6th ESACP Congress, Heidelberg, April 7-11, 1999

A035
PROOXIDANT ACTIVITY OF ANTHRAQUINONE CAUSE CYTOTOXICITY AND APOPTOSIS IN PRIMARY CULTURES OF RAT HEPATOCYTES
Bironaite D

Institute of Biochemistry, Academy of Sciences, Vilnius, Lithuania

We have studied the hepatotoxicity and ability to induce apoptosis of three different anthraquinones, rhein (4,5-dihydroxy-anthraquinone-2-carboxylic acid), danthron (1,8-dihydroxy-anthraquinone) and chrysophanol (1,8-dihydroxy-3-methylanthraquinone). Among these tested quinoidal compounds, rhein was the most effective in producing free radicals and the only one to induce apoptosis. Rhein showed the highest enzymatic activity toward NADPH:ferredoxin reductase, as a single-electron reducer of quinoidal compounds and, as determined by the oxygen consumption rate, was better redox cycler (109µM 5±2.0 x 10-8 M/s and 60 ± 3.0 µM/min) than danthron (6.4 5 ± 0.5 x l0-8 M/s and 3.6 ± 3.0 µM/min ) or chrysophanol (1.3 ± 0.1 x l0-8 M/s and 1.2 ± 3.0 µM/min). Moreover, during the first 15 min of incubation of hepatocytes with 50 pM of rhein, the intracellular amount of GSH disappeared while the amount of ATP was still the same as in the control cells. During the next 15 min of exposure cells to rhein, the amount of GSH partly recovered whereas ATP started to deplete. It is known that rhein acts as a competitive inhibitor toward NADH of NADH:ubiquinone reductase with Ki = 50 µM and mixed-type inhibitor toward NADPH:glutathione reductase with uncompetitive Ki = 1.1 µM and competitive Ki = 8 µM. According to these results one may assume that rhein firstly initiated the generation of the reactive oxygen species with the subsequent lipid peroxidation and at the same time inhibited synthesis of GSH, which has been partly restored by the antioxidants. The depletion of ATP started together with the leaking out of the lipid peroxidation products. During the first 15 min of exposure of cells to rhein all these events initiated apoptosis. This was proven by DNA fragmentation, nuclear condensation and positive TUNEL reaction, which were most intensive after the additional 16 hours incubation of hepatocytes in the complete medium. According to these results one may assume that the compounds that produce free radicals and have strong autooxidation (rhein) capacity are able to initiate apoptosis during the short time of exposure.