Studies on the possible mechanisms of protective activity against alpha-amanitin poisoning by aucubin

Aucubin, an iridoid glucoside, was investigated to determine whether it has a stimulating effect on alpha-amanitin excretion in alpha-amanitin intoxicated rats, and whether there is binding activity to calf thymus DNA. High-performance liquid chromatography (HPLC) analysis of alpha-amanitin in rat urine allowed quantitative measurement of the alpha-amanitin concentration with a detection limit of 50 ng/ml. In this system, a group treated with both alpha-amanitin and aucubin showed that alpha-amanitin was excreted about 1.4 times faster than in the alpha-amanitin only treated group. Our previous results showed that the toxicity of alpha-amanitin is due to specific inhibition of RNA polymerase activity and the resultant blockage of the synthesis of certain RNA species in the nucleus. However, no significant activity change on RNA polymerase from Hep G2 cells was observed when aucubin was treated with alpha-amanitin at any concentration tested. Nevertheless, aucubigenin inhibited both DNA polymerase (IC50, 80.5 microg/ml) and RNA polymerase (IC50, 135.0 microg/ml) from the Hep G2 cells. The potential of both alpha-amanitin and aucubin to interact with DNA were examined by spectrophotometric analysis. Alpha-Amanitin showed no significant binding capacity to calf thymus DNA, but aucubin was found to interact with DNA, and the apparent binding constant (Kapp) and apparent number of binding sites per DNA phosphate (Bapp) were 0.45 x 10(4) M(-1) and 1.25, respectively.