Supporting Information. Multigenerational Disruption of Thyroid Endocrine System in Marine Medaka

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Supporting Information Multigenerational Disruption of Thyroid Endocrine System in Marine Medaka after A Life-Cycle Exposure to Perfluorobutane Sulfonate (PFBS) Lianguo Chen,, Chenyan Hu #, Mirabelle M. P. Tsui, Teng Wan, Drew R. Peterson, Qipeng Shi, Paul K. S. Lam, Doris W. T. Au, James C. W. Lam,, *, Bingsheng Zhou, * State Key Laboratory in Marine Pollution, City University of Hong Kong, Kowloon, Hong Kong SAR, China State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 4372, China # School of Chemistry and Environmental Engineering, Wuhan Institute of Technology, Wuhan 4372, China Department of Chemistry, City University of Hong Kong, Kowloon, Hong Kong SAR, China Department of Science and Environmental Studies, The Education University of Hong Kong, Hong Kong SAR, China * Corresponding author: Dr. James C. W. Lam Tel: 852-2948-8537 Fax: 852-2948-7676 E-mail: jameslam@eduhk.hk Dr. Bingsheng Zhou Tel: +86 27 687842 Fax: +86 27 6878123 E-mail: bszhou@ihb.ac.cn Number of pages: 1 Number of figures: 2 Number of tables: 4 S1

Figure captions Figure S1. Changes in brainsomatic index (BSI) values of adult marine medaka (A: F; B: F1) after a life-cycle exposure to various concentrations of PFBS (, 1., 2.9 and 9.5 µg/l). BSI = brain weight/body weight 1. Values represent the mean ± SEM of three replicates. *P <.5 and **P <.1 indicate significant difference between exposure groups and the corresponding control group. Figure S2. Changes in blood thyroxine-binding globulin (TBG) levels of adult marine medaka (A: F; B: F1) after a life-cycle exposure to various concentrations of PFBS (, 1., 2.9 and 9.5 µg/l). Values represent the mean ± SEM of three replicates. S2

(A) 1.5 Male Female BSI values% 1..5. 1. 2.9 Exposure concentrations (µg/l) 9.5 (B) 1.2.9 * ** Male Female * BSI values%.6.3. 1. 2.9 Exposure concentrations (µg/l) 9.5 Figure S1 S3

(A) 12 Male Female TBG contents in F blood (% relative to control) 9 6 3 1. 2.9 Exposure concentrations (µg/l) 9.5 (B) 24 Male Female TBG contents in F1 blood (% relative to control) 18 12 6 1. 2.9 Exposure concentrations (µg/l) 9.5 Figure S2 S4

Table S1. Primers of the selected genes examined in this study. a a Gene Sequence of primers (5 3 ) CRH F: CTGCAGCTGTTGGGAGTTTT R: CCAACTGTCTGGCAACCTTC CRHR F: CTTCACGGGTCAGCTTTCAC R: TCTCCAGAAAGTCCCTGTCG TSHβ F: TTTGGTCCCCGCTTCCTTAT R: AAGTCGAACTGGTTTGGTGC TSHR F: GGTGGTGTGCTTGTGTTACC R: TGTGAAGGTCATCAGCGGAT NIS F: GTGAGCAGCATTTCCAACCA R: TGGTCCTGTTGCATAGCTCA TPO F: GCGCTGCAGTTCCAATCATA R: TCCATCAGCCTCCAATGTGT TG F: TCTCTGGACCACAACCCTTC R: GAGAGCTCTGCGGATGAAGA TBG F: TTGATGGTGTTGACGCTGTC R: CTTCCGTAGCCTCCTCACAA TRα F: TGTCTCCACACACCACACAT R: CCAACGTTCAACAAGCAGGA TRβ F: GATCAGAGCGACGGGAAAAC R: ATGTGTGCGTCGATTCCTTG Dio1 F: AGCATCCTCGGGTTCATGAA R: TGCGCCTCAGTGATGTAGAT Dio2 F: CGTTGTTGTCCATGCAGTCA R: CCTGCTGGTTTACATCGACG Dio3 F: GAGGATGAAGGAGCAGGGAG R: CCAAAGTTGAGGATGAGCGG UGT1 F: CGGTACCCTCCCTTTGACAA R: GCCTCCTCAGCTATTACCCA SULT F: CGATTTGTTGGACCTGAGCC R: CAACTGTGGAGTCCTGGGAT MCT8 F: CGGCTGTTTTGGGCTACTTT R: CACTGCGCATTGAGGAATCA SLCO1c1 F: TTTCCACCCTCTGACTGCAT R: TTACCGCCCCATGAAGAAGT pax8 F: TGCCATTAACGAGGGACAGT R: TCGCTCAACCCAACTCTGAT hhex F: GTGGAGCTGGAGAGGAAGTT R: GGGTTCTCCTGCTTCAGTCT 18s (control) 1,2 F: GACAAATCGCTCCACCAACT R: CCTGCGGCTTAATTTGACCC Abbreviations: CRH, Corticotropin-releasing hormone; CRHR, S5

Corticotropin-releasing hormone receptor 1; TSHβ, Thyroid-stimulating hormone subunit beta; TSHR, Thyroid-stimulating hormone receptor; NIS, Sodium-iodide symporter; TPO, Thyroid peroxidase; TG, Thyroglobulin; TBG, Thyroxine-binding globulin; TR, Thyroid hormone receptor; Dio, 5'-Deiodinase; UGT1, UDP-glucuronosyltransferase 1A1; SULT, Thyroxine sulfotransferase; MCT8, Monocarboxylate transporter 8; SLCO1c1, Solute carrier organic anion transporter 1c1; pax8, Paired box protein 8; hhex, Hematopoietically-expressed homeobox protein. References (1) Chen, X.; Li, V. W. T.; Yu, R. M. K.; Cheng, S. H. Choriogenin mrna as a sensitive molecular biomarker for estrogenic chemicals in developing brackish medaka (Oryzias melastigma). Ecotoxicol. Environ. Saf. 28, 71 (1), 2 28. (2) Tse, A. C. K.; Lau, K. Y.; Ge, W.; Wu, R. S. A rapid screening test for endocrine disrupting chemicals using primary cell culture of the marine medaka. Aquat. Toxicol. 213, 144 145, 5 58. S6

Table S2. Changes in thyroid hormones of marine medaka (F, F1 and F2) after parental exposure to various concentrations of PFBS (, 1., 2.9 and 9.5 µg/l). a T3 (pg/mg protein) T4 (ng/mg protein) F/F1/F2 µg/l 1. µg/l 2.9 µg/l 9.5 µg/l µg/l 1. µg/l 2.9 µg/l 9.5 µg/l F blood 11.7±.5 11.±.7 12.3±1.5 13.8±1.8 1.5±.6 1.8±.4 1.7±.3 1.3±.2 F brain 11.6±1. 17.6±3.8 2.1±2.2 13.9±1.4 2.2±.5 2.2±.6 2.2±.5 1.7±.6 Male F1 blood 6.2±13.7 62.±6.3 57.5±9.3 74.9±1.6 12.5±3.3 8.4±2. 11.1±.7 1.2±1.4 F1 brain 36.2±5.2 46.7±7.2 42.7±8.1 46.8±3..3±.1.3±..4±.1.4±.1 Female F blood 11.7±2. 12.±.9 12.9±1.1 7.±.4 2.2±.6 2.4±.4 2.5±.2 3.±.4 F brain 12.6±2.3 13.1±2.2 2.8±6.1 13.8±1.4 1.±.3 2.5±.3* 1.3±.1 2.2±.3 F1 egg b 1.±..5±.2.4±.2.4±.2 7.5±5.2 6.5±4.5 6.1±3.1 6.2±3.2 F1 larvae 36.6±3.1 9.2±24.3* 45.7±4.4 45.6±7.2.4±.1.5±.2.5±.1.6±.2 F1 blood 43.8±3.4 28.4±.6 35.2±6.2 26.5±2.* 13.1±2.5 7.5±.8 7.8±1.5 12.9±2. F1 brain 36.6±1.4 4.3±4.1 48.9±11.9 37.5±1.8.3±.1.3±.1.5±..5±.1 F2 egg b.4±.2.3±.1.3±.1.4±.2 5.8±3.3 5.5±2.5 5.9±3.1 9.4±3.4 F2 larvae 66.9±8.6 65.5±1.6 52.±14.6 87.4±33..6±..5±..6±.1 1.5±.3** a Values represent the mean ± SEM of three replicates. b Contents of thyroid hormones in eggs are expressed as pg/egg. *P <.5 and **P <.1 indicate significant difference between exposure groups and the corresponding control group. S7

Table S3. Gene transcription changes in the brains of adult medaka (F and F1) after parental exposure to various concentrations of PFBS (, 1., 2.9 and 9.5 µg/l). a Brain F F1 Genes Male Female µg/l 1. µg/l 2.9 µg/l 9.5 µg/l µg/l 1. µg/l 2.9 µg/l 9.5 µg/l CRH 1.1±.4 1.2±.2 1.3±.3.9±.1 1.2±.5 1.1±.2 2.4±.7.8±.1 CRHR 1.1±.3.8±..9±.2.8±.2 1.1±.3.7±.2 1.2±.2 1.±.2 TSHβ 1.2±.6.6±.2.7±.2 1.2±.3 1.±.2 1.1±.2 1.7±.2.6±.1 TRα 1.3±.5 1.2±.2 1.1±.3.9±.3 1.4±.8.8±.3 2.7±.5.7±.1 TRβ 1.1±.3 1.3±.2 1.2±.2 1.1±.2 1.2±.5.8±.1 1.4±.1.7±.1 Dio1 1.1±.2 1.±.2 1.1±.3.6±.1 1.1±.3 1.±.3 1.7±.3.6±.1 Dio2 1.±.2.7±..5±.1*.6±.1* 1.1±.4.5±. 1.1±.2.5±. Dio3 1.2±.4 1.1±.2 1.2±.3 1.±.2 1.2±.4.8±.3 1.2±.2.7±. MCT8 1.3±.5 1.±.1.9±.2.8±.2 1.4±.8.8±.2 1.8±.4.7±.1 SLCO1c1 1.1±.4 1.2±.2 1.±.2.7±. 1.2±.5 1.±.1 1.4±.2.7±.1 CRH 1.1±.3 1.6±.1.9±.1.6±. 1.±. 1.3±.2.6±.*.6±.* CRHR 1.±.2 1.1±.1.8±.1.7±.1 1.±.1 1.2±.3.7±.1.8±.1 TSHβ 1.1±.3 4.9±1.6.9±.2.4±.1 1.±.1 1.2±.1.6±.1*.5±.* TRα 1.±.1 1.3±. 1.±.2.7±.1 1.±.1 1.±.1.3±.***.3±.*** TRβ 1.±.2 1.5±.* 1.±..9±.1 1.±. 1.2±.2.7±.*.7±. Dio1 1.1±.3 1.3±..7±..6±. 1.±..9±.1.4±.***.4±.*** Dio2 1.1±.2 1.2±..8±.1.6±.1 1.±. 1.1±.2.4±.**.4±.** Dio3 1.±.2 1.5±.1.9±.1.7±.1 1.±.1 1.1±.1.7±.1*.6±.1* MCT8 1.±.2 1.±.1.8±.1.7±.1 1.±.1 1.±.2.9±.1.9±. SLCO1c1 1.1±.3 1.4±.1.9±.1.7±. 1.±..9±..4±.***.5±.*** S8

a Values represent the mean ± SEM of three replicates. *P <.5, **P <.1 and ***P <.1 indicate significant difference between exposure groups and the corresponding control group. S9

Table S4. Gene transcription changes in the medaka offspring (F1 and F2) after parental exposure to various concentrations of PFBS (, 1., 2.9 and 9.5 µg/l). a Genes F1 offspring (15 dpf) F2 offspring (15 dpf) µg/l 1. µg/l 2.9 µg/l 9.5 µg/l µg/l 1. µg/l 2.9 µg/l 9.5 µg/l CRH 1.±..6±.***.9±..7±.*** 1.±..8±..6±.* 1.3±.2 CRHR 1.±.2.8±.1.7±..7±. 1.±.1 1.1±.2.7±. 1.1±. TSHβ 1.±.1.7±.1 1.±.1.9±.2 1.1±.3 1.2±.6.4±.1.8±.3 TSHR 1.±.1.6±.1*.7±.*.8±. 1.±.1 1.±.1.5±.1 1.1±.2 NIS 1.±..5±.1*.6±.* 1.±.1 1.±. 1.1±.1.8±. 1.3±.1 TPO 1.±..6±.1*.8±.*.7±.1* 1.±..8±.1*.5±.1*** 1.1±. TG 1.±.1.4±.***.6±.*.5±.** 1.±. 1.±.1.6±.* 1.1±.1 TBG 1.±..4±.***.6±.***.5±.*** 1.±. 1.±..6±.** 1.4±.1** TRα 1.±..8±.1.8±..5±.1** 1.±.1 1.2±.5.5±.1.9±.2 TRβ 1.±..8±.* 1.±. 1.±. 1.±..9±..6±.** 1.1±.1 Dio1 1.±.1.6±.***.3±.***.3±.*** 1.±. 1.±..7±.** 1.6±.2*** Dio2 1.±..9±.1.9±.1.9±.1 1.±.1 1.3±.5.6±.1.8±.2 Dio3 1.±.1.6±.***.5±.***.5±.*** 1.±..8±.1.6±.1* 1.3±.2 UGT1 1.±..6±.***.7±.*.8±.* 1.±.1.9±.1.9±.1 1.4±.1* SULT 1.±.1.5±.***.5±.***.4±.*** 1.±.1.9±..5±.*** 1.±.1 MCT8 1.±.1.7±.1.8±..9±.1 1.±.1 1.2±.2.8±.1.9±. SLCO1c1 1.±..8±.* 1.1±.*.8±.* 1.±..6±.*.5±.** 1.±.1 pax8 1.±.1.9±..8±..8±.1 1.±.1.8±..7±.1* 1.±. hhex 1.±.1.6±.**.7±.*.8±.* 1.±. 1.±.1.8±. 1.±.1 a Values represent the mean ± SEM of three replicates. *P <.5, **P <.1 and ***P <.1 indicate significant difference between exposure groups and the corresponding control group. S1

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