Jerome M. Goldman

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-induced alterations in the histological expression of angiogenic factors in pituitary and uterus ∗

Within the reproductive system, oestrogenic stimulation of uterine and pituitary tissue typically causes a proliferative response accompanied by an angiogenic induction of new blood vessels from existing ones, thereby providing nutrients and oxygen to the growing tissue. The pro-oestrogenic pesticide methoxychlor (MXC), however, has shown a differential effect on proliferative activity. An increase in uterine growth is present, while the pituitary undergoes a decrease in size, even though the effect is accompanied by a characteristic oestrogen-induced elevation in pituitary prolactin concentration. The focus of the current study was whether the observed differences in tissue growth between uterus and pituitary in response to MXC administration were paralleled by a corresponding disparity in the expression of those growth factors (members of the vascular endothelial growth factor (VEGF) and angiopoietin families and their receptors) that are involved in the angiogenic cascade. Ovariectomized adult Sprague–Dawley female rats were administered MXC (0–200 mg/kg, oral) for 1 or 3 weeks. Immunohistochemical staining of uteri and pituitaries was performed under strictly controlled conditions for VEGF and its receptor VEGFR2, Angiopoietin-1 (Ang1) and angiopoietin-2 and their tyrosine kinase receptor Tie2, and platelet endothelial adhesion factor (as an index of vascularity). Image acquisition and densitometric assessments of staining intensity were conducted under blind conditions. The results showed uterine MXC-induced increases in the expression of VEGFR2 and Ang1, changes consistent with a normal proliferative response to oestrogenic stimulation. For VEGF, staining tended to be most pronounced in the stromal region, although there did not appear to be a progressive increase with dose. VEGFR2 expression showed significant dose-related trends in luminal and glandular epithelia by 1 week. Similar effects at 1 week were evident for Ang1 in glandular epithelium. In the anterior pituitary, a dose-related increase in VEGF was present for the 1 and 3 week treatments, and the number of pituitary vessels per unit area was also increased after 3 weeks. The effects indicate that even though the insecticide has not been found to cause an augmentation in pituitary growth, a dose-related rise in the expression of at least one principal angiogenic factor is present that may be associated with an increase in vascular density.

Endocrine-Disrupting Chemicals: Prepubertal Exposures and Effects on Sexual Maturation and Thyroid Activity in the Female Rat. A Focus on the EDSTAC Recommendations

In 1996, the US Environmental Protection Agency was given a mandate by Congress to develop a screening program that would evaluate whether variously identified compounds could affect human health by mimicking or interfering with normal endocrine regulatory functions. Toward this end, the Agency chartered the Endocrine Disruptor Screening and Testing Advisory Committee in October of that year that would serve to recommend a series of in vitro and in vivo protocols designed to provide a comprehensive assessment of a chemicals potential endocrine-disrupting activity. A number of these protocols have undergone subsequent modification by EPA, and this review focuses specifically on the revised in vivo screening procedure recommended under the title Research Protocol for Assessment of Pubertal Development and Thyroid Function in Juvenile Female Rats. Background literature has been provided that summarizes what is currently known about pubertal development in the female rat and the influence of various forms of pharmaceutical and toxicological insult on this process and on thyroid activity. Finally, a section is included that discusses technical issues that should be considered if the specified pubertal endpoints are to be measured and successfully evaluated.

Effect of atrazine on ovarian function in the rat

The effect of the chlorotriazine herbicide, atrazine, on ovarian function was studied in Long-Evans hooded (LE-hooded) and Sprague-Dawley (SD) rats. Atrazine was administered by gavage for 21 d to females displaying regular 4-d estrous cycles. In both strains, 75 mg/kg/d disrupted the 4-d ovarian cycle; however, no distinct alteration (i.e., irregular cycles but not persistent estrus or diestrus) was apparent at this dose. At 150 mg/kg/d, atrazine induced repetitive pseudopregnancies in females of both strains. The highest dose tested (300 mg/kg/d) also induced repetitive pseudopregnancies in the SD females, while the ovaries of the LE-hooded female appeared regressed and the smear cytology was indicative of the anestrous condition. Although a NOAEL was not established, the doses employed in this experiment were in excess of those used in chronic feeding studies in which an early onset of mammary gland tumors was noted. These data demonstrate that atrazine can disrupt ovarian function and bring about major changes in the endocrine profile of the female.

Effects of low subchronic doses of methoxychlor on the rat hypothalamic-pituitary reproductive axis☆

The pesticide methoxychlor (MXC) is known to possess a weak estrogenic action and has been found to have a number of toxic effects on the rodent reproductive system, primarily at the gonadal level. The purpose of this study was to explore the influence of MXC on the pituitary and hypothalamic components of the male reproductive system at dose levels that were without detectable testicular effects. At 21 days, male Long-Evans rats were gavaged daily with 25 or 50 mg/kg MXC in corn oil. Controls received vehicle only. After 8 weeks of dosing, no significant changes were seen in serum LH, FSH, or prolactin, nor in the pituitary concentrations of LH or FSH. Pituitary prolactin was elevated for both doses, and pituitary fragments perifused in vitro released more prolactin than did controls. The concentration of gonadotropin-releasing hormone (GnRH) was higher in the mediobasal hypothalamus, but only for the 50-mg/kg group. At this dose, there was a corresponding increase in the KCl-stimulated release of GnRH. The data suggest that previously reported reproductive effects of MXC may be mediated, at least in part, through an elevation in prolactin concentration and release, which in turn is able to influence hypothalamic levels of GnRH. This prolactinemic effect may well represent an early component of the adverse action of MXC on the reproductive system.

Effect of lindane on hormonal control of reproductive function in the female rat.

The effect of the gamma isomer of 1,2,3,4,5,6-hexachlorocyclohexane, lindane, on reproductive function in the female rat was examined in two experiments. In the first experiment, chronic treatment with 0, 5, 10, 20, and 40 mg/kg lindane delayed vaginal opening and disrupted ovarian cyclicity until approximately 110 days of age. Thereafter, regular ovarian cycles were present in the majority of females in all dose groups. When killed on the day of vaginal proestrus, the females receiving the two higher doses of lindane had smaller pituitary and uterine weights, lower serum and pituitary luteinizing hormone (LH) and prolactin, and higher pituitary follicle stimulating hormone (FSH) concentrations than the oil-treated control females. Serum estrogen concentrations were not different from controls in the 5 and 20 mg/kg groups, significantly greater than the controls in the 10 mg/kg group, and significantly less than the controls in the group receiving 40 mg/kg. In a second experiment, the uterine weight and pituitary hormone response of 28-day-old, lindane-treated females to a 10-micrograms injection of estradiol benzoate (EB) were investigated. The uteri of the lindane-treated prepubertal females were smaller than controls at 30 hr after EB injection. Furthermore, the EB-induced increase in serum luteinizing hormone, observed at 30 hr after EB injection, was lower in the lindane-treated animals. Similarly, the reduction in pituitary LH, FSH, and prolactin induced by EB was not as great in the lindane-treated animals as in the controls. Serum estrogen concentrations in the lindane-treated animals were not different from controls. These data indicate that lindane may effectively block the response of estrogen-dependent tissues to this ovarian steroid hormone and that this apparent antiestrogenic effect of lindane is responsible for the disturbances observed in the neuroendocrine control of ovarian function in the rat.

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)

Neuroendocrine and reproductive effects of contemporary-use pesticides:

Work in our laboratory has focused on the hypothesis that certain environmental contaminants will interfere with reproductive function because they disrupt the neuroendocrine regulation of gonadal function. In this article, we review the evidence that certain classes of contemporary-use pesticides alter gonadotropin secretion through a disruption of hypothalamic mechanisms. Specifically, we discuss the effect of formamidine and dithiocarbamate pesticides on the noradrenergic control of pituitary hormone secretion, ovarian function, and pregnancy in the rat. This is followed by a review of studies evaluating the effect of a chlorotriazine herbicide, atrazine, on the hormonal control of ovulation and lactation. We also discuss the physiological consequences of these endocrine alterations in the female, how toxicant-induced endocrine alterations may differ in physiological outcome in the male and female, and the fact that the reproductive risk assessment of some pesticides that act on the central nervous system (CNS) may be influenced by the development of tolerance.

The dithiocarbamate fungicide thiram disrupts the hormonal control of ovulation in the female rat

Thiram has been reported to inhibit dopamine-beta-hydroxylase (D beta H), thereby affecting norepinephrine (NE) synthesis. Because NE is a neurotransmitter that is known to play an important role in the hypothalamic regulation of pituitary function, the acute effects of the thiram on the hormonal control of ovulation in the rat were investigated. Ovariectomized, estrogen-primed female rats were given a single injection of thiram (0, 6, 12, 25, 50, and 100 mg/kg, i.p.) at 1100 h and serum LH was measured in serial bleeds. Thiram at 100 and 50 mg/kg completely blocked the LH surge in all rats tested, while 12 and 25 mg/kg blocked the surge in 40 and 75% of the treated animals, respectively. Six mg/kg had no effect. Ovulation was then assessed in intact, proestrous females in response to thiram administration (0, 12, 25, or 50 mg/kg) at 0900, 1100, 1300, or 1800 h. Ovulation was blocked by 25 and 50 mg/kg at 1300 h in all rats, but when injected at 1100 h only the 50 mg/kg dose was effective. No such blockade was found with 50 mg/kg injected at 0900 and 1800 h. To assess the influence of thiram on the LH surge in intact rats, additional females were dosed at 1300 h on the day of proestrus and blood collected over that same day. Thiram at 50 mg/kg blocked the LH surge in all rats, while 25 mg/kg blocked the surge in 60% of the females tested. No effect occurred with 12 mg/kg.(ABSTRACT TRUNCATED AT 250 WORDS)