Claude L. Hughes

Dose-response characteristics of neonatal exposure to genistein on pituitary responsiveness to gonadotropin releasing hormone and volume of the sexually dimorphic nucleus of the preoptic area (SDN-POA) in postpubertal castrated female rats

Estrogen exposure during critical periods of development promotes androgenization of the brain, which is reflected in altered morphology, behavior, and cyclic hormone secretion in females. Previous work in our laboratory demonstrated that neonatal female rats injected with pharmaceutical or naturally occurring estrogens had decreased GnRH-induced LH secretion and increased volume of the SDN-POA as 42 day castrates. The current experiment defines the dose-response characteristics of neonatal exposure to the isoflavonoid phytoestrogen genistein (G) on pituitary sensitivity to GnRH and SDN-POA volume. Litters of rat pups received subcutaneous injections of either corn oil, 1, 10, 100, 200, 400, 500, or 1000 micrograms of G on days 1 to 10 of life. The litters were ovariectomized and weaned on day 21. On day 42 blood was drawn from right atrial catheters immediately prior to, 5, 10, 15, and 30 min following a single injection of 50 ng/kg of GnRH. Only the 10 micrograms dose of G was associated with increased pituitary response to GnRH, while progressive increases in exposure levels of G were associated with decreasing LH secretion. The SDN-POA volume was increased in only the 500 micrograms and 1000 micrograms exposure groups compared to controls. The results confirm that low doses of G have nonandrogenizing, pituitary-sensitizing effects, while higher doses of G mimic the more typical effects of estrogens. The use of both morphologic and physiologic end points more completely defines the reproductive consequences of environmental estrogen exposure during critical periods of CNS development.

Follicle-Stimulating Hormone Concentrations in Relation to Active and Passive Smoking

Objective To determine the association between various forms of tobacco exposure and ovarian status, as measured by FSH concentrations, in women 38–49 years old. Methods Two hundred ninety women between 38–49 years old, who had not had hysterectomy or oophorectomy, completed a self-administered questionnaire that included information on tobacco exposure and had serum FSH levels measured on days 2–4 of the menstrual cycle. Linear regression was used to assess the relation between FSH and tobacco exposure. Results Controlling for age and other factors, FSH concentrations were 66% higher among current smokers (geometric mean FSH 14.0 mIU/mL) and 39% higher among nonsmokers with passive smoke exposure (11.7 mIU/mL), compared to nonsmoking women without passive smoke exposure (8.4 mIU/mL). The estimated increase in FSH for each year of age was greater for current smokers than for nonsmokers (16 versus 6%, respectively). Ex-smokers did not have higher FSH concentrations, and there was no association between prenatal exposure to tobacco smoke and FSH. Conclusion Both active and passive smoking are associated with elevated FSH concentrations in women 38–49 years old. The effect, limited to women with current exposure, is consistent with a shorter duration of the menopausal transition period.

Acute and subacute effects of naturally occurring estrogens on luteinizing hormone secretion in the ovariectomized rat: Part 1

Acute pretreatment of ovariectomized rats with genistein (G) alters gonadotropin-releasing hormone-(GnRH)-induced LH secretion in a fashion comparable to estradiol (E2). In the present studies we wished to (A) determine whether G can acutely inhibit tonic LH secretion by oral (po) or intravenous (iv) routes, (B) compare GnRH-induced LH responses following higher iv dose pretreatments with G or E2, and (C) determine effects of G or E2 pretreatments on progesterone (P)-induced secretion of LH. Mature Charles River CD rats were ovariectomized, and 2 to 5 weeks later intraatrial cannulae were placed. Serial blood samples were drawn and LH was measured by RIA. In experiments 1 and 2, G or E2 was administered acutely by gavage or iv, while in experiment 3, G and E2 were given subcutaneously (sc) oil 3 days prior to cannulation and sampling. Acute po administration of vehicle or G (0.1, 1.0, and 10 mg/kg BW) had no effect on tonic LH, while E2 suppressed LH at all doses (0.1, 1.0, and 10 mg/kg BW). Acute iv administration of vehicle and higher doses of G (1 and 10 mg/kg BW) had no effect on tonic LH, while the lowest dose G (0.1 mg/kg BW) and all doses of E2 (0.1, 1, and 10 mg/kg BW) suppressed tonic LH. In the iv-treated rats, GnRH-induced LH secretion was more profoundly suppressed by G at all doses than by E2.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of neonatal exposure to des and o,p′-DDT on pituitary responsiveness to GnRH in adult castrated rats

While exposure of vertebrates to estrogens during early development has been shown to alter adult reproductive behavior, neuroanatomy, and neurophysiology, effects on gonadotropin secretion have not been studied. We conducted the present studies to assess the effects of neonatal exposure to xenobiotic estrogens on luteinizing hormone secretion in castrated adult rats. Rat pups were injected with either corn oil, 1 micrograms diethylstilbestrol (DES), or 0.5 mg o,p-DDT on postnatal days 1 to 10, and castration was performed on day 21. On day 42 of life, GnRH (50 ng/kg) was administered via right heart catheters, and blood was sampled for LH at 0, 5, 10, 15, and 30 min. Neonatal exposure to DES in both males and females significantly decreased basal and GnRH-induced LH secretion throughout the sampling period in castrated adults. o,p-DDT significantly suppressed initial LH levels and blunted GnRH-induced release in males at the 5 min interval, while in females it had no effect. These data show that early exposure to environmental estrogens alters adult pituitary response to GnRH. Our results suggest that sexually distinct effects of environmental estrogens occur and can be readily demonstrated in this experimental model.

Screening of potential reproductive toxicants by use of porcine granulosa cell cultures

While approximately 60 000 chemicals are in widespread use with 1000 new chemicals introduced into the environment each year, the biologic effects of these agents are poorly understood. With the specific goal of testing for potential reproductive toxicity, we have established methodology for the screening of compounds in vitro by measuring effects on progesterone production by porcine granulosa cells in culture. Granulosa cells were harvested by mechanical agitation, cryopreserved, and cells with known progesterone production capacity utilized for culture. Agents to be tested were added to cultures of 10(5) cells and the media assayed for progesterone by radioimmunoassay. Estradiol suppression of progesterone production was easily demonstrated in this system and utilized as a verification of responsiveness. The pesticide o,p-DDT and its isomer p,p-DDT produced dramatic suppression of progesterone production apparently with equal potencies to estradiol. By contrast, the pesticides malathion, parathion and dieldrin and the fungicide hexachlorobenzene were without effect in this test system.

Criteria for identifying and listing substances known to cause developmental toxicity under California's proposition 65☆

Because of the automatic restrictions and warning requirements imposed on substances identified by the state as known to cause developmental toxicity, the Expert Committee recommends the use of criteria that emphasize human relevancy, biological plausibility, and evidence in support of a selective, adverse developmental effect at non-maternally-toxic doses. In many instances, data for substances of public concern will be insufficient at present to meet these criteria. The fact that a substance is not listed as known to cause developmental toxicity does not create a presumption that the substance is safe. The Expert Committee, therefore, urges that these substances be recommended for further testing and that high priority be given to conducting the necessary tests. The Expert Committee reiterates its concern that substances listed by the SAP be identified according to the toxic endpoints (cancer, male reproductive toxicity, female reproductive toxicity, and/or developmental toxicity) that led to listing. Further, the Expert Committee recommends that the state Health and Welfare Agency institute education programs emphasizing appropriate courses of action for citizens informed of exposures to substances known to the state to cause cancer, birth defects, or reproductive toxicity.

Use of human cumulus granulosa cells for in vitro screening of reproductive toxicants

We investigated the possibility that human granulosa cells from the cumulus mass obtained during human in vitro fertilization/embryo transfer (IVF/ET) might be useful for screening of potential reproductive toxicants in vitro. The cumulus granulosa cells detached from the zona pellucida after fertilization were allowed to spontaneously adhere to the incubation dish following transfer (removal) of the embryo. These cumulus cells survived in culture for at least four additional days, appeared on simple inspection to be morphologically normal luteinized granulosa cells, and produced large amounts of progesterone (P) over the culture interval. Production gradually declined during culture in the absence of human chorionic gonadotropin (hCG); however, inclusion of hCG (100 ng/mL) in the medium maintained P production at control (day 1) levels. Introduction of estrogenic agents previously shown to suppress P production in porcine or human culture systems using mural granulosa cells showed comparable effects in this human cumulus cell system. 17 beta-estradiol (10(-5) M), clomiphene citrate (10(-5) M), and o,p-DDT (10(-5)) significantly inhibited hCG-supported P production by human cumulus cells in vitro. This system has the advantages that (1) human cumulus granulosa cells are readily available from IVF/ET programs, (2) the techniques for maintaining the cells in culture are extremely simple, (3) a marker of highly differentiated granulosa cell function (P production) can be reliably measured, and (4) the cells respond predictably like other comparable granulosa cell systems. We conclude that human cumulus cells are a readily available and useful resource for in vitro screening of potential female reproductive toxicants.

Effects of acetaminophen and nonsteroidal anti-inflammatory drugs on progesterone production by porcine granulosa cells in vitro

We investigated the effects of a group of pharmaceutical agents commonly ingested by reproductive-aged women, acetaminophen and the nonsteroidal anti-inflammatory drugs (NSAID), on progesterone (P) production by cultures of highly differentiated porcine granulosa cells. These compounds were added to cultures over a dose range of 10(-8) to 10(-5) M and P, and cell protein was measured after 24 hours. P production was suppressed by acetaminophen, fenoprofen, and sulindac to a maximum of 81%, 76%, and 71% of control, respectively. P production was enhanced by butazolidin at all doses tested to a maximum of 140% of control. Granulosa cell protein was suppressed by butazolidin and salicylic acid to a maximum of 81% of controls. These data imply that acetaminophen and several NSAID have the potential for clinical reproductive toxicity with differing individual effects on reproductive tract tissues, suggesting further selective testing in vivo.

Monitoring of ovulation in the assessent of reproductive hazards in the workplace

If women are exposed to reproductive hazards in the workplace, some disturbances of ovulation are expected. Assessment of ovulation requires monitoring to distinguish normal and abnormal ovarian cycles. Menstrual interval and basal body temperature charts are inadequate for identifying many abnormal cycles. A late-luteal endometrial biopsy or a single mid-luteal morning progesterone level each appears to be only 80% accurate in distinguishing normal and abnormal cycles. The complete cycle profile of daily ovarian ultrasonic scans and daily serum levels of reproductive hormones appears to be a definitive approach to characterizing ovarian cycles, but this demanding regimen would not be applicable to monitoring large populations in the workplace. An ovarian cycle monitoring protocol consisting of daily measurement of salivary or vaginal electrical resistance, mid-cycle urine testing for the luteinizing hormone surge, and daily luteal phase salivary progesterone levels would provide a practical comprehensive corroborative assessment of ovarian cycles in such populations of women. This monitoring of ovarian cycles would aid in the early detection of reproductive hazards and medical conditions that might present as an ovulatory disturbance.

Synthetic estrogens suppress granulosa cell progesterone production in vitro

We evaluated the effect of commonly used pharmaceutical estrogens and zeranol, an estrogenic growth-promoting agent used in livestock, on progesterone (P) production by cultures of highly differentiated porcine granulosa cells (GC). The compounds were added to GC cultures over a dose range of 10(-8) to 10(-5) M with P and cell protein measured after 24 h. P production was suppressed by estradiol (minimal suppressive dose: 10(-7) M; maximal suppression to 11% of control), ethinyl estradiol (10(-7) M, 15%), diethylstilbestrol (10(-5) M, 72%), clomiphene citrate (10(-6) M, 30%), nafoxidine (10(-7) M, 33%), tamoxifen (10(-6) M, 37%), and zeranol (10(-5) M, 83%). P production was not suppressed by mestranol. GC protein was suppressed by estradiol, ethinyl estradiol, nafoxidine, and zeranol. These data suggest that synthetic estrogens have the potential to suppress luteal P production by a mechanism unrelated to the usual measures of estrogenicity.