It is well-known that CBD patients with chronic inflammation have a high incidence of biliary tract cancer [1, 18, 19]. Genetic alterations in the biliary epithelium of CBD patients have been investigated [20–22], but the mechanism of carcinogenesis is still unknown. Additionally, few similar studies have been performed on the biliary epithelium of pediatric CBD patients. Our previous metabolomics analysis identified carcinogenesis-related metabolites in the bile of PBM patients . In this study, we investigated the possibility that these metabolites may cause DNA damage via ROS production in biliary epithelial cells, inducing activation of DNA damage repair pathways. Additionally, we have previously reported that biliary epithelial cells in pediatric CBD have carcinogenic potential  and have also investigated whether similar carcinogenic mechanisms occur in children and adults. The results showed that DNA damage markers and DNA damage repair response markers were significantly elevated in adult and pediatric CBD patients compared with controls, suggesting involvement of DNA damage repair pathways in the carcinogenic mechanisms of CBD. Furthermore, pediatric CBD patients also showed a significant increase in these DNA damage and DNA repair markers compared with controls, and the increase was comparable to that in adult CBD patients, suggesting that CBD has the same carcinogenic potential in children as in adults.
Chronic inflammation is generally thought to promote carcinogenesis due to oxidative stress . Frick et al. also reported that DSBs were a core molecular lesion of inflammation-induced carcinogenesis and that oxidative DNA damage due to inflammation was the driving force of mutagenesis . Under the chronic inflammatory conditions of inflammatory bowel disease, oxidative DNA damage is increased with the degree of inflammation; the expression of DSB repair proteins is likewise increased with the grade of inflammation and dysplasia, and elevated DNA damage with DNA damage repair is associated with the accumulation and spread of precancerous lesions . In other words, in chronic inflammatory conditions, there is increased oxidative stress on the intestinal epithelium, and this oxidative stress is enhanced not only with the degree of inflammation but also as the disease progresses toward carcinogenesis. DNA damage and repair responses are also enhanced during the carcinogenesis process, which is similar to our results and supports our findings. Furthermore, activation of the DNA damage repair pathway may promote cell survival and accumulate genetic alterations that lead to inflammation-induced carcinogenesis. Biliary epithelial cells in CBD under chronic inflammatory stress have a high proliferation rate and are constantly exposed to replicative stress, which may also contribute to increased DSBs [10, 25].
From a set of in vitro studies, Chang et al. reported that the addition of cytotoxic substances induced intracellular ROS production that led to DNA strand breaks, which in turn induced aggressive carcinogenesis through oncogenic RAS . It is possible that the expression and activation of KRAS that we previously observed in CBD patients  was one such accumulated change in cancer-related genes from the oxidative DNA damage repair response that we examined in this study of CBD patients.
In general, the DSB repair pathways include homologous recombination (HR) or non-homologous end joining (NHEJ). Compared with HR, NHEJ is considered to be more error-prone because it directly ligates truncated DNA ends without using a homologous template. NHEJ is active in all cell cycle states and is thought to be more effective than HR, but at the cost of creating more DNA mutations . Our findings suggested that NHEJ is activated in CBD biliary epithelial cells, as KU70, which binds to the ends of DSBs and is required for NHEJ, is elevated, suggesting this pathway may be a source of mutations in CBD.
In summary, our results suggest that in the chronic inflammatory condition of CBD, lysophosphatidylcholine and triacylglycerol in the bile induce ROS in the biliary epithelium, which leads to oxidative DNA damage and activation of DNA repair pathways. This process of chronic DNA damage and repair eventually induces the expression and activation of KRAS, which can lead to carcinogenesis.
In conclusion, the expression of γH2AX, MRE11, and KU70 in the biliary epithelium, which are also markers for activation of the DNA damage repair pathway, could influence carcinogenesis in CBD and predispose both children and adults with CBD to develop BTC. These findings suggested that both children and adults with CBD may develop BTC through overactivation of the DNA damage repair pathway in response to cellular ROS.