Giornata studio - "Salute e campi elettromagnetici" Carmela Marino ENEA C.R Casaccia. 23 giugno 2009

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1 Giornata studio - "Salute e campi elettromagnetici" Studi su modelli animali: il quadro delle conoscenze Carmela Marino ENEA C.R Casaccia Bari 23 giugno 2009

2 Attività sperimentali In vivo: carcinogenesi, tossicità sistemica, fisio-patologia; comportamento, memoria, effetti teratogeni;

3 IL CONCETTO DI MODELLO ANIMALE La maggior parte delle nostre conoscenze nel campo della biochimica generale, della fisiologia e dell endocrinologia origina dalla sperimentazione animale, e, idealmente, dovrebbe essere estrapolata all uomo. In molti esperimenti, infatti, l animale sostituisce l uomo e viene perciò considerato come modello animale. Una definizione possibile è la seguente: un modello è quella condizione che permette di studiare i processi biologici e comportamentali di base, o in cui può essere studiato un processo patologico indotto, e nel quale il fenomeno è simile, almeno sotto un certo punto di vista, allo stesso fenomeno nell uomo o in altre specie animali. La maggior parte dei modelli animali di laboratorio sono sviluppati e utilizzati per studiare la causa, la natura e la cura di malattie umane. L importanza dei risultati derivati dalla sperimentazione dipende dalla scelta di un adeguato modello. La possibilità di estendere l estrapolazione dei risultati dipende dal tipo di modello e dalla natura della ricerca.

4 I piccoli roditori, principalmente topi e ratti, sono le specie più utilizzate. Nei modelli animali indotti, una malattia o un processo patologico sono indotti sperimentalmente, sia chirurgicamente che somministrando sostanze biologicamente attive, per ottenere una condizione che somigli a quella che si verificherebbe nella specie bersaglio. A questo gruppo appartengono gli animali transgenici, tra cui i topi sono la specie di elezione. Per modelli animali spontanei si intendono quelli che portano mutazioni genetiche spontanee, e sono stati caratterizzati centinaia di ceppi e colonie animali che manifestano malattie spontanee simili all uomo. L estrapolazione dei risultati può essere qualitativa, vertente sui processi fisio-patologici dell animale e sulle sue reazioni a stimoli estrapolabili all uomo o ad altri animali, o quantitativa, che consiste nella determinazione del dosaggio di certi composti che potrebbero essere benefici o pericolosi per la specie bersaglio. L estrapolazione dall animale all uomo va sempre effettuata con riserva, e i risultati degli esperimenti dovranno essere, in ultima analisi, verificati con studi sull uomo.

5 La scelta di un modello richiede quindi una pianificazione meticolosa. Vanno definiti (1) il problema di base, (2) il substrato, ossia il tipo di cellule, tessuti, organi da studiare, (3) la specie o il ceppo opportuno, (4) i fattori decisivi, quali la disponibilità, l adattamento, l animal care, la strumentazione, la bibliografia, la perizia tecnica, il costo, la valutazione dei metodi alternativi; operare quindi considerazioni scientifiche, pratiche ed etiche. Sperimentazione animale, comitati bioetici, ruolo Ministero della Salute Principles of Laboratory Animal Sciences, LFM van Zutphen, V Baumans, AC Beynen, 1993, Elsevier

6 Rodent Models of Human Disease I - mouse Animal Model & Biomedical Problem Mouse Species & Genetic and developmental defects Mouse Species & Neoplastic disease Mouse Species & Metabolic/ nutritional disease Mouse Species & Degenerative Disease Mouse Species & Infectious disease Specific Disease Anemia, hereditary / Athymic / Autosomal trisomies / Chediak-Higashi syndrome Copper malabsorption, X-linked / Exencephaly / Hereditary asplenia / L call mutant / Megacolon, aganglionic Megaloblastic anemia / Polycystic kidney disease / Testicular feminization Adenocarcinoma, DES / Adenoma, salivary / Angiosarcoma, liver / Carcinoma, cervix Carcinoma, embryonal / Hodgkin's disease / Leukemia, myelogenous / Malignant tumor transplant Mammary tumor / Ovarian tumor / Preneoplastic lymphoid hyperplasia Teratoma and teratocarcinoma Amyloidosis / Diabetes mellitus / Gammopathies, monoclonal Gestational diabetes / Globoid cell leukodystrophy Glucose-6-phosphate dehydrogenase deficiency / Histidinemia Hyper- and hypotension Hypervitaminosis A / Hypophosphatemia (rickets) / Mast cell deficiency / Methylmercury poisoning Niemann-Pick Disease / Nonobese diabetic / Ochratoxicosis / Ornithine transcarbamylase deficiency Paraproteinemia, idiopathic / Thalassemia, alpha Adenosis, vagina-cervix / Autoimmune disease / Biliary obstruction Diverticulosis, oviduct / Dysbaric osteonecrosis / Proliferative glomerulonephritis/ Immunosuppression / Macroglobulinemic neuropathy / Menkes's disease / Motor neuron disease / Pulmonary fibrosis, bleomycin Pulmonary fibrosis, solvents/oxygen / Reye's syndrome / Salpingitis / Vitiligo Avian reovirus / Capillaria hepatica / Cytomegalovirus / Encephalomyocarditis Epstein-Barr virus-dependent / lymphoproliferative disease / Filariasis Giardiasis / Hepatitis, reovirus / Helicobacteriosis / Influenza B Listeriosis / Lymphocytic choriomeningitis / Meningoencephalitis, amebic Meningoencephalitis / Angiostrongylus / Scrapie / Theiler's encephalomyelitis Trypanosomiasis / Yersinia infection

7 Rodent Models of Human Disease II - rat Animal Model & Biomedical Problem Rat Species & Genetic/ developmental defect Rat Species & Neoplastic disease Rat Species & Metabolic/ nutritional disease Rat Species & Degenerative disease Rat Species & Infectious disease Specific Disease Amnionic fluid deficiency / Fetal colon implants / Fetal lung growth Hereditary / hyperbilirubinemia / Hydrocephalus / Hydronephrosis Intrauterine growth retardation / Megacolon, aganglionic Adenocarcinoma, colon / Adenocarcinoma, intestine Adenocarcinoma, prostate / Aflatoxin carcinogenesis / Angiosarcoma, hepatic Carcinoma, bladder / Carcinoma, esophagus / Carcinoma, pancreas Carcinoma, kidney / Carcinoma, squamous cell, lung / Carcinoma yolk sac Interstitial cell tumor / Lymphoblastic leukemia / Malignant histiocytoma Medullary carcinoma, thyroid / Neurogenic tumors, N-nitrosourea Osteosarcoma, Moloney sarcoma virus / Pituitary tumors Urothelial tumors Adrenal apoplexy / Alcoholic fatty liver / Anemia Cirrhosis / Diabetes insipidus / Diabetes mellitus Ethanol dependence / Fructose-induced lesions Hepatic necrosis, halothane induced / Hypervitaminosis A Hypothyroidism / Lead encephalopathy / Lipotrope deficiency Mucopolysaccharidosis / Obesity / Ochratoxicosis Osteopetrosis / Phenylketonuria / Skeletal muscle, defective glucose-glycogen Striatal lesions, kainic acid induced / Urolithiasis Vasculitis, pulmonary, glucan induced Aneurysm, cerebral / Arthritis / Autoimmune thyroiditis Duodenal ulcer / Hypertension, induced / Hypertension, spontaneous Hypertrophy, right ventricle / Immunosuppression / Ligation, cerebral artery Myocardial infarction / Optic disc swelling / Periodontitis Retinal degeneration / Silica-induced pulmonary lipoproteinosis Thromboembolism / Uterine vessel ligation Pneumocystis pneumonia / Venezuelan equine encephalitis

8 Rodent Models of Human Disease III other species Animal Model & Biomedical Problem Guinea Pig Species & Neoplastic disease Specific disease Transplantable leukemia Guinea Pig Species & Metabolic/ nutritional disease Guinea Pig Species & Degenerative disease Guinea Pig Species & Infectious disease Hamster Species & Genetic/ developmental defect Hamster Species & Neoplastic disease Hamster Species & Metabolic and nutritional disease Hamster Species & Degenerative disease Hamster Species & Infectious disease Gerbil Squirrel Woodchuck Opossum Hypervitaminosis A / Hypovitaminosis C Mannosidosis / Ulcerative colitis Allergic optic neuritis / Antitubular BM nephritis Inflammatory bowel disease / Optic disc swelling Genital herpes / Entamoeba histolytica Pichinde virus / Tuberculosis Autoimmunity Benzo(a)pyrene-induced tumors Carcinoma, larynx / Cholangiocarcinoma Pancreatic tumors / Spontaneous carcinoma, lung Tumors of respiratory tract Hypervitaminosis A / Diabetes mellitus Cardiomyopathy / Thrombosis, atrial Besnoitiosis, chronic Scrapie / Syphilis Transmissible mink encephalopathy Aural cholesteatoma / Lead neuropathy / Stroke Porphyria Hepatocellular carcinoma / Viral hepatitis Endocarditis

9 Perché è importante intraprendere studi con animali di laboratorio? Il cancro è una malattia multifattoriale con eziologia multipla. In laboratorio: - esistono protocolli sperimentali di cancerogenesi ben collaudati - l esposizione può essere controllata con estrema precisione - gli animali possono essere selezionati per uniformità e quindi la variabilità sperimentale può esser minimizzata - standardizzando i protocolli, gli esperimenti possono essere condotti contemporaneamente in più laboratori L. Gatta

10 Vainio H. et al. (Carcinogenesis 1985) Data on the carcinogenicity of chemicals in the IARC Monographs programme. (extracted from IARC Monographs volumes 1-38 ) C è una buona correlazione (84%) tra agenti che causano tumori nell uomo e quelli che causano tumori nel topo. La maggioranza dei chimici che causano leucemia nell uomo, causano leucemia anche nei roditori I topi o i ratti possono essere un buon modello per gli studi di cancerogenesi L. Gatta

11 Genes responsible for particular traits or disease susceptibility are chosen and extracted. Next they are injected into fertilized mouse eggs. Embryos are implanted in the uterus of a surrogate mother. The selected genes will be expressed by some of the offspring. Since the first gene transfers into mice were successfully executed in 1980, transgenic mice have allowed researchers to observe experimentally what happens to an entire organism during the progression of a disease. Transgenic mice have become models for studying human diseases and their treatments.

12 Figure 1.6: Generation of transgenic mice 1. Mouse development begins when the egg is fertilised by a sperm, creating a single cell which has the potential to form an entire organism. The single cell rapidly divides into two, each of which in turn divides into two and so forth, producing a mass of identical cells, each of which has the potential to form a foetus. 2. Eventually the mass of dividing cells begins to differentiate and forms a blastocyst, a hollow sphere of cells containing an inner cell mass. The outer layer goes on to form the placenta and other supporting tissue, while the inner cell mass goes on to form every type of body cell. These inner cells are termed embryonic stem cells. 3. Embryonic stem (ES) cells have the ability to replicate and divide indefinitely in vitro. Thus ES cell lines can be established by removing cells from the blastocyst using a micro-pipette and transferring them to suitable culture medium. 4. To generate transgenic mice, gene sequences are introduced into the genome of the ES cell in a process known as transformation. The gene can be introduced at a particular location by using various specialised techniques. Not all cells will contain the new sequence or will contain it at the right place, so those cells containing the gene at the correct location are selected and grown and other cells removed. Specific gene mutations can also be introduced into ES cells in this way. 5. Transformed ES cells containing the new gene sequences are transferred back into a blastocyst which is implanted into a surrogate mouse. 6. Progeny from the surrogate mouse are tested for the presence of the new gene or mutation, and mice heterozygous for the new gene are mated to generate homozygous mice

13 leukemia-prone and obesity-prone AKR mice Immune deficient or immune compromised models (e.g., nude or severe combined immunodeficiency (SCID) mice) are very susceptible to ordinarily non-pathogenic organisms such as Pasteurella spp. or Staphylococcus spp. Special caging and care procedures are vital to minimizing such infections C3H x DBA mice phenome.jax.org/.../details&stocknum=000648

14 Lymphoma Development in mice Chronically Exposed to UMTS_modulated Radiofrequency Electromagnetic Fields A.M. Sommer, A.K. Bitz, J. Strckert, V.W. Hansen, A. Lerchl Germany Female AKR/J mice 7 weeks old 160 mice exposed 160 mice sham exposed 160 mice cage-control Tattoo in one ear Animals were inspected daily for signs of morbidity and were weighed and palpated weekly: Starting at an age of 6 months, blood samples were taken twice montly from the tail and smeared on a slide Pathology: spleen, thymus, lympnodes; hematocrit; tumor infiltration (liver, lung, brain)

15 Field exposure 24 cages per unit Cone antenna, Black box, GHz cocktail based on technical features of the FDD-mode of UMTS 24h per day, 7 days per week 0.4 W/kg (imput power 15 W)

16 Results Body weight and Housing Survival: no effect Count of red and white cells: no changes related with the exposure AKR/J model had ben found to be a valuable model to chemicalcarcinogens or cocarcinogens with an early onset of leukemia.

17 DMBA induced tumors in Sprague-Dawley rats Terminal end buds (TEB; x77) located in Zone C of the mammary gland of a 55 day-old virgin rat become enlarged and darkly stained by 21 days post-dmba administration; they are called intraductal proliferations (IDP; x79), which by 35 days post-treatment grow and coalesce to form microtumors that are not palpable (x129). Whole mount preparations, toluidine blue Histological section of an intraductal proliferation (IDP; x110), which evolves to in situ carcinoma, either comedo or cribriform types. Both subtypes (x150) progress to invasive carcinoma (x150) Pathogenetic pathways of benign and malignant lesions induced in the virgin rat mammary gland by DMBA. The undifferentiated terminal end buds (TEB) originate adenocarcinomas progressing from intraductal proliferation (IDP), to carcinoma (Ca) in situ to develop several subtypes of in situ and invasive carcinomas. More differentiated alveolar buds (AB) and lobules (Lob) originate benign lesions that appear later than carcinomas

18 Research Projects: Perform A Two years exposure of mice (male and female) Two years exposure of rats (male and female) DMBA Transgenic mice CEMFEC

19 Status of PERFORM A Study type PERFORM -A 1 PERFORM -A 2 PERFORM -A 3 PERFORM -A 4 Two studies testing Two studies testing Long -term effects Co-carcinogenic for chronic toxicity for chronic toxicity on DMBA -induced effects on lymphoma and carcinogenicity and carcinogenicity mammary tumors induction in pim -1 in B6C3F1 mice in Wistar rats in SD rats transgenic mice Study laboratory Fraunhofer ITEM, Hannover, Germany RCC Ltd., Itingen, Switzerland ARCS, Seibersdorf, Austria LCG-RBM, Colleretto Giacosa, Italy EMF te sted Exposure 900 and 1,800 MHz (signal cocktail) 2 hours per day, 5 days per week, for 24 months 900 and 1,800 MHz (signal cocktail) 2 hours per day, 5 days per week, for 24 months 900 MHz (GSM basic signal) 4 hours per day, 5 days per week, for 6 months 900 MHz (GSM basic signal) 1 hour per day, 7 days per week, for 18 months Study start November 2001 July 2002 September 2002 November 2001

20 4 Ferris whell operanti a 902 MHz 4 Ferris Wheel operanti a 1747 MHz 1 per ogni dose considerata, possibile inserire fino a 65 animali, il posto vuoto viene riempito con un tubo con la giusta quantità di materiale equivalente

21 Hruby et al, Mut.Res. 649 (2008) 34 44

22 Tillman et al, Bioelectromagnetics 28: (2007)

23 Smith et al, Radiation Research 168, (2007)

24 Oberto et al, Radiation Research 168, , 2007

25 Long term exposure of Em-Pim1 Transgenic Mice to MHz Microwaves does not increase Lymphoma incidence Utteridge T.D., et al, Radiat Res Sep;158(3): Data on Tumor incidence for both Types of Mice Number L y m p h o - blastic Lymphoma s N o n l y m p h o - blastic Lymphoma s Neurological Tumors Other Tumors SAR W i l d Type Transgenic Wild Type Transgenic Wild Type Transgenic W i l d Type Transg enic Wild- Type Transgeni c Total

26 Referenze R. Hruby, G. Neubauer, N. Kuster, M. Frauscher, Study on potential effects of 902-MHz GSM-type Wireless communication Signals on DMBA-induced mammary tumours in Sprague Dawley rats, Mut.Res. 649 (2008) T. Tillmann, H. Ernst, S. Ebert, N. Kuster, W.Behnke, S. Rittinghausen, and C. Dasenbrock Carcinogenicity Study of GSM and DCSWireless Communication Signals in B6C3F1Mice, Bioelectromagnetics 28: (2007) P. Smith, N. Kuster, S. Ebert and H.Chevalier GSM and DCS Wireless Communication Signals: Combined Chronic Toxicity/Carcinogenicity Study in the Wistar Rat RAD. RES. 168, (2007) G. Oberto, K. Rolfo, P. Yu, M. Carbonato, S. Peano, N. Kuster, S. Ebert and S. Tofani. Carcinogenicity study of 217 Hz pulsed 900 MHz electromagnetic fields in Pim1 transgenic mice. Radiation Research 168, , 2007

27 Studies using combined exposure to RF fields with a known genotoxic/ carcinogenic agent Yu et al. (2006) DMBA (7,12-dimethyl-benz[a]-anthracene) female rats. SAR (0.44, 1.33 and 4 W/kg). No evidence for RF-induced effects on the development of mammary gland tumours. Huang et al. (2005) skin tumourigenesis using ICR mice single dose of DMBA. The SAR average of CDMA signal (849 MHz or GHz, 2 x 45 min/day for 19 wk) on freely moving animals was 0.4 W/kg. No effects of RF radiation on tumour development or epidermal thickness were observed. Heikkinen et al (2006) female rats by long-term treatment with 3-chloro-4- (dichloromethyl)-5-hydroxy-2(5h)-furanone in drinking water. 7 weeks of age, freely-moving animals, radial waveguide system to GSM 900 signals for 2 h d, 5 d/w for 2 ys at 0.3 and 0.9 W kg -1. complete histopathological examination, RF fields had not affected organ-specific incidence of any tumour type or total numbers of tumours. No significant effect on body weight gain or survival. Zook and Simmens (2006) ethylnitrosourea (ENU) female Spraque-Dawley rats 860 MHz pulsed RF signal 6 h per day, 5 days a week until they were killed between the ages of 171 and 325 days. No RF-related effects on the incidence, latency or any other characteristics of neurogenic tumours were seen. Shirai et al. (2007) RF radiation (1950 MHz, SAR 0.67 and 2.0 W/kg respectively; 90 minutes/day; 5 days/week; for 104 weeks) in Fischer344 rats N-ethyl-Nnitrosourea on gestational day 18. No significant increase in incidences or numbers of brain tumours was detected in the EMF-exposed groups.

28 Basal Cell Nevus Syndrome (BCNS) Individuals affected with the Gorlin syndrome inherit a germ-line mutation of the Patched (Ptch1) developmental gene. They show an increased susceptibility to spontaneous tumor development and radiation hypersensitivity. Predisposition to tumor development Medulloblastoma Soft tissue sarcomas Radiation hypersensitivity Basal cell carcinoma

29 BCNS mouse model pst mptc Ptch1 neo67/+ mice 650 bp pst neol 400 bp Survival Ptch1 +/- and wt mice Survival (%) Gy +- 0 Gy ++ Medulloblastoma (7.7%) Time (W eeks) Soft tissue sarcoma (40%) 59 versus 111 weeks P <

30 Ptch neo67/+ mice: radiation-induced tumorigenesis BCNS Generalized overgrowth Ptch1 heterozygous mice CNS tumors Soft tissue tumors

31 Ionizing radiation hypersensitivity X-rays irradiation of neonatal Ptch1 heterozygotes dramatically increases the incidence of medulloblastoma (81%) over the spontaneous rate (7%) and induce basal cell carcinoma development. Thus, newborn Ptch1 heterozygous mice constitute an extremely sensitive mouse model of radiation-induced tumorigenesis and represent a useful tool to evaluate the detrimental effects of exposure to potentially harmful agents. 250 kvp X Rays Gy 80 0 Gy 3 Gy neonatal 3 Gy adult Survival (%) Age (weeks)

32 Ptch neo67/+ mice: radiation-induced tumorigenesis High incidence of medulloblastoma following X-ray-irradiation of newborn Ptc1 heterozygous mice Medulloblastoma incidence /- 0 Gy +/- 3 Gy 60 +/+ 0GY and 3 Gy Age (weeks) Hystology of MBs developing in the posterior fossa of Ptch +/- mice Pazzaglia et al. Oncogene 21, , 2002

33 Protocol ϖ Newborn Ptch1 heterozygous and wild type mice (about 100 per genotype) were divided in sham and exposed groups at whole body exposure to GSM Basic 900 MHz, at 0.4 W/kg average, for 5 days, 30 twice a day. ϖ TEM cells and superficial body temperature has been monitored during exposure. ϖ Mice were observed daily for their whole life span. Upon decline of health or when tumors were visible, mice were killed and autopsied and processed for histologic examination. ϖ The experiments and the statistical evaluation were performed in blind mode with respect to the exposure conditions (sham or exposed).

34 Exposure system at 900 MHz The exposure system is constituted by two identical transverse electro-magnetic (TEM) cells of long length, to a uniform TEM plane wave at a frequency of 900 MHz. The set-up of two long TEM allows the simultaneous exposure of up to 12 mice per cell in blind way, i.e. the group of mice contained in one cell can be RF-exposed whereas the group in the other cell can be sham-exposed. The mice inside cylindrical plastic jigs, placed upon suitably sized polystyrene supports, for the positioning of mice at the same distance from the septum

35 RF protocol RF exposure for 30, twice a day, for 5 days 900 MHz, 0.4 W/kg average Room temperature, superficial body temperature, 100 Newborn Wild type 100 Newborn Heterozygotes sham-exposure, RF exposure

36 Survival of Ptch1 +/- mice exposed to RF Number of mice Median (weeks) Mean ± SD (weeks) Sham Male ± 32.9 Female ± 23.7 Total ± 28.0 Exposed Male ± 23.0 Female ± 20.6 Total ± 21.8 No difference in survival was observed between sham and exposed Ptch1 +/- mice

37 Tumorigenesis in Ptch1 +/- mice exposed to RF Number of autopsied mice Medulloblastoma Sarcoma Observed lesions (% ± SE) Basal cell carcinoma Preneoplastic skin lesions Sham Male 19-8 (42.1 ± 11.3) - 5 (29.4 ± 11.1) Female (55.0 ± 11.1) - 6 (31.6 ± 10.7) Total (48.7 ± 8.0) - 11 (30.6 ± 7.7) Exposed Male 22 2 (9.1 ± 6.1) 12 (54.6 ± 10.6) - 5 (25.0 ± 9.7) Female 31 2 (6.5 ± 4.4) 23 (74.2 ± 7.9) - 9 (32.1 ± 8.8) Total 53 4 (7.6 ± 3.6) 35 (66.0 ± 6.5) - 14 (29.2 ± 6.6) 50 µm 50 µ m No significant statistical differences in Ptch1-dependent tumorigenesis were observed between sham and exposed mice.

38 Summary ϖ Mortality of Ptch1 heterozygous and wild type mice showed a similar trend in the two groups. ϖ No significant statistical differences in Ptch1-dependent tumorigenesis were observed between sham and exposed mice. Conclusions The experimental data do not show a tumorigenic effect of exposure to GSM Basic 900 MHz in this highly radiationsusceptible mouse model. Effects of Exposure of Newborn Patched1 Heterozygous Mice to GSM, 900 MHz A. Saran, S. Pazzaglia, M. Mancuso, S. Rebessi, V. Di Majo, M. Tanori, G. A. Lovisolo, R. Pinto and C. Marino RADIATION RESEARCH 168, (2007)

39 Effects of 900MHz electromagnetic field on TSH and thyroid hormones in rats A. Koyu et al. / Toxicology Letters 157 (2005) Animals were randomly grouped as follows: cage control group (n = 10), sham-exposed group (n = 10) and 900MHz EMF (n = 10). They were exposed to 30 min/day radiation for a period of 5 days/week, 4 weeks, SAR 2 W/kg. Rats exposed to 900MHz EMF were compared to control and sham rats respect to serum TSH, T3 and T4. At the end of 4 weeks, the rats were sacrificed and blood samples were collected through a cardiac puncture. A special exposure device with five exposure antenna was used. The figure shows one of the antennas of the device. The exposure system consisted of a round plastic tube cage (length: 12 cm and diameter: 5.5 cm) and a dipole antenna. The whole body of the rats was positioned in close contact above the dipole antenna, and the tube was ventilated from head to tail in order to decrease the stress of the rat while in the tube. The 900 megahertz (MHz) continuous wave electromagnetic energy generator produced at the electromagnetic compatibility (EMC) Laboratory of Suleyman Demirel University was used in the study. The power density measurements were made using electromagnetic field meter (Holaday Industry Inc., Adapazari, Turkey). RESULTS:

40 Exposure set up 900 MHz, GSM-modulated SAR 0.4, 1, 2 W/Kg, whole body 2 hours/day, 1-4 weeks Exposure period Thymus Spleen Sera Bone marrow cells

41 Thymus Cell counts, cell subpopulations (CD4/CD8) Spleen Cell counts T cells Frequency of CD4 and CD8 Cell proliferation Cytokine production B cells Frequency Cell proliferation NO evidence of effect

42 BIOTEC-MED Bioelectromagnetic Group TEM cell setup: system Cellular phone Power meter Black box Amplifier Two jigs positioned Thermostatic bath Two non standard TEM cell (12 x 12 x 120 cm) operating at 900 MHz Three couple of mice on each side were placed with the caudal axis parallel to propagation direction and radiated in the bottom. Sham-exposed mice were placed in identical TEM cell in the same room. A black box was installed to perform the experimental procedure in blind way. To avoid temperature increase in the exposed mice a watercooling system has been set up with two external metallic jacket filled by circulating water fed through a thermostatic bath (at 20 C), and placed in contact with the bottom walls (difference between SARexposed and sham-exposed groups was <0.5 C).

43 IL-2 production (48 h stimulation) 1 week exposure 40 IL-2 (U/ml) Control Sham 1 W/Kg 2 W/Kg Anti-CD28 mab (mg/ml)

44 1 or 2 W / Kg, 2h / day, 7 consecutive days Antigen + adjuvant Lymph node cells Antige n Cell proliferation ( 3 H-TdR)

45 Lymph node cell number 30 LNC number ( 10 6 ) Ctrl Sham 1 W/Kg 2 W/Kg

46 Antigen-dependent cell proliferation H-TdR uptake ( cpm ) Ctrl Sham 1 W / Kg 2 W /Kg Ag (mg/ml)

47 Peripheral B cell differentiation

48 Antibody serum levels Ex vivo antibody production

49 Antigen-specific antibody response Anti-OVA Ab serum levels ( O.D. ) A IgM Control Sham 2 W/kg B IgG Log [dil] Log [dil]

50 RADIATION RESEARCH 170, 2008 Effects of GSM-Modulated Radiofrequency Electromagnetic Fields on Mouse Bone Marrow Cells Maria Grazia Prisco, Francesca Nasta, Maria Manuela Rosado, Giorgio Alfonso Lovisolo, Carmela Marino and Claudio Pioli X-rays Control mice Bone Marrow cells X-rays 3-6 weeks Sham-exposed mice Bone Marrow cells X-rays 3-6 weeks RF-exposed mice Bone Marrow cells 3-6 weeks X-ray-lethally-irradiated (9 Gy) mice were transplanted with bone marrow cells from either control, or sham-exposed or RF-exposed mice

51 Effects on BM hematopoietic stem cells Survival of X-ray-irradiated mice Survival (%) Control donors Sham-exposed donors RF-exposed donors no BM transplantation Time after BM transplantation (days) X-ray-irradiated mice received 5x10 6 bone marrow cells from either control, or sham-exposed, or RF-exposed mice

52 Effects on BM hematopoietic stem cells Thymus reconstitution - subpopulations DN Not-irradiated normal mice Control donors Sham-exposed donors RF-exposed donors DP Cell number (x10 6 ) CD CD Age (weeks) Time after BMT (weeks) Age (weeks) Time after BMT (weeks)

53 Effects on BM hematopoietic stem cells Thymus reconstitution - cell proliferation 3 H-TdR uptake (cpm x10 3 ) Not-irradiated normal mice 60 Control donors 50 Sham-exposed donors RF-exposed donors Age (weeks) 3 6 Time after BM transplantation (weeks)

54 Effects on BM hematopoietic stem cells Spleen reconstitution cell populations Cell number (x10 6 ) weeks post-bmt 6 weeks post-bmt Control donor Sham-exposed donor RF-exposed donor CD8 CD4 CD19 CD8 CD4 CD19 CD4 = Helper T cells CD8 = Cytotoxic T cells CD19 = B cells

55 Effects on BM hematopoietic stem cells Spleen reconstitution - B cell compartment B cell proliferation 3 H-TdR uptake (cpm x10 3 ) weeks after BMT 6 weeks after BMT Control donor Sham-exposed donor RF-exposed donor Not-irradiated normal mice LPS (µg/ml) LPS (µg/ml)

56 Referenze Gatta L, Pinto R, Ubaldi V, Pace L, Galloni P, Lovisolo GA, Marino C, Pioli C. Effects of In Vivo Exposure to GSM-Modulated 900 MHz Radiation on Mouse Peripheral Lymphocytes. Radiat Res Nov; 160(5): Francesca Nasta, Maria Grazia Prisco, Rosanna Pinto, Giorgio Alfonso Lovisolo, Carmela Marino, and Claudio Pioli. Effects of GSM-modulated RF radiation on B cell peripheral differentiation and antibody production. Radiat. Res, 165 (6): , M.G. Prisco, F. Nasta, M.M. Rosado, G. A. Lovisolo, C. Marino and C. Pioli, Effects of GSM-Modulated Radiofrequency Electromagnetic Fields on Mouse Bone Marrow Cells, Radiation Research 170, (2008)

57

58 Cochlear structure Human Ear Cochlea Rat s Cochlea Cross section of the whole cochlea SEM of Guinea Pig s Organ of Corti Schematic drawing of the Organ of Corti One single turn

59 a) Partial loss of outer hair cells 1 week of GM treatment * b) Complete loss of outer hair cells 4 weeks of GM treatment Healthy Organ of Corti * Figures a and b from Forge and Schacht, Audiol Neurootol 2000

60 DPOAE Recording Otoacoustic emissions Sound Sound Acoustic signal emitted by the cochlea in response to acoustic stimuli Example of DP-gram recorded from Sprague- Dawley rats

61 DPOAE recordings in rats treated with ototoxic drugs Before treatment After treatment Animals are injected i.m. with gentamicin 150 mg/kg/day for 7 days

62 Toxicology and Biomedical Science Bioelectromagnetism Lab Animali Ratti Sprague Dawley maschi Peso di circa 250 g all inizio del trattamento Minimo stress durante l esposizione Anestetizzati durante la misura dei DPOAE Anestesia gassosa N 2 O/O 2 (70%-30%) 2 3 % alotano

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