Cytochrome P4502C9 (CYP2C9) allele frequencies in Canadian Native Indian and Inuit populations.

CYP2C9 is the major P450 2C enzyme in human liver and contributes to the metabolism of a number of clinically important substrate drugs. This polymorphically expressed enzyme has been studied in Caucasian, Asian, and to some extent in African American populations, but little is known about the genetic variation in Native American populations. We therefore determined the 2C9*2 (Arg144Cys) and 2C9*3 (Ile359Leu) allele frequencies in 153 Native Canadian Indian (CNI) and 151 Inuit subjects by PCR-RFLP techniques. We also present genotyping data for two reference populations, 325 Caucasian (white North American) and 102 Chinese subjects. Genotyping analysis did not reveal any 2C9*4 alleles in the CNI, Inuit, Caucasian, or Chinese individuals. The 2C9*2 allele appears to be absent in Chinese and Inuit populations, but was present in CNI and Caucasian subjects at frequencies of 0.03 and 0.08-0.15, respectively. The 2C9*3 allele was not detected in the Inuit group, but occured in the CNI group (f = 0.06) at a frequency comparable to that of other ethnic groups. This group of Inuit individuals are the first population in which no 2C9*2 or *3 alleles have been detected so far. Therefore, these alleles may be extremely rare or absent, and unless other novel polymorphisms exist in this Inuit group one would not anticipate any CYP2C9 poor metabolizer subjects among this population.     

Cytochrome P450IIE1 (CYP2E1) is induced by skatole and this induction is blocked by androstenone in isolated pig hepatocytes

Skatole, a derivative of tryptophan, is produced in the hind-gut of pigs and is metabolised via hepatic cytochrome P4502E1 (CYP2E1). Excessive accumulation of skatole together with androstenone, a metabolite of testosterone, in adipose tissue in some pigs is a major cause of 'boar taint' and is associated with defective expression of CYP2E1. This phenomenon is not understood because factors regulating CYP2E1 expression in pig liver have not yet been characterised. Therefore effects of skatole and androstenone on CYP2E1 expression were studied using isolated pig hepatocytes as a model system. Skatole induced CYP2E1 protein expression to the same degree as did acetone, a known CYP2E1 inducer. Induction by skatole was maximum between 20 and 28 h and a half-maximum effect was obtained at a skatole concentration of 0.2 mM. Induction of CYP2E1 by skatole was protein-synthesis dependent. Androstenone antagonised the effect of skatole on CYP2E1 expression but did not affect the CYP2E1 protein level when added alone. These results suggest that defective expression of CYP2E1 in some pigs is due to excessive concentrations of androstenone which prevent CYP2E1 induction by its substrate skatole. As a result, skatole metabolism is reduced and skatole is accumulated in adipose tissue.     

Cytochrome P450(cin) (CYP176A), isolation,expression, and characterization

Cytochromes P450 are members of a superfamily of hemoproteins involved in the oxidative metabolism of various physiologic and xenobiotic compounds in eukaryotes and prokaryotes. Studies on bacterial P450s, particularly those involved in monoterpene oxidation, have provided an integral contribution to our understanding of these proteins, away from the problems encountered with eukaryotic forms. We report here a novel cytochrome P450 (P450(cin), CYP176A1) purified from a strain of Citrobacter braakii that is capable of using cineole 1 as its sole source of carbon and energy. This enzyme has been purified to homogeneity and the amino acid sequences of three tryptic peptides determined. By using this information, a PCR-based cloning strategy was developed that allowed the isolation of a 4-kb DNA fragment containing the cytochrome P450(cin) gene (cinA). Sequencing revealed three open reading frames that were identified on the basis of sequence homology as a cytochrome P450, an NADPH-dependent flavodoxin/ferrodoxin reductase, and a flavodoxin. This arrangement suggests that P450(cin) may be the first isolated P450 to use a flavodoxin as its natural redox partner. Sequencing also identified the unprecedented substitution of a highly conserved, catalytically important active site threonine with an asparagine residue. The P450 gene was subcloned and heterologously expressed in Escherichia coli at approximately 2000 nmol/liter of original culture, and purification was achieved by standard protocols. Postulating the native E. coli flavodoxin/flavodoxin reductase system might mimic the natural redox partners of P450(cin), it was expressed in E. coli in the presence of cineole 1. A product was formed in vivo that was tentatively identified by gas chromatography-mass spectrometry as 2-hydroxycineole 2. Examination of P450(cin) by UV-visible spectroscopy revealed typical spectra characteristic of P450s, a high affinity for cineole 1 (K(D) = 0.7 microm), and a large spin state change of the heme iron associated with binding of cineole 1. These facts support the hypothesis that cineole 1 is the natural substrate for this enzyme and that P450(cin) catalyzes the initial monooxygenation of cineole 1 biodegradation. This constitutes the first characterization of an enzyme involved in this pathway.     

Nerve growth factor mRNA in brain: localization by in situ hybridization

Nerve Growth Factor is a 118 amino acid polypeptide that plays an important role in the differentiation and survival of neurons. The recent discovery that a mRNA that encodes beta Nerve Growth Factor is present in brain suggests that the Nerve Growth Factor gene may not only regulate gene expression of peripheral but also of central neurons. To identify the site(s) of Nerve Growth Factor mRNA production in the brain and to determine which cells express the Nerve Growth Factor gene, the technique of in situ hybridization was employed. A 32P-labeled RNA probe complementary to Nerve Growth Factor mRNA hybridized to cells in the stratum granulosum of the dentate gyrus and the stratum pyramidale of the hippocampus. These observations identify for the first time cellular sites of Nerve Growth Factor gene expression in the central nervous system, and suggest that Nerve Growth Factor mRNA is produced by neurons.     

Localization of cytochrome P450 CYP2S1 expression in human tissues by in situ hybridization and immunohistochemistry.

CYP2S1 is a recently discovered dioxin-inducible member of the cytochrome P450 superfamily. It has been shown to be involved in the metabolism of some aromatic hydrocarbons as well as retinoic acid, suggesting a role in biotransformation of both exogenous and endogenous compounds. In this study, we used mRNA in situ hybridization and immunohistochemistry to investigate the cellular localization of CYP2S1 in various human tissues using tissue microarrays. High expression levels were observed mainly in epithelial cell types, especially in the epithelia frequently exposed to xenobiotics. In the respiratory tract, the expression was strong in nasal cavity, bronchi, and bronchioli, whereas it was low in the alveolar lining cells. Similarly, CYP2S1 was highly expressed in the epithelial cells throughout the gastrointestinal tract. Strong epithelial expression was also observed in uterine cervix, urinary bladder, and skin. In many exocrine glands (e.g., adrenal gland and pancreas), secretory epithelial cells showed moderate to strong expression levels. In the liver, the expression was low. CYP2S1 was highly expressed in epithelial cells that are major targets for carcinogen exposure and common progenitor cells to tumor development. Indeed, we found strong CYP2S1 expression in many tumors of epithelial origin.     

Ethanol Increases Cytochrome P4502E1 and Induces Oxidative Stress in Astrocytes

Abstract: We demonstrate the presence of cytochrome P4502E1 (CYP2E1) in astrocytes in primary culture, its induction by ethanol, and the concomitant generation of free radical species. Double immunofluorescence using anti-CYP2E1 and anti-glial fibrillary acidic protein showed that CYP2E1 was distributed over the cytoplasm and processes, although labeling was more pronounced over the nuclear membrane. Immunogold labeling confirmed this pattern of distribution. Addition of 25 m M ethanol to the astrocyte culture medium for 14 days resulted in an increase in the CYP2E1 content, as determined by confocal microscopy and dot blot. In addition, ethanol induced a dose-dependent increase in the formation of reactive oxygen species that was partially prevented by incubating the astrocytes with anti-CYP2E1. Alcohol also induced a dose-dependent increase in malonaldehyde and hydroxynonenal formation and a depletion of the glutathione (GSH) content. These results suggest that ethanol induces oxidative damage in astrocytes, which could explain some of the toxic effects of ethanol on these cells, such as cytoskeletal alterations. This assumption is supported here by the fact that an increase in GSH content prevents the deleterious effects of alcohol on the cytoskeleton of astrocytes. These results suggest that importance of oxidative stress as a mechanism involved in alcohol-induced neural and brain damage.     

Chromosomal localization of sixty autosomal loci in sheep (OVIS ARIES, 2n = 54) by fluorescence in situ hybridization and R-banding

Sixty autosomal loci (5 type I and 55 type II) from 24 bovine syntenic groups, and previously FISH-mapped to goat and river buffalo chromosomes, were localized by fluorescence in situ on sheep (OVIS ARIES, 2n = 54) chromosomes, thereby notably extending the cytogenetic map of this economically important species. Caprine BAC clones were hybridized to R-banded chromosome preparations. FITC-signals and RBPI- banding (R-banding by late BrdU-incorporation and propidium iodide staining) were simultaneously visualized and captured by a colour CCD-camera. All mapped loci were localized on homoeologous chromosomes and chromosome regions (bands) of sheep, goat and river buffalo, further supporting chromosome and genetic (loci) homoeologies among bovids.     

Cytochrome P450 aromatase (CYP19) in the non-human primate brain: distribution, regulation, and functional significance

In adult male primates, estrogens play a role in both gonadotropin feedback and sexual behavior. Inhibition of aromatization in intact male monkeys acutely elevates serum levels of luteinizing hormone, an effect mediated, at least partially, within the brain. High levels of aromatase (CYP19) are present in the monkey brain and regulated by androgens in regions thought to be involved in the central regulation of reproduction. Androgens regulate aromatase pretranslationally and androgen receptor activation is correlated with the induction of aromatase activity. Aromatase and androgen receptor mRNAs display both unique and overlapping distributions within the hypothalamus and limbic system suggesting that androgens and androgen-derived estrogens regulate complimentary and interacting genes within many neural networks. Long-term castrated monkeys, like men, exhibit an estrogen-dependent neural deficit that could be an underlying cause of the insensitivity to testosterone that develops in states of chronic androgen deficiency. Future studies of in situ estrogen formation in brain in the primate model are important for understanding the importance of aromatase not only for reproduction, but also for neural functions such as memory and cognition that appear to be modulated by estrogens.     

Rat brain thioltransferase: regional distribution, immunological characterization, and localization by fluorescent in situ hybridization

Thioltransferase (TTase) is a member of the family of thiol-disulfide oxidoreductases that are involved in the maintenance of sulfhydryl homeostasis in cells by catalyzing thiol-disulfide interchange reactions. One of the major consequences of oxidative stress in brain is the formation of protein-glutathione mixed disulfides (through oxidation of protein thiols), which can be reversed by TTase during the recovery of brain from oxidative stress. We therefore examined the presence of TTase in brain regions from rat. In the rat, TTase activity in the whole brain was comparable with the corresponding activity in liver, but significantly higher in hippocampus. The enzyme activity was significantly lower in striatum and cerebellum compared with activity in whole brain. Rat brain TTase shared immunological similarity with the human red blood cell enzyme, but not with the pig liver enzyme. The constitutive expression of the mRNA to TTase was demonstrable by northern blotting. Localization of the TTase mRNA in rat brain by fluorescent in situ hybridization showed the presence of high amounts of mRNA in the olfactory bulb, cortex, and hippocampus and its predominant localization in the neurons. TTase mRNA was also present in Purkinje cells in the cerebellum, in giant reticular neurons in the midbrain, and in the striatal and thalamic neurons. This study demonstrates the constitutive presence of a functional TTase system in brain and delineates the regional and cellular localization of the enzyme in rat brain.     

Cytochrome P4502E1, oxidative stress, JNK, and autophagy in acute alcohol-induced fatty liver

Binge alcohol drinking induces hepatic steatosis. Recent studies showed that chronic ethanol-induced fatty liver was, at least in part, CYP2E1 dependent. The mechanism of acute alcohol-induced steatosis and whether CYP2E1 plays any role are still unclear. Increasing oxidative stress by alcohol can activate the JNK MAP kinase signaling pathway, suggesting that JNK might be a target for prevention of alcohol-induced steatosis. We used CYP2E1 knockout (KO) mice, a JNK inhibitor, and JNK1 or JNK2 knockout mice to test the role of CYP2E1, JNK, and the individual role of JNK1 and JNK2 in acute alcohol-induced steatosis. In wild-type (WT) mice, acute alcohol activates CYP2E1 and increases oxidative stress, which reciprocally increases activation of the JNK signaling pathway. Acute alcohol-induced fatty liver and oxidative stress were blunted in CYP2E1 KO mice and by the JNK inhibitor in WT mice. The antioxidant N-acetylcysteine decreased the acute alcohol-induced oxidative stress, the activation of JNK, and the steatosis but not the activation of CYP2E1. Acute alcohol decreased autophagy and increased expression of SREBP, effects blocked by the JNK inhibitor. Acute alcohol-induced fatty liver was the same in JNK1 and JNK2 KO mice as in WT mice; thus either JNK1 or JNK2 per se is sufficient for induction of steatosis by acute alcohol. The results show that acute alcohol elevation of CYP2E1, oxidative stress, and activation of JNK interact to lower autophagy and increase lipogenic SREBP resulting in fatty liver.     

Cytochrome P450 (CYP) enzymes and the development of CYP biosensors

Cytochrome P450s (CYPs) are a large family of heme-containing monooxygenase enzymes involved in the first-pass metabolism of drugs and foreign chemicals in the body. CYP reactions, therefore, are of high interest to the pharmaceutical industry, where lead compounds in drug development are screened for CYP activity. CYP reactions in vivo require the cofactor NADPH as the source of electrons and an additional enzyme, cytochrome P450 reductase (CPR), as the electron transfer partner; consequently, any laboratory or industrial use of CYPs is limited by the need to supply NADPH and CPR. However, immobilizing CYPs on an electrode can eliminate the need for NADPH and CPR provided the enzyme can accept electrons directly from the electrode. The immobilized CYP can then act as a biosensor for the detection of CYP activity with potential substrates, albeit only if the immobilized enzyme is electroactive. The quest to create electroactive CYPs has led to many different immobilization strategies encompassing different electrode materials and surface modifications. This review focuses on different immobilization strategies that have been used to create CYP biosensors, with particular emphasis on mammalian drug-metabolizing CYPs and characterization of CYP electrodes. Traditional immobilization methods such as adsorption to thin films or encapsulation in polymers and gels remain robust strategies for creating CYP biosensors; however, the incorporation of novel materials such as gold nanoparticles or quantum dots and the use of microfabrication are proving advantageous for the creation of highly sensitive and portable CYP biosensors.     

Human constitutive androstane receptor mediates induction of CYP2B6 gene expression by phenytoin

Compared with its rodent orthologs, little is known about the chemical specificity of human constitutive androstane receptor (hCAR) and its regulation of hepatic enzyme expression. Phenytoin (PHY), a widely used antiepileptic drug, is a potent inducer of CYP2B6 in primary human hepatocytes, but does not activate human pregnane X receptor (PXR) significantly in cell-based transfection assays at the same concentrations associated with potent induction of CYP2B6. Based on this observation, we hypothesized that PHY may be a selective activator of hCAR. In primary human hepatocytes, expression of CYP2B6 reporter genes containing phenobarbital-responsive enhancer module (PBREM) or PBREM/xenobiotic-responsive enhancer module (XREM) response elements were activated up to 14- and 28-fold, respectively, by 50 microm PHY. By contrast, parallel experiments in HepG2 cell lines co-transfected with an hPXR expression vector did not show increased reporter activity. These results indicated that a PXR-independent pathway, which is retained in primary hepatocytes, is responsible for PHY induction of CYP2B6. Further experiments revealed that PHY effectively translocates hCAR from the cytoplasm into the nucleus in both primary human hepatocytes and CAR(-/-) mice. Compared with vehicle controls, PHY administration significantly increased CYP2B6 reporter gene expression, when this reporter construct was delivered together with hCAR expression vector into CAR(-/-) mice. However, PHY did not increase reporter gene expression in CAR(-/-) mice in the absence of hCAR vector, implying that CAR is essential for mediating PHY induction of CYP2B6 gene expression. Taken together, these observations demonstrate that, in contrast to most of the known CYP2B6 inducers, PHY is a selective activator of CAR in humans.     

Polymorphisms of the Cytochrome P450 (CYP1A1, CYP2E1) and Microsomal Epoxide Hydrolase (mEPHX) Genes in Cystic Fibrosis and Chronic Respiratory Disease

Frequencies of CYP1A1, CYP2E1, and mEPHX polymorphic variants were analyzed in cystic fibrosis, chronic obstructive lung disease, bronchiectatic disease, chronic nonobstructive bronchitis, and recurring bronchitis. Mutations in CYP1A1 and mEPHX were shown to modify the severity of respiratory disorders in cystic fibrosis, the combination of CYP1A1 genotype Val/Val with the very slow mEPHX phenotype being most unfavorable (odds ratio OR = 12.30). Heterozygosity at both CYP1A1 and CYP2E1 was associated with chronic obstructive lung disease and recurring bronchitis (OR = 4.08 and 11.72, respectively). The very slow phenotype of mEPHX was predisposing to chronic respiratory disorders regardless of the CYP1A1 or CYP2E1 alleles (OR = 4.06). Basing on the above correlations, a combination of the very slow mEPHX phenotype with elevated cytochrome P450 (CYP1A1 and CYP2E1) activities was assumed to expedite severe respiratory disorders.     

Choosing metaphases for biological dosimetry by fluorescence in situ hybridization (FISH)

Data are presented for a subset of lymphocytes characterized by FISH as missing painted chromosomal material. These lymphocytes occur in both control and irradiated subjects. These cells have a much greater frequency of one-way translocations than cells in which all of the painted chromosomal material is present. Their presence contributes to interindividual variability in control translocation yields. These cells do not appear to be more prevalent in persons exposed to high radiation doses. It is suggested that their exclusion when selecting cells for analysis may improve the sensitivity of FISH as a biological dosimeter at low doses. Mechanisms for the production of these one-way translocations in vivo are also discussed, with a proposal that their variable frequency in individuals may be consistent with exposure to chemical clastogens.     

Genetic polymorphism analysis of cytochrome P4502E1 (CYP2E1) in Chinese Han populations from four different geographic areas of Mainland China

CYP2E1 is one of a superfamily of enzymes that play a central role in activating and detoxifying many xenobiotics and endogenous compounds thought to be involved in the development of several human diseases. Among other factors, individual susceptibility to developing these pathologies relies on genetic polymorphisms, which are related to ethnic differences, since the frequency of mutant genotypes varies in different populations. The aim of this study was to investigate the genetic basis of CYP2E1 polymorphisms in the populations of four different geographical locations of China. Twenty-two different CYP2E1 polymorphisms, including six novel variants in promoter regions and a novel nonsense mutation, were identified. The frequencies of some polymorphisms and genotypes demonstrated significant differences among the four populations. Linkage disequilibrium analysis and tag SNP selection were performed. Haplotypes were analyzed within the selected tag SNPs. Tag SNP selection and haplotype distributions showed differences across the four populations.