| In this research, endocrine disruption of typical pesticides such as alachlor and atrazine will be studied in Carassius auratus as the test animal especially the low dose effect. The perturbation of reproduction, endocrine and immune system were evaluated. The most dose treatments of this experiment were setting at hormesis range. Juvenile male crucian carp Carassius auratus (33.5±7.6 g, 10.5±1.0 cm) were purchased from Shanghai Fisheries Research Institute. All fish were cultured in dechlorinated water (temp. 18±5℃, pH 7.2±0.3, DO 7.0±0.4 mg/L) with a 12-h light: 12-h dark regime for a minimum of 2 weeks prior for use, and fed with frozen adult brine shrimp very morning. Then fish were continuously exposed to alachlor and atrazine in a series of concentrations, 1/8,1/16,1/64,1/256,1/1024,1/4000 LC50, for 2 months, with an untreated group for control. The experiment was carried out using a static-renewal regimen, 50% of the exposure solution was replaced daily to ensure the stabilization of test substance concentrations. Alachlor and atrazine was measured by gas chromatograph with electron capture detector. The biochemical and molecular biology technique was applied to evaluate the toxicological effects caused by these environmental pollutants. This research will provided technological bases for improving the ecological and health risk assessment, in particular, the technique of pesticide safety evaluation, developing and applying the biomarkers of low-dose endocrine disruptor. The results are showed as following:1. The hepatosomatic index (HSI) and the gonadosomatic index (GSI) of the crucian carp was calculated after the 2 months exposure in atrazine and alachlor with different concentration levels. The result showed that: there is a depression to the development of the crucian carp's liver and spermary when the concentration of atrazine and alachlor was above their 1/4000 LC50, and the effect is performed as a non-monotonic dose-response relationship. The histology viewing of the crucian carp's liver and spermary was taken after the 2 months exposure in atrazine and alachlor with different concentration levels. The results showed that: there is the significant destroyed effect to the development of the crucian carp's liver when the concentration of these two pesticides was above their 1/4000 LC50, and hepatic lesions in liver were characterized by hepatocyte hypertrophy, nuclear atrophy and deformation, vacuolization and focal necrosis; two pesticides above theire 1/4000 LC50 concentration showed the effect of destroying the spermary's structures and despessing the forming of sperm, which making the crucian carp unprocreated. Pesticides EDCs showed the different toxicity in different concentration: it showed the endocrine disrupting toxicity in lower concentration and the acute toxicity in higher concentration.2. The hepatic antioxidant defense (GSH) and detoxifying enzyme (UDPGT and GST) activities were evaluated. The results shown that these enzyme activities were sensitive to alachlor and atrazine exposure. These index could reflect the harmfulness to the hepatic cell and should be used as biomarker for monitoring the aquatic pollution.3. Levels of serum E2 significantly (P<0.05) increased in the alachlor treatments (4, 16, 63, 250, 500μg/L) and in the atrazine treatments (23, 94, 1500, 3000μg/L). They had similar effects on serum E2 and both enhanced the levels of serum E2; No obvious effects on the concentrations of serum T in the alachlor treatments (1,0.004, 16, 63μg/L) or in all treatments were observed, but significantly (P<0.05) decline in the alachlor treatments (250, 500μg/L); E2/T was increased along with the doses of alachlor and atrazine raise, and significantly in all treatments of alachlor and in the atrazine treatments (1500, 3000μg/L). These indicated that both alachlor and atrazine had feminization effects on the fish; TSH was significantly (P<0.05) increased in the alachlor treatment (250μg/L) and significantly (P<0.05) depressed when fish were exposed to high doses of atrazine (94, 1500, 3000μg/L) and no significant difference in other treatments of both pesticide; T3 increased significantly (P<0.05) in the alachlor treatments (4,63,250, 500μg/L) and in the highest dose atrazine treatment (3000μg/L), but showed no significant differences in low-dose treatments (6,23,094,375,1500μg/L).These results suggested that both alachlor and atrazine have effects on the level of serum sex steroids, TSH and T3 in juvenile male Carassius auratus, and significant on E2, which can be a biomarker of evaluation of endocrine disruption.4. The E2 could not be metabolized when the hepatic GST and UDPGT. The result shown a negative correlation between E2 level and enzyme activities. So the GST and UDPGT should be the key enzyme for regulating serum sex hormone. And the GST activities played an important role in metabolized the T3. The change of hepatic enzyme activities could cause a series of biochemical effect, alternating the serum sex hormone, disturbing the endocrine system.5. To screen and establish a biomarker for monitoring water pollution is also job of mine. Crucian carp vitellogenin (VTG) induced by endocrine disrupters was a suitable biomarker in my research. It could be purified using an anion-exchange column. VTG was characterized by SDS-PAGE and Western blot. An efficient method for determining crucian carp VTG by ELISA was established. Further more, it could be easily applicable to the large-scale field monitoring of environmental estrogens contamination in aquatic ecosystems especially for sewage plants.6. The effect to crucian carp's DNA after the 2 months exposure in these two pesiticides in tested by random amplified polymorphism DNA (RAPD) experiment. The random primers were screened to show the details of this effect. The mainly performance of this effect is damage and it performed as a non-monotonic dose-response relationship.7. Lymphocytes separated from the blood of crucian carp were cultured and exposed to alachlor and atrazine in vitro. The activity of lymphocytes was significantly influenced by these two environmental hormones and dose-effect relationship was showed. The half inhibition concentration (IC50) of alachlor on lymphocytes was 27.7±7.6μg/L and IC50 of atrazine was 97.6±26.4μg/L. The lymphocyte proliferation test in vitro showed that lymphocyte proliferation was significantly inhibited when the concentration of alachlor was above 7μg/L or the concentration of atrazine was above 50μg/L.8. Macrophages separated from the head-kindey of crucian carp were cultured and exposed to alachlor and atrazine in vitro. IC50 of alachlor and atrazine on macrophages were 68.2±7.1μg/L and 153.9±23.5μg/L, respectively. Both alachor and atrazine can inhibit macrophages'productive activity of super oxide anion (O2-), but dose-effect relationship was not obvious.9. Alachlor and atrazine's effects on the enzyme activity of lymphocyte and macrophage were studied. The results showed that these two environmental hormones could inhibit the lactate dehydrogenase(LDH) activity of lymphocyte and both LDH and acid phosphatase(ACP) activity of macrophage and dose-effect relationship was showed. Alachor and atrazine's inhibition on the enzyme activity accorded with their effects on the activity and function of lymphocyte and macrophage.10. After 2 months exposure to alachlor and atrazine in water, the lymphocyte proliferative activity assay was made and the leucocytes of blood were counted. The results showed that exposure to alachlor at concentrations from 64 to 500μg/L and atrazine at concentrations from 6 to 3000μg/L could inhibit the proliferative activity of lymphocyte. And alachor of 250 and 500μg/L or atrazine of 1500 and 3000μg/L led to significant decrease of leucocytes of blood. The results of both in vitro and in vivo experiments showed that alachlor and atrazine could inhibit the activity and function of immune cells of crucian carp when their concentrations met a certain level. So these two environmental hormones might have potential immunotoxicity to crucian carp.
|
PDF Full Text Request