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6th ESACP Congress, Heidelberg, April 7-11, 1999 |
A035
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.
PROOXIDANT ACTIVITY OF ANTHRAQUINONE CAUSE CYTOTOXICITY AND APOPTOSIS IN
PRIMARY CULTURES OF RAT HEPATOCYTES
Bironaite D
Institute of Biochemistry, Academy of Sciences, Vilnius, Lithuania