Cell bioassays for detection of aryl hydrocarbon (AhR) and estrogen receptor (ER) mediated activity in environmental samples

In vitro cell bioassays are useful techniques for the determination of receptor-mediated activities in environmental samples containing complex mixtures of contaminants. The cell bioassays determine contamination by pollutants that act through specific modes of action. This article presents strategies for the evaluation of aryl hydrocarbon receptor (AhR)-(hereafter referred as dioxin-like) or estrogen receptor (ER)-mediated activities of potential endocrine disrupting compounds (EDCs) in complex environmental mixtures. Extracts from various types of environmental or food matrices can be tested by this technique to evaluate their 2,3,7,8-tetrachlorodibenzop-dioxin equivalents (TCDD-EQs) or estrogenic equivalents (E₂-EQs) and to identify contaminated samples that need further investigation using resource-intensive instrumental analyses. Fractionation of sample extracts exhibiting significant activities, and subsequent reanalysis with the bioassays can identify important classes of contaminants that are responsible for the observed activity. Effect-directed chemical analysis is performed only for the active fractions to determine the responsible compounds. Mass-balance estimates of all major compounds contributing to the observed effects can be calculated to determine if all of the activity has been identified, and to assess the potential for interactions such as synergism or antagonism among contaminants present in the complex mixtures. The bioassay approach is an efficient (fast and cost effective) screening system to identify the samples of interest and to provide basic information for further analysis and risk evaluation.     

Assessment of risk to humans of bisphenol A in marine and freshwater fish from Pearl River Delta, China

Bisphenol A (BPA) is a high production-volume chemical used in the manufacture of a wide variety of consumer products. However it is also a ubiquitous contaminant that can interfere with endocrine systems of wildlife and humans. China is the “world factory” and the Pearl River Delta is the major manufacturing center and is consequently polluted. Concentrations of BPA in meats of marketable fish had not been previously reported for this region. In the study upon which we report here concentrations of BPA were determined in 20 common species of freshwater and marine fish, collected from markets in Hong Kong, SAR, China. A comprehensive analytical method based on SPE extraction and liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) was developed, validated and applied. The method limit of detection (LOD) and limit of quantification (LOQ) were 0.5 and 1.25 ng g⁻¹ dw, respectively. BPA was detected in 19 species of fish at concentrations, ranging from 0.5 to 2.0 ng g⁻¹ ww. Average daily BPA intake per person ranged from 1.1 × 10² ng d⁻¹ for marine fish and 2.2 × 10² ng d⁻¹ for freshwater fish. Concentrations of BPA in fish from Hong Kong markets unlikely would be causing adverse population-level effects in humans.     

Effects of perinatal exposure to bisphenol A and di(2-ethylhexyl)-phthalate on gonadal development of male mice

Purpose

In this study, we investigated the effects of maternal transfer of bisphenol A (BPA) and di(2-ethylhexyl) phthalate (DEHP) during gestational and weaning periods on gonadal development of male offspring.

Methods

Pregnant CD-1 mice were administered by gavages in corn oil with 0.1, 1, or 10 mg/kg/day of BPA and DEHP from gestational days (GD1?C21) to the weaning period (postnatal days (PND) 1?C21).

Results

Our data indicated that the exposure significantly reduced the male-to-female sex ratio and the sizes of the gonads of male pups as recorded at PND15. The testes of the perinatally exposed male pups were developed less and the expression levels of testicular anti-mullerian hormone, androgen receptor, cyclin A, and StAR were significantly lesser than the control male pups. The less developed testes were accompanied with significant reductions in the expression levels of Gnrh and Fsh at the hypothalamic?Cpituitary levels. The negative effects were found to be persistent in the sexually mature pups at PND42.

Conclusion

Our data reveal that the maternal transfer of BPA and DEHP may impose negative influence on the development and functions of the reproductive system of male pups.     

The OECD validation program of the H295R steroidogenesis assay: Phase 3. Final inter-laboratory validation study

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 17??-estradiol (E2) in preparation for the development of an Organization for Economic Cooperation and Development (OECD) test guideline.

Methods

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.

Results

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.

Discussion and conclusions

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).

Perspectives

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.     

Enantioselective effects of alpha-hexachlorocyclohexane (HCH) isomers on androgen receptor activity in vitro

Alpha-hexachlorocyclohexane (alpha-HCH), a part of the HCH pesticide mixture, is one of the most widespread persistent organic pollutants. Interestingly, only limited number of studies addressed the toxicity of alpha-HCH and the effects of its individual optical isomers have not been investigated in detail. In the present study we separated two alpha-HCH enantiomers by preparative HPLC and studied their activities towards androgen receptor (AR) using the MDA-kb2 cell line stably transfected with the luciferase reporter gene under the control of AR. There was no direct effect of alpha-HCH on AR but both isomers significantly suppressed the activity of AR in co-exposure with the natural ligand dihydrotestosterone in a concentration-dependent manner. One of the enantiomers appeared to be more active at lower concentration, which was also supported by the molecular modeling calculations with AR that showed a slight difference in estimated free energy of binding and inhibition constant between two enantiomers. Although studies with other pesticides demonstrated strong enantioselective differences in toxicity, the present research shows rather minor differences in modulations of AR by both alpha-HCH enantiomers. For the first time, enantioselective effects of alpha-HCH were demonstrated and the results suggest interaction with multiple regulatory events controlling the AR activity. Full elucidation of the toxicity mechanism will require further research.     

Review of measured concentrations of triphenyltin compounds in marine ecosystems and meta-analysis of their risks to humans and the environment

The state of scientific knowledge regarding analytical methods, environmental fate, ecotoxicity and ecological risk of triphenyltin (TPT) compounds in marine ecosystems as well as their exposure and health hazard to humans was reviewed. Since the 1960s, TPT compounds have been commonly applied as biocides for diverse industrial and agricultural purposes. For instance, they are used as active ingredients in antifouling systems on marine vessels and mariculture facilities, and as fungicides in agriculture. Due to their intensive use, contamination of coastal waters by TPT and its products of transformation has become a worldwide problem. The proportion of quantified TPT to total phenyltin compounds in the marine environment provides evidence that TPT is photodegradable in water and sediment but resistant to biotransformation. Concentrations of TPT in marine biota are consistently greater than concentrations in water and sediment, which implies potential of TPT to bioaccumulate. TPT is toxic to both marine plants and animals. The predicted no effect concentration (PNEC) for TPT, as determined by use of the species sensitivity distribution approach, is 0.64 ng L⁻¹. In some parts of the world, concentrations of TPT in seawater exceed the PNEC, indicating that TPT can pose risks to marine life. Although there is negligible risk of TPT to average human consumers, TPT has been detected in blood of Finnish people and the concentration was greater in fishermen who ate more seafood. It is, therefore, advocated to initiate regular monitoring of TPT in blood and breast milk of populations that consume greater amounts of seafood.     

Effects of multigenerational exposures of D. magna to environmentally relevant concentrations of pentachlorophenol

The re-emergence of schistosomiasis has given rise to ubiquitous concentrations of the primary control agent pentachlorophenol (PCP) in the environment, especially in the surface waters of China. In this study, the effects of environmentally relevant concentrations of PCP, namely, 0.0002, 0.002, 0.02, 0.2, and 2 μmol/L on survival, age at first reproduction, fecundity, length of mothers, and number of molts of Daphnia magna were studied over three generations. The survival of D. magna exposed to 2 μmol/L was significantly affected in the three generations. Toxic effects were enhanced in later generations. Age at first reproduction of F ₁ and F ₂ D. magna was significantly slower than that of the controls. The total number of offspring per female exposed to concentrations of 0.002 μmol/L or greater was less (23.5 to 67.6, 9.4 to 73.7, and 3.6 to 83.7 %) than that of the controls in the F ₀, F ₁, and F ₂ generations, respectively. The body length of mothers significantly decreased (4.7 to 6.8, 9.6 to 15.1, and 13.3 to 23.2 %) after exposure to 0.002 μmol/L or greater than those of unexposed individuals in the F ₀, F ₁, and F ₂ generations, respectively. Dose–response relationships between concentrations of PCP and length and number of molts of D. magna were observed in the F ₀ to F ₂ generations. PCP concentrations on the surface waters of China caused adverse effects to D. magna, which increased over successive generations. Significant effects were observed in the third generation. The multigenerational studies were more sensitive than the single-generation experiments. Thus, multigenerational exposure may be more predictive of chronic exposure under field conditions.     

Toxicity of HC Orange No. 1 to Daphnia magna, zebrafish (Brachydanio rerio) embryos, and goldfish (Carassius auratus)

HC Orange No. 1 (HCO1; 2-nitro-4′-hydroxydiphenylamine) (CAS No. 54381-08-7) is used as a color additive in hair dyes and can be released into aquatic environments in wastewater. In this paper, the effects of HCO1 on aquatic organisms were studied using a battery of toxicological tests. These included measuring immobilization of Daphnia magna, inhibition of zebrafish embryo development, and acute lethality in zebrafish and goldfish, which are different species belonging to different trophic levels. HCO1 was toxic to all of the organisms studied. In our experiments, HCO1 remarkably restrained the mobility of D. magna, which may cause subsequent death. The EC₅₀ value for restrained the mobility of D. magna at 48 h was 1.54 mg HCO1 l⁻¹. In addition, HCO1 showed toxicity in zebrafish and goldfish, where LC₅₀values at 96 h were 4.04 and 5.37 mg l⁻¹, respectively. The results also indicated that HCO1 remarkably retarded the development of zebrafish embryos, which may cause embryo abnormality and even lethality. The most sensitive toxicological endpoint in the development of the embryos was failure to hatch, which had an EC₅₀ of 0.19 mg HCO1 l⁻¹. These results indicated that HCO1 is a potential teratogen to zebrafish embryos. In addition, as HCO1 concentrations increased, the outcomes of each of these toxicity tests changed in a concentration-dependent manner. Together, the results revealed that HCO1 appears to be toxic to multiple different species of aquatic organisms. The EC₅₀ (LC₅₀) values contain sufficient discriminatory power for risk assessment of HCO1 in aquatic environments. Based on the present results, more efficient risk assessment procedures for HCO1 will be designed in the future, integrating more flexible testing methods into the testing schemes that employ only the necessary tools for each case.     

Perfluorinated compounds in water, sediment, soil and biota from estuarine and coastal areas of Korea

Soil, sediment, water, and biota collected from the western coast of Korea were analyzed to determine occurrence and sources of perfluorinated compounds (PFCs). PFCs were significantly concentrations of PFCs were measured in some water and biological samples, while concentrations of PFCs in soils and sediments were relatively low. The most widely detected compound was found to be perfluorooctanesulfonate (PFOS), with a maximum concentration in water of 450 ng/L and in fish of 612 ng/g, dw. PFOS concentrations in water and biota were both less than those thought to cause toxicity. However, in both cases concentrations were within a factor of 10 of the toxicity threshold concentration. Concentrations of PFCs were significantly greater downstream than those upstream on the same river, suggesting point sources. Overall, the detection of PFCs at relatively great concentrations in various environmental matrixes from this region of Korea suggests that further studies characterizing PFCs and their potential risk to both humans and wildlife are needed.