The OECD program to validate the rat uterotrophic bioassay to screen compounds for in vivo estrogenic responses: phase 1

The Organisation for Economic Co-operation and Development has completed the first phase of an international validation program for the rodent uterotrophic bioassay. This uterotrophic bioassay is intended to identify the in vivo activity of compounds that are suspected agonists or antagonists of estrogen. This information could, for example, be used to help prioritize positive compounds for further testing. Using draft protocols, we tested and compared two model systems, the immature female rat and the adult ovariectomized rat. Data from 19 participating laboratories using a high-potency reference agonist, ethinyl estradiol (EE), and an antagonist, ZM 189,154, indicate no substantive performance differences between models. All laboratories and all protocols successfully detected increases in uterine weights using EE in phase 1. These significant uterine weight increases were achieved under a variety of experimental conditions (e.g., strain, diet, housing protocol, bedding, vehicle). For each protocol, there was generally good agreement among laboratories with regard to the actual EE doses both in producing the first significant increase in uterine weights and achieving the maximum uterine response. Furthermore, the Hill equation appears to model the dose response satisfactorily and indicates general agreement based on calculated effective dose (ED)(10) and ED(50) within and among laboratories. The feasibility of an antagonist assay was also successfully demonstrated. Therefore, both models appear robust, reproducible, and transferable across laboratories for high-potency estrogen agonists such as EE. For the next phase of the OECD validation program, both models will be tested against a battery of weak, partial estrogen agonists.     

Biological measurement of estrogenic activity in urine and bile conjugates with the in vitro ER-CALUX reporter gene assay

Although estrogens are excreted as biologically inactive conjugates, they can be reconverted to an active form, possibly by bacteria. A simple method was developed to deconjugate estrogen metabolites present in human urine and fish bile back to active estrogens by enzymatic hydrolysis with beta-glucuronidase or live Escherichia coli cells. Deconjugated extracts were tested for estrogenic activity in the in vitro stable estrogen receptor-mediated chemical-activated luciferase gene expression (ER-CALUX) assay. Estrogen glucuronides in urine obtained from human males and females were effectively converted to active forms after incubation with beta-glucuronidase or E. coli. The highest estrogenic activity was found in deconjugated metabolites from urine of a pregnant woman, in which levels up to 3,000 nmol estradiol equivalents per liter of urine were found after overnight incubation of urine with E. coli. Bile sampled from male bream and flounder from various freshwater and marine locations was also deconjugated and a good correlation was found between high biliary estrogenic activity and elevated levels of xenoestrogenic activity in surface water as well as in plasma vitellogenin. Therefore, the measurement of deconjugated bile could form a useful (indirect) biomarker for internal dose of xenoestrogens in male fish.     

A case of a laboratory animal feed with high estrogenic activity and its impact on in vivo responses to exogenously administered estrogens.

We recently noted that immature rats failed to exhibit a normal uterine response to exogenously administered estradiol as assessed by both biochemical (induction of gene expression) and morphological (altered uterine and vaginal histology and size) end points. An initial analysis suggested that this was due to a high degree of estrogenization from a dietary source which was producing a near maximal uterotrophic response prior to hormone treatment. Subsequent chemical analysis indicated that the feed in question contained high amounts of two well-known phytoestrogens, genistein (210 mg/kg) and daidzen (14 mg/kg), and the lot of feed in question produced a large uterotrophic effect when fed to immature ovariectomized rats. These findings illustrate that, despite increased awareness of phytoestrogens, some batches of animal feed contain very high amounts of estrogenic components which have marked effects on in vivo end points of hormone action. These observations have important implications for both basic research and screening methods that utilize in vivo approaches.     

AH receptor agonist activity in human blood measured with a cell-based bioassay: evidence for naturally occurring AH receptor ligands in vivo

In the present study, an aryl hydrocarbon receptor (AHR)-driven reporter gene bioassay was used to measure the activity, measured as an induction equivalent (IEQ) as compared to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), or IEQ concentration in human blood samples from 10 volunteers under different dietary regimens. Blood concentrations of polychlorinated dibenzo-p-dioxins/furans (PCDD/Fs) and polychlorinated biphenyls (PCBs), as determined by analytical chemistry (HR-GC/MS), and expressed as toxic equivalents (TEQs) with the use of TCDD equivalency factors (TEFs), were within a range that has been reported in the general US population, ranging from 0.022 to 0.119 ppt (whole blood basis). However, the human blood IEQ measured directly via bioassay ranged from 13.4 to 218 ppt (whole blood basis). These order of magnitude greater IEQs compared to the TEQs for dioxins, furans, and certain PCBs suggests that human blood contains a relatively high level of AHR agonists able to activate the CYP1A1 dioxin response element (DRE)-linked reporter gene bioassay and that this AHR activity is not accounted for by PCDDs/Fs and dioxin-like PCBs based on standard HR-GC/MS and TEF analysis. When study participants switched from a "baseline" to a high-vegetable diet, increases in bioassay IEQ were observed that were statistically significant (P<0.05). In addition, IEQ activity was elevated above levels observed following dietary intervention in two subjects given indole-3-carbinol (I3C) supplements. We conclude that a substantial portion of the IEQ activity occurred as a result of the increased intake of natural AHR agonists (NAHRAs) present in many fruits, vegetables. and herbs. Our findings also suggest that dietary NAHRAs constitute a substantial daily dietary intake of AHR-active compounds, and these NAHRAs could influence AHR status in humans and play a role in a basal level of AHR activation.     

Enzymatic and estrogenic responses in fish exposed to organic pollutants in the New York-New Jersey (USA) Harbor Complex

This study examines biochemical and hormonal responses in resident and migratory fish from the New York-New Jersey (USA) Harbor Complex (NY-NJHC) and those treated with sediment-associated organic contaminants. Following laboratory exposures to organic extracts of NY-NJHC sediments (injection), livers from adult male mummichogs, Fundulus heteroclitus, were analyzed for vitellogenin (VTG), cytochrome P4501A (CYPIA), CYP3A, and estradiol 2-hydroxylase (E2OHase) and ethoxyresorufin-O-deethylase (EROD) activities. Levels of CYP1A (311-391% of control) and EROD (267-361% of control) were elevated in mummichogs exposed to high doses of sediment extracts, while VTG, CYP3A, and E2OHase were unaffected. In field studies, reproductively mature male mummichogs collected from a highly contaminated area, Newark Bay (NJ, USA), did not have detectable levels of VTG but did exhibit elevated levels of CYP1A and EROD. Vitellogenin was also not detected in juvenile striped bass (Morone saxatilis) collected from the main stem of the lower Hudson River (NY, USA). Similar to results in the sediment extract-treated fish. CYP3A and E2OHase were unaltered in Newark Bay F. heteroclitus. The lack of response of CYP3A and E2OHase activities to contaminant mixtures, either environment or sediment derived, suggests that compounds in these mixtures either do not alter these enzymes, produce antagonistic effects in mixtures, are present at ineffective concentrations, or are regulated in a species-specific manner.     

Screening of environmental contaminants for ecdysteroid agonist and antagonist activity using the Drosophila melanogaster B(II) cell in vitro assay

The B(II) bioassay was developed as a rapid and reliable tool for detecting potential insect growth regulators acting as ecdysteroid receptor (ant)agonists. Based on an ecdysteroid-responsive cell line from Drosophila melanogaster, this microplate assay is ideally suited to the evaluation of environmental contaminants as potential endocrine disrupters. Data are presented for about 80 potential environmental contaminants, including industrial chemicals, pesticides, pharmaceuticals, phytoestrogens, and vertebrate steroids, and are compared with data for known (ant)agonists. Apart from androst-4-ene-3,17-dione (a weak antagonist), vertebrate steroids were inactive at concentrations up to 10(-3) M. The vast majority of xenobiotics also showed no (ant)agonist activity. Among the industrial chemicals, antagonistic activity was observed for bisphenol A median effective concentration (EC50) of 1.0 x 10(-4) M and diethylphthalate (EC50 of 2.0 x 10(-3) M). Some organochlorine compounds also showed weak antagonistic activity, including o,p'-dichlorodiphenyldichloroethylene (DDE), p,p'-DDE, dieldrin, and lindane (EC50 of 3.0 x 10(-5) M). For lindane, bisphenol A, and diethylphthalate, activity is not associated with impurities in the samples and, for lindane and bisphenol A at least, the compounds are able to compete with ecdysteroids for the ligand binding site on the receptor complex, albeit at concentrations very much higher than those found in the environment. The only pharmaceutical showing any detectable antagonist activity was 17alpha-ethynylestradiol. In the context of recent publications on potential endocrine disruption in marine and freshwater arthropods, these findings suggest that, for some compounds (e.g., diethylstilbestrol), ecdysteroid receptor-mediated responses are unlikely to be involved in producing chronic effects. The B(II) assay has a potentially valuable role to play in distinguishing between endocrine-mediated, which normally occur at submicromolar concentrations, and pharmacological effects in insects and crustaceans.     

Contaminant accumulation and biomarker responses in caged fish exposed to effluents from anthropogenic sources in the Karnaphuly River, Bangladesh

The possible ecotoxicological effects of a paper and pulp mill effluent were investigated by measuring selected contaminants and biomarkers in caged Nile tilapia (Oreochromis niloticus) and sharptooth catfish (Clarias gariepinus) from the Karnaphuly River, Bangladesh. Fish were caged for 28 d at two upstream reference sites (2 and 4 km) and four downstream sites (3, 5, 8, and 12 km) from the effluent outlets. Organochlorine contaminants and bile biomarkers were bioaccumulated to higher levels at downstream polluted sites than at the reference sites, including hexachlorobenzene (HCB), hexachlorocyclohexane (HCH), polychlorinated biphenyls (PCBs) in liver, and chlorophenolic compounds like 2,4-dichlorophenol and 2,4,6-trichlorophenol in bile. Levels of glucose, protein, and aspartate aminotransferase were analyzed in plasma, whereas cytochrome P4501A (CYP1A) levels were determined in S-12 supernatants of liver. The results, including CYP1A induction and bile biomarkers, clearly indicated that, in addition to the paper and pulp mill effluents, the downstream sites appear to receive other inputs of contaminants. This field assessment in a Bangladeshi river demonstrates biomarker measurements in caged fish as a promising approach for evaluating accumulation and effects of industrial effluents in fish of developing countries.     

The OECD program to validate the rat Hershberger bioassay to screen compounds for in vivo androgen and antiandrogen responses: phase 2 dose-response studies

OBJECTIVE: The Organisation for Economic Co-operation and Development (OECD) has completed phase 2 of an international program to validate the rodent Hershberger bioassay. DESIGN: The Hershberger bioassay is designed to identify suspected androgens and antiandrogens based on changes in the weights of five androgen-responsive tissues (ventral prostate, paired seminal vesicles and coagulating glands, the levator ani and bulbocavernosus muscles, the glans penis, and paired Cowper's or bulbourethral glands). Protocol sensitivity and reproducibility were tested using two androgen agonists (17alpha-methyl testosterone and 17beta-trenbolone), four antagonists [procymi-done, vinclozolin, linuron, and 1,1-dichoro-2,2-bis-(p-chlorophenyl)ethylene (p,p'-DDE)], and a 5alpha-reductase inhibitor (finasteride). Sixteen laboratories from seven countries participated in phase 2. RESULTS: In 40 of 41 studies, the laboratories successfully detected substance-related weight changes in one or more tissues. The one exception was with the weakest antiandrogen, linuron, in a laboratory with reduced sensitivity because of high coefficients of variation in all tissue weights. The protocols performed well under different experimental conditions (e.g., strain, diet, housing protocol, bedding, vehicle). There was good agreement and reproducibility among laboratories with regard to the lowest dose inducing significant effects on tissue weights. CONCLUSIONS: The results show that the OECD Hershberger bioassay protocol is reproducible and transferable across laboratories with androgen agonists, weak androgen antagonists, and a 5alpha-reductase inhibitor. The next validation phase will employ coded test substances, including positive substances and negative substances having no androgenic or antiandrogenic activity.     

Comparison of the sensitivities of common in vitro and in vivo assays of estrogenic activity: application of chemical toxicity distributions

A number of contaminants in municipal effluent discharges are estrogen agonists to fish. Whereas several in vitro and in vivo techniques have been developed to assess the estrogenic activity of these compounds or ambient environmental samples, previous comparisons of the relative sensitivities of these approaches remain inconclusive. We employed a probabilistic hazard assessment approach using chemical toxicity distributions (CTDs) to perform a novel evaluation of relative sensitivities of six common in vitro and in vivo assays. We predicted that there was an 8.3% (human breast ademocarcinoma cell line, MCF-7, assay), 6.3% (yeast estrogen screen assay), or 1.9% (fish hepatocyte vitellogenin, VTG, assay) probability of detecting a compound in aquatic systems that will elicit an estrogenic response at concentrations at or below 0.1 microg/L, suggesting that the MCF-7 assay was the most sensitive in vitro assay evaluated in this study. The probabilities of eliciting the estrogenic response of VTG induction at a concentration less than 0.1 microg/L in rainbow trout, fathead minnow, and Japanese medaka were determined at 29.9, 26.2, and 18.8%, respectively. Thus, rainbow trout VTG induction was the most sensitive in vivo assay assessed. Subsequently, CTDs may provide a useful technique for hazard assessment of chemical classes for which exposure data are limited and for chemicals with common toxicological mechanisms and modes of action.     

Phenotypic and functional characterization of ex vivo T cell responses to the live attenuated herpes zoster vaccine

To define the phenotypic characteristics and kinetics of T cell responses to a shingles vaccine in elderly individuals, 20 subjects > or =60 years of age received two doses of vaccine or placebo 6 weeks apart. VZV-specific T cell phenotypes and intracellular cytokines were determined by flow cytometry on blood mononuclear cells obtained pre-vaccination and up to 6 months after the second immunization. Results were compared with responses of five unvaccinated young adults. Pre-vaccination, elderly individuals had significantly lower VZV-specific effectors and cytokine-producing T cells compared with young adults. The vaccine significantly increased VZV-specific Th1, memory, early effector, and cutaneous homing receptor-bearing T cells.     

Equilibrium sampling of environmental pollutants in fish: comparison with lipid-normalized concentrations and homogenization effects on chemical activity

Equilibrium sampling of organic pollutants into the silicone polydimethylsiloxane (PDMS) has recently been applied in biological tissues including fish. Pollutant concentrations in PDMS can then be multiplied with lipid/PDMS distribution coefficients (D(Lipid,PDMS) ) to obtain concentrations in fish lipids. In the present study, PDMS thin films were used for equilibrium sampling of polychlorinated biphenyls (PCBs) in intact tissue of two eels and one salmon. A classical exhaustive extraction technique to determine lipid-normalized PCB concentrations, which assigns the body burden of the chemical to the lipid fraction of the fish, was additionally applied. Lipid-based PCB concentrations obtained by equilibrium sampling were 85 to 106% (Norwegian Atlantic salmon), 108 to 128% (Baltic Sea eel), and 51 to 83% (Finnish lake eel) of those determined using total extraction. This supports the validity of the equilibrium sampling technique, while at the same time confirming that the fugacity capacity of these lipid-rich tissues for PCBs was dominated by the lipid fraction. Equilibrium sampling was also applied to homogenates of the same fish tissues. The PCB concentrations in the PDMS were 1.2 to 2.0 times higher in the homogenates (statistically significant in 18 of 21 cases, p < 0.05), indicating that homogenization increased the chemical activity of the PCBs and decreased the fugacity capacity of the tissue. This observation has implications for equilibrium sampling and partition coefficients determined using tissue homogenates.     

Comet assay with the fish cell line rainbow trout gonad-2 for in vitro genotoxicity testing of xenobiotics and surface waters

The present study examines the potential of the comet assay using the rainbow trout gonad cell line-2 (RTG-2) as an in vitro indicator test for genotoxicity assessment of aquatic contaminants and native surface waters. Initially, the comet assay protocol was adapted to the RTG-2 cell line. An exposure period of 2 h was found to be optimal, because DNA damage decreased when exposure was prolonged. Then, the sensitivity of the comet assay with RTG-2 cells toward six genotoxic reference substances was evaluated. The lowest-observed-effect concentration values for the directly acting genotoxins, 4-nitroquinoline-N-oxide and N-methyl-N'-nitro-N-nitrosoguanidine, were in the low nanomolar range. The RTG-2 test system clearly was less sensitive for the indirectly acting genotoxins benzo[a]pyrene, nitrofurantoin, 2-acetylaminofluorene, and dimethylnitrosamine, despite the presence of xenobiotic metabolic capacities in RTG-2 cells. The two effect endpoints used, tail length (TL) and tail moment (TM), did not differ with respect to sensitivity, but the linearity of the concentration-response curve was better with TM than with TL. The overall reproducibility of the assay results was good. Finally, the applicability of the comet assay with RTG-2 cells for genotoxicity screening of native surface water samples was studied. The assay tolerated the use of nonsterile water samples and was able to detect genotoxic potentials in native water samples; that is, extraction and concentration of the samples were not needed. The results of the present study indicate the suitability of the comet assay with the fish cell line, RTG-2, as in vitro screen for detecting genotoxic potencies of xenobiotics and environmental samples.     

Quantitative assay for the transforming DNA activity in cell culture products for human use

DNA isolates from a number of established cell lines have been tested for their transforming capacity in the NIH3T3 transformation assay. It has been found that this assay can be used to quantify the amount of transforming DNA at a submicrogram to milligram level. This assay system has been applied to validate the reduction of transforming DNA in various purification steps of cell culture products. The NIH3T3 assay system has been shown useful for in-process control and release of cell culture derived vaccines or therapeutics.     

A comparison of the rabbit scarification technique with titrations in cell cultures for the potency assay of smallpox vaccine

Effective tissue culture methods have been developed for the titration of smallpox vaccines, and it seemed appropriate to study their application as methods for assaying the potency of commercial smallpox vaccines. Thirty-four commercial smallpox vaccines were assayed for potency by titration in both rabbit and monkey kidney cell test systems and by the traditional rabbit scarification method.     

A plant life-cycle bioassay for contaminated soil,with comparison to other bioassays: Mercury and zinc

Bioassays using rapid-cycling plants allow measurement of multiple endpoints and assessment of impacts on both growth and reproduction. Selections of Brassica rapa develop rapidly in a broad range of soils and are very consistent in production of flower and seed. Their sensitivity to variation in growth conditions was investigated to define the variables that most affect performance. Yield differences between soils were substantial, indicating the need for careful selection and use of control treatments. The sensitivity to contaminants was investigated with applications of mercury (Hg) and zinc (Zn) to three soils. In a sand soil, bloom initiation was slowed by <10 mg Hg kg^–1 soil and <50 mg Zn kg^–1 soil. In contrast, lettuce emergence and earthworm survival were less sensitive to these metals in this soil. Survival of Daphnia magna and the Microtox^® assay in soil extracts were more sensitive to Hg than bloom initiation, but less sensitive to Zn. A similar relationship among the bioassays was observed for two finer-textured soils, although for these, effects were usually apparent only at soil metal concentrations >200 mg kg^–1. Enzyme assays were included for comparison, but were not sensitive to Hg contamination. Rapid-cycling B. rapa selections are suitable for routine bioassays, and are representative of several widely distributed and utilized species.     

Environmental exposure to endocrine disruptors with estrogenic activity and the association with pubertal disorders in children

Endocrine disruptors are exogenous substances with adverse health effects in intact organisms or their progeny, secondary to changes in endocrine function. Recent years have witnessed constant reports of environmental factors with hormone-like effects causing pubertal or reproductive abnormalities in animals. The few cases proven to be associated with pubertal disorders in humans have been related to accidental exposure. Nevertheless, pediatricians and parents recommend suspending all possible estrogen-contaminated food, especially meat (poultry, beef) and soy products, when the child presents with a pubertal disorder. These recommendations, if not scientifically sound, may have deleterious consequences by eliminating sources of dietary protein and possibly delaying the investigation of other potential and treatable causes. On the other hand, not investigating potential side effects of these products could have similar harmful effects. The current article describes the main endocrine disruptors associated with pubertal disorders in humans and concludes that except for accidental exposure to high doses, more research is needed on the effects of chronic and low-dose exposures in altering human pubertal development.     

Antigen-specific responses assessment for the evaluation of Bordetella pertussis T cell immunity in humans

Measurement of antigen-specific T cell responses is an adjunctive parameter to evaluate protection induced by a previous Bordetella pertussis infection or vaccination. The assessment of T cell responses is technically complex and usually performed on fresh peripheral blood mononuclear cells (PBMC). The objective of this study was to identify simplified methods to assess pertussis specific T cell responses and verify if these assays could be performed using frozen/thawed (frozen) PBMC. Three read-outs to measure proliferation were compared: the fluorescent dye 5,6-carboxylfluorescein diacetate succinimidyl ester (CFSE) dilution test, the number of blast cells defined by physical parameters, and the incorporation of (3)H-thymidine. The results of pertussis-specific assays performed on fresh PBMC were compared to the results on frozen PBMC from the same donor. High concordance was obtained when the results of CFSE and blast read-outs were compared, an encouraging result since blast analysis allows the identification of proliferating cells and does not require any use of radioactive tracer as well as any staining. The results obtained using fresh and frozen PBMC from the same donor in the different T cell assays, including IFNγ and TNFα cytokine production, did not show significant differences, suggesting that a careful cryopreservation process of PBMC would not significantly influence T cell response evaluation. Adopting blast analysis and frozen PBMC, the possibility to test T cell responses is simplified and might be applied in population studies, providing for new instruments to better define correlates of protection still elusive in pertussis.     

Dioxinlike components in incinerator fly ash: a comparison between chemical analysis data and results from a cell culture bioassay.

Potent polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), and dioxinlike polychlorinated biphenyls (PCBs) are among the most relevant toxic emissions from incinerators. Induction of cytochrome P450 1A1-catalyzed 7-ethoxyresorufin O-deethylase (EROD) activity in mammalian cell culture (EROD bioassay) is thought to be a selective and sensitive parameter used for the quantification of dioxinlike compounds. Fly ash extracts from municipal waste incinerators (MWI), a crematorium, wood combustors, and a noble metal recycling facility were analyzed in the EROD bioassay using rat hepatocytes in primary culture. Fractions containing 2,3,7,8-substituted PCDDs/PCDFs, dioxinlike PCBs, and 16 major polycyclic aromatic hydrocarbons (PAHs) were isolated from the extract and analyzed by gas chromatography-mass spectrometry (GC-MS) and by the EROD bioassay. It was found that with MWI samples the bioassay of the extract resulted in a two- to fivefold higher estimate of TCDD equivalents (TEQ) than the chemical analysis of PCDDs/PCDFs and PCBs. However, the outcome of both methods was significantly correlated, making the bioassay useful as a rough estimate for the sum of potent PCDDs/PCDFs and dioxinlike PCBs in extracts from MWI fly ash samples and in a fly ash sample from a crematorium. In noble metal recycling facility and wood combustor samples, higher amounts of PAHs were found, contributing to more pronounced differences between the results of both methods. The remaining unexplained inducing potency in fly ash samples probably results from additional dioxinlike components including certain PAHs not analyzed in this study.The hypothesis that emissions from MWI of hitherto unidentified dioxinlike compounds are higher by orders of magnitude than emissions of potent PCDDs/PCDFs and dioxinlike PCBs could not be confirmed. We found no indication for a marked synergistic interaction of dioxinlike fly ash components in the bioassay.     

An accelerated vaccine schedule with a poly-antigenic hepatitis C virus MVA-based candidate vaccine induces potent, long lasting and in vivo cross-reactive T cell responses

We designed and evaluated in HLA-class I transgenic mouse models a hepatitis C virus (HCV) T cell-based MVA vectored vaccine expressing three viral antigens known to be targets of potent CD8+- and CD4+-mediated responses. An accelerated (3 week-based) vaccination induced specific CD8+ T cells harboring two effector functions (cytolytic activity - both in vitro and in vivo- and production of IFNgamma) as well as specific CD4+ T cells recognizing all three vaccine antigens. Responses were long lasting (6 months), boostable by a fourth MVA vaccination and in vivo cross-reactive as demonstrated in a surrogate Listeria-based challenge assay. This candidate vaccine has now moved into clinical trials.