N 6 -Formyllysine as a Biomarker of Formaldehyde Exposure: Formation and Loss of N 6 -Formyllysine in Nasal Epithelium in Long-Term, Low-Dose Inhalation Studies in Rats

Exposure to both endogenous and exogenous formaldehyde has been established to be carcinogenic, likely by virtue of forming nucleic acid and proteins adducts such as N6-formyllysine. To better assess N6-formyllysine as a biomarker of formaldehyde exposure, we studied accumulation of N6-formyllysine adducts in tissues of rats exposed by inhalation to 2 ppm [13C2H2]-formaldehyde for 7, 14, 21, and 28 days (6 h/day) and investigated adduct loss over a 7-day postexposure period using liquid chromatography-coupled tandem mass spectrometry. Our results showed formation of exogenous adducts in nasal epithelium and to some extent in trachea but not in distant tissues of lung, bone marrow, or white blood cells, with a 2-fold increase over endogenous N6-formyllysine over a 3-week exposure period. Postexposure analyses indicated a biexponential decay of N6-formyllysine in proteins extracted from different cellular compartments, with half-lives of ∼25 and ∼182 h for the fast and slow phases, respectively, in cytoplasmic proteins. These results parallel the behavior of DNA adducts and DNA-protein cross-links, with protein adducts cleared faster than DNA-protein cross-links, and point to the potential utility of N6-formyllysine protein adducts as biomarkers of formaldehyde

Determination of N2-hydroxymethyl-dG Adducts in Nasal Epithelium and Bone Marrow of Non-human Primates following 13CD2-Formaldehyde Inhalation Exposure

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

Effect of 90Sr internal emitter on gene expression in mouse blood

Background The radioactive isotope Strontium-90 (90Sr) may be released as a component of fallout from nuclear accidents, or in the event of a radiological incident such as detonation of an improvised nuclear device, and if ingested poses a significant health risk to exposed individuals. In order to better understand the response to 90Sr, using an easily attainable and standard biodosimetry sample fluid, we analyzed the global transcriptomic response of blood cells in an in vivo model system. Results We injected C57BL/6 mice with a solution of 90SrCl2 and followed them over a 30-day period. At days 4, 7, 9, 25 and 30, we collected blood and isolated RNA for microarray analyses. These days corresponded to target doses in a range from 1–5 Gy. We investigated changes in mRNA levels using microarrays, and changes in specific microRNA (miRNA) predicted to be involved in the response using qRT-PCR. We identified 8082 differentially expressed genes in the blood of mice exposed to 90Sr compared with controls. Common biological functions were affected throughout the study, including apoptosis of B and T lymphocytes, and atrophy of lymphoid organs. Cellular functions such as RNA degradation and lipid metabolism were also affected during the study. The broad down regulation of genes observed in our study suggested a potential role for miRNA in gene regulation. We tested candidate miRNAs, mmu-miR-16, mmu-miR-124, mmu-miR-125 and mmu-mir-21; and found that all were induced at the earliest time point, day 4. Conclusions Our study is the first to report the transcriptomic response of blood cells to the internal emitter 90Sr in mouse and a possible role for microRNA in gene regulation after 90Sr exposure. The most dramatic effect was observed on gene expression related to B-cell development and RNA maintenance. These functions were affected by genes that were down regulated throughout the study, suggesting severely compromised antigen response, which may be a result of the deposition of the radioisotope proximal to the hematopoietic compartment in bone

Formation, Accumulation, and Hydrolysis of Endogenous and Exogenous Formaldehyde-Induced DNA Damage

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. N2-hydroxymethyl-dG (N2-HOMe-dG) is the main formaldehyde-induced DNA mono-adduct, which together with DNA-protein crosslinks (DPCs) and toxicity-induced cell proliferation, play important roles in a mutagenic mode of action for cancer. In this study, N2-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 [13CD2]-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 (t1/2) of 7.1 days. Additionally, we examined artifact formation during DNA preparation to ensure the accuracy of nonlabeled N2-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

Pleiotropic effect of the proton pump inhibitor esomeprazole leading to suppression of lung inflammation and fibrosis

Background: The 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. Methods: Here, 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. Results: The 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-alpha) and interleukins (IL-1 beta 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 beta (TGF beta), 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). Conclusions: Overall, 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.Stanford School of Medicine [1049528-149- KAVFB]; Tobacco-Related Disease Research Program of the University of California [20FT-0090]; National Institutes of Health National Heart, Lung, and Blood Institute [5K01HL118683, P01HL114470]; Houston Methodist Research Institute [25150001]; Stanford SPARK Translational Research ProgramSCI(E)[email protected]

Pharmacokinetic and Pharmacodynamic Effects of Polyclonal Antibodies against SARS-CoV2 in Mice

Despite ongoing vaccination efforts to prevent SARS-CoV-2 infections, treatment tools are still necessary to address the ongoing COVID-19 pandemic. We report here that COVID-HIGIV, a human immunoglobulin product for treatment of COVID-19, provided a significant survival benefit in SARS-CoV-2 infected transgenic mice compared to controls. COVID-HIGIV also has similar pharmacokinetic profiles in healthy and SARS-CoV-2 infected mice over time after intravenous administration, with identical or comparable Tmax, Cmax, AUC0–∞ and Cl. AUC0–last increased and mean residence time, T1/2, and Vd reduced in infected animals compared to healthy animals. These data suggest that COVID-HIGIV may be an effective treatment for SARS-CoV-2 infection when given early after exposure