Janet M. Ferrell

A Dose-Response Analysis of Methoxychlor-Induced Alterations of Reproductive Development and Function in the Rat

In the present study rats were dosed from weaning, through puberty and gestation, to Day 15 of lactation with methoxychlor at 25, 50, 100, or 200 mg/kg/day. Morphological landmarks of puberty were measured, including the ages at vaginal opening, first estrus, and first estrous cycle in females and at preputial separation in males. In the female, estrous cyclicity, fertility, litter size, number of implantation sites, organ weights, and ovarian and uterine histology were also measured. The viability of the offspring (F1) and their fertility were evaluated using a continuous breeding protocol. Males were necropsied after breeding, the reproductive organs were weighed, and the cauda epididymal sperm counts were determined. One testis was used for histopathology, while the other was used to quantify interstitial fluid (IF) content, IF testosterone concentration, and testicular sperm production. Testosterone and androgen-binding protein were measured in the caput epididymis, and sperm motility and morphology were evaluated from a caudal sample. The serum and pituitary were saved for hormonal determinations. Methoxychlor accelerated the age at vaginal opening and first estrus, and the vaginal smears were cornified. Growth was retarded at 100 and 200 mg/kg/day and fertility was reduced when the females were bred with untreated or similarly treated males. In the highest-dose group, the mated females went from constant estrus into pseudopregnancy following mating, but they had no implants. In males, methoxychlor treatment markedly reduced growth, seminal vesicle weight, cauda epididymal weight, caudal sperm content, and pituitary weight. Puberty was delayed in the two highest-dosage groups. Testicular sperm measures were much less affected than caudal measures. Testis weight and histology were slightly affected, and testicular sperm production, sperm morphology, and motility were unaffected. Endocrine function of the testes and pituitary was altered by methoxychlor administration. Leydig cell testosterone production, in response to human chorionic gonadotropin challenge, was reduced and pituitary levels of prolactin, thyroid-stimulating hormone (TSH), and follicle-stimulating hormone (FSH) were altered. In contrast, serum levels of prolactin, FSH, and luteinizing hormone were unaffected. Serum TSH was reduced by 50% of control at 100 and 200 mg/kg/day, while pituitary levels were increased. Gonadotropin-releasing hormone concentration in the mediobasal hypothalamus was also elevated. In spite of the many reproductive alterations, the fertility of treated males was not reduced when they were mated with untreated females.(ABSTRACT TRUNCATED AT 400 WORDS)

Methoxychlor induces estrogen-like alterations of behavior and the reproductive tract in the female rat and hamster: Effects on sex behavior, running wheel activity, and uterine morphology

The current investigation was designed to determine if the pesticide methoxychlor (M) mimicked the effects of estrogen in the brain and on behavior. Running wheel activity (RWA) and sex behaviors were evaluated in this study because the role of estrogen in the regulation of these behaviors has been thoroughly established. M exposure at 400 mg/kg/day (90% pure) induced high levels of acyclic RWA and persistent vaginal estrus in the female rats. Following ovariectomy (ovx), RWA declined precipitously in controls but remained at high levels in M-treated-ovx females. M also produced estrogen-like alterations of the uterine endometrial epithelium, the ovary, and growth after ovx. In another study, ovx female rats were dosed with M at 200 mg/kg/day and then with progesterone (P). P acts as an antiestrogen and specifically suppresses estrogen-induced RWA. P blocks the synthesis of estrogen receptors in the CNS and reproductive tract but does not lower RWA induced by nonestrogenic mechanisms. After 14 days of M administration RWA was increased fourfold over the ovx-oil-treated females. Subsequently, P injections reduced RWA levels far below those seen when the ovx-M-treated rats were injected with oil. The P-induced decline represents a 95% inhibition of the M-induced increase in RWA. Subsequently, M-treated-ovx rats and hamsters were injected with P and tested for their ability to display reproductive behaviors when paired with a stud male. Female sexual behaviors are induced by the administration of estrogen followed by progesterone. In this study the M-treated females displayed reproductive behaviors, in contrast to the oil-treated rats and hamsters. The observation that the high levels of RWA induced by methoxychlor treatment in ovx rats can be suppressed by concurrent progesterone injections demonstrates that the increase in RWA is due to the estrogenic effects of methoxychlor on the CNS. The fact that methoxychlor, followed by P injections, induces behavioral estrus in the rat and hamster extends this estrogenicity to other areas in the CNS.

The development of a protocol to assess reproductive effects of toxicants in the rat.

The determination that a chemical poses a reproductive risk to man typically relies upon fertility studies using rodents. However, fertility in rodents is often difficult to disrupt and more sensitive indicators of reproductive function should be included in the risk assessment process. The present discussion compares the sensitivity of fertility to other endpoints following exposure to known reproductive toxicants. In our studies rats were dosed from weaning through puberty , gestation, and lactation. The reproductive function of the male, the female, and the offspring was assessed. The effects of methoxychlor, carbendazim (MBC), dibutyl phthalate (DBP), and lindane are discussed and compared to fertility. For each compound a ratio (SR = sensitivity ratio) of the lowest effect level (LEL) for infertility or reduced fecundity to the LEL for the most sensitive physiologic endpoint was calculated. The SR should be large when a compound produces effects over a wide range of doses, but should equal unity when the dose-response curve is very steep. For methoxychlor, which blocked implantation, pubertal landmarks and estrous cyclicity provided rapid and sensitive indicators of the subsequent reproductive failure. The SR = 8 (100/12) for methoxychlor using data from females. In contrast, DBP and MBC directly altered testicular function, and for these compounds, sperm and testicular measures provided sensitive indicators of toxicity. The SR for MBC was 2 (100/50), while DBP had a SR of 1 (500/500). In the lindane study, fertility was not reduced but most of the pups (F1) died shortly after birth. The SR for lindane is equal to 0.5 (10/20). At 20 mg/kg the treated females were larger and their estrous cycles were erratic.(ABSTRACT TRUNCATED AT 250 WORDS)

Chlorotriazine Herbicides and Metabolites Activate an ACTH-dependent Release of Corticosterone in Male Wistar Rats

Previously, we reported that atrazine (ATR) alters steroidogenesis in male Wistar rats resulting in elevated serum corticosterone (CORT), progesterone, and estrogens. The increase in CORT indicated that this chlorotriazine herbicide may alter the hypothalamic-pituitary-adrenal axis. This study characterizes the temporal changes in adrenocorticotropic hormone (ACTH), CORT, and P4 in male Wistar rats following a single dose of ATR (0, 5, 50, 100, and 200 mg/kg), simazine (SIM; 188 mg/kg), propazine (PRO; 213 mg/kg), or primary metabolites, deisopropylatrazine (DIA; 4, 10, 40, 80, and 160 mg/kg), deethylatrazine (DEA; 173 mg/kg), and diamino-s-chlorotriazine (DACT; 3.37, 33.7, 67.5, and 135 mg/kg). The maximum dose for each chemical was the molar equivalent of ATR (200 mg/kg). Significant increases in plasma ACTH were observed within 15 min, following exposure to ATR, SIM, PRO, DIA, or DEA. Dose-dependent elevations in CORT and progesterone were also observed at 15 and 30 min post-dosing with these compounds indicating an activation of adrenal steroidogenesis. Measurement of the plasma concentrations of the parent compounds and metabolites confirmed that ATR, SIM, and PRO are rapidly metabolized to DACT. Although DACT had only minimal effects on ACTH and steroid release, dosing with this metabolite resulted in plasma DACT concentrations that were 60-fold greater than that observed following an equimolar dose of ATR and eightfold greater than equimolar doses of DIA or DEA, indicating that DACT is not likely the primary inducer of ACTH release. Thus, the rapid release of ACTH and subsequent activation of adrenal steroidogenesis following a single exposure to ATR, SIM, PRO, DIA, or DEA may reflect chlorotriazine-induced changes at the level of the brain and/or pituitary.

Postnatal developmental alterations following prenatal exposure to the herbicide 2,4-dichlorophenyl-p-nitrophenyl ether: A dose response evaluation in the mouse

Although nitrofen, 2,4-dichlorophenyl-p-nitrophenyl ether, is a relatively nontoxic herbicide, prenatal exposure to doses considerably less than the LD50 value for adult rats and mice produces numerous developmental defects that become apparent as the animals mature. In the present study postnatal development was observed following prenatal exposure during Days 7 to 17 of gestation at doses of 0, 6.25, 12.5, 25, 50, 100, 150, and 200 mg/kg/day. These doses did not cause maternal toxicity as indicated by the viability of the dams or maternal weight gain during pregnancy. By 3 days of age all pups in the two highest dose groups were dead and 50% had died in the 100 mg/kg/day dose group. Some of the dead and moribund pups from the 200 mg/kg/day exposure group necropsied at three days of age had cleft palate (15%) or diaphragmatic hernia (6%). In addition, about 22% of the pups at 200 mg/kg/day developed a distended abdomen from gasping and swallowing air. These pups did not suckle and eventually died. Body weights of offspring were reduced at birth in the 150 and 200 mg/kg/day groups and at 3 days of age in the 100 mg/kg/day group. Growth rates were subsequently retarded at 12.5, 25, 50, and 100 mg/kg. The Harderian glands were reduced or absent in 97, 65, and 4% of the mice in the 100, 50, and 25 mg/kg dosage groups, respectively, and the gland weights were reduced at all dosages, including the lowest dose of 6.25 mg/kg/day. Weights of other organs including lung and liver (at 6.25 and above), seminal vesicle (at 12.5 and above), and testes (at 100 mg/kg/day) were also reduced by prenatal nitrofen exposure. In addition, prenatal treatment with nitrofen produced functional deficits of the reproductive system; puberty was delayed in females and litter sizes were reduced at 50 and 100 mg/kg/day. A cross-fostering experiment with 100 mg/kg/day of nitrofen demonstrated that the effects noted in the present study were produced solely by prenatal exposure; pups exposed to nitrofen in the milk alone as a consequence of any accumulation of nitrofen in the dam during gestation were unaffected.

Alteration of behavioral sex differentiation by exposure to estrogenic compounds during a critical neonatal period: Effects of zearalenone, methoxychlor, and estradiol in hamsters☆

The present study was designed to determine if neonatal exposure to the estrogenic mycotoxin zearalenone or the weakly estrogenic pesticide methoxychlor could masculinize and/or defeminize the behavior of female hamsters. Neonatal hamsters were given a single sc injection of either zearalenone (1 mg/pup), methoxychlor (1 mg/pup), 17 beta-estradiol (E2) (40 micrograms/pup), or the vehicle 2 days after birth. After puberty, behavioral estrous cyclicity was measured. The females were then ovariectomized, treated with the male hormone testosterone, and tested for their ability to mount a receptive female (a behavior not normally displayed by female hamsters). Females treated neonatally with estradiol or zearalenone were masculinized but not defeminized, an effect consistent with perinatal exposure to low doses of sex hormones. Females in these two treatment groups displayed normal 4-day behavioral estrous cycles, but following ovariectomy and testosterone treatment they mounted a sexually receptive female at a frequency comparable to the males. Methoxychlor-treated females did not differ from controls. The mounting behavior of similarly treated males was unaffected by any of the chemicals. However, males receiving estradiol treatment had smaller testes, seminal vesicles, and cauda epididymides and 57% had epididymal cysts. These results demonstrate that a single exposure to a weakly estrogenic chemical like zearalenone during a critical developmental period can cause the brain to differentiate in a manner inconsistent with the females genetic sex. This enables the female to respond to the activational influence of testosterone as an adult and readily mount a sexually receptive female. The failure of methoxychlor to alter reproductive development in the current study may be due to an inability of the neonatal hamster to convert methoxychlor to estrogenic metabolites.

Prenatal dinocap exposure alters swimming behavior in mice due to complete otolith agenesis in the inner ear.

Exposure to the fungicide dinocap during gestation produces behavioral abnormalities in the house mouse that are not apparent at birth but become obvious at weaning. Pregnant mice (CD-1) were exposed on Days 7 to 16 of gestation to dinocap at 0, 6, 12, or 25 mg/kg/day and the postnatal behavioral development of the offspring was assessed. Torticollis (head-tilting) appears in the treated offspring at 3 weeks of age (4.4% at 12 and 25.3% at 25 mg/kg/day) and, during a test of swimming ability, many of the mice (6.8% at 12 and 47.2% at 25 mg/kg/day) sink below the surface or are unstable and swim on their side in the water. These behavioral abnormalities are the result of agenesis of the otoliths in the inner ears. These were the only developmental defects noted in the 12 mg/kg/day dosage group. In this group 4.4% of the mice displayed torticollis, 9.2% did not swim normally, 19% were missing one or more whole otoliths (7.7% were missing all four otoliths), and partial agenesis of the crystalline material was seen in an additional 11.6% of the mice. The frequency of behavioral and inner ear defects increased in the higher dosage group, but the order of sensitivity of the effects did not change.

The postnatal effects of prenatal exposure to low doses of nitrogen (2,4-dichlorophenyl-p-nitrophenyl ether) in Sprague-Dawley rats☆

Nitrofen was administered to pregnant Sprague-Dawley rats by gavage on days 8-16 of gestation at 5 different dose levels--0, 0.46, 1.39, 4.17 and 12.5 mg/kg/day. Diaphragmatic hernias were found in pups that died immediately after birth at the 3 highest dose levels. At the 1.39-mg/kg dose level 3 of the 4 pups examined had diaphragmatic hernias, at the 4.17-mg/kg dose level 2 out of 3 pups had diaphragmatic hernias, and at the 12.5-mg/kg dose level all 5 pups found dead had diaphragmatic hernias. Locomotor activity of the offspring was measured on postnatal days 17 and 24, and hyperactivity was evident at the 3 highest dose levels. However, when the rats were later tested at 45, 49 and 90 days of age they had apparently recovered from this earlier hyperactivity. In the female rat, nitrofen did not delay the onset of puberty as measured by the age of vaginal opening or the age at first estrus. At necropsy of the offspring which began on postnatal day 133, Harderian gland weight reduction and hydronephrosis were seen at the 4.17- and 12.5-mg/kg dose levels, while no effects were found in body, liver, testes, seminal vesicle, kidney, or lung weights. Results of the present study and earlier studies demonstrate that rats are more sensitive than mice to the teratogenic effect of nitrofen (Gray et al., Science, 215 (1982) 293 and Gray et al., Toxicol. Appl. Pharmacol., 67 (1983) 1). In general, nitrofen affects the same organ systems in rats as it does in mice, but the rank order of sensitivity of these effects differs from those described earlier in the mouse by Gray et al. (Toxicol. Appl. Pharmacol., 67 (1983) 1).

The use of cultured ovarian fragments to assess toxicant alterations in steroidogenesis in the sprague-dawley rat

This study was conducted to determine the utility of using steroid production by cultured ovarian fragments to assess toxicant-induced alterations in ovarian steroidogenesis in Sprague-Dawley rats. To this end, serum steroid concentration and steroid production (progesterone (P4), testosterone (T), estradiol (E2)) by cultured ovarian fragments is described during a normal 4-day estrous cycle. This culture system was then used to profile the effects of aminoglutethimide shown to have two sites of steroidogenic inhibition, side chain cleavage enzyme and aromatase. LH, FSH, P4, and E2 concentrations in serum during the 4-day estrous cycle confirmed that described in the literature for untreated rats. All of the steroids measured had peak production levels during proestrus. The patterns of P4 and E2 production by the ovaries in an unstimulated culture mimics that seen in serum. Stimulation with hCG (100 mIU/mL) after the initial 1 h culture tends to even out the production of P4, while T production rises faster and peaks earlier. The pattern and levels of estradiol production in hCG-stimulated cultures are very similar to those in the unstimulated culture, both in pattern and in production levels. When cultured ovarian fragments from proestrous rats were treated in vitro with aminoglutethimide (1 to 16 microM), the pattern of steroid production that characterized the inhibitory effects were similar to those reported in the literature using isolated cell culture procedures. This pattern showed a rapid decrease in E2 production (IC50 of 2.43 microM), a concurrent rise in T production, and a decrease in P4 production (IC50 of 15.5 microM). This culture system is an appropriate system to rapidly assess toxicant effects on ovarian steroidogenesis following in vivo or in vitro exposure.