| Environmental thyroid hormone disruptors were defined as "an exogenous substance that changes thyroid function and causes adverse effects at the level of the organism, its progeny and/or (sub) populations of organisms". It is of great importance to establish a system to screen and test Endocrine Disrupting Chemicals (EDCs) from thousands of environmental chemicals, which could help to constitute prophylactic measures, protect environment and human health. Up to now, however, the only accepted reasonable screening and testing method for thyroid hormone disruptors is the in vivo test on levels of serum thyroid hormones in rodents after exposure to certain chemicals. The international tendency of establishing such a screening and testing methods system is to develop an in vitro screening system with the characteristic of fast, economical and reliable.In the world, an in vitro testing model based on FRTL-5 cell line for thyroglobulin disruptors has been first established in our laboratory. Based on the work of Doctor SUN Ding, we explored the feasibility of TPO exoression in FRTL-5 cells as an observing index inscreening environmental thyroid hormone disruptors. At the mean time, we used thecDNA microarrays technology to shudy the in vitro mechanism of environmental thyroid hormone disruptors.It is now widely accepted that thyroid hormones (TH) play predominantly a nuclear role and function in the growth and differentiation of many organs, including the central nervous system. Perinatal exposure to environmental thyroid hormone disruptors may lead to TH deficiency in fetal and neonate. The other objective of this research is to observe the effect of perinatal exposure to environmental thyroid hormone disruptors on the thyroid function and development of rats, and to explore its mechanisms.Part one Study on in vitro mechanism of environmental thyroid hormone disruptorsFor the first time, cDNA microarray technique was introduced into the research field on thyroid hormone disruptors to explore in vitro mechanism. We also choose different thyroid hormone disruptors to observe their effect on TPO expression in FRTL-5 cells, in order to explore their mechanism of disrupting Tg synthesis, and to explore the feasibility of TPO exoression in FRTL-5 cells as an observing index inscreening environmental thyroid hormone disruptors.1. cDNA microarrays are capable of profiling generxpression patterns of thens of thousands of genes in a sigle experiment. In this section, cDNA microarray technique was used to analyze the gene expression of FRTL-5 cells treated by 2.0 U g/ml Sulphamethazine for 24 h. There were 679 genes (679/9753, 7%) exhibiting different expression, in which 395 genes up-regulated and 284 genes down-regulated. These genes relate toregulation of gene expression, regulation of cell cycle, metabolism in cells, and so on. It suggested that the effect of Sulphamethazine on FRTL-5 cells might be related with a series of genes, which provided important foundation for themolecular mechanism of thyroid hormone disruptors.2. To establish the in vitro screening method and to explore the mechanism of thyroid hormone disruptors, four chemicals were chosen to be tested their effect on TPO expression in FRTL-5 cells. They are N'N-methylene-bis, propylthiouracil, sulfamethazine, and malathion. We found no effect of N'N-methylene-bis and propylthiouracil on TPO exprssion in FRTL-5 cells, while sulfamethazine and malathion decreased TPO exprssion. It suggested that sulfamethazine and malathion may disrupt Tg synthsis by suppressing TPO avtivity, on the contrary, N'N-methylene-bis and propylthiouracil maybe by other mechanism. The results were consistent with other related in vivo and in vitro studies, so this method could be used in screening thyroid hormone disruptors together with other methods.Part two Developmental toxicity of environmental thyroid hormone disruptorsThe thyroid hormones (TH) play crucial roles in development. Abnormal structure and function of brain were found under thyroid hormone deficiency state. In human, thyroid hormone deficiency during development will result in cretinism. Perinatal exposure to environmental thyroid hormone disruptors may lead to TH deficiency in fetal and neonate by some way, and make a great impact on the growth of central nervous system. The objective of this section is to observe the effect of perinatal exposure to sulfamethazine on the thyroid function and development of rats,and to explore its mechanisms.1. Pregnant female rats were fed variours doses of sulfamethazine (0, 50, 100 and 200 mg/kg body weight) from gestational day 7 to weaning on postpartum day 20, at dose of respectively. We found that serum FT4 decreased from postnatal 10 to 20 day. Hyperplasia and increase of PCNA positive cell number were observed in thyroid gland in pups of experimental group on postnatal day 20. It indicated that perinatal exposure to thyroid hormone disruptors might result in thyroid disfunction in pups.2. Perinatally exposed to sulfamethazine, body weights of pups were significantly reduced compared to controls. The delay of eye opening and the deficiency on learning and remembering, which is dose-related, were observed too, especially in 100 and 200 mg/kg group. The result suggested perinatal exposure tosulfamethazine may lead to stunt and learning trouble in pups by disrupting thyroid function.3. Brain histology on postnatal day 20 showed abnormal change in hippocampus. Shrinkage occurred in pyramidal cell in CA3 of hippocampus while decrease in cells number was found in CA1 of hippocampus. The effect on brain development may be attributable, at least in part, to its ability to reduce circulating levels of thyroid hormone in pups. It could not exclude the possibility of direct toxicity of large dose of sulfamethazine on the brain.4. Neurotrophs are very important to survival, differentiation and function of neural cells. We observed NGF and BDNF expression in hippocampus on postnatal day 20 by immunohistochemistry. And we found the expression of NGF and BDNF weaken in CA1 and CA3 of hippocampus, number of positive cells was significantly decreased in pups of perinatal exposure to sulfamethazine. It indicated that sulfamethazinedown-regulated NGF and BDNF expression by reducing TH level, and affected brain development.5. Choline acetyltransferase (ChAT) is a specific phenotypic marker of cholinergic neurons, which is related to learning and memery. Immunohistochemistry was used to observe ChAT expression in brain on postnatal day 20. We found the number of ChAT positive cells were significantly decreased in cerebral cortex and hippocampus of pups in experimental groups. It suggested that thyroid hormone disruptors may reduce TH level, then down-regulated neurotrophs and ChAT, and at last affect pups' capacity for spatial learning.Part three The effect of perinatal exposure to environmental thyroid hormone disruptors on the thyroid function of damsPregnant female rats were fed various doses of (0, 50, 100 and 200 mg/kg body weight) from gestational day 7 to weaning on postpartum day 20, at dose of respectively. At postpartum day 20, dams were killed by decapitation for measurement of serum FT4 and TSH. Although we could not detect the difference of serum hormones between experimental and control groups, hyperplasia and increase of PCNA positive cell number were observed in thyroid gland of dams exposed to sulfamethazine. It indicated that sulfamethazine made an impact on the function of thyroid in dams.
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