Thyroid Disruption Induced By Sodium Chlorate And Pentachlorophenol (Sodium Pentachlorophenol)

Sodium chlorate (NaC103) and pentachlorophenol (sodium pentachlorophenol) are both common pollutants in drinking water. NaC103 derives from disinfection treatment with chlorine dioxide as disinfectant during the treatment process of drinking water. PCP(Na) which comes from raw water, can't be removed completely by regular water treatment. Therefore, human can be exposed to both chemicals through drinking water in a long time. Recent research found that, chlorate disrupts thyroid hormone synthesis through competitive inhibition of iodine intake by sodium iodide sympoter (NIS).PCP interferes the transportation of THs and TH receptors (TRs)-mediated downstream gene transcription that affect the levels of THs and its functions in the body. These results suggest that, NaClO3 and PCP may have the effects of thyroid disruption. However, few studies systematically assess their thyroid disruption.Restricted to limitation of cognition and evaluated methods in early times, current Standard for drinking water quality (GB5749-2006) did not take the effect of thyroid disruption into consideration when they were established. Now it still remains unknown whether chemicals at current standard value will produce thyroid disruption. Moreover, standard-setting process does not take into account the combined effects between pollutants. So it is important to study single and joint effects of NaC103 and PCP on the effect of thyroid disruption that will recognize the joint effects of exogenous chemicals on thyroid system and its function.In the present study, OECD TG 407 was referred to establish the assessment model for thyroid disruption.Meanwhile, primary culture of thyroid follicular cells was used to study single and joint effects of NaClO3 and PCP on functional genes and specific protein expression to explore the sensitive biomarkers of thyroid disruption. Part 1 Thyroid disruption induced by sodium chlorate and sodium pentachlorophenol in vivoTG 407 recommended by OECD for thyroid disruption assessment was used to set up animal model of thyroid disruption. Healthy and adult SD rats were administrated of NaClO3(30 mg/kg·bw,120 mg/kg·bw), PCP-Na (0.33 mg/kg·bw,30 mg/kg·bw), and NaClO3+PCP-Na (30 mg/kg·bw+0.33 mg/kg·bw,120 mg/kg·bw+30 mg/kg·bw) for continuously 28 days by oral gavage.24 hours after final dose, rats were sacrificed and THs (TT3,TT4, FT3,FT4) and TSH in serum were determined by radioimmunoassay. The expression levels of deiodinases(dio1,dio2) and TRs (thra, thrb) mRNA in liver were analyzed by Real Time RT-PCR. Histological changes of thyroid were observed by hemotoxylin-eosin stain. Results showed that THs didn't change significantly in both dose groups of NaC103 (30mg/kg·bw and 120 mg/kg·bw) (P>0.05).Liver dio2 mRNA expression influenced by NaC103 exhibited differences between doses and sexes, i.e.,up-regulated in 30mg/kg·bw dose group and down-regulated in 120 mg/kg-bw dose group in male rat, while down-regulated in both dose groups in female rats (P<0.05).Moreover, histological changes of mild thyroid hyperplasia and increase in small follicles presented in the group of NaC103 (120 mg/kg·bw).THs levels and Dios/TRs mRNA levels in liver didn't change significantly, compared to control group (P>0.05),but mild lymphocytic infiltration and increase in heights of follicular cells were observed in PCP-Na (0.33 mg/kg·bw) dose group. In the higher dose group of PCP-Na (30 mg/kg·bw), TSH increased, and FT4 decreased significantly (P<0.05), but TT4 decreased,and dio2 expression down-regulated only in male rats, while TT3 and FT3 increased, and diol expression up-regulated only in female rats (P<0.05).In the same dose group, hyperplasia of the follicular epithelium cells, and focal lymphocytic infiltration were significant. In the dose group of NaC103 (30 mg/kg·bw)+PCP-Na (0.33 mg/kg·bw), dio1 expression down-regulated significantly in male rats (P<0.05),and histological changes included hyperplasia of epithelial cells and depletion of colloid. In a higher dose group of NaClO3(120 mg/kg·bw)+PCP-Na (30 mg/kg·bw),TSH increased, FT4 decreased and diol expression up-regulated significantly (P<0.05),but TT4 decreased only in male rats, while TT3 increased, and dio2 expression down-regulated only in female rats (P<0.05).In the same dose group, solid buds, and focal lymphocytic infiltration were induced.This study also investigated thyroid function biomarkers and specific gene expression with the variation of time after exposure to NaC103 and PCP-Na for 3,7,14 and 28 days, to explore and find sensitive biomarkers of thyroid disruption as early as possible.Adult SD rats were administrated of NaClO3(120 mg/kg·bw),PCP-Na (30 mg/kg·bw),and NaC103(120 mg/kg·bw)+PCP-Na (30 mg/kg·bw) for continuously 3, 7,14,and 28 days by oral gavage.24 hours after final dose, rats were sacrificed and serum THs and TSH levels, liver mRNA expression levels, and thyroid histological changes were analyzed as before.The results showed that TSH increased at dosing time of 7 days (DT7), and FT3 decreased at DT14 in male rats, and TT4 increased at DT3 in female rats significantly at the dose group of NaClO3 (120 mg/kg·bw) (P<0.05).Histological examination found that reduction of colloid depletion and increase of follicular cell heights at DT7, and obvious hyperplasia of follicular cells at DT14, but histological changes at DT28 were mainly small follicular. There are gender differences in gene expression. In the same group, dio2 mRNA down-regulated at DT28 in male rats significantly (P<0.05);diol mRNA down-regulated at DT7, dio2 mRNA up-regulated at DT3 and DT7, and down-regulated at DT14, Thra mRNA up-regulated at DT3 in female rats significantly (P<0.05).In the dose group of PCP-Na (30mg/kg·bw), TSH increased at DT3 and DT28,TT3 increased at DT3,FT3 decreased at DT7 and DT14,TT4 decreased at DT7,14 and 28,and FT4 decreased at all DTs in male rat significantly (P<0.05);TSH increased at DT28,TT3 increased since DT7,FT3 increased at DT28, TT4 decreased at DT7, and FT4 decreased at DT28 in female rats significantly (P<0.05).Histological examination found that small follicular increased at DT3, sporadic lymphocytic infiltration happened at DT14, and focal lymphocytic infiltration at DT28.In the same dose group, diol mRNA up-regulated at DT14, dio2 mRNA down-regulated at DT3,7 and 28,thrb mRNA up-regulated at DT3 in male rats significantly (P<0.05);diol mRNA up-regulated at DT7,14 and 28,dio2 mRNA up-regulated at DT3 and 7 in female rats significantly (P<0.05).In the dose group of NaClO3(120 mg/kg·bw)+PCP-Na (30 mg/kg·bw),TSH increased at DT3,7, and 28, FT3 decreased at DT7 and 14, TT4 and FT4 decreased at all DTs in male rats significantly(P<0.05); TSH increased at DT28,TT3 increased at DT14 and 28,FT3 increased at DT14, TT4 decreased at DT7 and FT4 decreased at DT28 in female rats significantly (P<0.05).Histological examination found that colloid depleted at DT7, hyperplasia spots appeared at DT14, and lymphocytic infiltration and hyperplasia were both significant at DT28.In the same dose group, diol mRNA up-regulated at DT7,14 and 28, dio2 mRNA down-regulated at DT3 and 7, but up-regulated at DT14, thrb mRNA up-regulated at DT3 in male rats significantly (P<0.05);diol mRNA up-regulated at DT3,14 and 28,dio2 mRNA up-regulated at DT 7, but down-regulated at DT28, and thra mRNA up-regulated at DT3 in female rats significantly (P<0.05).The results suggested that TG 407 was a sensitive method for detection of thyroid function changes. Histological change was the sensitive endpoint for thyroid disruption which happened before other endpoints.In this study, disruption on thyroid system was within compensation induced by NaC103 at 30 mg/kg-bw and 120 mg/kg-bw. However, PCP-Na disrupted THs and TSH levels significantly, and induced thyroid histological changes.The joint effects by NaC103 and PCP-Na were relatively independent, which were inhibition of iodide uptake and gene transcription mediated by TR, respectively.Part 2 Thyroid disruption induced by sodium chlorate and pentachlorophenol in vitroPrimary culture of rat thyroid follicular cells was applied to investigate thyroid disruption induced by NaC103 and PCP. Thyroid follicular cells expressed TPO and Tg protein, secret Tg into culture media, and also expressed 4 specific genes (tpo,tg, tshr and nis) after cultured in complete F12 media for 1 week. The thyroid follicular cells cultured maintained regular morphology and function.Given the stability and reliability of this method, a 42 orthogonal experiment was designed with doses of NaClO3(0,5,10,20μg/ml)and PCP (0,0.25,0.5,1μg/ml). The endpoints observed included thyroid follicular cell proliferation, expression of dio1,tg and tpo mRNA, and secretion of Tg. The results showed that tg mRNA expression down-regulated at single dose 10μg/ml of NaC103,while Tg secretion decreased and tpo mRNA expression down-regulated at single dose 1μg/ml of PCP significantly(P<0.05).In joint dose groups of NaC103 (5μg/ml,20μg/ml)+PCP (0.25μg/ml), all groups with 0.5μg/ml PCP, and all groups with 1μg/ml PCP,Tg secretion decreased significantly (P<0.05).In joint groups of NaC103(10μg/ml, 20μg/ml)+PCP (0.25μg/ml), NaClO3(20μg/ml)+PCP(0.5μg/ml), and all groups with 1μg/ml PCP, tpo mRNA down-regulated significantly (P<0.05). In joint groups of NaC103(10μg/ml,20μg/ml)+PCP (0.25μg/ml), NaC103(5μg/ml)+PCP (0.5μg/ml), and all groups with 1μg/ml PCP, tg mRNA expression down-regulated significantly (P<0.05).According to two factors analysis of variance, PCP inhibited Tg secretion (P<0.05), while NaC103 didn't present this effect, but strengthened PCP's inhibition; PCP also inhibited expression of diol and tpo mRNA (P<0.05),and the joint effects of NaC103 and PCP were independently; NaClO3 and PCP didn't influence tg mRNA expression (P>0.05).The results showed that, PCP inhibited Tg secretion, but NaClO3 didn't. However, NaC103 could strengthen PCP's inhibition on Tg secretion. Meanwhile, PCP inhibited expression of diol and tpo mRNA in thyroid follicular cells, and influenced synthesis of THs. The possible health hazards induced by coexist chemicals may be greater than single ones.