Integration of positive and negative growth signals during ras pathway activation in vivo.

Expression of RAS proteins can have either positive or negative effects on cell growth, differentiation and death. New technologies are being developed for the generation of animal models to address the questions of where, when and how much Ras is expressed during tumorigenesis, and how these disparate signals are integrated during multistage carcinogenesis.

A GSK3-binding peptide from FRAT1 selectively inhibits the GSK3-catalysed phosphorylation of axin and beta-catenin.

The Axin-dependent phosphorylation of beta-catenin catalysed by glycogen synthase kinase-3 (GSK3) is inhibited during embryogenesis. This protects beta-catenin against ubiquitin-dependent proteolysis, leading to its accumulation in the nucleus, where it controls the expression of genes important for development. Frequently rearranged in advanced T-cell lymphomas 1 (FRAT1) is a mammalian homologue of a GSK3-binding protein (GBP), which appears to play a key role in the correct establishment of the dorsal-ventral axis in Xenopus laevis. Here, we demonstrate that FRATtide (a peptide corresponding to residues 188-226 of FRAT1) binds to GSK3 and prevents GSK3 from interacting with Axin. FRATtide also blocks the GSK3-catalysed phosphorylation of Axin and beta-catenin, suggesting a potential mechanism by which GBP could trigger axis formation. In contrast, FRATtide does not suppress GSK3 activity towards other substrates, such as glycogen synthase and eIF2B, whose phosphorylation is independent of Axin but dependent on a 'priming' phosphorylation. This may explain how the essential cellular functions of GSK3 can continue, despite the suppression of beta-catenin phosphorylation.

GSK-3 inhibition by adenoviral FRAT1 overexpression is neuroprotective and induces Tau dephosphorylation and beta-catenin stabilisation without elevation of glycogen synthase activity.

Glycogen synthase kinase 3 (GSK-3) has previously been shown to play an important role in the regulation of apoptosis. However, the nature of GSK-3 effector pathways that are relevant to neuroprotection remains poorly defined. Here, we have compared neuroprotection resulting from modulation of GSK-3 activity in PC12 cells using either selective small molecule ATP-competitive GSK-3 inhibitors (SB-216763 and SB-415286), or adenovirus overexpressing frequently rearranged in advanced T-cell lymphomas 1 (FRAT1), a protein proposed as a negative regulator of GSK-3 activity towards Axin and beta-catenin. Our data demonstrate that cellular overexpression of FRAT1 is sufficient to confer neuroprotection and correlates with inhibition of GSK-3 activity towards Tau and beta-catenin, but not modulation of glycogen synthase (GS) activity. By comparison, treatment with SB-216763 and SB-415286 proved more potent in terms of neuroprotection, and correlated with inhibition of GSK-3 activity towards GS in addition to Tau and beta-catenin.

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.

Prone positioning for acute respiratory distress syndrome in the surgical intensive care unit: who, when, and how long?

BACKGROUND: We evaluated the effects of prone positioning (PP) on surgery and trauma patients with acute respiratory distress syndrome (ARDS). METHODS: Patients with ARDS were studied. Exclusion criteria were contraindications to PP. Patients were evaluated in the supine position and after being turned to the PP. After 6 hours, patients were returned to the supine position for 3 hours. One hour after each position change, arterial and mixed venous blood was drawn and analyzed for blood gases and pH, and hemodynamics were measured. RESULTS: Over 20 months, 27 patients met the criteria, and 20 of the patients were entered into the study. On day 1, 18 of 20 patients (90%) responded with an increase in PaO(2) during PP. On day 2, 16 of 17 patients (94%) responded; on day 3, 15 of 16 patients responded (94%); on day 4, 11 of 13 patients responded (85%); on day 5, 8 of 8 patients responded (100%); and on day 6, 4 of 5 patients responded (80%). Pao(2)/Fio(2) and Qs/Qt were significantly improved (P<.05) during PP. There were 91 periods of PP, lasting 10.3+/-1.2 hours. Of 91 changes to PP, 78 changes (86%) resulted in an improvement in Pao(2)/Fio(2) of more than 20%. CONCLUSIONS: PP improves oxygenation in ARDS for 6 days with few complications.

Positive end-expiratory pressure and response to inhaled nitric oxide: changing nonresponders to responders.

BACKGROUND: Inhaled nitric oxide (INO) has been shown to improve oxygenation in two thirds of patients with acute respiratory distress syndrome (ARDS). Failure to respond to INO is multifactorial. We hypothesized that the addition of positive end expiratory pressure (PEEP) might modify the response to INO in patients who had previously failed to respond to INO. METHODS: Patients with ARDS who failed to respond to INO at 1 ppm (PaO2 increase of < 20%) were selected. Each patient underwent a PEEP trial using an improvement in static lung compliance as the end point. One hour after the new PEEP level was reached, hemodynamic and blood gas values were obtained. INO was then reinstituted at 1 ppm, and hemodynamic and blood gas variables were obtained 1 hour later. RESULTS: Six of nine patients demonstrated an increase in PaO2/FIO2 (161 +/- 27 to 186 +/- 29) with a mean increase in PEEP of 3.7 cm H2O. Each patient responding to PEEP further improved PaO2/FIO2 (186 +/- 29 to 223 +/- 36) with INO at 1 ppm. The three patients who failed to improve after the PEEP increase also failed to respond to a second trial of INO. There were no changes in cardiac output or systemic vascular resistance. Pulmonary artery pressures decreased slightly (39 +/- 5 vs 38 +/- 7 vs 35 +/- 9 mm Hg). Pulmonary vascular resistance decreased significantly after reintroduction of INO (298 +/- 131 vs 310 +/- 122 vs 249 +/- 105 dynes/sec/cm-5) in patients who responded positively. CONCLUSIONS: The response of ARDS patients to INO can be improved if optimum alveolar recruitment is achieved by the addition of PEEP. PEEP and INO have a synergistic effect on PaO2/FIO2. Patients who fail to respond to INO may benefit from an optimum PEEP trial.

Zone I retroperitoneal hematoma identified by computed tomography scan as an indicator of significant abdominal injury.

OBJECTIVE: All zone I retroperitoneal hematomas (Z1RPHs) identified at laparotomy for blunt trauma traditionally require exploration. The purpose of this study was to correlate patient outcome after blunt abdominal trauma with the presence of Z1RPH diagnosed on admission computed tomography (CT) scan. METHODS: This is a retrospective review of patients with blunt trauma who were admitted to a Level 1 trauma center and who underwent CT scan during a 40-month period. All scans with a traumatic injury were reviewed to identify and grade Z1RPH as mild, moderate, or severe. Patients requiring operative treatment were compared with those who were observed. Statistical analysis was performed with Student's t test and chi-square test, with P < .05 considered significant. RESULTS: Eighty-five (15.5%) of the CT scans were positive for Z1RPH. None of the 50 patients with a mild Z1RPH had their treatment altered. Of the 29 patients with a moderate or severe Z1RPH, 8 required celiotomy. The patients requiring celiotomy had significant elevations of solid viscus score (SVS) (4.9 +/- 1.6 versus 1.8 +/- 0.3), abdominal Abbreviated Injury Scale (3.8 +/- 0.3 versus 2.6 +/- 0.3), and transfusion requirements (13 +/- 4 versus 2 +/- 1). All patients (N = 4) with an SVS >4 required operative treatment. Seventy-two percent of patients with more than 1 intra-abdominal injury required abdominal exploration. CONCLUSIONS: The presence of a moderate or severe Z1RPH and more than 1 intra-abdominal injury or an SVS >4 on admission CT scan is an important radiographic finding. This injury pattern should be considered a contraindication for nonoperative treatment of the associated solid organ injury.

The risk assessment profile score identifies trauma patients at risk for deep vein thrombosis.

BACKGROUND: The identification of trauma patients at risk for the development of deep venous thrombosis (DVT) at the time of admission remains difficult. The purpose of this study is to validate the risk assessment profile (RAP) score to stratify patients for DVT prophylaxis. METHODS: All patients admitted from November 1998 thru May 1999 were evaluated for enrollment. We prospectively assigned patients as low risk or high risk for DVT using the RAP score. High-risk patients received both pharmacologic and mechanical prophylaxis. Low-risk patients received none. Surveillance duplex Doppler scans were performed each week of hospitalization or if symptoms developed. Hospital charges for prophylaxis were used to determine the savings in the low-risk group. Statistical differences between the risk groups for each factor of the RAP and development of DVT were determined by the chi-squared test, with significance at a probability value of less than .05. RESULTS: There were 102 high-risk (64%) and 58 low-risk (36%) individuals studied. Eleven of the high-risk group (10.8%) experienced the development of DVT (asymptomatic, 64%). None of the low-risk group was diagnosed with DVT. Five of the 16 RAP factors were statistically significant for DVT. Eliminating prophylaxis and Doppler scans in low-risk patients resulted in a total savings of $18,908 in hospital charges. CONCLUSIONS: The RAP score correctly identified trauma patients at increased risk for the development of DVT. Despite prophylaxis, the high-risk group warrants surveillance scans. Withholding prophylaxis in low-risk patients can reduce hospital charges without risk.

Dynamic helical computed tomography scan accurately detects hemorrhage in patients with pelvic fracture.

BACKGROUND: The purpose of this study was to evaluate the use of dynamic helical computed tomography (CT) scan for screening patients with pelvic fractures and hemorrhage requiring angiographic embolization for control of bleeding. METHODS: Patients admitted to the trauma service with pelvic fractures were identified from the trauma registry. Data retrieval included demographics, hemodynamic instability, Injury Severity Score, blood transfusion requirement, length of stay, and mortality. CT scans obtained during the initial evaluation were reviewed for the presence of contrast extravasation and correlated with angiographic findings. Data are reported as mean +/- SEM, with P<.05 considered significant. RESULTS: Seven thousand seven hundred eighty-one patients were admitted from June 1994 to May 1999. A pelvic fracture was diagnosed in 660 (8.5%). Two hundred ninety (44.0%) dynamic helical CT scans were performed, of which 13 (4.5%) identified contrast extravasation. Nine (69%) were hemodynamically unstable and had pelvic arteriography performed. Arterial bleeding was confirmed in all and controlled by embolization. Patients with contrast extravasation had significantly greater Injury Severity Score, blood transfusion requirement and length of stay. Sensitivity, specificity, and accuracy of CT scan for identifying patients requiring embolization were 90.0%, 98.6%, and 98.3%, respectively. CONCLUSIONS: Early use of dynamic helical CT scanning in the multiply injured patient with a pelvic fracture accurately identifies the need for emergent angiographic embolization.

Time- and concentration-dependent increases in cell proliferation in rats and mice administered vinyl acetate in drinking water.

Chronic administration of vinyl acetate (VA) in drinking water to rats and mice has produced upper digestive tract neoplasms. These tumors were believed to arise from the intracellular metabolism of VA by carboxylesterases to cytotoxic and genotoxic compounds. We hypothesized that prolonged VA exposure at high concentrations would induce cytotoxicity and a restorative cell proliferation (CP). These endpoints were measured in F-344 rats and BDF1 mice administered drinking water containing 0, 1000, 5000, 10,000, or 24,000 ppm VA for 92 days. On test days, Days 1, 8, 29, and 92, upper digestive tract histopathology and oral cavity CP (pulsed 5-bromodeoxyuridine [BrdU] to measure S-phase DNA synthesis) were evaluated. Analysis of test solutions showed that VA spontaneously hydrolyzed, slowly releasing acetic acid and thereby lowering pH. Statistically significant, concentration-related increases in CP occurred in basal cells of the mandibular oral cavity mucosa of mice at 10,000 and 24,000 ppm but only after 92 days. CP increases were approximately 2.4- and 3.4-fold above controls and were considered to be toxicologically significant. Some statistically significant increases in CP were also measured in the oral cavity mucosa of rats; however, these changes were considered to be of equivocal biological relevance. No histopathological evidence of mucosal injury was seen in either species. The absence of cytotoxicity in the upper digestive tract mucosa suggests that the increased CP at high administered VA concentrations may be due to a mitogenic response, ostensibly from the loss of cell growth controls in oral cavity mucosa.

Sensitivity of a Tier I screening battery compared to an in utero exposure for detecting the estrogen receptor agonist 17 beta-estradiol.

A Tier I screening battery for detecting endocrine active compounds (EACs) has been evaluated for its ability to identify 17 beta-estradiol, a pure estrogen receptor agonist. In addition, the responses obtained with the Tier I battery were compared to the responses obtained from F1 generation rats from a 90-day/one-generation reproduction study with 17 beta-estradiol in order to characterize the sensitivity of the Tier I battery against the sensitivity of an in utero exposure for detecting EACs. The Tier I battery incorporates two short-term in vivo tests (5-day ovariectomized female battery; 15-day intact male battery) and an in vitro yeast transactivation system (YTS) for identifying compounds that alter endocrine homeostasis. The Tier I female battery consists of traditional uterotrophic endpoints coupled with biochemical and hormonal endpoints. It is designed to identify compounds that are estrogenic/antiestrogenic or modulate dopamine levels. The Tier I male battery consists of organ weights coupled with microscopic evaluations and a comprehensive hormonal assessment. It is designed to identify compounds that have the potential to act as agonists or antagonists to the estrogen, androgen, progesterone, or dopamine receptor; steroid biosynthesis inhibitors (aromatase, 5 alpha-reductase, and testosterone biosynthesis); or compounds that alter thyroid function. The YTS is designed to identify compounds that bind to steroid hormone receptors (estrogen, androgen, and progesterone) and activate gene transcription. The profile generated for 17 beta-estradiol was characteristic of the responses expected with a pure estrogen receptor agonist. In the female battery, responses to 17 beta-estradiol included increases in uterine fluid imbibition, uterine weight, estrus conversion, uterine stromal cell proliferation, uterine epithelial cell height, uterine progesterone receptor content, serum prolactin and estradiol levels, and decreases in uterine estrogen receptor content and follicle stimulating hormone and luteinizing hormone levels. In the male battery, responses to 17 beta-estradiol included decreases in absolute testis and epididymides weights, decreases in relative weights for androgen-dependent tissues (prostate, seminal vesicles, and accessory sex gland unit), hormonal alterations (decreased serum testosterone, dihydrotestosterone, and LH and increased serum prolactin levels), and microscopic alterations of the testis and epididymides. In the YTS for the estrogen receptor, 17 beta-estradiol had an EC50 value of 7.2 x 10(-9) M, while DHT and progesterone had little cross-activation. The androgen and progesterone receptor systems were less selective in that 17 beta-estradiol activated these systems within 3 orders of magnitude of the primary ligand. In the 90-day/one-generation reproduction study, responses to dietary administration of 17 beta-estradiol included alterations in organ weights, developmental landmarks, and hormonal levels. Comparison of the responses obtained with our Tier I battery and an in utero exposure demonstrates that the Tier I screening battery is as sensitive as an in utero exposure for detecting 17 beta-estradiol-induced alterations in hormonal homeostasis.

Analysis of vinyl acetate metabolism in rat and human nasal tissues by an in vitro gas uptake technique.

Physiologically based pharmacokinetic (PBPK) models require estimates of catalytic rate constants controlling the metabolism of xenobiotics. Usually, these constants are derived from whole tissue homogenates wherein cellular architecture and enzyme compartmentation are destroyed. Since the nasal cavity epithelium is composed of a heterogeneous cell population measurement of xenobiotic metabolizing enzymes using homogenates could yield artifactual results. In this article a method for measuring rates of metabolism of vinyl acetate, a metabolism-dependent carcinogen, is presented that uses whole-tissue samples and PBPK modeling techniques to estimate metabolic kinetic parameters in tissue compartments. The kinetic parameter estimates were compared to those derived from homogenate experiments using two methods of tissue normalization. When the in vitro gas uptake constants were compared to homogenate-derived values, using a normalization procedure that does not account for tissue architecture, there was poor agreement. Homogenate-derived values from rat nasal tissue were 3- to 23-fold higher than those derived using the in vitro gas uptake method. When the normalization procedure for the rat homogenate-derived values took into account tissue architecture, a good agreement was observed. Carboxylesterase activity in homogenates of human nasal tissues was undetectable. Using the in vitro gas uptake technique, however, carboxylesterase activity was detected. Rat respiratory carboxylesterase and aldehyde dehydrogenase activities were about three and two times higher than those of humans, respectively. Activities of the rat olfactory enzymes were about equivalent to those of humans. K(m) values did not differ between species. The results suggest that the in vitro gas uptake technique is useful for deriving enzyme kinetic constants where effects of tissue architecture are preserved. Furthermore, the results suggest that caution should be exercised when scaling homogenate-derived values to whole-organ estimates, especially in organs of cellular heterogeneity.

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.

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.

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 dietary 17 beta-estradiol exposure on serum hormone concentrations and testicular parameters in male Crl:CD BR rats.

A 90-day/one-generation reproduction study was conducted in male and female Crl:CD BR rats using dietary levels of 0, 0.05, 2.5, 10, and 50 ppm 17 beta-estradiol. The goals of this study were to set dose levels and evaluate several mechanistic endpoints for inclusion in multigeneration reproduction and combined chronic toxicity/oncogenicity studies with 17 beta-estradiol. In this report we discuss the effects of dietary 17 beta-estradiol exposure on serum hormonal levels and sperm parameters from P1 and F1 male rats. Sperm parameters were also evaluated in recovery P1 and F1 male rats that were fed control diets for 105 and 103 days, respectively, following 97 and 86-94 days of estradiol exposure, respectively. Measurement of Sertoli cell number from F1 male rats was performed to test the hypothesis that in utero exposure to estrogens will decrease Sertoli cell number and sperm production. Other findings from this 90-day/one-generation reproduction study are summarized elsewhere. 17 beta-Estradiol produced a dose-dependent decrease in body weight in P1 male rats at > or = 2.5 ppm and in the F1 male rats at 2.5 ppm. This decrease in body weight was due to a combination or reduced food consumption and food efficiency. In the recovery P1 males, body weight increased in the affected groups, albiet not to control levels, due to food consumption returning to control levels accompanied by an increase in food efficiency. However, in F1 males there was no corresponding rebound in body weight. In the P1 rats, exposure to 17 beta-estradiol decreased testis and epididymis weights in the 10 and 50 ppm groups, while no effects were seen in the P1 2.5 ppm group. In contrast, epididymis weights in the F1 and F1 recovery 2.5 ppm groups were statistically decreased; however, there were no histopathological effects observed. The decreases in testis weights in the P1 generation correlated with histopathologic evidence of interstitial cell atrophy and seminiferous tubule degeneration and reduced sperm production. Correlative changes in the epididymides of P1 rats were characterized by oligospermia or aspermia, the presence of germ cell debris in the lumen of tubules, and atrophy of epididymal tubules. 17 beta-Estradiol decreased testicular spermatid numbers, epididymal sperm numbers, and sperm motility in the P1 males in the 10 and 50 ppm groups, but not in the 2.5 ppm group. Following a 105-day recovery period in the P1 males, all sperm parameters and reproductive organ weights returned to control values except for the epididymal sperm count. Overall, the decline in testicular spermatid and epididymal sperm numbers in the P1 rats correlated with the reduced organ weights and the observed histopathological changes and appeared primarily related to the decrease in serum testosterone levels. In the F1 rats, no significant decreases were noted in the testicular spermatid number but a slight decrease in epididymal sperm number was seen in the 2.5 ppm group, which showed no evidence of recovery. Using morphometric analysis, no change was seen in the number of Sertoli cell nuclei per testis in F1 males. The pattern of hormonal responses seen in this study was characteristic of an estrogen receptor agonist such as 17 beta-estradiol: increased serum prolactin and decreased testosterone, luteinizing hormone, and follicle stimulating hormone levels. The data demonstrate that in utero and postnatal dietary administration of 17 beta-estradiol at levels which increased serum estradiol levels to approximately 400% of control and decreased testosterone levels to 33% of control did not reduce the number of Sertoli cell nuclei per testis.

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.

90-day feeding and one-generation reproduction study in Crl:CD BR rats with 17 beta-estradiol.

Over the past several years, there has been increasing concern that chemicals and pesticides found in the environment may mimic endogenous estrogens, potentially producing adverse effects in wildlife and human populations. Because estrogenicity is one of the primary concerns, a 90-day/one-generation reproduction study with 17 beta-estradiol was designed to set dose levels for future multigenerational reproduction and combined chronic toxicity/oncogenicity studies. The purpose of these studies is to evaluate the significance of a range of responses as well as to provide benchmark data for a risk assessment for chemicals with estrogen-like activities. This 90-day/one-generation reproduction study was conducted in male and female Crl:CD BR rats using dietary concentrations of 0, 0.05, 2.5, 10, and 50 ppm 17 beta-estradiol. Endpoints were chosen in order to evaluate both subchronic and reproductive toxicity. In addition, several mechanistic/biochemical endpoints were evaluated for their usefulness in follow-up studies. In the P1 generation, dietary administration of 2.5, 10, and 50 ppm 17 beta-estradiol produced dose-dependent decreases in body weight, body weight gain, food consumption, and food efficiency. At 10 and 50 ppm 17 beta-estradiol, minimal to mild nonregenerative anemia, lymphopenia, decreased serum cholesterol (50 ppm only), and altered splenic lymphocyte subtypes were also observed in the P1 generation. Additionally, at these concentrations, there were changes in the weights of several organs. Evidence of ovarian malfunction, characterized by reduced numbers of corpora lutea and large antral follicles, was observed at 2.5 ppm 17 beta-estradiol and above. Other pathologic changes in males and females fed 10 and 50 ppm 17 beta-estradiol included centrilobular hepatocellular hypertrophy; diffuse hyperplasia of the pituitary gland; feminization of the male mammary glands; mammary gland hyperplasia in females; increased number of cystic follicles in the ovary; hypertrophy of the endometrium and endometrial glands in the uterus; degeneration of seminiferous epithelium; and atrophy of the testes and the accessory sex glands. In the reproduction portion of this study, rats fed 10 or 50 ppm 17 beta-estradiol did not produce litters. While there was no evidence that the 50 ppm treated rats mated, 33.3% of the rats fed 10 ppm mated but did not produce litters. No effects on mating and fertility indices were observed in rats fed 0.05 and 2.5 ppm 17 beta-estradiol. Pup weights at birth were statistically decreased relative to control in the groups fed 0.05 and 2.5 ppm 17 beta-estradiol. Weights of the rats in the 0.05 ppm group recovered by postnatal day 4 and remained similar to control throughout the remainder of the study. The mean gestation length of the 0.05 ppm group was slightly, albeit not statistically significantly, shorter (0.5 days) than that of the control group, which may have contributed to the decrease in birth weight of the 0.05 ppm group. In contrast, the weights of the F1 generation rats fed 2.5 ppm 17 beta-estradiol remained decreased relative to the control group throughout the study. Parental administration of 17 beta-estradiol did not alter anogenital distance in male or female pups. The onset of sexual maturation, as measured by day of preputial separation in males and day of vaginal opening in females, was delayed in male rats fed 2.5 ppm (by 8.2 days) and was hastened in female rats fed 0.05 and 2.5 ppm (by 1.6 and 8.8 days, respectively). The age at vaginal opening ranged from 26 to 37, 26 to 35, and 21 to 25 days for rats fed 0, 0.05, and 2.5 ppm 17 beta-estradiol, respectively. Hence, the range of age at vaginal opening was similar between the control and 0.05 ppm group. The organ weight and pathologic alterations observed in the adult F1 generation rats were similar to those observed in the P1 generation rats. (ABSTRACT TRUNCATED)

Single-dose cefotetan or cefoxitin versus multiple-dose cefoxitin as prophylaxis in patients undergoing appendectomy for acute nonperforated appendicitis.

OBJECTIVE: A prospective, double-blind study was performed to examine the effects of prophylactic cefotetan and cefoxitin in postoperative wound infection for patients with nonperforated acute appendicitis. METHODS: One hundred thirty-six of 179 patients with a clinical diagnosis of appendicitis were evaluated and divided into three groups: group 1 received 2 g cefotetan preoperatively, group 2 received 2 g cefoxitin preoperatively, and group 3 received 2 g cefoxitin preoperatively followed by three postoperative doses. RESULTS: The overall wound infection rate was 4.6 percent. Group 2 (single-dose cefoxitin) had a significantly higher wound infection rate (11.1 percent) than group 1 (single-dose cefotetan) (zero percent) or group 3 (multiple-dose cefoxitin) (1.9 percent). CONCLUSIONS: Single-dose cefotetan and multiple-dose cefoxitin are equally effective. However, because of the greater convenience and markedly decreased cost, single-dose cefotetan is the prophylaxis of choice in appendectomy for nonperforated appendicitis.