n-(1-3-benzodioxol-5-ylmethyl)-2-6-dichlorobenzamide and Disease-Models--Animal

Aldehyde dehydrogenase 2 (ALDH2) plays important role in ethanol metabolism, and also serves as an important shield from the damage occurring under oxidative stress. A special inactive variant was found carried by 35-45% of East Asians. The variant carriers have recently been found at the higher risk for the diseases related to the damage occurring under oxidative stress, such as cardiovascular and cerebrovascular diseases. As a result, ALDH2 activators may potentially serve as a new class of therapeutics. Herein, N-benzylanilines were found as novel allosteric activators of ALDH2 by computational virtual screening using ligand-based and structure-based screening parallel screening strategy. Then a structural optimization was performed and has led to the discovery of the compound C6. It has good activity in vitro and in vivo, which could reduce infarct size by ∼70% in ischemic stroke rat models. This study provided good lead compounds for the further development of ALDH2 activators.

Topics: Aldehyde Dehydrogenase, Mitochondrial; Allosteric Regulation; Animals; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Discovery; Humans; Infarction, Middle Cerebral Artery; Ischemic Stroke; Molecular Structure; Rats; Rats, Sprague-Dawley; Small Molecule Libraries; Structure-Activity Relationship

Chronic hyperglycemia and hyperlipidemia hamper beta cell function, leading to glucolipotoxicity. Mitochondrial aldehyde dehydrogenase 2 (ALDH2) detoxifies reactive aldehydes, such as methylglyoxal (MG) and 4-hydroxynonenal (4-HNE), derived from glucose and lipids, respectively. We aimed to investigate whether ALDH2 activators ameliorated beta cell dysfunction and apoptosis induced by glucolipotoxicity, and its potential mechanisms of action. Glucose-stimulated insulin secretion (GSIS) in MIN6 cells and insulin secretion from isolated islets in perifusion experiments were measured. The intracellular ATP concentrations and oxygen consumption rates of MIN6 cells were assessed. Furthermore, the cell viability, apoptosis, and mitochondrial and intracellular reactive oxygen species (ROS) levels were determined. Additionally, the pro-apoptotic, apoptotic, and anti-apoptotic signaling pathways were investigated. We found that Alda-1 enhanced GSIS by improving the mitochondrial function of pancreatic beta cells. Alda-1 rescued MIN6 cells from MG- and 4-HNE-induced beta cell death, apoptosis, mitochondrial dysfunction, and ROS production. However, the above effects of Alda-1 were abolished in

Topics: Adenosine Triphosphate; Aldehyde Dehydrogenase, Mitochondrial; Aldehydes; Animals; Apoptosis; Benzamides; Benzodioxoles; Cell Death; Disease Models, Animal; Glucose; Humans; Insulin Secretion; Insulin-Secreting Cells; Lipids; Metabolic Detoxication, Phase I; Mitochondria; Oxidative Stress; Reactive Oxygen Species

The use of doxorubicin (DOX) as an antineoplastic drug is compromised by its cardiotoxicity risk. Although several mechanisms have been proposed for DOX-induced cardiac dysfunction, there is still increased interest in assessing its effects. Likewise, it is important to find protocols that can prevent or minimize the side effects of DOX without hindering its antitumor activity. Thus, this study was designed to investigate the molecular mechanisms underlying DOX cardiotoxicity, with a special focus on cardiac energy metabolism and the ability of Alda-1 (ALDH2 agonist) to prevent DOX-induced cardiac alterations. We explored the effects of DOX on the histological morphology of the myocardium, on lipid profile, and on the expression of genes related to fatty acid metabolism, in the presence and absence of Alda-1 (8 mg/kg body weight; b.wt.). Two DOX treatment protocols were used: a single dose of DOX (4 mg/kg b.wt.); four doses of DOX (4 mg/kg b.wt.), one dose/week, for 4 weeks. Treatment with DOX caused a progressive injury in the cardiac tissue and an increase in the blood total cholesterol, high-density lipoproteins, very low-density lipoproteins and triglyceride, as well as an up-regulation of FABP4 (DOX and DOX + Alda-1 groups) and Slc27a2 (in DOX-treated animals). Alda-1 administration promoted reduction in the severity of the histopathological injuries (after single dose of DOX) and Slc27a2 overexpression was restored. In conclusion, the study revealed novel insights regarding the development of DOX-mediated cardiomyopathy, indicating a relationship between DOX exposure and FABP4 and Slc27a2 overexpression, and confirmed the cardioprotective effect of Alda-1.

Topics: Aldehyde Dehydrogenase, Mitochondrial; Animals; Benzamides; Benzodioxoles; Cardiotoxicity; Coenzyme A Ligases; Disease Models, Animal; Doxorubicin; Energy Metabolism; Fatty Acid-Binding Proteins; Gene Expression Profiling; Heart Diseases; Lipid Metabolism; Lipids; Male; Myocytes, Cardiac; Rats, Wistar; Transcriptome

Retinitis pigmentosa (RP) is a kind of inherited retinal degenerative diseases characterized by the progressive loss of photoreceptors. RP has been a conundrum without satisfactory countermeasures in clinic until now. Acetaldehyde dehydrogenase 2 (ALDH2), a major enzyme involved in aldehyde detoxification, has been demonstrated to be beneficial for a growing number of human diseases, such as cardiovascular dysfunction, diabetes mellitus and neurodegeneration. However, its protective effect against RP remains unknown. Our study explored the impact of ALDH2 on retinal function and structure in N-methyl-N-nitrosourea (MNU)-induced RP rats.. Rats were gavaged with 5 mg/kg Alda-1, an ALDH2 agonist, 5 days before and 3 days after MNU administration. Assessments of retinal function and morphology as well as measurement of specific proteins expression level were conducted.. Electroretinogram recordings showed that Alda-1 administration alleviated the decrease in amplitude caused by MNU, rendering protection of retinal function. Mitigation of photoreceptor degeneration in MNU-treated retinas was observed by optical coherence tomography and retinal histological examination. In addition, Western blotting results revealed that ALDH2 protein expression level was upregulatedwith increased expression of SIRT1 protein after the Alda-1 intervention. Besides, endoplasmic reticulum stress (ERS) was reduced according to the significant downregulation of GRP78 protein, while apoptosis was ameliorated as shown by the decreased expression of PARP1 protein.. Together, our data demonstrated that ALDH2 could provide preservation of retinal function and morphology against MNU-induced RP, with the underlying mechanism at least partly related to the modulation of SIRT1, ERS and apoptosis.

Topics: Aldehyde Dehydrogenase, Mitochondrial; Alkylating Agents; Animals; Benzamides; Benzodioxoles; Blotting, Western; Dark Adaptation; Disease Models, Animal; Electroretinography; Endoplasmic Reticulum Chaperone BiP; Heat-Shock Proteins; Injections, Intraperitoneal; Male; Methylnitrosourea; Photic Stimulation; Poly (ADP-Ribose) Polymerase-1; Rats; Rats, Sprague-Dawley; Retina; Retinitis Pigmentosa; Sirtuin 1; Tomography, Optical Coherence

Achilles tendinopathy has a high re-injury rate and poor prognosis. Development of effective therapy for Achilles tendinopathy is important. Excessive accumulation of ROS and resulting oxidative stress are believed to cause tendinopathy. Overproduction of hydrogen peroxide (H

Topics: Achilles Tendon; Aldehyde Dehydrogenase, Mitochondrial; Animals; Benzamides; Benzodioxoles; Cell Survival; Cells, Cultured; Disease Models, Animal; Dose-Response Relationship, Drug; Hydrogen Peroxide; Mice; Mice, Inbred C57BL; Tendinopathy; Tenocytes

Hepatic ischemia/reperfusion injury (HIRI) is a complex pathophysiological process that often leads to poor clinical prognosis. Clinically, the effective means to alleviate HIRI are limited. The aim of the present study was to investigate whether Alda‑1, an activator of mitochondrial aldehyde dehydrogenase 2 (ALDH2), had a protective effect on HIRI and to investigate the mechanisms underlying this protective effect. Sprague‑Dawley rats were treated with Alda‑1 or Daidzin, an ALDH2 inhibitor, 30 min before partial (70%) warm liver ischemia to induce HIRI. The 48 rats were randomly divided into four groups: Sham, ischemia injury (IR), IR‑Alda‑1, and IR‑Daidzin. After 6 and 24 h of reperfusion, serum and liver tissue samples were collected and stored for further experiments. Alanine aminotransferase, aspartate aminotransferase and hematoxylin & eosin staining was used to evaluate the liver damage. Western blotting and reverse transcription‑quantitative PCR were used to detect the expression of related proteins and mRNA. TUNEL staining was used to observe the apoptosis of liver cells. Transmission electron microscopy was used to detect the mitochondrial injuries. Alda‑1 pretreatment ameliorated the HIRI‑induced damage to the liver function and reduced histological lesions. Alda‑1 also increased ALDH2 activity after HIRI. Moreover, the pretreatment with Alda‑1 reduced the accumulation of toxic aldehyde 4‑hydroxy‑2‑nonenal, decreased the production of reactive oxygen species and malondialdehyde, reversed the damage to the liver mitochondria, attenuated hepatocyte apoptosis and inhibited the HIRI‑induced inflammatory response, including high‑mobility group box 1/toll‑like receptor 4 signaling. Alda‑1 also induced autophagy by upregulating autophagy‑related 7 and Rab7 increasing the microtubule associated protein 1 light chain 3 αII/I ratio and inhibiting p62 expression. ALDH2‑induced autophagy was dependent on the activation of the AKT/mammalian target of rapamycin (mTOR) and AMP‑activated protein kinase (AMPK) signaling pathways. In conclusion, the findings of the present study suggested that Alda‑1 may protect the liver against HIRI‑induced damage, including hepatic enzyme injury, acetaldehyde accumulation, oxidative stress, hepatocyte apoptosis and inflammation. Alda‑1 may confer this protection by inducing autophagy through the AKT/mTOR and AMPK signaling pathways. Therefore, ALDH2 could represent a potential pharmacological target in the clinical treatment

Topics: Aldehydes; Animals; Autophagy; Benzamides; Benzodioxoles; Disease Models, Animal; Gene Expression Regulation; Liver Diseases; Liver Function Tests; Male; Oxidative Stress; Random Allocation; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Reperfusion Injury

Topics: Aldehyde Dehydrogenase, Mitochondrial; Aldehydes; Animals; Benzamides; Benzodioxoles; Disease Models, Animal; Epithelium; Gene Expression Regulation; Humans; Lung; Lung Injury; Occludin; Rats; Shock, Hemorrhagic; Tumor Necrosis Factor-alpha; Zonula Occludens-1 Protein

Liver fibrosis is a leading cause of mortality worldwide. Oxidative stress is a key component in the pathogenesis of liver fibrosis. We investigated the role of aldehyde formation resulting from lipid peroxidation in cholestatic liver injury and fibrosis.. C57Bl/6J mice underwent bile duct ligation (BDL) or sham operation. One hour after surgery and daily thereafter, animals were given Alda-1 (20 mg/kg, s.c.), an aldehyde dehydrogenase-2 activator, or equivalent volume of vehicle. Blood and livers were collected after 3 and 14 days.. Serum alanine aminotransferase (ALT) increased from 39.8 U/L after sham operation to 537 U/L 3 days after BDL, which Alda-1 decreased to 281 U/L. Biliary infarcts with a periportal distribution developed with an area of 7.8% at 14 days after BDL versus 0% area after sham operation. Alda-1 treatment with BDL decreased biliary infarcts to 1.9%. Fibrosis detected by picrosirius red staining increased from 1.6% area in sham to 7.3% after BDL, which decreased to 3.8% with Alda-1. Alda-1 suppression of fibrosis was additionally confirmed by second harmonic generation microscopy. After BDL, collagen-I mRNA increased 12-fold compared to sham, which decreased to 6-fold after Alda-1 treatment. Smooth muscle α-actin expression in the liver, a marker of activated stellate cells, increased from 1% area in sham to 18.7% after BDL, which decreased to 5.3% with Alda-1. CD68-positive macrophages increased from 33.4 cells/field in sham to 134.5 cells/field after BDL, which decreased to 64.9 cells/field with Alda-1. Lastly, 4-hydroxynonenal adduct (4-HNE) immunofluorescence increased from 2.5% area in sham to 14.1% after BDL. Alda-1 treatment decreased 4-HNE to 2.2%.. Accelerated aldehyde degradation by Alda-1 decreases BDL-induced liver necrosis, inflammation, and fibrosis, implying that aldehydes play an important role in the pathogenesis of cholestatic liver injury and fibrosis.

Topics: Aldehyde Dehydrogenase, Mitochondrial; Animals; Benzamides; Benzodioxoles; Bile Ducts; Disease Models, Animal; Humans; Ligation; Liver; Liver Cirrhosis; Mice; Necrosis; Oxidative Stress

Post-MI depression is a critical clinical problem, the comorbidity of which complicates depression treatment and worsens cardiovascular outcomes. However, which antidepressant is the best to lower the risk of cardiovascular events in persons with depression was still unknown. Recently, it has been proposed that the activation of ALDH2 by Alda-1 can effectively reduce depressive-like behaviors and improve the prognosis of coronary heart disease. In the present study, we investigated the effect of Alda-1 on the expression of VEGF in the hippocampus of a rat model with post-MI depression, as well as the potential treatment mechanism. Alda-1 administration significantly decreased the immobility time and increased the swimming time of the post-MI depression rats in the forced swim test. Moreover, treatment of post-MI depression rats with Alda-1 significantly increased the sucrose preference ratio, as assessed by a sucrose preference test. These behaviors were associated with an increase 5-HT and DA neurotransmitter content, as well as an increase of VEGF levels in the hippocampus of the post-MI depression rats. These results suggest that Alda-1 improves depressive-like behavior in rats after MI by increasing VEGF expression in the hippocampus of rats.

Topics: Aldehyde Dehydrogenase, Mitochondrial; Animals; Antidepressive Agents; Behavior, Animal; Benzamides; Benzodioxoles; Depression; Disease Models, Animal; Dopamine; Hippocampus; Male; Myocardial Infarction; Rats, Sprague-Dawley; Serotonin; Vascular Endothelial Growth Factor A

Aldehyde dehydrogenase 2 (ALDH2) is a key enzyme for metabolism of reactive aldehydes, but its role during liver ischemia-reperfusion injury (IRI) remains unclear. In the present study, we investigated the effects of the ALDH2 activator, Alda-1, in liver IRI and elucidated the underlying mechanisms. Mice were pretreated with Alda-1 and subjected to a 90 min hepatic 70% ischemia model, and liver tissues or serum samples were collected at indicated time points after reperfusion. We demonstrated that Alda-1 pretreatment had a hepatoprotective role in liver IRI as evidenced by decreased liver necrotic areas, serum ALT/AST levels, and liver inflammatory responses. Mechanistically, Alda-1 treatment enhanced ALDH2 activity and subsequently reduced the accumulation of reactive aldehydes and toxic protein adducts, which result in decreased hepatocyte apoptosis and mitochondrial dysfunction. We further demonstrated that Alda-1 treatment could activate AMPK and autophagy and that AMPK activation was required for Alda-1-mediated autophagy enhancement. These findings collectively indicate that Alda-1-mediated ALDH2 activation could be a promising strategy to improve liver IRI by clearance of reactive aldehydes and enhancement of autophagy.

Topics: Aldehyde Dehydrogenase, Mitochondrial; AMP-Activated Protein Kinases; Animals; Anti-Inflammatory Agents; Autophagy; Benzamides; Benzodioxoles; Disease Models, Animal; Liver; Male; Mice; Mice, Inbred C57BL; Reperfusion Injury

The etiology of depression remains still unclear. Recently, it has been proposed, that mitochondrial dysfunction may be associated with development of mood disorders, such as depression, bipolar disorder and anxiety disorders. Mitochondrial aldehyde dehydrogenase (ALDH2), an enzyme responsible for the detoxification of reactive aldehydes, is considered to exert protective function in mitochondria. We investigated the influence of Alda-1, a small-molecule activator of ALDH2, on depressive- and anxiety-like behaviors in an animal model of depression - the prenatally stressed rats - using behavioral, molecular and proteomic methods. Prolonged Alda-1 administration significantly increased the climbing time, tended to reduce the immobility time and increased the swimming time of the prenatally stressed rats in the forced swim test. Moreover, treatment of prenatally stressed rats with Alda-1 significantly increased number of entries into the open arms of the maze and the time spent therein, as assessed by elevated plus-maze test. Such actions were associated with reduction of plasma 4-HNE-protein content, decrease of TNF-α mRNA and increase of PGC-1α (regulator of mitochondrial biogenesis) mRNA level in the frontal cortex and hippocampus of the prenatally stressed rats as well as with normalization of peripheral immune parameters and significant changes in expression of 6 and 4 proteins related to mitochondrial functions in the frontal cortex and hippocampus, respectively. Collectively, ALDH2 activation by Alda-1 led to a significant attenuation of depressive- and anxiety-like behaviors in the prenatally stressed rats. The pattern of changes suggested mitoprotective effect of Alda-1, however the exact functional consequences of the revealed alterations require further investigation.

Topics: Aldehyde Dehydrogenase, Mitochondrial; Animals; Anxiety; Apoptosis; Benzamides; Benzodioxoles; Cell Proliferation; Cytokines; Depressive Disorder; Disease Models, Animal; Female; Frontal Lobe; Hippocampus; Lymphocytes; Male; Mitochondria; Motor Activity; Pregnancy; Prenatal Exposure Delayed Effects; Rats; Rats, Sprague-Dawley; RNA, Messenger; Stress, Psychological

This study was designed to examine the impact of mitochondrial aldehyde dehydrogenase 2 (ALDH2) on transverse aorta constriction (TAC)-induced cardiac hypertrophy and related molecular mechanisms using an ALDH2 knockout (ALDH2-/-) murine model.. Male wild-type and ALDH2-/- mice were subjected to TAC or sham operation (n=6-8 for each group). After two weeks, cardiac function was assessed by echocardiography and hemodynamic measurements. Myocardial phosphorylated and total PI3K, the catalytic subunit of PI3Ks (p110α and p110γ), Akt, and total PTEN levels were detected by Western blotting. Cardiomyocytes were stretched for 6h in vitro in the presence or absence of Alda-1 (a small-molecule activator of ALDH2) prior to assessment of phosphorylated PI3K, Akt and total PTEN expressions by Western blot.. Heart to body weight ratio and left ventricular posterior wall thickness as well as the cross-sectional area of cardiomyocyte were significantly lower in ALDH2-/- mice than in wild-type mice following TAC. Western blot analysis showed p110γ was upregulated post TAC in both wild-type mice and ALDH2-/- mice, phosphorylation of Akt was disrupted, PTEN expression was upregulated in ALDH2-/- mice post TAC while phosphorylated PI3K, p110α and p110γ expression was similar between ALDH2-/- and wild-type mice post TAC. In vitro, phosphorylation of Akt was significantly accentuated and PTEN expression was reduced while PI3K phosphorylation remained unchanged in stretched cardiomyocytes pretreated by Alda-1 compared to stretched cardiomyocytes treated by saline.. Our results show that ALDH2 deficiency attenuates compensatory cardiac hypertrophy through regulating Akt but not PI3K phosphorylation early after TAC in mice.

Topics: Aldehyde Dehydrogenase, Mitochondrial; Animals; Aorta; Benzamides; Benzodioxoles; Cardiomegaly; Cells, Cultured; Constriction; Disease Models, Animal; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Myocytes, Cardiac; Phosphatidylinositol 3-Kinases; Phosphorylation; Proto-Oncogene Proteins c-akt; PTEN Phosphohydrolase

Many studies have shown that mitochondrial aldehyde dehydrogenase 2 (ALDH2) functions as a cellular protector against oxidative stress by detoxification of cytotoxic aldehydes. Within dopaminergic neurons, dopamine is metabolized by monoamine oxidase to yield 3,4-dihydroxyphenylacetaldehyde (DOPAL) then converts to a less toxic acid product by ALDH. The highly toxic and reactive DOPAL has been hypothesized to contribute to the selective neurodegeneration in Parkinson's disease (PD). In this study, we investigated the neuroprotective mechanism and therapeutic effect of ALDH2 in rotenone models for parkinsonism. Overexpression of wild-type ALDH2 gene, but not the enzymatically deficient mutant ALDH2*2 (E504K), reduced rotenone-induced cell death. Application of a potent activator of ALDH2, Alda-1, was effective in protecting against rotenone-induced apoptotic cell death in both SH-SY5Y cells and primary cultured substantia nigra (SN) dopaminergic neurons. In addition, intraperitoneal administration of Alda-1 significantly reduced rotenone- or MPTP-induced death of SN tyrosine hydroxylase (TH)-positive dopaminergic neurons. The attenuation of rotenone-induced apoptosis by Alda-1 resulted from decreasing ROS accumulation, reversal of mitochondrial membrane potential depolarization, and inhibition of activation of proteins related to mitochondrial apoptotic pathway. The present study demonstrates that ALDH2 plays a crucial role in maintaining normal mitochondrial function to protect against neurotoxicity and that Alda-1 is effective in ameliorating mitochondrial dysfunction and inhibiting mitochondria-mediated apoptotic pathway. These results indicate that ALDH2 activation could be a neuroprotective therapy for PD.

Topics: Aldehyde Dehydrogenase; Animals; Benzamides; Benzodioxoles; Cell Line; Disease Models, Animal; Enzyme Activation; Enzyme-Linked Immunosorbent Assay; Humans; Immunoblotting; Membrane Potential, Mitochondrial; Mice, Inbred C57BL; Microscopy, Confocal; MPTP Poisoning; Neurons; Parkinsonian Disorders; Rats; Reactive Oxygen Species; Rotenone; Uncoupling Agents

Effective therapies for alcoholic liver disease are currently unavailable. The present study tested the efficacy of Alda-1, a specific aldehyde dehydrogenase 2 (ALDH2) activator, in treating alcoholic liver disease.. Male C57BL/6J mice were exposed to alcohol for a time-course study on aldehyde metabolism. The specificity and efficacy of Alda-1 on activating hepatic ALDH2 and aldehyde clearance were determined by acute treatments. Then, mice were fed alcohol for 8 weeks with Alda-1 administration for the last 10 days to test the therapeutic potential of Alda-1. Lastly, H4IIEC3 cells were treated with ethanol, acetaldehyde, or 4-hydroxynonenal to define the link between aldehydes and hepatotoxicity.. Alcohol feeding for 8 weeks induced hepatic ALDH2 dysfunction and aldehyde accumulation. One dose of Alda-1 administration elevated hepatic ALDH activity, which was blocked by the specific ALDH2 inhibitor, daidzin. Alda-1 accelerated acetaldehyde clearance after acute alcohol intoxication. Alda-1 treatment in the 8-week alcohol feeding model reversed liver damage along with reduction of hepatic aldehydes. Alda-1 re-activated transcription factors, upregulated fatty acid oxidation enzymes, and reversed steatosis. Alcohol-induced endoplasmic reticulum stress and apoptotic cell death were also attenuated by Alda-1. Acetaldehyde or 4-hydroxynonenal treatment to H4IIEC3 cells inactivated transcription factors and induced endoplasmic reticulum stress and apoptosis, while ethanol per se showed limited effects.. Pharmacological activation of ALDH2 by Alda-1 reversed alcoholic steatosis and apoptosis through accelerating aldehyde clearance. This study indicates that ALDH2 is a promising molecular target and Alda-1 has therapeutic potential for treating alcoholic liver disease.

Topics: Aldehyde Dehydrogenase; Aldehyde Dehydrogenase, Mitochondrial; Aldehydes; Animals; Apoptosis; Benzamides; Benzodioxoles; Cell Death; Cell Line; Disease Models, Animal; Endoplasmic Reticulum Stress; Enzyme Activation; Fatty Liver, Alcoholic; Lipid Metabolism; Liver; Male; Metabolic Clearance Rate; Mice; Mice, Inbred C57BL; Rats

Hypoglycemic encephalopathy (HE) is caused by a lack of glucose availability to neuronal cells, and no neuroprotective drugs have been developed as yet. Studies on the pathogenesis of HE and the development of new neuroprotective drugs have been conducted using animal models such as the hypoglycemic coma model and non-coma hypoglycemia model. However, both models have inherent problems, and establishment of animal models that mimic clinical situations is desirable. In this study, we first developed a short-term hypoglycemic coma model in which rats could be maintained in an isoelectric electroencephalogram (EEG) state for 2 min and subsequent hyperglycemia without requiring anti-seizure drugs and an artificial ventilation. This condition caused the production of 4-hydroxy-2-nonenal (4-HNE), a cytotoxic aldehyde, in neurons of the hippocampus and cerebral cortex, and a marked increase in neuronal death as evaluated by Fluoro-Jade B (FJB) staining. We also investigated whether N-(1,3-benzodioxole-5-ylmethyl)-2,6-dichlorobenzamide (Alda-1), a small-molecule agonist of aldehyde dehydrogenase-2, could attenuate 4-HNE levels and reduce hypoglycemic neuronal death. After confirming that EEG recordings remained isoelectric for 2 min, Alda-1 (8.5 mg/kg) or vehicle (dimethyl sulfoxide; DMSO) was administered intravenously with glucose to maintain a blood glucose level of 250 to 270 mg/dL. Fewer 4-HNE and FJB-positive cells were observed in the cerebral cortex of Alda-1-treated rats than in DMSO-treated rats 24 h after glucose administration (P = 0.002 and P = 0.020). Thus, activation of the ALDH2 pathway could be a molecular target for HE treatment, and Alda-1 is a potentially neuroprotective agent that exerts a beneficial effect on neurons when intravenously administered simultaneously with glucose.

Topics: Aldehyde Dehydrogenase; Aldehyde Dehydrogenase, Mitochondrial; Aldehydes; Animals; Benzamides; Benzodioxoles; Cell Death; Cerebral Cortex; Coma; Disease Models, Animal; Glucose; Hippocampus; Hypoglycemia; Injections, Intravenous; Male; Mitochondrial Proteins; Neurons; Neuroprotective Agents; Rats; Rats, Sprague-Dawley

Exogenous aldehydes can cause pain in animal models, suggesting that aldehyde dehydrogenase-2 (ALDH2), which metabolizes many aldehydes, may regulate nociception. To test this hypothesis, we generated a knock-in mouse with an inactivating point mutation in ALDH2 (ALDH2*2), which is also present in human ALDH2 of ~540 million East Asians. The ALDH2*1/*2 heterozygotic mice exhibited a larger response to painful stimuli than their wild-type littermates, and this heightened nociception was inhibited by an ALDH2-selective activator (Alda-1). No effect on inflammation per se was observed. Using a rat model, we then showed that nociception tightly correlated with ALDH activity (R(2) = 0.90) and that reduced nociception was associated with less early growth response protein 1 (EGR1) in the spinal cord and less reactive aldehyde accumulation at the insult site (including acetaldehyde and 4-hydroxynonenal). Further, acetaldehyde- and formalin-induced nociceptive behavior was greater in the ALDH2*1/*2 mice than in the wild-type mice. Finally, Alda-1 treatment was even beneficial when given after the inflammatory agent was administered. Our data in rodent models suggest that the mitochondrial enzyme ALDH2 regulates nociception and could serve as a molecular target for pain control, with ALDH2 activators, such as Alda-1, as potential non-narcotic, cardiac-safe analgesics. Furthermore, our results suggest a possible genetic basis for East Asians' apparent lower pain tolerance.

Topics: Acetaldehyde; Acute Pain; Aldehyde Dehydrogenase; Aldehyde Dehydrogenase, Mitochondrial; Animals; Behavior, Animal; Benzamides; Benzodioxoles; Disease Models, Animal; Enzyme Activation; Formaldehyde; Heterozygote; Hyperalgesia; Inflammation; Mice, Inbred C57BL; Mitochondrial Proteins; Nociception; Rats

Mitochondrial dysfunction has been shown to play an important role in the development of atherosclerosis and nonalcoholic fatty liver disease (NAFLD). Mitochondrial aldehyde dehydrogenase (ALDH2), an enzyme responsible for the detoxification of reactive aldehydes, is considered to exert protective function in mitochondria. We investigated the influence of Alda-1, an activator of ALDH2, on atherogenesis and on the liver steatosis in apolipoprotein E knockout (apoE(-/-)) mice.. Alda-1 caused decrease of atherosclerotic lesions approximately 25% as estimated by "en face" and "cross-section" methods without influence on plasma lipid profile, atherosclerosis-related markers of inflammation, and macrophage and smooth muscle content in the plaques. Plaque nitrotyrosine was not changed upon Alda-1 treatment, and there were no changes in aortic mRNA levels of factors involved in antioxidative defense, regulation of apoptosis, mitogenesis, and autophagy. Hematoxylin/eosin staining showed decrease of steatotic changes in liver of Alda-1-treated apoE(-/-) mice. Alda-1 attenuated formation of 4-hydroxy-2-nonenal (4-HNE) protein adducts and decreased triglyceride content in liver tissue. Two-dimensional electrophoresis coupled with mass spectrometry identified 20 differentially expressed mitochondrial proteins upon Alda-1 treatment in liver of apoE(-/-) mice, mostly proteins related to metabolism and oxidative stress. The most up-regulated were the proteins that participated in beta oxidation of fatty acids.. Collectively, Alda-1 inhibited atherosclerosis and attenuated NAFLD in apoE(-/-) mice. The pattern of changes suggests a beneficial effect of Alda-1 in NAFLD; however, the exact liver functional consequences of the revealed alterations as well as the mechanism(s) of antiatherosclerotic Alda-1 action require further investigation.

Topics: Aldehyde Dehydrogenase; Aldehyde Dehydrogenase, Mitochondrial; Animals; Aorta; Aortic Diseases; Apolipoproteins E; Atherosclerosis; Benzamides; Benzodioxoles; Biomarkers; Disease Models, Animal; Enzyme Activation; Enzyme Activators; Female; Gene Expression Regulation; Hep G2 Cells; Humans; Liver; Mice, Inbred C57BL; Mice, Knockout; Mitochondria, Liver; Non-alcoholic Fatty Liver Disease; Signal Transduction

Reactive aldehydes can initiate protein oxidative damage which may contribute to heart senescence. Sirtuin 1 (SIRT1) is considered to be a potential interventional target for I/R injury management in the elderly. We hypothesized that aldehyde mediated carbonyl stress increases susceptibility of aged hearts to ischemia/reperfusion (I/R) injury, and elucidate the underlying mechanisms with a focus on SIRT1. Male C57BL/6 young (4-6 mo) and aged (22-24 mo) mice were subjected to myocardial I/R. Cardiac aldehyde dehydrogenase (ALDH2), SIRT1 activity and protein carbonyls were assessed. Our data revealed that aged heart exhibited increased endogenous aldehyde/carbonyl stress due to impaired ALDH2 activity concomitant with blunted SIRT1 activity (P<0.05). Exogenous toxic aldehydes (4-HNE) exposure in isolated cardiomyocyte verified that aldehyde-induced carbonyl modification on SIRT1 impaired SIRT1 activity leading to worse hypoxia/reoxygenation (H/R) injury, which could all be rescued by Alda-1 (ALDH2 activator) (all P<0.05). However, SIRT1 inhibitor blocked the protective effect of Alda-1 on H/R cardiomyocyte. Interestingly, myocardial I/R leads to higher carbonylation but lower activity of SIRT1 in aged hearts than that seen in young hearts (P<0.05). The application of Alda-1 significantly reduced the carbonylation on SIRT1 and markedly improved the tolerance to in vivo I/R injury in aged hearts, but failed to protect Sirt1(+/-) knockout mice against myocardial I/R injury. This was verified by Alda-1 treatment improved postischemic contractile function recovery in ex vivo perfused aged but not in Sirt1(+/-) hearts. Thus, aldehyde/carbonyl stress is accelerated in aging heart. These results provide a new insight that impaired cardiac SIRT1 activity by carbonyl stress plays a critical role in the increased susceptibility of aged heart to I/R injury. ALDH2 activation can restore this aging-related myocardial ischemic intolerance.

Topics: Adaptation, Physiological; Age Factors; Aging; Aldehyde Dehydrogenase; Aldehyde Dehydrogenase, Mitochondrial; Aldehydes; Animals; Benzamides; Benzodioxoles; Disease Models, Animal; Enzyme Activation; Hypoxia; Male; Mice; Mice, Knockout; Myocardial Ischemia; Myocardial Reperfusion Injury; Myocytes, Cardiac; Sirtuin 1; Stress, Physiological

Although premenopausal females have a lower risk for cardiovascular disease, the mechanism(s) are poorly understood.. We tested the hypothesis that cardioprotection in females is mediated by altered mitochondrial protein levels and/or posttranslational modifications.. Using both an in vivo and an isolated heart model of ischemia and reperfusion (I/R), we found that females had less injury than males. Using proteomic methods we found that female hearts had increased phosphorylation and activity of aldehyde dehydrogenase (ALDH)2, an enzyme that detoxifies reactive oxygen species (ROS)-generated aldehyde adducts, and that an activator of ALDH2 reduced I/R injury in males but had no significant effect in females. Wortmannin, an inhibitor of phosphatidylinositol 3-kinase, blocked the protection and the increased phosphorylation of ALDH2 in females, but had no effect in males. Furthermore, we found an increase in phosphorylation of alpha-ketoglutarate dehydrogenase (alphaKGDH) in female hearts. alphaKGDH is a major source of ROS generation particularly with a high NADH/NAD ratio which occurs during I/R. We found decreased ROS generation in permeabilized female mitochondria given alphaKGDH substrates and NADH, suggesting that increased phosphorylation of alphaKGDH might reduce ROS generation by alphaKGDH. In support of this hypothesis, we found that protein kinase C-dependent phosphorylation of purified alphaKGDH reduced ROS generation. Additionally, myocytes from female hearts had less ROS generation following I/R than males and addition of wortmannin increased ROS generation in females to the same levels as in males.. These data suggest that posttranslational modifications can modify ROS handling and play an important role in female cardioprotection.

Topics: Aldehyde Dehydrogenase; Aldehyde Dehydrogenase, Mitochondrial; Androstadienes; Animals; Benzamides; Benzodioxoles; Blotting, Western; Disease Models, Animal; Electrophoresis, Gel, Two-Dimensional; Enzyme Activation; Enzyme Activators; Estradiol; Female; Indoles; Ketoglutarate Dehydrogenase Complex; Male; Maleimides; Mitochondria, Heart; Mitochondrial Proteins; Myocardial Infarction; Myocardial Reperfusion Injury; Myocytes, Cardiac; NAD; Ovariectomy; Oxidative Stress; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Protein Kinase C; Protein Kinase Inhibitors; Protein Processing, Post-Translational; Proteomics; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Sex Factors; Ventricular Function, Left; Ventricular Pressure; Wortmannin