Studi su modelli animali: ilquadro delle conoscenze

Carmela MarinoENEA C.R Casaccia

Bari23 giugno 2009

Giornata studio - "Salute e campielettromagnetici"

Attività sperimentali

•In vivo:carcinogenesi,tossicità sistemica,fisio-patologia;comportamento,memoria,effetti teratogeni;

IL CONCETTO DI MODELLO ANIMALEIL CONCETTO DI MODELLO ANIMALE

La maggior parte delle nostre conoscenze nel campo della biochimicagenerale, della fisiologia e dell’endocrinologia origina dallasperimentazione animale, e, idealmente, dovrebbe essere estrapolataall’uomo. In molti esperimenti, infatti, l’animale sostituisce l’uomo e vieneperciò considerato come modello animale. Una definizione possibile è laseguente: un modello è quella condizione che permette di studiare iprocessi biologici e comportamentali di base, o in cui può esserestudiato un processo patologico indotto, e nel quale il fenomeno èsimile, almeno sotto un certo punto di vista, allo stesso fenomenonell’uomo o in altre specie animali. La maggior parte dei modelli animalidi laboratorio sono sviluppati e utilizzati per studiare la causa, la naturae la cura di malattie umane.L’importanza dei risultati derivati dalla sperimentazione dipende dallascelta di un adeguato modello. La possibilità di estenderel’estrapolazione dei risultati dipende dal tipo di modello e dalla naturadella ricerca.

I piccoli roditori, principalmente topi e ratti, sono le specie più utilizzate.

Nei modelli animali indotti, una malattia o un processo patologico sonoindotti sperimentalmente, sia chirurgicamente che somministrandosostanze biologicamente attive, per ottenere una condizione che somiglia quella che si verificherebbe nella specie bersaglio. A questo gruppoappartengono gli animali transgenici, tra cui i topi sono la specie dielezione.

Per modelli animali spontanei si intendono quelli che portano mutazionigenetiche spontanee, e sono stati caratterizzati centinaia di ceppi ecolonie animali che manifestano malattie spontanee simili all’uomo.

L’estrapolazione dei risultati può essere qualitativa, vertente suiprocessi fisio-patologici dell’animale e sulle sue reazioni a stimoliestrapolabili all’uomo o ad altri animali, o quantitativa, che consiste nelladeterminazione del dosaggio di certi composti che potrebbero esserebenefici o pericolosi per la specie bersaglio. L’estrapolazionedall’animale all’uomo va sempre effettuata con riserva, e i risultati degliesperimenti dovranno essere, in ultima analisi, verificati con studisull’uomo.

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, lastrumentazione, la bibliografia, la perizia tecnica, il costo, la valutazionedei 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

Avian reovirus / Capillaria hepatica / Cytomegalovirus / EncephalomyocarditisEpstein-Barr virus-dependent / lymphoproliferative disease / FilariasisGiardiasis / Hepatitis, reovirus / Helicobacteriosis / Influenza BListeriosis / Lymphocytic choriomeningitis / Meningoencephalitis, amebicMeningoencephalitis / Angiostrongylus / Scrapie / Theiler's encephalomyelitisTrypanosomiasis / Yersinia infection

Mouse Species &Infectious disease

Adenosis, vagina-cervix / Autoimmune disease / Biliary obstructionDiverticulosis, oviduct / Dysbaric osteonecrosis / Proliferative glomerulonephritis/Immunosuppression / Macroglobulinemic neuropathy / Menkes's disease / Motor neurondisease / Pulmonary fibrosis, bleomycinPulmonary fibrosis, solvents/oxygen / Reye's syndrome / Salpingitis / Vitiligo

Mouse Species &Degenerative Disease

Amyloidosis / Diabetes mellitus / Gammopathies, monoclonalGestational diabetes / Globoid cell leukodystrophyGlucose-6-phosphate dehydrogenase deficiency / Histidinemia Hyper- and hypotensionHypervitaminosis A / Hypophosphatemia (rickets) / Mast cell deficiency / MethylmercurypoisoningNiemann-Pick Disease / Nonobese diabetic / Ochratoxicosis / Ornithine transcarbamylasedeficiencyParaproteinemia, idiopathic / Thalassemia, alpha

Mouse Species &Metabolic/ nutritional disease

Adenocarcinoma, DES / Adenoma, salivary / Angiosarcoma, liver / Carcinoma, cervixCarcinoma, embryonal / Hodgkin's disease / Leukemia, myelogenous / Malignant tumortransplantMammary tumor / Ovarian tumor / Preneoplastic lymphoid hyperplasiaTeratoma and teratocarcinoma

Mouse Species &Neoplastic disease

Anemia, hereditary / Athymic / Autosomal trisomies / Chediak-Higashi syndromeCopper malabsorption, X-linked / Exencephaly / Hereditary asplenia / L call mutant /Megacolon, aganglionicMegaloblastic anemia / Polycystic kidney disease / Testicular feminization

Mouse Species &Genetic and developmental defects

Specific DiseaseAnimal Model &

Biomedical Problem

Rodent Models of Human Disease I - mouse

Specific DiseaseAnimal Model &

Biomedical Problem

Pneumocystis pneumonia / Venezuelan equine encephalitisRat Species & Infectious disease

Aneurysm, cerebral / Arthritis / Autoimmune thyroiditisDuodenal ulcer / Hypertension, induced / Hypertension, spontaneousHypertrophy, right ventricle / Immunosuppression / Ligation, cerebral arteryMyocardial infarction / Optic disc swelling / PeriodontitisRetinal degeneration / Silica-induced pulmonary lipoproteinosisThromboembolism / Uterine vessel ligation

Rat Species & Degenerative disease

Adrenal apoplexy / Alcoholic fatty liver / AnemiaCirrhosis / Diabetes insipidus / Diabetes mellitusEthanol dependence / Fructose-induced lesionsHepatic necrosis, halothane induced / Hypervitaminosis AHypothyroidism / Lead encephalopathy / Lipotrope deficiencyMucopolysaccharidosis / Obesity / OchratoxicosisOsteopetrosis / Phenylketonuria / Skeletal muscle, defective glucose-glycogenStriatal lesions, kainic acid induced / UrolithiasisVasculitis, pulmonary, glucan induced

Rat Species & Metabolic/ nutritional disease

Adenocarcinoma, colon / Adenocarcinoma, intestineAdenocarcinoma, prostate / Aflatoxin carcinogenesis / Angiosarcoma, hepaticCarcinoma, bladder / Carcinoma, esophagus / Carcinoma, pancreasCarcinoma, kidney / Carcinoma, squamous cell, lung / Carcinoma yolk sacInterstitial cell tumor / Lymphoblastic leukemia / Malignant histiocytomaMedullary carcinoma, thyroid / Neurogenic tumors, N-nitrosoureaOsteosarcoma, Moloney sarcoma virus / Pituitary tumorsUrothelial tumors

Rat Species & Neoplastic disease

Amnionic fluid deficiency / Fetal colon implants / Fetal lung growthHereditary / hyperbilirubinemia / Hydrocephalus / HydronephrosisIntrauterine growth retardation / Megacolon, aganglionic

Rat Species &Genetic/ developmental defect

Rodent Models of Human Disease II - rat

Specific diseaseAnimal Model &

Biomedical Problem

EndocarditisOpossum

Hepatocellular carcinoma / Viral hepatitisWoodchuck

PorphyriaSquirrel

Aural cholesteatoma / Lead neuropathy / StrokeGerbil

Besnoitiosis, chronicScrapie / SyphilisTransmissible mink encephalopathy

Hamster Species & Infectious disease

Cardiomyopathy / Thrombosis, atrialHamster Species & Degenerative disease

Hypervitaminosis A / Diabetes mellitusHamster Species & Metabolic and nutritional disease

Benzo(a)pyrene-induced tumorsCarcinoma, larynx / CholangiocarcinomaPancreatic tumors / Spontaneous carcinoma, lungTumors of respiratory tract

Hamster Species & Neoplastic disease

AutoimmunityHamster Species & Genetic/ developmental defect

Genital herpes / Entamoeba histolyticaPichinde virus / Tuberculosis

Guinea Pig Species & Infectious disease

Allergic optic neuritis / Antitubular BM nephritisInflammatory bowel disease / Optic disc swelling

Guinea Pig Species & Degenerative disease

Hypervitaminosis A / Hypovitaminosis CMannosidosis / Ulcerative colitis

Guinea Pig Species & Metabolic/ nutritional disease

Transplantable leukemiaGuinea Pig Species & Neoplastic disease

Rodent Models of Human Disease III – other species

www.montauc.org/Tech/rodentmod.htm

Perché è importante intraprendere studi con animali dilaboratorio?

Il cancro è una malattia multifattoriale con eziologia multipla.

In laboratorio:

- esistono protocolli sperimentali di cancerogenesi bencollaudati

- 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 laboratoriL. Gatta

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 checausano 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 studidi cancerogenesi

L. Gatta

Genes responsible for particular traits or disease susceptibility arechosen and extracted. Next they are injected into fertilized mouse eggs.Embryos are implanted in the uterus of a surrogate mother. The selectedgenes will be expressed by some of the offspring.Since the first gene transfers into mice were successfully executed in1980, transgenic mice have allowed researchers to observeexperimentally what happens to an entire organism during theprogression of a disease. Transgenic mice have become models forstudying human diseases and their treatments.

www.accessexcellence.org/AB/GG/transgenic.html

Figure 1.6: Generation of transgenic mice1. 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 hasthe 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. Theouter 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. Theseinner 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 cellsfrom 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 genecan be introduced at a particular location by using various specialised techniques. Not all cells will contain the new sequence or will contain it atthe right place, so those cells containing the gene at the correct location are selected and grown and other cells removed. Specific genemutations 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 aremated to generate homozygous mice

www.bseinquiry.gov.uk

leukemia-prone and obesity-prone AKR mice

phenome.jax.org/.../details&stocknum=000648

www.i-dna.biz/products/Biolasco

C3H x DBA mice

Immune deficient or immune compromised models (e.g.,nude or severe combined immunodeficiency (SCID) mice)are very susceptible to ordinarily non-pathogenicorganisms such as Pasteurella spp. or Staphylococcusspp. Special caging and care procedures are vital tominimizing such infections

www.ccac.ca/.../ETCC/Module05/06.html

Lymphoma Development in mice Chronically Exposed toUMTS_modulated Radiofrequency Electromagnetic Fields

A.M. Sommer, A.K. Bitz, J. Strckert, V.W. Hansen, A. LerchlGermany

• 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 takentwice montly from the tail and smeared on a slide

• Pathology: spleen, thymus, lympnodes; hematocrit;tumor infiltration (liver, lung, brain)

Field exposure

• 24 cages per unit

• Cone antenna, Black box,

• 1.966 GHz cocktail based on technicalfeatures of the FDD-mode of UMTS

• 24h per day, 7 days per week

• 0.4 W/kg (imput power 15 W)

Results

• Body weight and Housing

• Survival: no effect

• Count of red and white cells: no changes relatedwith the exposure

AKR/J model had ben found to be a valuablemodel to chemicalcarcinogens or co-carcinogens with an early onset of leukemia.

Terminal end buds (TEB; x77) located in Zone C of themammary gland of a 55 day-old virgin rat become enlarged anddarkly stained by 21 days post-DMBA administration; they arecalled intraductal proliferations (IDP; x79), which by 35 dayspost-treatment grow and coalesce to form microtumors that arenot palpable (x129). Whole mount preparations, toluidine blue

Histological section of an intraductal proliferation (IDP;x110), which evolves to in situ carcinoma, either comedo orcribriform types. Both subtypes (x150) progress to invasivecarcinoma (x150)

www.ehponline.org/.../1996/104-9/russo.html

Pathogenetic pathways of benign and malignant lesions inducedin the virgin rat mammary gland by DMBA. The undifferentiatedterminal end buds (TEB) originate adenocarcinomas progressingfrom intraductal proliferation (IDP), to carcinoma (Ca) in situ todevelop several subtypes of in situ and invasive carcinomas.More differentiated alveolar buds (AB) and lobules (Lob)originate benign lesions that appear later than carcinomas

DMBA induced tumors in Sprague-Dawley rats

• Research Projects:

• Perform A Two years exposure of mice (male and female) Two years exposure of rats (male and female) DMBA Transgenic mice

• CEMFEC

Status of PERFORM A

PERFORM -A 1 PERFORM-A 2 PERFORM -A 3 PERFORM-A 4 Study type

Two studies testing for chronic toxicity and carcinogenicity in B6C3F1 mice

Two studies testing for chronic toxicity and carcinogenicity in Wistar rats

Long-term effects on DMBA -induced mammary tumors in SD rats

Co-carcinogenic effects on lymphoma induction in pim-1 transgenic mice

Study laboratory

Fraunhofer ITEM, Hannover, Germany

RCC Ltd., Itingen, Switzerland

ARCS, Seibersdorf, Austria

LCG-RBM, Colleretto Giacosa, Italy

EMF te sted

900 and 1,800 MHz (signal cocktail)

900 and 1,800 MHz (signal cocktail)

900 MHz (GSM basic signal)

900 MHz (GSM basic signal)

Exposure

2 hours per day, 5 days per week, for 24 months

2 hours per day, 5 days per week, for 24 months

4 hours per day, 5 days per week, for 6 months

1 hour per day, 7 days per week, for 18 months

Study start November 2001 July 2002 September 2002 November 2001

4 Ferris whell operanti a 902 MHz4 Ferris Wheel operanti a 1747 MHz

1 per ogni dose considerata,possibile inserire fino a 65 animali, ilposto vuoto viene riempito con untubo con la giusta quantità dimateriale equivalente

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

Tillman et al, Bioelectromagnetics 28:173 -187 (2007)

Smith et al, Radiation Research 168, 480–492 (2007)

Oberto et al, Radiation Research 168, 316-326, 2007

Long term exposure of Em-Pim1 Transgenic Mice to 898.4Long term exposure of Em-Pim1 Transgenic Mice to 898.4MHz Microwaves does not increase Lymphoma incidenceMHz Microwaves does not increase Lymphoma incidence

Utteridge T.D., et al, Utteridge T.D., et al, RadiatRadiat Res. 2002 Res. 2002 SepSep;158(3):357-64;158(3):357-64

Data on Tumor incidence for bothTypes of Mice

1615962400198557600598Total

112 984361501201184

252108442921201202

320157835821201201

614178040801201200.25

4511174351531201200

Transgenic

Wild-Type

Transgenic

W i l dType

TransgenicWild TypeTransgenicWild TypeTransge-nic

W i l dType

SAR

TumorsOtherTumorsNeurolo-gical

Lymphomas

N o nl y m p h o -blastic

Lymphomas

L y m p h o -blastic

Number

Referenze• R. Hruby, G. Neubauer, N. Kuster, M. Frauscher, Study on potential

effects of 902-MHz GSM-type Wireless communication Signals” onDMBA-induced mammary tumours in Sprague–Dawley rats,Mut.Res. 649 (2008) 34–44

• T. Tillmann, H. Ernst, S. Ebert, N. Kuster, W.Behnke, S.Rittinghausen, and C. Dasenbrock Carcinogenicity Study of GSMand DCSWireless Communication Signals in B6C3F1Mice,Bioelectromagnetics 28:173-187 (2007)

• P. Smith, N. Kuster, S. Ebert and H.Chevalier GSM and DCSWireless Communication Signals: Combined ChronicToxicity/Carcinogenicity Study in the Wistar Rat RAD. RES. 168,480–492 (2007)

• G. Oberto, K. Rolfo, P. Yu, M. Carbonato, S. Peano, N. Kuster, S.Ebert and S. Tofani. Carcinogenicity study of 217 Hz pulsed 900MHz electromagnetic fields in Pim1 transgenic mice. RadiationResearch 168, 316-326, 2007

Studies using combined exposure to RF fields with aknown 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 developmentof 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 1.763 GHz, 2 x 45 min/day for 19wk) on freely moving animals was 0.4 W/kg. No effects of RF radiation ontumour 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, 5d/w for 2 ys at 0.3 and 0.9 W kg-1. complete histopathological examination, RFfields had not affected organ-specific incidence of any tumour type or totalnumbers of tumours. No significant effect on body weight gain or survival.

• Zook and Simmens (2006) ethylnitrosourea (ENU) female Spraque-Dawley rats860 MHz pulsed RF signal 6 h per day, 5 days a week until they were killedbetween 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-N-nitrosourea on gestational day 18. No significant increase in incidences ornumbers of brain tumours was detected in the EMF-exposed groups.

Individuals affected with the Gorlin syndrome inherit a germ-linemutation of the Patched (Ptch1) developmental gene. They showan increased susceptibility to spontaneous tumor development andradiation hypersensitivity.

Basal Cell Nevus Syndrome (BCNS)

Predisposition totumor development

Radiation hypersensitivity

Medulloblastoma Soft tissue sarcomas

Basal cell carcinoma

BCNS mouse model

Ptch1neo67/+ mice

0 25 50 75 100 125 1500

25

50

75

1000 Gy +-

0 Gy ++

Time (Weeks)

Su

rviv

al

(%)

Survival Ptch1+/- and wt mice

59 versus 111 weeksP < 0.0001

Medulloblastoma (7.7%)

Soft tissue sarcoma (40%)

ESONE 6 ESONE 7

NEO C

650 bp

400 bp

ESONE 6 ESONE 7ESONE 6ESONE 6 ESONE 7ESONE 7

NEO CNEO C

pst neoL

pst mptc

ESONE 6 ESONE 7ESONE 6ESONE 6 ESONE 7ESONE 7

NEO CNEO C

650 bp650 bp

400 bp400 bp

ESONE 6ESONE 6 ESONE 7ESONE 7ESONE 6EXON 6 ESONE 7EXON 7

NEO CNEO C

pstpst neoLneoL

pstpst mptcmptc

Generalized overgrowth

Soft tissue tumorsCNS tumors

Ptch1 heterozygous mice

BCNS

Ptchneo67/+ mice: radiation-induced tumorigenesis

250 kVp X Rays

3 Gy

0 20 40 60 80 100 1200

20

40

60

80

100

0 Gy

3 Gy neonatal

3 Gy adult

Age (weeks)

Su

rviv

al

(%)

Ionizing radiation hypersensitivityX-rays irradiation of neonatal Ptch1 heterozygotes dramatically increases theincidence of medulloblastoma (81%) over the spontaneous rate (7%) and inducebasal cell carcinoma development. Thus, newborn Ptch1 heterozygous miceconstitute an extremely sensitive mouse model of radiation-inducedtumorigenesis and represent a useful tool to evaluate the detrimental effects ofexposure to potentially harmful agents.

0

20

40

60

80

100

0 4 8 12 16 20 24 28 32 36 40Age (weeks)

Med

ullo

bla

sto

ma in

cid

en

ce

+/- 0 Gy

+/- 3 Gy

+/+ 0GY and 3 Gy

Hystology of MBs developing in the posteriorfossa of Ptch+/- mice

Pazzaglia et al. Oncogene 21, 7588-84, 2002

Ptchneo67/+ mice: radiation-induced tumorigenesis

High incidence of medulloblastoma following X-ray-irradiation of newborn Ptc1 heterozygous mice

ϖ Newborn Ptch1 heterozygous and wild type mice (about 100per genotype) were divided in sham and exposed groups atwhole body exposure to GSM Basic 900 MHz, at 0.4 W/kgaverage, for 5 days, 30’ twice a day.

ϖ TEM cells and superficial body temperature has beenmonitored during exposure.

ϖ Mice were observed daily for their whole life span. Upondecline of health or when tumors were visible, mice werekilled and autopsied and processed for histologic examination.

ϖ The experiments and the statistical evaluation wereperformed in blind mode with respect to the exposureconditions (sham or exposed).

Protocol

The exposure system is constituted by two identical transverseelectro-magnetic (TEM) cells of long length, to a uniform TEM planewave at a frequency of 900 MHz. The set-up of two long TEM allowsthe 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 whereasthe group in the other cell can be sham-exposed.

Exposure system at 900 MHz

The mice inside cylindricalplastic jigs, placed uponsuitably sized polystyrenesupports, for thepositioning of mice at thesame distance from theseptum

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 NewbornHeterozygotes

sham-exposure, RF exposure

Number of mice

Median (weeks)

Mean ± SD (weeks)

Sham

Male 23 33.0 48.9 ± 32.9 Female 27 46.0 48.0 ± 23.7

Total 50 43.0 48.4 ± 28.0 Exposed

Male 27 47.0 50.0 ± 23.0 Female 36 38.5 42.6 ± 20.6

Total 63 41.0 45.8 ± 21.8

Survival of Ptch1+/- mice exposed to RF

No difference in survival was observedbetween sham and exposed Ptch1+/- mice

Observed lesions (% ± SE)

Number of autopsied

mice Medulloblastoma Sarcoma Basal cell carcinoma

Preneoplastic skin lesions

Sham

Male 19 - 8 (42.1 ± 11.3) - 5 (29.4 ± 11.1) Female 20 - 11 (55.0 ± 11.1) - 6 (31.6 ± 10.7)

Total 39 - 19 (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)

Tumorigenesis in Ptch1+/- mice exposed to RF

50 µm

50 µm

No significant statisticaldifferences in Ptch1-dependenttumorigenesis were observed

between sham and exposed mice.

ϖ Mortality of Ptch1 heterozygous and wild type mice showed asimilar trend in the two groups.

ϖ No significant statistical differences in Ptch1-dependenttumorigenesis were observed between sham and exposed mice.

Summary

ConclusionsThe experimental data do not show a tumorigenic effect ofexposure to GSM Basic 900 MHz in this highly radiation-

susceptible mouse model.

Effects of Exposure of Newborn Patched1 Heterozygous Mice to GSM, 900MHzA. Saran, S. Pazzaglia, M. Mancuso, S. Rebessi, V. Di Majo, M. Tanori, G. A.Lovisolo, R. Pinto and C. Marino

RADIATION RESEARCH 168, 733–740 (2007)

A. Koyu et al. / Toxicology Letters 157 (2005) 257–262

Animals were randomly grouped as follows: cage controlgroup (n = 10), sham-exposed group (n = 10)and 900MHz EMF (n = 10). They were exposed to 30 min/dayradiation for a period of 5 days/week, 4 weeks, SAR 2 W/kg.Rats exposed to 900MHz EMF were compared to controland sham rats respect to serum TSH, T3 and T4. At the endof 4 weeks, the rats were sacrificed and blood sampleswere 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 dipoleantenna. The whole body of the rats was positioned in close contact above the dipole antenna, and the tube wasventilated 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) Laboratoryof Suleyman Demirel University was used in the study. The power densitymeasurements were made using electromagnetic field meter (Holaday Industry Inc., Adapazari, Turkey).

RESULTS:

Effects of 900MHz electromagnetic field onTSH and thyroid hormones in rats

Exposure set up

• 900 MHz, GSM-modulated• SAR 0.4, 1, 2 W/Kg, whole body• 2 hours/day, 1-4 weeks

• Thymus

• Spleen

• Sera

• Bone marrow cells

Exposure period

• 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 evidenceof effect

TEM cell setup: systemBIOTEC-MEDBioelectromagnetic Group

Two jigs positioned

Black box

Cellularphone

Amplifier

Thermostatic bath

Power meter

Two non standard TEM cell (12 x 12 x 120 cm) operating at 900MHz

Three couple of mice oneach side were placed withthe caudal axis parallel topropagation direction andradiated in the bottom.Sham-exposed mice wereplaced in identical TEM cellin the same room. A blackbox was installed to performthe experimental procedurein blind way. To avoidtemperature increase in theexposed mice a water-cooling system has beenset up with two externalmetallic jacket filled bycirculating water fed througha thermostatic bath (at 20°C), and placed in contactwith the bottom walls(difference between SAR-exposed and sham-exposedgroups was <0.5 °C).

IL-2 production(48 h stimulation)

1 week exposure

IL-2

(U

/ml)

40

30

20

10

00 0.25 0.50 0.75 1.00

Anti-CD28 mAb (mg/ml)

Control

Sham

1 W/Kg

2 W/Kg

Antigen + adjuvant

1 or 2 W / Kg, 2h / day, 7 consecutive days

Lymph node cells

Cell proliferation(3H-TdR)

Antigen

LNC

num

ber

( 10

6 )

30

20

10

0

Ctrl 1 W/Kg 2 W/KgSham

Lymph node cell number

Ag (mg/ml)

3 H-T

dR u

ptak

e (

cpm

)

3000

2000

0

4000

1001 100.10

CtrlSham1 W / Kg2 W /Kg

Antigen-dependent cell proliferation

PeripheralB cell

differentiation

Antibodyserum levels

Ex vivo antibodyproduction

IgG

2 3 4

B

Log [dil]

1.2

1.0

0.8

0.6

0.4

0.2

0.0

IgM

0.6

0.5

0.4

0.3

0.2

0.1

0.0

Log [dil]

A

Ant

i-OV

A A

b se

rum

leve

ls (

O.D

. )

2 3 4

Control

2 W/kgSham

Antigen-specific antibody response

Bone Marrowcells

Control mice

Sham-exposed mice

RF-exposed mice

Bone Marrowcells

Bone Marrowcells

X-ray-lethally-irradiated (9 Gy) micewere transplanted with bone marrow cells from

either control, or sham-exposed or RF-exposed mice

3-6 weeks

3-6 weeks

3-6 weeks

X-rays

X-rays

X-rays

RADIATION RESEARCH 170, 2008Effects of GSM-Modulated Radiofrequency Electromagnetic Fields on Mouse BoneMarrow CellsMaria Grazia Prisco, Francesca Nasta, Maria Manuela Rosado, Giorgio Alfonso Lovisolo, Carmela Marino and Claudio Pioli

Effects on BM hematopoietic stemcells

Survival of X-ray-irradiated mice

X-ray-irradiated mice received 5x106 bone marrow cellsfrom either control, or sham-exposed, or RF-exposed mice

Control donorsSham-exposed donorsRF-exposed donorsno BM transplantation

0

20

40

60

80

100

0 10 20 30

Time after BM transplantation (days)

Sur

viva

l (%

)

Cel

l num

ber

(x10

6)

Effects on BM hematopoietic stem cellsThymus reconstitution - subpopulations

0.0

2.5

5.0

7.5

10.0

0

5

10

15

20

25

30

0

25

50

75

100

125

150

0.0

2.5

5.0

7.5

10.0

Control donorsSham-exposed donorsRF-exposed donors

Not-irradiated normal mice

Age(weeks)

12

Time after BMT(weeks)

3 6

Age(weeks)

12

Time after BMT(weeks)

3 6

DN

CD4 CD8

DP

Effects on BM hematopoietic stem cells

Thymus reconstitution - cell proliferation

0

10

20

30

40

50

60 Control donorsSham-exposed donorsRF-exposed donors

Not-irradiated normal mice

Age(weeks)

12

Time after BM transplantation(weeks)

3 63 H-T

dR u

ptak

e (c

pm x

103)

Effects on BM hematopoietic stem cellsSpleen reconstitution – cell populations

Control donorSham-exposed donorRF-exposed donor

Cel

l num

ber

(x10

6 )

150

125

100

75

50

25

0

CD8 CD4 CD19 CD8 CD4 CD19

3 weeks post-BMT 6 weeks post-BMT

CD4 = Helper T cellsCD8 = Cytotoxic T cellsCD19 = B cells

Effects on BM hematopoietic stem cells

Spleen reconstitution - B cell compartment

10

8

6

4

2

03 H-T

dR u

ptak

e (c

pm x

103)

10.50.25

LPS (µg/ml)

10.50.25

LPS (µg/ml)

Control donor

RF-exposed donorSham-exposed donor

Not-irradiated normal mice

B cell proliferation

3 weeks after BMT 6 weeks after BMT

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 MHzRadiation on Mouse Peripheral Lymphocytes”. Radiat Res. 2003 Nov;160(5): 600-605

• Francesca Nasta, Maria Grazia Prisco, Rosanna Pinto, Giorgio AlfonsoLovisolo, Carmela Marino, and Claudio Pioli. Effects of GSM-modulatedRF radiation on B cell peripheral differentiation and antibody production.Radiat. Res, 165 (6): 664-670, 2006.

• M.G. Prisco, F. Nasta, M.M. Rosado, G. A. Lovisolo, C. Marino and C.Pioli, Effects of GSM-Modulated Radiofrequency Electromagnetic Fieldson Mouse Bone Marrow Cells, Radiation Research 170, 803–810 (2008)

Cochlear structure

Human Ear Rat’s CochleaCross section of thewhole cochlea

One single turnSchematic drawing ofthe Organ of Corti

SEM of Guinea Pig’sOrgan of Corti

Cochlea

Healthy Organ of Corti

a) Partial loss of outer hair cells 1 week of GM treatment *

b) Complete loss of outer hair cells4 weeks of GM treatment

* Figures a and b fromForge and Schacht, Audiol Neurootol 2000

DPOAE Recording

Sound

Otoacoustic emissions

Sound

Otoacoustic emissions

Example of DP-gramrecorded from Sprague-

Dawley rats

Acoustic signal emitted by the cochleain response to acoustic stimuli

DPOAE recordings in rats treated with ototoxic drugs

Before treatment After treatment

Animals are injected i.m. with gentamicin150 mg/kg/day for 7 days

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

N2O/O2 (70%-30%)

2 – 3 % alotano

Toxicology and Biomedical ScienceBioelectromagnetism Lab

P. M.

G

AP. M.

Blind procedure

c1

c2

c3D

Attenuator

?

Thermometer

BIOTEC-MEDBioelectromagnetic Group

Black box

EXPOSURE SYSTEM

BIOTEC-MEDBioelectromagnetic Group

Room conditions (50 Hz):

B1= 0.05 microT E1 =1.7 V/mB2= 0.07 microT E2 =1.13V/mB3= 0.08 microT E3 =0.7 V/m

1

2

3

Interference among levels:

A close to zero external field has beenmonitored verifying the level ofinterference generated by the otherarrays (E˜0 V/m).

The same results have been obtainedinside the head of the rat phantoms (seefig: H2O, NaCl, Agar=230g; ε’=75.4,ε’’=23.0) by isotropic sensor.

RF setup:Generator: Motorola 8900 (f= 1745.8 MHz; pw 1212 cod)Bi-directional coupler: Narda 3022 (f=1.0-4.0 GHz)Power meter: HP 438A and two sensor HP 8481HRF divider: Miteq 0.5-4.0 GHz and Sama 0.9-1.9 GHzE probe: Speag ET3DV6 (isotropic)Multimeter: HP three channels

SAR= 1,8W/kg

SAR= 0,5 W/kg

Test (localized exposure): 1.7 Winput at array; measures in phantomat “head” and “body” levels

Experimental room

ENEA Exposure System

¬ 3 set of four loop antennas

¬ EM field: 900 or 1800 MHz, CW orGSM modulated; 2 or 4 W/kg of SAR

¬ Localized exposure (the right ear)

¬ Rats were inserted in perspexindividual jigs

Splitter

Loopantenna

RESULTS I

L1/L2=70/65

L1/L2=70/65

RESULTS II

F2=8346 Hz

Session

131211109876543210

DP

OA

E A

mpl

itude

(dB

SP

L)

50

45

40

35

30

25

20

15

10

5

0

-5-10

GROUP

SHAM

UMTS

F2=9747 Hz

Session

131211109876543210

DP

OA

E A

mpl

itude

(dB

SP

L)

50

45

40

35

30

25

20

15

10

5

0

-5-10

GROUP

SHAM

UMTS

F2=8346 Hz

Session

14131211109876543210

DP

OA

E A

mpl

itude

(dB

SP

L)50

45

40

35

30

25

20

15

10

5

0

-5

-10

-15-20

GROUP

RF+KM

KM

F2=9747 Hz

Session

14131211109876543210

DP

OA

E A

mpl

itude

(dB

SP

L)50

45

40

35

30

25

20

15

10

5

0

-5

-10

-15-20

GROUP

RF+KM

KM

Results

No effect of UMTS exposure was evidenced; ototoxic effect of kanamycin was notmodified by UMTS co-exposure

INSERM Exposure System

¬ RF generator¬ 8 channel divider¬ Holding box¬ Loop antenna¬ Guinea pig¬ PVC stand for antennafixation screw (nylon)

¬ Resting platform for theguinea pig

Array of eight loop antennas in INSERMlaboratories (scheme)

Mean Control LE

0.5 1 2 4 8 16 32-20

-10

0

10

20

30

40 T0 T1 T2

n = 6

tone burst frequency: kHz

AB

R t

hres

hold

: d

B n

RL

Mean LE GM + Exposure

0.5 1 2 4 8 16 32-20

-10

0

10

20

30

40 T0 T1 T2

n = 8

tone burst frequency: kHz

AB

R t

hres

hold

: d

B n

RL

0 bef 1w 2w 3w 4w 1d1w af -15-10

-505

10152025303540

ExposedSham

EMF Exposure

F2 = 6348 Hz

Session

dB SPL

No effects of EM field exposure on ABR measurements in Guinea Pigs

No effects of EM field exposure onDPOAE measurements in rats

Bibliografia e siti web

¬http://web.jrc.ec.europa.eu/emf-net/

Reports on laboratory studies and projects related to effects onBlood Brain Barrier, Auditory system, Behaviour,Cardiovascular system and Nervous System

Effects on immune function and the endocrine system

Reports on cancer- related projects (bioassay, transgenic study,promotion study)

Report on Effects on reproduction and development