DSBs are the most lethal type of DNA damage and result from a variety of events, including ionizing radiation, ultraviolet light, drugs, and inflammatory attacks . γ-H2AX foci have been reported to be produced very soon after treatment with chemicals. Because this damage is undetectable by the neutral comet assay, γ-H2AX is considered the most sensitive and specific marker of DNA damage caused by various agents . The present study revealed that the expression of γ-H2AX was more prominent in gallbladder epithelium specimens from PBM patients in comparison to controls, indicating that DSBs occurred more frequently in PBM. In PBM, pancreatic juice is constantly refluxed into the biliary duct to produce activated pancreatic enzymes and secondary bile acid. Since these substances are extremely harmful to the biliary epithelium, the tissue is subjected to long-term inflammatory damage  . Chronic inflammation can produce active oxygen and nitrogen, which cause cell cycle acceleration, DNA damage, and ultimately DSBs  . In a previous study, the immunohistochemical expression of 8-hydroxy-2’-deoxyguanosine, a marker of oxidative damage, confirmed that oxidative injury occurred more prominently in the gallbladder epithelium of subjects with PBM than in those without . In addition, several reports have used immunohistochemical methods to describe the proliferative hyperactivity of the mucosal epithelium in PBM    . Ki-67, a common marker of cell proliferation activity, was reportedly overexpressed in the gallbladder epithelium of PBM patients. Thus, the longstanding inflammatory injury in PBM induces various undesirable cell changes, such as oxidative stress and cell proliferation, which elicit repeated DSBs and other serious DNA damage.
In our study, the expression of γ-H2AX was significantly more prominent in the background epithelium of carcinoma in PBM patients in comparison to non-carcinoma cases, indicating that DSBs exist more abundantly in the PBM mucosa surrounding cancer. The background mucosa of cancers is suspected to be more susceptible to oncogenic transformation. The present study showed a close association between DSB formation and PBM peri-cancerous regions, suggesting that a DSB increase could contribute to carcinogenesis in PBM. PBM patients have a reported 200-fold increased risk of biliary duct cancer in comparison to the general population . In a nationwide Japanese survey of 2561 adult PBM patients , biliary tract cancers were found in 21.6% of patients with biliary dilation and in 42.4% of those without. Specifically, bile duct and gallbladder carcinomas were respectively detected in 6.9% and 13.4% of PBM patients with biliary dilation and in 3.1% and 37.4% of those without. The incidence of gallbladder cancer was notably high in PBM patients. Genomic instability is a common characteristic of most cancers and plays a critical role in carcinogenesis . DSBs have been related to the early stage of carcinogenesis in various cancers, and tissues exposed to DSBs are suspected as precancerous lesions . Chronic inflammation of the gallbladder epithelium caused by refluxed pancreatic juice induces DNA damage in PBM patients. In the present study, a large amount of DSBs were specifically detected in the gallbladder mucosa surrounding cancers, implying that DSBs may be related to the early process of gallbladder cancer development.
This investigation immunohistochemically evaluated the expression of p53 as a representative tumor suppressor mechanism in the PBM mucosa. However, no cases exhibited detectable p53 expression. A human cell line experiment  found that most γ-H2AX foci disappeared within 24 hours after irradiation, indicating that the DDR is immediately activated for DNA repair of DSBs in the nucleus. Indeed, the DDR is an essential protective mechanism against malignant cell transformation  , and its failure to repair DSBs leads to cancer development. Several studies have found that while p53 overexpression was frequently noted in the PBM gallbladder cancerous epithelium, it was largely negative noncancerous lesions, in agreement with our findings  . The mutation of p53 is widely recognized as a late event in the adenoma-carcinoma sequence of colorectal cancer. Similarly, in gallbladder cancer with PBM, p53 mutation may be a relatively late event in the carcinogenic process. This study indicated that while DSBs may trigger cancer development as an early event in PBM carcinogenesis, DSBs alone could not initiate oncogenesis. The underlying mechanism of cancer development after DSBs remains unclear, and further study is required to discern the processes that precipitate DDR dysfunction.
PBM is well known to occur in both dilated or non-dilated extrahepatic bile duct forms . The site of cancer development in these two types is quite different; PBM patients without biliary dilation have a high incidence of gallbladder carcinoma, whereas those with biliary dilation display a high incidence of both of gallbladder and bile duct carcinoma. The prevalence of gallbladder adenocarcinoma is considerably higher in PBM patients without biliary dilation . According to our findings, DSBs occurred significantly more frequently in the PBM gallbladder mucosa of patients without biliary dilation. Considering the higher prevalence of gallbladder cancer in non-dilated PBM, our result that DSB occurrence is more common in cases without biliary dilation is convincing. In PBM with biliary dilation, the refluxed pancreatic juice is likely to stagnate in the dilated bile duct and gallbladder, whereas in PBM without biliary dilation, the stasis is likely to occur selectively in the gallbladder, where long-term inflammatory damage to the mucosa may induce the accumulation of DSBs, resulting in a high prevalence of gallbladder cancer.
This study suggests that DSBs induced by persistent inflammation may be involved in the development of gallbladder cancers in PBM at an early stage of carcinogenesis. It has been reported that tissue samples of ulcerative colitis  and chronic hepatitis , both widely known as chronic inflammatory diseases predisposing patients to cancer, overexpressed γ-H2AX. γ-H2AX was found to be a marker for predicting carcinogenesis in those diseases. Furthermore, γ-H2AX has been reported as a prognostic factor in several other types of cancers, including breast cancer , hepatocellular carcinoma , lung cancer , and colorectal cancer , in which γ-H2AX overexpression has been associated with poor patient survival. Taking the above into consideration, γ-H2AX may have a role in early cancer screening as a prognostic indicator in PBM patients. Larger studies are required to elucidate the detailed role of this marker.