Division, National Toxicology Program, NIEHS, Durham, North Carolina, USA.
Molecular Genomics Core Laboratory, Signal Transduction Laboratory, Division of Intramural Research, NIEHS, Durham, North Carolina, USA.
J Appl Toxicol. 2023 Sep;43(9):1293-1305. doi: 10.1002/jat.4463. Epub 2023 Mar 28.
We recently developed a rat whole exome sequencing (WES) panel and used it to evaluate early somatic mutations in archival liver tissues from F344/N rats exposed to the hepatocarcinogen, Aflatoxin B1 (AFB1), a widely studied, potent mutagen and hepatocarcinogen associated with hepatocellular carcinoma (HCC). Rats were exposed to 1-ppm AFB1 in feed for 14, 90, and 90 days plus a recovery 60-day, non-exposure period (150-day) timepoint. Isolated liver DNA was exome sequenced. We identified 172 sequence variants across all timepoints, of which 101 were non-synonymous variants. Well-annotated genes carried a diverse set of 29 non-synonymous mutations at 14 days, increasing to 39 mutations at 90 days and then decreasing to 33 mutations following the 60-day recovery. Gene Set Enrichment Analysis conducted on previously reported, available RNA expression data of the same exome sequenced archival samples identified altered transcripts in pathways associated with malignant transformation. These included HALLMARK gene sets associated with cell proliferation (MYC Targets Version 1 and Version 2, E2F targets), cell cycle (G2M checkpoint, mitotic spindle), cell death (apoptosis), and DNA damage (DNA repair, UV response Up, Reactive oxygen species) pathways. DriverNet Impact analysis integrated exome-seq and expression data to reveal somatic mutations in Mcm8, Bdp1, and Cct6a that may drive cancer formation. Connectivity with transcript expression changes identified these genes as the top-ranked candidate driver genes associated with hepatocellular transformation. In conclusion, exome sequencing revealed early somatic mutations that may play a role in cancer cell transformation that are translatable to aflatoxin-induced HCC.
我们最近开发了一个大鼠全外显子组测序 (WES) 面板，并将其用于评估暴露于黄曲霉毒素 B1 (AFB1) 的 F344/N 大鼠存档肝组织中的早期体细胞突变，AFB1 是一种广泛研究的、有效的致突变剂和肝癌致癌物，与肝细胞癌 (HCC) 相关。大鼠以 1ppm 的 AFB1 饲料喂养 14、90 和 90 天，加 60 天的非暴露恢复期（150 天）时间点。分离的肝 DNA 进行外显子组测序。我们在所有时间点都鉴定了 172 个序列变异，其中 101 个是非同义变异。在 14 天时，经过良好注释的基因携带了一组多样化的 29 个非同义突变，在 90 天时增加到 39 个突变，在 60 天恢复期后减少到 33 个突变。对相同外显子组测序存档样本的先前报道的可用 RNA 表达数据进行基因集富集分析，鉴定出与恶性转化相关途径中改变的转录本。这些包括与细胞增殖（MYC 靶标版本 1 和版本 2、E2F 靶标）、细胞周期（G2M 检查点、有丝分裂纺锤体）、细胞死亡（细胞凋亡）和 DNA 损伤（DNA 修复、UV 反应 Up、活性氧）途径相关的 HALLMARK 基因集。DriverNet Impact 分析整合外显子组测序和表达数据，揭示了 Mcm8、Bdp1 和 Cct6a 中的体细胞突变，这些突变可能导致癌症形成。与转录表达变化的连接性确定这些基因为与肝细胞转化相关的排名最高的候选驱动基因。总之，外显子组测序揭示了早期的体细胞突变，这些突变可能在癌症细胞转化中发挥作用，可转化为黄曲霉毒素诱导的 HCC。
