| Part1:Biomarkers of Expoure to Microcystin-LR in MiceObjective:In order to investigate the response indices to toxic microcystin-LR in blood of mice, concentrations of free and total microcystin-LR in blood and tissues, accompanied by serous parameters in series, including some enzymatic activities, hematology and the function of leukocytes, were determined.Methods:SPF (specific pathogen free) Kunming male mice, weighting20～25g, were randomly divided into5groups,7mice for each group. The mice in5groups were exposed to microcystin-LR through intraperitoneal injection at0.000,3.125,6.250,12.500and25.000μg/kg body weight respectively for7days. Then the blood samples were automatically analyzed by the instrument for the serum biochemical indices and the hematological investigations; cytokine levels in the serum were measured by radioimmunoassay; the measurement of DNA-protein crosslinks was performed using the SDS/KC1precipitation technique; the phagocytosis and reactive oxygen species of leukocytes was detected by flow cytometry; and the concentrations of free and total microcystin-LR in blood and tissues were determined by the liquid chromatography-mass spectrometry method.Results:The positive and sensitive findings in this experiment could be divided into four groups. The most sensitive indices were total microcystin-LR in the liver, phagocytic index and reactive oxygen species. The toxin in the liver was reliable and powerful, phagocytic index was affected by some subject factors and reactive oxygen species lacked a dose-effect relationship. The second sensitive indices were interleukin-6, alkaline phosphatase and lactate dehydrogenase, all of which showed good dose-effect results. The third were alanine aminotransferase and free microcystin-LR in the blood, of which the former was relatively less sensitive and specific, and the latter was specific and reliable but less sensitive compared with the toxins in the liver. The least sensitive indices were aspartate transaminase and tumor necrosis factor-a. The total microcystin-LR in the four dose-treated groups were0.1529±0.0296,0.3047±0.0648,0.6006±0.1105and0.9899±0.2261μg/g liver weight, respectively. The reactive oxygen species levels of lymphocytes (3299.37±120.54,3281.38±58.34,3308.06±136.12and3346.92±108.69, respectively) in the4treated groups were significantly lower than those in the control group (P<0.05); and the reactive oxygen species levels of monocytes (3271.51±140.79,3270.05±117.92,3326.90±114.39and3292.49±145.97, respectively) in the4treated groups also decreased significantly compared with the control group (P<0.05). The levels of interleukin6(346.837±25.536,360.847±37.886and434.245±35.858pg/mL, respectively) in the6.250,12.500and25.000μg/(kg·d) dose group rose in proportion, higher than those in the control group (P<0.05); while the levels of tumor necrosis factor-alpha (10.782±0.966fmol/mL) in25.000μg/(kg·d) dose group were statistically lower than those in the control group (P<0.05). The levels of alanine aminotransferase (187.99±53.00and381.54±89.23U/L, respectively) in the12.500and25.000μg/(kg-d) dose group were on the significant rise, higher than those in the control group (P<0.05). The levels of aspartate transaminase (473.10±168.07U/L) in the25.000μg/(kg·d) dose group were significantly higher than those in the control group (P<0.05).The levels of alkaline phosphatase (128.54±21.46,170.43±22.11and223.26±66.35U/L, respectively) and lactate dehydrogenase (690.50±112.11U/L,769.00±136.92U/L and952.53±169.73U/L, respectively) in the6.250,12.500and25.000μg/(kg·d) dose group were all above the control group, showing statistical differences (P<0.05).Conclusion:The results of the studies suggest that measurement of microcystin-LR in blood is powerful and clear evidence to indicate that the subjects have been exposed to microcystin-LR and can be used to discriminate from other causes leading to hepatic lesions although the microcystin-LR in blood is not as sensitive as other indices that are usually less specific but are useful complements to reflect the liver function. Part2:The Role of Microcystin-LR in Inducing Apoptosis of Liver Cells and its Relationship with the mRNA Expressions of Caspase-3, Apoptosis Inducing Factor and Endonuclease GObjective:The main purpose of this study was to investigate the apoptotic effects caused by microcystin-LR and its possible influence on the mRNA expressions of caspase-3, apoptosis inducing factor and Endonuclease G in mice liver.Methods:SPF (specific pathogen free) Kunming male mice, weighting20-25g, were randomly divided into5groups,7mice for each group. The mice in5groups were exposed to microcystin-LR through intraperitoneal injection at0.000,3.125,6.250,12.500and25.000μg/kg body weight respectively for7days. HE staining, TUNEL and real time PCR were used to detect the histopathological changes, cell apoptosis as well as the mRNA expression of caspase-3, apoptosis inducing factor and Endonuclease G in mice liver.Results:Compared to the control group, severe histopathological changes and significant differences in cell apoptosis were observed in3.125,6.250,12.500and25.000μg/(kg-d) dose groups. And further examinations showed that the mRNA expressions of caspase-3and apoptosis inducing factor were decreased in a dose-response relationship. There was no significant difference in the mRNA expressions of Endonuclease G among the5dose groups. The levels of apoptosis index by TUNEL assay(46.51±4.69,11.08±3.18,10.72±3.36and10.44±3.48, respectively) in the3.125,6.250,12.500and25.000μg/(kg-d) dose group were significantly higher than those in the control group (P<0.05). And the mRNA expressions of Caspase-3(0.8214±0.1150,0.5594±0.1800,0.3842±0.0745and0.3158±0.1307, respectively) in the3.125,6.250,12.500and25.000μg/(kg·d) dose group were significantly lower than those in the control group (P<0.05).Conclusion:The present study has demonstrated that microcystin-LR has relevance to the abnormal mRNA expressions of caspase-3and apoptosis inducing factor in the liver. |
PDF Full Text Request