Effect of Oral Administration of Methylene Blue MMx® Tablets on Double-Stranded DNA Damage Assessed by γH2AX Analysis of Colon Biopsy Samples- A Single Center, Open Label, Safety, Phase II Study

Background: Colonoscopy with methylene blue (MB) or other dyes (i.e. chromoendoscopy) is a technique that has been shown to improve adenoma detection and characterize colonic surface and polyp pit patterns. However, safety concerns have been raised regarding the use of MB for endoscopic procedures, as some studies have suggested MB can induce DNA damage when exposed to white light (in colorectal cancer cell lines), and in vivo (Barrett's oesophagus and ulcerative colitis biopsy samples), (Davies 2007, Olliver 2003). In the past, measurement of the amount of DNA double strand breaks has been performed with assays such as pulsed field gel electrophoresis and comet assays. Recently, detection of γH2AX has been shown to be considerably more sensitive, efficient, and reproducible compared to the previously reported techniques (Sedelnikova & Bonner 2006) in detection of DNA breaks. Aim: This study investigated if oral administration of Methylene Blue MMX®, followed by white light exposure of the mucosa during colonoscopy, may lead to DNA damage by the detection of γH2A. XMethods: Subjects undergoing 2 colonoscopies 2 weeks apart, for medical reasons, were prospectively enrolled for this study. During the initial standard white light colonoscopy, 5 control biopsies were obtained from the normal appearing mucosa - 1 biopsy each from the 5 bowel regions; cecum, ascending, transverse, descending, sigmoidrectum. The second colonoscopy was preceded by the oral administration of 200 mg Methylene Blue MMX® tablets during the bowel prep time. Five biopsies were once again obtained from the same sites during the second colonoscopy-1 biopsy each in each from the 5 bowel regions; cecum, ascending, transverse, descending, sigmoid-rectum. γH2AX activity in colonic cells of all biopsy specimens taken before and after exposure to MB was evaluated by using γH2AX antibody staining with flow cytometry. Results: A total of 10 subjects were prospectively enrolled; mean age 58.7 years; 6 males. The mean number of days between the 1st and 2nd procedure was 11.4 days (range: 7-14days). All the tested samples did not show any signs of DNA damage and there were no differences between samples before and after MB mmx administration (Figures). Conclusion: In conclusion, , exposure of the colonic mucosa to MB and white light during standard colonoscopy did not show any evidence of DNA damage as evaluated by detection of γH2AX, that has been shown to be a more sensitive, efficient, and reproducible measure of DNA damage.