Researches On The Apoptosis Mechanisms Induced By Microcystins

Microcystins (MCs) produced from alga blooms have been one of the most important toxins in the aquatic ecological systems. Mechanisms and the toxic effects of MCs were still the most focused issues.In this paper a simple SPE-HPLC method for the determination of microcystins in water samples was optimized and a HPLC-ESI-MS method for the determination of trace microcystins in fish tissues was established. Based on these methods, we tried to find the relationships between microcystins and environmental factors in the aquatic environements and we analyzed the bioaccumulation effects and organ distribution of microcystins in phytoplanktivorous fish Hypophthalmichthys. molitrix. We also found microcystins can induce sensitive apoptosis of fish lymphocytes in vitro and the mitochondrial damage was the main apoptosis mechanism. Finally a new cytotoxic index of EC50 was used to evaluate the lose-dose exposed cytotoxicities of microcystins and other benzenes. The main results are shown as follows:(1) The determination method for MCLR and MCRR in water samples was established by the Oasis HLB SPE cartridge and Agillent 1100 HPLC systems;The lowest determination limitation of MCLR and MCRR were 2.0ng and 0.5ng, respectively, with the average recoveries of 79.5% and 81.4%;By the established method, 22ug/L MCRR was detected from samples of the Baoyang River in Taihu Watershed and 0.71-0.96ug/L MCLR of the Qiantang River.(2) The determination method for MCLR and MCRR in fish tissue samples was established by a HPLC-ESI-MS system;The lowest determination limitation of MCLR and MCRR were 70pg and 40pg, respectively, with the average recoveries of 74.2% and 88.4%;By the established method, 0.51~2.4ug/g MCLR and 0.92-6.4u.g/g MCRR were detected in the intestine, liver, kidney and muscle samples of phytoplanktivorous fish Hypophthalmichthys. molitrix in Tiesha River;both MCLR and MCRR were bioaccumulated in the intestine and there were no microcystins detected in the lipid tissues. Oral uptake may be the main exposure pathway for microcystins uptaked in Hypophthalmichthys. molitrix. We also calculated the daily MCs intakes of consuming Hypophthalmichthys. molitrix in theRiver were 0.03 ug/kg body weight which was very close to the WHO standards (0.04 ug/kg body weight).(3) According to the results of the relationship researches between the concentrations of microcystins and environmental factors, NO2-N and NO3-N may be the different nitrogen resources for the biosynthesis of MCLR and MCRR, respectively. TP also may be related with the production of MCLR.(4) MCLR and MCRR can induce sensitive apoptosis of Carassius auratus lymphocytes at the exposured levels of l~10nmol/L in vitro and this apoptosis was time and dose dependent manner. We further confirmed that the mitochondrial pathway was involved in the sensitive apoptosis in Carassius auratus lymphocytes in vitro induced by both MCLR and MCRR. A new index of apoptosis EC50 can be used to evaluate the cytotoxic effects of MCs and benzeness, and we found the cytotoxicities of MCs were deeply infacted by the dipole manner.