Abstract
The Aphanizomenon flos-aquae DC-1, frequently appeares in freshwater, is a dominant species in DianchiLake of Yunnan Province, China, in winter and spring. During the past 20 years, blooms of Aphanizomenon flos-aquaeDC-1 have occurred in the Dianchi Lake per year because of heavy pollution and the dominant species overgrowth. Theblooms always sustained for six months in a year, even throughout a year in some water of Dianchi Lake. And theblooms could also give off distasteful smell into the air. As a result, visitors are more likely to breathe the odorous airthan fresh air today, and see a bad scenery of green-mud water than clear blue waters. Especially the dominant speciescan produce aphantoxins, which is attributed to the paralytic shellfish poisons (PSPs) and influences aquatic ecosystemand damages the nerve system of animals and human. However, little research has been carried out on the toxin, in particularthe effects on central nervous system (CNS) of animals and human. Thus the present research is to investigateabnormity of apoptotic gene expression, ultrastructural damage of brain induced by aphantoxin, to reveal mechanism ofresponse 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 daysof acclimatization, were randomly assigned into control and treated groups (45 fish per group). The two groups werereceived 30μL 0.01 mol/L acetic acid solution (control) and 30 μL 0.01 mol/L acetic acid solution contained aphantoxins(5.3μg STXeq/kg bodyweight, treated) through intraperitoneal injections (i.p.). At each time point (1, 3, 6, 9, 12 and24h), five zebrafish in each group were sacrificed by cold shock (embedding into crushed ice at -8℃), and cerebra wereremoved from whole brain and washed at once in 0.86% ice-cold physiological saline, prefixed in 2.5% glutaraldehydesolution 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) weredehydrated in graded ethanol and propylene oxide, embedded in Epon 812, and cut into the ultra-thin sections usingglass knives on an ultramicrotome (Leica, Germany). Sections were stained with uranyl acetate and lead citrate beforeelectron microscopy (JEM-1230, Japan). The 45mg frozen (-70℃) whole brain (five fish each timepoint) of zebrafishwere 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 50 μL as follows: Reactions contained 25μL of 2× AccessQuick TMMaster Mix, 5μL oligo-dT, 10 ?mol/L upstream and downstream primers, 1 ?g template RNA, and RNase-free MilliQwater to 50 μL. AMV reverse transcriptase (1 ?L) was added and incubated at 45oC for 45 min for reverse transcription.Samples were then heated at 95oC for 5min before PCR; amplification conditions were 40 cycles of 95oC for 30s, 58oCfor 30s, 72oC for 45s, followed by a final extension step (72oC for 10min). RT-PCR amplicons were analyzed by electrophoresison 1.5%—2.0% agarose gels (60—100 V, 40 A, 30min), ethidium bromide staining, visualization on a BioimagingSystem (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 analysisof variance (ANOVA) combined with least significant difference (LSD) post-hoc tests to express significances ofdifferences between groups (P/i> P/i> p53,bax, caspase-3 and c-jun genes were increased and followed with time-dependent manner whose level of expressionupreguation were 1.92, 1.55, 1.6 and 1.55 times that of control respectively. These results displayed that aphantoxinscould lead to apoptotic ultrastructural damage of brain tissues through increasing expression of genes. And the braincells could activate the mechnism of response to aphantoxins through the mitochondrial route which was involved in thecascade of p53→bax→caspase-3 genes. Therefore the toxin could result in death of brain cells by apoptotic way andexhibited the neurotoxicity of damage brain. These findings provide direct evidence that freshwater cyanobacterialaphantoxins can exert toxic effects on brain tissue. This is also strong evidences that find the mechanism of response onmolecular level from brain cells to cyanobacterial neurotoxins of freshwater.
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