DNA-AP sites generation by etoposide in whole blood cells.
BACKGROUND
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. 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.


METHODS
To 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.


RESULTS
Alkaline (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.


CONCLUSIONS
Our 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.
Rojas
Emilio
E
Departamento de Medicina Genómica y Toxicología Ambiental Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México D,F, C,P, 04510, México. emilior@servidor.unam.mx
Mussali
Patricia
P
Tovar
Efrain
E
Valverde
Mahara
M
D000972:Antineoplastic Agents, Phytogenic / Q000633:toxicity*; D001773:Blood Cells / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D020552:Comet Assay; D004247:DNA / Q000187:drug effects*; D004249:DNA Damage / Q000187:drug effects*; D004586:Electrophoresis; D005047:Etoposide / Q000633:toxicity*; D006801:Humans
D000972:Antineoplastic Agents, Phytogenic; D005047:Etoposide; D004247:DNA