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1 Supplementary Information Metabolomics reveals trichloroacetate as a major contributor to trichloroethylene-induced metabolic alterations in mouse urine and serum Zhong-Ze Fang, Kristopher W Krausz, Naoki Tanaka, Fei Li, Aijuan Qu, Jeffrey R. Idle, Frank J Gonzalez 1
2 Supplement table 1: Primer pairs used for qpcr Gene Primer sequence Ppara F 5 -AGTTCGGGAACAAGACGTTG-3 R 5 -CAGTGGGGAGAGAGGACAGA-3 Acot1 F 5 -CTGGCGCATGCAGGATC-3 R 5 -GGCACTTTTCTTGGATAGCTCC-3 Acox1 F 5 -TCGCAGACCCTGAAGAAATC-3 R 5 -CCTGATTCAGCAAGGTAGGG-3 Acadm F 5 -AGCTCTAGACGAAGCCACGA-3 R 5 -GCGAGCAGAAATGAAACTCC-3 Acadl F 5 -TCTTGCGATCAGCTCTTTCA-3 R 5 -GGTACATGTGGGAGTACCCG-3 Ehhadh F 5 -CTATGATCCGCCTCTGCAA-3 R 5 -TGGCTCTAACCGTATGGTCC-3 Acaa1a F 5 -GTGGCATCAGAAATGGGTCT-3 R 5 -CGGGAAGAGATATTCCCAGG-3 Cpt1a F 5 -GCCCATGTTGTACAGCTTCC-3 R 5 -AGTGGCCTCACAGACTCCAG-3 Cpt2 F 5 -ATGCACTACCAGGACAGGCT-3 R 5 -TGGCTGTCATTCAAGAGAGG-3 Cyp4a1 F 5 -GATGGACGCTCTTTACCCAA-3 R 5 -AAGGGTCAAACACCTCTGGA-3 2
3 Supplementary figure 1. Body weight change and food intake in control and TCE-treated mice. The data are given as mean±sem (n=5). body weight (g) TCE-8 TCE Time (day) Food intake (g per 24 h) TCE-8 TCE Time (day) 3
4 Supplementary figure 2. The influence of TCE exposure on alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP) activities in serum, and triglyceride (TG) levels in liver. *p<.5, **p<.1, ***p<.1. 4 ** 6 *** ALT(U/L) AST(U/L) 4 2 TCE-8 TCE-16 TCE-8 TCE ALP(U/L) 2 1 TG (mg/g liver) 1 5 TCE-8 TCE-16 Control TCE-8 TCE-16 4
5 5
6 Supplementary figure 3. The influence of DCA exposure on alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP) activities in serum, and triglyceride (TG) levels in liver. *p<.5, **p<.1, ***p< ALT (U/L) AST(U/L) 2 1 DCA DCA ** 3 3 ALP(U/L) 2 1 TG (mg/g liver) 2 1 DCA DCA 6
7 Supplementary figure 4. The influence of TCA exposure on alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP) activities in serum, and triglyceride (TG) levels in liver. 3 4 ALT (U/L) 2 1 AST(U/L) TCA TCA 2 15 ALP(U/L) TG (mg/g liver) 1 5 TCA TCA 7
8 Supplementary figure 5. Histopathology (H&E staining) of livers from C57BL/6 treated with TCE. (A) TCE-ctrl; (B) TCE-8 mg/kg/day; (C) TCE-16 mg/kg/day; (D) TCE-ctrl; (E) TCE- 8 mg/kg/day; (F) TCE-16 mg/kg/day. 1 X magnification. (A) (B) (C) (D) (E) (F) 8
9 Supplementary figure 6. Histopathology (H&E staining) of livers after exposure of C57BL/6 mice to DCA and TCA. (A) DCA-ctrl; (B) DCA-treat; (C) TCA-ctrl; (D) TCA-treat. (A) (B) (C) (D) 9
10 Supplementary figure 7A. UPLC-ESI-QTOFMS comparison of the authentic standard of hippuric acid with ions derived from urine obtained from C57BL/6 mice. Comparison of retention time Time Comparison of MS/MS fragmentation
11 Supplementary figure 7B. UPLC-ESI-QTOFMS comparison of authentic standard of cinnamoylglycine with ions derived from urine obtained from C57BL/6 mice. Comparison of retention time Time Comparison of MS/MS fragmentation Cinnamoyl-glycine urine sample-c
12 Supplementary figure 7C. UPLC-ESI-QTOFMS comparison of the authentic standard of phenylacetylglycine with ions derived from urine obtained from C57BL/6 mice. Comparison of retention time Time Comparison of MS/MS fragmentation Phenylacetylglycine urine sample-c
13 Supplementary figure 7D. UPLC-ESI-QTOFMS comparison of the authentic standard of hexanoylglycine with ions derived from urine obtained from C57BL/6 mice. Comparison of retention time Time Comparison of MS/MS fragmentation Hexanoyl-glycine urine sample-c
14 Supplementary figure 7E. UPLC-ESI-QTOFMS comparison of the authentic standard of phenylpropionylglycine with ions derived from urine obtained from C57BL/6 mice. Phenylpropionyl-glycine Time
15 Supplementary figure 7F. UPLC-ESI-QTOFMS comparison of authentic standard of pantothenic acid with ions derived from urine obtained from C57BL/6 mice. Comparison of retention time Time Comparison of MS/MS fragmentation Pantothenic acid urine sample-c
16 Supplementary figure 7G. UPLC-ESI-QTOFMS comparison of authentic standard of carnitine with ions derived from urine obtained from C57BL/6 mice. Comparison of retention time Time Comparison of MS/MS fragmentation Carnitine urine sample-c
17 Supplementary figure 7H. UPLC-ESI-QTOFMS comparison of authentic standards of acetylcarnitine with ions derived from urine obtained from C57BL/6 mice. Comparison of retention time Time Comparison of MS/MS fragmentation Acetylcarnitine urine sample-c
18 Supplementary figure 7I. UPLC-ESI-QTOFMS comparison of authentic standard of 1- octadecanoyl-sn-glycero-3-phosphocholine (LPC (18:) with ions derived from serum obtained serum from C57BL/6 mice. Comparison of retention time 1 Serum Time Comparison of MS/MS fragmentation LPC(18:) serum sample-16-5 Serum
19 Supplementary figure 7J. UPLC-ESI-QTOFMS comparison of authentic standard of 1-oleoyl- 2-hydroxy-sn-glycero-3-phosphocholine (LPC(18:1, 9Z) with ions derived from serum obtained from C57BL/6 mice. Comparison serum of retention sample-16-5 time 1 Serum Time Comparison of MS/MS fragmentation LPC(18:1,9Z) serum sample-16-5 Serum
20 Supplementary figure 8. Relative quantitation of PC components altered in serum from mice treated with TCE. The relative abundance (peak area of metabolites/internal standard) is given. The results were given as mean ±SEM. *, p<.5; **, p<.1; ***, p<.1. 5 (MS=81.64,R t =8.4 min) *** *** 4 (MS=834.65,R t =8.29 min) *** *** 8 (MS=76.588,R t =8.47 min) *** ** Relative abundance Relative abundance Relative abundance TCE-8 TCE-16 (MS= ,R t =8.2 min) ** 8 ** TCE-8 2 TCE-16 (MS=88.589,R t =8.4 min) *** *** TCE-8 TCE-16 Relative abundance Relative abundance TCE-8 TCE-16 TCE-8 TCE-16 2
21 Supplementary figure 9. Comparison of the alteration of pantothenic acid and phenylacetylglycine in urine of mice treated with TCA and DCA. Pantothenic acid (µmol/mmol Creatinine) TCA days phenylacetyl-glycine (mmol/mmol Creatinine) TCA days Pantothenic acid (µmol/mmol Creatinine) DCA days phenylacetyl-glycine (mmol/mmol Creatinine) DCA days 21
Supplementary Figure 1
Supplementary Figure 1 A B mir-141, human cell lines mir-2c, human cell lines mir-141, hepatocytes mir-2c, hepatocytes Relative RNA.1.8.6.4.2 Relative RNA.3.2.1 Relative RNA 1.5 1..5 Relative RNA 2. 1.5
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