Impact of 1.8-GHz radiofrequency radiation (RFR) on DNA damage and repair induced by doxorubicin in human B-cell lymphoblastoid cells

In the present in vitro study, a comet assay was used to determine whether 1.8-GHz radiofrequency radiation (RFR, SAR of 2 W/kg) can influence DNA repair in human B-cell lymphoblastoid cells exposed to doxorubicin (DOX) at the doses of 0 μg/ml, 0.05 μg/ml, 0.075 μg/ml, 0.10 μg/ml, 0.15 μg/ml and 0.20 μg/ml. The combinative exposures to RFR with DOX were divided into five categories. DNA damage was detected at 0 h, 6 h, 12 h, 18 h and 24 h after exposure to DOX via the comet assay, and the percent of DNA in the tail (% tail DNA) served as the indicator of DNA damage. The results demonstrated that (1) RFR could not directly induce DNA damage of human B-cell lymphoblastoid cells; (2) DOX could significantly induce DNA damage of human B-cell lymphoblastoid cells with the dose–effect relationship, and there were special repair characteristics of DNA damage induced by DOX; (3) E–E–E type (exposure to RFR for 2 h, then simultaneous exposure to RFR and DOX, and exposure to RFR for 6 h, 12 h, 18 h and 24 h after exposure to DOX) combinative exposure could obviously influence DNA repair at 6 h and 12 h after exposure to DOX for four DOX doses (0.075 μg/ml, 0.10 μg/ml, 0.15 μg/ml and 0.20 μg/ml) in human B-cell lymphoblastoid cells.     

DNA damage induced by mutagens in plant and human cell nuclei in acellular comet assay

Higher plant cells have a long tradition of use in the studies on environmental mutagenesis in situ, especially in relation to human health risk determination. The studies on the response of plant and human cells to physical and chemical mutagens showed differences in their sensitivity. The differences in the presence of cell components in plants and humans could influence such response. Additionally, the level of the organization of the employed material could influence DNA-damaging effect: leukocytes are isolated cells and plant--an intact organism. To preclude these obstacles, the effects of direct treatment of isolated nuclei with genotoxic agents were determined to compare the sensitivity of plant and human cells. In the present study, we have determined the DNA-damaging effects of two chemical mutagens: maleic acid hydrazide (MH) and N-methyl-N-nitroso-urea (MNU) applied to isolated nuclei of both plant and human cells. In order to compare the sensitivity of the nuclei of Nicotiana tabacum var. xanthi and the nuclei of leukocytes, the acellular Comet assay was carried out. The results showed higher sensitivity of the nuclei of leukocytes as compared to the nuclei of plant cells to mutagenic treatment with the applied doses of MH and MNU.     

Detection of alkylation damage in human lymphocyte DNA with the comet assay

The enzyme 3-methyladenine DNA glycosylase II (AlkA) is a bacterial repair enzyme that acts preferentially at 3-methyladenine residues in DNA, releasing the damaged base. The resulting baseless sugars are alkali-labile, and under the conditions of the alkaline comet assay (single cell gel electrophoresis) they appear as DNA strand breaks. AlkA is no t lesion-specific, but has a low activity even w ith undamagedbases. We have tested the enzyme at different concentrations to find conditions that maximise detection of alkylated bases with minimal attack on normal, undamaged DNA. AlkA detects damage in the DNA of cells treated with low concentrations of methyl methanesulphonate. We also find low background levels of alkylated bases in normal human lymphocytes.     

Transient DNA damage induced by high-frequency electromagnetic fields (GSM 1.8 GHz) in the human trophoblast HTR-8/SVneo cell line evaluated with the alkaline comet assay

One of the most controversial issue regarding high-frequency electromagnetic fields (HF-EMF) is their putative capacity to affect DNA integrity. This is of particular concern due to the increasing use of HF-EMF in communication technologies, including mobile phones. Although epidemiological studies report no detrimental effects on human health, the possible disturbance generated by HF-EMF on cell physiology remains controversial. In addition, the question remains as to whether cells are able to compensate their potential effects. We have previously reported that a 1-h exposure to amplitude-modulated 1.8 GHz sinusoidal waves (GSM-217 Hz, SAR = 2 W/kg) largely used in mobile telephony did not cause increased levels of primary DNA damage in human trophoblast HTR-8/SVneo cells. Nevertheless, further investigations on trophoblast cell responses after exposure to GSM signals of different types and durations were considered of interest. In the present work, HTR-8/SVneo cells were exposed for 4, 16 or 24 h to 1.8 GHz continuous wave (CW) and different GSM signals, namely GSM-217 Hz and GSM-Talk (intermittent exposure: 5 min field on, 10 min field off). The alkaline comet assay was used to evaluate primary DNA damages and/or strand breaks due to uncompleted repair processes in HF-EMF exposed samples. The amplitude-modulated signals GSM-217 Hz and GSM-Talk induced a significant increase in comet parameters in trophoblast cells after 16 and 24 h of exposure, while the un-modulated CW was ineffective. However, alterations were rapidly recovered and the DNA integrity of HF-EMF exposed cells was similar to that of sham-exposed cells within 2 h of recovery in the absence irradiation. Our data suggest that HF-EMF with a carrier frequency and modulation scheme typical of the GSM signal may affect the DNA integrity.     

In vitro effects of low-level, low-frequency electromagnetic fields on DNA damage in human leucocytes by comet assay

The sources for the effects of electromagnetic fields (EMFs) have been traced to time-varying as well as steady electric and magnetic fields, both at low and high to ultra high frequencies. Of these, the effects of low-frequency (50/60 HZ) magnetic fields, directly related to time-varying currents, are of particular interest as exposure to some fields may be commonly experienced. In the present study, investigations have been carried out at low-level (mT) and low-frequency (50 Hz) electromagnetic fields in healthy human volunteers. Their peripheral blood samples were exposed to 5 doses of electromagnetic fields (2,3,5,7 and 10mT at 50 Hz) and analysed by comet assay. The results were compared to those obtained from unexposed samples from the same subjects. 50 cells per treatment per individual were scored for comet-tail length which is an estimate of DNA damage. Data from observations among males were pooled for each flux density for analysis. At each flux density, with one exception, there was a significant increase in the DNA damage from the control value. When compared with a similar study on females carried out by us earlier, the DNA damage level was significantly higher in the females as compared to the males for each flux density.     

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.     

Effects of 1.8 GHz radiofrequency field on DNA damage and expression of heat shock protein 70 in human lens epithelial cells

To investigate the DNA damage, expression of heat shock protein 70 (Hsp70) and cell proliferation of human lens epithelial cells (hLEC) after exposure to the 1.8 GHz radiofrequency field (RF) of a global system for mobile communications (GSM). An Xc-1800 RF exposure system was used to employ a GSM signal at 1.8 GHz (217 Hz amplitude-modulated) with the output power in the specific absorption rate (SAR) of 1, 2 and 3 W/kg. After 2 h exposure to RF, the DNA damage of hLEC was accessed by comet assay at five different incubation times: 0, 30, 60, 120 and 240 min, respectively. Western blot and RT-PCR were used to determine the expression of Hsp70 in hLECs after RF exposure. The proliferation rate of cells was evaluated by bromodeoxyuridine incorporation on days 0, 1 and 4 after exposure. The results show that the difference of DNA-breaks between the exposed and sham-exposed (control) groups induced by 1 and 2 W/kg irradiation were not significant at any incubation time point (P > 0.05). The DNA damage caused by 3 W/kg irradiation was significantly increased at the times of 0 and 30 min after exposure (P < 0.05), a phenomenon that could not be seen at the time points of 60, 120 or 240 min (P > 0.05). Detectable mRNA as well as protein expression of Hsp70 was found in all groups. Exposure at SARs of 2 and 3 W/kg for 2 h exhibited significantly increased Hsp70 protein expression (P < 0.05), while no change in Hsp70 mRNA expression could be found in any of the groups (P > 0.05). No difference of the cell proliferation rate between the sham-exposed and exposed cells was found at any exposure dose tested (P > 0.05). The results indicate that exposure to non-thermal dosages of RF for wireless communications can induce no or repairable DNA damage and the increased Hsp70 protein expression in hLECs occurred without change in the cell proliferation rate. The non-thermal stress response of Hsp70 protein increase to RF exposure might be involved in protecting hLEC from DNA damage and maintaining the cellular capacity for proliferation.     

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.     

Application of the comet assay to measure DNA damage induced by UV radiation in the hydrophyte, Spirodela polyrhiza

The single-cell gel electrophoresis or comet assay is now widely used to detect DNA damage in animal cells induced by radiation or chemicals. Here, we apply the comet assay to measure ultraviolet (UV)-B-induced DNA damage in plant cells. The accepted animal cell protocol for the comet assay was modified to adapt it to plant cells. The major modifications were conversion of the plant cells to protoplasts and the use of T4 endonuclease V. As a positive control hydrogen peroxide was applied. Significant DNA damage was detected at 100 μ M H₂O₂. This type of DNA damage was not affected by T4 endonuclease V treatment, which implies that the mechanism of H₂O₂-induced DNA damage was different from UV-B-induced DNA damage. Our results also indicate that both UV-A and UV-B radiation can induce DNA single-strand breaks in plant cells, while UV-B was more effective than UV-A for inducing pyrimidine dimer formation.     

Evaluation of DNA damage in agricultural workers exposed to pesticides using single cell gel electrophoresis (comet) assay

BACKGROUND:

Pesticides are used in agriculture to protect crops, but they pose a potential risk to farmers and environment. The aim of the present study is to investigate the relation between the occupational exposure to various pesticides and the presence of DNA damage.

MATERIALS AND METHODS:

Blood samples of 210 exposed workers (after a day of intense spraying) and 50 control subjects belonging to various districts of Punjab (India) were evaluated using Comet assay. Sixty workers who showed DNA damage were selected for follow up at 5-6 months after the first sampling during a low or null spraying period.

RESULTS:

Significant differences were found in DNA damage between freshly exposed workers and controls and freshly exposed and followed up cases. There was significant increase in the comet parameters viz. mean comet tail length and frequency of cells showing migration in exposed workers as compared to controls (72.22 ± 20.76 vs. 46.92 ± 8.17, P<0.001; 31.79 vs. 5.77, P<0.001). In the second samples, followed up cases showed significant decrease in frequency of damaged cells as compared to freshly exposed workers of first sampling (P<0.05). The confounding factors such as variable duration of pesticide exposure, age, smoking, drinking and dietary habits etc which were expected to modulate the damage, were instead found to have no significant effect on DNA fragmentation.

CONCLUSION:

The evidence of a genetic hazard related to exposure resulting from the intensive use of pesticides stresses the need for educational programs for agricultural workers to reduce the use of chemicals in agriculture.     

Evaluation of DNA damage in leukocytes of G6PD-deficient Iranian newborns (Mediterranean variant) using comet assay

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common inherited disease, which causes neonatal hemolytic anemia and jaundice. Recent studies of our group showed that the Mediterranean variant of this enzyme (Gd-Md) is the predominant G6PD in Iranian male infants suffering from jaundice; this variant is classified as severe G6PD deficiency. Considering the importance of G6PD reaction and its products NADPH and glutathione (GSH) against oxidative stress, we hypothesized the failure of detoxification of H(2)O(2) in G6PD-deficient white blood cells that could probably induce primary DNA damage. For the evaluation of DNA damage, we analyzed mononuclear leukocytes of 36 males suffering from the Gd-Md deficiency using alkaline single cell gel electrophoresis (SCGE) or comet assay. The level of DNA damage was compared with the level of basal DNA damage in control group represented by healthy male infant donors (of the same age group). Visual scoring was used for the evaluation of DNA damages. The results showed that the mean level of the DNA strand breakage in mononuclear leukocytes of 36 male G6PD-deficient (Gd-Md) infants was significantly higher (P < 0.001) than those observed in the normal lymphocytes. In conclusion, this investigation indicates that the mononuclear leukocytes of the Gd-Md samples may be exposed to DNA damage due to oxidative stress. This is the first report using comet assay for evaluation of DNA damage in severe G6PD deficiency samples.     

DNA damage and repair induced in vitro by nitrilotriacetic acid (NTA) in human lymphocytes

In cultured human lymphocytes we determined the ability of nitrilotriacetic acid (NTA) to inhibit DNA replication and to stimulate DNA repair synthesis (UDS), as well as to influence the UDS induced by UV irradiation. In phytohemagglutinin-stimulated lymphocytes a strong inhibition of DNA replication was induced by NTA concentrations above 10(-3) M, which was accompanied by a marked cell lethality, whereas at lower doses the incorporation of tritiated thymidine (3H-TdR) into DNA or treated cells was slightly increased in comparison to untreated cells. When, after NTA pretreatment, UDS was determined by scintillation spectrometry or autoradiography in unstimulated G0 lymphocytes, UV-irradiated or unirradiated, an increased incorporation of 3H-TdR was observed, positively correlated with the NTA doses. This effect was only partially due to the expansion of the intracellular TdR pool as a consequence of the stimulation of 3H-TdR uptake by NTA. Even after normalization of the scintillometric data by the radioactivities of the soluble nucleotide fraction, significant increase of DNA repair synthesis was detected after treatment with 7.5 x 10(-3)-10(-2) M NTA.     

Evaluation of DNA damage in workers occupationally exposed to pesticides using single-cell gel electrophoresis (SCGE) assay. Pesticide genotoxicity revealed by comet assay

The comet assay, also called the single-cell gel electrophoresis (SCGE) assay, is a rapid and sensitive method for the detection of DNA damage (strand breaks and alkali-labile sites) in individual cells. The assay is based on the embedding of cells in agarose, their lysis in alkaline buffer and finally subjection to an electric current. In the present study, alkaline SCGE was used to evaluate the extent of primary DNA damage and DNA repair in peripheral blood lymphocytes of workers employed in pesticide production. After the period of high pesticide exposure, lymphocytes of the occupationally exposed workers manifested increased tail length and tail moment compared to the control group. After the workers spent 6 months out of the pesticide exposure zone, both endpoints were still above that of the control but significantly decreased as compared to the results of the first analysis.     

SIN-1-induced DNA damage in isolated human peripheral blood lymphocytes as assessed by single cell gel electrophoresis (comet assay)

Human lymphocytes were exposed to increasing concentrations of SIN-1, which generates superoxide and nitric oxide, and the formation of single-strand breaks (SSB) in individual cells was determined by the single-cell gel electrophoresis assay (comet assay). A dose- and time-dependent increase in SSB formation was observed rapidly after the addition of SIN-1 (0.1-15 mM). Exposure of the cells to SIN-1 (5 mM) in the presence of excess of superoxide dismutase (0.375 mM) increased the formation of SSB significantly, whereas 1000 U/ml catalase significantly decreased the quantity of SSB. The simultaneous presence of both superoxide dismutase and catalase before the addition of SIN-1 brought the level of SSB to that of the untreated cells. Moreover, pretreatment of the cells with the intracellular Ca(2+)-chelator BAPTA/AM inhibited SIN-1-induced DNA damage, indicating the involvement of intracellular Ca(2+) changes in this process. On the other hand, pretreatment of the same cells with ascorbate or dehydroascorbate did not offer any significant protection in this system. The data suggest that H2O2-induced changes in Ca(2+) homeostasis are the predominant pathway for the induction of SSB in human lymphocytes exposed to oxidants.     

Detection of oxidative DNA damage in isolated marine bivalve hemocytes using the comet assay and formamidopyrimidine glycosylase (Fpg)

Organisms in polluted areas can be exposed to complex mixtures of chemicals; however, exposure to genotoxic contaminants can be particularly devastating. DNA damage can lead to necrosis, apoptosis, or heritable mutations, and therefore has the potential to impact populations as well as individuals. Single cell gel electrophoresis (the comet assay) is a simple and sensitive technique used to examine DNA damage in single cells. The lesion-specific DNA repair enzyme formamidopyrimidine glycoslyase (Fpg) can be used in conjunction with the comet assay to detect 8-oxoguanine and other damaged bases, which are products of oxidative damage. Fpg was used to detect oxidative DNA damage in experiments where isolated oyster (Crassostrea virginica) and clam (Mercenaria mercenaria) hemocytes were exposed to hydrogen peroxide. Standard enzyme buffers used with Fpg and the comet assay produced unacceptably high amounts of DNA damage in the marine bivalve hemocytes used in this study necessitating a modification of existing methods. A sodium chloride based reaction buffer was successfully used. Oxidative DNA damage can be detected in isolated oyster and clam hemocytes using Fpg and the comet assay when the sodium chloride reaction buffer and protocols outlined here are employed. The use of DNA repair enzymes, such as Fpg, in conjunction with the comet assay expands the usefulness and sensitivity of this assay, and provides important insights into the mechanisms of DNA damage.     

Detection of oxidative DNA damage in isolated marine bivalve hemocytes using the comet assay and formamidopyrimidine glycosylase (Fpg)

Organisms in polluted areas can be exposed to complex mixtures of chemicals; however, exposure to genotoxic contaminants can be particularly devastating. DNA damage can lead to necrosis, apoptosis, or heritable mutations, and therefore has the potential to impact populations as well as individuals. Single cell gel electrophoresis (the comet assay) is a simple and sensitive technique used to examine DNA damage in single cells. The lesion-specific DNA repair enzyme formamidopyrimidine glycoslyase (Fpg) can be used in conjunction with the comet assay to detect 8-oxoguanine and other damaged bases, which are products of oxidative damage. Fpg was used to detect oxidative DNA damage in experiments where isolated oyster (Crassostrea virginica) and clam (Mercenaria mercenaria) hemocytes were exposed to hydrogen peroxide. Standard enzyme buffers used with Fpg and the comet assay produced unacceptably high amounts of DNA damage in the marine bivalve hemocytes used in this study necessitating a modification of existing methods. A sodium chloride based reaction buffer was successfully used. Oxidative DNA damage can be detected in isolated oyster and clam hemocytes using Fpg and the comet assay when the sodium chloride reaction buffer and protocols outlined here are employed. The use of DNA repair enzymes, such as Fpg, in conjunction with the comet assay expands the usefulness and sensitivity of this assay, and provides important insights into the mechanisms of DNA damage.     

A simplified chromatin dispersion (nuclear halo) assay for detecting DNA breakage induced by ionizing radiation and chemical agents

Methods for visualizing DNA damage at the microscopic level are based on treatment of cell nuclei with saline or alkaline solutions. These procedures for achieving chromatin dispersion produce halos that surround the nuclear remnants. We improved the fast halo assay for visualizing DNA breakage in cultured cells to create a simplified method for detection and quantitative evaluation of DNA breakage. Nucleated erythrocytes from chicken blood were selected as a model test system to analyze the production of nuclear halos after treatment with X-rays or H(2)O(2). After staining with ethidium bromide or Wright's methylene blue-eosin solution, nuclear halos were easily observed by fluorescence or bright-field microscopy, respectively, which permits rapid visualization of DNA breakage in damaged cells. By using image processing and analysis with the public domain ImageJ software, X-ray dose and H(2)O(2) concentration could be correlated well with the size of nuclear halos and the halo:nucleus ratio. Our results indicate that this simplified nuclear halo assay can be used as a rapid, reliable and inexpensive procedure to detect and quantify DNA breakage induced by ionizing radiation and chemical agents. A mechanistic model to explain the differences between the formation of saline or alkaline halos also is suggested.     

A simplified chromatin dispersion (nuclear halo) assay for detecting DNA breakage induced by ionizing radiation and chemical agents

Abstract

Methods for visualizing DNA damage at the microscopic level are based on treatment of cell nuclei with saline or alkaline solutions. These procedures for achieving chromatin dispersion produce halos that surround the nuclear remnants. We improved the fast halo assay for visualizing DNA breakage in cultured cells to create a simplified method for detection and quantitative evaluation of DNA breakage. Nucleated erythrocytes from chicken blood were selected as a model test system to analyze the production of nuclear halos after treatment with X-rays or H₂O₂. After staining with ethidium bromide or Wright's methylene blue-eosin solution, nuclear halos were easily observed by fluorescence or bright-field microscopy, respectively, which permits rapid visualization of DNA breakage in damaged cells. By using image processing and analysis with the public domain ImageJ software, X-ray dose and H₂O₂ concentration could be correlated well with the size of nuclear halos and the halo:nucleus ratio. Our results indicate that this simplified nuclear halo assay can be used as a rapid, reliable and inexpensive procedure to detect and quantify DNA breakage induced by ionizing radiation and chemical agents. A mechanistic model to explain the differences between the formation of saline or alkaline halos also is suggested.     

Isolation of mature spinal motor neurons and single-cell analysis using the comet assay of early low-level DNA damage induced in vitro and in vivo.

We developed an isolation technique for motor neurons from adult rat spinal cord. Spinal cord enlargements were discretely microdissected into ventral horn tissue columns that were trypsin-digested and subjected to differential low-speed centrifugation to fractionate ventral horn cell types. A fraction enriched in alpha-motor neurons was isolated. Motor neuron enrichment was verified by immunofluorescence for choline acetyltransferase and prelabeling axon projections to skeletal muscle. Adult motor neurons were isolated from na?ve rats and were exposed to oxidative agents or were isolated from rats with sciatic nerve lesions (avulsions). We tested the hypothesis, using single-cell gel electrophoresis (comet assay), that hydrogen peroxide, nitric oxide, and peroxynitrite exposure in vitro and axotomy in vivo induce DNA damage in adult motor neurons early during their degeneration. This study contributes three important developments in the study of motor neurons. It demonstrates that mature spinal motor neurons can be isolated and used for in vitro models of motor neuron degeneration. It shows that adult motor neurons can be isolated from in vivo models of motor neuron degeneration and evaluated on a single-cell basis. This study also demonstrates that the comet assay is a feasible method for measuring DNA damage in individual motor neurons. Using these methods, we conclude that motor neurons undergoing oxidative stress from reactive oxygen species and axotomy accumulate DNA damage early in their degeneration.     

Estimates of DNA damage by the comet assay in the direct-developing frog Eleutherodactylus johnstonei (Anura, Eleutherodactylidae)

The aim of this study was to use the Comet assay to assess genetic damage in the direct-developing frog Eleutherodactylus johnstonei. A DNA diffusion assay was used to evaluate the effectiveness of alkaline, enzymatic and alkaline/enzymatic treatments for lysing E. johnstonei blood cells and to determine the amount of DNA strand breakage associated with apoptosis and necrosis. Cell sensitivity to the mutagens bleomycin (BLM) and 4-nitro-quinoline-1-oxide (4NQO) was also assessed using the Comet assay, as was the assay reproducibility. Alkaline treatment did not lyse the cytoplasmic and nuclear membranes of E. johnstonei blood cells, whereas enzymatic digestion with proteinase K (40 μg/mL) yielded naked nuclei. The contribution of apoptosis and necrosis (assessed by the DNA diffusion assay) to DNA damage was estimated to range from 0% to 8%. BLM and 4NQO induced DNA damage in E. johnstonei blood cells at different concentrations and exposure times. Dose-effect curves with both mutagens were highly reproducible and showed consistently low coefficients of variation (CV ≤ 10%). The results are discussed with regard to the potential use of the modified Comet assay for assessing the exposure of E. johnstonei to herbicides in ecotoxicological studies.