Original scientific paper
A multi-biomarker approach to study the genotoxic effects of irinotecan on human peripheral blood lymphocytes 'in vitro'
Nevenka Kopjar
; Institute for Medical Research and Occupational Health, Zagreb, Croatia
Nina Pleša
; Institute for Medical Research and Occupational Health, Zagreb, Croatia
Snježana Ramić
; Institute for Medical Research and Occupational Health, Zagreb, Croatia
Vesna Pavlica
; University Hospital for Tumors, Zagreb, Croatia
Marija Gamulin
; University Hospital Zagreb, Zagreb, Croatia
Abstract
In the present study a multi-biomarker approach was used to evaluate genotoxic effects of irinotecan administered in vitro in its therapeutic dose (350 mg/m2) on non-target cells, peripheral blood lymphocytes. The levels of primary DNA damage in lymphocyte genome and the dynamics of its removal were assessed using the alkaline and neutral comet assay. Lymphocyte viability and the induction of apoptosis following exposure to irinotecan were studied by simultaneous use of a fluorescent assay with ethidium bromide and acridine orange. The levels of residual DNA damage were assessed by SCE assay, while the possible influences of treatment on the progression through the mitotic cycles were studied by analyzing lymphocyte proliferative kinetics. We observed that the percentage of apoptotic cells was higher as compared to necrotic ones in all time-points when irinotecan-treated samples were analyzed. Positive results obtained using both modifications of the comet assay indicate that in lymphocyte DNA following treatment with irinotecan a lot of single and double strand breaks are induced. Dynamics of damage infliction as observed both in alkaline and neutral modification of the comet assay clearly reflects the ’poisoning’ of the topoisomerase I, reported as the main mechanism of the irinotecan cytotoxicity. After treatment with irinotecan we observed an almost 7-fold increase of SCE frequency in exposed as compared to untreated lymphocytes that was obviously caused by topoisomerase poisoning in S-phase. Considering the results obtained we can conclude that irinotecan caused a delay of in vitro cell proliferation in first mitotic cycle. Despite their limitations, the results of our study indicate that irinotecan in its therapeutic concentrations is able to cause significant amount of primary and residual DNA damage in human peripheral blood lymphocytes. We could assume that the actual levels of DNA damage produced in actively divided cells of patients treated with irinotecan are much higher as compared to those estimated in vitro, since DNA damaging potential of irinotecan in vivo is up to one thousand times higher due to effectively conversion to its more potent metabolite SN-38. Our results point to the significance of biomarker studies in non-target cells of cancer patients after successful chemotherapy since they could be a good predictive factor to detect sensitive subpopulations of patients with genome instability that have an increased risk for developing of secondary malignancies.
Keywords
irinotecan; lymphocytes; genotoxicity; DNA damage and repair
Hrčak ID:
281571
URI
Publication date:
20.12.2004.
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