Genotoxic, epigenetic, and transcriptomic effects of tamoxifen in mouse liver

Tamoxifen is a non-steroidal anti-estrogenic drug widely used for the treatment and prevention of breast cancer in women; however, there is evidence that tamoxifen is hepatocarcinogenic in rats, but not in mice. Additionally, it has been reported that tamoxifen may cause non-alcoholic fatty liver disease (NAFLD) in humans and experimental animals. The goals of the present study were to (i) investigate the mechanisms of the resistance of mice to tamoxifen-induced hepatocarcinogenesis, and (ii) clarify effects of tamoxifen on NAFLD-associated liver injury. Feeding female WSB/EiJ mice a 420 p.p.m. tamoxifen-containing diet for 12 weeks resulted in an accumulation of tamoxifen-DNA adducts, (E)-alpha-(deoxyguanosin-N2-yl)-tamoxifen (dG-TAM) and (E)-alpha-(deoxyguanosin-N2-yl)-N-desmethyltamoxifen (dG-DesMeTAM), in the livers. The levels of hepatic dG-TAM and dG-DesMeTAM DNA adducts in tamoxifen-treated mice were 578 and 340 adducts/108 nucleotides, respectively, while the extent of global DNA and repetitive elements methylation and histone modifications did not differ from the values in control mice. Additionally, there was no biochemical or histopathological evidence of NAFLD-associated liver injury in mice treated with tamoxifen. A transcriptomic analysis of differentially expressed genes demonstrated that tamoxifen caused predominantly down-regulation of hepatic lipid metabolism genes accompanied by a distinct over-expression of the lipocalin 13 (Lcn13) and peroxisome proliferator receptor gamma (Pparg), which may prevent the development of NAFLD. The results of the present study demonstrate that the resistance of mice to tamoxifen-induced liver carcinogenesis may be associated with its ability to induce genotoxic alterations only without affecting the cellular epigenome and an inability of tamoxifen to induce the development of NAFLD. Female and male WSB/EiJ mice were fed diet containing 420 p.p.m. tamoxifen for 12 weeks, and then tamoxifen-DNA adduct formation and gene expression profiles in the livers from four control and four tamoxifen-treated mice were investigated.他莫昔芬（Tamoxifen）是一类非甾体抗雌激素药物，广泛应用于女性乳腺癌的治疗与预防；但现有研究证据表明，他莫昔芬可诱发大鼠肝癌，却不会对小鼠产生此类致癌效应。此外，已有文献报道他莫昔芬可能在人类及实验动物中引发非酒精性脂肪性肝病（non-alcoholic fatty liver disease, NAFLD）。本研究旨在达成两个目标：(i) 探究小鼠对他莫昔芬诱导肝癌发生产生抗性的分子机制；(ii) 阐明他莫昔芬对NAFLD相关肝损伤的影响效应。给雌性WSB/EiJ小鼠喂食含有420 ppm（百万分比浓度）他莫昔芬的饲料12周后，其肝脏中可检测到两类他莫昔芬-DNA加合物的累积：(E)-α-(脱氧鸟苷-N2-基)-他莫昔芬（dG-TAM）与(E)-α-(脱氧鸟苷-N2-基)-N-去甲基他莫昔芬（dG-DesMeTAM）。经他莫昔芬处理的小鼠肝脏中，dG-TAM与dG-DesMeTAM DNA加合物的水平分别为每10^8个核苷酸578个和340个加合物，而全基因组DNA、重复元件甲基化及组蛋白修饰水平与对照组小鼠无显著差异。此外，经他莫昔芬处理的小鼠未表现出NAFLD相关肝损伤的生化或组织病理学证据。对差异表达基因的转录组分析结果显示，他莫昔芬主要下调肝脏脂质代谢相关基因的表达，同时显著上调脂质运载蛋白13（lipocalin 13, Lcn13）与过氧化物酶体增殖物激活受体γ（peroxisome proliferator receptor gamma, Pparg）的转录水平，这一表达谱变化或可阻止NAFLD的发生发展。本研究结果表明，小鼠对他莫昔芬诱导的肝癌发生产生抗性，可能与其仅能诱发基因毒性改变、不影响细胞表观基因组，以及无法诱导NAFLD发生的特性相关。本实验将雌性与雄性WSB/EiJ小鼠喂食含420 ppm他莫昔芬的饲料12周，随后对4只对照组小鼠与4只他莫昔芬处理组小鼠的肝脏组织中的他莫昔芬-DNA加合物形成情况及基因表达谱进行了检测。