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
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
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) 24 22 2 18 TCE-8 TCE-16 16 2 4 6 8 Time (day) Food intake (g per 24 h) 4.5 4. 3.5 3. TCE-8 TCE-16 2.5 2 4 6 8 Time (day) 3
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) 3 2 1 AST(U/L) 4 2 TCE-8 TCE-16 TCE-8 TCE-16 3 15 ALP(U/L) 2 1 TG (mg/g liver) 1 5 TCE-8 TCE-16 Control TCE-8 TCE-16 4
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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<.1. 5 4 4 3 ALT (U/L) 3 2 1 AST(U/L) 2 1 DCA DCA ** 3 3 ALP(U/L) 2 1 TG (mg/g liver) 2 1 DCA DCA 6
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) 3 2 1 TCA TCA 2 15 ALP(U/L) 15 1 5 TG (mg/g liver) 1 5 TCA TCA 7
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
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
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 1 2. 4. 6. 8. Time Comparison of MS/MS fragmentation 1 15.332 1 15.323 1 2. 4. 6. 8. 77.395 77.393 16.368 1.17 76.9899 18.656 197.7952 51.293 76.9958 78.425 16.373 162.565 18.643 2 4 6 8 1 12 14 16 18 2 22 2 4 6 8 1 12 14 16 18 2 1
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 1 1 1. 2. 3. 4. 5. 6. 7. 8. 9. 1. 2. 3. 4. 5. 6. 7. 8. 9. Time Comparison of MS/MS fragmentation Cinnamoyl-glycine urine sample-c4 1 131.49 1 131.482 13.541 13.539 1.216 77.392 51.249 133.1566 26.85 132.537 25 5 75 1 125 15 175 2 225 1.264 77.389 51.235 132.53 133.556 26.814 2 4 6 8 1 12 14 16 18 2 22 11
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 1 1 1. 2. 3. 4. 5. 6. 7. 1. 2. 3. 4. 5. 6. 7. Time Comparison of MS/MS fragmentation Phenylacetylglycine urine sample-c4 1 91.547 1 91.547 76.44 92.58 65.393 12.79 176.714 194.813 2 4 6 8 1 12 14 16 18 2 76.41 92.583 65.49 3.394 12.811 176.735 194.817 2 4 6 8 1 12 14 16 18 2 12
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 1 1 2. 4. 6. 8. 2. 4. 6. 8. Time Comparison of MS/MS fragmentation Hexanoyl-glycine urine sample-c4 1 76.43 1 76.47 71.859 99.89 71.869 99.814 174.1138 3.46 43.544 113.9865 149.219 175.1147 2 4 6 8 1 12 14 16 18 2 174.1134 3.388 43.591 1.845 156.133 175.1194 2 4 6 8 1 12 14 16 18 2 13
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 1 1 15.71 1 15.71 1 1. 2. 3. 4. 5. 6. 7. 8. 9. 1. 2. 3. 4. 5. 6. 7. 8. 9. Time 76.41 16.74 133.649 28.962 3.391 2 4 6 8 1 12 14 16 18 2 76.44 16.741 133.652 28.979 48.452 25 5 75 1 125 15 175 2 14
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 1 1 1. 2. 3. 4. 5. 6. 7. 1. 2. 3. 4. 5. 6. 7. Time Comparison of MS/MS fragmentation Pantothenic acid urine sample-c4 1 9.553 22.1172 1 9.547 22.1182 22.174 184.974 85.654 124.757 221.1227 142.858 3.388 222.1238 25 5 75 1 125 15 175 2 225 85.653 124.753 142.864 184.967 22.176 221.124 223.1356 25 5 75 1 125 15 175 2 225 25 15
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 1 1. 2. 3. 4. 5. 6. 7. 8. 9. 1 1. 2. 3. 4. 5. 6. 7. 8. 9. Time Comparison of MS/MS fragmentation Carnitine urine sample-c4 1 162.191 1 162.1115 13.385 85.287 163.1169 1.2582 14.43 6.827 164.1188 25 5 75 1 125 15 175 2 225 13.382 166.729 161.1322 1.1373 85.278 6.812 123.431 166.1178 2 4 6 8 1 12 14 16 18 16
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 1 1 1. 2. 3. 4. 5. 6. 7. 8. 9. 1. 2. 3. 4. 5. 6. 7. 8. 9. Time Comparison of MS/MS fragmentation Acetylcarnitine urine sample-c4 1 85.284 1 24.1279 85.285 24.1155 86.338 145.482 25.1288 29.43 6.826 146.547 25 5 75 1 125 15 175 2 225 25 71.572 158.1279 145.455 25.1263 26.1184 25 5 75 1 125 15 175 2 225 25 17
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 1 2. 4. 6. 8. 2. 4. 6. 8. Time Comparison of MS/MS fragmentation LPC(18:) serum sample-16-5 Serum 1 184.74 1 184.729 14.17 14.166 86.964 185.764 524.3654 341.361 525.3763 447.289 1 2 3 4 5 6 86.965 185.759 524.3692 56.364 525.3746 341.337 447.2891 1 2 3 4 5 6 18
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 1 1. 2. 3. 4. 5. 6. 7. 8. 9. 1. 2. 3. 4. 5. 6. 7. 8. 9. Time Comparison of MS/MS fragmentation LPC(18:1,9Z) 1 184.734 serum sample-16-5 Serum 14.17 522.353 1 184.736 14.171 522.3541 523.3588 86.961 15.117 185.773 54.3454 524.3611 339.293 445.2716 1 2 3 4 5 6 523.3598 54.3474 185.773 339.291 445.275 524.364 1 2 3 4 5 6 19
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 4 3 2 1 Relative abundance 3 2 1 Relative abundance 6 4 2 TCE-8 TCE-16 (MS=784.589,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 6 4 2 Relative abundance 15 1 5 TCE-8 TCE-16 TCE-8 TCE-16 2
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) 2 15 1 5 TCA 2 4 6 8 days phenylacetyl-glycine (mmol/mmol Creatinine) 15 1 5 TCA 2 4 6 8 days Pantothenic acid (µmol/mmol Creatinine) 2 15 1 5 DCA 2 4 6 8 days phenylacetyl-glycine (mmol/mmol Creatinine) 15 1 5 DCA 2 4 6 8 days 21