The formation of Charon's red poles from seasonally cold-trapped volatiles.

A unique feature of Pluto's large satellite Charon is its dark red northern polar cap. Similar colours on Pluto's surface have been attributed to tholin-like organic macromolecules produced by energetic radiation processing of hydrocarbons. The polar location on Charon implicates the temperature extremes that result from Charon's high obliquity and long seasons in the production of this material. The escape of Pluto's atmosphere provides a potential feedstock for a complex chemistry. Gas from Pluto that is transiently cold-trapped and processed at Charon's winter pole was proposed as an explanation for the dark coloration on the basis of an image of Charon's northern hemisphere, but not modelled quantitatively. Here we report images of the southern hemisphere illuminated by Pluto-shine and also images taken during the approach phase that show the northern polar cap over a range of longitudes. We model the surface thermal environment on Charon and the supply and temporary cold-trapping of material escaping from Pluto, as well as the photolytic processing of this material into more complex and less volatile molecules while cold-trapped. The model results are consistent with the proposed mechanism for producing the observed colour pattern on Charon.

Design of a neutron microscope based on Wolter mirrors.

The predominant geometry for a neutron imaging experiment is that of a pinhole camera. This is primarily due to the difficulty in focusing neutrons due to the weak refractive index, which is also strongly chromatic. Proof of concept experiments demonstrated that neutron image forming lenses based on reflective Wolter mirrors can produce quantitative, high spatial resolution neutron images while also increasing the time resolution compared to the conventional pinhole camera geometry. Motivated by these results, we report the design of a neutron microscope where two Wolter mirrors replace condensing and objective lenses, in direct analogy with typical visible light microscopes. Ray tracing results indicate that this system will yield 3 µm spatial resolution images with an acquisition time of order <1 s (104 faster than currently possible at this spatial resolution) with a field of view of about 5 mm in diameter.

Color, composition, and thermal environment of Kuiper Belt object (486958) Arrokoth.

The outer Solar System object (486958) Arrokoth (provisional designation 2014 MU69) has been largely undisturbed since its formation. We studied its surface composition using data collected by the New Horizons spacecraft. Methanol ice is present along with organic material, which may have formed through irradiation of simple molecules. Water ice was not detected. This composition indicates hydrogenation of carbon monoxide-rich ice and/or energetic processing of methane condensed on water ice grains in the cold, outer edge of the early Solar System. There are only small regional variations in color and spectra across the surface, which suggests that Arrokoth formed from a homogeneous or well-mixed reservoir of solids. Microwave thermal emission from the winter night side is consistent with a mean brightness temperature of 29 ± 5 kelvin.

Halohydrocarbon synthesis by bromoperoxidase.

An enzyme extracted from marine red algae, Bonnemaisonia hamifera, is capable of incorporating bromine into a number of organic substrates in the pH range 5 to 8. At pH 7.3, incubation of partially purified preparations of bromoperoxidase with hydrogen peroxide, bromide ion, and 3-oxooctanoic acid leads to the formation of three volatile brominated hydrocarbons: dibromomethane, bromoform, and 1-pentyl bromide. The presence of significant quantities of halometabolites including volatile halohydrocarbons in marine organisms, ocean waters, and the upper atmosphere may result from peroxidase-catalyzed halogenation reactions.

Quasielastic neutron scattering study of hydrogen motion in NbC(0.71)H(0.28).

In order to study the mechanism and parameters of H jump motion in the nonstoichiometric Nb carbides, we have performed quasielastic neutron scattering (QENS) measurements for NbC(0.71)H(0.28) over the temperature range 11- 475 K. Our results indicate that about 30% of H atoms in this system participate in a fast diffusive motion. The temperature dependence of the corresponding H jump rate in the range 298-475 K follows the Arrhenius law with an activation energy of 328 ± 9 meV. The Q dependence of the QENS data suggests that the observed jump motion corresponds to long-range diffusion of H atoms along chains of the off-centre sites in carbon vacancies.

Detection of ammonia on Pluto's surface in a region of geologically recent tectonism.

We report the detection of ammonia (NH3) on Pluto's surface in spectral images obtained with the New Horizons spacecraft that show absorption bands at 1.65 and 2.2 µm. The ammonia signature is spatially coincident with a region of past extensional tectonic activity (Virgil Fossae) where the presence of H2O ice is prominent. Ammonia in liquid water profoundly depresses the freezing point of the mixture. Ammoniated ices are believed to be geologically short lived when irradiated with ultraviolet photons or charged particles. Thus, the presence of NH3 on a planetary surface is indicative of a relatively recent deposition or possibly through exposure by some geological process. In the present case, the areal distribution is more suggestive of cryovolcanic emplacement, however, adding to the evidence for ongoing geological activity on Pluto and the possible presence of liquid water at depth today.

Surface compositions across Pluto and Charon.

The New Horizons spacecraft mapped colors and infrared spectra across the encounter hemispheres of Pluto and Charon. The volatile methane, carbon monoxide, and nitrogen ices that dominate Pluto's surface have complicated spatial distributions resulting from sublimation, condensation, and glacial flow acting over seasonal and geological time scales. Pluto's water ice "bedrock" was also mapped, with isolated outcrops occurring in a variety of settings. Pluto's surface exhibits complex regional color diversity associated with its distinct provinces. Charon's color pattern is simpler, dominated by neutral low latitudes and a reddish northern polar region. Charon's near-infrared spectra reveal highly localized areas with strong ammonia absorption tied to small craters with relatively fresh-appearing impact ejecta.

The small satellites of Pluto as observed by New Horizons.

The New Horizons mission has provided resolved measurements of Pluto's moons Styx, Nix, Kerberos, and Hydra. All four are small, with equivalent spherical diameters of ~40 kilometers for Nix and Hydra and ~10 kilometers for Styx and Kerberos. They are also highly elongated, with maximum to minimum axis ratios of ~2. All four moons have high albedos (~50 to 90%) suggestive of a water-ice surface composition. Crater densities on Nix and Hydra imply surface ages of at least 4 billion years. The small moons rotate much faster than synchronous, with rotational poles clustered nearly orthogonal to the common pole directions of Pluto and Charon. These results reinforce the hypothesis that the small moons formed in the aftermath of a collision that produced the Pluto-Charon binary.

Measurement of health-related quality of life for people with dementia: development of a new instrument (DEMQOL) and an evaluation of current methodology.

OBJECTIVES: To develop and validate a psychometrically rigorous measure of health-related quality of life (HRQoL) for people with dementia: DEMQOL. DATA SOURCES: Literature review. Expert opinion. Interviews and questionnaires. REVIEW METHODS: Gold standard psychometric techniques were used to develop DEMQOL and DEMQOL-Proxy. A conceptual framework was generated from a review of the literature, qualitative interviews with people with dementia and their carers, expert opinion and team discussion. Items for each component of the conceptual framework were drafted and piloted to produce questionnaires for the person with dementia (DEMQOL) and carer (DEMQOL-Proxy). An extensive two-stage field-testing was then undertaken of both measures in large samples of people with dementia (n = 130) and their carers (n = 126) representing a range of severity and care arrangements. In the first field test, items with poor psychometric performance were eliminated separately for DEMQOL and DEMQOL-Proxy to produce two shorter, more scientifically robust instruments. In the second field test, the item-reduced questionnaires were evaluated along with other validating measures (n = 101 people with dementia, n = 99 carers) to assess acceptability, reliability and validity. RESULTS: Rigorous evaluation in two-stage field testing with 241 people with dementia and 225 carers demonstrated that in psychometric terms: (1) DEMQOL is comparable to the best available dementia-specific HRQoL measures in mild to moderate dementia, but is not appropriate for use in severe dementia [Mini Mental State Examination (MMSE) <10]; and (2) DEMQOL-Proxy is comparable to the best available proxy measure in mild to moderate dementia, and shows promise in severe dementia. In addition, the DEMQOL system has been validated in the UK in a large sample of people with dementia and their carers, and it provides separate measures for self-report and proxy report, which allows outcomes assessment across a wide range of severity in dementia. CONCLUSIONS: The 28-item DEMQOL and 31-item DEMQOL-Proxy provide a method for evaluating HRQoL in dementia. The new measures show comparable psychometric properties to the best available dementia-specific measures, provide both self- and proxy-report versions for people with dementia and their carers, are appropriate for use in mild/moderate dementia (MMSE >/= 10) and are suitable for use in the UK. DEMQOL-Proxy also shows promise in severe dementia. As DEMQOL and DEMQOL-Proxy give different but complementary perspectives on quality of life in dementia, the use of both measures together is recommended. In severe dementia, only DEMQOL-Proxy should be used. Further research with DEMQOL is needed to confirm these findings in an independent sample, evaluate responsiveness, investigate the feasibility of use in specific subgroups and in economic evaluation, and develop population norms. Additional research is needed to address the psychometric challenges of self-report in dementia and validating new dementia-specific HRQoL measures.

Increase in thyroid follicular cell tumors in nelfinavir-treated rats observed in a 2-year carcinogenicity study is consistent with a rat-specific mechanism of thyroid neoplasia.

The carcinogenic potential of nelfinavir mesylate (nelfinavir) was evaluated in a 2-year oral (gavage) study on Sprague-Dawley rats at dose levels of 0 (control), 0 (vehicle control), 100, 300 and 1000 mg/kg per day. At the end of the treatment, increased incidences of thyroid follicular cell hyperplasia and neoplasms were observed at 300 (males) and 1000 mg/kg per day (both sexes). There were no other treatment-related effects and no tumors at other sites. Results from previous studies indicated a number of effects in the liver and thyroid, as well as metabolic profiles that suggested nelfinavir might cause thyroid hyperplasia/neoplasia secondary to hormone imbalance by altering thyroid hormone disposition. To investigate this hypothesis, the effects of nelfinavir on gene expression in rat hepatocytes and liver slices (in vitro), thyroxine plasma clearance, and thyroid gland function were evaluated. Compared to controls, gene expression analyses demonstrated an increased expression of glucuronyltransferase (UDPGT) and CYP450 3A1 in nelfinavir-treated rat hepatocytes and liver slices. In rats treated with nelfinavir (1000 mg/kg per day) for 4 weeks, liver weights and centrilobular hepatocellular hypertrophy were increased and minimal to mild diffuse thyroid follicular cell hypertrophy and follicular cell hyperplasia were evident in the thyroid gland. Thyroid-stimulating hormone (TSH) levels were significantly increased (three-fold), while tri-iodothyronine (T3)/tetra-iodothyronine (T4) and reverse T3(rT3) levels were unchanged, indicating that a compensated state to maintain homeostasis of T3/T4 had been achieved. Plasma 125I-thyroxine clearance was increased and the plasma thyroxine AUC0-48 was decreased (24%) compared to control. In conclusion, these data indicate that thyroid neoplasms observed in the nelfinavir-treated rats were secondary to thyroid hormone imbalance. Increased thyroxine clearance contributes to the effects of nelfinavir on thyroid gland function and is probably a result of UDPGT induction that leads to elevated TSH levels in the rat and eventual thyroid neoplasia. These results are consistent with a well-recognized rat-specific mechanism for thyroid neoplasms.

The Pluto system: Initial results from its exploration by New Horizons.

The Pluto system was recently explored by NASA's New Horizons spacecraft, making closest approach on 14 July 2015. Pluto's surface displays diverse landforms, terrain ages, albedos, colors, and composition gradients. Evidence is found for a water-ice crust, geologically young surface units, surface ice convection, wind streaks, volatile transport, and glacial flow. Pluto's atmosphere is highly extended, with trace hydrocarbons, a global haze layer, and a surface pressure near 10 microbars. Pluto's diverse surface geology and long-term activity raise fundamental questions about how small planets remain active many billions of years after formation. Pluto's large moon Charon displays tectonics and evidence for a heterogeneous crustal composition; its north pole displays puzzling dark terrain. Small satellites Hydra and Nix have higher albedos than expected.

Purification and partial characterization of ubiquitin-activating enzyme from Saccharomyces cerevisiae.

Ubiquitin-activating enzyme was purified from the yeast Saccharomyces cerevisiae by covalent affinity chromatography on ubiquitin-Sepharose followed by HPLC anion-exchange chromatography. Enzyme activity was monitored by the ubiquitin-dependent ATP: 32PPi exchange assay. The purified enzyme has a specific activity of 1.5 mumol 32PPi incorporated into ATP.min-1.mg-1 at 37 degrees C and pH 7.0 under standard conditions for substrate concentrations as described by Ciechanover et al. (1982) J. Biol. Chem. 257, 2537-2542. The catalytic activity showed a maximum at pH 7.0. Its molecular weight both in non-denaturing and in SDS-gel electrophoresis was estimated to be 115 kDa, suggesting a monomeric form. The isoelectric point determined by gel electrofocusing was approximately 4.7. Two protein bands differing slightly in electrophoretic mobility could be distinguished when SDS gels were loaded with very small amounts of purified E1 and immunoblotted, the one with higher molecular weight being clearly predominant. The same two bands were also found in anti-E1 immunoblots of crude yeast lysates prepared under broad protease inhibition.

Cytochrome P-450 induction by 3-methylcholanthrene and its antagonism by 2,2-dimethyl-5-t-butyl-1,3-benzodioxole.

Previous studies in this laboratory have shown 2,2-dimethyl-5-t-butyl-1,3-benzodioxole (DBBD) to antagonize 3-methylcholanthrene induction of cytochrome P-450 in Dub:ICR mice yet have no effect on phenobarbital induction. In the present experiments, C57BL/6 mice, an Ah responsive strain, produced a similar response under the same experimental conditions. The hypothesis that DBBD, although not a cytochrome P-450 inducer, competes with 3-methylcholanthrene for binding to the Ah receptor was tested. Using sucrose density gradients, the Ah receptor was measured in hepatic cytosol from Dub:ICR and C57BL/6 male mice. DBBD was unable to displace either 2,3,7,8-tetra-chlorodibenzo-p-dioxin or 3-methylcholanthrene from the Ah receptor, in vitro. However, in in vivo experiments, DBBD treatment of Dub:ICR mice caused Ah receptor depression at 6 and 24 hr with complete recovery in between, while 3-methylcholanthrene treatment caused a 2-fold Ah receptor reduction at 2 hr followed by complete recovery after 12 hr. When 3-methylcholanthrene and DBBD were coadministered, the depression of the Ah receptor was additive. DBBD-pretreated mice had a 2.25-fold reduction in Ah receptor level, effectively blocking the ability of 3-methylcholanthrene to increase the cytochrome P-450 content and either benzo[a]pyrene hydroxylase or ethoxyresorufin O-deethylase activities. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis confirmed that 3-methylcholanthrene induction of cytochrome P-450 was inhibited by DBBD pretreatment. Hence, although DBBD does not displace 3-methylcholanthrene from the Ah receptor in vitro, it does antagonize 3-methylcholanthrene induction of cytochrome P-450 and also reduces the amount of available receptor in vivo. This interaction may be due either to antagonism or to downregulation of the Ah receptor.

2,2-Dimethyl-5-t-butyl-1,3-benzodioxole: an unusual inducer of microsomal enzymes.

Our previous studies have shown that 2,2-dimethyl-5-t-butyl-1,3-benzodioxole (DBBD), a methylenedioxyphenyl (MDP) analog in which the methylene hydrogens have been replaced by methyl groups, does not form an inhibitory complex with cytochrome P-450 nor induce this cytochrome. However, in the present experiments, DBBD-treated male Dub:ICR mice showed an increase in NADPH-dependent cytochrome c (P-450) reductase and epoxide hydrolase activity. This separation of cytochrome P-450 induction from the induction of epoxide hydrolase and NADPH-dependent cytochrome c (P-450) reductase appears to be unique among inducers of xenobiotic metabolizing enzymes. In similar experiments, mice were treated with phenobarbital + DBBD or 3-methylcholanthrene + DBBD and the following parameters were measured: cytochrome P-450 content; NADPH-dependent reduction of cytochrome c; ethylmorphine and benzphetamine N-demethylase; 7-ethoxycoumarin O-deethylase; benzo[a]pyrene hydroxylase; and ethoxyresorufin O-deethylase. The microsomal proteins were examined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE). Phenobarbital + DBBD treatment gave results which did not differ significantly from those obtained with phenobarbital alone. In contrast, cytochrome P-450 content and benzo[a]pyrene hydroxylase and ethoxyresorufin O-deethylase activities were less in mice treated with 3-methylcholanthrene + DBBD than in animals treated with 3-methylcholanthrene alone. SDS-PAGE confirmed that induction of cytochrome P-450 by 3-methylcholanthrene was reduced by DBBD, suggesting that the latter compound may be an antagonist to the Ah cytosolic receptor.

Ah receptor: relevance of mechanistic studies to human risk assessment.

Studies of the toxic actions of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in numerous animal models and in human and animal cells in culture have established that the most characteristic pathologic lesions produced by this compound result from events initiated by the interaction of TCDD with a specific intracellular receptor protein, the Ah receptor. Although most research on the interaction of TCDD with the Ah receptor has focused on establishing involvement of this receptor complex in specific toxic responses, recent application of modern cell and molecular biology techniques is yielding new insights into the mechanism(s) of signal transduction. Elucidation of these mechanisms is essential for understanding the molecular basis of the cell and species specificity which is a hallmark of TCDD toxicity. This knowledge should provide the framework for development of a more toxicologically relevant risk assessment model.

Mechanisms of extrahepatic tumor induction by peroxisome proliferators in male CD rats.

Wyeth-14,643 (WY) and ammonium perfluorooctanoate (C8) belong to a diverse class of compounds which have been shown to produce hepatic peroxisome proliferation in rodents. From previous work, WY, but not C8, has been shown to produce hepatocellular carcinoma in rats, while C8 has been shown to produce Leydig cell adenomas. In addition, based on a review of bioassay data a relationship appears to exist between peroxisome-proliferating compounds and Leydig cell adenoma and pancreatic acinar cell hyperplasia/adenocarcinoma formation. To further investigate the relationship between peroxisome-proliferating compounds and hepatic, Leydig cell, and pancreatic acinar cell tumorigenesis, a 2-year feeding study in male CD rats was initiated to test the hypothesis that peroxisome proliferating compounds induce a tumor triad (liver, Leydig cell, pancreatic acinar cell), and to examine the potential mechanism for the Leydig cell tumors. The study was conducted using 50 ppm WY and 300 ppm C8. The concentration of WY in the diet was decreased to 25 ppm on test day 301 due to increased mortality. In addition to the ad libitum control, a second control was pair-fed to the C8 group. Interim sacrifices were performed at 1- or 3-month intervals. Peroxisome proliferation measured by beta-oxidation activity and cell proliferation were measured in the liver and testis at all time points and in the pancreas beginning at the 9-month time point (cell proliferation only). Serum hormone concentrations (estradiol, testosterone, LH, FSH, and prolactin) were also measured at each time point. Increased relative liver weights and hepatic beta-oxidation activity were observed in both the WY- and C8-treated rats at all time points. In contrast, hepatic cell proliferation was significantly increased only in the WY-treated group. Neither WY nor C8 significantly altered the rate of Leydig cell beta-oxidation or Leydig cell proliferation when compared to the control groups. Moreover, the basal rate of beta-oxidation in Leydig cells was approximately 20 times less than the rate of hepatic beta-oxidation. There were no biologically meaningful differences in serum testosterone, FSH, prolactin, or LH concentrations in the WY- and C8-treated rats when compared to their respective controls. There were, however, significant increases in serum estradiol concentrations in the WY- and C8-treated rats at 1, 3, 6, 9, 15, 18, and 21 months. At 12 months, only the C8-treated rats had elevated serum estradiol concentrations when compared to the pair-fed control. Histopathological evaluation revealed compound-related increases in liver, Leydig cell, and pancreatic acinar cell tumors in both WY- and C8-treated rats. The data support the hypothesis that the peroxisome-proliferating compounds induce the previously described tumor triad. In addition, both C8 and WY produced a sustained increase in serum estradiol concentrations that correlated with the potency of the 2 compounds to induce Leydig cell tumors (i.e., WY caused a more consistent sustained increase in serum estradiol throughout the entire study, and more specifically at the end of the study, than did C8). This study suggests that estradiol may play a role in enhancement of Leydig cell tumors in the rat, and that peroxisome proliferators may induce tumors via a non-LH type mechanism.

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.

Detection of the environmental antiandrogen p,p-DDE in CD and long-evans rats using a tier I screening battery and a Hershberger assay.

In this report, p,p'-DDE, a weak androgen receptor (AR) antagonist, has been examined in a Tier I screening battery designed to detect endocrine-active compounds (EACs). The screening battery that was used to examine p,p'-DDE was an abbreviated version of a proposed Tier I screening battery (Cook et al., 1997, Regul. ToxicoL Pharmacol. 26, 60-68) that consisted of a 15-day intact male in vivo battery and an in vitro yeast transactivation system (YTS). In addition, strain sensitivity differences were evaluated using male Crl:CDIGS BR (CD) and Long-Evans (LE) rats. Finally, p,p'-DDE was examined in a Hershberger assay designed to detect AR agonists. In the in vivo male battery using CD rats, responses to p,p'-DDE included organ weight changes (increased relative liver weight and decreased absolute epididymis weight) and hormonal alterations (increased serum estradiol [E2] levels and decreased serum FSH and T4 levels). Responses to p,p'-DDE in LE rats included organ weight changes (increased relative liver weight, absolute epididymis weight, relative accessory sex gland [ASG] unit weight, as well as the individual component weights of the ASG [prostate and seminal vesicles]), and hormonal alterations (increased serum testosterone [T], E2, dihydrotestosterone [DHT], thyroid-stimulating hormone [TSH], and decreased T4 levels). These data demonstrate that there are considerable strain-sensitivity differences to p,p'-DDE exposure. The described in vivo male battery using CD rats did not identify p,p'-DDE as an EAC. In contrast, the in vivo male battery using LE rats identified p,p'-DDE as a EAC. Evaluation of the data for the LE rats demonstrate that p,p'-DDE appears to be acting as an AR antagonist whose primary effects are more potent centrally than peripherally. In the YTS for the AR, p,p'-DDE had an EC50 value of 3.5 x 10(-4) M; however, in the AR YTS competition assay, p,p'-DDE did not inhibit DHT binding to the AR. p,p'-DDE was inactive in the YTS containing the estrogen receptor or progesterone receptor at the concentrations evaluated. In the Hershberger assay, p,p'-DDE administration caused antiandrogen-like effects characterized by attenuation of the testosterone propionate-induced increases in reproductive-organ weights. In summary, these data suggest that strain selection will affect the ability to detect certain weak EACs. However, a Tier I screening battery consisting of both in vivo and in vitro endpoints would reduce the chance that weak-acting compounds such as p,p'-DDE would not be identified as potential EACs.

Detection of thyroid toxicants in a tier I screening battery and alterations in thyroid endpoints over 28 days of exposure.

Phenobarbital (PB), a thyroid hormone excretion enhancer, and propylthiouracil (PTU), a thyroid hormone-synthesis inhibitor, have been examined in a Tier I screening battery for detecting endocrine-active compounds (EACs). The Tier I battery incorporates two short-term in vivo tests (5-day ovariectomized female battery and 15-day intact male battery using Sprague-Dawley rats) and an in vitro yeast transactivation system (YTS). In addition to the Tier I battery, thyroid endpoints (serum hormone concentrations, liver and thyroid weights, thyroid histology, and UDP-glucuronyltransferase [UDP-GT] and 5'-deiodinase activities) have been evaluated in a 15-day dietary restriction experiment. The purpose was to assess possible confounding of results due to treatment-related decreases in body weight. Finally, several thyroid-related endpoints (serum hormone concentrations, hepatic UDP-GT activity, thyroid weights, thyroid follicular cell proliferation, and histopathology of the thyroid gland) have been evaluated for their utility in detecting thyroid-modulating effects after 1, 2, or 4 weeks of treatment with PB or PTU. In the female battery, changes in thyroid endpoints following PB administration, were limited to decreased serum tri-iodothyronine (T3) and thyroxine (T4) concentrations. There were no changes in thyroid stimulating hormone (TSH) concentrations or in thyroid gland histology. In the male battery, PB administration increased serum TSH and decreased T3 and T4 concentrations. The most sensitive indicator of PB-induced thyroid effects in the male battery was thyroid histology (pale staining and/or depleted colloid). In the female battery, PTU administration produced increases in TSH concentrations, decreases in T3 and T4 concentrations, and microscopic changes (hypertrophy/hyperplasia, colloid depletion) in the thyroid gland. In the male battery, PTU administration caused thyroid gland hypertrophy/hyperplasia and colloid depletion, and the expected thyroid hormonal alterations (increased TSH, and decreased serum T3 and T4 concentrations). The dietary restriction study demonstrated that possible confounding of the data can occur with the thyroid endpoints when body weight decrements are 15% or greater. In the thyroid time course experiment, PB produced increased UDP-GT activity (at all time points), increased serum TSH (4-week time point), decreased serum T3 (1-and 2-week time points) and T4 (all time points), increased relative thyroid weight (2- and 4-week time points), and increased thyroid follicular cell proliferation (1- and 2-week time points). Histological effects in PB-treated rats were limited to mild colloid depletion at the 2- and 4-week time points. At all three time points, PTU increased relative thyroid weight, increased serum TSH, decreased serum T3 and T4, increased thyroid follicular cell proliferation, and produced thyroid gland hyperplasia/hypertrophy. Thyroid gland histopathology, coupled with decreased serum T4 concentrations, has been proposed as the most useful criteria for identifying thyroid toxicants. These data suggest that thyroid gland weight, coupled with thyroid hormone analyses and thyroid histology, are the most reliable endpoints for identifying thyroid gland toxicants in a short-duration screening battery. The data further suggest that 2 weeks is the optimal time point for identifying thyroid toxicants based on the 9 endpoints examined. Hence, the 2-week male battery currently being validated as part of this report should be an effective screen for detecting both potent and weak thyroid toxicants.

Effects of 17 beta-estradiol on serum hormone concentrations and estrous cycle in female Crl:CD BR rats: effects on parental and first generation rats.

The recently passed Food Quality Protection Act of 1996 requires the U.S. EPA to implement screening strategies for endocrine active compounds (EACs) within the next 2 years. Interpreting results from screening tests is complicated by the absence of traditional dietary rodent bioassay data with model estrogenic compounds such as 17 beta-estradiol. Thus, a 90-day/one-generation reproduction study with 17 beta-estradiol was designed to: (1) provide such baseline data; (2) set dose levels for multigeneration reproduction and combined chronic toxicity/oncogenicity studies; and (3) evaluate various mechanistic/biochemical endpoints for inclusion in these follow-up studies. The current article describes the effects of dietary administration of 0, 0.05, 2.5, 10, and 50 ppm 17 beta-estradiol on the serum hormone concentrations and estrous cyclicity of female Crl:CD BR rats and evaluates a sampling strategy for measuring serum hormone levels in cycling female rats. Serum hormones were measured at three time points during a 90-day dietary exposure (1 week, 28 days, and 90 days) and in the F1 generation rats on postnatal day 98. Over the course of the 90-day feeding study for the P1 generation and from postnatal days 21 to 98 for the F1 generation, the estrous cycle was monitored daily in 10 rats/group. In P1 generation rats, dietary administration of 2.5, 10, and 50 ppm 17 beta-estradiol produced a dose-dependent increase in serum estradiol (E2) concentrations at all time points. In contrast, administration of 0.05, 2.5, 10, and 50 ppm 17 beta-estradiol produced a dose-dependent decrease in serum progesterone (P4) concentrations on test day 90, which correlated with an absence of corpora lutea and ovarian atrophy. At 10 and 50 ppm 17 beta-estradiol, serum luteinizing hormone (LH) concentrations were consistently decreased at all time points and were decreased at 2.5 ppm on test day 90. Serum prolactin (PRL) concentrations were increased at 50 ppm 17 beta-estradiol on test day 90. Serum follicle stimulating hormone (FSH) concentrations were either similar to the control levels or minimally changed at all time points. No F1 generation rats were produced at 10 or 50 ppm 17 beta-estradiol. In F1 generation rats, serum E2 concentrations were increased and P4 concentrations were decreased at a dietary concentration of 2.5 ppm 17 beta-estradiol, while serum concentrations of LH, FSH, and PRL were similar to the control. Dietary administration of 17 beta-estradiol at concentrations of 2.5 (both generations) and 10 and 50 ppm (P1 generation only) produced marked effects on the estrous cycle: decreased number of cycles, increased mean cycle length, and decreased number of normally cycling rats. The estrous cyclicity of rats fed 2.5 ppm 17 beta-estradiol appeared more severely affected in rats of the F1 generation than in rats of the P1 generation. Whether this increase in severity is related to an in utero exposure and/or greater mean daily intake of 17 beta-estradiol in the F1 generation rats in the postnatal period is unclear. Another goal of this study was to evaluate whether a single time point sampling strategy using cycling female rats could be used to detect compound-related changes in serum hormone concentrations. In evaluating a sampling strategy for measuring serum hormone levels, it appears that detection of compound-related alterations in serum hormone concentrations can be best detected by sampling during diestrus. Since the stage of the cycle dramatically influences hormone concentrations, large sample sizes (n = 50) are needed if serum hormone measurements are not matched with the stage of the cycle. The data indicate that this strategy of measuring serum hormone concentrations has utility in detecting compound-related effects within the confines of a traditional guideline study (subchronic, chronic, or multigenerational reproduction study).