A microelectric cell sensing technique for in vitro assessment of ocular irritation.

The animal-based Draize test remains the gold standard for assessment of ocular irritation. However, subjective scoring methods, species differences, and animal welfare concerns have spurred development of alternative test methods. In this study, a novel in vitro method for assessing ocular irritancy was developed using a microelectric cell sensing technology, real-time cell analysis (RTCA). The cytotoxicity of sixteen compounds was assessed in two cell lines: ARPE-19 (human retina) and SIRC (rabbit cornea). In vitro inhibitory (IC50 and AUC50) values were determined at 6, 12, 24, 48, 72, and 96 h exposure, with a subset of values confirmed with MTT testing. The values displayed comparable predictivity of in vivo ocular irritation on the basis of a linear regression between the calculated values and each compounds' corresponding Draize-determined modified maximum average score (MMAS), but the ARPE-19 derived values were more strongly correlated than those from SIRC cells. Hence, IC50 values derived from ARPE-19 cells were used to predict the UN GHS/EU CLP classification of each test compound. The method was determined to have sensitivity of 90%, specificity of 50%, and overall concordance of 75%. Thus, RTCA testing may be best incorporated into a top-down tiered testing strategy for identification of ocular irritants in vitro.

A hidden risk: Survival and resuscitation of Escherichia coli O157:H7 in the viable but nonculturable state after boiling or microwaving.

We report the existence and resuscitation of viable but nonculturable (VBNC) Escherichia coli O157:H7 cells in drinking water induced by the common point-of-use disinfection treatments of boiling or microwaving. Tap water and saline samples containing E. coli O157:H7 culturable cells from a bovine isolate or two clinical isolates were boiled (1, 10, or 15 min) on a hot plate or microwaved (1.5 min) to reach boiling. No culturable E. coli O157:H7 cells were observed in the treated samples using conventional plating methods. In samples boiled for 1 or 10 min, two viability assays separately detected that 2-5.5% of the cells retained an intact membrane, while 28 to 87 cells out of the initial 108 cells retained both measurable intracellular esterase activity and membrane integrity. In samples boiled for 15 min, no viable cells were detected. The microwaved samples contained 6-10% of cells with an intact membrane, while 21 to 108 cells out of the initial 108 cells retained both membrane integrity and esterase activity. The number of viable cells retaining both metabolic activity and membrane integrity were consistent in all samples, supporting the survival of a small number of E. coli O157:H7 cells in the VBNC state after boiling for 1 or 10 min or microwaving. Furthermore, the VBNC E. coli O157:H7 cells regained growth at 37 °C in culture media containing autoinducers produced by common non-pathogenic E. coli, commonly present in the human intestine, and norepinephrine. The resuscitated cells were culturable on conventional plates and expressed mRNA encoding the E. coli O157 lipopolysaccharide gene (rfbE) and the H7 flagellin gene (fliC). This study highlights potential concerns for public health risk management of VBNC E. coli O157:H7 in drinking water disinfected by heat treatment at point-of-use. The public health significance of these concerns warrants further investigation.

Cytotoxicity of Halogenated Tyrosyl Compounds, an Emerging Class of Disinfection Byproducts.

Halogenated amino acids and peptides are an emerging class of disinfection byproducts (DBPs), having been detected in drinking water and in washed food products. However, the toxicological significance of these emerging DBPs remains unclear. In this study, the cytotoxicity of eight halogenated tyrosyl compounds was investigated in Chinese hamster ovary (CHO) cells using real-time cell analysis (RTCA). Dihalogenated tyrosyl compounds are more cytotoxic than their monohalogenated analogues. The cytotoxicity of the dihalogenated compounds is associated with their ability to induce intracellular reactive oxygen species (ROS), suggesting that oxidative stress is an important toxicity pathway of these compounds. Pearson correlation analysis of the cytotoxicity (IC50 values) of these compounds with eight physicochemical parameters showed strong associations with their lipophilicity (logP) and reactivity (polarizability, ELUMO). Finally, cytotoxicity testing of the concentrated extracts of a chloraminated mixture of eight dipeptides with bromide or iodide showed the cytotoxicity of these mixtures in the order: iodinated peptides > brominated peptides ≥ chlorinated peptides. These results demonstrate that halogenated peptide DBPs are toxicologically relevant, and further research is needed to understand the implications of long-term exposure for human health.

Halobenzoquinone-Induced Developmental Toxicity, Oxidative Stress, and Apoptosis in Zebrafish Embryos.

The developmental toxicity of water disinfection byproducts remains unclear. Here we report the study of halobenzoquinone (HBQ)-induced in vivo developmental toxicity and oxidative stress using zebrafish embryos as a model. Embryos were exposed to 0.5-10 µM of individual HBQs and 0.5-5 mM haloacetic acids for up to 120 h postfertilization (hpf). LC50 values of the HBQs at 24 hpf were 4.6-9.8 µM, while those of three haloacetic acids were up to 200 times higher at 1900-2600 µM. HBQ exposure resulted in significant developmental malformations in larvae, including failed inflation of the gas bladder, heart malformations, and curved spines. An increase in reactive oxygen species was observed, together with a decrease in superoxide dismutase activity and glutathione content. Additionally, the antioxidant N-acetyl-l-cysteine significantly mitigated all HBQ-induced effects, supporting that oxidative stress contributes to HBQ toxicity. Further experiments examined HBQ-induced effects on DNA and genes. HBQ exposure increased 8-hydroxydeoxyguanosine levels, DNA fragmentation, and apoptosis in larvae, with apoptosis induction related to changes in the gene expression of p53 and mdm2. These results suggest that HBQs are acutely toxic, causing oxidative damage and developmental toxicity to zebrafish larvae.

Halobenzoquinone-Induced Alteration of Gene Expression Associated with Oxidative Stress Signaling Pathways.

Halobenzoquinones (HBQs) are emerging disinfection byproducts (DBPs) that effectively induce reactive oxygen species and oxidative damage in vitro. However, the impacts of HBQs on oxidative-stress-related gene expression have not been investigated. In this study, we examined alterations in the expression of 44 genes related to oxidative-stress-induced signaling pathways in human uroepithelial cells (SV-HUC-1) upon exposure to six HBQs. The results show the structure-dependent effects of HBQs on the studied gene expression. After 2 h of exposure, the expression levels of 9 to 28 genes were altered, while after 8 h of exposure, the expression levels of 29 to 31 genes were altered. Four genes ( HMOX1, NQO1, PTGS2, and TXNRD1) were significantly upregulated by all six HBQs at both exposure time points. Ingenuity pathway analysis revealed that the Nrf2 pathway was significantly responsive to HBQ exposure. Other canonical pathways responsive to HBQ exposure included GSH redox reductions, superoxide radical degradation, and xenobiotic metabolism signaling. This study has demonstrated that HBQs significantly alter the gene expression of oxidative-stress-related signaling pathways and contributes to the understanding of HBQ-DBP-associated toxicity.

Formation of water disinfection byproduct 2,6-dichloro-1,4-benzoquinone from chlorination of green algae.

We report that green algae in lakes and rivers can serve as precursors of halobenzoquinone (HBQ) disinfection byproducts (DBPs) produced during chlorination. Chlorination of a common green alga, Chlorella vulgaris, produced 2,6-dichloro-1,4-benzoquinone (2,6-DCBQ), the most prevalent HBQ DBP in disinfected water. Under varying pH conditions (pH6.0-9.0), 2,6-DCBQ formation ranged from 0.3 to 2.1µg/mg C with maximum formation at pH8.0. To evaluate the contribution of organic components of C. vulgaris to 2,6-DCBQ formation, we separated the organics into two fractions, the protein-rich fraction of intracellular organic matter (IOM) and the polysaccharide-laden fraction of extracellular organic matter (EOM). Chlorination of IOM and EOM produced 1.4µg/mg C and 0.7µg/mg C of 2,6-DCBQ, respectively. The IOM generated a two-fold higher 2,6-DCBQ formation potential than the EOM fraction, suggesting that proteins are potent 2,6-DCBQ precursors. This was confirmed by the chlorination of proteins extracted from C. vulgaris: the amount of 2,6-DCBQ produced is linearly correlated with the concentration of total algal protein (R2=0.98). These results support that proteins are the primary precursors of 2,6-DCBQ in algae, and control of green algal bloom outbreaks in source waters is important for management of HBQ DBPs.

Metabolism of a Phenylarsenical in Human Hepatic Cells and Identification of a New Arsenic Metabolite.

Environmental contamination and human consumption of chickens could result in potential exposure to Roxarsone (3-nitro-4-hydroxyphenylarsonic acid), an organic arsenical that has been used as a chicken feed additive in many countries. However, little is known about the metabolism of Roxarsone in humans. The objective of this research was to investigate the metabolism of Roxarsone in human liver cells and to identify new arsenic metabolites of toxicological significance. Human primary hepatocytes and hepatocellular carcinoma HepG2 cells were treated with 20 or 100 µM Roxarsone. Arsenic species were characterized using a strategy of complementary chromatography and mass spectrometry. The results showed that Roxarsone was metabolized to more than 10 arsenic species in human hepatic cells. A new metabolite was identified as a thiolated Roxarsone. The 24 h IC50 values of thiolated Roxarsone for A549 lung cancer cells and T24 bladder cancer cells were 380 ± 80 and 42 ± 10 µM, respectively, more toxic than Roxarsone, whose 24 h IC50 values for A549 and T24 were 9300 ± 1600 and 6800 ± 740 µM, respectively. The identification and toxicological studies of the new arsenic metabolite are useful for understanding the fate of arsenic species and assessing the potential impact of human exposure to Roxarsone.

Multidrug Resistance Protein 4 (MRP4/ABCC4) Protects Cells from the Toxic Effects of Halobenzoquinones.

Halobenzoquinones (HBQs) are frequently detected disinfection byproducts (DBPs) in treated water. Recent studies have demonstrated that HBQs are highly cytotoxic and capable of inducing the generation of reactive oxygen species (ROS) and depleting cellular glutathione (GSH). Multidrug resistance proteins (MRPs/ABCCs) are known to play a critical role in the elimination of numerous drugs, carcinogens, toxicants, and their conjugated metabolites. In general, little is known about the roles of transporters in DBP toxicity. Here, we hypothesize that MRPs may play roles in the detoxication of HBQs. To test this hypothesis, we used human embryonic kidney 293 (HEK293) cells stably expressing MRPs (MRP1, 3, 4, and 5) and HEK293 cells with empty vector (HEK-V) to examine the comparative cytotoxicity of four HBQs: 2,6-dichloro-1,4-benzoquinone (2,6-DCBQ), 2,6-dibromo-1,4-benzoquinone (2,6-DBBQ), 2,6-dichloro-3-methyl-1,4-benzoquinone (DCMBQ), and 2,3,6-trichloro-1,4-benzoquinone (TriCBQ). The cytotoxicity (IC50) of the four HBQs in HEK-MRP1, -MRP3, -MRP4, and -MRP5 cells and the control HEK-V cells clearly showed that MRP4 had the most significant effect on reducing the toxicity of the four HBQs. To further support MRP4-mediated detoxication of HBQs, we examined the HBQ-induced ROS levels in HEK-MRP4 and HEK-V cells. ROS levels were significantly reduced in HEK-MRP4 cells compared with HEK-V cells after HBQ treatment. Furthermore, it was found that MRP4-mediated detoxication of the HBQs was GSH dependent, as the cytotoxicity of the HBQs was increased in GSH-depleted HEK-MRP4 cells in comparison to HEK-MRP4 cells. The GSH-dependent protection of cells from HBQs supports the possibility of HBQ-GSH conjugate efflux by MRP4. This study demonstrates a role for MRP4 in cellular protection against HBQ DBP-induced toxicity and oxidative stress.

Effects of halobenzoquinone and haloacetic acid water disinfection byproducts on human neural stem cells.

Human neural stem cells (hNSCs) are a useful tool to assess the developmental effects of various environmental contaminants; however, the application of hNSCs to evaluate water disinfection byproducts (DBPs) is scarce. Comprehensive toxicological results are essential to the prioritization of DBPs for further testing and regulation. Therefore, this study examines the effects of DBPs on the proliferation and differentiation of hNSCs. Prior to DBP treatment, characteristic protein markers of hNSCs from passages 3 to 6 were carefully examined and it was determined that hNSCs passaged 3 or 4 times maintained stem cell characteristics and can be used for DBP analysis. Two regulated DBPs, monobromoacetic acid (BAA) and monochloroacetic acid (CAA), and two emerging DBPs, 2,6-dibromo-1,4-benzoquinone (2,6-DBBQ) and 2,6-dichloro-1,4-benzoquinone (2,6-DCBQ), were chosen for hNSC treatment. Both 2,6-DBBQ and 2,6-DCBQ induced cell cycle arrest at S-phase at concentrations up to 1µmol/L. Comparatively, BAA and CAA at 0.5µmol/L affected neural differentiation. These results suggest DBP-dependent effects on hNSC proliferation and differentiation. The DBP-induced cell cycle arrest and inhibition of normal hNSC differentiation demonstrate the need to assess the developmental neurotoxicity of DBPs.

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.

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.

Emerging Disinfection Byproducts, Halobenzoquinones: Effects of Isomeric Structure and Halogen Substitution on Cytotoxicity, Formation of Reactive Oxygen Species, and Genotoxicity.

Halobenzoquinones (HBQs) are a structurally diverse class of water disinfection byproducts. Here, we report a systematic study on the effects of isomeric structure and the type and number of halogen substitutions of HBQs on their cytotoxicity, formation of reactive oxygen species (ROS), and genotoxicity. Dynamic responses and IC50 histograms were obtained using real-time cell analysis, clearly ranking the cytotoxicity of the HBQs in Chinese hamster ovary (CHO-K1) cells. Strong isomeric structure effects were shown with 2,5-HBQ isomers inducing greater cytotoxicity than their corresponding 2,6-HBQ isomers (P < 0.05). HBQ-halogen substitution groups also influence cytotoxicity, as cytotoxicity increases across the dihalogenated HBQs: iodo- > bromo- > chloro-HBQs (P < 0.05). Determination of HBQ-induced ROS further supports isomeric structure and halogen substitution effects. HBQ-induced genotoxicity was shown as increased levels of 8-hydroxy-2'-deoxyguanosine and p53 protein. Pearson correlation analysis of the HBQ toxicity measurements with their physicochemical parameters demonstrates that dipole moment and the lowest unoccupied molecular orbital energy are two major structural influences on toxicity (r = -0.721 or -0.766, P < 0.05). Dipole moment also correlates with isomer toxicity. This study suggests that formation and occurrence of highly toxic iodo-HBQs and 2,5-HBQs warrant further investigation to fully assess the impact of HBQs in drinking water.

Chemical and toxicological characterization of halobenzoquinones, an emerging class of disinfection byproducts.

Halobenzoquinones (HBQs), a new class of disinfection byproducts (DBPs), occur widely in treated drinking water and recreational water. The main concern regarding human exposure to DBPs stems from epidemiological studies that have consistently linked the consumption of chlorinated drinking water with an increased risk of developing bladder cancer. The U.S. Environmental Protection Agency and Health Canada have set regulations on the amount of DBPs in drinking water to minimize the risk. However, these regulated DBPs do not account for the increased risk of bladder cancer because they have different target organs or lower magnitudes of risk based on animal carcinogenesis studies. Because of the pervasive exposure to DBPs, identification of DBPs relevant to human health has become one of the important research targets to address DBP-associated health concerns. Quantitative structure-toxicity relationship (QSTR) analysis has predicted HBQs to be potential bladder carcinogens. Therefore, this perspective focuses on the chemical and toxicological characterization of HBQs. In vitro cytotoxicity experiments have shown that HBQs induce greater cytotoxicity and/or greater developmental toxicity than most of the regulated DBPs. Cellular mechanistic studies indicate that HBQs are capable of producing reactive oxygen species (ROS) either within cells or in solution, depleting cellular glutathione levels, and influencing cellular antioxidant enzymes, which further induces oxidative stress and oxidative damage to cellular proteins and DNA. Oxidative damage to DNA was demonstrated in the form of significant increases in cellular levels of 8-hydroxydeoxyguanosine (8-OHdG), DNA strand breaks, and apurinic/apyrimidinic (AP) sites. HBQs can also form DNA adducts, affect genome-wide DNA methylation, and inhibit DNA repair enzymes. These findings demonstrate that HBQs are highly cytotoxic and potentially genotoxic and carcinogenic, although in vivo data corroborating this is not available. To fully understand the potential adverse health effects and cancer risk due to HBQ exposure, multidisciplinary research is required regarding human exposure, health risk assessment, and toxicological mechanisms of HBQs.

Analytical and toxicity characterization of halo-hydroxyl-benzoquinones as stable halobenzoquinone disinfection byproducts in treated water.

Exposure to chlorination disinfection byproducts (DBPs) is potentially associated with an increased risk of bladder cancer. Four halobenzoquinones (HBQs) have been detected in treated drinking water and have shown potency in producing reactive oxygen species and inducing damage to cellular DNA and proteins. These HBQs are unstable in drinking water. The fate and behavior of these HBQs in drinking water distribution systems is unclear. Here we report the high-resolution mass spectrometry identification of the transformation products of HBQs as halo-hydroxyl-benzoquinones (OH-HBQs) in water under realistic conditions. To further examine the kinetics of transformation, we developed a solid-phase extraction with ultrahigh-performance liquid chromatography tandem mass spectrometry (SPE-UHPLC-MS/MS) method to determine both the HBQs and OH-HBQs. The method provides reproducible retention times (SD < 0.05 min), limits of detection (LODs) at subnanogram per liter levels, and recoveries of 68%-96%. Using this method, we confirmed that decrease of HBQs correlated with increase of OH-HBQs in both the laboratory experiments and several distribution systems, supporting that OH-HBQs were more stable forms of HBQ DBPs. To understand the toxicological relevance of the OH-HBQs, we studied the in vitro toxicity with CHO-K1 cells and determined the IC50 of HBQs and OH-HBQs ranging from 15.9 to 72.9 µM. While HBQs are 2-fold more toxic than OH-HBQs, both HBQs and OH-HBQs are substantially more toxic than the regulated DBPs.

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.

Cytotoxicity and oxidative damage induced by halobenzoquinones to T24 bladder cancer cells.

Four halobenzoquinones (HBQs), 2,6-dichloro-1,4-benzoquinone (DCBQ), 2,6-dichloro-3-methyl-1,4-benzoquinone (DCMBQ), 2,3,6-trichloro-1,4-benzoquinone (TCBQ), and 2,6-dibromobenzoquinone (DBBQ), have been recently confirmed as disinfection byproducts (DBPs) in drinking water; however, their toxicological information is scarce. Here, we report that HBQs are cytotoxic to T24 bladder cancer cells and that the IC50 values are 95 µM for DCBQ, 110 µM for DCMBQ, 151 µM for TCBQ, and 142 µM for DBBQ, after a 24-h exposure. The antioxidant N-acetyl-l-cysteine (NAC) significantly reduces the cytotoxicity induced by the four HBQs, supporting the hypothesis that oxidative stress contributes to the cytotoxicity of HBQs. To further explore the oxidative mechanisms of cytotoxicity, we examined HBQ-induced production of reactive oxygen species (ROS) in T24 cells, and measured 8-hydroxydeoxyguanosine (8-OHdG), protein carbonyls, and malondialdehyde (MDA) adducts of proteins, markers of oxidative damage to DNA, proteins, and lipids, respectively. All four HBQs generated intracellular ROS in T24 cells in a concentration-dependent manner. HBQs also produced 8-OHdG in genomic DNA of T24 cells, with the highest levels of 8-OHdG induced by DCMBQ. Protein carbonylation was significantly increased in T24 cells that were incubated with each of the four HBQs for 24 h. However, MDA adduct formation, a marker of lipid peroxidation, was not affected by any of the four HBQs tested. These results suggest that the ROS-induced oxidative damage to DNA and protein carbonylation are involved in the observed toxicity of HBQs in T24 cells.

A cell-microelectronic sensing technique for profiling cytotoxicity of chemicals.

A cell-microelectronic sensing technique is developed for profiling chemical cytotoxicity and is used to study different cytotoxic effects of the same class chemicals using nitrosamines as examples. This technique uses three human cell lines (T24 bladder, HepG2 liver, and A549 lung carcinoma cells) and Chinese hamster ovary (CHO-K1) cells in parallel as the living components of the sensors of a real-time cell electronic sensing (RT-CES) method for dynamic monitoring of chemical toxicity. The RT-CES technique measures changes in the impedance of individual microelectronic wells that is correlated linearly with changes in cell numbers during t log phase of cell growth, thus allowing determination of cytotoxicity. Four nitrosamines, N-nitrosodimethylamine (NDMA), N-nitrosodiphenylamine (NDPhA), N-nitrosopiperidine (NPip), and N-nitrosopyrrolidine (NPyr), were examined and unique cytotoxicity profiles were detected for each nitrosamine. In vitro cytotoxicity values (IC(50)) for NDPhA (ranging from 0.6 to 1.9 mM) were significantly lower than the IC(50) values for the well-known carcinogen NDMA (15-95 mM) in all four cell lines. T24 cells were the most sensitive to nitrosamine exposure among the four cell lines tested (T24>CHO>A549>HepG2), suggesting that T24 may serve as a new sensitive model for cytotoxicity screening. Cell staining results confirmed that administration of the IC(50) concentration from the RT-CES experiments inhibited cell growth by 50% compared to the controls, indicating that the RT-CES method provides reliable measures of IC(50). Staining and cell-cycle analysis confirmed that NDPhA caused cell-cycle arrest at the G0/G1 phase, whereas NDMA did not disrupt the cell cycle but induced cell death, thus explaining the different cytotoxicity profiles detected by the RT-CES method. The parallel cytotoxicity profiling of nitrosamines on the four cell lines by the RT-CES method led to the discovery of the unique cytotoxicity of NDPhA causing cell-cycle arrest. This study demonstrates a new approach to comprehensive testing of chemical toxicity.