Multitarget Antioxidant NO-Donor Organic Nitrates: A Novel Approach to Overcome Nitrates Tolerance, an Ex Vivo Study

Chronic use of glyceryl trinitrate (GTN) is limited by serious side effects, such as tolerance and endothelial dysfunction of coronary and resistance arteries. Although GTN is used as a drug since more than 130 years, the mechanisms of the vasodilatory effects and of tolerance development to organic nitrates are still incompletely elucidated. New synthesized organic nitrates with and without antioxidant properties were characterized for their ex vivo tolerance profile, in order to investigate the oxidative stress hypothesis of nitrate tolerance. The organic nitrates studied showed different vasodilation and tolerance profiles, probably due to the ability or inability of the compounds to interact with the aldehyde dehydrogenase-2 enzyme (ALDH-2) involved in bioactivation. Furthermore, nitrooxy derivatives endowed with antioxidant properties did not determine the onset of tolerance, even if bioactivated by ALDH-2. The results of this study could be further evidence of the involvement of ALDH-2 in the development of nitrate tolerance. Moreover, the behavior of organic nitrates with antioxidant properties supports the hypothesis of the involvement of ROS in inactivating ALDH-2.

Activation of PKCε-ALDH2 Axis Prevents 4-HNE-Induced Pain in Mice

Protein kinase Cε (PKCε) is highly expressed in nociceptor neurons and its activation has been reported as pro-nociceptive. Intriguingly, we previously demonstrated that activation of the mitochondrial PKCε substrate aldehyde dehydrogenase-2 (ALDH2) results in anti-nociceptive effects. ALDH2 is a major enzyme responsible for the clearance of 4-hydroxy-2-nonenal (4-HNE), an oxidative stress byproduct accumulated in inflammatory conditions and sufficient to induce pain hypersensitivity in rodents. Here we determined the contribution of the PKCε-ALDH2 axis during 4-HNE-induced mechanical hypersensitivity. Using knockout mice, we demonstrated that PKCε is essential for the nociception recovery during 4-HNE-induced hypersensitivity. We also found that ALDH2 deficient knockin mice display increased 4-HNE-induced nociceptive behavior. As proof of concept, the use of a selective peptide activator of PKCε (ΨεHSP90), which favors PKCε translocation to mitochondria and activation of PKCε-ALDH2 axis, was sufficient to block 4-HNE-induced hypersensitivity in WT, but not in ALDH2-deficient mice. Similarly, ΨεHSP90 administration prevented mechanical hypersensitivity induced by endogenous production of 4-HNE after carrageenan injection. These findings provide evidence that selective activation of mitochondrial PKCε-ALDH2 axis is important to mitigate aldehyde-mediated pain in rodents, suggesting that ΨεHSP90 and small molecules that mimic it may be a potential treatment for patients with pain.

Low-Dose Pesticides Alter Primary Human Bone Marrow Mesenchymal Stem/Stromal Cells through ALDH2 Inhibition

(1) Background: The impact of occupational exposure to high doses of pesticides on hematologic disorders is widely studied. Yet, lifelong exposure to low doses of pesticides, and more particularly their cocktail effect, although poorly known, could also participate to the development of such hematological diseases as myelodysplastic syndrome (MDS) in elderly patients. (2) Methods: In this study, a cocktail of seven pesticides frequently present in water and food (maneb, mancozeb, iprodione, imazalil, chlorpyrifos ethyl, diazinon and dimethoate), as determined by the European Food Safety Authority, were selected. Their in vitro effects at low-doses on primary BM-MSCs from healthy volunteers were examined. (3) Results: Exposure of normal BM-MSCs to pesticides for 21 days inhibited cell proliferation and promoted DNA damage and senescence. Concomitantly, these cells presented a decrease in aldehyde dehydrogenase 2 (ALDH2: mRNA, protein and enzymatic activity) and an increase in acetaldehyde levels. Pharmacological inhibition of ALDH2 with disulfiram recapitulated the alterations induced by exposure to low doses of pesticides. Moreover, BM-MSCs capacity to support primitive hematopoiesis was significantly altered. Similar biological abnormalities were found in primary BM-MSCs derived from MDS patients. (4) Conclusions: these results suggest that ALDH2 could participate in the pathophysiology of MDS in elderly people long exposed to low doses of pesticides.

E-cigarette aerosol exacerbates cardiovascular oxidative stress in mice with an inactive aldehyde dehydrogenase 2 enzyme

Aims: E-cigarette aerosol containing aldehydes, including acetaldehyde, are metabolized by the enzyme aldehyde dehydrogenase 2 (ALDH2). However, little is known how aldehyde exposure from e-cigarettes, when coupled with an inactivating ALDH2 genetic variant, ALDH2*2 (present in 8% of the world population), affects cardiovascular oxidative stress. The aim of this study was to determine how e-cigarette aerosol exposure, when coupled with genetics, impacts cardiovascular oxidative stress in wild type ALDH2 and ALDH2*2 knock-in mice. Methods and Results: Using selective ion flow mass spectrometry, we determined that e-cigarette aerosol contains acetaldehyde that are 10-fold higher than formaldehyde or acrolein. Next, using wild type ALDH2 and ALDH2*2 knock-in rodents, we identified organ-specific differences in ALDH2 with the heart having 1.5-fold less ALDH2 enzyme activity relative to the liver and lung. In isolated cardiac myocytes, acetaldehyde exposure (30seconds, 0.1-1μM) caused a 4-fold greater peak in calcium levels for ALDH2*2 relative to ALDH2 cardiomyocytes. ALDH2*2 cardiomyocytes exposed to acetaldehyde also demonstrated a 2-fold increase in ROS production and 2.5-fold increase in 4HNE protein adducts relative to ALDH2 cardiomyocytes. For intact rodents, ALDH2*2 knock-in mice exposed to e-cigarette aerosol had an increased heart rate beginning 5 days after exposure compared to wild type ALDH2 mice (775±30bpm versus 679±33bpm, respectively, *p<0.01, n=7-8 per group). E-cigarette aerosol exposure also exacerbated oxidative stress in ALDH2*2 heart homogenates, including a 1.3-fold higher protein carbonyl level, a 1.7-fold higher lipid peroxide level and 1.5-fold greater phosphorylation of NF-κB relative to wild type ALDH2 homogenates. Conclusions: The increased oxidative stress from e-cigarette aerosol aldehydes triggers the proinflammatory NF-κB pathway. As ALDH2 expression and activity is lower in the heart relative to the lung, the heart could be more susceptible to increases in cardiovascular oxidative stress from e-cigarette aerosol; particularly for those carrying an ALDH2*2 genetic variant which limits acetaldehyde metabolism.