Micro Nucleus Assay and Lymphocyte Mitotic Activity in Occupational Exposure to Microwaves

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A positive comet assay and the presence of micronuclei are a clear indication of genetic damage to cells. This is in fact a catastrophic event that precedes the appearance of cancer in human and animal tissues. The fact that lymphocytes are also suffering genetic damages is at least alarming. This finding supports the research done by Soviet medical science more than 50 years ago that described this finding in workers exposed to microwave radiation. The need to vanish cell phone telephony from the planet has become a historical necessity in front of the the giant amount of scientific evidence from all over the planet that emphasizes the damaging action of its microwaves. This is an international medical fraud sponsored by the World Health Organisation which holds lackeys of the cell phone industry within its scientific committees on the subject, and particularly, inside the Electromagnetic Fields Project.
  Pergamon Chemosphere, Vol. 39, No. 13, pp. 2301-2312, 199~© 1999 Elsevier Science Ltd. All rights reserved0045-6535/99/$ - see front matter PII: S0045-6535(99)00139-3 MICRONUCLEUS ASSAY AND LYMPHOCYTE MITOTIC ACTIVITYIN RISK ASSESSMENT OF OCCUPATIONAL EXPOSURE TOMICROWAVE RADIATION Vera Garaj- VrhovacInstitute for Medical Research and Occupational Health, Ksaverska c. 2, HR-10001 Zagreb, CroatiaCorresponding author: Tel.: ++385 1 4673 188; Fax.: ++385 1 4673 303;E-mall: vgaraj@mimi.imi.hr (Received in Germany 26 November 1998; accepted 22 March 1999) Abstract The effects of radiofrequency electromagnetic radiation (RFR) on the cellkinetics and genome damages in peripheral blood lymphocytes were determinedin lymphocytes of 12 subjects occupationally exposed to microwave radiation.Results showed an increase in frequency of micronuclei (MN) as well asdisturbances in the distribution of cells over the first, second and third mitoticdivision in exposed subjects compared to controls. According to previousreports micronucleus assay can serve as a suitable indicator for the assessmentof exposure to genotoxic agents (such as RFR ) and the analysis of mitoticactivity as an additional parameter for the efficient biomonitoring. ©1999 Elsevier Science Ltd. All rights reserved KEY WORDS: microwave radiation, mitotic activity, micronucleus assay,occupational exposure, human lymphocytes2301  2302 Introduction Widely used police and military radars are very potent sources ofradiofrequency electromagnetic radiation - RFR (in the non-ionizing range 10kHz -300 GHz). Biological and possible efects of such exposure have longbeen a subjects of scientific interest. It is known that exposure to microwaveradiation had different biological effects on the eye, the nervous system and itsfunction, circulatory and the reproductive system [1,2,3 and 4]. Resultsobtained on the level of the cell and results of animal and epidemiologicalstudies proved of great importane in the assessment of potential biological andcarcinogenic effects induced by microwave radiation [5,6,7]. Available data oncytogenetic conseqences of microwave exposure on the induction ofchromosome damage are sometimes contradictory, mostly because of differentexperimental conditions of ill vitro and in vivo studies [8,9,10 and 11 ].The aim of this study was to investigate the effects of microwaveradiation on induction and frequency of micronuclei in peripheral bloodlymphocytes of occupationally exposed subjects. The mitotic activity oflymphocytes was also studied as an additional parameter tbr the evaluation ofeffects in the biomonitoring of microwave radiation exposure. MATERIALS AND METHODSSubjects The study was carried out on 12 clinically healthy workers, nonsmokers,employed on radar equipment and antenna system service. All subjects weremale. The subjects worked within a microwave field of 101-tW/cm -  20mW/cm2 with frequency range of 1250-1350 MHz. Their average age was40.6 yr (21-55 yrs) and employment duration 13.3 yrs (10-19 yrs). The controlgroup consisted of 12 nonsmoking subjects of the similar age. They had neverworked with any source of ionizing or nonionizing radiation. For bothexperimental groups, data were collected on previous diagnostic or therapeuticexaminations including exposure to radiation sources or chemical agents.2303 Methods Cytogenetic damage in peripheral blood lymphocytes of subjectsoccupationally exposed to microwave was assessed through micronucleus assayand the analysis of mitotic activity. Both test procedures were carried out onperipheral blood lymphocytes cultivated in in vitro conditions.Micronucleus assay on 72-hours lymphocyte culture was used tomeasure the risk of occupational exposure to non-ionizing radiation.. Wholeblood samples were cultivated in vitro in F-10 medium (Gibco) supplementedwith fetal bovine serum (Biological Industries, Israel) and phytohaemagglutinin(Murex). After 44 hours of incubation, the lymphocyte culture was addedcytochalasin B (Sigma) in final concentration of 3 ktg/ml. Preparations weremade according to modified method of Fenech and Morley [ 12]. Lymphocyteswere fixed in 3:1 methanol-acetic acid without hypotonic treatment and thenstained with Giemsa. For each subject 500 binucleated cells were analysed andthe total number and distribution of micronuclei were determined. Thesignificance of differences between the experimental groups was calculated byanalysis of variance.Lymphocyte mitotic activity was determined on slides prepared for sisterchromatid exchange (SCE) analysis. Bromodeoxyuridine (BrdUrd) in a finalconcentration of 10 lag/rnl was added to the culture medium. Preparations were  2304made according to Perry [13]. Cell kinetics was investigated by scoringnumber of cells in the first (MI), second (M2) and third mitosis (M3). Thosecells are easy to distinguish as they differ in the color of chromatides. For eachsubject 500 cells were analysed. Figure 1 shows results of 72- hours oflymphocyte cultivation for the first, second and third mitosis. RESULTS Table 1 shows the frequency of micronuclei and their distribution inbinucleated cells for both experimental groups. Maximal number of micronucleiin some subjects of control group was up to 7MN per 500 analysed cells. Themajority of binucleated cells contained only one micronucleus, while twosubjects had binucleated cells with two micronuclei. The exposed groupsubjects showed an increase in the number of micronuclei (up to 23MN per500 analysed cells). The majority of binucleated cells contained 2 or 3micronuclei. One-way analysis of variance showed significant differencesbetween control and exposed group (F-ratio 17.395).The cell cycle progression was measured as a percentage of first (M1), second(M2) or third (M3) metaphases following growth of cells in the presence of 5-bromodeoxyuridine(Figure 1).
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