RESUMEN
Polychlorinated biphenyls (PCBs) as a group of persistent organic pollutants are confirmed human carcinogens; however, their mutagenicity remains mostly unknown. We have reported the mutagenicity of some PCBs with one to four chlorines in mammalian cells expressing human CYP2E1. To further explore the structural requirements for the mutagenicity of PCBs, eight tri- and tetrachlorobiphenyls untested before were investigated for the induction of gene mutations and micronuclei in a V79-derived cell line expressing both human CYP2E1 and sulfotransferase (SULT) 1A1 (V79-hCYP2E1-hSULT1A1), with SULT1A1 activity inhibited by pentachlorophenol, a potent SULT1 inhibitor. 2,2',6-Tri-, 2,3',6-tri, 2,4',6-tri-, and 2,2',5-trichlorobiphenyls (PCBs 19, 27, 32, and 18, respectively) induced micronuclei and gene mutations in V79-hCYP2E1-hSULT1A1 cells, at potencies slightly higher than 2,6-dichlorobiphenyl, but one order of magnitude below that by 2,3,3'- and 2,3,4'-trichlorobiphenyls as reported recently; in the parental V79-Mz cells, they were nonmutagenic and weak in micronuclei induction. Among the four tetrachlorobiphenyls with varying number of ortho chlorines, 2,3,3',4'-tetrachlorobiphenyl (PCB 56) induced both micronuclei and gene mutations in V79-hCYP2E1-hSULT1A1 cells with a potency greater than the above compounds; however, 2,2',3,3'-tetrachlorobiphenyl was equivocal and 2,2',3,6'-tetra- and 2,2',6,6'-tetrachlorobiphenyls inactive in V79-hCYP2E1-hSULT1A1 cells. Immunofluorescent staining of micronuclei formed by PCBs 32 and 56 in V79-hCYP2E1-hSULT1A1 cells with centromere protein B antibodies indicated that they were predominantly whole chromosomes, implying aneugenic potentials. This study suggests that tri- and tetrachlorobiphenyls with a single ortho chlorine can be most mutagenic under activation by human CYP2E1, and greater numbers of ortho chlorines may cause a drastic decline in the activity, especially for tetrachlorobiphenyls.
Asunto(s)
Citocromo P-450 CYP2E1/toxicidad , Mutágenos/química , Mutación , Bifenilos Policlorados/metabolismo , Animales , Arilsulfotransferasa/metabolismo , Línea Celular , Cricetinae , Cricetulus , Citocromo P-450 CYP2E1/metabolismo , Inhibidores del Citocromo P-450 CYP2E1/farmacología , Halogenación , Humanos , Mutagénesis/efectos de los fármacos , Relación Estructura-ActividadRESUMEN
Pyrazole treatment to induce cytochrome P-450 2E1 (CYP2E1) was recently shown to cause liver injury in ob/ob mice but not in lean mice. The present study investigated the effects of S-adenosyl-l-methionine (SAM) on the CYP2E1-dependent liver injury in ob/ob mice. Pyrazole treatment of ob/ob mice for 2 days caused necrosis, steatosis, and elevated serum transaminase and triglyceride levels compared with saline ob/ob mice. Administration of SAM (50 mg/kg body wt ip every 12 h for 3 days) prevented the observed pathological changes as well as the increase of apoptotic hepatocytes, caspase 3 activity, and serum TNF-alpha levels. SAM administration inhibited CYP2E1 activity but not CYP2E1 content. The pyrazole treatment increased lipid peroxidation, 4-hydroxynonenal and 3-nitrotyrosine protein adducts, and protein carbonyls. These increases in oxidative and nitrosative stress were prevented by SAM. Treatment of ob/ob mice with pyrazole lowered the endogenous SAM levels, and these were elevated after SAM administration. Mitochondrial GSH levels were very low after pyrazole treatment of the ob/ob mice; this was associated with elevated levels of malondialdehyde and 4-hydroxynonenal and 3-nitrotyrosine protein adducts in the mitochondria. All these changes were prevented with SAM administration. SAM protected against pyrazole-induced increase in serum transaminases, necrosis, triglyceride levels, caspase-3 activity, and lipid peroxidation even when administered 1 day after pyrazole treatment. In the absence of pyrazole, SAM lowered the slightly elevated serum transaminases, triglyceride levels, caspase-3 activity, and lipid peroxidation in obese mice. In conclusion, SAM protects against and can also reverse or correct CYP2E1-induced liver damage in ob/ob mice.
Asunto(s)
Citocromo P-450 CYP2E1/toxicidad , S-Adenosilmetionina/farmacología , Animales , Enfermedad Hepática Inducida por Sustancias y Drogas , Fragmentación del ADN/efectos de los fármacos , Hígado/efectos de los fármacos , Hepatopatías/prevención & control , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Obesos , Mitocondrias Hepáticas/efectos de los fármacos , Óxido Nítrico Sintasa de Tipo II/antagonistas & inhibidores , Estrés Oxidativo/efectos de los fármacos , PirazolesRESUMEN
Cytochrome P450 2E1 (CYP2E1) can mediate reactive oxygen species (ROS) induced cell death through its catalytic processes. Heat shock protein 90 (Hsp90) is an important molecular chaperone which is essential for cellular integrity. We previously showed that inhibition of Hsp90 with Geldanamycin (GA), an inhibitor of Hsp90 increased CYP2E1 mediated toxicity in CYP2E1 over-expressing HepG2 cells (E47 cells) but not in C34-HepG2 cells devoid of CYP2E1 expression. The aim of the present study was to test the hypothesis that the potentiation of CYP2E1 toxicity in E47 cells with GA may involve changes in mitogen activated protein kinase signal transduction pathways. GA was toxic to E47 cells and SB203580, an inhibitor of p38 MAPK prevented this decrease in viability. The protective effects of SB203580 were effective only when SB203580 was added before GA treatment. GA activated p38 MAPK in E47 cells and this activation was an early and a sustained event. GA elevated ROS levels and lipid peroxidation and lowered GSH levels in E47 cells and these changes were blunted or prevented by treatment with SB203580. Apoptosis was increased by GA and prevented by pre-treatment with SB203580. The loss in mitochondrial membrane potential in E47 cells after GA treatment was also decreased significantly with SB203580 treatment. The activity and expression of CYP2E1 and Hsp90 levels were not altered by SB203580. In conclusion, the inhibition of Hsp90 with GA increases the toxicity of CYP2E1 in HepG2 cells through an early and sustained activation of the p38 MAPK pathway.
Asunto(s)
Benzoquinonas/farmacología , Citocromo P-450 CYP2E1/fisiología , Citocromo P-450 CYP2E1/toxicidad , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Lactamas Macrocíclicas/farmacología , Hígado/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/fisiología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Línea Celular Tumoral , Activación Enzimática/efectos de los fármacos , Humanos , Hígado/enzimologíaRESUMEN
The objective of this work was to evaluate the possible role of PI3-kinase/AKT as a survival pathway against CYP2E1-dependent toxicity. E47 cells (HepG2 cells transfected with human CYP2E1 cDNA) exposed to 25 microM iron-nitrilotriacetate+5 microM arachidonic acid (AA+Fe) developed higher toxicity than C34 cells (HepG2 cells transfected with empty plasmid). Toxicity was associated with increased oxidative stress and activation of calcium-dependent hydrolases calpain and phospholipase A2. Treatment of E47, but not C34 cells, with arachidonic acid and iron (AA+Fe) led to a decrease in the phosphorylation state of AKT. 2-(4-Morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride (LY294002), a specific inhibitor of PI3-kinase, produced a further decrease of phosphorylated AKT in AA+Fe-treated E47 cells. LY294002 and down-regulation of endogenous AKT with small interference RNAs increased the toxicity of AA+Fe in E47 cells. Toxicity of AA+Fe in rat hepatocytes was also increased by LY294002. LY294002 did not affect phospholipase A2 or calpain activation, CYP2E1 activity, or lipid peroxidation elicited by AA+Fe. alpha-Tocopherol prevented both AA+Fe and AA+Fe+LY294002-induced toxicity and decrease of phosphorylated AKT. LY294002 potentiated AA+Fe-induced loss of mitochondrial membrane potential and ATP, whereas overexpression of constitutively active AKT partially prevented mitochondrial impairment and toxicity. Mitochondrial permeability transition inhibitors prevented both AA+Fe and AA+Fe+LY294002-induced toxicity and decrease of mitochondrial membrane potential. These results suggest that: i) AA+Fe+CYP2E1-induced oxidative stress decreases AKT activation; ii) AKT inactivation induces mitochondrial impairment associated with opening of the permeability transition pore but is not dependent on the activation state of bad, glycogen synthase kinase-3beta, mammalian target of rapamycin, or bcl-xL; and iii) PI3-kinase/AKT may serve as a survival pathway against CYP2E1-dependent toxicity.
Asunto(s)
Citocromo P-450 CYP2E1/toxicidad , Fosfatidilinositol 3-Quinasas/fisiología , Proteínas Proto-Oncogénicas c-akt/fisiología , Transducción de Señal/fisiología , Animales , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Células Cultivadas , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Humanos , Masculino , Inhibidores de las Quinasa Fosfoinosítidos-3 , Ratas , Ratas Sprague-Dawley , Transducción de Señal/efectos de los fármacosRESUMEN
The involvement of cytochrome P450 2E1 (CYP2E1) in the metabolism of N-methyl-2-pyrrolidone (NMP) was studied with three experimental approaches: in the rat, in vitro in human microsomes, and in human volunteers. NMP was administered dermally (40 mg/kg) to OFA rats to examine the influence of CYP2E1 inhibition (5 mg/kg diethyldithiocarbamate, DETC, 30 min before) and CYP2E1 induction (after 4 days of fasting). The main NMP metabolite 5-hydroxy- N-methylpyrrolidone (5HNMP) in the urine fractions collected during the following 48 h was analysed by gas chromatography-mass spectrometry. CYP2E1 inhibition led to a statistically significant retardation of 5HNMP excretion in urinary fractions collected during the first 12 h. In the group of fasted rats, a two-fold increase of CYP2E1 activity was observed in comparison with the control group. During the first 6 h after dermal administration of NMP to fasted rats, about 33% of the dose was excreted in urine versus 22% in controls. In vitro, NMP (15 mM) was incubated (up to120 min) with human liver microsomes and the formation of 5HNMP followed Michaelis-Menten kinetics with V(max) of 1.1 nmol/min per mg protein and K(m) of 2.4 mM. The formation of 5HNMP was inhibited by 35% in the presence of a monoclonal antibody against CYP2E1, but not by CYP1A2 antibody. In a dermal application experiment, 12 humans volunteers were exposed by means of a dermal patch to 300 mg NMP; five urine fractions were collected during the 48 h following the onset of application in order to measure the major metabolites 5HNMP and 2-hydroxymethylsuccinimide (2HMSI). Before NMP application, a blood sample was collected for the quantification of CYP2E1 mRNA in peripheral blood lymphocytes (PBLs). The mean dermal absorption of NMP was 67.9%. The highest amount of 5HNMP was excreted in urine in the fraction collected between 6-12 h (12.6% of dose), while 2HMSI peaked in fractions 12-24 h and 36-48 h (3.3 and 3.2% of dose, respectively). A significant relationship was found between CYP2E1 mRNA content in PBLs and the amount of both the metabolites excreted in urine within 24 h ( r(2)=0.54, P<0.01). It is concluded that CYP2E1 is involved in the first steps of NMP metabolism in the rat and, to a lesser extent, in humans. Since large variations in CYP2E1 activity exist in the human population (at least 5-fold range), it seems justified to take into account the activity of this enzyme in an individual for an accurate interpretation of biological monitoring of exposure to NMP when relying on 5HNMP and/or 2HMSI determination in urine.
Asunto(s)
Citocromo P-450 CYP2E1/metabolismo , Contaminantes Ambientales/farmacocinética , Pirrolidinonas/farmacocinética , Teratógenos/farmacocinética , Administración Cutánea , Adulto , Animales , Citocromo P-450 CYP2E1/toxicidad , Inhibidores del Citocromo P-450 CYP2E1 , Ditiocarba/farmacología , Inhibidores Enzimáticos/farmacología , Femenino , Cromatografía de Gases y Espectrometría de Masas , Humanos , Técnicas In Vitro , Masculino , Microsomas Hepáticos/efectos de los fármacos , Microsomas Hepáticos/enzimología , Pruebas del Parche , Pirrolidinonas/administración & dosificación , Ratas , Ratas Sprague-Dawley , Absorción Cutánea/efectos de los fármacosRESUMEN
Induction of cytochrome P450 2E1 (CYP2E1) by ethanol appears to be one of the central pathways by which ethanol generates a state of oxidative stress. Glutathione (GSH) is critical in preserving the proper cellular redox balance and for its role as a cellular protectant. The goal of the present study was to characterize the GSH homeostasis in human hepatocarcinoma cells (HepG2-E47 cells) that overexpress CYP2E1. Toxicity in the E47 cells was markedly enhanced after GSH depletion by buthionine sulfoximine (BSO) treatment. The antioxidant trolox partially prevented the apoptosis and necrosis, while diallylsulfide, a CYP2E1 inhibitor, was fully protective. Damage to mitochondria appears to play a role in the CYP2E1- and BSO-dependent toxicity. CYP2E1-overexpressing cells showed increases in total GSH levels, GSH synthetic rate and in gamma-glutamylcysteine synthetase (GCS) mRNA. This GCS increase was due to transcriptional activation of the GCS gene and could be blocked by certain antioxidants. Activity, protein and mRNA levels for other antioxidants such as catalase, alpha- and microsomal glutathione transferases were also increased in the E47 cells. Up-regulation of these antioxidant genes may reflect an adaptive mechanism to remove CYP2E1-derived oxidants. These oxidants are diffusable and were able to elevate collagen type I protein in a co-culture system consisting of the E47 cells + rat hepatic stellate cells. Such interactions between CYP2E1, mitochondria and altered GSH homeostasis, and elevation of collagen levels, may play a role in alcohol-induced liver injury.
Asunto(s)
Antioxidantes/metabolismo , Citocromo P-450 CYP2E1/toxicidad , Glutamato-Cisteína Ligasa/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Animales , Antimetabolitos/farmacología , Apoptosis/efectos de los fármacos , Butionina Sulfoximina/farmacología , Técnicas de Cocultivo , Colágeno/efectos de los fármacos , Colágeno/metabolismo , Citocromo P-450 CYP2E1/genética , Inhibidores del Citocromo P-450 CYP2E1 , Genes Reporteros , Glutamato-Cisteína Ligasa/genética , Glutatión/efectos de los fármacos , Glutatión/metabolismo , Humanos , ARN Mensajero/efectos de los fármacos , Ratas , Transfección , Células Tumorales Cultivadas , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Two Hep G2 subclones overexpressing CYP2E1 were established with the use of transfection and limited dilution screening techniques. The Hep G2-CI2E1-43 and -47 (E47) cells (transduced Hep G2 subclones that overexpress CYP2E1) grew at a slower rate than parental Hep G2 cells or control subclones that do not express CYP2E1, but remained fully viable. When GSH synthesis was inhibited by treatment with buthionine sulfoximine, GSH levels rapidly declined in E47 cells but not control cells, which is most likely a reflection of CYP2E1-catalyzed formation of reactive oxygen species. Under these conditions of GSH depletion, cytotoxicity and apoptosis were found only with the E47 cells. Low levels of lipid peroxidation were found in the E47 cells, which became more pronounced after GSH depletion. The antioxidants vitamin E, vitamin C, or trolox prevented the lipid peroxidation as well as the cytotoxicity and apoptosis, as did transfection with plasmid containing antisense CYP2E1 or overexpression of Bcl-2. Levels of ATP were lower in E47 cells because of damage to mitochondrial complex I. When GSH was depleted, oxygen uptake was markedly decreased with all substrates in the E47 extracts. Vitamin E completely prevented the decrease in oxygen uptake. Under conditions of CYP2E1 overexpression, two modes of CYP2E1-dependent toxicity can be observed in Hep G2 cells: a slower growth rate when cellular GSH levels are maintained and a loss of cellular viability when cellular GSH levels are depleted. Elevated lipid peroxidation plays an important role in the CYP2E1-dependent toxicity and apoptosis. This direct toxicity of overexpressed CYP2E1 may reflect the ability of this enzyme to generate reactive oxygen species even in the absence of added metabolic substrate.