The effect of BPDE on induced pluripotent stem cells of a Nijmegen Breakage Syndrome patient and healthy donors.

Benzo[a]pyrene diol epoxide (BPDE) is a highly carcinogenic metabolite of the environmental contaminant Benzo[a]pyrene, which is commonly found as a by-product of incomplete combustion of organic matter. BPDE reacts with the DNA to form BPDE-DNA bulky adducts which, if not removed, can lead to mutations due to DNA base-pair substitution and consequently to tumorigenesis. Due to its lipophilic nature, B[a]p can cross the placenta to reach the developing fetus and the placenta of mice, rats and humans can metabolize B[a]p into BPDE, which can lead to fetal genotoxic exposure. Besides direct embryonic exposure, BPDE-DNA adducts are detectable in the sperm and ovarian cells of cigarette smokers, and these DNA modifications can be paternally transmitted through the spermatozoa to the embryo. Human induced pluripotent stem cells (hiPSCs) can be used as a model of human embryonic stem cells (hESCs), an with that in mind, we treated hiPSCs with BPDE and performed a microarray analysis to evaluate for the first time the effects of BPDE exposure in pluripotent stem cells. Furthermore, hiPSCs derived from patients suffering from Nijmegen Breakage Syndrome, a chromosomal instability disorder characterized by microcephaly, defective DNA repair and increased risk of malignancies, were also investigated in the context of BPDE exposure and compared to their healthy counterparts. Transcriptomics analysis, coupled with protein content analysis through immunostaining and western blots, revealed that hiPSCs have a robust reaction to BPDE exposure, with an enhanced expression of several targets related to the p53 mediated DNA damage response, including DNA lesion bypass, cell cycle checkpoints and extrinsic apoptosis. Additionally, hiPSC harbouring an NBS mutation reacted differently to BPDE treatment than WT cells, showing less apoptotic response, no p53 or MDM2 protein increase, increased transcription of cancer-related targets and the repression of DNA-repair pathways transcription. The transcriptomes of iPSCs derived from healthy donors and Nijmegen Breakage Syndrome patients, with and without BPDE treatment, were compared by microarray analysis (PrimeView Human Gene Expression Array, Affymetrix, Thermo Fisher Scientific).

苯并[a]芘二醇环氧化物（Benzo[a]pyrene diol epoxide, BPDE）是环境污染物苯并[a]芘（Benzo[a]pyrene）的高致癌性代谢产物，而苯并[a]芘通常作为有机物不完全燃烧的副产物广泛存在。BPDE可与DNA结合形成BPDE-DNA大体积加合物，若未被机体清除，该加合物可因引发DNA碱基替换突变，进而导致肿瘤发生。由于苯并[a]芘具有亲脂特性，其可穿过胎盘屏障抵达发育中的胎儿；小鼠、大鼠及人类的胎盘均可将苯并[a]芘代谢为BPDE，从而造成胎儿的遗传毒性暴露。

除直接的胚胎暴露外，在吸烟者的精子与卵巢细胞中也可检测到BPDE-DNA加合物，这类DNA修饰可通过精子完成父系传递至胚胎。人类诱导多能干细胞（human induced pluripotent stem cells, hiPSCs）可作为人类胚胎干细胞（human embryonic stem cells, hESCs）的替代研究模型，基于此，本研究团队使用BPDE处理hiPSCs并开展微阵列分析，首次评估了BPDE暴露对多能干细胞的影响。此外，本研究还针对尼曼根断裂综合征（Nijmegen Breakage Syndrome, NBS）患者来源的hiPSCs展开了BPDE暴露相关研究，并与健康对照细胞进行对比——该综合征是一类以小头畸形、DNA修复功能缺陷及恶性肿瘤患病风险升高为特征的染色体不稳定紊乱症。

转录组学分析结合免疫染色与蛋白质印迹的蛋白质含量检测结果显示，hiPSCs对BPDE暴露存在强烈的应答反应，与p53介导的DNA损伤应答相关的多个靶基因表达均显著上调，包括DNA损伤旁路修复、细胞周期检验点及外源性凋亡相关通路。进一步研究发现，携带尼曼根断裂综合征突变（NBS突变）的hiPSCs与野生型细胞（wild-type, WT）对BPDE处理的应答存在显著差异：其凋亡反应更弱，未出现p53与MDM2蛋白水平的升高，癌症相关靶基因的转录水平上调，而DNA修复通路的转录则受到抑制。

本研究通过微阵列分析，采用PrimeView人类基因表达芯片（PrimeView Human Gene Expression Array, Affymetrix, Thermo Fisher Scientific），对经BPDE处理与未处理的健康供者及尼曼根断裂综合征患者来源的iPSCs转录组进行了对比分析。