Identification of repressor element 1 in cytochrome P450 genes and their negative regulation by RE1 silencing transcription factor/neuron-restrictive silencer factor.

RE1 silencing transcription factor/neuron-restrictive silencing factor (REST/NRSF) mediates transcriptional repression in many neuron-specific genes by interaction with the repressor element 1/neuron-restrictive silencing element (RE1/NRSE). This element has been identified at least in 20 neuron specific genes. REST/NRSF is highly expressed in non-neuronal tissues, where it is thought to repress gene transcription. We performed a BLAST search to look for the presence of RE1/NRSE elements in the rat cytochrome P450 genes. We identified the presence of RE1/NRSE element in the cytochrome P450 genes CYP1A1, 2A2, 2E1 and 3A2. Electrophoretic mobility shift assay and supershift assays were carried out to prove functionality of these sites and detect the interaction of REST/NRSF with this sequence. Cotransfection studies in PC12 cells with a plasmid containing the RE1 element of the CYP genes, cloned upstream of the minimal type II sodium channel promoter, in the presence of REST/NRSF, showed a marked expression inhibition of the CAT reporter gene. These data suggest that the RE1 elements that exist in these four CYP genes might be a target for the REST/NRSF transcription factor and such an interaction might play a role in the negative regulation of these genes.

Establishment and characterization of immortal hepatocytes derived from various transgenic mouse lines.

The potential of three genetic changes introduced into mice by the transgenic or knockout technology aimed at immortalizing hepatocytes in vitro and concomitantly preserving their differentiated hepatic functions was analyzed. Six hepatocyte lines were isolated from neonatal and adult transgenic mice expressing either IgEGF (a secretable variant of hEGF) or SV40 T antigen in the liver and from neonatal and adult p53 knockout (KO) mice and have been subcultured >150 times in serum-free, arginine-deficient medium. Only in SV40 T antigen transgenic lines profiles of mRNAs encoding serum proteins, transcription factors, and liver-specific enzymes were similar to those found in livers and primary hepatocytes. Accordingly, these cells displayed basal and inducible expression of CYP proteins as well as testosterone metabolizing activities. Thus, either knockout of the p53 gene or expression of SV40 T antigen or of IgEGF imparts immortality to hepatocytes in vitro, but only SV40 T antigen expression is compatible with the concomitant long-term preservation of differentiated liver functions.

Estudio preliminar de la hepatotoxicidad del anticonvulsionante DL-4-hidroxi, 4-etil, 4-fenil butiramida (HEPB) y sus homólogos inferiores HEPP y HEPA / Preliminary study of the hepatotoxicity of the anticonvulsant DL-4-hydroxy, 4-ethyl, 4-phenyl butyramide (HEPB) and its inferior homologues HEPA and HEPP

En este trabajo los efectos hepatotóxicos del anticonvulsionantes 4-hidroxi,, 4-etil, 4-fenil butiramida (HEPB) y sus homólogos HEPA y HEPP fueron evaluados y comparados con los efectos del valproato de sodio y del fenobarbital en cultivos de hepatocitos de larga sobrevivencia. Los hepatocitos se sembraron sobre una monocapa alimentadora de células 3T3 letalmente tratadas con mitomicina C y fueron expuestos por una o dos semanas a los anticonvulsionantes (50-500 mg/ml). Se seterminó en el medio de cultivo la liberación de las enzimas citoplásticas transaminasa glutámico oxalacética (GOT). Transaminasa glutámico pirúvica (GPT) y deshidrogenasa láctica (LDH), se determinó también el contenido de triclicéridos (TG) mediante tinción con rojo oleoso O. HEPB produjo gotas intracitoplásmicas de lípidos, ensanchamiento de los espacios intercelulares y retracción de las células. HEPA y HEPP produjeron efectos similares pero en menor grado. El valproato de sodio causó retracción de las células, ensanchamiento de los espacios intracelulares y también vacuolización de las células. Después de una exposición de una semana el valproato de sodio produjo la liberación más alta de TGO, TGP y LDH. Los cultivos tratados con los otros anticonvulsionantes mostraron poca diferencia con los cultivos control. Después de dos semanas de exposición los efectos fueron menores. El mayor contenido de TG lo produjo el HEPB, mientras que el valpoatro de sodio produjo una disminución en el contenido de TG. Nuestros resultados muestran que el rango de toxicidad de los anticonvulsionantes probados es valproato de sodio >HEPB>HEPA>HEPP>fenobarbital