Primary Neuronal-astrocytic Co-culture Platform For Neurotoxicity Assessment Of Di-(2-ethylhexyl) Phthalate

Plastics are widely used in modern life,the industrial plasticizer di-(2-ethylhexyl)phthalate(DEHP)which is the largest amount used phthalate esters(PAEs)in manufacturing of a wide variety of polyvinyl chloride(PVC),and their application as a chemical additive agent,phthalates can leach out into the surrounding environment,and it always take long time to degradation.Meanwhile,PAEs are also existed in our indoor built around us.The main risk of DEHP is reproductive and developmental toxicity.The mechanism always been considered that it has the characteristics of endocrine disruptors.DEHP may also have estrogenic and anti-androgenic activity,and can affect the enzyme activities on catalyzing steroids synthesis.The central nerve system(CNS)in development process is very sensitive to many hormones such as the endocrine disruptors like DEHP.Epidemiological data shows that DEHP had the risk of neurotoxic;it could also be a probable carcinogen.Meanwhile,the neural effects of DEHP were not very certain.Also limited information reported concerning neurotoxic effects of DEHP in animals following DEHP exposure by any route.DEHP has recently attracted the attention of the scientific community due to their neural toxic effects as well as possible reproductive/developmental effects to human.There is therefore a need to investigate the effects and to explore the potential risks to human.This study focused on DEHP’s effect in the central nervous system by determining the precise DEHP content in mice brain tissue after exposure to the chemical,to evaluate the specific exposure range.In vivo studys detectable DEHP in brain homogenate samples was analysed using Gas chromatography-mass spectrometry assay GC-MS quantification showing DEHP had crossed the blood-brain barrier(BBB).In the highest exposure group 10mg/kg/day,there was 22.3μmol/L DEHP in the brain.Histological studies hematoxylin and eosin staining(H&E)and nissl staining also gave us the information that not only the DEHP could cross BBB and also caused the nerve injury.In vitro models,primary neuronal-astrocyte co-culture systems were used for chemical hazard identification of DEHP.Oxidative stress was hypothesized as a probable involved mechanism and thus the total reactive oxygen species(ROS)concentration was determined as a biomarker of oxidative stress.Vitamin C might serve as a useful biomarker of decreases in oxidative stress effect.In addition,NeuriteTracer,a neurite tracing plugin with ImageJ was used to develop an assay for neurotoxicity to provide quantitative measurements of neurological parameters,such as neuronal number,neuron count and neurite length,all of which could indicate neurotoxic effects.The results showed that with 1 nmol/L DEHP exposure,there was a significant increase in ROS concentrations,indicating that the neuronal-astrocytic cultures were injured due to exposure to DEHP.In response,astrocyte proliferation(gliosis)was initiated in the two exposure groups 100nmol/L,1 pmol/L,serving as a mechanism in maintaining a homeostatic environment for neurons and protecting neurons from toxic chemicals.At high concentrations(10μmol/L),DEHP’s toxic effects had resulted in harm to astrocyte ability to form a protective system,evidenced by large areas of astrocyte damage.In fact,with 10μmol/L DEHP,all neuronal parameters in these cultures were down regulated producing severe neurological damage to the co-culture system.There is a need to assess cumulative effects of DEHP in animal body to evaluate the possible uptake and effects on human neuronal system due to exposure to DEHP in indoor environment.