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Methoxychlor (MXC) is a widely used organochlorine pesticide primarily targeting pests. However, MXC has been found to negatively impact the reproductive system of both humans and animals, triggering oxidative stress and apoptosis. Melatonin (MLT), an endogenous hormone, possesses various benefits, including circadian rhythm regulation and anti-inflammatory and antioxidative stress effects. Moreover, MLT plays a crucial role in the development of animal germ cells and embryos. This study aimed to investigate the impact of MLT on porcine oocytes exposed to MXC. The experimental findings revealed that 200 µM MXC had detrimental effects on the maturation of porcine oocytes. However, the addition of 10-8 M MLT mitigated the toxic effects of MXC. MXC induced oxidative stress in porcine oocytes, leading to an increase in reactive oxygen species and impairing mitochondrial function. Consequently, oocyte quality was affected, resulting in elevated levels of early apoptosis and DNA damage, ultimately negatively impacting subsequent embryonic development. However, the addition of MLT showed the potential to ameliorate the damage caused by MXC. In conclusion, our results suggest that MLT exhibits a protective effect against MXC-induced damage to porcine oocyte maturation.

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Multi-element compound-specific stable isotope analysis (ME-CSIA) allows monitoring the environmental behavior and transformation of most common and persistent contaminants. Recent advancements in analytical techniques have extended the applicability of ME-CSIA to organic micropollutants, including pesticides. Nevertheless, the application of this methodology remains unexplored concerning harmful insecticides such as methoxychlor, a polar organochlorine pesticide usually detected in soil and groundwater. This study introduces methods for dual carbon and chlorine compound-specific stable isotope analysis (δ13C-CSIA and δ37Cl-CSIA) of both methoxychlor and its metabolite, methoxychlor olefin, with a sensitivity down to 10 and 100 mg/L, and a precision lower than 0.3 and 0.5 ‰ for carbon and chlorine CSIA, respectively. Additionally, three extraction and preconcentration techniques suitable for ME-CSIA of the target pesticides at environmentally relevant concentrations were also developed. Solid-phase extraction (SPE) and liquid-solid extraction (LSE) effectively extracted methoxychlor (107 ± 27 % and 87 ± 13 %, respectively) and its metabolite (91 ± 27 % and 106 ± 14 %, respectively) from water and aquifer slurry samples, respectively, with high accuracy (Δδ13C and Δδ37Cl ≤ ± 1 ‰). Combining CSIA with polar organic chemical integrative samplers (POCISs) for the extraction of methoxychlor and methoxychlor olefin from water samples resulted in insignificant fractionation for POCIS-CSIA (Δδ13C ≤ ± 1 ‰). A relevant sorption of methoxychlor was detected within the polyethersulfones membranes of the POCISs resulting in temporary carbon isotope fractionation depending on the sorbed mass fraction during the first deployment days. This highlights the critical role of the interactions of polar analytes with POCIS sorbents and membranes in the performance of this method. Altogether, this study proposes a proof of concept for ME-CSIA of methoxychlor and its metabolites, opening the door for future investigations of their sources and transformation processes in contaminated sites.

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Laccase immobilized and cross-linked on Fe3S4/earthworm-like mesoporous SiO2 (Fe3S4/EW-mSiO2) was used to degrade methoxychlor (MXC) in aqueous environments. The effects of various parameters on the degradation of MXC were determined using free and immobilized laccase. Immobilization improved the thermal stability and reuse of laccase significantly. Under the conditions of pH 4.5, temperature 40 °C, and reaction time 8 h, the degradation rate of MXC by immobilized laccase reached a maximum value of 40.99% and remained at 1/3 of the original after six cycles. The excellent degradation performance of Fe3S4/EW-mSiO2 was attributable to the pyrite (FeS2) impurity in Fe3S4, which could act as an electron donor in reductive dehalogenation. Sulfide groups and Fe2+ reduced the activation energy of the system resulting in pyrite-assisted degradation of MXC. The degradation mechanism of MXC in aqueous environments by laccase immobilized on Fe3S4/EW-mSiO2 was determined via mass spectroscopy of the degradation products. This study is a new attempt to use pyrite to support immobilized laccase degradation.

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Introduction: Methoxychlor (MXC) is an organochlorine pesticide (OCP) that was formerly used worldwide as an insecticide against pests and mosquitoes. However, MXC is not biodegradable and has lipophilic characteristics; thus, it accumulates in organisms and affects reproductive function. MXC, as an estrogenic compound, promotes oxidative stress, induces oxidative stress damage to ovarian follicles, and causes miscarriages and stillbirths in females. In this research endeavor, our primary objective was to explore the ramifications of MXC regarding the developmental processes occurring during the initial stages of embryogenesis in pigs. Methods: In this study, we counted the blastocyst rate of early embryos cultured in vitro. We also examined the reactive oxygen species level, glutathione level, mitochondrial membrane potential, mitochondrial copy number and ATP level in four-cell stage embryos. Finally, apoptosis and DNA damage in blastocyst cells, as well as pluripotency-related and apoptosis-related genes in blastocyst cells were detected. The above experiments were used to evaluate the changes of MXC damage on early parthenogenetic embryo development. Results and Discussion: The results showed that early embryos exposed to MXC had a significantly lower cleavage rate, blastocyst rate, hatching rate, and total cell count compared with the control group. It was also of note that MXC not only increased the levels of reactive oxygen species (ROS), but also decreased the mitochondrial membrane potential (ΔΨm) and mitochondrial copy number during the development of early embryos. In addition, after MXC treatment, blastocyst apoptosis and DNA damage were increased, decreased cell proliferation, and the expression of pluripotency-related genes SOX2, NANOG, and OCT4 was down-regulated, while the expression of apoptosis-related genes BAX/BCL-2 and Caspase9 was up-regulated. Our results clearly show that MXC can have deleterious effects on the developmental processes of early porcine embryos, establishing the toxicity of MXC to the reproductive system. In addition, the study of this toxic effect may lead to greater concern about pesticide residues in humans and the use of safer pesticides, thus potentially preventing physiological diseases caused by chemical exposure.

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The presence of methoxychlor (MXC) in soil and wastewater is considered a nonnegligible environmental threat. Herein, Fe3O4@Fe(0) was obtained by NaBH4 reduction of Fe3O4 nanoparticles and served as a carrier for laccase to construct catalyst. The catalyst was evaluated for the degradation of MXC in treated wastewater and soil with 2, 2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) being used as cocatalyst. The removal rate of MXC in wastewater and soil was found to be 89% and 88% in optimum conditions, and the influences of initial MXC concentration, pH, and temperature on the degradation rate were evaluated. The metabolites including 2-methylpentane, 3-methylpentane, and n-pentane of MXC were identified, and possible degradation mechanisms were proposed. Overall, this work successfully demonstrates not only the ability to degrade MXC in different circumstances but also provides a new idea for environmental remediation in the future.

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In this paper, we investigated the effects of neonatal exposure to methoxychlor (MXC), a synthetic organochlorine used as an insecticide with estrogenic, antiestrogenic, and antiandrogenic activities on ovarian follicles of adult pigs. Piglets were injected with MXC (20 µg/kg body weight) or corn oil (controls) from postnatal Day 1 to Day 10 (n = 5 per group). Then, mRNA expression, protein abundance and immunolocalization of growth and differentiation factor 9 (GDF9), bone morphogenetic protein 15 (BMP15), anti-Müllerian hormone (AMH) and cognate receptors (ACVR1, BMPR1A, BMPR1B, TGFBR1, BMPR2, and AMHR2), as well as FSH receptor (FSHR) were examined in preantral and small antral ovarian follicles of sexually mature gilts. The plasma AMH and FSH levels were also assessed. In preantral follicles, neonatal exposure to MXC increased GDF9, BMPR1B, TGFBR1, and BMPR2 mRNAs, while the levels of AMH and BMP15 mRNAs decreased. In addition, MXC also decreased BMP15 and BMPR1B protein abundance. Regarding small antral follicles, neonatal exposure to MXC upregulated mRNAs for BMPR1B, BMPR2, and AMHR2 and downregulated mRNAs for AMH, BMPR1A, and FSHR. MXC decreased the protein abundance of AMH, and all examined receptors in small antral follicles. GDF9 and BMP15 were immunolocalized in oocytes and granulosa cells of preantral follicles of control and treated ovaries. All analyzed receptors were detected in the oocytes and granulosa cells of preantral follicles, and in the granulosa and theca cells of small antral follicles. The exception, however, was FSHR, which was detected only in the granulosa cells of small antral follicles. In addition, MXC decreased the plasma AMH and FSH concentrations. In conclusion, the present study may indicate long-term effects of neonatal MXC exposure on GDF9, BMP15, AMH, and FSH signaling in ovaries of adult pigs. However, the MXC effects varied at different stages of follicular development. It seems that neonatal MXC exposure may result in accelerated initial recruitment of ovarian follicles and impaired cyclic recruitment of antral follicles.

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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.

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This study investigated the effects of neonatal exposure to methoxychlor (MXC), a synthetic organochlorine used as an insecticide with estrogenic, antiestrogenic, and antiandrogenic activities, on luteal function in pigs. Piglets were injected subcutaneously with MXC (20 µg/kg body weight) or corn oil (control) between postnatal Days 1 and 10 (N = 5/group). Corpora lutea from sexually mature gilts were examined for luteal steroid and prostaglandin concentrations and processed for total RNA isolation and subsequent RNA sequencing. Intra-luteal concentrations of androstenedione and prostaglandin E2 were greater, while that of estrone was lower when compared to control. Fifty-three differentially expressed (DE) microRNAS (miRNAs) (p-adjusted <.05 and log2(fold change) ≥.5) and 359 DE genes (p-adjusted <.05 and log2(fold change) ≥1) were identified in luteal tissue in response to neonatal MXC treatment. MXC was found to affect the expression of genes related to lipogenesis, steroidogenesis, membrane transport, immune response, cell signaling and adhesion. These results suggest an earlier onset of structural luteolysis in pigs caused by MXC actions in neonates. Since negative correlation analysis showed the potential interactions of miRNAs with specific messenger RNAs, we propose that these miRNAs are potential mediators of the long-term MXC effect on the CL function in pigs.

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Being the largest agriculture country in the Arab world, Egypt was one of the major consumer of organochlorine pesticides (OCPs) in this area, continued to have a heavy burden of OCPs in the environment. There is growing concern that OCPs could pass from the maternal circulation through the placenta to the fetal circulation and pose several health risks to their fetuses. The current study was intended to identify OCPs residue exposure in healthy pregnant women and to justify the potential impacts of these residues on their fetuses. In this study, the prevalence of 18 OCPs was estimated in 81 maternal and cord blood samples, using Agilent 7890, gas chromatograph equipped with micro-electron capture detector (GC-µECD). Our data signposted that the heptachlor epoxide has the highest detection rate among all residues in both maternal (32%) and cord blood serum (27.16%). DDTs were still quantifiable, but with the lowest quantifiable percentage. More than 85% of mothers' serum with detectable residues transfer OCPs residue to their fetuses in a statistically significant manner (x = 42.9, p value < 0.001). The present findings showed no significant growth retardation, or preterm delivery induced by in utero exposure to the most abundant residues. There is growing evidence that exposure to OCPs residue has profound impact on sex ratio. Methoxychlor, in this study be deemed as testosterone triggers which yields high boys ratio (x = 4.37, p < 0.05). In conclusion, Egypt continued to have a heavy burden of OCPs residues, and fetuses and infants are especially the most vulnerable groups to their adverse health effects. Exposure to OCPs may disrupt the maternal hormones, which regulate the offspring gender, but these results need to be validated in larger sample sizes.

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There is an ample epidemiological evidence to support the role of environmental contaminants such as bisphenol A (BPA) in breast cancer development but the molecular mechanisms of their action are still not fully understood. Therefore, we sought to analyze the effects of three common contaminants (BPA; 4-tert-octylphenol, OP; hexabromocyclododecane, HBCD) on mammary epithelial cell (HME1) and MCF7 breast cancer cell line. We also supplied some data on methoxychlor, MXC; 4-nonylphenol, NP; and 2-amino-1-methyl-6-phenylimidazo [4-b] pyridine, PhIP. We focused on testing the prolonged (two months) exposure to low nano-molar concentrations (0.0015-0.0048 nM) presumed to be oncogenic and found that they induced DNA damage (evidenced by upregulation of pH2A.X, pCHK1, pCHK2, p-P53) and disrupted the cell cycle. Some agents induced epigenetic (methylation) changes of tumor suppressor genes TIMP3, CHFR, ESR1, IGSF4, CDH13, and GSTP1. Obviously, the accumulation of these molecular alterations is an essential base for cancer development. Consistent with this, we observed that these agents increased cellular invasiveness through collagen. Cellular abilities to form colonies in soft agar were increased for MCF7. Toxic agents induced phosphorylation of protein kinase such as EGFR, CREB, STAT6, c-Jun, STAT3, HSP6, HSP27, AMPKα1, FAK, p53, GSK-3α/ß, and P70S6 in HME1. Most of these proteins are involved in potential oncogenic pathways. Overall, these data clarify the molecular alterations that can be induced by some common environmental contaminants in mammary epithelial cells which could be a foundation to understand environmental carcinogenesis.

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Chiral mesoporous silica (SiO2) with helical structure was synthesized by using anionic surfactants as template. Pre-prepared graphene oxide (GO) was then loaded onto SiO2 to synthesize composite carrier chial-meso-SiO2@GO for the immobilization of laccase. The enzyme activity, thermostability, acid stability, and repeatability of the immobilized enzyme were significantly improved after immobilization. The chial-meso-SiO2@GO-immobilized laccase was then used for the degradation of MXC in aqueous phase. The degradation conditions, including temperature, time, pH, MXC concentration, and the dose of immobilized enzyme for cellulosic hydrolysis, were optimized. The optimum conditions for degradation of methoxychlor were selected as pH 4.5, MXC concentration 30 mg/L, immobilized enzyme dose 0.1 g, the maximum MXC removal of over 85% and the maximum degradation rate of 50.75% were achieved after degradation time of six h at temperature of 45 °C. In addition, the immobilized cellulase was added into the immobilized laccase system to form chial-meso-SiO2@GO-immobilized compound enzyme with the maximum MXC degradation rate of 59.58%, higher than that of 50.75% by immobilized laccase. An assessment was made for the effect of chial-meso-SiO2@GO-immobilized compound enzyme on the degradation of MXC in soil phase. For three contaminated soils with MXC concentration of 25 mg/kg, 50 mg/kg, and 100 mg/kg, the MXC removals were 93.0%, 85.8%, and 65.1%, respectively. According to the GC-MS analyses, it was inferred that chial-meso-SiO2@GO-immobilized compound enzyme had a different degradation route with that of chial-meso-SiO2@GO-immobilized laccase. The hydrolysis by immobilized cellulase might attack at a weak location of the MXC molecule with its free radical OH and ultimately removed three chlorine atoms from MXC molecule, leading to generating small molecular amount of degradation product.

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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.

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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.

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Methoxychlor is primarily used as an insecticide and it is widely present in the environment. The objective of the present study was to investigate the direct effects of methoxychlor and its metabolite hydroxychlor (HPTE) on rat neurosteroidogenic 3α-hydroxysteroid dehydrogenase (AKR1C14) and retinol dehydrogenase 2 (RDH2) activities. Rat AKR1C14 and RDH2 were cloned and expressed in COS-1 cells, and the effects of methoxychlor and HPTE on these enzymes were measured. HPTE was more potent to inhibit AKR1C14 and RDH2 activities than methoxychlor, with IC50 values of 2.602 ± 0.057 µM and 20.473 ± 0.049 µM, respectively, while those of methoxychlor were over 100 µM. HPTE competitively inhibited AKR1C14 and RDH2 when steroid substrates were used, while it showed a mode of mixed inhibition on these enzymes when NADPH/NAD+ were used. We elucidated the binding mode of methoxychlor and HPTE to the crystal structure of AKR1C14 by molecular docking and found that HPTE had higher affinity with the enzyme than methoxychlor. In conclusion, HPTE is more potent than methoxychlor to inhibit both AKR1C14 and RDH2.

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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.

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The association between genetic variations in the cytochrome P450 (CYP) family genes and pathological conditions related to long-term exposure to organochlorine compounds (OCs) deserves further elucidation. OCs are persistent organic pollutants with bioaccumulative and lipophilic characteristics. They can act as endocrine disruptors and perturb cellular mechanisms. Prolonged exposure to OCs has been associated with different pathological manifestations. CYP genes are responsible for transcribing enzymes essential in xenobiotic metabolism. Therefore, polymorphisms in these genetic sequences a. alter the metabolic pathways, b. induce false cellular responses, and c. may provoke pathological conditions. The main aim of this review is to define the interaction between parameters a, b and c at a mechanistic/molecular level, with references in clinical cases.

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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.

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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.

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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.

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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.

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