Thyroidal Hormone Disrupting Effects and Histopathological Examination in an Amphibian Metamorphosis Assay

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1 Thyroidal Hormone Disrupting Effects and Histopathological Examination in an Amphibian Metamorphosis Assay Sumitomo Chemical Co., Ltd. Environmental Health Science Laboratory Kaori MYATA Keiko OSE The interference of a chemical with the thyroidal hormone system, that is the normal function of the hypothalamic-pituitary-thyroid (HPT) axis, is considered as an endocrine disrupting effect. The amphibian metamorphosis assay (AMA), which is an OECD and EPA guideline study, represents a generalized vertebrate model to evaluate such a chemical action not only on wildlife but also mammals. This review deals with the state-of-the-art of frog metamorphosis processes and thyroid hormone regulation, and introduces our attempts, especially the histopathological examination, for more precise evaluation of AMA. HPT OECDEPA Xenopus. laevisfig. ), ) Fig. Fig. A larva and a froglet of African clawed frogs (Xenopus laevis)

2 Feedback Hypothalamus TRH CRH GnRH Pituitary TSH Thyroid gland nhibition of T4 synthesis Decreased iodine uptake Peroxidase inhibition Disrupting proteolysis odine Thyroglobulin T4 T3 Competitive displacement from carrier proteins (TTR) Fig. Arrows indicate thyroid glands in Stage 5 T4 T3Fig. 3T4 T3 TTR 3) HPT Fig. 4 T4T3 T4T3 (a) O HO O CH OH NH Liver Excretion Conjugation T4 glucuronate T3 sulfonate Fig. 4 TTR ncreased thyroid hormone elimination Peripheral tissues T4 T3 Deiodination Altered local deiodination Overview of the thyroid hormone pathway and regulation Fig. 4 T3T4 4), 5) 6) P45 B 7) TTR T4T3 6), 8) TTR 9) ) TTR (b) HO Fig. 3 O CH NH Thyroxine: T4 (a), and 3,3,5- triiodothyronine: T3 (b) O OH 3 premetamorphosisprometamorphosismetamorphic climaxfig. 5

3 Stage 46 Premetamorphosis Head (Brain) Stage566 53/54 Prometamorphosis 57/58 ntestine & lung Metamorphic climax 66 Froglet Developmental stages are according to Nieuwkoop and Faber Fig. 5 The stage of metamorphosis. The premetamorphosis is the period until the appearance of the hind limbs, and the prometamorphosis is from their appearance to that of the forelimbs. During the period of metamorphic climax, resorption of the tail and gills and development of lungs occur. 3) 4) TSH 5) corticotrophin-releasing hormonecrh gonadotropin- releasing hormonegnrh TRHTSH 6) 8) luteinizing hormone-releasing hormonelhrh PRL 7), 9) ) ) 5) TSH Perchlorate 6), 7) polychlorinated biphenyls PCB 8) bisphenol ABPA 9) polybrominated diphenyl ethersbde47 pentabromodiphenyl etherde-7 3) methoxychlor 3), 3) acetochlor 33) 34) Athabasca Oil Sands 35)

4 4-methylbenzylidene camphor4-mbc3-benzylidene camphor3- BCUV 36) atrazine 37) Triclosan Veldhoen 38) FortGLP 39) 4) 44) HPT TSH HPT 45) amphibian metamorphosis assayama HPT TSH O Connor 46) 47) AMATable NieuwkoopFaber 48) AMA ), 5) Table 3 Table nitial larval stage Exposure period Larvae selection criteria Test concentrations Exposure regime Test system flow rate Larval density Test solution/ test vessel Replication Acceptable mortality rate in controls Water temperature Lighting Thyroid fixation ph Dissolved oxygen concentration Table Apical Endpoints Mortality Developmental stage Hind limb length Snout-vent length Wet body weight Thyroid gland histology Snout-vent length Protocol of the AMA Nieuwkoop and Faber stage 5 days Developmental stage and total length (optional) Minimum of 3 concentrations spanning approximately one order of magnitude Flow-through (preferred) and/ or static-renewal 5 ml/min larvae/test vessel (5 larvae/l) 4 L ( 5 cm minimum water)/ glass or stainless steel test vessel (e.g.,.5 cm 4 cm 6.5 cm) 4 replicate test vessels/ test concentration and control % per replicate test vessel ± C h Light : h dark, 6 to lux Davidson s fixative > 3.5mg/L ( > 4% air saturation) Observation time points for primary endpoints in the AMA Observation time points Daily Day 7 and, comply with N&F Stage Day 7 and Day 7 and Day 7 and Day, comply with guidance for histopathology Hind limb length Fig. 6 AMA

5 Table 3 Diagnostic criteria, severity and grading for histopathology in AMA Core criteria (severity graded) Thyroid gland hypertrophy/atrophy Follicular cell hypertrophy Follicular cell hyperplasia Additional criteria (severity graded and/or qualitatively described) Follicular lumen area: reduced or increased Colloid quality: homogeneous, heterogeneous, lacy or granular Follicular cell height/shape: squamous, cuboidal, low/high columnar Grading (For multifocal or diffusely-distributed alteration, the percentage of tissue area involved should be considered.) Grade (not remarkable to minimal, less than %) Grade (mild, 3-5%) Grade (moderate, 6-8%) Grade 3 (severe, over 8%) Fixation by Davidson s solution Trimming Dehydration Embedding by paraffin Slicing serial sections (5 7μm) Staining (Hematoxylin & Eosin) Observation & Picking suitable specimen up Observation & Evaluation Trim Embed 5), 5) AMA 53) AMA AMA T4 PropylthiouracilPTU Fig. 7PTU T4T3 TSH T4 Fig. 6 Histology procedure T4 TSH Fig. 8 (a) Control (b) PTU 5 ppm (c) T4 5. ppb PTU induces thyroid hypertrophy, follicular cell hyperplasia and altered colloid quality. T4 causes follicular atrophy. Fig. 7 Effect on thyroid histopathology

6 Group G: Control G: PTU.5ppm G3: PTU 5ppm G4: T4 ppb G5: T4 5.ppb G G G3 G4 G5 Thyroid hypertrophy G G G3 G4 G5 Thyroid atrophy G G G3 G4 G5 G G G3 G4 G5 G G G3 G4 G5 Follicular cell hyperplasia Follicular cell hypertrophy Reduced lumen area The scores show the mean of grade. Grade: ~3 Fig. 8 Result of histopathological examination TSH T4 53) HPT Fig. 9b 6 Fig. 9cPTU Fig. 7 Triclosan 4), 4) AMA (a) Control (b) Restricted feeding 6% (c) PTU 5ppm Restricted feeding induced thyroid atrophy (b). There is considerable morphological differences among individuals affected (c). Fig. 9 Effect of food intake and individual difference

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