Induction of rat hepatic mixed-function oxidases by acetone and other physiological ketones: Their role in diabetes-induced changes in cytochrome P450 proteins

1. To evaluate the role of ketone bodies in diabetes-induced changes in hepatic cytochrome P450 composition, rats were treated with acetone, 3-hydroxybutyrate or 1,3-butanediol.

2. Treatment with acetone enhanced the rat hepatic O-dealkylations of ethoxyresorufin and methoxyresorufin, and the hydroxylation of p-nitrophenol, but had no effect on lauric acid hydroxylation and ethylmorphine N-demethylation. Neither 3-hydroxybutyrate nor 1,3-butanediol modulated the metabolism of the above substrates.

3. Immunoblot analysis of hepatic microsomal proteins revealed that treatment with acetone increased the apoprotein levels of P4501A2, P4502B1/2 and P4502E1.

4. It is concluded that acetone is responsible, at least partly, for the diabetes-induced increase in hepatic microsomal P4501A2, P4502B1/2 and P4502E1 proteins but does not mediate the increases in the P4503A1 and P4504A1 proteins. On the basis of work from our own and other laboratories a mechanism for the diabetes-induced changes in hepatic cytochrome P450 proteins is proposed.     

Anti-peptide antibodies to the P4502D subfamily in rat,dog and man

1. In order to obtain specific antibodies of the P4502D subfamily, we generated two anti-peptide antibodies against synthetic peptides, DPAQPPRD (peptide A) and DPTQPPRH (peptide B). The sequence of peptide A occurs in rat P4502D2, P4502D4 and human P4502D6, whereas the sequence of peptide B occurs in the dog P4502D subfamily. These sequences are closely related to an epitope of liver/kidney microsomal autoimmune hepatitis.

2. In immunoblotting studies, the anti-peptide antibody peptide A recognized a 49-KDa protein in microsomes derived from human lymphoblasts expressing P4502D6 and rat liver. It showed no crossreactivity with microsomes from dog liver. In contrat, the anti-peptide antibody against peptide B recognized a 49-KDa protein only in microsomes of dog liver. These indicate that each anti-peptide antibody has the specificity for the respective sequences of the members of P4502D subfamily, with the species investigated herein.

3. In immunoinhibition studies, the anti-peptide antibodies against peptide B inhibited bunitrolol 4-hydroxylation and propranolol 4, 5-hydroxylation, which are mediated by the dog P4502D subfamily. These data suggest that the anti-peptide antibodies against peptide B bind to the native and denatured forms of the P4502D subfamily.

4. The present study has demonstrated that the anti-peptide antibodies against this region are useful for studying the members of the P4502D subfamily.     

Multiple forms of cytochrome P450 and associated monooxygenase activities in human brain mitochondria

We have investigated cytochrome P450 (P450) and associated monooxygenase activities in human brain mitochondria isolated from eight regions of four human brain samples obtained at autopsy. P450-associated monooxygenase activities including aminopyrine N-demethylase (APD), 7-ethoxycoumarin O-deethylase (ECD), p-nitrophenol hydroxylase (PNPH), and N-nitrosodimethylamine N-demethylase (ND-MAD) were detectable in the mitochondria from human brain regions. Immunoblot experiments using antisera to purified rat liver microsomal P450, namely P4502B1/2, P4501A1/2, and P4502E1, revealed immunoreactive bands in isolated mitochondria from different regions of the human brain. The antibody to P4502B1/2 and P4501A1/2 inhibited the human brain mitochondrial APD and ECD activities, respectively. The addition of antiserum to microsomal NADPH cytochrome P450 reductase did not affect the mitochondrial P450-associated monooxygenase activities, although it completely inhibited the corresponding activities in brain microsomes. Overall, the present study demonstrates, in human brain mitochondria, the presence of multiple forms of P450 belonging to the 1A, 2B, and 2E subfamilies that are involved in xenobiotic metabolism.     

Induction of rat hepatic mixed-function oxidases by acetone and other physiological ketones: their role in diabetes-induced changes in cytochrome P450 proteins.

1. To evaluate the role of ketone bodies in diabetes-induced changes in hepatic cytochrome P450 composition, rats were treated with acetone, 3-hydroxybutyrate or 1,3-butanediol. 2. Treatment with acetone enhanced the rat hepatic O-dealkylations of ethoxyresorufin and methoxyresorufin, and the hydroxylation of p-nitrophenol, but had no effect on lauric acid hydroxylation and ethylmorphine N-demethylation. Neither 3-hydroxybutyrate nor 1,3-butanediol modulated the metabolism of the above substrates. 3. Immunoblot analysis of hepatic microsomal proteins revealed that treatment with acetone increased the apoprotein levels of P4501A2, P4502B1/2 and P4502E1. 4. It is concluded that acetone is responsible, at least partly, for the diabetes-induced increase in hepatic microsomal P4501A2, P4502B1/2 and P4502E1 proteins but does not mediate the increases in the P4503A1 and P4504A1 proteins. On the basis of work from our own and other laboratories a mechanism for the diabetes-induced changes in hepatic cytochrome P450 proteins is proposed.     

Concurrent induction of rat hepatic microsomal cytochrome P450 and haem oxygenase by 2,2'-dipyridyl ketone: comparison with the effect of 2,2'-dipyridyl amine

1. The effect of 2,2'-dipyridyl ketone and 2,2'-dipyridyl amine on the induction of hepatic microsomal cytochrome P450 (P450) and heme oxygenase was compared, and their effects on five different P450 isoforms (P4501A1, 3A2, 2B1, 2E1 and 2C11) in rat were examined. 2. Treatment of rat with 2,2-dipyridyl amine resulted in the marked induction of haem oxygenase to about seven-fold of the controls with a decrease in P450 content. 2,2-'Dipyridyl ketone produced concomitant induction of both P450 and haem oxygenase activity in a dose- and time-dependent manner without showing any sex differences. 3. Immunoblot analysis revealed that 2,2'-dipyridyl ketone slightly increased CYP2E1 and CYP3A2 at low doses, but not at high dose levels. There was no effect on P4502C11. P4502B1 was induced by the treatment with 2,2'-dipyridyl ketone in a dose-dependent manner. 4. These results indicate that dipyridyl compounds having different bridges between two aromatic moieties act as differential inducers of hepatic microsomal P450s and haem oxygenase.     

Concurrent induction of rat hepatic microsomal cytochrome P450 and haem oxygenase by 2,2'-dipyridyl ketone: comparison with the effect of 2,2'-dipyridyl amine

1. The effect of 2,2'-dipyridyl ketone and 2,2'-dipyridyl amine on the induction of hepatic microsomal cytochrome P450 (P450) and heme oxygenase was compared, and their effects on five different P450 isoforms (P4501A1, 3A2, 2B1, 2E1 and 2C11) in rat were examined. 2. Treatment of rat with 2,2'-dipyridyL amine resulted in the marked induction of haem oxygenase to about seven-fold of the controls with a decrease in p450 content. 2,2'-Dipyridyl ketone produced concomitant induction of both P450 and haem oxygenase activity in a dose- and time-dependent manner without showing any sex differences. 3. Immunoblot analysis revealed that 2,2'-dipyridyl ketone slightly increased CYP2E1 and CYP3A2 at low doses, but not at high dose levels. There was no effect on P4502C11. P4502B1 was induced by the treatment with 2,2'-dipyridyl ketone in a dose-dependent manner. 4. These results indicate that dipyridyl compounds having different bridges between two aromatic moieties act as differential inducers of hepatic microsomal P450s and haem oxygenase.     

Oxidation of methyl- and ethyl- tertiary-butyl ethers in rat liver microsomes: role of the cytochrome P450 isoforms

Methyl t-butyl ether (MTBE) and ethyl t-butyl ether (ETBE) are commonly used in unleaded gasoline to increase the oxygen content of fuel and to reduce carbon monoxide emissions from motor vehicles. This study was undertaken to investigate: (1) the effect of administration to rats of ETBE and its metabolite, t-butanol, on the induction and/or inhibition of hepatic P450 isoenzymes; (2) the oxidative metabolism of MTBE and ETBE by liver microsomes from rats pretreated with selected P450 inducers and purified rat P450(s), (2B1, 2E1, 2C11, 1A1). ETBE administration by gavage at a dose of 2 ml/kg for 2 days induced hepatic microsomal P4502E1-linked p-nitrophenol hydroxylase and the P4502B1/2-associated PROD and 16β-testosterone hydroxylase, verified by immunoblot experiments. t-Butanol treatments at doses of 200 and 400 mg/kg i. p. for 4 days did not alter any liver microsomal monoxygenases. Both MTBE and ETBE were substrates for rat liver microsomes and were oxidatively dealkylated to yield formaldehyde and acetaldehyde, respectively. The dealkylation rates of both MTBE and ETBE were increased c. fourfold in phenobarbital (PB)-treated rats. In rats pretreated with pyrazole, an inducer of 2E1, only the demethylation of MTBE was increased (c. twofold). When the oxidations of MTBE and ETBE were investigated with purified P450(s) in a reconstituted system, it was found that P4502B1 had the highest activities towards both solvents, whereas 1A1 and 2C11 were only slightly active; P4502E1 had an appreciable activity on MTBE but not against ETBE. Metyrapone, a potent inhibitor of P450 2B, consistently inhibited both the MTBE and ETBE dealkylations in microsomes from PB-treated rats. Furthermore, 4-methylpyrazole (a probe inhibitor of 2E1) and anti-P4502E1 IgG showed inhibition, though modest, only on MTBE demethylation, but not on ETBE deethylation. Inhibition experiments have also suggested that rat 2A1 may exert an important role in MTBE and ETBE oxidation. Taken together, these results indicate that 2B1, when expressed, is the major enzyme involved in the oxidation of these two solvents and that 2E1 may have a role, although minor, in MTBE demethylation. The implications of these data for MTBE and ETBE toxicity remain to be established. Received: 5 August 1997 / Accepted: 29 October 1997     

Hyperinsulinaemia causes a preferential increase in hepatic P4501A2 activity.

Male NEDH (New England Deaconess Hospital) rats were transplanted with a radiation-induced tumour from a donor male rat and were killed 18 days following transplantation. At the time of killing the insulinoma-bearing animals were severely hypoglycaemic but plasma ketone levels were normal. Insulinoma-bearing animals exhibited higher hepatic O-deethylation of ethoxyresorufin and N-demethylation of ethylmorphine activities when compared to control animals. Similarly, hepatic microsomal preparations from insulinoma-bearing rats were more efficient than control animals in converting the promutagen 2-amino-6-methyldipyrido[1,2-a:3',2']imidazole (Glu-P-1) to mutagenic intermediates in the Ames test. Immunoblot analysis employing polyclonal antibodies against the P4501A and P453A families revealed that insulinoma-bearing rats had higher hepatic P4501A2 apoprotein levels. No major differences in P4503A1 apoprotein levels between insulinoma-bearing and control rats were noted. Subcutaneous administration of insulin to male Wistar rats gave rise to a modest increase in ethoxyresorufin O-deethylase activity and in the ability to activate Glu-P-1 to mutagens in the Ames test. Immunoblot analysis revealed an increase in hepatic P4501A2 apoprotein levels following the treatment with insulin. It is concluded that insulinoma-bearing rats display high P4501A2 activity and the hyperinsulinaemia that characterize this condition is responsible for the effect. Moreover, administration of insulin to other strains of rat, such as Wistar, also enhances P4501A2 activity, presumably as a result of hyperinsulinaemia.     

Regioselectivity and Enantioselectivity of Metoprolol Oxidation by Two Variants of cDNA-Expressed P4502D6

Purpose. The oxidative metabolism of metoprolol was investigated in two human lymphoblastoma cell-lines transfected with variants of cDNA for cytochrome P4502D6. Methods. The regioselective and enantioselective features of the oxidations of deuterium-labeled pseudoracemic metoprolol were characterized by GC/MS analysis of the substrate and products. Results. There were significant differences between the two P4502D6 variants in the formation kinetics of O-demethylmetoprolol and -hydroxymetoprolol. The h2D6-Val microsomes highly favored the formation of the O-demethylmetoprolol regioisomer 6.3:1 and 2.8:1, respectively from (R)-metoprolol-d₀ and (S)-metoprolol-d₂, while the corresponding ratios for h2D6v2 microsomes were much lower. For both variants, O-demethylmetoprolol formation favored the (R)-substrate 1.5 to 2-fold, while -hydroxymetoprolol formation was non-enantioselective. Similar Km values of metoprolol oxidation, 10-20 µM, were observed for the two microsomal preparations. Conclusions. The regioselectivity, enantioselectivity, and Km values for the h2D6-Val microsomes resemble those observed for the native P4502D6 in human liver microsomes, whereas the h2D6v2 microsomes deviated remarkably in regioselectivity.     

Deuterium isotope effect on the metabolism of N-nitrosodimethylamine and related compounds by cytochrome P4502E1.

1. Deuteration of N-nitrosodimethylamine (NDMA) decreases its carcinogenicity, and produces an isotope effect on its metabolism in vivo. Consistent with these results are the observations that deuteration caused a 5-fold increase in the apparent Km, but not the Vmax for the demethylation and denitrozation of NDMA in acetone-induced rat liver microsomes. These microsomes are a good source of cytochrome P4502E1. 2. For demethylation of Z-[2H3]NDMA and E-[2H3]NDMA, the Km values were indistinguishable, and were between the values for those of NDMA and [2H6]NDMA. Almost all the formaldehyde formed was derived from the non-deuterated methyl group, indicating a lack of stereoselectivity in the demethylation of NDMA. 3. NDMA and [2H6]NDMA displayed apparent Ki values of 59 and 441 microM, respectively, for N-nitrosodiethylamine deethylase, showing an apparent isotope effect of 0.13, and displayed an isotope effect of 0.21 in the Ki values for p-nitrophenol hydroxylase. 4. With acetone and deuterated acetone as inhibitors for p-nitrophenol hydroxylase, the isotope effect on the Ki was 0.11. Similar deuterium isotope effects were also observed with acetone and dimethylformamide as competitive inhibitors for NDMA demethylase. 5. In the microsomal oxidation of ethanol, a deuterium isotope effect of about five was observed in the Vmax/Km when carbon-1 was deuterated, but was not observed in the Vmax. 6. Results illustrate a unique deuterium isotope effect on the Km values of reactions catalysed by P4502E1.     

Effects of flupyrazofos on liver microsomal cytochrome P450 in the male Fischer 344 rat

1. The effects of flupyrazofos on liver microsomal cytochrome P450 were investigated in the male Fischer 344 rat.When rats were treated intraperitoneally with flupyrazofos for 3 consecutive days, the activities of ethoxyresorufin O-deethylase and testosterone 2 β-hydroxylase were significantly reduced, whereas the activities of pentoxyresorufin O-depentylase and testosterone 6 β- and 7 α-hydroxylases were induced in liver microsomes. 2. Within 24 h after treatment with 50 m kg^?1 flupyrazofos, most enzyme activities were decreased, indicating the interaction of flupyrazofos with cytochrome P450. 3. In Western immunoblotting, cytochrome P4502B1/2 proteins were clearly induced by treatment with flupyrazofos, whereas P4501A1/2 and 2C6 proteins were reduced in liver microsomes. 4. The present results indicate that flupyrazofos modulates the expression of cytochrome P450 in rat.     

Deuterium isotope effect on the metabolism of N-nitrosodimethylamine and related compounds by cytochrome P4502E1

1. Deuteration of N-nitrosodimethylamine (NDMA) decreases its carcinogenicity, and produces an isotope effect on its metabolism in vivo. Consistent with these results are the observations that deuteration caused a 5-fold increase in the apparent K_m, but not the V_max the demethylation and denitrozation of NDMA in acetone-induced rat liver microsomes. These microsomes are a good source of cytochrome P4502E1.

2. For demethylation of Z-[²H₃]NDMA and E-[²H₃)NDMA, the K_m values were indistinguishable, and were between the values for those of NDMA and [²H₆]NDMA. Almost all the formaldehyde formed was derived from the non-deuterated methyl group. indicating a lack of stereoselectivity in the demethylation of NDMA.

3. NDMA and [²H₆]NDMA displayed apparent K_i values of 59 and 441 μM, respectively, for N-nitrosodiethylamine deethylase, showing an apparent isotope effect of 0.13, and displayed an isotope effect of 0.21 in the K_i values for p-nitrophenol hydroxylase.

4. With acetone and deuterated acetone as inhibitors for p-nitrophenol hydroxylase, the isotope effect on the K_i was 0.11. Similar deuterium isotope effects were also observed with acetone and dimethylformamide as competitive inhibitors for NDMA demethylase.

5. In the microsomal oxidation of ethanol, a deuterium isotope effect of about five was observed in the V_max/K_m when carbon-1 was deuterated, but was not observed in the V_max.

6. Results illustrate a unique deuterium isotope effect on the K_m values of reactions catalysed by P4502E1.     

Effects of flupyrazofos on liver microsomal cytochrome P450 in the male Fischer 344 rat

1. The effects of flupyrazofos on liver microsomal cytochrome P450 were investigated in the male Fischer 344 rat. When rats were treated intraperitoneally with flupyrazofos for 3 consecutive days, the activities of ethoxyresorufin O-deethylase and testosterone 2 beta-hydroxylase were significantly reduced, whereas the activities of pentoxyresorufin beta-depentylase and testosterone 6beta- and 7 alpha-hydroxylases were induced in liver microsomes. 2. Within 24 h after treatment with 50 m kg(-1) flupyrazofos, most enzyme activities were decreased, indicating the interaction of flupyrazofos with cytochrome P450. 3. In Western immunoblotting, cytochrome P4502B1/2 proteins were clearly induced by treatment with flupyrazofos, whereas P4501A1/2 and 2C6 proteins were reduced in liver microsomes. 4. The present results indicate that flupyrazofos modulates the expression of cytochrome P450 in rat.     

Molecular modelling of members of the P4502A subfamily: application to studies of enzyme specificity

1. Using the recently published crystal structure of a bacterial P450, namely 102 (also termed P450_bm3), as a template molecular models of mammalian 2A1, 2A4, 2A5 and 2A6 were constructed.

2. Substrate interaction studies demonstrated that in keeping with known catalytic activities the putative binding sites of mouse hepatic P4502A4 and 2A5 oriented testosterone for 15α-hydroxylation and coumarin for 7-hydroxylation respectively.

3. Substrate interaction studies with the putative binding site of human liver P4502A6 demonstrated that coumarin was oriented for 7-hydroxylation. However, in keeping with previous site-directed mutagenesis studies with P4502A4 and 2A5, changing a single phenylalanine residue to leucine in 2A6 gave rise to a mutant enzyme, which could bind testosterone as a substrate for 15α-hydroxylation rather than coumarin.

4. Substrate interaction studies with the putative binding site of rat hepatic P4502A1 suggested that this isoenzyme would hydroxylate coumarin at the 3- rather than at the 7-position.

5. The results of these molecular modelling studies demonstrate that apparently minor modifications to P4502A subfamily amino acid sequences can result in major alterations in enzyme specificity.

6. Molecular modelling is thus a useful technique that can aid in elucidating substrate specificities of P450 isoenzymes and species differences in xenobiotic metabolism. The technique can also be utilized to complement site-directed mutagenesis studies in order to identify critical structural features of P450s and other enzymes.     

Extrahepatic expression of P450 proteins in insulin-dependent diabetes mellitus

1. Male Wistar rats were rendered diabetic by the administration of a single intraperitoneal dose of streptozotocin; the levels of the xenobiotic-inducible P450 proteins were determined in the lung and kidney using diagnostic substrates and immunoblotting employing polyclonal antibodies. The glutathione conjugation system in the cytosol of these tissues was also investigated.

2. The onset of insulin-dependent diabetes did not influence the O-dealkylations of methoxyresorufin, ethoxyresorufin and pentoxyresorufin in either kidney or lung.

3. Lauric acid hydroxylase activity, however, was induced in the kidney but no activity was detectable in the lung. Immunoblot analysis of kidney microsomes using antibodies to P4504A1 revealed the presence of two bands, both of which were clearly inducible in diabetes. In pulmonary microsomes a single faint band was detected which also appeared to be higher in the diabetic rats.

4. Aniline p-hydroxylase activity was not detectable in the kidney, but activity was measurable in the lung and was suppressed in diabetes. Immunoblot analysis of pulmonary microsomes using antibodies to P4502E1 immunodetected a single band which was suppressed in diabetes. In the kidney microsomes a single band was also detected which was, however, markedly elevated in diabetes.

5. Glutathione S-transferase activity was modestly higher in the kidney, but not lung, of the diabetic animals. Glutathione reductase and total glutathione levels were not influenced by the presence of diabetes.

6. It is concluded that streptozotocin-induced insulin-dependent diabetes modulates extrahepatic P450 proteins, the effect being both tissue- and isoform-specific.     

Immunochemical detection of cytochrome P450 isozymes induced in rat liver byn-hexane, 2-hexanone and acetonyl acetone

Cytochrome P450 isozymes induced in rat liver by treatment withn-hexane, 2-hexanone and acetonyl acetone (given intraperitoneally 5 mmol/kg for 4 days) were investigated using enzyme assays (benzene, toluene, 7-ethoxyresorufin and 7-pentoxyresorufin metabolism) and monoclonal antibodies (anti-P450IA1/2, anti-P450IIB1/2, anti-P450IIC11/6, anti-P450IIE1(91) and anti-P450IIE1(98)).n-Hexane treatment enhanced the activities of low-K _m benzene aromatic hydroxylase and toluene side-chain oxidase, but not 7-ethoxyresorufin O-deethylase or 7-pentoxyresorufin O-depentylase. 2-Hexanone or acetonyl acetone treatment enhanced the activities of low-and high-K _m benzene aromatic hydroxylases, toluene side-chain oxidase and 7-pentoxyresorufin O-depentylase, but not of 7-ethoxyresorufin O-deethylase. Immunoblot analysis showed that anti-P450IA1/2 did not bind liver microsomal protein from either control and treated rats in the region of cytochrome P450s, whereas with anti-P450IIE1(98) a clear-cut band was seen in liver microsomes from control and treated rats, with intensities in the following order: 2-hexanone=acetonyl acetone n-hexane > control > phenobarbital. With anti-P450IIB1/2, a band was detected in microsomes from phenobarbital-treated rats, and to a lesser extent, in microsomes from 2-hexanone-and acetonyl acetone-treated rats. Like the immunoblot analysis, anti-P450IIE1(91) inhibited toluene side-chain hydroxylase activity in all microsomes, except in preparations from phenobarbital-treated rats and anti-P450IIB1 in microsomes from phenobarbital-, 2-hexanone- and acetonyl acetone-treated rats. Anti-P450IIC11/6 also inhibited toluene side-chain hydroxylase activity: the inhibited activity in the five different microsome preparations was as follows:n-hexane=control > acetonyl acetone=2-hexanone=phenobarbital. These results indicate thatn-hexane induces only quantitative alterations in the constitutive cytochrome P450 isozyme (P450IIE1), whereas its metabolites 2-hexanone and acetonyl acetone induce not only quantitative changes in constitutive cytochrome P450 (P450IIE1 and P450IIC11/6) but also a different type of isozyme (P450IIB1/2).     

Sex-related differences in rat hepatic cytochromes P450 expression following treatment with phenobarbital or 3-methylcholanthrene

The induction of hepatic cytochromes P450 and metabolic effects have been examined in male and female Sprague-Dawley rats following treatment with either phenobarbital or 3-methylcholanthrene. Hepatic cytochrome P450 levels were higher in males than in females by ≈40%. Treatment of male and female rats with phenobarbital or 3-methylcholanthrene resulted in an ≈1.6- and 2-fold increase, respectively, in heptic microsomal cytochrome P450 levels in both sexes, relative to untreated animals. Immunoblot analyses were performed to compare sex-related changes in P450 levels. Hepatic P450IIB1 levels in males were greater than those in females following phenobarbital treatment. 3-Methylcholanthrene-induced male hepatic microsomes exhibited greater levels of P450 IA1 and IA2 than female microsomes, whereas uninduced microsomes from males or females failed to exhibit a band. Mab PCN 2-13-1 against P450IIIA recognized an intense band in uninduced hepatic microsomes from males whereas no band was recognized in uninduced microsomes from female rats. The levels of P450IIIA in males were increased 2 to 3-fold following treatment with phenobarbital. while the increase of IIIA levels in females by phenobarbital was minimal, as monitored by immunoblot analysis. Solid phase radioimmunoassay using monoclonal antibodies supported the results of immunoblot analysis. Phenobarbital treatment caused a 6.5-fold increase in the monoclonal antibody binding to IIB1 in males, whereas treatment of females with phenobarbital resulted in a 12-fold increase of IIB1 binding, relative to respective controls. The relative increase of IA levels by 3-methylcholanthrene was also greater in females than in males (10-vs. 8-fold) although the levels of induced IA were comparable in both sexes, as assessed by radioimmunoassay. Radioimmunoassay also showed that hepatic IIE1 level was 1.5-fold higher in males than in females and that either phenobarbital or 3-methylcholanthrene treatment caused 80% to 40% decrease in IIE1 levels, relative to control, in both sexes. Sex-related metabolic activities were examined in hepatic microsomes. Hexobarbital hydroxylase activity was 2- to 3-fold higher in uninduced microsomes from males than that from females. This hydroxylase activity was increased 2- and 3-fold in males and females, respectively, following phenobarbital treatment, as compared to controls. Addition of anti-P450IIB1 antibody to phenobarbital-induced hepatic microsomes from males and females produced 64% and 84% inhibition of hexobarbital oxidation, respectively. Aryl hydrocarbon hydroxylase activity was increased ≈12- and 26-fold in males and females. respectively, following 3-methylcholanthrene treatment relative to controls. The anti-P450IA antibody inhibitable rate of aryl hydrocarbon hydroxylase activity was comparable in both sexes following 3-methylcholanthrene treatment (≈70%). These results demonstrate that levels of hepatic P450IIB1 or P450IA are greater in male than in female for untreated, phenobarbital- or 3-methylcholanthrene treated rats. In addition, the relative increase of P450IIB1 or IA by phenobarbital or 3-methylcholanthrene is more significant in females.     

Spectral and kinetic studies of the interaction of imidazole anti-fungal agents with microsomal cytochromes P-450

1. The imidazole antifungal agents, ketoconazole, miconazole and clotrimazole have been shown to be potent inhibitors of the phenobarbital-induced cytochromes P-450 and the 3-methylcholanthrene-induced cytochromes P-448-dependent rat hepatic microsomal mixed-function oxidases.

2. All three drugs were more potent inhibitors of the phenobarbital-induced O-deethylation of ethoxycoumarin than of the 3-methylcholanthrene-induced activity indicating selective inhibition of the phenobarbital-induced cytochromes P-450. In both types of microsomes ketoconazole was always the weakest inhibitor.

3. All three compounds elicited type II spectral interactions with both types of microsomes, and had similar K_s values. Miconazole and clotrimazole, and to a lesser extent ketoconazole, also interacted with the substrate binding sites of both phenobarbital-induced cytochromes P-450 and to a lesser extent with the 3-methylcholanthrene-induced cytochrome P-448.

4. It is concluded that at least part of the inhibitory effect of these antifungal agents may reflect competitive inhibition at the substrate binding site.     

Differential effects of B- and T-lymphocyte mitogens on cytochrome P450 in mice

Lipopolysaccharide (LPS) is widely used as a B-lymphocyte mitogen and is known to depress expression of the cytochrome P450 (P450). However, there have been no studies regarding to the effects of the other mitogens on the expression of P450. This study investigated the effects of mitogens on the constitutive and inducible expression of mouse hepatic P450. Following treatment with B-lymphocyte mitogens, such as LPS and pokeweed mitogen (PWM), hepatic P450 content was reduced. LPS and PWM also suppressed activities of microsomal ethoxyresorufin O-deethylase, pentoxyresorufin O-dealkylase and aniline hydroxylase, a representative activity of P4501A1/2, P4502B1/2 and P4502E1, respectively, in both constitutive and P450 induced mice. However, there was no effect when treated with T-lymphocyte mitogens, such as concanavalin A and phytohemagglutinin. Suppression of P450 expression in the LPS- or PWM-treated mice occurred and was shown to involve a decrease in P450 protein and mRNA levels in liver. These results suggest that suppressive effects of mitogens on the expression of P450 might be different and that B-lymphocyte mitogens selectively depress the expression of P450.     

Effects of tebuconazole on cytochrome P450 enzymes,oxidative stress,and endocrine disruption in male rats

The major objective of the present study was to determine the ability of a triazole fungicide tebuconazole to induce cytochrome P450‐dependent monooxygenases, oxidative stress, and endocrine‐disrupting activity using male rats treated with tebuconazole at 10, 25, and 50 mg/kg p.o. once daily for 28 days. In liver, tebuconazole dose‐dependently increased microsomal contents of cytochrome P450 and cytochrome b₅ and the activities of NADPH‐cytochrome P450 reductase, 7‐ethoxyresorufin O‐deethylase, methoxyresorufin O‐demethylase, pentoxyresorufin O‐dealkylase, 7‐ethoxycoumarin O‐deethylase, aniline hydroxylase, and erythromycin N‐demethylase. In kidney, tebuconazole increased 7‐ethoxycoumarin O‐deethylase activity without affecting other monooxygenase activities. In marked contrast to liver and kidney, tebuconazole decreased testicular 7‐ethoxyresorufin O‐deethylase, methoxyresorufin O‐demethylase, 7‐ethoxycoumarin O‐deethylase, aniline hydroxylase, and erythromycin N‐demethylase activities. The results of immunoblot analysis of liver microsomes of controls and tebuconazole‐treated rats revealed that tebuconazole induced CYP1A1/2, CYP2B1/2, CYP2E1, and CYP3A proteins in liver. Additions of tebuconazole to liver microsomes inhibited microsomal 7‐ethoxycoumarin O‐deethylase activity in vitro (IC₅₀ = 1.50–1.69 µM). Treatment of rats with tebuconazole decreased glutathione content and increased glutathione S‐transferase, superoxide dismutase, catalase, and glutathione peroxidase activities in liver; increased superoxide dismutase activities in kidney and testis; but decreased glutathione S‐transferase activity in testis. Treatments with tebuconazole decreased serum testosterone concentration and cauda epididymal sperm count. The present study demonstrates that tebuconazole induces a multiplicity of CYPs and oxidative stress in liver; inhibits testicular P450 and glutathione S‐transferase activities; and produces anti‐androgenic effects in male rats.