James P. McNamee

Photophysical Properties of Fluorescent DNA-dyes Bound to Single- and Double-stranded DNA in Aqueous Buffered Solution¶

Abstract The absorption and fluorescence spectra, fluorescence quantum yields, lifetimes and time-resolved fluorescence spectra are reported for nine different fluorescent DNA-dyes. The work was initiated in search of a quantitative method to detect the ratio of single-to-double stranded DNA (ssDNA/dsDNA) in solution based on the photophysics of dye–DNA complexes; the result is a comprehensive study providing a vast amount of information for users of DNA stains. The dyes examined were the bisbenzimide or indole-derived stains (Hoechst 33342, Hoechst 33258 and 4′,6-diamidino-2-phenylindole), phenanthridinium stains (ethidium bromide and propidium iodide) and cyanine dyes (PicoGreen, YOYO-1 iodide, SYBR Green I and SYBR Gold). All were evaluated under the same experimental conditions in terms of ionic strength, pH and dye–DNA ratio. Among the photophysical properties evaluated only fluorescence lifetimes for the cyanine stilbene dyes allowed a convenient differentiation between ssDNA and dsDNA. The bisbenzimide dyes showed multiexponential decays when bound to either form of DNA, making lifetime-based analysis cumbersome with inherent errors. These dyes also presented biexponential decay when free in aqueous buffered solutions at different pH. A mechanism for their deactivation is proposed based on two different conformers decaying with different kinetics. The phenanthridinium dyes showed monoexponential decays with ssDNA and dsDNA, but there was no discrimination between them. High dye–DNA ratios (e.g. 1:1) resulted in multiexponential decays for cyanine dyes, resulting from energy transfer or self-quenching deactivation. Shifts in both absorption and fluorescence maxima for both ssDNA and dsDNA DNA–cyanine dye complexes were small. Broadening of dye–ssDNA absorption and fluorescence bands for the cyanine dyes relative to dye–dsDNA bands was detected and attributed to higher degrees of rotational freedom in the former.

Mobile phones, mobile phone base stations and cancer: a review.

There have been reports in the media and claims in the courts that radiofrequency (RF) emissions from mobile phones are a cause of cancer, and there have been numerous public objections to the siting of mobile phone base antennas because of a fear of cancer. This review summarizes the current state of evidence concerning whether the RF energy used for wireless communication might be carcinogenic. Relevant studies were identified by searching MedLine with a combination of exposure and endpoint terms. This was supplemented by a review of the over 1700 citations assembled by the Institute of Electrical and Electronics Engineers (IEEE) International Committee on Electromagnetic Safety as part of their updating of the IEEE C95.1 RF energy safety guidelines. Where there were multiple studies, preference was given to recent reports, to positive reports of effects and to attempts to confirm such positive reports. Biophysical considerations indicate that there is little theoretical basis for anticipating that RF energy would have significant biological effects at the power levels used by modern mobile phones and their base station antennas. The epidemiological evidence for a causal association between cancer and RF energy is weak and limited. Animal studies have provided no consistent evidence that exposure to RF energy at non-thermal intensities causes or promotes cancer. Extensive in vitro studies have found no consistent evidence of genotoxic potential, but in vitro studies assessing the epigenetic potential of RF energy are limited. Overall, a weight-of-evidence evaluation shows that the current evidence for a causal association between cancer and exposure to RF energy is weak and unconvincing. However, the existing epidemiology is limited and the possibility of epigenetic effects has not been thoroughly evaluated, so that additional research in those areas will be required for a more thorough assessment of the possibility of a causal connection between cancer and the RF energy from mobile telecommunications.

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.

QUICKSCAN DICENTRIC CHROMOSOME ANALYSIS FOR RADIATION BIODOSIMETRY

The dicentric chromosome assay (DCA) is the gold-standard assay for accurately estimating unknown radiological doses to individuals following radiological or nuclear accidents. However in a mass-casualty scenario, this assay is not well suited for providing timely dose estimates due to its time- and expertise-intensive nature. In Canada, two approaches are being developed in an attempt to increase triage-quality biological dosimetry throughput. These are 1) increasing the number of trained personnel capable of conducting the DCA, and 2) evaluating alternative biodosimetry approaches or DCA variations. In a recent exercise, a new scoring technique (termed DCA QuickScan) was evaluated as an alternative rapid-scoring approach. Triage-quality conventional DCA and DCA QuickScan analysis were based upon scoring a minimum of 50 metaphase cells or 30 dicentrics by 9–15 scorers across four laboratories. Dose estimates for the conventional DCA were found to be within 0.5 Gy of the actual dose for 83% of the unknown samples, while DCA QuickScan dose estimates were within 0.5 Gy for 80% of the samples. Of the dose estimates falling 0.5 Gy or more outside the actual dose, the majority were dose over-estimates. It was concluded that the DCA QuickScan approach can provide critical dose information at a much faster rate than the conventional DCA without sacrificing accuracy. Future studies will further evaluate the accuracy of the DCA QuickScan method.

Validation of the cytokinesis-block micronucleus (CBMN) assay for use as a triage biological dosimetry tool

Traditionally, the dicentric chromosome assay (DCA) has been used to derive biological dose estimates for unknown radiological exposures. While sensitive, this assay requires highly trained evaluators and is extremely time consuming. The cytokinesis-block micronucleus (CBMN) assay has been suggested as an alternative to the DCA, as it is much faster to evaluate samples and requires less technical expertise. In order to validate this assay for triage biodosimetry, dose-response curves were generated for six donors at eight doses of gamma-radiation (0-4.0 Gy). Each sample was evaluated by 12 individuals, among three different laboratories and the incidence of micronuclei was determined after counting 50-500 binucleated cells. This study demonstrated that the CBMN assay was capable of detecting radiation doses >or=1 Gy after scoring only 200 binucleated cells. As such, the CBMN assay may provide a sensitive and reliable technique for deployment as an initial screening tool in a large-scale radiological emergency where large numbers of biological dose estimates are required.

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.