Biomedical subjects

J P Sumpter

Publications and source records attributed to J P Sumpter.

At least 19 recordsLinked to original sources

PubMed2003

Cloning and gene expression of P450 17alpha-hydroxylase,17,20-lyase cDNA in the gonads and brain of the fathead minnow Pimephales promelas.

P450 17alpha-hydroxylase,17,20-lyase (P450c17) is a key steroidogenic enzyme in the production of androgens and, therefore, is also indispensable for the production of oestrogens (that are produced from the aromatisation of androgens). In this study, P450c17 cDNA was cloned from the ovary of the fathead minnow (FHM) and its gene expression was examined in the gonads and brains of male and female FHM at different stages of gonadal development with a view to developing an understanding of its involvement in the reproductive physiology in this species. The FHM-P450c17 cDNA sequence cloned was 1812 bp in length, with an open reading frame of 1554 nucleotides encoding a protein of 518 amino acids. Amino acid identity of FHM-P450c17 with P450c17s in other animals was up to 81.8% in other teleosts (channel catfish), 62% in elasmobranches (spiny dogfish), 64% in birds (chicken), and up to 48.8% in mammals (human). FHM-P450c17 gene expression occurred in the ovary, testis, and also in the brain (both male and female) at all stages of sexual development studied. Expression in the brain was at least 30-fold lower than in the gonads, but consistent in all fish life stages studied. In the testis, FHM-P450c17 gene expression was negatively correlated with gonadal development, but there was no obvious association between P450c17 gene expression and sexual development in the ovary, or brain (in both males and females). The results from this study demonstrate the expression of P450c17 in the brain for the first time in fish. Enzymatic studies are now needed to investigate the possible role of P450c17 in neurosteroid production in teleosts.

Amino Acid Sequence↗

PubMed2003

Overwinter fasting and re-feeding in rainbow trout: plasma growth hormone and cortisol levels in relation to energy mobilisation.

This study investigated the roles of cortisol and growth hormone (GH) during a period of fasting in overwintering salmonid fish. Indices of carbohydrate (plasma glucose, liver glycogen), lipid (plasma free fatty acids (FFAs)) and protein metabolism (plasma protein, total plasma amino acids) were determined, together with plasma GH, cortisol and somatolactin (SL) levels at intervals in three groups of rainbow trout (continuously fed; fasted for 9 weeks then fed; fasted for 17 weeks). In fasted fish, a decline in body weight and condition factor was accompanied by reduced plasma glucose and hepatic glycogen and increased plasma FFA. No consistent elevation of plasma GH occurred until after 8 weeks of fasting when plasma GH levels increased ninefold. No changes were observed in plasma total protein and AA until between weeks 13 and 17 when both were reduced significantly. When previously fasted fish resumed feeding, plasma glucose and FFA, and hepatic glycogen levels rapidly returned to control values and weight gain resumed. No significant changes in plasma cortisol levels, related to feeding regime, were evident at any point during the study and there was no evidence that SL played an active role in the response to fasting. The results suggest that overwinter fasting may not represent a significant nutritional stressor to rainbow trout and that energy mobilisation during fasting may be achieved without the involvement of GH, cortisol or SL.

Amino Acids↗

PubMed2002

Exposure to exogenous 17beta-oestradiol disrupts p450aromB mRNA expression in the brain and gonad of adult fathead minnows (Pimephales promelas).

Oestrogens are key regulators in sexual differentiation and development in higher vertebrates. P450 aromatase (p450arom) is the steroidogenic enzyme responsible for the synthesis of oestrogens from aromatisable androgens. Effects of endocrine disrupting chemicals on steroidogenic enzyme gene expression have received little attention so far, yet it is potentially a major pathway for sexual disruption. In this 14-day study the effects of exogenous 17beta-oestradiol (E2) at environmentally relevant concentrations were assessed on gene expression of p450aromB in the gonad and brain of maturing male and female fathead minnows (FHM). Exposure to E2 resulted in an oestrogenic response as shown by a dose-dependent induction of plasma vitellogenin (VTG) in female and male fish and a dose-dependent inhibition of testis growth. There was an effect of exposure to E2 on p450aromB mRNA expression in the gonads; E2 up-regulated p450aromB mRNA expression in the testis and ovary in a dose-response manner after 14 days of exposure. In male brain, p450aromB mRNA concentrations were significantly reduced in fish exposed to 100 and 320 ng E2/l on day 4, but on day 14 were elevated in males exposed to both 32 and 100 ng E2/l. No effects of E2 on p450aromB mRNA expression occurred in the brain of females. The results of this study show that concentrations of E2 found in the environment can have disruptive effects on key steroidogenic enzyme pathways that control sexual development in fish.

Animals↗

PubMed2002

Altered sexual maturation and gamete production in wild roach (Rutilus rutilus) living in rivers that receive treated sewage effluents.

Disruption in gonadal development of wild roach living in U.K. rivers receiving large volumes of treated sewage effluent is manifest in a variety of ways, ranging from malformation of the germ cells and/or reproductive ducts to altered gamete production. Intersex fish were also found to have an altered endocrine status and an elevated concentration of plasma vitellogenin. Gonadal growth was inhibited only in severely intersex fish, whereas progression of spermatogenesis was delayed in a large proportion of all intersex and exposed male fish. In contrast to the effects observed in the intersex and exposed male fish, the maturation of ovaries in female fish inhabiting effluent-contaminated rivers appeared to be less obviously affected, although a higher incidence of oocyte atresia was found in the effluent-exposed fish compared with the reference fish. A positive correlation was found between the proportion of female tissue in the gonads of intersex fish and their plasma vitellogenin concentration, suggesting that vitellogenin can be an indicator for the level of gonadal disruption in intersex roach. The estradiol-17beta concentration in intersex fish was intermediate between the concentration found in males and females, and the plasma testosterone was between 2- and 3-fold higher in intersex fish compared with male fish. These data suggest a link between altered endocrine status in intersex and female fish and gonadal disruption. Spermiation was also affected in roach living in effluent-impacted rivers: a lower proportion of fish were found releasing sperm, and in those intersex fish that were spermiating, a reduced milt volume and a reduced sperm density were found. All intersex fish had malformations of the reproductive duct(s), and in severely affected fish, the ducts were occluded, thus preventing release of gametes. In view of the widespread occurrence of intersexuality in wild fish populations in rivers throughout the United Kingdom, assessment of the reproductive capabilities of these intersex roach is clearly needed to understand the impact of this phenomenon on roach fertility.

Animals↗

PubMed2002

Wild intersex roach (Rutilus rutilus) have reduced fertility.

Endocrine-disrupting chemicals, known to be present in the environment, have great potential for interfering with reproductive health in wildlife and humans. There is, however, little direct evidence that endocrine disruption has adversely affected fertility in any organism. In freshwater and estuarine fish species, for example, although a widespread incidence of intersex has been reported, it is not yet known if intersexuality influences reproductive success. The purpose of this study was, therefore, to determine gamete quality in wild intersex roach (Rutilus rutilus) by assessing sperm characteristics, fertilization success, and ability to produce viable offspring. The results clearly demonstrate that gamete production is reduced in intersex roach. A significantly lower proportion of moderately or severely feminized fish (17.4% and 33.3%, respectively) were able to release milt compared with normal male fish from contaminated rivers (in which 97.6% of the males were able to release milt), reference male fish (97.7%), or less severely feminized intersex fish (experiment 1: 85.8%, experiment 2: 97%). Intersex fish that did produce milt produced up to 50% less (in terms of volume per gram of testis weight) than did histologically normal male fish. Moreover, sperm motility (percentage of motile sperm and curvilinear velocity) and the ability of sperm to successfully fertilize eggs and produce viable offspring were all reduced in intersex fish compared with normal male fish. Male gamete quality (assessed using sperm motility, sperm density, and fertilization success) was negatively correlated with the degree of feminization in intersex fish (r = -0.603; P < 0.001) and was markedly reduced in severely feminized intersex fish by as much as 50% in terms of motility and 75% in terms of fertilization success when compared with either less severely feminized intersex fish or unaffected male fish. This is the first evidence documenting a relationship between the morphological effects (e.g., intersex) of endocrine disruption and the reproductive capabilities of any wild vertebrate. The results suggest that mixtures of endocrine-disrupting substances discharged into the aquatic environment could pose a threat to male reproductive health.

Animals↗

PubMed2001

Removal of endocrine-disrupting chemicals in activated sludge treatment works.

The release of endocrine-disrupting chemicals into the aquatic environment has raised the awareness of the central role played by sewage treatment in lowland water quality. This review focuses on the activated sludge process, which is commonly used to treat sewage in large towns and cities and which successfully removes the bulk of the organic compounds that enter the works. However, not all compounds are completely broken down or converted to biomass. For example, the estrogenic alkylphenols and steroid estrogens found in effluent are the breakdown products of incomplete breakdown of their respective parent compounds. Batch microcosm studies have indicated that estrone, ethinylestradiol, and alkylphenols will not be completely eliminated in activated sludge over typical treatment times. Field data suggest that the activated sludge treatment process can consistently remove over 85% of estradiol, estriol, and ethinylestradiol. The removal performance for estrone appears to be less and is more variable. Because of its relatively high hydrophobicity, the accumulation of alkylphenol in sludge has been observed. Although it has not been examined, accumulation of ethinylestradiol in sludge is a possibility due to its recalcitrance and hydrophobicity. A comparison between the concentrations of some of the major endocrine-active chemicals in effluents and their biological potencies has been made, to direct attention to the chemicals of most concern. While water purification techniques such as UV or activated charcoal could significantly remove these microorganic contaminants, the high costs involved suggest that research into the potential for treatment optimization should receive more attention.

Animals↗

PubMed2001

Reproductive effects of long-term exposure to Bisphenol A in the fathead minnow (Pimephales promelas).

Bisphenol A (BPA), a high-volume chemical used to make polycarbonate plastic, epoxy resins, and other chemicals has been reported to be weakly estrogenic. To investigate the effects of long-term exposure to Bisphenol A, a multigeneration study was conducted in which fathead minnows (Pimephales promelas) were exposed to water concentrations of BPA in the range from 1 to 1280 micrograms/L. In this paper, we report the growth and reproductive effects of BPA on sexually mature adults in the F0 generation (after 43, 71, and 164 d of exposure) and the effects on hatchability in the F1 generation. Mean measured concentrations of BPA in the water for all doses, over a 164-d exposure period, were between 70% and 96% of nominal. An inhibitory effect of BPA on somatic growth (length and weight) occurred in adult male fish exposed to 640 and 1280 micrograms/L (after 71 and 164 d). BPA induced vitellogenin synthesis (VTG; a biomarker for estrogen exposure) in males at concentrations of 640 and 1280 micrograms/L after 43 d and 160 micrograms/L after 71 d. In females, plasma VTG concentrations were elevated above controls only after 164-d exposure to 640 micrograms/L. Inhibition of gonadal growth (as measured by the gonadosomatic index) occurred in both males and females at concentrations of 640 and 1280 micrograms/L after 164 d. In males, a concentration of 16 micrograms/L altered the proportion of sex cell types in the testis, suggesting inhibition of spermatogenesis. Concentrations of BPA that induced VTG synthesis and affected gonadal development were lower than those that resulted in discernible effects on reproductive output. Egg production was inhibited at a BPA concentration of 1280 micrograms/L, and hatchability in the F1 generation was reduced at a BPA concentration of 640 micrograms/L (there were not enough eggs spawned in the 1280 micrograms/L group for hatchability studies to be conducted). The results demonstrate that BPA acts as a weak estrogen to fish when administered via the water, with effects on breeding at and above 640 micrograms/L.

Animals↗

PubMed2001

Nonylphenol affects gonadotropin levels in the pituitary gland and plasma of female rainbow trout.

Female rainbow trout (Oncorhynchus mykiss) were exposed to 4-nonylphenol (NP) at (mean measured) concentrations of 0.7, 8.3, and 85.6 micrograms/L for 18 weeks, during early ovarian development. Fish were sampled sublethally every six weeks, and terminal samples were taken at 18 weeks. NP induced an estrogenic effect (the synthesis of vitellogenin) at concentrations of 8.3 and 85.6 micrograms/L. An effect on gonadotropin synthesis and secretion was also observed. Plasma follicle stimulating hormone (FSH) levels and FSH gene expression in the pituitary were the most sensitive endpoints assessed, being reduced at the lowest dose employed (0.7 microgram NP/L). Pituitary gland luteinizing hormone (LH) content was significantly lower in fish exposed to 85.6 micrograms NP/L, and LH gene expression was suppressed in fish exposed to 8.3 and 85.6 micrograms NP/L. In contrast, plasma LH concentration increased in these fish, but by a very minor absolute amount, and returned to control levels by the final sampling time. Gonadal development ceased in the fish exposed to 85.6 micrograms NP/L, and steroidogenesis in these fish was also markedly inhibited. Although the mechanisms underlying these responses are unknown, this study demonstrates that NP has adverse effects on pituitary function that can result in inhibition of ovarian development.

Animals↗

PubMed2001

Assessing the biological potency of binary mixtures of environmental estrogens using vitellogenin induction in juvenile rainbow trout (Oncorhynchus mykiss).

Experiments were conducted to assess the in vivo potency of binary mixtures of estrogenic chemicals using plasma vitellogenin (VTG) concentrations in juvenile rainbow trout (Oncorhynchus mykiss) as the endpoint. The estrogenic potencies of estradiol-17beta (E2), 4-tertnonylphenol (NP), and methoxychlor (MXC) were determined following 14 day exposures to the individual chemicals and binary mixtures of these chemicals. E2, NP, and MXC all induced concentration dependent increases in plasma VTG, with lowest observed effect concentrations of 4.7 and 7.9 ng L(-1) for E2, 6.1 and 6.4 microg L(-1) for NP, and 4.4 and 6.5 microg L(-1) for MXC. Concentration-response curves for fixed ratio binary mixtures of E2 and NP (1:1000), E2 and MXC (1:1000), and NP and MXC (1:1) were compared to those obtained for the individual chemicals, using the model of concentration addition. Mixtures of E2 and NP were additive at the concentrations tested, but mixtures of E2 and MXC were less than additive. This suggests that while NP probably acts via the same mechanism as E2 in inducing VTG synthesis, MXC may be acting via a different mechanism(s), possibly as a result of its conversion to HPTE which is an estrogen receptor alpha agonist and an estrogen receptor beta antagonist. It was not possible to determine whether mixtures of MXC and NP were additive using VTG induction, because the toxicity of MXC restricted the effect range forwhich the expected response curve forthe binary mixture could be calculated. The data presented illustrate that the model of concentration addition can accurately predict effects on VTG induction, where we know that both chemicals act via the same mechanism in mediating a vitellogenic response.

Animals↗

PubMed2001

Exposure of juvenile roach (Rutilus rutilus) to treated sewage effluent induces dose-dependent and persistent disruption in gonadal duct development.

Wild roach (Rutilus rutilus) have been found with intersex gonads in rivers throughout the United Kingdom. The incidence of intersexuality is strongly correlated with discharges of estrogenic treated sewage effluent into those rivers, and this has led to the hypothesis that estrogenic chemicals in effluents are feminizing wild male fish. In this study, early-life stage roach (50 days post hatch, dph) were exposed for 150 days to a graded concentration (0%, 12.5%, 25%, 50%, and 100%) of treated sewage (primarily domestic) effluent to examine the effects of exposure on sexual differentiation and development. Measurement of steroid estrogens and alkylphenolic chemicals in the effluent and a resulting dose-dependent induction of vitellogenin (VTG; a female-specific, estrogen-dependent plasma protein) confirmed that the fish had been exposed and responded to "estrogen" in the effluent. Exposure to treated sewage effluent induced feminization of the reproductive ducts in "male" roach in a dose-dependent manner (in full-strength effluent, 100% of the fish had feminized ducts), indicating that the disruption of the gonad ducts seen in wild roach is the result of exposure to treated sewage effluents during early-life stages. There were no effects of treated sewage effluent exposure on germ cell development; therefore, no oocytes occurred in the testes of the feminized male roach. Subsequent, depuration of the effluent exposed fish in "clean" water for 150 days resulted in a reduction in plasma VTG but no alteration of the feminized ducts, indicating that the effect of the treated sewage effluent on reproductive duct development was permanent. The causality of oocytes in the testes of wild male roach therefore remains to be elucidated.

Animals↗

PubMed2001

Estrogenic activity of phenolic additives determined by an in vitro yeast bioassay.

We used a recombinant yeast estrogen assay to assess the activity of 73 phenolic additives that are used as sunscreens, preservatives, disinfectants, antioxidants, flavorings, or for perfumery. Thirty-two of these compounds displayed activity: 22 with potencies relative to 17beta-estradiol, ranging from 1/3,000 to < 1/3,000,000, and 10 compounds with an impaired response that could not be directly compared with 17beta-estradiol. Forty-one compounds were inactive. The major criteria for activity appear to be the presence of an unhindered phenolic OH group in a para position and a molecular weight of 140-250 Da.

Dose-Response Relationship, Drug↗

PubMed2001

Effects of the synthetic estrogen 17 alpha-ethinylestradiol on the life-cycle of the fathead minnow (Pimephales promelas).

A fish full life-cycle (FFLC) study was conducted for 17 alpha-ethinylestradiol (EE2) using the fathead minnow, Pimephales promelas. Newly fertilized embryos (< 24 h old) were exposed to five concentrations of EE2 (0.2, 1.0, 4.0, 16, and 64 ng/L nominal) in continuous flow-through conditions for 305 d at 25 +/- 1 degrees C. Exposure concentrations were verified by 14C-EE2 radiochemistry, supported by radioimmunoassay, and mean measured values were > or = 70% of nominal. For the F0 adult phase until 301 d posthatch, the no-observed-effect concentrations (NOECs) for growth, survival, and reproduction (as egg production) were all > or = 1.0 ng/L. The NOEC values for F1 embryo hatching success and larval survival (at 28 d posthatch) were both > or = 1.0 ng/L. While statistically detectable changes in F1 growth were evident at 0.2 ng/L, these were not considered to be biologically significant when compared with historical control data. Male fish exposed to EE2 at 4.0 ng/L failed to develop normal secondary sexual characteristics; on the other hand, assumed females exposed to this level of EE2 were able to breed when paired with males that had not been exposed to EE2. Histology of F0 control, 0.2-, and 1-ng/L exposed fish at 56 d posthatch indicated an approximate female-to-male (F:M) sex ratio of 50:50 (with no ovatestes observed in the control), while fish exposed to EE2 at 4.0 ng/L for 56 d posthatch had a F:M sex ratio of 84:5 (with ovatestes in 11% of fish). After 172 d posthatch, no testicular tissue was observed in any fish exposed to EE2 at 4.0 ng/L. At the same time point, plasma vitellogenin levels were significantly higher in fish exposed to EE2 at 16 ng/L. A lack of sexual differentiation occurred in males at concentrations > or = 4.0 ng/L. Taking into account these data, the overall no-observed-adverse-effect concentration was considered to be 1.0 ng/L.

Animals↗

PubMed2001

Assessment of the effects of metabolism on the estrogenic activity of xenoestrogens: a two-stage approach coupling human liver microsomes and a yeast estrogenicity assay.

Concern that the reproductive health of humans is being affected by exposure to xenoestrogens has led to the development of various in vitro and in vivo screening assays for the identification of suspected xenoestrogens. However, the estrogenic activity of a chemical determined in vitro may not necessarily predict its activity in vivo if the chemical is metabolized during the assay and/or in vivo. Therefore, to investigate the role of metabolism in modulating the estrogenic activity of suspected xenoestrogens, we have devised a two-stage approach coupling incubations with either human or rat hepatic microsomes with a yeast estrogenicity (transcription) assay. We have assessed the activity of the proestrogenic pesticide 99.5% methoxychlor [1,1,1-trichloro-2,2-bis-(4-methoxyphenyl)ethane, MXC] (EC(50) = 4.45 +/- 1.9 ,icroM, n = 6) and a structural analog, methoxybisphenol A [2,2-bis-(4-methoxyphenyl) propane, MBPA], in the yeast estrogenicity assay and also established that yeast (Saccharomyces cerevisiae), unlike human liver microsomes, are not able to demethylate MXC or MBPA to estrogenic metabolites. This indicates that the proestrogen MXC has weak intrinsic estrogenic activity. Using 99.5% MXC and 17beta-estradiol as paradigms, we have demonstrated how metabolism can enhance or suppress, respectively, estrogenic activity. The effect of metabolism on the activities of the weak xenoestrogens 3,17beta-bisdesoxyestradiol [1,3,5(10)-estratriene] and 6-hydroxytetralin (5,6,7,8-tetrahydro-2-naphthol) was also assessed. This two-stage approach can distinguish the estrogenic activity of a suspect chemical from the activity due to its more, or less, active metabolites and will aid in the evaluation of novel xenoestrogens and, more importantly, proestrogens.

Animals↗

PubMed2001

Sexual disruption in a second species of wild cyprinid fish (the gudgeon, Gobio gobio) in United Kingdom freshwaters.

To establish whether the intersex condition seen in the roach (Rutilus rutilus) in United Kingdom (UK) rivers was species specific or a more general phenomenon in fish, evidence for sexual disruption was sought in a second cyprinid species, the gudgeon (Gobio gobio). Gudgeon were collected from the Rivers Aire and Lea (at locations that receive high-volume discharges of sewage treatment works [STW] effluent and that contain intersex roach) and from two still waters, and their gonads were examined histologically for evidence of intersexuality (the simultaneous presence of oocytes and testicular tissue). Intersex gonads were found at all sites, with the highest incidences occurring at one of the still waters (Lakeside Fisheries: 15%) and at sites on the River Aire (Thwaite Weir, Silsden Bridge, and Knostrop: 14, 13, and 12%, respectively). In the River Lea and Longton Park Lake, the incidence of intersexuality in gudgeon was 6%. In most cases, intersex gonads were characterized by a few primary oocytes/gonad section in an otherwise normal testis. However, at some sites on the River Aire (Thwaite Weir and Knostrop), the intersex condition was more severe. At Thwaite Weir, for example, more than half of the gonad in 40% of the intersex fish was comprised of ovarian tissue. Elevated concentrations of plasma vitellogenin both in male and in intersex fish indicated that fish had been exposed to estrogen(s). Some of the gudgeon were found at sites several kilometers downstream of any point discharge of STW effluent; therefore, the results likely are representative of this species in wild populations found in typical UK river ecosystems. Together with the findings in the roach, these data on the gudgeon confirm that sexual disruption in fish in UK rivers is not species specific.

Animals↗

PubMed2000

Differential effects of xenoestrogens on coactivator recruitment by estrogen receptor (ER) alpha and ERbeta.

It has been proposed that tissue-specific estrogenic and/or antiestrogenic actions of certain xenoestrogens may be associated with alterations in the tertiary structure of estrogen receptor (ER) alpha and/or ERbeta following ligand binding; changes which are sensed by cellular factors (coactivators) required for normal gene expression. However, it is still unclear whether xenoestrogens affect the normal behavior of ERalpha and/or ERbeta subsequent to receptor binding. In view of the wide range of structural forms now recognized to mimic the actions of the natural estrogens, we have assessed the ability of ERalpha and ERbeta to recruit TIF2 and SRC-1a in the presence of 17beta-estradiol, genistein, diethylstilbestrol, 4-tert-octylphenol, 2',3',4', 5'-tetrachlorobiphenyl-ol, and bisphenol A. We show that ligand-dependent differences exist in the ability of ERalpha and ERbeta to bind coactivator proteins in vitro, despite the similarity in binding affinity of the various ligands for both ER subtypes. The enhanced ability of ERbeta (over ERalpha) to recruit coactivators in the presence of xenoestrogens was consistent with a greater ability of ERbeta to potentiate reporter gene activity in transiently transfected HeLa cells expressing SRC-1e and TIF2. We conclude that ligand-dependent differences in the ability of ERalpha and ERbeta to recruit coactivator proteins may contribute to the complex tissue-dependent agonistic/antagonistic responses observed with certain xenoestrogens.

Air Pollutants, Occupational↗

PubMed2000

Obstacles to the prediction of estrogenicity from chemical structure: assay-mediated metabolic transformation and the apparent promiscuous nature of the estrogen receptor.

Information on structure-activity relationships (SAR) and pathways of metabolic activation would facilitate the preliminary screening of chemicals for estrogenic potential. Published crystallographic studies of the estrogen receptor (ER) imply an essential role of the two hydroxyl groups on estradiol (17beta-E(2)) for its binding to ER. The influence of these hydroxyl groups on ER binding and estrogenicity was evaluated by the study of 17beta-E(2) with one or both of these hydroxyl groups removed (17beta-desoxyestradiol and 3, 17beta-bisdesoxyestradiol, respectively). 6-Hydroxytetralin (17beta-E(2) with its C- and D-rings removed) and other synthetic estrogens were also studied. The estrogenicity assays comprised a yeast ER-mediated transcription assay, mammalian cell transcription assays incorporating either ER alpha or ER beta, and the immature rat uterotrophic assay. With the exception of 6-hydroxytetralin in the uterotrophic assay, all the chemicals were active in all the assays. Hydroxylation of the two desoxy compounds to estradiol was shown to occur in immature female rats, but metabolism was not implicated in the responses observed in the ER-binding and yeast systems. It is concluded that the 3-hydroxyl and 17beta-hydroxyl groups of 17beta-E(2) are not absolute requirements for estrogenicity. It would therefore be of value to the derivation of SAR for estrogenicity were the crystal structure of the bisdesoxy-E(2)/ER complex to be evaluated.

Animals↗

PubMed2000

Re-evaluation of the first synthetic estrogen, 1-keto-1,2,3, 4-tetrahydrophenanthrene, and bisphenol A, using both the ovariectomised rat model used in 1933 and additional assays.

1-Keto-1,2,3,4-tetrahydrophenanthrene (THP-1) was reported by Cook et al. in 1933 as the first synthetic estrogen. Estrogenic activity was assessed by the induction of vaginal cornification in ovariectomised rats. The corresponding 4-isomer (THP-4) was shown to be inactive. Both chemicals have been re-synthesised and assessed for hormonal activity. Each chemical bound weakly and to the same extent to isolated estrogen receptors, but only at high concentrations. However, they each lacked estrogenic or anti-estrogenic activity when evaluated in vitro using a yeast hER assay, and both failed to induce vaginal cornification or uterotrophic effects in ovariectomised rats. THP-1, and to a lesser extent THP-4, were shown to possess weak androgenic and anti-androgenic activity in vitro when evaluated using an hAR yeast assay. Estrogenic activity for bisphenol A (BPA) was subsequently demonstrated by [Dodds and Lawson, Synthetic, oestrogenic agents without the phenanthrene nucleus, Nature 137, (1936)] using the same ovariectomised rat protocol, and this activity has been confirmed and supplemented by positive uterotrophic effects for BPA in the same bioassays. The present results illustrate the complexity of deriving conclusions regarding the hormonal activities of chemicals. First, some activities observed in isolated hormonal receptor binding assays may not be expressed in functional hormonal assays. This indicates the need for functional hormonal assays in any screening programme. Second, that activities observed for a chemical in one hormonal assay may not be reflected in related hormonal assays. This indicates the need to define assay protocols with some precision when incorporating them into screening batteries. Finally, that some literature reports of hormonal activity for chemicals may not be capable of independent confirmation under apparently identical conditions of test. This illustrates the need to use lists of hormonally active chemicals with care.

Androgen Antagonists↗

PubMed2000

Issues arising when interpreting results from an in vitro assay for estrogenic activity.

Concern about possible adverse effects caused by the inadvertent exposure of humans and wildlife to endocrine-active chemicals, has led some countries to develop an in vivo-in vitro screening program for endocrine effects. In this paper, a previously described estrogen-inducible recombinant yeast strain (Saccharomyces cerevisiae) is used to investigate a number of issues that could potentially lead to the mislabeling of chemicals as endocrine disruptors. The chemicals studied were: 17beta-estradiol, dihydrotestosterone, testosterone, estradiol-3-sulfate, 4-nonylphenol, 4-tert-octylphenol, 4-tert-butylphenol, bisphenol-A, methoxychlor, 2,2-bis(p-hydroxyphenyl)-1,1,1-trichloroethane, butyl benzyl phthalate, 4-hydroxytamoxifen, and ICI 182,780. Alterations in assay methodology (for example, incubation time, initial yeast cell number, and the use of different solvents) did not affect the potency of bisphenol-A and 4-nonylphenol relative to 17beta-estradiol, but did alter the apparent potency of butyl benzyl phthalate. Other issues (including the metabolic activation of methoxychlor, the chemical purity of a steroid metabolite and unusual chemical artifacts observed with alkylphenolic chemicals) which affect data interpretation are described. Many of the issues raised will also affect other in vitro assays for endocrine activity, and some will be relevant to the interpretation of data from in vivo assays. These examples illustrate that considerable care and thought must be applied when interpreting results derived from any single assay. Only by using a suite of assays will we minimize the chances of wrongly labeling chemicals as endocrine disruptors.

Biological Assay↗