title: Apoptotic responses of Carassius auratus lymphocytes to nodularin exposure in vitro.
abstract: Nodularin, a metabolite of Nodularin spumigena, is widely detected in water blooms worldwide and causes serious negative effects on fish. The apoptosis-related cytotoxic effects and mechanisms of nodularin on Carassius auratus lymphocytes were investigated. Transmission electron microscopy results showed that nodularin-treated lymphocytes display a series of morphological changes, including condensed cytoplasm, nuclear chromatin agglutination and marginalization. DNA fragmentation was verified by the DNA-ladder and formation of sub-G1 DNA peaks. These cell characteristics confirmed the occurrence of apoptosis in lymphocytes. Flow cytometric results showed that the percentages of apoptotic cells incubated with 1, 5, 10, and 100 μg/L nodularin for 12 h reached 15.76%, 17.36%, 20.34% and 44.21%, respectively; controls showed low rates of apoptosis (2.4%). The mechanism of apoptosis induced by nodularin was determined, and results showed that nodularin exposure caused a significant increase in intracellular reactive oxygen species (ROS), loss of mitochondrial transmembrane potential in a dose-dependent manner, upregulation of intracellular Ca²⁺, downregulation of Bcl-2 and upregulation of Bax expression at the mRNA and protein levels, and activation of caspase-3 and caspase-9 without caspase-8. In summary, all the results suggest that nodularin induces lymphocyte apoptosis via the mitochondrial apoptotic pathway and destroys the immune response of fish.
authors.affiliation: Department of Environmental Sciences, Hangzhou Normal University, Xuelin Road 16#, Xiasha Gaojiao Dongqu, Hangzhou, Zhejiang Province 310036, China. zhanghangjun@gmail.com
authors.forename: Hangjun
authors.initials: H
authors.lastname: Zhang
authors.forename: Dandan
authors.initials: D
authors.lastname: Shao
authors.forename: Yingzhu
authors.initials: Y
authors.lastname: Wu
authors.forename: Chenchen
authors.initials: C
authors.lastname: Cai
authors.forename: Ciming
authors.initials: C
authors.lastname: Hu
authors.forename: Xiaolu
authors.initials: X
authors.lastname: Shou
authors.forename: Bingru
authors.initials: B
authors.lastname: Dai
authors.forename: Binhui
authors.initials: B
authors.lastname: Ye
authors.forename: Mengdi
authors.initials: M
authors.lastname: Wang
authors.forename: Xiuying
authors.initials: X
authors.lastname: Jia
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D002118:Calcium / Q000378:metabolism; D053938:DNA Fragmentation / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D005434:Flow Cytometry / Q000662:veterinary; D006054:Goldfish; D066298:In Vitro Techniques; D008214:Lymphocytes / Q000187:drug effects* / Q000648:ultrastructure; D008387:Marine Toxins / Q000633:toxicity*; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D046529:Microscopy, Electron, Transmission / Q000662:veterinary; D046932:Nodularia / Q000737:chemistry*; D010456:Peptides, Cyclic / Q000633:toxicity*; D017382:Reactive Oxygen Species / Q000378:metabolism
chemical_list: D008387:Marine Toxins; D010456:Peptides, Cyclic; D017382:Reactive Oxygen Species; C063998:nodularin; D002118:Calcium