RESPONSE OF APOPTOTIC GENES TO APHANTOXIN-PARALYTIC SHELLFISH POISON IN FRESHWATER EXTRACTED FROM THE APHANIZOMENON FLOS-AQUAE DC-1 IN CELLS OF BRAIN ON ZEBRAFISH (DANIO RERIO)

The Aphanizomenon flos-aquae DC-1,frequently appeares in freshwater,is a dominant species in Dianchi Lake of Yunnan Province,China,in winter and spring.During the past 20 years,blooms of Aphanizomenon flos-aquae DC-1 have occurred in the Dianchi Lake per year because of heavy pollution and the dominant species overgrowth.The blooms always sustained for six months in a year,even throughout a year in some water of Dianchi Lake.And the blooms could also give off distasteful smell into the air.As a result,visitors are more likely to breathe the odorous air than fresh air today,and see a bad scenery of green-mud water than clear blue waters.Especially the dominant species can produce aphantoxins,which is attributed to the paralytic shellfish poisons(PSPs) and influences aquatic ecosystem and damages the nerve system of animals and human.However,little research has been carried out on the toxin,in particular the effects on central nervous system(CNS) of animals and human.Thus the present research is to investigate abnormity of apoptotic gene expression,ultrastructural damage of brain induced by aphantoxin,to reveal mechanism of response of brain on zebrafish to aphantoxins.Virus-free male zebrafish(Danio rerio),110 days old or so,average bodyweight of(0.4±0.01) g,after 7—10 days of acclimatization,were randomly assigned into control and treated groups(45 fish per group).The two groups were received 30μL 0.01 mol/L acetic acid solution(control) and 30 μL 0.01 mol/L acetic acid solution contained aphantoxins(5.3 μg STXeq/kg bodyweight,treated) through intraperitoneal injections(i.p.).At each time point(1,3,6,9,12 and 24h),five zebrafish in each group were sacrificed by cold shock(embedding into crushed ice at -8℃),and cerebra were removed from whole brain and washed at once in 0.86% ice-cold physiological saline,prefixed in 2.5% glutaraldehyde solution for electron microscopy.In another 5 fish at each timepoint,whole brains were removed as described above,frozen in liquid nitrogen,and stored at either -70℃ for subsequent RNA analysis.The prefixed cerebra(at 9h) were dehydrated in graded ethanol and propylene oxide,embedded in Epon 812,and cut into the ultra-thin sections using glass knives on an ultramicrotome(Leica,Germany).Sections were stained with uranyl acetate and lead citrate before electron microscopy(JEM-1230,Japan).The 45mg frozen(-70 ℃) whole brain(five fish each timepoint) of zebrafish were homogenized(IKA Werke,Germany) in ice-cold TRIzoL reagent(w:v,1:20,30s) to extract total RNA.Gene-specific RNA levels were measured using an RT-PCR kit(Promega,USA) according to the protocol of manufacturer.The system of reactions was carried out into 50μL as follows:Reactions contained 25μL of 2× AccessQuick TM Master Mix,5 μL oligo-dT,10 μmol/L upstream and downstream primers,1 μg template RNA,and RNase-free MilliQ water to 50μL.AMV reverse transcriptase(1μL) was added and incubated at 45℃ for 45 min for reverse transcription.Samples were then heated at 95℃ for 5min before PCR;amplification conditions were 40 cycles of 95℃ for 30s,58℃ for 30s,72℃ for 45s,followed by a final extension step(72℃ for 10min).RT-PCR amplicons were analyzed by elec-trophoresis on 1.5%—2.0% agarose gels(60—100 V,40 A,30min),ethidium bromide staining,visualization on a Bio-imaging System(M-20X,UVP,USA),and densitometry using analytical software(Quantity One v4.62,Bio-Rad,USA).All statistical analyses employed commercial software(SPSS 13.0,Chicago,IL,USA) and assessed by one-way analy-sis of variance(ANOVA) combined with least significant difference(LSD) post-hoc tests to express significances of differences between groups(P0.05 and P0.01).The results showed that aphantoxins could induce the apoptosis of brain cells in ultrastructure,such as the membrane blebbing and apoptotic-like body.And it was further analyzed on molecular level to reveal that the expressions of p53,bax,caspase-3 and c-jun genes were increased and followed with time-dependent manner whose level of expression upreguation were 1.92,1.55,1.6 and 1.55 times that of control respectively.These results displayed that aphantoxins could lead to apoptotic ultrastructural damage of brain tissues through increasing expression of genes.And the brain cells could activate the mechnism of response to aphantoxins through the mitochondrial route which was involved in the cascade of p53→bax→caspase-3 genes.Therefore the toxin could result in death of brain cells by apoptotic way and exhibited the neurotoxicity of damage brain.These findings provide direct evidence that freshwater cyanobacterial aphantoxins can exert toxic effects on brain tissue.This is also strong evidences that find the mechanism of response on molecular level from brain cells to cyanobacterial neurotoxins of freshwater.