BACKGROUND Currently, many emerging polycyclic aromatic hydrocarbons (PAHs) have been found to be widely present in the environment. However, little has been reported about their toxicity, particularly in relation to CYP1A1. OBJECTIVES This study aimed to explore the toxicity of naphtho[2,1-a]pyrene (N21aP) and elucidate the mechanism underlying N21aP-induced expression of CYP1A1. METHODS The concentration and sources of N21aP were detected and analyzed by gas chromatography-triple quadrupole mass spectrometry (GC-MS/MS) and diagnostic ratio analysis. Then the effects of CYP1A1 on the toxicity of N21aP were conducted in male wild-type (WT) and Cyp1a1 knockout mice exposed to N21aP (0.02, 0.2, and 2mg/kg) through intratracheal instillation. Further, the aryl hydrocarbon receptor (AhR) pathway was examined through luciferase and chromatin immunoprecipitation (ChIP) assays. N6-methyladenosine (m6A) modification levels were measured on global RNA and specifically on CYP1A1 mRNA using dot blotting and methylated RNA immunoprecipitation-quantitative real-time polymerase chain reaction (MeRIP qRT-PCR), with validation by m6A inhibitors, DAA and SAH. m6A sites on CYP1A1 were identified by bioinformatics and luciferase assays, and CYP1A1 mRNA's interaction with IGF2BP3 was confirmed by RNA pull-down, luciferase, and RNA binding protein immunoprecipitation (RIP) assays. RESULTS N21aP was of the same environmental origin as benzo[a]pyrene (BaP) but was more stably present in the environment. N21aP could be metabolically activated by CYP1A1 to produce epoxides, causing DNA damage and further leading to lung inflammation. Importantly, in addition to the classical AhR pathway (i.e., BaP), N21aP also induced CYP1A1 expression with a posttranscriptional modification of m6A in CYP1A1 mRNA via the METTL14-IGF2BP3-CYP1A1 axis. Specifically, in the two recognition sites of METTL14 on the CYP1A1 mRNA transcript (position at 2700 and 5218), a methylation site (position at 5218) in the 3'-untranslated region (UTR) was recognized by IGF2BP3, enhanced the stability of CYP1A1 mRNA, and finally resulted in an increase in CYP1A1 expression. DISCUSSION This study systematically demonstrated that in addition to AhR-mediated transcriptional regulation, N21aP, had a new additional mechanism of m6A-mediated posttranscriptional modification, jointly contributing to CYP1A1 expression. Given that PAHs are the metabolic substrates of CYP1A1, this study not only helps to understand the significance of environment-genetic interactions for the toxicity of PAHs but also helps to better understand the health risks of the emerging PAHs at environmental exposure levels. https://doi.org/10.1289/EHP14055.

中文翻译：

---

萘并[2,1-a]芘暴露对 CYP1A1 表达的影响：探索 m6A 转录后修饰作用的体内外机制研究。


背景技术目前，已发现许多新兴的多环芳烃(PAH)广泛存在于环境中。然而，关于其毒性的报道很少，特别是与 CYP1A1 相关的毒性。目的 本研究旨在探讨萘并[2,1-a]芘 (N21aP) 的毒性并阐明 N21aP 诱导 CYP1A1 表达的机制。方法采用气相色谱-三重四极杆质谱（GC-MS/MS）和诊断比分析对N21aP的浓度和来源进行检测和分析。然后通过气管内滴注暴露于N21aP（0.02、0.2和2mg/kg）的雄性野生型（WT）和Cyp1a1敲除小鼠中进行CYP1A1对N21aP毒性的影响。此外，通过荧光素酶和染色质免疫沉淀 (ChIP) 测定检查芳基碳氢化合物受体 (AhR) 通路。使用斑点印迹和甲基化 RNA 免疫沉淀-定量实时聚合酶链反应 (MeRIP qRT-PCR) 测量整体 RNA 上的 N6-甲基腺苷 (m6A) 修饰水平，特别是 CYP1A1 mRNA，并通过 m6A 抑制剂、DAA 和 SAH 进行验证。通过生物信息学和荧光素酶测定鉴定了 CYP1A1 上的 m6A 位点，并通过 RNA Pull-down、荧光素酶和 RNA 结合蛋白免疫沉淀 (RIP) 测定证实了 CYP1A1 mRNA 与 IGF2BP3 的相互作用。结果 N21aP 与苯并[a]芘 (BaP) 具有相同的环境来源，但在环境中更稳定地存在。 N21aP 可以被 CYP1A1 代谢激活，产生环氧化物，造成 DNA 损伤，进一步导致肺部炎症。重要的是，除了经典的 AhR 途径（即N21aP 还通过 METTL14-IGF2BP3-CYP1A1 轴对 CYP1A1 mRNA 中的 m6A 进行转录后修饰，从而诱导 CYP1A1 表达。具体来说，METTL14在CYP1A1 mRNA转录本上的两个识别位点（位置2700和5218）中，3'-非翻译区（UTR）的甲基化位点（位置5218）被IGF2BP3识别，增强了CYP1A1的稳定性mRNA，最终导致 CYP1A1 表达增加。讨论本研究系统地证明，除了 AhR 介导的转录调控之外，N21aP 还具有 m6A 介导的转录后修饰的新机制，共同促进 CYP1A1 的表达。鉴于PAHs是CYP1A1的代谢底物，这项研究不仅有助于了解环境-遗传相互作用对PAHs毒性的重要性，而且有助于更好地了解环境暴露水平下新兴PAHs的健康风险。 https://doi.org/10.1289/EHP14055。