Oxidative Stress Balance Research Articles

Redox Balance in the Pathogenesis of Nonalcoholic Fatty Liver Disease: Mechanisms and Therapeutic Opportunities

Nonalcoholic fatty liver disease (NAFLD) is currently the most common liver disease in the world. It encompasses a histological spectrum, ranging from simple, nonprogressive steatosis to nonalcoholic steatohepatitis (NASH), which may progress to cirrhosis and hepatocellular carcinoma. While liver-related complications are confined to NASH, emerging evidence suggests both simple steatosis and NASH predispose to type 2 diabetes and cardiovascular disease. The pathogenesis of NAFLD is currently unknown, but accumulating data suggest that oxidative stress and altered redox balance play a crucial role in the pathogenesis of steatosis, steatohepatitis, and fibrosis. We will examine intracellular mechanisms, including mitochondrial dysfunction and impaired oxidative free fatty acid metabolism, leading to reactive oxygen species generation; additionally, the potential pathogenetic role of extracellular sources of reactive oxygen species in NAFLD, including increased myeloperoxidase activity and oxidized low density lipoprotein accumulation, will be reviewed. We will discuss how these mechanisms converge to determine the whole pathophysiological spectrum of NAFLD, including hepatocyte triglyceride accumulation, hepatocyte apoptosis, hepatic inflammation, hepatic stellate cell activation, and fibrogenesis. Finally, available animal and human data on treatment opportunities with older and newer antioxidant will be presented.

Evaluation of How Gene-Job Strain Interaction Affects Blood Pressure in the PAMELA Study

To see whether there is interaction between single nucleotide polymorphisms (SNPs) belonging to candidate genes for hypertension and job strain and its components (job demand and job control) in an accurately phenotyped Italian cohort (Pressioni Arteriose Monitorate E Loro Associazioni). From the Pressioni Arteriose Monitorate E Loro Associazioni cohort, we sampled 924 employed men and women who had completed a modified version of the Karasek questionnaire and for whom deoxyribonucleic acid information was available. We selected 1,510 common tagging and putative functional SNPs in 92 candidate autosomal genes involved in sympathetic nervous system, oxidative stress, renin-angiotensin-aldosterone system, and sodium balance. The interaction of each candidate SNP with the perceived job stress scores on ambulatory systolic and diastolic blood pressures was evaluated by adding product terms to a multiple regression linear model, adjusted for sex, age, total cholesterol, and body mass index. Among all the SNPs tested, the interaction of SNP rs11210278 located on the endothelin 2 gene and job control was statistically significant after controlling for multiple testing, using false discovery rate (unadjusted p = .00000661; p adjusted for false discovery rate = .0085), indicating an inverse association with systolic blood pressure for the homozygous wild-type allele (C/C) and a positive association for the variant genotypes (C/T and T/T). We show that the hypertensive response to job strain is present only in carriers of the rs11210278 wild-type genotype. The phenomenon is discussed in relationship to the controversial results of field studies on job strain and hypertension.

Assessment of Anti-Aging Effects of Fish Products Peptide in Middle-Aged Subjects

Objective: In this study, the antioxidant properties of peptide from boiled fish paste (fish products peptide; FPP) supplement were investigated in middle-aged subjects. Methods: The subjects who participated in this study were men and premenopausal women, aged between 30 and 50 years (39.6 ± 5.5 years). The subjects consumed 10 tablets/day (containing 0.9 g of total fish peptide) for 60 days. Pre- and post-supplementation, subjects underwent analysis by physical measurement, completed the Anti-Aging QOL Common Questionnaire, and were examined in terms of vascular features, blood biochemical and uric characteristics, and hormone and oxidative stress markers. Results: After 60 days of supplementation with FPP, significant improvement was observed in serum lipid peroxide (LPO) (–16.2 %, p = 0.001). In terms of oxidative markers, 8-hydroxy-2’-deoxyguanosine (8-OHdG) showed a tendency to decrease; in addition, potential antioxidant (PAO) and serum total antioxidant status (STAS), as antioxidant properties, showed slight increases. Furthermore, highsensitivity C-reactive protein (hs-CRP) also exhibited a tendency to decrease. Discussion: The results show that FPP might contribute to the prevention of lifestyle-related disease by regulating the balance of oxidative stress and improving arteriosclerosis with high oxidative stress.

The Oxidative Stress Balance Measured in Humans with Different Markers, Following a Single Oral Antioxidants Supplementation or a Diet Poor of Antioxidants

Four different markers of oxidative stress [OS] (8-OHdG in urine, 8-Iso-PGF, hydroperoxides and carbonylated proteins in plasma, a new marker of antioxidant capacity (AC) in plasma/urine/saliva, and hs-CRP were determined concomitantly in twelve apparently healthy volunteers. All the markers were determined at 8 am, 10 am, 12 am in three different moments: after a week of normal diet (baseline), after an acute supplementation with an antioxidant pool, and finally following a week of a diet poor in antioxidant. The supplementation of antioxidants determined a significant (t test p < 0.05) decrease up to 12% of 8-OHdG in urine and up to 46% of carbonylated proteins in plasma, whereas hydroperoxides and 8-Iso-PGF were unmodified; the antioxidant capacity increased significantly (t test p < 0.05) up to 19%, 78%, and 67%, respectively in plasma, urine and saliva. Hs-CRP was unchanged.The diet poor in antioxidant caused significant increases (t test p< 0.01) of hydroperoxides (up to 24%), 8-Iso-PGF 23 (up to 69%), carbonylated proteins (up to 76%) and 8-OHdG (up to 16%): hs-CRP increase reached 72% despite the levels were still within the normal range. Any reduction of soluble antioxidants activity in plasma was detected, whereas in urine and saliva a reduction of 45% and 38% respectively was shown. In conclusion, the antioxidant surplus determined by a single antioxidants pool administration seems to protect DNA and proteins from oxidation. On the contrary the shortage of antioxidant intake increases all the markers of OS, particularly those related to lipids and proteins, whereas the DNA seems to be protected more efficiently. The AC in plasma tends to be constant, and the limitation of antioxidants intake is followed by reduction of AC in urine and saliva.

Spatio-temporal variation in territory quality and oxidative status: a natural experiment in the Seychelles warbler (Acrocephalus sechellensis)

1.Fluctuations in the quality of the habitat in which an animal lives can have major consequences for its behaviour and physiological state. In poor-quality habitat with low food availability, metabolically intensive foraging activity is likely to result in increased generation of reactive oxygen species, while scarcity of food can lead to a weakening of exogenously derived antioxidant defences. The consequent oxidant/antioxidant imbalance may lead to elevated oxidative stress.2.Although the link between food availability and oxidative stress has been studied in the laboratory, very little is known about this relationship in the wild. Here, we investigate the association between territory quality (measured through food availability) and oxidative stress in the Seychelles warbler (Acrocephalus sechellensis).3.Seychelles warblers are insectivorous birds that inhabit a fixed feeding territory year round. Individuals experience profound and rapid local fluctuations in territory quality within these territories, owing to changing patterns of vegetation defoliation resulting from seasonal changes in prevailing wind direction and wind-borne salt spray.4.As expected, oxidant generation (measured as reactive oxygen metabolites; ROMs) was higher when territory quality was low, but there was no correlation between territory quality and antioxidant capacity (OXY). The negative correlation between territory quality and ROMs was significant between individuals and approached significance within individuals, indicating that the pattern resulted from individual responses to environmental variation.5.ROMs and OXY levels within individuals were positively correlated, but the relationship between territory quality and ROMs persisted after including OXY as a covariate, implying that oxidative stress occurs in low territory quality conditions.6.Our results indicate that the oxidative stress balance of an individual is sensitive to relatively short-term changes in territory quality, which may have consequences for the birds’ fitness.

Critical role of the nitric oxide/reactive oxygen species balance in endothelial progenitor dysfunction.

Endothelial injury and dysfunction are critical events in the pathogenesis of cardiovascular disease. During these processes, an impaired balance of nitric oxide bioavailability and oxidative stress is mechanistically involved. Circulating angiogenic cells (including early and late outgrowth endothelial progenitor cells (EPC)) contribute to formation of new blood vessels, neovascularization, and homeostasis of the vasculature, and are highly sensitive for misbalance between NO and oxidative stress. We here review the role of the endothelial nitric oxide synthase and oxidative stress producing enzyme systems in EPC during cardiovascular disease. We also focus on the underlying molecular mechanisms and potential emerging drug- and gene-based therapeutic strategies to improve EPC function in cardiovascular diseased patients.

Proteomic Profile of Uterine Luminal Fluid from Early Pregnant Ewes.

Embryonic development is a time-sensitive period that requires a synchronized uterine environment, which is created by the secretion of proteins from both the embryo and uterus. Numerous studies have identified uterine luminal proteins and related these to specific adaptations during early pregnancy (EP). However, no study has yet utilized LC-MS/MS to identify the signature profile of proteins in the uterine lumen during EP. In this study, uterine luminal fluid from nonpregnant (NP; n = 3) and EP (n = 3; gestational day 16) ewes were analyzed by LC-MS/MS and validated by Western immunoblotting. We identified a unique signature profile for EP luminal fluid; 15 proteins related to specific aspects of embryonic development including growth and remodeling, immune system regulation, oxidative stress balance, and nutrition were significantly altered (up to 65-fold of NP) in EP profile. Specific uterine remodeling proteins such as transgelin (P = 0.008) and placental proteins like PP9 (P = 0.02) were present in EP luminal fluid but were barely detectable in the NP flushings. Direct correlations (R(2) = 0.84, P = 0.01) were observed between proteomics and immunoblotting. These data provide information on dynamic physiological processes associated with EP at the level of the uterus and conceptus and may potentially demonstrate a signature profile associated with embryonic well-being.

Proteomic Profile of Uterine Luminal Fluid from Early Pregnant Ewes

Embryonic development is a time-sensitive period that requires a synchronized uterine environment, which is created by the secretion of proteins from both the embryo and uterus. Numerous studies have identified uterine luminal proteins and related these to specific adaptations during early pregnancy (EP). However, no study has yet utilized LC-MS/MS to identify the signature profile of proteins in the uterine lumen during EP. In this study, uterine luminal fluid from nonpregnant (NP; n = 3) and EP (n = 3; gestational day 16) ewes were analyzed by LC-MS/MS and validated by Western immunoblotting. We identified a unique signature profile for EP luminal fluid; 15 proteins related to specific aspects of embryonic development including growth and remodeling, immune system regulation, oxidative stress balance, and nutrition were significantly altered (up to 65-fold of NP) in EP profile. Specific uterine remodeling proteins such as transgelin (P = 0.008) and placental proteins like PP9 (P = 0.02) were present in EP luminal fluid but were barely detectable in the NP flushings. Direct correlations (R(2) = 0.84, P = 0.01) were observed between proteomics and immunoblotting. These data provide information on dynamic physiological processes associated with EP at the level of the uterus and conceptus and may potentially demonstrate a signature profile associated with embryonic well-being.

Experimental Guidelines for Studies Designed to Investigate the Impact of Antioxidant Supplementation on Exercise Performance

Research interest in the effects of antioxidants on exercise-induced oxidative stress and human performance continues to grow as new scientists enter this field. Consequently, there is a need to establish an acceptable set of criteria for monitoring antioxidant capacity and oxidative damage in tissues. Numerous reports have described a wide range of assays to detect both antioxidant capacity and oxidative damage to biomolecules, but many techniques are not appropriate in all experimental conditions. Here, the authors present guidelines for selecting and interpreting methods that can be used by scientists to investigate the impact of antioxidants on both exercise performance and the redox status of tissues. Moreover, these guidelines will be useful for reviewers who are assigned the task of evaluating studies on this topic. The set of guidelines contained in this report is not designed to be a strict set of rules, because often the appropriate procedures depend on the question being addressed and the experimental model. Furthermore, because no individual assay is guaranteed to be the most appropriate in every experimental situation, the authors strongly recommend using multiple assays to verify a change in biomarkers of oxidative stress or redox balance.

Plasma Protein Oxidation and Triglyceride Levels in Sickle Cell Anemia.

Abstract 2578Poster Board II-555 Introduction:A mutant and unstable hemoglobin is characteristic in sickle cell anemia (SCA) with an increased formation of methemoglobin, hemichrome and haemin. Increased oxidative stress and altered lipid balance in the plasmas of SCA patients have been documented previously. Lower total plasma cholesterol (TC) and higher-than-avarage triglyceride (TG) levels have also been reported in SCA patients compared to healthy controls. Plasma phospholipids (PL) were normal or increased according to various studies. However, to our knowledge only a few reports documented the plasma protein oxidation in SCA patients. In this study protein oxidation, lipid levels, bilurubin levels and their probable correlation were investigated in the plasmas of SCA patients, carriers and healthy controls. Patient and Methods:15 SCA patients, 10 carriers and 10 healthy controls were included in the study. The patients had not been transfused and hospitalized and had no crisis for the last three months. The oxidation status of plasma and its proteins were detirmined by total sulfhydryl (T-SH) and protein carbonly levels respectively. TC, TG and bilurubin levels were detirmined in the plasma and PL levels were detirmined in the lipid extract from plasma. Results:SCA patients had lower T-SH levels compared to controls (p=0.003) and higher protein carbonyl levels compared to carriers (p=0.001) and healthy children (p=0.032). T-SH values were 417.41±77.90 μM, 457.46±106.26 μM, 522.59±77.18 μM and protein carbonyl levels were 0.93±0.15 nmol/mg protein, 0.74±0.08 nmol/mg protein , 0.82±0.05 nmol/mg protein for patients, carriers and controls respectively. Significantly lower mean TC levels than controls (p<0.0001) and higher TG levels than carriers (p=0.001) were observed in SCA patients. TC levels were 116.67±23.60, 127.53±22.40, 151.62±16.53 and TG levels were 89.31±23.50 mg/dl, 60.94±9.18 mg/dl, 82.96±21.06 mg/dl for patients, carriers and controls respectively. Phospholipid levels were not different between the groups. TG levels were positively correlated to protein carbonyl levels (p=0.002) and to bilirubin levels (p=0.033). Bilurubin levels and protein carbonyl levels are also positively correlated (p<0,0001). Conclusions:Alteration of lipid balance besides protein oxidation and hemolysis were observed in the plasmas of SCA patients compared to the carriers and healthy group. The positive correlation between total bilurubin, TG and carbonyl levels indicates that oxidative stress may affect the SCA plasma extensively, ie. lipids, proteins and erythrocytes are all affected. Disclosures:No relevant conflicts of interest to declare.

Effect of diet, energy balance and milk production on oxidative stress in early-lactating dairy cows grazing pasture

The aim of this study was to determine the effect of diet, energy balance and milk production on oxidative stress in early-lactating, Holstein-Friesian dairy cows fed to produce either low or high levels of milk. Indicators of energy balance (non-esterified fatty acids, β-hydroxybutyrate, glucose and insulin-like growth factor-1) and indicators of oxidative stress (reactive oxygen metabolites and biological antioxidants) were measured in the first 5 weeks of lactation. Energy balance indicators showed that high producing animals had a lower degree of negative energy balance. Diet was found to have an indirect effect on the level of oxidative stress. Factors associated with a high level of oxidative stress were severe negative energy balance (mean -71 ± 6.85 27 MJ/cow/day, P < 0.05) and lower levels of milk production (mean 26.4 ± 0.07 28 L/cow/day, P < 0 .05). Further studies will be required to more precisely determine the specific effects of diet, energy balance and milk production on such stress in dairy cows and to establish normal ranges for these biomarkers.

Vitamin C and Resveratrol Supplementation to Rat Dams Treated with Di(2-ethylhexyl)phthalate: Impact on Reproductive and Oxidative Stress End Points in Male Offspring

This study was carried out to assess the influence of di(2-ethylhexyl)phthalate (DEHP) alone or associated with antioxidants on the male reproductive system in newborn rats, emphasizing the implications of oxidative stress and hormonal balance during prenatal and early postnatal periods. Wistar females were exposed by oral route to DEHP alone or associated with antioxidants from gestational day 7 to lactational day 2 according to the following treatment regimens: (C) vehicle control (canola oil + 1% Tween-80); (V) vitamin C (200 mg/kg) + canola oil; (R) resveratrol (10 mg/kg) + canola oil; (D) DEHP (500 mg/kg) + 1% Tween-80; (DV) DEHP (500 mg/kg) + vitamin C (200 mg/kg); and (DR) DEHP (500 mg/kg) + resveratrol (10 mg/kg). Two male pups per litter were randomly selected and necropsied on postnatal day 2. The brain and liver were removed and weighed and anogenital distance (AGD) was measured. Additionally, the testes were removed for assessment of intratesticular testosterone levels and histopathology; the liver was used to measure biomarkers of oxidative stress. Vitamin C and resveratrol alone did not affect the reproductive end points and did not induce oxidative stress. Exposure of dams to DEHP alone and associated with antioxidants resulted in hepatomegaly in offspring and significantly increased the incidence of multinucleated gonocytes in seminiferous cords. Testosterone and AGD presented a trend to decrease in DEHP-exposed groups. Catalase activity increased only in groups exposed to DEHP associated with antioxidants, although GST (gluthatione-S-transferase) activity decreased in all DEHP-exposed groups. The levels of hydroperoxides increased only in group exposed to DEHP associated with vitamin C. These results indicate that the association of DEHP with antioxidants was unable to ameliorate DEHP-induced reproductive changes, and the coadministration of DEHP and these antioxidants might even contribute to an overall increase in oxidative stress.

Tipping the redox balance of oxidative stress in fibrogenic pathways in chronic kidney disease

Patients with moderate to advanced chronic kidney disease or end-stage renal disease have a greatly increased cardiovascular risk that cannot be explained entirely by traditional cardiovascular risk factors. An increase in oxidative stress and inflammation have been proposed as nontraditional cardiovascular risk factors in this patient population. Oxidative stress reflects the redox balance between oxidant generation and antioxidant mechanisms. The generation of reactive oxygen species is not simply a random process that oxidizes nearby macromolecules, but, in many instances, the oxidants target particular amino acid residues or lipid moieties. Oxidant mechanisms are now recognized to be intimately involved in cell signaling and to be vital components of the immune response. This is equally true for antioxidant mechanisms as well. In the progression of chronic kidney disease, the redox balance is not in equilibrium and is tipped toward oxidation, resulting in the dysregulation of cellular process and subsequent tissue injury. In this review we discuss the major oxidant and antioxidant pathways and the biomarkers to assess redox status. We also review the data linking the pathogenesis of oxidative stress, inflammation, and the progressive loss of kidney function in chronic kidney disease.

Sex-dependent differences in aged rat brain mitochondrial function and oxidative stress

Females show lower incidences of several neurodegenerative diseases related to oxidative stress and mitochondrial dysfunction than males. In addition, female rats show more differentiated mitochondria than males in several tissues. The aim of this work was to investigate the existence of sex-dependent differences in brain mitochondrial bioenergetics and oxidative balance in aged rats. Results showed that aged female rat brain had a lower mitochondria content than aged male brain but with a greater differentiation degree given the higher mitochondrial protein content and mitochondrial complex activities in females. Female rat brain also showed a better oxidative balance than that of males, reflected by the fact that higher mitochondrial respiratory chain function is accompanied by a similar ROS production and greater antioxidant enzyme activities, which could be responsible for the lesser oxidative damage observed in proteins and lipids in this sex. Interestingly, levels of UCP4 and UCP5—proteins related to a decrease in ROS production—were also higher in females. In conclusion, aged female rat brain had more differentiated mitochondria than male brain and showed a better control of oxidative stress balance, which could be due, in part, to the neuroprotective effect of UCPs.

Adrenaline modulates the global transcriptional profile of Salmonella revealing a role in the antimicrobial peptide and oxidative stress resistance responses

BackgroundThe successful interaction of bacterial pathogens with host tissues requires the sensing of specific chemical and physical cues. The human gut contains a huge number of neurons involved in the secretion and sensing of a class of neuroendocrine hormones called catecholamines. Recently, in Escherichia coli O157:H7, the catecholamines adrenaline and noradrenaline were shown to act synergistically with a bacterial quorum sensing molecule, autoinducer 3 (AI-3), to affect bacterial virulence and motility. We wished to investigate the impact of adrenaline on the biology of Salmonella spp.ResultsWe have determined the effect of adrenaline on the transcriptome of the gut pathogen Salmonella enterica serovar Typhimurium. Addition of adrenaline led to an induction of key metal transport systems within 30 minutes of treatment. The oxidative stress responses employing manganese internalisation were also elicited. Cells lacking the key oxidative stress regulator OxyR showed reduced survival in the presence of adrenaline and complete restoration of growth upon addition of manganese. A significant reduction in the expression of the pmrHFIJKLM antimicrobial peptide resistance operon reduced the ability of Salmonella to survive polymyxin B following addition of adrenaline. Notably, both phenotypes were reversed by the addition of the β-adrenergic blocker propranolol. Our data suggest that the BasSR two component signal transduction system is the likely adrenaline sensor mediating the antimicrobial peptide response.ConclusionSalmonella are able to sense adrenaline and downregulate the antimicrobial peptide resistance pmr locus through the BasSR two component signalling system. Through iron transport, adrenaline may affect the oxidative stress balance of the cell requiring OxyR for normal growth. Both adrenaline effects can be inhibited by the addition of the β-adrenergic blocker propranolol. Adrenaline sensing may provide an environmental cue for the induction of the Salmonella stress response in anticipation of imminent host-derived oxidative stress. However, adrenaline may also serve in favour of the host defences by lowering antimicrobial peptide resistance and hence documenting for the first time such a function for a hormone.

Endothelin Receptor Antagonism

The endothelins are a family (ET-1, ET-2, and ET-3) of naturally occurring polypeptides, with ET-1 being the most clinically pertinent isoform. ET-1 was formally identified in 1988, at which time it was viewed mainly as a potent vasoconstrictor; however, the biological understanding of endothelin has since rapidly evolved, and it is now appreciated that ET-1 is not only a potent vasoconstrictor but also that it has a prominent role in fibrogenesis, inflammatory states, oxidative stress, atherosclerosis, salt and water balance, and pulmonary artery hypertension.1–3 ET-1 binds to 2 receptors: ETA and ETB. In general, stimulation of these receptors has opposing actions. For example, ETA stimulation is vasoconstricting, as well as being profibrotic and proinflammatory, whereas ETB stimulation is vasodilatory, antiproliferative, and natriuretic. The ETB receptor also subserves a clearance role for ET-1. The varying distribution of the endothelin receptors in different tissues is responsible for the mixture of actions attributed to endothelin. For example, the renal medulla contains the highest concentration of endothelin receptors in the body.4 The renal cortex is also heavily populated with endothelin receptors (50% of the density of the medulla). The ETB receptor predominates in the kidney, composing 70% of the receptors in both locations. Thus, the important roles of ET-1 and the ETB receptor are in regulating renal hemodynamics and tubular transport processes, as well as mesangial and vascular smooth muscle cell proliferation and mitogenesis.3 Likewise, ETA and ETB receptors are highly expressed in smooth muscle cells and foamy macrophages in atherosclerotic models. Stimulation of receptors in these locations is the basis of the proinflammatory and (pro)atherosclerotic actions of …

The plant Apolipoprotein D ortholog protects Arabidopsis against oxidative stress

BackgroundLipocalins are a large and diverse family of small, mostly extracellular proteins implicated in many important functions. This family has been studied in bacteria, invertebrate and vertebrate animals but little is known about these proteins in plants. We recently reported the identification and molecular characterization of the first true lipocalins from plants, including the Apolipoprotein D ortholog AtTIL identified in the plant model Arabidopsis thaliana. This study aimed to determine its physiological role in planta.ResultsOur results demonstrate that the AtTIL lipocalin is involved in modulating tolerance to oxidative stress. AtTIL knock-out plants are very sensitive to sudden drops in temperature and paraquat treatment, and dark-grown plants die shortly after transfer to light. These plants accumulate a high level of hydrogen peroxide and other ROS, which causes an oxidative stress that is associated with a reduction in hypocotyl growth and sensitivity to light. Complementation of the knock-out plants with the AtTIL cDNA restores the normal phenotype. On the other hand, overexpression enhances tolerance to stress caused by freezing, paraquat and light. Moreover, this overexpression delays flowering and maintains leaf greenness. Microarray analyses identified several differentially-regulated genes encoding components of oxidative stress and energy balance.ConclusionThis study provides the first functional evidence that a plant lipocalin is involved in modulating tolerance to oxidative stress. These findings are in agreement with recently published data showing that overexpression of ApoD enhances tolerance to oxidative stress and increases life span in mice and Drosophila. Together, the three papers strongly support a similar function of lipocalins in these evolutionary-distant species.

AtOSA1, a Member of the Abc1-Like Family, as a New Factor in Cadmium and Oxidative Stress Response

The analysis of gene expression in Arabidopsis (Arabidopsis thaliana) using cDNA microarrays and reverse transcription-polymerase chain reaction showed that AtOSA1 (A. thaliana oxidative stress-related Abc1-like protein) transcript levels are influenced by Cd2+ treatment. The comparison of protein sequences revealed that AtOSA1 belongs to the family of Abc1 proteins. Up to now, Abc1-like proteins have been identified in prokaryotes and in the mitochondria of eukaryotes. AtOSA1 is the first member of this family to be localized in the chloroplasts. However, despite sharing homology to the mitochondrial ABC1 of Saccharomyces cerevisiae, AtOSA1 was not able to complement yeast strains deleted in the endogenous ABC1 gene, thereby suggesting different function between AtOSA1 and the yeast ABC1. The atosa1-1 and atosa1-2 T-DNA insertion mutants were more affected than wild-type plants by Cd2+ and revealed an increased sensitivity toward oxidative stress (hydrogen peroxide) and high light. The mutants exhibited higher superoxide dismutase activities and differences in the expression of genes involved in the antioxidant pathway. In addition to the conserved Abc1 region in the AtOSA1 protein sequence, putative kinase domains were found. Protein kinase assays in gelo using myelin basic protein as a kinase substrate revealed that chloroplast envelope membrane fractions from the AtOSA1 mutant lacked a 70-kD phosphorylated protein compared to the wild type. Our data suggest that the chloroplast AtOSA1 protein is a new factor playing a role in the balance of oxidative stress.

Disruption of aldo-keto reductase genes leads to elevated markers of oxidative stress and inositol auxotrophy in Saccharomyces cerevisiae

A large family of aldo-keto reductases with similar kinetic and structural properties but unknown physiological roles is expressed in the yeast Saccharomyces cerevisiae. Strains with one or two AKR genes disrupted have apparently normal phenotypes, but disruption of at least three AKR genes results in a heat shock phenotype and slow growth in inositol-deficient culture medium (Ino−). The present study was carried out to identify metabolic or signaling defects that may underlie phenotypes that emerge in AKR deficient strains. Here we demonstrate that pretreatment of a pentuple AKR null mutant with the anti-oxidative agent N-acetyl-cysteine rescues the heat shock phenotype. This indicates that AKR gene disruption may be associated with defects in oxidative stress response. We observed additional markers of oxidative stress in AKR-deficient strains, including reduced glutathione levels, constitutive nuclear localization of the oxidation-sensitive transcription factor Yap1 and upregulation of a set of Yap1 target genes whose function as a group is primarily involved in response to oxidative stress and redox balance. Genetic analysis of the Ino− phenotype of the null mutants showed that defects in transcriptional regulation of the INO1, which encodes for inositol-1-phosphate synthase, can be rescued through ectopic expression of a functional INO1. Taken together, these results suggest potential roles for AKRs in oxidative defense and transcriptional regulation.

Intracellular balance of oxidative stress and cytoprotective molecules in damaged interlobular bile ducts in autoimmune hepatitis and primary biliary cirrhosis: In situ detection of 8‐hydroxydeoxyguanosine and glutathione‐S‐transferase‐pi

Bile duct injury has been thought to be absent in autoimmune hepatitis (AIH), but recent studies have indicated that AIH patients do have bile duct injury. In this study, the intracellular balance of oxidative stress and cytoprotection in biliary epithelial cells was investigated to clarify the pathogenesis of bile duct injury in AIH. The intracellular status of oxidative DNA damage caused by oxidative stress and glutathione, an endogenous cytoprotective molecule, were examined in patients with AIH, primary biliary cirrhosis (PBC), and normal controls by immunostaining of 8-hydroxydeoxyguanosine (8-OHdG) and glutathione-S-transferase-pi. Immunohistochemically, 8-OHdG expression was detected as abundantly in the damaged bile ducts of AIH patients as in PBC patients. Moreover, in AIH, 8-OHdG expression was detected in damaged bile ducts more than in undamaged bile ducts. Glutathione-S-transferase-pi expression was relatively preserved in the damaged bile ducts of AIH patients compared to PBC patients, reflecting preservation of intracellular glutathione. In AIH, oxidative stress and DNA damage may be involved in the pathogenesis of bile duct injury in a manner similar to that found in PBC. However, relatively preserved intracellular glutathione may play a key role in preventing progressive bile duct loss following bile duct injury in AIH.