Key words: BCL-2/J H translocation; biomarker; healthy; lymphocytesThe incidence of Non-Hodgkin's lymphoma (NHL) presents a 6 -8% increase since the 1970s for largely unknown reasons, even if AIDS in the USA and modification of diagnosis could play a role. 1,2 NHL are among the tumor localizations for which an increased risk has been associated with farm-related activities, including use of pesticides. 3 With the aim to further assess the risk of cancer associated to occupational exposure to pesticides, we are currently conducting an epidemiological and biological prospective study among farmers in Calvados (Normandy, France) using various biomarkers. Considered as an early step in lymphomagenesis, the translocation t(14;18)(q32;q21) between the BCL-2 protooncogene and the J H immunoglobulin gene region 4,5 will be used to assess the effect of pesticide exposure. Occurring in 85% of human follicular lymphoma and in 20% of diffuse large cell lymphoma, 6 it is also present in peripheral blood lymphocytes of healthy individuals. [7][8][9][10][11][12] Before studying the effect of pesticide exposure among farmers, it is necessary to evaluate the influence of potential confounding factors. Pesticide use follows a temporal cycle with potential effects on biomarkers of season-related environmental factors. We determined the characteristics of BCL-2/J H rearrangements in peripheral blood lymphocytes collected in winter (lowest pesticide exposure period) and at the end of spring (peak pesticide exposure period) from healthy individuals living in the same region and not involved in farming or pesticide handling.
MATERIAL AND METHODS
Study populationPeripheral blood lymphocytes were obtained, after authorization of the local ethical committee, from 33 healthy men recruited among white collar workers, laboratory or radiology staff in January 1999 and at the end of spring (May 1999). Information on age, workplace and smoking habits were recorded.
Sample preparationMononuclear cells were isolated from peripheral blood by Ficoll-Hypaque gradient centrifugation. The cells were washed 2ϫ in PBS and then the samples were then cryopreserved in 20% FCS and 10% DMSO. DNA was isolated using standard procedure. 13 Briefly, cells were thawed at 37°C and washed in PBS. After centrifugation, they were resuspended in TEN-SDS 10% (10 mM Tris-HCl pH ϭ 7.4, 25 mM EDTA, 100 mM NaCl) with 1 mg/ml of proteinase K and incubated overnight at 56°C. DNA was extracted 3ϫ in phenol/chloroform/isoamylalcohol (25:24:1) and precipitated by sodium acetate 0.3 M/absolute ethanol at Ϫ20°C. The concentration and purity of the DNA was determined by spectrophotometry at 260 and 280 nm.