB concentrations in breast muscle (26–523 μg/g lipid weight) were 50–1,000 times greater than human consumption guidelines for edible poultry in Canada (0.5 μg/g lipid weight) and 9–176 times greater than consumption guidelines for edible poultry in the United States (3.0 μg/g lipid weight). Additionally, PCB concentrations in carcass and breast muscle exceeded the threshold of the Great Lakes Sport Fish Consumption Advisory ‘do not eat’ category (1.9 μg/g wet weight) by day 20 and day 50, respectively. Hepatic cytochrome P450-associated monooxygenases including BROD (benzyloxyresorufin-O-dealkylase), EROD (ethoxyresorufin-O-dealkylase), and PROD (pentoxyresorufin-O-dealkylase) were induced over 5-fold compared to reference mallards. BROD, EROD, and PROD were each significantly correlated to total PCBs and to the toxicity of selected PCB congeners, relative to 2,3,7,8-tetrachlorodibenzo-p-dioxin.

Mechanism-based inactivation of hepatic ethoxyresorufin O-dealkylation activity by naturally occurring coumarins.

Several naturally occurring coumarins contained in the human diet have been found to be effective inhibitors and inactivators of murine hepatic ethoxyresorufin O-dealkylase (EROD) and pentoxyresorufin O-dealkylase in vitro [Cai, Y., Bennett, D., Nair, R.V., Ceska, O., Ashwood-Smith, M., and DiGiovanni, J. (1993) Chem. Res. Toxicol. 6, 872-879]. In the present study, these same coumarins decreased the content of cytochrome P450 (P450) in either 3-methylcholanthrene (MC)- or phenobarbital-induced murine hepatic microsomes but did not have a major effect on heme content. Detailed in vitro studies with [14C]coriandrin, which selectively inhibits and inactivates P450 1A1-mediated EROD activity, demonstrated that it covalently bound, in a preferential manner, to hepatic microsomal protein from MC-pretreated mice. A linear relationship was observed between covalent binding and loss of EROD activity. The inclusion of electrophile trapping agents in the incubations significantly inhibited the covalent binding of [14C]coriandrin to microsomal protein. In addition, the covalent binding of [14C]coriandrin was decreased 46% by 7,8-benzoflavone (7,8-BF), 58% by a monoclonal antibody with specificity toward MC-induced form(s) of P450, and 60% by ethoxyresorufin, implicating the bioactivation of coriandrin by P450 1A1. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of [14C]coriandrin-bound microsomal protein from MC-pretreated mice showed that [14C]coriandrin bound covalently to a protein with an approximate molecular mass of 49 kDa. Again, addition of 7,8-BF or polyclonal antibody against P450 1A1 reduced the covalent binding of [14C]coriandrin to this specific protein band. Interestingly, coriandrin was also found to be a potent inhibitor and inactivator of purified human P450 1A1. These results demonstrate that certain coumarins to which humans are exposed in the diet are bioactivated by P450 1A1 to reactive intermediates that subsequently form covalent adducts with the apoprotein, effectively destroying enzyme activity. Thus, certain naturally occurring coumarins may have a significant effect on human health.

Catalytic and Immunochemical Characterization of Cytochrome P450 Isozyme Induction in Dog Liver

The purpose of this study was to characterize hepatic cytochrome P450 induction in the dog by phenobarbital, β-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), β-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 β-naphthoflavone (186%), while it slightly decreased after treatment with isoniazid (54%) and dexamethasone (71%). Dog liver microsomes hydroxylated testosterone mainly at the 6β and 16α positions but also at the 6α-, 15β-, 15α-, 16β-, 18-, 2β-, 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 6β- (184%), 16α- (379%), 16β- (210%), 18- (195%), and 2β-testosterone (203%) hydroxylase and pentoxyresorufin O-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 6β-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 β-naphthoflavone produced increases that were 689 and 357% of control in ethoxyresorufin O-deethylase and chlorzoxazone hydroxylase activities, respectively. β-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.

Modulation of microsomal cytochrome P450 by Scutellariae Radix and Gentianae scabrae Radix in rat liver.

The present study has determined the effects of Scutellariae Radix (Huangqin) and Gentianae scabrae Radix (Longdan) on liver microsomal cytochrome P450 (P450)-dependent mono-oxygenases using rats pretreated with crude extracts of medicinal herbs. Scutellariae Radix resulted in a 53% decrease of pentoxyresorufin O-dealkylase activity in liver microsomes. In contrast, Gentianae scabrae Radix caused a 50% increase of benzo(a)pyrene hydroxylase activity. Immunoblotting analysis of liver microsomes revealed that Scutellariae Radix induced and suppressed the levels of P450 1A and 2B proteins, respectively. Scutellariae and Gentianae scabrae Radixes had no effects on microsomal aniline hydroxylase activity and P450 2E1 protein.

Structure-dependent induction of CYP2B by polychlorinated biphenyl congeners in female Sprague-Dawley rats

The dose-response induction of hepatic microsomal pentoxyresorufin O-dealkylase (PROD) activity by phenobarbital (PB) and several polychlorinated biphenyl (PCB) mixtures and congeners was determined in the immature female Sprague-Dawley rat. At a dose of 75 mg/kg/day of PB for 3 days, the microsomal PROD activity was 2154 pmol/min/mg protein. Aroclors 1260, 1254, 1242, and 1016 did not induce maximal PROD activity at doses up to 500 mg/kg, and only Aroclor 1016 induced > a half-maximal response at the 500 mg/kg dose. The relative potencies of eighteen different PCB congeners were also determined, and the structures of these compounds differed with respect to the degree of chlorination (tri- to octochloro) and substitution patterns. The relative potencies of these compounds were estimated by comparing their induced activities at the high dose (150 or 100 mg/kg) with that of PB. The most potent inducers were 2,3,3′,4′,5,6-hexaCB and 2,2′,3,4′,5,5′,6-heptaCB; at a dose of 150 mg/kg, the PROD activity induced by 2,2′,3,4′,5,5′,6-heptaCB was comparable to that observed for PB. 2,3,3′,4′,5,6-HexaCB was the most potent inducer, and hepatic PROD activity in rats treated with 150 mg/kg was 4202 pmol/min/mg; this value was higher than that observed for PB at a dose of 75 mg/kg. A second group of congeners including 2,2′,3,4,4′,5,5′-heptaCB, 2,2′,4,4′,5,5′-hexaCB, 2,2′,3,3′,4,4′,5,5′-oc-taCB, 2,2′,4,4′-tetraCB, 2,2′,4,5,5′-pentaCB, 2,2′,3,4,4′,5′,6-heptaCB, 2,2′,4,4′,5-pentaCB and 2,2′,3,3′,4′,5,5′,6-octaCB induced PROD activity ⩾ 1090 pmol/min/mg at the 150 mg/kg dose, and this value was > 50% of the maximal response observed for PB. The remaining compounds, namely 2,4,4′-triCB, 2,2′,3,4′-tetraCB, 2,2′,5,5′-tetraCB, 2,3′,4,4′,5-pentaCB, 2,3,3′,4,4′-pentaCB, 2,2′,4,4′,5,6′-hexaCB, 2,3,3′,4,4′,5,5′-heptaCB and 2,2′,3,-3′,4,4,5-heptaCB were all relatively weak inducers of hepatic microsomal PROD activity (< 450 pmol/min/mg). In parallel experiments, western blot analysis of immunoreactive CYP2B1 and CYP2B2 proteins showed that PB, the PCB mixtures, and congeners induced both proteins. Previous studies have identified a cis-acting DNA element that plays a role in regulating CYP2B1/B2 gene expression and binds nuclear trans-acting factor(s) induced by PB. The results of gel electrophoretic mobility shift assays with nuclear extracts showed that both PB and 2,2′,3,4′,5,5′,6-heptaCB induce formation of a common retarded band using a 32P-labeled oligonucleotide corresponding to the cis-acting DNA promoter sequence. Both PB and PCBs appear to induce CYP2B1/B2 via a common mechanism. Although the results of this study do not define structure-induction (CYP2B1/B2) relationships for PCBs, two compounds, namely 2,3,3′,4′,5,6-hexaCB and 2,2′,3,4′,5,5′,6-heptaCB, were identified as highly potent inducers.

Production of a Form-Specific, Inhibitory Antibody Against Rat Cytochrome P450 2B1 Using a Synthetic Peptide Antigen against a Putative Substrate Binding Site

Rat cytochrome P450 2B1 antipeptide antibodies were produced by immunizing rabbits with a synthetic peptide antigen. The anti-CYP2B1 IgG obtained did not cross-react with CYP2B2, which has 97% identity in primary sequence of CYP2B1. This result demonstrates that a difference of 2 amino acid residues among 12 is sufficient to produce a form-specific antibody. The CYP2B1 antipeptide IgG inhibited pentoxyresorufin O-dealkylase activity of microsomes obtained from phenobarbital-treated rats in a dose-dependent manner, whereas it did not inhibit ethoxyresorufin O-deethylase activity of microsomes obtained from 3-methylcholanthrene-treated rats. These results suggest that the selected amino acid sequence, which coincides with one of the substrate binding sites of Pseudomonas putida CYP101A (P450cam) and one of the putative substrate binding sites of CYP2B2, is located on the surface of the CYP2B1 molecule, as opposed to inside the molecule or in the lipid bilayer of microsomes.

Induction of Liver Cytochrome-P450 2B1 by β-Ionone in Sprague-Dawley Rats

Induction of liver cytochrome P450 2B1 by β-ionone was investigated in male and female Sprague Dawley rats. Administration of β-ionone subcutaneously 72 and 48 hr before sacrificing the animals not only significantly induced the liver microsomal activity of pentoxyresorufin O-dealkylase, but also clearly increased in the level of cytochrome P450 2B1 protein. The induction of cytochrome P450 2B1 by β-ionone was much greater in male rats than in female rats. A slot blot analysis showed that the mRNA level was increased from 6 hr after treatment with β-ionone in male rats and from 12 hr after treatment in female rats. Taken together, the present results indicate for the first time that the induction of cytochrome P450 2B1 by β-ionone might be regulated by the accumulation of mRNAs.

Environmental polychlorinated biphenyls: acute toxicity of landfill soil extract to female prepubertal rats.

Subsurface soil from a National Priorities List landfill containing about 2.5% polychlorinated biphenyls (PCBs) was extracted and the extract cleaned by Florisil slurry and alumina column chromatography. The refined extract contained 48 mg/mL PCB, mainly trichlorobiphenyls and tetrachlorobiphenyls, traces of polychlorinated naphthalenes, 125 micrograms/mL 2,2-bis-p-chlorophenyl-1,1-dichloroethylene (DDE), and low levels of chlorinated dibenzofurans. The refined extract was dissolved in corn oil and administered intraperitoneally to weanling (day 20) female rats on days 20 and 21; rats were terminated on day 22. Limited data indicated possible hematopoietic effects, including neutrophilia. There were no changes in relative uterus, kidney, or adrenal gland weights between total doses of 3 to 96 mg/kg total PCB. Relative liver weights increased significantly at 36 mg/kg and activities of P450s 1A1 (as ethoxyresorufin O-dealkylase) and 2B (as pentoxyresorufin O-dealkylase) increased at 12 mg/kg and plateaued at 36 (P450 1A1) or 48 (P450 2b) mg/kg. Serum total thyroxine (T4) declined significantly at doses of 36 mg/kg and greater; thyroid follicular epithelial cells were significantly larger within the same dose range, but the follicular colloid area decreased to less than 60% control values at 12 mg/kg and remained at this size through 72 mg/kg. Maximum mobilization of T4 apparently occurred at 12 mg/kg and attenuated measured declines in circulating levels. Even though a large proportion of proven and probable estrogenic chlorobiphenyls (CBs) were present, the lower amounts of more potent antiestrogenic aryl hydrocarbon (Ah) receptor agonists and/or decreased responsiveness because of low serum T4 levels may have antagonized the uterotropic response.

Inhibitory effects of H2-receptor antagonists on cytochrome P450 in male ICR mice

1. The present study was undertaken to examine the effects of H2-receptor antagonists including newly developed mifentidine derivatives, IY-80843 and IY-80845, on cytochrome P450(P450) in vitro and in vivo. 2. Initially, 3-methylcholanthrene-, phenobarbital-, ethanol- and dexamethasone-induced liver microsomes were prepared from male ICR mice to study in vitro effects of above chemicals on ethoxyresorufin O-deethylase(EROD), pentoxyresorufin O-dealkylase(PROD), p-nitrophenol hydroxylase and erythromycin N-demethylase(ERDM) activities, respectively. It was found that histamine, cimetidine and famotidine were not inhibitory to four enzyme activities. Meanwhile, mifentidine slightly inhibited EROD and PROD activities and its derivatives IY-80843 and IY-80845 strongly inhibited PROD, EROD and ERDM activities. 3. Prolongation of hexobarbital-induced sleeping time was determined in male ICR mice to confirm in vitro inhibitory effects of mifentidine and its derivatives in vivo. It was observed that cimetidine, mifentidine, IY-80843 and IY-80845 caused dose-dependent increases in the sleeping time, indicating the inhibition of P450 responsible for hexobarbital metabolism. 4. It was concluded that mifentidine and its derivatives are P450 inhibitors and that our newly synthesized IY-80843 is most inhibitory. 5. The present results indicate that mifentidine and its derivatives not only antagonise the H2-receptor but also inhibit P450 enzymes.