Methoxychlor Research Articles

Estrogenic potencies of several environmental pollutants, as determined by vitellogenin induction in a carp hepatocyte assay.

Estrogenic potencies of several xenoestrogens were determined in vitro, using cultured hepatocytes from a genetically uniform male carp strain (Cyprinus carpio). Estrogenicity was measured as induction of the yolk protein precursor vitellogenin (Vtg), and compared to Vtg induction by 17beta-estradiol (E2). The order of estrogenic potency was: methoxychlor (MXCL) > o,p-DDT > chlordecone approximately/= bisphenol-A approximately/= 4-t-pentylphenol. Estrogenic potencies of these compounds varied from 1 x 10(-3) to 1 x 10(-4) relative to E2. The synthetic estrogen DES had a relative estrogenic potency of 0.5, whereas dieldrin, beta-endosulfan, o,p-DDE, and toxaphene (technical mixture) did not induce vitellogenesis at concentrations up to 100 microM. Experiments in which cells were simultaneously exposed to E2 and these xenoestrogens showed that the Vtg-inducing activities of E2 and 4-t-pentylphenol or bisphenol-A were (partially) additive, whereas E2 antagonized the estrogenic effects of MXCL and o,p-DDT. The effect of cytochrome P4501A (CYP1A)-induction on the estrogenicity of MXCL was studied by co-exposing cells to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). TCDD (10 pM) caused a greater than 50-fold induction of CYP1A, measured as ethoxyresorufin O-deethylase (EROD) activity, but Vtg induction by MXCL was not significantly affected. This indicates that CYP1A is not involved in the bioactivation of MXCL to more potent estrogenic metabolites in carp. The CARP-HEP (hepatocyte) assay can detect xenoestrogens with a potency > or = 2 x 10(-5) relative to E2. It allows simultaneous testing of more than 10 compounds for both estrogenic and antiestrogenic effects, which makes it a promising tool for the screening of suspected xenoestrogens.

Inhibitory effects of organochlorine pesticides on intercellular transfer of lucifer yellow in cultured bovine oviductal cells

The present study investigated the effects of dichlorodiphenyltrichloroethane (DDT), methoxychlor (MXC), and γ-hexachlorocyclohexane (γHCH, lindane) on gap junctional intercellular communication (GJIC) in cultured bovine oviductal cells. GJIC was evaluated by microinjecting fluorescent dye Lucifer Yellow and observing the inhibition of the spreading of dye into adjacent cells. After incubation for 1 h at 37°C, a dose-dependent inhibition of GJIC was observed over a concentration range of 16 to 128 μ m DDT, MXC, or γHCH compared with nonexposed controls. A significant inhibition began at 32 μ m DDT, MXC, or γHCH. After incubation for 5 h, a dose-dependent inhibition of GJIC was obtained in the concentration range from 8 to 64 μ m of the pesticides. The first significant inhibitory effect on GJIC was caused by 8 μ m DDT, 16 μ m MXC, and 32 μ m γHCH. The 128 μ m concentration of the pesticides was toxic. At pesticide concentration of 64 μ m, the decrease in dye-coupling observed was not due to lethal cell injury, as is indicated by the use of trypan blue dye exclusion. After removal of 64 μ m DDT from the culture medium, intercellular communication was reestablished within 3 h. Measurement of cytosolic free Ca 2+ concentration [Ca 2+] i in fura-2/AM-loaded oviductal cells showed that the inhibition of GJIC by addition of DDT, MXC, or γHCH was not associated with a detectable increase in [Ca 2+] i. Coincubation of the DDT with dibutyryl-cAMP prevented the 64 μ m DDT-induced inhibition of intercellular communication in adherent oviduct cells. It is suggested that organochlorine pesticides can influence cells responsible for reproduction.

Effects of Methoxychlor on Directional and Fluctuating Asymmetry of Mandible Characters in Mice

Two kinds of asymmetries are common in bilateral characters: fluctuating asymmetry (FA), a type of nondirectional variation between left and right sides, and directional asymmetry (DA), in which one side is consistently larger than the other. FA has been extensively used as a measure of developmental instability and is presumed to increase as stress from toxins or other sources increases. In this paper we tested the hypothesis that exposure of pregnant female mice to two different dosage levels of methoxychlor (MXC), an insecticide derived from DDT, would increase FA in one or more of 10 mandible characters measured in the offspring of these females. The data showed that exposure of pregnant females to MXC produced no detectable effect on FA in their offspring, but at the higher dosage level (10 μg/g body weight), did change FA to DA in one of the characters. MXC also had a significant effect on the mean size of five of the mandible dimensions, all of which were located primarily in the anterior portion of the mandible.

Neonatal exposure to technical methoxychlor alters pregnancy outcome in female mice

This study was designed to determine the ability of female mice who were exposed neonatally to the pesticide methoxychlor (MXC) to mate, ovulate, and become pregnant upon reaching sexual maturity. One-day-old female mice (5 to 8/group) were exposed daily by intraperitoneal (ip) injection for 14 d to either sesame oil or 10 μg estradiol-17β or 0.1, 0.5 or 1.0 mg MXC suspended in sesame oil. The MXC exposures corresponded to 14 to 71, 68 to 357, or 135 to 714 mg/kg body weight, respectively. Three months later, female mice were placed with proven breeder males and checked daily for vaginal plugs. Mated female mice were sacrificed 18 d after the appearance of a vaginal plug to evaluate pregnancy. Uteri were examined for the presence of living fetuses and/or resorption sites. Ovaries were removed and prepared for histologic evaluation and tabulation of corpora lutea. All mice from all three MXC-treated groups did in fact mate, in comparison with only one of those exposed neonatally to estradiol. Increasing the dose of MXC produced a decreased number of pregnant animals at 18 d following mating. The mean number of live fetuses/litter was reduced in the 0.5 and 1.0 mg MXC-treated groups. Corpora lutea were significantly reduced in ovaries from only the 1.0 mg MXC group and the estradiol group. No effects of treatment were seen at 0.1 mg MXC. It is concluded that neonatal exposure to MXC does not interfere with mating. Instead, significant alterations are seen in initiating and/or maintaining pregnancy. The deleterious effects on pregnancy may be due to the influence of neonatal MXC treatments on the hypothalamic–pituitary–ovarian axis as well as on possible alteration of the uterine environment.

Influence of organochlorine pesticides on maturation and postfertilization development of bovine oocytes in vitro

The aim of this study was to perform a dose–response test to determine whether bovine oocytes exposed to dichlorodiphenyltrichloroethane (DDT), hexachlorocyclohexane (γHCH), or methoxychlor (MXC) in vitro would exhibit changes in maturation rates, cleavage rates at Day 2, or blastocyst rates at Day 7 to 8 after fertilization in vitro (IVF). All three pesticides affected maturation and degeneration rates in a dose-dependent manner, but to different extents. Higher concentrations of pesticides were associated with higher rates of chromatin degeneration. Because the maturation of bovine oocytes was depressed in a dose-dependent manner, the fertilizability and further embryonic development of in vitro matured oocytes was studied at the lowest previously tested concentration (7.25 μg/mL) only. No significant difference in fertilization rates was seen between unexposed control and treated groups. The cleavage rates did not differ among groups 48 h after IVF. The number of morulae and blastocysts on Day 7 to 8 after IVF, which is commonly used as a parameter for normal development, was significantly different between control and DDT- and γHCH-treated groups, but not between the control and MXC groups. The pesticides did not differ significantly among themselves. These results show that the tested pesticides decrease the rate of normal oocyte maturation in vitro in a dose-dependent manner. The effect of the lowest concentration of pesticides is seen only after Day 7 of embryo development.

Biotransformation and estrogenic activity of Methoxychlor and its metabolites in channel catfish ( Ictalurus punctatus)

Methoxychlor (MXC) has been shown to possess estrogenic activity in mammals and fish. Although MXC does not appear to appreciably bind the mammalian estrogen receptor, its demethylated metabolites have been shown to be significantly more potent agonists and are believed responsible for estrogenic effects in mammals following exposure to this pesticide. To determine whether catfish were capable of MXC demethylotion, and, hence, activation to a more estrogenic compound, in vitro biotransformation studies were carried out using hepatic microsomes from mature male channel catfish. Hepatic microsomes catalyzed the NADPH-dependent formation of monodemethylated (mono-MXC) and bisdemethylated (bis-MXC) metabolites of MXC. Treatment with mono-MXC at 40% of the MXC dose in catfish significantly induced serum vitellogenin (Vg) levels compared to MXC. Estrogen receptor binding studies in catfish liver cytosol showed that a racemic mixture of the mono-MXC had approximately 43 times the affinity for the receptor than MXC, but was still over 1000-fold less potent that 17β-estradiol. These results demonstrate that catfish are capable of biochemically activating MXC to a more potent hepatic estrogen receptor agonist.

Xenobiotic Interaction with and Alteration of Channel Catfish Estrogen Receptor

In teleostean in vivostudies, the vitellogenin response to environmental estrogens is not completely predicted by mammalian literature. One possible explanation for differences is heterogeneity of the estrogen receptor (ER) structure between species. Therefore, ER from channel catfish ( Ictalurus punctatus) hepatic tissue was characterized by binding affinity for several compounds. Affinity was indirectly measured as potency of the chemical for inhibiting binding of radiolabeled estradiol (E2) to specific binding sites. The order of potency among therapeutic chemicals was ethinylestradiol > unlabeled E2 = diethylstilbestrol > mestranol > tamoxifen ⪢ testosterone. Unlabeled E2 had an IC50 of 2.2 n m. Several environmentally relevant chemicals were evaluated in a similar manner and the order of potency established was the o-demethylated metabolite of methoxychlor (MXC) > nonylphenol (NP) > chlordecone > MXC > o,p′-DDT > o,p′-DDE > β-hexachlorocyclohexane. Demethylated MXC had an IC50 1000-fold greater than that of E2. Of the most potent inhibitors, NP appeared to be a competitive inhibitor for the same binding site as E2, while o-demethylated MXC had a more complex interaction with the receptor protein. ER from nonvitellogenic females was determined to have a K d value of 1.0 to 1.3 n m. Because E2 has been reported to up-regulate teleostean ER, the hepatic ER population following in vivoxenobiotic exposure was assessed. NP significantly increased ER per milligram hepatic protein almost to the same extent as E2, but did not increase K d to the same extent as E2.

The effects of perinatal/juvenile methoxychlor exposure on adult rat nervous, immune, and reproductive system function.

In order to address data gaps identified by the NAS report Pesticides in the Diets of Infants and Children, a study was performed using methoxychlor (MXC). Female rats were gavaged with MXC at 0, 5, 50, or 150 mg/kg/day for the week before and the week after birth, whereupon the pups were directly dosed with MXC from postnatal day (pnd) 7. Some dams were killed pnd7 and milk and plasma were assayed for MXC and metabolites. For one cohort of juveniles, treatment stopped at pnd21; a modified functional observational battery was used to assess neurobehavioral changes. Other cohorts of juveniles were dosed until pnd42 and evaluated for changes to the immune system and for reproductive toxicity. Dose-dependent amounts of MXC and metabolites were present in milk and plasma of dams and pups. The high dose of MXC reduced litter size by approximately 17%. Ano-genital distance was unchanged, although vaginal opening was accelerated in all treated groups, and male prepuce separation was delayed at the middle and high doses by 8 and 34 days, respectively. In the neurobehavioral evaluation, high-dose males were more excitable, but other changes were inconsistent and insubstantial. A decrease in the antibody plaque-forming cell response was seen in males only. Adult estrous cyclicity was disrupted at 50 and 150 MXC, doses which also showed reduced rates of pregnancy and delivery. Uterine weights (corrected for pregnancy) were reduced in all treated pregnant females. High-dose males impregnated fewer untreated females; epididymal sperm count and testis weight were reduced at the high, or top two, doses, respectively. All groups of treated females showed uterine dysplasias and less mammary alveolar development; estrous levels of follicle stimulating hormone were lower in all treated groups, and estrus progesterone levels were lower at 50 and 150 MXC, attributed to fewer corpora lutea secondary to ovulation defects. These data collectively show that the primary adult effects of early exposure to MXC are reproductive, show that 5 mg/kg/day is not a NO(A)EL in rats with this exposure paradigm (based on changes in day of vaginal opening, pubertal ovary weights, adult uterine and seminal vesicle weights, and female hormone data) and imply that the sites of action are both central and peripheral.

Effect of methoxychlor on implantation and embryo development in the mouse

Methoxychlor (MXC) has been shown to have adverse effects on reproductive functions. However, it has not been fully determined whether the effects of MXC on reproduction are due to its estrogenic or antiestrogenic effects. Therefore, to further elucidate the estrogenic action of this pesticide in the mouse, we studied the effect of MXC on implantation and embryo development. MXC was found to initiate implantation in most delayed implanting mice at 400 μg/g body weight. However, at the higher dose of 800 μg/g body weight, MXC initiated implantation in only 50% of animals and the number of embryos implanting was significantly decreased ( P < 0.05). It was determined that MXC inhibited implantation in intact pregnant mice only when given on Day 1 or Day 2 at 800 μg/g but not at lower doses or later in the preimplantation period. Embryonic development and transport were delayed on Days 3 and 4 in these animals. Finally, reciprocal embryo transfers with donor embryos recovered from MXC-treated animals (800 μg/g body weight on Day 1) transferred to untreated recipients resulted in no implantation compared to 79% implantation when donor embryos were treated with vehicle. These data indicate that MXC acts as an estrogen agonist at the level of the uterus and oviduct but as an antiestrogen in the ovary. In addition, MXC appears to alter normal preimplantation embryonic development. These results suggest the need for further studies to assess the mechanism of action of MXC in preimplantation embryos.

The Effects of Methoxychlor on Early Sea Urchin Development

Methoxychlor (MXC) is a widely used pesticide which has been found in water sources near agricultural sites. Embryos of aquatic organisms are likely to encounter MXC due to land runoff. The sea urchin embryo (Strongylocentrotus purpuratus) was used as a model system to document the effects of MXC on early development up to the pluteus stage. Fertilized eggs and embryos were exposed to several concentrations (0.1, 1.0, 10, and 100 ppm) of the pesticide in both chronic and acute exposure regimens. With chronic exposure, percentages of embryos completing normal first cleavage decreased with increased concentrations of MXC, and subsequent cleavages became even more irregular in that blastomeres divided asymmetrically and asynchronously. Ten parts per million MXC allowed development through the hatched blastula stage, whereas embryos in 100 ppm MXC did not hatch. In acute exposure trials, fertilized eggs were pulsed (i.e., exposed for brief durations) to MXC for 30, 60, or 90 min. The MXC was then washed out. Recovery of normal development was proportional to the amount and duration of MXC exposure. Development was delayed in embryos exposed to 100 ppm MXC for 30 or 60 min. The embryos exposed to 100 ppm MXC for 90 min were abnormal as early as the four-cell stage, and by 72 hr more than 90% had abnormal gut development, indicating disruption of gastrulation. These data show that MXC exposure resulted in retardation of cleavage and abnormal gastrulation, basic morphogenetic processes.

Decreased superovulation in adult mice following neonatal exposures to technical methoxychlor

To examine the effects of technical methoxychlor (MXC) on superovulation, neonatal mice received intraperitoneal (i.p.) injections of either sesame oil, 10 μg of estradiol 17β, or 0.1, 0.5, or 1 mg of technical MXC. At 2 and 4 months, half of the mice received a superovulatory regimen of 10 IU pregnant mare's serum gonadotropin followed by 10 IU human chorionic gonadotropin. The mice were sacrificed 15 to 20 h later, the number of ovulated oocytes were counted, and the ovaries were removed for histology. In the lowest MXC dose, the ovaries appeared normal and at 2 months, ovulated the same number of oocytes as controls. Estradiol or the highest two MXC closes induced ovarian atrophy. Following gonadotropin injections, these ovaries also ovulated oocytes. However, the number of oocytes recovered from experimental mice exhibited a time- and dose-dependent decline, and by 4 months, their number was significantly reduced. Neonatal exposures to MXC reduces ovulatory rates and ovarian functions in adults.

Methoxychlor mimics the action of 17β-estradiol on induction of uterine epidermal growth factor receptors in immature female rats

Epidermal growth factor (EGF) and its receptor (EGF-R) have been implicated as mediators for estrogen induced cellular growth. This study examines whether the action of the estrogenic pesticide methoxychlor (MXC) parallels the action of 17β-estradiol (E 2) on uterine EGF-R. Administration of 20 μg E 2/sexually immature female rat increased 125I-EGF binding to membranes extracted from whole uteri 175% over endogenous levels, while 500 mg MXC/kg led to a 156% increase. E 2 in both 20 and 40 μg/rat doses and 500 mg MXC/kg led to maximal stimulation over endogenous levels, 12-h posttreatment. Rats were treated with E 2, MXC, or vehicle plus 100 μg actinomycin-D (ACT-D) or 100 μg cycloheximide (CYCLO) per rat to determine if mRNA transcription and translation are involved in the increased EGF-R binding following estrogenic treatment. Only ACT-D inhibited the estrogenic stimulation of EGF-R binding, resulting in a 44% decrease when given concurrently with E 2 or MXC, suggesting transcription is required. Additionally, ACT-D decreased endogenous receptor levels by 55%. No other differences were detected. When EGF-R binding data were analyzed by the method of Scatchard, both E 2 and MXC, at maximal dosages, elevated uterine EGF-R binding sites by over 200% after 12 h as measured by maximal binding ( B max) with no significant difference in dissociation constant ( K d) values. These results demonstrate that both E 2 and MXC can stimulate the number of EGF-R binding sites without significantly altering the receptor binding affinity ( K d). Further, this stimulation is time dependent and is affected by dose.

Estradiol or methoxychlor stimulates estrogen receptor (ER) expression in uteri

The estrogenic pesticide methoxychlor (MXC) abnormally alters reproduction in rodents. The influence of MXC on expression of the estrogen receptor (ER) gene was investigated in the uterus of BALB/c mice and compared to the effect of estradiol. Uterine epithelium in control mice is devoid of ER until 5 d of life. Sesame oil, 10.0 μg estradiol 17β or 1.0 mg of base-washed 95% MXC were injected into neonates (days 1 to 4) and immature mice (days 10 to 14), after which they were sacrificed. By day 5, E or MXC induced production of nuclear ER mRNA and its translation in uterine epithelium. The mRNA was detected by in situ hybridization and ER protein by immunocytolocalization. In controls, ER was present only in stroma and myometrium. By day 15, the nuclei of uterine epithelium, stroma, and myometrium expressed the ER gene, with no apparent influence of E or MXC. Both E and MXC induce premature nuclear expression of the ER gene in neonatal uterine epithelium, without apparent change in ER gene expression between days 10 and 14 of life.

Influence of organochlorine pesticides on development of mouse embryos in vitro

The effect of DDT, methoxychlor (MXC), and γ-hexachlorocyclohexane (γHCH) on preimplantation mouse embryos in culture was evaluated by the proportion of eight-cell stage embryos developing to expanded and hatched blastocysts. A concentration of 29 μg/mL DDT or γHCH led to a complete degeneration of exposed embryos. The addition of 29 μg/mL MXC showed blastocyst formation (72.3%), but fewer than half of the blastocysts were intact and only 4.3% hatched from the zona pellucida. A concentration of 14.5 μg/mL DDT led to 57.5% blastocyst formation and to 6.8% hatched blastocysts; the same concentration of γHCH and MXC showed 66.7 and 95.9% blastocyst formation and 22.9 and 49% hatched blastocysts, respectively. The addition of 7.25 μg/mL led to 85.9% (DDT), 77.6% (γHCH), and 81% (MXC) blastocysts in total, and to 28.2, 35.5, and 49.4% hatched blastocysts, respectively. Compared with untreated controls, only the 3.625 μg/mL concentration for all pesticides showed no significant differences in blastocyst formation and hatching. These results show different toxicity of these three pesticides for the preimplantation mouse embryo in the order MXC < γHCH ⩽ DDT).

Influence of organochlorine pesticides on physiological potency of cultured granulosa cells from bovine preovulatory follicles

In the present study the direct effect of Dichlorodiphenyltrichloroethane (DDT), Methoxychlor (MXC) and Hexachlorocyclohexane (γHCH) was investigated on the DNA synthesis (measured by [ 3H]thymidine incorporation); proliferation (determined by colorimetric MTT[ 3-[4,5-dimethylthiazol-2yl]-2,5-diphenyl tetrazolium bromide] assay); and steroidogenesis (estradiol 17β, progesterone determination) of granulosa cells during in vitro culture. The cells were aspirated from preovulatory follicles in PMSG-treated heifers. They were cultivated in phenol red and serum (0.1 % FCS) RPMI 1640 medium without or with the 3 pesticides in different concentrations. In summary, the results showed differential inhibition of parameters named above by these pesticides (γHCH <MXC<DDT) in comparison with vehicle-treated control. Progesterone synthesis was more inhibited than estradiol 17β production by 3 chemicals, which resulted from the loss of viability of cells indicated by estimation of cell-shape-factor and viability of cells. Besides the inhibitory effect, there was a stimulatory effect of low concentrations of DDT and MXC on [ 3H]thymidine incorporation and proliferation.

Effects of Estrogen, Progesterone, and Methoxychlor on Surgically Induced Endometriosis in Rats

Effects of Estrogen, Progesterone, and Methoxychlor on Surgically Induced Endometriosis in Rats. Cummings, A. M., and Metcalf, J. L. (1995). Fundam. Appl. Toxicol. 27, 287-290. Endometriosis is a disease of women where endometrial tissue is found growing at ectopic sites. While evidence suggesting a role for the ovarian hormones in endometriosis exists, no complete studies of the roles of estrogen and progesterone have heretofore been performed. Also, if estrogen has a role in the growth and/or maintenance of endometriosis, it is likely that the proestrogenic pesticide, methoxychlor (MXC), might also have such an effect. Sixty rats underwent surgery on Day 0 to induce endometriosis. On Day 21, all rats were ovariectomized. During surgery, the diameters of all endometriotic implants (which were fully developed) were measured. Starting on Day 21, groups of rats were treated daily, for 3 weeks, with (a) vehicle; (b) estrone, 1 μg/rat, E; (c) progesterone, 2 mg/rat, P; (d) E + P, 1 μg + 2 mg; (e) MXC, 250 mg/kg; or (f) MXC + P, 250 mg/kg + 2 mg/rat. On Day 42, all rats were killed, and the diameters of all endometriotic sites were measured. While no differences in diameter were found across groups prior to ovariectomy, ovariectomy plus treatment altered the growth of endometriotic tissue. Progesterone and vehicle treatments produced results that were identical: regression of endometriotic sites. Both estrogen and MXC treatments maintained endometriotic site size at a level greater than that in the vehicle-treated group. The combination of progesterone with either estrone or MXC did not alter the effect of either chemical. We conclude that while estrogen promotes the growth of endometriosis, progesterone either produces regression or fails to maintain the sites. MXC, at a relatively high dose, supports the development of endometriosis. Concurrent progesterone treatment does not modulate the effects of estrone or MXC. These results suggest that exposure of women to high doses of MXC may exacerbate the development of endometriosis or contribute to its recurrence.

Neonatal exposures to technical methoxychlor alters ovaries in adult mice

Technical grade methoxychlor (MXC) is an estrogenic pesticide currently used for pest control in the US. To determine the long-term effects of technical MXC on ovaries and estrous cycles, neonatal mice received 14 daily intraperitoneal injections of sesame oil only, 10.0 μg estradiol-17β, or 0.05 mg, 0.1 mg, 0.5 mg, or 1.0 mg of technical MXC. At 3, 6, and 12 months, vaginal smears were examined for 12 d and ovaries collected. All technical MXC doses and estradiol increased the duration of vaginal cornification but only estradiol, 0.5, or 1.0 mg technical MXC induced ovarian atrophy, relative ovarian weight loss, and depletion of corpora lutea. Technical MXC doses of 0.05 or 0.1 mg produced the opposite effects; the ovaries remained heavy, large, and filled with corpora lutea. On the other hand, follicular cysts were recorded in all groups, except the 1.0 mg MXC group. These augmentary and inhibitory effects of MXC at low and high doses mimic the estrogen effects at low and high doses, and were probably due to the altered hypothalamic-hypophyseal function.

Methoxychlor Regulates Rat Uterine Estrogen-Induced Protein

Methoxychlor (MXC), a proestrogenic pesticide, has adverse effects on fertility and uterine function in rodents. MXC is converted to an estrogenic substance, 2,2-bis-(p-hydroxyphenyl)-1,1,1-trichloroethane (HPTE), which binds to the estrogen receptor. We examined the similarities in mechanism between MXC (500 mg/kg) and estrogen (10 micrograms/rat) actions using the estrogen-induced protein, IP, also known as creatine kinase. Immature, female rats were treated with MXC or estradiol (E2). Concurrent treatment included actinomycin D (100 micrograms), cycloheximide (100 micrograms), or progesterone (0.5 mg). Uterine proteins were labeled in vitro with 3H for treated rats and with 14C for controls. The uteri were combined, cytosol was isolated, non-denaturing (ND) gels were run, and dpm/gel slice were plotted. In a follow-up study, labeled cytosols from MXC- and E2-treated rats were immunoprecipitated with a monoclonal antibody to creatine kinase. The immunoprecipitate was run on SDS gels. The data show that both MXC and E2 treatments result in ND gels with peaks in (a) induced protein and (b) the 3H/14C ratio, in the same position. The induction of IP by MXC is time- and dose-dependent. Concurrent treatment with MXC plus actinomycin D or cycloheximide blocked IP synthesis, a result parallel to E2 action signifying the necessity of RNA and protein synthesis for IP induction. Progesterone did not block either MXC or E2 induction of IP synthesis. Immunoprecipitation of creatine kinase revealed a single peak at a molecular weight of approximately 49,000. SDS gels of cytosol after MXC or E2 treatment also yielded protein and ratio peaks at molecular weights of approximately 49,000. This estimate is near the published estimated molecular weight of creatine kinase of 46,000. We conclude that MXC action parallels that of estradiol on the induction and regulation of the estrogen-induced protein in immature rat uterus.

Effect of environmental pollutants on maturation of bovine oocytes in vitro

A study was designed to evaluate ameliorative effect of propolis against methoxychlor (MXC) induced ovarian toxicity in rat. The organochlorine pesticide (MXC) is a known endocrine disruptor with estrogenic, anti-estrogenic, and anti-androgenic properties. To investigate whether chronic exposure to MXC could cause ovarian dysfunction, two groups of Sprague–Dawley adult female rats were exposed to MXC alone in a dose of 200 mg/kg, twice/weekly, orally or MXC dose as previous plus propolis in a dose of 200 mg/l/day, in drinking water for 10 months. Another two groups of rat were given corn oil (control) or propolis. Multiple reproductive parameters, ovarian weight, serum hormone levels, ovarian oxidative status and ovarian morphology were examined. In MXC-exposed group, there is a significant decrease in body and ovarian weight vs. control. MXC decreases serum estradiol and progesterone levels. A significant increase in the levels of lipid peroxidation was obtained while a significant decrease of the total antioxidant was recorded. Ovarian histopathology showed primary, secondary and vesicular follicles displaying an atretic morphology. Increase in the ovarian surface epithelium height accompanied with vacuolated, pyknotic oocytes were obtained. The previous toxic effects were neutralized by the administration of propolis in MXC + propolis group. The present results suggest that propolis may be effective in decreasing of MXC-induced ovarian toxicity in rat.

Mechanisms of the stimulation of rat uterine peroxidase activity by methoxychlor

Methoxychlor (MXC) has adverse effects on fertility and rat uteri via its active metabolite HPTE (2,2-bis( p-hydroxyphenyl)-1,1,1-trichloroethane). Uterine peroxidase, a marker of estrogen action, was used to probe potential mechanisms of MXC's adverse effects. Specifically, our objective was to compare the regulation of the effects of estrogen and MXC on uterine peroxidase. Immature female rats were treated with MXC (250 mg/kg; gavage) 24 h prior to the measurement of uterine peroxidase activity, with or without concurrent treatment with actinomycin D, cycloheximide, progesterone, or tamoxifen. MXC alone produced an increased in peroxidase activity. The prior and/or concurrent treatment with the compounds listed blocked the MXC-induced stimulation of peroxidase. These data show similarities between the mechanisms of estrogen MXC action. Both estrogen and MXC act to stimulate uterine peroxidase activity via increased RNA and protein synthesis and this stimulation can be blocked by progesterone and tamoxifen.