The Immunotoxic Effects And Mechanisms Induced By Perfluorooctanoic Acid On Zebrafish(Danio Rerio)

Perfluorooctanoic acid(PFOA), a prominent perfluorinated compound(PFC), has been widely detected in natural water bodies worldwide. PFOA is a threat to aquatic organisms. However, few studies have focused on PFOA-induced immunotoxicity in aquatic organisms; thus, knowledge on the potential mechanisms of PFOA-induced immunotoxicity should be enhanced. In this study, the model organism zebrafish(Danio rerio) were exposed to nominal concentrations of 0.05, 0.1, 0.5, and 1 mg/L PFOA for 7, 14, and 21 d. After that, the fish were decapitated, and the spleens and kidneys were removed to observe morphological change via transmission electron microscopy(TEM), and detect the mRNA expression patterns of immunoglobulin gene, rel A transcription factor, myeloid differentiation 88(myd88), Toll-Like Receptor 2(TLR2) and relative cytokines by quantitative real-time polymerase chain reaction(qRT-PCR). Furthermore, use the function of third-order polynomial fitting in MATLAB R2013 a software to analyze the experimental data, triangle plots in Excel2007 to treat the relative expression of immunoglobulins, and analyze the correlation between all the indicators in this study, in order to research the potential mechanisms of immunotoxicity in aquatic organisms.TEM results showed that PFOA could not only damage the structure of spleen and kidney, but also interference lipid transport and metabolism in spleen immune cells, which would cause the lipofuscin pigment granules accumulation. Meanwhile, the mitochondria and endoplasmic reticulum vacuolization have been observed in kidneys and spleens immune cells, which would further induce proliferation, differentiation and maturation inhibition, prevent protein synthesis, and even lead to apoptosis. qRT-PCR results showed that the expression of cytokines and immunoglobulins are mediate by TLR/NF-κB pathway, and the expression level will alter at different PFOA exposure dose or exposure time, so it is a dynamic process. In addition, the cytokines in spleen cells are more vulnerable than the cytokines in kidneys after exposed to PFOA, especially for interleukine-1β(IL-1β) in spleen. The data showed that the relative expression of IL-1β in spleen sharply increased to 8.1 times of the control after 0.5 mg/L PFOA exposure for 14 days; however, when the dose reached to 1 mg/L and exposure time extended to 21 d, the relative expression of IL-1β decreased and the level was just 15% of the control group. The results of immunoglobulins triangle plots showed that, when zebrafish exposed to low PFOA concentration, the immune system in spleen is more stable than the system in kidney at the beginning of the exposed(7d). But the immune system in spleen and kidneys would face collapse after increasing exposure dose and prolong exposure time. The results of linear correlation showed that the cytokines in kidney are mainly regulated by TLR/myd88/NF-κB pathway, while the regulatory capacity of above pathway is weak in mediating the cytokines in spleen. Besides, it was also discovered that B cell activating factor(BAFF) is an important role in regulating the immuneglobulin expression level, no matter in spleen or kidney. MATLAB analysis results showed that TLR/myd88/NF-κB pathway can not only mediate BAFF level, but also interfere with IL-1β, interferon(IFN), IL-4 and IL-21 expression level in zebrafish kidney, and even affect the immunoglobulins expression level in kidney. Moreover, we found that the expression level of relA in spleen is controlled by its upstream protein myd88, but the ability of NF-κB pathway in regulating cytokines is inadequate compared to the kidneys. Based on the above results, we discovered that the reaction speed of cytokines and immunoglobulins in kidney lag behind spleen. So, we concluded that spleen is the target organ of PFOA, which will further make the immune system of the kidney unbalance, and eventually led the immune system in zebrafish to overall collapse. In conclusion, we try to demonstrate the underlying mechanism of PFOA-induced immunotoxicity in zebrafish by integration of specific responses of immunoglobulins and cytokines, and this study will provide guidance on prevention and control of persistent organic pollutant PFOA.