Mechanism And Joint Neurotoxicity Of Typical Persistent Toxic Substances In Zebrafish

Persistent toxic substances(PTS)have become an important environmental issue,which affect human health and survival in 21st Century.E-waste dismantling activities are primary source of PTS pollution in the environment.Long term and large amouts of raw dismantling activities have led to a serious PTS pollution including lead(Pb),Polycyclic Aromatic Hydrocarbons(PAHs)and flame retardant.The aquatic ecosystem is an important place for the enrichment of PTS.Different kinds of PTS entered into water individual or together would finally result in mixture pollution in water,which poses a potential threat to the aquatic ecosystem.Jiulong River Estuary(JRE)is a classic subtropic estuary.The frequently dismantling activities of Xiamen and Zhangzhou and oil spill from submarine oil pipelines or ship have caused serious contamination of PTS(including PBDEs,heavy metals and PAHs)in the water and sediments of JRE.However,currently the reports about the pollution levels of new flame retardant Dechlorane Plus(DP)and A-PAHs in JRE are not clear.Animal experiments demonstrated that DP is a kind of potential of the nerve poison,but the neurotoxicity of DP,and joint neurotoxicity of DP with other inorganic or organic pollutants are still unkown.In this study,the pollution levels of DP and A-PAHs in water and sediment of Jiulong river estuary have been investigated,and then three typical PTS,including DP,Pb and A-Phen,were selected as research targets.The mechanism and neurotoxicity of DP and joint neurotoxicity of co-exposure with DP and Pb or A-Phen have been investigated.The main research findings are as following:1.Firstly,the pollution characteristics of 4 kinds of DP and related compounds and 40 kinds of PAHs(? PAHs,including 21 kinds of A-PAHs and 19 kinds of P-PAHs)were inverstiged in water and sediment of JRE.The results showed that the mixed pollution of DP and A-PAHs is very common in water and sediment of JRE.Total dechloranes concentration ranged from 1.4 to 4.1 ng/L and 9.3-36.2 ng/g in the water and sediment samples,respectively.The distribution pattern of dechloranes in water and sediment were both dominated by DP with 100%detection rate.The concentration of ?PAHs ranged from 26.2-398 ng/L and 2905-5449 ng/g in the water and sediment samples,respectively.The mean levels of A-PAHs in water and sediment accounted for 31%and 24%of the concentration of ?PAHs and the most important components of A-PAHs were alkyl phenanthrene(A-Phen).There was no significant correlation between the levels of PAHs and suspended particle matters(SPM)and total organic carbon(TOC)in water from JRE.However,the correlations between the concentration of DP and SPM and TOC in water from the JRE were both significant,suggesting SPM and TOC played important roles in the distribution of DP in water from JRE.In comparison to other estuaries and coasts,the pollution levels of DP and PAHs in water and sediment from Jiulong River Estuary were both at moderate pollution levels.2.Secondly,using zebrafish as animal model,the neurotoxic effect of DP exposure on zebrafish embryos were investigated.The results showed that DP exposure significantly altered embryonic spontaneous movement,reduced touch-induced movement and free-swimming speed and decreased swimming speed of larvae in response to dark stimulation,indicating that DP has the neurobehavioral toxicity.Intriguingly,in accord with these behavioral anomalies,DP exposure significantly inhibited axonal growth of primary motoneuron,and upregulated expression of nerve related gene gap-43 and al-tubulin,which may be a compensatory response to inhibit the growth of axons.Furthermore,DP exposure at 30 ?g/L and 60 ?g/L significantly increased reactive oxygen species(ROS)and lipid oxygen(MDA)levels,upregulated the expression of apoptosis related genes Caspase-3 and Bax,and caused the cell apoptosis and muscle fiber injury in zebrafish.Together,these results demonstrated that DP induced neurobehavioral deficits may result from the combined effects of altered the neuronal connectivity and muscle injuries.3.Thirdly,based on the study of DP neurotoxicity in zebrafish and the phenomenon of mixed pollution of DP and Pb in JRE and other e-waste areas,the combined neurotoxicity of DP and Pb were further investigated.The results showed that Pb(5,20 ?g/L)and DP(15,60 ?g/L)exposure alone altered the spontaneous movements,significantly decreased the motor activity and reduced the touch response and light/dark stimulation response ability,suggesting DP and Pb exposure elict neurotoxicity in zebrafish.Further study showed that DP co-exposure with Pb has an antagonistic effect on spontaneous movement,touch response and free swimming activity.Immunohistochemistry and AO staining results showed that individual DP or Pb exposure significant inhibited the axonal growth of primary motorneuro and increased the number of cell apoptosis in the muscle of tail.However,in comparision with single exposure,DP co-exposure with Pb can obviously impair the inhibitor effect on axonal grow of primary motorneuro,and reduce the cell apoptosis in the tail.Together,these findings indicated that DP co-exposure with Pb also has an an an antagonistic effect on axonal growth of neuro and cell apoptosis.Chemical content analysis showed that DP did not affect the absorption of Pb,but Pb significantly inhibited DP accumulation and promote the transformation of anti-DP in zebrafish,which may be the main cause of mixture exposure elicited the antagonistic effect in zebrafish.4.Finally,based on the study of DP neurotoxicity in zebrafish and the phenomenon of combined pollution of DP and A-Phen in JRE,3-Methylphenylene(3-MP)was selected as representive chemical in A-Phen.The combined neurotoxicity of DP and 3-MP were further investigated.The results showed that exposure to 3-MP(5,20 ?g/L)alone or in combination with(15,60?g/L)DP induced motor behavior deficiency in zebrafish larvae.Co-exposure with DP and 3-MP observed an additive or synergistic effect on spontaneous movement,touch response and free swimming activity.In accordance with these behavioral anomalies,3-MP alone or co-exposure with DP significantly inhibited axonal growth of secondary motoneuron,altered intracellular Ca2+ homeostasis and induced apoptotic cell death in the muscle fiber of zebrafish.Furthermore,3-MP alone or co-exposure significantly increased reactive oxygen species(ROS)and the mRNA transcript levels of apoptosis related genes Caspase-3 and Bax.Together,these findings indicated that 3-MP alone or co-exposure with DP induced neurobehavioral deficits may result from combined effects of altered neuronal connectivity and muscle function.Chemical analyses revealed significant increase of 3-MP and DP bioaccumulation in zebrafish larvae co-exposed to the high dose 3-MP and DP,which seems to be the likely cause for heightened effects observed in this treatment group.Elevated bioaccumulation resulting from mixture exposure may represent a significant contribution of additive or synergistic effects observed in some combined chemical exposures.Taken together,these results demonstrated that DP and A-Phe co-exist in water and sediment of JRE,which may pose a potential threat to aquatic organisms;Further behavioral assessment of DP have confirmed that DP has neurobehavioral toxicity,and its mechanism of action includes muscle oxidative injury and neuron damage.Studies on the mixure exposure manifested that DP co-exposure with Pb has an antagonistic effect on the neural behavioral toxicity in zebrafish,while DP and 3-MP co-exposure showed an additive effect.Therefore,the combined effect of DP with inorganic or organic pollutants should be considered comprehensively in the DP environmental risk assessment in future.