Study On Establishment Of OECD Enhanced Guideline407Test And Its Application In Evaluation Of Endocrine Disruption Of Cadmium

Objectives:1. To investigate and establish sub-acute animal tests for detecting antithyroid effects, estrogen-like effects and antiestrogen effects, and to evaluate applicability and sensitiveness of additional parameters in detection of endocrine-mediated effects following OECD guidelines and methods for evaluation of endocrine disruption, i.e. the enhanced TG407test.2. To detect the endocrine disrupting of Cd by the enhanced TG407test, and compare the endocrine disrupting with renal toxicity to explore sensitive endpoint for health hazard evaluation of Cd.Methods:1. Select antithyroid drug propylthiouraci(FTU),synthetic estrogen170-ethinylestradiol(17β--EE2) and non-steroidal antiestrogen tamoxifen (TAM) as positive substances to investigate and establish enhanced TG407test in detecting antithyroid effects, estrogen-like effects and antiestrogen effects. Dosage of PTU was0.1,0.5,1,5,10mg/kg BW,17β-EE2was10,25,50,200,300μg/kg BW, and TAM was5,15,30,200,300μg/kg BW. Control group was given corn oil (vehicle control). All of these three experiments chose six-week old healthy SD rats, randomized into five treatment groups and one control group (10male and10female each group, gavage once a day for28days) by body weight. Results included:body weight, food consumption, haematology, clinical biochemistry, serum thyroid hormones (T3, T4) and TSH, serum sex hormone, the estrous cycle, endocrine organs weight and histopathology, etc.2. In evaluating the endocrine disrupting of Cd by enhanced TG407test, six-week old healthy SD rats were selected and randomized into six groups (8female and8male at each group) by body weight. Rats in treatment groups were administered1,2.5,5,10,20mg Cd /kg BW by gavage once a day for28days, respectively. Control group was given the vehicle distilled water. Body weight, food consumption, haematology, clinical biochemistry, serum thyroid hormones (T3,T4) and TSH, serum sex hormone, urine creatinine and urine β2-microglobulin, endocrine organs weight and histopathology, the estrous cycle were detected.Results:1. Applicability of the enhanced TG407test to evaluation of antithyroid effectsThe positive substance, PTU, could significantly reduce animal body weight, food intake and food consumption rate (P<0.05). In some treatment groups, increased RBC, HCT, HGB and decreased PLT as well as increased BUN, CRE and decreased TG were observed. Female serum TSH of1-10mg/kg BW dosage groups, male TSH of5-10mg/kg BW dosage groups significantly increased, while female serum T4at5-10mg/kg BW dosage groups and male at10mg/kg BW dosage group significantly reduced (P<0.05). Absolute and relative weight of thyroid of female at all treatment groups and that of male at0.5-10mg/kg BW dosage groups significantly increased, with low columnar epithelium of thyroid follicular, decreased lumens or occasionally thyroid follicular full of colloid. The absolute weight of ovary, pituitary, adrenal gland in relative high dosage groups significantly reduced(P<0.05), but no change of other organs was observed. All female rats in treatment groups showed normal estrous cycle.2. Applicability of the enhanced TG407test to evaluation of estrogen-like effectsThe positive substance,17β-EE2,could significantly decrease animal body weight, food intake and food consumption rate (P<0.05). In some treatment groups, increased RBC, HCT, HGB of male and decreased RBC, HCT, HGB of female were observed, and increased TG, ALP and decreased CHO were also observed. Increased serum LH, E2and decreased serum P were observed in female at25-300μg/kg BW,50-300μg/kg BW and50-300μg/kg BW dosage groups, respectively (P<0.05). Absolute and relative weight of uterus at all treatment groups significantly increased(P<0.05), with myometrium thickening and endometrial hyperplasia in high dosage groups. At200-300μg/kg BW dosage groups, absolute weight of female thyroid, testis, epididymis significantly reduced, while absolute weight of male adrenal gland significantly increased(P<0.05). At50-300μg/kg BW dosage groups, absolute weight of prostate+seminal vesicle glands significantly decreased,while relative weight of male adrenal gland significantly increased(P<0.05). Testis convolutedtubule atrophy, epididymis sperm reducing or disappearing, prostate gland and seminal vesicle gland atrophy were observed in some treatment groups.Most female animals at50-300μg/kg BW dosage groupswere in continuous estrogen stage with vaginal epithelial cutinization or thickening, reduced or degraded ovarian corpus luteum with mitochondria reduction were also observed;female pituitary acidophil reducing became serious with dosage increased.3. Applicability of the enhanced TG407test to evaluation of antiestrogen effectsThe positive substance, TAM, could significantly decrease animal body weight, food intake and food consumption rate (P<0.05). Increased RBC, HCT, HGB, serum ALT and decreased CHO were observed in some treatment groups. Female serum P and male serum E2at200-300|ig/kg BW dosage groups as well as male serum LH at30-300μg/kg BW dosage groups significantly increased(P<0.05). At200-300μg/kg BW dosage groups, absolute and relative weight of uterus, ovary and prostate+seminal vesicle glands significantly reduced, while relative weight of testis, epididymis significantly increased(P<0.05). All female rats in treatment groups showed normal estrous cycle. Female pituitary acidophil reducing and reduced or disappeared glycogen in vaginal epithelial cells became serious as dosage increased.5(5/5) ovarian corpus luteum degradation with rich heterochromatin and less organelles were observed at high dosage groups Endonmetrial showed atrophy by microvilli fracture or disappear and abnormal organelles. Seminal vesicle gland atrophy and testis abnormal were observed at high dosage groups.4. Application of the enhanced TG407test to evaluating endocrine disruption of CdCd could significantly decrease animal body weight, total food intake and total food consumption rate (P<0.05). In some treatment groups, increased RBC, HGB and decreased PLT of male were observed, and increased ALT, TG, GLU and decreased ALB, AST, TP, BUN were also observed. No change was found in urine β2-microglobulin (P>0.05). Decreased serum LH at10-20mg/kg BW dosage groups, serum T at2.5and10mg/kg BW dosage group and increased serum E2at20mg/kg BW dosage group were observed (P<0.05) Absolute and relative weight of uterus at10-20mg/kg BW dosage groups significantly increased, and endometrial epithelial cells and interstitial cell hyperplasia, rich organelles in endometrial epithelial cells were also observed. Absolute and relative weight of prostate+seminal vesicle glands at2.5,10,20mg/kg BW dosage groups significantly reduced (P <0.05). Absolute weight of thyroid at2.5-20mg/kg BW dosage groups significantly reduced (P<0.05). No histopathological change was observed in other organs, such as kidney.Conclusions:1) OECD enhanced TG407test could efficiently detect endocrine-mediated effects ingcluding antithyroid effects, estrogen-like effects and antiestrogen effects, and the additional parameters allowed reliably and sensitively detection of endocrine-mediated effects.2) Results of the enhanced TG407test showed that cadmium had endocrine disrupting effects, which was more sensitive than renal toxicity in this study.