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Title: HEPATOTOXICITY OF ALCOHOL-INDUCED POLAR RETINOID METABOLITES INVOLVES APOPTOSIS VIA LOSS OF MITOCHONDRIAL MEMBRANE POTENTIAL

Author
item DAN, ZILI - UNIV ERLANGEN GERMANY
item POPOV, YURY - UNIV ERLANGEN GERMANY
item PATSENKER, ELEONORA - UNIV ERLANGEN GERMANY
item PREIMEL, DOROTHEE - UNIV ERLANGEN GERMANY
item Liu, Chun
item Wang, Xiang-Dong
item SEITZ, HELMUT - UNIV HEIDELBERG GERMANY
item SCHUPPAN, DETLEF - UNIV ERLANGEN GERMANY
item STICKEL, FELIX - UNIV ERLANGEN GERMANY

Submitted to: Journal of Federation of American Societies for Experimental Biology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/18/2004
Publication Date: 2/24/2005
Citation: Dan, Z., Popov, Y., Patsenker, E., Preimel, D., Liu, C., Wang, X., Seitz, H.K., Schuppan, D., Stickel, F. 2005. Hepatotoxicity of alcohol-induced polar retinoid metabolites involves apoptosis via loss of mitochondrial membrane potential. Journal of Federation of American Societies for Experimental Biology. [FJ Express 10.1096/fj.04-2809fje, published online February 24, 2005] Available at http://www.fasebj.org.

Interpretive Summary: Chronic alcohol consumption decreases liver vitamin A level and vitamin A supplementation has been recommended for correction of this deficiency. However, high-dose vitamin A supplementation is hepatotoxic. It was suggested that polar vitamin A metabolites induced by alcohol aggravate liver damage. However, direct experimental evidence supporting this hypothesis is still lacking. In the present study, we investigated effects of polar vitamin A metabolites on cultured liver cell toxicity. Polar vitamin A metabolites were extracted from liver tissues of rats fed either an alcoholic or control diet for one month. Cultured liver cell toxicity was evaluated using six different methods. Results show that polar metabolites of vitamin A caused marked cytotoxicity in a concentration- and time-dependent manner. This toxicity involved apoptosis via loss of mitochondrial membrane potential. In conclusion, the present data demonstrates that polar vitamin A metabolites cause marked hepatocyte death through the induction of apoptosis.

Technical Abstract: Chronic alcohol consumption depletes hepatic vitamin A stores and vitamin A supplementation has been recommended for correction of this deficiency. However, high-dose vitamin A supplementation is hepatotoxic which is further potentiated by concomitant alcohol consumption. The mechanisms for this phenomenon are poorly understood. Recently, it was suggested that polar retinoid metabolites generated by alcohol-inducible cytochrome P4502E1 aggravate liver damage. However, direct experimental evidence supporting this hypothesis is still lacking. Our aim was to elucidate the effects of polar retinoid metabolites on cultured HepG2 cells and primary rat hepatocytes. Polar retinoid metabolites were extracted from liver tissues of Sprague-Dawley rats fed either an alcoholic or isocaloric control Lieber-DeCarli liquid diet for one month. Cell culture toxicity assays included morphology assessment, trypan blue exclusion test, and LDH/AST leakage. Staining for DAPI and acridine orange, FACS analysis and Western Blot for cleaved caspase-9 and -3 were used to detect apoptosis. Polar retinoid metabolites caused marked cytotoxicity in a concentration- and time-dependent manner in both cell types reflected by morphological changes, a dramatic increase in trypan blue positive cells and LDH/AST leakage. This toxicity involved apoptosis, as demonstrated by a time-dependent increase of sub-G1 cellular events, a rapid loss of mitochondrial membrane potential and a time-dependent activation of caspase-9 and -3. No toxicity was found with equivalent doses of the control extract from non-alcoholic rats. The present data demonstrate that polar retinoid metabolites cause marked hepatocyte death through the induction of apoptosis.