Endpoint Bioassays Research Articles

Toxicological characteristics of drinking water in two large-scale municipal water supply systems of a metropolitan city in Central China.

Drinking water safety is threatened by numerous toxic organic pollutants difficult to chemically monitor. This study aimed to determine the toxicological profiles of organic extracts (OEs) of water samples from source to tap in two drinking water supply systems in a metropolitan city, Central China, during different hydrological periods. Mortality, DNA damage, growth, and development of Caenorhabditis elegans were evaluated following exposure to OEs. The median lethal doses of OEs of drinking water samples (n = 48) ranged from 266 REF (relative enrichment factor) to > 1563 REF. When tested at a dose of 100 REF, 56.25% (27/48) of OEs induced genotoxicity, 4.17% (2/48) inhibited the growth, and 45.83% (22/48) decreased the offspring number in C. elegans. No clear temporal-spatial variation patterns of the OEs toxicity indicators were observed. The correlations among the toxicity indicators were generally poor. The observed toxicities were not closely related to the level of dissolved organic carbon in drinking water. These findings support using multiple endpoint bioassays, such as C. elegans-based approaches, as complementary tools to conventional chemical analysis for drinking water quality monitoring.

Characterization of microalgal toxicants in the sediments from an industrial area: Application of advanced effect-directed analysis with multiple endpoint bioassays

Microalgal toxicants in sediments from an industrialized area (Ulsan Bay) in South Korea were identified using effect-directed analysis (EDA) with full-scan screening analysis (FSA) and microalgal bioassays with multiple endpoints. The growth rate and cell viability of three microalgae (Isochrysis galbana, Dunaliella tertiolecta, and Phaeodactylum tricornutum) were strongly inhibited following exposure to raw organic extracts of sediments from Site D5 (Woehang River). The polar fraction separated using a silica gel column significantly inhibited growth rate, esterase activity, cell membrane intensity, and chlorophyll a autofluorescence. In comparison, non- and mid-polar fractions induced non-toxic or esterase inhibition. Target toxicants, such as polycyclic aromatic hydrocarbons, styrene oligomers, and alkylphenols, were detected at low concentrations (450, 79, and 98 ng g−1 dw, respectively) in the sediment of D5, indicating the presence of unmonitored toxicants. FSA was performed for the polar fraction using LC-QTOFMS, and 31 candidates of toxicants were selected. Toxicological confirmation was conducted for 7 candidates for which standards are available. Out of these, 2-nitrophenol, 3-nitrophenol, and 4-nitrophenol showed significant microalgal toxicity; however, these compounds did not fully explain the induced toxicity. Overall, combining EDA and FSA with multiple endpoint bioassays demonstrated the benefits of characterizing the microalgal toxicants in the environments.

Omics to address the opportunities and challenges of nanotechnology in agriculture

Increasing global food demand, and risks associated with climate change and agrochemicals demand novel and sustainable agricultural practices to improve crop yield and quality. Various strategies using nanotechnology have been explored widely to provide solutions. However, their application in the food industry is constrained by limited understanding of nanomaterial safety. Growing interest in the potential of engineered nanomaterials (ENMs) in agricultural applications has also resulted in increased crop exposure, leading to unknown risks to plants, animals and humans. Recently, considerable research has been carried out on overt plant response to ENMs; however, conclusive mechanistic information is lacking. With advancements in hyphenated analytical techniques, research on ENM-biota interactions has witnessed a paradigm shift from low-throughput, single end-point bioassays to high-throughput, discovery-oriented omic tools, including transcriptomics, proteomics and metabolomics. This review summarizes the pioneering studies utilizing omics in plants exposed to ENMs to explain phenotypic expressions and elucidate associated biological pathways. Advantages and challenges of current analytical techniques employed in proteomics and metabolomics are also discussed. We acknowledge the need for integration and application of multiomics to identify sensitive biomarkers in plants in response to ENMs, and provide mechanistic insights, in order to design enhanced and safer nano-enabled products for agriculture.

Development of a 2-chamber culture system for impedimetric monitoring of cell-cell interaction

In cancer research, study of cell-cell interaction is important to understand tumor initiation, progression, metastasis, and therapeutic resistance. Conventionally, transwell system was adopted and cell proliferation was quantified by end-point bio-assays. The operations are labor-intensive and time-consuming while studying of the dynamic cellular responses of cell-cell interaction. Although impedance measurement was suggested to be a promising technique to monitor cellular responses, electrodes cannot be integrated into the transwell for the measurement purpose. In this work, a 2-chamber culture system incorporated with impedance measurement technique was developed to quantitatively study cell-cell interaction. The chamber was composed of 2 sub-chambers separated with a barrier. By this design, two types of cells could be independently cultured and concurrently monitored under common medium supplied. Cell-cell interaction was demonstrated by aberrant cell proliferation induced by the EGF secreted from the transfected cells cultured on another sub-chamber. Real-time and non-invasive monitoring of cell-cell interaction was successfully demonstrated. This work provides a practical solution for monitoring the dynamic cellular responses of cell-cell interaction during the culture course. It is a reliable and convenient platform and facilitate more quantitative assessments in cancer research.

Integrated biomarker response: An approach to assess antioxidant response in nesting green turtles

To extend the current understanding of the mercury-mediated cytotoxic effect, five neural cell lines established from different animal species were comparatively analyzed using three different endpoint bioassays: thiazolyl blue tetrazolium bromide, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay (MTT), neutral red uptake assay (NRU), and Coomassie blue assay (CB). Following a 24-hr exposure to selected concentrations of mercury chloride (HgCl2) and methylmercury (II) chloride (MeHgCl), the cytotoxic effect on test cells was characterized by comparing their 50% inhibition concentration (IC50) values. Experimental results indicated that both these forms of mercury were toxic to all the neural cells, but at very different degrees. The IC50 values of MeHgCl among these cell lines ranged from 1.15 ± 0.22 to 10.31 ± 0.70 μmol/L while the IC50 values for HgCl2 were much higher, ranging from 6.44 ± 0.36 to 160.97 ± 19.63 μmol/L, indicating the more toxic nature of MeHgCl. The IC50 ratio between HgCl2 and MeHgCl ranged from 1.75 to 96.0, which confirms that organic mercury is much more toxic to these neural cells than inorganic mercury. Among these cell lines, HGST-BR and TriG44 derived from marine sea turtles showed a significantly high tolerance to HgCl2 as compared to the three mammalian neural cells. Among these neural cells, SK-N-SH represented the most sensitive cells to both chemical forms of mercury.

In vitro evaluation of inorganic and methyl mercury mediated cytotoxic effect on neural cells derived from different animal species

To extend the current understanding of the mercury-mediated cytotoxic effect, five neural cell lines established from different animal species were comparatively analyzed using three different endpoint bioassays: thiazolyl blue tetrazolium bromide, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay (MTT), neutral red uptake assay (NRU), and Coomassie blue assay (CB). Following a 24-hr exposure to selected concentrations of mercury chloride (HgCl2) and methylmercury (II) chloride (MeHgCl), the cytotoxic effect on test cells was characterized by comparing their 50% inhibition concentration (IC50) values. Experimental results indicated that both these forms of mercury were toxic to all the neural cells, but at very different degrees. The IC50 values of MeHgCl among these cell lines ranged from 1.15±0.22 to 10.31±0.70μmol/L while the IC50 values for HgCl2 were much higher, ranging from 6.44±0.36 to 160.97±19.63μmol/L, indicating the more toxic nature of MeHgCl. The IC50 ratio between HgCl2 and MeHgCl ranged from 1.75 to 96.0, which confirms that organic mercury is much more toxic to these neural cells than inorganic mercury. Among these cell lines, HGST-BR and TriG44 derived from marine sea turtles showed a significantly high tolerance to HgCl2 as compared to the three mammalian neural cells. Among these neural cells, SK-N-SH represented the most sensitive cells to both chemical forms of mercury.

Scenario-targeted toxicity assessment through multiple endpoint bioassays in a soil posing unacceptable environmental risk according to regulatory screening values.

Lanestosa is a chronically polluted site (derelict mine) where the soil (Lanestosa (LA) soil) exceeds screening values (SVs) of regulatory policies in force (Basque Country; Europe) for Zn, Pb and Cd. A scenario-targeted toxicity assessment was carried out on the basis of a multi-endpoint bioassay approach. Acute and chronic toxicity bioassays were conducted with selected test species (Vibrio fischeri, Dictyostelium discoideum, Lactuca sativa, Raphanus sativus and Eisenia fetida) in combination with chemical analysis of soils and elutriates and with bioaccumulation studies in earthworms. Besides, the toxicity profile was compared with that of the mine runoff (RO) soil and of a fresh artificially polluted soil (LAAPS) resembling LA soil pollutant profile. Extractability studies in LA soil revealed that Pb, Zn and Cd were highly available for exchange and/or release into the environment. Indeed, Pb and Zn were accumulated in earthworms and LA soil resulted to be toxic. Soil respiration, V. fischeri, vegetative and developmental cycles of D. discoideum and survival and juvenile production of E. fetida were severely affected. These results confirmed that LA soil had unacceptable environmental risk and demanded intervention. In contrast, although Pb and Zn concentrations in RO soil revealed also unacceptable risk, both metal extractability and toxicity were much lower than in LA soil. Thus, within the polluted site, the need for intervention varied between areas that posed dissimilar risk. Besides, since LAAPS, with a high exchangeable metal fraction, was the most toxic, ageing under in situ natural conditions seemingly contributed to attenuate LA soil risk. As a whole, combining multi-endpoint bioassays with scenario-targeted analysis (including leaching and ageing) provides reliable risk assessment in soils posing unacceptable environmental risk according to SVs, which is useful to optimise the required intervention measures.

Toxicity assessment through multiple endpoint bioassays in soils posing environmental risk according to regulatory screening values.

Toxicity profiles of two soils (a brownfield in Legazpi and an abandoned iron mine in Zugaztieta; Basque Country) contaminated with several metals (As, Zn, Pb and Cu in Legazpi; Zn, Pb, Cd and Cu in Zugaztieta) and petroleum hydrocarbons (in Legazpi) were determined using a multi-endpoint bioassay approach. Investigated soils exceeded screening values (SVs) of regulatory policies in force (Basque Country; Europe). Acute and chronic toxicity bioassays were conducted with a selected set of test species (Vibrio fischeri, Dictyostelium discoideum, Lactuca sativa, Raphanus sativus and Eisenia fetida) in combination with chemical analysis of soils and elutriates, as well as with bioaccumulation studies in earthworms. The sensitivity of the test species and the toxicity endpoints varied depending on the soil. It was concluded that whilst Zugaztieta soil showed very little or no toxicity, Legazpi soil was toxic according to almost all the toxicity tests (solid phase Microtox, D. discoideum inhibition of fruiting body formation and developmental cycle solid phase assays, lettuce seed germination and root elongation test, earthworm acute toxicity and reproduction tests, D. discoideum cell viability and replication elutriate assays). Thus, albeit both soils had similar SVs, their ecotoxicological risk, and therefore the need for intervening, was different for each soil as unveiled after toxicity profiling based on multiple endpoint bioassays. Such a toxicity profiling approach is suitable to be applied for scenario-targeted soil risk assessment in those cases where applicable national/regional soil legislation based on SVs demands further toxicity assessment.

Evaluation of mercury mediated in vitro cytotoxicity among cell lines established from green sea turtles

In vitro cell cultures are currently tested for their application as a biological tool for enhanced monitoring and field evaluation of environmental toxic chemical pollution. Here cell lines established from green sea turtles (GSTs) were comparatively tested for their cytotoxic responses to mercury chloride (HgCl2) exposure and also their potential use as a biological tool for effective monitoring and screening of mercury contamination in environmental waters. Following a 24-h exposure to different concentrations of mercury solution, marine turtle cells were evaluated for their cytotoxic responses using three different endpoint bioassays: tetrazolium salt reduction (MTT), neutral red uptake (NR), and Coomassie blue (CB) methods. Cytotoxic sensitivities of GST cell lines to HgCl2 were determined and compared basing on their 50% inhibition concentration (IC50) values calculated from these tests. These marine turtle cells share a very different pattern of cytotoxic sensitivities and reactions to inorganic Mercury. Among these nine turtle cell lines, turtle liver cells (GST-LV) appear to be the most tolerant one to mercury exposure while turtle lung cells (GST-LG) exhibit to be the most sensitive one. Results from this in vitro study correlate well with in vivo examination of mercury concentration in the tissues of marine turtles and are also validated and ascertained by calculated regression equations showing a significant correlation (P<0.01) between these test methods. This study also reveals the cytotoxic effect of inorganic mercury on in vitro green turtle cells and also shows GST-LG to be a cell line with potential application in field monitoring and assessing mercury contamination as a bioindicator.

Further examination of toxicity profiles in contaminated soils posing potential risk to the environment: multiple endpoint bioassays

Further examination of toxicity profiles in contaminated soils posing potential risk to the environment: multiple endpoint bioassays

In vitro acute cytotoxicity of neonicotinoid insecticide imidacloprid to gill cell line of flounder Paralichthy olivaceus

In vitro acute cytotoxicity of neonicotinoid insecticide imidacloprid (IMI) to the gill cell line of flounder (FG) that collected in the gill of Paralichthys olivaceus, was examined by 3 widely used endpoint bioassays: NR (neutral red), MTT (3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) and TCP (total cell protein). The result shows that the IMI increased at concentrations ≥0.5 μg/ml. The IC50 value of NR. MTT, and TCP was 41.86, 38.46, and 39.08 μg/ml, respectively. The ultrastructural observation revealed that the mitochondria of the cells exposed to 60 μg/ml IMI for 48 h were severely damaged, swollen or disrupted, while their nuclei and rough endoplasmic reticulum (RER) remained normal. This would suggest that the mitochondria are probably the primary target of IMI.

Kinase receptor activation (KIRA): a rapid and accurate alternative to end-point bioassays

We have developed a novel strategy for a rapid bioassay that is accurate, precise, sensitive, and high capacity. It is capable of quantifying ligand bioactivity by measuring ligand-induced receptor tyrosine kinase activation in terms of receptor phosphorylation. The assay, termed a `kinase receptor activation' or KIRA, utilizes two separate microtiter plates, one for ligand stimulation of intact cells, and the other for receptor capture and phosphotyrosine ELISA. The assay makes use of either endogenously expressed receptors or stably transfected receptors with a polypeptide flag. KIRA assays for the ligands IGF-I and NGF were compared to their corresponding endpoint bioassays (3T3 cell proliferation for IGF-I and PC12 cell survival for NGF). The KIRA assays showed excellent correlation with the more classical endpoint bioassays. Further, they were highly reproducible, minimizing the requirement for repeat assays. The KIRA assay format has great potential as a rapid, accurate and precise bioassay, both for potency determination as well as stability-indicating analyses.

Waxes enhance Plutella xylostella oviposition in response to sinigrin and cabbage homogenates

AbstractOviposition by the diamondback moth, Plutella xylostella (Lepidoptera: Plutellidae), on substrates treated with host stimuli (cabbage homogenate or sinigrin) and/or waxes (paraffin or a mixture of 10 single chain n‐alkanes) was quantified using continuous observations and endpoint bioassays. Paraffin or an n‐alkane mixture applied over cabbage homogenate or sinigrin caused an increase in oviposition compared to that on any single stimulus in choice tests. Sinigrin alone at 10−5 M to 10−2 M is an ovipositional stimulant; addition of alkane over sinigrin made all sinigrin concentrations (10−6 M to 10−2 M) significantly more stimulatory than controls. Waxes alone do not stimulate oviposition. In choice tests, insect movement between sinigrin/alkane treatment combinations was random, however, once encountered, visit duration was significantly longer on sinigrin with alkane than on sites treated with either stimulus alone. Given the ubiquity of waxes on plant surfaces and the interaction between waxes and host‐specific chemical stimuli, waxes should be included when considering factors that significantly influence herbivore host acceptance.