Involvement of proliferative and apoptotic factors in the development of hindgut in rat fetuses with ethylenethiourea-induced anorectal malformations.

BACKGROUND: Anorectal malformations (ARMs) are common congenital malformations of the terminal digestive tract, but little is known regarding their pathogenesis. Aberrant cell proliferation/apoptosis are believed to be involved in ARMs. However, there are no studies on proliferation/apoptosis-related genes. PURPOSE: We aimed to investigate the spatiotemporal expression patterns of two proliferation/apoptosis-related genes (MYC proto-oncogene and tumor protein p53) and explore their potential functions in the hindguts of ethylene thiourea-induced ARMs rat fetuses. METHODS: MYC and p53 expression was evaluated using immunohistochemical staining, western blotting, and quantitative real-time polymerase chain reaction (RT-qPCR). Terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) and p53 costaining were performed to assay the colocalization of apoptotic and p53-expressing cells. RESULTS: Rat fetuses with ARMs displayed fusion failure of the urogenital septum and cloacal membrane. In the control group, MYC was persistently expressed from gestational day (GD)14 to GD16 and distributed throughout the hindgut, while p53 was weakly detected in the terminal segment of the urethra and hindgut; in the ARMs group, MYC expression was obviously reduced, while p53 was widely and highly expressed in the urethra and hindgut. Western blotting and RT-qPCR confirmed the decrease in MYC and increase in p53 expression in ARMs. TUNEL and p53 co-staining revealed considerable overlap between apoptotic and p53-expressing cells. CONCLUSION: The expression patterns of c-myc and p53 were disrupted in ARMs rat embryos, and the downregulation of c-myc and upregulation of p53 might be related to the development of ARMs at the key time points of ARMs morphogenesis.

The Expression of Shh, Ptch1, and Gli1 in the Developing Caudal Spinal Cord of Fetal Rats With Anorectal Malformations.

BACKGROUND: Postoperative incontinence and constipation still remain the major complications of anorectal malformations (ARMs), despite improvements in their treatment. One of the most important factors that affect postoperative anorectal function is malformations in the lumbosacral spinal cord. However, far too little attention has been paid to the underlying mechanism that produces these malformations. MATERIALS AND METHODS: The levels of sonic hedgehog (Shh), patched homolog 1 (Ptch1), and zinc finger-containing transcription factors 1 (Gli1) expression were investigated in the lumbosacral spinal cord in ethylenethiourea-exposed rat fetus with ARMs, and Shh, Ptch1, and Gli1 expression was confirmed with immunohistochemical staining, quantitative real-time polymerase chain reaction, and western blot analyses during lumbosacral spinal cord development both in the ARMs and normal rat embryos. RESULTS: Our results have shown that Shh, Ptch1, and Gli1 expression in the lumbosacral spinal cord of rat embryos with ARMs was decreased at both the messenger RNA and protein levels, when compared with their expression levels in normal tissues (P < 0.05). CONCLUSIONS: This study demonstrated that the expression of Shh, Ptch1, and Gli1 in lumbosacral spinal cord was remarkably reduced during late developmental stages in fetal rats with ARMs. These findings offered some important insights into the involvement of the Shh-Ptch1-Gli1 signaling pathway in the pathogenesis of lumbosacral spinal cord maldevelopment in rat fetus with ARMs, which leads to complications after procedures for ARMs.

MSX2 and BCL2 expressions in the development of anorectal malformations in ethylenethiourea-induced rat embryos.

BACKGROUND: This study aimed to determine Msh homeobox 2 (MSX2) and B cell lymphoma-2 (BCL2) expression patterns during anorectal development in anorectal malformations (ARM) and normal rat embryos, with the goals of determining the role of MSX2 and BCL2 in ARM pathogenesis. METHODS: ARM was induced in rat embryos with ethylenethiourea administered to dams on gestational day 10 (GD10). Embryos were harvested by cesarean deliveries from GD14 to GD16. MSX2 and BCL2 expression was evaluated via immunohistochemical staining, immunofluorescence, western blotting and quantitative real-time polymerase chain reaction (qRT-PCR). RESULTS: Immunohistochemical staining of ARM embryos revealed that MSX2 was mainly expressed in the epithelium of the hindgut and urorectal septum (URS) on GD14. On GD15 and GD16, MSX2-immunolabeled cells were noted in the epithelium of the rectum, fistula and URS. However, in normal embryos, faint immunopositivity for MSX2 was demonstrated in the epithelium of the rectum and URS from GD14 to GD16. As for BCL2, in normal embryos, BCL2-immunopositive cells were extensively expressed in the epithelium of the hindgut and URS on GD14 and GD15. In ARM embryos, weak immunopositivity for BCL2 was detected in the epithelium of hindgut and URS on GD14 and GD15. Immunofluorescence revealed that MSX2 and BCL2 colocalized in the hindgut. In ARM embryos, we observed more MSX2-positive than BCL2-positive cells on GD14; the normal embryos had the opposite pattern. Analyses by western blot and qRT-PCR showed that MSX2 protein and mRNA expression was significantly increased in ARM embryos compared with the normal embryos on GD15 and GD16 (p < 0.05). However, BCL2 protein and mRNA expression was significantly decreased in ARM embryos compared with the normal embryos on GD14 (p < 0.05). The MSX2/BCL2 ratio of protein and mRNA expression level in the ARM group was the highest on GD15. CONCLUSION: These results indicate that upregulation of MSX2 and downregulation of BCL2 during cloacal development into the rectum and urethra might be related to the ARM development, and MSX2 promoted apoptosis through reduction of BCL2 expression during the development of anorectal development in ARM.

Pesticide toxicogenomics across scales: in vitro transcriptome predicts mechanisms and outcomes of exposure in vivo.

In vitro Omics analysis (i.e. transcriptome) is suggested to predict in vivo toxicity and adverse effects in humans, although the causal link between high-throughput data and effects in vivo is not easily established. Indeed, the chemical-organism interaction can involve processes, such as adaptation, not established in cell cultures. Starting from this consideration we investigate the transcriptomic response of immortalized thyrocytes to ethylenthiourea and chlorpyrifos. In vitro data revealed specific and common genes/mechanisms of toxicity, controlling the proliferation/survival of the thyrocytes and unrelated hematopoietic cell lineages. These results were phenotypically confirmed in vivo by the reduction of circulating T4 hormone and the development of pancytopenia after long exposure. Our data imply that in vitro toxicogenomics is a powerful tool in predicting adverse effects in vivo, experimentally confirming the vision described as Tox21c (Toxicity Testing in the 21st century) although not fully recapitulating the biocomplexity of a living animal.

Determinación de etilentiourea en acelgas tratadas con Zineb: Efecto del calor húmedo por ebullición / Determination of ethylenethiourea in chard treated with Zineb: Effect of wet heat by boiling

El Zineb es un plaguicida perteneciente a la familia de los etilenbisditiocarbamatos (EBDC) ampliamente utilizado en la provincia de Jujuy (Argentina). La toxicidad de este fungicida está dada por la etilentiourea (ETU) que es el principal producto de su degradación. La ETU tiene efectos mutagénicos, teratogénicos y cancerígenos en animales. La EPA la clasifica como probable cancerígeno humano. El objetivo del trabajo fue determinar la transformación del Zineb a ETU en acelgas cultivadas en Jujuy, por efecto del calor húmedo por tratamiento térmico. El método se basó en la extracción de la ETU con agua y su posterior reextracción con diclorometano a pH alcalino. El extracto se purificó por cromatografía en columna de alúmina y la ETU se cuantificó por HPLC con un detector UV-Visible a 232 nm. Para cumplir con el objetivo planteado se realizó un cultivo experimental de acelgas a las que se aplicó el fungicida Zineb, el cual luego se cuantificó a diferentes periodos de tiempo. La ETU se cuantificó con y sin aplicación de calor húmedo. La concentración de ETU en las acelgas sometidas a tratamiento térmico fue de 8,07 mg/kg y transcurridos 30 días disminuyó a 0,05 mg/kg, lo que representa una disminución de más del 99 % respecto a la concentración inicial. Simultáneamente, se determinó la concentración de Zineb en las acelgas y se obtuvo, inicialmente, 44 mg/kg y luego de 36 días la concentración de Zineb disminuyó a 3,83 mg/kg, lo que representa una disminución del 91 % respecto a la concentración del plaguicida al inicio. La detección de ETU en acelgas sometidas a tratamientos térmicos confirma la transformación del fungicida Zineb a un producto de degradación clasificado según el IARC en el grupo 3. Esto pone en discusión la reglamentación Argentina vigente, por cuanto al finalizar los tiempos de carencia no se evalúan la presencia de metabolitos o productos de degradación potencialmente tóxicos.

Zineb is a pesticide belonging to the family of ethylenebisdithiocarbamates (EBDC) widely used in the province of Jujuy, Argentina. The toxicity of this fungicide is given by the ethylenethiourea (ETU) which is the main product of degradation. The ETU has mutagenic, teratogenic and carcinogenic effects in animals. The EPA classifies it as a probable human carcinogen. The aim of the study was to determine the transformation of Zineb to ETU in cultivated chards in Jujuy, by treatment with humid heat. The method is based on extraction of ETU with water and subsequent extraction of the aqueous phase with dichloromethane. The extract was cleaned by alumina column chromatography and the ETU was quantified by HPLC with a UV-Visible detector at 232 nm. An experimental cultivation of chards was carried out and the Zineb fungicide was applied and then quantified at different time periods. The ETU was quantified with and without application of moist heat. The concentration of ETU in heat treated chards was 8.07 mg/kg and after 30 days decreased to 0.05 mg/kg, representing a decrease of more than 99 % over the initial concentration. Simultaneously, Zineb concentration in chard was initially determined as 44 mg/kg and, 36 days later, the Zineb concentration decreased to 3.83 mg/kg, representing a 91% decrease with respect to the concentration of the pesticide at the beginning. Detection of ETU in chards subjected to heat treatment confirms the transformation of the fungicide Zineb to a degradation product classified by the IARC as group 3. This puts into discussion the current Argentine regulations since, at the end of the deficiency times the presence of potentially toxic metabolites or degradation products is not evaluated.

Hepatic damage in newborns from female rats exposed to the pesticide derivative ethylenethiourea / Lesões hepáticas em recém-nascidos de ratas expostas ao derivado de agrotóxico etilenotioureia

PURPOSE: To evaluate hepatic morphological-histological abnormalities in newborns from female rats exposed to ethylenethiourea. METHODS: A randomized study was conducted on fifty-five newborn Wistar rats were studied: 34 in the experimental group, whose mothers had been exposed to 1% ethylenethiourea; and 21 in the control group, whose mothers had received 0.9% physiological solution. The solution was administered via gavage on the 11th day of gestation. Cesarean section was performed on the 20th day of gestation. The newborns' livers were examined and any morphological-histological abnormalities were registered. The presence of megakaryocytes was quantified in 50 microscope fields, as the total number of these cells per mm². RESULTS: The entire experimental group presented abnormalities of embryonic formation, with musculoskeletal anomalies, digestive system anomalies, hepatic congestion and friability, hydrops and delayed intrauterine growth. The histopathological analysis showed that morphological-histological hepatic destructuring had occurred in all entire experimental with removal of the hepatic trabeculae and severe hepatic megakaryocytosis. The mean megakaryocyte density ranged from 107.9 to 114.2 per mm², and it was eight times greater than in the control group, thus characterizing a situation of extramedullary hematopoiesis. CONCLUSION: The fetal exposure to ethylenethiourea caused hepatic damage characterized by severe extramedullary hematopoiesis.

OBJETIVO: Avaliar alterações hepáticas morfohistológicas em recém-nascidos de ratas prenhes expostas à etilenotioureia. MÉTODOS: Realizado ensaio randomizado em animais de experimentação, onde foram estudados 55 recém-nascidos de ratas Wistar, 34 do Grupo Experimento, expostas a etilenotioureia 1% e 21 do Grupo Controle, em que a rata prenhe recebeu solução fisiológica 0,9%, ambos por gavagem no 11º dia de gestação. Realizada no 20º dia de gestação cesariana, analisados os fígados dos recém-nascidos e registradas as alterações morfohistológicas. Realizou-se a quantificação dos megacariócitos em 50 campos microscópicos, avaliando a quantidade total destas células por mm². RESULTADOS: Todos os recém-nascidos do Grupo Experimento apresentaram alterações na formação embrionária, com anomalias musculoesqueléticas, anormalidades do sistema digestório, congestão e friabilidade hepática, hidropisia e crescimento intrauterino retardado. A análise histopatológica mostrou desestruturação hepática morfohistológica em todos os recém-nascidos expostos à etilenotioureia, com destrabeculação dos hepatócitos e intensa megacariocitose hepática, apresentando média da densidade de megacariócitos de 107,9 até 114,2 por mm² sendo cerca de oito vezes maior que no Grupo Controle, caracterizando hematopoese extramedular. CONCLUSÃO: A exposição fetal a etilenotioureia provocou danos hepáticos caracterizados pela intensa hematopoese extramedular.

Abnormalities of digestive tract innervation in rat fetus treated with ethylenethiourea / Anomalias da inervação do trato digestório de fetos de ratas expostas à etilenotioureia

PURPOSE: The pathophysiology of abnormalities associated with myenteric plexus lesions remains imperfectly understood. Such abnormalities have been correlated with subocclusive intestinal conditions in children with Hirschsprung's disease, cases of chronic constipation and, postoperatively, in cases of anorectal anomalies. This study evaluated abnormalities of the myenteric plexus in fetus from female rats that received ethylenethiourea. METHODS: Female rats were exposed to ethylenethiourea on the 11th day of pregnancy (experimental group) or to 0.9 percent physiological solution (control group). Abnormalities were only found in the experimental group. The digestive tract muscle layer was analyzed morphometrically and changes to the frequencies of nerve plexus cells and interstitial cells of Cajal were evaluated, using hematoxylin-eosin, S-100 protein, neuron-specific enolase and C-Kit, respectively. RESULTS: Muscle and skeletal abnormalities were observed in 100 percent, anorectal anomalies in 86 percent, absent tail in 71 percent, short tail in 29 percent, duodenal atresia in 5 percent, esophageal atresia in 5 percent and persistent omphalomesenteric duct in 5 percent. Histopathological analysis showed a thinner muscle layer associated with lower frequencies of ganglion cells and interstitial cells of Cajal, in all gastrointestinal tract. CONCLUSION: Severe nerve plexus abnormalities associated with muscle layer atrophy were observed throughout the gastrointestinal tract in newborn rats exposed to ethylenethiourea.

OBJETIVO: As anomalias associadas a lesões dos plexos mioentéricos permanecem sem plena compreensão da sua fisiopatologia. Alterações nos plexos nervosos têm sido correlacionadas com quadros suboclusivos intestinais em crianças portadoras de doença de Hirschsprung, em constipação crônica e no pós-operatório de anomalias anorretais. Este estudo avaliou as anomalias do plexo mioentérico em fetos de ratos fêmea que ingeriram etilenotioureia (ETU). MÉTODOS: Ratos fêmea foram expostos no 11º dia de gestação a ETU 1 por cento no Grupo Experimento e a solução fisiológica 0,9 por cento no Grupo Controle. Foram observadas anomalias apenas no Grupo experimento, sendo realizada morfometria da camada muscular e avaliadas alterações da frequência celular nos gânglios do plexo mioentérico e nas células intersticiais de Cajal (CIC) utilizando hematoxilina-eosina, P S-100, Enolase Neurônio Específica e C-KIT. RESULTADOS: Foram observadas anomalias musculoesqueléticas (100 por cento), anorretais (86 por cento), ausência de cauda (71 por cento), cauda curta (29 por cento), atresia duodenal (5 por cento), atresia esofágica (5 por cento) e conduto onfalomesentérico persistente (5 por cento). A análise histopatológica mostrou adelgaçamento da camada muscular associada às alterações da frequência das células ganglionares e das CIC em todos os segmentos do trato gastrointestinal. CONCLUSÃO: Foram observadas alterações graves nos plexos nervosos associadas ao adelgaçamento da camada muscular de todo o trato gastrointestinal nos fetos expostos a ETU.

The effect of vitamin A deficiency during pregnancy on anorectal malformations.

OBJECTIVE: The aim of this study was to study the effect of vitamin A deficiency (VAD) on the embryological development of anorectal malformations (ARMs) and the enteric nervous system. MATERIALS AND METHODS: Female Sprague-Dawley rats were divided into 3 groups: VAD group, normal group (negative control), and ethylene thiourea (ETU) group (positive control) with a normal diet. On day 20 of pregnancy, cesarean section was performed on all rats. The incidence of ARMs in the fetal rats and Protein gene product 9.5 (PGP9.5) and S-100 protein expression by immunohistochemistry were determined. RESULTS: The incidence of ARMs in VAD and ETU groups was 64.8% (59/91) and 45.9% (61/133), respectively (P > .05). Anorectal malformations were not found in the normal group. Protein gene product 9.5 and S-100 protein expression in the non-ARM rectums of the VAD group was lower than the ETU (P = .0156 vs P = .0105) and normal groups (P = .0091 vs P = .0024). There was no significant difference in PGP9.5 and S-100 protein expression between ETU and normal groups. In the ARM rectums, PGP9.5 and S-100 protein expression in the VAD group was lower than the ETU group (P < .0001). Protein gene product 9.5 and S-100 protein expression was also lower in ARM than non-ARM rectums in the VAD and ETU groups (P < .0001, P = .0203, and P = .0122, respectively). CONCLUSION: Vitamin A deficiency during pregnancy may result in the embryological development of ARMs. Enteric nervous system development may be related to ARMs.

Avaliação de resíduos de etilenotiouréia (ETU) em frutas comercializadas na cidade de São Paulo / Evaluation of ethylene thiourea (ETU) in fruits traded in the city of São Paulo

Etilenotiouréia (ETU) é uma substância tóxica, formada pela degradação e/ou biotransformação dos fungicidas etilenobisditiocarbamatos (EBDC). Seus resíduos podem ser encontrados em plantas e no ambiente, após o uso de EBDC na agricultura, ou em animais no ser humano, quando expostos a esses produtos. Comprovadamente, a ETU tem a capacidade de induzir tumor na tireóide de roedores e no fígado de camundongo, apresentando evidência suficiente para carcinogenicidade em animais e evidência inadequada para carcinogenicidade em seres humanos. Os objetivos destes estudos foram: validar método analítico para determinação de resíduos de ETU nas matrizes estudadas; verificar a presença de resíduos de ETU em amostras de frutas (mamão, maça e morango), coletadas em diferentes pontos de comercialização da cidade de São Paulo; avaliar os resultados e a contribuição de risco à saúde da população consumidora. Foram analisadas 90 amostras, sendo 30 de cada fruta (maça, mamão e morango), adquiridas em diferentes pontos de comercialização da cidade São Paulo, no período de dezembro de 2005 a dezembro de 2006, distribuídas nas diferentes regiões do referido município e durante as estações do ano. Os parâmetros de validação avaliados foram: seletividade, linearidade, limite de quantificação, limite de detecção, exatidão e precisão. Os estudos de recuperação foram realizados com fortificações em três níveis (1,2 e 10 LQ) em amostras controle. A determinação de resíduos de ETU foi feita por cromatografia a líquido de alto desempenho, com detector de absorção no ultravioleta (HPLC-UV) e cromatografia a líquido acoplada à espectrometria de massas em "tandem" (LC/MS/MS). Como todos os resultados obtidos por HPLC-UV apresentaram níveis de ETU abaixo do LQ limite de quantificação (10,0 micro grama/Kg), as quantificações foram realizadas no LC/MS/MS, com limite de quantificação e de detecção respectivamente de 1,0 micro grama/Kg e 0,5 micro grama/Kg. O método por LC/MS/MS mostrou-se adeq...

DNA fragmentation and DNA repair synthesis induced in rat and human thyroid cells by chemicals carcinogenic to the rat thyroid.

Five chemicals that are known to induce in rats thyroid follicular-cell adenomas and carcinomas were assayed for their ability to induce DNA damage and DNA repair synthesis in primary cultures of human thyroid cells. Significant dose-dependent increases in the frequency of DNA single-strand breaks and alkali-labile sites, as measured by the same Comet assay, were obtained after a 20-h exposure to the following subtoxic concentrations of the five test compounds: methimazole from 2.5 to 10mM; nitrobenzene, potassium bromate, N,N'-diethylthiourea and ethylenethiourea from 1.25 to 5mM. Under the same experimental conditions, DNA repair synthesis, as evaluated by quantitative autoradiography, was present in potassium bromate-exposed thyroid cells from all the three donors and in those from two of three donors with either nitrobenzene or ethylenethiourea, but did not match the criteria for a positive response in thyroid cells from any of the donors with methimazole and N,N'-diethylthiourea. Consistently with their ability to induce thyroid tumors, all the five test compounds, administered p.o. in rats in a single dose corresponding to 1/2 LD50, induced a statistically significant degree of DNA fragmentation in the thyroid. These findings suggest that the five test compounds might be carcinogenic to thyroid in humans.

Evaluation of histological and molecular endpoints for enhanced detection of thyroid system disruption in Xenopus laevis tadpoles.

Amphibian metamorphosis represents a promising model for the identification of thyroid system-disrupting chemicals due to the pivotal role played by thyroid hormones for the initiation and regulation of metamorphosis. An important aspect of bioassay development is the identification and evaluation of sensitive and diagnostic endpoints. In this study, several morphological, histological, and molecular endpoints were evaluated for their utility to detect alterations in thyroid system function after exposure of stage 51 Xenopus laevis tadpoles to various concentrations (1.0, 2.5, 10, 25, and 50 mg/l) of the anti-thyroidal compound ethylenethiourea (ETU). Analysis of developmental stages on exposure day 20 and monitoring of time to fore limb emergence (FLE) revealed retardation and complete arrest of tadpole development at 25 mg/l and 50 mg/l ETU, respectively. Development was not affected by 1.0, 2.5, and 10 mg/l ETU. Histological alterations in the thyroid gland were observed in FLE-displaying tadpoles after exposure to 2.5, 10, and 25 mg/l ETU, as well as in developmentally arrested tadpoles exposed to 50 mg/l ETU. Prevalence and severity of histological changes increased in a concentration-dependent manner. Semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) showed increased mRNA expression of the alpha- and beta-subunits of thyroid-stimulating hormone (TSHalpha, TSHbeta) in pituitary tissue of tadpoles exposed to 25 and 50 mg/l ETU. Results demonstrate the successful detection of anti-thyroidal effects of ETU in Xenopus laevis tadpoles using various endpoints and highlight the particular sensitivity of thyroid gland histology to detect thyroid system disruption in tadpoles.

Molecular mechanisms underlying mancozeb-induced inhibition of TNF-alpha production.

Mancozeb, a polymeric complex of manganese ethylenebisdithiocarbamate with zinc salt, is widely used in agriculture as fungicide. Literature data indicate that ethylenebisdithiocarbamates (EBDTCs) may have immunomodulatory effects in humans. We have recently found in agricultural workers occupationally exposed to the fungicide mancozeb a statistically significant decrease in lipopolysaccharide (LPS)-induced tumor necrosis factor-alpha (TNF) production in leukocytes. TNF is an essential proinflammatory cytokine whose production is normally stimulated during an infection. The purpose of this work was to establish an in vitro model reflecting in vivo data and to characterize the molecular mechanism of action of mancozeb. The human promyelocytic cell line THP-1 was used as in vitro model to study the effects of mancozeb and its main metabolite ethylenthiourea (ETU) on LPS-induced TNF release. Mancozeb, but not ETU, at non-cytotoxic concentrations (1-100 microg/ml), induced a dose- and time-dependent inhibition of LPS-induced TNF release, reflecting in vivo data. The modulatory effect observed was not limited to mancozeb but also other EBDTCs, namely zineb and ziram, showed similar inhibitory effects. Mancozeb must be added before or simultaneously to LPS in order to observe the effect, indicating that it acts on early events triggered by LPS. It is known that nuclear factor-kappaB (NF-kappaB) tightly regulates TNF transcription. We could demonstrate that mancozeb, modulating LPS-induced reactive oxygen species generation, prevented IkappaB degradation and NF-kappaB nuclear translocation, which in turn resulted in decreased TNF production. To further understand the mechanism of the effect of mancozeb on TNF transcription, THP-1 cells were transfected with NF-kappaB promoter-luciferase construct, and the effect of mancozeb on luciferase activity was measured. Cells transfected with promoter constructs containing kappaB site showed decreased LPS-induced luciferase activity relative to control after mancozeb treatment, confirming NF-kappaB binding as an intracellular target of mancozeb. Overall, this study contributes to our understanding of the mechanism underlying mancozeb-induced immunotoxicity.

Studies on the goitrogenic mechanism of action of N,N,N',N'-tetramethylthiourea.

N,N,N',N'-Tetramethylthiourea (TMTU) is a rat goitrogen inducing thyroid hyperplasia, hypertrophy, and tumor formation. Little is known about the exact underlying mechanism of action. As thyroid peroxidase (TPO) and type I iodothyronine deiodinase (ID-I) have been established as targets of goitrogenic thiourea derivatives, we investigated interactions of TMTU with target enzymes using a partially purified fraction from hog thyroids or solubilized hog thyroid microsomes and 10,000g supernatant from rat liver homogenate, respectively, as enzyme sources. For comparison, comprehensively characterized goitrogenic thiourea derivatives were studied as well. In contrast to propylthiouracil (PTU), and like ethylenethiourea (ETU), TMTU only marginally affected TPO-catalyzed oxidation of guaiacol. TMTU, like ETU, concentration-dependently suppressed TPO-catalyzed iodine formation with concomitant oxidative metabolism. Suppression ceased upon consumption of thiourea derivatives, the rate of the reappearing iodine formation was similar to that of controls. TMTU, like ETU, also suppressed non-enzymatic and TPO-catalyzed monoiodination of l-tyrosine with a stoichiometry of 2:1, i.e., one molecule of thiourea derivative suppressed two times monoiodination. TMTU and ETU were unable to irreversibly inhibit TPO. In contrast to PTU, TMTU did not inhibit ID-I. These findings provide evidence that TMTU interferes with thyroid hormone synthesis at the level of iodination and demonstrate a metabolic route for the oxidative detoxification of TMTU in the thyroid suggesting that low-level or intermittent exposure to TMTU would have only minimal effects on thyroid hormone synthesis. Finally, it can be concluded that meaningful toxicological studies on TPO inhibition can be performed without a need for highly purified TPO.

[Using the FRTL-5 cell to screen the thyroxine disrupting effects of the two pesticides-ethylenethiourea and pendimethalin].

OBJECTIVE: In order to study the effects of ethylenethiourea(ETU) and pendimethalin on synthesis and secretion of thyroglobulin(Tg) in FRTL-5 cells in vitro. METHODS: MTT test was used to investigate the cytotoxicity of the two chemicals to FRTL-5 cell. When the concentration of pendimethalin exceeded 5 ng/microliter, and the concentration of ethylenethiourea exceeded 360 ng/microliter, there was distinct cytotoxicity to FRTL-5 cell. The radioimmunoassay was also used to detect the level of thyroglobulin in culture. The exposure doses in radioimmunoassay was based on the MTT test. RESULTS: The concentration of Tg was decreased in medium and high dose of pendimethalin and ethylenethiourea. CONCLUSION: It is likely, therefore, that the two pesticides might disrupt FRTL-5 cell of synthesizing and secreting Tg.

Human flavin-containing monooxygenase form 2 S-oxygenation: sulfenic acid formation from thioureas and oxidation of glutathione.

Thioureas are oxygenated by flavin-containing monooxygenases (FMOs), forming reactive sulfenic and/or sulfinic acids. Sulfenic acids can reversibly react with GSH and drive oxidative stress through a redox cycle. For this reason, thiourea S-oxygenation is an example of FMO-dependent bioactivation of a xenobiotic. Functional FMO2 is expressed in the lung of 26% of individuals of African descent and 5% of Hispanics but not in Caucasians or Asians. We have previously demonstrated that human FMO2.1 protein expressed in Sf9 microsomes has high activity toward a series of thioureas that are known or suspected lung toxicants including thiourea, 1-phenylthiourea, and ethylenethiourea. We now show by HPLC and LC-MS that 1-phenylthiourea and alpha-naphthylthiourea are converted to their sulfenic acids. GSH in the incubations at concentrations of 0.5-1.0 mM completely eliminated the sulfenic acid with resultant production of GSSG. These results indicate that individuals with the FMO21 allele may be at enhanced risk of pulmonary damage upon exposure to thioureas.

Normal and abnormal embryonic development of the anorectum in rats.

BACKGROUND: The embryologic and pathologic aspect of anorectal malformation (ARM) remains poorly understood. There is no universally accepted theory to explain anorectal embryology and the abnormal development that produces ARM. The aim of this study was to observe the developmental processes of anorectum in rats and to explore the abnormal embryonic development that leads to ARM. METHODS: Rat embryos with ARM were obtained by treating pregnant rats with administration of ethylenethiourea (ETU). Normal rat embryos and embryos with ARM from gestational days 12.5 to 20 were sectioned serially and sagittally and stained with H & E. The relevant structure including cloaca and urorectal septum (URS) were examined in a temporospatial sequence. RESULTS: Characteristics of anorectum development in ARM rat embryos treated by ETU were as follows: (1) URS never fused with cloaca membrane. (2) Dorsal cloacal membrane was maldeveloped. (3) Cloacal configuration was abnormal. (4) Tail groove never appeared. All type of ARM was the rectourethal fistula and common cloaca in ETU-treated rat embryos and was discernible on gestation day 16. CONCLUSIONS: Absence of the tail groove and maldevelopment of the dorsal cloacal membrane, cloacal configuration, and urorectal septum are likely to be responsible for the formation of ARM. Failure of fusion of the URS with cloacal membrane is the immediate reason for rectourethral fistula or common cloaca in ETU-treated rats.

Initiating and promoting effects of concurrent oral administration of ethylenethiourea and sodium nitrite on uterine endometrial adenocarcinoma development in Donryu rats.

The effects of oral administration of ethylenethiourea (ETU) and sodium nitrite on endometrial adenocarcinoma development was investigated in female Donryu rats. At 10 week of age, groups 1 and 3 received a single dose of polyethylene glycol (PEG) into the uterine cavity, while groups 2 and 4 were given N-ethyl-N-nitrosourea (ENU) (15 mg/kg) solution, dissolved in PEG, in the same manner. ETU (80 mg/kg) and sodium nitrite (56 mg/kg) dissolved in distilled water were orally given to animals in groups 3 and 4 once a week from 11 to 51 weeks of age. Groups 1 and 2 received the vehicle alone. At termination (52 weeks of age), endometrial adenocarcinomas were observed in 29, 13 and 57% of rats in groups 2, 3 and 4, respectively, but not in group 1. Persistent estrus appeared earlier in groups 3 and 4 than in group 1. In groups 3 and 4, hyperplastic and neoplastic lesions of the digestive tract, especially the forestomach, were also observed. The results indicate that N-nitroso ETU formed in the stomach by concurrent oral administration of ETU and sodium nitrite, itself induces endometrial adenocarcinomas by its mutagenic action, as well as promoting their development after ENU-initiation, presumably by influencing the hormonal balance.