Malignant mesothelioma is an aggressive cancer that often originates in the pleural and peritoneal mesothelium. Exposure to asbestos is a frequent cause. However, studies in rodents have shown that certain multiwalled carbon nanotubes (MWCNTs) can also induce malignant mesothelioma. The exact mechanisms are still unclear. To gain further insights into molecular pathways leading to carcinogenesis, we analyzed tumors in Wistar rats induced by intraperitoneal application of MWCNTs and amosite asbestos. Using transcriptomic and epigenetic approaches, we compared the tumors by inducer (MWCNTs or amosite asbestos) or by tumor type (sarcomatoid, epithelioid, or biphasic). Genome-wide transcriptome datasets, whether grouped by inducer or tumor type, showed a high number of significant differentially expressed genes (DEGs) relative to control peritoneal tissues. Bioinformatic evaluations using Ingenuity Pathway Analysis (IPA) revealed that while the transcriptome datasets shared commonalities, they also showed differences in DEGs, regulated canonical pathways, and affected molecular functions. In all datasets, among highly- scoring predicted canonical pathways were Phagosome Formation, IL8 Signaling, Integrin Signaling, RAC Signaling, and TREM1 Signaling. Top-scoring activated molecular functions included cell movement, invasion of cells, migration of cells, cell transformation, and metastasis. Notably, we found many genes associated with malignant mesothelioma in humans, which showed similar expression changes in the rat tumor transcriptome datasets. Furthermore, RT-qPCR revealed downregulation of Hrasls, Nr4a1, Fgfr4, and Ret or upregulation of Rnd3 and Gadd45b in all or most of the 36 tumors analyzed. Bisulfite sequencing of Hrasls, Nr4a1, Fgfr4, and Ret revealed heterogeneity in DNA methylation of promoter regions. However, higher methylation percentages were observed in some tumors compared to control tissues. Lastly, global 5mC DNA, m6A RNA and 5mC RNA methylation levels were also higher in tumors than in control tissues. Our findings may help better understand how exposure to MWCNTs can lead to carcinogenesis. This information is valuable for risk assessment and in the development of safe-by-design strategies.

中文翻译：

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多壁碳纳米管和铁石棉诱导的大鼠恶性腹膜间皮瘤：转录组和表观遗传谱

恶性间皮瘤是一种侵袭性癌症，通常起源于胸膜和腹膜间皮。接触石棉是一个常见原因。然而，对啮齿动物的研究表明，某些多壁碳纳米管（MWCNT）也可以诱发恶性间皮瘤。确切的机制仍不清楚。为了进一步了解导致致癌的分子途径，我们分析了 Wistar 大鼠腹腔内应用多壁碳纳米管和铁石棉诱发的肿瘤。使用转录组学和表观遗传学方法，我们通过诱导物（MWCNT 或铁石棉）或肿瘤类型（肉瘤样、上皮样或双相）对肿瘤进行比较。全基因组转录组数据集，无论是按诱导物还是肿瘤类型分组，都显示相对于对照腹膜组织有大量显着差异表达的基因（DEG）。使用 Ingenuity Pathway Analysis (IPA) 进行的生物信息学评估表明，虽然转录组数据集具有共同点，但它们也显示出差异表达基因、调节的规范途径和影响的分子功能的差异。在所有数据集中，高分预测的经典途径包括吞噬体形成、IL8 信号传导、整合素信号传导、RAC 信号传导和 TREM1 信号传导。得分最高的激活分子功能包括细胞运动、细胞侵袭、细胞迁移、细胞转化和转移。值得注意的是，我们发现了许多与人类恶性间皮瘤相关的基因，这些基因在大鼠肿瘤转录组数据集中表现出类似的表达变化。此外，RT-qPCR 显示在所分析的 36 个肿瘤的全部或大部分中，Hrasls、Nr4a1、Fgfr4 和 Ret 下调，或者 Rnd3 和 Gadd45b 上调。Hrasls、Nr4a1、Fgfr4 和 Ret 的亚硫酸氢盐测序揭示了启动子区域 DNA 甲基化的异质性。然而，与对照组织相比，在一些肿瘤中观察到更高的甲基化百分比。最后，肿瘤中的整体 5mC DNA、m6A RNA 和 5mC RNA 甲基化水平也高于对照组织。我们的研究结果可能有助于更好地了解接触多壁碳纳米管如何导致致癌。这些信息对于风险评估和制定安全设计策略非常有价值。