The Developmental Toxicity Of Triphenyltin On Xenopus Tropicalis Embryo

In recent decades, the global amphibian population declines seriously. A lot of investigation and research show that environmental pollutants are highly involved in this phenomenon. Triphenyltin (TPT) has been widely used in crop fungicides, wood preservatives, ship anti fouling paint and so on. It’s a typical environmental endocrine disruptor. However, the overall impact of TPT on the toxicity of amphibians has not been fully studied. In this thesis, Xenopus tropicalis embryo was used as a model species for the study of the teratogenic effects and possible mechanisms of TPT by solution exposure and in ovo exposure. The results will provide a basis for the effect of TPT on amphibian populations.Based on our previous results in the frog embryo teratogenesis assay-Xenopus (FETAX), we exposed X. tropicalis embryos to 10 μgSn/L TPT solution from S8 (blastocyst stage) for four seperate 12-h periods. The results showed the survival rate declined and whole body length reduced in TPT treatment groups. TPT also induced malformations, such as narrow fin, enlarged proctodaeum, hypopigmentation and turbid lens. Narrow fin was the most obviously malformation. Among the four 12-h periods, embryos were the most sensitive to TPT during a 24-36 h exposure period (about stage32 to stage40) based on the value of the score of malformations.24-36 h exposure period was the sensitive window of TPT.After that, we further explored the possible affected pathway of TPT using the whole genome expression microarray chip. The results showed that 452 genes significantly up-regulated or down regulated in X. tropicalis after exposure to 1-10 μgSn/L TPT during sensitive windows (Fold Change ≥2, P< 0.05). These genes were mainly enriched in ATP binding cassette transporters, vascular smooth muscle contraction and PPAR pathway. Quantitative PCR results verified that TPT inhibited the expression of PPARa and PPARy, and greatly induced their downstream genes mmpl and scdl.Based on the results above, since apoptosis plays an important role in amphibian metamorphosis and tissue reconstruction, we detected the in situ apoptosis cell through TDT mediated dUTP nick Nick-End labeling asaay. The results showed that 1-10 μgSn/L TPT significantly increased the apoptosis level in fin during the sensitive windows. The maximum apoptosis density in fin was occurred in 5 μgSn/L TPT group, and it was 3.22 times of the control group. We detected the expression levels of 16 apoptosis related genes using quantitative PCR, and 13 of them had significantly changes when compared to the control groups. Genes like caspase9 and p53 play important roles in this apoptosis pathway. Apoptosis induced by TPT may be the main reason for the narrow fin malformation.To explore the developmental toxicity of TPT to X. tropicalis embryos through maternal transfer, we injected TPT into embryo at stage 2. This in ovo exposure advanced the starting exposure point in FETAX (stage 8) and also simulated the maternal transfer toxicity of TPT in environment. Survival rate and the whole body length of embryos were affected after TPT (1-5 ng) injection. The most featured malformations caused by TPT in ovo exposure were microcephaly and small eye (even no eye). Different from the FETAX assay results, fin was not affected through in ovo exposure. On the other hand, we detected the spatial expression of the head and eye marker genes in TPT injected embryos (at stage 20 and stage 25) through whole mount in situ hybridization. The expression region of bfl (forebrain marker), en2 (midbrain marker), krox20 (hindbrain marker) and pax6 (eye marker) were significantly reduced, and some of the signals were dispersed. The sensitive stage properties of TPT in two exposure methods were different.At the same time, we also conducted the PPAR gamma agonist (rosiglitazone) and antagonist (T0070907) in ovo exposure experiments. The results showed that rosiglitazone was less toxic to the embryos, but T0070907 (5 ng) in ovo exposure induced the similar malformations of TPT (e.g. microcephaly and small eye). The combined injection of T0070907 and TPT also showed an obviously synergistic effect. These results provide important clues for us to study the relationship between TPT and PPAR.To sum up, no matter through the TPT in water or maternal transferred TPT, it has a strong developmental toxicity to the amphibian embryos. PPAR pathway and apoptosis played an important role in the toxicological mechanism of TPT. This paper has enriched the research on the toxicity of TPT to vertebrate embryos, and has made some contribution to the toxicological mechanism of TPT.