Cytotoxicity of Tirapazamine (3-Amino-1,2,4-benzotriazine-1,4-dioxide)-Induced DNA Damage in Chicken DT40 Cells

Abstract

Tirapazamine (TPZ) is an anticancer drug with highly selective cytotoxicity toward hypoxic cells. TPZ is converted to a radical intermediate under hypoxic conditions, and this intermediate interacts with intracellular macromolecules, including DNA. TPZ has been reported to indirectly induce DNA double-strand breaks (DSBs) through the formation of various intermediate DNA lesions under hypoxic conditions. Although the topoisomerase II-DNA complex has been identified as one of these intermediates, other lesions have not yet been defined. In order to obtain a deeper understanding of the mechanisms responsible for the selective cytotoxicity of TPZ toward hypoxic cells, its cellular sensitivity was systematically examined with genetically isogenic DNA-repair-deficient mutant DT40 cell lines. Our results showed that tdp1, tdp2, parp1, and aptx1 cells displayed hypersensitivity to TPZ only under hypoxic conditions. These results strongly suggest that the accumulation of the topoisomerase I-trapped DNA complex, topoisomerase II-trapped DNA complex, and abortive ligation products with 5'-AMP are the potential causes of TPZ-induced hypoxic cell death. Furthermore, our genetic analysis revealed that under normoxic conditions (as well as hypoxic conditions), TPZ exhibited significant cytotoxicity toward cell lines deficient in homologous recombination, nonhomologous end joining, base excision repair, and translesion synthesis. Ascorbic acid, a radical scavenger, suppressed TPZ-induced cytotoxicity toward normoxic cells. These results suggest the involvement of oxidative DNA damage and DSBs produced by reactive oxygen species generated from superoxide, a byproduct of the oxidation of TPZ radical intermediates in normoxic cells. Collectively, our results demonstrate that TPZ induces oxidative DNA damage under normoxic and hypoxic conditions and selectively introduces abortive topoisomerase-DNA complexes and unligatable DNA ends under hypoxic conditions.

Macrophage‐Mediated Delivery of Hypoxia‐Activated Prodrug Nanoparticles

“…Despite its increased activity in hypoxic environments, TPZ still produces some cytotoxic effects in normoxia as a consequence of superoxide radical production by the back‐oxidation of the TPZ radical intermediates . While these effects may pose little threat at lower concentrations, the drug concentrations achieved inside carrier macrophages could be as high as 2.0 × 10 4 µg mL −1 , which gives superoxide radicals the potential to cause premature toxicity to the carrier cells (Table S2, Supporting Information).…”

“…Upon entering cells, a cytochrome P450 reductase‐driven one‐electron reduction converts TPZ to a radical intermediate. In normoxic conditions, the oxygen rapidly reoxidizes the TPZ radical to its original form . However, low oxygen conditions stabilize this radical long enough to allow for its protonation, leading to the production of hydroxyl and benzotriazinyl radicals, which result in extensive DNA damage .…”

A Simple Computational Tool for Accurate, Quantitative Prediction of One–Electron Reduction Potentials of Hypoxia–Activated Tirapazamine Analogues

“…Of these compounds, tirapazamine (TPZ; Fig.1) is a benzotriazine di-Noxide (BTDO) that has been subjected to extensive preclinical and clinical studies as a radiosensitizer [10]. TPZ requires a single-electron bioreductive activation [11][12][13] to produce free radicals which in turn induce single and double strand breaks in DNA, exploiting the hypoxic microenvironment to be selectively cytotoxic to solid hypoxic tumors ( Fig. 1) [13][14][15][16][17].…”

Selective cytotoxicity of the anti-diabetic drug, metformin, in glucose-deprived chicken DT40 cells

“…In addition, the microenvironment of tumors may modify the activities of other drugs. We previously reported that tirapazamine (3-amino-1,2,4-benzotriazine 1,4-dioxide), which is a well-known hypoxic cytotoxic drug, preferentially induced lethal DNA damage under hypoxic conditions [ 9 ]. Thus, the selective toxicity of metformin may be attributable to the tumor-specific microenvironment.…”