Volume 23, Issue 5, Pages 555-560 (May 2016) Component of Caramel Food Coloring, THI, Causes Lymphopenia Indirectly via a Key Metabolic Intermediate  Mamoru Ohtoyo, Nobuo Machinaga, Ryotaku Inoue, Katsunobu Hagihara, Hiroshi Yuita, Masakazu Tamura, Ryuji Hashimoto, Jun Chiba, Fumihito Muro, Jun Watanabe, Yoshimasa Kobayashi, Koji Abe, Yasuo Kita, Miyuki Nagasaki, Takaichi Shimozato  Cell Chemical Biology  Volume 23, Issue 5, Pages 555-560 (May 2016) DOI: 10.1016/j.chembiol.2016.04.007 Copyright © 2016 Elsevier Ltd Terms and Conditions

Cell Chemical Biology 2016 23, 555-560DOI: (10. 1016/j. chembiol. 2016 Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 1 Discovery of A6770 and Its Lymphopenic Activity (A) Chemical structure of A6770. (B and C) A6770 induces reductions in peripheral lymphocyte number in rats. A6770 was orally administered to rats at indicated doses under a fed condition. Peripheral blood was collected at the indicated time points and the plasma concentrations of A6770 were quantified by LC-MS/MS (B), and the lymphocyte was counted via a hematology system (C). The relative lymphocyte number compared with vehicle was calculated at each point. Data represent the mean ± SD (n = 3 rats per group). Cell Chemical Biology 2016 23, 555-560DOI: (10.1016/j.chembiol.2016.04.007) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 2 S1PL Inhibition by A6770 (A and B) A6770 induces a reduction in dhS1P degradation activity and an increase in S1P in rat thymus. A6770 was orally administered at indicated doses under a fed condition. (A) Thymus homogenates derived from rats 3 hr after administration were incubated with [3H]dhS1P, followed by alkaline-chloroform extraction and the measurement of radioactivity of aqueous phase. The results are shown as the amounts of degraded [3H]dhS1P compared with vehicle. (B) Thymus was collected at the indicated time points and the concentrations of S1P were measured by LC-MS/MS. Data represent the mean ± SD (n = 3 rats per group). (C) A6770 leads to S1PL inhibition rather than SPHK activation or SPP inhibition. Cells pre-treated with FB1 were incubated with the indicated concentrations of A6770 or vehicle (V), followed by labeling with [3H]dhSph under normal (left panel) or VB6-deficient (right panel) condition. Extracted lipids were then analyzed by TLC and autoradiography. Cer, ceramide; GlcCer, glycosylceramide; PE, phosphatidylethanolamine; PS, phosphatidylserine; PI, phosphatidylinositol; PC, phosphatidylcholine; SM, sphingomyelin. Cell Chemical Biology 2016 23, 555-560DOI: (10.1016/j.chembiol.2016.04.007) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 3 Investigation of S1PL Inhibition Mechanism of A6770 (A) A6770 is phosphorylated by PDXK. A6770 was incubated with or without recombinant PDXK, followed by an HPLC analysis. Chemically synthesized A6770-P was analyzed for reference. (B and C) A6770-P is detected in rat thymus after oral administration of A6770. Thymus was collected at the indicated time points and the concentrations of A6770 (B) or A6770-P (C) were measured by LC-MS/MS. Data represent the mean ± SD (n = 3 rats per group). (D) A6770-P but not A6770 inhibits S1PL in a cell-free assay. Test compounds were added to the apoenzyme-enriched MF and then incubated prior to the addition of 5 μM PLP (except for PLP(−)). [3H]dhS1P was then added to initiate an enzymatic reaction. Data represent the relative amount of degraded [3H]dhS1P compared with the total (mean ± SD, n = 3). Cell Chemical Biology 2016 23, 555-560DOI: (10.1016/j.chembiol.2016.04.007) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 4 In Vivo Conversion from THI to A6770 (A–C) THI was orally administered to rats at 50 mg/kg under a fed condition, with or without pre-administration of antibiotics. At the indicated time points, plasma concentrations of THI (A) or A6770 (B) were measured by LC-MS/MS, and the peripheral lymphocyte number was counted via a hematology system (C). The relative lymphocyte number compared with vehicle treatment was calculated at each point. Data are presented as mean ± SD (n = 3 rats per group). Cell Chemical Biology 2016 23, 555-560DOI: (10.1016/j.chembiol.2016.04.007) Copyright © 2016 Elsevier Ltd Terms and Conditions