有机磷阻燃剂TDCIPP诱导的m6A修饰引发雄性小鼠生殖毒性

目的 探讨TDCIPP染毒致雄性C57BL/6小鼠生殖毒性的机制。 方法 将24只4周龄C57BL/6小鼠根据体重随机分为四组，即5、25、125 mg/(kg·d) TDCIPP暴露组和玉米油对照组，灌胃染毒28 d后，HE染色观察小鼠睾丸组织形态学结构、附睾尾精子形态，并计算精子数量和畸形率；评估m6A修饰水平及m6A甲基转移酶（Mettl3、Mettl14、Wtap基因）、去甲基化酶（Alkbh5、Fto基因）mRNA水平；采用m6A - seq测序技术分析125mg/(kg·d) TDCIPP暴露后睾丸组织发生了m6A甲基化的基因差异表达情况；Western blot法检测凋亡相关蛋白Caspase - 3、Bax、Bcl - 2的表达。 结果 与对照组相比，TDCIPP暴露组小鼠睾丸组织结构均发生破坏；25和125 mg/(kg·d) TDCIPP暴露组附睾尾精子数量下降（P＜0.01）、精子畸形率上升（P＜0.01），存在剂量 - 效应关系。125 mg/(kg·d) TDCIPP暴露组小鼠睾丸组织的RNA m6A修饰水平和Mettl14基因mRNA表达水平显著增加（P＜0.01）；m6A - seq测序发现差异表达基因主要富集在凋亡、RAP1、PI3K/AKT、MAPK等信号通路；Caspase - 3蛋白水平在各暴露组均上升（P＜0.001），Bax/Bcl - 2比值在25和125 mg/(kg·d) TDCIPP暴露组上升（P＜0.001），均存在剂量 - 效应关系。 结论 TDCIPP暴露可诱导小鼠睾丸组织内甲基转移酶METTL14介导的m6A甲基化修饰异常，进而引发雄性小鼠生殖毒性。

Organophosphorus flame retardant TDCIPP-induced m6A modification leads to reproductive toxicity in male mice

Objective To investigate the reproductive toxicity of male mice induced by TDCIPP. Methods Twenty-four male C57BL/6 mice were randomly divided into four groups according to body weight, which exposed to corn oil (control) and TDCIPP (5, 25, 125mg/(kg·d)). After 28 days of intragastric exposure, the testicular structure and sperm morphology of caudal epididymis was observed using HE staining assay, and the number and abnormality rate of sperm were calculated. Total m6A modification level and m6A methyltransferase (Mettl3, Mettl14, Wtap), demethylase (Alkbh5, Fto) mRNA expression were measured. The differential expression of m6A methylated genes in testicular tissues after exposure to 125 mg/(kg·d) TDCIPP was analyzed by m6A-seq sequencing. Western blot was used to detect the expression of apoptosis related proteins such as Caspase-3 and Bax/Bcl-2. Results Compared with the control group, the testicular structure of mice exposed to TDCIPP was damaged. The number of sperm in caudal epididymis of mice exposed to 25 and 125 mg/(kg·d) TDCIPP decreased (P<0.01), and the rate of sperm deformity increased (P<0.01) in a dose dependent manner. In 125 mg/(kg·d) TDCIPP exposure group, the mRNA expression level of Mettl14 and RNA m6A modification level were significantly increased (P<0.01). Differentially expressed genes were mainly enriched in apoptosis, RAP1, PI3K/AKT, MAPK and other signaling pathways by m6A-seq analysis. Caspase-3 protein level increased in all exposure groups (P<0.001), and Bax/Bcl-2 ratio increased in 25 and 125 mg/(kg·d) TDCIPP exposure groups (P<0.001) in a dose dependent manner. Conclusion Exposure to TDCIPP can induce abnormal m6A methylation modification mediated by methyltransferase METTL14 in testes, leading to reproductive toxicity in male mice.