Approach to Investigate Estrogen Receptor-Dependent and. Independent Effects of o,p -DDT in the Uterus and Brain of Immature Mice

Transcription

1 Metabolomics Approach to Investigate Estrogen Receptor-Dependent and Independent Effects of o,p -DDT in the Uterus and Brain of Immature Mice Dezhen Wang, Wentao Zhu, Yao Wang, Yin Yan, Miaomiao Teng, Jiyan Miao, Zhiqiang Zhou * Beijing Advanced Innovation Center for Food Nutrition and Human Health, Department of Applied Chemistry, and Department of Applied Chemistry, China Agricultural University, Yuanmingyuan west road 2, Beijing , P. R. China * zqzhou@cau.edu.cn Number of Figures: 9 Number of Tables: 2

2 Figure S1. EE and DDT mediated uterine growth responses. Photomicrographs showing an increase of luminal epithelial cell height in EE (B) and DDT (D) treated groups compared to vehicle (A) group. ICI can reverse the effects of EE (C) and DDT (E) on uterine growth.

3 Figure S2. Representative 600 MHz 1 H NMR spectra of uterus from CK (A), EE (B) and DDT (C) groups. Key: 1, Lipid; 2-4, Isoleucine, Leucine, Valine; 5, Ethanol; 6, Lactate; 7, Alanine; 8, Lysine; 9, Acetate 10, Glutamate; 11, Glutamine; 12, Acetoacetate; 13, Choline; 14, Phosphorylcholine (PC); 15, Glycerophosphocholine (GPC); 16, Succinate; 17, Citrate; 18, Creatine; 19, Taurine; 20 Betaine; 21, Scyllo-inositol; 22, Glycerol; 23, Glycine; 24, Fumarate; 25, Tyrosine; 26, Phenylalanine; 27, Histidine; 28, Formate; 29, Uracil; 30, Inosine; 31, Hypoxanthine; 32, Uridine; 33, Nicotinamide.

4 t[2] 60 CK EE EE+ICI Figure S3. PCA score plot constructed using 1 H NMR data from uterine samples of the CK, EE and EE+ICI groups. t[1] SIMCA-P /3/25 21:48:44

5 t[2] CK DDT DDT+ICI Figure S4. PCA score plot constructed using 1 H NMR data from uterine samples of the CK, DDT and DDT+ICI groups. t[1] SIMCA-P /3/25 22:03:02

6 Figure S5. DDT and EE effects on immature mice uterine metabolites as visualized by one-way ANOVA with Tukey post. Hoc test (P<0.05 vs. CK, noted as *; P<0.05 vs EE, noted as #; P<0.05 vs DDT, noted as $).

7 Figure S6. The new reconstructed PCA model using the data subset of identified metabolites. The score plot showed a successful separation between CK group and DDT group and between CK group and EE group and between CK group and DDT+ICI group.

8 Figure S7. Representative 600 MHz 1 H NMR spectra of brain from CK (A), EE (B) and DDT (C) groups. Key: 1-3, Isoleucine, Leucine, Valine; 4, Ethanol; 5, Lactate; 6, Alanine; 7, Lysine; 8, Acetate 9, Glutamate; 10, Glutamine; 11, GABA (γ-aminobutyrate); 12, N-Acetylaspartate; 13, Choline; 14, Phosphorylcholine (PC); 15, Glycerophosphocholine (GPC); 16, Creatine; 17, Aspartate; 18, Taurine; 19 Betaine; 20, Scylloinositol; 21, Glycerol; 22, Glycine; 23, Fumarate; 24, Tyrosine; 25, Histidine; 26, Phenylalanine; 27, Formate; 28, Uracil; 29, Inosine; 30, Hypoxanthine; 31, Uridine.

9 t[2] CK DDT DI Figure S8. PCA score plot constructed using 1 H NMR data from brain samples of the CK, DDT and DDT+ICI groups. t[1] SIMCA-P /3/25 22:13:54

10 Figure S9. Summary of pathway analysis with MetaboAnalyst 3.0. Key: 1, Phenylalanine, tyrosine and tryptophan biosynthesis; 2, Glutamine and glutamate metabolism; 3, Glycerophospholipid metabolism; 4, Phenylalanine metabolism; 5, Citrate cycle (TCA cycle); 6, Pyruvate metabolism.

11 Table S1. Parameters of PLS-DA Models. Groups compared CK, EE and EE+ICI CK, DDT, DDT+ICI CK, DDT, DDT+ICI CK, EE, Tissue R 2 (Y) Q 2 (cumulative) R intercept Q intercept Uterus Uterus Brain EE+ICI a Brain Note: R 2 Y are the cumulative modelled variation in Y matrix, and Q 2 Y (cumulative) is the cumulative predicted variation in Y matrix. R and Q intercepts were obtained after permutation test (n=200). a Comparisons among these groups failed to generated a PLS-DA model.

12 Table S2. Estrogenic Chemicals and Their Interrupted Metabolites by Metabolomics Approach. Chemicals Animal and Samples Instruments Concentr ation Increased Metabolites Decreased Metabolites Nilestriol 1 Rat(ovariectomised) Serum 1 H NMR 1.5mg/kg 2-Oxoglutarate, Acetate, Citrate, Fumarate, Glutamate, Lactate, Methionine, Succinate, Taurine, DMA, TMAO 3-indoxylsulfate, Allantoin, Betaine, Creatine, Creatinine, Glutamine, Glycine, Hippurate, Lysine, MethyHisitinine, TMA, Urea, Beta-Alanine Acetate, Betaine, Urine Alanine, Formate, Glutamine, Isoleucine, Lactate, Threonine Carnitine, Choline, Creatine, Glucose, Glycine, Histidine, Lysine, Ornithine, Proline,Urea Eicosapentaenoic acid Genistein 2 17β-estradiol 2 Rat(ovariectomised) Serum UPLC- QTOF-MS 50μg/kg 30μg/kg (EPA), Ergocalciferol, Cholecalciferol Eicosapentaenoic acid (EPA), Ergocalciferol arachidonic acid (AA) arachidonic acid (AA) 2-oxoglutarate, Methoxyclor 3 Rat(ovariectomised) Urine 1 H NMR 50/100/2 00 mg/kg Allantoin,Citrate,Form ate Acetate,Alanine,Lactate, Glycine, Phenylacetate, Benzoate HPLC- 17β- estradiol 4, 5 Cow Serum LTQ- Orbitrap 2 mg Dipeptide Glucosamine, Fructose, 17αethinylestradiol 6 Mussel(Lampsilis fasciola) Gill UPLC- LTQ/GC- DSQ 1000ng/L (Day 12) N-acetylmethionine, Proline, Glycerophospholipids Glycerate, Fatty acid, Myo-inositol, Hypoxanthine, Inosine, 2'-Deoxyinosine, Glycerophospholipids

13 Bisphenol A 7 Mussel(Mytilus galloprovincialis) Gonad 1 H NMR 1 μg/l 10 μg/l Betaine, Hypotaurine, Choline, BCAA BCAA, Proline, β- alanine,histine Threonine, Glycogen, Homarine Glycogen, Homarine 17α- Ethinylestradiol 8 Rainbow trout Plasma 1 H NMR 10 ng/l Vitellogenin, UFA,PUFA Alanine, Cholesterol Cholesterol, Cortisol, Arg-Pro, Asn-Ser, Isoleucine, Phenylalanine, Estradiol, Progesterone, Isoflavones 9 Rat follicul arfluid HPLC-MS 50,100,2 00mg/kg Methionine, Myristic acid, Linoleic acid, Stearic acid, L-alanine,Cysteine, Lysine,Glutamine, Palmitic acid, Linolenic Arachidonic acid, Citric acid, Vidarabine acid, Niacinamide, Lipoamide, Homogentisate Tamoxifen 10 Cunner fish Ovary GC-Tof 2,20mg/k g None reported Endosulfan 11 Mice Liver Plasma 1 H NMR 30 μg/kg Glucose Lactate,Betaine,Taurine VLDL/LDL,Choline/P Cho/GPCho Glucose, Glutathione(oxidized) 1. Liu, Y. R.; Huang, R. Q.; Xiao, B. K.; Yang, J. Y.; Dong, J. X., (1)H NMR metabolic profiling analysis offers evaluation of Nilestriol treatment in ovariectomised rats. Mol Cell Endocrinol 2014, 387, Zhu, X.; Liu, X.; He, P.; Cao, B.; Lv, Y.; Zhang, W.; Ni, X., Metabolomics in serum of ovariectomised rats and those exposed to 17beta-oestradiol and genistein. Gynecol Endocrinol 2010, 26, Kim, K. B.; Kim, S. H.; Um, S. Y.; Chung, M. W.; Oh, J. S.; Jung, S. C.; Kim, T. S.; Moon, H. J.; Han, S. Y.; Oh, H. Y.; Lee, B. M.; Choi, K. H., Metabolomics approach to risk assessment: methoxyclor exposure in rats. J Toxicol Environ Health A 2009, 72, Regal, P.; Anizan, S.; Antignac, J. P.; Le Bizec, B.; Cepeda, A.; Fente, C., Metabolomic approach based on liquid chromatography coupled to high resolution mass spectrometry to screen for the illegal use of estradiol and progesterone in cattle. Anal Chim Acta 2011, 700,

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