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John Ashby - One of the best experts on this subject based on the ideXlab platform.
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Articles Confirmation of Uterotrophic Activity of 3-(4-Methylbenzylidine)camphor in the Immature Rat
2013Co-Authors: Helen Tinwell, George Orphanides, Jenny Odum, P A Lefevre, Angie Burns, T D Spurway, Graeme J. Moffat, John AshbyAbstract:In this study we found that the ultraviolet sunscreen component 3-(4-methylbenzylidine)camphor (4MBC) is Uterotrophic in immature rats when administered by either subcutaneous injection or oral gavage. These data confirm earlier reports of Uterotrophic activity for this agent when administered to immature rats in the diet or by whole-body immersion; however, they are in contrast to negative unpublished immature rat Uterotrophic assay results. Data also indicate that 4MBC binds to isolated rat uterine estrogen receptors and shows activity in a human estrogen receptor yeast transactivation assay; however, we considered both of these effects equivocal. In this study, we confirmed the original observation that 4MBC was active as a mitogen to MCF-7 breast cancer cells. We evaluated and discounted the possibility that the estrogenic activity of 4MBC is related to its bulky camphor group, which is of similar molecular dimensions to that of the weak estrogen kepone. Uncertainty remains regarding the mechanism of the Uterotrophic activity of 4MBC. Key words: estrogenic activity, ER binding, immature rat Uterotrophic assay, MCF-7 cell proliferation, 3-(4-methylbenzylidine)camphor, yeast ER transactivation. Environ Health Perspect 110:533–536 (2002). [Online 4 April 2002
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The reported in vitro anti-estrogen pentachloronitrobenzene enhances the estrogenic activity of estradiol in vivo in the rat.
Environmental toxicology and pharmacology, 2005Co-Authors: John Ashby, J. Odum, Angela Burns, Paul LefevreAbstract:Pentachloronitrobenzene (PCNB) has been shown to inhibit foci-formation for MCF-7 cells in vitro (Zou, E., Hatakeyama, M., Matsumra, F., 2002. Foci-formation of MCF-7 cells as an in vitro screening method for estrogenic chemicals. Environ. Toxicol. Pharmacol. 11, 71) This effect was referred to as representing an anti-estrogenic property of PCNB. However, we have found no evidence that PCNB acts as either an estrogen or an anti-estrogen, either in vitro or in vivo. The assays conducted were binding to human and rat estrogen receptors (ER), a hER yeast trans-activation assay, the immature rat Uterotrophic assay and a pubertal female rat assay. Nonetheless, when PCNB was evaluated as a possible anti-estrogen against estradiol in the immature rat Uterotrophic assay, it enhanced, rather than reduced the activity of estradiol. Absence of an effect by PCNB on the Uterotrophic activity of diethylstilbestrol suggests that the effect with estradiol was related to alteration of its metabolism. However, PCNB was not hepatotoxic and failed to inhibit cytochrome P450 or estradiol sulphotransferase. Pentachlorophenol, a major metabolite of PCNB, was inactive as an estrogen and failed to enhance the Uterotrophic activity of estradiol.
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Gene Expression Changes in the Immature Rat Uterus: Effects of Uterotrophic and Sub-Uterotrophic Doses of Bisphenol A
Toxicological sciences : an official journal of the Society of Toxicology, 2004Co-Authors: John Ashby, J. OdumAbstract:J. C. Gould et al., 1998, Mol. Cell Endocrinol. 142, 203-214, have reported that administration of 5-150 mg/kg/day BPA to immature rats leads to increases in uterine peroxidase activity and progesterone receptor (PR) protein levels in the absence of a Uterotrophic response. These observations are of interest given current concerns regarding the adequacy of the Uterotrophic assay to act as a sentinel for the estrogenic activity of chemicals in vivo. Therefore, the Uterotrophic activity of BPA to the immature rat has been re-evaluated over the dose range 2 microg/kg-800 mg/kg/day. Expression levels of three estrogen responsive uterine genes were determined using real-time RT-PCR--namely, complement component 3, lipocalin 2, and PR. 18S rRNA and RNA polymerase II large subunit acted as control genes. Observations of gene expression were made 4 h and 72 h after the first of three daily po administrations of BPA. Increases in gene expression were observed over the Uterotrophic dose range (approximately 200-800 mg/kg BPA). Over the dose range 2 microg/kg-20 mg/kg BPA there was no Uterotrophic response and no increase in gene expression. We conclude that BPA does not produce reproducible changes in gene expression in the uterus of immature rats at dose levels that are not also Uterotrophic. Therefore, in the present study, the no effect level for Uterotrophic activity for BPA coincided with the no transcriptional effect level for uterine genes.
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the need to decide if all estrogens are intrinsically similar
Environmental Health Perspectives, 2004Co-Authors: Jonathan G. Moggs, Fei Ling Lim, John Ashby, Helen Tinwell, Ian Kimber, David Moore, George OrphanidesAbstract: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 17β-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.
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Sensitivity of the immature rat Uterotrophic assay to mixtures of estrogens.
Environmental health perspectives, 2004Co-Authors: Helen Tinwell, John AshbyAbstract: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...
William Owens - One of the best experts on this subject based on the ideXlab platform.
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the oecd program to validate the rat Uterotrophic bioassay phase 2 dietary phytoestrogen analyses
Environmental Health Perspectives, 2003Co-Authors: William Owens, John Ashby, Jenny Odum, Lesley OnyonAbstract: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.
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The intact immature rodent Uterotrophic bioassay: possible effects on assay sensitivity of vomeronasal signals from male rodents and strain differences.
Environmental health perspectives, 2003Co-Authors: John Ashby, William Owens, J. Odum, Helen TinwellAbstract: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.
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The OECD program to validate the rat Uterotrophic bioassay. Phase 2: coded single-dose studies.
Environmental health perspectives, 2003Co-Authors: Jun Kanno, John Ashby, Lesley Onyon, Shyamal D. Peddada, Elard Jacob, William OwensAbstract: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.
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The OECD program to validate the rat Uterotrophic bioassay: an overview.
Environmental health perspectives, 2003Co-Authors: William Owens, Herman B W M KoëterAbstract:The Organisation for Economic Co-operation and Development has undertaken an international validation program for the rodent Uterotrophic bioassay. This validation program comprised two major parts. The first part was the development of a detailed background review document compiling the existing data on the bioassay's history, the molecular and physiologic basis for the bioassay's mechanistic relevance to detect estrogen agonists and antagonists, a review of important bioassay protocol parameters, and a review of the data generated by in vitro assays, previous Uterotrophic bioassays, and developmental and reproductive assays to assess and support the overall predictivity of the Uterotrophic bioassay. The second part was an extensive multiyear effort managed by a validation management group to demonstrate the operating characteristics of four protocols. The effort was conducted in two phases. The phase 1 results with the reference agonist ethinyl estradiol (EE) and antagonist ZM 189,154 has been published previously. This Environmental Health Perspectives mini-monograph is devoted to the phase 2 work using five weak estrogen agonists, bisphenol A, genistein, methoxychlor, nonylphenol, and o,p -DDT, as well as the negative substance dibutylphthalate. These data show that all protocols successfully detected increases in uterine weights when a sufficient dose level of the weak agonists was administered, whether the substances were known or provided as coded doses to the laboratory. The data with both the reference EE and all five weak agonists are reproducible over time and under a variety of different experimental conditions (e.g., animal strain, diet, housing, bedding, vehicle, animal age). In conclusion, all protocols now have sufficient data to support their validity.
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The OECD program to validate the rat Uterotrophic bioassay. Phase 2: Dose-response studies
Environmental health perspectives, 2003Co-Authors: Jun Kanno, John Ashby, Lesley Onyon, Shyamal D. Peddada, Elard Jacob, William OwensAbstract: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.
Lesley Onyon - One of the best experts on this subject based on the ideXlab platform.
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the oecd program to validate the rat Uterotrophic bioassay phase 2 dietary phytoestrogen analyses
Environmental Health Perspectives, 2003Co-Authors: William Owens, John Ashby, Jenny Odum, Lesley OnyonAbstract: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.
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The OECD program to validate the rat Uterotrophic bioassay. Phase 2: coded single-dose studies.
Environmental health perspectives, 2003Co-Authors: Jun Kanno, John Ashby, Lesley Onyon, Shyamal D. Peddada, Elard Jacob, William OwensAbstract: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.
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The OECD program to validate the rat Uterotrophic bioassay. Phase 2: Dose-response studies
Environmental health perspectives, 2003Co-Authors: Jun Kanno, John Ashby, Lesley Onyon, Shyamal D. Peddada, Elard Jacob, William OwensAbstract: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.
Helen Tinwell - One of the best experts on this subject based on the ideXlab platform.
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Articles Confirmation of Uterotrophic Activity of 3-(4-Methylbenzylidine)camphor in the Immature Rat
2013Co-Authors: Helen Tinwell, George Orphanides, Jenny Odum, P A Lefevre, Angie Burns, T D Spurway, Graeme J. Moffat, John AshbyAbstract:In this study we found that the ultraviolet sunscreen component 3-(4-methylbenzylidine)camphor (4MBC) is Uterotrophic in immature rats when administered by either subcutaneous injection or oral gavage. These data confirm earlier reports of Uterotrophic activity for this agent when administered to immature rats in the diet or by whole-body immersion; however, they are in contrast to negative unpublished immature rat Uterotrophic assay results. Data also indicate that 4MBC binds to isolated rat uterine estrogen receptors and shows activity in a human estrogen receptor yeast transactivation assay; however, we considered both of these effects equivocal. In this study, we confirmed the original observation that 4MBC was active as a mitogen to MCF-7 breast cancer cells. We evaluated and discounted the possibility that the estrogenic activity of 4MBC is related to its bulky camphor group, which is of similar molecular dimensions to that of the weak estrogen kepone. Uncertainty remains regarding the mechanism of the Uterotrophic activity of 4MBC. Key words: estrogenic activity, ER binding, immature rat Uterotrophic assay, MCF-7 cell proliferation, 3-(4-methylbenzylidine)camphor, yeast ER transactivation. Environ Health Perspect 110:533–536 (2002). [Online 4 April 2002
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the need to decide if all estrogens are intrinsically similar
Environmental Health Perspectives, 2004Co-Authors: Jonathan G. Moggs, Fei Ling Lim, John Ashby, Helen Tinwell, Ian Kimber, David Moore, George OrphanidesAbstract: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 17β-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.
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Sensitivity of the immature rat Uterotrophic assay to mixtures of estrogens.
Environmental health perspectives, 2004Co-Authors: Helen Tinwell, John AshbyAbstract: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...
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The intact immature rodent Uterotrophic bioassay: possible effects on assay sensitivity of vomeronasal signals from male rodents and strain differences.
Environmental health perspectives, 2003Co-Authors: John Ashby, William Owens, J. Odum, Helen TinwellAbstract: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.
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confirmation of Uterotrophic activity of 3 4 methylbenzylidine camphor in the immature rat
Environmental Health Perspectives, 2002Co-Authors: Helen Tinwell, George Orphanides, Jenny Odum, P A Lefevre, Graeme Moffat, Angie Burns, T D Spurway, John AshbyAbstract:In this study we found that the ultraviolet sunscreen component 3-(4-methylbenzylidine)camphor (4MBC) is Uterotrophic in immature rats when administered by either subcutaneous injection or oral gavage. These data confirm earlier reports of Uterotrophic activity for this agent when administered to immature rats in the diet or by whole-body immersion; however, they are in contrast to negative unpublished immature rat Uterotrophic assay results. Data also indicate that 4MBC binds to isolated rat uterine estrogen receptors and shows activity in a human estrogen receptor yeast transactivation assay; however, we considered both of these effects equivocal. In this study, we confirmed the original observation that 4MBC was active as a mitogen to MCF-7 breast cancer cells. We evaluated and discounted the possibility that the estrogenic activity of 4MBC is related to its bulky camphor group, which is of similar molecular dimensions to that of the weak estrogen kepone. Uncertainty remains regarding the mechanism of the Uterotrophic activity of 4MBC.
J. Odum - One of the best experts on this subject based on the ideXlab platform.
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The reported in vitro anti-estrogen pentachloronitrobenzene enhances the estrogenic activity of estradiol in vivo in the rat.
Environmental toxicology and pharmacology, 2005Co-Authors: John Ashby, J. Odum, Angela Burns, Paul LefevreAbstract:Pentachloronitrobenzene (PCNB) has been shown to inhibit foci-formation for MCF-7 cells in vitro (Zou, E., Hatakeyama, M., Matsumra, F., 2002. Foci-formation of MCF-7 cells as an in vitro screening method for estrogenic chemicals. Environ. Toxicol. Pharmacol. 11, 71) This effect was referred to as representing an anti-estrogenic property of PCNB. However, we have found no evidence that PCNB acts as either an estrogen or an anti-estrogen, either in vitro or in vivo. The assays conducted were binding to human and rat estrogen receptors (ER), a hER yeast trans-activation assay, the immature rat Uterotrophic assay and a pubertal female rat assay. Nonetheless, when PCNB was evaluated as a possible anti-estrogen against estradiol in the immature rat Uterotrophic assay, it enhanced, rather than reduced the activity of estradiol. Absence of an effect by PCNB on the Uterotrophic activity of diethylstilbestrol suggests that the effect with estradiol was related to alteration of its metabolism. However, PCNB was not hepatotoxic and failed to inhibit cytochrome P450 or estradiol sulphotransferase. Pentachlorophenol, a major metabolite of PCNB, was inactive as an estrogen and failed to enhance the Uterotrophic activity of estradiol.
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Gene Expression Changes in the Immature Rat Uterus: Effects of Uterotrophic and Sub-Uterotrophic Doses of Bisphenol A
Toxicological sciences : an official journal of the Society of Toxicology, 2004Co-Authors: John Ashby, J. OdumAbstract:J. C. Gould et al., 1998, Mol. Cell Endocrinol. 142, 203-214, have reported that administration of 5-150 mg/kg/day BPA to immature rats leads to increases in uterine peroxidase activity and progesterone receptor (PR) protein levels in the absence of a Uterotrophic response. These observations are of interest given current concerns regarding the adequacy of the Uterotrophic assay to act as a sentinel for the estrogenic activity of chemicals in vivo. Therefore, the Uterotrophic activity of BPA to the immature rat has been re-evaluated over the dose range 2 microg/kg-800 mg/kg/day. Expression levels of three estrogen responsive uterine genes were determined using real-time RT-PCR--namely, complement component 3, lipocalin 2, and PR. 18S rRNA and RNA polymerase II large subunit acted as control genes. Observations of gene expression were made 4 h and 72 h after the first of three daily po administrations of BPA. Increases in gene expression were observed over the Uterotrophic dose range (approximately 200-800 mg/kg BPA). Over the dose range 2 microg/kg-20 mg/kg BPA there was no Uterotrophic response and no increase in gene expression. We conclude that BPA does not produce reproducible changes in gene expression in the uterus of immature rats at dose levels that are not also Uterotrophic. Therefore, in the present study, the no effect level for Uterotrophic activity for BPA coincided with the no transcriptional effect level for uterine genes.
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The intact immature rodent Uterotrophic bioassay: possible effects on assay sensitivity of vomeronasal signals from male rodents and strain differences.
Environmental health perspectives, 2003Co-Authors: John Ashby, William Owens, J. Odum, Helen TinwellAbstract: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.
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comparative activities ofp nonylphenol and diethylstilbestrol in noble rat mammary gland and Uterotrophic assays
Regulatory Toxicology and Pharmacology, 1999Co-Authors: J. Odum, I T G Pyrah, John R Foster, Ronald L Joiner, J. P. Miller, John AshbyAbstract:Abstract Colerangle and Roy (1996,Endocrine4, 115–122) have described the apparent ability of both diethylstilbestrol (DES) andp-nonylphenol (NP) to cause extensive cell proliferation and lobular development in the mammary glands of young adult Noble rats. The chemicals were administered over 11 days via subcutaneously implanted minipumps. The dose level of DES used (0.076 mg/kg/day) was about 70 times higher than its minimum detection level in rodent Uterotrophic and reproductive toxicology studies. In contrast, the lowest active dose level of NP (0.073 mg/kg/day) in the Noble rat mammary gland study was about 600 times lower than its minimum detection level in rat Uterotrophic and multigeneration studies. The apparent enhanced sensitivity of the Noble rat mammary gland to the estrogenic activity of NP was considered worthy of further study. Ovariectomized Noble rat Uterotrophic assays with NP (minimum detection level ∼40 mg/kg/day, 3 or 11 days, oral gavage) revealed similar assay sensitivity to that observed for earlier immature and ovariectomized Alderley Park (AP) rat Uterotrophic assays of this chemical. The response of the ovariectomized Noble rat Uterotrophic assay to DES and estradiol was also as expected from earlier immature AP rat assays. It is concluded that the general sensitivity to estrogens of the Noble rat and the AP rat is similar. A repeat of the Noble rat mammary gland study with DES (11 × 0.076 mg/kg/day) and NP (11 × either 0.073 or 53.2 mg/kg/day), as originally reported by Colerangle and Roy (1996), revealed a strong positive response to DES and no response to NP. It is concluded that the minimum detection level of NP as a weakly estrogenic material in the rat should be based on the results of rat Uterotrophic and multigeneration studies and therefore be set at ∼40 mg/kg/day. It is also concluded that induced S-phase in the rodent mammary gland is best monitored using BRDU, as opposed to PCNA staining, and that use of subcutaneously implanted minipumps/pellets is inappropriate for risk/hazard assessment studies of chemicals already established as estrogenicin vitroandin vivo,as are NP and DES.
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Activity of Raloxifene in Immature and Ovariectomized Rat Uterotrophic Assays
Regulatory toxicology and pharmacology : RTP, 1997Co-Authors: John Ashby, J. Odum, J.r. FosterAbstract:Abstract Raloxifene is generally regarded as a tissue-selective estrogen agonist, being capable of selectively counteracting both the loss of bone density and the increase in serum cholesterol that occur in rats following ovariectomy, without the induction of a trophic effect on the rat uterus. An implication of this activity profile is that reliance cannot be placed on the rat Uterotrophic assay for the detection and assessment of xenobiotic estrogens. Within that context the estrogenic activity of raloxifene has been reevaluated in immature and ovariectomized rat Uterotrophic assays. Four separate experiments were conducted. In the first two a reproducible increase (1.7-fold) was observed in the uterus wet weights of immature rats administered three daily doses of raloxifene. The maximum Uterotrophic response observed over the dose range 0.01–2 mg/kg was for 0.1 mg/kg raloxifene. Further experiments utilized three daily doses of 0.1 mg/kg raloxifene. In the third experiment the Uterotrophic response elicited by raloxifene in immature rats was abolished by coadministration of the estrogen receptor blocking agent Faslodex (ICI 182,780). This confirmed the direct involvement of estrogen receptors in the Uterotrophic response elicited by raloxifene. Two further indications of the estrogenicity of raloxifene were obtained in this experiment. First, dry uterus weights were also shown to be increased by raloxifene administration, thereby eliminating water retention as the sole cause of the observed increases in uterus weights. Second, the height of the surface epithelium was increased by 1.7-fold in the raloxifene-treated animals, an effect that was accompanied by increases in mitotic activity and glandular formation in the stromal endometrium. The endometrial epithelium of the treated rats also showed evidence of vacuolation and, occasionally, the presence of degenerating cells. Raloxifene did not, however, cause premature vaginal opening in immature rats, unlike estradiol. In the fourth experiment the Uterotrophic activity of raloxifene was confirmed in ovariectomized rats, although the response was less (1.2-fold) than in immature rats. In contrast to the effects seen for the positive control agent estradiol, the Uterotrophic responses observed for raloxifene in ovariectomized animals were not accompanied by cornification of the vaginal epithelium. Premature vaginal opening and vaginal cornification may be less sensitive markers of estrogenic activity than the Uterotrophic response. These collected observations confirm that raloxifene exerts a genuine trophic effect on the rat uterus, and as a consequence, the Uterotrophic assay can be relied upon to detect estrogens with only a marginal effect on the uterus.