In this study, both endogenous and exogenous N2-hydroxymethyl-dG adducts in nasal DNA of rats exposed to 0.7, 2, 5.8, 9.1 or 15.2 ppm [13CD2] formaldehyde for 6 h were quantified by a highly sensitive nano-UPLC-MS/MS method. Our data clearly demonstrated that exogenous formaldehyde DNA adducts form in a highly nonlinear fashion, with a 21.7-fold increase in exposure causing a 286-fold increase in exogenous adducts. The ratio of exogenous/endogenous DNA adducts demonstrated that endogenous DNA adducts dominated at low exposures, comprising more than 99%. In contrast, exogenous adducts were not detectable in bone marrow of rats exposed to 15.2 ppm [13CD2] formaldehyde.

BackgroundThe beneficial outcome associated with the use of proton pump inhibitors (PPIs) in idiopathic pulmonary fibrosis (IPF) has been reported in retrospective studies. To date, no prospective study has been conducted to confirm these outcomes. In addition, the potential mechanism by which PPIs improve measures of lung function and/or transplant-free survival in IPF has not been elucidated.MethodsHere, we used biochemical, cell biological and preclinical studies to evaluate regulation of markers associated with inflammation and fibrosis. In our in vitro studies, we exposed primary lung fibroblasts, epithelial and endothelial cells to ionizing radiation or bleomycin; stimuli typically used to induce inflammation and fibrosis. In addition, we cultured lung fibroblasts from IPF patients and studied the effect of esomeprazole on collagen release. Our preclinical study tested efficacy of esomeprazole in a rat model of bleomycin-induced lung injury. Furthermore, we performed retrospective analysis of interstitial lung disease (ILD) databases to examine the effect of PPIs on transplant-free survival.ResultsThe cell culture studies revealed that esomeprazole controls inflammation by suppressing the expression of pro-inflammatory molecules including vascular cell adhesion molecule-1, inducible nitric oxide synthase, tumor necrosis factor-alpha (TNF-α) and interleukins (IL-1β and IL-6). The antioxidant effect is associated with strong induction of the stress-inducible cytoprotective protein heme oxygenase-1 (HO1) and the antifibrotic effect is associated with potent inhibition of fibroblast proliferation as well as downregulation of profibrotic proteins including receptors for transforming growth factor β (TGFβ), fibronectin and matrix metalloproteinases (MMPs). Furthermore, esomeprazole showed robust effect in mitigating the inflammatory and fibrotic responses in a murine model of acute lung injury. Finally, retrospective analysis of two ILD databases was performed to assess the effect of PPIs on transplant-free survival in IPF patients. Intriguingly, this data demonstrated that IPF patients on PPIs had prolonged survival over controls (median survival of 3.4 vs 2 years).ConclusionsOverall, these data indicate the possibility that PPIs may have protective function in IPF by directly modulating the disease process and suggest that they may have other clinical utility in the treatment of extra-intestinal diseases characterized by inflammatory and/or fibrotic phases.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-015-0614-x) contains supplementary material, which is available to authorized users.

The presence of endogenous and exogenous N2-hydroxymethyl-dG adducts in DNA from nasal mucosa and bone marrow of cynomolgus macaques exposed to 1.9 and 6.1 ppm of [13CD2]-formaldehyde for 6 hours a day for 2 consecutive days was investigated using a highly sensitive nano-UPLC-MS/MS method with a Limit of Detection of 20 amol. Both exogenous and endogenous adducts were readily detected and quantified in the nasal tissues of both exposure groups, with an exposure dependent increase in exogenous adducts observed. In contrast, only endogenous adducts were detectable in the bone marrow, even though ~10 times more DNA was analyzed.

Formaldehyde is not only a widely used chemical with well-known carcinogenicity but is also a normal metabolite of living cells. It thus poses unique challenges for understanding risks associated with exposure. N 2-hydroxymethyl-dG (N 2 -HOMedG) is the main formaldehyde-induced DNA mono-adduct, which together with DNA-protein crosslinks (DPCs) and toxicityinduced cell proliferation, play important roles in a mutagenic mode of action for cancer. In this study, N 2 -HOMe-dG was shown to be an excellent biomarker for direct adduction of formaldehyde to DNA and the hydrolysis of DPCs. The use of inhaled [ 13 CD 2 ]-formaldehyde exposures of rats and primates coupled with ultrasensitive nano ultra performance liquid chromatography-tandem mass spectrometry permitted accurate determinations of endogenous and exogenous formaldehyde DNA damage. The results show that inhaled formaldehyde only reached rat and monkey noses, but not tissues distant to the site of initial contact. The amounts of exogenous adducts were remarkably lower than those of endogenous adducts in exposed nasal epithelium. Moreover, exogenous adducts accumulated in rat nasal epithelium over the 28-days exposure to reach steady-state concentrations, followed by elimination with a half-life (t 1/2 ) of 7.1 days. Additionally, we examined artifact formation during DNA preparation to ensure the accuracy of nonlabeled N 2 -HOMe-dG measurements. These novel findings provide critical new data for understanding major issues identified by the National Research Council Review of the 2010 Environmental Protection Agency's Draft Integrated Risk Information System Formaldehyde Risk Assessment. They support a data-driven need for reflection on whether risks have been overestimated for inhaled formaldehyde, whereas underappreciating endogenous formaldehyde as the primary source of exposure that results in bone marrow toxicity and leukemia in susceptible humans and rodents deficient in DNA repair.

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