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PubMed2006

Calbindin-D9k mRNA expression in the rat uterus following exposure to methoxychlor: a comparison of oral and subcutaneous exposure.

Calbindin-D(9k) (CaBP-9k) is a cytosolic calcium-binding protein that is induced by estrogenic compounds possibly through estrogen receptors. We compared CaBP-9k mRNA expression in the uterus with uterotrophic response in immature rats exposed to methoxychlor (MC), an environmental chemical with estrogenic activity. MC was orally or subcutaneously administered to 3-week-old female Sprague-Dawley rats for 3 days. The weights of the uterus and vagina significantly increased in the oral treatment group at a dose of 50, 100 and 200 mg/kg, but those of the subcutaneous (SC) treatment group only increased at 200 mg/kg. Northern blot analysis showed that CaBP-9k mRNA expression was significantly induced in a dose-dependent manner at doses of 50, 100 and 200 mg/kg/day in the oral treatment group. SC administration of MC induced significant expression at only a dose of 200 mg/kg/day; this was similar to the uterotrophic response. MC has an estrogenic effect on the uterus as shown by the increase in weight and induction of CaBP-9k mRNA expression, which were much greater following exposure via oral gavage than via the SC route. The strong correlation between the results of in vivo uterotrophic assay and CaBP-9k mRNA expression suggests that CaBP-9k mRNA expression in the rat uterus may be used as an early gene marker for detection of the estrogenic effects of putative environmental chemicals.

Administration, Oral↗

PubMed2004

Effects of methoxychlor exposure during perinatal period on reproductive function after maturation in rats.

Methoxychlor (MXC) is a non-steroidal pesticide that is known to possess estrogenic activity, and therefore may be potentially hazardous to the development and/or reproduction. The present study assessed the effects of perinatal exposure to MXC on reproductive function after maturation in both male and female rats. Pregnant rats were fed a phytoestrogen-free diet containing different doses of MXC (24, 240, and 1200 ppm) from day 15 of gestation to day 10 after parturition, and reproductive functions of offspring were examined after maturation. In males, MXC exposure during the perinatal period decreased serum LH and FSH, but not testosterone levels, but it did not affect copulatory behavior. In females, MXC exposure prolonged the days exhibiting cornified vaginal smears during the estrous cycle. In addition, both the lordosis reflex and preovulatory LH surge on the presumptive proestrous evening were suppressed in MXC-exposed females. These results suggest that perinatal exposure to MXC exerts permanent effects on several aspects of the reproductive function in both male and female rats.

Animals↗

PubMed2006

Early postnatal methoxychlor exposure inhibits folliculogenesis and stimulates anti-Mullerian hormone production in the rat ovary.

Methoxychlor [1,1,1-trichloro-2,2-bis(4-methoxyphenyl) ethane; MXC] is a chlorinated hydrocarbon pesticide commonly used in the United States as a replacement for DDT [1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane]. While MXC is a weak estrogenic compound, its more active, major metabolite [2,2-bis(p-hydroxyphenyl)-1,1,1-trichloroethane; HPTE] shows estrogenic, anti-estrogenic, or anti-androgenic properties depending on the receptor subtype with which it interacts. Anti-Mullerian hormone (AMH) is a paracrine factor that suppresses initial follicle recruitment in the ovary. Studies have shown the effects of exposure to MXC on adult ovarian morphology and function. However, the effect of exposure to MXC at an early postnatal stage on pre-pubertal follicular development and ovarian AMH production has not been studied. Around postnatal day (P) 4, most of the primordial follicular assembly in rats is complete, and a large number of primordial follicles transition into the primary follicle stage, a process that is inhibited by estrogen. The objective of this study was to examine the effect of early postnatal (P3-P10) MXC exposure on ovarian morphology and size, follicle number, and AMH production in the pre-pubertal (P20) rat ovary and to investigate the effect of HPTE on AMH production in immature rat granulosa cells in vitro. Female rats were injected (s.c.) daily with vehicle (control) or 1, 10, 50, 100, or 500 mg MXC/kg per day (referred to here as 1MXC, 10MXC, and so forth.) between P3 and P10. On P20, uterine and ovarian weights were determined, ovarian histology was examined, and follicles were counted and classified into primordial, primary, secondary, pre-antral, or antral stages using the two largest serial sections at the center of the ovary. Ovarian AMH production was examined using immunohistochemistry and western blot analysis. The effect of HPTE (0.5-25 microM) on AMH production in cultured immature rat granulosa cells was determined by western blot analysis. Ovarian weight was reduced by 50, 100, and 500MXC (P < 0.01). MXC treatment inhibited folliculogenesis. Both 100 and 500MXC had a reduced number of antral follicles (P < 0.05) with a concomitant increase in pre-antral follicles (P < 0.05). Follicle numbers were not significantly affected by 1, 10, or 50MXC. Total follicle number and the number of primordial, primary, or secondary stage follicles were not significantly different in all treatment groups. Immunohistochemistry showed that MXC-treated ovaries had more AMH-positive follicles with stronger AMH immunostaining. Western blot analysis showed that AMH production was 1.6 +/- 0.2, 1.85 +/- 0.6, and 2.2 +/- 0.5 times higher in the 50, 100, and 500MXC ovaries as compared with the control ovaries respectively (P < 0.05). Granulosa cells treated with 1 or 5 microM HPTE had significantly greater AMH production (P < 0.05). These results demonstrate that MXC inhibits early ovarian development and stimulates AMH production directly in the rat ovary. In addition, HPTE was shown to stimulate AMH production in rat granulosa cells. Endocrine disruptors are widespread in the environment, and MXC represents a model endocrine disruptor due to the multiple actions of its metabolites. This study confirms that the endocrine disruptor MXC inhibits follicular development and demonstrates for the first time that MXC and HPTE directly stimulate AMH production in the ovary. This novel finding suggests that elevated AMH may play a role in MXC's inhibitory effect in the ovary.

Animals↗

PubMed2004

Estrogenic and toxic effects of methoxychlor on zebrafish (Danio rerio).

Although zebrafish (Danio rerio) have been suggested as a good candidate for screening potential endocrine disruptors, little information is available on the effects of weak estrogens on this species. We investigated the sensitivity of different life stages of zebrafish toward toxic and estrogenic properties of methoxychlor (MXC). Short-term tests with adults resulted in a sex-specific 96-h lethal concentration for 50% (LC50) of the test animals of 36 microg/L for males and 129 microg/L for females. To determine the estrogenic capacity of MXC, adult zebrafish were exposed to 0, 0.5, 5, and 50 microg MXC/L for 14 d. Induction of vitellogenin ([VTG] measured with protein electrophoresis and Western blot) in males was detected at 5 and 50 microg MXC/L. Females, however, did not exhibit higher blood VTG concentrations at the tested MXC concentrations. In a second series of experiments, juvenile zebrafish were exposed to 0, 0.05, 0.5, and 5 microg MXC/L for 33 d. Survival, length, weight, and condition of larvae were examined as indicators of toxic stress and the VTG content in whole body homogenates of juveniles was measured to determine xenoestrogenic effects. No effects of the tested concentrations of MXC were observed. Finally, the effect of MXC on zebrafish eggs, exposed to 0, 1, 10, and 32 microg MXC/L, was examined. Hatching and survival of hatched zebrafish were affected at 10 and 32 microg MXC/L. This study demonstrated that adult male zebrafish are sensitive toward the estrogenic effects of MXC. However, the use of VTG induction to detect effects of (xeno)estrogens in early life stages has to be further investigated, as low concentrations of VTG were detectable in exposed as well as unexposed juvenile fish.

Animals↗

PubMed2003

Methoxychlor modifies the ultradian secretory pattern of prolactin and affects its TRH response.

BACKGROUND: Methoxychlor (MTX) is an insecticide characterized by both estrogenic and antiandrogenic properties that may modify the secretory pattern of prolactin. The objective of the present work was to analyze MTX's effects on changes in the episodic secretion of prolactin induced by thyrotropin-releasing hormone (TRH). MATERIAL/METHODS: Adult male Sprague-Dawley rats (400-420 g body weight) were administered 25 or 50 mg/kg/day of MTX s.c. in sesame oil for 30 days. Control animals received the vehicle only. RESULTS: In animals treated with 50 mg/kg/day of MTX, mean serum prolactin levels and absolute pulse amplitude increased (p< or = 0.05 and p< or = 0.001 respectively). TRH administration (two pulses of 2 Kg) increased both mean serum prolactin levels and the absolute amplitude of the prolactin peaks in all groups studied (p< or = 0.001). The response to TRH was greatly increased in the animals treated with 50 mg/kg/day. TRH reduced the mean life of the hormone in control rats (p< or = 0.01) and in animals treated with 50 mg/kg/day (p< or = 0.001). After TRH administration, the duration of prolactin peaks was increased in the MTX-treated animals (p< or = 0.05 and p< or = 0.001 for 25 and 50 mg/kg/day respectively). TRH reduced the frequency of prolactin pulses in the animals treated with 50 mg/kg/day (p< or = 0.05). CONCLUSIONS: These data suggest that MTX sensitizes the pituitary to TRH, indicating a direct effect of MTX on the pituitary gland.

Activity Cycles↗

PubMedDate not supplied

The histological changes in some organs of the laboratory mice after intragastrically given bromfenvinphos and mixture of bromfenvinphos with methoxychlor.

Bromfenvinphos (0.0-diethyl-0-1-(2.4-dichlorphenyl)-2-bromvinyl phosphate), BrV, 12.33 mg/kg/day, alone and in combination with methoxychlor ( (1,1,1-trichlor-2.2-)4-methoxyphenyl ethane), MeOCl, 24.66 mg/kg/day, were administered to laboratory mice in the olive-oil suspension through the stomach gauge once a day for 6 weeks. The histological changes developed in liver and kidneys of mice were investigated. BrV and BrV + MeOCl caused the hyperemia and fine-grained inflammatory infiltration in liver tissue. The small histological changes were observed in kidneys of animals which received the pesticides: the stellate shape lumen of the proximal convoluted kidney tubules and the vacuolar degeneration changes in the wall cells of these tubules. There occurred degeneration changes in the wall cells of these tubules. No pathological changes appeared in stomach, small and large intestine, heart, spleen and in gonads of the experimental mice.

Animals↗

PubMed1982

Influence of bromfenvinfos alone and in mixture with methoxychlor on the blood indices of laboratory mice.

Bromfenvinfos (O,O-diethyl-O-1-(2,4-dichlorphenyl)-2-bromvinyl phosphate, BrV, 12.23 mg/kg/day) alone and in combination with methoxychlor (1,1,1-trichlor-2,2-(4-methoxyphenyl) ethane, MeOCl, 24.66 mg/kg/day) were administered per os daily for 6 weeks to male and female laboratory mice in the olive-oil suspension. Total leukocyte and erythrocyte count, hemoglobin level, the percentage of leukocytes and the plasma proteins were determined. BrV and BrV + MeOCl depressed the blood hemoglobin level; BrV and BrV + MeOCl treatment reduced also the erythrocyte diameter.

Animals↗

PubMedDate not supplied

Influence of bromfenvinphos alone, and in mixture with methoxychlor, on levels of gamma-glutamyl transpeptidase, ceruloplasmin and cholesterol in the blood plasma of laboratory mice.

Bromfenvinphos [0,0-diethyl-1-(2,4-dichlorophenyl)-2-bromovinyl phosphate], (BrV), 12.33 mg/kg/day alone and in combination with methoxychlor [1,1,1-trichloro-2,2-bis(4-methoxyphenyl)ethane], (MeOCl), 24.66 mg/kg/day were administered daily per os (intragastrically) in pure olive-oil for 6 weeks to male laboratory mice. The activity of gamma-glutamyl transpeptidase (GGTP), ceruloplasmin (CRP), and cholesterol in the blood plasma were determined. The hematocrit value, the erythrocyte count and the weights of the liver, spleen, heart, kidney and gonads were also measured after the end of treatment. BrV and BrV + MeOCl increased the activity of gamma-glutamyl transpeptidase. The activity of ceruloplasmin and the level of cholesterol in the blood plasma as well as the erythrocyte count in the blood were also similar in the control and experimental mice. The hematocrit value decreased after BrV and MeOCl administration. The relative weight of liver was higher in mice receiving both drugs in combination, than in control and in mice receiving BrV alone. The relative weight of spleen was significantly higher in animals receiving BrV alone, than in other groups.

Animals↗

PubMed2002

A comparison of the estrogenic potencies of estradiol, ethynylestradiol, diethylstilbestrol, nonylphenol and methoxychlor in vivo and in vitro.

Five natural, pharmaceutical, or xenobiotic chemicals [17beta-estradiol (E2), ethynylestradiol (EE2), diethystilbestrol (DES), methoxychlor (MXC), nonylphenol (NP)] were tested in two in vitro assays [yeast estrogen screen (YES), MCF-7 breast tumor cell proliferation (E-Screen)], and compared with previously reported results from two in vivo male sheepshead minnow vitellogenin (VTG) production studies. The purpose of this investigation was to determine how accurately the two in vitro assays predicted responses observed in live animals. EC50 values for all five chemicals were approximately one order of magnitude less sensitive in the YES assay than in the MCF-7 assay. Based on the EC50 values, DES was 1.1 (YES) to 2.5 (MCF-7) times more potent in these receptor binding assays than was E2, while EE2 was slightly less potent than E2 in the YES assay (0.7) and nearly twice as potent (1.9) as E2 in the MCF-7 assay. EE2 and DES were of approximately equal potency in the 13-day sheepshead minnow VTG production bioassay. Both MXC and NP were 10(7) times less potent than E2 in the YES assay, MXC was 10(5) times less estrogenic than E2 in the MCF-7 assay, while both were approximately 100 times less potent than E2 in the live animal bioassay. The in vitro tests were substantially less sensitive (at least 1000 times) than the sheepshead minnow VTG assay for estimating estrogenic potency of the two xenobiotic chemicals, which suggests that in vitro-based, large-scale screening programs could potentially result in many false negative evaluations.

Animals↗

PubMed2006

Toxic effects of methoxychlor on the episodic prolactin secretory pattern: possible mediated effects of nitric oxide production.

BACKGROUND: This work addresses the issue of whether methoxychlor (MTX) exposure may modify the ultradian secretion of prolactin through changes in the synthesis of nitric oxide (NO) induced by Nomega-nitro-L-arginine methyl ester (L-NAME) in the hypothalamic-pituitary axis. Associated changes in dopamine (DA) content in the anterior (AH), mediobasal (MBH) and posterior hypothalamus (PH) and median eminence (ME) were evaluated. METHODS: Two groups of animals (MTX and MTX+L-NAME treated) received subcutaneous (sc) injections of MTX at a dose of 25 mg/kg/day for one month. The other two groups of animals (control and L-NAME treated) received sc vehicle injections (0.5 mL/day of sesame oil), during the same period of time to be used as controls. Forty hours before the day of the experiment, animals were anaesthetized with intrapritoneal injections of 2.5% tribromoethanol in saline and atrial cannulas were implanted through the external jugular vein. Plasma was continuously extracted in Hamilton syringes coupled to a peristaltic bomb in tubes containing phosphate-gelatine buffer (to increase viscosity). The plasma was obtained by decantation and kept every 7 minutes for the measurement of plasma prolactin levels through a specific radioimmnunoassay and DA concentration by high-pressure liquid chromatography (HPLC). RESULTS: Prolactin release in animals from all experimental groups analyzed was episodic. Mean plasma prolactin levels during the bleeding period, and the absolute pulse amplitude were increased after MTX or Nomega-nitro-L-arginine methyl ester (L-NAME) administration. However MTX and L-NAME did not modify any other parameter studied with the exception of relative pulse amplitude in MTX treated rats. L-NAME administration to rats treated with the pesticide reduced mean plasma prolactin levels and the absolute amplitude of prolactin peaks. Peak duration, frequency and relative amplitude of prolactin peaks were not changed in the group of rats treated with MTX plus L-NAME as compared to either control or MTX treated rats. Whereas MTX decreased DA content in the ME and increased it in the AH, its content did not change in the MBH or PH, as compared to the values found in controls. Also, L-NAME administration decreased DA content in the ME as compared to controls. However, L- NAME administration to MTX exposed rats, markedly increased DA content in the ME as compared to either MTX treated or control rats. L-NAME administration increased DA content in the AH as compared to the values found in non-treated rats. However L-NAME administration to MTX exposed rats did not modify DA content as compared to either MTX treated or control rats. L-NAME administration did not modify DA content at the MBH nor in saline treated nor in MTX treated rats. However, the values of DA in the MBH in MTX plus L-NAME treated animals were statistically decreased as compared to L-NAME treated rats. In the PH, L-NAME administration increased DA content as compared to the values found in non-treated animals. L-NAME administration to MTX exposed rats also increased DA content as compared to either MTX treated or control rats. CONCLUSION: The results suggest the existence of an interaction between MTX and L-NAME in the modulation of the ultradian prolactin secretion at the pituitary levels. The possibility of an indirect effect mediated by changes in DA content at the ME requires further examination.

Journal Article↗

PubMed1988