| Simazine is a widely used chlorotriazine herbicide in agriculture and in recreational park and garden areas when it has been commercially available since 1955. The breakdown of simazine can be through chemical, photochemical or biological processes. Degradation studies have shown DT50 values (Time required for concentration to decline by 50%) of simazine in both soil and water, vary between a few days and 150 days. Temperature and humidity can affect the half-life of simazine. Low temperatures and drought may prolong the dissipation time by a factor of two or more. In 2009, simazine was tested in an endocrine disruptor screening program (EDSP) by the US EPA due to its multiple exposure pathways and high production volume. In China, simazine is one of the most commonly detected pesticides in underground and surface waters.Simazine belongs to the group of environmental endocrine disrupting chemicals (EDCs) which are defined as "exogenous agents that interferes with the synthesis, secretion, transport, binding, action, or elimination of natural hormones that are responsible for the maintenance of homeostasis, reproduction, development, and behavior". To date, studies have shown that in vitro simazine has a stimulatory effect on the antiandrogen screen and in vivo simazine produces adverse effects on the growth, development, immune and endocrine systems in a wide range of species. Simazine was also found to affect the reproductive system in mammals and amphibians species.Simazine is the second most commonly detected pesticide in surface and ground waters in the US, Europe, and Australia, presumably due to relatively high persistence in soil and water. Concentrations in both surface and ground water are variable, since they are influenced by season and the extent of simazine-based herbicide use in the location investigated. In US simazine levels can reach values up to 1300 μg/L in surface water, and 800 μg/L in ground water. According to the water purity standards of the World Health Organization, tap water that is contaminated with 2 μg/L of simazine is considered harmful. This study aims to address whether some environmentally-relevant concentrations of simazine might affect the physiological parameters of amphibians and how simazine induce the toxicity effect.Amphibians have a complex life cycle and permeable skin and can be exposed to environmental contaminants through multiple routes on land and in the water, which make them to be ideal model organisms for testing EDCs exposure and resultant effects. We aimsto explore how xenoestrogen-induced changes in gene expression relate to conventional physiological and toxicological endpoints.The study respectively selected amphibians Bufo bufo gargarizans Cantor and Xenopus laevis as animal models, to study the effects of simazine on the growth and development of the two species, as well as the related genes (vtga2, ar, er,gr, hsp70 and hsp90) expression in livers of X. laevis, and microarray expression profile of male gonad tissue. The study explored the molecular pathways and cellular processes which mediate the adverse responses to simazine. The study aims to speculate the molecular mechanisms of simazine on amphibians for providing theoretical basis for the further research in molecular level.At stage 25/26, B. bufo gargarizans tadpoles from the same pair of brood stock were treated with simazine at four concentrations (0.1,1,10 and 100 μg/L). Stock solutions of simazine were made with the reagent vehicle DMSO (0.05%). Control groups were treated with the reagent vehicle DMSO (0.01%) only. Each group had 144 tadpoles which were divided into 8 replicate tanks (25×20×20cm3) with 10 L of water each at 22 ± 2℃ and pH 7.5. Animals were maintained on 12-h light/12-h dark cycles and treated for 85 days. On day 49, tadpoles from each tank were rapidly transferred and weighed. The body and tail length were measured. Animals were returned to their home tank immediately after the measurements. On day 85, all experimental animals were euthanized for body weight and length measurements, morphological assessment and tissue collection. The dorsal wall of the abdominal cavity with kidneys and gonads from males and females were collected and fixed in Bouin’s solution for histopathological examination. The sections were cut in 5 μm and stained with hematoxylin and eosin (HE) for light-microscopic examination. Sex and gonadal morphology of B. bufo gargarizans from the treatments and control groups were determined by direct visual inspection on day 85. Sex ratios were expressed as percentage of male and female in each group. Intersex was referred to as gonadal abnormality.In the results of B. bufo gargarizanse exposed to simazine, the mortality increased significantly when the tadpoles were exposed to 10 and 100 u.g/L simazine (p< 0.01). No significant difference in the size and weight of tadpoles was observed between the control and treatment groups on day 49 and day 85 respectively (p> 0.05). Our results showed there was no significant effect of simazine on the percentage of B. bufo gargarizans reaching FLE on day 46,48,50 and 52 (p> 0.05), but the hindlimb extension of animals exposed to 10 and 100μg/L simazine was inhibited significantly, respectively (p< 0.05 and p< 0.01). Simazine affects the development and metamorphosis of B. bufo gargarizans, when all the tadpoles in the control group just had completed metamorphosis, the gross abnormalities were still at stage 38-40 in treatments. Ovarian tissuesfrom simazine-treated groups were unnormal including necrotic part, disrupted histological architectures and vacuolization, germ cells reduced. Obvious changes were found in testes from toads treated with simazine including undeveloped testes without germ cells, testicular oocytes. The effects of simazine on sex ratio of B. bufo gargarizans was not significant (p> 0.05). Gonadal morphology abnormality was not found in any of the simazine-treated animals.At Nieuwkoop-Faber (NF) stage 46, X. laevis tadpoles (n= 600) from the same pair of brood stock were randomly divided into five groups. Each group (n= 120) was divided into 8 replicate tanks (25×20×20 cm3), each containing 10 L water. The tadpoles were exposed to simazine dissolved in solvent vehicle DMSO (0.01%) at designed dosages of 0.1,1,10 and 100 μg/L for 100 days. The control tadpoles were treated with 0.01% DMSO only. Larvae were observed daily for monitoring their morphological changes and health status. Dead or moribund animals were removed and recorded. The testicular tissues and liver tissues of ten males from each group were respectively isolated and fixed in Bouin’s solution and 10% formalin, and then paraffin-embedded. The sections were cut at 5 μm serially and stained with HE. The specimens were examined microscopically to assess the gonad development.Tissues from the remaining males were flash-frozen in liquid nitrogen, and stored at -80℃ for microarray expression analysis. On the basis of the results of the toxic endpoints six male control frogs, six male 100 μg/L simazine-treated frogs were selected for gene expression analysis using Agilent(?) Xenopus 4×44K Gene Expression Microarrays. Differentially expressed genes were identified by the value of fold-change (FC) (≥ 2) and t-test was used in comparison with those in the control group. We further validated the data from microarray analysis using Q-RT-PCR. Gene ontology (GO) and pathway analysis were used to translate complex gene expression profiling data. The tools able to associategene expression changes with alterations inconventional indicators of phenotypic and toxicologic changes.In the results of X. laevis exposed to simazine, the mortality increased significantly in the 10 and 100 μg/L simazine treatment groups (p< 0.05). In this study, significant changes in the body weight and length of frogs after exposure to simazine were not observed (p> 0.05). The effects of simazine on sex ratio of X. laevis was not observed (p>0.05). Gonadal morphology abnormality was found in any of the simazine-treated animals. Interestingly, the gonad weight and GSI were significantly reduced in male frogs exposed to simazine at 10 and 100 μg/L compared to the controls (p<0.05), but the gonad weight and GSI in females were not affected (p> 0.05). Meanwhile, histologic structure of ovaries was not changed, but the histologic structure of the testes was significantly changed in X. laevis treated with simazine over the entire dose range. Irregular shape of seminiferous lobules, hypertrophic spermatogonias and large empty spaces were observed in the frogs from the simazine treatment groups. Particularly, in testes of frogs from the 100 μg/L simazine treatment group, spermatogonias were hypertrophied and parts of the seminiferous lobules appeared pycnotic, a process involving necrosis in which the cell nuclei were characterized by condensation with hyperchromatic staining or pycnosis and sheet structure. The days of X. laevis from 10 and 100 μg/L simazine treatments completing metamorphosis were delayed (p< 0.01). Liver weight and HSI did not show significant changes after treatments (p>0.05). Besides atrophy and necrosis of hepatocytes in all simazine treatments, vacuolization was also observed in the liver of X. laevis treated by simazine. RT-PCR analysis did not showed a statistically significant changes in vtga2, ar, er, gr, hsp90 gene expression in livers from X. laevisexposed to simazine except for hsp70 in livers from female X. laevis exposed to 0.1 μg/L simazine.The study selected the 100 μg/L which was the typically effective concentration of male reproductive toxicity to the further study on the mechanisms of simazine effects. The raw dataset of microarray was submitted into the NCBI Gene Expression Omnibus database (# 17214940). I then analyzed gene expressions using microarray technology and found that the 1315 genes were significantly altered (454 upregulated, 861 downregulated) in frogs exposed to 100μg/L simazine. The results of genes expression were validated by Q-RT-PCR analysis. These results of Q-RT-PCR were consistent with the microarray data. The results of GO analysis showed respectively 188,37,33 GO terms which associated with the biological processes, cellular components and molecular functions. Based on KEGG pathway analysis, five signaling pathways were obviously interrupted by simazine. These pathways include arginine/proline, alanine/aspartate/glutamate, riboflavin, tyrosine metabolism pathway and cell cycle pathway.In conclusion, our results demonstrated that simazine has potential impairment on male reproduction of X. laevis and caused irregular shape of seminiferous lobules, hypertrophic spermatogonias and large empty spaces in testes. By microarray analysis, expression of 1315 genes was altered by at least 2 fold in the testes of X. laevis after simazine exposure. GO analysis elucidated the predominant gene functions and biological pathways that are affected by simazine exposure in a given experimental system. KEGG pathway analysis showed that these genes were enriched in five pathways. However, our study found genes and pathways that are linked to mechanisms of toxicity and adverse effects. What is clear is that simazine did not induce aromatase activity and interrupt er and ar signal pathway in adult male X. laevis exposed to 100 μg/L simazine. It is concluded that simazine is likely to act through a more general mechanism such as increase in metabolic activity rather than specifically targetingestrogen synthesis. |
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