由+3价砷甲基转移酶介导的三氧化二砷甲基化代谢途径对砷剂所致急慢性肝损的影响及机制研究

Arsenic trioxide (ATO) plays an important role in the treatment of acute promyelocytic leukemia (APL) combined with all-trans retinoic acid （ATRA） by multiple mechanisms, including degradation of PML-RARα fusion protein and elimination of leukemia initiating cells, making APL curable. It has been focused by many clinical trials that ATO is a promising medication in the front-line therapy for APL. However, various acute and chronic adverse effects and potential carcinogenicity caused by ATO are the main concerns preventing it from becoming front-line therapy. Studies in chemistry and toxicology has proved that orally ingested inorganic arsenic in +3 oxidation state (iAsⅢ) and +5 oxidation state (iAsⅤ) can be metabolized in the liver through methylation catalyzed by arsenite (+3 oxidation state) methyltransferase (AS3MT), which is considered as detoxification of arsenicals. Nevertheless, little systematic investigation is taken in the metabolism and toxicity of intravenous ATO. Our previous work showed that acute and chronic hepatotoxicity induced by ATO was the main problems among patients off ATO therapy, which is correlated to arsenic methylation index. On the basis of previous studies, this project is aimed to compare the difference of hepatotoxicity under oxidative stress between inorganic and organic arsenicals, to elucidate the relationship among AS3MT mRNA, arsenic methylation index, and hepatotoxicity, to evaluate the risk of chronic hepatic injury by monitoring dynamic changes in arsenic methylation index, and to discover the protective effect of recombinant human AS3MT for acute and chronic hepatotoxicity induced by ATO, thus introducing new sight into ATO as front-line therapy for APL.

三氧化二砷(ATO)通过联合全反式维甲酸(ATRA)降解PML-RARα融合蛋白、清除白血病起始细胞等机制使急性早幼粒细胞白血病(APL)成为可被治愈的疾病。最近诸多临床研究聚焦于ATO进入APL的一线用药，但各种急慢性毒副作用和潜在的致肿瘤作用是阻碍其成为一线用药的主要顾虑。化学、毒理学等研究表明，经消化道摄入的+3价(iAsⅢ)和+5价(iAsⅤ)无机砷在肝脏中经+3价砷甲基转移酶(AS3MT)甲基化代谢解毒，但有关静脉ATO(iAsⅢ)的代谢和毒性尚无系统研究。我们前期工作发现ATO在人体内致急慢性肝损问题较突出，与砷甲基化指数相关。本研究拟在前期工作的基础上比较无机和有机砷对肝细胞氧化应激损伤的差异，研究AS3MT mRNA、砷甲基化指数和肝细胞损伤之间的相关性，监测砷甲基化指数以判断发生慢性肝损的危险性，及探索重组人AS3MT的保护作用，为推动ATO成为APL一线用药提供新依据。

三氧化二砷(ATO)通过联合全反式维甲酸(ATRA)使急性早幼粒细胞白血病(APL)成为可被治愈的疾病。最近欧美诸多临床研究聚焦于ATO成为低中危APL的一线用药。但ATO的急慢性毒副作用和潜在的致肿瘤风险也是当前学者的主要顾虑。化学、毒理学等研究已证实无机砷是在肝脏中经+3价砷甲基转移酶(AS3MT)甲基化代谢解毒，但有关静脉ATO的代谢和毒性尚无系统研究。. 我们前期工作发现，112例患者中有42.9%的肝脂肪变性发生率，以及17.2%的慢性肝功能不全发生率。并且这部分患者的尿液一甲基化指数（PMI）均明显降低（P值分别为0.027和0.047）。ATO在人体内致急慢性肝损问题较突出，与砷甲基化指数相关。. 在前期工作的基础上，我们首先延续了APL患者的砷检测工作，发现停药6个月以上的患者体内无明显TAs蓄积，但患者尿液中毒性较高的无机砷所占比例明显高于健康对照（P值<0.001）。提示停用ATO之后，患者对砷的甲基化解毒代谢能力已明显低于健康对照，导致相对较多的iAs蓄积。同时体外研究表明，无机砷引起肝细胞氧化应激相关代谢产物的升高和肝细胞凋亡远大于有机砷。. 进一步研究发现，AS3MT基因的单核苷酸多态性（SNPs）可影响个体对砷的甲基化代谢能力。在18个报道的SNPs中，发现2个SNPs（A9749G和A27215G），野生型（AA）的尿液二甲基指数（SMI）均明显低于突变型（AG和GG）。说明突变型的砷二甲基化能力提高。这两个SNPs都位于AS3MT基因的内含子上，目前尚缺乏文献报道，本研究或可帮助确立这两个SNPs的功能。. 最后，通过给AS3MT基因敲除小鼠模型喂药，我们再次证明了缺乏AS3MT酶的小鼠，砷解毒代谢能力大大降低（PMI明显降低，P值=0.004），同时转氨酶检测和肝脏病理也证实了更易发生急慢性肝损。短期给药-停药后加入重组人AS3MT可逆转慢性肝脂肪变性，但对急性肝损无明显改善。说明AS3MT或可弥补SNPs差异带来的代谢能力不足，但短期高浓度药物暴露引起的急性损伤仍难以避免。. 本研究证实了AS3MT SNPs、砷甲基化指数和肝损之间的相关性，临床中可将PMI、SMI作为监测指标，指导早期评估和干预，推动ATO成为APL治疗的一线用药。