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In the original article wrong unites were quoted in Table 3 (page 508) and Table 4 (page 510) as well as in the paragraph 3.2 Core chemical exposure experiments on page 509. Also in paragraph 2.3 Selection and testing of chemicals the link to the Supplemental Materials (ESM) was missing.

Although the past two decades have witnessed a significant increase in the number of studies investigating effects of estrogenic chemicals on amphibians, to date little is known about specific molecular interactions of estrogens with the hypothalamus–pituitary–gonadal–hepatic axis in developing amphibians. Here, tissue-specific functional sets of genes, derived previously from studies of fishes exposed to endocrine active chemicals, were evaluated in Xenopus laevis exposed to 17α-ethynylestradiol (EE2) throughout their early development. Specifically, transcriptional responses of X. laevis exposed to 0.09, 0.84, or 8.81 µg EE2/L were characterized during sexual differentiation [31 day post hatch (dph)] and after completion of metamorphosis during the juvenile stage (89 dph). While at 31 dph there were no consistent effects of EE2 on abundances of transcripts,at 89 dph X. laevis exhibited significant alterations in expression of genes involved in steroid signaling and metabolism, synthesis of cholesterol, and vitellogenesis. Specifically, expression of androgen receptor, farnesyl diphosphate synthase, estrogen receptor α, and vitellogenin A2 was significantly greater (>2-fold) than in controls while expression of farnesoid x-activated receptors α and β was significantly less (>2-fold reduction) than in controls. These results support the hypothesis that sets of genes derived from studies in teleost fish can be extrapolated for use in amphibians during the juvenile stage but not in sexually undifferentiated individuals. Furthermore, changes in abundances of transcripts of the here utilized sets of genes in animals sampled post sexual differentiation were in accordance with developmental effects and alterations of gonadal histology reported in a parallel study. This set of genes might be useful for predicting potential adverse outcomes at later life-stages.

The white sturgeon (Acipenser transmontanus) population that resides in the Columbia River in British Columbia (BC), Canada, has suffered recruitment failures for more than three decades. During the summers of 2008 and 2009, studies were performed to determine whether exposure to water downstream of a metal smelter in Trail, BC affected survival or growth of early life-stages of white sturgeon through 60+ days post-fertilization (dpf). In both years, there were no significant differences in survival of fish that were exposed to water from downstream compared to water from upstream of the smelter. At 20-21dpf, average mortality was 2.4 percent and 12 percent in upstream water for 2008 and 2009, respectively, which was similar to the average mortality of 3.8 percent and 7.2 percent in downstream water for 2008 and 2009, respectively. Relatively great mortality after 20-21dpf complicated analysis of the subchronic exposure, but use of a survival analysis indicated that the average fish died at 25-29dpf, regardless of whether the water to which they were exposed came from upstream or downstream of the smelter. In addition, measured concentrations of metals in river water were less than the threshold for adverse effects on early life stages of white sturgeon. Based upon these analyses, it is not likely that current concentrations of metals in the Columbia River in southern BC are adversely affecting survival of early life stages of white sturgeon larvae.

Exposure to contaminants in the environment has been suggested as a contributing cause of ongoing declines in populations of amphibians reported in certain locations around the world. In the current study, responses of the wood frog (Lithobates sylvaticus) to exposure to triphenyltin (TPT), a commonly used fungicide, during the larval period were characterized. Exposure of L. sylvaticus to 0.1, 1.0, or 5.0 μg TPT/L significantly affected survival, growth, days to metamorphosis (DTM), and abundances of transcripts of genes of interest. After seven days of exposure there were no significant effects on survival, but masses and snout-ventral length (SVL) of larvae exposed to 5.0 μg TPT/L were significantly lesser than controls. Mortality of larvae after exposure to 5.0 μg TPT/L was 100% nine days after initiation of the experiment. Larvae exposed to 0.1 or 1.0 μg TPT/L were allowed to grow for 100 days or until they reached metamorphic climax, whichever occurred earlier. Mortality of wood frogs exposed to 1.0 μg TPT/L was 80%. The LC20 or LC50 after 100 days of exposure was 0.12 or 0.34 μg TPT/L, respectively. However, DTM of larvae that survived exposure to 1.0μgTPT/L was significantly less than that of controls. Abundances of transcripts of retinoid-X-receptor (rxr) and perixosomal proliferation receptor gamma (pparγ) were significantly lesser in larvae exposed to either concentration of TPT for seven days. Also, abundances of transcripts of stearoyl-CoA desaturase-1 (scd1), fatty acid synthase (fas), lipoprotein lipase (lpl), and β-hydroxybutyrate dehydrogenase (β-hb-m) were lesser in larvae exposed to 5.0 μg TPT/L, which suggested that disruption of lipid metabolism might have affected survival in this exposure group. However, in larvae that survived to metamorphic climax during exposure to TPT for as long as 100 days, abundances of transcripts of perixosomal proliferation receptor alpha (pparα), pparγ, cytochrome p4504B1 (cyp4b1), fas, and lpl were greater than in controls, suggesting that an up-regulation of processes related to metabolism of lipids might have been important for survival and development of these animals. Overall, concentrations of TPT that are found in the environment had a significant effect on the survival and development of L. sylvaticus, and this might have been due, in part, to effects on metabolism of lipids.

Triphenyltin chloride (TPTC), which has been extensively used in industry and agriculture, can occur at concentrations in the environment sufficient to be toxic. Here, potency of TPTC to modulate genes in a library containing 1,820 modified green fluorescent protein (GFP)-expressing promoter reporter vectors constructed from Escherichia coli K12 strains was determined. Exposure to TPTC resulted in 22 (fold change > 2) or 71 (fold change > 1.5) differentially expressed genes. The no observed transcriptional effect (NOTEC) and median transcriptional effect concentrations (TEC50) were determined to be 0.036 and 0.45 mg/L in E. coli. These responses were 1,230 and 97 times more sensitive than the acute median effect concentration (EC50) required to inhibit growth of cells, which demonstrated that this live cell array represents a sensitive method to assess toxic potency of chemicals. The 71 differentially expressed genes could be classified into seven functional groups. Of all the altered genes, three groups which encoded for catalytic enzymes, regulatory proteins, and structural proteins accounted for 28 %, 18 %, and 14 % of all altered genes, respectively. The pattern of differential expression observed during this study was used to elucidate the mechanism of toxicity of TPTC. To determine potential relationships among genes that were changed greater than 2.0-fold by exposure to TPTC, a correlation network analysis was constructed, and four genes were related to aroH, which is the primary target for metabolic regulation of aromatic biosynthesis by feedback inhibition in bacteria. The genes rnC, cld, and glgS were selected as potential biomarkers for TPTC, since their expression was more than 2.0-fold greater after exposure to TPTC.

Several studies have shown that exposure of amphibians, including the African clawed frog (Xenopus laevis), to potent estrogens at critical times during development results in feminization and/or demasculinization. However, genotyping of X. laevis has only recently become possible, so studies performed in the past were rarely able to make explicit linkages between genetic and phenotypic sex. Therefore, to further characterize this relationship, X. laevis tadpoles were exposed during development to 0.09, 0.84, or 8.81 μg/L 17α-ethynylestradiol (EE2), which is the estrogen analog commonly used in oral contraceptives. Exposure to all concentrations of EE2 tested resulted in significant delays in time to metamorphosis. Genotyping showed that genetic sex ratios were similar among treatments. However, morphological evaluation revealed that a significant number of individuals with a male genotype displayed mixed sex and abnormal phenotypes. Additionally, both genetic males and females exposed to EE2 exhibited greater presence of vitellogenin protein relative to the respective controls. Since estrogens function downstream of the initial molecular signals of sexual differentiation, it is likely that genetic male animals received mixed endogenous male and exogenous female signals that caused disordered sexual development. The production of vitellogenin was probably temporally separated and independent from primary effects on sexual differentiation, and might have contributed to delays in metamorphosis observed in individuals exposed to EE2.

Effect-directed analysis (EDA) can be useful in identifying and evaluating potential toxic chemicals in matrixes. Previous investigations of extracts of sediments from the upper Danube River in Germany revealed acute nonspecific and mechanism-specific toxicity as determined by several bioassays. In the present study, EDA was used to further characterize these sediments and identify groups of potentially toxic chemicals. Four extracts of sediments were subjected to a novel fractionation scheme coupled with identification of chemicals to characterize their ability to disrupt steroidogenesis or cause mutagenic and/or teratogenic effects. All four whole extracts of sediment caused significant alteration of steroidogenesis and were mutagenic as well as teratogenic. The whole extracts of sediments were separated into 18 fractions and these fractions were then subjected to the same bioassays as the whole extracts. Fractions 7 to 15 of all four extracts were consistently more potent in both the Ames fluctuation and H295R assays. Much of this toxicity could be attributed to polycyclic aromatic hydrocarbons, sterols, and in fraction 7-naphthoic acids. Because the fraction containing polychlorinated biphenyls, polychlorodibenzodioxin/furan, dichlorodiphenyltrichloroethane, and several organophosphates did not cause any observable effects on hormone production or a mutagenic response, or were not detected in any of the samples, these compounds could be eliminated as causative agents for the observed effects. These results demonstrate the value of using EDA, which uses multiple bioassays and new fractionation techniques to assess toxicity. Furthermore, to our knowledge this is the first study using the recently developed H295R assay within EDA strategies. Environ. Toxicol. Chem. 2012; 31: 1053–1062. © 2012 SETAC

Selenomethionine (Se-Met) is the major dietary form of selenium (Se). Detrimental effects have been associated with exposure to elevated dietary selenium. Previous studies have demonstrated effects of Se on the endocrine system, in particular effects on cortisol and thyroid hormones. However, no information is available regarding effects of Se on sex steroid hormones. In the present study, effects of dietary exposure to an environmentally relevant concentration (4.54 mg/kg wet weight (ww)) of Se-Met for 126 days on concentrations of sex steroid hormones in blood plasma of female rainbow trout were determined. Furthermore, the molecular basis for effects of Se-Met on plasma sex steroid hormone concentrations was investigated. Concentrations of androstenedione (A), estrone (E1), and estradiol (E2) were 39.5-, 3.8-, and 12.7-fold greater in plasma of treated females than the untreated controls, respectively. Testosterone (T) was detected only in plasma of treated females. The greater E2 concentration stimulated greater transcript abundance of vitellogenin (vtg) and zona-radiata protein (zrp). Female rainbow trout exposed to Se-Met had greater transcript abundance of key steroidogenic proteins and enzymes, including peripheral benzodiazepine receptor (pbr), cytochrome P450 side-chain cleavage (P450scc), and 3β-hydroxysteroid dehydrogenase (3β-hsd). Exposure to Se-Met did not affect transcript abundance of luteinizing hormone (lh) or follicle stimulating hormone (fsh). Similarly, there was no change in transcript abundance of luteinizing hormone receptor (lhr) or follicle stimulating hormone receptor (fshr). Long-term exposure to dietary Se-Met has the potential to stimulate vitellogenesis in female rainbow trout by directly stimulating ovarian tissue steroidogenesis. This is the first study to report effects of Se on sex steroid hormone production in fish.

BACKGROUND, GOALS, AND SCOPE: In response to increasing concerns regarding the potential of chemicals to interact with the endocrine system of humans and wildlife, various national and international programs have been initiated with the aim to develop new guidelines for the screening and testing of these chemicals in vertebrates. Here, we report on the validation of an in vitro assay, the H295R steroidogenesis assay, to detect chemicals with the potential to inhibit or induce the production of the sex steroid hormones testosterone (T) and 17β-estradiol (E2) in preparation for the development of an Organization for Economic Cooperation and Development (OECD) test guideline.A previously optimized and pre-validated protocol was used to assess the potential of 28 chemicals of diverse structures and properties to validate the H295R steroidogenesis assay. These chemicals are comprised of known endocrine-active chemicals and "negative" chemicals that were not expected to have effects on the targeted endpoints, as well as a number of test chemicals with unknown modes of action at the level of the steroidogenic pathway. A total of seven laboratories from seven countries participated in this effort. In addition to effects on hormone production, confounding factors, such as cell viability and possible direct interference of test substances with antibody-based hormone detection assays, were assessed. Prior to and during the conduct of exposure experiments, each laboratory had to demonstrate that they were able to conduct the assay within the margin of predefined performance criteria.With a few exceptions, all laboratories met the key quality performance parameters, and only 2% and 7% of all experiments for T and E2, respectively, were excluded due to exceedance of these parameters. Of the 28 chemicals analyzed, 13 and 14 tested affected production of T and E2, respectively, while 11 and 8 did not result in significant effects on T and E2 production, respectively. Four and six chemicals produced ambiguous results for effects on T and E2 production, respectively. However, four of these cases each for T and E2 were associated with only one laboratory after a personnel change occurred. Significant interference of test chemicals with some of the antibody-based hormone detection systems occurred for four chemicals. Only one of these chemicals, however, significantly affected the ability of the detection system to categorize the chemical as affecting E2 or T production.With one exception, the H295R steroidogenesis assay protocol successfully identified the majority of chemicals with known and unknown modes of interaction as inducers or inhibitors of T and E2 production. Thus it can be considered a reliable screen for chemicals that can alter the production of sex steroid hormones. One of the remaining limitations associated with the H295R steroidogenesis assay protocol is the relatively small basal production of E2 and its effect on quantifying the decreased production of this hormone with regard to the identification of weak inhibitors. An initial comparison of the data produced in this study with those from in vivo studies from the literature demonstrated the potential of the H295R steroidogenesis assay to identify chemicals affecting hormone homeostasis in whole organisms. Particularly promising was the lack of any false negatives during the validation and the very low number of false positives (1 out of 28 chemicals for each T and E2).Based on the results obtained during this validation study and the accordingly revised test protocols, an OECD draft test guideline was developed and submitted to the OECD working group of the national coordinators of the test guidelines program (WNT) for comments in December 2009.

The present study was part of a comprehensive weight-of-evidence approach with the goal of identifying potential causes for the declines in fish populations, which have been observed during the past decades in the Upper Danube River.The specific goal was the investigation of the endocrine disrupting potential of sediment extracts from different sites along the Danube River. Parallel to the identification and quantification of target estrogens, two in vitro bioassays were employed to assess the estrogenic potential (yeast estrogen screen, YES) of the sediment samples and to evaluate their effects on the production of testosterone (T) and E2 (H295R Steroidogenesis Assay). Using a potency balance approach, the contribution of the measured compounds (Chem-EEQs) to the total endocrine activity measured by the YES (YES-EEQs) was calculated.Of the nine sediment extracts tested five extracts exhibited significant estrogenic activities in the YES, which suggested the presence of ER agonists in these samples. The xenoestrogens nonylphenol (NP) and bisphenol A (BPA) and the natural estrogen estrone (E1) were detected while concentrations of 17β-estradiol (E2) and ethinylestradiol (EE2) were less than their respective limits of quantification in all sediment extracts. A comparison of the measured YES-EEQs and the calculated Chem-EEQs revealed that as much as 6% of estrogenic activity in extracts of most sediments could be explained by two xeno- and one natural estrogen. Exposure of H295R cells to sediment extracts from four different locations in the Danube River resulted in significantly increased concentrations of E2, but only slight inhibition of T synthesis. Furthermore, application of the H295R Steroidogenesis Assay provided evidence for endocrine disrupting potencies in sediment samples from the Upper Danube River, some of which were not detectable with the YES. In conclusion, differential endocrine activities were associated with several sediments from the Upper Danube River. Further investigations will have to show whether the observed activities are of biological relevance with regard to declines in fish populations in the Upper Danube River.

In response to concerns about chemical substances that can alter the function of endocrine systems and may result in adverse effects on human and ecosystem health, a number of in vitro tests have been developed to identify and assess the endocrine disrupting potential of chemicals and environmental samples. One endpoint that is frequently used in in vitro models for the assessment of chemical effects on the endocrine system is the alteration of aromatase activity (AA). Aromatase is the enzyme responsible for converting androgens to estrogens. Some commonly used aromatase assays, including the human microsomal assay that is a mandatory test in US-EPA’s endocrine disruptor screening program (EDSP), detect only direct effects of chemicals on aromatase activity and not indirect effects, including changes in gene expression or transcription factors. This can be a problem for chemical screening initiatives such as the EDSP because chemicals can affect aromatase both indirectly and directly. Here we compare direct, indirect, and combined measurements of AA using the H295R cell line after exposure to seven model chemicals. Furthermore, we compare the predictability of the different types of AA measurements for 17β-estradiol (E2) and testosterone (T) production in vitro. H295R cells were exposed to forskolin, atrazine, letrozole, prochloraz, ketoconazole, aminoglutethimide, and prometon for 48 h. Direct, indirect, and combined effects on aromatase activity were measured using a tritiated water-release assay. Direct effects on aromatase activity were assessed by exposing cells only during the conduct of the tritium-release assay. Indirect effects were measured after exposing cells for 48 h to test chemicals, and then measuring AA without further chemical addition. Combined AA was measured by exposing cells prior and during the conduction of the tritium-release assay. Estradiol and testosterone were measured by ELISA. Exposure to the aromatase inhibitors letrozole, prochloraz, ketoconazole, and aminoglutethimide resulted in greater indirect aromatase activity after a 48-h exposure due to presumed compensatory mechanisms involved in aromatase activity regulation. Forskolin and atrazine caused similar changes in hormone production and enzyme profiles, and both chemicals resulted in a dose-dependent increase in E2, T, and indirect AA. Neither of these two chemicals directly affected AA. For most of the chemicals, direct and combined AA and E2 were good predictors of the mechanism of action of the chemical, with regard to AA. Indirect aromatase activity was a less precise predictor of effects at the hormone level because of presumed feedback loops that made it difficult to predict the chemicals’ true effects, mostly seen with the aromatase inhibitors. Further, it was found that direct and indirect AA measurements were not reliable predictors of effects on E2 for general inducers and inhibitors, respectively. Differential modulation of AA and hormone production was observed in H295R cells after exposure to seven model chemicals, illustrating the importance of measuring multiple endpoints when describing mechanisms of action in vitro. For future work with the H295R, it is recommended that a combination of direct and indirect aromatase measurements is used because it was best in predicting the effects of a chemical on E2 production and its mechanism of action. Further, it was shown that direct AA measurements, which are a common way to measure AA, must be used with caution in vitro.

Hintergrund und Ziel der Studie Die Fischbestande in der Donau sind seit Ende der 1980er-Jahre stark rucklaufig. Trotz intensiver bestandsstutzender Masnahmen und einer verbesserten Wasserqualitat entlang der Donau seit den 1970er-Jahren konnte diese Entwicklung nicht gestoppt werden. Ziel der umfassenden Weight-of-Evidence-Studie (WOE) mit verschiedenen Lines-of-Evidence war es, 1) mogliche kausale Verbindungen zwischen molekularen und zellbiologischen Endpunkten sowie okologisch relevanten Effekten zu analysieren, und 2) zu uberprufen, ob die okotoxikologischen Effekte fur den Ruckgang der Fische verantwortlich sein konnten. Dazu wurden Sedimente und Fische an verschiedenen Standorten der Donau entnommen und in einem integrierten Ansatz untersucht. Das Ziel dieses Beitrages ist es, die Untersuchungsstrategie und den Stand der seit 2003 laufenden Studie vorzustellen. Wie kurzlich von Chapman und Hollert (2006) vorgestellt, wurde als Untersuchungsstrategie eine WOE-Studie mit zahlreichen Lines-of-Evidence eingesetzt. 1) Mit einer Biotestbatterie wurde die akute und mechanismusspezifische Toxizitat untersucht, 2) histologische Untersuchungen und der Mikrokerntest mit Fischzellen wurden als In-situ-Studien durchgefuhrt. 3) Die Diversitat und Abundanz von Makroinvertebraten und Fischen sowie 4) die Konzentrationen von ausgewahlten persistenten organischen Verbindungen, endokrinen Disruptoren und Schwermetallen wurde bestimmt. 5) Um die Substanzen zu identifizieren, die fur die toxischen Effekte in den Sedimenten verantwortlich sind, wurden effektdirigierte Analysen durchgefuhrt.

The presence of a variety of pollutants in the aquatic environment that can potentially interfere with the production of sex steroid hormones in wildlife and humans has been of increasing concern. The aim of the present study was to investigate the effects of extracts from Hong Kong marine waters, and influents and effluents from wastewater treatment plants on steroidogenesis using the H295R cell bioassay. After exposing H295R cells to extracts of water, the expression of four steroidogenic genes and the production of three steroid hormones were measured.Water samples were collected during the summer of 2005 from 24 coastal marine areas and from the influents and effluents of two major waste water treatment plants (WWTPs) in Hong Kong, China. Samples were extracted by solid phase extraction (SPE). H295R cells were exposed for 48 h to dilutions of these extracts. Modulations of the expression of the steroidogenic genes CYP19, CYP17, 3betaHSD2, and CYP11beta2 were determined by measuring mRNA concentrations by real-time polymerase chain reaction (Q-RT-PCR). Production of the hormones progesterone (P), estradiol (E2), and testosterone (T) was quantified using enzyme linked immunosorbent assays (ELISA).Extracts from samples collected in two fish culture areas inhibited growth and proliferation of H295R cells at concentrations greater or equal to 10(5) L equivalents. The cells were exposed to the equivalent concentration of active substances in 10,000 L of water. Thus, to observe the same level of effect as observed in vitro on aquatic organisms would require a bioaccumulation factor of this same magnitude. None of the other 22 marine samples affected growth of the cells at any dilution tested. Twelve of the marine water samples completely inhibited the expression of CYP19 without affecting E2 production; inhibition of CYP17 expression was observed only in one of the samples while expression of CYP11beta2 was induced as much as five- and ninefold after exposure of cells to extracts from two locations. The expression of the progesterone gene 3betaHSD2 was not affected by any of the samples; only one sample induced approximately fourfold the production of E2. Although more than twofold inductions were observed for P and T production, none of these values were statistically significant to conclude effects on the production of these two hormones. While influents from WWTPs did not affect gene expression, an approximately 30% inhibition in the production of E2 and a 40% increase in P occurred for the exposure with influents from the Sha Tin and Stonecutters WWTPs, respectively. Effluents from WWTPs did not affect the production of any of the studied hormones, but a decrement in the expression of the aldosterone gene CYP11beta2 was observed for the Sha Tin WWTP exposure. No direct correlation could be established between gene expression and hormone production.Observed cytotoxicity in the two samples from fish culture areas suggest the presence of toxic compounds; chemical analysis is required for their full identification. Although effluents from WWTPs did not affect hormone production, other types of endocrine activity such as receptor-mediated effects cannot be ruled out. Interactions due to the complexity of the samples and alternative steroidogenic pathways might explain the lack of correlation between gene expression and hormone production results.Changes observed in gene expression and hormone production suggest the presence in Hong Kong coastal waters of pollutants with endocrine disruption potential and others of significant toxic effects. The aromatase and aldosterone genes seem to be the most affected by the exposures, while E2 and P are the hormones with more significant changes observed. Results also suggest effectiveness in the removing of compounds with endocrine activity by the WWTPs studied, as effluent samples did not significantly affect hormone production. The H295R cell showed to be a valuable toll in the battery required for the analysis of endocrine disrupting activities of complex environmental samples.Due to the intrinsic complexity of environmental samples, a combination of analytical tools is required to realistically assess environmental conditions, especially in aquatic systems. In the evaluation of endocrine disrupting activities, the H295R cell bioassay should be used in combination with other genomic, biological, chemical, and hydrological tests to establish viable modes for endocrine disruption and identify compounds responsible for the observed effects.

The H295R cell bioassay was used to evaluate the potential endocrine disrupting effects of 18 of the most commonly used pharmaceuticals in the United States. Exposures for 48 h with single pharmaceuticals and binary mixtures were conducted; the expression of five steroidogenic genes, 3betaHSD2, CYP11beta1, CYP11beta2, CYP17 and CYP19, was quantified by Q-RT-PCR. Production of the steroid hormones estradiol (E2), testosterone (T) and progesterone (P) was also evaluated. Antibiotics were shown to modulate gene expression and hormone production. Amoxicillin up-regulated the expression of CYP11beta2 and CYP19 by more than 2-fold and induced estradiol production up to almost 3-fold. Erythromycin significantly increased CYP11beta2 expression and the production of P and E2 by 3.5- and 2.4-fold, respectively, while production of T was significantly decreased. The beta-blocker salbutamol caused the greatest induction of CYP17, more than 13-fold, and significantly decreased E2 production. The binary mixture of cyproterone and salbutamol significantly down-regulated expression of CYP19, while a mixture of ethynylestradiol and trenbolone, increased E2 production 3.7-fold. Estradiol production was significantly affected by changes in concentrations of trenbolone, cyproterone, and ethynylestradiol. Exposures with individual pharmaceuticals showed the possible secondary effects that drugs may exert on steroid production. Results from binary mixture exposures suggested the possible type of interactions that may occur between drugs and the joint effects product of such interactions. Dose-response results indicated that although two chemicals may share a common mechanism of action the concentration effects observed may be significantly different.

Goals and Scope. In response to concerns that have been raised about chemical substances that may alter the function of endocrine systems and result in adverse effects on human health, an OECD initiative was undertaken to develop and validate in vitro and in vivo assays to identify chemicals that may interfere with endocrine systems of vertebrates. Here we report on studies that were conducted to develop and standardize a cell-based screening assay using the H295R cell line to prioritize chemicals that may act on steroidogenic processes in humans and wildlife. These studies are currently ongoing as part of the 'Special Activity on the Testing and Assessment of Endocrine Disruptors' within the OECD Test Guidelines Program to review, develop, standardize, and validate a number of in vitro and in vivo toxicological assays for testing and assessment of chemicals concerning their potential to interact with the endocrine system of vertebrates. -: Study Design. Six laboratories from five countries participated in the pre-validation studies. Each laboratory tested the effects of three model chemicals on the production of testosterone (T) and estradiol (E2) using the H295R Steroidogenesis Assay. Chemicals tested were well described inducers or inhibitors of steroidogenic pathways (forskolin, prochloraz and fadrozole). All experiments were conducted in 24 well plates following standard protocols. Six different doses per compound were analyzed in triplicate per plate. A quality control (QC) plate was run in conjunction with the chemical exposure plate to account for inter-assay variation. Each chemical exposure was conducted two or three times.All laboratories successfully detected increases and/or decreases in hormone production by H295R cells after exposure to the different model compounds and there was good agreement in the pattern of response for all groups. Forskolin increased both T and E2 while fadrozole and prochloraz decreased production of both hormones. All chemicals affected hormone production in a dose-response manner with the exception of fadrozole which caused maximum inhibition of E2 at the two least concentrations tested. Some inter-laboratory differences were noted in the alteration of hormone production measured in chemically exposed cells. However, with the exception of the production of T measured at one laboratory in cells exposed to forskolin, the EC50s calculated were comparable (coefficients of variation 34-49%) for all hormones.and Perspectives. The results indicated that the H295R Steroidogenesis Assay protocol was robust, transferable and reproducible across all laboratories. However, in several instances that were primarily related to one laboratory there were unexplained minor uncertainties related to the inter-laboratory hormone production variation. Based on the findings from this Phase 2 pre-validation study, the H295R Steroidogenesis Assay protocol is currently being refined. The next phase of the OECD validation program will test the refined protocol across the same group of laboratories using an extended set of chemicals (~30) that will include positive and negative chemical controls as well as a broad spectrum of different potential inducers and inhibitors of steroidogenic pathways.

The present studies were undertaken to evaluate the utility of the H295R system as an in vitro assay to assess the potential of chemicals to modulate steroidogenesis. The effects of four model chemicals on the expression of ten steroidogenic genes and on the production of three steroid hormones were examined. Exposures with individual model chemicals as well as binary mixtures were conducted. Although the responses reflect the known mode of action of the various compounds, the results show that designating a chemical as "specific inducer or inhibitor" is unwise. Not all changes in the mixture exposures could be predicted based on results from individual chemical exposures. Hormone production was not always directly related to gene expression. The H295R system integrates the effects of direct-acting hormone agonists and antagonists as well as chemicals affecting signal transduction pathways for steroid production and provides data on both gene expression and hormone secretion which makes this cell line a valuable tool to examine effects of chemicals on steroidogenesis.

The novel brominated flame retardants (NBFRs), 2-ethylhexyl-2,3,4,5-tetrabromobenzoate (TBB), Bis(2-ethylhexyl)-2,3,4,5-tetrabromophtalate (TBPH), and 1,2,5,6-tetrabromocyclooctane (TBCO) are components of flame retardant mixtures including Firemaster 550 and Saytex BC-48. Despite the detection of these NBFRs in environmental and biotic matrices, studies regarding their toxicological effects are poorly represented in the literature. The present study examined endocrine disruption by these three NBFRs using the yeast YES/YAS reporter assay and the mammalian H295R steroidogenesis assay. Activation of the aryl hydrocarbon receptor (AhR) was also assessed using the H4IIE reporter assay. The NBFRs produced no TCDD-like effects in the H4IIE assay or agonistic effects in the YES/YAS assays. TBB produced a maximal antiestrogenic effect of 62% at 0.5mgL(-1) in the YES assay while TBPH and TBCO produced maximal antiandrogenic effects of 74% and 59% at 300mgL(-1) and 1500mgL(-1), respectively, in the YAS assay. Significant effects were also observed in the H295R assay. At 0.05mgL(-1), 15mgL(-1), and 15mgL(-1) TBB, TBPH, and TBCO exposures, respectively resulted in a 2.8-fold, 5.4-fold, and 3.3-fold increase in concentrations of E2. This is one of the first studies to demonstrate the in vitro endocrine disrupting potentials of TBB, TBPH, and TBCO.

Exposure to estrogens during the period of sexual differentiation is known to adversely affect the development of testes in African clawed frogs (Xenopus laevis), but little is known about molecular changes that coincide with the development of altered phenotypes. Therefore, the transcriptome-level effects of exposure to 17α-ethynylestradiol (EE2) during sexual differentiation of X. laevis were evaluated by use of Illumina sequencing coupled with RNA-Seq expression analysis. Overall, a number of processes were affected by 17α-ethynylestradiol, including steroid biosynthesis, thyroid hormone signaling and metabolism, testicular development, and spermatogenesis. Some of the altered pathways, such as thyroid hormone signaling and testicular development, could be linked with biological effects on metamorphosis and gonadal phenotypes, respectively, that were observed in frogs that were exposed to 17α-ethynylestradiol throughout metamorphosis and the early postmetamorphic period. Thus, early changes at the transcriptome-level were predictive of pathologies that did not manifest until later in development. To validate the quantitative capacity of RNA-Seq, a subset of transcripts identified to have altered abundances in individuals exposed to 17α-ethynylestradiol was also evaluated by use of quantitative polymerase chain reaction (qPCR). While small sample sizes (n = 3) limited the ability to draw conclusions pertaining to differences in qPCR-derived abundances of transcripts between control and exposed tadpoles, there was a significant relationship (r(2) = 0.78) between fold-changes for RNA-Seq and qPCR.

PLoS one 8(10), e75596 (2013). doi:10.1371/journal.pone.0075596, Published by PLoS [u.a.], Lawrence, Kan.

Populations of amphibians are in decline in certain locations around the world, and the possible contribution of environmental contaminants, including estrogenic compounds, to these declines is of potential concern. In the current study, responses of the wood frog (Lithobates sylvaticus) to exposure to 17α-ethynylestradiol (EE2), the synthetic estrogen used in oral contraceptives, during the larval period were characterized. Exposure of L. sylvaticus to 1.08, 9.55, or 80.9 μg EE2/L had no effects on survival, growth, or metamorphic endpoints monitored in the current study. However, there were significant effects of exposure to EE2 on phenotypic sex ratios. In general, lesser proportions of L. sylvaticus developed as phenotypic males and greater proportions developed as phenotypic females or with mixed sex phenotypes at all concentrations of EE2 tested. Utilizing the data collected in the current study, the EC(50) for complete feminization of L. sylvaticus was determined to be 7.7 μg EE2/L, and the EC(50) for partial feminization was determined to be 2.3 μg EE2/L. In addition, after chronic exposure, abundances of transcripts of vitellogenin A2, high density lipoprotein binding protein, and 7-dehydrocholesterol reductase were 1.8-280-fold greater in livers from L. sylvaticus exposed to EE2 compared to controls. Overall, there were significant effects of exposure to all concentrations of EE2 tested, the least of which was within about 2-fold of estrogen equivalent concentrations previously measured in the environment.

There is increasing concern over the risk of environmentally relevant doses of bisphenol A (BPA) on human endocrine systems. Effects of BPA on steroidogenesis and the related molecular mechanisms were investigated in H295R human adenocarcinoma cells. This immortal cell line is unique in expressing all the enzymes of the steroidogenic pathways. The effects of BPA on steroidogenesis, 17β-estradiol (E2) metabolism, and aromatase activity were examined in H295R cells exposed to BPA from 3.0 × 10(-1) to 3.0 × 10(3) ng/ml. Concentrations of BPA in basic cell culture materials were verified. Stable CYP17A-knockdown H295R cells were developed to verify the mechanism of inhibited steroidogenesis by BPA. Background concentrations of BPA in control cell culture media ranged from 0.03 to 0.38 ng/ml. Significantly lesser concentrations of androstenedione, testosterone, cortisol, and cortisone were caused by exposure to 30-3000 ng BPA/ml. In contrast, sconcentrations of estrone (E1) and E2 were significantly greater in BPA-exposed H295R cells. Lesser production of androstenedione and testosterone by H295R cells exposed to BPA was the most sensitive endpoint (no observable effect concentrations < 30 ng BPA/ml). CYP17A knockdown in H295R cells resulted in less production of both 17α hydroxyprogesterone and androstenedione. The results are consistent with the hypothesis that in H295R cells, BPA selectively inhibits 17,20-lyase but not 17α-hydroxylase. The primary mechanism causing increased E2 in the medium was inhibition of E2 metabolism rather than greater aromatase (CYP19) activity. These results suggest that BPA has the potential to interfere with cellular steroidogenesis in humans through multiple molecular mechanisms.

Concentration-dependent response relationships provide essential information on the characteristics of chemical-induced effects on toxicological end points, which include effect (inhibition or induction), potency, and efficacy of the chemical. Recent developments in systems biology and high throughputtechnologies have allowed simultaneous examination of many chemicals at multiple end point levels. While this increase in the quantity of information generated offers great potential, it also poses a significant challenge to environmental scientists to efficiently manage and interpret these large data sets. Here we present a novel method, ToxClust, that allows clustering of chemicals on the basis of concentration-response data derived with single or multiple end points. This method utilizes a least distance-searching algorithm (LDSA) to measure the pattern dissimilarity of concentration-response curves between chemicals and their relative toxic potency. ToxClust was tested using simulated data and chemical test data collected from the human H295R cell-based in vitro steroidogenesis assay. ToxClust effectively identified similar patterns of simulated data and responses to the exposure with the five model chemicals and separated them into different groups on the basis of their dissimilarities. These observations demonstrate that ToxClust not only provides an effective data analysis and visualization tool, but also has value in hypothesis generation and mechanism-based chemical classification.

Chlorinated and brominated polycyclic aromatic hydrocarbons (CIPAHs and BrPAHs) occur as pollutants in the environment. Nevertheless, there is little information available regarding the toxic effects of CIPAHs and BrPAHs. The potencies of 19 individual ClPAHs and 11 individual BrPAHs to induce aryl hydrocarbon receptor (AhR)-mediated activities (i.e., dioxin-like toxicity), relative to the potency of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), were determined in vitro by use of a recombinant rat hepatoma cell (H4IIE-luc) assay. Several CIPAHs elicited AhR-mediated activity; the relative potencies (RePs) of 6-monochlorochrysene, and 7-monochlorobenz[a]anthracene were 2.6 x 10(-5) and 6.3 x 10(-6), respectively. Among BrPAHs, 7-monobromobenz[a]anthracene and 4,7-dibromobenz[a]anthracene had the highest RePs, 2.1 x 10(-5) and 2.3 x 10(-5), respectively. None of the chlorinated or brominated anthracene or fluorene compounds elicited AhR-mediated activity atthe concentrations tested. We developed a structure-activity relationship for AhR-mediated potencies of CIPAHs.The RePs of ClPhe and ClFlu (low-molecular-weight ClPAHs) were directly proportional to the compounds' degrees of chlorination. The RePs of higher-molecular-weight ClPAHs (or = 4-rings) were lower than those of the corresponding parent PAHs. The RePs of BrPAHs were higher than the RePs of the corresponding ClPAHs. For instance, 6-BrBaP was more potent than 6-ClBaP and 7-BrBaA was more potent than 7-ClBaA. The RePs determined in this study were applied to literature concentrations of Cl- and Br-PAHs in environmental samples, to calculate dioxin-like toxicities, as toxic equivalents (TEQs). The TEQs of ClPAHs calculated using the concentrations of individual ClPAHs were 4.6 pg-TEQ/g in fly ash, 0.015 fg-TEQ/m3 in automobile exhaust, and 0.085 fg-TEQ/m3 in urban air. 6-ClChr accounted for 80% of the total ClPAHs-TEQs in fly ash. This is the first in vitro study to report AhR-mediated activities of Cl- and Br-PAHs relative to the activity of TCDD.

Dose- and time-dependent effects of environmentally relevant concentrations of 2,3,7,8-tetrachlorodibenzo-p-dioxin equivalents (TEQ) of 2,3,7,8-tetrachlorodibenzofuran (TCDF), 2,3,4,7,8-pentachlorodibenzofuran (PeCDF), or a mixture of these two congeners on hepatic P450 enzyme activity and tissue morphology, including jaw histology, of adult ranch mink were determined under controlled conditions. Adult female ranch mink were fed either TCDF (0.98, 3.8, or 20 ng TEQ(TCDF)/kg bw/day) or PeCDF (0.62, 2.2, or 9.5 ng TEQ(PeCDF)/kg bw/day), or a mixture of TCDF and PeCDF (4.1 ng TEQ(TCDF)/kg bw/day and 2.8 ng TEQ(PeCDF)/kg bw/day, respectively) for 180 days. Doses used in this study were approximately eight times greater than those reported in a parallel field study. Activities of the cytochrome P450 1A enzymes, ethoxyresorufin O-deethylase (EROD) and methoxyresorufin O-deethylase (MROD) were significantly greater in livers of mink exposed to TCDF, PeCDF, and a mixture of the two congeners; however, there were no significant histological or morphological effects observed. It was determined that EROD and MROD activity can be used as sensitive biomarkers of exposure to PeCDF and TCDF in adult female mink; however, under the conditions of this study, the response of EROD/MROD induction occurred at doses that were less than those required to cause histological or morphological changes.

An in vitro steroidogenesis assay using H295R human adenocarcinoma cells has been suggested as a possible alternative to gonad explant assays for use as a Tier I screening assay to detect endocrine active chemicals capable of modulating steroid hormone synthesis. This study is one of the first to investigate the utility of the H295R assay for predicting effects and/or understanding mechanisms of action across species and tissues. Six chemicals, including one selective aromatase inhibitor (fadrozole), four fungicides (fenarimol, ketoconazole, prochloraz, and vinclozolin), and one herbicide (prometon), were tested in both the H295R steroidogenesis assay, and an in vitro steroidogenesis assay using fathead minnow ovary explants. All six chemicals caused significant alterations in 17beta-estradiol (E2) and/or testosterone (T) production in vitro. Effects of ketoconazole, prochloraz, and prometon were similar in both assays. However, there were differences in the profile of responses for T for fadrozole and fenarimol, and for T and E2 for vinclozolin. In terms of sensitivity, steroid production in the H295R assay was most sensitive for detecting the effects of fadrozole, fenarimol, and prochloraz, but was less sensitive than the fathead minnow ovary explant assay to the effects of ketoconazole and vinclozolin. The H295R assay was consistently less variable (among replicates) than the fathead minnow ovary explant assay. However, the ovary explant assay was more predictive of in vivo effects of the six chemicals on fathead minnows than the H295R system. Further characterization of autoregulatory capacities, interaction of steroid-hormone receptor pathways with steroidogenesis, and metabolic capabilities of each system are needed for either system to provide clear and informative insights regarding a chemical's mechanism of action. Overall, however, results of this study suggest that both the H295R and fathead minnow ovary explant assays have utility for identifying endocrine-active chemicals in screening-type applications.

To identify and prioritize chemicals that may alter steroidogenesis, an in vitro screening assay based on measuring alterations in hormone production was developed using the H295R human adrenocortical carcinoma cell line. Previous studies indicated that this cell line was useful to screen for effects on gene expression of steroidogenic enzymes. This study extended that work to measure the integrated response on production of testosterone (T), estradiol (E2), and progesterone/pregnenolone (P) using an ELISA. Under optimized culture and experimental conditions, the basal release of P, T and E2 into the medium was 7.0 ± 1.2 ng/ml, 1.6 ± 0.4 ng/ml, and 0.51 ± 0.13 ng/ml, respectively. Model chemicals with different modes of action on steroidogenic systems were tested. Exposure to forskolin resulted in dose-dependent increases in all three hormones with the greatest relative increase being observed for E2. This differed from cells exposed to prochloraz or ketoconazole where P concentrations increased while T and E2 concentrations decreased in a dose-dependent manner. In cells exposed to fadrozole, E2 decreased in a dose-dependent manner while T and P only decreased at the greatest dose tested. Aminoglutethimide decreased P and E2 concentrations but increased T concentrations. Vinclozolin reduced both P and T but resulted in a slight increase in E2. The alteration in the patterns of hormone production in the H295R assay was consistent with the modes of action of the chemicals and was also consistent with observed effects of these chemicals in animal models. Based on these results, the H295R in vitro system has potential for high throughput screening to not only characterize the effects of chemicals on endocrine systems but also to prioritize chemicals for additional testing.

The triazine herbicide atrazine has been hypothesized to disrupt sexual development in frogs by up-regulating aromatase activity, resulting in greater estradiol (E2) concentrations and causing feminization in males. The goal of this study was to collect native ranid frogs from atrazine-exposed ponds and determine whether relationships exist between measured atrazine concentrations and the gonadosomatic index (GSI), plasma concentrations of testosterone (T), E2 or 11-ketotestosterone (KT), or with aromatase activity. In the summer of 2002 and 2003, adult and juvenile green frogs (Rana clamitans), bullfrogs (R. catesbeiana) and Northern leopard frogs (R. pipiens) were collected from areas with extensive corn cultivation and areas where there was little agricultural activity in south-central Michigan. Atrazine concentrations were below the limit of quantification at non-agricultural sites. Atrazine concentrations did not exceed 2 microg/L at most agricultural sites, but a concentration of 250 microg atrazine/L was measured in one sample from one site in 2002. Plasma steroid concentrations varied among locations. Aromatase activity was measurable in less than 11% of testes in adult males, and in less than 4% of testes in juvenile males. Median aromatase activities in ovaries of adult females ranged from 3 to 245 pmol/h/mg protein, and maximum activities were 2.5-fold greater in juveniles than in adults. Atrazine concentrations were not significantly correlated with any of the parameters measured in this study. These results indicate that atrazine does not up-regulate aromatase in green frogs in the wild, and does not appear to affect plasma steroid hormone concentrations.

Effects of the non-steroidal aromatase inhibitor letrozole on ovarian function in cattle were determined. The hypothesisthat letrozole would arrest growth of thedominant follicle, resultinginemergence ofa new follicular waveat a predictable post-treatment interval, was tested. Heifers were assigned randomly to four groups 4 days after follicular ablation (,2½ days after wave emergence) and given intravenous doses of 500 (n ¼9), 250 (n ¼10), or 125mgkg � 1 (n ¼10) letrozole or phosphate-buffered saline (controls; n ¼10). Blood was collected and ovarian structures were monitored daily by transrectal ultrasonography. Plasma concentrations of LH and FSH were measured by radioimmuno- assay; plasma concentrations of letrozole were determined by high-performance liquid chromatography tandem mass spectrometry. A single intravenous dose of letrozole did not induce regression of the dominant follicle present at the time of treatment, nor did it directly affect FSH release. Conversely, treatment with letrozole increased endogenous concentrations of LH and extended the lifespan of the dominant follicle, which delayed the next FSH surge and subsequent follicular wave emergence. Letrozole continues to have potential as a non-steroidal treatment for controlling ovarian function in cattle. Additional keywords: bovine reproduction, oestradiol, ovarian synchronisation.