Key Lab of Modern Toxicology of Ministry of Education, Center for Global Health, School of Public Health, Nanjing Medical University, Nanjing, Jiangsu, China.
State Key Lab of Reproductive Medicine and Offspring Health, Institute of Toxicology, Nanjing Medical University, Nanjing, Jiangsu, China.
Environ Health Perspect. 2024 Aug;132(8):87003. doi: 10.1289/EHP14055. Epub 2024 Aug 12.
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.
This study aimed to explore the toxicity of naphtho[2,1-]pyrene (N21aP) and elucidate the mechanism underlying N21aP-induced expression of CYP1A1.
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 knockout mice exposed to N21aP (0.02, 0.2, and ) through intratracheal instillation. Further, the aryl hydrocarbon receptor (AhR) pathway was examined through luciferase and chromatin immunoprecipitation (ChIP) assays. -methyladenosine () modification levels were measured on global RNA and specifically on mRNA using dot blotting and methylated RNA immunoprecipitation-quantitative real-time polymerase chain reaction (MeRIP qRT-PCR), with validation by inhibitors, DAA and SAH. sites on were identified by bioinformatics and luciferase assays, and mRNA's interaction with IGF2BP3 was confirmed by RNA pull-down, luciferase, and RNA binding protein immunoprecipitation (RIP) assays.
N21aP was of the same environmental origin as benzo[]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 in mRNA via the METTL14-IGF2BP3-CYP1A1 axis. Specifically, in the two recognition sites of METTL14 on the 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 mRNA, and finally resulted in an increase in CYP1A1 expression.
This study systematically demonstrated that in addition to AhR-mediated transcriptional regulation, N21aP, had a new additional mechanism of -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.
目前，许多新兴多环芳烃（PAHs）已被发现广泛存在于环境中。然而，关于它们的毒性，特别是与 CYP1A1 的关系，报道甚少。
本研究旨在探讨萘并[2,1-j]苊（N21aP）的毒性，并阐明 N21aP 诱导 CYP1A1 表达的机制。
采用气相色谱-三重四极杆质谱联用（GC-MS/MS）和诊断比分析检测和分析 N21aP 的浓度和来源。然后，通过气管内滴注，在雄性野生型（WT）和敲除（KO）小鼠中研究 CYP1A1 对 N21aP 毒性的影响，浓度分别为 N21aP（0.02、0.2 和 ）。进一步通过荧光素酶和染色质免疫沉淀（ChIP）实验研究芳烃受体（AhR）途径。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）分别在全 RNA 和特定的 mRNA 上测量 -甲基腺苷（）修饰水平，并通过 DAA 和 SAH 抑制剂进行验证。通过生物信息学和荧光素酶实验鉴定 mRNA 上的 位点，通过 RNA 下拉、荧光素酶和 RNA 结合蛋白免疫沉淀（RIP）实验验证 IGF2BP3 与 mRNA 的相互作用。
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 可被 CYP1A1 代谢激活生成环氧化物，导致 DNA 损伤，进而导致肺部炎症。重要的是，除了经典的 AhR 途径（即 BaP）外，N21aP 还通过 METTL14-IGF2BP3-CYP1A1 轴，以外转录修饰 mRNA 的 来诱导 CYP1A1 表达。具体来说，在 mRNA 转录本的 METTL14 两个识别位点（位置 2700 和 5218）处，IGF2BP3 识别 3'-非翻译区（UTR）中 5218 位的一个甲基化位点，增强了 mRNA 的稳定性，最终导致 CYP1A1 表达增加。
本研究系统地表明，除了 AhR 介导的转录调节外，N21aP 还具有新的 METTL14 介导的 -介导的转录后修饰机制，共同导致 CYP1A1 表达。鉴于 PAHs 是 CYP1A1 的代谢底物，本研究不仅有助于理解环境-遗传相互作用对 PAHs 毒性的意义，还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.
