Peripheral T3 signaling is the target of pesticides in zebrafish larvae and adult liver.

The intra-tissue levels of thyroid hormones (THs) regulate organ functions. Environmental factors can impair these levels by damaging the thyroid gland and/or peripheral TH metabolism. We investigated the effects of embryonic and/or long-life exposure to low-dose pesticides, ethylene thiourea (ETU), chlorpyrifos (CPF) and both combined on intra-tissue T4/T3 metabolism/signaling in zebrafish at different life stages. Hypothyroidism was evident in exposed larvae that showed reduced number of follicles and induced tshb mRNAs. Despite that, we found an increase in free T4 (fT4) and free T3 (fT3) levels/signaling that was confirmed by transcriptional regulation of TH metabolic enzymes (deiodinases) and T3-regulated mRNAs (cpt1, igfbp1a). Second-generation larvae showed that thyroid and TH signaling was affected even when not directly exposed, suggesting the role of parental exposure. In adult zebrafish, we found that sex-dependent damage of hepatic T3 level/signaling was associated with liver steatosis, which was more pronounced in females, with sex-dependent alteration of transcripts codifying the key enzymes involved in 'de novo lipogenesis' and ß-oxidation. We found impaired activation of liver T3 and PPARα/Foxo3a pathways whose deregulation was already involved in mammalian liver steatosis. The data emphasizes that the intra-tissue imbalance of the T3 level is due to thyroid endocrine disruptors (THDC) and suggests that the effect of a slight modification in T3 signaling might be amplified by its direct regulation or crosstalk with PPARα/Foxo3a pathways. Because T3 levels define the hypothyroid/hyperthyroid status of each organ, our findings might explain the pleiotropic and site-dependent effects of pesticides.

Exposure to pesticide mixtures and DNA damage among rice field workers.

This study describes the use of pesticides mixtures and their potential association with comet assay results in 223 rice field workers in Colombia. Thirty-one pesticides were quantified in blood, serum, and urine (15 organochlorines, 10 organophosphorus, 5 carbamates, and ethylenethiourea), and the comet assay was performed. Twenty-four (77.42%) pesticides were present in the workers. The use of the maximum-likelihood factor analysis identified 8 different mixtures. Afterwards, robust regressions were used to explore associations between the factors identified and the comet assay. Two groups of mixtures--α-benzene hexachloride (α-BHC), hexachlorobenzene (HCB), and ß-BHC (ß: 1.21, 95% confidence interval [CI]: 0.33-2.10) and pirimiphos-methyl, malathion, bromophos-methyl, and bromophos-ethyl (ß: 11.97, 95% CI: 2.34-21.60)--were associated with a higher percentage of DNA damage and comet tail length, respectively. The findings suggest that exposure to pesticides varies greatly among rice field workers.

The expression analysis of Notch-1 and Jagged-2 during the development of the hindgut in rat embryos with ethylenethiourea induced anorectal malformations.

BACKGROUND: This study was designed to investigate the expression of Notch-1 and Jagged-2 in the terminal hindgut in ethylenethiourea (ETU)-exposed rat embryos with anorectal malformations (ARMs) and its potential association with the maldevelopment of the terminal hindgut in ARMs. MATERIAL AND METHODS: ETU-exposed ARMs model was introduced to investigate the expression pattern of Notch-1 and Jagged-2 during the hindgut development using immunohistochemical staining, reverse transcriptase polymerase chain reaction (RT-PCR) and Western blot analysis. RESULTS: Immunostaining revealed that the expression of Notch-1 and Jagged-2 showed changes in the developing terminal hindgut of ARMs. The expression of Notch-1 and Jagged-2 in the terminal hindgut of ARMs rat embryos decreased at both mRNA level and protein level (P < 0.05) compared with normal tissues. CONCLUSION: This study demonstrated that the expression of Notch-1 and Jagged-2 in ARMs of ETU-exposed rat embryos was remarkably reduced, which implied its potential role in the pathogenesis of the terminal hindgut maldevelopment in ARMs.

The development of satellite cells and their niche in striated muscle complex of anorectal malformations rat embryos.

BACKGROUND: It has been demonstrated that different degrees of pelvic floor muscle (PFM) maldevelop in anorectal malformations (ARMs); yet the development of satellite cells, the myogenic stem cells responsible for muscle growth, repair, and maintenance remains elusive during the embryogenesis of PFM. Striated muscle complex (SMC) is one of the most important components of PFM. The objective of this study was to observe the development pattern of satellite cells and their niche of SMC and investigate its possible role in PFM dysplasia in ARMs. METHODS: Immunohistochemistry, cell culture, transmission electron microscopy (TEM), real-time quantitative PCR, and Western blot were performed to trace the dynamic development pattern of satellite cells during the morphogenesis of PFM in ethylenethiourea (ETU)-induced ARMs rat embryos. RESULTS: In ARMs rat embryos, earlier presentation and higher number of Pax7-expressing cell were observed in SMC. The expression of Pax7 and vimentin were up-regulated, while the expression of myogenin, vWF, and neurofilament were down-regulated. Ultrastructure analysis of SMC was characterized by increased amount of nuclear heterochromatin of satellite cell nuclei, thickened basal lamina, widened gap between satellite cell and myofiber, and disarrangement of muscle fibers. The satellite cells demonstrated abnormal differentiation after they were isolated and cultured in vitro. CONCLUSIONS: Our results suggest that premature origination of satellite cell from myogenic progenitor or precursor may result in the depletion of myogenic precursor and cessation of muscle growth; intrinsic defect in satellite cell structure, and extrinsic impairment of microenvironment compromised the myogenic competence of satellite cell, which might contribute substantially to the hypoplastic SMC in ARMs.

Sonic hedgehog, BMP4, and Hox genes in the development of anorectal malformations in Ethylenethiourea-exposed fetal rats.

BACKGROUND: shh signaling pathway has been shown to be involved in the morphogenesis of many organ systems. In this study, we investigated the expression of shh and its targets, BMP4 and Hox genes, in the development of anorectal malformations in Ethylenethiourea (ETU)-exposed embryos. METHODS: We used ETU murine model of the vertebral, anal, cardiac, tracheoesophageal, renal, and limb association. Ethylenethiourea 1% (125 mg/kg) was given to the pregnant females via gavage feeding on gestational day (gD) 10 and saline to control animals. Embryos were collected at gD12 to gD16 and gD21; hindguts were dissected and snap frozen. Highly purified RNA was isolated, and expression of shh, BMP4, Hoxa13, and Hoxd13 genes was confirmed with RT-PCR. Relative quantitative expression of shh and target genes at each time point was done with SYBR Green I qPCR. Normalized gene of interest expression was calculated by geNorm, and data analysis was done with 2-tail Student t test. RESULTS: shh, BMP4, Hoxa13, and Hoxd13 transcripts were detected in all samples, confirming that shh cascade is active during the process of hindgut development in fetal rats. Relative quantitation demonstrated that shh cascade expression shows time-dependent changes in the developing hindgut. CONCLUSION: This study shows that ETU disturbs the expression of shh signaling pathway during the development of hindgut. We provide evidence that shh plays a pivotal role in the hindgut morphogenesis, and its misexpression affect the expression of targets, BMP4 and Hox genes.

Abnormalities of the vertebral column and ribs associated with anorectal malformations.

Lumbosacral vertebral abnormalities are a common association of anorectal malformations (ARMs) and are one of the determinants of the eventual level of fecal continence that can be achieved. This study used a fetal rat model to investigate the spectrum of axial skeletal maldevelopment that may occur with ARMs. Time-mated pregnant rats received 125 mg/kg of 1% ethylenethiourea (ETU) (experimental group) or vehicle only (control). Their fetuses were examined for external malformations and prepared for staining of their skeletons using Alcian blue and Alizarin red S. ARMs developed in 67/68 (98%) of ETU-exposed fetuses, of which 28 (42%) also developed rachischisis, mainly involving the lumbosacral vertebrae. No skeletal abnormality was found in control fetuses. ETU-exposed fetuses with ARMs and rachischisis had abnormal ossification of the vertebral centrum, abnormal fusion between the neural arches of vertebrae, localized narrow or interrupted thoracic vertebral canal, a widely open vertebral canal in the lumbosacral area (rachischisis), and absence of the lower two sacral and coccygeal vertebrae. Rib abnormalities included absence of two to three floating ribs, abnormal fusion of adjacent proximal segments, and abnormal ramification, irregularity, and angulation of their distal segments. The vertebral and rib abnormalities found in ETU-exposed fetuses with ARMs but no rachischisis were much less severe. In addition to the lumbosacral anomalies that are common with ARMs, severe abnormalities of the thoracic vertebrae and their corresponding ribs may occur also. Fetuses with both ARM and rachischisis tend to have more extensive and severe vertebral and rib anomalies. These observations imply a possible common aetiology for ARMs and vertebral anomalies and are consistent with our understanding of the perceived role of the notochord in axial development.

Ethylenebisdithiocarbamates and ethylenethiourea: possible human health hazards.

Humans are exposed to ethylenebisdithiocarbamates (EBDCs) from environmental sources. Exposure to EBDCs is chronic for workers in a variety of industries, where EBDCs are used for their properties as slimicides, vulcanization accelerators, antioxidants, and scavengers in waste-water treatment. EBDCs, and particularly the EBDC metabolite ethylenethiourea, have clearly defined, important toxic effects in various animal species, and there is reason to suspect they are carcinogenic in humans. In the absence of definitive information regarding human risk, further studies need to be done. In the interim, regular surveillance of workers with high levels of exposure to EBDCs, with specific attention to markers of thyroid and hepatic pathology, should be considered.

Effects of thyroid antagonists on rat embryos cultured in vitro.

A literature review of individual pregnancies and recent surveys involving large cohorts reveal an association between congenital malformation and maternal hyperthyroidism, suggesting that some aspect of hyperthyroidism or its treatment might compromise the development of the fetus. Experiments have shown that the thyroid antagonist, ethylenethiourea (ETU), causes fetal malformations when administered to pregnant rats, but it is not known whether it is ETU or the imbalance in maternal thyroid hormone which it causes which is the teratogenic agent. Here we employ in vitro culture to determine the possible direct effects on rat embryos of two thyroid antagonists, ETU and methimazole (MMI), the latter being one which is used for treatment of thyrotoxicosis in humans. It was found that ETU can compromise the development of rat embryos in vitro, confirming that ETU has a direct effect on the rat embryo. It was also found that MMI can cause abnormal development of rat embryos in vitro, although the concentration at which MMI disturbs rat embryogenesis is higher than that which is reached in hyperthyroid patients treated with clinical doses of MMI or carbimazole.

Environmental and biological monitoring of exposure to ethylenebisdithiocarbamate fungicides and ethylenethiourea.

Exposure of workers to ethylenebisdithiocarbamates (EBDCs; maneb or mancozeb) in 29 potato farms was evaluated during the control of potato late blight. Concentrations of EBDCs and ethylenethiourea (ETU), an impurity and degradation product in EBDC formulations, in ambient air were evaluated during pesticide application. Biological monitoring of exposure to EBDCs was carried out by measuring the concentrations of ETU, a metabolite of EBDCs, in urine for 22 days after the end of the exposure. The estimated inhaled doses of ETU and EBDCs during the average four hour application period were 0.07 and 1.8 micrograms/kg, respectively. Only 1-10% of ETU on the clothes reached the skin. The creatinine corrected concentrations of ETU in urine were 0.1-2.5 micrograms/mmol creatinine 24 hours after exposure ended. The estimated half life for eliminating ETU through the kidneys was close to 100 hours. These results indicate that the measurement of ETU in urine is suitable for biological monitoring of exposure to EBDCs.

Ethylene thiourea: thyroid function in two groups of exposed workers.

Ethylene thiourea is manufactured at one factory in the United Kingdom and is mixed into masterbatch rubber at another. Clinical examinations and thyroid function tests were carried out over a period of three years on eight process workers and five mixers and on matched controls. The results show that the exposed mixers, but not exposed process workers, have significantly lower levels of total thyroxine (T4) than the controls. One mixer had an appreciably raised level of thyroid stimulation hormone (TSH).

Allergic contact dermatitis from ethylene thiourea.

A case of allergic contact dermatitis to the rubber additive material ethylene thiourea (ETU) is presented. Testing for cross-sensitivity gave a positive reaction to the fungicide Maneb, manganous ethylenebisdithiocarbamate, but this reaction was probably due to ETU which was detected in Maneb by thin-layer chromatography. Different sources of ETU are reported.

Teratogenic effects of combined administration of ethylenethiourea and nitrite in mice.

Teratogenic potential of ethylenethiourea (ETU) was investigated in SLC-ICR mice after its reaction with sodium nitrite. ETU was given orally in doses of 400 mg/kg on various days of pregnancy in combination with 200 mg/kg NaNO2 at varied intervals. When NaNO2 was given to females immediately after their treatment with ETU on day 6 or 8 of pregnancy, fetal survival was significantly decreased. Various types of malformations were observed in the living fetuses from mothers treated on day 6, 8, or 10 of pregnancy, but not on day 12. The teratogenicity disappeared when NaNO2 was given 2 h after the treatment with ETU.