| Objectives1. Screen sensitive endpoints and exposure duration of thyroid disruption.2. Study the dose-response relationship of PTU, PCBs, and AP on thyroid function in OVX rats respectively. Acquire toxicological reference points based on NOAEL/LOAEL method and benchmark dose analysis.3. Study the combined effect of PTU, PCBs, and AP on thyroid function in OVX rats based on3×2factorial designs and "dose addition" theory.MethodsThe study consists of three parts:1. Time-effect study of PTU on thyroid function of OVX rats Fifty healthy SD rats were randomly allocated into six groups based on body weight. The six groups were:sham control, OVX control, OVX4-day, OVX8-day, and OVX12-day group.4-day,8-day, and12-day groups were treated with5mg/kg bw PTU by gavage for4,8, and12days respectively. Sham and OVX controls were treated with corn oil. Endpoints examined include:serum T3, T4, and TSH, liver5’-DI and ME activity, thyroid organ/body weight ratio, epithelium/collid ratio.2. Study on dose-response relationships of PTU, PCBs, and AP on thyroid function in OVX rats One hundred-forty healthy SD rats were randomly allocated into fourteen groups based on body weight. They were:sham control, OVX control, PTU4groups(0.1,0.5,1.0,5.0mg/kg bw), PCBs4groups(0.1,1.0,5.0,10.0mg/kg bw), and AP4groups (50,100,250,500mg/kg bw). All dosed groups were treated with TDCs accordingly and controls were treated with corn oil for8consecutive days. Endpoints examined include:serum T3, T4, and TSH, liver5’-DI activity, thyroid organ/body weight ratio, epithelium/collid ratio. NOAEL/LOAEL values were analysed based on the experiment data. BMDS was employed to analyze dose-response relationships and generate BMDs and BMDLs for each TDC.3. Study on the combined effect of PTU, PCBs, and AP on thyroid function of OVX ratsSixty healthy SD rats were randomly allocated into six groups based on body weight. They were:OVX control, PTU+PCBs, PTU+AP, PCBs+AP and PTU+PCBs+AP based on LOAEL of each TDC, PTU+PCBs+AP based on BMDLs of serum T4(0.02+0.02+28.0mg/kg bw). Rats in all groups were removed with their ovaries bilaterally. All dosed groups were treated with combination of TDCs accordingly and OVX control was treated with corn oil for8days. Endpoints examined include:serum T3and T4, liver5’-DI activity, thyroid organ/body weight ratio, and epithelium/collid ratio.3x2factorial designs and "dose addition" method were employed to analyze the combined effect of the tested TDCs.Results1. Time-effect study of PTU on thyroid function in OVX ratsBody weight of rats in OVX groups were significantly higher than those in sham control (P<0.01) and no significance of such was observed among OVX groups. All PTU treated groups showed significant changes in serum T3, T4, TSH,5’-DI activity, thyroid organ/body weight ratio, and epithelium/colloid ratio (P<0.05), while no significant change in ME activity. Histopathological observation showed different level of hyperplasia epithelium cells, depletion of colloid, and congestion of red blood cells in thyroid vessels in different groups. The sensitive exposure duration will be analysed based on the experiment data. Despite ME activity, all other endpoints were significantly changed at day8. Comparing to those in12-d group, some endpoint including5’-DI, T3, T4, and thyroid epithelium/colloid ratio showed no significant changes.2. Dose-response relationship study of PTU, PCBs, and AP on thyroid function in OVX ratsBody weight of rats in OVX groups were significantly higher than those in OVX control (P<0.01) and no significance of body weight change was observed among OVX groups. All PTU treated groups showed significant changes in serum T3, T4, and epithelium/colloid ratio (P<0.05), while only PTU0.5,1.0, and5.0mg/kg bw groups showed significant changes in serum TSH,5’-DI activity, thyroid organ/body weight ratio. Histopathological observation showed different level of changes of thyroid tissue in different groups.All PCBs treated groups had significantly higher serum T4level (P<0.01) while showed no statistical difference in5’-DI activity and thyroid/body weight ratio. Only PCBs10.0mg/kg bw group showed significant decrease of serum T3(P<0.05); PCBs5.0and10.0mg/kg bw groups had significant higher serum TSH levels (P<0.05); PCBs1.0,5.0, and10.0groups showed significant changes in thyroid epithelium/colloid ratio (P<0.05). In PCBs O.lmg/kg bw groups, only minor histological change was observed. In other PCBs treated groups, different levels of histopathological changes can be seen.All AP treated groups showed significant changes in serum T4, TSH, thyroid/body weight ratio, and epithelium/colloid ratio (P<0.05), while no significant change in5’-DI activity. Only AP250and500mg/kg bw groups showed significant lower serum T3levels comparing with OVX control (P<0.05). In all AP treated groups, different levels of thyroid histopathological changes can be observed.Summarizing from all endpoints, only LOAELs were observed for PTU, PCBs, and AP, which were0.1,0.1, and50mg/kg bw. Form BMD analysis, sets of BMD and BMDL for PTU, PCBs, and AP were0.03and0.01,0.06and0.02,22and15mg/kg bw respectively.3. Study on the combined effect of PTU, PCBs, and AP on thyroid function in OVX ratsNo significance of body weight change was observed among OVX groups. All TDCs treated groups showed significant changes in serum T4, thyroid/body weight ratio, and epithelium/colloid ratio (P<0.05). Different levels of histopathological changes can be observed in all TDCs treated groups. ANOVA analysis for factorial design indicated:PTU and PCBs affected serum T3of OVX rats synergistically, serum T4and thyroid/body weight additively, epithelium/colloid ratio antagonistically; PTU and AP affected serum T3, T4, and thyroid/body weight ratio additively, epithelium/colloid ratio synergistically; PCBs and AP affected serum T3additively, serum T4, thyroid/body weight ratio, and epithelium/colloid ratio synergistically; PTU, PCBs, and AP affected serum T3and T4synergistically, thyroid epithelium/colloid ratio antagonistically, thyroid/body weight ratio additively. Results of "dose addition" method showed that all combined exposure groups act synergistically with each other on serum T4of OVX rats.Conclusion1. PTU can disrupt thyroid function of OVX rats with obvious time-effect mode. The sensitive exposure duration is8days and sensitive endpoints are serum T3, T4, and TSH, liver5’-DI activity, thyroid organ/body weight ratio, and epithelium/collid ratio.2. PTU, PCBs, and AP affect thyroid function of OVX rats with significant dose-response relationship. The LOAELs for PTU, PCBs and AP are0.1,0.1, and50mg/kg bw and BMDLs are0.01,0.02, and15mg/kg bw.3. The main modes of combined effect of PTU, PCBs, and AP are additivity and synergism. However, due to restrictions of different mechanisms and analyzing methods, further researches on combined effects of TDCs are needed. |
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