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.
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