Mass spectrometric determination of N7-HPTE-dG and N7-HPTE-Gua in mammalian cells and mice exposed to methoxychlor, an emergent persistent organic pollutant.

Methoxychlor (MXC) is an organopesticide classified as a "Proposed Persistent Organic Pollutant" in the Stockholm Convention, and recent studies revealed that MXC could induce DNA strand breaks, whereas its underlying mechanisms were underinvestigated. Here, we first reported that hydroxymethoxychlor (HPTE), one of MXC's active metabolites, could be oxidized in vivo to form quinone intermediate, which attacked N7 position of 2'-deoxyguanosine to form N7-HPTE-deoxyguanosine (N7-HPTE-dG), followed by depurination to produce N7-HPTE-guanine (N7-HPTE-Gua) in MXC-treated mammalian cells and tissues from mice fed with MXC, employing an ultra-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UPLC-ESI-MS/MS) method. We observed a positive correlation between the doses of MXC exposure and the levels of N7-HPTE-Gua and N7-HPTE-dG in cytoplasm and genomic DNA, respectively. Furthermore, after removal of exogenous MXC, the amount of genomic N7-HPTE-dG was significantly decreased during 24 h, while the level of cytoplasmic N7-HPTE-Gua was elevated during first 12 h, indicating the accumulation of the N7-HPTE-Gua in cells. Additionally, for animal experiment, genomic N7-HPTE-dG was observed in livers and cortexes from female C57BL/6 mice fed with MXC, suggesting a potential mechanism of its hepatoxicity and neurotoxicity. Overall, our study provides new understanding about the formation of MXC-induced DNA adducts in mammalian cells and animal models.

Effects of neonatal methoxychlor exposure on the ovarian transcriptome in piglets.

Methoxychlor (MXC) is a man-made organochlorine insecticide capable of disrupting endocrine functions due to its mixed steroidal properties (estrogenic, anti-estrogenic and/or anti-androgenic). Retarded follicle development was recently reported in neonatal pigs treated with MXC. The goal of the current study was to better understand the mechanism of MXC action in the ovary of newborn piglets. By employing RNA-Seq we studied the expression of protein coding (mRNA) and long non-coding RNA (lncRNA) transcripts in the ovary of the MXC-treated piglets. Piglets were injected (sc) daily with MXC (100 mg/kg body weight) or corn oil (controls) between postnatal Days 1 and 10 (n = 3 piglets/group). The ovaries excised from 11-day-old piglets were processed for total RNA isolation and subsequent RNA sequencing. Four hundred sixty differentially expressed genes (DEGs) and 143 differentially expressed lncRNAs (DELs) were identified in the ovaries of MXC-treated piglets (P-adjusted < 0.05; abs(log2FC) > 1). Functional enrichment analysis showed that MXC altered the expression of genes associated with intracellular and membrane transport, intra-ovarian signaling as well as cell-cell junction and communication. Moreover, positive and negative correlations determined between the identified DEGs and DELs suggest that some lncRNAs may mediate the MXC action in the ovary. The results support the hypothesis that MXC-induced changes in the expression of genes involved in neonatal ovarian folliculogenesis increase the risk of fertility problems in adults.

N-acetyl-l-cysteine mediated regulation of DNA fragmentation, an apoptotic event, against methoxychlor toxicity in the granulosa cells of ovarian antral follicles.

Methoxychlor (MXC), an organo-chlorine insecticide, is a reproductive toxicant in females, causing apoptosis-mediated follicular atresia. To elucidate the potentials of Methoxychlor as a geno-toxicant, granulosa cells of healthy antral follicles, exposed to MXC and antioxidant, N-acetyl-l-cysteine, were studied by the terminal deoxynucleotidyltransferase-dUTP nick end-labelling and single-cell gel electrophoresis (comet) assays. MXC caused DNA fragmentation, as revealed by the increased incidence of dark brown condensed TUNEL positive cells in contrast with lightly brown TUNEL negative cells with maximum TUNEL positive cells were observed in 100 µg/mL MXC treated groups. Quantitatively, maximum geno-toxicity was exhibited at highest MXC treatment with percent tail DNA as 17.87 ± 0.85, 41.16 ± 3.94, and 47.73 ± 3.71 in comparison with control (0.65 ± 0.03, 2.91 ± 0.27, and 7.16 ± 1.39) after 24, 48 and 72 h exposure duration, respectively. MXC treated groups exhibited Type 1-Type 3 comets as compared to Type 0 comets in control groups. Supplementation of NAC led to significant (p < 0.05) decline in geno-toxicity in MXC treated groups with maximum amelioration observed at 5 and 10 mM. Consequently, increased DNA damage attributed to the granulosa cells apoptosis in response to Methoxychlor exposure was significantly combated by NAC supplementation, preventing the geno-toxicity induced cyto-toxicity in GCs.

Assessing the Impacts of Methoxychlor Exposure on the Viability, Reproduction, and Locomotor Behavior of the Seminole Ramshorn Snail (Planorbella duryi).

In the present study, the effects of short-term methoxychlor exposure on the viability, reproduction, and locomotor behavior of adult seminole ramshorn snails (Planorbella duryi) was assessed. To examine impacts on viability and behavior, individuals were exposed to a water control, vehicle control, or 12.5, 50, 100, 250, 500, or 1000 µg/L of methoxychlor for 48 h; and differences in mortality and locomotor behavior assessed using the freely available ToxTrac software. To determine impacts on reproduction, pairs of snails were exposed to a vehicle control and 12.5, 25, 50, 100, and 250 µg/L of methoxychlor for 9 d; and the number of clutches and eggs laid quantified every 24 h. Methoxychlor concentrations in treatments were determined using gas chromatography. Complete mortality was observed in the 500 µg/L and 1000 µg/L treatments after 48 h and in the 250 µg/L treatment after 9 d. Decreases in the number of egg clutches were observed in all treatments, and the number of eggs laid decreased starting in the 25 µg/L treatment. Decreases in average speed, mobile speed, and total distance traveled, as well as a significant increase in frozen events, were also observed. Our results suggest that methoxychlor exposure causes detrimental effects on several nonlethal endpoints in a nonmodel aquatic invertebrate species and that the analysis of locomotor behaviors serves as a reliable, sensitive endpoint for ecotoxicology testing. Environ Toxicol Chem 2019;39:220-228. © 2019 SETAC.

Development of a Generic Physiologically Based Kinetic Model to Predict In Vivo Uterotrophic Responses Induced by Estrogenic Chemicals in Rats Based on In Vitro Bioassays.

The present study assessed the potential of a generic physiologically based kinetic (PBK) model to convert in vitro data for estrogenicity to predict the in vivo uterotrophic response in rats for diethylstibestrol (DES), ethinylestradiol (EE2), genistein (GEN), coumestrol (COU), and methoxychlor (MXC). PBK models were developed using a generic approach and in vitro concentration-response data from the MCF-7 proliferation assay and the yeast estrogen screening assay were translated into in vivo dose-response data. Benchmark dose analysis was performed on the predicted data and available in vivo uterotrophic data to evaluate the model predictions. The results reveal that the developed generic PBK model adequate defines the in vivo kinetics of the estrogens. The predicted dose-response data of DES, EE2, GEN, COU, and MXC matched the reported in vivo uterus weight response in a qualitative way, whereas the quantitative comparison was somewhat hampered by the variability in both in vitro and in vivo data. From a safety perspective, the predictions based on the MCF-7 proliferation assay would best guarantee a safe point of departure for further risk assessment although it may be conservative. The current study indicates the feasibility of using a combination of in vitro toxicity data and a generic PBK model to predict the relative in vivo uterotrophic response for estrogenic chemicals.

Bioaccumulation and toxicity of methoxychlor on Chinese mitten crab (Eriocheir sinensis).

Chinese mitten crab, a featured macrobenthos, has been one of the most important economical aquatic species in China. This study assessed the accumulation of an organochlorine pesticide methoxychlor (MXC) in Chinese mitten crab during exposure to 1 mg/L of MXC. The results showed the residual concentration of MXC in the ovary and hepatopancreas reached 55.07 ±â€¯2.64 ng/g and 34.51 ±â€¯2.35 ng/g, respectively. After exposure, tubular vacuolization of epithelial tissues, condensed egg cells and obvious intervals between egg cell wall and stroma were observed in the hepatopancreas and ovary, respectively. Significant changes of three key metabolic enzymes in hepatopancreas were observed upon exposure to MXC. Compared to the control, acetylcholinesterase level was significantly higher at day 7 (0.15 ±â€¯0.01 vs. 0.06 ±â€¯0.00 U/mgprot); glutathione S-transferase level was elevated at both day 4 (12.01 ±â€¯0.48 vs. 3.20 ±â€¯0.44 U/mgprot) and day 7 (12.84 ±â€¯1.01 vs. 8.22 ±â€¯0.81 U/mgprot); superoxide dismutase was sharply increased at day 4 (21.20 ±â€¯0.24 vs. 3.66 ±â€¯0.60 U/mgprot) but decreased at day 7 (3.74 ±â€¯0.12 vs. 9.44 ±â€¯0.85 U/mgprot). Overall, dissolved MXC accumulated in lipid-rich tissues could cause damages on epithelial cells and egg cells and change metabolic activities of enzymes involved in antioxidative stress and detoxification processes.

Effect of prenatal exposure to combined immunosuppressive agrochemicals in a mouse model of allergic airway inflammation.

The aim of this study is to identify the combined effect of multiple chemicals to the development of allergy. In this study, the effect of prenatal exposure to an organochlorine agent methoxychlor (MXC) and/or an organophosphate agent parathion (PARA) on trimellitic anhydride-induced allergic airway inflammation was examined in mice. Eosinophil infiltration in the bronchoalveolar lavage fluid (BALF) was significantly enhanced by MXC + PARA exposure compared to that of the control, MXC, and PARA groups. In the hilar lymph node, only slight increases in B-cell infiltration, as well as IL-6 and IL-9 secretions were observed in MXC + PARA group, and no effect was observed in the individual treatment groups. Our findings imply that prenatal exposure to some combinations of multiple chemicals may exacerbate the allergic inflammatory responses including eosinophils and cytokine production.

Methoxychlor metabolite HPTE alters viability and differentiation of embryonic thymocytes from C57BL/6 mice.

Endocrine-disrupting chemicals (EDC) are widespread in the built and natural environments. Heightened public awareness of their potential danger has led to concern about whether EDC and their metabolites have significant negative biological effects. Studies have shown that EDC like DDT and other organochlorine pesticides, such as methoxychlor (MXC), have adverse effects on immune cells, but no studies have addressed the impact of HPTE, the primary metabolite of MXC. To elucidate the presence and significance of HPTE adverse effects, this study explored the impact of HPTE on a critical window and component of immune system development, embryonic T-cell development. Lesions at this phase of development can lead to lifelong immune dysfunction and increased incidence of immune disease, such as autoimmunity. Embry-onic thymocytes (GD 16-18) from C57BL/6 mice were subjected to an in vitro differentiation culture that mimicked early steps in thymocyte development in the presence of 0.005, 0.05, 0.5, 5, or 50 µM HPTE, or a model endocrine disruptor, DES. The results indicated that compared to the vehicle control, HPTE- and DES-induced death of thymocytes. Annexin-V staining and Caspase 8, markers of programed cell death, revealed that the loss of cells was due at least in part to induction of apoptosis. Moreover, HPTE-induced cell death not only resulted in selective loss of double positive thymocytes, but also loss of developing CD4 intermediate cells (post-double positive partially differentiated thymocyte population). Phenotypic analysis of thymocyte maturation (T-cell receptor, TCR) and TCR ligation (CD5) surface markers revealed that surviving embryonic thymocytes expressed low levels of both. Taken together these data demonstrate that immature embryonic thymocytes are sensitive to HPTE exposure and that HPTE exposure targets thymocyte populations undergoing critical differentiation steps. These findings suggest HPTE may play a pivotal role in MXC exposure-induced immune dysfunction.

The effects of environmentally relevant concentrations of aldrin and methoxychlor on the testes and sperm of male Clarias gariepinus (Burchell, 1822) after short-term exposure.

The organochlorine pesticides aldrin (0.14 µg/L) and methoxychlor (0.23 µg/L) were both present in the Albasini Dam, Limpopo Province, South Africa, during a field survey in 2014. The use of aldrin has been banned in the USA since 1987 and restricted in South Africa since 1992. The use of methoxychlor, however, remains undefined with little information available about its registration in South Africa despite being banned in Europe (2002) and USA (2003). The aim of this study was to determine the potential effects of environmentally relevant concentrations of aldrin and methoxychlor on the reproductive system of male catfish, Clarias gariepinus. Males were exposed for 96 h to the two pesticides under controlled laboratory conditions. Following exposure, each fish was weighed and measured, and a necropsy performed to determine any macroscopic abnormalities and the general health of the fish. The fish were killed and dissected and the testes removed, weighed and measured to determine the gonadosomatic index (GSI). The right testis of each fish was sectioned for histopathological assessment and to calculate the testes index (IT). The left testis was used for computer-assisted sperm analysis (CASA). The histopathological assessment of the testes showed histopathological changes such as of melano-macrophage centres (MMCs) and vacuolation of spermatogonia and spermatocytes. However, the classification of these changes indicated that the testes tissue structure was normal with slight histological changes. No statistically significant differences (p > 0.05) were found in the CASA parameters between exposure groups. The results of this study showed that the environmentally relevant concentrations of aldrin and methoxychlor did not have a negative effect on the motility of the mature sperm, but adverse effects were noted in the early stages of spermatogenesis, indicating possible effects over longer exposure periods.

Effects of methoxychlor on IGF-I signaling pathway in rat ovary.

Follicular development and other ovarian functions are regulated by growth factors that can be affected by exogenous agents. Methoxychlor (MXC) is an organochloride pesticide that causes female infertility. We investigated how MXC affects the distribution of developing ovarian follicles in adult rats after treatment between embryonic day (E) 18 and postnatal day (PND) 7. We also measured insulin-like growth factor-I (IGF-I) and its receptor, IGF-IR, expressions in ovarian follicles and investigated whether MXC changed the levels of IGF-I and IGF-IR in the ovary. Using immunohistochemical (IHC) staining, we detected IGF-I expression in oocytes and granulosa cells of the follicles, luteal cells, interstitial cells, theca externa and theca interna, and the smooth muscle of ovarian vessels. IGF-IR was co-localized with IGF-I in the ovary except for the theca externa. IGF-I expression was decreased in granulosa cells of preantral and antral follicles after treatment with MXC compared to granulosa cells of preantral and antral follicles of the control group. We also observed that oocytes of secondary follicles and granulosa cells of secondary and preantral follicles of the MXC treated groups showed increased IGF-IR expression compared to oocytes of secondary follicles and granulosa cells of secondary and preantral follicles of the control group. We also detected more secondary and preantral follicles, and fewer primordial and antral follicles after MXC administration compared to controls. Therefore, the IGF signaling pathway may participate in MXC induced ovary dysfunction and female infertility.

N-acetyl cysteine-mediated effective attenuation of methoxychlor-induced granulosa cell apoptosis by counteracting reactive oxygen species generation in caprine ovary.

Methoxychlor (MXC), an organochloride insecticide, is a potent toxicant-targeting female reproductive system and known to cause follicular atresia by inducing apoptosis within granulosa cells. Oxidative stress plays a pivotal role in apoptosis; thus, this study focuses on the ameliorative action of N-acetyl cysteine (NAC) on MXC-induced oxidative stress and apoptosis within granulosa cell of caprine ovary. Classic histology, fluorescence assay, and biochemical parameters were employed to evaluate the effect of varied concentration of NAC (1, 5, and 10 mM) on granulosa cell apoptosis after 24, 48, and 72 h exposure duration. Histomorphological studies revealed that NAC diminished the incidence of apoptotic attributes like condensed or marginated chromatin, pyknosis, crescent-shaped nucleus, empty cell spaces, and degenerated cellular structure along with the presence of cytoplasmic processes within granulosa cells in dose- and time-dependent manner. NAC significantly downregulated the percentage of MXC-induced granulosa cell apoptosis within healthy ovarian follicle with its increasing dose, maximum at 10 mM concentration. It also significantly (p < 0.05) upregulated the activity of antioxidant enzymes, namely catalase, superoxide dismutase, and glutathione-s-transferase, along with ferric reducing antioxidant power further declining lipid peroxidation in the MXC-treated caprine ovary. The results revealed a negative correlation between apoptosis frequency and antioxidant enzymes' activity (rCAT = -0.67, rSOD = -0.56, rGST = -0.31; p < 0.05) while a positive correlation was observed with lipid peroxidation (r = 0.63; p < 0.05) after NAC supplementation. Thus, NAC supplementation reduces the MXC-generated oxidative stress that perhaps declines the ROS generating signal transduction pathway of apoptosis, thereby preventing MXC-induced granulosa cell apoptosis and follicular atresia. © 2015 Wiley Periodicals, Inc. Environ Toxicol 32: 156-166, 2017.

In utero methoxychlor exposure increases rat fetal Leydig cell number but inhibits its function.

The objective of the present study is to determine whether in utero exposure to methoxychlor (MXC) affects rat fetal Leydig cell number, cell size, or functions. Pregnant Sprague Dawley dams were gavaged with corn oil (control, 0mg/kg/day MXC) or MXC at doses of 10, 50, or 100mg/kg/day from gestational day (GD) 12 to 21. The results show that MXC increased fetal Leydig cell numbers dose-dependently from 95±8×103 cells/testis (control, mean±SEM) to 101±6, 148±22, and 168±21×103 cells/testis, at the doses of 10, 50, and 100mg/kg, respectively. The increase of Leydig cell number by MXC was contributed by the increase of single cell population of Leydig cells, which increased from 21±2% of the control to 31±4%, 39±3%, or 40±4% at the doses of 10, 50 or 100mg/kg, respectively. Quantitative PCR results show that MXC increased Lhcgr expression at dose of 10mg/kg and Scarb1 and Cyp11a1 mRNA levels at doses of 50 and 100mg/kg. Immunohistochemical staining demonstrated the increase of CYP11A1 protein level from the dose of 10mg/kg. However, at the highest dose (100mg/kg) MXC reduced the testicular testosterone level and MXC (1µM) in vitro treatment also inhibited androgen production from isolated fetal Leydig cells. In conclusion, our findings indicate that at low dose MXC may increase fetal Leydig cell numbers and the expressions of some steroidogenic enzymes, but at high dose it reduces the testicular testosterone level leading to reproductive tract malformations in the male offspring.

Methoxychlor and Vinclozolin Induce Rapid Changes in Intercellular and Intracellular Signaling in Liver Progenitor Cells.

Methoxychlor (MXC) and vinclozolin (VIN) are well-recognized endocrine disrupting chemicals known to alter epigenetic regulations and transgenerational inheritance; however, non-endocrine disruption endpoints are also important. Thus, we determined the effects of MXC and VIN on the dysregulation of gap junctional intercellular communication (GJIC) and activation of mitogen-activated protein kinases (MAPKs) in WB-F344 rat liver epithelial cells. Both chemicals induced a rapid dysregulation of GJIC at non-cytotoxic doses, with 30 min EC50 values for GJIC inhibition being 10 µM for MXC and 126 µM for VIN. MXC inhibited GJIC for at least 24 h, while VIN effects were transient and GJIC recovered after 4 h. VIN induced rapid hyperphosphorylation and internalization of gap junction protein connexin43, and both chemicals also activated MAPK ERK1/2 and p38. Effects on GJIC were not prevented by MEK1/2 inhibitor, but by an inhibitor of phosphatidylcholine-specific phospholipase C (PC-PLC), resveratrol, and in the case of VIN, also, by a p38 inhibitor. Estrogen (ER) and androgen receptor (AR) modulators (estradiol, ICI 182,780, HPTE, testosterone, flutamide, VIN M2) did not attenuate MXC or VIN effects on GJIC. Our data also indicate that the effects were elicited by the parental compounds of MXC and VIN. Our study provides new evidence that MXC and VIN dysregulate GJIC via mechanisms involving rapid activation of PC-PLC occurring independently of ER- or AR-dependent genomic signaling. Such alterations of rapid intercellular and intracellular signaling events involved in regulations of gene expression, tissue development, function and homeostasis, could also contribute to transgenerational epigenetic effects of endocrine disruptors.

Transcriptional networks associated with the immune system are disrupted by organochlorine pesticides in largemouth bass (Micropterus salmoides) ovary.

Largemouth bass (Micropterus salmoides) inhabiting Lake Apopka, Florida are exposed to high levels of persistent organochlorine pesticides (OCPs) and dietary uptake is a significant route of exposure for these apex predators. The objectives of this study were to determine the dietary effects of two organochlorine pesticides (p, p'-dichlorodiphenyldichloroethylene; p, p' DDE and methoxychlor; MXC) on the reproductive axis of largemouth bass. Reproductive bass (late vitellogenesis) were fed one of the following diets: control pellets, 125ppm p, p'-DDE, or 10ppm MXC (mg/kg) for 84days. Due to the fact that both p,p' DDE and MXC have anti-androgenic properties, the anti-androgenic pharmaceutical flutamide was fed to a fourth group of largemouth bass (750ppm). Following a 3 month exposure, fish incorporated p,p' DDE and MXC into both muscle and ovary tissue, with the ovary incorporating 3 times more organochlorine pesticides compared to muscle. Endpoints assessed were those related to reproduction due to previous studies demonstrating that these pesticides impact the reproductive axis and we hypothesized that a dietary exposure would result in impaired reproduction. However, oocyte distribution, gonadosomatic index, plasma vitellogenin, and plasma sex steroids (17ß-estradiol, E2 and testosterone, T) were not different between control animals and contaminant-fed largemouth bass. Moreover, neither p, p' DDE nor MXC affected E2 or T production in ex vivo oocyte cultures from chemical-fed largemouth bass. However, both pesticides did interfere with the normal upregulation of androgen receptor that is observed in response to human chorionic gonadotropin in ex vivo cultures, an observation that may be related to their anti-androgenic properties. Transcriptomics profiling in the ovary revealed that gene networks related to cell processes such as leukocyte cell adhesion, ossification, platelet function and inhibition, xenobiotic metabolism, fibrinolysis, and thermoregulation were altered by p, p' DDE, MXC, and flutamide. Interestingly, immune-related gene networks were suppressed by all three chemicals. The data suggest that p, p' DDE and flutamide affected more genes in common with each other than either chemical with MXC, consistent with studies suggesting that p, p' DDE is a more potent anti-androgen than MXC. These data demonstrate that reproductive health was not affected by these specific dietary treatments, but rather the immune system, which may be a significant target of organochlorine pesticides. The interaction between the reproductive and immune systems should be considered in future studies on these legacy and persistent pesticides.

Hepatocytes as in vitro test system to investigate metabolite patterns of pesticides in farmed rainbow trout and common carp: Comparison between in vivo and in vitro and across species.

In vitro tools using isolated primary fish hepatocytes have been proposed as a useful model to study the hepatic metabolism of xenobiotics in fish. In order to evaluate the potential of in vitro fish hepatocyte assays to provide information on in vivo metabolite patterns of pesticides in farmed fish, the present study addressed the following questions: Are in vitro and in vivo metabolite patterns comparable? Are species specific differences of metabolite patterns in vivo reflected in vitro? Are metabolite patterns obtained from cryopreserved hepatocytes comparable to those from freshly isolated cells? Rainbow trout and common carp were dosed orally with feed containing the pesticide methoxychlor (MXC) for 14days. In parallel, in vitro incubations using suspensions of freshly isolated or cryopreserved primary hepatocytes obtained from both species were performed. In vivo and in vitro samples were analyzed by thin-layer chromatography with authentic standards supported by HPLC-MS. Comparable metabolite patterns from a qualitative perspective were observed in liver in vivo and in hepatocyte suspensions in vitro. Species specific differences of MXC metabolite patterns observed between rainbow trout and common carp in vivo were well reflected by experiments with hepatocytes in vitro. Finally, cryopreserved hepatocytes produced comparable metabolite patterns to freshly isolated cells. The results of this study indicate that the in vitro hepatocyte assay could be used to identify metabolite patterns of pesticides in farmed fish and could thus serve as a valuable tool to support in vivo studies as required for pesticides approval according to the EU regulation 1107.

Regulation of arcuate genes by developmental exposures to endocrine-disrupting compounds in female rats.

Developmental exposure to endocrine-disrupting compounds (EDCs) alters reproduction and energy homeostasis, both of which are regulated by the arcuate nucleus (ARC). Little is known about the effects of EDC on ARC gene expression. In Experiment #1, pregnant dams were treated with either two doses of bisphenol A (BPA) or oil from embryonic day (E)18-21. Neonates were injected from postnatal day (PND)0-7. Vaginal opening, body weights, and ARC gene expression were measured. Chrm3 (muscarinic receptor 3) and Adipor1 (adiponectin receptor 1) were decreased by BPA. Bdnf (brain-derived neurotropic factor), Igf1 (insulin-like growth factor 1), Htr2c (5-hydroxytryptamine receptor), and Cck2r (cholescystokinin 2 receptor) were impacted. In Experiment #2, females were exposed to BPA, diethylstilbestrol (DES), di(2-ethylhexyl)phthalate, or methoxychlor (MXC) during E11-PND7. MXC and DES advanced the age of vaginal opening and ARC gene expression was impacted. These data indicate that EDCs alter ARC genes involved in reproduction and energy homeostasis in females.

Methoxychlor and fenvalerate induce neuronal death by reducing GluR2 expression.

GluR2, an α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor subunit, plays important roles in neuronal survival. We previously showed that exposure of cultured rat cortical neurons to several chemicals decreases GluR2 protein expression, leading to neuronal toxicity. Methoxychlor, the bis-p-methoxy derivative of dichlorodiphenyltrichloroethane, and fenvalerate, a synthetic pyrethroid chemical, have been used commercially as agricultural pesticides in several countries. In this study, we investigated the effects of long-term methoxychlor and fenvalerate exposure on neuronal glutamate receptors. Treatment of cultured rat cortical neurons with 1 or 10 µM methoxychlor and fenvalerate for 9 days selectively decreased GluR2 protein expression; the expression of other AMPA receptor subunits GluR1, GluR3, and GluR4 did not change under the same conditions. Importantly, the decreases in GluR2 protein expression were also observed on the cell surface membrane where AMPA receptors typically function. In addition, both chemicals decreased neuronal viability, which was blocked by pretreatment with 1-naphtylacetylspermine, an antagonist of GluR2-lacking AMPA receptors, and MK-801, an N-methyl-d-aspartate (NMDA) receptor antagonist. These results suggest that long-term exposure to methoxychlor and fenvalerate decreases GluR2 protein expression, leading to neuronal death via overactivation of GluR2-lacking AMPA and NMDA receptors.

Exposure Duration-Dependent Ovarian Recovery in Methoxychlor-Treated Mice.

The pesticide methoxychlor (MXC) is known to target ovarian antral follicles in the mouse. In previous in vivo studies, MXC administration for 20 days increased atresia, but did not affect female fertility immediately after dosing. Thus, we hypothesized that perhaps not enough time had elapsed between the onset of MXC-induced atresia and actual follicle loss to result in reduced fertility. The current study was undertaken to determine whether MXC treatment for 20 days results in reduced antral follicle numbers and fertility at 30 and 60 days after dosing. To test this hypothesis, adult CD-1 female mice were dosed with vehicle control or MXC (64 mg/kg/day) for 20 days. At 30 and 60 days postdosing, the mice were either subjected to fertility tests or their ovaries were collected and subjected to histological evaluation of follicle numbers and atresia. The results indicate that at 30 days after the completion of dosing, MXC significantly increased atresia and reduced primordial and total follicle numbers, but did not affect fertility compared to controls. At 60 days after completion of dosing, MXC did not significantly affect fertility, follicle numbers, or atresia compared to controls. Collectively, these data indicate that the ovary may be able to recover from MXC treatment for 20 days.

Genome-Wide Locations of Potential Epimutations Associated with Environmentally Induced Epigenetic Transgenerational Inheritance of Disease Using a Sequential Machine Learning Prediction Approach.

Environmentally induced epigenetic transgenerational inheritance of disease and phenotypic variation involves germline transmitted epimutations. The primary epimutations identified involve altered differential DNA methylation regions (DMRs). Different environmental toxicants have been shown to promote exposure (i.e., toxicant) specific signatures of germline epimutations. Analysis of genomic features associated with these epimutations identified low-density CpG regions (<3 CpG / 100bp) termed CpG deserts and a number of unique DNA sequence motifs. The rat genome was annotated for these and additional relevant features. The objective of the current study was to use a machine learning computational approach to predict all potential epimutations in the genome. A number of previously identified sperm epimutations were used as training sets. A novel machine learning approach using a sequential combination of Active Learning and Imbalance Class Learner analysis was developed. The transgenerational sperm epimutation analysis identified approximately 50K individual sites with a 1 kb mean size and 3,233 regions that had a minimum of three adjacent sites with a mean size of 3.5 kb. A select number of the most relevant genomic features were identified with the low density CpG deserts being a critical genomic feature of the features selected. A similar independent analysis with transgenerational somatic cell epimutation training sets identified a smaller number of 1,503 regions of genome-wide predicted sites and differences in genomic feature contributions. The predicted genome-wide germline (sperm) epimutations were found to be distinct from the predicted somatic cell epimutations. Validation of the genome-wide germline predicted sites used two recently identified transgenerational sperm epimutation signature sets from the pesticides dichlorodiphenyltrichloroethane (DDT) and methoxychlor (MXC) exposure lineage F3 generation. Analysis of this positive validation data set showed a 100% prediction accuracy for all the DDT-MXC sperm epimutations. Observations further elucidate the genomic features associated with transgenerational germline epimutations and identify a genome-wide set of potential epimutations that can be used to facilitate identification of epigenetic diagnostics for ancestral environmental exposures and disease susceptibility.

Fetal and neonatal exposure to the endocrine disruptor, methoxychlor, reduces lean body mass and bone mineral density and increases cortical porosity.

Endogenous estrogen has beneficial effects on mature bone and negatively affects the developing skeleton, whereas the effect of environmental estrogens is not known. Methoxychlor (MXC) is a synthetic estrogen known as a persistent organochlorine and used as a pesticide. Methoxychlor and its metabolites display estrogenic, anti-estrogenic and anti-androgenic activity and may therefore influence bone. Fifty-eight male fetal and neonatal rats were exposed to either: a negative control (DMSO), 0.020, 100 mg/kg MXC, or 1 mg/kg ß-estradiol-3-benzoate (EB; positive control). Rats were treated daily for 11 days, from embryonic day 19 to postnatal day (PND) 7 or for 4 days during the postnatal period (PND 0-7). All rats were analyzed at PND-84. Total body, femur, spine, and tibia areal bone mineral density (BMD) and content (BMC), lean body mass (LBM) and fat were measured by dual energy X-ray absorptiometry. Bone geometry and volumetric (v) BMD were measured using micro-computed tomography and biomechanical properties using three-point bending were assessed. Rats exposed to EB or MXC (at either the high and/or low dose), independent of exposure interval showed lower body weight, LBM, tibia and femur BMD and length, and total body BMD and BMC than DMSO control group (p ≤ 0.05). Methoxychlor and EB exposure increased cortical porosity compared to DMSO controls. Trabecular vBMD, number and separation, and cortical polar moment of inertia and cross-sectional area were lower due to EB exposure compared to control (p < 0.05). Early MXC exposure compromises cortical porosity and bone size at maturity, and could ultimately increase the risk of fracture with aging.