To achieve this objective two studies are planned to take place in the next three years

(1) TPA (12-O-tetradecanoyl-phorbol-13-acetate) promotion study;
(2) MF promotion study.

The first study will both validate the applicability of the two-stage theory of carcinogenesis to neurogenic tumors in the Fischer rats and quantitate the promoting effect of TPA on macrotumors, microtumors and early proliferative lesions. We are also planing to identify stable and reproducible changes that play an important role in ENU-tumor promotion at the biochemical (ornithine decarboxylase activity) and molecular (dominant oncogene and tumor suppressor gene expression) levels. This quantitative study is crucial to establish a laboratory baseline for the evaluation of brain-tumor promotion in Fischer rats so as to allow the estimation of the bio- equivalence of MF promoting effect. In the MF promotion study, in addition to establishing whether 60 Hz MF could promote neurogenic tumors in the Fischer rats, this study will quantitate the promoting effect of MF on macrotumors, microtumors and early proliferative lesions.

The present research program will provide direct answers to several important questions concerning the possible tumor promoting effect of 60 Hz linear, sinusoidal, continuous-wave MF:

(1) Can chronic exposure to MF promote the development of neurogenic tumors initiated by a chemical carcinogen (ENU) in Fischer rats?
(2) In the case of tumor promotion, is there a dose-response effect (2, 20, 200, 2000 µT)?
(3) What is the bio-equivalence to a classical promoter, TPA ?
(4) Can we identify stable and reproducible changes that play an important role in tumor promotion?

Accordingly, the aims for our efforts is to define both the robustness of MF promoting effect and determine the biological significance of such an effect.

THE MAGNETIC FIELDS BRAIN TUMOUR PROMOTION STUDY: Eight groups of 50 female F1 rats will be used in this study. This is based on two considerations: First, sample size calculation determining that 50 animals per dose group will provide 90% power to detect an increase in cancer incidence of 25%-30% Second, we have elected to use only females in this study mainly because data generated from our initial study (ENU #P90) have shown no statistically significant difference in ENU-induced tumors between gender. Moreover, because of the big difference in size between males and females at 14 months of age, only 1 male could be housed in our custom made cages. Pregnant females will be listed in numerical order based on Day 1 of gestation and assigned a random number corresponding to ENU (treatment or controls) and field intensity (color code). This is a computer assisted randomization program developed specifically for these MF studies. In the selection of the 50 females we will use the largest possible number of litter. Ideally we will take only two females from each litter.

FIELDS SPECIFICATIONS: Animals will be exposed to 60 Hz linear, sinusoidal, continuous-wave MF of different intensities ranging from <0.02 µT (Sham exposure) to 2, 20, 200, and 2000 µT. Exposure will be performed 20 hours per day (10 hours in the dark and 10 hours in the light). It will start in the prenatal period (18th day of gestation) and will continue until sacrifice (60 weeks of age). Animals will be assigned at random to treatment and control groups. This study will be performed under stringently controlled environmental conditions with a constant light cycle (12/12 hrs dark/light cycle).

ENVIRONMENTAL MONITORING: Environmental monitoring tests will be conducted in both holding and exposure rooms, these tests will include: (1) temperature; (2) humidity; (3) air flow; (4) photo period; (5) noise; (6) room access and (7) AC fields. We will also monitor ammonia and CO2 levels after housing animals in units for a period of seven days. Moreover, brief animal exposures to magnetic fields will help monitor the effect of magnetic fields on: (a) exposure environment; (b) stray fields from the exposure units and (c) stability of the fields.

Blinding and Field Rotation: In order to limit the effect of any potential environmental confounder in this study, we have elected to develop a rotation strategy that is completely randomized. This rotation strategy is based on the colour light coding provided in the exposure room to designate the exposure level assigned to each of the five groups of modules. The lights are red, green, yellow, blue and white. The position of the module inside the room will change. In this rotation, care has been taken so as not to have Sham (0) and 2 µT fields besides 2000 µT.

THE MAGNETIC FIELDS BRAIN TUMOUR PROMOTION STUDY: This study was started earlier than originally planed in order to shorten our study period and to be able to complete the study and the final reporting before the end of 1997. Animals were received on the 21st of June, 1995 and they are now in the period of acclimatization. Exposure will start August 1995 and will end in October 1996.

Immunohistochemical Profile of Early Stages of Neoplasia: We have purchased a panel of antibodies from Boehringer-Mannheim Biochemica. This panel of antibodies has been evaluated on a number of permanent cell lines of neurogenic origin and is presently been studied on the normal brain of adult rats (frozen sections and paraffin embedded materials).

Effect on Ornithine Decarboxylase Activity: An in vivo/in vitro model was recently developed in our laboratory in order to study ODC activity and expression of ODC protein in brain tumour cells. Pregnant females are injected with ENU (20 mg/kg body weight) and F1 offsprings are sacrificed three days after birth. The brains are removed and cells seeded into petri dishes. These cells are exposed to different concentrations of TPA. The antigenic expression of ODC is evaluated using the cytofluorometer and anti-ODC antibody.

Effect on Dominant Oncogenes and Tumor Suppressor Genes: We have elected to start this section by studying the expression and deletion of p53 gene mainly because deletions of p53 could play a role in the genesis and progression of human gliomas. Two different monoclonal antibodies against p53 oncogenes were recently purchased from Oncogene science (Ab-5 recognises the wild type and Ab-3, the mutated type). We are also developing the methods and technologies of PCR SSCP (single-strand conformation polymorphism): These 2 primers will be used in these studies:

Reverse: 5'-TCC-TCT-GTC-CGA-CGG-TCT-CTC- 3'
Forward: 5'-GTG-CAG-TTG-TGG-GTC-ACC-TCC- 5'

Mandeville R. Mercier G, Oth D, Descoteaux J-P, Lis M et Franco E. "A unique model for the study of 60 Hz magnetic fields on brain tumor promotion in Fischer rats." The Annual Review of Research on Biological Effects of Electric and Magnetic Fields from the Generation, Delivery and Use of Electricity. Albuquerque, New Mexico, November 6-10, 1994.