Detection of double-stranded DNA breaks and apoptosis induced by bleomycin in mouse intestine
Togo Ikuta1, Aki Koike2, Manabu Koike2
1Research Institute for Clinical Oncology, Saitama Cancer Center.
Abstract:
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.
Insights
This study demonstrates how DNA damage response and apoptosis occur in the mouse intestine after bleomycin treatment. This provides a new in vivo intestinal toxicity assay for studying DNA damage and repair.
Area of Science:
- Gastrointestinal health
- DNA damage response
- Cancer research
Background:
- The gastrointestinal tract faces constant mutagen exposure, necessitating robust DNA damage response (DDR) for intestinal homeostasis.
- Understanding DDR mechanisms is crucial for diseases like colorectal cancer, often studied using in vivo models.
- Double-stranded DNA breaks (DSBs) are highly toxic, leading to cell death via apoptosis if unrepaired.
Purpose of the Study:
- To investigate the in vivo DNA damage response (DDR) and apoptosis in the mouse intestine following bleomycin administration.
- To establish a reliable in vivo intestinal toxicity assay for evaluating bioactive compounds' effects on DDR.
Main Methods:
- C57BL mice were intraperitoneally injected with bleomycin, an antitumor agent.
- Western blotting and immunostaining were used to detect γH2AX expression (a DDR marker) in ileum, cecum, and colon tissues.
- Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay and active caspase-3 immunofluorescence identified apoptotic cells.
Main Results:
- Significant increase in γH2AX expression observed 2-6 hours post-bleomycin treatment in mouse intestinal tissues.
- Apoptotic cells, detected via TUNEL and active caspase-3, appeared 6-24 hours after treatment.
- Both γH2AX expression and apoptotic cells were localized to the lower crypt regions.
Conclusions:
- Bleomycin induces DNA damage response and apoptosis in the mouse intestine in a time- and location-dependent manner.
- The described protocol serves as a simple and effective in vivo intestinal toxicity assay.
- This method facilitates the study of various compounds' impact on DDR, crucial for understanding intestinal homeostasis and disease.
