Evaluation Of Toxicity Levels And Cytotoxicity Mechanisms Of Corexit 9500

The Gulf of Mexico oil spill(DWH)incident on April 20,2010 was the largest oil spill in the history of the United States.The accident was caused by the explosion of the well MC252,which was located at a depth of 1,500 meters below sea level and 84 kilometers away from Louisiana.It took 87 days for the damaged well to be completely closed.It is estimated that 780000 m³of oil was released into the ocean.Crude oil is a complex mixture containing thousands of chemicals with a density ranging from 800 to 900 kg/m³.The density of seawater is about 1200 kg/m;therefore,crude oil will float on seawater.Crude oil contains a variety of toxic compounds,including polycyclic aromatic hydrocarbons(PAHs),accounting for 0.2%to 7%of its weight.Several of these polycyclic aromatic hydrocarbons have been identified as environmental toxins,and studies have shown that exposure to these compounds poses a considerable risk to human health.Dispersants act to reduce the interfacial tension between water and oil,allowing the oil to break into small globules which are distributed over a larger volume of water.This enhances the removal of oil through volatilization,photo-oxidation,and biodegradation.As an emergency measure,BP predominately used Corexit9500A to disperse crude oil in the Gulf of Mexico to prevent shoreline contamination.The use of Corexit is a concern since the impacts of Corexit on human health and environment are unclear.This study attempts to quantify the in-vitro toxicity of Corexit by using cell lines from different tissues,and also provides data regarding toxicity mechanisms.The study provides indirect evidence on the effects of Corexit on human health effects.A3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide(MTT)assay was used to study the cytotoxicity effects.Reactive oxygen species(ROS)and lipid peroxidase(LPO)were measured by fluorometric and colorometric methods.Also glutathione(GSH)content and superoxide dismutase(SOD)and catalase activity were measured to develop a better understanding of toxicological pathways.In our study,we found that different mammalian cells have variable ability to resist Corexit toxicity effects.Cells were found to be more defenseless under serum-free states when compared to serum-fed states.In the serum-free state,the order of sensitivity towards Corexit induced cell death was found to be:B16/BL6>1321N1>H19-7>HEK293>HK2.The average value of LC50 for each cell line was found to be:16 ppm for BL16/B6;33 ppm for 1321N1;70 ppm for H19-7 cell;93 ppm for HEK-293 and 95 ppm for HK-2cell.These results imply that highly proliferative cells are more susceptible to Corexit induced cell death,as compared to normally transformed adult cells of neuronal and kidney tissues.Previous studies have reported the LC50 of Corexit in two human liver cell lines(Hep G2 and Hep G2/C3A)to be 120 ppm and 250 ppm,respectively.In the current study,our aim was to develop a fundamental understating of Corexit toxicity at the cellular level.The data obtained from this study will be useful for quantifying the environmental and human health risks posed by Corexit.Furthermore,the result obtained from this study will help to understand the underlying toxicity mechanisms which can be used to develop novel drug therapies to protect against Corexit-induced toxicity effects.Mechanistically,the electron transportation chain serves as the means of oxidative phosphorylation in eukaryotes.Through electron transportation chain,ATP is produced to provide the energy for living cells and this procedure occurs on the membrane of the mitochondria.The decreased activity of Complex-I can lead to the dysfunction of mitochondria.Previous studies have mapped the potential relation between Complex-I and ROS generation.ROS includes free radicals such as hydroxyl(·OH),alkoxyl(RO),and peroxyl(ROO)ions,it also includes non-radicals such as hydrogen peroxide(H₂O₂).ROS are the by-products of electron transportation chain reactions in the mitochondria.And it is believed that the generation of superoxide anion is related within Complex-I and Complex-III¹.When superoxide anion is formed,the superoxide dismutase will be activated and convert superoxide anion to hydrogen peroxide.Glutathione and catalase are the two antioxidants which can eliminate the deleterious effects of hydrogen peroxide by converting it to water and oxygen.If formed in excess,Hydrogen peroxide being a stable chemical will be rapidly degraded to OH·.The hydroxyl radical thus formed attacks cell membrane and initiate lipid peroxidation,causing DNA cleavage,and protein degradation.Our results show decreased Complex-I activity in cells treated with Corexit(80ppm)which indicated mitochondria dysfunction.ROS concentrations increased significantly after cells were treated with Corexit(80ppm)for 48 hours,indicating that the cells were under stressful conditions.Meanwhile,the antioxidants also responded to the stress triggered by Corexit.The SOD activity was elevated indicating a self-protection mechanism of the cell,while catalase activity was also increased.However,another antioxidant GSH showed decreased activity in cells treated with high concentration of Corexit(80ppm).It has been reported in the published literature that the depletion of GSH may be the reason for accumulation of ROS within cells.Our data is consistent with this literature data since we also observed increased level of ROS and decreased level of GSH,indicating Corexit induced depletion of GSH.The significant increased LPO activity in cells treated with Corexit(80ppm)suggests the lipids on the surface of the cell membrane were degraded;and this could explain cell death.The apoptotic maker Caspase-3 and Bax was also shown to increase in response to toxic levels of Corexit(at 80 ppm).Overall,our experimental data support our hypothesis that Corexit induced cell death was caused by apoptosis.