The Study On Endocrine Disruption Of Di-Butyltin Dilaurate And Phylogenetic Relationship Of Alga-Toxins

Organotin compounds belong to the most widely used organometallic compounds, which are widely used as stabilizers in polyvinyl chloride (PVC), antifouling paints, pesticides and fungicides. Organotin compounds showed serious harm to aquatic organisms. Tributyltin (TBT) has been reported to cause imposex in female marine mollusks, even resulted in population decline or even mass extinction. As the serious impact of TBT on nontarget organisms, consequently, the International Maritime Organization (IMO) decided in October 2001 to ban the application of TBT-based paints on all boats by January 2003 and the presence on ship hulls by January 2008. Despite the large amount of toxicological data on invertebrates, only recently have the effects of organotin compounds on mammalian reproduction been reported. Studies of the reproductive toxic effects of organotin compounds have been focused on TBT and triphenyltin (TPT). Much less attention has been paid to reproductive effects of other organotin compounds in mammal.No works were performed on the study of the androgen disruption of dibutyltin dilaurate (DBTD), a widely used organtin additive, on rat. Laboratory experiments have demonstrated that exposure of rodents to sex hormones during prenatal or early postnatal life can cause permanent and irreversible alteration of the reproductive organs and reproductive functions in both sexes, which suggests that prenatal or early postnatal exposure period might be a critical period for environmental endocrine disruptors. In this study, the endocrine disruption of DBTD to mammal and whose mechanisms were studied after exposure of rodents to DBTD during the critical period in the Wistar rat model.To investigate the consequences of exposure to dibutyltin dilaurate in the Wistar rat model, pregnant rats were given a daily dose of vehicle DBTD at 0, 10, 20 or 30 mg/kg by gavage during the sex differentiation period of fetuses (gestational day, GD12-GD19). Dams were killed on GD20 to detect the effect of DBTD on the number of corpora lutea, live and dead fetuses, weights and sizes of fetuses. The results showed that exposure to DBTD resulted in embryonic toxicity for rats. Delayed ossification of fetal phalanges was observed in all DBTD groups, but no visible malformation or disfigurement in other skeleton after Alizarin Red S staining. It was the first report that a significant increase in anogenital distances being found in female fetuses from the dams treated with DBTD, to our best knowledge. This result suggests DBTD may exert a masculinizing effect on female fetuses.A major issue regarding endocrine disruptors in the environment is whether their effects are permanent or reversible. There are few studies on the reproductive toxicology of DBTD on the mammal. Permanent effects of DBTD on reproductive system of mammal have not being reported at present. In order to provide evidences that the endocrine defects of DBTD was in permanent on mammal, the following experiments were done.Neonatal male rats were treated by hypodermic injection at doses of 0, 0.01, 0.1 or 1 mg/kg DBTD for 5 days, respectively and were breed to puberty. The development of rats was mensurated during this period. Animals were decapitated on postnatal day 49 to analyze organ index, testicular and hepatic histological evaluation. These results showed that the testicular organ index significantly increased in 1mg/kg DBTD group, while these rats did not show general toxicity (the hepatic, nephritic organ index in all DBTD groups compared to the control group, histological evaluation of the testes and livers showing no obvious histological changes in them) in all DBTD group. It suggests that testis is one target organ for DBTD.Further studies were done on the concentration of serum testosterone (T) and the activities assays of enzymes, which are relative with the process of spermatogenesis, including succinate dehydrogenase (SDH), lactate dehydrogenase (LDH) and glucose-6-phosphatase-dehydrogenase (G-6-PD). These results showed that DBTD exposure increase the concentration of serum T and the activity of SDH, which presents conclusive evidences of DBTD possessing permanent endocrine disruption on rats following the exposure rats to DBTD.The present study provides, to our knowledge, the first direct evidence that di-butyltin dilaurate impairs male rat reproduction by increasing circulatory testosterone. Controversial results have also been reported which demonstrated a decrease in serum T concentration in mammalians when treated rats or mouse with organotin compounds in postweaning or adult period. After comparing with them, we suggested there are critical exposure periods for organotin compounds disrupting the endocrine system of mammalian.The mechanisms of DBTD exposure resulting in rats' serum T concentration were as following.The mRNAs expression of key steroidogenic enzymes including steroidogenic acute regulatory protein (StAR), cytochrome P450 cholesterol side-chain-cleavage enzyme (P450scc), cytochrome P450 17α-hydroxylase (Cyp17) and 3β-hydroxysteroid dehydrogenase (3β-HSD) and the 5αreductase which responsible for dihydrotestosterone (DHT) forming from testosterone, the P450 aromatase(CYP19) which catalyzes the conversion of the testosterone, to the estradiol, were detected by reverse transcription polymerase chain reaction (RT-PCR).The relative mRNA expression of StAR, P450scc, 3β-HSD, 5αreductase and CYP19 did not change. There is an increase of Cyp17 mRNA relative expression in all DBTD groups, even a significant increase in 0.1mg/kg DBTD group compared to the control group. The Cyp17 might be a sensitive factor for rat exposure to DBTD. The transcription process of Cyp17 was accelerated by the disruption of DBTD, which promoted the conversion of pregnenolone or progesterone to dehydroepiandrosterone (DHEA) or androstenedione, as a result, the serum T level increased.Serum concentration of luteinizing hormone (LH), follicle-stimulating hormone (FSH) was detected by Radioimmunoassay (RIA). The increases in serum LH, FSHconcentrations observed in the DBTD groups, occurring in the presence of elevated serum T concentration, imply that regulatory pathways in the hypothalamo-pituitary-testicular axis were disrupted. The disrupt affectes the regulation and control of hypothalamo, pituitary hormone on sexual gland.The report of JI Guibin showed that organtin pollution might play a pivotal role in blooms of cyanobacteria. The alga toxin produced by bloom-forming cyanobacteria has been the cause of human and animal health hazards. There are two main groups of toxins in cyanobacteria, named microcystins and nodularins. In this study, the relationship of the both toxins was analyzed genetically.Gene sequences of conserved functional motifs of polyketide synthases (PKS) from 2 microcystins-producing cyanobacteria were sequenced by us, and the relevant target gene from 14 microcystins-producing cyanobacteria and 4 nodularins-producing cyanobacteria were derived from GenBank. Whose sequences identity and phylogenetic relationships of corresponding proteins were analyzed by the software of DNAStar and Phylip. The result showed that target gene sequences of microcystin synthetases and nodularin synthetases share high identity. Phylogenetic analysis suggests that the phylogenetic relationship between Anabaena, Nostoc and Nodularia were closer than that between Anabaena, Nostoc and Microcystis. The Nodularia NSOR10 and the Anabaena 202A1/35 were chosen as the PKS representative of microcystins and nodularins, respectively to anysis the proteins secondary structure of them. The secondary structure of target proteins was predicted by the methods of Garnier-Robson and Karplus-Schulz. Hydrophilicity plot and surface probability plot were obtained by the methods of Kyte-doolittle and Emini respectively. The results showed that there are significant similarities in the secondary structure of the analyzed proteins. The two synthetases share nearly identical hydrophilicity and surface probability characteristics. From the results, it is apparent that the nodularins might belong to microcystins, genetically, which offers a new basis to study the prevention and cure of eutrophication and the toxicity mechanism of bloom-toxin.