2 research outputs found
Istraživanje genotoksiÄnih uÄinaka irinotekana na ljudskim limfocitima periferne krvi primjenom razliÄitih 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 āpoisoningā 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 razliÄitih biomarkera u uvjetima in vitro istraženi su genotoksiÄni uÄinci terapijske koncentracije irinotekana (350 mg/m2) na limfocite periferne krvi. Razine primarnih oÅ”teÄenja 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 oÅ”teÄenja DNA procijenjene su i s pomoÄu testa izmjena sestrinskih kromatida, a moguÄi uÄinci tretmana na progresiju mitotiÄkog ciklusa istraženi su analizom proliferacijske kinetike limfocita. Utvr|eno je da je postotak apoptoza u svim vremenima uzorkovanja i analize bio veÄi od postotka nekroza. Pozitivni rezultati dobiveni s obje modifikacije komet testa pokazuju da se u limfocitnoj DNA nakon tretmana irinotekanom inducira mnoÅ”tvo jednolanÄanih i dvolanÄanih lomova. Dinamika nastanka oÅ”teÄenja uoÄena primjenom obje modifikacije komet testa, jasno upuÄuje na disfunkciju enzima topoizomeraze I, koje se navodi kao glavni mehanizam citotoksiÄnosti irinotekana. Nakon tretmana irinotekanom uoÄili smo gotovo sedmerostruki porast uÄestalosti SCE u izloženim limfocitima u odnosu na kontrolu, Å”to upozorava na poremeÄenu funkciju topoizomeraze u S-fazi. Na osnovi rezultata zakljuÄujemo da irinotekan uzrokuje zastoj proliferacije stanica u prvom mitotskom ciklusu in vitro. Dobiveni rezultati pokazuju da terapijske koncentracije irinotekana uzrokuju znaÄajan porast oÅ”teÄenja DNA i kromosoma u ljudskim limfocitima periferne krvi. BuduÄi da su uÄinci irinotekana u uvjetima in vivo znatno pojaÄani metaboliÄkom pretvorbom u aktivni metabolit SN-38, možemo pretpostaviti da su razine oÅ”teÄenja DNA u aktivno dijeleÄim stanicama u pacijenata lijeÄenih primjenom irinotekana znaÄajno viÅ”e nego one utvr|ene u uvjetima in vitro. Rezultati upuÄuju i na važnost istraživanja biomarkera u ne-tumorskim stanicama pacijenata koji su lijeÄeni primjenom kemoterapije jer takvi biomarkeri mogu biti dobri pretkazatelji u otkrivanju osjetljivih populacija pacijenata s nestabilnim genomom u kojih je prisutan veÄi rizik za razvoj sekundarnih karcinoma
Istraživanje genotoksiÄnih uÄinaka irinotekana na ljudskim limfocitima periferne krvi primjenom razliÄitih 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 āpoisoningā 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 razliÄitih biomarkera u uvjetima in vitro istraženi su genotoksiÄni uÄinci terapijske koncentracije irinotekana (350 mg/m2) na limfocite periferne krvi. Razine primarnih oÅ”teÄenja 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 oÅ”teÄenja DNA procijenjene su i s pomoÄu testa izmjena sestrinskih kromatida, a moguÄi uÄinci tretmana na progresiju mitotiÄkog ciklusa istraženi su analizom proliferacijske kinetike limfocita. Utvr|eno je da je postotak apoptoza u svim vremenima uzorkovanja i analize bio veÄi od postotka nekroza. Pozitivni rezultati dobiveni s obje modifikacije komet testa pokazuju da se u limfocitnoj DNA nakon tretmana irinotekanom inducira mnoÅ”tvo jednolanÄanih i dvolanÄanih lomova. Dinamika nastanka oÅ”teÄenja uoÄena primjenom obje modifikacije komet testa, jasno upuÄuje na disfunkciju enzima topoizomeraze I, koje se navodi kao glavni mehanizam citotoksiÄnosti irinotekana. Nakon tretmana irinotekanom uoÄili smo gotovo sedmerostruki porast uÄestalosti SCE u izloženim limfocitima u odnosu na kontrolu, Å”to upozorava na poremeÄenu funkciju topoizomeraze u S-fazi. Na osnovi rezultata zakljuÄujemo da irinotekan uzrokuje zastoj proliferacije stanica u prvom mitotskom ciklusu in vitro. Dobiveni rezultati pokazuju da terapijske koncentracije irinotekana uzrokuju znaÄajan porast oÅ”teÄenja DNA i kromosoma u ljudskim limfocitima periferne krvi. BuduÄi da su uÄinci irinotekana u uvjetima in vivo znatno pojaÄani metaboliÄkom pretvorbom u aktivni metabolit SN-38, možemo pretpostaviti da su razine oÅ”teÄenja DNA u aktivno dijeleÄim stanicama u pacijenata lijeÄenih primjenom irinotekana znaÄajno viÅ”e nego one utvr|ene u uvjetima in vitro. Rezultati upuÄuju i na važnost istraživanja biomarkera u ne-tumorskim stanicama pacijenata koji su lijeÄeni primjenom kemoterapije jer takvi biomarkeri mogu biti dobri pretkazatelji u otkrivanju osjetljivih populacija pacijenata s nestabilnim genomom u kojih je prisutan veÄi rizik za razvoj sekundarnih karcinoma