Effect of neonatal exposure to endosulfan on myometrial adaptation during early pregnancy and labor in rats.

Proper myometrial adaptation during gestation is crucial for embryo implantation, pregnancy maintenance and parturition. Previously, we reported that neonatal exposure to endosulfan alters uterine development and induces implantation failures. The present work investigates the effects of endosulfan exposure on myometrial differentiation at the pre-implantation period, and myometrial activation during labor. Newborn female rats were s.c. injected with corn oil (vehicle) or 600 µg/kg/day of endosulfan (Endo600) on postnatal days (PND) 1, 3, 5 and 7. On PND90, the rats were mated to evaluate: i) the myometrial differentiation on gestational day 5 (GD5, pre-implantation period), by assessment myometrial histomorphology, smooth muscle cells (SMCs) proliferation, and expression of proteins involved in myometrial adaptation for embryo implantation (steroid receptors, Wnt7a and Hoxa10); ii) the timing of parturition and myometrial activation during labor by determining the uterine expression of contraction-associated genes (oxytocin receptor, OTXR; prostaglandin F2α receptor, PTGFR and connexin-43, Cx-43). Endosulfan decreased the thickness of both myometrial layers, with a concomitant decrease in the collagen remodeling. Blood vessels relative area in the interstitial connective tissue between muscle layers was also decreased. Endo600 group showed lower myometrial proliferation in association with a downregulation of Wnt7a and Hoxa10. Although in all females labor occurred on GD23, the exposure to endosulfan altered the timing of parturition, by inducing advancement in the initiation of labor. This alteration was associated with an increased uterine expression of OTXR, PTGFR and Cx-43. In conclusion, neonatal exposure to endosulfan produced long-term effects affecting myometrial adaptation during early pregnancy and labor. These alterations could be associated with the aberrant effects of endosulfan on the implantation process and the timing of parturition.

Development and removal ability of non-toxigenic Aspergillus section Flavi in presence of atrazine, chlorpyrifos and endosulfan.

This study evaluated the in vitro effect of three concentrations of atrazine, chlorpyrifos and endosulfan on the growth parameters of four non-toxigenic Aspergillus section Flavi strains. The ability of the strains to remove these pesticides in a synthetic medium was also determined. Growth parameters were measured on soil extract solid medium supplied with 5, 10 and 20mg/l of each pesticide, and conditioned to -0.70, -2.78, -7.06 and -10.0 water potential (MPa). Removal assays were performed in Czapek Doc medium (CZD) supplied with 20mg/l of each pesticide under optimal environmental conditions (-2.78 of MPa and 25°C). The residual levels of each pesticide were detected by the reversed-phase HPLC/fluorescence detection system. The lag phases of the strains significantly decreased in the presence of the pesticides with respect to the control media. This result indicates a fast adaptation to the conditions assayed. Similarly, the mycelial growth rates in the different treatments increased depending on pesticide concentrations. Aspergillus oryzae AM 1 and AM 2 strains showed high percentages of atrazine degradation (above 90%), followed by endosulfan (56 and 76%) and chlorpyrifos (50 and 73%) after 30 days of incubation. A significant (p<0.001) correlation (r=0.974) between removal percentages and growth rate was found. This study shows that non-toxigenic Aspergillus section Flavi strains from agricultural soils are able to effectively grow in the presence of high concentrations of atrazine, chlorpyrifos and endosulfan under a wide range of MPa conditions. Moreover, these strains have the ability to remove high levels of these pesticides in vitro in a short time.

Total and differential white blood cell counts in Caiman latirostris after in ovo and in vivo exposure to insecticides.

Agricultural activities associated mainly with soybean crops affect the natural environment and wildlife by habitat destruction and the extensive use of agrochemicals. The aim of this study was to evaluate immunotoxic effects of the insecticides cypermethrin (CYP) and endosulfan (END) in Caiman latirostris analyzing total blood cell count (TWBC) and differential white blood cell count (DWBC) after in ovo and in vivo exposure. Eggs (in ovo) and hatchlings (in vivo) from nests harvested in natural habitats were artificially incubated and reared under controlled conditions in the Proyecto Yacaré (Gob.Santa Fe/MUPCN) facilities. Exposure of embryos was performed by topication on the eggshell during the first stage of development. The treatments were distilled water (negative control; NC), ethanol (vehicle control; VC), four groups treated with different concentrations of CYP and four groups with END. In vivo exposure was performed by immersion; treatments were NC, VC, two groups exposed to CYP and two to END. After embryonic exposure to the insecticides, no differences were found in TWBC or DWBC among the neonates exposed to pesticides versus controls. In the in vivo scenario, similar results were obtained for TWBC, but DWBC data showed differences between NC hatchlings and CYP-1 hosts for heterophil, lymphocyte and monocyte levels, and between NC and END-1 hosts for lymphocyte and monocyte levels. Research on the effects of pesticide exposure on this species is of special interest not only to assess the impact on caiman populations, but also to further characterize the species as a potential sentinel of ecosystem health.

Chronic exposure to endosulfan induces inflammation in murine colon via ß-catenin expression and IL-6 production.

Endosulfan (ENDO) is a widely used organochlorine (OC) pesticide and persistent organo-pollutant. Epidemiological studies have shown that high levels of OC exposure were related to colorectal cancer (CRC) incidence. The objectives of the present study were to evaluate histological changes in the colon, as well as in in situ expression of ß-catenin and P-selectin, and serum levels of select pro-inflammatory cytokines in mice administered ENDO; there is a relationship between increased serum IL-6 and P-selectin levels in CRC patients and aberrant ß-catenin signaling is important in initiation/maintenance of most CRCs. Mice were exposed to ENDO (at dose < LD50) orally once a week for up to 24 weeks, and monitored (inclusive) for a total of 42 weeks. The experiment was comprised of three groups, one that did not receive ENDO (olive oil vehicle), one administered 2 mg ENDO/kg/week and a positive control (for induction of CRC) given a weekly 20 mg 1,2-dimethylhydrazine (DMH)/kg injection. The results indicated that oral administration of ENDO provoked moderate inflammation starting at six weeks, and severe colonic inflammation with an appearance of dysplastic formations (aberrant crypts) in mice treated with ENDO (or DMH) for 12 weeks or longer. Serum IL-6 levels significantly increased starting at six weeks and rose to a peak of 15-fold higher than in controls at 42 weeks; TNFα levels likewise significantly increased, with a later peak (≈four-fold higher than controls) at 30-42 weeks. Immunohistochemical analysis of the colon also showed that expression of ß-catenin and P-selectin increased with length of exposure to ENDO. Taken together, the results indicate that continued repeated oral exposure to ENDO induces increased expression of ß-catenin and P-selectin, inflammation in the colon, and, ultimately, local tissue dysplasia.

Endosulfan affects uterine development and functional differentiation by disrupting Wnt7a and ß-catenin expression in rats.

Neonatal exposure to a low dose of endosulfan may disrupt the expression of Wnt7a and ß-catenin during uterine development leading to the failure of uterine functional differentiation during implantation. New-born female Wistar rats were treated with vehicle, endosulfan (600 µg/kg/d, E600) or diethylstilbestrol (0.2 µg/kg/d, DES) on postnatal days (PNDs) 1, 3, 5 and 7. Subsequently, uterine histomorphology and the protein expression of Wnt7a and ß-catenin were evaluated on PND8, PND21 and gestational day (GD) 5 (pre-implantation period). In the E600 rats, Wnt7a and ß-catenin protein expression was increased in the epithelium on PND8, and Wnt7a expression was decreased in the endometrial glands on PND21. On GD5, the number of uterine glands was decreased in the E600-and DES-treated rats. In addition, Wnt7a expression was decreased in all uterine compartments, and ß-catenin expression was increased in the luminal and glandular epithelia of the E600-and DES-treated rats. Disruption of Wnt7a and ß-catenin uterine expression in the prepubertal and adult females altered the uterine preparation for embryo implantation, which could be associated with the subfertility triggered by endosulfan.

Neonatal exposure to low doses of endosulfan induces implantation failure and disrupts uterine functional differentiation at the pre-implantation period in rats.

We investigated whether neonatal exposure to low doses of endosulfan affects fertility and uterine functional differentiation at pre-implantation in rats. Newborn female rats received the vehicle, 0.2 µg/kg/d of diethylstilbestrol (DES), 6 µg/kg/d of endosulfan (Endo6) or 600 µg/kg/d of endosulfan (Endo600) on postnatal days (PND) 1, 3, 5, and 7. On PND90, the rats were mated to evaluate their reproductive performance on gestational day (GD) 19 and their ovarian steroid serum levels, endometrial proliferation and implantation-associated proteins on GD5. DES and endosulfan decreased the pregnancy rate and the number of implantation sites. On GD5, DES and endosulfan did not change the serum levels of 17ß-estradiol (E2) and progesterone (P); the endometrial proliferation decreased, which was associated with silencing of Hoxa10 in the Endo600-treated rats. Both doses of endosulfan increased the progesterone receptor (PR) expression, whereas the higher dose led additionally to an increase in estrogen receptor alpha (ERα). In the Endo600-treated rats, the down-regulation of Hoxa10 was associated with a deregulation of the steroid receptor coregulators. Alterations in endometrial proliferation and the endocrine pathway of Hoxa10/steroid receptors/coregulators might be the mechanism of endosulfan-induced implantation failure.

Assessment of the sublethal toxicity of organochlorine pesticide endosulfan in juvenile common carp (Cyprinus carpio).

This study is aimed at evaluating the sublethal effects of endosulfan (EDS) in juvenile common carp (Cyprinus carpio). For this purpose, fish were exposed for 15 days to the technical EDS (95% pure) diluted in dimethyl sulfoxide (DMSO) 0.1% of the total volume in water solution in a semi-static system at sublethal concentration (1 µg/L). Subsequently, the liver somatic index (LSI) and factor condition (K) were determined. The total cytocrome P450 (CYP), CYP1A isoform, and the ethoxyresorufin-O-deethylase (EROD) activity were determined from the hepatic microsomal fraction as well as the activity of the oxidative stress enzyme system such as superoxide dismutase (SOD), catalase (CAT), glutathione-S-transferase (GST), glutathione peroxidase (GP(X)), glutathione reductase (GR), and glucose-6-phosphate dehydrogenase (G6PDH). Among the parameters assessed, EDS at the sublethal concentration in subchronic exposure caused significant changes in liver somatic indices as well as induction of the phase I biotransformation system and oxidative stress in juvenile common carp (Cyprinus carpio). Thus, it is seen that the use of biochemical biomarkers of environmental contamination in this study proved to be an extremely important tool for detecting the adverse effects of xenobiotics in the aquatic environment, even at low concentration.

Neonatal exposure to low doses of endosulfan disrupts the expression of proteins regulating uterine development and differentiation.

This study investigates the effects of neonatal exposure to low doses of endosulfan on the expression of proteins regulating uterine development and differentiation. Female pups received vehicle, endosulfan (Endo6: 6 µg/kg, Endo600: 600 µg/kg) or diethylstilbestrol (DES: 0.2 µg/kg) from postnatal day 1 (PND1) to PND7. The uterine expression of estrogen receptor alpha (ERα), progesterone receptor (PR), Hoxa10 and alpha smooth muscle actin (α-SMA) was detected by immunohistochemistry on PND8 (neonatal period) and PND21 (prepubertal period), to evaluate acute and short-term responses. ERα, Hoxa10 and α-SMA were induced in the Endo600 group in both ages, while a striking decrease in PR expression was detected in the prepubertal rats following each dose of endosulfan. DES treatment deregulated ERα and Hoxa10 uterine expression at each age. Studies are currently underway to investigate whether the dysregulation of steroid receptors, Hoxa10 and α-SMA observed following neonatal exposure to endosulfan affect uterine functions in adulthood.

Endosulfan affects health variables in adult zebrafish (Danio rerio) and induces alterations in larvae development.

Adult zebrafish (Danio rerio) were exposed to 0 (control), 0.16 or 0.48µg/L of the insecticide, endosulfan, for 28days. Haematology, whole body ions, thiobarbituric acid reactive substances (TBARS), Na(+)K(+)-ATPase, organ histology and reproduction were assessed in adults. The resulting offspring were examined for latent effects on development (heart rate and morphometrics). On day 14, adult fish exposed to 0.16µg/L endosulfan showed significantly lower red blood cell counts than those exposed to 0.48µg/L endosulfan; adult fish exposed to 0.16 ug/L also showed elevated TBARS compared to controls. Both concentrations of endosulfan caused a 4.0 fold increase in Na(+)K(+)-ATPase activity compared to controls (ANOVA, p<0.05). On day 14, the livers of fish exposed to endosulfan had fewer, enlarged hepatocytes, with cell diameters greater than the controls (ANOVA, p<0.05). Morphological alterations in the progeny of fish exposed to endosulfan were observed. Heart beat frequency was significantly lower in larvae from exposed adults to 0.16 µg/L compared to the control (ANOVA, p<0.05). These findings show that sublethal exposure to endosulfan causes adverse sublethal effects in adult D. rerio, and effects on the development of their offspring.