Assessment of the in vitro developmental toxicity of diethylstilbestrol and estradiol in the zebrafish embryotoxicity test.

The present study investigated the developmental toxicity of diethylstilbestrol (DES) in the zebrafish embryotoxicity test (ZET). This was done to investigate whether the ZET would better capture the developmental toxicity of DES than the embryonic stem cells test (EST) that was previously shown to underpredict the DES-induced developmental toxicity as compared to in vivo data, potentially because the EST does not capture late events in the developmental process. The ZET results showed DES-induced growth retardation, cumulative mortality and dysmorphisms (i.e. induction of pericardial edema) in zebrafish embryos while the endogenous ERα agonist 17ß-estradiol (E2) showed only growth retardation and cumulative mortality with lower potency compared to DES. Furthermore, the DES-induced pericardial edema formation in zebrafish embryos could be counteracted by co-exposure with ERα antagonist fulvestrant, indicating that the ZET captures the role of ERα in the mode of action underlying the developmental toxicity of DES. Altogether, it is concluded that the ZET differentiates DES from E2 with respect to their developmental toxicity effects, while confirming the role of ERα in mediating the developmental toxicity of DES. Furthermore, comparison to in vivo data revealed that, like the EST, in a quantitative way also the ZET did not capture the relatively high in vivo potency of DES as a developmental toxicant.

High-content screening imaging and real-time cellular impedance monitoring for the assessment of chemical's bio-activation with regards hepatotoxicity.

Testing hepatotoxicity is a crucial step in the development and toxicological assessment of drugs and chemicals. Bio-activation can lead to the formation of metabolites which may present toxicity for the organism. Classical cytotoxic tests are not always appropriate and are often insufficient, particularly when non metabolically-competent cells are used as the model system, leading to false-positive or false-negative results. We tested over 24 h the effects of eight reference compounds on two different cell models: primary cultures of rat hepatocytes and FAO hepatoma cells that lack metabolic properties. We performed inter-assay validation between three classical cytotoxicity assays and real-time cell impedance data. We then complemented these experiments with high-content screening (HCS) to determine the cell function disorders responsible for the observed effects. Among the different assays used, the neutral red test seemed to be well suited to our two cell models, coupled with real-time cellular impedance which proved useful in the detection of bio-activation. Indeed, impedance monitoring showed a high sensitivity with interesting curve profiles yet seemed unsuitable for evaluation of viability on primary culture. Finally, HCS in the evaluation of hepatotoxicity is likely to become an essential tool for use in parallel to a classical cytotoxic assay in the assessment of drugs and environmental chemicals.

Transferability and inter-laboratory variability assessment of the in vitro bovine oocyte fertilization test.

The dramatic increase in the number of animals required for reproductive toxicity testing imposes the validation of alternative methods to reduce the use of laboratory animals. As we previously demonstrated for in vitro maturation test of bovine oocytes, the present study describes the transferability assessment and the inter-laboratory variability of an in vitro test able to identify chemical effects during the process of bovine oocyte fertilization. Eight chemicals with well-known toxic properties (benzo[a]pyrene, busulfan, cadmium chloride, cycloheximide, diethylstilbestrol, ketoconazole, methylacetoacetate, mifepristone/RU-486) were tested in two well-trained laboratories. The statistical analysis demonstrated no differences in the EC50 values for each chemical in within (inter-runs) and in between-laboratory variability of the proposed test. We therefore conclude that the bovine in vitro fertilization test could advance toward the validation process as alternative in vitro method and become part of an integrated testing strategy in order to predict chemical hazards on mammalian fertility.

Toxicity assessment on trophoblast cells for some environment polluting chemicals and 17ß-estradiol.

The identification of reproductive toxicants is a major scientific challenge for human health. We investigated the effects of a selected group of environmental polluting chemicals mostly provided with estrogenic activity on the human trophoblast cell lines BeWo and HTR-8/SVneo. Cells were exposed for 24h to various concentrations (from 0.1 pM to 1 mM) of atrazine (ATR), diethylstilbestrol (DES), para-nonylphenol (p-NP), resveratrol (RES) and 17 ß-estradiol (E2) and assayed for cell viability and human beta-Chorionic Gonadotropin (ß-hCG) secretion. Decrease of cell viability as respect to control, vehicle-treated, cultures was obtained for all chemicals in the concentration range of 1 µM-1 mM in both cell types. A parallel decrease of ß-hCG secretion was observed in BeWo cells, at 1 µM-1 mM concentrations, with the only exception of ATR which caused an increase at concentrations up to 1mM. ß-hCG release was also unexpectedly inhibited by ATR, DES, p-NP and RES at non-toxic (pM-nM) concentrations. These findings raise concern about the negative, potential effects of various environmental polluting chemicals on pregnancy success and fetal health.

Is the current product safety assessment paradigm protective for epigenetic mechanisms?

INTRODUCTION: The emerging field of epigenetics has revealed a new layer of gene regulation that is only now being fully explored. Concomitant with the increase in our understanding of epigenetic regulation are questions as to the role environmental factors may play in altering the epigenome. As these correlations between epigenetic changes and toxicity are made, the natural next question is if the current safety assessment paradigm utilizing a no-observed-adverse-effect level (NOAEL) is protective of public health for an epigenetic mechanism. METHODS: To begin to answer this question, several case studies were examined where apical end point dose response curves were compared to dose response data on epigenetic end points for 1,3-butadiene, arsenic, and diethylstilbesterol. RESULTS: This limited examination of the available literature for these three molecules revealed that epigenetic alterations largely fell within the dose response curve for apical effects. Perhaps more importantly, this analysis also revealed some key data gaps that should be addressed such as incongruent study designs and limited epigenetic dose response data for only a small subset of known epigenetic marks. Taken together, the answer to the question of whether the current product safety assessment paradigm is protective of epigenetic alterations is "yes, based on our current understanding of epigenetics". That is, this paradigm would be protective of any mechanism that resulted in adverse effects typically observed in guideline studies, because product safety assessment is based upon observed apical effects to drive an overall NOAEL that is the basis to set reference doses for a risk assessment. DISCUSSION: These adverse apical effects are the culmination of all molecular events, regardless of mechanism and may include alterations in the epigenome secondary to the actions of those mechanism(s). The epigenome is in a constant state of flux throughout cellular growth and development, and this dynamic variability is not completely characterized. Thus given the state of our current scientific understanding, a change in itself cannot be contextualized as adverse in the absence of a phenotypic anchor. Clearly, more research is needed in this area to perform additional epigenetic studies that include apical end points with full dose response curves in order to gain a more comprehensive understanding of adverse health outcomes that could be causally linked to epigenetic changes.

Assessment of estrogenic contamination and biological effects in Lake Taihu.

Lake Taihu is the third largest freshwater lake in China and is contaminated with xenoestrogens associated with high population density, intensive livestock and aquatic breeding activities. A field study in Lake Taihu was conducted using the goldfish (Carassius auratus) as an indicator organism. Several biological markers were selected to assess the extent of estrogenic contamination. Changes in serum vitellogenin (VTG), and gill 7-Ethoxyresorufin-O-deethylase (EROD), glutathione-S-transferase (GST) and reduced glutathione (GSH) were measured in caged juvenile goldfish for 28 days in seven locations in northern Lake Taihu. Bioassay showed VTG increased 0.64-2.42 folds over time in goldfish collected from five stations and GSH decreased in samples from all seven stations after 7 days of exposure. EROD levels increased continually in fish collected at all the seven stations and the highest concentrations occurred at day 21. GST activity increased significantly at 7 days. The concentration of the target estrogens estrone (E(1)), 17ß-estradiol (E(2)), ethinylestradiol (EE(2)), octylphenol (OP), diethylstilbestrol (DES), nonylphenol (NP) and bisphenol A (BPA) were determined in lake water at the sampling stations. Each individual estrogen concentration measured was multiplied by its relative potency to gain the estradiol equivalent (EEQ). There was an obvious correlation between the concentration of VTG and the total EEQ for all seven locations (P < 0.001). The biomarker VTG, EROD, GST and GSH assays and chemical analysis might be used to illustrate the potential risk in Lake Taihu.

Comparison of different methods for an accurate assessment of cytotoxicity in the in vitro micronucleus test without cytokinesis block.

A European collaborative study was conducted in order to investigate the different measurements of cytotoxicity assessment in the in vitro micronucleus test. Relative population doubling and relative increase in cell counts were the measurements of cytotoxicity assessed in this study. The test chemicals, etoposide, benzo[a]pyrene, cytosine arabinoside and diethylstilboestrol were tested in this in vitro micronucleus assay, in mouse lymphoma L5178Y cells, without cytokinesis block. This study was conducted in support of the toxicity measurements recommended in the draft OECD Test Guideline 487. Etoposide, benzo[a]pyrene and cytosine arabinoside produced positive responses at concentrations where approximately 50% toxicity was observed measured by relative population doubling and relative increase in cell counts. Diethylstilboestrol was negative at all concentrations tested. These results are in concordance with the principles of the draft OECD Test Guideline 487.

In ovo exposure quail assay for risk assessment of endocrine disrupting chemicals.

Although there are in vivo assays using various organisms for the risk assessment of chemicals with endocrine disrupting properties, effective experimental methods for avian species are still under debate. We have developed an in ovo exposure assay using Japanese quail eggs, aimed at assessing disrupting effects on avian reproductive development and function. Hybrid eggs from Brazilian Brown male and White Egg female quails, which can be genetically sexed by their plumage color after hatching, were prepared, and test materials dissolved in olive oil were injected into the air-chamber on day 10 of incubation. After sexual maturation of hatched chicks, we observed egg production by females and the egg quality and male-typical reproductive behavior, and then examined reproductive system morphology and serum steroid concentrations in both sexes. Treatment with a synthetic estrogen, diethylstilbestrol (DES, 0.5-50 ng/g egg), dose-dependently reduced the eggshell thickness and strength of eggs. A few females treated with 5 ng/g DES per egg produced soft-shelled/ unmarked eggs, and all laying females treated with 50 ng/g egg produced eggs completely lacking shells. DES also induced shortening of the left oviduct and abnormal development of the right oviduct in a dose-dependent manner, while testis weight was reduced symmetrically. In addition, 2,2',4',6'-tetrachlorobiphenyl-4-ol (10-1,000 ng/g egg), which previously showed relatively high estrogenic activity in vitro, caused dose-dependent shortening of the left oviduct and reduction in testis weight. The methods for evaluating endocrine disrupting effects and preparing experimental birds proposed in the present study are expected to facilitate assays for avian reproductive toxicology.

Measuring mouse sperm parameters using a particle counter and sperm quality analyzer: a simple and inexpensive method.

This study examined a method for analyzing the count, motility, and morphology of mouse epididymal sperm, optimizing the diluent, incubation time, sample concentration, and temperature, using a particle counter (CDA-500) to count and size sperm and a sperm quality analyzer (SQA-IIC) to measure sperm motility, quantified as the sperm motility index (SMI). The optimal conditions consisted of a 30-min incubation in D-MEM (Dulbecco's modified Eagle's medium; considering cost and availability) at 37 degrees C, with 5 x 10(6)cells mL(-1) in the original solution. Furthermore, the influence of formalin fixation, and the correlation between the automated counter and a manual method were investigated. The sample fixation had no marked effect on the sperm count or morphology assessment. A linear correlation was observed between the manual and automated methods (y=0.920x +0.276; r(2)=0.571; p<0.001; range: (3-6) x 10(6)). The suitability of the proposed method was confirmed using spermatozoa prepared from mice treated with the reproductive toxin diethylstilbestrol (DES). Using sperm from the cauda epididymidis on one side per mouse, we confirmed that measurement of these sperm parameters using the two devices was simple, rapid, inexpensive, and reproducible.

DES awareness and exposure: the 1994 California Behavioral Risk Factor Survey.

INTRODUCTION: Diethylstilbestrol (DES), a drug used in millions of pregnancies between 1938 and 1971, is the first known human transplacental carcinogen. DES is also associated with other serious health problems for those exposed to it either in utero or while pregnant; however, many men and women are unaware of their exposure or how to protect their health. This first population-based study of DES awareness is part of the National Cancer Instututes's National DES Education Program. METHODS: In 1994, 2,077 women and 1,625 men 23 years of age and over responded to the California Behavioral Risk Factor Survey (BRFS). These subjects were either born during the years DES was in use (men and women 23-53 years old in 1994) or could have been pregnant during those years (women 39 years or older). RESULTS: Analyses weighted to the 1994 California age and ethnicity distribution indicate that only 43% of women and 22% of men had over heard of DES (P < .001). Although 44% of Caucasians had heard of DES, only 10% of Hispanics, 27% of African Americans, and 24% of other races had heard of DES. Within each group, women had heard of DES significantly more often than men. Only 17% of women and 5% of men had ever tried to confirm whether they were exposed to DES in utero, and 8% of women whether they were exposed while pregnant. CONCLUSIONS: Given the serious health consequences of DES exposure and available prevention strategies, this lack of awareness warrants an immediate educational effort.

Analysis of the six additional chemicals for in vitro assays of the European Economic Communities' EEC aneuploidy programme using Saccharomyces cerevisiae D61.M and the in vitro porcine brain tubulin assembly assay.

We tested six additional chemicals (acetaldehyde, benomyl, diethylstilboestrol, diethylstilboestrol dipropionate, griseofulvin, and mercaptoethanol) for in vitro systems of the coordinated programme to study aneuploidy induction sponsored by the Commission of the European Communities in two in vitro test systems. Using Saccharomyces cerevisiae D61.M (mitotic chromosomal malsegregation assay), benomyl showed a dose-dependent increase in the frequency of chromosomal malsegregation with a lowest effective dose tested (LEDT) of 30 micrograms/ml (0.1 mM). Diethylstilboestrol (DES) showed solvent-dependent effects. DES dissolved in ethanol induced an increase in chromosomal malsegregation as well as in the frequency of total resistant colonies (mutations and recombinations) with a LEDT around 13 micrograms/ml (0.048 mM). Using dimethylsulfoxide as the solvent, no increases were observed with DES up to 333 micrograms/ml (1.24 mM). Acetaldehyde induced an increase in chromosomal malsegregation with the cold treatment protocol (LEDT: 1.25 microliters/ml (21 mM) and 0.75 microliters/ml (13 mM), respectively) but no increase with the overnight protocol (highest dose tested (HDT): 1.75 microliters/ml; 30 mM). Concerning the frequency of total cycloheximide-resistant colonies (mutations and recombinations) increases were obtained with both protocols. The other three compounds were negative when tested up to toxic doses (survival below 10%), up to the maximum solubility in the solvent used or up to heavy precipitation in the incubation mix. The HDT were 333 micrograms/ml (0.88 mM) for diethylstilboestrol dipropionate, 1,600 micrograms/ml (4.5 mM) for griseofulvin and 0.5 microliters/ml (7 mM) for mercaptoethanol. Concerning effects on porcine brain tubulin assembly in vitro, diethylstilboestrol and griseofulvin inhibited the assembly process. The IC30% (30% inhibition concentration) values were 12.5 microM and 100 microM for DES and griseofulvin, respectively. Mercaptoethanol showed no effects up to 50 mM.

Questionnaire study of cancer etiology in 503 children.

Usefulness of an etiologic questionnaire was examined in an interview study of 503 children with cancer. The medical records of the children were abstracted, and their parents responded to a questionnaire-interview to identify genetic and environmental causes of cancer. Among 1,123 siblings of the index patients, 10 developed cancer as compared with 2 expected on the basis of cancer rates for the general population. Cancer risk factors were identified in individual patients with predisposing genetic and congenital disorders: neurofibromatosis (brain tumor), hereditary immunodeficiency (lymphoma), Down's syndrome (leukemia), XY gonadal dysgenesis (germ cell tumor), giant nevus (melanoma), and meningocele (sacral teratocarcinoma). Environmental causes of childhood cancer were difficult to discern because prior exposures were numerous, diverse, and usually ill defined. The questionnaire yielded more data than the medical record on gestational and family history and helped identify patients with exceptionally high cancer risk for additional investigation. Although the findings provide anecdotal confirmation of several associations, few original etiologic hypotheses were generated for formal testing with conventional epidemiologic techniques.

Assessment of myelotoxicity caused by environmental chemicals.

Potential antineoplastic agents must be screened for the delayed toxicity that occurs in many cases of drug-induced bone marrow aplasia. In vitro clonal assays for hematopoietic progenitor cells have been developed to assess the degree of myelotoxicity. This adverse side effect is often the limiting factor in the development of new cancer chemotherapeutics. In addition, many environmental chemicals are cytotoxic to rapidly proliferating cells, but a systematic assessment of their myelotoxicity has not been performed. We have used clonal marrow assays to investigate a panel of chemicals including 2,3,7,8-tetrachlorodibenzo-p-dioxin, polybrominated biphenyls, diethylstilbestrol, benzo(a)pyrene and indomethacin. All were immunotoxic, some to pleuripotent hemopoetic stem cells and other to granulocyte-macrophage progenitors, and at concentrations below those causing other toxic manifestations. This shows that these bone marrow clonal assays, and hopefully future one for erythroid, B- and T-lymphocytes, and megakaryocytes, will provide the specificity and sensitivity necessary to delineate the myelotoxicity of a broad spectrum of environmental chemicals.