Stephan Gabos

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.

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 μg (range, 0.0078–3.5 μg). 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 μg (range, 0.011–0.41 μg). 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 μg, which is lower than the average daily intake of arsenic from water and food.

Characteristics of PAHs, PCDD/Fs and PCBs in sediment following forest fires in northern Alberta

Extensive forest fires occurred in northern Alberta, Canada, in 1998. Polynuclear aromatic hydrocarbons (PAHs), polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs) and polychlorinated biphenyls (PCBs) were studied following these forest fires in order to explore natural and anthropogenic influences on the affected forest fire area. Specifically, concentrations and profiles of these compounds were determined in sediment samples. A predominance of alkylated PAH derivatives over parent PAHs were observed at the burned and reference sites. Naturally dervied PAHs were abundant at all sites. A high proportion and concentration of retene was observed at the totally burned site and indicates some inputs from forest fires. Very low concentrations of PCDD/Fs and PCBs were observed at all sites, and the profiles were very similar. This likely reflects common atmospheric contributing sources to the study area.

Pre-analytical and analytical procedures for the detection of enteric viruses and enterovirus in water samples

Practical pre-analytical and analytical procedures were developed and validated for detection of enteric viruses in three water matrices. Both RNA viruses (norovirus, coxsackievirus, echovirus, and rotavirus) and DNA virus (adenovirus 41) were included in the study. The NanoCeram 90mm laminated disc with electropositive filter and procedures of filtration, elution and flocculation were utilized to concentrate known amount of viruses in different water matrices. Real time quantitative PCR was used to evaluate the recovery of virus and cell culture to assess viral infectivity. There was no PCR inhibition using various concentrations and pH of beef extract eluting buffer. A good recovery of the viruses spiked in 10L of deionized water was achieved for serial dilutions of coxsackievirus (41-67%), echovirus (22-90%), norovirus (23-44%) and rotavirus (24-46%). Relatively lower recovery was observed for adenovirus 41 (24-35%). There was no significant difference in viral recovery from deionized, tap and river water samples. The infectivity of recovered adenovirus, coxsackievirus and echovirus was demonstrated using in vitro cell culture. The pre-analytical and analytic procedures attained consistent recovery of RNA and DNA viruses both as infectious viral particles and viral genome, provided effective removal of inhibitory substances, achieved reliable reproducibility, and were relatively inexpensive for monitoring viruses in water.

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.

Biomonitoring of Arsenic in Urine and Saliva of Children Playing on Playgrounds Constructed from Chromated Copper Arsenate-Treated Wood

Children may be exposed to arsenic during contact with structures treated with chromated copper arsenate (CCA). A high frequency of hand-to-mouth activity may increase their risk of ingesting arsenic. Previous work showed that arsenic concentrations in the hand-wash samples of children playing on CCA playgrounds were four times higher than those playing on non-CCA playgrounds. It is not clear whether playing on CCA playgrounds results in elevated overall exposure to arsenic. The objective of this study was to perform arsenic biomonitoring in children to determine whether playing on CCA-treated playgrounds substantially contributes to their overall exposure to arsenic. One hundred and twenty five saliva samples from 61 children and 101 urine samples from 45 children were collected after children played on 8 CCA and 8 non-CCA playgrounds. Arsenic speciation analysis was conducted using high performance liquid chromatography combined with inductively coupled plasma mass spectrometry. The arsenic species detected in the urine and saliva samples from children playing on CCA and non-CCA playgrounds were similar. Dimethylarsinic acid and arsenobetaine were the main arsenic species found in urine samples. The sum of inorganic trivalent and pentavalent arsenic, monomethylarsonic acid, and dimethylarsinic acid in urine was 15 +/- 28 microg/L in the CCA group and 12 +/- 23 microg/L in the non-CCA group (p = 0.60). The sum of these species in saliva was 1.1 +/- 2.1 microg/L in the CCA group and 1.4 +/- 1.1 microg/L in the non-CCA group (p = 0.32). These results show that there is no significant difference in the concentration or speciation of arsenic between the samples from children playing on CCA and non-CCA playgrounds. Contact with CCA playgrounds is not likely to significantly contribute to the overall arsenic exposure in children; other sources such as dietary arsenic may be a main contributor to their overall exposure.

Determination of vanadium in biological fluids using HR-ICP-MS

A simple, fast and sensitive method is described for the determination of vanadium in biological fluids using high resolution inductively coupled plasma mass spectrometry (HR-ICP-MS). Samples were diluted 20-fold in 0.3% HNO3. Spectral interference from 35Cl16O+, present in urine or serum matrices, was completely resolved from the vanadium major isotope of 51V+ at medium resolution of 4000. Quantitation of V in urine and serum was achieved by the method of additions using standards prepared in a pooled urine or serum sample. Mass offset and lock mass (40Ar16O+) options were employed for more accurate and precise results. Better than 2.0% RSD was obtained with a 200 pg ml−1 V spike added to a 20-fold diluted urine or serum matrix, reflecting one day stability. Vanadium concentrations in the range of <10 to 1500 pg ml−1 and <10 to 760 pg ml−1 were determined in the urine and serum samples, respectively. Good day-to-day precision of less than 4.0% RSD was typical for both sample matrices. Spike recoveries of 99 ± 2 and 98 ± 3% (one standard deviation, n = 3) were obtained in the urine and serum, respectively. An in-house CRM (SLRS-4: river water) was analyzed daily, yielding a V concentration of 0.337 ± 0.010 ng ml−1 (one standard deviation, n = 35), in good agreement with the certified value of 0.32 ± 0.03 ng ml−1. A detection limit (3 s) of 10 pg ml−1 was estimated for V in reconstituted urine and serum samples.

Cytotoxicity assessment based on the AUC50 using multi-concentration time-dependent cellular response curves

Although many indices have been developed to quantify chemical toxicity, substantial shortcoming is inherent in most of them, such as observation time dependence, insufficient robustness, and no comparison with the negative control. To assess the extent of exposure of the tested substance, a cytotoxicity assay named AUC(50) was developed to describe the time and concentration-dependent cellular responses. By monitoring the dynamic cytotoxicity response profile of living cells via the xCELLigence real-time cell analysis high-throughput (RTCA HT) system, changes in cell number (named cell index, CI) were recorded and analyzed subsequently. A normalized cell index (NCI) is introduced to reduce the influence of inter-experimental variations. The log-phase of cellular growth is considered, which alleviates the cells spontaneous effect. The area between the control line and the assessed time-dependent cellular response curve (TCRC) of the tested substance was calculated, and the corresponding exponential kill model (concentration-response curve) was developed along with exploiting the concept of AUC(50). The validation of the proposed method is demonstrated by exposing HepG2 cell line to seven chemical compounds. Our findings suggested that the proposed AUC-based toxicity assay could be an alternative to the traditional single time-point assay, and it has potential to become routine settings for evaluating the cell-based in vitro assay. Furthermore, the AUC(50) combined with RTCA HT assay can be used to achieve a high-throughput screening that conventional cellular assay cannot achieve.

Research article: In vitro cytotoxicity assessment based on KC50 with real-time cell analyzer (RTCA) assay

Technological advances in cytotoxicity analysis have now made it possible to obtain real time data on changes in cell growth, morphology and cell death. This type of testing has a great potential for reducing and refining traditional in vivo toxicology tests. By monitoring the dynamic response profile of living cells via the xCELLigence real-time cell analyzer for high-throughput (RTCA HT) system, cellular changes including cell number (cell index, CI) are recorded and analyzed. A special scaled index defined as normalized cell index (NCI) is used in the analysis which reduces the influence of inter-experimental variations. To assess the extent of exposure of the tested chemicals, a two-exponent model is presented to describe rate of cell growth and death. This model is embodied in the time and concentration-dependent cellular response curves, and the parameters k1 and k2 in this model are used to describe the rate of cell growth and death. Based on calculated k2 values and the corresponding concentrations, a concentration-response curve is fitted. As a result, a cytotoxicity assessment named KC50 is calculated. The validation of the proposed method is demonstrated by exposing six cell lines to 14 chemical compounds. Our findings suggest that the proposed KC50-based toxicity assay can be an alternative to the traditional single time-point assay such as LC50 (the concentration at which 50% of the cells are killed). The proposed index has a potential for routine evaluation of cytotoxicities. Another advantage of the proposed index is that it extracts cytotoxicity information when CI fails to detect the low toxicity.

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.