Early life exposure to environmental chemicals can cause developmental neurotoxicity (DNT). The impairment of key neurodevelopmental processes such as neurite outgrowth inhibition can be used as endpoints for screening of DNT effects. We quantified neurite-specific effects using the ratio of effect concentrations for cytotoxicity and neurite outgrowth inhibition (SRcytotoxicity). Baseline cytotoxicity, the minimal toxicity of any chemical, was used to quantify enhanced cytotoxicity (toxic ratio, TR) and neuronal-specific toxicity (SRbaseline) by comparing baseline cytotoxicity with the effects on cell viability and neurite outgrowth, respectively. The effects on cell viability and neurite length were measured based on image analysis in human neuroblastoma SH-SY5Y cells. Baseline cytotoxicity was predicted from hydrophobicity descriptors using a previously published model for SH-SY5Y cells. Enhanced cytotoxicity and neuronal-specific toxicity were more often observed for hydrophilic chemicals, which indicates that they are more likely to act through specific modes of action (MOA) on cell viability and neurite outgrowth. Hydrophobic chemicals showed a tendency to act through baseline toxicity without showing specific or enhanced toxicity, but were highly potent considering their low effect concentrations for both cytotoxicity and neurite outgrowth inhibition. The endpoint-specific controls (narciclasine, colchicine, cycloheximide, and rotenone), two carbamates (3-hydroxycarbofuran and carbaryl), and two redox cyclers (diquat and paraquat) showed distinct neurite-specific effects (SRcytotoxicity > 4). By comparing neurite-specific effects with enhanced cytotoxicity, one can explain whether the observed effects involve specific inhibition of neurite outgrowth, other specific MOAs, or merely baseline toxicity arising from hydrophobicity.
Benzophenones (BPs) have been widely used in personal care products (PCPs) such as UV protectants. Sex endocrine-disrupting effects have been documented for some BPs, but, significant knowledge gaps are present for their thyroid-disrupting effects. To investigate the thyroid-disrupting potential of BPs, a rat pituitary (GH3) and thyroid follicle (FRTL-5) cell line were employed on six BPs, i.e., benzophenone (BP), benzophenone-1 (BP-1), benzophenone-2 (BP-2), benzophenone-3 (BP-3), benzophenone-4 (BP-4), and benzophenone-8 (BP-8). Subsequently, zebrafish ( Danio rerio) embryo exposure was conducted for three potent BPs that were identified based on the transcriptional changes observed in the cells. In GH3 cells, all BPs except BP-4 down-regulated the Tshβ, Trhr, and Trβ genes. In addition, some BPs significantly up-regulated the Nis and Tg genes while down-regulating the Tpo gene in FRTL-5 cells. In zebrafish embryo assay conducted for BP-1, BP-3, and BP-8, significant decreases in whole-body T4 and T3 level were observed at 6 day postfertilization (dpf). The up-regulation of the dio1 and ugt1ab genes in the fish suggests that decreased thyroid hormones are caused by changing metabolism of the hormones. Our results show that these frequently used BPs can alter thyroid hormone balances by influencing the central regulation and metabolism of the hormones.
Cell-based assays covering environmentally relevant modes of action are widely used for water quality monitoring. However, no high-throughput assays are available for testing developmental neurotoxicity of water samples. We implemented an assay that quantifies neurite outgrowth, which is one of the neurodevelopmental key events, and cell viability in human neuroblastoma SH-SY5Y cells using imaging techniques. We used this assay for testing of extracts of surface water collected in agricultural areas during rain events and effluents from wastewater treatment plants (WWTPs), where more than 200 chemicals had been quantified. Forty-one chemicals were tested individually that were suspected to contribute to the mixture effects among the detected chemicals in environmental samples. Sample sensitivity distributions indicated higher neurotoxicity for surface water samples than for effluents, and the endpoint of neurite outgrowth inhibition was six times more sensitive than cytotoxicity in the surface water samples and only three times more sensitive in the effluent samples. Eight environmental pollutants showed high specificity, and those ranged from pharmaceuticals (mebendazole and verapamil) to pesticides (methiocarb and clomazone), biocides (1,2-benzisothiazolin-3-one), and industrial chemicals (N-methyl-2-pyrrolidone, 7-diethylamino-4-methylcoumarin, and 2-(4-morpholinyl)benzothiazole). Although neurotoxic effects were newly detected for some of our test chemicals, less than 1% of the measured effects were explained by the detected and toxicologically characterized chemicals. The neurotoxicity assay was benchmarked against other bioassays: activations of the aryl hydrocarbon receptor and the peroxisome proliferator-activated receptor were similar in sensitivity, highly sensitive and did not differ much between the two water types, with surface water having slightly higher effects than the WWTP effluent. Oxidative stress response mirrored neurotoxicity quite well but was caused by different chemicals in the two water types. Overall, the new cell-based neurotoxicity assay is a valuable complement to the existing battery of effect-based monitoring tools.
The acetylcholinesterase (AChE) inhibition assay has been frequently applied for environmental monitoring to capture insecticides such as organothiophosphates (OTPs) and carbamates. However, natural organic matter such as dissolved organic carbon (DOC) co‐extracted with solid‐phase extraction from environmental samples can produce false‐negative AChE inhibition in free enzyme‐based AChE assays. We evaluated whether disturbance by DOC can be alleviated in a cell‐based AChE assay using differentiated human neuroblastoma SH‐SY5Y cells. The exposure duration was set at an optimum of 3 h considering the effects of OTPs and carbamates. Because loss to the airspace was expected for the more volatile OTPs (chlorpyrifos, diazinon, and parathion), the chemical loss in this bioassay setup was investigated using solid‐phase microextraction followed by chemical analysis. The three OTPs were relatively well retained (loss <34%) during 3 h of exposure in the 384‐well plate, but higher losses occurred on prolonged exposure, accompanied by slight cross‐contamination of adjacent wells. Inhibition of AChE by paraoxon‐ethyl was not altered in the presence of up to 68 mgc/L Aldrich humic acid used as surrogate for DOC. Binary mixtures of paraoxon‐ethyl and water extracts showed concentration‐additive effects. These experiments confirmed that the matrix in water extracts does not disturb the assay, unlike purified enzyme‐based AChE assays. The cell‐based AChE assay proved to be suitable for testing water samples with effect concentrations causing 50% inhibition of AChE at relative enrichments of 0.5–10 in river water samples, which were distinctly lower than corresponding cytotoxicity, confirming the high sensitivity of the cell‐based AChE inhibition assay and its relevance for water quality monitoring. Environ Toxicol Chem 2022;41:3046–3057. © 2022 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.
Background Location-specific patterns of regulated and non-regulated disinfection byproducts (DBPs) were detected in tap water samples of the Barcelona Metropolitan Area. However, it remains unclear if the detected DBPs together with undetected DPBs and organic micropollutants can lead to mixture effects in drinking water. Objective To evaluate the neurotoxicity, oxidative stress response and cytotoxicity of 42 tap water samples, 6 treated with activated carbon filters, 5 with reverse osmosis and 9 bottled waters. To compare the measured effects of the extracts with the mixture effects predicted from the detected concentrations and the relative effect potencies of the detected DBPs using the mixture model of concentration addition. Methods Mixtures of organic chemicals in water samples were enriched by solid phase extraction and tested for cytotoxicity and neurite outgrowth inhibition in the neuronal cell line SH-SY5Y and for cytotoxicity and oxidative stress response in the AREc32 assay. Results Unenriched water did not trigger neurotoxicity or cytotoxicity. After up to 500-fold enrichment, few extracts showed cytotoxicity. Disinfected water showed low neurotoxicity at 20- to 300-fold enrichment and oxidative stress response at 8- to 140-fold enrichment. Non-regulated non-volatile DBPs, particularly (brominated) haloacetonitriles dominated the predicted mixture effects of the detected chemicals and predicted effects agreed with the measured effects. By hierarchical clustering we identified strong geographical patterns in the types of DPBs and their association with effects. Activated carbon filters did not show a consistent reduction of effects but domestic reverse osmosis filters decreased the effect to that of bottled water. Impact statement Bioassays are an important complement to chemical analysis of disinfection by-products (DBPs) in drinking water. Comparison of the measured oxidative stress response and mixture effects predicted from the detected chemicals and their relative effect potencies allowed the identification of the forcing agents for the mixture effects, which differed by location but were mainly non-regulated DBPs. This study demonstrates the relevance of non-regulated DBPs from a toxicological perspective. In vitro bioassays, in particular reporter gene assays for oxidative stress response that integrate different reactive toxicity pathways including genotoxicity, may therefore serve as sum parameters for drinking water quality assessment.
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