Use of a standardized JaCVAM in vivo rat comet assay protocol to assess the genotoxicity of three coded test compounds; ampicillin trihydrate, 1,2-dimethylhydrazine dihydrochloride, and N-nitrosodimethylamine.

As part of the Japanese Center for the Validation of Alternative Methods (JaCVAM)-initiative international validation study of the in vivo rat alkaline comet assay (comet assay), our laboratory examined ampicillin trihydrate (AMP), 1,2-dimethylhydrazine dihydrochloride (DMH), and N-nitrosodimethylamine (NDA) using a standard comet assay validation protocol (v14.2) developed by the JaCVAM validation management team (VMT). Coded samples were received by our laboratory along with basic MSDS information. Solubility analysis and range-finding experiments of the coded test compounds were conducted for dose selection. Animal dosing schedules, the comet assay processing and analysis, and statistical analysis were conducted in accordance with the standard protocol. Based upon our blinded evaluation, AMP was not found to exhibit evidence of genotoxicity in either the rat liver or stomach. However, both NDA and DMH were observed to cause a significant increase in % tail DNA in the rat liver at all dose levels tested. While acute hepatoxicity was observed for these compounds in the high dose group, in the investigators opinion there were a sufficient number of consistently damaged/measurable cells at the medium and low dose groups to judge these compounds as genotoxic. There was no evidence of genotoxicity from either NDA or DMH in the rat stomach. In conclusion, our laboratory observed increased DNA damage from two blinded test compounds in rat liver (later identified as genotoxic carcinogens), while no evidence of genotoxicity was observed for the third blinded test compound (later identified as a non-genotoxic, non-carcinogen). This data supports the use of a standardized protocol of the in vivo comet assay as a cost-effective alternative genotoxicity assay for regulatory testing purposes.

Analysis of chromosome damage for biodosimetry using imaging flow cytometry.

The dicentric chromosome assay (DCA), which involves counting the frequency of dicentric chromosomes in mitotic lymphocytes and converting it to a dose-estimation for ionizing radiation exposure, is considered to be the gold standard for radiation biodosimetry. Furthermore, for emergency response, the DCA has been adapted for triage by simplifying the scoring method [1]. With the development of new technologies such as the imaging flow cytometer, it may now be possible to adapt this microscope-based method to an automated cytometry method. This technology allows the sensitivity of microscopy to be maintained while adding the increased throughput of flow cytometry. A new protocol is being developed to adapt the DCA to the imaging cytometer in order to further increase the rapid determination of a biological dose. Peripheral blood mononuclear cells (PBMC) were isolated from ex vivo irradiated whole blood samples using a density gradient separation method and cultured with PHA and Colcemid. After 48h incubation, the chromosomes were isolated, stained for DNA content with propidium iodide (PI) and labelled with a centromere marker. Stained chromosomes were then analyzed on the ImageStream(×) (EMD-Millipore, Billerica, MA). Preliminary results indicate that individual chromosomes can be identified and mono- and dicentric chromosomes can be differentiated by imaging cytometry. A dose response curve was generated using this technology. The details of the method and the dose response curve are presented and compared to traditional microscope scoring. Imaging cytometry is a new technology which enables the rapid, automated analysis of fluorescently labelled chromosomes. Adapting the dicentric assay to this technology has the potential for high throughput analysis for mass casualty events.

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.

Radiofrequency radiation and gene/protein expression: a review.

Mobile telecommunications have developed considerably in recent years. With the proliferation of wireless technologies, there is much public anxiety about the potential health impact associated with exposure to radiofrequency (RF) radiation from these novel products. Contradictory scientific evidence, often reported in the popular media, has further fueled public concern. Some epidemiological studies have reported that ipsilateral use of a mobile phone is associated with an increased risk for brain tumors, while other studies have reported an association between brain tumor risk and mobile phone use for longer than 10 years. However, other large epidemiological studies have failed to find similar associations. Despite the existence of national and international RF-radiation exposure guidelines, there are increasing public demands for precaution with respect to human exposure to RF radiation. Since current epidemiological evidence is insufficient to make a definitive judgment on the health risks of low-level RF radiation exposure, laboratory evidence assessing biological plausibility and theoretical mechanisms of interaction are important. A number of studies have reported that RF radiation may induce alterations in gene/protein expression in a variety of cells/tissues that may be associated with potentially harmful health outcomes, while other studies have shown no clear effects related to RF radiation. This review focuses on the current scientific evidence related to changes in protein and gene expression induced by low-level RF radiation.

Evaluating the biological effects of intermittent 1.9 GHz pulse-modulated radiofrequency fields in a series of human-derived cell lines.

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 degrees 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 (G(2)/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.

Microarray gene expression profiling of a human glioblastoma cell line exposed in vitro to a 1.9 GHz pulse-modulated radiofrequency field.

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 degrees 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.

Relative biological effectiveness of 280 keV neutrons for apoptosis in human lymphocytes.

The relative biological effectiveness (RBE) of neutrons varies from unity to greater than ten depending upon neutron energy and the biological endpoint measured. In our study, we examined apoptosis in human lymphocytes to assess the RBE of low energy 280 keV neutrons compared to Cs gamma radiation and found the RBE to be approximately one. Similar results have been observed for high energy neutrons using the same endpoint. As apoptosis is a major process that influences the consequences of radiation exposure, our results indicate that biological effect and the corresponding weighting factors for 280 keV neutrons may be lower in some cell types and tissues.

Gene expression analysis of a human lymphoblastoma cell line exposed in vitro to an intermittent 1.9 GHz pulse-modulated radiofrequency field.

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.

Increased FITC fluorescence on LPS stimulated neutrophils cultured in whole blood.

Recently, it has been observed that Annexin V labelling of phosphatidylserine (PS) on non-apoptotic cells can vary in different leukocyte populations and with the activation of cells, due to differences in the absolute level of exposed PS. We have also observed changes in the absolute level of Annexin V-FITC intensity, but under conditions where absolute PS expression did not change. In the present study, we have explored the effect of neutrophil cell activation on Annexin V-FITC fluorescence intensity by comparing alternatively labelled matched antibodies against Annexin V. Human venous whole blood was cultured with and without stimulation with lipopolysaccharide (LPS). Apoptosis in the neutrophil and lymphocyte populations was analyzed by flow cytometry and the intensity of FITC labelling was compared to matched fluorochromes conjugated to the same cell surface markers. There was an increase in the intensity of Annexin V-FITC in non-apoptotic neutrophils when stimulated with LPS, which did not correlate with increased apoptosis. Furthermore, CD65-FITC intensity also increased on activated neutrophils. Activated neutrophils exhibited higher amounts of FITC fluorescence that were not associated with changes in extracellular PS expression. This effect appears to be fluorochrome related, likely due to an increase in the pH surrounding activated neutrophils.

Response to pulsed dose rate and low dose rate irradiation with and without mild hyperthermia using human breast carcinoma cell lines.

The purpose of this study was to establish whether a pulsed dose rate (PDR) treatment of 1.5 Gy given every 3 h in combination with 41 degrees C mild hyperthermia or a continuous low dose rate (LDR) treatment with mild hyperthermia could radiosensitize two isogenic human breast carcinoma cell lines in comparison to pulsed dose rate or low dose rate irradiation alone. The radiation resistant cell line was derived from the parental cell line and was transfected to over-express DNA polymerase beta. The end-points assessed were the survival of the cells using the clonogenic assay, the amount of residual DSB(s) using the comet assay and gene expression of polymerase beta using RT-PCR. Results showed that the PDR and LDR treatments combined with mild hyperthermia caused significant radiosensitization when compared to PDR and LDR irradiation alone in terms of the clonogenic and comet assays with both cell lines. RT-PCR results showed that polymerase beta levels of expression were not elevated in response to these treatments, implying that this polymerase may not be involved in sub-lethal damage repair or thermal radiosensitization. These results suggest a potential clinical advantage when combining LDR or PDR with hyperthermia, since they indicate that hyperthermia is an effective radiosensitizer.

Evaluating DNA damage in rodent brain after acute 60 Hz magnetic-field exposure.

In recent years, numerous studies have reported a weak association between 60 Hz magnetic-field exposure and the incidence of certain cancers. To date, no mechanism to explain these findings has been identified. The objective of the current study was to investigate whether acute magnetic-field exposure could elicit DNA damage within brain cells from both whole brain and cerebellar homogenates from adult rats, adult mice and immature mice. Rodents were exposed to a 60 Hz magnetic field (0, 0.1, 1 or 2 mT) for 2 h. Then, at 0, 2 and 4 h after exposure, animals were killed humanely, their brains were rapidly removed and homogenized, and cells were cast into agarose gels for processing by the alkaline comet assay. Four parameters (tail ratio, tail moment, comet length and tail length) were used to assess DNA damage for each comet. For each species, a significant increase in DNA damage was detected by each of the four parameters in the positive control (2 Gy X rays) relative to the concurrent nonirradiated negative and sham controls. However, none of the four parameters detected a significant increase in DNA damage in brain cell homogenates from any magnetic-field exposure (0- 2 mT) at any time after exposure. The dose-response and time-course data from the multiple animal groups tested in this study provide no evidence of magnetic-field-induced DNA damage.

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.

Effect of pro-inflammatory cytokines on spontaneous apoptosis in leukocyte sub-sets within a whole blood culture.

Pro-inflammatory cytokines are known to affect apoptosis in human peripheral blood cells. Neutrophils, which are an essential component of the immune response and usually undergo apoptosis rapidly, are greatly affected by these cytokines. In this study, the effect of varying concentrations of TNF-alpha, IL-1beta and IL-6 on the apoptotic response of leukocytes and their sub-sets in cultured whole blood were studied over a 48 h culture period. At clinically relevant concentrations, it was found that these pro-inflammatory cytokines reduced the amount of spontaneous apoptosis in neutrophils in culture, but had little effect on the lymphocyte population. Distinct differences in the sensitivity of neutrophils to cytokine-mediated protection against spontaneous apoptosis were apparent when compared to previous studies conducted using purified or enriched neutrophil cultures. IL-1beta, at a dose of 0.01 pg/mL, was observed to significantly inhibit spontaneous neutrophil apoptosis by approximately 90% and 65% at 24 and 48 h of culturing, respectively. This concentration used in whole blood is dramatically lower than that required to elicit similar protection in neutrophil-enriched cell cultures. Higher concentrations of TNF-alpha (1.0 pg/mL) and IL-6 (125 pg/mL) were also found to significantly inhibit neutrophil apoptosis, at levels much lower than previously published using neutrophil-enriched cultures. Furthermore, each cytokine displayed a unique signature with respect to the optimal applied doses required to elicit maximal protection against spontaneous neutrophil apoptosis. These results demonstrate the dramatic differences in cellular responses that exist between neutrophil-enriched cultures and whole blood culture systems, where multiple blood cell types provide a much more complex environment.

Sustained antiproliferative mechanisms by RB24, a targeted precursor of multiple inhibitors of epidermal growth factor receptor and a DNA alkylating agent in the A431 epidermal carcinoma of the vulva cell line.

Recently, with the purpose of enhancing the potency of epidermal growth factor receptor (EGFR)-based therapies, we designed a novel strategy termed 'Cascade-release targeting' that seeks to develop molecules capable of degrading to multiple tyrosine kinase (TK) inhibitors and highly reactive electrophiles, in a stepwise fashion. Here we report on the first prototype of this model, RB24, a masked methyltriazene, that in addition to being an inhibitor on its own was designed to degrade to RB14, ZR08, RB10+a DNA alkylating methyldiazonium species. The cascade degradation of RB24 requires the generation of two reactive electrophiles: (a) an iminium ion and (b) a methyldiazonium ion. Thus, we surmise that these species could alkylate the active site of EGFR, thereby irreversibly blocking its action and that DNA damage could be induced by the methyldiazonium. Using the EGFR-overexpressing human epidermoid carcinoma of the vulva cell line, A431, we demonstrate herein that (a) RB24 and its derived species (e.g. RB14, ZR08) irreversibly inhibit EGFR autophosphorylation, (b) RB24 induced significant levels of DNA strand breaks, (c) sustained inhibition of EGFR by RB24 was associated with blockade of MAPK activation and c-fos gene expression, (d) RB24 induced irreversible cell growth inhibition with a 100-fold greater potency than Temodaltrade mark, a clinical methyltriazene. The pronounced growth inhibitory potency of RB24 was attributed to its ability to simultaneously damage DNA and irreversibly block EGFR TK activity.

No evidence for genotoxic effects from 24 h exposure of human leukocytes to 1.9 GHz radiofrequency fields.

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 degrees 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.

A 60 Hz magnetic field does not affect the incidence of squamous cell carcinomas in SENCAR mice.

Two groups of SENCAR mice were treated with a single dose of carcinogen and then, for 23 weeks, with a chemical tumor promoter to induce skin tumors. During this period, one group was coexposed to a 2 mT power frequency (60 Hz) magnetic field, while the other was exposed to sham conditions. Application of the tumor promoter ceased after 23 weeks, but the exposure to sham conditions or magnetic fields continued for an additional 29 weeks. No difference was found between the two groups of mice in terms of the incidence of total tumors (P =.297) or squamous cell carcinomas (SSC) (P =.501). In summary, there was no evidence to support the hypotheses that 60 Hz magnetic fields (MF) can influence the development of either papillomas or SSC under our defined experimental conditions. The overall results add to previous animal studies that find no association between exposure to 60 Hz MF and the incidence of benign or malignant tumors.

Cylindrical waveguide applicator for in vitro exposure of cell culture samples to 1.9-GHz radiofrequency fields.

An applicator for in vitro cell culture exposure was developed based on a circularly polarized, cylindrical waveguide for the 1.9-GHz frequency band used by Personal Communications Services (PCS) in Canada. The applicator consists of two coaxial Petri dishes that sit on the open end of the cylindrical waveguide. The inner 60-mm Petri dish contains the cell culture while the outer 150-mm dish contains coolant water, which is circulated from a pump. A dosimetric evaluation was made using thermometric and E-field probe techniques. The latter allowed the entire inner dish to be scanned to determine the range of specific absorption rates (SARs) pertinent to the expected position of the cells. A representative SAR rate (SAR per unit of input power) of 8.6 +/- 2.1 W/kg/W (95th percentile) was determined 1 mm from the bottom, for a 10 ml sample volume of standard medium. Evaluation of the cooling system demonstrated that following an initial 0.3 degrees C temperature increase, a constant temperature was maintained for 24 h when the waveguide was energized to achieve an average sample SAR of 10 W/kg. These properties enable both acute and sub-acute in vitro bio-effect studies to be performed on a variety of cell culture samples.

DNA damage and micronucleus induction in human leukocytes after acute in vitro exposure to a 1.9 GHz continuous-wave radiofrequency field.

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 degrees C. Concurrent negative (incubator) and positive (1.5 Gy (137)Cs gamma 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.

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

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 degrees 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.

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.