P450 Protein Levels Research Articles

Downregulation of the gene expression of Cyp2c29 and Cyp3a11 by cecal ligation and puncture-induced sepsis is associated with interleukin-6.

Sepsis is a pathological condition that affects the metabolism of administered drugs, leading to changes in the duration and intensity of their intended efficacies. Proinflammatory cytokines downregulate the expression of cytochrome P450s (P450s). The effects of P450 expression under inflammatory conditions have been studied using prophlogistic substances such as lipopolysaccharide; however, few studies have focused on clinical models of sepsis. Here, we show that cecal ligation and puncture (CLP), an approach for the study of human polymicrobial sepsis, leads to the expression of interleukin-1β (IL-1β), IL-6, and tumor necrosis factor α (TNFα) at 24h after the CLP operation. Following CLP, IL-6-/- mice exhibited markedly lower survival than WT mice. In addition, CLP led to the significant downregulation of Cyp2c29 and Cyp3a11 gene expression in IL-1α-/-/β-/- (IL-1-/-) and TNFα-/- mice as well as in WT mice. In contrast, CLP elicited no significant effect on Cyp3a11 expression in IL-6-/- mice. Although CLP reduced the Cyp2c29 expression level in IL-6-/- mice, the expression of Cyp2c29 was lower in CLP-operated WT mice than in CLP-operated IL-6-/- mice. The reduction in the respective P450 protein levels and activities due to CLP-induced sepsis, reflected in the mRNA expression levels, was abolished by IL-6 depletion. Thus, CLP-induced sepsis downregulates P450 gene expression, particularly Cyp2c expression, and this effect is associated with IL-6 without affecting resistance to CLP-induced sepsis. These findings demonstrate the usefulness of CLP for studying the regulation of P450s and highlight IL-6 as a potential indicator of drug-metabolizing capacity under septic conditions.

Progesterone receptor membrane component 1 (PGRMC1) binds and stabilizes cytochromes P450 through a heme-independent mechanism

Progesterone receptor membrane component 1 (PGRMC1) is a heme-binding protein implicated in a wide range of cellular functions. We previously showed that PGRMC1 binds to cytochromes P450 in yeast and mammalian cells and supports their activity. Recently, the paralog PGRMC2 was shown to function as a heme chaperone. The extent of PGRMC1 function in cytochrome P450 biology and whether PGRMC1 is also a heme chaperone are unknown. Here, we examined the function of Pgrmc1 in mouse liver using a knockout model and found that Pgrmc1 binds and stabilizes a broad range of cytochromes P450 in a heme-independent manner. Proteomic and transcriptomic studies demonstrated that Pgrmc1 binds more than 13 cytochromes P450 and supports maintenance of cytochrome P450 protein levels posttranscriptionally. In vitro assays confirmed that Pgrmc1 KO livers exhibit reduced cytochrome P450 activity consistent with reduced enzyme levels. Mechanistic studies in cultured cells demonstrated that PGRMC1 stabilizes cytochromes P450 and that binding and stabilization do not require PGRMC1 binding to heme. Importantly, Pgrmc1-dependent stabilization of cytochromes P450 is physiologically relevant, as Pgrmc1 deletion protected mice from acetaminophen-induced liver injury. Finally, evaluation of Y113F mutant Pgrmc1, which lacks the axial heme iron-coordinating hydroxyl group, revealed that proper iron coordination is not required for heme binding, but is required for binding to ferrochelatase, the final enzyme in heme biosynthesis. PGRMC1 was recently identified as the causative mutation in X-linked isolated pediatric cataract formation. Together, these results demonstrate a heme-independent function for PGRMC1 in cytochrome P450 stability that may underlie clinical phenotypes.

PGRMC1 Stabilizes Cytochromes P450 And Is Required for Activity In Vivo

Progesterone receptor membrane component 1 (PGRMC1) is a membrane‐bound hemoprotein with many ascribed functions that binds cytochromes P450 in yeast and human cells. In this study, we generated a whole‐body Pgrmc1 KO mouse to examine the function of PGRMC1 in cytochrome P450 biology. Here, we report that Pgrmc1 is a novel post‐translational stabilizer of cytochromes P450 required for cytochrome P450 activity in vivo. In mouse liver, Pgrmc1 bound 13 cytochromes P450 of diverse families (Cyp1, 2, 3, 8, and 51) and functioned to maintain liver cytochrome P450 levels post‐transcriptionally. In the absence of Pgrmc1, cytochrome P450 protein levels were 22–70% of wild‐type. These reductions in P450 enzymes were shown to be physiologically important. Pgrmc1 promoted Cyp1a2 activity in vitro as the Vmax for the Cyp1a2‐dependent O‐deethylation of 7‐ethoxycoumarin was 40% lower in the absence of Pgrmc1 in liver membranes. Mechanistically, PGRMC1 stabilized CYP1A2 post‐translationally, doubling the half‐life of the cytochrome P450 in transfected human cells. The stabilization of cytochromes P450 and promotion of cytochrome P450 activity by Pgrmc1 were the result of a protein‐protein interaction between Pgrmc1 and the cytochrome P450 that did not require Pgrmc1 to bind heme. Reductions in Cyp2e1 protein in Pgrmc1 KO mice protected animals against hepatotoxicity during Cyp2e1‐dependent acetaminophen overdose, the leading cause of acute liver failure in the U.S. This study establishes PGRMC1 as a central component controlling cytochrome P450 activity thus making PGRMC1 the first new player in the liver cytochrome P450 system in 50 years as it joins cytochrome P450 oxidoreductase and cytochrome b5. This study also establishes PGRMC1 as a clinically relevant effector of cytochrome P450 activity, which has direct clinical implications related to the efficacy of pharmacotherapies and will be of interest to pharmacologists, clinicians, and the pharmaceutical industry.Support or Funding InformationM.M. was supported by NIH 5T32GM007445, NIH F31HL131185, and American Heart Association 15PRE25250008. P.E. was supported by NIH R01HL077588 and NIH R21HL094774.

In vitro metabolism of benzo[a]pyrene and dibenzo[def,p]chrysene in rodent and human hepatic microsomes

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous and often carcinogenic contaminants released into the environment during natural and anthropogenic combustion processes. Benzo[a]pyrene (B[a]P) is the prototypical carcinogenic PAH, and dibenzo[def,p]chrysene (DBC) is a less prevalent, but highly potent transplacental carcinogenic PAH. Both are metabolically activated by isoforms of the cytochrome P450 enzyme superfamily to form reactive carcinogenic and cytotoxic metabolites. Metabolism of B[a]P and DBC was studied in hepatic microsomes of male Sprague-Dawley rats, naïve and pregnant female B6129SF1/J mice, and female humans, corresponding to available pharmacokinetic data. Michaelis–Menten saturation kinetic parameters including maximum rates of metabolism (VMAX, nmol/min/mg microsomal protein), affinity constants (KM, μM), and rates of intrinsic clearance (CLINT, ml/min/kg body weight) were calculated from substrate depletion data. CLINT was also estimated from substrate depletion data using the alternative in vitro half-life method. VMAX and CLINT were higher for B[a]P than DBC, regardless of species. Clearance for both B[a]P and DBC was highest in naïve female mice and lowest in female humans. Clearance rates of B[a]P and DBC in male rat were more similar to female human than to female mice. Clearance of DBC in liver microsomes from pregnant mice was reduced compared to naïve mice, consistent with reduced active P450 protein levels and elevated tissue concentrations and residence times for DBC observed in previous in vivo pharmacokinetic studies. These findings suggest that rats are a more appropriate model organism for human PAH metabolism, and that pregnancy's effects on metabolism should be further explored.

Determination of the Dominant Arachidonic Acid Cytochrome P450 Monooxygenases in Rat Heart, Lung, Kidney, and Liver: Protein Expression and Metabolite Kinetics

Cytochrome P450 (P450)-derived arachidonic acid (AA) metabolites serve pivotal physiological roles. Therefore, it is important to determine the dominant P450 AA monooxygenases in different organs. We investigated the P450 AA monooxygenases protein expression as well as regioselectivity, immunoinhibition, and kinetic profile of AA epoxygenation and hydroxylation in rat heart, lung, kidney, and liver. Thereafter, the predominant P450 epoxygenases and P450 hydroxylases in these organs were characterized. Microsomes from heart, lung, kidney, and liver were incubated with AA. The protein expression of CYP2B1/2, CYP2C11, CYP2C23, CYP2J3, CYP4A1/2/3, and CYP4Fs in the heart, lung, kidney, and liver were determined by Western blot analysis. The levels of AA metabolites were determined by liquid chromatography-electrospray ionization mass spectroscopy. This was followed by determination of regioselectivity, immunoinhibition effect, and the kinetic profile of AA metabolism. AA was metabolized to epoxyeicosatrienoic acids and 19- and 20-hydroxyeicosatetraenoic acid in the heart, lung, kidney, and liver but with varying metabolic activities and regioselectivity. Anti-P450 antibodies were found to differentially inhibit AA epoxygenation and hydroxylation in these organs. Our data suggest that the predominant epoxygenases are CYP2C11, CYP2B1, CYP2C23, and CYP2C11/CYP2C23 for the heart, lung, kidney, and liver, respectively. On the other hand, CYP4A1 is the major ω-hydroxylase in the heart and kidney; whereas CYP4A2 and/or CYP4F1/4 are probably the major hydroxlases in the lung and liver. These results provide important insights into the activities of P450 epoxygenases and P450 hydroxylases-mediated AA metabolism in different organs and their associated P450 protein levels.

Cytochrome P450 Dysregulations in Thioacetamide-Induced Liver Cirrhosis in Rats and the Counteracting Effects of Hepatoprotective Agents

Dysregulations of cytochromes P450 (P450s) under liver injury have been extensively studied. However, little is known about the possible reversing effects of hepatoprotective agents, the understanding of which is of great importance in guiding clinical dosage adjustment for patients with liver injury. This study aims to investigate the dysregulation patterns of major P450s in thioacetamide (TAA)-induced liver cirrhosis in rats and the potential counteracting effects of hepatoprotective agents schisandra lignans extract (SLE) and dimethyl diphenyl bicarboxylate (DDB). TAA intoxications for 6 weeks induced apparent liver injury and dramatically reduced the hepatic protein expressions of CYP1A2, CYP2C6, CYP2E1, and CYP3A2 to 18, 71, 30, and 21% of that in the normal control, respectively. Both SLE and DDB treatments could significantly reverse the TAA-induced loss of P450 protein levels, which may be ascribed to their hepatoprotective effects and direct P450-inducing effects that have been confirmed in healthy rats. However, the recovery of enzyme activities of most P450s by SLE and DDB treatment was less evident than that for the protein expression levels. TAA exhibited NADPH-, time-, and concentration-dependent inactivating effects on all of the four major P450 isozymes; both DDB and GSH showed little effects on counteracting such an inactivation efficacy. These findings provided a good explanation on the disproportional effects of hepatoprotective agents in recovering the protein levels and enzyme activities of TAA-induced dysregulated P450s.

Use of isogenic strains indicates CYP9M10 is linked to permethrin resistance in Culex pipiens quinquefasciatus

Previous studies on a strain of Culex pipiens quinquefasciatus from Saudi Arabia indicated permethrin resistance was a result of cytochrome P450 mediated detoxification and kdr. The P450 detoxification was found to be larval specific and associated with a fitness cost in certain environments. The P450 responsible for resistance (and the fitness cost) has not been identified, but recently two candidate P450s (CYP4H34 and CYP9M10) have been found. We measured cytochrome P450 and cytochrome b₅ content as well as the expression levels of CYP4H34 and CYP9M10 in a susceptible (SLAB) and two isogenic strains (isolated by repeated backcrossing and selection) of mosquito (ISOP450 and ISOJPAL) resistant to permethrin. Cytochrome P450 protein levels of the resistant strains were significantly higher (1.5-fold) than SLAB, but were not significantly different from one another. Expression of CYP4H34 in the larvae and adults of the resistant (ISOP450 and ISOJPAL) and susceptible (SLAB) strains were not statistically different. CYP9M10 was found to be significantly over-expressed in larvae of both permethrin-resistant isogenic strains (1800-fold in ISOP450 and 870-fold in ISOJPAL) when compared to SLAB. Partial sequence analysis of CYP9M10 revealed eight polymorphic sites that distinguished the susceptible allele from the resistant allele. We conclude that CYP9M10 is linked to permethrin resistance in these strains of C. p. quinquefasciatus, and is likely to be the P450 gene responsible for resistance in these strains.

Ex vivo carbon monoxide prevents cytochrome P450 degradation and ischemia/reperfusion injury of kidney grafts

Renal ischemia/reperfusion injury is a major complication of kidney transplantation. We tested if ex vivo delivery of carbon monoxide (CO) to the kidney would ameliorate the renal injury of cold storage that can complicate renal transplantation. Orthotopic syngeneic kidney transplantation was performed in Lewis rats following 24 h of cold preservation in University of Wisconsin solution equilibrated without or with CO (soluble CO levels about 40 microM). Ischemia/reperfusion injury in control grafts resulted in an early upregulation of inflammatory mediator mRNAs and progressive deterioration of graft function. In contrast, the grafts preserved with CO had significantly less oxidative injury and this was associated with improved recipient survival compared to the control group. Renal injury in the control group showed considerable degradation of cytochrome P450 heme proteins, active heme metabolism and increased detrimental intracellular free heme levels. Kidney grafts preserved in CO-equilibrated solution maintained their cytochrome P450 protein levels, had normal intracellular heme levels and had less lipid peroxidation. Our results show that CO-mediated suppression of injurious heme-derived redox reactions offers protection of kidney grafts from cold ischemia/reperfusion injury.

Induction of P450 1A by 3-methylcholanthrene protects mice from aristolochic acid-I-induced acute renal injury

Cytochrome P450 1A, an enzyme known to metabolize polycyclic aromatic hydrocarbons (PAHs), participates in the metabolism of aristolochic acid I (AAI) in liver and kidney microsomes isolated from humans and rodents. This study was designed to investigate whether P450 1A plays a role in AAI-induced renal injury in C57BL/6 mice. Separate groups of mice were given AAI (10 mg/kg and 20 mg/kg) or pretreatment with 3-methylcholanthrene (3-MC, an agent known to induce P450 1A expression in many species including rodents) at 60 mg/kg given at 24 h before AAI injection. Renal function and histopathology were determined at the 3rd day following the high dose of AAI and at the 14th day following the low dose of AAI treatment. For both doses, we determined in vivo AAI clearances and pharmacokinetic parameters. We also determined in vitro P450 1A1/2 activity and the ability of liver microsomes from 3-MC-treated and vehicle-treated mice to metabolize AAI. Finally, the effect of 3-MC on protein levels of P450 1A1/2 in both liver and kidney was measured by western blotting. Pretreatment with 3-MC greatly protected mice against renal failure induced by AAI. In vivo AAI clearance was more rapid in 3-MC-pretreated mice than in the vehicle-pretreated mice. In addition, the P450 1A1/2 activity and the ability to metabolize AAI in hepatic microsomes isolated from 3-MC-treated mice were much greater than in vehicle-treated mice. Western blotting showed that protein levels of hepatic P450 1A1/2 were greatly increased in 3-MC-treated mice than in vehicle-treated mice. These results demonstrated that the induction of hepatic P450 1A1/2 protected against AAI-induced kidney injury through faster in vivo clearance of AAI and suggested an important role for hepatic P450s in the detoxification of AAI-induced renal injury.

Influence of CYP2C19*18 and CYP2C19*19 Alleles on Omeprazole 5‐Hydroxylation: In vitro Functional Analysis of Recombinant Enzymes Expressed in Saccharomyces cerevisiae

Omeprazole is one of the most widely used proton pump inhibitors for the treatment of gastric acid-related disorders. The major metabolic pathway of omeprazole is 5-hydroxylation, which is catalysed by CYP2C19. In this study, the effect of CYP2C19*18 and CYP2C19*19 alleles on omeprazole 5-hydroxylation was studied using recombinant CYP2C19 enzymes of wild-type (CYP2C19.1B having Ile331Val) and variants (CYP2C19.18 having Arg329His/Ile331Val and CYP2C19.19 Ser51Gly/Ile331Val) expressed in yeast cells. The K(m) value for omeprazole 5-hydroxylation of CYP2C19.1B was 1.46 microM. The K(m) value of CYP2C19.19 was significantly higher (1.5-fold) than that of CYP2C19.1B. V(max) and V(max)/K(m) values for omeprazole 5-hydroxylation of CYP2C19.1B on the basis of cytochrome P450 protein level were 8.09 pmol/min./pmol CYP and 5.45 microl/min./pmol CYP, respectively. The V(max) value of CYP2C19.19 was significantly higher (1.8-fold) than that of CYP2C19.1B, whereas the V(max)/K(m) value was comparable to that of CYP2C19.1B. In contrast, K(m), V(max) and V(max)/K(m) values of CYP2C19.18 were similar to those of CYP2C19.1B. These results suggest that CYP2C19*19 allele decreases the affinity between CYP2C19 enzyme and the substrate in omeprazole metabolism.

Portal Blood Flow Regulates Volume Recovery of the Rat Liver after Partial Hepatectomy: Molecular Evaluation

Background/Aim: Liver regeneration is a finely tuned process that is closely regulated by multiple cell cycle steps. Although the portal blood flow affects liver regeneration, the molecular mechanism by which the blood flow regulates gene expression and liver function is largely unknown. The aim of this study was to investigate the molecular effect of portal blood flow on hepatocyte proliferation and gene regulation during liver regeneration. Materials and Methods: We developed a simple surgical rat model to investigate the relation between portal blood flow and liver regeneration by partially ligating the portal trunk with 8-0 Proline sutures under microscopy to reduce the blood flow by 40%. We investigated recovery of liver volume, DNA synthesis, and gene expression associated with cell cycle regulators, comparing partially hepatectomized (PH) rats without (PH group; n = 30) and with partial portal ligation (PHPL group; n = 30) for 7 days after the operation. Results: The hepatic tissue blood flow and the recovery ratio between liver weight and body weight in the PHPL group were significantly lower than in the PH group after hepatectomy. The peak 5-bromo-2′-deoxyuridine labeling index in the PHPL group was delayed and weak compared with the PH group. The expression of CT-1 and cyclin D, E, and B mRNAs indicated that the liver regeneration in the PHPL group was delayed and weak. In addition, there was reciprocal expression of C/EBPα and C/EBPβ mRNAs, an observation supported by their nuclear protein levels. Furthermore, the cytochrome P-450 protein level in the PHPL group was higher than that in the PH group 1 day after hepatectomy. Conclusion: The portal blood flow regulates the activity of liver regeneration and the gene expression associated with cell cycle regulators, while the functions are maintained.

Alkane-induced expression, substrate binding profile, and immunolocalization of a cytochrome P450 encoded on the nifD excision element of Anabaena 7120

BackgroundAlkanes have been hypothesized to act as universal inducers of bacterial cytochrome P450 gene expression. We tested this hypothesis on an unusual P450 gene (cyp110) found on a conserved 11 kilobase episomal DNA element of unknown function found in filamentous cyanobacteria. We also monitored the binding of potential substrates to the P450 protein and explored the distribution of P450 protein in vegetative cells and nitrogen-fixing heterocysts using immuno-electron microscopy.ResultsHexadecane treatments resulted in a two-fold increase in mRNA, and a four-fold increase in P450 protein levels relative to control cultures. Hexane, octane and dodecane were toxic and induced substantial changes in membrane morphology. Long-chain saturated and unsaturated fatty acids were shown to bind the CYP110 protein using a spectroscopic spin-shift assay, but alkanes did not bind. CYP110 protein was detected in vegetative cells but not in differentiated heterocysts where nitrogen fixation occurs.ConclusionHexadecane treatment was an effective inducer of CYP110 expression in cyanobacteria. Based on substrate binding profiles and amino acid sequence similarities it is hypothesized that CYP110 is a fatty acid ω-hydroxylase in photosynthetic cells. CYP110 was found associated with membrane fractions unlike other soluble microbial P450 proteins, and in this regard CYP110 more closely resembles eukarytotic P450s. Substrate stablization is an unlikely mechanism for alkane induction because alkanes did not bind to purified CYP110 protein.

Brief undernutrition in late-gestation sheep programs the hypothalamic-pituitary-adrenal axis in adult offspring.

Reduced size at birth in humans has been associated with altered function of the hypothalamic-pituitary-adrenal (HPA) axis in childhood and adult life. Experimentally, maternal undernutrition has also been associated with altered fetal HPA function. However, the relationship between birth size, fetal nutrition, and adult pathophysiology is not clear. We recently have reported that glucose tolerance, blood pressure, and IGF-I levels in adult sheep were more closely associated with birth weight than with nutritional insult in late gestation or with current weight. Here, we report adult HPA function in the same group of animals. Pregnant ewes were severely undernourished for 10 d (UN10) or 20 d (UN20) from 105 d gestation (term, 146 d), or were ad libitum-fed controls. At 30 months, female offspring were subjected to an insulin tolerance test and a CRH plus arginine vasopressin (AVP) challenge. UN20 lambs were lighter at birth, but there were no significant differences in weight at 30 months. Adult UN10 ewes had an increased ACTH response to both CRH+AVP challenge and insulin tolerance test, but no differences in cortisol response. UN10 ewes also demonstrated elevated 11-deoxycortisol concentrations, but lower progesterone concentrations, in response to CRH+AVP challenge. In contrast, the responses of UN20 ewes to these challenges were not different from ad libitum controls. Protein levels of P450(c17) and P450(11beta1) were not significantly different among groups. We conclude that brief maternal undernutrition for 10 d, but not 20 d, in late gestation alters HPA function in adult offspring. In contrast to our previous findings, these HPA effects are independent of birth weight and current weight, suggesting that different mechanisms may be involved in programming different physiological axes.

Effects of anesthesia (tricaine methanesulfonate, MS222) on liver biotransformation in rainbow trout ( Oncorhynchus mykiss)

The effect of tricaine methanesulfonate (MS222) on rainbow trout liver biotransformation rates was investigated with a microsomal model; an in vitro preparation that can be employed with or without the use of an anaesthetic. Two experimental sets of rainbow trout microsomes were tested; one representing in vivo or surgical tricaine exposures and the other representing in vitro tissue/organ collection tricaine exposures. Microsomal incubations were performed on these two experimental groups with phenol as substrate to assess the effects of tricaine on Phase I (ring-hydroxylation) and II (glucuronidation) liver biotransformation by monitoring production of hydroquinone (HQ), catechol (CAT), and phenylglucuronide (PG). The use of a 2-h 100 mg/l exposure of tricaine for surgical anesthesia with or without 24-h recovery did not significantly ( P≤0.05) affect rates of phenol (Phase I and II) biotransformation rates; nor, did the 5-min 300 mg/l tricaine exposure for isolated organ/tissue collection significantly ( P≤0.05) affect phenol (Phase I and II) biotransformation rates. There were also no significant statistical differences ( P≤0.05) in P450 protein levels, or 7-ethoxyresorufin- O-deethylase (EROD) activity in these microsomal assays between any of the tricaine treated rainbow trout and controls.

The contribution of hepatic inactivation of testosterone to the lowering of serum testosterone levels by ketoconazole.

Hepatic biotransformation processes can be modulated by chemical exposure and these alterations can impact the biotransformation of endogenous substrates. Furthermore, chemically mediated alterations in the biotransformation of endogenous steroid hormones have been implicated as a mechanism by which steroid hormone homeostasis can be disrupted. The fungicide ketoconazole has been shown to lower serum testosterone levels and alter both gonadal synthesis and hepatic inactivation of testosterone. The present study examined whether the effects of ketoconazole on the hepatic biotransformation of testosterone contribute to its lowering of serum testosterone levels. Results also were used to validate further the use of the androgen-regulated hepatic testosterone 6alpha/15alpha-hydroxylase ratio as an indicator of androgen status. Male CD-1 mice were fed from 0 to 160 mg/kg ketoconazole in honey. Four h after the initial treatment, serum testosterone levels, gonadal testosterone secretion, and hepatic testosterone hydroxylase activity decreased, and the hepatic testosterone 6alpha/15alpha-hydroxylase ratio increased in a dose-dependent manner. Immunoblot analysis indicated that the transient decline in hepatic biotransformation was not due to reduced P450 protein levels. Rather, hepatic testosterone biotransformation activities were found to be differentially susceptible to direct inhibition by ketoconazole. Differential inhibition was also responsible for the increase seen in the 6alpha/15alpha-hydroxylase ratio. The changes in serum testosterone levels could be explained by decreased gonadal synthesis of testosterone and were not impacted by decreased hepatic biotransformation of testosterone. These results demonstrate that changes in the hepatic hydroxylation of testosterone by ketoconazole, and perhaps other chemicals, have little or no influence serum testosterone levels.

Induction of Rat Hepatic Cytochrome P450 Enzymes by Myristicin

The effect of myristicin on the expression of liver cytochrome P450s and its mRNA levels was examined in rats. Treatment of rats with myristicin (i.p., 500 μmol/kg) caused 2-20 fold increases in fiver P450 1A1/2, 2B1/2, and 2E1 enzyme activities relative to controls. Immunoblot analysis using anti-rat liver P450 1A, 2B, and 2E1 showed that the increases in each of P450 protein levels by myristicin were consistent with those in enzyme activity levels. When increased levels of P450 mRNA by myristicin were examined by Northern blot analysis, levels of mRNA of P450 1A1/2 and 2B1/2 also increased. However, the level of P4502E1 mRNA did not increase. The total amount of spectrally detectable P450, also increased about 1.5-fold following treatment of myristicin. These results demonstrate that myristicin is an inducer of rat liver P450 1A1/2, 2B1/2, and 2E1, and that the induction involves increases in mRNA levels except in the case of P4502E1.

Changes in content of P450 isozymes in hepatic and renal microsomes of the male rat treated with cis-diamminedichloroplatinum.

1. The changes in the activity of drug-metabolizing enzymes and in the content of P450 isozymes in renal and hepatic microsomes after treatment of the male Sprague-Dawley rat with cis-diamminedichloroplatinum (Cisplatin, CDDP) were examined. 2. NADPH-P450 reductase activity in renal microsomes was significantly increased by treatment with CDDP, but lauric acid omega- and (omega-1)-hydroxylation activities of renal microsomes were not increased. 3. The level of P4502C23 was increased significantly and levels of P4504A2 and 4A8 tended to increase in renal microsomes. 4. In hepatic microsomes, lauric acid omega-hydroxylation activity was increased, but (omega-1)-hydroxylation activity was not. Levels of P4502C11 and 3A2, which are male-specific forms, were decreased, whereas levels of P4502A1, 2C7 and 2E1 were increased in hepatic microsomes. The levels of P4504A2 and 4A3 were increased by CDDP and the level of P4504A1 was not changed. Changes in the protein levels of P450 by CDDP were consistent with those in the mRNA levels reported previously (LeBlanc et al. 1992). 5. Male-specific forms in rat liver such as P4502C11 were decreased by CDDP, but those in the kidney such as P4504A2 was not. Therefore, CDDP has different influences on the regulation of hepatic and renal P450s.

Mechanism of induction of cytochrome P-450 by phenobarbital.

Treatment of rats with phenobarbital (PB) leads to a substantial increase in the hepatic levels of translatable polysomal poly(A) + cytochrome P-450 mRNA. An enriched fraction of P-450 mRNA was obtained by agarose gel electrophoresis and used to prepare a cDNA probe by differential hybridization to total mRNA from control and PB-treated rats. The majority of the sequences within the probe hybridized to recombinant DNA plasmids which contained a bona fide P-450 cDNA insert identified by positive hybridization selection and in vitro translation. The cDNA probe was used to demonstrate that PB treatment leads to a 30-fold increase in polysomal P-450 mRNA, which is not due to more efficient utilization of previously existing mRNA but to the appearance of new messenger in the cytoplasm. The induction of cytoplasmic P-450 mRNA by PB was rapid, with increases detected within 3 h of PB injection and steady state levels reached in approximately 20 h. The data suggest that the increase in cytoplasmic P-450 protein levels observed after PB treatment may be totally accounted for by an enhanced rate of synthesis resulting from translation of higher cytoplasmic levels of its specific mRNA. The P-450 mRNA was almost exclusively segregated into the membrane-bound polysome compartment was expected for an mRNA coding for an integral membrane protein of the endoplasmic reticulum.