Abstract
BackgroundEtoposide is currently one of the most commonly used antitumor drugs. The mechanisms of action proposed for its antitumor activity are based mainly on its interaction with topoisomerase II. Etoposide effects in transformed cells have been described previously. The aim of the present study was to evaluate the genotoxic effects of this drug in non-transformed whole blood cells, such as occurs as collateral damage induced by some chemotherapies.MethodsTo determine etoposide genotoxicity, we employed Comet assay in two alkaline versions. To evaluate single strand breaks and delay repair sites we use pH 12.3 conditions and pH >13 to evidence alkali labile sites. With the purpose to quantified apurinic or apyrimidine (AP) sites we employed a specific restriction enzyme. Etoposide effects were determined on whole blood cells cultured in absence or presence of phytohemagglutinin (PHA) treated during 2 and 24 hours of cultured.ResultsAlkaline (pH > 13) single cell gel electrophoresis (SCGE) assay experiments revealed etoposide-induced increases in DNA damage in phytohemaglutinine (PHA)-stimulated blood and non-stimulated blood cells. When the assay was performed at a less alkaline pH, 12.3, we observed DNA damage in PHA-stimulated blood cells consistent with the existence of alkali labile sites (ALSs). In an effort to elucidate the molecular events underlying this result, we applied exonuclease III (Exo III) in conjunction with a SCGE assay, enabling detection of DNA-AP sites along the genome. More DNA AP-sites were revealed by Exo III and ALSs were recognized by the SCGE assay only in the non-stimulated blood cells treated with etoposide.ConclusionOur results indicate that etoposide induces DNA damage specifically at DNA-AP sites in quiescent blood cells. This effect could be involved in the development of secondary malignancies associated with etoposide chemotherapy.
Highlights
Etoposide is currently one of the most commonly used antitumor drugs
Single cell gel electrophoresis single cell gel electrophoresis (SCGE) at pH >13, which reveals single-strand breaks (SSBs), delayed repair sites (DRSs) and alkali labile sites (ALSs), revealed a dose-dependent effect of etoposide treatment (2.07, 20.7 and 207 μM) on cells treated for 2 h and 24 h (Figure 1A, B respectively)
SCGE assay performed at pH 12.3, which detect SSBs and DRSs, but not ALSs, showed genotoxic effects in the PHA-stimulated whole blood cells only at both treatment durations (Figure 2)
Summary
Etoposide is currently one of the most commonly used antitumor drugs. The mechanisms of action proposed for its antitumor activity are based mainly on its interaction with topoisomerase II. Rather than blocking the activity of this essential enzyme, etoposide kills cells by increasing the concentration of topoisomerase II-DNA cleavage complexes [7,12,14,15,16] This action converts topoisomerase II into a potent cellular toxin that fragments the genome. Etoposide has been deemed a topoisomerase II poison, distinct from drugs that inhibit the overall catalytic activity of an enzyme [7,12,14,15,16,17,18] It has been known for more than a decade that etoposide stabilizes topoisomerase II-associated DNA breaks, thereby abolishing the ability of the enzyme to ligate cleaved nucleic acid molecules [7,12,16,19,20,21]. When etoposide interacts with topoisomerase IIα, it traps the enzyme in a covalently bound form with its DNA substrate [5,22]
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