Istraživanje genotoksičnih učinaka irinotekana na ljudskim limfocitima periferne krvi primjenom različitih biomarkera u uvjetima in vitro

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.Primjenom različitih biomarkera u uvjetima in vitro istraženi su genotoksični učinci terapijske koncentracije irinotekana (350 mg/m2) na limfocite periferne krvi. Razine primarnih oštećenja DNA u limfocitnoj DNA i dinamika njihovog popravka istraženi su primjenom komet testa u alkalnim i neutralnim uvjetima. Preživljenje limfocita i indukcija apoptoze nakon izlaganja stanica irinotekanu istraženi su istodobnom primjenom fluorescencijskog bojenja etidij-bromidom i akridin oranžom. Razine oštećenja DNA procijenjene su i s pomoću testa izmjena sestrinskih kromatida, a mogući učinci tretmana na progresiju mitotičkog ciklusa istraženi su analizom proliferacijske kinetike limfocita. Utvr|eno je da je postotak apoptoza u svim vremenima uzorkovanja i analize bio veći od postotka nekroza. Pozitivni rezultati dobiveni s obje modifikacije komet testa pokazuju da se u limfocitnoj DNA nakon tretmana irinotekanom inducira mnoštvo jednolančanih i dvolančanih lomova. Dinamika nastanka oštećenja uočena primjenom obje modifikacije komet testa, jasno upućuje na disfunkciju enzima topoizomeraze I, koje se navodi kao glavni mehanizam citotoksičnosti irinotekana. Nakon tretmana irinotekanom uočili smo gotovo sedmerostruki porast učestalosti SCE u izloženim limfocitima u odnosu na kontrolu, što upozorava na poremećenu funkciju topoizomeraze u S-fazi. Na osnovi rezultata zaključujemo da irinotekan uzrokuje zastoj proliferacije stanica u prvom mitotskom ciklusu in vitro. Dobiveni rezultati pokazuju da terapijske koncentracije irinotekana uzrokuju značajan porast oštećenja DNA i kromosoma u ljudskim limfocitima periferne krvi. Budući da su učinci irinotekana u uvjetima in vivo znatno pojačani metaboličkom pretvorbom u aktivni metabolit SN-38, možemo pretpostaviti da su razine oštećenja DNA u aktivno dijelećim stanicama u pacijenata liječenih primjenom irinotekana značajno više nego one utvr|ene u uvjetima in vitro. Rezultati upućuju i na važnost istraživanja biomarkera u ne-tumorskim stanicama pacijenata koji su liječeni primjenom kemoterapije jer takvi biomarkeri mogu biti dobri pretkazatelji u otkrivanju osjetljivih populacija pacijenata s nestabilnim genomom u kojih je prisutan veći rizik za razvoj sekundarnih karcinoma