Thousands of organic micropollutants and their transformation products occur in water. Although often present at low concentrations, individual compounds contribute to mixture effects. Cell-based bioassays that target health-relevant biological endpoints may therefore complement chemical analysis for water quality assessment. The objective of this study was to evaluate cell-based bioassays for their suitability to benchmark water quality and to assess efficacy of water treatment processes. The selected bioassays cover relevant steps in the toxicity pathways including induction of xenobiotic metabolism, specific and reactive modes of toxic action, activation of adaptive stress response pathways and system responses. Twenty laboratories applied 103 unique in vitro bioassays to a common set of 10 water samples collected in Australia, including wastewater treatment plant effluent, two types of recycled water (reverse osmosis and ozonation/activated carbon filtration), stormwater, surface water, and drinking water. Sixty-five bioassays (63%) showed positive results in at least one sample, typically in wastewater treatment plant effluent, and only five (5%) were positive in the control (ultrapure water). Each water type had a characteristic bioanalytical profile with particular groups of toxicity pathways either consistently responsive or not responsive across test systems. The most responsive health-relevant endpoints were related to xenobiotic metabolism (pregnane X and aryl hydrocarbon receptors), hormone-mediated modes of action (mainly related to the estrogen, glucocorticoid, and antiandrogen activities), reactive modes of action (genotoxicity) and adaptive stress response pathway (oxidative stress response). This study has demonstrated that selected cell-based bioassays are suitable to benchmark water quality and it is recommended to use a purpose-tailored panel of bioassays for routine monitoring.

Hyalella azteca is a cryptic species complex of epibenthic amphipods of interest to ecotoxicology and evolutionary biology. It is the primary crustacean used in North America for sediment toxicity testing and an emerging model for molecular ecotoxicology. To provide molecular resources for sediment quality assessments and evolutionary studies, we sequenced, assembled, and annotated the genome of the H. azteca U.S. Lab Strain. The genome quality and completeness is comparable with other ecotoxicological model species. Through targeted investigation and use of gene expression data sets of H. azteca exposed to pesticides, metals, and other emerging contaminants, we annotated and characterized the major gene families involved in sequestration, detoxification, oxidative stress, and toxicant response. Our results revealed gene loss related to light sensing, but a large expansion in chemoreceptors, likely underlying sensory shifts necessary in their low light habitats. Gene family expansions were also noted for cytochrome P450 genes, cuticle proteins, ion transporters, and include recent gene duplications in the metal sequestration protein, metallothionein. Mapping of differentially expressed transcripts to the genome significantly increased the ability to functionally annotate toxicant responsive genes. The H. azteca genome will greatly facilitate development of genomic tools for environmental assessments and promote an understanding of how evolution shapes toxicological pathways with implications for environmental and human health.

A genetic transformation system with similarities to those reported for gram-negative bacteria was found to be associated with membrane vesicles of the ruminal cellulolytic genus Ruminococcus. Double-stranded DNA was recovered from the subcellular particulate fraction of all the cellulolytic ruminococci examined. Electron microscopy revealed that the only particles present resembled membrane vesicles. The likelihood that the DNA was associated with membrane vesicles (also known to contain cellulosomes) was further supported by the adherence of the particles associated with the subcellular DNA to cellulose powder added to culture filtrates. The particle-associated DNA comprised a population of linear molecules ranging in size from <20 kb to 49 kb (Ruminococcus sp. strain YE73) and from 23 kb to 90 kb (Ruminococcus albus AR67). Particle-associated DNA from R. albus AR67 represented DNA derived from genomic DNA of the host bacterium having an almost identical HindIII digestion pattern and an identical 16S rRNA gene. Paradoxically, particle-associated DNA was refractory to digestion with EcoRI, while the genomic DNA was susceptible to extensive digestion, suggesting that there is differential restriction modification of genomic DNA and DNA exported from the cell. Transformation using the vesicle-containing fraction of culture supernatant of Ruminococcus sp. strain YE71 was able to restore the ability to degrade crystalline cellulose to two mutants that were otherwise unable to do so. The ability was heritable and transferred to subsequent generations. It appears that membrane-associated transformation plays a role in lateral gene transfer in complex microbial ecosystems, such as the rumen.The rumen ecosystem comprises a complex of dense microbial communities of bacteria, archaea, protozoans, fungi, and bacteriophages (18). The fermentation effected by this complex microbiota is responsible for the conversion of plant feedstuffs to compounds that can be utilized by the animal. Hence, the fermentations and interactions of the microbes are central to ruminant digestion and nutrition. Of considerable interest are the exchange of genetic material between ruminal bacteria and the mechanisms that may enable this process to occur. While extrachromosomal elements, such as plasmids, transposons, and bacteriophages, are well known from ruminal bacteria, few examples of genetic transfer have been documented, and such transfers are largely inferred from the acquisition of antibiotic resistance genes (21). In addition to the transfer of extrachromosomal genetic elements, the presence of transformation systems, in which the chromosomal DNA of the host bacterium is "broken up" and exported from the cell, is known for a variety of bacterial species but has not been reported for ruminal bacteria. Naturally occurring transformation systems are associated with the release of DNA-containing particles from the cell and comprise two main types, phage-like systems based on particles with a viral appearance (4, 7, 25) and cell wall/membrane...

Changes in the endocrine potency of municipal wastewater at 3 wastewater treatment plants (WWTPs) in Australia were investigated using a panel of in vitro receptor-driven transactivation assays. The assays were based on human estrogen receptor α, androgen receptor, progesterone receptor, glucocorticoid receptor, and peroxisome proliferator-activated receptor γ2. Total removal efficiencies for estrogenic activity in the dissolved phase were 79.8% to 99.4%. Chemical analysis of 17β-estradiol, estrone, and 17α-ethinylestradiol levels showed that they accounted for the majority of the observed in vitro estrogenic activity in the final effluents but only 18% to 70% of estrogenic activity in the influents. Removal efficiency for androgenic activity was 97.5% to 100%. Endocrine activity levels were low in the final effluent of the WWTP with the lowest catchment population, with only estrogenic activity detected. In the final effluent of the WWTP with an intermediate catchment population, estrogenic, glucocorticoid, and peroxisome proliferator activities were detected. Estrogenic, antiandrogenic, progestagenic, glucocorticoid, and peroxisome proliferator activities were detected in the final effluent of the WWTP with the highest catchment population. The present study confirms the efficacy of secondary and tertiary treatment in reducing the concentrations of endocrine-active compounds in municipal wastewater. Further work is required to determine the possible health risks to aquatic biota posed by multiple hormonal activities present at low levels.

Cyanobacteria are capable of producing metabolites that are in some cases toxic to humans and other animals. Of these metabolites, the toxin cylindrospermopsin (CYN) is produced by a number of species of cyanobacteria including Cylindrospermopsis raciborskii, and its toxicity has been documented. The CYN analog deoxycylindrospermopsin (deoxyCYN) is commonly produced in varying proportions by the cyanobacteria that produce CYN. The toxicological profile of CYN suggests that it is primarily a hepatotoxin, but with the capacity to damage other organs and tissues. Limited in vivo information is available on the toxicity of deoxyCYN and suggests it to be of low potency. The aim of this research was to determine the comparative toxicology of deoxyCYN using in vitro systems. Using cell viability assays, it was shown that deoxyCYN had inhibitory effects on cell viability and proliferation of a similar magnitude to that of CYN. Morphological changes in deoxyCYN-treated cells were similar to those of CYN. Investigation of protein synthesis inhibition demonstrated that deoxyCYN was of similar potency to CYN. Inhibition of protein synthesis is an acknowledged mechanism of toxicity for CYN, and the results produced here suggest that deoxyCYN operates by similar toxicological mechanisms to CYN and that in vivo animal testing should be undertaken to clarify the potential for risk to humans from this toxin.

Cylindrospermopsin (CYN) is a cyanobacterial toxin that induces a range of genotoxic indicators in a variety of models. The possible involvement of the tumor suppressor protein p53 in cylindrospermopsin-induced gene expression was examined in cultured human dermal fibroblasts and the human hepatocellular carcinoma cell line HepG2. After 6 h of exposure to CYN, concentration-dependent increases in mRNA levels were observed for the p53 target genes CDKN1A, GADD45alpha, BAX, and MDM2, indicating an early activation of p53. After 24 h, relative mRNA levels for these genes remained elevated. Accumulation of p53 protein occurred after longer exposures in the HepG2-derived cell line C3A. Data suggest that cylindrospermopsin induces stress responses that result in the activation of the p53 transcription factor.

No abstract

Synthetic progestins act as endocrine disrupters in fish but their risk to the environment is not sufficiently known. Here, we focused on an unexplored antiandrogenic progestin, chlormadinone acetate (CMA), and the antiandrogenic progestin cyproterone acetate (CPA). The aim was to evaluate whether their in vitro interaction with human and rainbowfish (Melanotaenia fluviatilis) sex hormone receptors is similar. Furthermore, we investigated their activity in zebrafish (Danio rerio) eleuthero-embryos. First, we studied agonistic and antagonistic activities of CMA, CPA, and 17α-ethinylestradiol (EE2), in recombinant yeast expressing either the human progesterone (PGR), androgen (AR), or estrogen receptor. The same compounds were also investigated in vitro in a stable transfection cell system expressing rainbowfish nuclear steroid receptors. For human receptors, both progestins exhibited progestogenic, androgenic and antiestrogenic activity with no antiandrogenic or estrogenic activity. In contrast, interactions with rainbowfish receptors showed no progestogenic, but antiandrogenic, antiglucocorticoid, and some antiestrogenic activity. Thus, interaction with and transactivation of human and rainbowfish PGR and AR were distinctly different. Second, we analyzed transcriptional alterations in zebrafish eleuthero-embryos at 96 and 144h post fertilization after exposure to CPA, CMA, EE2, and binary mixtures of CMA and CPA with EE2, mimicking the use in oral contraceptives. CMA led to slight down-regulation of the ar transcript, while CPA down-regulated ar and pgr transcripts. EE2 exposure resulted in significant transcriptional alterations of several genes, including esr1, pgr, vtg1, cyp19b, and gonadotropins (fshb, lhb). The mixture activity of CMA and EE2 followed the independent action model, while CPA and EE2 mixtures showed additive action in transcriptional alterations. Third, we analyzed the interactions of binary mixtures of CMA and CPA, and of CMA and EE2 for their joint activity in vitro and in eleuthero-embryos. Both mixtures behaved according to the concentration addition model in their in vitro interaction with human and rainbowfish receptors, often showing antagonism. In zebrafish eleuthero-embryos, binary mixtures of CMA and EE2 showed the same expression patterns as EE2 alone, indicating an independent action in vivo. Our study demonstrates that CMA and CPA interact distinctly with human and rainbowfish receptors, suggesting that activities of these and possibly additional environmental steroids determined with yeast expressing human receptors cannot simply be translated to fish. The lack of agonistic activities of both progestins to rainbowfish PGR and AR is the probable reason for the low activity found in zebrafish eleuthero-embryos.