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J C Cook

Publications and source records attributed to J C Cook.

At least 19 recordsLinked to original sources
PubMed1999

The L1 major capsid protein of human papillomavirus type 11 recombinant virus-like particles interacts with heparin and cell-surface glycosaminoglycans on human keratinocytes.

The L1 major capsid protein of human papillomavirus (HPV) type 11, a 55-kDa polypeptide, forms particulate structures resembling native virus with an average particle diameter of 50-60 nm when expressed in the yeast Saccharomyces cerevisiae. We show in this report that these virus-like particles (VLPs) interact with heparin and with cell-surface glycosaminoglycans (GAGs) resembling heparin on keratinocytes and Chinese hamster ovary cells. The binding of VLPs to heparin is shown to exhibit an affinity comparable to that of other identified heparin-binding proteins. Immobilized heparin chromatography and surface plasmon resonance were used to show that this interaction can be specifically inhibited by free heparin and dextran sulfate and that the effectiveness of the inhibitor is related to its molecular weight and charge density. Sequence comparison of nine human L1 types revealed a conserved region of the carboxyl terminus containing clustered basic amino acids that bear resemblance to proposed heparin-binding motifs in unrelated proteins. Specific enzymatic cleavage of this region eliminated binding to both immobilized heparin and human keratinocyte (HaCaT) cells. Removal of heparan sulfate GAGs on keratinocytes by treatment with heparinase or heparitinase resulted in an 80-90% reduction of VLP binding, whereas treatment of cells with laminin, a substrate for alpha6 integrin receptors, provided minimal inhibition. Cells treated with chlorate or substituted beta-D-xylosides, resulting in undersulfation or secretion of GAG chains, also showed a reduced affinity for VLPs. Similarly, binding of VLPs to a Chinese hamster ovary cell mutant deficient in GAG synthesis was shown to be only 10% that observed for wild type cells. This report establishes for the first time that the carboxyl-terminal portion of HPV L1 interacts with heparin, and that this region appears to be crucial for interaction with the cell surface.

Amino Acid Sequence↗
PubMed1999

Purification of virus-like particles of recombinant human papillomavirus type 11 major capsid protein L1 from Saccharomyces cerevisiae.

Recombinant major capsid protein, L1 (M(r) = 55,000), of human papillomavirus type 11 was expressed intracellularly at high levels in a galactose-inducible Saccharomyces cerevisiae expression system by an HPV6/11 hybrid gene. The capsid protein self-assembled into virus-like particles (VLPs) and accounted for 15% of the total soluble protein. A purification process was developed that consisted of two main steps: microfiltration and cation-exchange chromatography. The purified VLPs were 98% homogeneous, and the overall purification yield was 10%. The final product was characterized by several analytical methods and was highly immunogenic in mice.

Amino Acids↗
PubMed1999

Rodent Leydig cell tumorigenesis: a review of the physiology, pathology, mechanisms, and relevance to humans.

Leydig cells (LCs) are the cells of the testis that have as their primary function the production of testosterone. LCs are a common target of compounds tested in rodent carcinogenicity bioassays. The number of reviews on Leydig cell tumors (LCTs) has increased in recent years because of its common occurrence in rodent bioassays and the importance in assessing the relevance of this tumor type to humans. To date, there have been no comprehensive reviews to identify all the compounds that have been shown to induce LCTs in rodents or has any review systematically evaluated the epidemiology data to determine whether humans were at increased risk for developing LCTs from exposure to these agents. This review attempts to fill these deficiencies in the literature by comparing the cytology and ontogeny of the LC, as well as the endocrine and paracrine regulation of both normal and tumorigenic LCs. In addition, the pathology of LCTs in rodents and humans is compared, compounds that induce LC hyperplasia or tumors are enumerated, and the human relevance of chemical-induced LCTs is discussed. There are plausible mechanisms for the chemical induction of LCTs, as typified by agonists of estrogen, gonadotropin releasing hormone (GnRH), and dopamine receptors, androgen receptor antagonists, and inhibitors of 5alpha-reductase, testosterone biosynthesis, and aromatase. Most of these ultimately involve elevation in serum luteinizing hormone (LH) and/or LC responsiveness to LH as proximate mediators. It is expected that further work will uncover additional mechanisms by which LCTs may arise, especially the role of growth factors in modulating LC tumorigenesis. Regarding human relevance, the pathways for regulation of the hypothalamo-pituitary-testis (HPT) axis of rats and humans are similar, such that compounds that either decrease testosterone or estradiol levels or their recognition will increase LH levels. Hence, compounds that induce LCTs in rats by disruption of the HPT axis pose a risk to human health, except for possibly two classes of compounds (GnRH and dopamine agonists). Because GnRH and prolactin receptors are either not expressed or are expressed at very low levels in the testes in humans, the induction of LCTs in rats by GnRH and dopamine agonists would appear not to be relevant to humans; however, the potential relevance to humans of the remaining five pathways of LCT induction cannot be ruled out. Therefore, the central issue becomes what is the relative sensitivity between rat and human LCs in their response to increased LH levels; specifically, is the proliferative stimulus initiated by increased levels of LH attenuated, similar, or enhanced in human vs. rat LCs? There are several lines of evidence that suggest that human LCs are quantitatively less sensitive than rats in their proliferative response to LH, and hence in their sensitivity to chemically induced LCTs. This evidence includes the following: (1) the human incidence of LCTs is much lower than in rodents even when corrected for detection bias; (2) several comparative differences exist between rat and human LCs that may contribute, at least in part, to the greater susceptibility of the rat to both spontaneous and xenobiotic-induced LCTs; (3) endocrine disease states in man (such as androgen-insensitivity syndrome and familial male precocious puberty) underscore the marked comparative differences that exist between rats and man in the responsiveness of their LC's to proliferative stimuli; and (4) several human epidemiology studies are available on a number of compounds that induce LCTs in rats (1,3-butadiene, cadmium, ethanol, lactose, lead, nicotine) that demonstrate no association between human exposure to these compounds and induction of LC hyperplasia or adenomas. (ABSTRACT TRUNCATED)

Animals↗
PubMed1999

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.

Animals↗
PubMed1999

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.

Animals↗
PubMed1999

Mutations that confer resistance to 2-deoxyglucose reduce the specific activity of hexokinase from Myxococcus xanthus.

The glucose analog 2-deoxyglucose (2dGlc) inhibits the growth and multicellular development of Myxococcus xanthus. Mutants of M. xanthus resistant to 2dGlc, designated hex mutants, arise at a low spontaneous frequency. Expression of the Escherichia coli glk (glucokinase) gene in M. xanthus hex mutants restores 2dGlc sensitivity, suggesting that these mutants arise upon the loss of a soluble hexokinase function that phosphorylates 2dGlc to form the toxic intermediate, 2-deoxyglucose-6-phosphate. Enzyme assays of M. xanthus extracts reveal a soluble hexokinase (ATP:D-hexose-6-phosphotransferase; EC 2.7.1.1) activity but no phosphotransferase system activities. The hex mutants have lower levels of hexokinase activities than the wild type, and the levels of hexokinase activity exhibited by the hex mutants are inversely correlated with the ability of 2dGlc to inhibit their growth and sporulation. Both 2dGlc and N-acetylglucosamine act as inhibitors of glucose turnover by the M. xanthus hexokinase in vitro, consistent with the finding that glucose and N-acetylglucosamine can antagonize the toxic effects of 2dGlc in vivo.

Acetylglucosamine↗
PubMed1998

Expression and purification of recombinant tick anticoagulant peptide (Y1W/D10R) double mutant secreted by Saccharomyces cerevisiae.

A double mutant of tick anticoagulant peptide (TAP) was cloned as a chimeric fusion with the yeast alpha-mating factor pre-proleader peptide. Expression in yeast (Saccharomyces cerevisiae) resulted in the secretion of the TAP mutein into the culture medium. An HPLC-based assay was used to screen yeast strains to find those giving highest expression levels. Efficiency of cleavage at the junction of the leader-TAP mutein varied from strain to strain, and a rapid purification method followed by N-terminal sequence analysis was used to identify a host strain that minimized undesirable cleavage products. A purification scheme was developed which separated the TAP mutein from improperly processed peptides present in the medium. This scheme employed cation-exchange chromatography and reversed-phase HPLC. Scale-up of the process was successful and produced 100 mg of fully functional TAP mutein of >96% homogeneity from a 50-L yeast culture.

Amino Acid Sequence↗
PubMed1998

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)

Animals↗
PubMed1998

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).

Animals↗
PubMed1998

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.

Animals↗
PubMed1998

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.

Animals↗
PubMed1998

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.

Animals↗
PubMed1998

Evaluation of the primary humoral immune response following exposure of male rats to 17beta-estradiol or flutamide for 15 days.

There is a concern that certain industrial chemicals found in the environment may mimic or antagonize endogenous hormones and adversely affect the endocrine as well as the immune system. The objective of this study was to determine if exposure of Crl:CD (SD)BR male rats to 17beta-estradiol (17beta-E2), an estrogen receptor agonist, or flutamide (FLUT), an androgen receptor antagonist, would significantly alter the primary IgM humoral immune response to sheep red blood cells (SRBC). This study was conducted in the context of a male in vivo Tier I battery designed to identify endocrine-active compounds (EACs). The Tier I male battery consists of organ weights coupled with a comprehensive hormonal assessment. Rats were dosed by the intraperitoneal route for 15 days with vehicle or 0.001, 0.0025, 0.0075, or 0.050 mg/kg/day 17beta-E2 or 0.25, 1, 5, or 20 mg/kg/day FLUT. Six days prior to termination, selected rats were injected intravenously with SRBC for assessment of humoral immune function. Spleen cell number and spleen and thymus weights were obtained. Serum was analyzed for anti-SRBC IgM antibody by using an enzyme-linked immunosorbent assay. At 0.050 mg/kg/day 17beta-E2, mean final body and absolute thymus weights were significantly decreased to 84 and 65% of control, respectively. 17beta-E2 did not significantly alter spleen weight, spleen cell number, or the primary IgM humoral immune response to SRBC. The no-observed-adverse-effect level (NOAEL) for immune system alteration was 0.050 mg/kg/day 17beta-E2 since the decrease in absolute thymus weight was judged to be secondary to the decrements in body weight. In the Tier I male battery, responses to 17beta-E2 included decreased absolute testis and epididymis weights, decreased relative accessory sex gland unit weights, hormonal alterations (decreased serum testosterone (T), dihydrotestosterone (DHT), and luteinizing hormone (LH), and increased serum prolactin and E2 levels). The lowest-observed-adverse-effect level (LOAEL) for the reproductive indices was 0.001 mg/kg/day 17beta-E2 based on the hormonal alterations seen at this level; no NOAEL was established. Exposure to FLUT did not significantly alter mean final body, spleen, or absolute thymus weights, spleen cell number, or the primary IgM humoral immune response to SRBC. A significant increase (118% of control) in relative thymus weight was observed at 20 mg/kg/day FLUT. The NOAEL for immune system alteration was 5 mg/kg/day FLUT based on the increased relative thymus weights that were judged to be compound-related. In the Tier I male battery, responses to FLUT included decreased absolute epididymis and relative accessory sex gland unit weights and hormonal alterations (increased serum T, DHT, E2, and LH, and decreased follicle stimulating hormone levels). The LOAEL for the reproductive indices was 0.25 mg/kg/day FLUT based on the hormonal alterations seen at this level; no NOAEL was established. Based on these data, the reproductive and not the immune system appears to be the primary target organ of toxicity in young adult male rats treated with either 17beta-E2 or FLUT.

Androgen Antagonists↗
PubMed1998

Ex vivo and in vitro testis and ovary explants: utility for identifying steroid biosynthesis inhibitors and comparison to a Tier I screening battery.

Testis and ovary explants have been proposed as in vitro screens for identifying potential inhibitors of steroid biosynthesis. The goals of the current study were to optimize the conditions of the two assays, to characterize these assays using several compounds with well-defined endocrine activity, and to compare the responses from the explant assays with an in vivo male battery currently undergoing validation using the Crl:CD BR rat in order to evaluate their utility as test systems for screening unknown compounds for possible steroid biosynthesis inhibition activity. There were two components to the testis/ovary assays: ex vivo and in vitro. The ex vivo component used testes/ovaries from animals dosed with the test compounds in vivo, and the in vitro component used testes/ovaries from control animals. For the testis assays, decapsulated testis explants (50 mg) were placed into glass scintillation vials, +/-1.0 IU/ml hCG for 3 h in a shaking water bath (34 degrees C). Following the incubation period, medium was removed, centrifuged, and frozen until assayed for hormone concentrations. A similar procedure was used for the ovary explant assay except that each ovary was incubated separately. The testis explants were evaluated using the following compounds: ketoconazole (KETO), a testosterone biosynthesis inhibitor; aminoglutethimide (AG) (only in vitro) and anastrozole (ANA), aromatase inhibitors; finasteride (FIN), a 5alpha-reductase inhibitor; 17beta-estradiol (17beta-E2), an estrogen receptor agonist; flutamide (FLUT), an androgen receptor antagonist; ICI-182,780 (ICI), an estrogen receptor antagonist; haloperidol (HALO), a D2 receptor antagonist; and reserpine (RES), a dopamine depletor. In the ovary assay, AG (only in vitro), ANA, ICI, and HALO (only in vitro) were evaluated. Addition of fetal calf serum to the medium allowed measurement of estradiol (E2) in the testis assay, but production was not inhibited by ANA or AG. In the ovary explant assay, only AG was identified as inhibiting E2 production in vitro. Hence, both the testis and ovary explant assays appear to have limited utility for detecting aromatase inhibitors. Screening of these nine diverse endocrine-active compounds resulted in all of them being identified as altering the endocrine system when assessed by ex vivo and in vitro testis explants. Using only the in vitro assessment with the criteria of steroid biosynthesis inhibition, four of nine compounds were correctly identified in the testis explant assay (17beta-E2, KETO, FLUT, and HALO). The predictability of both the in vitro and ex vivo ovary assay was 50%, suggesting a 50% false positive or negative rate with unknown compounds. However, of the seven compounds assessed to date (17beta-E2, ICI, ANA, KETO, FLUT, HALO, and RES), all were correctly identified using an in vivo male battery, which also has the capability to detect other endocrine activities. Therefore, the testis and ovary explant assay would not be necessary if one were using an in vivo male battery, since this screen would identify steroid biosynthesis inhibitors and would also identify several other endocrine activities. Because of the difficulties in assessing cytotoxicity and the high false positive/negative rates, the ovary and testis explant assays are not useful as routine screening procedures for detecting steroid biosynthesis inhibitors; however, they may have utility in confirming in vivo findings.

Animals↗
PubMed1998

EAA receptors in the dorsomedial hypothalamic area mediate the cardiovascular response to activation of the amygdala.

The role of excitatory amino acid (EAA) receptors in the dorsomedial hypothalamus (DMH) in mediating the cardiovascular response to activation of the basolateral amygdala (BLA) was examined using conscious rats. Microinjection of the nonselective EAA receptor antagonist kynurenic acid (0.1-10 nmol) into the DMH blocked or reversed the increases in heart rate and arterial pressure resulting from injection of the GABAA receptor antagonists bicuculline methiodide (BMI; 100 pmol) and picrotoxin (100 pmol) into the BLA. Similar injections of kynurenic acid at sites lateral or dorsal to the DMH or injection of the inactive analog xanthurenic acid into the DMH were less effective in blocking the cardiovascular changes resulting from intra-amygdalar injection of BMI. Hypothalamic injection of the NMDA receptor antagonist 3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (10 pmol) or the DL-alpha-amino-3-hydroxy-5-methylisoxazole-propionic acid receptor antagonist 1,2,3,4-tetrahydro-6-nitro-2, 3-dioxo-benzo[f]quinoxaline-7-sulfonamide (50 pmol) at doses shown to be selective for their respective EAA receptor subtypes attenuated the cardiovascular changes associated with intra-amygdalar injection of BMI. Therefore, EAA receptors in the area of the DMH appear to be involved in mediating the cardiovascular changes resulting from activation of the amygdala.

Amygdala↗