Genotoxic effect of cigarette smoking detected by comet assays using high throughput scanning of lymphocytes
aRadGenomics Project, Frontier Research Center, National Institute of Radiological Sciences, , bResearch Center for Charged Particle Therapy, National Institute of Radiological Sciences, , cChiba Cancer Center, , dToyonaka Municipal Hospital, , eKawakami Breast Thyroid Clinic
Aim To examine the genotoxic effect of cigarette smoking on DNA damage among patients with breast cancer, we applied alkaline comet assay as genotoxicity test in a high throughput fashion. Materials and methods Between October 2001 and January 2003, 302 female patients with breast cancer who underwent radiotherapy after breast conserving surgery were recruited from 5 collaborating Japanese institutions. Of these, we recruited 50 patients whose clinical information was complete and who were followed up for over 6 months after receiving uniform doses of radiotherapy. Among 227 healthy volunteers, 66 healthy women also participated in the study. Physicians in collaborating institutions obtained the medical history, family history of cancer, complications of diabetes mellitus, numbers of children and clinical details of the patients, including information about the tumor, therapy, and side effects of radiation therapy from patient records after review by each institutional ethical committee. Self-administered questionnaires given to the healthy volunteers revealed smoking history, alcohol consumption, family history of breast cancer and medical history. Adverse effects on the skin after radiotherapy for breast conserving surgery were clinically scored according to the RTOG/EORTC system. The alkaline comet assay measured X-ray induced DNA damage, quantified by the mean tail moment as a parameter of initial damage (ID) and residual damage (RD) at 15 min after irradiation, in individual B lymphocyte cell lines established from the peripheral blood of 71 patients with breast cancer and of 70 healthy controls. Two hundred and one cells per area on the slides were evaluated by fluorescence microscopy (Axioplan 2 Imaging, Carl Zeiss, Tokyo, Japan) and image analysis software (Metafar CometScan and Comet Imager, MetaSystems, Altlussheim, Germany). To estimate the discriminative ability of the comet assay as a predictor of radiosensitivity, we constructed receiver operator characteristic curves (ROC). A value for RD from ROC curves was 16 %. Thus, a cut- off RD level of 16 % for the alkaline comet assay might distinguish radiosensitive patients. Results: Residual damage was significantly higher in patients with grades 2+3 than in those with grades 0+1, or in age-matched healthy controls. The inter-individual variation in RD was large among breast cancer patients with grades 2+3. However, there was no correlation between cigarette smoking and genotoxicic effects, and this approach did not reveal significant differences between smokers and non-smokeres. Conclusions: Although more data are needed for a final concusion, the obvious inconsistency in the detection of smoking-related genotoxic effects of smoking-related genotoxic effects indicate some limitations of the comet assay with regard to the detection of DNA-damage induced by environmental mutagens in peripheral blood cells.
Paper presented at the International Symposium on Predictive Oncology and Intervention Strategies; Nice, France; February 7 - 10, 2004; in oral session 996 (Genetic & environmental interactions).