Effetti biologici rilevati su roditori esposti a campi magnetici - Biological effects induced by magnetic field exposure in rodents

During the first half of the XVII century, engineers promoted the industrial use of electromagnetic energy following the discovery of electromagnetism. Since then, the use of electromagnetic fields has steadily increased industry and telecommunications (mobile phones, radio-navigation, radar), at home (electrical household appliances, computers) and in devices for health assessment. Therefore, concern has arisen with regards to security and tolerance in humans. The exposure to ELF magnetic fields has been correlated with cancer induction, including breast and brain cancer, but overall, with leukaemia in children. A major incidence of neurological diseases such as Alzheimer’s, Parkinson’s and amyotrophic lateral sclerosis has been shown in exposed workers. Several studies have already suggested a possible association between ELF magnetic fields and non-neoplastic effects together with cardiac disorders, headaches, depression syndromes and suicide tendencies. In order to define the genotoxic potential of electromagnetic fields, different frequencies and intensities have been analysed. In vitro studies have been conducted at the cellular, molecular and genetic levels and in non-cellular systems. In vivo studies have been carried out in vertebrates, non-vertebrates, plants and bacteria. Taken together, so far the studies have not fully clarified the mechanisms of interaction among electromagnetic waves and living organisms. However, suggestions exist about the genotoxic potential of the magnetic field. As genotoxic damage can be a first step for carcinogenesis, the goal of this thesis has been to evaluate the exposure effects to electromagnetic fields in laboratory mice The current in vivo investigation assesses the potential genotoxic damage induced by long-term exposure to magnetic fields in two cell types (whole blood and brain cells) of laboratory mice measured with the Comet assay. The experiment was performed at 50 Hz. The features of the magnetic fields selected (0,65 mT) are in the range of those found in the working and domestic environment. The alkaline Comet assay reveals DNA damage generated by single strand breaks, double strand breaks and/or alkali labile sites caused by genotoxic insults. It is a rapid and highly sensitive assay when compared with other well known genotoxic techniques (micronuclei, chromosome aberrations, SCE, etc.). Moreover, it allows all types of cells/tissues to be analysed. The magnetic field was produced by solenoids (80 cm length, 25 cm diameter, 552 coils (690 coils/m), 3 resistance of copper wire, 5-6 V supply). 4 Swiss CD-1 male and female mice, two months old, and pregnant females, four months old, were placed in the centre of the solenoids and were exposed for 22-23 days, 24 hours per day. Newborns were kept in the solenoids for 72 h after birth together with the mothers. Control mice were placed in the same environment (exposure room) as the exposed mice although solenoids were switched off. The room was maintained at a temperature of 23 + 1 °C, and humidity of 45 + 5%. A time-controlled system provided a daily 08-16 light and 16-08 dark cycle. Food and tap water were available ad libitum. At the end of the exposure, animals were sacrificed and brain and whole blood were collected within 24 h. After removal, brain was washed in PBS and cut in little pieces. Tissue pieces were then dispersed into single-cell suspension using a pipette. The blood and brain suspensions were processed in the same manner in the Comet assay. Cells were embedded in low-melting-point agarose on a microscope slide. The cells were lysed in a lysis buffer and electrophoresed in a highly alkaline (pH>13) condition. After staining, slides were scored on a fluorescence microscope connected to a computer. Images of comets were analysed with appropriate image analysis software. Tail Moment, %DNA in the tail and Tail Length were the parameters selected to evaluate DNA damage in the Comet assay. Furthermore, treatment with proteinase K was performed in order to assess the presence of crosslink in the DNA. The results obtained showed that exposure to electromagnetic fields cause DNA damage in the brain cells of adult (Figg. 13, 14) and newborn (Figg. 21, 22) mice, such damage being significantly higher than in control groups. In addition, even though basal levels of DNA damage were higher in adults than in new-borns, the increase of damage due to exposure was higher in new-born mice (Figg. 26, 27, 28). DNA damage in the brain cells of young mice after exposure was 4-fold higher than controls, whereas it was 2-fold higher in the adult group. No evidence of cross-links in brain cells following exposure was found in new-born or adult mice. The results obtained in peripheral blood showed that the mean values of TM, % DNA and TL were higher in control groups than in the exposed, both in adult (Figg. 32, 33) and in young (Figg. 40, 41) mice. These results are difficult to explain. Bearing in mind that higher levels of DNA damage in the control group could suggest a mechanism of DNA cross-linking, these results have to be considered as controversial, even considering results from proteinase K treatment. In fact, in new-born mice the TM, %DNA and TL values of exposed group treated with proteinase K were significantly higher than in non-treated exposed group, but this was not observed in adults. Recent studies lead to the consideration that Fe2+ ions play an important role in the biological effects of magnetic fields. Fe2+ catalyses the Fenton reaction and triggers a cascade of secondary 5 events that, in a second step, lead to an increase of nitric oxide synthesis. The latter one may be responsible for DNA damage revealed after exposure. The properties of blood are known as a buffer, thereby, quenching a variety of insults. Normal human plasma contains high and low molecular mass redox-active molecules, such as transferrin and caeruloplasmin, that offer considerable protection against organic and inorganic oxygen radicals generated by ions. Such features of blood may explain the lack of responses found after exposure. In conclusion, this study highlights harmful genotoxic effects in brain cells of both adult and new-born mice due to long exposure to magnetic fields of 0,65 mT intensity (50 Hz frequency). Nevertheless, the results obtained in this investigation require further research to enhance its consistency. Studies should be performed to assess the biological consequences after exposure in other target organs. A wider variety of endpoints will allow a better understanding of the effects of magnetic fields in living organisms, as well as provide a more accurate and reliable data in terms of risk assessment for humans. As a model of oxidative stress, the Comet assay was also used to investigate the effect of hydrogen peroxide, oestrogen and progesterone on blood lymphocytes from vitiligo patients (treated and untreated) and healthy donors in presence and absence of catalase. Dose-response curves were obtained for the three compounds used (Figg. 48, 49, 50). Statistically significant difference result when DNA damage in the control group was compared with different doses of H2O2 on lymphocytes from treated vitiligo patients and healthy donors. Oestrogen and progesterone, at the higher doses used, damage DNA in both control and vitiligo treated and untreated groups. Results also confirmed the capacity of H2O2 to induced severe DNA damage in lymphocytes from the control group and treated vitiligo group in a dose-dependent manner. Catalase abolished DNA damage induced by hydrogen peroxide and oestrogen in lymphocytes from healthy donors and treated vitiligo patients but not in lymphocytes from untreated vitiligo patients. This last part of the study was carried out in the Department of Biomedical Science of the University of Bradford (UK) with Prof. D. Anderson and Prof. K. Schallreuter.