Multi Species Analyses Reveal Testicular T3 Metabolism and Signalling as a Target of Environmental Pesticides

Thyroid hormones (THs) regulate many biological processes in vertebrates, including reproduction. Testicular somatic and germ cells are equipped with the arrays of enzymes (deiodinases), transporters, and receptors necessary to locally maintain the optimal level of THs and their signalling, needed for their functions and spermatogenesis. Pesticides, as chlorpyrifos (CPF) and ethylene thiourea (ETU), impair the function of thyroid and testis, affecting male fertility. However, their ability to disarrange testicular T3 (t-T3) metabolism and signalling is poorly considered. Here, a multi-species analysis involving zebrafish and mouse suggests the damage of t-T3 metabolism and signalling as a mechanism of gonadic toxicity of low-doses CPF and ETU. Indeed, the developmental exposure to both compounds reduces Dio2 transcript in both models, as well as in ex-vivo cultures of murine seminiferous tubules, and it is linked to alteration of steroidogenesis and germ cell differentiation. A major impact on spermatogonia was confirmed molecularly by the expression of their markers and morphologically evidenced in zebrafish. The results reveal that in the adopted models, exposure to both pesticides alters the t-T3 metabolism and signalling, affecting the reproductive capability. Our data, together with previous reports suggest zebrafish as an evaluable model in assessing the action of compounds impairing locally T3 signalling.

Influence of different curing regimes on properties of new copolymer of chloroprene and acrylonitrile

The curing system plays a vital role in designing rubber compounds for various industrial applications. Therefore, it is paramount to establish viable curing strategies for any new elastomer to explore its application potentials and commercial significance. Impacts of different curing regimes on the properties of a recently developed copolymer of chloroprene and acrylonitrile (acrylonitrile-chloroprene rubber, NCR) are reported here. Several primary accelerators (four from thiourea- and one from thiazolene product families) were used for curing the new rubber along with fixed loadings of zinc oxide (5 phr) and magnesium oxide (4 phr). Besides, curatives based on sulfur and peroxide were also evaluated. The influence of different curing systems on the rheological and physical properties of the copolymer was explored. It has been seen that the properties of the copolymer are considerably influenced by the different curing systems used. While ethylene thiourea (ETU) and propylene thiourea (PTU), as primary accelerators, provide the highest state of cure but may cause scorch. The use of trimethyl thiourea (TMU), on the other hand, results in the fastest rate and the most stable state of cure, good scorch safety, bin stability, and an overall good balance of properties. The sulfur-based crosslinking system induces good mechanical properties but causes limited bin stability, poor high-temperature compression set, and impaired heat resistance properties. As a curing agent, peroxide delivers the best bin stability in the rubber stocks but yields higher stiffness and limited aging resistance in the vulcanizates.

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 β-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.

Electrochemical Characterization of Mancozeb Degradation for Wastewater Treatment Using a Sensor Based on Poly (3,4-ethylenedioxythiophene) (PEDOT) Modified with Carbon Nanotubes and Gold Nanoparticles

Mancozeb is a worldwide fungicide used on a large scale in agriculture. The active component and its main metabolite, ethylene thiourea, has been related to health issues. Robust, fast, and reliable methodologies to quantify its presence in water are of great importance for environmental and health reasons. The electrochemical evaluation of mancozeb using a low-cost electrochemical electrode modified with poly (3,4-ethylene dioxythiophene), multi-walled carbon nanotubes, and gold nanoparticles is a novel strategy to provide an in-situ response for water pollution from agriculture. Additionally, the thermal-, electrochemical-, and photo-degradation of mancozeb and the production of ethylene thiourea under controlled conditions were evaluated in this research. The mancozeb solutions were characterized by electrochemical oxidation and ultraviolet-visible spectrophotometry, and the ethylene thiourea concentration was measured using ultra-high-performance liquid chromatography high-resolution mass spectrometry. The degradation study of mancozeb may provide routes for treatment in wastewater treatment plants. Therefore, a low-cost electrochemical electrode was fabricated to detect mancozeb in water with a robust electrochemical response in the linear range as well as a quick response at a reduced volume. Hence, our novel modified electrode provides a potential technique to be used in environmental monitoring for pesticide detection.

Methods for the Identification of Outliers and Their Influence on Exposure Assessment in Agricultural Pesticide Applicators: A Proposed Approach and Validation Using Biological Monitoring

The “patch” approach for skin exposure assessment can easily be combined with biological monitoring in real-life pesticide studies. Nevertheless, this approach is sensitive to outliers, with values markedly deviating from other members of the sample, which can result in a gross overestimation of exposure. This study aimed at developing methods for outlier identification and validating them while using biological monitoring. Twenty-seven workers applying mancozeb in Italian vineyards participated in this study. Their skin exposure was estimated while using the patch methodology, while ethylene-thiourea (ETU) was measured in the 24-h post-exposure urine as a biomarker of exposure. The outliers were detected using methods that were based on the multiplication of the median, the median absolute deviation, and boxplots. The detection rate varied between 2.3% and 17.3%. The estimated median skin exposure of 3.2 μg was reduced to 1.2 μg when the modified Z score was used. The highest reduction in the skin exposure was above 54 μg. The use of the modified Z score for outlier detection resulted in an increase in the correlation coefficient between the skin exposure and the urine ETU levels from 0.46 to 0.71, which suggested the validity of the approach. Future studies should standardize and improve the methods for pesticide exposure and risk assessment.

FOXD3/FOXD4 is required for the development of hindgut in the rat model of anorectal malformation

Congenital anorectal malformation is the most common digestive tract malformation in newborns. It has been reported that FOXD3/FOXD4, a forkhead transcription factor, regulates the generation, migration, and differentiation of neural crest cells. However, whether FOXD3/FOXD4 takes part in anorectal malformation remains unclear. In the present study, we used ethylene thiourea to induce the animal models of anorectal malformation in rat embryos and to interrogate the role of FOXD3/FOXD4 in anorectal malformation pathogenesis. Hindgut samples of the animal models were collected at E15, E17, E19, and E21 days of age. The expression of FOXD3/FOXD4 was detected by immunohistochemistry, western blot, and quantitative real-time fluorescence PCR. By immunohistochemical staining, FOXD3/FOXD4 was observed in epithelial cells of the rectum and the anus both in normal and rat embryos with anorectal malformation. Expression level analysis by western blot indicated that FOXD3/FOXD4 expression increased in ethylene thiourea-induced anorectal malformation groups. mRNA expression as determined by quantitative real-time fluorescence PCR analysis was consistent with the western blot results. Tentative conclusions were drawn that FOXD3/FOXD4 is expressed in the hindgut in rat embryos and is upregulated in anorectal malformation. FOXD3/FOXD4 is required for the development of the hindgut, and its aberrant expression may be an important factor leading to the incidence of anorectal malformation. Impact statement Congenital anorectal malformation (ARM) is the most common digestive tract malformation in newborns. The pathophysiological ground remains unclear. In this study, we used animal models of ARM for the first time to interrogate the role of FOXD3/FOXD4 in ARM pathogenesis. The animal models of ARM were successfully induced by ethylene thiourea (ETU) in rat embryos providing a strong basis for pathogenesis study of this disease. Expression analysis of FOXD3/FOXD4 was carried out in these models, and the results shape a deeper understanding of FOXD3/FOXD4 being required for the normal development of the hindgut. The aberrant expression of FOXD3/FOXD4 may be an important factor leading to ARM incidence.