Genomic instability induced by radiation-mimicking chemicals is not associated with persistent mitochondrial degeneration.

Ionizing radiation has been shown to cause induced genomic instability (IGI), which is defined as a persistently increased rate of genomic damage in the progeny of the exposed cells. In this study, IGI was investigated by exposing human SH-SY5Y neuroblastoma cells to hydroxyurea and zeocin, two chemicals mimicking different DNA-damaging effects of ionizing radiation. The aim was to explore whether IGI was associated with persistent mitochondrial dysfunction. Changes to mitochondrial function were assessed by analyzing mitochondrial superoxide production, mitochondrial membrane potential, and mitochondrial activity. The formation of micronuclei was used to determine immediate genetic damage and IGI. Measurements were performed either immediately, 8 days, or 15 days following exposure. Both hydroxyurea and zeocin increased mitochondrial superoxide production and affected mitochondrial activity immediately after exposure, and mitochondrial membrane potential was affected by zeocin, but no persistent changes in mitochondrial function were observed. IGI became manifested 15 days after exposure in hydroxyurea-exposed cells. In conclusion, immediate responses in mitochondrial function did not cause persistent dysfunction of mitochondria, and this dysfunction was not required for IGI in human neuroblastoma cells.

Pilot study on the therapeutic potential of radiofrequency magnetic fields: growth inhibition of implanted tumours in mice.

The present study investigated possible therapeutic effects of radiofrequency or hypomagnetic fields on the growth rate of two types of implanted tumours. To this end, mice with implanted fibrosarcoma and pancreatic tumours were exposed continuously to a 2 µT, 10 MHz radiofrequency magnetic field (MF) perpendicular to a 45 µT static MF or to a hypomagnetic (~0.4-1 µT) field. The reasoning for a 10 MHz treatment was based on a current theoretical explanation for MF effects, which predicts a resonance phenomenon in this frequency range. Radiofrequency MFs reduced consistently the growth rate of two implanted tumour types (by ~30% in both cases). Also, hypomagnetic field hindered tumour growth in both tumour types, but the observation was not statistically significant with fibrosarcoma tumours. In conclusion, although experiments included a limited number of animals, the results indicate that MFs may offer a novel therapeutic strategy in the treatment of cancer.

Magnetocarcinogenesis: is there a mechanism for carcinogenic effects of weak magnetic fields?

Extremely low-frequency (ELF) magnetic fields have been classified as possibly carcinogenic, mainly based on rather consistent epidemiological findings suggesting a link between childhood leukaemia and 50-60 Hz magnetic fields from power lines. However, causality is not the only possible explanation for the epidemiological associations, as animal and in vitro experiments have provided only limited support for carcinogenic effects of ELF magnetic fields. Importantly, there is no generally accepted biophysical mechanism that could explain such effects. In this review, we discuss the possibility that carcinogenic effects are based on the radical pair mechanism (RPM), which seems to be involved in magnetoreception in birds and certain other animals, allowing navigation in the geomagnetic field. We review the current understanding of the RPM in magnetoreception, and discuss cryptochromes as the putative magnetosensitive molecules and their possible links to cancer-relevant biological processes. We then propose a hypothesis for explaining the link between ELF fields and childhood leukaemia, discuss the strengths and weaknesses of the current evidence, and make proposals for further research.

Genotoxicity of intermediate frequency magnetic fields in vitro and in vivo.

We assessed genotoxic effects of intermediate frequency magnetic fields (MF) in vitro and in vivo. Rat primary astrocytes were exposed for 24 h to a 7.5 kHz MF at a magnetic flux density of 30 or 300 µT. Male C57BL/6 J mice were exposed continuously for 5 weeks to a 7.5 kHz MF at 12 or 120 µT, and blood samples were collected for the genotoxicity assays. To evaluate possible co-genotoxicity, the in vitro experiments included combined exposure with menadione (an agent that induces mitochondrial superoxide production and DNA damage) and methyl methanesulfonate (an alkylating agent). DNA damage and DNA repair (in vitro) were measured using the alkaline Comet assay and formation of micronuclei was assessed microscopically (in vivo) or using flow cytometry (in vitro). The results did not support genotoxicity or co-genotoxicity of 7.5 kHz MFs at magnetic flux densities up to 300 µT in vitro or in vivo. On the contrary, there was some evidence that exposure to 7.5 kHz MFs might reduce the level of genetic damage. Strongest indication of any biological effects was obtained from measurements of relative cell number, which was significantly and consistently increased after MF exposure in all in vitro experiments. Health implications of this finding are unclear, but it suggests that 7.5 kHz MFs may stimulate cell proliferation or suppress cell death.

Induction of genomic instability, oxidative processes, and mitochondrial activity by 50Hz magnetic fields in human SH-SY5Y neuroblastoma cells.

Epidemiological studies have suggested that exposure to 50Hz magnetic fields (MF) increases the risk of childhood leukemia, but there is no mechanistic explanation for carcinogenic effects. In two previous studies we have observed that a 24-h pre-exposure to MF alters cellular responses to menadione-induced DNA damage. The aim of this study was to investigate the cellular changes that must occur already during the first 24h of exposure to MF, and to explore whether the MF-induced changes in DNA damage response can lead to genomic instability in the progeny of the exposed cells. In order to answer these questions, human SH-SY5Y neuroblastoma cells were exposed to a 50-Hz, 100-µT MF for 24h, followed by 3-h exposure to menadione. The main finding was that MF exposure was associated with increased level of micronuclei, used as an indicator of induced genomic instability, at 8 and 15d after the exposures. Other delayed effects in MF-exposed cells included increased mitochondrial activity at 8d, and increased reactive oxygen species (ROS) production and lipid peroxidation at 15d after the exposures. Oxidative processes (ROS production, reduced glutathione level, and mitochondrial superoxide level) were affected by MF immediately after the exposure. In conclusion, the present results suggest that MF exposure disturbs oxidative balance immediately after the exposure, which might explain our previous findings on MF altered cellular responses to menadione-induced DNA damage. Persistently elevated levels of micronuclei were found in the progeny of MF-exposed cells, indicating induction of genomic instability.

50 Hz magnetic field influences caspase-3 activity and cell cycle distribution in ionizing radiation exposed SH-SY5Y neuroblastoma cells.

PURPOSE: Earlier evidence suggests that extremely low frequency magnetic fields (ELF MFs) can modify the effects of carcinogenic agents. However, the studies conducted so far with ionizing radiation as the co-exposure agent are sparse and have provided inconclusive results. We investigated whether 50 Hz MFs alone, or in combination with ionizing radiation alter cell biological variables relevant to cancer and the biological effects of ionizing radiation. MATERIALS AND METHODS: Human SH-SY5Y neuroblastoma cells were sham exposed or exposed to 100 or 500 µT MF for 24 h either before or after ionizing radiation exposure (0, 0.4 or 2 Gy). After the exposures, cells were assayed for viability, clonogenicity, reactive oxygen species, caspase-3 activity, and cell cycle distribution. Cell cycle distribution was assayed with propidium iodide staining followed by flow cytometry analysis and ROS levels were assayed together with cell viability by double staining with DeepRed and Sytox Blue followed by flow cytometry analysis. RESULTS: Increased caspase-3 activity was observed in cells exposed to 500 µT MF before or after ionizing radiation. Furthermore, exposure to the 500 µT MF after the ionizing radiation decreased the percentage of cells in S-phase. No changes in the ROS levels, clonogenicity, or viability of the cells were observed in the MF exposed groups compared to the corresponding sham exposed groups, and no MF effects were observed in cells exposed at 100 µT. CONCLUSIONS: Only the 500 µT magnetic flux density affected SH-SY5Y cells significantly. The effects were small but may nevertheless help to understand how MFs modify the effects of ionizing radiation. The increase in caspase-3 activity may not reflect effects on apoptosis, as no changes were observed in the subG1 phase of the cell cycle. In contrast to some earlier findings, 50 Hz MF exposure after ionizing radiation was not less effective than MF treatment given prior to ionizing radiation.

Static or 50 Hz magnetic fields at 100 µT do not modify the clonogenic survival of doxorubicin-treated MCF-7 cancer cells.

The objective of this study was to evaluate whether static or 50 Hz magnetic fields (MFs) modify responses to the chemotherapeutic agent doxorubicin in human MCF-7 breast cancer cells. To this end, cells were exposed to static or 50 Hz MFs at 100 µT with or without doxorubicin for 3 h. Following the exposures, cytosolic and mitochondrial superoxide levels, DNA damage levels, and the clonogenic survival of the cells were evaluated. It was found that static MFs decreased the DNA damage level induced by doxorubicin treatment (p = 0.023), but no effects were observed for either cytosolic and mitochondrial superoxide levels or the clonogenic survival of the cells. On the other hand, 50 Hz MF increased doxorubicin-induced cytosolic superoxide levels (p = 0.016), while the mitochondrial superoxide level, DNA damage level, and clonogenic survival were unaffected. In conclusion, we found that static and 50 Hz MFs may modify responses to doxorubicin treatment, but the subsequent survival of the doxorubicin-treated cancer cells was unaffected by both types of MFs. Therefore, the present results suggest that static or 50 Hz MFs for 3 h do not modify the efficacy of doxorubicin in MCF-7 cancer cells.

Do 50/60 Hz magnetic fields influence oxidative or DNA damage responses in human SH-SY5Y neuroblastoma cells?

PURPOSE: We investigated the possible effects of 50 and 60 Hz magnetic fields (MFs) on reactive oxygen species (ROS) production, DNA damage, DNA damage repair rate, as well as gene expression related to oxidative stress and DNA damage signaling. MATERIALS AND METHODS: Human SH-SY5Y neuroblastoma cells were sham-exposed or exposed to 100 µTRMS MFs for 24 h, then assayed or further treated with 100 µM menadione for 1 h before the assay. The levels of ROS and cytosolic superoxide anion (O2•-) were assayed fluorometrically. DNA damage and gene expression were assayed by comet assay and RT-qPCR, respectively. To examine whether MFs affected DNA damage repair rate, cells were allowed to repair their DNA for 1 or 2 h after menadione treatment and then assayed for DNA damage. RESULTS: There was suggestive evidence of a general low-magnitude increase in the expression of ROS-related genes (primarily genes with antioxidant activity) when quantified immediately after MF exposure, suggesting a response to a small increase in ROS level. The possible upregulation of ROS-related genes is supported by the finding that the level of menadione-induced ROS was consistently decreased by 50 Hz MFs (not significantly by 60 Hz MFs) in several measurements 30-60 min after MF exposure. MF exposures did not affect cytosolic O2•- levels, DNA damage, or its repair rate. Changes in the expression of DNA damage-signaling genes in the MF-exposed cells did not exceed the expected rate of false-positive findings. No firm evidence was found for differential effects from 50 vs. 60 Hz MFs. CONCLUSIONS: While only weak effects were found on the endpoints measured, the results are consistent with MF effects on ROS signaling.

Development of an adverse outcome pathway for radiation-induced microcephaly via expert consultation and machine learning.

BACKGROUND: Brain development during embryogenesis and in early postnatal life is particularly complex and involves the interplay of many cellular processes and molecular mechanisms, making it extremely vulnerable to exogenous insults, including ionizing radiation (IR). Microcephaly is one of the most frequent neurodevelopmental abnormalities that is characterized by small brain size, and is often associated with intellectual deficiency. Decades of research span from epidemiological data on in utero exposure of the A-bomb survivors, to studies on animal and cellular models that allowed deciphering the most prominent molecular mechanisms leading to microcephaly. The Adverse Outcome Pathway (AOP) framework is used to organize, evaluate and portray the scientific knowledge of toxicological effects spanning different biological levels of organizations, from the initial interaction with molecular targets to the occurrence of a disease or adversity. In the present study, the framework was used in an attempt to organize the current scientific knowledge on microcephaly progression in the context of ionizing radiation (IR) exposure. This work was performed by a group of experts formed during a recent workshop organized jointly by the Multidisciplinary European Low Dose Initiative (MELODI) and the European Radioecology Alliance (ALLIANCE) associations to present the AOP approach and tools. Here we report on the development of a putative AOP for congenital microcephaly resulting from IR exposure based on discussions of the working group and we emphasize the use of a novel machine-learning approach to assist in the screening of the available literature to develop AOPs. CONCLUSION: The expert consultation led to the identification of crucial biological events for the progression of microcephaly upon exposure to IR, and highlighted current knowledge gaps. The machine learning approach was successfully used to screen the existing knowledge and helped to rapidly screen the body of evidence and in particular the epidemiological data. This systematic review approach also ensured that the analysis was sufficiently comprehensive to identify the most relevant data and facilitate rapid and consistent AOP development. We anticipate that as machine learning approaches become more user-friendly through easy-to-use web interface, this would allow AOP development to become more efficient and less time consuming.

The duration of exposure to 50 Hz magnetic fields: Influence on circadian genes and DNA damage responses in murine hematopoietic FDC-P1 cells.

We investigated the effects of 50 Hz extremely low-frequency magnetic fields (MFs) on gene expression related to the circadian rhythm or DNA damage signaling and whether these fields modify DNA damage repair rate after bleomycin treatment. Murine FDC-P1 hematopoietic cells were exposed for different durations (15 min, 2 h, 12 h, and 24 h) to either 200 µT MFs or sham-exposures. Cells were then collected for comet assay or real-time PCR to determine immediate DNA damage level and circadian rhythm gene expression, respectively. To assess DNA-damage signaling and DNA repair rate, the cells were subsequently treated with 20 µg/mL bleomycin for 1 h and then either assayed immediately or allowed to repair their DNA for 1 or 2 h. We found that circadian rhythm-related genes were upregulated after 12 h of MF exposure and downregulated after 24 h of MF exposure, but none of the affected genes were core genes controlling the circadian rhythm. In addition, we found that the repair rate for bleomycin-induced damage was only decreased after MF exposure for 24 h. In conclusion, our findings suggest that the effects of MFs are duration-dependent; they were observed predominantly after long exposures.

Combined effects of 872 MHz radiofrequency radiation and ferrous chloride on reactive oxygen species production and DNA damage in human SH-SY5Y neuroblastoma cells.

The aim of the present study was to investigate possible cooperative effects of radiofrequency (RF) radiation and ferrous chloride (FeCl(2)) on reactive oxygen species (ROS) production and DNA damage. In order to test intracellular ROS production as a possible underlying mechanism of DNA damage, we applied the fluorescent probe DCFH-DA. Integrity of DNA was quantified by alkaline comet assay. The exposures to 872 MHz RF radiation were conducted at a specific absorption rate (SAR) of 5 W/kg using continuous waves (CW) or a modulated signal similar to that used in Global System for Mobile Communications (GSM) phones. Four groups were included: (1) Sham exposure (control), (2) RF radiation, (3) Chemical treatment, (4) Chemical treatment, and RF radiation. In the ROS production experiments, human neuroblastoma (SH-SY5Y) cells were exposed to RF radiation and 10 microg/ml FeCl(2) for 1 h. In the comet assay experiments, the exposure time was 3 h and an additional chemical (0.015% diethyl maleate) was used to make DNA damage level observable. The chemical treatments resulted in statistically significant responses, but no effects from either CW or modulated RF radiation were observed on ROS production, DNA damage or cell viability.

Mitochondrial DNA damage in the hair bulb: can it be used as a noninvasive biomarker of local exposure to low LET ionizing radiation?

Purpose: Our aim was to evaluate whether mitochondrial DNA (mtDNA) damage in hair bulbs could be a suitable biomarker for the detection of local exposure to ionizing radiation.Materials and methods: Mouse hair was collected 4 and 24 hours, 3 and 10 days after single whole-body exposure to 0, 0.1, and 2 Gy radiation. Pubic hair (treated area) and scalp hair (control area) were collected from 13 prostate cancer patients before and after fractioned radiotherapy with an average total dose of 2.7 Gy to follicles after five fractions. Unspecified lesion frequency of mtDNA was analyzed with long PCR, large mtDNA deletion levels were tested with real-time PCR.Results: Unspecified lesion frequency of mtDNA significantly increased in mouse hair 24 hours after irradiation with 2 Gy, but variance among samples was high. No increase in lesion frequency could be detected after 0.1 Gy irradiation. In prostate cancer patients, there was no significant change in either the unspecified lesion frequency or in the proportion of 4934-bp deleted mtDNA in pubic hair after radiotherapy. The proportions of murine 3860-bp common deletion, human 4977-bp common deletion and 7455-bp deleted mtDNA were too low to be analyzed reliably.Conclusions: Our results suggest that the unspecified lesion frequency and proportion of large deletions of mtDNA in hair bulbs are not suitable biomarkers of exposure to ionizing radiation.

Enhancement of chemically induced reactive oxygen species production and DNA damage in human SH-SY5Y neuroblastoma cells by 872 MHz radiofrequency radiation.

The objective of the study was to investigate effects of 872 MHz radiofrequency (RF) radiation on intracellular reactive oxygen species (ROS) production and DNA damage at a relatively high SAR value (5 W/kg). The experiments also involved combined exposure to RF radiation and menadione, a chemical inducing intracellular ROS production and DNA damage. The production of ROS was measured using the fluorescent probe dichlorofluorescein and DNA damage was evaluated by the Comet assay. Human SH-SY5Y neuroblastoma cells were exposed to RF radiation for 1 h with or without menadione. Control cultures were sham exposed. Both continuous waves (CW) and a pulsed signal similar to that used in global system for mobile communications (GSM) mobile phones were used. Exposure to the CW RF radiation increased DNA breakage (p<0.01) in comparison to the cells exposed only to menadione. Comparison of the same groups also showed that ROS level was higher in cells exposed to CW RF radiation at 30 and 60 min after the end of exposure (p<0.05 and p<0.01, respectively). No effects of the GSM signal were seen on either ROS production or DNA damage. The results of the present study suggest that 872 MHz CW RF radiation at 5 W/kg might enhance chemically induced ROS production and thus cause secondary DNA damage. However, there is no known mechanism that would explain such effects from CW RF radiation but not from GSM modulated RF radiation at identical SAR.

Proliferation, oxidative stress and cell death in cells exposed to 872 MHz radiofrequency radiation and oxidants.

Human SH-SY5Y neuroblastoma and mouse L929 fibroblast cells were exposed to 872 MHz radiofrequency (RF) radiation using continuous waves (CW) or a modulated signal similar to that emitted by GSM mobile phones at a specific absorption rate (SAR) of 5 W/kg in isothermal conditions. To investigate possible combined effects with other agents, menadione was used to induce reactive oxygen species, and tert-butylhydroperoxide (t-BOOH) was used to induce lipid peroxidation. After 1 or 24 h of exposure, reduced cellular glutathione levels, lipid peroxidation, proliferation, caspase 3 activity, DNA fragmentation and viability were measured. Two statistically significant differences related to RF radiation were observed: Lipid peroxidation induced by t-BOOH was increased in SH-SY5Y (but not in L929) cells, and menadione-induced caspase 3 activity was increased in L929 (but not in SH-SY5Y) cells. Both differences were statistically significant only for the GSM-modulated signal. The other end points were not significantly affected in any of the experimental conditions, and no effects were observed from exposure to RF radiation alone. The positive findings may be due to chance, but they may also reflect effects that occur only in cells sensitized by chemical stress. Further studies are required to investigate the reproducibility and dose response of the possible effects.

TCDD-induced mitochondrial superoxide production does not lead to mitochondrial degeneration or genomic instability in human SH-SY5Y neuroblastoma cells.

Several genotoxic and non-genotoxic agents have been reported to cause delayed genetic damage in the progeny of the exposed cells. Such induced genomic instability (IGI) may be a driving force in carcinogenesis, and it is thus highly important to understand the cellular events accompanying it. The aim of this study was to investigate whether 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) affects mitochondrial integrity and can consequently induce genomic instability. Mitochondrial integrity was evaluated by measuring mitochondrial superoxide production, mitochondrial membrane potential, and mitochondrial activity. Micronucleus formation was used to assess immediate genetic damage and IGI. The assays were performed either immediately, 8 or 15d after the exposure. Mitochondrial superoxide production was increased by TCDD immediately after the exposure. No consistent effects on mitochondrial integrity were observed at later time points, although slightly decreased mitochondrial membrane potential at 8d and increased mitochondrial superoxide potential production at 15 after exposure were observed in the TCDD-exposed cells. TCDD did not cause immediate genetic damage, and significant IGI was not observed. In conclusion, the present results suggest that immediate TCDD-induced increase in mitochondrial superoxide level does not lead to persistent loss of mitochondrial integrity or IGI in human SH-SY5Y neuroblastoma cells.

Cellular detection of 50 Hz magnetic fields and weak blue light: effects on superoxide levels and genotoxicity.

PURPOSE: We tested the hypothesis that the effects of 50 Hz magnetic fields (MFs) on superoxide levels and genotoxicity depend on the presence of blue light. MATERIALS AND METHODS: Human SH-SY5Y neuroblastoma cells were exposed to a 50 Hz, 100 µT MF with or without non-phototoxic level of blue light for 24 h. We also studied whether these treatments alter responses to menadione, an agent that induces mitochondrial superoxide (O2• -) production and DNA damage. Micronuclei, proliferation, viability, cytosolic and mitochondrial O2• - levels were assessed. RESULTS: MF (without blue light) increased cytosolic O2• - production and blue light suppressed this effect. Mitochondrial O2• - production was reduced by both MF and blue light, but these effects were not additive. Micronucleus frequency was not affected by blue light or MF alone, but blue light (significantly when combined with MF) enhanced menadione-induced micronuclei. CONCLUSIONS: The original simple hypothesis (blue light is needed for MF effects) was not supported, but interaction of MF and blue light was nevertheless observed. The results are consistent with MF effects on light-independent radical reactions.

Modification of p21 level and cell cycle distribution by 50 Hz magnetic fields in human SH-SY5Y neuroblastoma cells.

PURPOSE: In our previous studies, exposure to extremely low frequency (ELF) magnetic fields (MF) altered responses to DNA damage caused by menadione. The aim of this study was to evaluate possible ELF MF induced changes in proteins involved in DNA damage responses and in cell cycle distribution. MATERIALS AND METHODS: Based on our previous studies, the exposure protocol included pre-exposure of human SH-SY5Y neuroblastoma cells to a 50 Hz, 100 µT MF for 24 h prior to a 3-h menadione treatment. As DNA damage responses are relatively fast processes, a 1-h menadione treatment was also included in the experiments. The menadione concentrations used were 1, 10, 15, 20, and 25 µM. Immunoblotting was used to assess the levels of DNA damage response-related proteins (γ-H2AX, Chk1, phospho-Chk1, p21, p27, and p53), while the level of DNA damage was assessed by the alkaline Comet assay. Cell cycle distribution was assayed by SYTOX Green staining followed by flow cytometry analysis. RESULTS: The main findings in MF-exposed cells were decreased p21 protein level after the 1-h menadione treatment, as well as increased proportion of cells in the G1 phase and decreased proportion of S phase cells after the 3-h menadione treatment. These effects were detectable also in the absence of menadione. CONCLUSIONS: The results indicate that MF exposure can alter the G1 checkpoint response and that the p21 protein may be involved in early responses to MF exposure.

Induction of micronuclei and superoxide production in neuroblastoma and glioma cell lines exposed to weak 50 Hz magnetic fields.

Extremely low-frequency (ELF) magnetic fields (MF) have been associated with adverse health effects in epidemiological studies. However, there is no known mechanism for biological effects of weak environmental MFs. Previous studies indicate MF effects on DNA integrity and reactive oxygen species, but such evidence is limited to MFs higher (greater than or equal to 100 µT) than those generally found in the environment. Effects of 10 and 30 µT fields were studied in SH-SY5Y and C6 cells exposed to 50-Hz MFs for 24 h. Based on earlier findings, menadione (MQ) was used as a cofactor. Responses to MF were observed in both cell lines, but the effects differed between the cell lines. Micronuclei were significantly increased in SH-SY5Y cells at 30 µT. This effect was largest at the highest MQ dose used. Increased cytosolic and mitochondrial superoxide levels were observed in C6 cells. The effects on superoxide levels were independent of MQ, enabling further mechanistic studies without co-exposure to MQ. The micronucleus and mitochondrial superoxide data were consistent with a conventional rising exposure-response relationship. For cytosolic superoxide, the effect size was unexpectedly large at 10 µT. The results indicate that the threshold for biological effects of ELF MFs is 10 µT or less.

Genomic instability induced by 50Hz magnetic fields is a dynamically evolving process not blocked by antioxidant treatment.

Increased level of micronuclei was observed in SH-SY5Y cells in a previous study at 8 and 15 days after exposure to extremely low frequency (ELF) magnetic fields (MF), indicating possible induction of genomic instability in the progeny of the exposed cells. The aim of this study was to further explore the induction of genomic instability by ELF MFs by increasing the follow-up time up to 45 days after exposure. Human SH-SY5Y neuroblastoma cells were exposed to a 50Hz, 100µT MF for 24h with or without co-exposure to menadione (MQ), a chemical agent that increases cellular superoxide production. Micronuclei, reactive oxygen species (ROS) and lipid peroxidation (LPO) were measured at 15, 30 and 45 days after exposure. To study the possible causal role of ROS in the delayed effects of MF, the antioxidant N-acetylcysteine (NAC) was administered before MF exposure. Consistently with the previous study, the level of micronuclei was statistically significantly elevated 15 days after exposure. A similar effect was observed at 30 days, but not at 45 days after exposure. The level of LPO was statically significantly decreased 30 and 45 days after exposure. Consistently with our previous findings, the MF effect did not depend on co-exposure to MQ. Treatment with NAC effectively decreased cellular ROS level and suppressed the effect of MQ on ROS, but it did not block the MF effect, indicating that increase in ROS is not needed as a causal link between MF exposure and induction of delayed effects. The results presented here are consistent with genomic instability that persists in the progeny of MF-exposed cells up to at least 30 days after exposure. Changes in LPO observed at 30 and 45 days after exposure indicates that the MF-initiated process may continue up to at least 45 days after exposure.