Formation of (2E)-4-Hydroxy-2-nonenal and (2E)-4-Hydroxy-2-hexenal by Plant Enzymes: A Review Suggests a Role in the Physiology of Plants

It is demonstrated that (3Z)-nonenal (NON) and (3Z)-hexenal (HEX) are oxidized in a cascade by lipoxygenase (LOX) and hydroperoxide peroxygenase (HP peroxygenase) into (2E)-4-hydroxy-2- nonenal (HNE) and (2E)-4-hydroxy-2-hexenal (HHE), respectively. In turn, HNE inactivates LOX terminating the cascade. The hydroxy-alkenals produced serve to inhibit plant pathogens, which initiated the cascade. In addition to LOX, other unknown oxygenases may be involved in the cascade.

Role of Cathepsin G in the Degradation of Glyceraldehyde-3-Phosphate Dehydrogenase Triggered by 4-Hydroxy-2-Nonenal in U937 Cells

Degradation of oxidized or oxidatively modified proteins is an essential part of the cellular antioxidant defense system. 4-Hydroxy-2-nonenal (HNE), a major reactive aldehyde formed by lipid peroxidation, causes many types of cellular damage. HNE-modified proteins are degraded by the ubiquitin-proteasome pathway or the lysosomal pathway. However, our previous studies using U937 cells showed that HNE-modified glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is degraded by cathepsin G. In the present study, we examined whether GAPDH in U937 cells treated with HNE in culture is degraded similarly to that incubated with HNE and U937 cell extract. Treatment with HNE for 10 min in culture decreased GAPDH activity in a concentration dependent manner, but did not affect GAPDH degradation. The proteasome activities were not affected by HNE, but culturing with HNE decreased cathepsin G activity and protein level in a concentration dependent manner. These results suggest that HNE-induced oxidative stress leads to decreased cathepsin G activity and results in the loss of GAPDH degradation. Taken together, our findings indicate that cathepsin G has an important role in the degradation of oxidatively modified GAPDH in U937 cells.

Determination of trans-4-hydroxy-2-alkenals in thermally treated soybean oil by SPE-HPLC

Trans-4-hydroxy-2-hexenal(4-HHE) and trans-4-hydroxy-2-nonenal(4-HNE) are secondary lipid peroxidation products in edible oils, which are cytotoxic and genotoxic. They could covalently bind with protein, phospholipids and DNA, further disrupting the normal function of liver, lung and brain.Derivation process was generally conducted during pretreatment before detection and quantification of 4-HHE and 4-HNE. However, the derivation procedures were time consuming and chemical degradation may occur during the process. Hence, this paper aims to establish a simple solid phase extractionhigh performance liquid chromatography(SPE-HPLC) method to determine the 4-HHE and 4-HNE contents in thermally treated soybean oil. C18 solid phase extraction was applied in the pretreatment process. Firstly, the reliability of the method was evaluated. Good linearity was observed in the range of 0.1–0.5 μg/m L and 0.5–10 μg/m L for 4-HHE and 4-HNE. The limit of detection(LOD) of 4-HHE and 4-HNE were 0.0486 and 0.0129 μg/m L, respectively. And the limit of quantitation(LOQ) of4-HHE and 4-HNE were 0.1458 and 0.0431 μg/m L, respectively. Recovery rate were in the range of89.11%–91.58% and 71.83%–79.40% for 4-HHE and 4-HNE, respectively. The method achieved the extraction, purification and detection of 4-HHE and 4-HNE simultaneously and had the advantages of simple operation, effectiveness, high precision, good repeatability. Then, the method was applied to monitor the concentrations of 4-HHE and 4-HNE in soybean oil heated at 180 °C for 40 h. The contents of 4-HHE and 4-HNE were 0–0.32 μg/g and 0–6.97 μg/g, respectively, which provided guidance for evaluating health risks of thermally treated soybean oil during heating.

Simultaneous determination of typical toxic aldehydes formed during food frying and digestion using isotope dilution UHPLC-QqQ-MS/MS

An isotope dilution ultra-performance liquid chromatography-triple quadrupole mass spectrometry method was developed to simultaneously detect two typical kinds ofα,β-unsaturated aldehydes,namely 4-hydroxy-2-hexenal(4-HHE)and 4-hydroxy-2-nonenal(4-HNE),in foods.The proposed method exhibited a linear range of 10-1000 ng/mL with a limit of detection of 0.1-2.0 ng/g and a limit of quantification of 0.3-5.0 ng/g.The recovery rates of these typical toxic aldehydes(i.e.,4-HHE,4-HNE)and their d3-labeled analogues were 91.54%-105.12%with a low matrix effect.Furthermore,this proposed method was successfully applied to a real frying system and a simulated digestion system,wherein the contents of 4-HHE and 4-HNE were determined for both.Overall,the obtained results provide strong support for further research into the production of 4-HHE and 4-HNE resulting from foods during oil digestion and frying.

Role of aldehyde dehydrogenase 2 in ischemia reperfusion injury:An update

Aldehyde dehydrogenase 2(ALDH2) is best known for its critical detoxifying role in liver alcohol metabolism. However, ALDH2 dysfunction is also involved in a wide range of human pathophysiological situations and is associated with complications such as cardiovascular diseases, diabetes mellitus, neurodegenerative diseases and aging. A growing body of research has shown that ALDH2 provides important protection against oxidative stress and the subsequent loading of toxic aldehydes such as 4-hydroxy-2-nonenal and adducts that occur in human diseases, including ischemia reperfusion injury(IRI). There is increasing evidence of its role in IRI pathophysiology in organs such as heart, brain, small intestine and kidney; however, surprisingly few studies have been carried out in the liver, where ALDH2 is found in abundance. This study reviews the role of ALDH2 in modulating the pathways involved in the pathophysiology of IRI associated with oxidative stress, autophagy and apoptosis. Special emphasis is placed on the role of ALDH2 in different organs, on therapeutic "preconditioning" strategies, and on the use of ALDH2 agonists such as Alda-1, which may become a useful therapeutic tool for preventing the deleterious effects of IRI in organ transplantation.

Neuroprotective effects of Alda-1 mitigate spinal cord injury in mice:involvement of Alda-1-induced ALDH2 activation-mediated suppression of reactive aldehyde mechanisms

Spinal cord injury(SCI)is associated with high production and excessive accumulation of pathological 4-hydroxy-trans-2-nonenal(4-HNE),a reactive aldehyde,formed by SCI-induced metabolic dysregulation of membrane lipids.Reactive aldehyde load causes redox alteration,neuroinflammation,neurodegeneration,pain-like behaviors,and locomotion deficits.Pharmacological scavenging of reactive aldehydes results in limited improved motor and sensory functions.In this study,we targeted the activity of mitochondrial enzyme aldehyde dehydrogenase 2(ALDH2)to detoxify 4-HNE for accelerated functional recovery and improved pain-like behavior in a male mouse model of contusion SCI.N-(1,3-benzodioxol-5-ylmethyl)-2,6-dichlorobenzamide(Alda-1),a selective activator of ALDH2,was used as a therapeutic tool to suppress the 4-HNE load.SCI was induced by an impactor at the T9–10 vertebral level.Injured animals were initially treated with Alda-1 at 2 hours after injury,followed by once-daily treatment with Alda-1 for 30 consecutive days.Locomotor function was evaluated by the Basso Mouse Scale,and pain-like behaviors were assessed by mechanical allodynia and thermal algesia.ALDH2 activity was measured by enzymatic assay.4-HNE protein adducts and enzyme/protein expression levels were determined by western blot analysis and histology/immunohistochemistry.SCI resulted in a sustained and prolonged overload of 4-HNE,which parallels with the decreased activity of ALDH2 and low functional recovery.Alda-1 treatment of SCI decreased 4-HNE load and enhanced the activity of ALDH2 in both the acute and the chronic phases of SCI.Furthermore,the treatment with Alda-1 reduced neuroinflammation,oxidative stress,and neuronal loss and increased adenosine 5′-triphosphate levels stimulated the neurorepair process and improved locomotor and sensory functions.Conclusively,the results provide evidence that enhancing the ALDH2 activity by Alda-1 treatment of SCI mice suppresses the 4-HNE load that attenuates neuroinflammation and neurodegeneration,promotes the neurorepair process,and improves functional outcomes.Consequently,we suggest that Alda-1 may have therapeutic potential for the treatment of human SCI.Animal procedures were approved by the Institutional Animal Care and Use Committee(IACUC)of MUSC(IACUC-2019-00864)on December 21,2019.

4-HNE Induces Apoptosis of Human Retinal Pigment Epithelial Cells by Modifying HSP70

The role of heat shock protein 70 (HSP70) in apoptosis of human retinal pigment epithelial cells (ARPE-19) induced by 4-hydroxy-2-nonenal (4-HNE) was explored. Different concentrations of 4-HNE were used to stimulate ARPE-19 cells, and apoptosis was measured by flow cytometry. The expression of apoptotic-related proteins, HSP70, X-linked inhibitorof- apoptosis (XIAP), Bcl-2, and Bax were quantified by Western blotting. HSP70 and XIAP overexpression plasmids, or their corresponding siRNAs were transfected into ARPE-19 cells using Lipofectamine. 2000. Co-immunoprecipitation and Western blotting were used to detect the effect of 4-HNE on the expression of HSP70 and the binding level between 4-HNE and HSP70. The results showed that 4-HNE induced late apoptosis in ARPE-19 cells, accompanied by elevated levels of 4-HNE-modified IISP70, but it did not affect HSP70 protein expression. 4-HNE-modified HSP70 down-regulated the expression of the apoptosis inhibitory protein XIAP. Overexpression of HSP70 or XIAP inhibited 4-HNE-induced apoptosis of ARPE-19 cells. It was suggested that 4-HNE could promote XIAP degradation by modification of HSP70 to induce late apoptosis of human retinal pigment epithelial cells.

Chronic stress causes protein kinase C epsilon-aldehyde dehydrogenase 2 signaling pathway perturbation in the rat hippocampus and prefrontal cortex,but not in the myocardium

Chronic stress is strongly associated with the occurrence and development of depression and cardiovascular disease.Stress can induce altered mitochondrial function and activation of apoptosis in the cardio-cerebral system.However,it is unknown whether the protein kinase C ε（PKCε）-aldehyde dehydrogenase 2（ALDH2） pathway is altered under chronic stress,and this study sought to address this question.A rat model of depression was established using a chronic unpredictable mild stress（CUMS） protocol.After experiencing CUMS for 4 weeks,the sucrose preference test and the forced swim test verified depressive-like behaviors.Enzyme linked immunosorbent assays showed that ALDH2 activity was decreased in the rat hippocampus and prefrontal cortex,but was not altered in the myocardium.Western blot assays demonstrated reduced levels of ALDH2 and PKCε,but increased levels of 4-hydroxy-2-nonenal（4 HNE） adducts.Caspase-3 expression did not obviously alter,but active forms of caspase-3 were increased in the hippocampus and prefrontal cortex.In the myocardium,expression of ALDH2,PKCε and 4 HNE adducts did not remarkably alter;while caspase-3 expression was reduced and the active forms of caspase-3 were upregulated.Pearson＇s correlation test demonstrated that expression of 4 HNE adducts was positively correlated with levels of the active forms of caspase-3 in the hippocampus and prefrontal cortex,but not in the myocardium.In conclusion,chronic stress can damage the PKCε-ALDH2 signaling pathway in the hippocampus and prefrontal cortex,but not in the myocardium.Moreover,4 HNE is associated with active forms of caspase-3 in the hippocampus and prefrontal cortex.

Hydroxyproline alleviates 4-hydroxy-2-nonenal-induced DNA damage and apoptosis in porcine intestinal epithelial cells

Oxidative stress has been confirmed in relation to intestinal mucosa damage and multiple bowel diseases.Hydroxyproline (Hyp) is an imino acid abundant in sow's milk. Compelling evidence has beengathered showing the potential antioxidative properties of Hyp. However, the role and mechanism ofHyp in porcine intestinal epithelial cells in response to oxidative stress remains unknown. In this study,small intestinal epithelial cell lines of piglets (IPEC-1) were used to evaluate the protective effects of Hypon 4-hydroxy-2-nonenal (4-HNE)-induced oxidative DNA damage and apoptosis. IPEC-1 pretreated with0.5 to 5 mmol/L Hyp were exposed to 4-HNE (40 mmol/L) in the presence or absence of Hyp. Thereafter,the cells were subjected to apoptosis detection by Hoechst staining, flow cytometry, and Western blot orDNA damage analysis by comet assay, immunofluorescence, and reverse-transcription quantitative PCR(RT-qPCR). Cell apoptosis and the upregulation of cleaved-caspase-3 induced by 4-HNE (40 mmol/L) wereinhibited by 5 mmol/L of Hyp. In addition, 5 mmol/L Hyp attenuated 4-HNE-induced reactive oxygenspecies (ROS) accumulation, glutathione (GSH) deprivation and DNA damage. The elevation in transcriptionof GADD45a (growth arrest and DNA-damage-inducible protein 45 alpha) and GADD45b(growth arrest and DNA-damage-inducible protein 45 beta), as well as the phosphorylation of H2AX(H2A histone family, member X), p38 MAPK (mitogen-activated protein kinase), and JNK (c-Jun N-terminalkinase) in cells treated with 4-HNE were alleviated by 5 mmol/L Hyp. Furthermore, Hyp supplementationincreased the protein abundance of Krüppel like factor 4 (KLF4) in cells exposed to 4-HNE.Suppression of KLF4 expression by kenpaulone impeded the resistance of Hyp-treated cells to DNAdamage and apoptosis induced by 4-HNE. Collectively, our results indicated that Hyp serves to protectagainst 4-HNE-induced apoptosis and DNA damage in IPEC-1 cells, which is partially pertinent with theenhanced expression of KLF4. Our data provides an updated explanation for the nutritional values ofHyp-containing animal products.

Biliverdin Reductase-A correlates with inducible nitric oxide synthasein in atorvastatin treated aged canine brain

Alzheimer’s disease is a neurodegenerative disorder characterized by progressive cognitive impairment and neuropathology. Recent preclinical and epidemiological studies proposed statins as a possible therapeutic drug for Alzheimer’s disease, but the exact mechanisms of action are still unknown. Biliverdin reductase-A is a pleiotropic enzyme involved in cellular stress responses. It not only transforms biliverdin-IX alpha into the antioxidant bilirubin-IX alpha but its serine/threonine/ tyrosine kinase activity is able to modulate cell signaling networks. We previously reported the beneficial effects of atorvastatin treatment on biliverdin reductase-A and heme oxygenase-1 in the brains of a well characterized pre-clinical model of Alzheimer’s disease, aged beagles, together with observed improvement in cognition. Here we extend our knowledge of the effects of atorvastatin on inducible nitric oxide synthase in parietal cortex, cerebellum and liver of the same animals. We demonstrated that atorvastatin treatment （80 mg/day for 14.5 months） to aged beagles selectively increased inducible nitric oxide synthase in the parietal cortex but not in the cerebellum. In contrast, inducible nitric oxide synthase protein levels were significantly decreased in the liver. Significant positive correlations were found between biliverdin reductase-A and inducible nitric oxide synthase as well as heme oxygenase-1 protein levels in the parietal cortex. The opposite was observed in the liver. Inducible nitric oxide synthase up-regulation in the parietal cortex was positively associated with improved biliverdin reductase-A functions, whereas the oxidative-induced impairment of biliverdin reductase-A in the liver negatively affected inducible nitric oxide synthase expression, thus suggesting a role for biliverdin reductase-A in atorvastatin-dependent inducible nitric oxide synthase changes. Interestingly, increased inducible nitric oxide synthase levels in the parietal cortex were not associated with higher oxidative/nitrosative stress levels. We hypothesize that biliverdin reductase-A-dependent inducible nitric oxide synthase regulation strongly contributes to the cognitive improvement observed following atorvastatin treatment.