Differences in the expression and inducibility of cytochrome P450 2B isoenzymes in cultured rat brain neuronal and glial cells

Studies initiated to investigate the distribution of cytochrome P450 2B (CYP2B) isoenzymes in rat brain cells revealed significant activity of CYP2B-dependent 7-pentoxyresorufin-O-dealkylase (PROD) in microsomes prepared from both, cultured rat brain neuronal and glial cells. Neuronal cells exhibited 2-fold higher activity of PROD than the glial cells. RT-PCR and immunocytochemical studies demonstrated significant constitutive mRNA and protein expression of CYP2B in cultured neuronal and glial cells. Induction studies with phenobarbital (PB), a known CYP2B inducer, revealed significant concentration dependent increase in the activity of PROD in cultured brain cells with glial cells exhibiting greater magnitude of induction than the neuronal cells. This difference in the increase in enzyme activity was also observed with RT-PCR and immunocytochemical studies indicating differences in the induction of CYP2B1 and 2B2 mRNA as well as protein expression in the cultured brain cells. Furthermore, a greater magnitude of induction was observed in CYP2B2 than CYP2B1 in the brain cells. Our data indicating differences in the expression and sensitivity of the CYP2B isoenzymes in cultured rat brain cells will help in identifying and distinguishing xenobiotic metabolizing capability of these cells and understanding the vulnerability of the specific cell types toward neurotoxins.     

Quantification of cytochrome P450 reductase gene expression in human tissues.

We have isolated and sequenced cDNA clones that code for a variant of human cytochrome P450 reductase. An RNase protection assay was used to quantify the corresponding mRNA in adult and fetal tissues. The results demonstrate that, in the samples analyzed, the cytochrome P450 reductase gene displays very little inter-individual variation in its expression in adult liver and is subject to little developmental or tissue-specific regulation.     

Regulation of cytochrome P450 expression by sphingolipids

Sphingolipids modulate many aspects of cell function, including the expression of cytochrome P450, a superfamily of heme proteins that participate in the oxidation of a wide range of compounds of both endogenous (steroid hormones and other lipids) and exogenous (e.g. alcohol, drugs and environmental pollutants) origin. Cytochrome P450-2C11 (CYP 2C11) is down-regulated in response to interleukin-1beta (IL-1beta), and this response involves the hydrolysis of sphingomyelin to ceramide as well as ceramide to sphingosine, and phosphorylation of sphingosine to sphingosine 1-phosphate. Activation of ceramidase(s) are a key determinant of which bioactive sphingolipid metabolites are formed in response to IL-1beta. Ceramidase activation also appears to account for the loss of expression of CYP 2C11 when hepatocytes are placed in cell culture, and the restoration of expression when they are plated on Matrigel; hence, this pathway is influenced by, and may mediate, interactions between hepatocytes and the extracellular matrix. Recent studies using inhibitors of sphingolipid metabolism have discovered that sphingolipids are also required for the induction of CYP1A1 by 3-methylcholanthrene, however, in this case, the requirement is for de novo sphingolipid biosynthesis rather than the turnover of complex sphingolipids. These findings illustrate how changes in sphingolipid metabolism can influence the regulation of at least several isoforms of cytochrome P450.     

Transfection by genomic DNA of cytochrome P1-450 enzymatic activity and inducibility.

An aryl hydrocarbon hydroxylase (AHH)-deficient gene A- mutant of the mouse line Hepa-1 was treated with calcium phosphate precipitates of DNA from Hepa-1, the rat line H4IIEC3, or an A- -human hybrid in which the A- mutation is complemented by the corresponding human gene. AHH+ transfectants were isolated by selection with benzo[ghi]perylene plus near UV. In addition, a gene A- mutant which also carries a mutation for hypoxanthine phosphoribosyltransferase deficiency was treated with the above genomic DNAs together with pSV2-gpt DNA, and cotransfectants were isolated after treatment with both benzo[ghi]pereylene and HAT. All transfectants and cotransfectants were inducible for AHH by 2,3,7,8-tetrachlorodibenzo-p-dioxin. Both transfectants and cotransfectants were unstable during culture, rapidly losing AHH activity. Rat DNA-derived transfectants were probed in Southern blots with a cDNA probe to mouse cytochrome P1-450 that cross-hybridizes to the corresponding rat gene. All rat DNA-derived transfectants contained the rat P1-450 gene. In half of the transfectants, the rat gene was amplified four- to sevenfold. In one transfectant, the rat gene was truncated at the 3' end. The proportion of rat DNA in different transfectants, as determined by hybridization to a rat repetitive sequence, ranged from less than 1% to 5%. AHH activity and the rat P1-450 gene segregated together in subclones of one of the transfectants. These results demonstrate that the A gene is either the structural gene for cytochrome P1-450, or another very closely linked gene. Previous results (O. Hankinson et al., J. Biol. Chem. 260:1790-1795, 1985) favor the former alternative.     

Improved cloning and expression of cytochrome P450s and cytochrome P450 reductase in yeast

Combination of the pYeDP60 yeast expression system with a modified version of the improved uracil-excision (USER) cloning technique provides a new powerful tool for high-throughput expression of eukaryotic cytochrome P450s. The vector presented is designed to obtain an optimal 5' untranslated sequence region for yeast (Kozak consensus sequence), and has been tested to produce active P450s and NADPH-cytochrome P450 oxidoreductase (CPR) after 5' end silent codon optimization of the cDNA sequences. Expression of two plant cytochrome P450s, Sorghum bicolor CYP79A1 and CYP71E1, and S. bicolor CPR2 using the modified pYeDP60 vector in all three cases produced high amounts of active protein. High-throughput functional expression of cytochrome P450s have long been a troublesome task due to the workload involved in cloning of each individual P450 into a suitable expression vector. The redesigned yeast P450 expression vector (pYeDP60u) offers major improvements in cloning efficiency, speed, fidelity, and simplicity. The modified version of the USER cloning system provides great potential for further development of other yeast vectors, transforming these into powerful high-throughput expression vectors.     

Induction of cytochrome P450 1A1 expression in captive river otters fed Prudhoe Bay crude oil: evaluation by immunohistochemistry and quantitative RT-PCR

Numerous studies have explored the relationships between exposure to a variety of environmental contaminants, such as polycyclic aromatic hydrocarbons, and induction of cytochrome P450 1A (CYP1A) in different vertebrates. Few controlled studies, however, simulated chronic long-term exposure with repeated non-lethal sampling of the same individuals, which should better represent repeated exposure incidents in animals inhabiting polluted areas. In this study, we investigated the effects of chronic exposure to crude oil on levels of CYP1A1 in endothelial cells of skin biopsies and peripheral mononuclear blood cells in captive river otters (Lontracanadensis) using repeated sampling of the same individuals. We hypothesized that ingestion of oil would result in an increase in levels of CYP1A1 in both targets, and predicted that the relationship between prolonged exposure and expression of CYP1A1 would reach a plateau indicative of continuous detoxification of hydrocarbons. Fifteen wild-caught male otters were acclimated to captivity, and then fed diets containing no oil (control) or diets containing weathered crude oil at 5 mg day^-1 kg^-1 body weight (low-dose) and 50 mg day^-1 kg^-1 body weight (high-dose), at the Alaska Sealife Center in Seward, Alaska, USA. Expression of CYP1A1 was assessed with immunohistochemical analysis of CYP1A1 protein in skin biopsies and by quantitative RT-PCR analysis of CYP1A1 mRNA in mononuclear blood cells. Both assays revealed a decrease between capture and the transfer to captivity, indicating that the enclosure at the Alaska Sealife Center, and the food we offered to the otters were free of potential inducers of CYP1A1. During the exposure period, increases in CYP1A1 expression were registered by both techniques, followed by a decline in CYP1A1 after oil administration ended. Levels of endothelial CYP1A1 in the high-dose group were comparable to those recorded for wild river otters in PWS in 1996 and 1997. Levels of CPY1A1 mRNA in mononuclear blood cells, however, were well below levels recorded for river otters in Prince William Sound, and no correlation was detected between values obtained from the two methods. Thus, our results from this longitudinal study with repeated sampling of the same individuals provide support for the use of cytochrome P450 1A1 as a biomarker for hydrocarbon exposure. Nonetheless, our results also suggest that the induction process of CYP1A1 may be complicated and interacting with other processes in vivo. Such interactions may obscure our ability to describe specific, quantitative, predictable, dose-response relationships between exposure to hydrocarbons and induction of CYP1A1, which are required of reliable biomarkers. Evaluations of such interactions based on theoretical physiological models in live-animals merit further investigation.     

Organic nitrate tolerance is induced by degradation of some cytochrome P450 isoforms

The mechanism of nitrate tolerance is poorly defined. We studied the rat P450 (CYP)-catalyzed conversion of organic nitrate to nitric oxide (NO) by purified CYP isoforms and the relationship between P450 expression and nitrate tolerance following continuous infusion of organic nitrates in rats. CYP1A2 effectively formed NO from isosorbide dinitrate and nitroglycerine (NTG). The hypotensive effect of an NTG bolus injection was abolished in rats which had been previously given a continuous 48 h infusion of NTG. Nitrate tolerance was reversible to control levels 2 days after cessation of the continuous infusion. At 48 h after infusion, NTG-induced NO generation of the vessels increased in acetone (a P450 inducer)-pretreated rats, and nitrite and nitrate levels were markedly greater than in normal rats. The appearance and disappearance of P450 isoforms paralleled the conversion of organic nitrates to NO as assessed by immunohistochemistry and Western blotting. Our observations indicate that nitrate tolerance is in large part the result of decreased P450 expression and activity. Interventions that maintain or increase P450 activity may be a useful strategy to provide sustained relief from ischemic conditions in humans.     

Zonation of cytochrome P450 isozyme expression and induction in rat liver.

The regional expression of six different cytochrome P450 (CYP) forms in rat liver under constitutive and induced conditions was compared using immunological techniques. Immunostaining of consecutive thin sections from control liver revealed that the same hepatocytes, forming a 6-8 cells thick layer surrounding the terminal hepatic venules, were stained for CYP2B1/2, CYP2E1 and CYP3A1. Staining of CYP2A1 extended further into the midzonal region, whereas all cells of the acinus stained for CYPEtOH2. These results were supported by Western blot analysis of cell lysates from the periportal or perivenous region obtained by zone-restricted digitonin treatment during in situ perfusion. The data suggest three distinct patterns of constitutive P450 expression: perivenous-restricted (CYP2B1/2, CYP2E1 and CYP3A1); perivenous-dominated (CYP2A1) and panacinar (CYPEtOH2). Chronic exposure to ethanol caused induction of CYP2E1 in the same cells already being constitutively expressed, whereas CYPEtOH2 was more induced in the periportal area. The relative induction of CYP2B1/2, CYP3A1 and CYPEtOH2 after treatment with phenobarbital was stronger in periportal hepatocytes, resulting in levelling out of the initial perivenous dominance of CYP2B1/2 and CYP3A1, whereas CYPEtOH2 became periportal-dominated. Acetone induced CYP2E1, CYP2C11 and CYP3A1 selectively in the perivenous area. These studies indicate that a particular P450 isozyme is generally induced in the same cells where it is constitutively expressed, and that this regional selectivity is independent of the kind of inducer. The data suggest that, during maturation, the hepatocytes acquire various phenotypes in the periportal and perivenous region, to respond differently to endogenous and exogenous signals in the control of P450 expression.     

Sex differences in androgen-regulated expression of cytochrome P450 aromatase in the rat brain

The basis of functional gender differences in adult responsiveness to testosterone (T) is not yet understood. Conversion of T to estradiol by cytochrome P450 aromatase in the medial preoptic area is required for the full expression of male sexual behavior in rats. High levels of aromatase are found in the medial preoptic nucleus (MPN) and in an interconnected group of sexually dimorphic nuclei which mediate masculine sexual behavior. Within this neural circuit, aromatase is regulated by T, acting through an androgen receptor (AR)-mediated mechanism. This arrangement constitutes a feedforward system because T is both the regulator and the major substrate of aromatase. Preoptic aromatase is thus more active in adult males than in females because of normal sex differences in circulating androgen levels. However, the mechanism of enzyme induction also appears to be sexually dimorphic because equivalent physiological doses of T stimulate aromatase to a greater extent in males than in females. Dose-response studies indicate that the sex difference is apparent over a range of circulating T concentrations and constitute a gender difference in T efficacy, but not potency. Sex differences in aromatase correlate with sex differences in nuclear AR concentrations in most regions of the sexually dimorphic neural circuit, but not in MPN. These results suggest that males may have larger populations of target cells in which aromatase is regulated by androgen, but the lack of a gender difference in AR levels in the MPN suggests that differences in post-receptor mechanisms could also be involved. Measurements of aromatase mRNA in androgen-treated gonadectomized rats demonstrate that sex difference in regulation is exerted pretranslationally. Taken together these results demonstrate a sexually dimorphic mechanism that could potentially limit the action of T in females, and may relate to the enhanced expression of T-stimulated sexual behaviors in males.     

The influence of age on the inducibility of rat liver cytochrome P450IA1 (CYPIA1) and P450IA2 (CYPIA2) mRNAs

The levels of the messenger RNAs for the cytochromes P450IA1 (CYPIA1) and P450IA2 (CYPIA2) were determined in liver cytoplasmic RNA of rats of various ages after maximal induction with either 3-methylcholanthrene or isosafrole and in untreated rats. An increase in the CYPIA1 mRNA levels was observed only after treatment with 3-methylcholanthrene, whereas both 3-methylcholanthrene and isosafrole were able to induce the levels of CYPIA2 mRNA. The study presented here shows that the maximal induction of these 2 mRNAs did not change with age when 3-methylcholanthrene was used as the inducing agent. Isosafrole induction did not yield higher CYPIA1 mRNA levels in young rats but reduced the amount of this mRNA in old animals to levels below the detection limit of our assay. After induction with isosafrole the levels of the CYPIA2 mRNAs in the older age groups were lower than those observed in young rats. It is concluded that with age the responsiveness to cytochrome P450 inducers may change. This change is different for the various cytochrome P450 enzymes and depends on which inducer is used.     

Modulations of cytochrome P450 expression in diabetic mice by berberine

Berberine, an isoquinoline alkaloid isolated from medicinal plants such as Berberis aristata, Coptis chinesis, Coptis japonica, Coscinium fenestatun, and Hydrastis Canadensis, is widely used in Asian countries for the treatment of diabetes, hypertension, and hypercholesterolemia. Interaction between berberine and the cytochrome P450 enzymes (CYPs) has been extensively reported, but there are only a few reports of this interaction in the diabetic state. In this study, the effect of berberine on the mRNA of the CYPs in primary mouse hepatocytes and in streptozotocin (STZ)-induced diabetic mice was investigated. In primary mouse hepatocytes, berberine suppressed the induction of Cyp1a1, Cyp1a2, Cyp2e1, Cyp3a11, Cyp4a10, and Cyp4a14 mRNA expression by their prototypical inducers in a concentration-dependent fashion. However, berberine treatment alone increased the expression of Cyp2b9 and Cyp2b10 mRNA. In vivo, berberine showed the same hypoglycemic activity as metformin, an established hypoglycemic drug. The hepatic mRNA levels of Cyp1a1, Cyp2b9, Cyp2b10, Cyp3a11, Cyp4a10, and Cyp4a14 were increased in STZ-induced diabetic mice. Interestingly, berberine itself suppressed the expression of Cyp2e1, an adverse hepatic event-associated enzyme, while the expression of Cyp3a11, Cyp4a10, and Cyp4a14 were restored to normal levels by berberine. In conclusion, berberine has the potential to modify the expression of CYPs by either suppression or enhancement of CYPs' levels. Consumption of berberine as an anti-hyperglycemic compound by diabetic patients might provide an extra benefit due to its potential to restore the expression of Cyp2e1, Cyp3a, and Cyp4a to normal levels. However, an herb-drug interaction might be of concern since any berberine-containing product would definitely cause pronounced interactions based on CYP3A4 inhibition.     

Occurrence and inducibility of cytochrome P450IIIA in maternal and fetal rats during prenatal development

The purpose of this study was to quantify cytochrome P450IIIA1 in fetal and maternal livers of uninduced and pregnenolone-16 alpha-carbonitrile (PCN) induced rats during the course of prenatal development. The activities and levels of P450IIIA in hepatic microsomes from maternal rats and fetuses at 15-21 days of gestation were measured by triacetyloleandomycin (TAO) inhibited debenzylation of (benzyloxy)phenoxazone and by immunoassay with defined antiserum specific for P450IIIA. P450IIIA was not detectable (less than 10 pmol/mg for maternal microsomes and less than 2 pmol/mg for fetal microsomes) by immunoassay in uninduced maternal or fetal livers. In hepatic microsomes from PCN-induced dams, values ranged from 59.3 to 116 micrograms P450IIIA1/mg of protein during the same gestational period. Changes in debenzylase activity of 15.9-46.5 pmol of resorufin (mg of protein)-1 min-1 were consistent with these findings as were the changes in TAO-inhibitable debenzylase activity. In the transplancentally induced fetal liver, debenzylase activity increased steadily from 0.19 pmol of resorufin mg-1 min-1 at day 15 to 9.34 pmol of resorufin mg-1 min-1 at day 21 and was paralleled by the TAO-inhibitable activity that ranged from 0.09 pmol of resorufin mg-1 min-1 at day 15 to 3.33 pmol of resorufin mg-1 min-1 at day 21. The amount of immunoreactive P450IIIA1 also increased from 0.5 to 28.7 micrograms/mg of microsomal protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vitro metabolism of alachlor by human liver microsomes and human cytochrome P450 isoforms.

Alachlor (2-chloro-N-methoxymethyl-N-(2,6-diethylphenyl)acetamide) is a widely used pre-emergent chloroacetanilide herbicide which has been classified by the USEPA as a probable human carcinogen. The putative carcinogenic metabolite, 2,6-diethylbenzoquinone imine (DEBQI), is formed through a complex series of oxidative and non-oxidative steps which have been characterized in rats, mice, and monkeys but not in humans. A key metabolite leading to the formation of DEBQI is 2-chloro-N-(2,6-diethylphenyl)acetamide (CDEPA). This study demonstrates that male human liver microsomes are able to metabolize alachlor to CDEPA. The rate of CDEPA formation for human liver microsomes (0.0031 +/- 0.0007 nmol/min per mg) is significantly less than the rates of CDEPA formation for rat liver microsomes (0.0353+/-0.0036 nmol/min per mg) or mouse liver microsomes (0.0106 +/- 0.0007). Further, we have screened human cytochrome P450 isoforms 1A1, 1A2, 2B6, 2C8, 2C9, 2C18, 2C19, 2D6, 2E1, and 3A4 and determined that human CYP 3A4 is responsible for metabolism of alachlor to CDEPA. Further work is necessary to determine the extent to which humans are able to metabolize CDEPA through subsequent metabolic steps leading to the formation of DEBQI.     

Regulation of rat hepatic cytochrome P-450: age-dependent expression, hormonal imprinting, and xenobiotic inducibility of sex-specific isoenzymes

The influence of age, sex, and hormonal status on the expression of eight rat hepatic cytochrome P-450 (P-450) isoenzymes was evaluated by both catalytic and immunochemical methods. The male specificity of P-450 2c(male)/UT-A, the major microsomal steroid 16 alpha-hydroxylase of uninduced rat liver [Waxman, D.J. (1984) J. Biol. Chem. 259, 15481-15490], was shown to reflect its greater than or equal to 30-fold induction at puberty in male but not in female rats. The female specificity of P-450 2d(female)/UT-I was shown to reflect its developmental induction in females. P-450 PB-2a/PCN-E was shown to mediate greater than or equal to 85% of microsomal steroid 6 beta-hydroxylase activity; the male specificity of this P-450 largely reflects its developmental suppression in female rats. Neonatal gonadectomy and hormonal replacement experiments established that neonatal androgen "imprints" or programs the male rat for developmental induction of P-450 2c(male)/UT-A, for maintenance of P-450 PB-2a/PCN-E, and for suppression of P-450 2d(female)/UT-I, all of which occur in male rats at puberty. By contrast, the expressed levels of P-450 isoenzymes PB-1/PB-C, 3/UT-F, PB-4/PB-B, ISF-G, and beta NF-B were mostly unaffected by the rats' age, sex, and hormonal status. Studies on the sex specificity of P-450 induction established that the response of these latter five isoenzymes to the P-450 inducers phenobarbital, beta-naphthoflavone, pregnenolone-16 alpha-carbonitrile, and isosafrole is qualitatively and quantitatively equivalent in females as in males.     

Inactivation of the hepatic cytochrome P450 system by conditional deletion of hepatic cytochrome P450 reductase

Cytochrome P450 (CYP) monooxygenases catalyze the oxidation of a large number of endogenous compounds and the majority of ingested environmental chemicals, leading to their elimination and often to their metabolic activation to toxic products. This enzyme system therefore provides our primary defense against xenobiotics and is a major determinant in the therapeutic efficacy of pharmacological agents. To evaluate the importance of hepatic P450s in normal homeostasis, drug pharmacology, and chemical toxicity, we have conditionally deleted the essential electron transfer protein, NADH:ferrihemoprotein reductase (EC, cytochrome P450 reductase, CPR) in the liver, resulting in essentially complete ablation of hepatic microsomal P450 activity. Hepatic CPR-null mice could no longer break down cholesterol because of their inability to produce bile acids, and whereas hepatic lipid levels were significantly increased, circulating levels of cholesterol and triglycerides were severely reduced. Loss of hepatic P450 activity resulted in a 5-fold increase in P450 protein, indicating the existence of a negative feedback pathway regulating P450 expression. Profound changes in the in vivo metabolism of pentobarbital and acetaminophen indicated that extrahepatic metabolism does not play a major role in the disposition of these compounds. Hepatic CPR-null mice developed normally and were able to breed, indicating that hepatic microsomal P450-mediated steroid hormone metabolism is not essential for fertility, demonstrating that a major evolutionary role for hepatic P450s is to protect mammals from their environment.