Evidence for cytochrome P450 2B1/2B2 isoenzymes in freshly prepared peripheral blood lymphocytes

Cytochrome P450 2B1 and 2B2, the major hepatic drug metabolizing enzymes belonging to CYP2 family and associated constitutive androstane receptor (CAR) were found to be expressed in peripheral blood lymphocytes (PBL) isolated from rats. As observed in liver, pretreatment of phenobarbital (PB) or phenytoin were found to increase the expression of CYP2B1, CYP2B2 and associated enzyme activity in PBL. Like in liver, blood lymphocyte CYP2B1/2B2 catalyzed the activity of 7-pentoxyresorufin O-dealkylase (PROD). The present data, demonstrating similarities in the regulation of blood lymphocyte CYP2B-isoenzymes with the liver enzymes, suggests that blood lymphocyte CYP2B-isoenzymes could be used as a biomarker to monitor tissue levels.     

Evidence for cytochrome P450 2B1/2B2 isoenzymes in freshly prepared peripheral blood lymphocytes

Cytochrome P450 2B1 and 2B2, the major hepatic drug metabolizing enzymes belonging to CYP2 family and associated constitutive androstane receptor (CAR) were found to be expressed in peripheral blood lymphocytes (PBL) isolated from rats. As observed in liver, pretreatment of phenobarbital (PB) or phenytoin were found to increase the expression of CYP2B1, CYP2B2 and associated enzyme activity in PBL. Like in liver, blood lymphocyte CYP2B1/2B2 catalyzed the activity of 7-pentoxyresorufin O-dealkylase (PROD). The present data, demonstrating similarities in the regulation of blood lymphocyte CYP2B-isoenzymes with the liver enzymes, suggests that blood lymphocyte CYP2B-isoenzymes could be used as a biomarker to monitor tissue levels.     

Phosphorylation/Dephosphorylation steps are crucial for the induction of CYP2B1 and CYP2B2 gene expression by phenobarbital.

The effects of several protein kinase activators and protein phosphatase inhibitors on the phenobarbital (PB)-induced gene expression of CYP2B1 and CYP2B2 (CYP2B1/2B2) in adult rat hepatocytes were investigated. Insulin, epidermal growth factor, interleukin 6, cAMP, phorbol 12-myristate 13-acetate, tumor necrosis factor alpha, vanadate, and okadaic acid were found to suppress the induction of CYP2B1/2B2 at mRNA and protein levels in hepatocytes. cAMP and vanadate completely suppressed the induction of CYP2B1/2B2 gene expression in both rat hepatocytes and liver. The addition of genistein to vanadate-treated hepatocytes partially recovered the induction of CYP2B1/2B1 gene expression by PB. These results of the present study demonstrate that phosphorylation/dephosphorylation steps are crucial for the induction of CYP2B1/2B2 gene expression by PB.     

Ascorbic acid deficiency decreases hepatic cytochrome P-450, especially CYP2B1/2B2, and simultaneously induces heme oxygenase-1 gene expression in scurvy-prone ODS rats

The mechanisms underlying the decrease in hepatic cytochrome P-450 (CYP) content in ascorbic acid deficiency was investigated in scurvy-prone ODS rats. First, male ODS rats were fed a diet containing sufficient ascorbic acid (control) or a diet without ascorbic acid (deficient) for 18?days, with or without the intraperitoneal injection of phenobarbital. Ascorbic acid deficiency decreased hepatic microsomal total CYP content, CYP2B1/2B2 protein, and mitochondrial cytochrome oxidase (COX) complex IV subunit I protein, and simultaneously increased heme oxygenase-1 protein in microsomes and mitochondria. Next, heme oxygenase-1 inducers, that is lipopolysaccharide and hemin, were administered to phenobaribital-treated ODS rats fed sufficient ascorbic acid. The administration of these inducers decreased hepatic microsomal total CYP content, CYP2B1/2B2 protein, and mitochondrial COX complex IV subunit I protein. These results suggested that the stimulation of hepatic heme oxygenase-1 expression by ascorbic acid deficiency caused the decrease in CYP content in liver.     

Age-related changes in the mRNA levels of CYP1A1, CYP2B1/2 and CYP3A1 isoforms in rat small intestine

It has been established beyond doubt that, as well as the liver, the small intestine is an important site of first-pass metabolism of numerous drugs, food components and toxic xenobiotics. However, there is not much information available about age-dependent changes of intestinal biotransformation pathways. In the present paper, we evaluated the relationships between intestinal cytochrome P450 complex activity and the age of animals. The study was carried out on male Sprague–Dawley rats (n = 5) from 5 age series: 0.5-, 2-, 4-, 20-, and 28 months old. Animals at every age series were divided into 4 groups: control and three groups of rats treated with the CYP450 specific inducers: phenobarbital, β-naphtoflavone and dexamethasone, respectively. RNA was isolated from intestinal mucosa, and then standard RT-PCR was used for the analysis of CYP1A1, CYP2B1/2 and CYP3A1 mRNA expression. Additionally, the activities of NADPH-cytochrome P450 and NADH-cytochrome b₅ reductases in the microsomal fraction were biochemically estimated. The constitutive intestinal CYP1A1 mRNA expression changes during maturation and aging. Inducibility of CYP1A1 gene was evident in intestinal mucosa at 2-, 4- and 20-month-old rats. A similar pattern of changes was observed for CYP2B1/2 isoforms. CYP3A1 mRNA expression was not detected in small intestine of 2-week-old rats. In matured rats, constitutive intestinal CYP3A1 expression was low, although after induction, significant increases in CYP3A1 mRNA amount were noted in aged individuals. Intestinal activity of both analyzed reductases was lowest in immature rats and highest in 28-month-old animals. In conclusion, the activity of cytochrome P450 complex in rat small intestine was not decreased by the aging processes, so the high rate of oxidative metabolic reactions in intestinal mucosa can be maintained till the advanced life stage.     

Comparison of genomic and cDNA sequences of guinea pig CYP2B18 and rat CYP2B2: absence of a phenobarbital-responsive enhancer module in the upstream region of the CYP2B18 gene

Potential mechanisms were investigated whereby CYP2B18, a cytochrome P450 gene exhibiting high constitutive expression but only low levels of phenobarbital-inducibility in the guinea pig liver, may be differentially regulated versus the highly inducible rat CYP2B2 gene. To comparatively assess potential regulatory sequences associated with CYP2B18, a guinea pig genomic library was screened enabling isolation of the CYP2B18 gene. The genomic screening process resulted in the identification of at least four closely-related CYP2B18 genes, designated here as CYP2B18A-D. Of these isolates, CYP2B18A exhibited sequence identical to that of the CYP2B18 cDNA. Further, the deduced amino acid sequence of the CYP2B18 cDNA was identical to that of N-terminal and internally-derived peptide sequences obtained in this investigation from CYP2B18 protein isolated from guinea pig liver. Genomic structural sequences were derived for CYP2B18A, together with the respective 5'-upstream and intronic regions of the gene. Comparison of the CYP2B18A and CYP2B2 gene sequences revealed the lack of repetitive LINE gene sequences in CYP2B18A, putative silencing elements that effect neighboring genes, although these sequences were present in both 5'-upstream and 3'-downstream regions of CYP2B2. We determined that the phenobarbital-responsive enhancer module was absent from the 5'-upstream region as well as the intronic regions of CYP2B18A gene. We hypothesize that the compromised phenobarbital inducibility of CYP2B18A stems from its lack of a functional phenobarbital responsive enhancer module.     

Repression of CAR-mediated transactivation of CYP2B genes by the orphan nuclear receptor, short heterodimer partner (SHP)

The induction of CYP2B gene expression by phenobarbital (PB) is mediated by the translocation of the constitutive androstane receptor (CAR) from the cytoplasm to the nucleus. The CAR/RXR heterodimer binds to two DR-4 sites in a complex phenobarbital responsive unit (PBRU) in the CYP2B gene. The short heterodimer partner (SHP), an orphan nuclear receptor that lacks a conventional DNA binding domain, was initially identified by its interaction with CAR. We have examined the role of SHP in CAR-mediated transactivation of the CYP2B gene. Coexpression of SHP inhibited the transactivation of the CYP2B gene by CAR in cultured hepatoma cells and the p160 coactivator GRIP1 reversed the inhibition. The interaction of CAR with SHP was confirmed by GST pulldown experiments. SHP did not block the binding of either CAR/RXR to the PBRU or binding of GRIP1 to the CAR/RXR complex in gel mobility shift assays, but slightly increased CAR/RXR binding and slightly altered the mobility of the CAR/RXR/GRIP1 complex, suggesting an interaction of SHP with these complexes. The presence of SHP in the complexes, however, could not be detected in an antibody supershift assay. Recombinant corepressors mSin3A, SMRT, and HDAC1, but not NCoR1, interacted with GST-SHP but each of these corepressors in liver nuclear extracts bound to GST-SHP. SMRT and NCoR1 inhibited CAR-mediated activation independent of SHP, but mSin3A and HDAC1 had little effect alone, and were additive with SHP. These studies demonstrate that SHP does not inhibit CAR-mediated trans-activation by interfering with DNA binding or by competition with GRIP1. Instead, SHP may either inhibit recruitment of other coactivators by GRIP1 or actively recruit corepressors directly to the CAR/RXR/PBRU complex.     

Hepatic cytochrome P450 2B induction by ethyl/ phenyl-substituted congeners of phenobarbital in the B6C3F1 mouse

The abilities of structural congeners of phenobarbital to induce immunoreactive hepatic cytochrome P450 2B (CYP2B) protein and associated catalytic activity (benzyloxyresorufin O-dealkylation) in the male B6C3F1 mouse were examined. Interspecies differences in inducing ability were examined through comparison of the results with induction data obtained previously with the male F344/NCr rat. The congeners were administered in the diet for 2 weeks at concentrations equimolar to 500 ppm of the prototype CYP2B inducer, phenobarbital. Of the series of compounds tested, phenobarbital was the most effective inducer of benzyloxyresorufin O,-dealkylation and immunoreactive CYP2B protein, with 2-ethyl-2-phenylsuccinimide, 5-ethyl-5-phenylhydantoin, primidone, and glutethimide being only 19–42% as effective. 5-Ethyl-5-phenyloxazolidinedione and the ring-opened and decarboxylated congeners, N-(2-ethyl-2-phenylacetyl)urea and 2-ethyl-2-phenyl-malonamide, displayed minimal induction of these catalytic activities. Dose-response experiments performed with 5-ethyl-5-phenylhydantoin indicated that the intrinsic CYP2B-inducing activity of this congener was as great as that of phenobarbital in the mouse, although a fourfold greater dietary concentration of this hydantoin (2000 ppm) was required to elicit a response equivalent to that caused by 500 ppm phenobarbital. When extent of induction was related to serum total xenobiotic concentration rather than to administered dietary concentration, the potencies of the two congeners were determined to be more similar (58 vs. ≥78 μ M for phenobarbital and 5-ethyl-5-phenylhydantoin, respectively).     

Modulation of murine phenobarbital-inducible CYP2A5, CYP2B10 and CYP1A enzymes by inhibitors of protein kinases and phosphatases.

Phenobarbital causes a multitude of effects in hepatocytes, including increased cell proliferation, inhibition of apoptosis and upregulation of xenobiotic and endobiotic metabolizing enzymes. In this study, the involvement of several protein kinase and phosphatase pathways on constitutive and phenobarbital-induced activities of CYP2A5, CYP2B10 and CYP1A1/2 in primary mouse hepatocytes was determined using well-defined chemical modulators of intracellular protein phosphorylation and desphosphorylation events. A 48-h treatment of the hepatocytes with 2-aminopurine, a nonspecific serine/threonine kinase inhibitor, elicited dose-dependent increases in both basal and phenobarbital-induced CYP2A5 catalytic activity (assayed as coumarin 7-hydroxylation), the maximal induction being 60-fold greater than the control value upon cotreatment with 1.5 mM phenobarbital and 10 mM 2-aminopurine. In contrast, phenobarbital induction of CYP2B10 (pentoxyresorufin O-deethylase) and CYP1A1/2 (ethoxyresorufin O-deethylase) activities were blocked by 2-aminopurine. Increases in CYP2A5 activity were also observed after exposure of the hepatocytes to other protein kinase inhibitors affecting the cell cycle, i.e. roscovitine, K-252a and rapamycin. Inhibitors of protein kinases A and C, as well as tyrosine kinases, did not appreciably affect CYP2A5 activity levels. The serine/threonine phosphatase inhibitors tautomycin, calyculin A and okadaic acid all reduced both basal and phenobarbital-induced CYP2A5, CYP2B10 and CYP1A1/2 activities. These results further strengthen the concept that hepatic CYP2A5 is regulated in a unique way compared with CYP2B10 and CYP1A.     

Expression of cytochrome CYP2B1/2 in nonpregnant, pregnant and fetal rats exposed to tobacco smoke

Four-month-old female Wistar rats were exposed for 20 days to tobacco smoke obtained from non-filter cigarettes. During the exposure, concentration of tobacco smoke was monitored indirectly by measuring the CO level (1500 mg/m3 air). The efficacy of exposure was assessed by measuring urine nicotine and cotinine levels. Cigarette smoke did not change total cytochrome P450 and b5 protein levels in any of the organs studied, and most of these organs did not show any changes in the activity of reductases associated with these cytochromes. Following exposure to tobacco smoke, fetal rat liver expressed CYP2B1/2 protein; in newborns (day 1) both liver and lung showed CYP2B1/2 protein expression and very low pentoxyresorufin O-dealkylase activity. Western blot analysis of adult liver, lung, heart, but not of brain microsomes, showed that tobacco smoke induced CYP2B1/2 in both nonpregnant and pregnant rats, though its expression was lower in the livers and hearts of pregnant females. In the rat and human placenta, neither rat CYP2B1/2 nor human CYP2B6 showed basal or tobacco smoke-induced expression at the protein level. This study shows clearly that the expression of CYP2B1/2, which metabolizes nicotine and some drugs and activates carcinogens, is controlled in rats by age-, pregnancy-, and tissue-specific regulatory mechanisms.