Inducible nitric oxide synthase (iNOS; encoded by nitric oxide synthase isoform 2 [NOS2]) is the major enzyme for nitric oxide synthesis in airways. As such, measurement of fractional concentration of exhaled nitric oxide (Feno) provides an in vivo assessment of iNOS activity. Short-term exposure to air pollution, haplotypes, and DNA methylation in the NOS2 promoter has been associated independently with iNOS expression, Feno levels, or both.

We aimed to examine the effects of ambient air pollutants, NOS2 promoter haplotypes, and NOS2 promoter methylation on Feno levels in children.

We selected 940 participants in the Children’s Health Study who provided buccal samples and had undergone Feno measurement on the same day. DNA methylation was measured with a bisulfite-PCR Pyrosequencing assay. Seven single nucleotide polymorphisms captured the haplotype diversity in the NOS2 promoter. Average particulate matter with an aerodynamic diameter of 2.5 μm or less (PM_2.5) and 10 μm (PM₁₀) or less and ozone and nitrogen dioxide levels 7 days before Feno measurement were estimated based on air pollution data obtained at central monitoring sites.

We found interrelated effects of PM_2.5, NOS2 promoter haplotypes, and iNOS methylation on Feno levels. Increased 7-day average PM_2.5 exposure was associated with lower iNOS methylation (P = .01). NOS2 promoter haplotypes were globally associated with NOS2 promoter methylation (P = 6.2 × 10⁻⁸). There was interaction among 1 common promoter haplotype, iNOS methylation level, and PM_2.5 exposure on Feno levels (P_interaction = .00007).

Promoter variants in NOS2 and short-term PM_2.5 exposure affect iNOS methylation. This is one of the first studies showing contributions of genetic and epigenetic variations in air pollution–mediated phenotype expression.