The Dynamic Distribution And Toxicology Of Nodularin In Carassius Auratus

Nodularin is a cyclic pentapeptide cyanobacterial hepatotoxinproduced by Nodularia spumigena. It is synthetized by different molecular weight of multi-enzyme complex. It has negative effects on humans, animals, plants and eukaryotic microorganisms.A method using reversed phase high performance liquid chromatography with UVdetector has been developed for the determination of nodularincontent in C.auratus muscle, plasma, hepatopancreas and kidney. Homogenized or cut up tissues were extracted with 90% methanol solution through ultrasonic prior to centrifugation. The supermatants were defatted with hexane. After centrifugation, abandon the upper hexane phase and the underlayers were evaporated nearly to dryness at 40℃ with nitrogen.The residues were dissolved with methanol and diluted to the methanol content to 10% with water followed by purification by CNW Bond C18 column. The eluent were evaporated to dryness at 40℃ with nitrogen. The residues were dissolved with methanol and then the solution were filtrated with 0.45μm filter and then 20μL filtrates were detected by reverse phase high performance liquid chromatography with 238 nm detection wavelength. The results showed that the detection limits of NOD in muscles and plasma were 11.30μg/kg and 24.00μg/kg. The concentration of NOD in four crucian tissues showed good linearity with the peak area and all the correlation coefficients（R2） were greater than 0.99 in the concentration range of 0.05⁵.00mg/kg in crucian muscles, hepatopancreas, kidney and 0.05⁵.00mg/L in crucian plasma. In differenttissues,the average within-batch standard deviation of this methodwere（1.12±0.99）%^3.50±1.88% respectively, with an average recovery of（87.93±14.38）%^98.77±11.56%. This method could be applied to dynamic distribution and residues determination of NOD in C.auratus, providing an effective tool for the detection of NOD in other aquatic products and for the safety control of aquatic products quality.Dynamic distribution of nodularin in C.auratus was carried out following a single intraperitoneal injection at dose of 0.25mg/kg and 0.10mg/kg. The results demonstrated that at two doses, the content of NOD ranged from 0.600mg/kg to 4.029mg/kg and 0.229mg/kg to 0.753mg/kg in C.auratus hepatopancreas;range from 0.138mg/kg to 1.193mg/kg and 0.160mg/kg to 0.725mg/kg in C.auratus kidney; range from 0.118mg/kg to 0.406mg/kg and 0.035mg/kg to 079mg/kg in C.auratus plasma; range from 0.035mg/kg to 0.074mg/kg and 0.032mg/kg to 0.050mg/kg in C.auratus muscles. Consequently, nodularin were both accumulated in four tissues of crucian and the content of nodularin in hepatopancreas and kidney were higher than the initial injection doses. The ability of accumulation in four tissues of C.auratus were hepatopancreas>kidney>plasma>muscle. As a result, crucian hepatopancreas and kidney were the main accumulate library of nodularin.The preliminary inquiry of dynamic distribution of nodularin in C.auratus layed foundation for the detection of dynamic distribution of NOD in other freshwater omnivorous fish.The effects of nodularin on parameter of blood cell in C.auratus was studied following a single intraperitoneal administration. The dose of C, D and E were 0.05mg/kg, 0.10mg/kg and 0.25mg/kg respectively.Group B was administrated with pure methanol and group A was set as the control. All injection volume were in accordance with the actual weight of C.auratus. The result showed that white blood cell count（WBC） was not affected by NOD. The blood lymphocytes count（Lymph） and blood mononuclear（Mon） cell count had increased, Meanwhile, the blood neutrophils count（Gran） had decreased. The blood lymphocytes are main immune cells of fish and the bloodmononuclear cells play a role as transferring antigen in fish immune function. The main reason for this phenomenon may be that NOD stimulated the specific immune system in crucian making the number of immune cells increase. The number of red blood cells, hemoglobin（HGB） content and hematocrit（HCT） level are three significance indexes of red blood cell parameter. Other indexes including meancorpuscular volume（MCV）, mean corpuscular hemoglobin（MCH）, mean corpuscular hemoglobin concentration（MCHC） and red cell distribution width coefficient of wariation（RDW） are associated with RBC, HGB and HCT. The results showed that the count of RBC and the content of HGB had reduced in a short time. The possible reason may be that NOD could cause abnormal hemorrhagic anemia or hemorrhage of organs following by damages of organsin C.auratus.The effects of nodularin on the physiological and biochemical characteristic features of liver in C.auratus was studied following a signal intraperitoneal administration.The dose of C, D and E were 0.05mg/kg, 0.10mg/kg and 0.25mg/kg respectively.Group B was administrated with pure methanol and group A was set as the control. All injection volume were in accordance with the actual weight of crucian. The study found that the CAT activity in group C, D and E were induced by nodularin.In group C with the low dose, the CAT activity was very significantly increased when administrated NOD after 3h, 24 h and 192 h and significantly increased when administrated NOD after 0.5h and 12 h. In group D with the middle dose, the CAT activity was very significantly increased when administrated NOD after 3h, 6h and 24 h and significantly increased when administrated NOD after 0.5h and 192 h. In group E with the high dose, the CAT activity was very significantly increased when administrated NOD after 24 h and 48 h and significantly increased when administrated NOD after 0.5h and 1h. As a result, there were excess of reactive oxygen radicals in crucian under the stress of nodularin. The T-SOD activity of crucian hepatopancreas was not obviously affected by nodularin. The GPX activity in group C with the low dose and group D with the middle dose were induced. Meanwhile, it was inducedfirst and then obviously inhibited in group E with the high dose. The possible reasons for this phenomenon were the high dose of nodularin could cause oxidative damage in crucian to some extent making the antioxidant capacity of crucian depress. The reduced glutathione（GSH） as the substrate of GPX, the content of GSH in group C with the low dose and group E with the high dose were lower than the control group while it had increased first and then decreased in group D with the middle dose. It indicated that crucian had the ability of detoxification to NOD after sometime of intraperitoneal administration. However, the content of GSH in group E with the high dose had kept at a low level illustrating that the oxidative damage had increased and the antioxidant capacity had depressed in crucian. The conclusion of GSH was consistent with the GPX activity. The content of malonaldehyde（MDA） in groups C, D and E were greater than the control group as a whole. There was significant difference between the group E with the high dose and the control group about the content of MDA after 24h¹92h of intraperitoneal administration. The result showed that lipid peroxidation had happened in crucian under the stress of nodularin. As a consequent, NOD could cause injury of crucian cells and there was no obvious dose-dependent effects.