Study Of Combined Effect Caused By Combined Exposure Of TCDD And PCBs Using Metabonomics Technology

Endocrine disruptors(EDs) are widely dispersed environmental and food contaminants in our country that poses a great risk to the public health.They have been one of the widespread current subjects for concern in health safety throughout the world,especially their combined effects of combined exposure.2,3,7,8-Tetrachlorodibenzo-p-dioxin(TCDD) and polychlorinated biphenyls(PCBs) are typical EDs.At present,they are one of the hot spots in the field of environmental toxicology because of their extensive disposition and coexistence in the environment,persistence,bioaccumulation,migration and high toxicity.But most researches on the toxicity and human health hazards of these EDs have focused primarily on toxicology studies conducted on individual compound,and traditional toxicity evaluating techniques remain mainstream,but the possible interactions of combined exposure to EDs have not been investigated systematically.However,considering the facts that humans are ubiquitously simultaneously exposed daily to complex mixtures of environmental contaminants in our country, assessment of the mixture effects of multiple pollutants with the new technique is of more theoretical and actual significance.Nuclear magnetic resonance(NMR)-based metabonomic approach is a newly developed technology that has been extensively applied in the studies of toxicology.Therefore,the present study was designed to assess the potential combined toxic effects of combined exposure to TCDD and PCBs with metabonomics combined with conventional toxicologic technologies,so as to explore the possible modes and mechanisms of combined effects and select the possible biomarkers of combined toxicity.The potential application of metabonomics in the combined effects study of EDs was explored.First,the platform,based on metabonomics technique,for EDs toxicity study was established.Twenty Sprague-Dawley rats were subjected to 2×2 factorial experimental design and assigned randomly into four groups of five rats each group,including the control group,the PCBs exposure group,the TCDD exposure group,and the combined-exposure group.Rats were intragastrically administered vehicle(olive oil),PCBs(Aroclor1254,10 mg/kg),TCDD(10μg/kg),or the combination(10μg/kg TCDD+10 mg/kg Aroclor1254),once a day for consecutive 3 days.Rats were housed in metabolism cages and the 24 hour urinary samples of each rat were collected before and after exposure and its hydrogen nuclear magnetic resonance (~1H NMR) spectra were measured,spectral characteristics for each group were summarized and the data were analyzed by principal component analysis(PCA).Based on the results from PCA in all groups,the samples were primarily distributed in four regions of the scoring oval(95% confidence intervals).The urine metabonomic trajectory of all exposure groups animals could obviously separated with that of control animals,and there were separated between the exposure to TCDD or PCBs group and the combined-exposure group,which suggested that all groups had a distinct metabonomic spectra.The results of this partial study suggest that metabonomic techniques can be used to characterize the metabolic consequence combined exposure to TCDD and PCBs,to elucidate the combined toxic effects and mechanism,that the platform based on metabonomics technique has been successfully established.Metabonomics technology can be proved more sensitive than traditional method for toxicity evaluation.Metabonomics is a promising new technology for the combined effects study of EDs.Secondly,using the metabonomics technique combined with conventional toxicologic technologies to investigate the potential combined toxic effects of combined exposure to TCDD and PCBs,to observe the relationship of the metabonomic pattern of rat urine with blood biochemical indices and histopathology and select the possible biomarker candidates of combined toxicity,to explore the toxic effect mechanism of combined exposure.SD rats were intragastrically administered TCDD,PCBs,or the combination daily for consecutive 6 days.The 24 hour urinary samples of each rat were collected before and after exposure and its nuclear magnetic resonance spectra were measured,the serum biochemical analysis,histopathology examination and tissue oxidative damage detection were taken.The results showed that all treatment groups produced different degree toxicities,as characterized by losses in body weight, changes in organ coefficients and serum biochemistry parameters,histological changes and tissue oxidative damage.Most of these effects were more remarkable in the combined-exposure group.The ~1H NMR spectroscopic and pattern recognition methods were used to detect alteration of the urine metabolite expression profile of combined exposure to TCDD and PCBs. Results showed that all treatment groups could be readily distinguished in the PCA analysis diagram without overlap,and just after exposure,the urine metabonomics trajectory biased from that of the controls or pre-exposure.It was well in agreement with the toxic injury differentia of differently treated rats that the metabonomic spectra of urine from these rats could also be distinguished easily from each other.The result of rat urine metabonomic pattern change was in accordance with that of traditional toxicity evaluation.The PCA analysis of urine ~1H NMR data showed that the levels of 2-oxoglutarate,citrate,succinate,creatine,lactate, trimethylamine-N-oxide(TMAO),hippurate,2-hydroxy-isovaleric acid,taurine,dimethylamine (DMA),creatinine and glucose of rats urine changed after exposure,and most of these changes were more conspicuous in the combined-exposure group.These changed metabolites may be considered biomarker candidates of the combined toxicity.Furthermore,the increase of the toxic effects induced by combined exposure to TCDD and PCBs are related to the injury of mitochondrial function,reductions energy metabolism in tricarboxylic acid cycle(TAC) and perturbations in the metabolism of glucose,lipid and amino acidsFinally,the possible modes and mechanisms of combined effects of TCDD and PCBs were investigated.SD rats were intragastrically administered TCDD,PCBs,or the combination daily for consecutive 12 days.After sacrificed rats,serum was used for detecting blood biochemical parameters,the tissues were used to histopathologic study,and tissue lipid peroxidation and the protein expression of cytochrome P450 1A1(CYP1A1),heat shock protein 70(HSP70) were determined.The consequence suggested that all rats of treatment groups showed significant toxicities,as evidenced by significant decreases in body weight,changes in organ coefficients and serum biochemistry parameters,and marked histological changes and tissue oxidative damage,furthermore,high expression of CYP1A1 and HSP70 in tissues.Most of these effects were more conspicuous in the combined-exposure group.These findings indicated that aryl hydrocarbon receptor-mediated the high expression of CYP1A1 elicited oxidative injuries may play an important role in the combined toxicity of TCDD and PCBs,and suggested that malondialdehyde(MDA),superoxide dismutase(SOD),glutathione peroxide(GSH-Px), CYP1A1 and HSP70 in tissue may be considered molecular biomarker candidates of the combined toxicity.In addition,the result of the two-way analysis of variance revealed that the combined effects of TCDD and PCBs were complicated,being additive,synergistic or antagonistic depending on the selection of toxicity endpoints under the present experimental condition.Based on the results of this study,the following conclusions could be made under the present experimental condition:①The metabonomics spectra of urine could be distinguished readily by different toxic mechanism using metabonomics technique.Metabonomics is a promising new technology for the combined effects study of EDs.②It was in accordance with traditional measurements for evaluating toxicity,but metabonomics method was more sensitive than traditional measurements.③Exposure alone or combined exposure to TCDD and PCBs produced significant toxicities,and the toxic effects of combined exposure were more conspicuous.④The mode of combined effects of TCDD and PCBs were complicated,and showed primarily to be additive or synergistic.⑤Using the metabonomics technique combined with conventional toxicologic technologies had selected the possible biomarkers of combined toxicity.⑥The aryl hydrocarbon receptor-mediated the high expression of CYP1A1 elicited oxidative injuries may play an important role in the combined toxicity of TCDD and PCBs.In addition,the combined toxic effects are also related to the injury of mitochondrial function, reductions energy metabolism in TAC and perturbations in the metabolism of glucose,lipid and amino acids.