In Vitro Cytotoxicity and Adaptive Stress Responses to Selected Haloacetic Acid and Halobenzoquinone Water Disinfection Byproducts.

The process of disinfecting drinking water inadvertently leads to the formation of numerous disinfection byproducts (DBPs). Some of these are mutagenic, genotoxic, teratogenic, and cytotoxic, as well as potentially carcinogenic both in vivo and in vitro. We investigated the in vitro biological activity of five DBPs: three monohaloacetic acids (monoHAAs) [chloroacetic acid (CAA), bromoacetic acid (BAA), and iodoacetic acid (IAA)] and two novel halobenzoquinones (HBQs) [2,6-dichloro-p-benzoquinone (DCBQ) and 2,6-dibromo-p-benzoquinone]. We focused particularly on cytotoxicity and induction of two adaptive stress response pathways: the oxidative stress responsive Nrf2/ARE and DNA-damage responsive p53 pathways. All five DBPs were cytotoxic to the Caco-2 cell line after a 4 h exposure, and all DBPs induced both of the adaptive stress response pathways, Nrf2/ARE and p53, in the micromolar range, as measured by two ß-lactamase-based reporter gene assays. The decreasing order of potency for all three endpoints for the five DBPs was IAA ∼ BAA > DCBQ ∼ DBBQ > CAA. Induction of oxidative stress was previously proposed to be the molecular initiating event (MIE) for both classes of DBPs. However, comparing the levels of activation of the two pathways uncovered that the Nrf2/ARE pathway was the more sensitive endpoint for HAAs, whereas the p53 pathway was more sensitive in the case of HBQs. Therefore, the DNA damage-responsive p53 pathway may be an important piece of information to fill in a gap in the adverse outcome pathway framework for the assessment of HBQs. Finally, we cautiously compared the potential risk of the two novel HBQs using a benchmarking approach to that of the well-studied CAA, which suggested that their relative risk may be lower than that of BAA and IAA.

Benchmarking organic micropollutants in wastewater, recycled water and drinking water with in vitro bioassays.

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.

Global Transcriptional Analysis of Nontransformed Human Intestinal Epithelial Cells (FHs 74 Int) after Exposure to Selected Drinking Water Disinfection By-Products.

BACKGROUND: Drinking water disinfection inadvertently leads to the formation of numerous disinfection by-products (DBPs), some of which are cytotoxic, mutagenic, genotoxic, teratogenic, and potential carcinogens both in vitro and in vivo. OBJECTIVES: We investigated alterations to global gene expression (GE) in nontransformed human small intestine epithelial cells (FHs 74 Int) after exposure to six brominated and two chlorinated DBPs: bromoacetic acid (BAA), bromoacetonitrile (BAN), 2,6-dibromo-p-benzoquinone (DBBQ), bromoacetamide (BAM), tribromoacetaldehyde (TBAL), bromate (BrO3-), trichloroacetic acid (TCAA), and trichloroacetaldehyde (TCAL). METHODS: Using whole-genome cDNA microarray technology (Illumina), we examined GE in nontransformed human cells after 4h exposure to DBPs at predetermined equipotent concentrations, identified significant changes in gene expression (p≤0.01), and investigated the relevance of these genes to specific toxicity pathways via gene and pathway enrichment analysis. RESULTS: Genes related to activation of oxidative stress-responsive pathways exhibited fewer alterations than expected based on prior work, whereas all DBPs induced notable effects on transcription of genes related to immunity and inflammation. DISCUSSION: Our results suggest that alterations to genes associated with immune and inflammatory pathways play an important role in the potential adverse health effects of exposure to DBPs. The interrelationship between these pathways and the production of reactive oxygen species (ROS) may explain the common occurrence of oxidative stress in other studies exploring DBP toxicity. Finally, transcriptional changes and shared induction of toxicity pathways observed for all DBPs caution of additive effects of mixtures and suggest further assessment of adverse health effects of mixtures is warranted. https://doi.org/10.1289/EHP4945.

Histopathology, vitellogenin and chemical body burden in mosquitofish (Gambusia holbrooki) sampled from six river sites receiving a gradient of stressors.

There are over 40,000 chemical compounds registered for use in Australia, and only a handful are monitored in the aquatic receiving environments. Their effects on fish species in Australia are largely unknown. Mosquitofish (Gambusia holbrooki) were sampled from six river sites in Southeast Queensland identified as at risk from a range of pollutants. The sites selected were downstream of a wastewater treatment plant discharge, a landfill, two agricultural areas, and two sites in undeveloped reaches within or downstream of protected lands (national parks). Vitellogenin analysis, histopathology of liver, kidney and gonads, morphology of the gonopodium, and chemical body burden were measured to characterize fish health. Concentrations of trace organic contaminants (TrOCs) in water were analyzed by in vitro bioassays and chemical analysis. Estrogenic, anti-estrogenic, anti-androgenic, progestagenic and anti-progestagenic activities and TrOCs were detected in multiple water samples. Several active pharmaceutical ingredients (APIs), industrial compounds, pesticides and other endocrine active compounds were detected in fish carcasses at all sites, ranging from <4-4700ng/g wet weight, including the two undeveloped sites. While vitellogenin protein was slightly increased in fish from two of the six sites, the presence of micropollutants did not cause overt sexual endocrine disruption in mosquitofish (i.e., no abnormal gonads or gonopodia). A correlation between lipid accumulation in the liver with total body burden warrants further investigation to determine if exposure to low concentrations of TrOCs can affect fish health and increase stress on organs such as the liver and kidneys via other mechanisms, including disruption of non-sexual endocrine axes involved in lipid regulation and metabolism.

Assessment of urban stream sediment pollutants entering estuaries using chemical analysis and multiple bioassays to characterise biological activities.

Stormwater contaminants are a major source of often neglected environmental stressors because of the emphasis placed on the management of municipal and industrial wastewaters. Stormwater-derived pollutants in sediments from two New Zealand estuaries was characterised by analytical chemistry and bioassays. Contaminants were extracted from sediment using accelerated solvent extraction (ASE), recovered and concentrated by solid phase extraction (SPE), and analysed for polycyclic aromatic hydrocarbons (PAHs), selected metals, and musk fragrances. The concentrations of PAHs were below the ANZECC Interim Sediment Quality Guideline values while those of lead and zinc exceeded them in some samples. The sediment extracts containing organic contaminants exhibited acute toxicity in the zebrafish fish embryo toxicity (FET) and teratogenicity, induction of biotransformation (EROD activity), and genotoxicity (comet assay) in zebrafish. The potential of the extracts to interact with endocrine signalling processes was assessed by GeneBLAzer reporter gene bioassays and they exhibited estrogenic, androgenic, and anti-progestagenic activities.

In vitro bioassays to evaluate complex chemical mixtures in recycled water.

With burgeoning population and diminishing availability of freshwater resources, the world continues to expand the use of alternative water resources for drinking, and the quality of these sources has been a great concern for the public as well as public health professionals. In vitro bioassays are increasingly being used to enable rapid, relatively inexpensive toxicity screening that can be used in conjunction with analytical chemistry data to evaluate water quality and the effectiveness of water treatment. In this study, a comprehensive bioassay battery consisting of 36 bioassays covering 18 biological endpoints was applied to screen the bioactivity of waters of varying qualities with parallel treatments. Samples include wastewater effluent, ultraviolet light (UV) and/or ozone advanced oxidation processed (AOP) recycled water, and infiltrated recycled groundwater. Based on assay sensitivity and detection frequency in the samples, several endpoints were highlighted in the battery, including assays for genotoxicity, mutagenicity, estrogenic activity, glucocorticoid activity, arylhydrocarbon receptor activity, oxidative stress response, and cytotoxicity. Attenuation of bioactivity was found to be dependent on the treatment process and bioassay endpoint. For instance, ozone technology significantly removed oxidative stress activity, while UV based technologies were most efficient for the attenuation of glucocorticoid activity. Chlorination partially attenuated genotoxicity and greatly decreased herbicidal activity, while groundwater infiltration efficiently attenuated most of the evaluated bioactivity with the exception of genotoxicity. In some cases, bioactivity (e.g., mutagenicity, genotoxicity, and arylhydrocarbon receptor) increased following water treatment, indicating that transformation products of water treatment may be a concern. Furthermore, several types of bioassays with the same endpoint were compared in this study, which could help guide the selection of optimized methods in future studies. Overall, this research indicates that a battery of bioassays can be used to support decision-making on the application of advanced water treatment processes for removal of bioactivity.

Assessment of the application of bioanalytical tools as surrogate measure of chemical contaminants in recycled water.

The growing use of recycled water in large urban centres requires comprehensive public health risk assessment and management, an important aspect of which is the assessment and management of residual trace chemical substances. Bioanalytical methods such as in vitro bioassays may be ideal screening tools that can detect a wide range of contaminants based on their biological effect. In this study, we applied thirteen in vitro assays selected explicitly for their ability to detect molecular and cellular effects relevant to potential chemical exposure via drinking water as a means of screening for chemical contaminants from recycled water at 9 Australian water reclamation plants, in parallel to more targeted direct chemical analysis of 39 priority compounds. The selected assays provided measures of primary non-specific (cytotoxicity to various cell types), specific (inhibition of acetylcholinesterase and endocrine receptor-mediated effects) and reactive toxicity (mutagenicity and genotoxicity), as well as markers of adaptive stress response (modulation of cytokine production) and xenobiotic metabolism (liver enzyme induction). Chemical and bioassay analyses were in agreement and complementary to each other: the results show that source water (treated wastewater) contained high levels of biologically active compounds, with positive results in almost all bioassays. The quality of the product water (reclaimed water) was only marginally better after ultrafiltration or dissolved air floatation/filtration, but greatly improved after reverse osmosis often reducing biological activity to below detection limit. The bioassays were able to detect activity at concentrations below current chemical method detection limits and provided a sum measure of all biologically active compounds for that bioassay, thus providing an additional degree of confidence in water quality.