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- MATERIALS AND METHODS
Pentachlorophenol (PCP) is a broad-spectrum biocide that has been widely used in wood preservatives, pesticides, and disinfectants . Because of its toxicity, PCP has been restricted or banned in many countries since the 1980s; however, it is still used as a wood preservative . In 2002, approximately 11 million pounds of PCP were produced in the United States , and in China, the annual production of PCP increased to 3000 tons in 2003 after the re-emergence of schistosomiasis in several provinces . The continued use of PCP throughout the world means it is still likely to result in environmental contamination, with potential risks to human health and the environment.
Environmental monitoring has demonstrated that PCP exists in various environmental media (e.g., water, soil, sediment, and aquatic organisms) and human samples [5-7], and it is a particularly common contaminant in water [8, 9]. For instance, the PCP level of surface waters is typically in the range of 0.1 µg/L to 1.0 µg/L, whereas an elevated PCP concentration can be found in groundwater (3–23 µg/L) and surface water (0.07–31.9 µg/L) in the United States, where the treatment of wood products with PCP has been conducted . In China, PCP contamination in the Yangtze River was most severe among 7 of its watersheds and 3 drainage areas, with a median value of 0.06 µg/L (up to 0.59 µg/L) , and greater concentrations of PCP (up to 103.7 µg/L) were detected in Dongting Lake . High concentrations of PCP in water may lead to a high risk of exposure and negative biological effects in aquatic organisms as well as humans.
The impact of environmental chemicals on the thyroid endocrine system has received much attention in recent years . Endocrine system disrupting chemicals can have a direct impact on thyroid hormone synthesis, transport, binding, catabolism, and clearance of circulating thyroid hormones. Limited information shows that PCP might disrupt thyroid endocrine functions. Epidemiological studies have reported that exposure to low levels of PCP in the environment is associated with decreased thyroid hormone levels in human neonates . A link between PCP exposure and disrupted thyroid hormone signaling has been experimentally demonstrated in rats [13-15], ewes , and Fischer rat thyroid cell line FRTL-5 . For instance, a pronounced decrease in circulating thyroxine (T4) and 3, 5, 3'-triiodothyronine (T3) levels were observed in female rats treated with 3 mg PCP/kg and 30 mg PCP/kg body weight for 28 d . Similarly, oral administration of PCP during development significantly increased the messenger RNA (mRNA) expression of thyroid hormone receptor β1 and synapsin I in the brain of rats, and reduced plasma levels of total T4 (TT4) in dams and 3-wk-old pups . Treatment with pentachlorophenate sodium (PCP-Na) significantly affected the thyroid endocrine system and altered mRNA expression of thyroid hormone receptors and deiodinases in the rat liver. In fish, PCP exposure decreases serum T4 levels .
The thyroid hormones play a crucial role in the regulation of development, growth, immunity, metabolism, reproduction, and behavior in vertebrates . In fish, thyroid homeostasis is controlled primarily by the hypothalamic-pituitary-thyroid (HPT) axis . Recently, zebrafish (Danio rerio) thyroid system has become a very popular vertebrate model to screen for thyroid-disrupting chemical pollutants [18, 21-23]. Several studies have suggested that PCP exposure may affect the expression level of genes associated with thyroid hormone metabolism and signaling in rats [14, 15] and the HPT axis in zebrafish larvae ; however, the thyroid hormone endocrine disruption and environmental risk of lower concentration of PCP for adult fish remains unclear. As such, in the present study we have evaluated the effects of lower concentration of PCP exposure on thyroid hormone homeostasis in zebrafish, using plasma thyroid hormone levels and gene expression involved in the HPT axis, to gain an improved understanding of PCP's effect on thyroid hormone function.
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- MATERIALS AND METHODS
Our results demonstrate that PCP significantly alters plasma thyroid hormone levels, as well as the expression level of selected genes associated with thyroid hormone metabolism and signaling in the HPT axis and liver. The concentrations of PCP we used in the present study (0.1 μg/L, 1 μg/L, and 9 μg/L) have been reported to be prevalent in the aquatic environment ; thus, our results suggest that environmental concentrations of PCP might have an adverse effect on fish thyroid systems in an aquatic environment and could affect the development of both adult fish and their offspring.
Although the mortality rate of adult zebrafish was not significantly affected by PCP exposure, a significant decrease in body weight was observed in males exposed to 27 µg/L PCP. These data are in agreement with a previous study, which observed significant effects on growth (total length and body wt) in Japanese medaka (Oryzias latipes) with a 28-d exposure to 200 µg/L PCP . In the present study, the inhibition of growth in males might be partially explained by the significant decrease in plasma T3 levels, as T3 plays an essential role in the regulation of development and growth in fish . In females, gonadal-somatic index was significantly reduced in the highest exposure group (27 µg/L PCP). The low gonadal-somatic index might be related to disruption of the endocrine pathways regulating reproduction, generally as the HPG-axis signaling. Our results indicate that low concentrations of PCP have significant effects on growth of zebrafish and could be endocrine-disrupting chemicals.
We observed an increase in plasma T4 levels after PCP exposure in fish. In contrast, most studies have found reduced T4 levels with treatment at higher doses of PCP in fish , rats [13-15], ewes , and cell lines . Pentachlorophenol has twice the affinity of T4 to thyroid hormone serum binding transthyretin (TTR) , and also affects thyroid hormone metabolism by competitively inhibiting iodothyronine sulfation in vitro . The increases in circulating T4 levels observed in the present study may be attributable to different mechanisms or reflect toxicity of long-term low-dose exposure to PCP. Decreased T3 in males and unaltered T3 in females on exposure to PCP were observed in our study. Although the reason for the sex difference is unclear, a previous study also reported a similar result, in which significant increased TT3 levels and unaltered TT3 levels were observed in PCP-Na–treated female and male rats, respectively . The significant decrease in T3 observed in our study is consistent with previous studies [13, 14], and the observed decrease in T3, at least in part, can be attributed to the thyroid hormone metabolism-disrupting properties of PCP. Indeed, this observation corresponds with the PCP-induced downregulation of dio2 and upregulation of sult1st5 observed in the liver of male fish.
In fish, TSH secretions function as common regulators of the thyroidal axis as feedback mechanisms triggered by changes in the concentration of circulating thyroid hormones . Interestingly, previous studies have reported that changes in tshβ mRNA levels may be related to alterations in T4 levels in fish . In addition, downregulation of tshβ gene transcription has been related to increased levels of T4 in fish after exposure to PBDEs . Consistent with these results, our study revealed that the reduction in tshβ expression could be explained as a negative feedback response to increased levels of T4. Physiological actions of thyroid hormone are usually mediated through interaction with nuclear receptors, and thyroid hormone receptor β (trβ) is one of the main thyroid hormone receptor isoforms. In the present study, trβ gene transcription was significantly downregulated in the male brain and unaltered in female brain. The downregulation of trβ gene expression in the male brain might result from the PCP-induced decline in plasma T3 levels, as the mRNA transcription of trβ was previously reported to be autoinduced in the fish brain by T3 .
The TTR is a key thyroid hormone carrier protein that maintains extra thyroidal stores of thyroid hormone, regulates the supply of thyroid hormone to various target tissues, and plays an important role in the thyroid axis in fish . Previous work has demonstrated that phenol compounds such as PCP, halogenated phenols, and tetrabromobisphenol A have strong affinities for TTR [28, 33, 34]. In the present study, ttr gene expression levels were significantly downregulated in the male liver and were upregulated in the female. Our results confirmed that PCP strongly influences TTR expression, as previously reported [28, 33]. Interestingly, the effect of PCP on ttr expression was different in female and male livers, which might be associated with the different T3 levels observed in male and female fish. In addition to TTR, albumin and thyroxine-binding globulin can also bind to thyroid hormones in fish plasma . As such, further studies are needed to better understand the different response to PCP exposure between the sexes.
Hepatic deiodinases are important regulators of circulating and peripheral thyroid hormone levels in vertebrates. In fish, there are 3 dio genes: dio1, dio2, and dio3. Dio1 has a considerable influence on iodine recovery and thyroid hormone degradation . Dio2 is responsible for the conversion of T4 to T3, allowing adequate availability of local and systemic T3 , and dio3 is a purely inactivating enzyme . Our results demonstrate that dio1 was significantly downregulated in the liver of both females and males, and the expression of dio2 was upregulated in females but downregulated in males. Therefore, we suggest that the downregulation of dio1 is partially responsible for the increased T4 concentrations we observed. Similarly, Yu et al.  demonstrated that exposure to PBDEs downregulated mRNA expression of dio1 and increased plasma T4 concentrations in adult zebrafish . Conversely, the decrease in dio2 expression in males may, at least partly, be associated with the reduced levels of circulating T3, while the upregulation of dio2 expression in females was associated with an increasing trend in T3 levels. The different expression levels of dio2 induced by PCP in male and female fish is consistent with a previous report, which showed that PCP-Na exposure inhibited the dio2 expression of male rats by 79.2% but did not affect the dio2 expression of female rats . In addition, previous studies also provide evidence for sex differences in the magnitude of T3-induced relative mRNA responses for dio3 in liver, as well as dio2 and dio3 in the fish brain . Thus, the cause of the sex differences in dio2 expression may be attributable to the different levels of T3 in females and males. Interestingly, the significant changes in expression of dio1 and dio2 expression may indicate a regulatory role in response to altered thyroid hormone levels and confirm that dio2 is the major contributor to thyroid hormone activation in fish .
Uridinediphosphate glucoronosyltransferases and sulfotransferases (SULTs) play important roles in thyroid hormone homeostasis via the major pathway for T4 conjugation [37, 38]. Uridinediphosphate glucoronosyltransferases play a role in decreasing circulating thyroid hormones, and upregulation of ugt gene expression or enzyme activities have generally been observed in rats and zebrafish exposed to different chemicals [23, 39]. In the present study, increased ugt1ab expression could possibly be explained as an autoregulatory response to increased T4 levels, by increased biliary elimination of the conjugated hormone within the thyroid axis. Sulfonation has been viewed as a key step in thyroid hormone metabolism, and it may increase the hydrophilicity and the biliary excretion of the hormone . Sulfonation reactions are catalyzed by SULTs . In zebrafish, sult1 st5 appears to be the only known enzyme that displays substrate specificity exclusively for thyroid hormones and their metabolites . In the present study, we found that sult1 st5 expression was downregulated in female livers and upregulated in male livers, in response to PCP exposure. These results are consistent with a previous study in which the expression of SULT in female and male rare minnow liver were different after PCP treatment . We suggest that downregulation of sult1 st5 in females may be partially responsible for the increased T4 concentrations and unchanged T3 levels, and that the upregulation of sult1 st5 in the male liver might play a role in reducing the circulating levels of T3.
In summary, our results demonstrate that PCP can affect the thyroid endocrine system at lower concentrations in fish. The thyroid hormone disrupting effects of environmental levels of PCP was probably associated with altered thyroid hormone metabolism, as evidenced by marked alterations in the expression levels of dio1, dio2, sult1 st5, and ugt1ab. Interestingly, sex-specific effects on gene expression and plasma TT3 were observed. The causes of these sex-specific differences are unclear and require further study. Nevertheless, our results suggest that analysis of the HPT axis might be suitable for determining thyroid endocrine disruption after PCP exposure.