Developmental toxicity of triphenyltin to Xenopus tropicalis embryo
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摘要: 有机锡的外暴露能引起脊椎动物胚胎的独特畸形，但通过母体传递引起的内暴露毒性还缺乏研究.本文通过显微注射的方式研究了三苯基锡（TPT）、过氧化物酶体增殖体激活受体γ（PPARγ）的激动剂罗格列酮（Rosi）和抑制剂T0070907（C12H8ClN3O3）对热带爪蟾胚胎的发育毒性.将3种化合物注入S1-S2期的胚胎后，胚胎的存活率显著下降，其中5 ng TPT，80 ng Rosi和10 ngT0070907，注射组存活率分别为46.9%、42.7%和54.2%.胚胎的体长也受到不同程度的影响5 ng TPT，80 ng Rosi和20 ng T0070907注射组与对照相比体长分别减少了27%、22%和57%.3种化合物还引起了多样的畸形效应，尤其是头部变小及眼睛畸形.说明，PPARγ在热带爪蟾早期胚胎的发育特别是头、眼发育中扮演非常重要的角色.TPT与相近剂量T0070907引起的畸形非常相似，说明TPT的致畸机制可能与PPARγ存在某种关系.另一方面，利用整胚原位杂交检测了注射TPT后S20及S25期胚胎的头、眼部标志基因的空间表达，结果表明bf1、en2、krox20及pax6的表达信号均随TPT剂量增大而逐渐变弱且区域变小，定量PCR进一步验证了TPT能在神经胚及早期尾牙期之前影响胚胎头、眼标志基因的表达.研究结果表明TPT的内暴露对脊椎动物胚胎具有较强的致畸效应和神经毒性.
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关键词:
- 三苯基锡 (TPT) /
- 发育毒性 /
- 显微注射 /
- 热带爪蟾 /
- 胚胎
Abstract: Organotin compounds can lead to the unique malformations in vertebrate embryos after waterborne exposure, but the toxicity of organotin compounds to embryos through maternal transfer is still lack. In the present study, Xenopus tropicalis embryos were exposed to triphenyltin (TPT), the agonist (rosiglitazone, Rosi) and antagonist (T0070907) of peroxisome proliferator activated receptor gamma (PPARγ) through microinjection. Compared with the control, the survival rates and body length of embryos were significantly decreased in treatment groups. The survival rates were 46.9% (5 ng TPT), 42.7% (80 ng Rosi) and 54.2% (10 ng T0070907). The whole body lengths were reduced by 27% (5 ng TPT), 22% (80 ng Rosi) and 57% (20 ng T0070907). Three chemicals caused a variety of malformations including microcephaly, turbid lens of eyes and small eyes. These results indicated that PPARγ played an important role in embryonic development especially for eyes and brain development of Xenopus tropicalis. The phenotypes of malformation induced by TPT and T0070907 groups were highly identical, which suggested that the toxic mechanism of TPT might be related to PPARγ. After TPT treatment, brain and eye marker gene expression in embryos at stage20 and stage25 was detected using whole mount in situ hybridization. The results showed that en2, bf1, krox20 and pax6 expression regions were gradually decreased with the increase of the TPT doses. Quantitative PCR results further confirmed that TPT could affect the head and eye marker gene expression in neural and early tailbud stages. All the results indicated that organotin compounds showed high teratogenicity and neurotoxicity to vertebrate embryos.-
Key words:
- triphenyltin /
- developmental toxicity /
- microinjection /
- Xenopus tropicalis /
- embryo
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图 1 TPT、Rosi及T0070907内暴露对热带爪蟾胚胎存活、体长及畸形级数的影响
注: 各标准差均由3次平行实验结果得出; 暴露组与对照组相比* $P < $ 0.05, ** $P < $ 0.01, *** $P < $ 0.001
Fig. 1 Effects of TPT, Rosi and T0070907 in ovo exposure on the survival rate, whole body length and malformation degree in Xenopus tropicalis embryos
图 2 TPT、Rosi及T0070907内暴露对热带爪蟾胚胎的致畸效应
注: b头, bn脊柱弯曲, bt尾部弯曲, dcg唾液腺下移, e眼, eg唾液腺, eh围心腔水肿, elp泄殖腔膨大, hpo色素减少, mcp小头畸形, p泄殖腔, sp皮肤色素, st躯干拉长, t尾, te晶状体浑浊; 标尺=0.5 mm
Fig. 2 Teratogenic effects of TPT, Rosi and T0070907 in ovo exposure on Xenopus tropicalis embryos
图 3 TPT内暴露对热带爪蟾胚胎头部及眼部标志基因表达的影响
注: 红色箭头所示蓝紫色区域为整胚原位杂交信号; bf1, en2, krox20, pax6分别为热带爪蟾前脑、中脑、后脑、眼睛的标志基因; A-C组胚胎为S20期, 照片为背面观; D组胚胎为S25期, 照片为侧面观
Fig. 3 Effects of TPT in ovo exposure on brain and eye marker genes expression in Xenopus tropicalis embryos
图 4 TPT内暴露对热带爪蟾胚胎头部及眼部标志基因表达量的影响
注: 各标准差均由3次平行实验结果计算得出; 暴露组与对照组相比* $P < $ 0.05, ** $P < $ 0.01, *** $P < $ 0.001
Fig. 4 Effects of TPT in ovo exposure on brain and eye marker gene expression levels in Xenopus tropicalis embryos
表 1 热带爪蟾头眼部标志基因定量PCR引物序列表
Tab. 1 Oligonucleotide primers for real-time RT-PCR analysis of marker genes in Xenopus tropicalis
$\textbf{Genes}$ Sequence of forward primer (5'-3') Sequence of reverse primer (5'-3') Accession number bf1 CACTGGGGAGCAACCATTCT TTAAGGGAGTAGGTGCCCGA NW_004668241.1 en2 GAGATGAGTCCAACAGGGGG TCCTTCTTTCCTCCTCCCGA NW_004668239.1 krox20 TACTCGGCTCTCTTCCCTCC AGTTATGGGGGCTGTAAGCG NW_004668240.1 pax6 TTCCCAGGATCCAGACACCC TGTGCGCCTAGTGATTTCCC NW_004668236.1 β-actin TGCTGTTTTCCCATCCATTGT CTGTCCCATTCCAACCATGAC NM_213719.1 
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