Assessment of DNA double-strand breaks and gammaH2AX induced by the topoisomerase II poisons etoposide and mitoxantrone

Abstract

Double-strand breaks (DSBs) are highly deleterious DNA lesions as they lead to chromosome aberrations and/or apoptosis. The formation of nuclear DSBs triggers phosphorylation of histone H2AX on Ser-139 (defined as gammaH2AX), which participates in the repair of such DNA damage. Our aim was to compare the induction of gammaH2AX in relation to DSBs induced by topoisomerase II (TOPO II) poisons, etoposide (ETOP) and mitoxantrone (MXT), in V79 cells. DSBs were measured by the neutral comet assay, while gammaH2AX was quantified using immunocytochemistry and flow cytometry. Stabilized cleavage complexes (SCCs), lesions thought to be responsible for TOPO II poison-induced genotoxicity, were measured using a complex of enzyme-DNA assay. In the case of ETOP, a no observed adverse effect level (NOAEL) and lowest observed effect level (LOEL) for genotoxicity was determined; gammaH2AX levels paralleled DSBs at all concentrations but significant DNA damage was not detected below 0.5 microg/ml. Furthermore, DNA damage was dependent on the formation of SCCs. In contrast, at low MXT concentrations (0.0001-0.001 microg/ml), induction of gammaH2AX was not accompanied by increases in DSBs. Rather, DSBs were only significantly increased when SCCs were detected. These findings suggest MXT-induced genotoxicity occurred via at least two mechanisms, possibly related to DNA intercalation and/or redox cycling as well as TOPO II inhibition. Our findings also indicate that gammaH2AX can be induced by DNA lesions other than DSBs. In conclusion, gammaH2AX, when measured using immunocytochemical and flow cytometric methods, is a sensitive indicator of DNA damage and may be a useful tool in genetic toxicology screens. ETOP data are consistent with the threshold concept for TOPO II poison-induced genotoxicity and this should be considered in the safety assessment of chemicals displaying an affinity for TOPO II and genotoxic/clastogenic effects.

Fig. 1

Formation of SCCs in V79 cells treated with TOPO II poisons: (A) ETOP and (B) MXT.

Fig. 2

Induction of DSBs (measured as % tail DNA; solid line) by ETOP in V79 cells as assessed by the neutral comet assay (control; 2.7±0). * P < 0.05. RCC (dashed line) are also shown as a measure of cytotoxicity (control; 100%).

Fig. 3

Induction of DSBs (measured as % tail DNA; solid line) by MXT in V79 cells as assessed by the neutral comet assay (control; 2.5±0.9). *P < 0.05, **P < 0.01. RCC (dashed line) are also shown as a measure of cytotoxicity (control; 100%).

Fig. 4

γH2AX mean fluorescence (±S.E.M.) in V79 cells treated with ETOP (control; 111±5) or MXT (control; 123±17).