Toxicity of a serotonin-derived neuromelanin

Postoperative Cognitive Dysfunction (POCD) is associated with increased mortality in the elderly and may occur from lipid peroxidation in aging. We previously showed that sevoflurane sequesters acrolein, which promotes the formation of a novel species of a putative neuromelanin. The current study examined the properties of this serotonin-derived melanoid (SDM). The interaction of SDM with unilamellar vesicles (ULVs) was examined using lipid membrane probes. Vesicle disruption was investigated by leakage of dye from calcein-loaded ULVs. We observed that SDM decreased diphenyl-hexatriene fluorescence anisotropy and increased the temperature-dependent change in anisotropy. SDM changed the absorbance of merocyanin-bound ULVs. SDM increased detergent-mediated calcein leakage. SDM structure was dramatically altered upon interaction with ULVs. We also observed that SDM enhanced detergent-mediated leakage of loaded ULVs, suggesting that SDM may be neurotoxic. We propose that inhalational agents, which sequester acrolein, may promote the production of certain species of neuromelanin that depletes local serotonin and enhances neuronal vulnerability.     

Carnosine Protects Against the Neurotoxic Effects of a Serotonin-Derived Melanoid

Anesthesia-related postoperative cognitive dysfunction (POCD) leads to morbidity in the elderly. Lipid peroxidative byproducts (i.e. acrolein) accumulate in aging and may play a role. Sevoflurane, an inhaled anesthetic, sequesters acrolein and enhances the formation of a serotonin-derived melanoid (SDM). SDM may be a biologically relevant polymeric melanoid that we previously showed exhibits redox activity and disrupts lipid bilayers. In this study, we examined the toxicity of SDM in cell culture and looked at protection using L-carnosine. SDM’s toxic effects were tested on neuronal-like SH-SY5Y cells, causing an exponential decrease in viability, while human dermal fibroblasts were completely resistant to the toxic effects. SDM brought about morphological changes to differentiated SH-SY5Y cells, particularly to neuronal processes. Co- but not pre-treatment with L-carnosine protected differentiated SH-SY5Y cells exposed to SDM. Our mechanism suggests focal sevoflurane-induced sequestration of age-related acrolein leading to SDM synthesis and neuronal impairment, which is prevented by L-carnosine.     

Trifluoroethanol increases albumin's susceptibility to chemical modification

Acrolein-dependent chemical modification is implicated in the etiology of postoperative cognitive dysfunction (POCD). We examined this process further using human serum albumin (HSA), which is a target of acrolein modification and contains anesthetic binding sites. We tested whether trifluoroethanol (TFE), which mimics inhaled anesthetics, affects the susceptibility of HSA to modification by acrolein. We observed that acrolein promoted the formation of fluorescent adducts. TFE (10%) increased the amount of acrolein-HSA adducts. TFE (40%) caused a 5-fold increase in adduct formation. Acrolein also increased tryptophan anisotropy of HSA, which was further increased by TFE (10%). Acrolein-induced protein cross-linking was also increased in the presence of TFE (40%). These observations suggest that TFE promotes acrolein-induced modification of HSA, supporting a putative mechanism for POCD.     

Acute effects of acrolein on breathing: role of vagal bronchopulmonary afferents.

Spontaneous inhalation of acrolein vapor (350 ppm, 1 ml/100 g body wt) elicited an immediate and transient inhibitory effect on breathing in anesthetized rats, characterized by a prolongation of expiratory duration and accompanied by a bradycardia; ventilation was reduced by 47 +/- 6%, which returned to baseline after three to seven breaths. When both vagi were cooled to 6.6 +/- 0.1 degrees C, the reflex apneic response to lung inflation was completely abolished but the bradypneic response to acrolein was not affected. After perineural capsaicin treatment of both cervical vagi to selectively block the capsaicin-sensitive C-fiber afferents, acrolein no longer evoked an inhibitory effect on breathing; conversely, an augmented inspiration was consistently elicited with the first breath of acrolein inhalation, which was subsequently abolished by cooling both vagi to 6.5 degrees C. The inhibitory effect of inhaling acrolein at a lower concentration (200 ppm) was not detectable, whereas that of a higher concentration (600 ppm) was more intense and prolonged. All these responses were completely eliminated by bilateral vagotomy. These results suggest that inhaled acrolein activated both vagal C-fiber endings and rapidly adapting irritant receptors in the airways, but the acrolein-induced inhibitory effect on breathing was elicited primarily by the C-fiber afferent stimulation.     

Mechanism of action of adenosylcobalamin: 3-fluoro-1,2-propranediol as substrate for propanediol dehydrase--mechanistic implications.

3-Fluro-1,2-propanediol has been found to be a substrate for propanediol dehydrase and has very similar binding and catalytic constants compared to the natural substrate. The only isolable products of the reaction are acrolein and inorganic fluoride; with 3-fluoro-3,3-dideuterio-1,2-propanediol as substrate, only 3,3-dideuterioacrolein is obtained. These results indicate that the primary product of the reaction is 3-fluoropropionaldehyde which spontaneously loses hydrogen fluoride to yield acrolein. The similar kinetic parameters for the fluorinated as compared to the normal substrate suggest that significant charge does not develop on the fluorinated or, by implication, the natural substrate during any rate-limiting steps of the reaction. These results support a radical, as contrasted to an ionic pathway for reactions involving adenosylcobalamin and diol dehydrase.     

Positive Allosteric Modulation of Kv Channels by Sevoflurane: Insights into the Structural Basis of Inhaled Anesthetic Action

Inhalational general anesthesia results from the poorly understood interactions of haloethers with multiple protein targets, which prominently includes ion channels in the nervous system. Previously, we reported that the commonly used inhaled anesthetic sevoflurane potentiates the activity of voltage-gated K⁺ (Kv) channels, specifically, several mammalian Kv1 channels and the Drosophila K-Shaw2 channel. Also, previous work suggested that the S4-S5 linker of K-Shaw2 plays a role in the inhibition of this Kv channel by n-alcohols and inhaled anesthetics. Here, we hypothesized that the S4-S5 linker is also a determinant of the potentiation of Kv1.2 and K-Shaw2 by sevoflurane. Following functional expression of these Kv channels in Xenopus oocytes, we found that converse mutations in Kv1.2 (G329T) and K-Shaw2 (T330G) dramatically enhance and inhibit the potentiation of the corresponding conductances by sevoflurane, respectively. Additionally, Kv1.2-G329T impairs voltage-dependent gating, which suggests that Kv1.2 modulation by sevoflurane is tied to gating in a state-dependent manner. Toward creating a minimal Kv1.2 structural model displaying the putative sevoflurane binding sites, we also found that the positive modulations of Kv1.2 and Kv1.2-G329T by sevoflurane and other general anesthetics are T1-independent. In contrast, the positive sevoflurane modulation of K-Shaw2 is T1-dependent. In silico docking and molecular dynamics-based free-energy calculations suggest that sevoflurane occupies distinct sites near the S4-S5 linker, the pore domain and around the external selectivity filter. We conclude that the positive allosteric modulation of the Kv channels by sevoflurane involves separable processes and multiple sites within regions intimately involved in channel gating.     

Tissue distribution of lipid peroxidation product acrolein in human colon carcinogenesis

Lipid peroxidation product acrolein, well-known pollutant in tobacco and automotive smoke, accumulates in vivo bound to proteins. It suppresses p53 synthesis acting as potent carcinogenic factor for oral, respiratory and bladder carcinomas, while its possible association with colon carcinogenesis was not studied so far. We used genuine monoclonal antibody to evaluate immunohistochemical distribution of acrolein–protein adducts in 113 human colon tumours. The presence of acrolein–protein adducts was increasing with respect to colon carcinogenesis, from moderate appearance in tubular and villotubular low-grade adenomas to abundant and diffuse distribution in high-grade villotubular adenomas and Dukes A carcinomas. However, in advanced Dukes B and C carcinomas acrolein was hardly noticed, although, its protein adducts were found abundant in non-malignant colon epithelium of these patients. There was no relationship between p53 and acrolein distribution. According to these findings, acrolein seems to be lipid peroxidation product associated with transition from benign into malignant colon tumours.     

Old yellow enzymes protect against acrolein toxicity in the yeast Saccharomyces cerevisiae

Acrolein is a ubiquitous reactive aldehyde which is formed as a product of lipid peroxidation in biological systems. In this present study, we screened the complete set of viable deletion strains in Saccharomyces cerevisiae for sensitivity to acrolein to identify cell functions involved in resistance to reactive aldehydes. We identified 128 mutants whose gene products are localized throughout the cell. Acrolein-sensitive mutants were distributed among most major biological processes but particularly affected gene expression, metabolism, and cellular signaling. Surprisingly, the screen did not identify any antioxidants or similar stress-protective molecules, indicating that acrolein toxicity may not be mediated via reactive oxygen species. Most strikingly, a mutant lacking an old yellow enzyme (OYE2) was identified as being acrolein sensitive. Old yellow enzymes are known to reduce alpha,beta-unsaturated carbonyl compounds in vitro, but their physiological roles have remained uncertain. We show that mutants lacking OYE2, but not OYE3, are sensitive to acrolein, and overexpression of both isoenzymes increases acrolein tolerance. Our data indicate that OYE2 is required for basal levels of tolerance, whereas OYE3 expression is particularly induced following acrolein stress. Despite the range of alpha,beta-unsaturated carbonyl compounds that have been identified as substrates of old yellow enzymes in vitro, we show that old yellow enzymes specifically mediate resistance to small alpha,beta-unsaturated carbonyl compounds, such as acrolein, in vivo.     

七氟烷全身麻醉对失血性休克复苏小鼠血流动力学及记忆功能的影响机制

摘要 目的：探讨七氟烷全身麻醉对失血性休克复苏小鼠血流动力学及记忆功能的影响机制。方法：将建立成功的42只失血性休克复苏小鼠按照随机数字表法平分为3组-模型组、氧气组、七氟烷组，对照组吸入空气4 h，氧气组吸入100.0 %氧气4 h，七氟烷组吸入100.0 %氧气和2.5 %七氟烷4 h，监测与记录小鼠血流动力学及记忆功能变化情况。结果：氧气组、七氟烷组麻醉后第3 d与第7 d的Morris水迷宫逃避潜伏期少于模型组，穿越原平台次数多于模型组(P<0.05)，氧气组与七氟烷组对比差异有统计学意义(P<0.05)。氧气组、七氟烷组麻醉后第3 d与第7 d的去甲肾上腺素(Norepinephrine，NE)pD₂与Emax值高于模型组(P<0.05)，七氟烷组高于氧气组(P<0.05)。氧气组、七氟烷组麻醉后第3 d与第7 d的脑组织凋亡指数低于模型组(P<0.05)，七氟烷组低于氧气组(P<0.05)。氧气组、七氟烷组麻醉后第3 d与第7 d的海马组织Akt、Caspase-3蛋白相对表达水平低于模型组(P<0.05)，七氟烷组低于氧气组(P<0.05)。结论：七氟烷全身麻醉在失血性休克复苏小鼠的应用能抑制Akt、Caspase-3蛋白的表达，可抑制脑组织神经细胞凋亡，可改善血流动力学状况，促进小鼠记忆能力的恢复。     

Sevoflurane modulates the activity of glyceraldehyde 3-phosphate dehydrogenase

The mechanism of inhalation anesthesia remains to be fully elucidated. While certain neuronal membrane proteins are considered sites of action, cytosolic proteins may also be targets. We hypothesize that inhaled anesthetics may act via glyceraldehyde 3-phosphate dehydrogenase (GAPDH), which has recently been shown to participate in neuronal inhibition. We examined the effects of sevoflurane, a halogenated ether anesthetic, on the catalytic and fluorescence properties of GAPDH. Initial rates of oxidoreductase activity decreased approximately 30% at saturating levels of sevoflurane. NADH-stimulated oxidoreductase activity (25 microM NADH; 0.8mM NAD+) increased with sevoflurane. Sevoflurane quenched tryptophan fluorescence emission and increased polarization. Additionally, sevoflurane increased the susceptibility of GAPDH to thermal denaturation suggesting an effect on conformation. Our findings warrant further research on sevoflurane's effect on GAPDH and indicate that this approach may lead to delineation of a novel contribution to the mechanism of anesthesia.     

Tissue distribution of lipid peroxidation product acrolein in human colon carcinogenesis

Lipid peroxidation product acrolein, well-known pollutant in tobacco and automotive smoke, accumulates in vivo bound to proteins. It suppresses p53 synthesis acting as potent carcinogenic factor for oral, respiratory and bladder carcinomas, while its possible association with colon carcinogenesis was not studied so far. We used genuine monoclonal antibody to evaluate immunohistochemical distribution of acrolein-protein adducts in 113 human colon tumours. The presence of acrolein-protein adducts was increasing with respect to colon carcinogenesis, from moderate appearance in tubular and villotubular low-grade adenomas to abundant and diffuse distribution in high-grade villotubular adenomas and Dukes A carcinomas. However, in advanced Dukes B and C carcinomas acrolein was hardly noticed, although, its protein adducts were found abundant in non-malignant colon epithelium of these patients. There was no relationship between p53 and acrolein distribution. According to these findings, acrolein seems to be lipid peroxidation product associated with transition from benign into malignant colon tumours.     

Sevoflurane modulates the activity of glyceraldehyde 3-phosphate dehydrogenase

The mechanism of inhalation anesthesia remains to be fully elucidated. While certain neuronal membrane proteins are considered sites of action, cytosolic proteins may also be targets. We hypothesize that inhaled anesthetics may act via glyceraldehyde 3-phosphate dehydrogenase (GAPDH), which has recently been shown to participate in neuronal inhibition. We examined the effects of sevoflurane, a halogenated ether anesthetic, on the catalytic and fluorescence properties of GAPDH. Initial rates of oxidoreductase activity decreased approximately 30% at saturating levels of sevoflurane. NADH-stimulated oxidoreductase activity (25 μM NADH; 0.8 mM NAD⁺) increased with sevoflurane. Sevoflurane quenched tryptophan fluorescence emission and increased polarization. Additionally, sevoflurane increased the susceptibility of GAPDH to thermal denaturation suggesting an effect on conformation. Our findings warrant further research on sevoflurane's effect on GAPDH and indicate that this approach may lead to delineation of a novel contribution to the mechanism of anesthesia.     

Increase in putrescine,amine oxidase,and acrolein in plasma of renal failure patients

Since polyamines have been suggested to be one of the uremic "toxins," the levels of each polyamine, its oxidized product, acrolein, and amine oxidase in plasma of patients with renal failure were investigated. The level of putrescine was increased, whereas the level of spermine was decreased in the plasma of patients with renal failure. The patients also had increased serum amine oxidase activity leading to increased degradation of spermine. Both levels of free and protein-conjugated acrolein were also increased in plasma of patients with renal failure. The accumulated acrolein found as protein conjugates was equivalent to 180 microM, which was 6-fold higher than in plasma of normal subjects. It was found that acrolein is mainly produced by polyamine oxidase in plasma. A cell lysate containing polyamine oxidase was cytotoxic in the presence of spermine. Our results indicate that the level of acrolein is well correlated with the degree of seriousness of chronic renal failure.     

Old Yellow Enzymes Protect against Acrolein Toxicity in the Yeast Saccharomyces cerevisiae

Acrolein is a ubiquitous reactive aldehyde which is formed as a product of lipid peroxidation in biological systems. In this present study, we screened the complete set of viable deletion strains in Saccharomyces cerevisiae for sensitivity to acrolein to identify cell functions involved in resistance to reactive aldehydes. We identified 128 mutants whose gene products are localized throughout the cell. Acrolein-sensitive mutants were distributed among most major biological processes but particularly affected gene expression, metabolism, and cellular signaling. Surprisingly, the screen did not identify any antioxidants or similar stress-protective molecules, indicating that acrolein toxicity may not be mediated via reactive oxygen species. Most strikingly, a mutant lacking an old yellow enzyme (OYE2) was identified as being acrolein sensitive. Old yellow enzymes are known to reduce α,β-unsaturated carbonyl compounds in vitro, but their physiological roles have remained uncertain. We show that mutants lacking OYE2, but not OYE3, are sensitive to acrolein, and overexpression of both isoenzymes increases acrolein tolerance. Our data indicate that OYE2 is required for basal levels of tolerance, whereas OYE3 expression is particularly induced following acrolein stress. Despite the range of α,β-unsaturated carbonyl compounds that have been identified as substrates of old yellow enzymes in vitro, we show that old yellow enzymes specifically mediate resistance to small α,β-unsaturated carbonyl compounds, such as acrolein, in vivo.     

Induction of COX-2 by acrolein in rat lung epithelial cells

Acrolein is a highly reactive alpha, beta-unsaturated aldehyde, and a product of lipid peroxidation reactions. Acrolein is also an environmental pollutant and a key component of cigarette smoke, and has been implicated in multiple respiratory diseases. Lung tissue is a primary target for acrolein toxicity in smokers and may lead to chronic lung inflammation and lung cancer. Chronic inflammation, associated with expression of cyclooxygenase-2 (COX-2) and prostaglandins, are predisposing factors for malignancy. In this study, we investigated the induction of COX-2 by acrolein in rat lung epithelial cells and its related signaling cascade. Induction of COX-2 by acrolein was significant at 6 h post-treatment and was dependent upon NFκB activation. The activation of NFκB by acrolein was induced as a result of degradation of IκBα over the time of treatment. In addition, the upstream signaling cascade involved Raf-1/ERK activation by acrolein in the COX-2 induction and was inhibited by GW5074 (a Ras/Raf-1/ERK inhibitor), thereby providing evidence for the role of this cascade in this process. The results of these studies offer an explanation for the mechanism of COX-2 induction by acrolein in rat lung epithelial cells.     

Glutathione elevation and its protective role in acrolein-induced protein damage in synaptosomal membranes: relevance to brain lipid peroxidation in neurodegenerative disease.

Oxidative stress may be a hallmark of several neurodegenerative disorders, including Alzheimer's disease (AD) Huntington's, and Parkinson's diseases as well as amyotrophic lateral sclerosis. Acrolein is a highly reactive product of lipid peroxidation that is elevated in the brains of persons with AD. This alkenal potentially can react with proteins by Michael addition to alter their structure and function. In the present study, we used electron paramagnetic resonance in conjunction with a protein-specific spin label to monitor synaptosomal membrane protein conformational alterations induced by acrolein. A dose-dependent increased conformational alteration was observed. Consistent with this finding, protein carbonyl levels from protein-bound acrolein were significantly elevated. However, pretreatment of synaptosomes with glutathione ethyl ester (GEE) significantly ameliorated both the conformational alterations and protein carbonyls induced by acrolein. Based on this success, we tested the hypothesis that elevated levels of endogenous glutathione (GSH) would offer protection against acrolein-induced oxidative stress. In-vivo elevation of GSH (215% over control, P<0.04) was produced by i.p. injection of N-acetylcysteine (NAC), a known precursor of GSH. Synaptosomes were treated with vehicle or 2 nM acrolein, the level of this alkenal found in AD brain. In contrast to synaptosomes from control animals, which had significantly increased protein carbonyl levels following addition of 2 nM acrolein, synaptosomes that were isolated from NAC-treated rodents and treated with 2 nM acrolein showed no increased carbonyl levels compared to untreated controls. These results demonstrate protection by increased in-vivo GSH levels against acrolein-induced oxidative stress at levels found in AD brain and are consistent with the notion that methods to increase endogenous GSH levels in neurodegenerative diseases associated with oxidative stress may be promising.     

Induction of thioredoxin reductase as an adaptive response to acrolein in human umbilical vein endothelial cells

Acrolein is a highly electrophilic alpha,beta-unsaturated aldehyde to which humans are exposed in a variety of environment situations and is also a product of lipid peroxidation. Increased unsaturated aldehyde levels and reduced antioxidant status play an important role in the pathogenesis of a number of human diseases such as Alzheimer's, atherosclerosis, and diabetes. Mammalian thioredoxin reductase (TR), a central antioxidant enzyme, is a selenoprotein that catalyzes the reduction of oxidized thioredoxin. The findings reported here show that low concentrations of acrolein rapidly inactivate TR, both in vitro and in vivo. These data suggest that acrolein may directly inactivate TR, resulting in an increase in oxidative cellular damage. In addition, we also found that the initial inactivation of TR molecules by acrolein triggers a compensatory signal for inducing TR gene expression in human umbilical vein endothelial cells (HUVEC). The results of the present study suggest that HUVEC may have a protective system against cell damage by acrolein via the upregulation of TR, which is an adaptive response to oxidative stress.     

Proteomic profiling of acrolein adducts in human lung epithelial cells

Acrolein (2,3-propenal) is a major indoor and outdoor air pollutant originating largely from tobacco smoke or organic combustion. Given its high reactivity, the adverse effects of inhaled acrolein are likely due to direct interactions with the airway epithelium, resulting in altered epithelial function, but only limited information exists to date regarding the primary direct cellular targets for acrolein. Here, we describe a global proteomics approach to characterize the spectrum of airway epithelial protein targets for Michael adduction in acrolein-exposed bronchial epithelial (HBE1) cells, based on biotin hydrazide labeling and avidin purification of biotinylated proteins or peptides for analysis by LC-MS/MS. Identified protein targets included a number of stress proteins, cytoskeletal proteins, and several key proteins involved in redox signaling, including thioredoxin reductase, thioredoxin, peroxiredoxins, and glutathione S-transferase π. Because of the central role of thioredoxin reductase in cellular redox regulation, additional LC-MS/MS characterization was performed on purified mitochondrial thioredoxin reductase to identify the specific site of acrolein adduction, revealing the catalytic selenocysteine residue as the target responsible for enzyme inactivation. Our findings indicate that these approaches are useful in characterizing major protein targets for acrolein, and will enhance mechanistic understanding of the impact of acrolein on cell biology.