Pascale V. Bellier

Comet assay: rapid processing of multiple samples

The present study describes modifications to the basic comet protocol that increase productivity and efficiency without sacrificing assay reliability. A simple technique is described for rapidly preparing up to 96 comet assay samples simultaneously. The sample preparation technique allows thin layers of agarose-embedded cells to be prepared in multiple wells attached to a flexible film of Gelbond, which improves the ease of manipulating and processing samples. To evaluate the effect of these modifications on assay sensitivity, dose-response curves are presented for DNA damage induced by exposure of TK6 cells to low concentrations of hydrogen peroxide (0-10 microM) and for exposure of human lymphocytes to X-irradiation (0-100 cGy). The limit of detection of DNA damage induced by hydrogen peroxide in TK6 cells was observed to be 1 uM for all parameters (tail ratio, tail moment, tail length and comet length) while the limit of detection of DNA damage in human lymphocytes was 10 cGy for tail and comet length parameters, but 50 cGy for tail ratio and tail moment parameters. These results are similar to those previously reported using the conventional alkaline comet assay. The application of SYBR Gold for detection of DNA damage was compared to that of propidium iodide. Measurements of matching samples for tail length and comet length were similar using both stains. However, comets stained with SYBR Gold persisted longer and were much brighter than those obtained with propidium iodide. SYBR Gold was found to be ideal for measuring tail length and comet length but, under present assay conditions, impractical for measuring tail ratio or tail moment due to saturation of staining in the head region of the comets.

DNA Damage and Micronucleus Induction in Human Leukocytes after Acute In Vitro Exposure to a 1.9 GHz Continuous-Wave Radiofrequency Field

Abstract McNamee, J. P., Bellier, P. V., Gajda, G. B., Miller, S. M., Lemay E. P., Lavallée, B. F., Marro, L. and Thansandote, A. DNA Damage and Micronucleus Induction in Human Leukocytes after Acute In Vitro Exposure to a 1.9 GHz Continuous-Wave Radiofrequency Field. Radiat. Res. 158, 523–533 (2002). Human blood cultures were exposed to a 1.9 GHz continuous-wave (CW) radiofrequency (RF) field for 2 h using a series of six circularly polarized, cylindrical waveguides. Mean specific absorption rates (SARs) of 0.0, 0.1, 0.26, 0.92, 2.4 and 10 W/kg were achieved, and the temperature within the cultures during a 2-h exposure was maintained at 37.0 ± 0.5°C. Concurrent negative (incubator) and positive (1.5 Gy 137Cs γ radiation) control cultures were run for each experiment. DNA damage was quantified immediately after RF-field exposure using the alkaline comet assay, and four parameters (tail ratio, tail moment, comet length and tail length) were used to assess DNA damage for each comet. No evidence of increased primary DNA damage was detected by any parameter for RF-field-exposed cultures at any SAR tested. The formation of micronuclei in the RF-field-exposed blood cell cultures was assessed using the cytokinesis-block micronucleus assay. There was no significant difference in the binucleated cell frequency, incidence of micronucleated binucleated cells, or total incidence of micronuclei between any of the RF-field-exposed cultures and the sham-exposed controls at any SAR tested. These results do not support the hypothesis that acute, nonthermalizing 1.9 GHz CW RF-field exposure causes DNA damage in cultured human leukocytes.

No Evidence for Genotoxic Effects from 24 h Exposure of Human Leukocytes to 1.9 GHz Radiofrequency Fields

Abstract McNamee, J. P., Bellier, P. V., Gajda, G. B., Lavallée, B. F., Marro, L., Lemay, E. and Thansandote, A. No Evidence for Genotoxic Effects from 24 h Exposure of Human Leukocytes to 1.9 GHz Radiofrequency Fields. Radiat. Res. 159, 693–697 (2003). The current study extends our previous investigations of 2-h radiofrequency (RF)-field exposures on genotoxicity in human blood cell cultures by examining the effect of 24-h continuous-wave (CW) and pulsed-wave (PW) 1.9 GHz RF-field exposures on both primary DNA damage and micronucleus induction in human leukocyte cultures. Mean specific absorption rates (SARs) ranged from 0 to 10 W/kg, and the temperature within the cultures was maintained at 37.0 ± 1.0°C for the duration of the 24-h exposure period. No significant differences in primary DNA damage were observed between the sham-treated controls and any of the CW or PW 1.9 GHz RF-field-exposed cultures when processed immediately after the exposure period by the alkaline comet assay. Similarly, no significant differences were observed in the incidence of micronuclei, incidence of micronucleated binucleated cells, frequency of binucleated cells, or proliferation index between the sham-treated controls and any of the CW or PW 1.9 GHz RF-field-exposed cultures. In conclusion, the current study found no evidence of 1.9 GHz RF-field-induced genotoxicity in human blood cell cultures after a 24-h exposure period.

Microarray Gene Expression Profiling of a Human Glioblastoma Cell Line Exposed In Vitro to a 1.9 GHz Pulse-Modulated Radiofrequency Field

Abstract Qutob, S. S., Chauhan, V., Bellier, P. V., Yauk, C. L., Douglas, G. R., Berndt, L., Williams, A., Gajda, G. B., Lemay, E., Thansandote, A. and McNamee, J. P. Microarray Gene Expression Profiling of a Human Glioblastoma Cell Line Exposed In Vitro to a 1.9 GHz Pulse-Modulated Radiofrequency Field. Radiat. Res. 165, 636–644 (2006). The widespread use of mobile phones has led to public concerns about the health effects associated with exposure to radiofrequency (RF) fields. The paramount concern of most persons relates to the potential of these fields to cause cancer. Unlike ionizing radiation, RF fields used for mobile telecommunications (800–1900 MHz) do not possess sufficient energy to directly damage DNA. Most rodent bioassay and in vitro genotoxicity/mutation studies have reported that RF fields at non-thermal levels have no direct mutagenic, genotoxic or carcinogenic effects. However, some evidence has suggested that RF fields may cause detectable postexposure changes in gene expression. Therefore, the purpose of this study was to assess the ability of exposure to a 1.9 GHz pulse-modulated RF field for 4 h at specific absorption rates (SARs) of 0.1, 1.0 and 10.0 W/kg to affect global gene expression in U87MG glioblastoma cells. We found no evidence that non-thermal RF fields can affect gene expression in cultured U87MG cells relative to the nonirradiated control groups, whereas exposure to heat shock at 43°C for 1 h up-regulated a number of typical stress-responsive genes in the positive control group. Future studies will assess the effect of RF fields on other cell lines and on gene expression in the mouse brain after in vivo exposure.

DNA damage in human leukocytes after acute in vitro exposure to a 1.9 GHz pulse-modulated radiofrequency field.

Abstract McNamee, J. P., Bellier, P. V., Gajda, G. B., Lavallée, B. F., Lemay, E. P., Marro, L. and Thansandote, A. DNA Damage in Human Leukocytes after Acute In Vitro Exposure to a 1.9 GHz Pulse-Modulated Radiofrequency Field. Radiat. Res. 158, 534–537 (2002). Blood cultures from human volunteers were exposed to an acute 1.9 GHz pulse-modulated radiofrequency (RF) field for 2 h using a series of six circularly polarized, cylindrical waveguides. Mean specific absorption rates (SARs) ranged from 0 to 10 W/kg, and the temperature within the cultures during the exposure was maintained at 37.0 ± 0.5°C. DNA damage was quantified in leukocytes by the alkaline comet assay and the cytokinesis-block micronucleus assay. When compared to the sham-treated controls, no evidence of increased primary DNA damage was detected by any parameter for any of the RF-field-exposed cultures when evaluated using the alkaline comet assay. Furthermore, no significant differences in the frequency of binucleated cells, incidence of micronucleated binucleated cells, or total incidence of micronuclei were detected between any of the RF-field-exposed cultures and the sham-treated control at any SAR tested. These results do not support the hypothesis that acute, nonthermalizing 1.9 GHz pulse-modulated RF-field exposure causes DNA damage in cultured human leukocytes.

Differential apoptotic response to ionizing radiation in subpopulations of human white blood cells

The purpose of this paper is to characterize the apoptotic response of various subpopulations of human white blood cells after in vitro exposure to ionizing radiation using the modified neutral comet assay (MNCA). White blood cells, isolated from human whole blood, were fractionated into granulocytes and mononuclear cells which were further separated into B-cells, natural killer (NK) cells, and CD4(+) and CD8(+) T-cells. The separated fractions were exposed to low doses of X-rays and then MNCA was used to measure the apoptotic fraction (AF) at different time points in irradiated and unirradiated aliquots of sorted cultures. The spontaneous AF in unirradiated control cells was the most critical determinant of whether an apoptotic response could be detected in irradiated cells. When cultured in isolation granulocytes and B-cells had the highest background AF, with NK cells having the next highest. CD4(+) and CD8(+) T-cells had a low, stable, spontaneous AF which gave them the highest signal-to-noise ratio. Although B-cells demonstrated the highest radiation-induced apoptotic response to 1Gy of X-rays, CD8(+) T-cells were the most radiation-responsive lymphocytes due to their low spontaneous AF. By generating dose response curves for CD4(+) and CD8(+) T-cells, the sensitivity of the MNCA for detecting apoptosis in these two cell types was also examined.

Gene Expression Analysis of a Human Lymphoblastoma Cell Line Exposed In Vitro to an Intermittent 1.9 GHz Pulse-Modulated Radiofrequency Field

Abstract Chauhan, V., Mariampillai, A., Bellier, P. V., Qutob, S. S., Gajda, G. B., Lemay, E., Thansandote, A. and McNamee, J. P. Gene Expression Analysis of a Human Lymphoblastoma Cell Line Exposed In Vitro to an Intermittent 1.9 GHz Pulse-Modulated Radiofrequency Field. Radiat. Res. 165, 424–429 (2006). This study was designed to determine whether radiofrequency (RF) fields of the type used for wireless communications could elicit a cellular stress response. As general indicators of a cellular stress response, we monitored changes in proto-oncogene and heat-shock protein expression. Exponentially growing human lymphoblastoma cells (TK6) were exposed to 1.9 GHz pulse-modulated RF fields at average specific absorption rates (SARs) of 1 and 10 W/kg. Perturbations in the expression levels of the proto-oncogenes FOS, JUN and MYC after exposure to sham and RF fields were assessed by real-time RT-PCR. In addition, the transcript levels of the cellular stress proteins HSP27 and inducible HSP70 were also monitored. We demonstrated that transcript levels of these genes in RF-field-exposed cells showed no significant difference in relation to the sham treatment group. However, concurrent positive (heat-shock) control samples displayed a significant elevation in the expression of HSP27, HSP70, FOS and JUN. Conversely, the levels of MYC mRNA were found to decline in the positive (heat-shock) control. In conclusion, our study found no evidence that the 1.9 GHz RF-field exposure caused a general stress response in TK6 cells under our experimental conditions.

DNA damage and apoptosis in the immature mouse cerebellum after acute exposure to a 1 mT, 60 Hz magnetic field

Several recent studies have reported that whole-body exposure of rodents to power frequency magnetic fields (MFs) can result in DNA single- and double-strand breaks in the brains of these animals. The current study was undertaken to investigate whether an acute 2h exposure of a 1 mT, 60 Hz MF could elicit DNA damage, and subsequently apoptosis, in the brains of immature (10-day-old) mice. DNA damage was quantitated at 0, 2, 4, and 24h after exposure using the alkaline comet assay. Apoptosis was quantitated in the external granule cell layer (EGCL) of the immature mouse cerebellum at 0 and 24h after exposure to MF by the TdT-mediated dUTP nick-end labeling (TUNEL) assay. Four parameters (tail ratio, tail moment, comet length and tail length) were used to assess DNA damage for each comet. While increased DNA damage was detected by tail ratio at 2h after MF exposure, no supporting evidence of increased DNA damage was detected by the other parameters. In addition, no similar differences were observed using these parameters at any of the other post-exposure times. No increase in apoptosis was observed in the EGCL of MF-exposed mice, when compared to sham mice. Taken together, these results do not support the hypothesis that acute MF exposure causes DNA damage in the cerebellums of immature mice.

Evaluating the Biological Effects of Intermittent 1.9 GHz Pulse-Modulated Radiofrequency Fields in a Series of Human-Derived Cell Lines

Abstract Chauhan, V., Mariampillai, A., Kutzner, B., Wilkins, R. C., Ferrarotto, C., Bellier, P. V., Marro, L., Gajda, G. B., Lemay, E., Thansandote, A. and McNamee, J. P. Evaluating the Biological Effects of Intermittent 1.9 GHz Pulse-Modulated Radiofrequency Fields in a Series of Human-Derived Cell Lines. Radiat. Res. 167, 87–93 (2007). Several recent studies have suggested that radiofrequency (RF) fields may cause changes in a variety of cellular functions that may eventually lead to potential long-term health effects. In the present study, we have assessed the ability of non-thermal RF-field exposure to affect a variety of biological processes (including apoptosis, cell cycle progression, viability and cytokine production) in a series of human-derived cell lines (TK6, HL60 and Mono-Mac-6). Exponentially growing cells were exposed to intermittent (5 min on, 10 min off) 1.9 GHz pulse-modulated RF fields for 6 h at mean specific absorption rates (SARs) of 0, 1 and 10 W/kg. Concurrent negative (incubator) and positive (heat shock for 1 h at 43°C) controls were included in each experiment. Immediately after the 6-h exposure period and 18 h after exposure, cell pellets were collected and analyzed for cell viability, the incidence of apoptosis, and alterations in cell cycle kinetics. The cell culture supernatants were assessed for the presence of a series of human inflammatory cytokines (TNFA, IL1B, IL6, IL8, IL10, IL12) using a cytometric bead array assay. No detectable changes in cell viability, cell cycle kinetics, incidence of apoptosis, or cytokine expression were observed in any of RF-field-exposed groups in any of the cell lines tested, relative to the sham controls. However, the positive (heat-shock) control samples displayed a significant decrease in cell viability, increase in apoptosis, and alteration in cell cycle kinetics (G2/M block). Overall, we found no evidence that non-thermal RF-field exposure could elicit any detectable biological effect in three human-derived cell lines.

The Genotoxicity of Mainstream and Sidestream Marijuana and Tobacco Smoke Condensates

While the prevalence of tobacco use has decreased in Canada over the past decade, that of marijuana use has increased, particularly among youth. However, the risks of adverse health effects from marijuana smoke exposure, specifically as compared to tobacco, are currently not well understood. The objectives of this study were to evaluate the relative ability of matched marijuana and tobacco condensates to induce (geno)toxic responses in three in vitro test systems. This study provides comparative data for matched sidestream and mainstream condensates, as well as condensates prepared under both a standard and an extreme smoking regime designed to mimic marijuana smoking habits. The results indicate that tobacco and marijuana smoke differ substantially in terms of their cytotoxicity, Salmonella mutagenicity, and ability to induce chromosomal damage (i.e., micronucleus formation). Specifically, the marijuana condensates were all found to be more cytotoxic and more mutagenic in the presence of S9 than the matched tobacco condensates. In contrast, the tobacco condensates appeared to induce cytogenetic damage in a concentration-dependent manner, whereas the matched marijuana condensates did not. In addition, when corrected for total particulate matter yield, little difference was observed in the mutagenic activity of samples smoked under the extreme vs the standard regime for both tobacco and marijuana condensates.