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.     

AMP-activated protein kinase mediates phenobarbital induction of CYP2B gene expression in hepatocytes and a newly derived human hepatoma cell line

Phenobarbital (PB) administration is known to trigger pleiotropic responses, including liver hypertrophy, tumor promotion, and induction of genes encoding drug-metabolizing enzymes. The induction of human CYP2B6 and the rat (CYP2B1) and mouse (Cyp2b10) homologues by PB is mediated by the nuclear receptor constitutive androstane receptor (CAR). The study of CYP2B gene regulation and CAR activity by PB has been difficult due to the lack of a cellular model. In this study, we describe a novel differentiated human hepatoma cell line (WGA), derived from HepG2, which expresses CYP2B6 and CAR. WGA cells represent a powerful system to study the regulation of CYP2B6 gene expression by PB. There is evidence that CAR activity is regulated by phosphorylation and that regulation of some CYP genes depends on the nutritional status of cells. The AMP-activated protein kinase (AMPK) functions as an energy sensor and is activated when cells experience energy-depleting stresses. In this report, we show that addition of 5-amino-imidazole carboxamide riboside, an AMPK activator, to WGA and human hepatocytes induces CYP2B6 gene expression. Expression of a constitutively active form of AMPK mimics the PB induction of CYP2B6 and CYP2B1 gene expression. Conversely, the expression of a dominant negative form of AMPK inhibits the induction of these genes by PB. Finally, we demonstrate, for the first time, that AMPK activity increases in cells cultured with PB. Our data strongly support a role for AMPK in the PB induction of CYP2B gene expression and provide new insights into the regulation of gene expression by barbiturate drugs.     

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.     

Receptor-mediated gene delivery approach demonstrates the role of 5'-proximal DNA region in conferring phenobarbitone responsiveness to CYP2B2 gene in rat liver in vivo

The phenobarbitone (PB) responsiveness of the 5'-proximal region of the CYP2B1/B2 gene was examined in detail with plasmid DNA constructs containing G-free cassette as reporter, using in vivo targeting of the same DNA constructs into rat liver as galactosylated-polylysine complexes. The contribution of the proximal region (-1 to -179 bp) and the positive element (-69 to -98 bp) identified earlier in this laboratory to PB responsiveness was assessed. The results obtained on PB treatment of rats subjected to receptor-mediated gene delivery to liver were conclusive and dramatic, with the control (saline-treated) rats manifesting very little expression of the reporter, reflecting the in vivo picture of CYP2B1/B2 gene expression. The positive element conferred PB responsiveness to homologous and heterologous promoters. Deletion of the positive element led to elimination of PB response. The entire -179 bp region was significantly more effective in responding to PB treatment than the region up to -98 bp, both containing one copy of the positive element. Thus, the positive element and its flanking sequences in the 5'-proximal region are involved in conferring PB responsiveness to the CYP2B1/B2 gene.     

L1-mediated retrotransposition of murine B1 and B2 SINEs recapitulated in cultured cells

SINEs are short interspersed nucleotide elements with transpositional activity, present at a high copy number (up to a million) in mammalian genomes. They are 80-400 bp long, non-coding sequences which derive either from the 7SL RNA (e.g. human Alus, murine B1s) or tRNA (e.g. murine B2s) polymerase III-driven genes. We have previously demonstrated that Alus very efficiently divert the enzymatic machinery of the autonomous L1 LINE (long interspersed nucleotide element) retrotransposons to transpose at a high rate. Here we show, using an ex vivo assay for transposition, that both B1 and B2 SINEs can be mobilized by murine LINEs, with the hallmarks of a bona fide retrotransposition process, including target site duplications of varying lengths and integrations into A-rich sequences. Despite different phylogenetic origins, transposition of the tRNA-derived B2 sequences is as efficient as that of the human Alus, whereas that of B1s is 20-100-fold lower despite a similar high copy number of these elements in the mouse genome. We provide evidence, via an appropriate nucleotide substitution within the B1 sequence in a domain essential for its intracellular targeting, that the current B1 SINEs are not optimal for transposition, a feature most probably selected for the host sake in the course of evolution.     

Discriminating activation of CYP2B9 expression in male C57BL/6 mouse liver by beta-estradiol

The inducible expression of the cytochrome P450 2B subfamily was investigated in male C57BL/6 (B6) and DBA/2 (D2) mice, as well as their hybrids, B6D2F1, at the mRNA level. The expression of hepatic CYP2B mRNAs in B6 was lightly induced by beta-estradiol (ES), while that by phenobarbital (PB) or 1,1,1-trichloro-2, 2-bis(p-chlorophenyl) ethane (DDT) was prominent. Discriminating analysis showed a novelty that ES markedly induced CYP2B9 mRNA expression, whereas PB and DDT increased CYP2B10 more than CYP2B9 expression: albeit both mRNA species responded to all three inducers. Furthermore, the specific induction by ES of CYP2B9 mRNA in B6 male mice, but not D2 male mice, suggests strain dependency in the regulatory pathway of CYP2B9 expression.     

Long interspersed repeated DNA (LINE) causes polymorphism at the rat insulin 1 locus.

The insulin 1, but not the insulin 2, locus is polymorphic (i.e., exhibits allelic variation) in rats. Restriction enzyme analysis and hybridization studies showed that the polymorphic region is 2.2 kilobases upstream of the insulin 1 coding region and is due to the presence or absence of an approximately 2.7-kilobase repeated DNA element. DNA sequence determination showed that this DNA element is a member of a long interspersed repeated DNA family (LINE) that is highly repeated (greater than 50,000 copies) and highly transcribed in the rat. Although the presence or absence of LINE sequences at the insulin 1 locus occurs in both the homozygous and heterozygous states, LINE-containing insulin 1 alleles are more prevalent in the rat population than are alleles without LINEs. Restriction enzyme analysis of the LINE-containing alleles indicated that at least two versions of the LINE sequence may be present at the insulin 1 locus in different rats. Either repeated transposition of LINE sequences or gene conversion between the resident insulin 1 LINE and other sequences in the genome are possible explanations for this.     

Insulin‐mediated modulation of cytochrome P450 gene induction profiles in primary rat hepatocyte cultures

In this investigation, we examined the effects of insulin on gene induction responsiveness in primary rat hepatocytes. Cells were cultured for 72 hours either in the absence or presence of 1 μM insulin and then exposed to increasing concentrations of phenobarbital (PB; 0.01–3.5 mM). Culturing in the absence of insulin produced 1.5–2‐fold increases in the induction magnitude of CYP2B1 and CYP2B2 mRNA expression resulting from PB exposures, without altering the bell‐shaped dose‐response curve characteristic of this agent. However, for the CYP3A1 gene, insulin removal led to a pronounced shift in both the PB‐induction magnitude and dose‐response relationships of the induction response, with higher levels of CYP3A1 expression resulting from exposures to lower concentrations of inducer. Insulin removal also reduced the time required to attain maximal induction of CYP2B1/2 and CYP3A1 gene expression. The insulin effects were not specific for PB induction, as insulin deprivation similarly enhanced both dexamethasone‐ and β‐naphthoflavone‐inducible CYP3A1 and CYP1A1 expression profiles, respectively. In contrast, the level of albumin mRNA expression was reduced considerably in cells deprived of insulin. We conclude that insulin is an important regulator of inducible and liver‐specific gene expression in primary rat hepatocytes. © 1998 John Wiley & Sons, Inc. J Biochem Toxicol 13: 1–9, 1999     

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.     

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.     

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 O-dealkylation of 7-pentoxyresorufin by cytochrome P450 2B1/2B2 isoenzymes in brain

O-dealkylation of 7-pentoxyresorufin (PR) was studied in rat brain to characterise the functional activity specific for cytochrome P450 2B1/2B2 isoenzymes in brain microsomes. Brain microsomes catalyzed the O-dealkylation of PR in the presence of NADPH. Pretreatment with phenobarbital (PB; 80 mg/kg body wt, i.p.× 5 days) resulted in 3-4 fold induction of pentoxyresorufin-O-dealkylase (PROD) activity while 3-methylcholanthrene (MC; 30 mg/kg body wt, i.p. × 5 days) did not produce any significant increase in enzyme activity. Kinetic studies revealed that the rate of velocity (Vmax) for the O-dealkylation of PR was significantly increased to 2.9 times higher in brain microsomes isolated from PB pretreated rats. In vitro studies using metyrapone, an inhibitor of P450 2B1/2B2 catalyzed reactions and antibody for hepatic PB inducible P450s (P450 2B1/2B2) significantly inhibited the activity of PROD in cerebral microsomes prepared from PB pretreated animals. These studies suggest that PB inducible isoenzymes of P450, i.e. P450 2B1/2B2 specifically catalyze the O-dealkylation of PR in brain microsomes.     

n-6 and n-3 polyunsaturated fatty acids down-regulate cytochrome P-450 2B1 gene expression induced by phenobarbital in primary rat hepatocytes

In mammals, polyunsaturated fatty acids (PUFAs) act not only as an important energy source, but also as substrates for cellular membrane and hormone formation. They also play key roles in cellular metabolism and gene regulation. The objective of the present study was to determine whether individual n-6 and n-3 PUFAs affect cytochrome P-450 2B1 (CYP 2B1) expression induced by phenobarbital (PB) in primary rat hepatocytes. We used 100-microM arachidonic acid (AA), linoleic acid, eicosapentaenoic acid and docosahexaenoic acid (DHA) to test this hypothesis. Phenobarbital-induced CYP 2B1 expression was down-regulated by n-6 and n-3 PUFAs, especially AA and DHA. Prostaglandin (PG) E2 but not PGE3 was found to down-regulate PB-induced CYP 2B1 expression. The cyclooxygenase inhibitor indomethacin (20 microM) attenuated the down-regulation of CYP 2B1 gene expression by n-6 and n-3 PUFAs induced by PB, and maximal attenuation was found in the AA-treated group. We also studied the PGE2 downstream cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) pathway to determine its role in the down-regulation of CYP 2B1 expression by AA with the use of 0.4 mM of the adenylate cyclase inhibitor 9-(tetrahydro-2'-furyl)adenine] (SQ22536) and 7.5 microM of the PKA inhibitor H-89. Both inhibitors attenuated the down-regulation of CYP 2B1 expression by AA. These results suggest that PB-induced CYP 2B1 expression is down-regulated by n-6 and n-3 PUFAs through different pathways. Prostaglandin E2 and the cAMP-dependent PKA pathway were involved in AA down-regulation of CYP 2B1 expression, whereas the down-regulation by n-3 PUFAs is not fully understood yet and the glucocorticoid receptor/constitutive androstane receptor/retinoid X receptor signal transduction cascade can be involved.     

Docosahexaenoic acid down-regulates phenobarbital-induced cytochrome P450 2B1 gene expression in rat primary hepatocytes via the sphingomyelinase/ceramide pathway

Docosahexaenoic acid (DHA) regulates the expression of cytochrome P450 2B1 (CYP 2B1) in rat primary hepatocytes in response to xenobiotics. Ceramide, a lipid signaling molecule, is involved in various physiological processes and can be generated by the hydrolysis of sphingomyelin via sphingomyelinase (SMase). DHA activates SMase and increases ceramide formation in vitro. Ceramides differentially enhance adenylyl cyclase activity in vitro depending on the chain length of their fatty acids. In addition, the cAMP-dependent PKA pathway down-regulates CYP 2B1 expression induced by phenobarbital (PB). In the present study, we determined the effect of DHA on SMase transactivation and the downstream pathway in CYP 2B1 expression induced by PB. SMase was activated by DHA 2 h after treatment, and D609 (an SMase inhibitor) attenuated the inhibition of PB-induced CYP 2B1 expression by DHA. Ceramide formation reached a maximum 3 h after DHA administration. C2-ceramide dose-dependently inhibited PB-induced CYP 2B1 expression and increased intracellular cAMP concentrations. SQ22536 (an adenylyl cyclase inhibitor) and H89 (a PKA-specific inhibitor) partially reversed the inhibition of PB-induced CYP 2B1 expression by C2-ceramide. These results suggest that stimulation of SMase, generation of ceramide and activation of the cAMP-dependent PKA pathway are involved in the inhibition exerted by DHA.     

Functional splice sites in a zebrafish LINE and their influence on zebrafish gene expression

Long interspersed elements (LINEs) are transposable elements that exist in many kinds of eukaryotic genomes, where they have a large effect on genome evolution. There are several thousands to hundreds of thousands of LINE copies in each eukaryotic genome. LINE elements are amplified by a mechanism called retrotransposition, in which a LINE-encoded protein reverse transcribes (copies) its own RNA. We previously isolated two retrotransposition-competent LINEs, ZfL2-1 and ZfL2-2, from zebrafish. Although it has generally been thought that LINEs do not have ‘introns’ (because the LINE RNA is used as the template during retrotransposition), we now show that these two LINEs contain multiple putative functional splice sites. We further show that at least one pair of these splice sites is actually functional in zebrafish cells. Moreover, some of these splice sites are coupled with the splicing signal of a host endogenous gene, thereby generating a new chimeric spliced mRNA variant for this gene. Our results suggest the possible role of these LINE splice sites in modulating retrotransposition and host gene expression.     

Temporal kinetics and concentration-response relationships for induction of CYP1A, CYP2B, and CYP3A in primary cultures of beagle dog hepatocytes

Compared to other species, little information is available on the xenobiotic-induced regulation of cytochrome P450 enzymes in the beagle dog. Dogs are widely used in the pharmaceutical industry for many study types, including those that will impact decisions on compound progression. The purpose of this study was (1) to determine the temporal kinetics of drug-induced changes in canine CYP1A, CYP2B, and CYP3A mRNA and enzymatic activity, and (2) to characterize concentration-response relationships for CYP1A2, CYP2B11, and CYP3A12 using primary cultures of canine hepatocytes treated with beta-naphthoflavone (BNF), phenobarbital (PB), and rifampin (RIF), respectively. CYP1A1 and CYP1A2 mRNA exhibited maximal expression (12,700-fold and 206-fold, respectively) after 36 h of treatment with BNF. PB treatment, but not RIF treatment, caused maximal induction of CYP2B11 mRNA (149-fold) after 48 h of treatment. CYP3A12 and CYP3A26 mRNA levels were increased maximally after 72 h of treatment with PB and RIF (CYP3A12, 35-fold and 18-fold, and CYP3A26, 72-fold and 22-fold with PB and RIF treatment, respectively). Concentration-response relationships for BNF induced 7-ethoxyresorufin O-dealkylation (EROD) (EC(50) = 7.8 +/- 4.2 microM), PB induced 7-benzyloxyresorufin O-dealkylation (BROD) (EC(50) = 123 +/- 30 microM), and PB and RIF induced testosterone 6beta-hydroxylation (EC(50) = 132 +/- 28 microM and 0.98 +/- 0.16 microM) resembled the relationship for human CYP induction compared to that of rodent. Interestingly, RIF had no effect on CYP2B11 expression, which represents a species difference overlooked in previous investigations. Overall, the induction of dog CYP1A, CYP2B, and CYP3A exhibits characteristics that are intermediate to those of rodent and human.