■目前,已经发现许多新兴的多环芳烃(PAHs)广泛存在于环境中。然而,关于它们毒性的报道很少,特别是与CYP1A1有关。
■本研究旨在探讨萘并[2,1-a]芘(N21aP)的毒性,并阐明N21aP诱导CYP1A1表达的潜在机制。
■通过气相色谱-三重四极杆质谱(GC-MS/MS)和诊断比率分析来检测和分析N21aP的浓度和来源。然后在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。Further,通过荧光素酶和染色质免疫沉淀(ChIP)测定检查了芳烃受体(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(MeRIPqRT-PCR),在全局RNA上和特异性地在CYP1A1mRNA上测量N6-甲基腺苷(m6A)修饰水平,通过m6A抑制剂的验证,DAA和SAH.通过生物信息学和荧光素酶测定鉴定CYP1A1上的M6A位点,CYP1A1mRNA与IGF2BP3的相互作用通过RNA下拉法得到证实,荧光素酶,和RNA结合蛋白免疫沉淀(RIP)测定。
■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代谢激活以产生环氧化物,导致DNA损伤并进一步导致肺部炎症。重要的是,除了经典的AhR途径(即,BaP),N21aP还通过METTL14-IGF2BP3-CYP1A1轴在CYP1A1mRNA中的m6A转录后修饰诱导CYP1A1表达。具体来说,在CYP1A1mRNA转录物上的METTL14的两个识别位点(位置2700和5218),IGF2BP3识别3'-非翻译区(UTR)中的甲基化位点(5218位),增强了CYP1A1mRNA的稳定性,并最终导致CYP1A1表达增加。
■这项研究系统地证明,除了AhR介导的转录调控外,N21aP,有一种新的m6A介导的转录后修饰机制,共同促进CYP1A1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新出现的PAHs在环境暴露水平下的健康风险。https://doi.org/10.1289/EHP14055.
UNASSIGNED: 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.
UNASSIGNED: This study aimed to explore the toxicity of naphtho[2,1-a]pyrene (N21aP) and elucidate the mechanism underlying N21aP-induced expression of CYP1A1.
UNASSIGNED: 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.
UNASSIGNED: 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.
UNASSIGNED: 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.