Real-Time Cell-Electronic Sensing of Coal Fly Ash Particulate Matter for Toxicity-Based Air Quality Monitoring.

The development of a unique bioassay for cytotoxicity analysis of coal fly ash (CFA) particulate matter (PM) and its potential application for air quality monitoring is described. Using human cell lines, A549 and SK-MES-1, as live probes on microelectrode-embedded 96-well sensors, impedance changes over time are measured as cells are treated with varying concentrations (1 µg/mL-20 mg/mL) of CFA samples. A dose-dependent impedance change is determined for each CFA sample, from which an IC50 histogram is obtained. The assay was successfully applied to examine CFA samples collected from three coal-fired power plants (CFPs) in China. The samples were separated into three size fractions: PM2.5 (<2.5 µm), PM10-2.5 (2.5 µm < x < 10 µm), and PM10 (>10 µm). Dynamic cell-response profiles and temporal IC50 histograms of all samples show that CFA cytotoxicity depends on concentration, exposure time (0-60 h), and cell-type (SK-MES-1 > A549). The IC50 values differentiate the cytotoxicity of CFA samples based on size fraction (PM2.5 ≈ PM10-2.5 ≫ PM10) and the sampling location (CFP2 > CFP1 ≈ CFP3). Differential cytotoxicity measurements of particulates in human cell lines using cell-electronic sensing provide a useful tool for toxicity-based air quality monitoring and risk assessment.

Comparative cytotoxicity of fourteen trivalent and pentavalent arsenic species determined using real-time cell sensing.

The occurrence of a large number of diverse arsenic species in the environment and in biological systems makes it important to compare their relative toxicity. The toxicity of arsenic species has been examined in various cell lines using different assays, making comparison difficult. We report real-time cell sensing of two human cell lines to examine the cytotoxicity of fourteen arsenic species: arsenite (AsIII), monomethylarsonous acid (MMAIII) originating from the oxide and iodide forms, dimethylarsinous acid (DMAIII), dimethylarsinic glutathione (DMAGIII), phenylarsine oxide (PAOIII), arsenate (AsV), monomethylarsonic acid (MMAV), dimethylarsinic acid (DMAV), monomethyltrithioarsonate (MMTTAV), dimethylmonothioarsinate (DMMTAV), dimethyldithioarsinate (DMDTAV), 3-nitro-4-hydroxyphenylarsonic acid (Roxarsone, Rox), and 4-aminobenzenearsenic acid (p-arsanilic acid, p-ASA). Cellular responses were measured in real time for 72hr in human lung (A549) and bladder (T24) cells. IC50 values for the arsenicals were determined continuously over the exposure time, giving rise to IC50 histograms and unique cell response profiles. Arsenic accumulation and speciation were analyzed using inductively coupled plasma-mass spectrometry (ICP-MS). On the basis of the 24-hr IC50 values, the relative cytotoxicity of the tested arsenicals was in the following decreasing order: PAOIII≫MMAIII≥DMAIII≥DMAGIII≈DMMTAV≥AsIII≫MMTTAV>AsV>DMDTAV>DMAV>MMAV≥Rox≥p-ASA. Stepwise shapes of cell response profiles for DMAIII, DMAGIII, and DMMTAV coincided with the conversion of these arsenicals to the less toxic pentavalent DMAV. Dynamic monitoring of real-time cellular responses to fourteen arsenicals provided useful information for comparison of their relative cytotoxicity.

Analysis of inter-/intra-E-plate repeatability in the real-time cell analyzer.

Over the past decade, the real-time cell analyzer (RTCA) has provided a good tool to the cell-based in vitro assay. Unlike the traditional systems that label the target cells with luminescence, fluorescence, or light absorption, RTCA monitors cell properties using noninvasive and label-free impedance measuring. However, realization of the maximum value of RTCA for applications will require assurance of within-experiment repeatability, day-to-day repeatability, and robustness to variations in conditions that might occur from different experiments. In this article, the performance and variability of RTCA is evaluated and a novel repeatability index (RI) is proposed to analyze the intra-/inter-E-plate repeatability of RTCA. The repeatability assay involves six cell lines and two media (water [H2O] and dimethyl sulfoxide [DMSO]). First, six cell lines are exposed to the media individually, and time-dependent cellular response curves characterized as a cell index (CI) are recorded by RTCA. Then, the variations along sampling time and among repeated tests are calculated and RI values are obtained. Finally, a discriminating standard is set up to evaluate the degree of repeatability. As opposed to the standardized methodologies, it is shown that the presented index can give the quantitative evaluation for repeatability of RTCA within E-plate and variation on different days.

High-throughput screening assay for the environmental water samples using cellular response profiles.

Chemical and physical analyses are commonly used as screening methods for the environmental water. However, these methods can only look for the targeted substance but may miss unexpected toxicants. Furthermore, the synergistic effects of mixture cannot be detected. In order to set up the assay criteria for determining various biological activities at a cellular level that could potentially lead to toxicity of environmental water samples, a novel test based on cellular response by using Real-Time Cellular Analyzer (RTCA) is proposed in this study. First, the water sample is diluted to a series of strengths (80%, 60%, 40%, 30%, 20% and 10%) to get the multi-concentration cellular response profile. Then, the area under the cellular response profile (AUCRP) is calculated. Comparing to the normal cell growth of negative control, a new biological activity index named Percentage of Effect (PoE) has been presented which reflects the cumulative inhibitory activity of cell growth over the log-phase. Finally, a synthetical index PoE50 is proposed to evaluate the intensity of biological activities in water samples. The biological experiment demonstrates the effectiveness of the proposed method.

High-throughput quantitative analysis with cell growth kinetic curves for low copy number mutant cells.

The mutation rate in cells induced by environmental genotoxic hazards is very low and difficult to detect using traditional cell counting assays. The established genetic toxicity tests currently recognized by regulatory authorities, such as conventional Ames and hypoxanthine guanine phosphoribosyl-transferase (HPRT) assays, are not well suited for higher-throughput screening as they require large amounts of test compounds and are very time consuming. In this study, we developed a novel cell-based assay for quantitative analysis of low numbers of cell copies with HPRT mutation induced by an environmental mutagen. The HPRT gene mutant cells induced by the mutagen were selected by 6-thioguanine (6-TG) and the cell's kinetic growth curve monitored by a real-time cell electronic sensor (RT-CES) system. When a threshold is set at a certain cell index (CI) level, samples with different initial mutant cell copies take different amounts of time in order for their growth (or CI accumulation) to cross this threshold. The more cells that are initially seeded in the test well, the faster the cell accumulation and therefore the shorter the time required to cross this threshold. Therefore, the culture time period required to cross the threshold of each sample corresponds to the original number of cells in the sample. A mutant cell growth time threshold (MT) value of each sample can be calculated to predict the number of original mutant cells. For mutagenesis determination, the RT-CES assay displayed an equal sensitivity (p > 0.05) and coefficients of variation values with good correlation to conventional HPRT mutagenic assays. Most importantly, the RT-CES mutation assay has a higher throughput than conventional cellular assays.

Maternal and child biomonitoring strategies and levels of exposure in western Canada during the past seventeen years: The Alberta Biomonitoring Program: 2005-2021.

The Alberta Biomonitoring Program (ABP) was created in 2005 with the initial goal of establishing baseline levels of exposure to environmental chemicals in specific populations in the province of Alberta, Canada, and was later expanded to include multiple phases. The first two phases focused on evaluating exposure in pregnant women (Phase One, 2005) and children (Phase Two, 2004-2006) by analyzing residual serum specimens. Phase Three (2013-2016) employed active recruitment techniques to evaluate environmental exposures using a revised list of chemicals in paired serum pools from pregnant women and umbilical cord blood. These three phases of the program monitored a total of 226 chemicals in 285 pooled serum samples representing 31,529 individuals. Phase Four (2017-2020) of the ABP has taken a more targeted approach, focusing on the impact of the federal legalization of cannabis on the exposure of pregnant women in Alberta to cannabis, as well as tobacco and alcohol using residual prenatal screening serum specimens. Chemicals monitored in the first three phases include herbicides, neutral pesticides, metals, metalloids, and micronutrients, methylmercury, organochlorine pesticides, organophosphate pesticides, parabens, phthalate metabolites, perfluoroalkyl substances (PFAS), phenols, phytoestrogens, polybrominated compounds, polychlorinated biphenyls (PCBs), dioxins and furans, polycyclic aromatic hydrocarbons (PAHs), and tobacco biomarkers. Phase Four monitored six biomarkers of tobacco, alcohol, and cannabis. All serum samples were pooled. Mean concentrations and 95% confidence intervals (CIs) were calculated for the chemicals detected in ≥25% of the sample pools. cross the first three phases, the data from the ABP has provided baseline exposure levels for the chemicals in pregnant women, children, and newborns across the province. Comparison within and among the phases has highlighted differences in exposure levels with age, geography, seasonality, sample type, and time. The strategies employed throughout the program phases have been demonstrated to provide effective models for population biomonitoring.

Real-time cell-impedance sensing assay as an alternative to clonogenic assay in evaluating cancer radiotherapy.

Intrinsic radiosensitivity of normal and tumour tissues has been shown to be an independent prognostic factor for patients' response to radiotherapy. This study compares the real-time cell-impedance sensing (RT-CES) assay with the conventional clonogenic assay in terms of in-vitro radiosensitivity. One objective in this study was to predict in-vivo response to gold nanoparticle (GNP) treatment on the basis of in-vitro RT-CES testing results. Four adenocarcinoma cancer cell lines were tested using both the RT-CES and the clonogenic assays. Cell-survival curves were plotted, and the mean SF2 values obtained by these two different assay methods were compared using ANOVA. Radiation sensitivities obtained in-vitro were then compared with the in-vivo results. On the basis of the measurement of cell colonies, the RT-CES assay has similar radiosensitivity to the clonogenic assay, but significantly shortens the testing time from 14-21 days to only 72 h. Intrinsic GNP enhanced radiation sensitivity using tumour volume (mm(3)) in vivo is comparable with that using RT-CES cell survival assay in vitro. Furthermore, the RT-CES system provides real-time information regarding the state of cell radiosensitivity that may give useful information towards personalizing radiotherapy. The RT-CES assay enables more reliable and time-efficient results in the evaluation of radiosensitivity.

A multi-function public health surveillance system and the lessons learned in its development: the Alberta Real Time Syndromic Surveillance Net.

OBJECTIVE: We describe a centralized automated multi-function detection and reporting system for public health surveillance--the Alberta Real Time Syndromic Surveillance Net (ARTSSN). This improves upon traditional paper-based systems which are often fragmented, limited by incomplete data collection and inadequate analytical capacity, and incapable of providing timely information for public health action. METHODS: ARTSSN concurrently analyzes multiple electronic data sources in real time to describe results in tables, charts and maps. Detected anomalies are immediately disseminated via alerts to decision-makers for action. RESULTS: ARTSSN provides richly integrated information on a variety of health conditions for early detection of and prompt action on abnormal events such as clusters, outbreaks and trends. Examples of such health conditions include chronic and communicable disease, injury and environment-mediated adverse incidents. DISCUSSION: Key advantages of ARTSSN over traditional paper-based methods are its timeliness, comprehensiveness and automation. Public health surveillance of communicable disease, injury, environmental hazard exposure and chronic disease now occurs in a single system in real time year round. Examples are given to demonstrate the public health value of this system, particularly during Pandemic (H1N1) 2009.

Reliability of using urinary and blood trichloroacetic acid as a biomarker of exposure to chlorinated drinking water disinfection byproducts.

This study was designed to analyse the reliability of using urinary and blood trichloroacetic acid (TCAA) as a biomarker of exposure. A total of 46 healthy women consumed supplied TCAA-containing tap water for 15 days and provided urine and blood samples for TCAA measurements. The findings revealed that the reliability of measurements was excellent by using measures of TCAA ingestion, blood concentration and urinary excretion (intraclass correlation coefficients (ICC) > 0.75, p < 0.001). Volume of tap water consumption (ICC = 0.69) and creatinine-adjusted urinary concentration (ICC = 0.72) were less reliable. This indicated that the intraindividual variability was small and the interindividual reliability was high by using these measures in this cohort study. Laboratory variability did not significantly contribute to total variance (ICC > 0.95, p < 0.001). Other possible sources of variation such as bathing, showering, dishwashing and physical activities were unlikely to contribute significantly to total variance. For sampling strategies, 1-day blood sampling and 2-day urine sampling are sufficient to achieve reliability for an epidemiological study if a quasi-steady-state TCAA level in the body is reached. The results suggest that TCAA ingestion, TCAA loading in blood and urinary TCAA excretion are reliable measures for use as biomarkers in epidemiological studies.

Validation of urinary trichloroacetic acid as a biomarker of exposure to drinking water disinfection by-products.

Disinfection by-products (DBPs) in drinking water represent a public health issue and a challenge for epidemiology to provide evidence towards the causation of various hypothesized health effects. Validation of a biomarker of exposure to DBPs is a strategy to achieve progress which has been advocated. The objective of this study was to validate urinary trichloroacetic acid (TCAA) excretion as a biomarker of exposure to DBPs in an experimental exposure cohort. A total of 52 healthy women participated in the study. Participants consumed supplied tap water for 15 d and provided urine and blood samples for TCAA measurements. The findings revealed that (1) background levels of TCAA in urine and blood were readily detectable, (2) TCAA levels in blood and urine increased with increased amounts of TCAA ingested, (3) the correlations between measurements of TCAA ingestion and urinary excretion were modest (r=0.66, p<0.001) based on one days' sampling and high (r=0.77-0.83, p<0.001) based on two to four days' sampling, (4) the correlations between measurements of TCAA ingestion and blood TCAA concentration were high (r=0.80, p<0.001) and (5) multiple days' urinary TCAA measures improved the prediction of TCAA ingestion through urinary TCAA excretion. TCAA can be a valid biomarker of exposure for DBPs in drinking water.

Modeling and simulation for toxicity assessment.

The effect of various toxicants on growth/death and morphology of human cells is investigated using the xCELLigence Real-Time Cell Analysis High Troughput in vitro assay. The cell index is measured as a proxy for the number of cells, and for each test substance in each cell line, time-dependent concentration response curves (TCRCs) are generated. In this paper we propose a mathematical model to study the effect of toxicants with various initial concentrations on the cell index. This model is based on the logistic equation and linear kinetics. We consider a three dimensional system of differential equations with variables corresponding to the cell index, the intracellular concentration of toxicant, and the extracellular concentration of toxicant. To efficiently estimate the model's parameters, we design an Expectation Maximization algorithm. The model is validated by showing that it accurately represents the information provided by the TCRCs recorded after the experiments. Using stability analysis and numerical simulations, we determine the lowest concentration of toxin that can kill the cells. This information can be used to better design experimental studies for cytotoxicity profiling assessment.

Chromium on the hands of children after playing in playgrounds built from chromated copper arsenate (CCA)-treated wood.

Children's exposure to arsenic and chromium from playground equipment constructed with chromated copper arsenate (CCA)-treated wood is a potential concern because of children's hand-to-mouth activity. However, there exists no direct measure of Cr levels on the hands of children after playing in such playgrounds. In this study we measured both soluble and total Cr on the hands of 139 children playing in playgrounds, eight of which were constructed with CCA-treated wood and eight of which were not. Children's age and duration of play were recorded. The hands of each child were washed after play with 150 mL deionized water, which was collected in a bag and subsequently underwent analysis of Cr and 20 other elements, using inductively coupled plasma mass spectrometry. Total average Cr on the hands of 63 children who played in CCA playgrounds was 1,112 +/- 1,089 ng (median, 688; range 78-5,875). Total average Cr on the hands of 64 children who played in non-CCA playgrounds was 652 +/- 586 ng (median, 492; range 61-3,377). The difference between the two groups is statistically significant (p < 0.01). Cr levels were highly correlated to both Cu (r = 0.672) and As (r = 0.736) levels in CCA playgrounds (p < or = 0.01), but not non-CCA playgrounds (r = 0.252 and 0.486 for Cu and As, respectively). Principal-component analysis indicates that Cr, Cu, and As are more closely grouped together in CCA than in non-CCA playgrounds. These results suggest that the elevated levels of Cr and As on children's hands are due to direct contact with CCA wood.

Microelectronic cell sensor assay for detection of cytotoxicity and prediction of acute toxicity.

This study reports in-house assessment of a real-time cell electronic sensing (RT-CES) system used as a test platform for both cytotoxicity assay and predicting acute toxicity. For cytotoxicity determination, the RT-CES assay displayed equal sensitivity and coefficients of variation values with good correlation to NRU assay. The IC50 values and the LD50 values for the cytotoxicity reference materials were compared in the context of the proposed prediction model for acute rodent toxicity. The results obtained from RT-CES assay fitted within the acceptance limits of the prediction model and showed that the RT-CES cytotoxicity assay met the qualification guidelines in NIH Publication #01-4500 to accurately predict acute toxicity. In addition to cell viability, the RT-CES assay provided dynamic information that can be used to identify maximum toxicity and reversibility of the toxic effects which are difficult to achieve by the endpoint assays and, therefore, the RT-CES assay is more accurate for assessment of cytotoxicity. The features of the RT-CES assay, such as labeling free, automatic detection, and easy operation, give this assay potential to replace BALB/c 3T3 NRU assay and be used as routine setting for drug monitoring in the toxicological laboratory.

Predicting GHS toxicity using RTCA and discrete-time Fourier transform.

In order to promote the acceptance of cell-based toxicity testings, the accuracy of cytotoxicity test must be determined when compared to in vivo results. Traditional methods of cytotoxicity analysis, such as LC[Formula: see text] (concentration where 50% of the cells are killed) can be problematic since they have been found to vary with time. Technological advances in cytotoxicity testing make it easy to record the dynamic data on changes in cell proliferation, morphology, and damage. To effectively and reasonably analyze the dynamic data, we present a new in vitro toxicity assessed method using the discrete-time Fourier transform (DTFT) which maps the measured cell index from the time domain to the frequency domain. The direct current (DC) component of the DTFT is extracted as a feature which reflects the intensity of cytotoxicity. The smaller the value, the higher the cytotoxicity. Then, a novel toxicity index, as expressed in terms of DC[Formula: see text], is calculated. Results generated with selected test chemicals are compared favorably with data obtained from The Interagency Coordinating Committee on the Validation of Alternative Method (ICCVAM) report concerning the prediction of acute systemic toxicity in rodents. The method can be applied with the standard and high throughput to estimate acute rodent oral toxicity which reduces the number of animals required in subsequent pharmacological/toxicological studies.

Machine learning algorithms for mode-of-action classification in toxicity assessment.

BACKGROUND: Real Time Cell Analysis (RTCA) technology is used to monitor cellular changes continuously over the entire exposure period. Combining with different testing concentrations, the profiles have potential in probing the mode of action (MOA) of the testing substances. RESULTS: In this paper, we present machine learning approaches for MOA assessment. Computational tools based on artificial neural network (ANN) and support vector machine (SVM) are developed to analyze the time-concentration response curves (TCRCs) of human cell lines responding to tested chemicals. The techniques are capable of learning data from given TCRCs with known MOA information and then making MOA classification for the unknown toxicity. A novel data processing step based on wavelet transform is introduced to extract important features from the original TCRC data. From the dose response curves, time interval leading to higher classification success rate can be selected as input to enhance the performance of the machine learning algorithm. This is particularly helpful when handling cases with limited and imbalanced data. The validation of the proposed method is demonstrated by the supervised learning algorithm applied to the exposure data of HepG2 cell line to 63 chemicals with 11 concentrations in each test case. Classification success rate in the range of 85 to 95 % are obtained using SVM for MOA classification with two clusters to cases up to four clusters. CONCLUSIONS: Wavelet transform is capable of capturing important features of TCRCs for MOA classification. The proposed SVM scheme incorporated with wavelet transform has a great potential for large scale MOA classification and high-through output chemical screening.

Development of a microelectronic chip array for high-throughput genotyping of Helicobacter species and screening for antimicrobial resistance.

A microelectronic array assay was developed to specifically genotype Helicobacter pylori versus Helicobacter heilmannii and to determine antimicrobial resistance. Helicobacter 16S rRNA and 23S rRNA genes were specifically generated with Helicobacter genus-specific primers, respectively. The single-nucleotide polymorphisms (SNPs) in 16S rRNA, 268T specific in the H. pylori sequence, and 263A specific in H. heilmannii were used as molecular markers for identification of H. pylori and H. heilmannii, respectively. A triple-base-pair resistant mutation, AGA965-967TTC in 16S rRNA, is known to be responsible for H. pylori tetracycline resistance and was detected to identify resistant strains. H. pylori macrolide resistance was determined by the identification of 3 defined mutations in the 23S rRNA gene using the same method. The assay could be directly used to detect H. pylori in feces. The assay performs multiple determinations, including identification of Helicobacter species and antibiotic resistances, on the same microelectronic platform and is highly amenable to the development of other DNA-based assays.

Potential factors that may affect acceptance of routine prenatal HIV testing.

BACKGROUND: Despite increasing advocacy for an "opt-out" strategy in routine prenatal HIV screening programs in Canada, no published studies have examined factors that may affect acceptance of prenatal HIV testing. METHODS: We included all pregnant women in Alberta who received prenatal care (N = 38,712) and their caregivers (N = 2,007) between January 1 and November 30, 2000. Factors associated with non-acceptance of HIV testing in both pregnant women and their caregivers were assessed using multivariate logistic regression. RESULTS: Overall, 1.5% of women declined HIV testing. First Nations women were about twice as likely to decline the test (adjusted odds ratio [OR(adj)] 1.91, 95% CI [1.42-2.58]) compared to non-First Nations women (p < 0.001). The proportion also increased with age (chi2 trend p < 0.001) in the general population. In First Nations women, however, most (3.2%) declined in the 20-24 year age group. No significant effect was seen for a socio-economic status marker or for the place of residence. The caregivers of women who declined HIV testing were more likely to be female (OR(adj) 1.56 [1.28-1.89]), midwives (OR(adj) 140.65 [58.61-337.49]), other non-obstetrical medical specialties (OR(adj) 4.92 [1.94-12.47]), and general practitioners (OR(adj) 3.44 [1.87-6.33]). CONCLUSION: In an "opt-out" routine prenatal HIV screening program, the characteristics of both the pregnant women and their caregivers may contribute to the non-acceptance of HIV testing. A higher likelihood of declining HIV testing among First Nations pregnant women and other pregnant women under the care of midwives and female physicians warrants further study.

Arsenic on the hands of children after playing in playgrounds.

Increasing concerns over the use of wood treated with chromated copper arsenate (CCA) in playground structures arise from potential exposure to arsenic of children playing in these playgrounds. Limited data from previous studies analyzing arsenic levels in sand samples collected from CCA playgrounds are inconsistent and cannot be directly translated to the amount of children's exposure to arsenic. The objective of this study was to determine the quantitative amounts of arsenic on the hands of children in contact with CCA-treated wood structures or sand in playgrounds. We compared arsenic levels on the hands of 66 children playing in eight CCA playgrounds with levels of arsenic found on the hands of 64 children playing in another eight playgrounds not constructed with CCA-treated wood. The children's age and duration of playtime were recorded at each playground. After play, children's hands were washed in a bag containing 150 mL of deionized water. Arsenic levels in the hand-washing water were quantified by inductively coupled plasma mass spectrometry. Our results show that the ages of the children sampled and the duration of play in the playgrounds were similar between the groups of CCA and non-CCA playgrounds. The mean amount of water-soluble arsenic on children's hands from CCA playgrounds was 0.50 microg (range, 0.0078-3.5 microg). This was significantly higher (p < 0.001) than the mean amount of water-soluble arsenic on children's hands from non-CCA playgrounds, which was 0.095 microg (range, 0.011-0.41 microg). There was no significant difference in the amount of sand on the children's hands and the concentration of arsenic in the sand between the CCA and non-CCA groups. The higher values of arsenic on the hands of children playing in the CCA playgrounds are probably due to direct contact with CCA-treated wood. Washing hands after play would reduce the levels of potential exposure because most of the arsenic on children's hands was washed off with water. The maximum amount of arsenic on children's hands from the entire group of study participants was < 4 microg, which is lower than the average daily intake of arsenic from water and food.

Mode of action classification of chemicals using multi-concentration time-dependent cellular response profiles.

In this paper, we present a new statistical pattern recognition method for classifying cytotoxic cellular responses to toxic agents. The advantage of the proposed method is to quickly assess the toxicity level of an unclassified toxic agent on human health by bringing cytotoxic cellular responses with similar patterns (mode of action, MoOA) into the same class. The proposed method is a model-based hierarchical classification approach incorporating principal component analysis (PCA) and functional data analysis (FDA). The cytotoxic cell responses are represented by multi-concentration time-dependent cellular response profiles (TCRPs) which are dynamically recorded by using the xCELLigence real-time cell analysis high-throughput (RTCA HT) system. The classification results obtained using our algorithm show satisfactory discrimination and are validated using biological facts by examining common chemical mechanisms of actions with treatment on human hepatocellular carcinoma cells (HepG2).

Real-time cell-microelectronic sensing of nanoparticle-induced cytotoxic effects.

We report a real-time cell analysis (RTCA) sensing method of 96 electronic microwells for profiling the cytotoxicity of nanoparticles on different cell lines. The method consists of 96 microwells embedded with microelectrodes (96x E-plate) to measure impedance changes of adherent cell lines. When the testing cells change in population, adhesion, and/or morphology, the impedance at the cell-electrode interface changes to provide real-time monitoring of overall cell status. To demonstrate this technique, we used three cell lines as sensing probes: two human lung carcinoma cell lines, A549 and SK-MES-1, and a normal mammalian cell line, CHO-K1. We tested two well-characterized nanoparticles: nano-titanium dioxide (nTiO2) and nano-silver (nAg). The three cell lines were separately seeded into 96x E-plates and treated with varying concentrations of nanoparticles (0.078-160 µg mL(-1)). This method provides dynamic cell response profiles and temporal IC50 histograms, showing concentration-, time-, particle-, and cell-dependent cytotoxicity. The 24 h and 48 h IC50 values of nAg obtained using both the RTCA and the neutral red uptake (NRU) assays were in good agreement, validating the RTCA technique. The RTCA assay does not suffer interference from nTiO2, whereas the NRU assay cannot be used due to severe interference from nTiO2. A cytostatic response was observed in CHO-K1 cells after 24 h exposure to 40 µg mL(-1) nTiO2, which was correlated with S-phase cell cycle arrest based on cell cycle analysis using flow cytometry. This suggests that the shapes of the response curves provide indicative information, directing further studies into the mode of action of the toxicant. Advantages of the RTCA technique over traditional colorimetric assays for screening the cytotoxicity of nanoparticles include minimizing interference, qualitative and quantitative cytotoxicity data, and the capability of real-time and high-throughput measurements.