Department of Occupational and Environmental Health, Faculty of Pharmaceutical Sciences, Tokyo University of Science, Noda 278-8510, Japan.
Evolutionary Medicine, Shiga Medical Center Research Institute, Moriyama 524-8524, Japan.
Toxicol Sci. 2019 Mar 1;168(1):137-148. doi: 10.1093/toxsci/kfy280.
1,2-dichloropropane (1,2-DCP) was reclassified recently by IARC as a Group 1 carcinogen based on epidemiological studies on an outbreak of cholangiocarcinoma in offset-printing workers exposed to 1,2-DCP in Japan. However, the underlying mechanism of 1,2-DCP-induced cholangiocarcinoma remains obscure. A previous whole-genome mutation analysis of cholangiocarcinoma of 4 cases exposed to 1,2-DCP suggested the involvement of activation-induced cytidine deaminase (AID), based on specific signatures of mutation patterns. The objective of the present study is to determine whether exposure to 1,2-DCP induces expression of AID in human cholangiocytes. Human MMNK-1 cholangiocytes, differentiated THP-1 macrophages, and co-cultures of MMNK-1/THP-1 cells were exposed to 1,2-DCP at different concentrations and time intervals. The mRNA expression levels of AID and related genes were quantified by real-time PCR. Protein expression was measured by immunostaining. Alkaline Comet assay was performed to examine DNA damage. The results showed that 1,2-DCP alone did not change AID expression in MMNK-1 cholangiocytes. 1,2-DCP significantly increased pro-inflammatory cytokine TNF-α expression in THP-1 macrophages. TNF-α treatment upregulated expression of AID, NF-κB, and IκB in MMNK-1 cholangiocytes. SN50, a NF-κB inhibitor, significantly downregulated TNF-α-induced AID expression, suggesting the involvement of NF-κB pathway in TNF-α-induced AID expression. Exposure to 1,2-DCP significantly increased AID expression in MMNK-1 cholangiocytes co-cultured with THP-1 macrophages. Comet assay showed that 1,2-DCP-induced DNA damage in MMNK-1 cholangiocytes, as indicated by increased tail DNA% and tail moment, was enhanced when co-cultured with macrophages. The results suggest that inflammatory response of macrophages and consequent aberrant AID expression or DNA damage in the cholangiocytes underlie the mechanism of 1,2-DCP-induced cholangiocarcinoma in humans.
1,2-二氯丙烷（1,2-DCP）最近被 IARC 重新分类为 1 组致癌物质，这是基于日本印刷工人因接触 1,2-DCP 而爆发胆管癌的流行病学研究。然而，1,2-DCP 诱导胆管癌的潜在机制仍不清楚。之前对 4 例接触 1,2-DCP 的胆管癌的全基因组突变分析表明，激活诱导胞嘧啶脱氨酶（AID）的参与是基于突变模式的特定特征。本研究的目的是确定暴露于 1,2-DCP 是否会诱导人胆管细胞中 AID 的表达。将人 MMNK-1 胆管细胞、分化的 THP-1 巨噬细胞以及 MMNK-1/THP-1 细胞共培养物暴露于不同浓度和时间间隔的 1,2-DCP 中。通过实时 PCR 定量测定 AID 和相关基因的 mRNA 表达水平。通过免疫染色测量蛋白质表达。进行碱性彗星试验以检查 DNA 损伤。结果表明，1,2-DCP 单独不会改变 MMNK-1 胆管细胞中的 AID 表达。1,2-DCP 可显著增加 THP-1 巨噬细胞中促炎细胞因子 TNF-α的表达。TNF-α处理上调了 MMNK-1 胆管细胞中 AID、NF-κB 和 IκB 的表达。NF-κB 抑制剂 SN50 显著下调 TNF-α诱导的 AID 表达，表明 NF-κB 途径参与 TNF-α诱导的 AID 表达。暴露于 1,2-DCP 可显著增加与 THP-1 巨噬细胞共培养的 MMNK-1 胆管细胞中的 AID 表达。彗星试验表明，1,2-DCP 诱导 MMNK-1 胆管细胞中的 DNA 损伤，如尾部 DNA%和尾部矩增加所表明的，与巨噬细胞共培养时增强。结果表明，巨噬细胞的炎症反应以及随后胆管细胞中异常 AID 表达或 DNA 损伤是 1,2-DCP 诱导人类胆管癌的机制。
