Because human sensory neurons (SN), or cell lines, are not available, the culture of SN from animal dorsal root ganglia is commonly used. Studies have highlighted the ability of iPS (induced pluripotent stem cells) or ES (embryonic stem cells) to differentiate into cells with SN characteristics. In most of these studies, cells were differentiated into cells expressing markers of neuronal precursors or cells from the neural crest. In a second step, the induction of Wnt and/or BMP pathways allowed differentiation into SN. Our work aimed to determine if it was possible to directly differentiate human SKP (skin-derived precursors), which are derived from the neural crest and extracted from the skin tissue, into SN. SKPs were extracted by enzymatic and mechanical dissociation of abdominal skin samples. These cells were grown in a DMEM/F12 medium containing FGF2 and EGF. After few weeks, the cells adhered spontaneously. To induce the Wnt pathway in SKP, we used CHIR99201 that activates the Wnt pathway by inhibiting GS3Kb. For the induction of BMP pathway, we added BMP4. We obtained 1) confirmation by PCR that our SKP cells expressed markers of neural crest and precursors (p75NTR, SOX9, AP2, PAX3) and 2) after differentiation, evidence that they acquired a SN phenotype. Part of the cells also acquired a bipolar neuronal morphology. In qPCR, we observed a 7-fold increase of Brn3a expression (SN marker) after 20 days with CHIR99021 and 8 days of BMP4 compared to undifferentiated cells. In immunochemistry, 100% of cells expressed the neuronal marker neurofilaments and p75NTR, whereas 78 and 75% of cells expressed respectively Brn3a and peripherin (a peripheral neuronal marker). The presence of the TRPV1 channel was also evidenced by immunochemistry and PCR. Altogether, these results demonstrate that SKP cells can be differentiated into cells with a sensory neuron phenotype.

CYP4F isoforms are involved in the oxidation of important cellular mediators such as leukotriene B-4 (LTB4) and Prostaglandins. The proinflammatory agent LTB4 and cytotoxic leukotoxins have been associated with several inflammatory diseases. We present evidence that the hydroxylation of Z9(10)-epoxyoctadecanoic, Z9(10)-epoxyoctadec-Z12-enoic, and Z12(13)-epoxyoctadec-Z9-enoic acids and that of monoepoxides from arachidonic acid [epoxyeicosatrienoic acid (EET)] is important in the regulation of leukotoxin and EET activity. These three epoxidized derivatives from the C18 family (C18-epoxides) were converted to 18-hydroxy-C18-epoxides by human hepatic microsomes with apparent K. values of between 27.6 and 175 muM. Among recombinant P450 enzymes, CYP4F2 and CYP4F3B catalyzed mainly the omega-hydroxylation of C18-epoxides with an apparent V-max of between 0.84 and 15.0 min(-1), whereas the apparent V-max displayed by CYP4F3A, the isoform found in leukocytes, ranged from 3.0 to 21.2 min(-1). The rate of omega-hydroxylation by CYP4A11 was experimentally found to be between 0.3 and 2.7 min(-1). CYP4F2 and CYP4F3 exhibited preferences for omega-hydroxylation of Z8(9)-EET, whereas human liver microsomes preferred Z11(12)-EET and, to a lesser extent, Z8(9)-EET. Moreover, vicinal diol from both C18-epoxides and EETs were omega-hydroxylated by liver microsomes and by CYP4F2 and CYP4F3. These data support the hypothesis that the human CYP4F subfamily is involved in the omega-hydroxylation of fatty acid epoxides. These findings demonstrate that another pathway besides conversion to vicinal diol or chain shortening by beta-oxidation exists for fatty acid epoxide inactivation.-Le Quere, V., E. Plee-Gautier, P. Potin, S. Madec, and J-P. Salaun. Human CYP4F3s are the main catalysts in the oxidation of fatty acid epoxides.

Alcohol and xenobiotics share the same oxidative microsomal pathway, which is mainly located in the endoplasmic reticulum of hepatocytes. This pathway involves enzymes that belong to the super family of cytochrome P450 and allows to explain a lot of pharmacokinetic or toxic interactions between alcohol and xenobiotics. Cytochrome P450 2E1 (CYP2E1) is the key enzyme of the microsomal pathway of ethanol oxidation. It is inducible by chronic ethanol consumption and its activity is increased by three to five fold in liver from alcoholics subjects. This induction involves to a lesser extent cytochromes P450 3A4 and 1A2 and contributes to the metabolic tolerance of alcohol and drugs observed in alcoholics. The metabolic tolerance persits several days after ethanol withdrawal. Furthermore, CYP2E1 has a high capacity to activate numerous xenobiotics into toxic or carcinogenic compounds. Drugs currently used such as paracetamol, anesthetics (enflurane, halothane), industrial solvents (benzene or its derivatives), halogenated solvents (CCl4, trichlorethylene) and nitrosamines which are present in food or tobacco smoke are included. Therefore, heavy consumption of alcohol, which results in CYP2E1 induction, increases individual susceptibility to the toxic or carcinogenic effects of these xenobiotics.

Alcohol and xenobiotics share the same oxidative microsomal pathway, which is mainly located in the endoplasmic reticulum of hepatocytes. This pathway involves enzymes that belong to the super family of cytochrome P450 and allows to explain a lot of pharmacokinetic or toxic interactions between alcohol and xenobiotics. Cytochrome P450 2E1 (CYP2E1) is the key enzyme of the microsomal pathway of ethanol oxidation. It is inducible by chronic ethanol consumption and its activity is increased by three to five fold in liver from alcoholics subjects. This induction involves to a lesser extent cytochromes P450 3A4 and 1A2 and contributes to the metabolic tolerance of alcohol and drugs observed in alcoholics. The metabolic tolerance persits several days after ethanol withdrawal. Furthermore, CYP2E1 has a high capacity to activate numerous xenobiotics into toxic or carcinogenic compounds. Drugs currently used such as paracetamol, anesthetics (enflurane, halothane), industrial solvents (benzene or its derivatives), halogenated solvents (CCl4, trichlorethylene) and nitrosamines which are present in food or tobacco smoke are included. Therefore, heavy consumption of alcohol, which results in CYP2E1 induction, increases individual susceptibility to the toxic or carcinogenic effects of these xenobiotics. (C) 2001 Editions scientifiques et medicales Elsevier SAS.

BACKGROUND:Differences in the regulatory region of the CYP2E1 gene could be responsible for the interindividual variation in the cytochrome P-450 2E1 (CYP2E1) involved in ethanol oxidation. Recently, a polymorphic repeat sequence in the human gene was described between -2178 and -1945 base pairs. Its frequency seemed to vary among different ethnic populations, and it was suspected to be related to an increased inducibility to further ethanol intake. In the study reported here, the frequency of this polymorphism was investigated in a white French population. Its relationship with the previously described PstI/RsaI or DraI CYP2E1 polymorphisms, alcoholism, alcoholic liver disease, and inducibility of CYP2E1 by ethanol was examined.METHODS:The polymorphic region was characterized by polymerase chain reaction in 103 controls, 148 alcoholic subjects without liver diseases, and 98 others with liver cirrhosis. By using in vivo chlorzoxazone (CHZ) metabolism, CYP2E1 phenotype was assessed in 36 non-ethanol-induced subjects (17 controls and 19 withdrawn alcoholics) and in 14 ethanol-induced subjects (10 controls after ingestion of 0.8 g/kg ethanol and four alcoholics with 100 g of daily intake). This phenotype was expressed as the 6-hydroxy CHZ/CHZ ratio.RESULTS:The rare allele frequency was found to be 1.58% in whites (n = 349). Neither significant association with alcoholism or alcoholic liver diseases, nor relationship with the PstI/RsaI polymorphism, was observed. But the DraI polymorphism was more frequent among the heterozygous subjects when compared with wild-type homozygous ones (p < 0.05). The CYP2E1 phenotype was similar in wild-type homozygotes and in heterozygotes at the constitutive level, as well as after induction with ethanol.CONCLUSIONS:Our data suggest that CYP2E1 repeat polymorphism does not seem to constitute a major factor for interindividual differences in CYP2E1 expression and susceptibility to alcohol-related disorders in whites.

Background Long-term ethanol consumption in laboratory animals is associated with histological alterations of liver cells and modifications of fatty acid metabolism.

We have studied the regulation of transaminase gene expression by hormones and drugs in humans and rodents. In rodents, most of the regulation is hormonal and is consistent with the role of these enzymes in gluconeogenesis. In humans, these genes are up-regulated by drugs such as fibrates, which may partially account for the increase in the activities in serum observed in some treated patients. Interestingly, fibrates down-regulate these genes in the rat, suggesting that there are important differences in the regulation of gene expression between rodents and humans.

Cytosolic aspartate aminotransferase (cAspAT) participates in gluconeogenesis in the liver and is expected to exert a glyceroneogenic function in the adipose tissue when the supply of glucose is limited. Here we demonstrate that adipose cAspAT messenger RNA (mRNA) is increased when rats are fed a low carbohydrate diet. In the 3T3-F442A, BFC-1 adipocyte cell lines and differentiated adipocytes in primary culture, a 24 h glucose deprivation induces approximately a 4-fold increase in cytosolic AspAT (cAspAT) mRNA, whereas mitochondrial AspAT mRNA remains unchanged. cAspAT activity is also increased in a weaker but reproducible manner. Addition of glucose within a physiological range of concentrations reverses the increase of cAspAT mRNA in 8 h (EC50 = 1.25 g/liter). Such a regulation requires protein synthesis and is specific for adipocytes differentiated in culture. It does not occur in Fao or H4IIE hepatoma cells, in C2 muscle cells, or in 293 kidney cells. 2-deoxyglucose mimicks glucose, while 3-orthomethyl-glucose has no effect, suggesting that glucose-6-phosphate is the effector. cAspAT mRNA stability is not affected by glucose deprivation. To ascertain the transcriptional nature of the glucose effect, we have stably transfected 3T3-F442A adipoblasts with constructs containing the chloramphenicol acetyltransferase reporter gene under the control of either 5'-deletions of the cAspAT gene promoter or internal fragments in an heterologous context. We demonstrate that a glucose response element(s) is present in the region between -1838 and -1702 bp relative to the translation start site. In this region, three DNA sequences bind nuclear proteins from adipocytes as shown by footprinting experiments. Our results indicate that cAspAT gene transcription is repressed by glucose selectively in adipocytes.

Stress controls the expression of a cohort of genes. Among these, the glucose-regulated protein (GRP) genes are specifically activated by glucose deprivation, reducing agents, glycosylation block, intracellular calcium or ex vivo incubations of tissues or cells. We demonstrate that these stimuli induce the expression of the cytosolic aspartate aminotransferase gene in adipocytes by a process involving the region of the promoter between -2405 and -26 bp. Therefore this transaminase is a new member of the GRP family.

Hormone-sensitive lipase (HSL) catalyses the rate-limiting step in adipocyte lipolysis. Short-term hormonal regulation of HSL activity is well characterized, whereas little is known about the control of HSL gene expression. We have measured HSL mRNA content of 3T3-F442A and BFC-1 adipocytes in response to the cAMP analogue 8-(4-chlorophenylthio)-cAMP (8-CPT-cAMP) and to the phorbol ester phorbol 12-myristate 13-acetate (PMA) by Northern blot, using a specific mouse cDNA fragment. Treatment of the cells for 12 or 6 h with, respectively, 0.5 mM 8-CPT-cAMP or 1 microM PMA produced a maximal decrease of about 60% in HSL mRNA. These effects were unaffected by the protein-synthesis inhibitor anisomycin, suggesting that cAMP and PMA actions were direct. The reduction in HSL mRNA was accompanied by a reduction in HSL total activity. The intracellular routes that cAMP and PMA follow for inducing such an effect seemed clearly independent. (i) After desensitization of the protein kinase C regulation pathway by a 24 h treatment of the cells with 1 microM PMA, PMA action was abolished whereas cAMP was still fully active. (ii) Treatment with saturating concentrations of both agents produced an additive effect. (iii) The synthetic glucocorticoid dexamethasone had no proper effect on HSL gene expression but potentiated cAMP action without affecting PMA action. cAMP inhibitory action on HSL is unexpected. Indeed, the second messenger of catecholamines is the main activator of HSL by phosphorylation. We envision that a long-term cAMP treatment of adipocytes induces a counter-regulatory process that reduces HSL content and, ultimately, limits fatty acid depletion from stored triacylglycerols.