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Randomized Controlled Trial
. 2013 Jun;121(6):670-5.
doi: 10.1289/ehp.1205963. Epub 2013 Apr 12.

MicroRNA Expression in Response to Controlled Exposure to Diesel Exhaust: Attenuation by the Antioxidant N-acetylcysteine in a Randomized Crossover Study

Free PMC article
Randomized Controlled Trial

MicroRNA Expression in Response to Controlled Exposure to Diesel Exhaust: Attenuation by the Antioxidant N-acetylcysteine in a Randomized Crossover Study

Masatsugu Yamamoto et al. Environ Health Perspect. .
Free PMC article


Background: Adverse health effects associated with diesel exhaust (DE) are thought to be mediated in part by oxidative stress, but the detailed mechanisms are largely unknown. MicroRNAs (miRNAs) regulate gene expression post-transcriptionally and may respond to exposures such as DE.

Objectives: We profiled peripheral blood cellular miRNAs in participants with mild asthma who were exposed to controlled DE with and without antioxidant supplementation.

Methods: Thirteen participants with asthma underwent controlled inhalation of filtered air and DE in a double-blinded, randomized crossover study of three conditions: a) DE plus placebo (DEP), b) filtered air plus placebo (FAP), or c) DE with N-acetylcysteine supplementation (DEN). Total cellular RNA was extracted from blood drawn before exposure and 6 hr after exposure for miRNA profiling by the NanoString nCounter assay. MiRNAs significantly associated with DEP exposure and a predicted target [nuclear factor (erythroid-derived 2)-like 2 (NRF2)] as well as antioxidant enzyme genes were assessed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) for validation, and we also assessed the ability of N-acetylcysteine supplementation to block the effect of DE on these specific miRNAs. 8-hydroxy-2'-deoxyguanosine (8-OHdG) was measured in plasma as a systemic oxidative stress marker.

Results: Expression of miR-21, miR-30e, miR-215, and miR-144 was significantly associated with DEP. The change in miR-144 was validated by RT-qPCR. NRF2 and its downstream antioxidant genes [glutamate cysteine ligase catalytic subunit (GCLC) and NAD(P)H:quinone oxidoreductase 1 (NQO1)] were negatively associated with miR-144 levels. Increases in miR-144 and miR-21 were associated with plasma 8-hydroxydeoxyguanosine 8-OHdG level and were blunted by antioxidant (i.e, DEN).

Conclusions: Systemic miRNAs with plausible biological function are altered by acute moderate-dose DE exposure. Oxidative stress appears to mediate DE-associated changes in miR-144.

Conflict of interest statement

The authors declare they have no actual or potential competing financial interests.


Figure 1
Figure 1
The normalized nCounter data (left panels) and the expression levels measured with RT-qPCR (right panels) of miR-144 (A), miR-21 (B), and miR-30e (C) are shown. Dot plots show 13 individual participants’ data, boxes delineate first and third quartile, whiskers represent minima and maxima respectively, solid lines within boxes indicate medians. *FDR < 0.05.
Figure 2
Figure 2
Scatter plots of miR-144 compared with GCLC, NQO1, and NRF2. The plots show log2-transformed levels measured by RT-qPCR in post-FAP and post-DEP. The p-values refer to the association analyzed using limma.
Figure 3
Figure 3
The effect of antioxidant on miRNAs and association with oxidative stress. (A,B) RT-qPCR plots of two miRNAs for three exposures. The normalized RT-qPCR data comparing FAP, DEP, and DEN are shown for miR-144 (A) and miR-21 (B). (C) Scatter plots of miR-144 compared with 8-OHdG of post-DEP and post-DEN; the x-axis represents log2-transformed expression, and the y-axis represents plasma 8-OHdG level on log2 axis. The p-value refers to the association analyzed with a linear mixed effect model. *FDR < 0.05.

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