Facoltà di Scienze Matematiche, Fisiche e Naturali ...... (1.0GHz, 0.5W power
input; SAR 0.9–3mW/kg) did not significantly alter heat shock reporter gene.
Università degli Studi di Milano - Bicocca Facoltà di Scienze Matematiche, Fisiche e Naturali Corso di Dottorato di Ricerca in Biologia XXIII ciclo
Negative effects on a bioindicator by electromagnetic field exposures alone, and in combination with UVC rays
A.A. 2009/2010
Ph.D. dissertation Matteo Sicolo
Supervisor Prof. Angela Santagostino
CONTENTS ABSTRACT
1
1. BIOMONITORING
3
1.1 – Introduction 1.2 – Biomarkers 1.2.1 – Markers of exposure 1.2.2 – Markers of effect 1.2.3 – Markers of susceptibility 1.2.4 – DNA damage as a biomarker 1.2.4.1 – DNA adducts 1.2.4.2 – DNA strand breakage 1.3 – Bioindicators 1.4 – Oligochetes 1.4.1 – Coelomocytes 1.4.2 – Immune system of earthworms 1.4.3 – Toxicology of earthworms 2. GENETIC TOXICOLOGY
3 4 5 6 6 7 8 9 10 12 13 14 15 17
2.1 – Typology of genetic damage 2.1.1 – Gene mutations 2.1.2 – Chromosome aberrations 2.1.3 – Aneuploidy and polyploidy 2.2 – Mechanisms of genetic alteration 2.2.1 – Ionizing radiation 2.2.2 – Ultraviolet radiation 2.2.3 – Chemicals 2.2.4 – Endogenous agents 2.3 – DNA repair
17 17 17 18 18 19 19 19 19 19
3. ELECTROMAGNETIC FIELD CHARACTERISTICS 3.1 – Electric field 3.2 – Magnetic field 3.3 – Maxwell’s equations 3.3.1 – Solutions of Maxwell equations according to frequency 3.4 – Interactions of fields with matter 3.4.1 – Electrical properties of biological tissues 3.4.2 – Planar absorption 3.4.3 – Specific absorption rate (SAR) 3.4.4 – Dielectric properties and field-generated force effects 3.5 – Human source of radiofrequency-microwave emissions 3.6 – Genetic effects of radiofrequency-microwave fields
4. AIM OF THE STUDY
23 23 23 23 24 25 26 27 28 29 31 34
39 i
5. MATERIALS AND METHODS
41
5.1 – Stripline configuration 5.2 – Comet assay 5.2.1 – Collection of cells 5.2.2 – Slide preparation 5.2.3 – Electrophoresis 5.2.4 – Slide scoring 5.3 – Oxidative damage 5.3.1 – Modified comet assay for oxidative damage 5.4 – UVR genotoxicity 5.4.1 – UVC exposure setting 5.4.2 – Repair enzyme-T4 Endonuclease V 5.4.3 – Modified comet assay for T4 Endonuclease V 5.5 – Field exposure
41 45 48 49 49 49 50 51 51 55 55 57 57
6. STATISTICAL ANALYSIS
61
7. RESULTS
63
7.1 – Results from laboratory exposure 7.1.1 – 900 MHz electromagnetic field 7.1.2 – Repair enzymes – Endonuclease III and FPG 7.1.3 – UV rays exposure 7.1.4 – Repair enzyme – T4 Endonuclease V 7.2 – Results from field exposure
63 63 64 69 70 72
8. DISCUSSION
75
9. REFERENCES
81
ii
Abstract
ABSTRACT Genotoxic effects of radiofrequency (RF)/microwave (MW) electromagnetic fields, by using s tandard protocol of single cell gel electrophoresis (SCGE) or comet assay, were investigated in the coelomocytes of the bioindicator Eisenia fetida exposed to both laboratory and field experiments. In particular, laboratory treatments were performed by a TEM microstrip (900MHz – 0.20mW/Kg) to reproduce the characteristics of the waves generated by RF anthropic sources found on field. In order to assess the potential oxidative damage caused by microwave electromagnetic exposure, two base excision repair enzymes, i.e. endonuclase III (Endo III) and formamidopyrimidine DNA glycosylase (FPG) were used in combination with a modified comet assay protocol. In addition, DNA fragmentation of combinative exposure of ultraviolet rays C (UVC) alone and in combination with microwaves was also studied; in order to assess the influence of electromagnetic fields on DNA repair mechanisms of UVC, T4 endonuclease V (T4PDG) enzyme, which specifically induces single-stranded breaks in ultraviolet-irradiated DNA, was used. Finally, a fieldwork was conducted in three electromagnetic hot-spots in the city of Milan, Italy; in addition, a negative control site with a low electromagnetic field intensity was considered. Loss of DNA integrity was detected by using two main comet assay parameters, i.e. Tail Moment (TM) and Tail Moment Olive (TMO). Data showed an initial increase in ∆TM and ∆TMO (expressed as differences between Tail Moment or Tail Moment Olive from exposed and respective controls averages) after EMF treatments, resulting the highest after the first minutes of recovery (∆TM: 6.63±0.70, immediately after exposure and ∆TMO: 4.43±0.38, after 30 minutes, respectively). However, a transient genotoxic damage was observed at 2 hours from exposure (p
