An In Vitro Study On Developmental Neurotoxicity Of DEHP And MEHP

Di-(2-ethylhexyl)-phthalate (DEHP) is the most frequently used plasticizer with an estimated annual world production around 3-4 million tons.Since DEHP is not held by chemical bond to the polymer, but rather by the weaker intermolecular forces, it can be released from plastics into the envioronment easily and later absorbed by people. As a ubiquitous environmental contaminant, DEHP shows a low acute toxicity on human beings while the consequences of chronic exposure have not been clarified. However, based on a large number of annimal experiments and some limited epidemiology studies, DEHP is suspected posing on human beings risk of respiratory allergic diseases,carcinogenesis, teratogenesis, hepatotoxicity and reproductive toxicity. Here we address the developmental neurotoxicity of DEHP in vitro because neurotoxicity is not adequately covered by current testing strategies and the developing brain is much more vulnerable to injury caused by different classes of chemicals than the adult brain. In this study, PC 12 cells and C6 cells were selected as models of neurons and glia cells, respectively. The developmental neurotoxicity of DEHP and MEHP are explored by evaluating their impact on proliferation and differentiation of PC 12 cells and C6 cells.Part I:The impact of DEHP,MEHP on proliferation and differentiation in PC 12 cellsObjectives:To explore the developmental toxicity of DEHP to neurons by evaluating its impact on proliferation and differentiation of PC 12 cells. Methods:PC 12 cells were cultured and exposed to DEHP or MEHP at a serial consentrations varied from 0.1mg/L to 100mg/L, along with blank and sovent control. The proliferation test panel including: Determining growth curve by an improved MTT method; Determing viable cells percentage by trypan blue exclusion test; Determining cell cycle by cytomytry; Determine DNA synthesis level by cytomytry and BrdU-ELISA method, respectively. The tests of cell differentiation was initiated by culturing the cells in medium containing 50ng/mL murine 2.5S NGF and DEHP or MEHP at different consentrtions, along with blank and sovent control. The differentiation test panel including:Counting and calculating percentage of neurite outgrowth-possitive cells by morphology exam under microscrope; Sequencial seperating and quantifying membrane and cytoskeletal proteins; Extracting total RNA in PC 12 cells and quantifying the levels of ChAT mRNA, TH mRNA and GAPDH mRNA by reverse transcription-real-time. quantitative-PCR(RT-qPCR) Results:DEHP and MEHP showed no cytotoxicity to undifferentiated PC 12 cells in concentration range 0.1～100mg/L; DEHP didn't alter cell viability, cell cycle and DNA synthesis level of undifferentiated PC 12 cells in concentration range 0.1～100mg/L; MEHP dose-dependantly decreased cell viability of PC12 cells in concentration range 1.6～100mg/L(P<0.05); MEHP dose-dependently increased the percentage of cells in G2 phase at expense of decreasing those in S phase as well as a sharp drop of DNA synthesis level in the concentration range 0.1～100mg/L(P<0.05). During the differentiation process of PC12 cells, DEHP and MEHP increased membrane and cytoskeletal proteins as well as the percentage of neurite outgrowth-possitive cells, while they decrease TH mRNA level(P<0.05) and keep the level of ChAT mRNA unchanged in the concentration range 1～100mg/L and 10～100mg/L, respectively. Conclusion:DEHP has no effect on cell proliferation of PC 12 cells in the concentration range 0.1～100mg/L,but it can dose-dependently promote differentiation of differentiating PC 12 cells in the concentration range 1～100mg/L. MEHP surpresses the proliferation of undifferentiated PC 12 cells dose-dependently in the concentration range 0.1～100mg/L. and promotes differentiation of differentiating PC 12 cells in the concentration range 10～100mg/L Part II:The impact of DEHP and MEHP on proliferation and differentiation in C6 cellsObjectives:To explore the developmental toxicity of DEHP to glia cell by evaluating its impact on proliferation and differentiation of C6 cells. Methods:C6 cells were cultured and the method for cell proliferation test is same as PartⅠ. The tests of cell differentiation was initiated by culturing the cells in medium containing 2mM sodium butyrate and DEHP (or MEHP) at different consentrations, along with blank and sovent control. The differentiation test panel including:Counting and calculating percentage of extend-possitive cells by morphology exam under microscrope; Sequencial seperating and quantifying of cytoskeletal proteins; Extracting total RNA in cells and quantifying the levels of GFAP mRNA and GAPDH mRNA by reverse transcription-real-time quantitative-PCR(RT-qPCR)Results:DEHP and MEHP showed no cytotoxicity to C6 cells in consentration range 0.1～100mg/L; 0.4mg/L of DEHP pulled the grow curve up without affecting cell cycle and DNA synthesis level; DEHP didn't affect cell growth curve at consentration abovel.6mg/L; DEHP didn't alter cell cycle and DNA synthesis level of C6 cells in concentration range 0.1～100mg/L; MEHP dose-dependantly decreased cell viability in concentration range 106～100mg/L(P<0.05); MEHP increased the percentage of cells in G2 phase at expense of decreasing those in S phase as well as a sharp drop of DNA synthesis level in the concentration range 0.1～100mg/L(P<0.05). During the differentiation process of C6 cells, DEHP and MEHP increased membrane and cytoskeletal proteins, and also increased the percentage of extending-possitive cells as well as the level of GFAP mRNA dose-dependantly in the concentration range 1～100mg/L and 10～100mg/L, respectively.in asconstant. Conclusion:DEHP has no effect on cell proliferation of C6 cells in the concentration range 0.1～100mg/L,but it can promote differentiation of differentiating C6 cells in the consentration range 10-100mg/L. MEHP surpresses the proliferation of undifferentiated C6 cells in the concentration range 0.1～100mg/L. and promotes differentiation of differentiating C6 cells in the concentration range 10-100mg/L.