Photocaged Dicarbonyl Probe Provides Spatiotemporal Control Over Protein Glycation

Protein glycation is a disease associated, non-enzymatic, posttranslational modification generated by endogenous dicarbonyl metabolites. Currently, there is a lack of chemical tools capable of studying protein adducts caused by this...

Quo vadis blood protein adductomics?

Chemicals are measured regularly in air, food, the environment, and the workplace. Biomonitoring of chemicals in biological fluids is a tool to determine the individual exposure. Blood protein adducts of xenobiotics are a marker of both exposure and the biologically effective dose. Urinary metabolites and blood metabolites are short term exposure markers. Stable hemoglobin adducts are exposure markers of up to 120 days. Blood protein adducts are formed with many xenobiotics at different sites of the blood proteins. Newer methods apply the techniques developed in the field of proteomics. Larger adducted peptides with 20 amino acids are used for quantitation. Unfortunately, at present the methods do not reach the limits of detection obtained with the methods looking at single amino acid adducts or at chemically cleaved adducts. Therefore, to progress in the field new approaches are needed.

Pharmacological Activation Of Aldehyde Dehydrogenase 2 Protects Against Heatstroke-Induced Acute Lung Injury by Modulating Oxidative Stress and Endothelial Dysfunction

Heatstroke (HS) can cause acute lung injury (ALI). Heat stress induces inflammation and apoptosis via reactive oxygen species (ROS) and endogenous reactive aldehydes. Endothelial dysfunction also plays a crucial role in HS-induced ALI. Aldehyde dehydrogenase 2 (ALDH2) is a mitochondrial enzyme that detoxifies aldehydes such as 4-hydroxy-2-nonenal (4-HNE) protein adducts. A single point mutation in ALDH2 at E487K (ALDH2*2) intrinsically lowers the activity of ALDH2. Alda-1, an ALDH2 activator, attenuates the formation of 4-HNE protein adducts and ROS in several disease models. We hypothesized that ALDH2 can protect against heat stress-induced vascular inflammation and the accumulation of ROS and toxic aldehydes. Homozygous ALDH2*2 knock-in (KI) mice on a C57BL/6J background and C57BL/6J mice were used for the animal experiments. Human umbilical vein endothelial cells (HUVECs) were used for the in vitro experiment. The mice were directly subjected to whole-body heating (WBH, 42°C) for 1 h at 80% relative humidity. Alda-1 (16 mg/kg) was administered intraperitoneally prior to WBH. The severity of ALI was assessed by analyzing the protein levels and cell counts in the bronchoalveolar lavage fluid, the wet/dry ratio and histology. ALDH2*2 KI mice were susceptible to HS-induced ALI in vivo. Silencing ALDH2 induced 4-HNE and ROS accumulation in HUVECs subjected to heat stress. Alda-1 attenuated the heat stress-induced activation of inflammatory pathways, senescence and apoptosis in HUVECs. The lung homogenates of mice pretreated with Alda-1 exhibited significantly elevated ALDH2 activity and decreased ROS accumulation after WBH. Alda-1 significantly decreased the WBH-induced accumulation of 4-HNE and p65 and p38 activation. Here, we demonstrated the crucial roles of ALDH2 in protecting against heat stress-induced ROS production and vascular inflammation and preserving the viability of ECs. The activation of ALDH2 by Alda-1 attenuates WBH-induced ALI in vivo.

The mechanism of cell death induced by silver nanoparticles is distinct from silver cations

Background Precisely how silver nanoparticles (AgNPs) kill mammalian cells still is not fully understood. It is not clear if AgNP-induced damage differs from silver cation (Ag+), nor is it known how AgNP damage is transmitted from cell membranes, including endosomes, to other organelles. Cells can differ in relative sensitivity to AgNPs or Ag+, which adds another layer of complexity to identifying specific mechanisms of action. Therefore, we determined if there were specific effects of AgNPs that differed from Ag+ in cells with high or low sensitivity to either toxicant. Methods Cells were exposed to intact AgNPs, Ag+, or defined mixtures of AgNPs with Ag+, and viability was assessed. The level of dissolved Ag+ in AgNP suspensions was determined using inductively coupled plasma mass spectrometry. Changes in reactive oxygen species following AgNP or Ag+ exposure were quantified, and treatment with catalase, an enzyme that catalyzes the decomposition of H2O2 to water and oxygen, was used to determine selectively the contribution of H2O2 to AgNP and Ag+ induced cell death. Lipid peroxides, formation of 4-hydroxynonenol protein adducts, protein thiol oxidation, protein aggregation, and activation of the integrated stress response after AgNP or Ag+ exposure were quantified. Lastly, cell membrane integrity and indications of apoptosis or necrosis in AgNP and Ag+ treated cells were examined by flow cytometry. Results We identified AgNPs with negligible Ag+ contamination. We found that SUM159 cells, which are a triple-negative breast cancer cell line, were more sensitive to AgNP exposure less sensitive to Ag+ compared to iMECs, an immortalized, breast epithelial cell line. This indicates that high sensitivity to AgNPs was not predictive of similar sensitivity to Ag+. Exposure to AgNPs increased protein thiol oxidation, misfolded proteins, and activation of the integrated stress response in AgNP sensitive SUM159 cells but not in iMEC cells. In contrast, Ag+ cause similar damage in Ag+ sensitive iMEC cells but not in SUM159 cells. Both Ag+ and AgNP exposure increased H2O2 levels; however, treatment with catalase rescued cells from Ag+ cytotoxicity but not from AgNPs. Instead, our data support a mechanism by which damage from AgNP exposure propagates through cells by generation of lipid peroxides, subsequent lipid peroxide mediated oxidation of proteins, and via generation of 4-hydroxynonenal (4-HNE) protein adducts. Conclusions There are distinct differences in the responses of cells to AgNPs and Ag+. Specifically, AgNPs drive cell death through lipid peroxidation leading to proteotoxicity and necrotic cell death, whereas Ag+ increases H2O2, which drives oxidative stress and apoptotic cell death. This work identifies a previously unknown mechanism by which AgNPs kill mammalian cells that is not dependent upon the contribution of Ag+ released in extracellular media. Understanding precisely which factors drive the toxicity of AgNPs is essential for biomedical applications such as cancer therapy, and of importance to identifying consequences of unintended exposures.

Pyrrolizidine Alkaloid-Induced Hepatotoxicity Associated with the Formation of Reactive Metabolite-Derived Pyrrole–Protein Adducts

Pyrrolizidine alkaloids (PAs) with 1,2-unsaturated necine base are hepatotoxic phytotoxins. Acute PA intoxication is initiated by the formation of adducts between PA-derived reactive pyrrolic metabolites with cellular proteins. The present study aimed to investigate the correlation between the formation of hepatic pyrrole–protein adducts and occurrence of PA-induced liver injury (PA-ILI), and to further explore the use of such adducts for rapidly screening the hepatotoxic potency of natural products which contain PAs. Aqueous extracts of Crotalaria sessiliflora (containing one PA: monocrotaline) and Gynura japonica (containing two PAs: senecionine and seneciphylline) were orally administered to rats at different doses for 24 h to investigate PA-ILI. Serum alanine aminotransferase (ALT) activity, hepatic glutathione (GSH) level, and liver histological changes of the treated rats were evaluated to assess the severity of PA-ILI. The levels of pyrrole–protein adducts formed in the rats’ livers were determined by a well-established spectrophotometric method. The biological and histological results showed a dose-dependent hepatotoxicity with significantly different toxic severity among groups of rats treated with herbal extracts containing different PAs. Both serum ALT activity and the amount of hepatic pyrrole–protein adducts increased in a dose-dependent manner. Moreover, the elevation of ALT activity correlated well with the formation of hepatic pyrrole–protein adducts, regardless of the structures of different PAs. The findings revealed that the formation of hepatic pyrrole–protein adducts—which directly correlated with the elevation of serum ALT activity—was a common insult leading to PA-ILI, suggesting a potential for using pyrrole–protein adducts to screen hepatotoxicity and rank PA-containing natural products, which generally contain multiple PAs with different structures.

Iron Replacement and Redox Balance in Non-Anemic and Mildly Anemic Iron Deficiency COPD Patients: Insights from a Clinical Trial

In COPD patients, non-anemic iron deficiency (NAID) is a common systemic manifestation. We hypothesized that in COPD patients with NAID, iron therapy may improve systemic oxidative stress. The FACE (Ferinject assessment in patients with COPD and iron deficiency to improve exercise tolerance) study was a single-blind, unicentric, parallel-group, placebo-controlled clinical trial (trial registry: 2016-001238-89). Sixty-six patients were enrolled (randomization 2:1): iron arm, n = 44 and placebo arm, n = 22, with similar clinical characteristics. Serum levels of 3-nitrotyrosine, MDA-protein adducts, and reactive carbonyls, catalase, superoxide dismutase (SOD), glutathione, Trolox equivalent antioxidant capacity (TEAC), and iron metabolism biomarkers were quantified in both groups. In the iron-treated patients compared to placebo, MDA-protein adducts and 3-nitrotyrosine serum levels significantly declined, while those of GSH increased and iron metabolism parameters significantly improved. Hepcidin was associated with iron status parameters. This randomized clinical trial evidenced that iron replacement elicited a decline in serum oxidative stress markers along with an improvement in GSH levels in patients with stable severe COPD. Hepcidin may be a surrogate biomarker of iron status and metabolism in patients with chronic respiratory diseases. These findings have potential clinical implications in the management of patients with severe COPD.