Levels Of Protein Adducts Research Articles

Abstract 428: Peptides Enrichment by Hydrophobic Interaction Chromatography for Detection of Oxidized Phospholipid-modified Protein Adducts

Oxidized phospholipids (oxPLs) accumulate at sites of oxidative stress and contribute significantly to atherosclerosis, acute inflammation, lung injury, and ischemia. Most oxPLs have electrophilic substituents and are highly likely to form covalent adducts with proteins, thus compromising protein function. Detection of covalent interaction between oxPLs and proteins could provide important information about the type of proteins preferentially modified by oxPLs and on the mechanism of interaction between proteins and oxPLs. These studies are very challenging due to low levels of oxPLs protein adducts. We have developed a highly efficient enrichment method for oxPLs-modified peptides that employs C18 solid phase extraction columns. This method takes advantage of the difference in polarity between oxPLs modified peptides and the corresponding unmodified peptides as well as other interfering compounds. Using this enrichment method, we have been able to detect very low levels of oxPLs modified peptides (< 2 ppm) from tryptic digestion mixture of cell lysate using LC-MS. To demonstrate the performance of the method, we studied the interaction of apolipoprotein A-I (apoA-I) in high density lipoprotein with a specific oxPL (1-palmitoyl-2-(5’-oxo-valeroyl)-sn-glycero-3-phosphocholine, POVPC) at pathophysiological concentrations. We found that Lys 94, Lys 96 and Lys 195 are covalently modified by POVPC. Thus, the present study is the first to detected oxPL adducts of apoA-I in HDL.

Loss of quadriceps muscle oxidative phenotype and decreased endurance in patients with mild-to-moderate COPD

Being well-established in advanced chronic obstructive pulmonary disease (COPD), skeletal muscle dysfunction and its underlying pathology have been scarcely investigated in patients with mild-to-moderate airflow obstruction. We hypothesized that a loss of oxidative phenotype (oxphen) associated with decreased endurance is present in the skeletal muscle of patients with mild-to-moderate COPD. In quadriceps muscle biopsies from 29 patients with COPD (forced expiratory volume in 1 s [FEV1] 58 ± 16%pred, body mass index [BMI] 26 ± 4 kg/m(2)) and 15 controls (BMI 25 ± 3 kg/m(2)) we assessed fiber type distribution, fiber cross-sectional areas (CSA), oxidative and glycolytic gene expression, OXPHOS protein levels, metabolic enzyme activity, and levels of oxidative stress markers. Quadriceps function was assessed by isokinetic dynamometry, body composition by dual-energy X-ray absorptiometry, exercise capacity by an incremental load test, and physical activity level by accelerometry. Compared with controls, patients had comparable fat-free mass index, quadriceps strength, and fiber CSA, but quadriceps endurance was decreased by 29% (P = 0.002). Patients with COPD had a clear loss of muscle oxphen: a fiber type I-to-II shift, decreased levels of OXPHOS complexes IV and V subunits (47% and 31%, respectively; P < 0.05), a decreased ratio of 3-hydroxyacyl-CoA dehydrogenase/phosphofructokinase (PFK) enzyme activities (38%, P < 0.05), and decreased peroxisome proliferator-activated receptor-γ coactivator-1α (40%; P < 0.001) vs. increased PFK (67%; P < 0.001) gene expression levels. Within the patient group, markers of oxphen were significantly positively correlated with quadriceps endurance and inversely with the increase in plasma lactate relative to work rate during the incremental test. Levels of protein carbonylation, tyrosine nitration, and malondialdehyde protein adducts were comparable between patients and controls. However, among patients, oxidative stress levels were significantly inversely correlated with markers of oxphen and quadriceps endurance. Reduced muscle endurance associated with underlying loss of muscle oxphen is already present in patients with mild-to-moderate COPD without muscle wasting.

The Effects of Chromium Picolinate and Chromium Histidinate Administration on NF-κB and Nrf2/HO-1 Pathway in the Brain of Diabetic Rats

The objective of this experiment was to investigate the effects of supplemental chromium picolinate (CrPic) and chromium histidinate (CrHis) on nuclear factor-kappa B (NF-κB p65) and nuclear factor (erythroid-derived 2)-like 2 (Nrf2) signaling pathway in diabetic rat brain. Nondiabetic (n = 45) and diabetic (n = 45) male Wistar rats were either not supplemented or supplemented with CrPic or CrHis via drinking water to consume 8μg elemental chromium (Cr) per day for 12weeks. Diabetes was induced by streptozotocin injection (40mg/kg i.p., for 2weeks) and maintained by high-fat feeding (40%). Diabetes was associated with increases in cerebral NF-κB and 4-hydroxynonenal (4-HNE) protein adducts and decreased in cerebral nuclear factor of kappa light polypeptide gene enhancer in B cells inhibitor, alpha (IκBα) and Nrf2 levels. Both Cr chelates were effective to decrease levels of NF-κB and 4-HNE protein adducts and to increase levels of IκBα and Nrf2 in the brain of diabetic rats. However, responses of these increases and decreases were more notable when Cr was supplemented as CrHis than as CrPic. In conclusion, Cr may play a protective role in cerebral antioxidant defense system in diabetic subjects via the Nrf2 pathway by reducing inflammation through NF-κB p65 inhibition. Histidinate form of Cr was superior to picolinate form of Cr in reducing NF-κB expression and increasing Nrf2 expression in the brain of diabetic rats.

Moringa oleifera Hydroethanolic Extracts Effectively Alleviate Acetaminophen-Induced Hepatotoxicity in Experimental Rats through Their Antioxidant Nature

The aim of the study was to investigate the in vitro antioxidant properties Moringa oleifera Lam. (MO) extracts and its curative role in acetaminophen (APAP)- induced toxic liver injury in rats caused by oxidative damage. The total phenolic content and antioxidant properties of hydroethanolic extracts of different MO edible parts were investigated by employing an established in vitro biological assay. In the antihepatotoxic study, either flowers or leaves extract (200 mg/kg or 400 mg/kg, i.p) were administered an hour after APAP administration, respectively. N-Acetylcysteine was used as the positive control against APAP-induced hepatotoxicity. The levels of liver markers such as alanine aminotransferase (ALT) and the levels of oxidative damage markers including malondialdehyde (MDA), 4-hydroxynonenal (4-HNE) protein adduct, reduced glutathione (GSH), superoxide dismutase (SOD) and catalase (CAT) were analysed and compared between experimental groups. Among MO edible parts the flower extracts contain the highest total phenolic content and antioxidant capacity, followed by leaves extract. The oxidative marker MDA, as well as 4-HNE protein adduct levels were elevated and GSH, SOD and CAT were significantly decreased in groups treated with hepatotoxin. The biochemical liver tissue oxidative markers measured in the rats treated with MO flowers and leaves hydroethanolic extracts showed a significant (p < 0.05) reduction in the severity of the liver damage. The results of this study strongly indicate the therapeutic properties of MO hydroethanolic extracts against acute liver injury and thereby scientifically support its traditional use.

Abstract 258: High-Efficiency Enrichment of Oxidized Phospholipid-Modified Peptides with C18 Solid Phase Extraction Column Facilitates Detection of Covalent Interaction Between Oxidized Phospholipids and Proteins

Oxidized phospholipids accumulate at sites of oxidative stress and contribute significantly to atherosclerosis, acute inflammation, lung injury and ischemia. Most of the oxidized phospholipids have electrophilic substituents and are highly likely to form covalent adducts with proteins thus compromising protein function. Detection of covalent interaction between oxidized phospholipids and proteins could provide important information about proteins preferentially modified by oxidized phospholipids in a relevant biological system, as well as, shed light on the mechanism of interaction between proteins and oxidized phospholipids. However, due to the relatively low levels of phospholipid protein adducts compared to unmodified proteins, such studies are challenging. We developed a high efficiency enrichment method for oxidized phospholipid-modified peptides that employs a C18 SPE column. Using this approach, in combination with LC-MS analysis, we studied the interaction of a synthetic oxidized phospholipid (9-keto-12-oxo-10-dodecenoic acid esters of 2-lysoPC, KODA-PC) with Apo-AI in HDL. We found that Lys45, His162, His193 and Lys208 are the major residues for covalent modification by oxidized phospholipids. According to the double superhelix model of HDL, these residues are located at both terminals of the double superhelix of Apo-AI. Thus the present study, for the first time, successfully detected oxidized phospholipids adducts of ApoA-I in HDL.

Identification of liver proteins altered by type 2 diabetes mellitus in obese subjects

Type 2 diabetes mellitus (T2DM) is a well-known factor risk for non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH) in obese patients. To better understand the association between T2DM and NAFLD, global changes in protein expression in diabetic and non-diabetic obese subjects were assessed by a proteomic approach. Liver samples were obtained from diabetic and non-diabetic morbid obese subjects (BMI>40 kg/m(2) ). Histological analysis was used to evaluate hepatic steatosis and the degree of anatomopathological alteration. Changes in protein expression were analysed by two-dimentional electrophoresis combined with MALDI-TOF mass spectrometry. Levels of glutathione, carbonyl and 4-HNE protein adducts were used to assess oxidative stress status. Of 850 proteins analysed, 33 were differentially expressed in T2DM obese subjects. Of these, 27 were unequivocally identified by mass spectrometry. Analysis of protein sets revealed patterns of decreased abundance in mitochondrial enzymes, proteins involved in methione metabolism, and oxidative stress response. Accordingly, T2DM subjects showed decreased levels of glutathione, the antioxidant byproduct of methionine metabolism via the transsulfuration pathway, and higher levels of protein and lipid oxidative damage. Changes in detoxyfing enzymes, carbohydrate metabolism, proteasome subunits and retinoic acid synthesis were also found. The results suggest alterations in mitochondrial function and methionine metabolism as potential contributing factors to increased oxidative stress in liver of obese diabetic patients which may be influencing the development of NAFLD and NASH.

Modulation of Aldehyde Dehydrogenase Activity Affects (±)-4-Hydroxy-2E-nonenal (HNE) Toxicity and HNE–Protein Adduct Levels in PC12 Cells

Oxidative stress is known to be one of the major factors underlying Parkinson's disease (PD). One of the consequences of oxidative stress is lipid peroxidation. A toxic product of lipid peroxidation, (±)-4-hydroxy-2E-nonenal (HNE) leads to membrane disruption and formation of HNE-protein adducts and such adducts have been detected in PD brain tissues. Aldehyde dehydrogenases (ALDHs) are involved in metabolizing HNE and other endogenous aldehydes. Interestingly, the cytosolic aldehyde dehydrogenase 1A1 (ALDH1A1) has been reported to be down-regulated in brain tissues affected in PD which could result in enhancement of HNE toxicity. We sought to first establish the role of ALDH1A1 in mediating HNE toxicity in PC12 cells by overexpressing ALDH1A1 and by using disulfiram, an ALDH inhibitor. Overexpression and inhibition of ALDH1A1 activity resulted in reduced and increased HNE toxicity, respectively. We then established conditions for detecting HNE-protein adducts following HNE treatment and showed that overexpression and inhibition of ALDH activity resulted in reduced and increased formation of HNE-protein adducts, respectively. We also show that 6-methyl-2-(phenylazo)-3-pyridinol, previously identified as an activator of ALDH1A1, can protect PC12 cells against HNE-mediated toxicity and can cause a small but significant decrease in levels of HNE-protein adducts. Our results should encourage identification of more potent ALDH activators and their testing in the PC12-HNE model. Such cytoprotective compounds could then be tested for their neuroprotective activity in in vivo models of oxidative stress-induced PD.

Aldehyde stress and up-regulation of Nrf2-mediated antioxidant systems accompany functional adaptations in cardiac mitochondria from mice fed n−3 polyunsaturated fatty acids

Diets replete with n-3 PUFAs (polyunsaturated fatty acids) are known to have therapeutic potential for the heart, although a specifically defined duration of the n-3 PUFA diet required to achieve these effects remains unknown, as does their mechanism of action. The present study was undertaken to establish whether adaptations in mitochondrial function and stress tolerance in the heart is evident following short- (3weeks) and long- (14weeks) term dietary intervention of n-3 PUFAs, and to identify novel mechanisms by which these adaptations occur. Mitochondrial respiration [mO2 (mitochondrial O2)], H2O2 emission [mH2O2 (mitochondrial H2O2)] and Ca2+-retention capacity [mCa2+ (mitochondrial Ca2+)] were assessed in mouse hearts following dietary intervention. Mice fed n-3 PUFAs for 14weeks showed significantly lower mH2O2 and greater mCa2+ compared with all other groups. However, no significant differences were observed after 3weeks of the n-3 PUFA diet, or in mice fed on an HFC (high-fat control) diet enriched with vegetable shortening, containing almost no n-3 PUFAs, for 14weeks. Interestingly, expression and activity of key enzymes involved in antioxidant and phase II detoxification pathways, all mediated by Nrf2 (nuclear factor E2-related factor 2), were elevated in hearts from mice fed the n-3 PUFA diet, but not hearts from mice fed the HFC diet, even at 3weeks. This increase in antioxidant systems in hearts from mice fed the n-3 PUFA diet was paralleled by increased levels of 4-hydroxyhexenal protein adducts, an aldehyde formed from peroxidation of n-3 PUFAs. The findings of the present study demonstrate distinct time-dependent effects of n-3 PUFAs on mitochondrial function and antioxidant response systems in the heart. In addition, they are the first to provide direct evidence that non-enzymatic oxidation products of n-3 PUFAs may be driving mitochondrial and redox-mediated adaptations, thereby revealing a novel mechanism for n-3 PUFA action in the heart.

Apocynin may limit total cell death following cerebral ischemia and reperfusion by enhancing apoptosis

The present study was designed to determine a dose–response relationship between apocynin and infarct volume as well as to provide a possible molecular mechanism mediating this effect. We tested the hypothesis that apocynin protects against cell death following stroke and reperfusion injury. Apocynin was administered 30min prior to, or immediately following removal of sutures used to occlude the middle cerebral artery (MCA) in male Sprague–Dawley rats. Following removal of the sutures, the MCA was allowed to undergo 5.5h of reperfusion. Pretreatment with apocynin 30min prior to occlusion resulted in a dose-dependent reduction in infarct volume by ∼50 %. Analysis of tissue from the ischemic cortex of apocynin-treated rats showed an increase in the level of glutathione (GSH), protein adducts (HNE-His), hydrogen peroxide (H2O2) and DNA fragmentation (apoptotic cell death) was also observed. This suggests that apocynin may increase antioxidant defense systems (GSH) to limit the degree of ischemia-induced cellular stress. In addition, this moderate cell stress results in more apoptotic vs necrotic cell death, and thus may limit the spreading depression and total cell death that occurs following ischemia/reperfusion. These effects may serve as a potential novel mechanism of action contributing to the apocynin-induced neuroprotection observed.

Acute liver failure after recommended doses of acetaminophen in patients with myopathies

To determine the likelihood that recommended doses of acetaminophen are associated with acute liver failure in patients with myopathies. Retrospective analysis. Level III pediatric intensive care unit. Two pediatric patients with myopathies and acute liver failure. We determined acetaminophen protein adduct levels, in combination with a literature review and systematic evaluation of the cases, using the Roussel Uclaf Causality Assessment Method for drug-induced liver injury to assess causality between recommended acetaminophen dosing and acute liver failure in two children with myopathies. The serum adduct levels were consistent with the values previously reported in children with acute liver injury following acetaminophen overdose. We found four similar cases of acute liver failure in pediatric and adult patients with myopathies following recommended acetaminophen doses in the literature (n = 3) and personal communication (n = 1). The Roussel Uclaf Causality Assessment Method suggested a probable relationship between acetaminophen use at recommended doses and acute liver failure in our myopathy patients. Our data suggest that some patients with myopathies who are receiving recommended doses of acetaminophen may be at increased risk for the development of toxicity resulting in acute liver failure. More studies are needed to corroborate these findings. In the meantime, we would advise physicians to be alert in these patients while taking acetaminophen, especially when critically ill or postoperative.

Flight restriction prevents associative learning deficits but not changes in brain protein-adduct formation during honeybee ageing

Honeybees (Apis mellifera) senesce within 2 weeks after they discontinue nest tasks in favour of foraging. Foraging involves metabolically demanding flight, which in houseflies (Musca domestica) and fruit flies (Drosophila melanogaster) is associated with markers of ageing such as increased mortality and accumulation of oxidative damage. The role of flight in honeybee ageing is incompletely understood. We assessed relationships between honeybee flight activity and ageing by simulating rain that confined foragers to their colonies most of the day. After 15 days on average, flight-restricted foragers were compared with bees with normal (free) flight: one group that foraged for ∼15 days and two additional control groups, for flight duration and chronological age, that foraged for ∼5 days. Free flight over 15 days on average resulted in impaired associative learning ability. In contrast, flight-restricted foragers did as well in learning as bees that foraged for 5 days on average. This negative effect of flight activity was not influenced by chronological age or gustatory responsiveness, a measure of the bees' motivation to learn. Contrasting their intact learning ability, flight-restricted bees accrued the most oxidative brain damage as indicated by malondialdehyde protein adduct levels in crude cytosolic fractions. Concentrations of mono- and poly-ubiquitinated brain proteins were equal between the groups, whereas differences in total protein amounts suggested changes in brain protein metabolism connected to forager age, but not flight. We propose that intense flight is causal to brain deficits in aged bees, and that oxidative protein damage is unlikely to be the underlying mechanism.

Acetaminophen hepatotoxicity and HIF-1α induction in acetaminophen toxicity in mice occurs without hypoxia

HIF-1α is a nuclear factor important in the transcription of genes controlling angiogenesis including vascular endothelial growth factor (VEGF). Both hypoxia and oxidative stress are known mechanisms for the induction of HIF-1α. Oxidative stress and mitochondrial permeability transition (MPT) are mechanistically important in acetaminophen (APAP) toxicity in the mouse. MPT may occur as a result of oxidative stress and leads to a large increase in oxidative stress. We previously reported the induction of HIF-1α in mice with APAP toxicity and have shown that VEGF is important in hepatocyte regeneration following APAP toxicity. The following study was performed to examine the relative contribution of hypoxia versus oxidative stress to the induction of HIF-1α in APAP toxicity in the mouse. Time course studies using the hypoxia marker pimonidazole showed no staining for pimonidazole at 1 or 2 h in B6C3F1 mice treated with APAP. Staining for pimonidazole was present in the midzonal to periportal regions at 4, 8, 24 and 48 h and no staining was observed in centrilobular hepatocytes, the sites of the toxicity. Subsequent studies with the MPT inhibitor cyclosporine A showed that cyclosporine A (CYC; 10 mg/kg) reduced HIF-1α induction in APAP treated mice at 1 and 4 h and did not inhibit the metabolism of APAP (depletion of hepatic non-protein sulfhydryls and hepatic protein adduct levels). The data suggest that HIF-1α induction in the early stages of APAP toxicity is secondary to oxidative stress via a mechanism involving MPT. In addition, APAP toxicity is not mediated by a hypoxia mechanism.

Increased levels of 4HNE-protein plasma adducts in Rett syndrome

Objective Rett syndrome (RTT) is a neurological disorder and a leading cause of mental retardation in females. It is caused by mutations in methyl-CpG-binding protein 2 (MeCP2) gene and more rarely in cyclin-dependent kinase-like 5 (CDKL5) and forkhead box protein G1 (FOXG1) genes. Increased oxidative stress (OS) has been documented in MeCP2-RTT patients. Here, we evaluated the levels of 4-hydroxynonenal plasma protein adducts (4HNE-PAs) in MeCP2-, CDKL5-, and FOXG1-RTT and in their clinical variants. Design and methods 4HNE-PAs were determined by Western blot in plasma from healthy subjects and RTT patients. Results 4HNE-PAs levels were increased in MeCP2- and CDKL5-related RTT but not in FOXG1-related RTT. Conclusion These results showed that OS is present in RTT clinical variants and could play a key role in RTT pathogenesis. Under the OS point of view FOXG1-related RTT appears to be distinct from the MeCP2/CDKL5, suggesting a distinct mechanism involved in its pathogenesis.

Anti-acrolein treatment improves behavioral outcome and alleviates myelin damage in experimental autoimmune enchephalomyelitis mouse

Oxidative stress is considered a major contributor in the pathology of multiple sclerosis (MS). Acrolein, a highly reactive aldehyde byproduct of lipid peroxidation, is thought to perpetuate oxidative stress. In this study, we aimed to determine the role of acrolein in an animal model of MS, experimental autoimmune enchephalomyelitis (EAE) mice. We have demonstrated a significant elevation of acrolein protein adduct levels in EAE mouse spinal cord. Hydralazine, a known acrolein scavenger, significantly improved behavioral outcomes and lessened myelin damage in spinal cord. We postulate that acrolein is an important pathological factor and likely a novel therapeutic target in MS.

Isoketals form cytotoxic phosphatidylethanolamine adducts in cells

Levuglandins and their stereo- and regio-isomers (termed isolevuglandins or isoketals) are gamma-ketoaldehydes (IsoK) that rapidly react with lysines to form stable protein adducts. IsoK protein adduct levels increase in several pathological conditions including cardiovascular disease. IsoKs can induce ion channel dysfunction and cell death, potentially by adducting to cellular proteins. However, IsoKs also adduct to phosphatidylethanolamine (PE) in vitro, and whether PE adducts form in cells or contribute to the effects of IsoKs is unknown. When radiolabeled IsoK was added to HEK293 cells, 40% of the radiolabel extracted into the chloroform lower phase suggesting the possible formation of PE adducts. We therefore developed methods to measure IsoK-PE adducts in cells. IsoK-PE was quantified by LC/MS/MS after hydrolysis to IsoK-ethanolamine by Streptomyces chromofuscus phospholipase D. In HEK293 and human umbilical vein endothelial cells (HUVEC), IsoK dose-dependently increased PE adduct concentrations to a greater extent than protein adduct. To test the biological significance of IsoK-PE formation, we treated HUVEC with IsoK-PE. IsoK-PE dose dependently induced cytotoxicity (LC(50) 2.2 muM). These results indicate that cellular PE is a significant target of IsoKs, and that formation of PE adducts may mediate some of the biological effects of IsoKs relevant to disease.

Human Recombinant Vascular Endothelial Growth Factor Reduces Necrosis and Enhances Hepatocyte Regeneration in a Mouse Model of Acetaminophen Toxicity

We reported previously that vascular endothelial growth factor (VEGF) was increased in acetaminophen (APAP) toxicity in mice and treatment with a VEGF receptor inhibitor reduced hepatocyte regeneration. The effect of human recombinant VEGF (hrVEGF) on APAP toxicity in the mouse was examined. In early toxicity studies, B6C3F1 mice received hrVEGF (50 microg s.c.) or vehicle 30 min before receiving APAP (200 mg/kg i.p.) and were sacrificed at 2, 4, and 8 h. Toxicity was comparable at 2 and 4 h, but reduced in the APAP/hrVEGF mice at 8 h (p < 0.05) compared with the APAP/vehicle mice. Hepatic glutathione (GSH) and APAP protein adduct levels were comparable between the two groups of mice, with the exception that GSH was higher at 8 h in the hrVEGF-treated mice. Subsequently, mice received two doses (before and 10 h) or three doses (before and 10 and 24 h) of hrVEGF; alanine aminotransferase values and necrosis were reduced at 24 and 36 h, respectively, in the APAP/hrVEGF mice (p < 0.05) compared with the APAP/vehicle mice. Proliferating cell nuclear antigen expression was enhanced, and interleukin-6 expression was reduced in the mice that received hrVEGF (p < 0.05) compared with the APAP/vehicle mice. In addition, treatment with hrVEGF lowered plasma hyaluronic acid levels and neutrophil counts at 36 h. Cumulatively, the data show that treatment with hrVEGF reduced toxicity and increased hepatocyte regeneration in APAP toxicity in the mouse. Attenuation of sinusoidal cell endothelial dysfunction and changes in neutrophil dynamics may be operant mechanisms in the hepatoprotection mediated by hrVEGF in APAP toxicity.

Phosphatidylethanolamine modification contributes to levuglandin/isoketal induced cytotoxicity

BackgroundLevuglandins and isoketals are γ‐ketoaldehyde isomers (IK) formed by non‐enzymatic rearrangement of prostaglandin H2 and H2‐isoprostanes, respectively. IK rapidly adduct to proteins and increases in IK protein adduct levels occur in several inflammatory conditions. IK induce cell death in cultured cells, potentially by adducting to cellular proteins. However, IK also adduct to other primary amines including phosphatidylethanolamine (PE) in vitro. Whether IK induces its biological activities by modifying PE has not been investigated.ObjectiveMeasure levels of IK‐PE in cells and determine whether IK‐ PE mediates the endothelial cell death induced by IsoK.Results40% radiolabeled IK added to HEK cells bound to phospholipids and only 8% with proteins. An LC/MS/MS assay using S. chromofuscus phospholipase D to convert IK‐PE to IK‐ethanolamine was used to measure IK‐PE adducts in IK treated endothelial cells (EC). IK‐PE added directly to EC dose‐dependently induced cytotoxicity (LC50 2.2 μM).ConclusionPE is a major cellular target of IK, and IK modified PE can mediate IK induced cytotoxicity.

Developmental Exposure to Manganese Increases Adult Susceptibility to Inflammatory Activation of Glia and Neuronal Protein Nitration

Chronic exposure to manganese (Mn) produces a neurodegenerative disorder affecting the basal ganglia characterized by reactive gliosis and expression of neuroinflammatory genes including inducible nitric oxide synthase (NOS2). Induction of NOS2 in glial cells causes overproduction of nitric oxide (NO) and injury to neurons that is associated with parkinsonian-like motor deficits. Inflammatory activation of glia is believed to be an early event in Mn neurotoxicity, but specific responses of microglia and astrocytes to Mn during development remain poorly understood. In this study, we investigated the effect of juvenile exposure to Mn on the activation of glia and production of NO in C57Bl/6J mice, postulating that developmental Mn exposure would lead to heightened sensitivity to gliosis and increased expression of NOS2 in adult mice exposed again later in life. Immunohistochemical analysis indicated that Mn exposure caused increased activation of both microglia and astrocytes in the striatum (St), globus pallidus (Gp), and substantia nigra pars reticulata (SNpr) of treated mice compared with controls. More robust activation of microglia was observed in juveniles, whereas astrogliosis was more prominent in adult mice preexposed during development. Co-immunofluorescence studies demonstrated increased expression of NOS2 in glia located in the Gp and SNpr. Additionally, greater increases in the level of 3-nitrotyrosine protein adducts were detected in dopamine- and cAMP-regulated phosphoprotein-32-positive neurons of the St of Mn-treated adult mice preexposed as juveniles. These data indicate that subchronic exposure to Mn during development leads to temporally distinct patterns of glial activation that result in elevated nitrosative stress in distinct populations of basal ganglia neurons.

Biotransformation of Benzo[a]pyrene in Ahr Knockout Mice Is Dependent on Time and Route of Exposure

Benzo[a]pyrene (BP) is an ubiquitous environmental pollutant with potent mutagenic and carcinogenic properties. The Ah receptor (Ahr) is important in the metabolic activation of BP and is therefore central to BP-induced carcinogenesis. Although Ahr(-/-) mice are refractory to BP-induced carcinogenesis, higher levels of BP-DNA and -protein adducts were formed in them than in wild-type mice. These results indicated the presence of an Ahr-independent and/or a slower biotransformation of BP in Ahr knockout mice. To address this issue further, we have now performed a time-course experiment, with mice receiving a single oral dose of BP (100 mg/kg). Wild-type mice have an effective clearance of BP metabolites, mainly through 3-hydroxybenzo[a]pyrene and 9-hydroxybenzo[a]pyrene in the feces with reduced levels of DNA and protein adducts in the examined tissues. On the other hand, the Ahr(-/-) mice appear to have a lower metabolic clearance of BP resulting in increased levels of DNA and protein adducts and of unmetabolized BP. In addition, we have performed an administration route experiment and found that skin-exposed Ahr(-/-) mice showed lower levels of protein adducts along with markedly reduced P450 1B1 expression, but only in the exposed area, as compared with the wild-type mice. In addition, the systemic uptake of BP is increased in the Ahr(-/-) mice as compared with the wild-type mice. Hence, the lack of a functional Ah receptor results in an Ahr-independent biotransformation of BP with a slower clearance of BP and higher levels of DNA and protein adducts, but the distribution and levels of BP and BP-protein adducts are clearly dependent on the route of exposure.

Time-dependent and ethanol-induced cardiac protection from ischemia mediated by mitochondrial translocation of ɛPKC and activation of aldehyde dehydrogenase 2

The cardioprotective effects of moderate alcohol consumption have been well documented in animal models and in humans. Protection afforded against ischemia and reperfusion injury (I/R) proceeds through an ischemic preconditioning-like mechanism involving the activation of epsilon protein kinase C (ɛPKC) and is dependent on the time and duration of ethanol treatment. However, the substrates of ɛPKC and the molecular mechanisms by which the enzyme protects the heart from oxidative damage induced by I/R are not fully described. Using an open-chest model of acute myocardial infarction in vivo, we find that intraperitoneal injection of ethanol (0.5 g/kg) 60 min prior to (but not 15 min prior to) a 30-minute transient ligation of the left anterior descending coronary artery reduced I/R-mediated injury by 57% (measured as a decrease of creatine phosphokinase release into the blood). Only under cardioprotective conditions, ethanol treatment resulted in the translocation of ɛPKC to cardiac mitochondria, where the enzyme bound aldehyde dehydrogenase-2 (ALDH2). ALDH2 is an intra-mitochondrial enzyme involved in the detoxification of toxic aldehydes such as 4-hydroxy-2-nonenal (4-HNE) and 4-HNE mediates oxidative damage, at least in part, by covalently modifying and inactivating proteins (by forming 4-HNE adducts). In hearts subjected to I/R after ethanol treatment, the levels of 4-HNE protein adducts were lower and JNK1/2 and ERK1/2 activities were diminished relative to the hearts from rats subjected to I/R in the absence of ethanol. Together, this work provides an insight into the mitochondrial-dependent basis of ethanol-induced and ɛPKC-mediated protection from cardiac ischemia, in vivo.