Constitutive expression and localization of cytochrome P-450 1A1 in rat and human brain: presence of a splice variant form in human brain

Cytochrome P-450 function as mono-oxygenases and metabolize xenobiotics. CYP1A1, a cytochrome P-450 enzyme, bioactivates polycyclic aromatic hydrocarbons to reactive metabolite(s) that bind to DNA and initiate carcinogenesis. Northern and immunoblot analyses revealed constitutive expression of Cyp1a1 and CYP1A1 in rat and human brain, respectively. CYP1A1 mRNA and protein were localized predominantly in neurons of cerebral cortex, Purkinje and granule cell layers of cerebellum and pyramidal neurons of CA1, CA2, and CA3 subfields of the hippocampus. RT-PCR analyses using RNA obtained from autopsy human brain samples demonstrated the presence of a splice variant having a deletion of 87 bp of exon 6. This splice variant was present in human brain, but not in the liver from the same individual, and was absent in rat brain and liver. Structural modeling indicated broadening of the substrate access channel in the brain variant. The study demonstrates the presence of a unique cytochrome P-450 enzyme in human brain that is generated by alternate splicing. The presence of distinct cytochrome P-450 enzymes in human brain that are different from well-characterized hepatic forms indicates that metabolism of xenobiotics including drugs could occur in brain by pathways different from those known to occur in liver.     

大鼠原代肝细胞培养方法的建立

目的：探索混合胶原凝胶培养肝细胞的方法,观察培养鼠肝细胞的功能与形态特征,用于评价中药十八反的作用机理。方法：两步法分离鼠肝细胞,与鼠尾胶原溶液混合接种于培养板,观察培养鼠肝细胞的形态学特征和生化指标。采用RT-PCR技术。检测药物对P450亚酶CYP3A1表达的影响,进一步确证该体系的可靠性。结果：双层胶原培养体系可观察到典型的肝细胞形态特征,肝细胞功能检测显示肝细胞合成分泌的尿素、白蛋白,而乳酸脱氢酶漏出量较少。药物对P450亚酶CYP3A1表达的影响呈良好的剂量依赖性,同时双层胶原具有保持肝细胞活性的优点。可作为原代肝细胞培养的条件。结论：混合胶原凝胶培养能保留体内的细胞功能和活性,特别是保留药物代谢酶的活性。     

Structural analysis of CYP2R1 in complex with vitamin D3

The activation of vitamin D to its hormonal form is mediated by cytochrome P450 enzymes. CYP2R1 catalyzes the initial step converting vitamin D into 25-hydroxyvitamin D. A CYP2R1 gene mutation causes an inherited form of rickets due to 25-hydroxylase deficiency. To understand the narrow substrate specificity of CYP2R1 we obtained the hemeprotein in a highly purified state, confirmed the enzyme as a vitamin D 25-hydroxylase, and solved the crystal structure of CYP2R1 in complex with vitamin D₃. The CYP2R1 structure adopts a closed conformation with the substrate access channel being covered by the ordered B′-helix and slightly opened to the surface, which defines the substrate entrance point. The active site is lined by conserved, mostly hydrophobic residues. Vitamin D₃ is bound in an elongated conformation with the aliphatic side-chain pointing toward the heme. The structure reveals the secosteroid binding mode in an extended active site and allows rationalization of the molecular basis of the inherited rickets associated with CYP2R1.     

微生物细胞色素P450与胆固醇的生物代谢

细胞色素P450(CYP450)是一类含亚铁血红素的单加氧酶,广泛存在于各类生物体内,参与多种外源物质的代谢和内源物质的转化,如甾类激素、胆汁酸、胆固醇等的代谢。胆固醇是一种环戊烷多氢菲的衍生物,也是人类重要的脂类物质和许多特殊生物活性物质的前体之一,当其过量时会导致高胆固醇血症、动脉粥样硬化、静脉血栓生成等,对机体产生不利的影响。微生物CYP450酶可催化胆固醇的生物代谢,特别是其中的CYP125酶是胆固醇分解代谢起始的关键酶,可用作调节胆固醇代谢的药物靶标。     

Regional and cellular expression of CYP2D6 in human brain: higher levels in alcoholics

Cytochrome P450 (CYP) 2D6 is expressed in liver, brain and other extrahepatic tissues where it metabolizes a range of centrally acting drugs and toxins. As ethanol can induce CYP2D in rat brain, we hypothesized that CYP2D6 expression is higher in brains of human alcoholics. We examined regional and cellular expression of CYP2D6 mRNA and protein by RT-PCR, Southern blotting, slot blotting, immunoblotting and immunocytochemistry. A significant correlation was found between mean mRNA and CYP2D6 protein levels across 13 brain regions. Higher expression was detected in 13 brain regions of alcoholics (n = 8) compared to nonalcoholics (n = 5) (anovap < 0.0001). In hippocampus this was localized in CA1-3 pyramidal cells and dentate gyrus granular neurons. In cerebellum this was localized in Purkinje cells and their dendrites. Both of these brain regions, and these same cell-types, are known to be susceptible to alcohol damage. For one case, a poor metabolizer (CYP2D6*4/*4), there was no detectable CYP2D6 protein, confirming the specificity of the antibody used. These data suggest that in alcoholics elevated brain CYP2D6 expression may contribute to altered sensitivity to centrally acting drugs and to the mediation of neurotoxic and behavioral effects of alcohol.     

Electrochemistry of cytochromes P450: Analysis of current-voltage characteristics of electrodes with immobilized cytochromes P450 for the screening of substrates and inhibitors

In the current study, an approach to elucidating the substrate specificity of cytochromes P450 based on the analysis of current-voltage characteristics of voltammograms and amperograms is proposed. Data on the electrochemical behavior of bioelectrodes with immobilized cytochromes P450 2B4, 1A2, 3A4, 11A1 (P450scc), and 51b1 (Mycobacterium tuberculosis sterol 14α-demethylase or CYP51 MT) in the presence of typical substrates and inhibitors for these hemoprotein forms are reported. Immobilization of the enzymes was accomplished by using graphite screen-printed electrodes modified with gold nanoparticles and with the synthetic membrane-like compound didodecyldimethylammonium bromide. The method of electro-analysis can be applied to the search of potential substrates and inhibitors of cytochromes P450 and to creation of multichannel electrochemical plates (chips, panels) with immobilized cytochromes P450. Published in Russian in Biokhimiya, 2009, Vol. 74, No. 4, pp. 542–549.     

The Hydroxyl Radical Generated by an Iron(II)/EDTA/Ascorbate System Preferentially Attacks Tryptophan Residues of the Protein

Iron(II)/EDTA/ascorbate-mediated oxidative damage to specific amino acid residues (tryptophan) of serum albumin was studied. The active species generated by Fe(II)/EDTA/ascorbate preferred to react with tryptophan residues rather than histidine or other amino acids. The observation of preferential damage to tryptophan residues of the protein was fully suported by a model experiment using a tryptophan analogue. The reaction of Fe(II)/EDTA/ascorbate to the protein was significantly suppressed by mannitol and dimethysulfoxide, suggesting the participation of the hydroxyl radical generated via Fenton’s reaction. The result was supported by the hydroxyl radical assay using 2-deoxyribose.     

Aflatoxin G1 induced TNF-α-dependent lung inflammation to enhance DNA damage in alveolar epithelial cells

Aflatoxin G₁ (AFG₁), a member of the AF family with cytotoxic and carcinogenic properties, could cause DNA damage in alveolar type II (AT-II) cells and induce lung adenocarcinoma. Recently, we found AFG₁ could induce chronic lung inflammation associated with oxidative stress in the protumor stage. Chronic inflammation plays a critical role in cigarette smoke or benzo[a]pyrene-induced lung tissues damage. However, it is unclear whether and how AFG₁-induced lung inflammation affects DNA damage in AT-II cells. In this study, we found increased DNA damage and cytochrome P450 (CYP2A13) expression in AFG₁-induced inflamed lung tissues. Furthermore, we treated the mice with a soluble tumor necrosis factor (TNF)-α receptor and AFG₁ and found that TNF-α neutralization inhibited the AFG₁-induced chronic lung inflammation in vivo, and then reversed the CYP2A13 expression and DNA damage in AT-II cells. The results suggest that AFG₁ induces TNF-α-dependent lung inflammation to regulate 2A13 expression and enhance DNA damage in AT-II cells. Then, we treated the primary mice AT-II cells and human AT-II like cells (A549) with AFG₁ and TNF-α and found that TNF-α enhanced the AFG₁-induced DNA damage in mice AT-II cells as well as A549 cells in vitro. In AFG₁-exposed A549 cells, TNF-α-enhanced DNA damage and apoptosis were reversed by CYP2A13 small interfering RNA. Blocking NF-κB pathway inhibited the TNF-α-enhanced CYP2A13 upregulation and DNA damage confirming that the CYP2A13 upregulation by TNF-α plays an essential role in the activation of AFG₁ under inflammatory conditions. Taken together, our findings suggest that AFG₁ induces TNF-α-dependent lung inflammation, which upregulates CYP2A13 to promote the metabolic activation of AFG₁ and enhance oxidative DNA damage in AT-II cells.