Detection of double-stranded DNA breaks and apoptosis induced by bleomycin in mouse intestine.

The gastrointestinal tract is exposed to a myriad of mutagens, making the DNA damage response (DDR) essential to maintain intestinal homeostasis. In vivo models to study DDRs are necessary to understand the mechanisms of disease development caused by genetic disorders such as colorectal cancer. A double-stranded break (DSB) in DNA is the most toxic type of DNA damage; it can be induced by either X-rays or chemicals, including anticancer agents. If DSBs in DNA cannot be repaired, cells can die by apoptosis to be removed from tissues. Here, we show that the DDRs observed as the phosphorylation of H2AX (γH2AX) and caspase-3-dependent apoptosis-induction are under critical control in the intestine of C57BL mice that were injected intraperitoneally with bleomycin, a natural glycopeptide used clinically as an antitumor agent. We found a significant increase in γH2AX expression 2-6 hr post-treatment in mouse ileum, cecum, and colon tissues by Western blotting and immunostaining. Apoptotic cells were observed after 6-24 hr by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay and immunofluorescence of active caspase-3. We observed that γH2AX expression and apoptotic cells were distributed in the lower part of the crypt. The experimental protocol described here is a simple procedure that can be used generally as an in vivo intestinal toxicity assay. Our experimental approach provides a useful method for examining the effects of various bioactive compounds on the DDR, which is essential for understanding intestinal homeostasis.