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Halogenated Hydrocarbon Solvent-Related Cholangiocarcinoma Risk: Biliary Excretion of Glutathione Conjugates of 1,2-dichloropropane Evidenced by Untargeted Metabolomics Analysis

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Halogenated Hydrocarbon Solvent-Related Cholangiocarcinoma Risk: Biliary Excretion of Glutathione Conjugates of 1,2-dichloropropane Evidenced by Untargeted Metabolomics Analysis

Yu Toyoda et al. Sci Rep.

Abstract

Recently, the International Agency for Research on Cancer issued a warning about the carcinogenicity of 1,2-dichloropropane (1,2-DCP) to humans based on an epidemiological study suggesting a relationship between the incidence of cholangiocarcinoma and occupational exposure to halogenated hydrocarbon solvent comprised mostly of 1,2-DCP. Although this dihaloalkane has been used in various industrial fields, there has been no biological evidence explaining the cholangiocarcinoma latency, as well as little understanding of general cholangiocarcinoma risk. In the present study, we explored the biliary excretion of 1,2-DCP metabolites by an untargeted metabolomics approach and the related molecular mechanism with in vitro and in vivo experiments. We hypothesized that the biliary excretion of carcinogens derived from 1,2-DCP contribute to the increased cholangiocarcinoma risk. We found that 1,2-DCP was conjugated with glutathione in the liver, and that the glutathione-conjugated forms of 1,2-DCP, including a potential carcinogen that contains a chloride atom, were excreted into bile by the bile canalicular membrane transporter, ABCC2. These results may reflect a risk in the backfiring of biliary excretion as a connatural detoxification systems for xenobiotics. Our findings would contribute to uncover the latent mechanism by which the chronic exposure to 1,2-DCP increases cholangiocarcinoma risk and future understanding of cholangiocarcinoma biology.

Figures

Figure 1
Figure 1. Proposed metabolic pathways of 1,2-DCP in the liver-biliary system.
As a result of untargeted metabolomics approach followed by differential analyses, 13 peaks of interest were selected (summarized in Supplementary Table S1). Based on the results of LC-MS/MS analysis (accurate mass, isotopic pattern, and fragment ions for target metabolites), we determined the putative chemical structures for each peak. According to the structural information, we finally selected nine compounds as the metabolites of 1,2-DCP. No. 12 and No. 10 are correspond to a half mustard and mercapturate form, respectively. The presence of chloride ion in the metabolite No. 12 was confirmed by the isotopic peak derived from 37Cl. Underlined compounds are GS-DCPs and include glutathione-derived chemical structure.
Figure 2
Figure 2. Comparison of biliary levels of each 1,2-DCP metabolite between 1,2-DCP-administered WT and Abcg2 KO mice.
Bile specimens of 1,2-DCP-administered WT and Abcg2 KO mice were analysed using LC-MS/MS. Relative intensity was calculated as the ratio of each metabolite level in the Abcg2 KO group to that of the WT group. This value is expressed as the mean ± S.E.M. n = 6 (WT), 7 (Abcg2 KO). Statistical analyses for significant differences were performed according to parametric, Student’s t test. There was no significant difference in the biliary level of each 1,2-DCP metabolite between WT and Abcg2 KO mice.
Figure 3
Figure 3. Comparison of biliary levels of each 1,2-DCP metabolite between 1,2-DCP-administered SD rats and Abcc2-deficient EHBRs.
Bile specimens of 1,2-DCP-administered SD rats and Abcc2-deficient EHBRs were analysed using LC-MS/MS. Relative intensity was calculated as the ratio of each metabolite level of 1,2-DCP in the EHBR group to that in the SD rat group. This value is expressed as the mean ± S.E.M. n = 9 (SD rat), 10 (EHBR). Statistical analyses for significant differences were performed according to a parametric Student’s t test or a nonparametric Mann-Whitney U test (**P < 0.01). N.D. not detected.
Figure 4
Figure 4. Comparison of liver levels of each 1,2-DCP metabolite between 1,2-DCP-administered SD rats and Abcc2-deficient EHBRs.
Liver tissues of 1,2-DCP-administered SD rats and Abcc2-deficient EHBRs were analysed using LC-MS/MS. Relative intensity was calculated as the ratio of each metabolite level in the SD rat group to that in the EHBR group. This value is expressed as the mean ± S.E.M. n = 6. Statistical analyses for significant differences were performed according to a parametric Student’s t test (P < 0.05; ††P < 0.01) or a nonparametric Mann-Whitney U test (**P < 0.01). N.D. not detected.
Figure 5
Figure 5. ABCC2-mediated transport of GSH-conjugated 1,2-DCP metabolites.
Transport of GS-DCPs was examined with human ABCC2-expressing vesicles. Incorporation activities of No. 12 (a) and No. 7 (b) in mock and ABCC2-expressing vesicles were shown with or without ATP. The incubation condition was 37 °C for 10 min. ATP-dependent transport was calculated by subtracting the transport activity in the presence of 5 mM AMP (ATP (−)) from the activity in the presence of 5 mM ATP. Data are expressed as the mean ± S.D. n = 3. Statistical analyses for significant differences were performed according to a parametric Student’s t test (P < 0.05) or a nonparametric Mann-Whitney U test (*P < 0.05).
Figure 6
Figure 6. Time course of biliary levels of each 1,2-DCP metabolite in 1,2-DCP-administered humanized-liver mice.
Bile specimens of 1,2-DCP-administered PXB mice were collected for 24 hours every 8 hours, and analysed using LC-MS/MS. The biliary levels of each metabolite at 0–8 h is defined as percent control (100%). Data are expressed as the mean ± S.E.M. n = 3. Statistical analyses for significant differences were performed according to Bartlett’s test, followed by Shirley-Williams’s multiple-comparison test (*P < 0.05; **P < 0.01 vs. control).
Figure 7
Figure 7. Schematic illustration of metabolism and elimination processes of 1,2-DCP and its metabolites in liver-biliary system.
In the liver, 1,2-DCP is metabolized to GS-DCPs via the reaction with GSH, followed by excretion of most glutathione conjugates into bile by ABCC2 localized on the bile canalicular membrane of hepatocytes. Harmful effect on biliary epithelial cells probably cause cholangiocarcinoma. However, a portion of 1,2-DCP metabolites are secreted into blood from the liver. ABCC3 would be involved in this process.

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