Expression of a truncated, kinase-defective TGF-beta type II receptor in mouse skeletal tissue promotes terminal chondrocyte differentiation and osteoarthritis.

Members of the TGF-beta superfamily are important regulators of skeletal development. TGF-betas signal through heteromeric type I and type II receptor serine/threonine kinases. When over-expressed, a cytoplasmically truncated type II receptor can compete with the endogenous receptors for complex formation, thereby acting as a dominant-negative mutant (DNIIR). To determine the role of TGF-betas in the development and maintenance of the skeleton, we have generated transgenic mice (MT-DNIIR-4 and -27) that express the DNIIR in skeletal tissue. DNIIR mRNA expression was localized to the periosteum/perichondrium, syno-vium, and articular cartilage. Lower levels of DNIIR mRNA were detected in growth plate cartilage. Transgenic mice frequently showed bifurcation of the xiphoid process and sternum. They also developed progressive skeletal degeneration, resulting by 4 to 8 mo of age in kyphoscoliosis and stiff and torqued joints. The histology of affected joints strongly resembled human osteo-arthritis. The articular surface was replaced by bone or hypertrophic cartilage as judged by the expression of type X collagen, a marker of hypertrophic cartilage normally absent from articular cartilage. The synovium was hyperplastic, and cartilaginous metaplasia was observed in the joint space. We then tested the hypothesis that TGF-beta is required for normal differentiation of cartilage in vivo. By 4 and 8 wk of age, the level of type X collagen was increased in growth plate cartilage of transgenic mice relative to wild-type controls. Less proteoglycan staining was detected in the growth plate and articular cartilage matrix of transgenic mice. Mice that express DNIIR in skeletal tissue also demonstrated increased Indian hedgehog (IHH) expression. IHH is a secreted protein that is expressed in chondrocytes that are committed to becoming hypertrophic. It is thought to be involved in a feedback loop that signals through the periosteum/ perichondrium to inhibit cartilage differentiation. The data suggest that TGF-beta may be critical for multifaceted maintenance of synovial joints. Loss of responsiveness to TGF-beta promotes chondrocyte terminal differentiation and results in development of degenerative joint disease resembling osteoarthritis in humans.

Failure of rotenone to interfere with 17 beta-estradiol action in the rat uterus.

The involvement of rotenone in rat mammary carcinogenesis has been suggested to occur through estrogenic effects. This hypothesis was tested by determining the extent of rotenone inhibition of 17 beta-estradiol binding to the estrogen receptor and of the 17 beta-estradiol-induced uterotrophic response in ovariectomized Sprague-Dawley rats. Estradiol binding to the uterine estrogen receptor in the presence of rotenone was determined by charcoal assay and Scatchard analysis. Additionally, 17 beta-estradiol-receptor interactions were assessed on sucrose density gradients. No inhibition of binding was observed in either assay with ratios of rotenone/17 beta-estradiol in excess of 10,000. Finally, an in vivo approach was used to extend the in vitro data. Silastic capsules containing rotenone or 17 beta-estradiol were implanted in various combinations into eight groups of ovariectomized Sprague-Dawley rats (four rats/group). After five days, uteri were removed and weighed. An analysis of variance revealed that rotenone neither interfered with 17 beta-estradiol-induced uterine weight gain nor displayed any uterotrophic properties by itself. Results from these three procedures demonstrate that rotenone does not act as an estrogen or as an estrogen antagonist. Additionally, there were no other effects attributable to rotenone.

Endometrial hyperplasia and apoptosis following neonatal diethylstilbestrol exposure and subsequent estrogen stimulation in both host and transplanted hamster uteri.

Prenatal exposure to the synthetic estrogen diethylstilbestrol (DES) causes morphogenetic alterations and neoplasia in the human reproductive tract. In the hamster, neonatal DES exposure alters early uterine morphogenesis and induces endometrial adenocarcinomas in adults. We now demonstrate that the preneoplastic stages of this phenomenon in the hamster reflect an abnormal uterotropic response to estrogen that is characterized by hyperplastic lesions in the endometrial epithelium and includes an immune and/or inflammatory component. Interestingly, biochemical and in situ analysis revealed that the hyperplastic epithelium is also an active site of cell death by apoptosis. To further probe the mechanism of this phenomenon, uteri from 7-day-old control or DES-exposed donors were transplanted into the cheek pouches of control or neonatally DES-exposed adult hosts, and both host groups were treated to provide high circulating levels of estradiol. Among the four ectopic scenarios, histopathological lesions (epithelial hyperplasia, dysplasia, and apoptosis), segregated almost exclusively to the two that consisted of neonatally DES-exposed uteri. The virtual absence of lesions in control uteri transplanted to DES hosts eliminated host systemic factors as causative agents. Therefore, we conclude that DES or its metabolites alter the cellular physiology and/or composition of the developing uterus (initiating event) in such a way that it thereafter responds abnormally to estrogenic stimulation (promoting event). These observations serve to further define a unique experimental system for probing: (a) various aspects of the clinical "DES Syndrome"; (b) how estrogen regulates normal uterine growth and morphogenesis; and (c) how this process can degenerate to the unregulated neoplastic state.

Effects of estrogen on gene expression in the chick oviduct. Isolation and fractionation of chromatin non-histone proteins.

Non-histones isolated from hen oviduct chromatin have been fractionated by a variety of methods. Chromatin was dissociated in 2 M NaC1, 5 M Urea, 0.1% beta-mercaptoethanol and 0.01 M Tris - HC1, pH 8.3, and the DNA removed by ultracentrifugation. After desalting by gel filtration the chromatin proteins were separated into three distinct fractions by stepwise elution with 0.10 M NaC1, 0.25 M Na C1 and 15% guanidine - HC1 from Bio-Rex 70 columns. Fractions I and II contain only non-histones and Fraction III contains histones plus a small amount of non-histones. Further fractionation of the non-histones was achieved by ammonium sulfate precipitation and DEAE-cellulose chromatography for Fraction I and phosphocellulose chromatography and gel filtration on Bio-Gel A-15 m for Fraction II. The histone and non-histones present in Fraction III were separated by gel filtration on Bio-Gel A-0.5 m. All fractionation methods have been used preparatively with reasonable recoveries of protein (greater than or equal to 60%). The fractions have been characterized by acrylamide gel electrophoresis. The integrity of the histones was maintained during the fractionation procedure indicating that proteolytic degradation was unlikely to have occurred. There was no selective loss of chromatin proteins during the ultracentrifugation and desalting steps and the non-histones were separated into distinct fractions with enrichment of some species not apparent prior to fractionation of the chromatin proteins.

Cell cycle-specific effects of sodium arsenite and hyperthermic exposure on incorporation of radioactive leucine and phosphate by stress proteins from mouse lymphoma cell nuclei.

Cultured mouse lymphoma cells incorporated [3H]leucine and [32P]phosphate into nuclear stress proteins within 3 h after exposure to either elevated temperature (45 degrees C) or sodium arsenite. Radiolabeled proteins were detected by autoradiography after two-dimensional polyacrylamide gel electrophoresis. To determine the cell cycle stage specificity of labeling, nuclei were isolated and sorted into two cell cycle phases using a fluorescent activated cell sorter. After either heat shock or sodium arsenite treatment, the majority of [3H]leucine incorporation into stress proteins occurred during the G0 + G1 phase with minimal labeling in the G2 phase. On the other hand, 32P labeling of stress proteins occurred in both the G0 + G1 and G2 phases after exposure to sodium arsenite, while incorporation of 32P was limited after heat stress. Following sodium arsenite treatment, a distinct set of four stress proteins (80-84 kDa) was detected with [3H]leucine only in G0 + G1 phase, but with [32P]phosphate these stress proteins were labeled in both G0 + G1 and G2. There was differential [32P]phosphate labeling between proteins of the 80-84 kDa set during cell cycling. Individual proteins of this set were isolated from gel plugs after sodium arsenite or heat-shock treatment. Coelectrophoresis of proteins from the two treatment groups showed that they had similar electrophoretic mobilities. All four proteins of the 80-84 kDa set (sodium arsenite induced) possessed similar polypeptide maps after digestion with V8 protease. Cytofluorometric analysis demonstrated a reduction in the number of nuclei in both S and G2 phases of the cell cycle two h after heat shock, but not following sodium arsenite treatment. However, there was a significant depression in the number of nuclei in S and G2 4 h after exposure to sodium arsenite and very modest labeling with 32P of stress proteins was observed at this time.

Diethylstilbestrol and estradiol binding to serum albumin and pregnancy plasma of rat and human.

The equilibrium binding of diethylstilbestrol (DES) and 17 beta-estradiol (E2) to plasma proteins has been characterized. DES exhibits a 10- to 20-fold greater binding affinity index for bovine serum albumin and rat plasma than E2. As expected, E2 gave high values for binding to plasma from pregnant mice or rats, reflecting the presence of alpha-fetoprotein. DES bound to these samples as it did to bovine albumin and rat plasma. These results suggested that DES ineracts weakly with alpha-fetoprotein. This was verified by Scatchard plots of DES and E2 binding to rat and human pregnancy plasma. High affinity, low capacity binding was demonstrated with E2 but not with DES. The significantly lower binding of DES suggests that increased delivery of DES to the fetus may be at least partially responsible for the transplacental toxicity and carcinogenicity of DES.

The postnatal ontogeny of rat uterine ornithine decarboxylase: acquisition of a second peak of estrogen-induced enzyme activity.

Uterine ornithine decarboxylase (ODC) activity is reported to increase after estrogen administration to fetal, neonatal, immature, and adult rats, suggesting that it may be a useful marker in studies of the development of estrogen responsiveness. Standard conditions were validated for enzyme assay of uterine cytosols from 5-day-old rats, and it was demonstrated that full activity was retained after freezing cytosol in liquid N2. Maximal activity, obtained 6 h after the injection of 10 micrograms estradiol (E2) to 5-day-old rats, was also elicited by the same dose of mestranol, ethynylestradiol, diethylstilbestrol, or moxestrol. Progesterone, testosterone, and low doses of the antiestrogens clomiphene and tamoxifen failed to alter background ODC levels, while high antiestrogen doses induced small increases in enzyme activity. The glucocorticoid prednisolone lowered ODC activity. Dose-response curves established that E2 was more effective in increasing adult ODC levels (ED50 = 0.2 micrograms/kg E2) than neonatal ODC levels (ED50 = 2 micrograms/kg E2). Time-course measurements were conducted over 24 h in control and E2-injected animals on postnatal days 5, 10, 14, 20, and 28 and in 60-day-old ovariectomized adults. While an age-dependent decrease in control and 6 h E2-induced ODC levels was observed, there was an unexpected progressive development by day 28 of a second peak of E2-induced ODC at 15-18 h. The 6 h neonatal and 6 and 15-18 h adult ODC peaks had apparent Km values for ornithine near 0.2 mM. The potential origin of the second peak and its relationship to other uterine events are discussed.

Estradiol-stimulated increases in uterine eosinophils and nuclear type II estrogen-binding sites are prevented by pertussis toxin.

Previous studies from several laboratories have demonstrated that estradiol treatment resulted in an increase in nuclear type II binding sites. Our previous data suggest that this increase was due to the estradiol-stimulated influx of circulating eosinophils. Therefore, we suggested that the uterine nuclear type II estrogen-binding sites were not of uterine origin. In this report we present further evidence to support this hypothesis. Treatment of immature rats with estradiol resulted in the stimulation of several uterine parameters, namely wet weight, protein synthesis, eosinophil number, peroxidase activity, nuclear type II binding sites, and the synthesis and secretion of a 180-kDa protein. The coadministration of pertussigen had no effect on the estradiol-stimulated increase in wet weight, protein synthesis, or the synthesis and secretion of the 180-kDa protein. However, pertussigen did prevent the estradiol-stimulated increase in eosinophils, peroxidase activity, and nuclear type II binding sites, demonstrating a coordinated response. Since peroxidase activity is known to be contained int he eosinophil, these data are consistent with our earlier demonstration that the type II sites are of eosinophil origin. These data also support and extend our previous findings in neonatal animals that estradiol can stimulate a growth response without a corresponding increase in the nuclear type II binding sites. These results further indicate that the estradiol-stimulated increase in eosinophils does not appear to play a key role in the control of uterine growth.

Uterine responses to estradiol in the neonatal rat.

Although single doses of estrogens are known to be ineffective in stimulating complete uterine responses in newborn rats, repeated doses elicit toxic responses that become evident in adulthood. In this study, neonates injected daily from birth with 10 micrograms 17 beta-estradiol (E2) demonstrated significant uterine wet weight gain by day 3 and near-maximum growth (230% of control) by day 5. Elevated uterine weight can be maintained by repeated daily injections at least through day 13. Other uterine growth responses after 5 days of E2 (as percent of control) are: dry weight, 163%; DNA content, 193%; protein content, 211%; and nuclear estrogen receptor, 890%. Ornithine decarboxylase activity increased to 520% of control when measured 6 h after a single E2 injection on day 5. Histological examination of uteri from animals treated for 5 days reveals an altered stroma with evidence of circular muscle differentiation, while the lumenal epithelium, which is cuboidal in controls, becomes columnar after E2 injections. These data suggest that estrogens act in fundamentally the same manner in the neonatal uterus as in the adult uterus, although the appearance of the complete response appears to e slower in the neonate. The estrogen-induced precocious development we describe suggests that an estrogen may be involved in the normal postnatal development of uterine estrogen responsiveness. Adult toxicity, resulting from repeated neonatal estrogen dosing, may partly be a consequence of continual hormone action inducing developmentally inappropriate responses.

QSAR models for binding of estrogenic compounds to estrogen receptor alpha and beta subtypes.

We have developed Quantitative Structure-Activity Relationship (QSAR) models based on Comparative Molecular Field Analysis (CoMFA) for 31 estrogenic chemicals whose relative binding affinity (RBA) is available for both ER-alpha and ER-beta. The models demonstrated a significant correlation (r2>0.95) between the CoMFA-calculated steric/electrostatic fields and corresponding RBA data and a good predictive capability (q2>0.6) based on cross-validation. The CoMFA models and contour plots obtained for ER-alpha and ER-beta suggest a close similarity between the receptors in terms of mode of binding and provide a rational basis for ligand selectivity.

The postnatal ontogeny of rat uterine glands and age-related effects of 17 beta-estradiol.

In the uterus of the newborn rat, only the luminal epithelium is differentiated. Differentiation of musculature and glandular epithelium occurs postnatally, the latter originating as invaginations of the luminal epithelium into the stroma. Using unambiguous criteria for quantification of uterine glands, we find that uterine glands first appear on postnatal day 9 after which the increase in the number of glands is rapid and synchronous, with approximately 4.4 glands per uterine section reached by day 15. Between days 15 and 35, the number of glands per uterine section varied in a cyclic manner with an amplitude of approximately one gland per uterine section and a period of 6-7 days. Although exogenous 17 beta-estradiol (E2) administered on postnatal days 1-5 induced slight premature gland genesis, the number of glands per uterine section was approximately 30% lower between days 15-26 compared to untreated animals. Administration of E2 during the period of normal gland genesis (days 10-14) induced a dose-related delay in the onset of appearance of glands. After this, gland genesis proceeded at a normal rate; however, the maximum levels reached were again generally below those observed in untreated controls. E2 administered after uterine glands were established (days 20-24) induced a small increase in gland number compared to controls. E2 also induced temporary hypertrophy, hyperplasia, and cellular degeneration in the luminal epithelium during each of the dosing periods without corresponding changes in the stroma or myometrium. These data demonstrate that uterine gland genesis occurs between postnatal days 9-15 and that exogenous estrogen can alter, in an age-specific manner, both uterine gland genesis and the number of glands per uterine section.

Estrogen regulation of an eosinophil chemotactic factor in the immature rat uterus.

Associated with the generalized uterine growth stimulated by estradiol in the rat are specific responses including messenger RNA (mRNA) synthesis, protein synthesis, and peroxidase activity. The increase in peroxidase activity, although sensitive to inhibitors of RNA and protein synthesis, results from an estradiol-stimulated influx of eosinophils into the uterus. We postulated the existence of an estradiol-regulated uterine chemotactic factor, testing this by an in vitro chemotactic assay with eosinophils isolated from mature rats. Treatment of immature rats with 1 microgram estradiol for 24 h resulted in a significant increase in eosinophil chemotaxis compared to uterine extracts of vehicle-treated rats. This increase was seen as early as 3 h after estradiol administration and was maximal at 24 h. The magnitude of the chemotactic response was dependent on the dose of estradiol administered (1-100 micrograms). Estrone or estriol treatment resulted in chemotactic activity greater than control but less than estradiol. Direct addition of estradiol to extracts of control animals did not increase chemotaxis. The estradiol-stimulated chemotaxis was blocked by in vivo treatment with the antiestrogen tamoxifen and by inhibitors of RNA and protein synthesis. Analysis of extracts from estradiol-treated uteri shows that the chemotactic factor is heat labile, pronase sensitive, and has a mass of approximately 20 kilodaltons (kDa). These data suggest that the estradiol-stimulated influx of eosinophils into the rat uterus is mediated by the synthesis, modification, or release of a protein whose synthesis is estradiol receptor mediated.

Inhibition of rat uterine gland genesis by tamoxifen.

We have previously shown that rat uterine gland genesis occurs rapidly and synchronously between postnatal days 9-15. Exogenous estrogens either stimulate or inhibit gland genesis depending on dose and age at administration. We therefore examined the developmental effects of the triphenylethylene antiestrogen tamoxifen, which exhibits both estrogen agonist and antagonist properties, in the postnatal rat uterus. Tamoxifen administered sc in oil on postnatal days 1-5 or days 10-14 caused dose-related inhibition of uterine gland genesis which persisted to day 26 or day 60, respectively. Tamoxifen administered on postnatal days 20-24, which is after the age of normal gland genesis, did not alter the number of preexisting glands. A 24-h exposure to tamoxifen inhibited 17 beta-estradiol (E2)-induced ornithine decarboxylase (ODC) activity measured 6 h after E2 administration in 14-day-old rats. Treatment with tamoxifen before or during the period of gland genesis also reduced uterine responsiveness to a single dose of E2 as measured by both uterine weight gain (after a 24-h exposure on days 14, 19, 22, and 26) and the pattern of E2-induced ODC activity in 26-day-old rats. Control rats respond to E2 with peaks of ODC activity at 6 and 18 h after administration. Treatment with tamoxifen on either postnatal days 1-5 or 10-14 reduced the 18-h peak to approximately half of controls but did not affect the 6-h E2-induced ODC peak. Analysis of both nuclear and translocatable cytosol estrogen receptor in uteri from 26-day-old rats indicate that neither the dissociation constant (KD) nor the number of binding sites was affected by tamoxifen treatment on postnatal days 1-5 or 10-14.

No threshold dose for estradiol-induced sex reversal of turtle embryos: how little is too much?

Risk assessments for nongenotoxic chemicals assume a threshold below which no adverse outcomes are seen. However, when an endogenous chemical, such as 17ss-estradiol (E2), occurs at a concentration sufficient to cause an effect, the threshold is already exceeded. Under these circumstances, exogenous estradiol is not expected to provide a threshold dose. This principle is demonstrated for E2 in the red-eared slider, a turtle with temperature-dependent sex determination. In this species, gonadal sex is determined by egg incubation temperature; female development requires endogenous estrogen produced by elevated temperature. While normal production of females by endogenous estrogens is not an adverse effect, exogenous estrogens can sex reverse presumptive males, which can be an adverse effect. A large dose-response study was conducted using seven doses and a vehicle control (starting n = 300/group); a single E2 dose was applied to the eggshell of recently laid eggs. Animals were sexed after hatching. The incubation temperature chosen, 28.6 degrees C, generates a minority of females. Thus, the criteria for testing the threshold hypothesis were met, i.e., there is evidence that there is endogenous estrogen and that it generates an irreversible response. The lowest E2 dose tested, 400 pg/egg (40 ng/kg), sex reversed 14.4% of the animals, demonstrating very low dose sensitivity. The data were fit with a modified Michaelis-Menten equation, which provided an estimate of 1.7 ng/egg for endogenous estradiol. The median effective dose (ED50) was 5.0 +/- 2.0 ng/egg (95% confidence limits), of which 1.7 ng/egg was endogenous estradiol and 3.3 ng/egg came from the applied estradiol. There was no apparent threshold dose for E2. A smaller replication confirmed these results. These results provide a simple biologically based dose-response model and suggest that chemicals which act mechanistically like E2 may also show no threshold dose. If so, even low environmental concentrations of such chemicals may carry risk for sex reversal.

Quantitative comparisons of in vitro assays for estrogenic activities.

Substances that may act as estrogens show a broad chemical structural diversity. To thoroughly address the question of possible adverse estrogenic effects, reliable methods are needed to detect and identify the chemicals of these diverse structural classes. We compared three assays--in vitro estrogen receptor competitive binding assays (ER binding assays), yeast-based reporter gene assays (yeast assays), and the MCF-7 cell proliferation assay (E-SCREEN assay)--to determine their quantitative agreement in identifying structurally diverse estrogens. We examined assay performance for relative sensitivity, detection of active/inactive chemicals, and estrogen/antiestrogen activities. In this examination, we combined individual data sets in a specific, quantitative data mining exercise. Data sets for at least 29 chemicals from five laboratories were analyzed pair-wise by X-Y plots. The ER binding assay was a good predictor for the other two assay results when the antiestrogens were excluded (r(2) is 0.78 for the yeast assays and 0.85 for the E-SCREEN assays). Additionally, the examination strongly suggests that biologic information that is not apparent from any of the individual assays can be discovered by quantitative pair-wise comparisons among assays. Antiestrogens are identified as outliers in the ER binding/yeast assay, while complete antagonists are identified in the ER binding and E-SCREEN assays. Furthermore, the presence of outliers may be explained by different mechanisms that induce an endocrine response, different impurities in different batches of chemicals, different species sensitivity, or limitations of the assay techniques. Although these assays involve different levels of biologic complexity, the major conclusion is that they generally provided consistent information in quantitatively determining estrogenic activity for the five data sets examined. The results should provide guidance for expanded data mining examinations and the selection of appropriate assays to screen estrogenic endocrine disruptors.

Quantitative structure-activity relationships (QSARs) for estrogen binding to the estrogen receptor: predictions across species.

The recognition of adverse effects due to environmental endocrine disruptors in humans and wildlife has focused attention on the need for predictive tools to select the most likely estrogenic chemicals from a very large number of chemicals for subsequent screening and/or testing for potential environmental toxicity. A three-dimensional quantitative structure-activity relationship (QSAR) model using comparative molecular field analysis (CoMFA) was constructed based on relative binding affinity (RBA) data from an estrogen receptor (ER) binding assay using calf uterine cytosol. The model demonstrated significant correlation of the calculated steric and electrostatic fields with RBA and yielded predictions that agreed well with experimental values over the entire range of RBA values. Analysis of the CoMFA three-dimensional contour plots revealed a consistent picture of the structural features that are largely responsible for the observed variations in RBA. Importantly, we established a correlation between the predicted RBA values for calf ER and their actual RBA values for human ER. These findings suggest a means to begin to construct a more comprehensive estrogen knowledge base by combining RBA assay data from multiple species in 3D-QSAR based predictive models, which could then be used to screen untested chemicals for their potential to bind to the ER. Another QSAR model was developed based on classical physicochemical descriptors generated using the CODESSA (Comprehensive Descriptors for Structural and Statistical Analysis) program. The predictive ability of the CoMFA model was superior to the corresponding CODESSA model.

Quantitative mechanistically based dose-response modeling with endocrine-active compounds.

A wide range of toxicity test methods is used or is being developed for assessing the impact of endocrine-active compounds (EACs) on human health. Interpretation of these data and their quantitative use in human and ecologic risk assessment will be enhanced by the availability of mechanistically based dose-response (MBDR) models to assist low-dose, interspecies, and (italic)in vitro(/italic) to (italic)in vivo(/italic) extrapolations. A quantitative dose-response modeling work group examined the state of the art for developing MBDR models for EACs and the near-term needs to develop, validate, and apply these models for risk assessments. Major aspects of this report relate to current status of these models, the objectives/goals in MBDR model development for EACs, low-dose extrapolation issues, regulatory inertia impeding acceptance of these approaches, and resource/data needs to accelerate model development and model acceptance by the research and the regulatory community.

Research needs for the risk assessment of health and environmental effects of endocrine disruptors: a report of the U.S. EPA-sponsored workshop.

The hypothesis has been put forward that humans and wildlife species adverse suffered adverse health effects after exposure to endocrine-disrupting chemicals. Reported adverse effects include declines in populations, increases in cancers, and reduced reproductive function. The U.S. Environmental Protection Agency sponsored a workshop in April 1995 to bring together interested parties in an effort to identify research gaps related to this hypothesis and to establish priorities for future research activities. Approximately 90 invited participants were organized into work groups developed around the principal reported health effects-carcinogenesis, reproductive toxicity, neurotoxicity, and immunotoxicity-as well as along the risk assessment paradigm-hazard identification, dose-response assessment, exposure assessment, and risk characterization. Attention focused on both ecological and human health effects. In general, group felt that the hypothesis warranted a concerted research effort to evaluate its validity and that research should focus primarily on effects on development of reproductive capability, on improved exposure assessment, and on the effects of mixtures. This report summarizes the discussions of the work groups and details the recommendations for additional research.

Threshold analysis of selected dose-response data for endocrine active chemicals.

Using a biologically relevant mathematical model, the Michaelis-Menten equation, we examined published data from endocrine active chemicals for evidence of no-threshold dose-response curves. Data were fit to a modified Michaelis-Menten equation which accounted for total background response. Subsequently, the data sets were analyzed using non-linear regression in order to estimate the four parameters of interest (non-hormone controlled background (Bnh), maximum response (Rmax), endogenous hormone level (D0), and the dose at which a half-maximal response was observed (ED50)) and to determine the fit to the fully modified Michaelis-Menten equation. Subsequently, response data were adjusted to account for Bnh and then normalized to Rmax, while dose data were adjusted to account for D0 and then normalized to the ED50. This data set was combined into a single, composite data set and fit to the fully modified Michaelis-Menten equation. We examined 31 data sets (24 endpoints) from studies on 9 different chemical/hormone treatments. Twenty-six of the data sets fit the modified Michaelis-Menten equation with high multiple correlation coefficients (r>0.90). The normalized data demonstrated a good fit to the modified Michaelis-Menten equation. These results indicate that a variety of biological responses fit the modified Michaelis-Menten equation, which does not have a threshold dose term.

Spleen cell phosphorylation of salt soluble nuclear protein from isoproterenol treated and sorted nuclei.

The effect of isoproterenol (IPR) on phosphorylation of acidic nuclear proteins was investigated by two-dimensional gel autoradiography. Mouse spleen cells stimulated to divide by the mitogen concanavalin A (Con A) were separated according to cell cycle stage by flow microfluorometric technique. Exposure of cells for 48 h to 4 micrograms IPR/ml culture medium produced no significant change in the proportion of S and G2 phase cells, while a cumulative dose of 8 micrograms IPR/ml caused a significant repression in DNA synthesis and a reduction in the number of nuclei in G2 + M phase. Four micrograms IPR/ml stimulated the greatest amount of G0 + G1 phosphorylation of nuclear protein. Several proteins from G0 + G1 and S nuclei incorporated 32P after Con A + IPR administration, and one protein from S phase nuclei revealed intensified labeling at the 8 micrograms cumulative IPR dose but not at the 4 micrograms dose. The isolated proteins (W, X, Y, and Z) were reassociated with homologous DNA, centrifuged in a sucrose gradient and shown to co-sediment with DNA. S phase nuclear protein X-S, which was found to be a mixture of proteins (X0 and X1), was the only exception. One component of X-S, X0 bound to DNA, while component X1 failed to bind. Chymotryptic and V8 protease digests of all isolated proteins were made and analyzed by autoradiography. Proteins X0 and X1, recovered from the sucrose gradient, possessed dissimilar fragment patterns. It is concluded that protein X-S is composed of two proteins (X0 and X1), one of which (X1) appears during S phase during the 8 micrograms IPR induced nuclear repression.