市原 佐保子

Abstract Objectives The roles of dichloromethane (DCM) and 1,2,3-trichloropropane (1,2,3-TCP) in occupational cholangiocarcinoma, first reported in 2012, remains elusive. This study aimed to determine the potential of 1,2-dichloropropane (1,2-DCP), DCM, and 1,2,3-TCP to induce DNA damage—a hallmark of carcinogenicity—in human cholangiocytes. Methods Mono-cultures of human MMNK-1 cholangiocytes and co-cultures of MMNK-1 cholangiocytes-human THP-1 monocyte-derived macrophages and monocytes were each exposed to 1,2-DCP, DCM or 1,2,3-TCP at 0, 0.1, and 0.4 mM for 24 hours. DNA double-strand break marker γ-H2AX-positive foci and γ-H2AX pan-nuclear staining-which is known to be induced during early or intermediate stage of apoptosis or under replicative stress/checkpoint abrogation-in cholangiocytes were counted in 100 and 200 cholangiocytes, respectively. Apoptosis was evaluated by TUNEL staining. Results The increase in dense (≥55) γ-H2AX foci induced by all three chemical compounds was significantly greater in co-cultures with macrophages, but not with monocytes, than in monocultures. However, only 1,2-DCP decreased the number of γ-H2AX pan-nuclear-positive and TUNEL-positive cholangiocytes in co-cultures with macrophages, but not in monocultured cholangiocytes. Co-treatment with a pan-caspase inhibitor decreased the number of γ-H2AX pan-nuclear-positive cholangiocytes in each group by about 50%, suggesting that apoptotic signaling is involved, at least in part, in the induction of γ-H2AX pan-nuclear-positive cholangiocytes. Conclusion Our study showed that co-culture with macrophages enhanced DNA double strand break induced by all three tested chemical compounds, while only 1,2-DCP exhibited a proliferative effect in monocultures and anti-apoptotic effects in co-cultures with macrophages, which are key characteristics of carcinogens, thus distinguishing 1,2-DCP from DCM and 1,2,3-TCP.