| Objective:DBDCT is a typical R2SnL2type organotin compound synthesized by our group. The central nervous system is the major toxic target organ of the organotin compounds, but the exact mechanism of their neurotoxicity still remains unclear. In the first part of this experiment, the detection method of tin in biological samples was established, the toxicokinetic parameters and the tissue distribution of DBDCT in a toxic dose were determined. And it was confirmed that DBDCT could penetrate through the blood-brain barrier and influence the central nerve system.The neurological effects in rats in vivo and the possible mechanisms of neurotoxicity of DBDCT in vitro were studied in the second and the third part of the experiment.Methods:(1) The atomic fluorescence spectrometry (AFS) assay was established to detect the concentration of tin in plasma or in tissue homogenates in rats. And the toxicokinetic parameters and tissue distributions of tin after single intravenous15mg/kg DBDCT in rats were detected using the AFS assay. These detections were used to preliminarily judge the possibility of DBDCT penetrating through the blood-brain barrier and influencing the central nerve system.(2) A series of biochemical indicators in serum and in brain tissues in rats after repeatedly intravenous injection of DBDCT were determined by UV spectrophotometric assay.(3) The changes of the rats’ body weights were observed during the exposure period of DBDCT, and the HE staining was used to observe the pathological changes in main organs after repeatedly intravenous injection of DBDCT. These detections were used to determine the main toxic target organ and possible causes of toxicity after exposure to DBDCT in vivo.(4) The apoptosis indexes (AI) and the expressions of apoptosis-related proteins in rats’ brains were detected after repeatedly intravenous injection of DBDCT using TUNEL and Western blot techniques, which were expected to explain the possible neurotoxic mechanism after exposure to DBDCT in vivo.(5) The PC12cell line was selected to study the neurotoxicity of DBDCT in vitro. Growth inhibitions of the PC12cells were analyzed by MTT method. Hoechst33258 and AO/EB staining, light and electron microscope were used to examine the nuclear changes after exposure to DBDCT. The apoptosis ratios were determined by PI/Annexin V-FITC double staining, and the specific DNA ladder-shaped strips were studied by agarose gel electrophoresis. The expression changes of apoptosis-related genes such as caspase-3, caspase-8, caspase-9, NF-κB, Fas, Fas-L and Cty-c were detected by RT-PCR. All the detection methods were used to confirm that the cytotoxicity and neurotoxicity exposure to DBDCT were caused by inducing apoptosis.(6) The changes of enzymatic activities of caspase-3and caspase-9in PC12cells before and after exposure to DBDCT were evaluated using UV spectrophotometric method. The changes of mitochondria transmembrane potential variance (ΔΨm) and reactive oxygen species (ROS) were measured using flow cytometry. Meanwhile, the protein expressions of Bax, Bcl-2, Cyt-c, caspase-3, caspase-9, p-JNK and p-p38were analyzed by western blot method to further confirm that the neuronal apoptosis induced by DBDCT was resulted through multiple signal transduction pathways.Results:(1) An AFS assay was established which was proved to be accurate quantitative, high precision, operated easily to detect the concentration of tin in plasma or in tissue homogenates in rats. After single intravenous injection of toxic dose to rats, the toxicokinetic parameters of DBDCT were showed below, the distribution half life t1/2α and the elimination half life t1/2β were49.977min and1.260min separately, the apparent volume of distribution Vc was8.201(μg/mL)/(mg/kg), the area under the curve was56.073(mg/kg)min, and the clearance rate Cl was0.297μg/mL/min/(mg/kg). After single intravenous injection of toxic dose to rats, DBDCT could be distributed quickly. The concentration of tin in adrenal gland was the highest, and the concentrations of tin in the rest tissues were similar. The tin concentrations in rats’brain were determined not less than0.10μg/g within the first two hours after intravenous injection.(2) The levels of AST, ALT, ALP, GGT, STB, BUN and CRE in serum after repeatedly intravenous injection of DBDCT were all elevated extremely obviously (p<0.01). The activities of SOD and GSH-Px in brain homogenate were increased, on the other hand, the MDA levels were decreased notably (p<0.01).(3) Although the body weights increased in the first three weeks, they dropped at the last week after repeatedly intravenous injection of DBDCT. After HE staining, the thickened liver capsules, eosinophilic change and karyopyknosis of liver cells were observed in liver, nodular hyperplasia of nerve cells and cell degenerations in cerebral cortex were also observed in brain.(4) After repeatedly intravenous injection of DBDCT, the nucleus of the neurons in the cerebral cortex appeared irregular shaped and were stained brown by TUNEL staining. The apoptosis index (AI) was significantly increased from8.26±1.25%to30.3±1.94%(p<0.01). After western blot assay, the protein expression of Bax increased, on the contrary, the protein expression of Bcl-2decreased, the ratio of Bax/Bcl-2increased from0.767to2.842. The cleaved caspase-3and cleaved caspase-9were detected.(5) The IC50of DBDCT in24h was4.110μmol/L. The classic nuclear morphology characteristics were observed in PC12cells exposed with DBDCT using fluorescent staining, light and electron microscope. The apoptosis rates determined by PI/Annexin V-FITC double staining method in PC12cells exposed with DBDCT for different period of time showed significant statistics difference (p<0.05) compared with the control group. In the agarose gel electrophoresis, DNA ladder-shaped strips were also clearly observed. The results of RT-PCR indicated that the mRNA expresses of apoptosis-related genes such as caspase-3, caspase-8, caspase-9, NF-κB, Fas, Fas-L and Cty-c in the PC12cells exposed with DBDCT were obviously changed (p<0.05, p<0.01) compared with the normal PC12cells.(6) The enzymatic activities of caspase-3and caspase-9of PC12cells exposed with different concentrations of DBDCT for different period of time increased significantly (p<0.05,P<0.01). The ΔΨm strikingly decreased which was detected by JC-1, and the generation of ROS increased exposed with different concentrations of DBDCT for different period of time. The results of western blot analysis showed the down-regulation of bcl-2and the up-regulation of Bax. The expression of Cyt-c decreased in mitochondria and elevated in the cytosol. The cleaved caspase-3and cleaved caspase-9were detected. The expressions of p-JNK and p-p38also increased. All the changes of protein expressions were dose-and time-dependent (p<0.05).Conclusions:(1) The AFS applied to the determination of the tin concentrations in the blood and tissue samples. The concentration changes of tin in blood over time met the two-compartment model. DBDCT distributed rapidly, but was not easy to accumulate in the major tissues and organs. It could penetrate through the blood-brain barrier, which indicates it may have neurotoxic effect.(2) DBDCT affected the increase of the body weights and the general growth and development of rats. The changes of a series of biochemical indicators in blood and in brain tissues in rats revealed that DBDCT had significant liver toxicity and kidney toxicity.(3) DBDCT reduced the activity of the antioxidant system and enhanced the lipid peroxidation in the brain tissue of rats, and to some extent, caused damage in central nerve system by inducing neuron apoptosis in vivo.(4) DBDCT was able to induce apoptosis in the PC12cells cultured in vitro, the mechanism of which might be oxidative stress and DNA damage. The Bcl-2family, mitochondrial apoptosis pathway and MAPK signal transduction pathway were all involved in the apoptosis in the PC12cells induced by DBDCT, which revealed that DBDCT could cause neurotoxicity by inducing apoptosis in neuron on multi-pathways. |
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