Jukka Luukkonen

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

Abstract Höytö, A., Luukkonen, J., Juutilainen, J. and Naarala, J. Proliferation, Oxidative Stress and Cell Death in Cells Exposed to 872 MHz Radiofrequency Radiation and Oxidants. Radiat. Res. 170, 235–243 (2008). 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.

Pre-exposure to 50 Hz magnetic fields modifies menadione-induced genotoxic effects in human SH-SY5Y neuroblastoma cells.

Background Extremely low frequency (ELF) magnetic fields (MF) are generated by power lines and various electric appliances. They have been classified as possibly carcinogenic by the International Agency for Research on Cancer, but a mechanistic explanation for carcinogenic effects is lacking. A previous study in our laboratory showed that pre-exposure to ELF MF altered cancer-relevant cellular responses (cell cycle arrest, apoptosis) to menadione-induced DNA damage, but it did not include endpoints measuring actual genetic damage. In the present study, we examined whether pre-exposure to ELF MF affects chemically induced DNA damage level, DNA repair rate, or micronucleus frequency in human SH-SY5Y neuroblastoma cells. Methodology/Principal Findings Exposure to 50 Hz MF was conducted at 100 µT for 24 hours, followed by chemical exposure for 3 hours. The chemicals used for inducing DNA damage and subsequent micronucleus formation were menadione and methyl methanesulphonate (MMS). Pre-treatment with MF enhanced menadione-induced DNA damage, DNA repair rate, and micronucleus formation in human SH-SY5Y neuroblastoma cells. Although the results with MMS indicated similar effects, the differences were not statistically significant. No effects were observed after MF exposure alone. Conclusions The results confirm our previous findings showing that pre-exposure to MFs as low as 100 µT alters cellular responses to menadione, and show that increased genotoxicity results from such interaction. The present findings also indicate that complementary data at several chronological points may be critical for understanding the MF effects on DNA damage, repair, and post-repair integrity of the genome.

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.

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.

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.

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

Abstract 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.

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

Abstract 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.

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.