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John Ashby

Publications and source records attributed to John Ashby.

At least 19 recordsLinked to original sources
PubMed2006

Induction of iron homeostasis genes during estrogen-induced uterine growth and differentiation.

We have previously used genome-wide transcript profiling to investigate the relationships between changes in gene expression and physiological alterations during the response of the immature mouse uterus to estrogens. Here we describe the identification of a functionally inter-related group of estrogen-responsive genes associated with iron homeostasis, including the iron-binding protein lactotransferrin, the ferroxidase ceruloplasmin, the iron delivery protein lipocalin 2 and the iron-exporter ferroportin. Quantitative real-time PCR revealed that the expression of these genes increases with time during the uterotrophic response, reaching maximal levels in the post-proliferative phase (between 48 and 72 h). In contrast, the heme biosynthesis genes aminolevulinic acid synthase 1 and 2 were maximally induced by estrogen at 2 and 4 h, respectively, prior to increased cell proliferation. Together, these data reveal that estrogen induces the temporally coordinated expression of iron homeostasis genes in the mouse uterus, and suggest an important role for iron metabolism during sex steroid hormone-induced uterine cell growth and differentiation.

Animals↗
PubMed2006

The OECD program to validate the rat Hershberger bioassay to screen compounds for in vivo androgen and antiandrogen responses. Phase 1: use of a potent agonist and a potent antagonist to test the standardized protocol.

The Organisation for Economic Cooperation and Development (OECD) has completed phase 1 of the Hershberger validation intended to identify in vivo activity of suspected androgens and antiandrogens. Seventeen laboratories from 7 countries participated in phase 1, and results were collated and evaluated by the OECD with the support of an international committee of experts. Five androgen-responsive tissues (ventral prostate, paired seminal vesicles and coagulating glands, levator ani and bulbocavernosus muscles, glans penis, and paired Cowper's or bulbourethral glands) were evaluated. The standardized protocols used selected doses of a reference androgen, testosterone propionate (TP), and an antiandrogen, flutamide (FLU). All laboratories successfully detected TP-stimulated increases in androgen-responsive tissue weight and decreases in TP-stimulated tissue weights when FLU was co-administered. The standardized protocols performed well under a variety of conditions (e.g., strain, diet, housing protocol, bedding). There was good agreement among laboratories with regard to the TP doses inducing significant increases in tissue weights and the FLU doses decreasing TP-stimulated tissue weights. Several additional procedures (e.g., weighing of the dorsolateral prostate and fixation of tissues before weighing) and serum component measurements (e.g., luteinizing hormone) were also included by some laboratories to assess their potential utility. The results indicated that the OECD Hershberger protocol was robust, reproducible, and transferable across laboratories. Based on this phase 1 validation study, the protocols have been refined, and the next phase of the OECD validation program will test the protocol with selected doses of weak androgen agonists, androgen antagonists, a 5alpha-reductase inhibitor, and chemicals having no androgenic activity.

Androgen Antagonists↗
PubMed2004

Sensitivity of the immature rat uterotrophic assay to mixtures of estrogens.

We have evaluated whether mixtures of estrogens, present in the mix at doses that are individually inactive in the immature rat uterotrophic assay, can give a uterotrophic response. Seven chemicals were evaluated: nonylphenol, bisphenol A (BPA), methoxychlor, genistein (GEN), estradiol, diethylstilbestrol, and ethinyl estradiol. Dose responses in the uterotrophic assay were constructed for each chemical. The first series of experiments involved evaluating binary mixtures of BPA and GEN at dose levels that gave moderate uterotrophic responses when tested individually. The mixtures generally showed an intermediate or reduced uterotrophic effect compared with when the components of the mixture were tested alone at the dose used in the mixture. The next series of experiments used a multicomponent (complex) mixture of all seven chemicals evaluated at doses that gave either weakly positive or inactive uterotrophic responses when tested individually in the assay. Doses that were nominally equi-uterotrophic ranged over approximately six orders of magnitude for the seven chemicals. Doses of agents that gave a weak uterotrophic response when tested individually gave a marginally enhanced positive response in the assay when tested combined as a mixture. Doses of agents that gave a negative uterotrophic response when tested individually gave a positive response when tested as a mixture. These data indicate that a variety of different estrogen receptor (ER) agonists, present individually at subeffective doses, can act simultaneously to evoke an ER-regulated response. However, translating these findings into the process of environmental hazard assessment will be difficult. The simple addition of the observed, or predicted, activities for the components of a mixture is confirmed here to be inappropriate and to overestimate the actual effect induced by the mixture. Equally, isobole analysis is only suitable for two- or three-component mixtures, and concentration addition requires access to dose-response data and EC50 values (concentration giving 50% of the maximum response) for the individual components of the mixture--requirements that will rarely be fulfilled for complex environmental samples. Given these uncertainties, we conclude that it may be most expedient to select and bioassay whole environmental mixtures of potential concern.

Animals↗
PubMed2004

Natural variability and the influence of concurrent control values on the detection and interpretation of low-dose or weak endocrine toxicities.

While defining the no effect level for the 5 alpha-reductase inhibitor finasteride in the Hershberger assay, we encountered an inverted-U low-dose trophic effect on the prostate gland of the rat. Two attempts to confirm this observation were unsuccessful, and we concluded that the positive effect initially observed was associated with normal biologic variability. During the same period we attempted, unsuccessfully, to repeat our own observation of weak uterotrophic activity in the rat for the sunscreen 3-(4-methylbenzylidene)camphor (4MBC). Further evaluation led us to conclude that 4MBC is uterotrophic only when the control uterine weights are at the low end of their normally encountered range. This led us to reevaluate our earlier mouse uterotrophic assay data for bisphenol A (BPA). Originally we had concluded that BPA gave irreproducible evidence of weak uterotrophic activity, but upon ordering the eight experiments we had conducted, according to decreasing control uterine weight, we confirmed reproducible weak uterotrophic activity for BPA when the control uteri were at the low end of their normal range. In this article, we describe these observations, together with a reanalysis of the data associated with several reported instances of weak or low-dose endocrine effects that have proven difficult to confirm in independent laboratories. These include the activity of BPA on the CF1 mouse prostate; the activities of BPA, octylphenol, and nonylphenol on the rat testis; and the effect of polycarbonate caging on control mouse uterine weight. In all of these cases, variability among controls provides a major obstacle to data interpretation and confirmation. Our recommendations on experimental design are also presented, with a view to ending the current impasse on the reality, or otherwise, of low-dose or weak endocrine toxicities.

Animals↗
PubMed2004

The need to decide if all estrogens are intrinsically similar.

We used gene expression profiling to investigate whether the molecular effects induced by estrogens of different provenance are intrinsically similar. In this article we show that the physiologic estrogen 17-beta-estradiol, the phytoestrogen genistein, and the synthetic estrogen diethylstilbestrol alter the expression of the same 179 genes in the intact immature mouse uterus under conditions where each chemical has produced an equivalent gravimetric and histologic uterotrophic effect, using the standard 3-day assay protocol. Data are also presented indicating the limitations associated with comparison of gene expression profiles for different chemicals at times before the uterotrophic effects are fully realized. We conclude that the case has yet to be made for regarding synthetic estrogens as presenting a unique human hazard compared with phytoestrogens and physiologic estrogens. Key words: diethylstilbestrol, estrogen, gene expression, genistein, microarray, phytoestrogen, toxicogenomics, uterus.

Animals↗
PubMed2004

Cumulative dietary energy intake determines the onset of puberty in female rats.

Laboratory animal diets for studies to determine the endocrine-disrupting potential of chemicals are under scrutiny because they can affect both assay control values and assay sensitivity. Although phytoestrogen content is important, we have previously shown that a phytoestrogen-rich diet and a phytoestrogen-free diet were equally uterotrophic to rats and advanced vaginal opening (VO) when compared with the standard diet RM1. Abolition of the effects by the gonadotrophin-releasing hormone antagonist Antarelix indicated that these effects were mediated through the hypothalamus-pituitary-reproductive organ axis. In the present study, we investigated the relationship between cumulative energy intake and sexual maturation in female rats. Infant formula (IF) at different concentrations and synthetic diets, with a wide range of metabolizable energy (ME) values, were used to modulate energy intake. Increasing energy intake was associated with an increase in uterine weight (absolute and adjusted for body weight) for both IF and the synthetic diets. In both cases, the increased uterine weight was directly proportional to energy intake. Body weight was unaffected by IF consumption but, in the case of the diets, was increased proportionally with energy consumption. Antarelix abolished the uterine weight increases with both formula and the diets, whereas body weight was unaffected. The mean day of VO was also advanced by high-ME diets and IF, whereas body weight at VO was unaffected. VO occurred at an energy intake of approximately 2,300 kJ/rat determined by measuring total food intake from weaning to VO, indicating that this cumulative energy intake was the trigger for puberty. ME is therefore a critical factor in the choice of diets for endocrine disruption studies.

Animal Feed↗
PubMed2004

Phenotypic anchoring of gene expression changes during estrogen-induced uterine growth.

A major challenge in the emerging field of toxicogenomics is to define the relationships between chemically induced changes in gene expression and alterations in conventional toxicologic parameters such as clinical chemistry and histopathology. We have explored these relationships in detail using the rodent uterotrophic assay as a model system. Gene expression levels, uterine weights, and histologic parameters were analyzed 1, 2, 4, 8, 24, 48, and 72 hr after exposure to the reference physiologic estrogen 17 beta-estradiol (E2). A multistep analysis method, involving unsupervised hierarchical clustering followed by supervised gene ontology-driven clustering, was used to define the transcriptional program associated with E2-induced uterine growth and to identify groups of genes that may drive specific histologic changes in the uterus. This revealed that uterine growth and maturation are preceded and accompanied by a complex, multistage molecular program. The program begins with the induction of genes involved in transcriptional regulation and signal transduction and is followed, sequentially, by the regulation of genes involved in protein biosynthesis, cell proliferation, and epithelial cell differentiation. Furthermore, we have identified genes with common molecular functions that may drive fluid uptake, coordinated cell division, and remodeling of luminal epithelial cells. These data define the mechanism by which an estrogen induces organ growth and tissue maturation, and demonstrate that comparison of temporal changes in gene expression and conventional toxicology end points can facilitate the phenotypic anchoring of toxicogenomic data.

Animals↗
PubMed2003

The leading role and responsibility of the international scientific community in test development.

The possibility that one or more environmental factors may be affecting adversely the endocrine systems of humans and wildlife has been the subject of international study for the past 7 years. Irrespective of which factors are the most important contributors to the perceived problem; the decision has been taken to evaluate synthetic chemicals for such chemical toxicities. This decision requires access to reliable and relevant endocrine disruption (ED) assays. However, few such tests currently exist, albeit many candidate assays are currently under development. Faced with this situation, the US Environmental Protection Agency has taken the lead in supporting the development and validation of appropriate ED assays. Two of these assays, the rodent uterotrophic assay and the Hershberger anti-androgen assay, are in the final stages of validation, a project conducted under the auspices of the Organisation for Economic and Commercial Development. This article describes the particular scientific issues associated with the validation of these two assays and alerts to the continuing need to consider new assay protocols as they become available.

Adenocarcinoma↗
PubMed2003

The effects of the phytoestrogen genistein on the postnatal development of the rat.

The present studies report the effects on neonatal rats of oral exposure to genistein during the period from birth to postnatal day (PND) 21 to generate data for use in assessing human risk following oral ingestion of genistein. Failure to demonstrate significant exposure of the newborn pups via the mothers milk led us to subcutaneously inject genistein into the pups over the period PND 1-7, followed by daily gavage dosing to PND 21. The targeted doses throughout were 4 mg/kg/day genistein (equivalent to the average exposure of infants to total isoflavones in soy milk) and a dose 10 times higher than this (40 mg/kg genistein). The dose used during the injection phase of the experiment was based on plasma determinations of genistein and its major metabolites. Diethylstilbestrol (DES) at 10 micro g/kg was used as a positive control agent for assessment of changes in the sexually dimorphic nucleus of the preoptic area (SDN-POA). Administration of 40 mg/kg genistein increased uterus weights at day 22, advanced the mean day of vaginal opening, and induced permanent estrus in the developing female pups. Progesterone concentrations were also decreased in the mature females. There were no effects in females dosed with 4 mg/kg genistein, the predicted exposure level for infants drinking soy-based infant formulas. There were no consistent effects on male offspring at either dose level of genistein. Although genistein is estrogenic at 40 mg/kg/day, as illustrated by the effects described above, this dose does not have the same repercussions as DES in terms of the organizational effects on the SDN-POA.

Administration, Oral↗
PubMed2003

The OECD program to validate the rat uterotrophic bioassay. Phase 2: dose-response studies.

The Organisation for Economic Co-operation and Development has completed phase 2 of an international validation program for the rodent uterotrophic bioassay. The purpose of the validation program was to demonstrate the performance of two versions of the uterotrophic bioassay, the immature female rat and the adult ovariectomized rat, in four standardized protocols. This article reports the dose-response studies of the validation program; the coded single-dose studies are reported in an accompanying paper. The dose-response study design used five selected weak estrogen agonists, bisphenol A, genistein, methoxychlor, nonylphenol, and o,p -DDT. These weak agonists were administered in a prescribed series of doses to measure the performance and reproducibility of the protocols among the participating laboratories. All protocols successfully detected increases in uterine weights when the weak agonists were administered. Within each protocol, there was good agreement and reproducibility of the dose response among laboratories with each substance. Substance-specific variations were observed in the influence of the route of administration on the uterine response, the potency as related to the dose producing the first statistically significant increase in uterine weights, and the maximum increase in uterine weight. Substantive performance differences were not observed between the uterotrophic bioassay versions or among the standardized protocols, and these were judged to be qualitatively equivalent. It is noteworthy that these results were reproducible under a variety of different experimental conditions (e.g., animal strain, diet, housing, bedding, vehicle, animal age), indicating that the bioassay's performance as a screen is robust. In conclusion, both the intact, immature, and adult OVX versions, and all protocols appear to be reproducible and transferable across laboratories and are able to detect weak estrogen agonists.

Age Factors↗
PubMed2003

The OECD program to validate the rat uterotrophic bioassay. Phase 2: coded single-dose studies.

The Organisation for Economic Co-operation and Development has completed phase 2 of an international validation program for the rodent uterotrophic bioassay. This portion of phase 2 assessed the reproducibility of the assay with a battery of positive and negative test substances. Positive agonists of the estrogen receptor included the potent reference estrogen 17-ethinyl estradiol (EE), and the weak estrogen agonists bisphenol A, genistein, methoxychlor, nonylphenol, and o,p -DDT. The negative test substance or nonagonist was n-dibutylphthalate. The test substances were coded, and prescribed doses of each test substance were administered in 16 laboratories. Two versions of the uterotrophic assay, the intact immature and the adult ovariectomized female rat, were tested and compared using four standardized protocols covering both sc and po administration. Assay reproducibility was compared using a) EE doses identical to those used in phase 1 and in parallel dose-response studies, b) single doses of the weak agonists identical to one of five doses from the dose-response studies, and c) a single dose of the negative test substance. The results were reproducible and in agreement both within individual laboratories and across the participating laboratories for the same test substance and protocol. The few exceptions are examined in detail. The reproducibility was achieved despite a variety of different experimental conditions (e.g., variations in animal strain, diet, housing protocol, bedding, vehicle, animal age). In conclusion, both versions of the uterotrophic bioassay and all protocols appear robust, reproducible, and transferable across laboratories and able to detect weak estrogen agonists. These results will be submitted along with other data for independent peer review to provide support for the validation of the uterotrophic bioassay.

Animal Feed↗
PubMed2003

The OECD program to validate the rat uterotrophic bioassay. Phase 2: dietary phytoestrogen analyses.

Many commercial laboratory diets have detectable levels of isoflavones (e.g., phytoestrogens such as genistein [GN]) that have weak estrogenic activity both in vitro and in vivo. During validation studies of the uterotrophic bioassay, diet samples from 20 participating laboratories were collected and analyzed for three major phytoestrogens: GN, daidzein (DN), and coumestrol (CM). Soy phytoestrogens GN and DN were found at total phytoestrogen levels from 100 to 540 microg/g laboratory diet; a forage phytoestrogen, CM, ranged from nondetectable to 4 microg/g laboratory diet. The phytoestrogen levels were compared with both baseline uterine weights of the control groups and with the relative uterine weight increase of groups administered two weak estrogen agonists: bisphenol A (BPA) and nonylphenol (NP). The comparison uses a working assumption of additivity among the phytoestrogens, despite several significant qualifications to this assumption, to estimate total genistein equivalents (TGE). Some evidence was found that phytoestrogen levels in the diet > 325-350 microg/g TGE could diminish the responsiveness of the uterotrophic bioassay to weak agonists. This was especially true for the case of the intact, immature female version of the uterotrophic bioassay, where higher food consumption relative to body weight leads to higher intakes of dietary phytoestrogens versus ovariectomized adults. This dietary level is sufficient in the immature female to approach a biological lowest observable effect level for GN of 40-50 mg/kg/day. These same data, however, show that low to moderate levels of dietary phytoestrogens do not substantially affect the responsiveness of the assay with weak estrogen receptor agonists such as NP and BPA. Therefore, laboratories conducting the uterotrophic bioassay for either research or regulatory purposes may routinely use diets containing levels of phytoestrogens < 325-350 microg/g TGE without impairing the responsiveness of the bioassay.

Administration, Oral↗
PubMed2003

The intact immature rodent uterotrophic bioassay: possible effects on assay sensitivity of vomeronasal signals from male rodents and strain differences.

The vomeronasal organ in rodents is an important social and sexual signaling pathway. We have investigated whether the housing of intact immature females in close proximity to mature males would interfere with the sensitivity of the immature rodent uterotrophic bioassay as the result of vomeronasal signals transmitted by male urinary proteins. The hypothesis was that the proximity of males might induce early puberty, thereby increasing mean uterine weight and reducing the responsiveness of the assay. The hypothesis was tested in both rats and mice by housing mature males above immature females, separated only by a wire screen, for 3 days and determining possible changes in uterine weight. The results were negative. Neither the mean uterine weight nor the group mean standard deviation of the uterine weights were changed in the uterotrophic bioassay. Given that the timing of sexual maturation may vary with the strain of mouse used, we also evaluated the sensitivity of the immature mouse uterotrophic assay to diethylstilbestrol (DES) using four strains of mice. Similar sensitivity was observed for the CD-1, C57Bl6, and Alpk strains, but B6CBF(1) mice were marginally less sensitive to DES than were the other strains. These findings add to earlier data indicating the robustness of the rodent uterotrophic assay protocol.

Age Factors↗
PubMed2002

Scientific issues associated with the validation of in vitro and in vivo methods for assessing endocrine disrupting chemicals.

The assays required to assess the potential of chemicals to act as endocrine disrupting (ED) agents are either in place or are under current development. However, the validation and utilisation of these assays is currently being hampered by uncertainties regarding their purpose and required sensitivity, and uncertainties as to the intrinsic variability of the parameters being measured. This article discusses these several sources of uncertainty and the intrinsic variability of many of the key assay parameters. It is concluded that current uncertainties regarding the use of ED assays, and the extrapolation of rodent effects to humans, are due to the absence of an extensive agreed rodent control database for the developmental parameters under study, coupled to the established intrinsic variability of these parameters between strains/species of test animals and test protocols. Only when these factors are generally accepted, well studied and controlled for, will it be possible to employ ED assays with confidence and to relate assay data to effects likely to be seen in humans.

Animals↗
PubMed2002

Detection of aromatase inhibitors in vitro using rat ovary microsomes.

Inhibition of aromatase activity in vitro is one of the Tier 1 screening assays proposed by the Endocrine Disrupter Screening and Testing Advisory Committee (EDSTAC) for the detection of potential endocrine disrupters. In this report a rat ovarian aromatase inhibition assay has been evaluated using the reference aromatase inhibitors anastrozole, fadrozole, letrozole and CGS 18320B. Rat ovary microsomes were used as the enzyme source, as endocrine disruption studies are most commonly carried out in this species. Aromatase activity was inhibited in vitro by anastrozole, fadrozole, letrozole and CGS 18320B with IC(50)s of 25, 7, 7 and 5 nM, respectively. This assay, therefore, appears to have good sensitivity to aromatase inhibitors and may be useful as a general screening assay and in mechanistic studies.

Animals↗
PubMed2002

Concept evaluation: androgen-stimulated immature intact male rats as an assay for antiandrogens.

The effects of the concomitant oral administration of a potent reference androgen (17alpha-methyltestosterone) and both a potent (flutamide) and a weak (p,p'-DDE) antiandrogen on intact weanling male rats are described. This protocol resulted in the inhibition by the antiandrogens of the increase in sex accessory tissue weights induced by coadministration of 17alpha-methyltestosterone. Although both flutamide and p,p'-DDE inhibited the androgen-induced growth of the levator ani/bulbocavernosus muscle complex, the Cowper's glands, and the seminal vesicles, the growth of the prostate gland was unaffected by either antiandrogen. The unresponsiveness of the prostate gland, a primary target tissue in the castrated rat antiandrogen assay, has yet to be fully explained. However, the ability of the assay to detect the activity of low dose levels of the weak antiandrogen DDE (at doses of 20 mg/kg body weight) makes the system worthy of further study as one of several alternatives. Given the rapid rate of assay/protocol exploration and refinement of this assay and its alternatives, there is the need for careful comparative studies before selecting a single bioassay for validation and regulatory use.

Administration, Oral↗
PubMed2002

Critical review and evaluation of the uterotrophic bioassay for the identification of possible estrogen agonists and antagonists: in support of the validation of the OECD uterotrophic protocols for the laboratory rodent. Organisation for Economic Co-operation and Development.

A current issue for regulatory agencies is endocrine-related modes of action such as those mediated by the estrogen, androgen, and thyroid nuclear receptors. At the national and international levels, the consensus recommendation for the assessment of such modes of action is a tiered series of in vitro and in vivo protocols. The tiered framework begins with screens for structural alerts and then moves to rapid, mechanistic in vitro screening assays, and then to in vivo screening bioassays. The objective of these screens is to identify substances that may warrant testing for endocrine-mediated adverse effects. The final framework tier as needed is to test these substances in long-term bioassays for adverse endocrine-mediated reproductive and/or developmental effects. The subject of this review, the rodent uterotrophic bioassay, is intended to be a rapid in vivo screening bioassay for possible estrogen agonists and based on the response of the estrogen-sensitive uterus. The central metric of bioassay is a statistically significant increase in the weight of the uterus after 3 consecutive days of test substance administration. The extensive background literature is summarized in this review on the mode of action underlying the bioassay and the uterine response to estrogens. The review includes the bioassay's history of development and how its employment has changed and evolved over time. The review describes two major uterotrophic bioassay versions, the intact, immature female and the mature, ovariectomized female, and the protocol factors likely to influence relevance, reproducibility, and reliability of bioassay. The emphasis of the review is the ability of the uterotrophic bioassay to identify the substances of current interest: weak estrogen agonists with binding affinities relative to the natural 17beta-estradiol in the log 0 to log -3 range. Using selected model substances having RBAs in this target range, the bioassay's performance in a hierarchical, tiered approach is evaluated, including the predictive capability of the uterotrophic bioassay based on available reproductive and developmental testing data. The review concludes that the uterotrophic bioassay is reliable and can identify substances that may act via an estrogen-mode of action, supporting the validity of the uterotrophic bioassay and its regulatory use as an in vivo mechanistic screening bioassay for estrogen agonists and antagonists.

Animals↗