Enhancement of the cell-killing effect of ultraviolet-C radiation
by short-term exposure to a pulsed magnetic field
MIGUEL J. RUIZ-GO
´ MEZ & MANUEL MARTI´NEZ-MORILLO
Laboratory of Radiobiology, Department of Radiology and Physical Medicine, Faculty of Medicine, University of Malaga, Spain
(Received 14 October 2004; accepted 18 May 2005)
Abstract
Purpose: To investigate whether low frequency pulsed magnetic field (PMF) exposures produce alterations in the cell killing induced by ultraviolet C (UVC) radiation.
Materials and methods: MCF-7 breast cancer cells of exponentially growing cultures were exposed to PMF (25 Hz, 0.75 mT) and UVC (from 6.6 J/m2to 59.4 J/m2) in two different protocols: (a) cells were exposed to PMF for 30 min and then exposed to UVC at different doses; (b) cells were exposed to PMF for 30 min. After 15 min of the PMF exposure they were exposed simultaneously to PMFþ different doses of UVC. After an additional time of 72 h of incubation, viability was measured by the neutral red stain cytotoxicity test.
Results: Both exposure protocols produced a significant decrease in the post UVC survival at 13.2 J/m2and 19.8 J/m2, as compared to controls. The simultaneous exposition of PMF and UVC produced an additional increment in cell killing at 26.4 J/m2, being the greater effects obtained for this second exposure protocol.
Conclusions: Results of the present study show that PMF in combination with UVC have the ability to augment the cell killing effects of UVC radiation. In addition, the effects appear to be greater when PMF and UVC are applied at the same time.
Keywords:Low frequency magnetic fields, ultraviolet, non-ionizing radiation, MCF-7, human breast cancer cells
Introduction
During evolution, cells of living organisms had to create protective mechanisms against physical and chemical agents to avoid cell killing. The mutagenic effect of ultraviolet (UV) radiation has played an important role in the development of terrestrial life.
It is well known that pre-exposure of a cell to a mild stressor can confer protection against a sub-sequent lethal agent. This effect is known as pre-conditioning, and can be achieved by pre-exposure to the same type of stimulus (auto-protection), or by exposure to an unrelated stimulus (cross-protection) (Dicarlo et al. 1999).
Electromagnetic fields (EMF) have caught public attention due to a possible association with cancer risk. There is fear that EMF may contribute to the development of cancer or have other undesirable biological effects. It has been reported by various authors that EMF can produce a variety of effects:
Apoptosis in different cell lines (Hisamitsu et al. 1997, Simko´ et al. 1998), variations in cell cycle distribution (Schimmelpfeng & Dertinger 1997), alterations in the potency of anticancer drugs (Liang et al. 1997, Miyagi et al. 2000) and changes in cellular Ca2þ regulation (Lindstrom et al. 1993), ornithine decarboxylase enzyme activity (Valtersson et al. 1997), RNA synthesis (Greene et al. 1991) and Naþ/Kþ– ATPase activities (Blank 1992). Moreover, Ishido et al. (2001) found that a 1.2 mT, 50 Hz sinusoidal magnetic field (MF) inhibits the antipro-liferative action of melatonin on MCF-7 cells. However, Loberg et al. (2000) published that MF exposure (60 Hz, 1 mT, 72 hours) has no effect on cell viability or growth in a battery of breast cancer cell lines, including MCF-7.
The combined effects of low frequency magnetic fields with other types of radiation such as UV have become a subject of interest during the recent years. Thus, it has been reported that MF exposure (60 Hz,
Correspondence: M. J. Ruiz-Go´mez. Laboratorio de Radiobiologı´a, Departamento de Radiologı´a y Medicina Fı´sica, Facultad de Medicina, Universidad de Malaga. Teatinos s/n, 29071 Malaga, Spain. Tel:þ34 95 2131631. Fax:þ34 95 2131630. E-mail: mjrg@uma.es
ISSN 0955-3002 print/ISSN 1362-3095 onlineÓ2005 Taylor & Francis DOI: 10.1080/09553000500196805
8 mT) induces protection against damage from UV light exposure (30 J/m2 and 45 J/m2), being the induction of the heat shock stress response, via the cross-protection mechanism, the possible pathway involved (Dicarlo et al. 1998, 1999). In addition to physiological effects, the mutagenic activities of MF have also been studied. Published results show that MF exposures (60 Hz, 1 mT) alone or in combina-tion with various ultraviolet C (UVC) exposures (2 J/m2– 50 J/m2) do not increase mutation, gene conversion or reciprocal mitotic crossing-over (Ager & Radul 1992).
Numerous studies have been published concern-ing the simultaneous effects of EMF and DNA damaging agents, including some studies that have used pulsed fields. For example, Cossarizza et al. (1989) exposed cells to pulsed magnetic fields (PMF) after gamma irradiation. They observed that exposure to PMF did not affect DNA synthesis or cell survival. Scarfi et al. (1991) published that PMF do not affect the number of micronuclei induced by mitomycin-C. Nevertheless, Miyakoshi et al. (1999) found that long-term exposure to more than 5 mT, 60 Hz MF may promote X-ray-induced mutations.
So far, there have been very few studies that investigate the potential effects of low frequency PMF below 50/60 Hz. Previousin vitrofindings from our laboratory indicate that 1 Hz and 25 Hz, 1.5 mT PMF can induce modulation of cytostatic agents, with an increased effect when the PMF is applied at the same time as the drug (Ruiz-Go´mez et al. 2002). Nevertheless, neither significant alteration in cell cycle phases nor induction of apoptosis has been observed for U-937 and HCA-2/1cch human tumor cells (Ruiz-Go´mez et al. 2001).
The aim of this work was to investigate whether PMF exposure (25 Hz, 0.75 mT) produce alterations in the cell killing induced by UVC radiation in MCF-7 human breast cancer cells.
Materials and methods
Cell culture
MCF-7 cells, a human breast cancer cell line, were supplied by Dr J. M. Ruiz de Almodo´var and Dr M. T. Valenzuela (Radiology and Physical Medicine Department, University of Granada, Spain). They were cultured in Dulbecco’s modified Eagle’s medium nutrient mixture F12-HAM (DME/ F12-HAM) (with L-glutamine and hepes) (Sigma Chemical, Co., St Louis, MO, USA), supplemented with sodium bicarbonate 7.5% (28 ml/L) (Panreac Quı´mica, S.A., Barcelona, Spain), 5% fetal bovine serum (PAA Laboratories GmbH, Linz, Austria) and 1% antibiotic-antimycotic solution (100X) (Penicil-lin (5000 units/ml), Streptomycin (5 mg/ml),
Neomycin (10 mg/ml) in 0.9% sodium chloride) (Sigma Chemical, Co., St Louis, MO, USA), at 378C in a 5% CO2/air atmosphere. These cells grow in monolayer and were subcultured with trypsin (0.05%) and Ethylenediaminetetraacetic acid (EDTA) (0.02%) in Dulbecco’s phosphate-buffered saline (PBS). Trypsin, EDTA and PBS were from Sigma Chemical, Co., St Louis, MO, USA.
Magnetic field exposure system
The equipment used (Pulsatro´n, CEM-84/J; J&J Electrome´dica; Ma´laga, Spain) generates rectangular voltage pulses (25 Hz) that feed two air core coils of 15 cm610.5 cm connected in series. Figure 1 shows the voltage waveform that consists of a group of 15 rectangular pulses, repeated at 25 Hz. The magnetic field associated with this low-frequency voltage waveform is proportional to the current intensity in the circuit (Law of Biot-Savart relating magnetic fields to currents) and its time derivative has the same waveform as the electromotive force in a pickup coil in air (Faraday’s Law of electromag-netic induction). The waveform of the time derivative of the current intensity in the circuit (the same waveform as the time derivative of the magnetic field) was observed with the help of a pickup coil in air, being checked that it was according to the temporal variation of the current intensity in an inductive circuit. It consisted of pulses decreasing exponentially towards zero; positive in those intervals in Figure 1 corresponding to the 180ms when voltage was applied and negative in the 20ms gaps.
The frequency (25 Hz) used in this study was the maximum value that can be obtained with this equipment, and the intensity (0.75 mT) was the maximum value obtained with one coil. Similar
Figure 1. Voltage source waveform generated by the ‘‘Pulsatro´n’’ equipment used. The number of rectangular pulses per group was 15 at 25 Hz group repetition rates.
intensities (1 mT) have been used previously by Loberg et al. (2000) with MCF-7 cells. The general characteristics of the voltage waveform applied to the coil and the electric field waveform associated to the generated magnetic field were the same as reported in a previous work (Ruiz-Go´mez et al. 2002), although in this previous report the coils were connected Helmholtz type. When the coils are arranged as Helmholtz coils (the two coils with a common axis, separated by a distance equal to their radius) the generated peak magnetic field at the midpoint is 1.5 mT, as calibrated by the manufacturer. The mag-netic field applied in this work was created by only one coil and therefore, the generated peak magnetic field in the axis at a distance equal to one half of the radius was 0.75 mT (Reitz et al. 1980).
The electric field (E) in each culture well of a 6-well dish was calculated by the equation that defines the electromotive force in a circuit:
x¼
I
~
E: ~dl ðFaraday’s Law of InductionÞ
where x¼induced electromotive force (V), E¼
electric field (V/m) and dl is an infinitesimal element of the circuit.
The value ofxwas measured by means of circular pickup coil of radius r located inside each well, where r¼radius of the well. The electric field was calculated by:
E¼ x
2pr
The electric field peak value fluctuated from 52 mV/m to 150 mV/m during the positive pulse (corres-ponding to the 180ms portion) and from 600 mV/m to 1000 mV/m in the negative pulse zone (correspond-ing to the 20ms between rectangular voltage pulses), depending on the spatial location of each well.
The induced electric current density (J) was cal-culated by means of the equation:
J ¼sE
where s (is the conductivity of the culture medium (s1.5S/m) (Bassen et al. 1992); the calculated values fluctuated from 78 mA/m2 to 225 mA/m2 in the positive pulse interval and from 900 mA/m2 to 1500 mA/m2in the negative pulse interval.
In relation to the spatial distribution of the magnetic field generated by a single coil, it is theoretically expected there will be the presence of a component parallel to the cell culture surface in addition to the main vertical component of the magnetic field. By measuring this parallel component in relation to the vertical component inside each well (by means of a
pickup coil) we can assure that its value was less than 1/10 of the vertical component. Well dimensions were 3.5 cm in diameter and 0.5 cm for the height of the culture medium.
Ultraviolet C exposure system
Cells were exposed to a germicidal fluorescent lamp (OSRAM S.A.; Sylvania G30W-T8, Japan) with 30 W of power, 8 W of UVC (253.7 nm) and 0.22 W/m2of intensity. The dose intensity was measured using a UV203 ultraviolet radiometer (Macam, Scotland, UK). The energy densities applied ranged from 6.6 J/m2to 59.4 J/m2. Control cells were sham-irradiated. Similar UVC dose levels have been used previously by Dicarlo et al. (1999) (30 J/m2and 45 J/m2) and Wang et al. (1999) (2.5 J/m2to 20 J/m2).
Cells exposure protocol
After trypsinization of an exponentially growing monolayer, 20,000 to 50,000 cells were seeded in each well of several 6-well dishes and were incubated for 24 h at 378C, 5% CO2, to allow the cells to attach.
Then, two different protocols were performed as following:
. Exposure to PMF for 30 min and then exposure to UVC at different doses;
. Exposure to PMF for 30 min. After 15 min of the PMF exposure they were exposed simultaneously to PMF plus different doses of UVC.
The number of cells seeded was higher in the dishes that received higher UVC doses so surviving cells could be obtained. The time of PMF exposure (30 min) has been chosen arbitrarily as a short-term exposure period. Similar exposure times have been used previously by Dicarlo et al. (1999).
In each type of experiment, four 6-well dishes were used per dosage point which corresponded to the following exposures: (i) the first dish was sham exposed; (ii) the second dish was exposed to UVC; (iii) the third was exposed to PMF, and (iv) the fourth dish was exposed to PMF and UVC.
The cells were treated according to protocols above and then they were incubated for an additional time of 72 h at 378C, 5% CO2. At the end of the incubation period, cell viability was measured by the neutral red stain cytotoxicity test (Babich et al. 1991; Morgan et al. 1991).
The main component of the magnetic field ran perpendicular to the cell culture surface as described above. Immediately following exposure to PMF, the control cells were put in place with no current passing through the coil. They were exposed in the
same place, but not at the same time. Therefore, there was no added artificial magnetic field at the control location. The same passage cells were used simultaneously for their matched sham and exposed experimental groups. There was no additional heating in the area where samples were located due to the activation of the coils, as measured directly by a conventional thermometer. Another possible source of heating would be the induced currents by PMF in the conductor liquid culture media. The absence of effects in the experiments with cells exposed only to PMF, in relation to sham-exposed samples indicate that this possible heating effect was not appreciable. A theoretical estimate of the energy deposition in the culture medium was performed with the expressions:
P ¼1
2 Z
v
sE2dv;W ¼
Z
P dt;
(P¼absorbed power (W); s¼electric conductivity (S/m); E¼electric field (V/m); v¼well volume with culture medium (m3); W¼absorbed energy ( J ); t¼time (s)) shows that, to a first approximation (taking E¼1V/m, the maximum peak electric field value measured with the pickup coil,s1.5 S/m and the exposure time to the PMF, t¼1800 s), results in a value for the energy deposition, W 7 mJ. The consequent temperature increment (assuming values for the density and specific heat of the culture medium were similar to water) would be less than 5 610748C.
A more accurate calculation (taking into account the waveform of the electric field) decreases this value for the temperature increment even further.
Two dosage points were performed within the same independent experiment performed the same day with the same initial sample of cells. UVC radiation was applied from the top after removing the plate cover. Several black opaque templates were set under the plate and surrounding the irradiated wells to protect the adjacent wells from scattered UVC irradiation.
In vitrocytotoxicity assay
The cytotoxicity test using neutral red stain (Morgan et al. 1991) was used to measure cell viability. This is a test based on the lysosomal uptake of supravital neutral red stain, which quantifies the number of viable cells after exposure to a physical or chemical agent. The quantification of the stain extracted from cultured cells has been shown to be linear with the number of viable cells through direct counting of cells (Babich et al. 1991). Although this test does not allow conclusions concerning toxicity mechanisms, it is possible to evaluate the number of viable cells after
a period of time in a similar way as other cytotoxicity tests, allowing the calculation of the surviving fraction of cells after a specific physical or chemical treatment.
For this assay, the culture medium was replaced by 1 ml of supplemented fresh medium with 40 mg/ml neutral red (Sigma Chemical, Co., St Louis, MO, USA). The new medium had been previously incubated 24 h at 378C and centrifuged at 2500 rpm for 10 min to get rid of crystalline stain precipitate. After 3 h of incubation of the plates in the presence of neutral red, the medium was removed and cells were washed in 1 ml of fixative (1% CaCl2: 0.5% formaldehyde). CaCl2 was from JRH Biosciences, Lenexa, KS, USA and formalde-hyde was from Probus, S.A., Barcelona, Spain. Then, 1 ml of 1% acetic acid: 50% ethanol solution was added to each well in order to extract the stain (Fautz et al. 1991). Acetic acid and ethanol were from Panreac Quı´mica, S.A., Barcelona, Spain. After 10 min at room temperature and subsequent shaking, the optical density (OD) was measured at 540 nm using a conventional spectrophotometer (Unicam, Cambridge, UK). The mean OD in the control cells determined after incubation was re-garded as 100%, and the percentage of survival at each UVC dose was calculated.
Statistical analyses
The Wilk-Shapiro rankit-plot test was used to assess the normal distribution of the data. Additional statistical analyses were made with the Student’s
t-test. Differences were considered significant when
p50.05. Data are expressed as mean+SD of two independent experiments with six replications.
Results
Exposure to PMF for 30 minutes and then exposure to different doses of UVC
The first exposure protocol assayed was the pre-exposure of MCF-7 breast cancer cells to PMF during 30 min and then the exposure to UVC radiation. The quantification of the neutral red stain, extracted from the viable cells, by measuring the OD at 540 nm ranged from 0.114 – 1.484. Figure 2 presents experimental data showing the effects of UVC radiation over a range of doses from 6.6 J/m2to 59.4 J/m2 (0.5 – 4.5 min of exposure). Decreased surviving fraction caused by the pre-exposure to PMF (25 Hz, 0.75 mT) was evidenced by a lower surviving fraction obtained for 13.2 J/m2 (1 min) (p50.05) and 19.8 J/m2(1.5 min) (p50.01) of UVC exposure. In contrast, no statistically significant effect was observed for the other UVC doses assayed.
Exposure to PMF for 30 minutes and meanwhile, at middle time, exposure to different doses of UVC, at the same time than PMF
The second exposure protocol assayed was the simultaneous exposition of MCF-7 cells to the PMF and UVC radiation. The cell cultures were exposed to PMF for a total of 30 min and but at 15 min of PMF exposure they were exposed to different UVC doses. The quantification of the neutral red stain, extracted from the viable cells, by measuring the OD at 540 nm ranged from 0.065 – 1.015. The results obtained show a significant decrease in surviving fraction after co-exposure to PMF (30 min, 25 Hz, 0.75 mT) and 13.2 J/m2 (1 min) (p50.01), 19.8 J/m2 (1.5 min) (p50.01) or 26.4 J/m2(2 min) (p50.001) of UVC (see Figure 3); the latter being the greatest effect for this simultaneous treatment protocol. In contrast, no effect statistically significant differences were observed for the other UVC doses assayed.
Discussion
Non-ionizing radiation includes electromagnetic energy distributed as ultraviolet, visible light, infra-red radiation, microwaves, radio frequencies, and very low frequency and extremely low frequency electric and magnetic fields.
The aim of this study was to evaluate the effects of PMF exposure on the cell killing induced by UVC radiation on MCF-7 breast cancer cells. We found that, under our experimental conditions, exposure to
PMF can produce a significant increase in the cell killing induced by UVC. This effect was even greater when PMF and UVC were administered at the same time. This result is reminiscent of the behavior of a typical radiosensitizing agent that sensitizes tumor and normal cells to ionizing radiation (Martı´nez Morillo et al. 2002).
The primary damage from exposure to germicidal lamp (UVC) is the damage induced to the DNA molecule due to its maximum light absorption in the range of 240 – 290 nm. In addition, UVC exposures can lead to the formation of reactive oxygen species (ROS), which are responsible for causing secondary damage to many cellular components (Dicarlo et al. 1999). In relation to this second damage mechanism, Roy et al. (1995) showed a relation between MF exposures and evolution of ROS in the presence of a chemical oxidative agent. They obtained a significant increased generation of free radical species when MF (0.1 mT, 60 Hz) were applied.
We have not found published results with similar field intensities and exposure conditions as in our experiments. For lower MF intensity values we can mention the work of Dicarlo et al. (1999). They reported that MF (8mT, 60 Hz) short-term exposures (20 min) induce protection against a subsequent damage from UVC light exposure (30 J/m2and 45 J/ m2). The hypothesis formulated by these authors was that MF can stimulate cellular repair processes such as the induction of the heat shock stress protein mediated by MF-induced ROS and also enhance anti-oxidant systems. These authors suggested that MF exposures might be beneficial in short doses. Figure 2. Effect of 25 Hz, 1.5 mT pulsed magnetic field exposure for 30 min and then exposure to ultraviolet C radiation at different doses on MCF-7 breast cancer cells. Mean+SD of two independent experiments with six replications. PMF: pulsed magnetic field; UVC: ultraviolet C radiation.*p50.05;**p50.01. Statistical analyses were made with the Student’st-test.
Our experiments, like those performed by Dicarlo et al. (1999), were short-term exposures but in our case the magnetic field did not induce protection against a subsequent damage from UVC radiation. In contrast, we found an increment in the cell killing induced by UVC when MCF-7 cells were previously exposed to magnetic fields. This cell killing was higher under the second exposure protocol (expo-sure to PMF and UVC simultaneously).
Another paper reported by Dicarlo et al. (2002), showed that long-term continuous or twice daily repeated (30 or 60 min) MF exposure (8mT, 60 Hz) for 4 days could reduce protection against a subsequent stressor. The MF intensity used in our work was 100-fold higher than the MF applied by these authors and it enhanced cell killing in contrast to the protective effect found by Dicarlo et al. (1999) for lower field intensities.
Currently, there is no single mechanism that explains the influence and interaction of EMF with biological systems although several hypotheses have been proposed. Ishido et al. (2001) hypothesized that magnetic fields (100 mT, 50 Hz sinusoidal) have an effect on the signal transduction pathway from melatonin receptors to adenylyl cyclase. Till et al. (1998) reported that magnetic fields can modify the recombination probability of pairs of free radicals even though they may be weaker than the local magnetic (hyperfine) interactions of the unpaired electrons. In addition, Timmel et al. (1998) pub-lished that even a feeble applied magnetic field can
boost the yield of free radicals and suppress recombination by between 10% and 40%. Moreover, Eveson et al. (2000) concluded that low field effects depend strongly on the local environment of the radical pair.
Nindl et al. (2002) using combinations of ultra-violet B radiation plus EMF found that 1 mT, 100 Hz decreased DNA synthesis of Jurkat cells, com-pared to ultraviolet B alone. In addition, these authors observed that the interaction between EMF and ultraviolet B appeared to be more than additive, suggesting that mechanistically they were synergistic in their relationship.
The results reported up to now are not in agreement, leaving the problem unresolved with the publication of different exposure protocols and combinations of different types of damaging agents. It could be useful to obtain additional results related to combined exposures of magnetic fields and UVC for the same cell line and complementary exposure protocols.
Conclusions
The data obtained in this investigation show that low frequency PMF (25 Hz, 0.75 mT) in combination with UVC have the ability to augment the cell killing effects of UVC radiation for only some of the UVC doses used (13.2 J/m2, 19.8 J/m2 and 26.4 J/m2). Moreover, the effects appear to be greater when PMF and UVC were applied at the same time. Figure 3. Effect of 25 Hz, 1.5 mT pulsed magnetic field exposure for 15 min followed by combined exposure to PMF plus different doses of UVC for an additional 15 min (total exposure time of 30 min) on MCF-7 breast cancer cells. Mean+SD of two independent experiments with six replications. PMF: pulsed magnetic field; UVC: ultraviolet C radiation.*p50.01;**p50.001. Statistical analyses were made with the Student’st-test.
Although in vitro experiments can never be a substitute for careful clinical studies, they are useful for initial screening. In this way, our results can be used as basic data for further studies about combined effects of radiation and magnetic fields.
Acknowledgments
The authors thank Prof. Dr J. M. Ruiz de Almodo´var and Dr M. T. Valenzuela (Radiology and Physical Medicine Department, University of Granada) for kindly providing the MCF-7 cell line; Prof. Dr J. M. Pastor (Radiology and Physical Medicine Department, University of Ma´laga) for the use of the pulsed electromagnetic field equipment; and Ms L. Gil Carmona for her technical assistance. We express our gratitude to Dr M. I. Prieto Barcia (Department of Communications Engineering, University of Malaga) for the magnetic field measurements and for critical review of the manuscript. We also greatly appreciate the kindness of Dr Jose´ Aguilera (Photo-biology Unit, Ecology Department; University of Ma´laga) for the use of the ultraviolet radiometer and the measurements he made. This work has been supported by the ‘‘Plan Andaluz de Investigacio´n, Junta de Andalucı´a’’, code CTS-181.
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