Primary Study On Shot-time In Vivo Screening And Testing Methods For Thyroid Hormone Disruptors

Thyroid hormone disruptors is a group of chemicals which alter thefunction and stability of thyroid hormones by affecting their synthesis,transportation, disassembly, etc. As thyroid hormone involves in individualgrowth, metabolism and has crucial importance in development of neuronalsystem, it is necessary to establish a valid method for discriminating thethyroid hormone disruptors. At present, the internationally accepteddiscriminating method for thyroid hormones is stratified, i.e. firstly, primaryscreening for verdicting whether certain chemical is a thyroid hormonedisruptor, then an identification of higher level may go on. Thus the primaryscreening calls for a quick, sensitive but economical technique, however,there is no such a method availble internationally at present.Some scholars proposed that the measure of concetration of FT4 andTSH combined with histological assessment of thyroid gland can be a potentdiscriminating method and the experimental period needs at least 2～6weeks. Apparently, the length of the time is the main barrier in establishinga primary screening system. The essential way of solve this problem isascertaining a stable and early onset morphological feature as the end pointfor observation, thereby shortening the time for screening and making the method feasible.Our present project uses PTU as the model drug and aims to find aneffective method for in vivo short-time standarised discriminating systemfor the thyroid hormone disruptors. The research involved in this projectincluiding: 1. Dose-effect relationship of PTU and thyroid glandhyperplasia, 2. Time-effect relationship of PTU and thyroid glandhyperplasia, 3. Histogenesis of thyroid gland hyperplasia caused by PTU, 4.Validating and consummating of method for in vivo short-time standariseddiscriminating system for the thyroid hormone disruptors using PTU asmodel drug.This present study aimes at the first and second parts, probes into thedose design principle of PTU, compares the sensitivity of male and femaleanimals, and provides the fundations for experimental design for time-effectresearch by studying dose-effect of PTU and thyroid gland hyperplasia.Validating the sensitive index and its reliability and providing experimentalguidelines for method for in vivo short-time standarised discriminatingsystem for the thyroid hormone disruptors using PTU as model drug.In dose-effect study, 100 health 7-week old SD rats were divided into 5groups (4 experimental and 1 control groups) randomly, 20 in each groupwith two sexes half and half. The treated rats were orally administeredvarious doses of PTU (i.e. 0.04, 0.2, 1.0 and 5.0 mg/kg.d). In experimentalgroup PTU was lavaged with corn oil 1ml/100g body weight for ten days,while control group lavaged with same amount of oil without PTU for thesame length of time. Changes in body weight and food intake were observedduring experimental period. We examined the viscera coefficients of thyroidgalnd and pathology changes of thyroid tissue by hematoxylin-eosin (HE) dyeing, and PAS after 10 days. The results showed that no significantchanges were observed in each group in terms of body weight, weightincrease and food intake either in male or female animals including thegroup with highest dose 5mg/(kg.d), whilst the viscera coefficients ofthyroid galnd in significantly high in 5mg/(kg.d) group comparing withcontrol group. Pathological observations found that thyroid follicularepithelium hyperplasia was aggravated gradually with dose increase in bothsexes, and in 5ml/(kg.d) group the hyperplasia almost suffused the wholevisual scale. The morphorlogical characteristics showed that in both sexespathological changes mainly manifested by hyperplastic patch in0.04mg/(kg.d) and 0.2mg/(kg.d) groups; solid bubble in 1.0mg/(kg.d) group;and solid sequential follicles and hollow sequential follicles in 5.0mg/(kg.d)group with visible hyperplasia of interstitial fibric cell and capillaryendothelium cell. PAS staining showed that in both sexes in 0.04mg/(kg.d),0.2mg/(kg.d) and 1 mg/(kg.d) groups thyroid gland follicle colloid was weakpositive, while in 5mg/(kg.d) group was negative. The above resultssuggested: 1. The dose for in vivo short-time discriminating test should usethe dose without affecting body weight and food intake of animals; 2. Thehyperplastic rules of thyroid follicle may be described as follow:stratification→hyperplastic patch→solid bubble→solid sequential follicle→hollow sequential follicle, whilst 5mg/(kg.d) was the most appropriatedose for time-effect study; 3. Both sexes of SD rats had the same sensitivityto thyroid gland hyperplastic response, single sex of SD rats will be neede inlater research.In time-effect study, 50 health 7-week old male SD rats were dividedinto 5 groups (4 experimental and 1 control groups) randomly, 10 in each group. The treated rats were orally administered 5mg/(kg.d) of PTU for 3, 6,9, and 12 days respectively, which was dissolved in corn oil and made asuspension liquid of 0.5mg/ml. In experimental group the PTU suspensionwas lavaged by 1ml/100g bw for a period of corresponding days, whilecontrol group lavaged with same amount of com without PTU for 12 days.Changes in serum T₃, T₄ and TSH concentration were detected bychemoluminescence method at the end of the experiment. We also examinedthe viscera coefficients of thyroid galnd and pathology changes in thyroidtissue by HE staining and changes in colloid component by PAS staining.Expression of PCNA protein was detected by immunohistochemistry andwestern blotting. The results showed that concentration of TT₃ decreasedfrom day 3 and reached its lowest point at day 12; concentration of TT₄started to decrease from day 6 and reached its lowest point at day 12;concentrartion of TSH increased from day 6 and arrived its peak at day 12.Viscera coefficients of thyroid galnd became significantly higher thancontrol group from day 6 and showed an increase trand with longerexposure. Pathological findings exhibited thyroid follicle epitheliumstratification in day 3 tissue, diffused hyperplasia and solid bubbles in day 6tissue, aggragated fibric cell and capillary endothelium cell hyperplasia,formation of intersected solid bubbles and solid sequential follicle in day 9tissue and mass of hollow sequential follicle in day 12 tissue. PAS stainingshowed weak positive in thyroid follicle colloid in day 3, 6, 9 tissue sectionsand negative in day 12 sections. Changes in numbers of PCNA positive cellsdetected by immunohistochemistry and expression ratio of PCNA/β-actindetected by western blotting showed a similar rule along with the time, i.e.increased from day 3 and reached its peak at day 6, started to decrease from day 9 and no significant difference comparing with the control group andback to normal at day 12. The results suggested: 1. TT₃ and TT₄ could notbe compensated even at 12day, as the difficulties in quality control of serumindex measurement, changes in serum thyroid hormones and concentraionof TSH can not be used as the only exclusive proof for thyroid hormonedisruptors; 2. It attested to the previous result that thyroid hyperplasiafollowed the herein rule "stratification→hyperplasitic patch→solid bubble→solid sequential follicle→hollow sequential follicle" and thyroid goiterfollowed the rule "hyperplasia→differentiation→re- hyperplasia→re-differentiation"; 3. The rules of time-course of PCNA expression wasobservable and reliable; hyperplastic patch and solid bubble used as earlyindex for hyperplasia was stable and relient; 4. The primary experimentalend point for short-time discriminating test for thyroid hormone disruptorswere serum concentration of TT3, TT4, TSH combined with pathologicalindications like hyperplastic patch and solid bubble and positve cell ofPCNA detected by immunohistochemistry.