Effects On Thyroid Receptor Signal Transduction And Neurotoxicity After Developmental Exposure To Hexabromocyclododecane

Hexabromocyclododecane as one kind of add flame retardant agent is the world's third largest brominated flame retardants in addition to bromine biphenyl ether (PBDE), tetrabromobis phenol A (TBBPA). It is widely used in polystyrene foam, interior decoration, textiles and electronics. As the use of PBDE in Europe and North America and other countries was banned, HBCD as substitute of PBDE is more extensively used. Currently, HBCD has become the ubiquity of pollutants in environment and have been detected in the environment and/or human body fluids, including those of children. It has been suggested that HBCD could have endocrine disruptive and neurotoxic effects. Impaired oligodendroglial development in the brain, impairment in learning and memory, and aberrant spontaneous behavior have also been reported. There are very few studies focused specifically on the effects of HBCD, as many of their effects are similar to PBDEs, which has a stronger presence effect in the environment and on human health (Ibhazehiebo et al.,2011).In order to study effects on thyroid receptor signal transduction and neurotoxicity after developmental exposure to HBCD, according to the characteristics of the high risk population of the development children exposure to HBCD, the new born 3 days SD rats (PND3) were selected. On the real content of HBCD in environment, exposure doses were divided into the control group and 10,50,100,300μg/kg groups. According to the characteristics of the rats development, exposed time was as for 21 days,42 days and 90 days three different brain development time node. By testing the indexes of animal behavior, thyroid hormone levels nerve, thyroid hormones nuclear receptor (TR) signal transmission process related gene expression, neurotransmitters and related enzyme activity and so on, try to reveal relation of the internal mechanism between thyroid hormone disturbance and neurotoxic effects from animal individual, biochemical and genetic levels.The results showed that: 1^ By the methods of Morris's Water maze and open field experiment, Neurobehavioral in rats are tested separately in 21 days and 90 days. The Results suggest, when the exposure time are 21 days, the 10μg/kg low-dose exposure group and the 30μg/kg high-dose exposure group used in rats can promote the capacity of study and memory, these capacity are shown in the index of escaping an incubation period and crossing a platform, and the influences are remarkable (P<0.05). And the two indexes that rats live in quadrant platform and escape the incubation period after changing another platform also show an apparent reaction. In contrast, when the exposure time become 90 days, results are consistent with the former test, the exposure group can impaired the capacity of learning and memory with 50μg/kg. According to the open field test in 21 days exposure time, the results are shown below. In the range of 50～300μg/kg exposure groups, the motor activity increased significantly (P<0.05) by total distance; and in the central distance area, exposure groups of rats appear the most significant psychological anxiety (P<0.05) with 10μg/kg. However, these effects are less clear with the growth of rats. So after the exposure time pass 90 days, it is useless in statistics.2,Using radioimmunoassay with 1251-anti-serum thyroid hormones to test related parameters on Serum thyroid hormone, indexes include:TT3,TT4,FT3,FT3,TSH and so on. the experiment results indicate the concentrations of TT3,TT4,FT3 and FT4 on a rise trend, and the exposed group of rats live in 21 days. Though the concentration of TSH declines, it is not important. In the condition of 42 exposed days, when the dosage is 10μg/kg, all the parameters'concentrations rise in the body of rats-group and the index of FT3 and FT4 increase apparently. Besides, in other exposed groups, the concentrations decline, thus, the results are not statistically significant (P> 0.05).in the condition of 90 exposed days, the concentrations of TT3,TT4,FT3 and FT4 rise in the body of all rats-group, and the index of TT4 increase apparently. What's more, when the dosage is 50μg/kg, the FT4's index is much higher, and the indexes of TT3 and FT3 both are improving in the condition of 50μg/kg and 100μg/kg exposed group. In addition, the 10μg/kg dose exposed group's TSH concentration drops. And in other groups this parameter Show the opposite trend.3,Using the measure of real-time PCR, the transfer process of rats' hippocampus TR are in a quantitative determination, the level of gene expressions are determined the expermeriments. There are three genes in configuration, the TR genes inclued TRα1,TRα2 and TRβ1; The complexes of Thyroid hormone in combination with TR and Xist genes of BTEB in Thyroid hormone response element (TRE); Neurological genes downstream controlled by TR single named RC3 and NGF; and the genes related to neurotransmitter-metabolizing enzymes function called MAOA,MAOB,ChAT and etc. The TR genes test results are shown as follown. In each exposure time,the expression levels of hippocampal TRα1 and mRNA are on a increasing trend (P<0.05). In these groups, the exposure group of 42 days with 50μg/kg and the exposure group of 90 days with 300μg/kg go up in the expression levels of TRα1 gene. And when the exposure time are 21 days and 90 days, the hippocampal TRα2 and mRNA levels emergence in all experiment groups. However, when the exposure time change to 42 days, the results are opposite, and this tendency Shows significantly in the groups of 10ug/kg,50ug/kg and 300ug/kg dosage (P<0.05); the expression level of hippocampal TRβ1 and mRNA rises in the exposure group of 21 days with lOug/kg (P<0.05) in according with 42 days and 90 days, in which the rising tendency are higher in 42 days at the range of 1100～300ug/kg groups and in 90 days the range is50～300ug/kg groups (P<0.05)The following are the BTEB gene's level test results. the expression levels of hippocampal BTEB mRNA in 90 exposure days are on a increasing trend (P<0.05),while the reduction in 21 days exposure group show much more significant than in 42 days exposure group,which is pointless; The NGF and RC3 genes test results indicate the expression levels of hippocampal NGF mRNA improved excepting that the 21 days and 90 days experiments are useless. And when the exposure time are 21 days and 90 days, the hippocampal T RC3 mRNA levels are in a dropping trend in all experiment groups, but in 42 days exposure group it is in a improving way, In these groups, the exposure group of 21 days with 10μg/kg reducesd obviously.The MAO-A, MAO-B, ChAT genes test results are shown that 21 days exposure group of the MAO-A mRNA genes express a downward trend, when exposed to 90 days, though the exposure group tended to increase, it is adjective. And when the exposure time are 21 days and 90 days, the hippocampal MAO-B mRNA levels are in a rising trend in all experiment groups, but in 42 days exposure group it is in a reducing trend, and in which the exposure group with 100μg/kg reducesd more quickly (P<0.05).in these groups, when the exposure time is 21 days, the ChAT mRNA genes express a upward trend in 100ug/kg (P<0.05); when the exposure time is 42 days, the ChAT mRNA genes express a rising trend, in addition, the groups with a range of 50-100ug/kg increase higher (P<0.05);while the exposure time turns to 90 days, thes genes express a upward trend in 10ug/kg (P<0.05)4,Spectrophotometric method, Fluorescent quantitative method and Kit method had been used to gauge the values of monoamines and cholinergic neurotransmitters in rats'brain and the related metabolic activity. The kinds of monoamines are DA, NE,5-HT neurotransmitter and MAO enzymes; while Ach and AchE, ChAT and other metabolic enzymes belong to cholinergic neurotransmitters. The analysis are discussed as below, In the monoaminergic system, the DA level decreased sharply in the exposure groups at the range of 100～300μg/kg, the range between 50μg/kg and 300μg/kg where the NE level decreased sharply in the body of the rats groups, and the 5-HT level shows the same tendency in the range of 300μg/kg, but the content of MAO's activity is enhanced in 50～300ug/kg. In contrast, when the time prolong to 42 days, the groups with 10μg/kg and 100μg/kg keep the similar law in the level of DA (P<0.05),but the level of NE change little in such rats. And in the range of 50～100μ/kg, the 5-HT level shows a different trend. Besides, MAO activity is on the rise, but this change is not statistically significant (P> 0.05). As the exposure time reaches to 90 days, the level of DA declined fast in the groups with 300μg/kg (P<0.05), the groups with 100μg/kg keep the similar law in the level of NE (P<0.05). On the contrary, the 5-HT levels increase quickly in 300μg/kg. MAO activity rise still but useless.In the cholinergic system, after a series of exposure times, the values of Ach in these experiments seem a downward trend, in which the exposure group of 21 days with 10～300μg/kg, the rats'Ach level reducesd apparently(P<0.05); And the group with 21 days and 42 days exposed are on a rise in the activity of AchE. What's more, this level increases much in the groups with 300μg/kg, but it is not helpful when the time comes to 90 days in AchE activity testing and so it is in ChAT activity testing as it comes to 20 days. However, in these groups, when the exposure time is 42 days, the ChAT activity improves notablly with 10ug/kg, and it reduces significantly in the range of 100～300μg/kg (P<0.05)Based on the above results conclusions can be drawn as following:1,In the experiment on studying animals'neurobehavioral, the method of HBCD exposure in rats can cause nervous excitement in 21 days and 42 days. And it plays a positive role in promoting rat's learning and memory ability. In addition, with a low dose of 10μg/kg and a high dose of 300μg/kg such influences appear more effective. Moreover, the HBCD exposure way can lead to improving the performance of activities in a short-term exposure of 21 days. Rats may also feel anxiety with the dose of 10μg/kg, but all these reactions would be weakened gradually with the growth of rats. Therefore,21 days of HBCD exposure on neurobehavioral effects in rats are significant.2,In the experiment of thyroid hormone levels, though the exposure time and doses are different, the changing trends are in consistence, that is the contents of TT3,TT4,FT3 and FT4 are on a rise and the TSH level is decline. A conclusion is that the effect of HBCD can promote the serum thyroid hormone levels. Among these factors the changes of FT3 and FT4 is adjective. So it is more easily for HBCD to affect FT3 and FT4 level of impacting the body's physiological processes. The results also showed the reaction is much more significant in 21-days exposure groups than that in 42-days and 90-days groups. The increasing levels of thyroid hormone mean that the HBCD method can bring out hyperthyroidism. And this conclusion can be used to explain the neurobehavioral behaviors mentioned above.3,In the test of gauging the level of gene expressions, the thyroid hormone disrupting effects and TR signaling processes may be one of the mechanisms from the neurotoxic effects of HBCD. Three different configurations of the TR gene expression is mainly controlled by the thyroid hormone T3, the way in HBCD exposure groups cause levels of TRα1 and TRβ1 mRNA express a rise trend and cause level of TR a 2 express a oppsite trend. The results mentioned above show that using the method of HBCD to change the expression of TR gene can interfere with thyroid hormone.BTEB gene is complexes involved in thyroid hormone and TR binding as well as Xist with TRE, controlled by thyroid hormone. The change of BTEB mRNA expression is also affected by the results of HBCD way interfering with thyroid hormones. In 21 days exposure time, the level of BTEB gene decreases while in 42says and 42 days the results are out of usage. As an important factor in nerve repair, the phenomenon of NGF expression can point the extent of damage of nerve. In 42 days exposure group this NGF gene display a sharp decline. RC3 plays a role on changing the Ca2+/calmodulin signaling, synaptic plasticity and cognitive function in region-specific attenuation of damage. In 42 days exposure group, the expression of 42d RC3 mRNA level decreased indicates that has neurotoxic effects on the brain.Related to the neurotransmitter-metabolizing enzymes, the functional genes of MAO-A affect less than MAO-B in the influence of HBCD, and it indicates that HBCD affects the expression of MAO-B more. However, the expression levels of ChAT mRNA decrease in 21 days but increase in 42 days. To conclude, the gene of MAO and ChAT are associated with brain cognition, learning and memory ability and neurological function. The interference of HBCD for thyroid hormone and TR signaling processes can produce neurotoxic effects, it is possible to damage brain, result in learning and memory impairments, cause cognitive impairment and nerve-related-nerve diseases. Besides, this may even lead to serious damages to the central nervous system development. The results found that the HBCD exposure in 21 days and 42 days for gene expression is more prominent.4,The content of neurotransmitters and the activity of enzyme are tested too. Biochemical markers show that the method of HBCD exposure can not only change the performance of neurotransmitter enzymes at the molecular level, but show the neurotoxic effects at the biochemical levels. In monoaminergic systems, when the exposure time is 21 days, HBCD play a role through inhibiting MAO gene's activity and promoting the content of DA, NE and 5-HT. and when the exposure time is 42 days and 90 days, HBCD exposure has little effect on neurotransmitter through MAO activity. Monoamine neurotransmitter is a kind of information-passing material, which is important in the central nervous system. It involve in pain, physical exercise, emotional activity, sleep, arousal, stress and other physiological processes, it also play a coordinating role in exciting or inhibiting the central nervous system. DA,NE,5-HT levels in rats can reveal neurobehavioral such as learning and memory capacity, activity performance, anxiety psychology and so on. In cholinergic systems, HBCD, reducing ChAT activity and promoting the content of AchE activity in the control of Ach mainly, impact the acetylcholine system a lack of functionality. Expect for Alzheimer's disease (AD), Ach cause disorders of learning and memory capacity as well. When the activity of ChAT decrease, it may affect the synthesis of Ach and the function of cholinergic system, so by synthesizing Ach, ChAT can control the transmission of information between synapses, regulate brain function and affect rat's learning and memory ability. Moreover, as a neurotrophic factor and a kind of related enzyme, AchE plays a vital role in the critical period of brain development, and if the level increased, it will bring about the brain barrier in learning and remembering.