Molecular Mechanisms Of Neurotoxicity Induced By Microcystin-LR In Zebrafish

Frequent outbreaks of cyanobacterial blooms around the world have aroused urgent concern at home and abroad,with microcystins?MCs?produced by cyanobacterial blooms posing a great threat to the growth and development of aquatic organisms.Abundant evidences have shown that MCs,which were regarded as typical hepatotoxins,can also exert obvious neurotoxicity and adverse impacts on the neurous system of fish.In order to systematically assess the effects of MCs-induced developmental neurotoxicity on fish,MCLR,the most toxic and widely distributed one of MCs,and model organism zebrafish?Danio rerio?were selected as experimental material and animal,respectively.On the one hand,the molecular mechanism of microcystin-LR?MCLR?-induced developmental neurotoxicity was revealed by acute exposure in vitro and subchronic exposure in vivo.On the other hand,nano-titanium dioxide?nano-TiO₂?was selected as a carrier of MCLR to further explore the mechanism of MCLR-induced neurotoxicity in zebrafish.In neurotoxicity testing,special attention should be given to developmental neurotoxicity because the developing nervous system is more vulnerable to chemicals than the nervous system of adult.Firstly,zebrafish embryos were exposed to MCLR in the acute exposure experiment and adult zebrafish were exposed to MCLR of environmentally relevant concentration in subchronic exposure.The accumulation of MCLR,the developmental end point of zebrafish embryo,the swimming behavior of zebrafish larvae,the content of neurotransmitters in larvae,the synthesis and secretion of neurotransmitters,and the neuronal development related gene expresssion were examined to systematically explore the molecular mechanism of MCLR-induced developmental neurotoxicity on zebrafish embryos and F1 embryos.Furthermore,nano-TiO₂ was selected as the carrier of MCLR.Adult zebrafish were exposed to MCLR or MCLR-co-nano-TiO₂ to reveal the molecular mechanism of brain damage induced by MCLR in zebrafish from the aspects of behavior,brain histopathology,and oxidative stress?enzyme activity and gene expression?level.Finally,in order to comprehensively evaluate the effects of single or combined exposure on ocular development of F1 embryos and further explore the toxic mechanism of MCLR-induced neurotoxicity,the swimming behavior,eye histology,cell apoptosis related enzyme activity,and gene expression in the co-exposure experiment of MCLR and nano-TiO₂ were evaluated comprehensively.These studies provide important theoretical basis for comprehensively and systematically understanding the neurotoxic effects of MCLR,effects of maternal transmission,and co-exposure toxic effects of MCLR and nano-TiO₂.The main results were as follows,1.In vitro acute exposure experiment,zebrafish embryos were exposed to 0,0.8,1.6,and 3.2 mg/L MCLR for 120 hours post-fertilization?hpf?.The results demonstrated that MCLR could accumulate in zebrafish larvae and cause serious hatching delay and body length decrease.The locomotor speed of zebrafish larvae was decreased.Furthermore,the dopamine and acetylcholine?ACh?contents were significantly decreased in MCLR exposure groups.In addition,the acetylcholinesterase?ACh E?activity was significantly increased after exposure to 1.6 and 3.2 mg/L MCLR.The expression levels of manf,chrna7,and ache gene related to the change of the dopamine content,ACh content and ACh E activity were significantly changed after exposure to MCLR.Gene expression involved in the development of neurons was also measured.The gene expression levels of ?1-tubulin,shha,mbp,and gap43 were abnormal.The above results indicated that MCLR-induced developmental neurotoxicity might attribute to the disorder of cholinergic system,dopaminergic signaling,and the development of neurons.2.In vivo subchronic exposure experiment,zebrafish of F0 were exposed to MCLR?0,1,5,and 25 ?g/L?for 60 days.The F1 zebrafish embryos were collected and incubated in clean water for 120 h.After examining the parental zebrafish and F1 embryos,MCLR was detected in the gonad of F0 adults and F1 embryos,indicating MCLR could potentially be transferred from parents to offspring.The parental transfer of MCLR can lead to the significant hypoactivity of F1 larvae.The neurotransmitter detection experiments found the contents of dopamine,dihydroxyphenylacetic acid?DOPAC?,and serotonin in F1 larvae were significantly reduced in the 5 and 25 ?g/L MCLR parental exposure groups.In addition,the acetylcholinesterase?ACh E?activity was remarkably decreased in MCLR parental exposure groups,while the expression levels?manf,bdnf,ache,htr1 ab,htr1b,htr2 a,htr1aa,htr5 a,DAT,TH1,and TH2 genes?coincided with the decreased content of neurotransmitters?dopamine,DOPAC and serotonin?and the activity of ACh E were remarkable changed.Neuronal development related genes??1-tubulin,syn2 a,mbp,gfap,elavl3,shha,and gap43?were also measured,but gap43 was the gene only up-regulated.Our results demonstrated MCLR could be transferred to offspring,and subsequently induced developmental neurotoxicity in F1 zebrafish larvae by disturbing the neurotransmitter systems and neuronal development.3.Adult zebrafish were exposed to?0,0.5,4,and 32 g/L?MCLR with and without nano-TiO₂?100 g/L?for 45 days.The results showed that the swimming behavior and social behavior of zebrafish were abnormal,and the presence of nano-TiO₂ further aggravated the abnormality of behavior.In addition,single MCLR exposure or co-exposure can cause brain tissue damage in zebrafish,and the damage is not gender-specific.In order to further explore the molecular mechanism of zebrafish behavior abnormality and brain injury,the activity of oxidative stress-related enzymes and gene expression levels in brain tissue were detected.It was found that MCLR could induce brain injury through inducing oxidative stress in zebrafish brain tissue,ultimately leading to the abnormality of swimming behavior and social behavior of zebrafish.The presence of nano-TiO₂ further aggravated the oxidative stress in zebrafish brain induced by MCLR,which was contributed to the abnormal behavior.4.In co-exposure experiment,wild-type adult zebrafish were exposed to various MCLR concentrations?0,0.5,4,and 32 ?g/L?with and without nano-TiO₂?100 ?g/L?for 45 days.F1 zebrafish embryos were collected and cultured in clean water for 120 h.The results showed that nano-TiO₂ could potentially increase the accumulation of MCLR content in F1 zebrafish embryos.In addition,co-exposure of MCLR and nano-TiO₂ further inhibited the embryonic development of F1 zebrafish embryos,slowed down the swimming speed of larvae,and aggravated the abnormal development of eye in F1 larvae.In this study,the potential molecular mechanisms were explored by means of cell apoptosis detection,protein expression detection,and enzyme activity detection.The results showed that MCLR could induce abnormal development of eye in F1 larvae through mitochondrial-mediated apoptotic pathway,ultimately interfering with the swimming behavior of F1 larvae.The existence of nano-TiO₂ can exacerbate parental transfer effect of MCLR through aggravating the abnormal development of eye and swimming behavior of F1 larvae.In summary,this research studied the bioaccumulation,parental transfer,and developmental neurotoxicity of MCLR and nano-TiO₂ to examine their interactions,revealing that the health risks of co-exposure should not be ignored.