Evaluation of a Tier I screening battery for detecting endocrine-active compounds (EACs) using the positive controls testosterone, coumestrol, progesterone, and RU486.

After previously examining 12 compounds with known endocrine activities, we have now evaluated 4 additional compounds in a Tier I screening battery for detecting endocrine-active compounds (EACs): a weak estrogen receptor (ER) agonist (coumestrol; COUM), an androgen receptor (AR) agonist (testosterone; TEST), a progesterone receptor (PR) agonist (progesterone; PROG), and a PR antagonist (mifepristone; RU486). The Tier I battery incorporates 2 short-term in vivo tests (5-day ovariectomized female battery; 15-day intact male battery) and an in vitro yeast transactivation system (YTS). The Tier I battery is designed to identify compounds that have the potential to act as agonists or antagonists to the estrogen, androgen, progesterone, or dopamine receptors; steroid biosynthesis inhibitors (aromatase, 5alpha-reductase, and testosterone biosynthesis); or compounds that alter thyroid function. In addition to the Tier I battery, a 15-day dietary restriction experiment was performed using male rats to assess confounding due to treatment-related decreases in body weight. In the Tier I female battery, TEST administration increased uterine weight, uterine stromal cell proliferation, and altered hormonal concentrations (increased serum testosterone [T] and prolactin [PRL]; and decreased serum FSH and LH). In the male battery, TEST increased accessory sex gland weights, altered hormonal concentrations (increased serum T, dihydrotestosterone [DHT], estradiol [E2], and PRL; decreased serum FSH and LH), and produced microscopic changes of the testis (Leydig cell atrophy and spermatid retention). In the YTS, TEST activated gene transcription in the yeast containing the AR or PR. In the female battery, COUM administration increased uterine weight, uterine stromal cell proliferation, and uterine epithelial cell height, and increased serum PRL concentrations. In the male battery, COUM altered hormonal concentrations (decreased serum T, DHT, E2; increased serum PRL) and, in the YTS, COUM activated gene transcription in the yeast containing the ER. In the female battery, PROG administration increased uterine weight, uterine stromal cell proliferation, and uterine epithelial cell height and altered hormonal concentrations (increased serum progesterone and decreased serum FSH and LH). In the male battery, PROG decreased epididymis and accessory sex gland weights, altered hormonal concentrations (decreased serum T, PRL, FSH, and LH; increased serum progesterone and E2), and produced microscopic changes of the testis (Leydig cell atrophy). In the YTS, PROG activated gene transcription in the yeast containing the AR or PR. In the female battery, RU486 administration increased uterine weight and decreased uterine stromal cell proliferation. In the male battery, RU486 decreased epididymis and accessory sex gland weights and increased serum FSH and LH concentrations. In the YTS, RU486 activated gene transcription in the yeast containing the ER, AR, or PR. Dietary restriction data demonstrate that confounding due to decrements in body weight are not observed when body weight decrements are 10% or less in the Tier I male battery. In addition, minimal confounding is observed at body decrements of 15% (relative liver weight, T3, and T4). Hence, compounds can be evaluated in this Tier I at levels that produce a 10% decrease in body weight without confounding of the selected endpoints. Using the responses obtained for all the endpoints in the Tier I battery, a distinct "fingerprint" was produced for each type of endocrine activity against which compounds with unknown activity can be compared. These data demonstrate that the described Tier I battery is useful for identifying EACs and they extend the compounds evaluated to 16.

An ongoing validation of a Tier I screening battery for detecting endocrine-active compounds (EACs).

After previously examining an estrogen receptor agonist (17beta-estradiol), several additional compounds have been evaluated in a Tier I screening battery for detecting endocrine-active compounds (EACs): an estrogen receptor antagonist (ICI-182,780, ICI), an androgen receptor antagonist (flutamide, FLUT), a testosterone biosynthesis inhibitor (ketoconazole, KETO), a 5alpha-reductase inhibitor (finasteride, FIN), and an aromatase inhibitor (anastrozole, ANA). The Tier I battery incorporates two short-term in vivo tests (a 5-day ovariectomized female battery and a 15-day intact male battery) and an in vitro yeast transactivation system (YTS). The Tier I battery is designed to identify compounds that have the potential to act as agonists or antagonists to the estrogen, androgen, progesterone, or dopamine receptors, steroid biosynthesis inhibitors (aromatase, 5alpha-reductase, and testosterone biosynthesis), or compounds that alter thyroid function. ICI administration decreased uterine estrogen and progesterone receptor number in the female battery, increased serum follicle-stimulating hormone (FSH) levels and caused spermatid retention in the male battery, and activated gene transcription in the YTS containing the estrogen receptor. FLUT administration increased uterine stromal cell proliferation in the female battery and decreased weights for all androgen-dependent tissues, induced Leydig cell hyperplasia, and caused hormonal alterations (increased testosterone (T), estradiol (E2), dihydrotestosterone (DHT), luteinizing hormone (LH), and FSH) in the male battery, and competed for binding to the androgen receptor in the YTS competition assay. In the male battery KETO decreased weights for all androgen-dependent tissues, caused hormonal alterations (decreased T and DHT and increased LH and FSH), and induced spermatid retention. FIN decreased seminal vesicle and accessory sex gland (ASG) unit weight and caused hormonal alterations (decreased DHT and increased LH, and PRL) in the male battery. KETO was judged not to affect any of the endpoints in the female battery. ANA decreased ASG unit weight and serum E2 levels in the male battery. Using the responses obtained for all the endpoints in the Tier I battery, a distinct "fingerprint" was produced for each type of endocrine activity against which compounds with unknown activity can be compared. These data demonstrate that the described Tier I battery is useful for identifying EACs.

Lung proliferative and clearance responses to inhaled para-aramid RFP in exposed hamsters and rats: comparisons with chrysotile asbestos fibers.

This study compared pulmonary effects of para-aramid respirable-sized, fiber-shaped particles (RFP) (p-aramid fibrils) and chrysotile asbestos fiber exposures in rats. Additional p-aramid inhalation studies were conducted in hamsters to compare species responses. The hamster results are preliminary. The parameters studied were clearance/biopersistence of inhaled p-aramid RFP or size-separated asbestos fibers as well as pulmonary cell proliferation and inflammation indices after 2-week inhalation exposures. Rats were exposed nose only to chrysotile asbestos fibers at concentrations of 459 and 782 fibers/ml or to p-aramid RFP at 419 or 772 fibrils/ml. Hamsters were exposed whole body to p-aramid RFP at concentrations of 358 and 659 fibrils/ml. Subsequently, animals were assessed immediately (time 0) as well as 5 days (10 days for hamsters), 1, 3, 6, and 12 months postexposure. Lung burdens for the p-aramid-exposed rats were 4.8 x 10(7) and 7.6 x 10(7) fibrils/lung, with similar numbers of chrysotile fibers > 5 microns recovered from the lungs of asbestos-exposed rats. In comparison, 1.4 x 10(6) fibrils/lung were recovered in the high-dose hamster group. Biopersistence studies in p-aramid-exposed rats and hamsters demonstrated an initial increase (relative to time 0) in retained p-aramid fibrils during the first month postexposure, which indicated breakage or shortening of inhaled fibrils. This result was associated with a progressive reduction, and increased residence time in the lung, in the mean lengths of the fibrils, which signified biodegradability of inhaled p-aramid fibrils in both species. In contrast, clearance of short chrysotile asbestos fibers was rapid, but clearance of the long chrysotile fibers was slow or insignificant, as evidenced by a progressive increase over time in the mean lengths of fibers recovered from the lungs of exposed rats. Two-week, high-dose exposures to p-aramid in both rats and hamsters produced transient increases in pulmonary inflammatory and cell proliferative responses. In contrast, inhalation of size-separated chrysotile asbestos fibers in rats produced persistent increases in cell labeling indices of airway, alveolar, and subpleural cells measured through a period of 1 to 3 months postexposure. These results suggest that inhaled p-aramid RFP are biodegradable in the lungs of exposed rats and hamsters. In contrast, exposures to chrysotile asbestos fibers in rats resulted in a selective pulmonary retention of long chrysotile fibers.

Pulmonary effects in rats inhaling size-separated chrysotile asbestos fibres or p-aramid fibrils: differences in cellular proliferative responses.

This study was designed to compare the pulmonary cellular proliferative effects of inhaled, size-separated preparations of chrysotile asbestos fibres with similar aerosol fibre concentrations of para-aramid fibrils. Following fibre preparation, rats were exposed for 2 weeks to aerosols of p-aramid fibrils or chrysotile asbestos fibres at design fibre concentrations of 750 and 400 f/cc. Two week exposures to p-aramid fibrils produced transient pulmonary inflammatory and cell labeling responses in terminal bronchiolar and subpleural regions. Similar to p-aramid, exposure to chrysotile produced a transient increase in neutrophils. In contrast, however, substantial increases compared to controls in pulmonary cell labeling indices were measured on terminal bronchiolar, parenchymal, subpleural, and mesothelial surfaces immediately after exposure, and some increases persisted for 3 months postexposure. In complementary studies we demonstrated that p-aramid is biodegradable in the lungs of exposed rats; in contrast, the clearance of long chrysotile fibres was slow or insignificant, resulting in a pulmonary retention of long chrysotile asbestos fibres. The dimensional changes of asbestos fibres as well as the pulmonary cell labeling data indicate that chrysotile asbestos fibres may produce greater long-term pulmonary effects when compared to inhaled para-aramid fibrils.