Immunotoxicity And Mechanisms Induced By Atrazine On The Frog(Pelophylax Nigromaculata)

Ecotoxicology researchs focused on the relationship between environment pollution of agriculture chemicals and species decline of amphibians. However, the effects of atrazine on immune system of frogs were not well known. Hence, Frogs Pelophylax nigromaculata were as subjects exposed to herbicide atrazine which was as the important tested pollutant.96h LC50value of atrazine on frogs was determined by acute exposure test. Then the study explored effects of atrazine on serum immune factors, such as ALP(alkaline phosphatase), TP(total protein) and IgM globulin in frogs exposed to atrazine via semi-static sub-chronic exposure test. Meanwhile, lymphocyte counts and viabilities were tested to determine the immunotoxicity of atrazine. Moreover, DNA damage and apoptosis morphological characteristics were detected as well as Bcl-2/Bax apoptosis protein and mRNA expression, which demonstrated the toxic mechanism of atrizine on frogs. The main results were as below:1. Certain concentration of atrazine caused toxicity effects on frogs(Pelophylax nigromaculata) executed by acute exposure test. LC50value of atrazine was49.249mg/L.2. The results proved that0.32-2.56mg/L atrazine exposure could induced increasing of total protein(TP) and IgM content and Alkaline phosphatase(ALP) activity in frogs serum. Compared with the control group, ALP activity increased siginificantly in0.32mg/L,0.64mg/L and1.28mg/L(P<0.05). In2.56mg/L group ALP activity was lower than that of other treatment groups, but also notably higher than control group(P<0.05). TP contents in treatment groups respectively increased significantly by22.40%,43.27%,55.16%and67.56%of control group(P<0.05). There was a dose-dependent relationship between TP content and different atrazine concentrations(r=0.9899, P<0.05). IgM contents in1.28mg/L and2.56mg/L groups rose with the increasing of atrazine concerntrations, and increments were39.46%and41.34%(P<0.05). There was a dose-dependent relationship between IgM contents and atrazine concerntrations(r=0.9630, P<0.05).3.Lymphocyte counts showed an increasing and then dropped with the increasing of atrazine concentration. It could be seen that in0.32mg/L,1.28and2.56mg/L groups lymphocytes counts decreased nonsignificantly. Whereas the number of lymphocytes was significantly increased in0.64mg/L groups(P<0.05). It proved that atrazine could lead to immune system hormesis in frogs. Meanwhile, atrazine could significantly reduced lymphocytes viabilities in treatment groups(.P<0.01), which in the group of highest atrazine concentration was down to the lowest level.4.DNA damages of lymphocytes were detected by the single nucleolus comet, it was found that the tail length, DNA in tail and tail moment notably increased with the rise of atrazine concentrations(P<0.01). There were dose-dependent manner between DNA damages and different treatment concentrations that with correlative coefficients of0.9841,0.9304and0.9157respectively. It indicated that atrazine could induced DNA damages of lymphocytes in frogs. DNA ladder was also observed in the groups of0.64,1.28and2.56mg/L that the DNA fragmentation length was from180bp to200bp. Moreover, lymphocytes had showed typically apoptosis morphological characteristics, which were showed as marginalization of chromatin in0.64mg/L, shrinkage and cytoplasm vacuolization in1.28mg/L, cytoplasm dissolution, dense granules and karyotheca disappears in2.56mg/L.5. Apoptosis protein and mRNA analysis revealed that pro-apoptotic protein Bax expression in the treatment groups was1.49times more than that in control group. Anti-apoptotic protein Bcl-2expression in treatment groups were only53.74%of control group expression. Meanwhile, the expression of Bax mRNA raised significantly with different atrazine concentrations(P<0.05), but the Bcl-2mRNA expression was opposite to Bax. It was obviously lower than control group(P<0.05).