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.
Discussion
This study systematically demonstrated that in addition to AhR-mediated transcriptional regulation, N21aP, had a new additional mechanism of m6Am6A<math><mrow><mrow><msup><mrow><mi>m</mi></mrow><mrow><mn>6</mn></mrow></msup></mrow><mi>A</mi></mrow></math>-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.
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/kg2mg/kg<math><mrow><mn>2</mn><mspace></mspace><mi>mg</mi><mo>/</mo><mi>kg</mi></mrow></math>) through intratracheal instillation. Further, the aryl hydrocarbon receptor (AhR) pathway was examined through luciferase and chromatin immunoprecipitation (ChIP) assays. N6N6<math><mrow><mrow><msup><mrow><mi>N</mi></mrow><mrow><mn>6</mn></mrow></msup></mrow></mrow></math>-methyladenosine (m6Am6A<math><mrow><mrow><msup><mrow><mi>m</mi></mrow><mrow><mn>6</mn></mrow></msup></mrow><mi>A</mi></mrow></math>) 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 m6Am6A<math><mrow><mrow><msup><mrow><mi>m</mi></mrow><mrow><mn>6</mn></mrow></msup></mrow><mi>A</mi></mrow></math> inhibitors, DAA and SAH. m6Am6A<math><mrow><mrow><msup><mrow><mi>m</mi></mrow><mrow><mn>6</mn></mrow></msup></mrow><mi>A</mi></mrow></math> 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 m6Am6A<math><mrow><mrow><msup><mrow><mi>m</mi></mrow><mrow><mn>6</mn></mrow></msup></mrow><mi>A</mi></mrow></math> 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.