Study On Molecular Toxicology Of Microcystins-LR To Fish

Microcystins are cyanobacterial toxins in water blooms that have received increasingattention as a public biohazard for human and animal health. Microcystins are a group ofcyanobacterial hepatotoxins and the most common and potently toxic one ismicrocystin-LR (MC-LR). It has been well documented that MC-LR may have adverseeffect on embryo development and growth of fish, and pathological damages in liver andkidney of fish, and oxidative damage in fish exposed to MC-LR. However, the moleculartoxicology of MC-LR on fish has not yet been investigated. The present study wastherefore designed to examine the molecular toxic effect of MC-LR on bighead carpAristichthys nobilis and zebrafish Danio rerio by using real-time polymerase chainreaction, molecular cloning and immunoblotting.Zebrafish embryos were collected from artificial fertilization. The transcriptions ofsix immune-related genes, i.e. Rag1, Rag2, Ikaros, TCRα, Lck, GATA1 and HSPs(HSP90,HSP70,HSP60,HSP27) were analyzed by real-time PCR at 12h, 24h, 48h,96h and 168h post immersing in 200μg/L and 800μg/L MC-LR, respectively. The increasein transcriptions of Ragl, Rag2, Ikaros, TCRα, Lck and GATA1 were observed, andsignificant higher transcriptions of those six genes were observed on fish exposed to800μg/L MC-LR. There were significant increases in transcriptions of HSPs, andespecially significant change on HSP70 of fish exposed to 800μg/L MC-LR at 168h.These results demonstrated that microcystin-LR exposure could affect the function ofzebrafish larval immune system, resulting in immunomodulation.The GPX and GR cDNA full sequence of Aristichthys nobilis has been cloned usingRACE-PCR and PCR.The GPX full length cDNA is 903 bp, including a 182 bp 5'UTR (untranslatedregion),a 426 bp open reading frame,and a 292 bp 3'UTR. The putative protein of GPX is142 aa. The molecular formula is C₇₄₀H₁₁₄₄N₁₉₆O₂₁₃S₄, and the molecular weight is16.3226KDa, and estimated pIs is 5.93. GPX protein is water solubility.Comparison of thededuced amnio acid sequences showed that GPX of Aristichthys nobilis shared 99.3%,97.9% and 93.0% identity with those of Ctenopharyngodon idella, Hypophthalmichthysmolitrix and Carassius auratus, respectively.The GR full length cDNA is 831 bp, including a 74 bp 5'UTR (untranslated region),a 717 bp open reading frame, and a 40 bp 3'UTR. The putative protein of GR is 239 aa. The molecular formula is C₁₁₃₁H₁₇₉₆N₃₂₄O₃₄₇S₇, and the molecular weight is 25.709KDa,and estimated pls is 7.71. GRprotein is water solubility. Comparison of the deduced amnioacid sequences showed that GR of Aristichthys nobilis shared 90.8% and 81.1%identitywith those of Danio rerio and Salmo salar, respectively.The organ distribution of GPX and GR was analyzed both in the transcription andprotein levels.By real time PCR analysis, the transcription products of the cloned geneswere detected in different tissues of healthy bighead carp, and the two genes had similiarpatterns of tissue distribution, with the highest expression level in liver. Western blottinganalyses revealed that GPX protein existed broadly in examined tissuesThe gene transcription of GPX and GR in liver, spleen, head kidney and intestine ofAristichthys nobilis were quantified by real-time PCR at 6h, 12h, 24h, 48h, 96h and 168hpost intraperitoneal injection with 50μg MC-LR/kg body weight. Significant increase ofGPX in liver, spleen and head kidney were observed. Also, significant increases of GR inintestine and head kidney were observed, and there were no significant changes of GR inliver and spleen. These results showed that the increased transcriptions of GPX and GR inAristichthys nobilis could be induced by MC-LR, leading to generating more antioxidaseand elevating the ability of detoxification on fish.