Induction Of Hepatic Cytochrome P-450 Research Articles

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.

Phenobarbital induction of cytochromes P-450. High-level long-term responsiveness of primary rat hepatocyte cultures to drug induction, and glucocorticoid dependence of the phenobarbital response

The induction of hepatic cytochromes P-450 by phenobarbital (PB) was studied in rat hepatocytes cultured for up to 5 weeks on Vitrogen-coated plates in serum-free modified Chee's medium then exposed to PB (0.75 mM) for an additional 4 days. Immunoblotting analysis indicated that P-450 forms PB4 (IIB1) and PB5 (IIB2) were induced dramatically (greater than 50-fold increase), up to levels nearly as high as those achieved in PB-induced rat liver in vivo. The newly synthesized cytochrome P-450 was enzymically active, as shown by the major induction of the P-450 PB4-dependent steroid 16 beta-hydroxylase and pentoxyresorufin O-dealkylase activities in the PB-induced hepatocyte microsomes (up to 90-fold increase). PB induction of these P-450s was markedly enhanced by the presence of dexamethasone (50 nM-1 microM), which alone was not an affective inducing agent, and was inhibited by greater than 90% by 10% fetal bovine serum. The PB response was also inhibited (greater than 85%) by growth hormone (250 ng/ml), indicating that this hormone probably acts directly on the hepatocyte when it antagonizes the induction of P-450 PB4 in intact rats. In untreated hepatocytes, P-450 RLM2 (IIA2), P-450 3 (IIA1) and NADPH P-450 reductase levels were substantially maintained in the cultures for 10-20 days. The latter two enzymes were also inducible by PB to an extent (3-4 fold elevation) that is comparable with that observed in the liver in vivo. Moreover, P-450c (IA1) and P-450 3 (IIA1) were highly inducible by 3-methylcholanthrene (5 microM; 48 h exposure) even after 3 weeks in culture. In contrast, the male-specific pituitary-regulated P-450 form 2c (IIC11) was rapidly lost upon culturing the hepatocytes, suggesting that supplementation of appropriate hormonal factors may be necessary for its expression. The present hepatocyte culture system exhibits a responsiveness to drug inducers that is qualitatively and quantitatively comparable with that observed in vivo, and should prove valuable for more detailed investigations of the molecular and mechanistic basis of the response to PB and its modulation by endogenous hormones.

Cytochrome P450 isozyme induction by methyl ethyl ketone and m-xylene in rat liver

The rat hepatic cytochrome P450 induction pattern caused by administration of a high peroral dose of methyl ethyl ketone (MEK, 1.4 ml/kg once daily for 3 consecutive days) and m-xylene (1.0 ml/kg × 3) was studied by catalytic activity and immunoblotting techniques. MEK caused a marked increase in the amount of P450 isozymes belonging to the phenobarbital- and ethanol-inducible P450 subfamilies P450IIB and P450IIE, respectively. Catalytic activities linked with these isozymes, pentoxyresorufin O-depentylase (P450IIB), aniline hydroxylase, and N-nitrosodimethylamine N-demethylase (P450IIE), were also increased (18.0-, 5.4-, and 2.4-fold, respectively). The activity of ethoxyresorufin O-deethylase, which is predominantly linked with the polycyclic aromatic hydrocarbon-inducible P450 isozymes, was also increased 2.3-fold without an apparent increase in the amount of the respective P450 protein (P450IA). m-Xylene caused a similar induction pattern with less effect on P450IIE. Simultaneous administration of MEK and m-xylene resulted in an additive or, in the case of pentoxyresorufin O-depentylase, a potentiating effect on P450-linked catalytic activities. These data indicate that MEK and m-xylene elicit a qualitatively similar induction of P450 isozymes, which may play a role in the metabolic interactions of these compounds.

Induction of hepatic cytochrome P-450 mediated alkoxyresorufin O-dealkylase activities in different species by prototype P-450 inducers

The induction of cytochrome P-450-mediated alkoxyresorufin O-dealkylase activities by various xenobiotics was examined in liver from a variety of animal species in order to gain insights into the substrate specificities of the induced P-450s. We found that forms of cytochrome P-450 capable of mediating the O-dealkylation of the short-chain phenoxazone ethers methoxy-, ethoxy- and propoxyresorufin were highly induced by 3-methylcholanthrenetype inducers and by Aroclor-1254 in all species tested, although there were species differences in the relative turnover rates for the various substrates. For example, in hamster liver the turnover rates for the short-chain resorufin ethers decreased in the following order: methoxy > ethoxy ⪢ propoxy, while in the rat liver almost the exact opposite order was observed: ethoxy = propoxy ⪢ methoxy. In contrast, the degree of induction by phenobarbital-type inducers of isozymes catalyzing the O-dealkylation of pentoxy- or benzyloxyresorufin was highly species-dependent. Thus, F344/NCr rats, B6C3F1 mice and NZB rabbits showed the greatest (> 20-fold) induction of these activities, either by phenobarbital or Aroclor-1254, while Mongolian gerbils showed intermediate levels of induction and Syrian golden hamsters exhibited very low induction. In the Japanese quail, phenobarbitalor DDT-treatment resulted in minimal induction of pentoxy- or benzyloxyresorufin O-dealkylase activity, although significant induction of the latter activity occurred following treatment with 5,6-benzoflavone or with Aroclor-1254. Since substrate specificities of most enzymes can be rationalized based upon differences in the steric requirements at the enzyme active site, we employed molecular modeling techniques to calculate the molecular dimensions of the alkoxyresorufins. Surprisingly, the minimal energy conformations in vacuo of each of the resorufin ethers examined are essentially planar. However, alternative configurations, especially for the pentoxy-and benzyloxy-ethers, having greater three-dimensional bulk are also energetically possible.

Induction of hepatic cytochrome p‐450 and xenobiotic metabolizing enzymes in rats gavaged with an alberta crude oil

Changes in body weight gain and in biochemical parameters of blood and liver were assessed in Sprague-Dawley rats after multiple oral administration of three test doses of an Alberta crude oil (ACO). Rats treated with ACO (1.25-5 ml/kg) did not show statistically significant (p greater than .05) differences from control, corn-oil treated (5 ml/kg) rats, in body weight gains, liver weight, and blood biochemical indicators of liver (alanine aminotransferase, gamma glutamyltransferase), kidney (blood urea nitrogen, creatinine), and erythrocyte (adenosine 5'-triphosphate, 2,3-diphosphoglyceric acid, reduced glutathione) cytotoxicity. Treatment with ACO, however, caused statistically significant (p less than .05) and dose-related increases from control in (1) microsomal protein and cytochrome P-450 content, and NADPH-cytochrome c reductase, aryl hydrocarbon hydroxylase (AHH), and 7-ethoxycoumarin-O-deethylase (7-ECOD) activities, and (2) cytosolic glutathione transferase activity of liver. The induction of hepatic cytochrome P-450 and xenobiotic-metabolizing enzymes in microsomes of ACO-treated rats was probably associated with dose-related changes in isozymic forms of cytochrome P-450, as evidenced by (1) appearance of a 448-nm spectral peak in microsomes of ACO-treated rats and (2) differences in the inhibition pattern of AHH and 7-ECOD activities in microsomes of control and ACO-treated rats upon treatment with metyrapone and 7,8-benzoflavone.

Attenuation of low dose phenobarbital induction of hepatic microsomal aminopyrine N-demethylase activity in rats by cimetidine

Cimetidine, a substituted imidazole, is an inhibitor of hepatic cytochrome P-450-mediated drug metabolism in rats and humans. We investigated the effect of cimetidine on phenobarbital induction of hepatic microsomal aminopyrine N-demethylase activity in the rat. Phenobarbital induction of aminopyrine N-demethylase was log-linear in the range of 1–6 mg/kg/day and the ED 50 was approximately 3 mg/kg/day. Cimetidine 75 mg/kg (four times a day) attenuated the induction of aminopyrine N-demethylase activity by 58% in low dose (3 mg/kg/day) but not in high dose (40 mg/kg/day) phenobarbital treated rats. This result could not be explained by residual inhibition of enzyme activity by cimetidine and suggests that cimetidine affects the induction of hepatic cytochrome P-450 by low dose phenobarbital.

Development of the cotton rat (Sigmodon hispidus) as a biomonitor of environmental contamination with emphasis on hepatic cytochrome P-450 induction and population characteristics.

Biological monitoring of exposure to various chemical pollutants has been employed for environmental toxic evaluation (Christian 1983; Rowley et al. 1983). The impact of pollutants can be predicted by biochemical responses of the exposed wild animals. Responses linked to biochemical detoxification processes would be especially meaningful because of their direct link to detoxification function. They are generally quite sensitive and precede the onset of more serious pathology at cellular and tissue levels.

Hepatic Ah-receptor levels and the effect of 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) on hepatic microsomal monooxygenase activities in a TCDD-susceptible and -resistant rat strain

Previous studies have shown that in two inbred strains of mice, straightforward correlations exist among the number of hepatic Ah-receptors, enzyme inducibility by TCDD, and lethality of TCDD. Here, studies were conducted in two strains of rats (Han/Wistar and Long-Evans) which differ widely in susceptibility to the lethal effects of TCDD, to determine if these are general phenomenona in TCDD toxicity. The total number of specific binding sites (Ah-receptors) for [ 3H]TCDD proved to be approximately equal in the livers of both rat strains. Likewise, no notable difference was detected in the effect of TCDD on the activities of 7-ethoxyresorufin O-deethylase, 7-ethoxycoumarin O-deethylase, and ethylmorphine N-demethylase or on the amount of cytochrome P-450 in hepatic microsomal fractions. Immunoblot analysis was carried out with monoclonal antibodies (Mabs). Mab 1-7-1 directed against rat liver 3-methylcholanthrene (MC)-inducible P-450 recognized forms P-450 c and P-450 d in TCDD-treated rats in a dose-dependent fashion and to a similar extent in both strains. In contrast, Mab 2-66-3 (against phenobarbital-inducible P-450) did not recognize any proteins in either strain, confirming the conclusion that TCDD elicites a MC-type induction of hepatic cytochrome P-450 in both strains of rats. Thus, it seems that the correlations observed in mice do not hold in rats and therefore should not be generalized. The parameters measured in the present study are causally unrelated to the mechanism of lethal action of TCDD in these rat strains.

High magnitude hepatic cytochrome P-450 induction by an N-substituted imidazole antimycotic, clotrimazole

A 4-fold induction of hepatic microsomal cytochrome P-450 following 3 days of treatment of rats with clotrimazole (75mg/kg), a potent monooxygenase inhibitor, greatly exceeded that evident from similar phenobarbital and dexamethasone treatment. The clotrimazole-induced microsomes exhibited a pattern of monooxygenase activities similar to that seen in microsomes from both phenobarbital- and dexamethasone-treated animals. Precautions were necessary to determine both monooxygenase activities and the full amount of cytochrome P-450 present in microsomes because of interference by residual clotrimazole in the microsomes.

Temporal effects on antipyrine metabolite kinetics in aroclor 1254-treated rats

The in vivo consequences of a single dose of Aroclor 1254 (50 mg/kg) on the drug metabolizing capacity of rats were investigated. A noninvasive method, employing [ N-methyl- 14C]-antipyrine where both 14CO 2 exhalation and urinary excretion of 4-hydroxy-, 3-hydroxymethyl-, and norantipyrine were monitored, was used. A group of rats were sequentially tested over a 3-week period to characterize temporal patterns. The antipyrine metabolite kinetic approach demonstrated that induction of hepatic cytochrome P-450 is maximal 3–6 days after Aroclor 1254 administration and the effects were apparent for at least a further 14–17 days. Evidence is presented to suggest selective effects of Aroclor 1254 on different cytochromes P-450 are apparent in vivo.

The effects of phenytoin dosage on the induction of α1-acid glycoprotein and antipyrine clearance in the dog

Doses of phenytoin from 90 to 800 mg/d were used to study induction of hepatic cytochrome P-450 and plasma alpha 1-acid glycoprotein in dogs. The antipyrine clearance was increased by 80%, which is equivalent to an increase in cytochrome P-450 of 140%, and the plasma glycoprotein concentration rose 200% at the highest dose of phenytoin used. Plasma concentrations of phenytoin were measured at each dose level to provide a definitive value for the amount of inducer present. These data were used to assess the concentration-response relationship for phenytoin inducing either cytochrome P-450 or the glycoprotein. A simple relationship between concentration and effect was not observed, suggesting a complex mechanism of induction.

Microsomal cytochrome P-450 “Handprints”: Five fractions from anion-exchange high-pressure liquid chromatography provide a rapid preliminary screen for selectivity in the induction and destruction of rat hepatic cytochrome P-450 subpopulations

Anion-exchange HPLC was used to monitor changes in rat hepatic cytochrome P-450 fractions resulting from animal exposure to several inducing and hepatotoxic agents. The column eluant was divided into five fractions and the cytochrome P-450 content in each was determined. Induction of hepatic cytochrome P-450 following 2,5-hexanedione and SKF 525-A pretreatment of animals showed an induction pattern in the five-fraction “handprint” similar to that produced by phenobarbital. The patterns of induction following β-naphthoflavone, clofibrate, and isosafrole pretreatments were unique in comparison to each other and also compared to that of phenobarbital. The pattern of cytochrome P-450 loss after allyl alcohol was similar to that seen after cobalt chloride treatment, but differed from that seen following acetaminophen administration. Five-fraction quantitation of the eluant from anion-exchange HPLC of microsomes appears to be a useful technique to screen for and convey the effects of previously uncharacterized inducers and the effects of hepatotoxic agents or heme synthesis inhibitors on rat hepatic cytochrome P-450 subpopulations.

Induction of hepatic cytochrome P-450 by natural steroids: Relationship to the induction of δ-aminolevulinate synthase and porphyrin accumulation in the avian embryo

Certain steroid hormones and their metabolites are potent inducers of δ-aminolevulinate synthase, the rate-limiting enzyme for heme biosynthesis in the liver. In this study we examined the influences of a number of natural steroids on the induction of liver cytochrome P-450, δ-aminolevulinate synthase, and protoporphyrin in the chick embryo in ovo, and the effects of “priming” with a small dose of 3,5-dicarbethoxy-1,4-dihydrocollidine (DDC), an inhibitor of ferrochelatase. There was a close relationship between the induction of cytochrome P-450 and δ-aminolevulinate synthase by steroids in experiments with, as well as in experiments without, DDC priming. In most instances, 5β steroids were more potent inducers than were the corresponding 5α epimers: 5β steroids more potently induced δ-aminolevulinate synthase and/or porphyrin accumulation for 11 of 15 steroid pairs tested and cytochrome P-450 for 10 of 14 pairs. With DDC priming, steroid induction of δ-aminolevulinate synthase and protoporphyrin was greatly accentuated, while the increase in cytochrome P-450 was partially prevented. This experimental model, is reminiscent of certain human porphyrias in which clinical expression is probably due in part to interactions of endogenous steroids with genetic deficiencies of heme pathway enzymes, and in which there is in the liver enhanced activity of δ-aminolevulinate synthase and accumulation of heme pathway intermediates, while hepatic cytochrome P-450 content is largely maintained. These features suggest that decreases in the regulatory “free” heme pool(s) in the liver can occur and produce pronounced δ-aminolevulinate synthase induction without the development of a marked deficiency of the major liver hemoprotein, cytochrome P-450.

The effect of inorganic lead on hepatic biochemical and ultrastructural changes produced by phenobarbital

Lead acetate ( 105 μmol kg , i.p.) decreased rat hepatic cytochrome P-450 to 57% and 63% of control values when measured 24 and 48 h after lead administration, respectively. A large increase in urinary delta-aminolevulinic acid (U-ALA) was observed after lead treatment, indicating a depression of heme synthesis. In addition, lead treatment produced dilated cisternae of the smooth endoplasmic reticulum (SER). Phenobarbital ( 100 mg kg , i.p.) produced an induction of cytochrome P-450, proliferation of the SER, and did not alter U-ALA content. Simultaneous lead and phenobarbital treatment produced a delayed but robust induction of cytochrome P-450, only a moderate rise in U-ALA, and a reduced proliferation of the SER of hepatocytes. Therefore, phenobarbital, an inducer of heme synthetic enzymes, is apparently capable of reversing lead-induced inhibition of heme synthesis as measured by hepatic cytochrome P-450 induction and U-ALA content.

Induction of hepatic cytochrome p-450 in fish and the indication of environmental induction in scup ( Stenotomus chrysops)

The responses of liver microsomal monooxygenases in three teleost species, croaker ( Micropogon undulatus), rainbow trout ( Salmo gairdneri) and scup ( Stenotomus chrysops) to polynuclear aromatic hydrocarbon (PAH) induccrs were compared. In all three species PAH induced higher raies of ethoxyresorufin and/or benzo[ a]pyrene (B[ a]P) metabolism and levels of cytochrome P-450, but the species differed in some other responses. The induction of P-450 appears to be associated with the synthesis of new (or additional) microsomal protein at 57000 daltons in trout and 54000 daltons in scup, identifiable by SDS-polyacrylamide gel electrophoresis. Further da.a suggest that ethoxyresorufin metabolism in scup may be catalyzed by a minor form of P-450 distinct from the major induced protein. All scur examined thus far from natural populations exhibit properties of hepatic microsomes characteristically associated with the induced state. B[ a]P is metabolized by control and untreated scup with a high regiospecificity normally associated with the production of reactive and mutagenic metabolites. which may have implications for animals in the field.

The induction of hepatic cytochrome P-450 in C57 BL/10 and DBA/2 mice by isosafrole and piperonyl butoxide. A comparative study with other inducing agents

The formation of cytochrome P-450 metabolite complexes with isosafrole and piperonyl butoxide in vivo in genetically 'responsive' C57 BL/10 mice and 'non-responsive' DBA/2 mice is described. Displacement of the isosafrole metabolite complex can be brought about by incubation with certain type I ligands. The capacity of isosafrole and piperonyl butoxide to induced cytochrome P-450 was evaluated by measurement of biphenyl 2- and 4-hydroxylase, ethoxyresofurin O-deethylase and ethylmorphine N-demethylase and by sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis and compared with results obtained for phenobarbitone, 3-methylcholanthrene and pregnenolone-16 alpha-carbonitrile. All four monooxygenase activities were elevated by isosafrole and piperonyl butoxide, as were cytochrome P-450 levels in both strains of mice. There was a large increase in biphenyl 2-hydroxylase in microsomes from isosafrole treated mice of both strains on displacement of the metabolite complex. SDS polyacrylamide gel electrophoresis demonstrated that isosafrole and piperonyl butoxide induce protein bands of mol. wt., 54000 in both the responsive and non-responsive strains. In addition, piperonyl butoxide induces a protein band of mol. wt. 49000 in both strains of mice. The changes in metabolic activities on pretreatment with isosafrole and piperonyl butoxide do not correspond to those seen with any single inducing agent. The differences in the inducing capabilities of isosafrole and 3-methylcholanthrene in the 'non-responsive' DBA/2 strain are discussed with reference to possible mechanisms of induction by benzodioxole (methylenedioxyphenyl) compounds.

Enhancement of hepatic microsomal drug metabolism in vitro following ethanol administration.

1. Administration of ethanol intraperitoneally at low dosages (10-25 mg/kg) to rats stimulates hepatic microsomal mixed-function oxidase activity in vitro. 2. Pretreatment with ethanol administered orally has no effect on in vivo drug metabolism as measured by pentobarbitone plasma half-life and has no effect on the excretion of ascorbic acid. Ethanol administration does not enhance its own binding to cytochrome P-450. 3. These observations suggest that the administration of ethanol, at moderate dosage, does not give rise to induction of hepatic cytochrome P-450. 4. Unwashed hepatic microsomes are contaminated with alcohol dehydrogenase, but pretreatment with ethanol does not increase microsomal generation of NADH. 5. Pretreatment with ethanol has no stimulatory effect on NADH-NADP+ transhydrogenation. 6. The stimulation of hepatic drug metabolism in vitro following administration of ethanol is not due to increased cytochrome P-450 nor to increased NADPH, per se, but appears to result from an increase in the activity of NADPH-cytochrome c reductase.

Studies on the induction of CO-binding pigments in liver microsomes by phenobarbital and 3-methylcholanthrene

Phenytoin (5,5-diphenylhydantoin; DPH) induces expression of cytochromes P450 (CYPs). Interactions between DPH and tacrolimus suggested that the persistence of CYP induction after discontinuation of DPH is dependent on the history of administration and dosing period of DPH. However, the relationship between the duration of DPH administration and expression of CYPs in the liver and small intestine of rats is not known. Alterations in levels of P-glycoprotein (P-gp; MDR1; ABCB1) as well as CYPs cause drug interactions in the small intestine. We examined the effects of the duration of DPH administration on expression of CYPs and P-gp in the liver and small intestine of rats. Rats were treated with DPH (100 mg/kg, peroral (p.o.) twice a day (b.d.)) for 2, 4, 8, and 16 d. mRNA levels of CYPs and P-gp were examined using the total RNA extracted from the liver and duodenum 2 h and 24 h after the final administration of DPH. CYP3A activities were determined using microsomes. DPH administration for 2 d and 4 d markedly increased mRNA levels of CYPs such as CYP3A1, CYP3A2, CYP2B1, and CYP2B2 in the liver. A relatively long duration of DPH administration (8 d and 16 d) resulted in abolition of the induction of hepatic CYP but increased CYP3A activities were maintained. These results suggest that the duration of DPH administration could be an important determinant of hepatic CYP induction.