Combined Exposure To MC-LR And PS-MPs/NPs Inhibits Early Cardiovascular Development In Zebrafish By Oxidative Stress And Disturbance Of Calcium Homeostasis

Background:Due to the increasingly serious problem of eutrophication of global water bodies,cyanobacteria blooms proliferate on the water surface,cyanobacteria toxins and other harmful substances will be released into the water bodies,which will seriously threaten the safety of water resources and human health.Microcystins(MCs)are a class of cyclic heptapeptide with more than 300 homologs,of which microcystins leucine-arginine(MCLR)is one of the most dangerous and common variants.Polystyrene(PS)is one of the most abundant types of plastic found in the ocean at a time when the growing global demand for plastic products is not only bringing convenience to people but also creating a large amount of plastic waste.After entering the water,these plastic wastes are decomposed into microplastics/nano-plastics(PS-MPs/NPs)with a smaller diameter under the comprehensive action of various factors.The accumulation of plastic pollution through the food chain layer by layer has become an important global problem,and become a cross-border threat to the natural environment and human health.In aquatic environments,MC-LR almost always co-exists with a variety of environmental pollutants.The coexistence of MC-LR and microplastics in freshwater has become inevitable due to the massive use of plastics and the generation of plastic waste.Previous studies have found that MC-LR is cardiotoxic and MPs/NPs can produce cumulative effects in organisms,causing oxidative stress and affecting growth and development.However,current studies focus more on the single toxic effects of MC-LR or PS-MPs/NPs on organisms.There are few studies on the effects of the combined toxic effects of MC-LR and PS-MPs/NPs on the cardiovascular system.Objective:To investigate the effects of MC-LR and PS-MPs/NPs on the cardiovascular development of zebrafish embryos/juveniles,168 hpf acute exposure was performed.We investigated the effects of combined exposure of MC-LR and PS-MPs/NPs on the oxidative and antioxidant systems of juvenile fish by detecting ROS,GSH,SOD,and MDA levels,and investigated the effects of combined exposure of MC-LR and PSMPs/NPs on the apoptosis of juvenile cardiocytes by AO and TUNEL staining.RT-PCR and Western blot were used to investigate the effects of combined exposure of MC-LR and PS-MPs/NPs on cardiovascular development,calcium ion pathway,and expression levels of genes and proteins related to inflammatory factors in juvenile fish.It provides a theoretical basis for elucidating the mechanism of cardiovascular toxicity caused by MCLR and PS-MPs/NPs combined exposure.Methods:1.The fertilized eggs were randomly divided into three groups: MC-LR alone exposure(0,1,10,100 μg/L),PS-MPs/NPs alone exposure(100 μg/L),(0,1,10,100 μg/L)MC-LR+100 μg/L PS-MPs/NPs combined exposure,and 30 n M astaxanthin intervention(100 μg/L MC-LR+100 μg/L PS-MPs/NPs+ASTA).The exposure time was 168 hpf.2.The characterization of PS-MPs/NPs particles in ultra-pure water and the adsorption and accumulation of microplastic particles in zebrafish embryos and larvae were detected.3.The morphological changes and deformities of continuous 168 hpf of zebrafish were observed,and the 96 hpf hatchability and weight,168 hpf survival rate,and body length of juvenile fish of F0 generation were measured.Heart rates of 24,96,and 168 hpf juveniles were measured,and behavioral data of 168 hpf juveniles were collected.4.The pericardial edema and cardiac morphological changes in 72 hpf juvenile fish were observed,as well as the occurrence of tail vascular embolism and absence.5.DCFH-DA method was used to detect the level of active oxygen in juvenile fish,and the contents and activities of SOD,MDA,and GSH in juvenile fish were detected by the kit.6.AO and TUNEL staining were used to observe the apoptosis of 72 hpf juvenile heart cells.7.The levels of genes related to heart development,vascular development,calcium signaling pathway,and inflammatory factors in zebrafish were measured by q RTPCR.The expression of the TNNT2 protein was determined by Western blot.ResultsPart 1 PS-MPs/NPs can accumulate in zebrafishWhen zebrafish embryos were exposed to PS-MPs/NPs and MC-LR+PS-MPs/NPs solutions,the embryonic chorionic membrane could adsorb PS-MPs/NPs particles when they developed to 48 hpf.When exposed to 120 hpf,PS-MPs/NPs particles would enter the intestine and colonize pores through the feeding of young fish.When exposed to 168 hpf,the embryonic chorionic membrane could adsorb PS-MPS/NPS particles.PSMPs/NPs particles can even make their way further into the blood circulation of young fish.Part 2 Combined exposure of MC-LR and PS-MPs/NPs affected the growth and development of juvenile zebrafishExposed to MC-LR and PS-MPs/NPs,zebrafish embryos began to hatch at 48 hpf.After 72 hpf,the larvae exhibited delayed incubation and multiple developmental deformities,including pericardial edema,yolk sac edema,spinal curvature,and tail curvature.The 96 hpf hatchability of embryos and 168 hpf survival rate of juvenile fish were inhibited in the exposed group(P<0.01),and there was a significant difference between 96 hpf body weight and 168 hpf body length reduction(P<0.05,P<0.01).The heart rate of juvenile fish was inhibited at 24,96,and 168 hpf,and the decrease was most significant in the MCLR+PS-MPs/NPs combined exposure group(P<0.01).In the 30 min light and dark period,the overall motor capacity of the 100 μg/L MC-LR+ PS-NPs group decreased,and the average swimming speed of 100 μg/L MC-LR+PS-MPs/NPs group decreased at 5min and 30s(P<0.01).Part 3 Combined exposure of MC-LR and PS-MPs/NPs results in abnormal cardiac and vascular development in juvenile zebrafishExposure to MC-LR and PS-MPs/NPs resulted in severe pericardial edema,increased SV-BA distance,elongated heart,changes in atrial and ventricular structure,reduced cardiac myocytes and thinning of the cell wall in 72 hpf 100 μg/L MC-LR+PS-MPs/NPs group.At the same time,blood embolism appeared in the tail vessels,dorsal longitudinal anastomosis ves,seals,and the absence of internode vessels.Part 4 Combined exposure of MC-LR and PS-MPs/NPs resulted in increased ROS and apoptosisExposure to MC-LR and PS-MPs/NPs resulted in significantly increased ROS in 72 hpf 100 μg/L MC-LR+PS-MPs/NPs group(P<0.01),GSH content and SOD activity decreased(P<0.01)and increased MDA content(P<0.01).At the same time,the 100 μg/LMC-LR+PS-MPs/NPs group exposed 72 hpf showed obvious apoptosis in the pericardial area,and astaxanthin reduced the number of apoptotic cells.Part 5 Combined exposure to MC-LR and PS-MPs/NPs affects cardiovascular development,calcium signaling pathways,transcription levels of inflammatory factorrelated genes,and expression levels of TNNT2 proteinsExposure to MC-LR and PS-MPs/NPs 168 hpf,compared with the corresponding MCLR alone exposure group,is associated with cardiac development(myh6、Nkx2.5、tnnt2a),vascular development(vegfaa、vegfr 1、vegfr 2、vegfr 3)and calcium ion pathways(cacna1ab、ryr2a、atp1a3b、atp1b2b、atp2a1l、atp2b1a、atp2b4).Related genes were significantly down-regulated in the MC-LR+PS-NPs combined exposure group(P<0.05,P<0.01),and significantly up-regulated in the MC-LR+PS-MPs combined exposure group(P<0.05,P<0.01).The transcription levels of inflammatory cytokines IL-6 and IL-8 were significantly increased in the MC-LR+PS-MPs/NPs group(P<0.01).Compared with the 100 μg/L MC-LR alone group,the 100 μg/L MC-LR +PS-NPs group had lower expression levels of TNNT2 protein associated with cardiac development(P<0.05).ConclusionCombined exposure of MC-LR and PS-MPs/NPs aggravated the cardiovascular toxicity of MC-LR induced zebrafish,which further inhibited the early growth and development of juvenile zebrafish.The underlying mechanism may be that the presence of PS-MPs/NPs aggravated the oxidative stress and inflammation induced by MC-LR.Thus,the calcium homeostasis regulation mechanism was further disturbed,and the apoptosis of cardiomyocytes and cardiac systolic function were induced.At the same time,PS-MPs/NPs particles can accumulate in blood vessels to form an obstructive effect and aggravate and inhibit the expression of genes related to vascular development,thus leading to vascular injury.