Modulation by SAMe of CYP2E1-dependent Effects in Liver.

描述(申请人提供)：有兴趣的作用，氧化应激和产生的反应自由基物种的机制(S)，乙醇是有毒的。乙醇产生氧化应激的途径之一就是诱导细胞色素P450酶2的表达。S-腺苷-L-蛋氨酸是细胞生长、分化和功能的调节因子。SAM合成障碍在包括酒精在内的多种因素所致的肝损伤中起重要作用。在MAT1A基因敲除小鼠中，细胞色素P450_2E_1水平升高，提示SAM可以调节或调节细胞色素P450_2E_1。本研究的目的是研究细胞色素P4502和环磷酰胺之间可能的相互作用/调节，并研究环磷酰胺对环磷酰胺依赖的毒性和活性氧产生的影响。目的1研究SAM及其代谢产物5-甲硫基腺苷(MTA)对吡唑诱导的大鼠肝细胞和高表达细胞色素P450-2E1(E47)的HepG2细胞的细胞毒性的影响。目的研究SAM和MTA在原代肝星状细胞与肝星状细胞共培养过程中对由细胞色素P450-EL衍生的扩散介质激活肝星状细胞的影响。 吡唑类肝细胞或E47细胞。目的3将评估SAM和MTA对细胞色素P450依赖的抗氧化剂基因激活的影响，这反映了对细胞色素P450依赖的氧化应激的适应性反应。SAM或MTA阻止细胞色素P2E1P38 MAP激活或其他应激信号通路的能力将被确定，因为这样的作用在SAM或MTA阻止细胞色素P2E1毒性的机制中可能是重要的。目的4研究SAM和MTA对细胞色素P450-2E1表达、含量和作用的影响。用SAM或MTA对正常大鼠或吡唑、乙醇诱导的高水平饥饿大鼠进行体内处理，观察SAM或MTA对基础或诱导的GSH和抗氧化剂上调的蛋白、活性、mRNA水平的影响，以及几种体内模型对细胞色素P450依赖毒性的影响。细胞色素P450_2E_1诱导对MAT1a和MAT1a基因表达的影响 将确定与合成SAM有关的MAT2A基因或酶的活性。目的5将评估SAM或MTA在体外抑制细胞色素P450 EL依赖的活性氧产生的能力。本研究利用肝细胞培养模型、体内模型和机制研究，将有助于明确SAM对CYP2E依赖毒性的影响，并有助于了解SAM在包括酒精性肝损伤在内的多种肝损伤模型中的保肝作用。

DESCRIPTION (provided by applicant): There is interest in the role of oxidative stress and generation of reactive radical species in the mechanism(s) by which ethanol is toxic. Induction of CYP2E1 is one pathway by which ethanol generates oxidative stress. S-Adenosyl- L-Methionine (SAM) is a regulator of cellular growth, differentiation and function. Impairment of SAM synthesis plays an important role in hepatic injury induced by various agents, including alcohol. CYP2E1 levels were increased in the MAT1A knockout mouse suggesting SAM could regulate or modulate CYP2E1. The goal of this application is to study possible interactions/modulation between CYP2E1 and SAM and to investigate the effects of SAM on CYP2E1- dependent toxicity and generation of reactive oxygen species. Aim 1 will evaluate the effect of SAM, and the SAM metabolite 5-methylthioadenosine (MTA) on CYP2E 1-dependent toxicity in cultured hepatocytes from pyrazole-treated rats and HepG2 cells overexpressing CYP2E1 (E47 cells). Aim 2 will study the effect of SAM and MTA on hepatic stellate cell activation by CYP2El-derived diffusible mediators in co-cultures of primary hepatic stellate cells with pyrazole hepatocytes or E47 cells. Aim 3 will assess the effect of SAM and MTA on CYP2El-dependent activation of antioxidants genes which reflect an adaptive response to CYP2El-dependent oxidative stress. The ability of SAM or MTA to prevent activation of P38 MAP kinasc or other stress kinascs by CYP2E1 will be determined, since such actions may be important in mechanisms by which SAM or MTA prevent CYP2E1 toxicity. Aim 4 will study in-vivo effects of SAM and MTA on CYP2E1 expression, content and actions. Control rats or rats induced by pyrazole, ethanol, starvation with high levels of CYP2E 1 will be treated with SAM or MTA in-vivo and the effect on basal or induced CYP2E1 protein, activity, mRNA level on up regulation of GSH and antioxidants and on CYP2El-dependent toxicity in several in-vivo models determined. The effect of CYP2E1 induction on expression of the MAT1A and MAT2A genes or enzyme activities responsible for the synthesis of SAM will be determined. Aim 5 will evaluate the ability of SAM or MTA, in-vitro, to inhibit CYP2El-dependent generation of reactive oxygen species. It is hoped that this study utilizing hepatocyte cell culture models, in-vivo models and mechanistic studies will help to define the effects of SAM on CYP2E 1-dependent toxicity and may prove valuable in understanding the hepatoprotective actions of SAM in many models of liver injury, including alcohol-induced liver injury.