Prostaglandin E2 down-regulation of cytochrome P-450 2B1 expression induced by phenobarbital is through EP2 receptor in rat hepatocytes

Cytochrome P-450 is an important bioactivation-detoxification system in vivo. Its expression is regulated by foreign chemicals and dietary factors, and lipids have been found to regulate its gene expression. We showed previously that prostaglandin E(2) (PGE(2)), a fatty acid metabolite, down-regulates cytochrome P-450 2B1 (CYP 2B1) expression induced by phenobarbital. The objective of the present study was to determine whether PGE(2) type 2 receptor (EP(2))-which is coupled to Gs-protein when bound by PGE(2), leading to cAMP production-is involved in this down-regulation. We also determined the possible roles of EP(2) downstream pathways in this down-regulation. We used a primary rat hepatocyte culture model in which EP(2) was shown to be present to study this question. The intracellular cAMP concentration in primary rat hepatocytes was significantly higher after treatment with 1microM PGE(2) than after treatment with 0, 0.01, or 0.1microM PGE(2). Butaprost, an EP(2) agonist, down-regulated CYP 2B1 expression in a dose-dependent manner. SQ22536, an adenylate cyclase inhibitor, reversed the down-regulation by PGE(2) as did H-89, a protein kinase A inhibitor. These results suggest that EP(2) and the downstream pathways of cAMP and protein kinase A are involved in the down-regulation of CYP 2B1 expression by PGE(2) in the presence of phenobarbital.     

Cloning, Sequencing, and Phylogenetic Analysis of Complementary DNA of Novel Cytochrome P-450 CYP1A in Japanese Eel (Anguilla japonica)

Complementary DNA of cytochrome P-450 CYP1A, in addition to CYP1A1, has been isolated from Japanese eel (Anguilla japonica) liver treated with 3-methylcholanthrene. The cDNA contained a 5′ untranslated region of 66 bp, an open reading frame of 1554 bp coding for 517 amino acids and a stop codon, and a 3′ untranslated region of 1166 bp. The predicted molecular weight of the Japanese eel CYP1A was approximately 58.5 kDa. The nucleotide sequence exhibited identities with the reported CYP1A1 sequences of 77% for Japanese eel, 75% for rainbow trout, 72% for scup, plaice, and butterfly fish, and 71% for toadfish. The deduced amino acid sequence exhibited identities with the reported CYP1A1 sequences of 78% for Japanese eel, 77% for rainbow trout, 75% for scup, 74% for toadfish, 73% for plaice, and 72% for butterfly fish. The novel eel CYP1A obtained had less similarity to the other teleost CYP1A1 proteins (72%–78%) than that of the eel CYP1A1 (74%–80%). When compared with mammalian CYP proteins, the novel eel CYP1A was more similar to the CYP1A1 proteins (54%–56%) than to the CYP1A2 proteins (50%–53%). The phylogenetic tree of the teleost CYP1A genes constructed using the maximum likelihood method suggested that the novel eel CYP1A is ubiquitous among the Anguilliformes. Received August 25, 2000; accepted November 30, 2000     

Role of cytochrome P450 2B sequence variation and gene copy number in facilitating dietary specialization in mammalian herbivores

Theory postulates that dietary specialization in mammalian herbivores is enabled by a specialized set of liver enzymes that process the high concentrations of similar plant secondary metabolites (PSMs) in the diets of specialists. To investigate whether qualitative and quantitative differences in detoxification mechanisms distinguish dietary specialists from generalists, we compared the sequence diversity and gene copy number of detoxification enzymes in two woodrat species: a generalist, the white‐throated woodrat (Neotoma albigula) and a juniper specialist, Stephens’ woodrat (N. stephensi). We focused on enzymes in the cytochrome P450 subfamily 2B (CYP2B), because previous research suggests this subfamily plays a key role in the processing of PSMs. For both woodrat species, we obtained and sequenced CYP2B cDNA, generated CYP2B phylogenies, estimated CYP2B gene copy number and created a homology model of the active site. We found that the specialist possessed on average ~5 more CYP2B gene copies than the generalist, but the specialist's CYP2B sequences were less diverse. Phylogenetic analysis of putative CYP2B homologs resolved woodrat species as reciprocally monophyletic and suggested evolutionary convergence of distinct homologs on similar key amino acid residues in both species. Homology modelling of the CYP2B enzyme suggests that interspecific differences in substrate preference and function likely result from amino acid differences in the enzyme active site. The characteristics of CYP2B in the specialist, that is greater gene copy number coupled with less sequence variation, are consistent with specialization to a narrow range of dietary toxins.     

Lateral segregation of anionic phospholipids in model membranes induced by cytochrome P450 2B1: bi-directional coupling between CYP2B1 and anionic phospholipid

The lateral segregation of anionic phospholipids phosphatidic acid (PA), phosphatidylinositol (PI), and phosphatidylserine (PS) was detected after addition of cytochrome P450 2B1 (CYP2B1). The tendency of lipid clustering was highly dependent on the type of anionic phospholipids examined. PA was the most highly clustered while PI and PS clustered to a lesser degree. Moreover, liposomes containing anionic phospholipids form anionic phospholipid-rich microdomains in the presence of CYP2B1. Anionic phospholipids (mostly notably PA) also increased the ability of CYP2B1 to bind to lipid monolayers. In addition to the ability of CYP2B1 to modulate the physical properties of the membrane, the membrane itself can have reciprocal effects on the activity and conformation of CYP2B1. The catalytic activity of CYP2B1 increased as a function of anionic phospholipid concentration and in the presence of 10 mol% PA, the activity increased by 85%. These results suggest a bi-directional coupling between the CYP2B1 and anionic phospholipids.     

Incorporation of the cytochrome P-450 monooxygenase system into large unilamellar liposomes using octylglucoside,especially for measurements of protein diffusion in membranes

Cytochrome P-450 and NADPH cytochrome P-450 reductase were incorporated into large unilamellar lipid vesicles (200–300 nm in diameter) removing octylglucoside from mixed micelles by dialysis. The large size of the protein-containing liposomes guarantees a negligibly small vesicle tumbling. Such large vesicles are better suited for studies of protein rotation in reconstituted membranes than vesicles prepared by use of bile salts. At present the octylglucoside reconstituted monooxygenase system seems to be the most appropriate model for studying protein-protein and protein-lipid interactions in liver microsomes due to the similarity with respect to the main structural and functional properties, including size.     

Long-term enhancement of cytochrome P450 2B1/2 expression in rat hepatocyte spheroids through adenovirus-mediated gene transfer

Tissue-like structures of cells organized in vitrohave a great potential for a number of clinical and biomedical applications. Cell functions may be modulated with gene delivery, improving the characteristics of these structures. Hepatocytes that self-assemble into spheroids can be transduced through adenovirus-mediated gene transfer. An adenoviral vector (AdGFP) was employed to deliver a gene encoding for green fluorescent protein (GFP) in rat hepatocyte spheroids. GFP fluorescence was detected for at least one month. Furthermore, the rat cytochrome P450 2B1 gene (CYP2B1) was transferred through infection with a recombinant adenovirus (AdCYP2B1) in hepatocyte spheroids cultured in suspension. The CYP2B1/2 mRNA and apoprotein levels were continuously higher for over 23 days compared to phenobarbital-induced and control cultures. P450-catalyzed pentoxyresorufin-O-dealkylation activity was also high in the AdCYP2B1-infected spheroids. In these spheroid cultures, albumin and urea levels were similar to those in uninfected spheroid cultures, indicating that expression of the CYP2B1transgene did not impair these liver-specific functions. Hepatocyte spheroids transduced by recombinant adenoviral vectors can be efficiently used for drug metabolism studies, in implantation, and in bioartificial liver devices. This revised version was published online in August 2006 with corrections to the Cover Date.     

Schisandrin B induced antioxidant response is partly mediated by cytochrome P-4502E1 catalyzed reaction in mouse liver

In order to explore the role of cytochrome P-450 (CYP) 2E1 in schisandrin B (Sch B)-induced antioxidant and heat shock responses, the effects of Sch B treatment on hepatic mitochondrial glutathione antioxidant status (mtGAS) and heat shock protein (Hsp)25/70 expression were compared between wild-type and cyp2e1 knock-out C57B/6N mice. Cyp2e1 knock-out mice exhibited a significantly smaller degree of Sch B-induced enhancement in hepatic mtGAS when compared with the wild-type counterpart. But Hsp25/70 expression induced by Sch B was not affected. Sch B-induced enhancement of mtGAS was corroborated by the increase in hepatic mitochondrial antioxidant capacity, as assessed by in vitro measurement of oxidant production, with the enhancing effect being slightly reduced in the knock-out mice. Using liver microsomes prepared from wild-type and knock-out mice as a source of CYP, Sch B was found to be a good co-substrate for the CYP-catalyzed reaction, with the rate of NADPH oxidation observable in microsomes prepared from knock-out mice being slower. The CYP-catalyzed reaction with Sch B was associated with a concomitant production of oxidant species, with the extent of oxidant production being reduced in cyp2e1 knock-out mouse microsomes. Taken together, the results indicate that CYP2E1 is partly responsible for the hepatic metabolism of Sch B that may trigger the antioxidant response in vivo.     

Induction of a cytochrome P-450-dependent fatty acid monooxygenase in Bacillus megaterium by a barbiturate analog, 1-[2-phenylbutyryl]-3-methylurea

Summary In previous publications from our laboratory, we reported that a soluble, cytochrome P-450-dependent fatty acid monooxygenase from Bacillus megaterium ATCC 14581 can be induced by phenobarbital and a variety of other barbiturates. The tested barbiturates showed an excellent correlation between increasing lipophilicity and increasing inducer potency (Kim BH, Fulco AJ; Biochem Biophys Res Commun 116: 843–850, 1983). The only exception proved to be mephobarbital (N-methylphenobarbital) which, although more lipophilic than phenobarbital, is not an inducer of fatty acid monooxygenase activity. We have now found that 1-[2-phenylbutyryl]-3-methylurea (PBMU), an acylurea that can be derived from mephobarbital by hydrolytic cleavage of the barbiturate ring, is an excellent inducer of this activity. Paradoxically, the addition of mephobarbital to the bacterial growth medium containing PBMU significantly enhances the apparent potency of the acylurea to induce fatty acid monooxygenase activity as measured in cell-free extracts. When cell-free extracts of cells grown separately in PBMU or mephobarbital are mixed no enhancement of activity is seen. This finding suggests that the effect of mephobarbital is to somehow increase the efficiency of PBMU as an inducer of the P-450-dependent fatty acid monooxygenase rather than to induce an activator of this enzyme or a rate-limiting component of the monooxygenase system. Finally, both mephobarbital and PBMU induce the synthesis of total cytochrome P-450 in B. megaterium although PBMU is a much more potent P-450 inducer. For cytochrome P-450 induction, however, there is no synergistic or even additive effect when mephobarbital and PBMU are used together in the bacterial growth medium.Abbreviations PBMU 1-[2-phenylbutyryl]-3-methylurea - M.P. melting point     

家蚕P450基因CYP18A1的克隆、序列分析及转录活性

蜕皮激素对昆虫生长、发育和繁殖有重要调控作用,尤其对蜕皮和变态过程。利用GenBank上登录的蜕皮激素C₂₆羟基化酶候选基因CYP18A1的氨基酸序列对家蚕Bombyx mori全基因组数据库进行BLASTP比对,发现了家蚕直向同源基因(ortholog),其完全编码序列经RT-PCR检测和克隆、测序验证后,再以此为信息探针检索家蚕表达序列标签(expressed sequence tags,EST)数据库进行拼接延伸,获得了一条包括5′非翻译区在内的长度为1 737 bp的cDNA序列,验证结果也表明与电子克隆序列完全一致(GenBank登录号为EF421988,P450命名委员会将其命名为CYP18A1)。该基因的开放阅读框为1 623 bp,编码541个氨基酸,含有包括P450s特征结构域在内的所有昆虫P450基因的5个保守结构域,其推定的分子量为61.67 kD,等电点为 8.54。将该基因cDNA序列与家蚕基因组序列进行比对,结果表明该基因具有6个外显子,5个内含子,外显子／内含子边界符合经典的GT-AG规则。同源性分析也发现家蚕CYP18A1与其他昆虫的直向同源基因具有较高相似性。用RT-PCR方法对家蚕主要发育变态时期与组织进行检测,显示出该基因的转录表达不仅具有时空特异性,而且在表达时期上与已报道的蚕体内蜕皮激素含量变化有紧密的一致性。该研究进一步证实了CYP18A1基因与昆虫体内蜕皮激素代谢平衡相关联。     

Analysis of ALDH1A2, CYP26A1, CYP26B1, CRABP1, and CRABP2 in human neural tube defects suggests a possible association with alleles in ALDH1A2

BACKGROUND: Vitamin A (retinol), in the form of retinoic acid (RA), is essential for normal development of the human embryo. Studies in the mouse and zebrafish have shown that retinol is metabolized in the developing spinal cord and must be maintained in a precise balance along the anteroposterior axis. Both excess and deficiency of RA can affect morphogenesis, including failures of neural tube closure. METHODS: We chose to investigate 5 genes involved in the metabolism or synthesis of RA, ALDH1A2, CYP26A1, CYP26B1, CRABP1, and CRABP2, for their role in the development of human neural tube defects, such as spina bifida. RESULTS: An association analysis using both allelic and genotypic single-locus tests revealed a significant association between the risk for spina bifida and 3 polymorphisms in the gene ALDH1A2; however, we found no evidence of a significant multilocus association. CONCLUSIONS: These results may suggest that polymorphisms in ALDH1A2 may influence the risk for lumbosacral myelomeningocele in humans.     

Molecular basis for the inability of an oxygen atom donor ligand to replace the natural sulfur donor heme axial ligand in cytochrome P450 catalysis. Spectroscopic characterization of the Cys436Ser CYP2B4 mutant

All cytochrome P450s (CYPs) contain a cysteinate heme iron proximal ligand that plays a crucial role in their mechanism of action. Conversion of the proximal Cys436 to Ser in NH₂-truncated microsomal CYP2B4 (ΔCYP2B4) transforms the enzyme into a two-electron NADPH oxidase producing H₂O₂ without monooxygenase activity [K.P. Vatsis, H.M. Peng, M.J. Coon, J. Inorg. Biochem. 91 (2002) 542–553]. To examine the effects of this ligation change on the heme iron spin-state and coordination structure of ΔC436S CYP2B4, the magnetic circular dichroism and electronic absorption spectra of several oxidation/ligation states of the variant have been measured and compared with those of structurally defined heme complexes. The spectra of the substrate-free ferric mutant are indicative of a high-spin five-coordinate structure ligated by anionic serinate. The spectroscopic properties of the dithionite-reduced (deoxyferrous) protein are those of a five-coordinate (high-spin) state, and it is concluded that the proximal ligand has been protonated to yield neutral serine (ROH-donor). Low-spin six-coordinate ferrous complexes of the mutant with neutral sixth ligands (NO, CO, and O₂) examined are also likely ligated by neutral serine, as would be expected for ferric complexes with anionic sixth ligands such as the hydroperoxo-ferric catalytic intermediate. Ligation of the heme iron by neutral serine vs. deprotonated cysteine is likely the result of the large difference in their acidity. Thus, without the necessary proximal ligand push of the cysteinate, although the ΔC436S mutant can accept two electrons and two protons, it is unable to heterolytically cleave the O–O bond of the hydroperoxo-ferric species to generate Compound I and hydroxylate the substrate.     

Tissue-specific expression of ALA synthase-1 and heme oxygenase-1 and their expression in livers of rats chronically exposed to ethanol

5-Aminolevulinic acid synthase-1 (ALAS1) and heme oxygenase-1 (HO-1) are the rate-controlling enzymes for heme biosynthesis and degradation, respectively. Expression of these two genes showed tissue-specific expression pattern at both mRNA and protein levels in selected non-treated rat tissues. In the livers of rats receiving oral ethanol for 10 weeks, ALAS1 mRNA levels were increased by 65%, and the precursor and mature ALAS1 protein levels were increased by 1.8- and 2.3-fold, respectively, while no changes were observed in HO-1 mRNA and protein levels, compared with pair-fed controls. These results provide novel insights into the effects of chronic ethanol consumption on hepatic heme biosynthesis and porphyrias.     

Chain length-dependent cooperativity in fatty acid binding and oxidation by cytochrome P450BM3 (CYP102A1)

Fatty acid binding and oxidation kinetics for wild type P450_BM3 (CYP102A1) from Bacillus megaterium have been found to display chain length-dependent homotropic behavior. Laurate and 13-methyl-myristate display Michaelis-Menten behavior while there are slight deviations with myristate at low ionic strengths. Palmitate shows Michaelis-Menten kinetics and hyperbolic binding behavior in 100 mmol/L phosphate, pH 7.4, but sigmoidal kinetics (with an apparent intercept) in low ionic strength buffers and at physiological phosphate concentrations. In low ionic strength buffers both the heme domain and the full-length enzyme show complex palmitate binding behavior that indicates a minimum of four fatty acid binding sites, with high cooperativity for the binding of the fourth palmitate molecule, and the full-length enzyme showing tighter palmitate binding than the heme domain. The first flavin-to-heme electron transfer is faster for laurate, myristate and palmitate in 100 mmol/L phosphate than in 50 mmol/L Tris (pH 7.4), yet each substrate induces similar high-spin heme content. For palmitate in low phosphate buffer concentrations, the rate constant of the first electron transfer is much larger than k_cat. The results suggest that phosphate has a specific effect in promoting the first electron transfer step, and that P450_BM3 could modulate Bacillus membrane morphology and fluidity via palmitate oxidation in response to the external phosphate concentration.     

Intratumoral activation of cyclophosphamide by retroviral transfer of the cytochrome P450 2B1 in a pancreatic tumor model. Combination with the HSVtk/GCV system

Background

Pancreatic cancer is one of the most aggressive human tumors and the development of new therapeutic approaches is particularly urgent since current therapies are not effective. The use of pro‐drug‐activating genes is a possible approach for cancer gene therapy.

Methods

The present study evaluated the efficiency of the cytochrome P4502B1 (CYP2B1) suicide gene that encodes the enzyme responsible for activating the pro‐drug cyclophosphamide (CPA), in pancreatic tumor cells invitro and in vivo. The effects on tumor growth of the combination of two suicide systems, CYP2B1/CPA and herpes simplex virus thymidine kinase gene/ganciclovir (HSVtk/GCV), were also studied.

Results

Retroviral CYP2B1 transfer followed by CPA treatment highly sensitized pancreatic tumor cells NP‐9, NP‐18, and NP‐31, and led to stabilization of tumor growth in a pancreatic tumor model. Differences in tumor volume at the end of the treatment were statistically significant when compared with animals injected with CPA alone. The combination of both suicide systems CYP2B1/CPA and HSVtk/GCV in vitro resulted in a potentiation of the killing effect. However, no potentiation was achieved in vivo, although retardation in tumor growth was evident.

Conclusions

The results show that in situ transduction of pancreatic tumor cells with the CYP2B1 gene by retroviral vectors clearly increases the sensitivity to CPA. Moreover, they suggest that in order to achieve a potentiation on cell killing when the two suicide systems HSVtk/GCV and CYP2B1/CPA are combined, co‐expression of both genes in the same tumor cell would be necessary. Copyright © 2002 John Wiley & Sons, Ltd.

     

The CYP2E1 inhibitor DDC up-regulates MMP-1 expression in hepatic stellate cells via an ERK1/2- and Akt-dependent mechanism

DDC (diethyldithiocarbamate) could block collagen synthesis in HSC (hepatic stellate cells) through the inhibition of ROS (reactive oxygen species) derived from hepatocyte CYP2E1 (cytochrome P450 2E1). However, the effect of DDC on MMP-1 (matrix metalloproteinase-1), which is the main collagen degrading matrix metalloproteinase, has not been reported. In co-culture experiments, we found that DDC significantly enhanced MMP-1 expression in human HSC (LX-2) that were cultured with hepatocyte C3A cells either expressing or not expressing CYP2E1. The levels of both proenzyme and active MMP-1 enzyme were up-regulated in LX-2 cells, accompanied by elevated enzyme activity of MMP-1 and decreased collagen I, in both LX-2 cells and the culture medium. H₂O₂ treatment abrogated DDC-induced MMP-1 up-regulation and collagen I decrease, while catalase treatment slightly up-regulated MMP-1 expression. These data suggested that the decrease in ROS by DDC was partially responsible for the MMP-1 up-regulation. ERK1/2 (extracellular signal-regulated kinase 1/2), Akt (protein kinase B) and p38 were significantly activated by DDC. The ERK1/2 inhibitor (U0126) and Akt inhibitor (T3830) abrogated the DDC-induced MMP-1 up-regulation. In addition, a p38 inhibitor (SB203580) improved MMP-1 up-regulation through the stimulation of ERK1/2. Our data indicate that DDC significantly up-regulates the expression of MMP-1 in LX-2 cells which results in greater MMP-1 enzyme activity and decreased collagen I. The enhancement of MMP-1 expression by DDC was associated with H₂O₂ inhibition and coordinated regulation by the ERK1/2 and Akt pathways. These data provide some new insights into treatment strategies for hepatic fibrosis.