| In the development of new drugs , about 90% candidates were eliminated, and 50% of them due to their toxicity. To protect human health , toxicity of new chemicals is assessed by industry and institution /agencies. Toxicity assessment mostly bases on the results of animal toxicological tests. The replacement of the in vivo tests by alternative in vitro assays would offer the opportunity to screen and assess numerous compounds at the same time, to predict acute oral toxicity and thus accelerate drug development.The advantages of in vitro systems in toxicity testing are numerous.In vitro tests are usually quicker and less expensive. Experimental conditions can be highly controlled and the results are easily quantified. Medium and high throughput screening in vitro introduced by pharmaceutical industries can greatly reduce the cost of analyzing lots of synthetic compounds and the number of animal studies. Because of these, pharmaceutical industries need to evaluate the toxicity of the candidates on an earlier stage with high throughput screening method which just need little chemical. This goal can be reached by the introduction of cell culture with cytotoxicity or target related toxicity assays.ObjectiveIn this study we set up a neuronal cell model by isolating and culturing primary rat cortical neurons and rat's pheochromocytoma (PC12) cells, a cardiotoxicity model using primarily cultured myocardial cells of neonatal rats and rat cardiomyocyte H9c2 cell, and a hepatotoxicity model by culturing three human hepatocyte cell strains, including human normal hepatocytes L-O2 cell, human liver cancer cell HepG2 cell and BEL-7402 cell. We employ these models to evaluate the acute toxic effect on nerve system, cardiac muscle and liver. Then we compared the results with the parameters of acute toxic effect in animal experiments, such as LD50, to analyze and determine the relationship between in vitro and in vivo safety assessment systems, and find out whether the assessment systems in vitro can replace or partly replace the animal toxic experiment. This study will provide evidences for establishing stable high-throughput screening methods to assessment system for the acute toxicity of drugs.Methods(1) Neurotoxical test of drug/compoundPrimary rat cortical neuron isolated from 24h-old Sprague-Dawley rats were cultured in 96 multiwell plates primarily coated with poly-lysine, and ready for use after 7 to 10 days. PC12 cells were cultured in 96 multiwell plates at a density of 5×105cells/ml. The cultures were incubated at 37℃in a 5% CO2/95% humidified atmosphere for 24h. 17 neurotoxicitive drugs or compounds at serial concentrations were co-cultured with primary neuron and PC12 cells for 24h. Cellular morphologic changes were observed and the MTT assay was used to assess toxicity. Inhibitory concentrations were extrapolated from concentration-effect curves after linear regression analysis. Seventeen chemicals were tested with each cell line and the IC50 was calculated by the curve, and was compared with the rat oral LD50.(2)Cardiotoxical tests of drug/compoundPrimary cardiac myocytes isolated from 24h-old Sprague-Dawley rats were cultured in 96 multiwell plates, and ready for experiment after 4 to 7 days. H9c2 cells were cultured in 96 multiwell plates at a density of 1×105cells/ml. The cultures were incubated at 37℃in a 5%CO2/95% humidified atmosphere for 24h. 20 drugs or compounds at serial concentrations were co-cultured with primary cardiac myocyte and H9c2 cells for 24h, and the MTT assay was used to assess toxicity. Cardiac pulsation and cellular morphologic changes were observed. Inhibitory concentrations were extrapolated from concentration-effect curves after linear regression analysis.Twenty chemicals were tested with each cell line and the cytotoxicity data were compared to rat oral LD50.(3) Hepatotoxcal tests of drug/compoundHepG2 (Human hepatocellular liver carcinoma cell line), BEL-7402(human hepatoma cell line), human liver cell line L-O2 were cultured at a density of 5×105cells/ml in 96 multiwell plates, incubated at 37℃in a 5%CO2/95% humidified atmosphere for 24h. 21 drugs or compounds were at serial concentrations were co-cultured with HepG2, BEL-7402, L-O2 cells 24h, cellular morphologic changes were observed and the MTT assay was used to assess toxicity. Inhibitory concentrations were extrapolated from concentration-effect curves after linear regression analysis. 21 chemicals were tested with each cell line and the cytotoxicity data were compared to rat oral LD50.Results(1)The experimental results showed significant correlation between IC50 values in cell lines and the LD50 of acute oral toxicity in rats,with R2 of 0.77 and 0.64for primary rat cortical neurons and PC12 cells respectively. The regression was determined by using the mean IC50 values obtained for each laboratory and LD50 data for the 17 chemicals that had rat oral LD50 values. Predicted LD50 values can be determined using the mean IC50 values obtained for each laboratory in the regression equation(Primary rat cortical neuron :lg(LD50)(mg/kg)=0.4575 lg(IC50)(mg/ml)+3.2826; PC12:lg(LD50)(mg/kg)=0.4715lg(IC50)(mg/ml)+3.1721). (2)Significant correlation was evident between IC50 values in cell lines and the LD50 of acute oral toxicity in rats,with R2 of 0.76 and 0.67 for primary cardiac myocyte and H9c2 cells respectively. The regression was determined by using the mean IC50 values obtained for each laboratory and LD50 data for the 20 chemicals that had rat oral LD50 values. Predicted LD50 values can be determined using the mean IC50 values obtained for each laboratory in the regression equation(Primary cardiac myocytes:lg(LD50)(mg/kg)=0.5177lg(IC50)(mg/ml)+ 3.1602; H9c2:lg(LD50)(mg/kg)=0.4362lg(IC50)(mg/ml)+ 3.2283).The cardiocytes beat arrhythmically and terminated along with increasing concentrations, and morphological changes were observed in the cells.(3)Significant correlation was evident between IC50 values in cell lines and theLD50 of acute oral toxicity in rats,with R2 of 0.81, 0.81 and 0.60for HepG2, L-O2 and BEL-7402 cells. The regression was determined by using the mean IC50 values obtained for each laboratory and LD50 data for the 21 chemicals that had rat oral LD50 values. Predicted LD50 values can be determined using the mean IC50 values obtained for each laboratory in the regression equation(HepG2:lg(LD50)(mg/kg)=0.569 lg(IC50)(mg/ml)+ 3.3062; L-O2:lg(LD50)(mg/kg)=0.5332lg(IC50)(mg/ml)+ 3.2913;BEL-7402:lg(LD50)(mg/kg)=0.5462lg(IC50)(mg/ml)+ 3.1746).ConclusionPrimary neuron, primary cardiac muscle cell and human normal hepatocyte cell line could be used for evaluating acute toxicity of compounds and drugs in vitro. Primary cells showed higher correlation between IC50 and oral LD50 than passage cells,such as PC12, HepG2,BEL-7402. For the passage cells, only ordinary cultural conditions and simple skills were needed. The experimental results demonstrated that the cellular cytotoxicitiy evaluating system was more desirable for high-through toxicological evaluation of compounds and drugs because of fair reproducibility and less expendicture of time and fund, and could reduce the quantity of animals or replace the zoopery partially. We have established the high-throughout selective toxicitiy evaluating system for different organ or tissue based on several kinds of relevant cells, and demonstrated the possibility of reducing or replacing the zoopery.
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