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The putative oxidation of hydroethidine (HE) has become a widely used fluorescent assay for the detection of superoxide in cultured cells. By covalently joining HE to a hexyl triphenylphosphonium cation (Mito-HE), the HE moiety can be targeted to mitochondria. However, the specificity of HE and Mito-HE for superoxide in vivo is limited by autooxidation as well as by nonsuperoxide-dependent cellular processes that can oxidize HE probes to ethidium (Etd). Recently, superoxide was shown to react with HE to generate 2-hydroxyethidium [Zhao, H., Kalivendi, S., Zhang, H., Joseph, J., Nithipatikom, K., Vasquez-Vivar, J. & Kalyanaraman, B. (2003) Free Radic. Biol. Med. 34, 1359–1368]. However, 2-hydroxyethidium is difficult to distinguish from Etd by conventional fluorescence techniques exciting at 510 nm. While investigating the oxidation of Mito-HE by superoxide, we found that the superoxide product of both HE and Mito-HE could be selectively excited at 396 nm with minimal interference from other nonspecific oxidation products. The oxidation of Mito-HE monitored at 396 nm by antimycin-stimulated mitochondria was 30% slower than at 510 nm, indicating that superoxide production may be overestimated at 510 nm by even a traditional superoxide-stimulating mitochondrial inhibitor. The rate-limiting step for oxidation by superoxide was 4 × 10 6 M −1 ·s −1 , which is proposed to involve the formation of a radical from Mito-HE. The rapid reaction with a second superoxide anion through radical–radical coupling may explain how Mito-HE and HE can compete for superoxide in vivo with intracellular superoxide dismutases. Monitoring oxidation at both 396 and 510 nm of excitation wavelengths can facilitate the more selective detection of superoxide in vivo .

Evidence suggests that mitochondrial dysfunction and oxidant production, in association with an accumulation of oxidative damage, contribute to the aging process. Regular physical activity can delay the onset of morbidity, increase mean lifespan, and reduce the risk of developing several pathological states. No studies have examined age-related changes in oxidant production and oxidative stress in both subsarcolemmal (SSM) and interfibrillar (IFM) mitochondria in combination with lifelong exercise. Therefore, we investigated whether long-term voluntary wheel running in Fischer 344 rats altered hydrogen peroxide (H2O2) production, antioxidant defenses, and oxidative damage in cardiac SSM and IFM. At 10–11 wk of age, rats were randomly assigned to one of two groups: sedentary and 8% food restriction (sedentary; n = 20) or wheel running and 8% food restriction (runners; n = 20); rats were killed at 24 mo of age. After the age of 6 mo, running activity was maintained at an average of 1,145 ± 248 m/day. Daily energy expenditure determined by doubly labeled water technique showed that runners expended on average ∼70% more energy per day than the sedentary rats. Long-term voluntary wheel running significantly reduced H2O2production from both SSM (−10.0%) and IFM (−9.6%) and increased daily energy expenditure (kJ/day) significantly in runners compared with sedentary controls. Additionally, MnSOD activity was significantly lowered in SSM and IFM from wheel runners, which may reflect a reduction in mitochondrial superoxide production. Activities of the other major antioxidant enzymes (glutathione peroxidase and catalase) and glutathione levels were not altered by wheel running. Despite the reduction in mitochondrial oxidant production, no significant differences in oxidative stress levels (4-hydroxy-2-nonenal-modified proteins, protein carbonyls, and malondialdehyde) were detected between the two groups. The health benefits of chronic exercise may be, at least partially, due to a reduction in mitochondrial oxidant production; however, we could not detect a significant reduction in several selected parameters of oxidative stress.

Mitochondrial dysfunction and the accumulation of oxidative damage to macromolecules are believed to play key roles in the aging process. Characterization of age-related changes to cardiac mitochondria has been complicated by the fact that two distinct populations of mitochondria exist in the myocardium: subsarcolemmal mitochondria (SSM) and interfibrillar mitochondria (IFM). We investigated whether differences in hydrogen peroxide production (H2O2) and oxidative stress existed between cardiac SSM and IFM isolated from young (6 mo) and old (24 mo) male Fischer-344 rats. There was a significant increase in oxidative stress levels (4-hydroxy-2-nonenal-modified proteins, protein carbonyls, and malondialdehyde) in IFM with age. In contrast, only protein carbonyls were elevated in SSM with age. Significant age-related increases in MnSOD, GPX, and CAT activities were detected in IFM, while in SSM, MnSOD, and GPX activities increased with age and CAT activity declined. These increases in antioxidant enzyme activity likely occurred in response to increased mitochondrial production of superoxide and hydrogen peroxide. Indeed, SSM produced more H2O2 with age, while the increase in IFM was not significant, but this may be due to the higher antioxidant enzyme activity observed in IFM compared with SSM. Finally, reduced glutathione levels were significantly lower in IFM compared with SSM in both young and old rats, while glutathione reductase activity was not different with age or mitochondrial subpopulations, indicating increased consumption of glutathione. The accumulation of oxidant-induced damage in IFM may be a major contributing factor to the age-related alterations in myocardial function. Our results emphasize the importance of studying both mitochondrial populations when attempting to elucidate the contribution of mitochondrial dysfunction to myocardial aging.

Glutathione (GSH) significantly declines in the aging rat liver. Because GSH levels are partly a reflection of its synthetic capacity, we measured the levels and activity of γ-glutamylcysteine ligase (GCL), the rate-controlling enzyme in GSH synthesis. With age, both the catalytic (GCLC) and modulatory (GCLM) subunits of GCL decreased by 47% and 52%, respectively ( P < 0.005). Concomitant with lower subunit levels, GCL activity also declined by 53% ( P < 0.05). Because nuclear factor erythroid2-related factor 2 (Nrf2) governs basal and inducible GCLC and GCLM expression by means of the antioxidant response element (ARE), we hypothesized that aging results in dysregulation of Nrf2-mediated GCL expression. We observed an ≈50% age-related loss in total ( P < 0.001) and nuclear ( P < 0.0001) Nrf2 levels, which suggests attenuation in Nrf2-dependent gene transcription. By using gel-shift and supershift assays, a marked reduction in Nrf2/ARE binding in old vs. young rats was noted. To determine whether the constitutive loss of Nrf2 transcriptional activity also affects the inducible nature of Nrf2 nuclear translocation, old rats were treated with ( R )-α-lipoic acid (LA; 40 mg/kg i.p. up to 48 h), a disulfide compound shown to induce Nrf2 activation in vitro and improve GSH levels in vivo . LA administration increased nuclear Nrf2 levels in old rats after 12 h. LA also induced Nrf2 binding to the ARE, and, consequently, higher GCLC levels and GCL activity were observed 24 h after LA injection. Thus, the age-related loss in GSH synthesis may be caused by dysregulation of ARE-mediated gene expression, but chemoprotective agents, like LA, can attenuate this loss.

Cardiac mitochondria are composed of two distinct subpopulations: one beneath the sarcolemma (subsarcolemmal mitochondria: SSM), and another along the myofilaments (interfibrillary mitochondria: IFM). Previous studies suggest a preferential loss of IFM function with age; however, the age-related changes in oxidative stress in these mitochondrial subpopulations have not been examined. To this end, the changes in mitochondrial antioxidant capacity, oxidant output, and oxidative damage to Complex IV in IFM and SSM from young and old rats were studied. Results show no apparent differences in any parameters examined between IFM and SSM from young rats. However, relative to young, only IFM from old rats had a significantly higher rate of oxidant production and a decline in mitochondrial ascorbate levels and GSH redox status. The age-related decline in mitochondrial antioxidant capacity in IFM was accompanied by a marked loss in glutaredoxin and GSSG reductase activities, suggesting a diminished reductive capacity in IFM with age. Moreover, the loss in Complex IV activity was limited to the IFM of old rats, which was accompanied by a 4-fold increase in 4-hydroxynonenal-modified Complex IV. Thus, mitochondrial decay is not uniform and further indicates that myofibrils may be uniquely under oxidative stress in the aging heart.

4-hydroxynonenal (HNE), a product of omega-6 polyunsaturated fatty acid peroxidation, impairs mitochondrial respiration in vitro by adducting the alpha-ketoglutarate dehydrogenase complex (KGDC) and inhibiting its activity. The present study seeks to define whether aging increases HNE adduction to rat heart KGDC, and whether such adduction impacts KGDC activity. We found that hearts from old rats exhibit significantly (por =0.01) higher HNE-modified mitochondrial proteins when compared with those from young rats. Among these proteins, dihydrolipoamide succinyltransferase, the E2k component of KGDC, was most markedly modified (por =0.01) by HNE with age. As opposed to that seen in vitro, no significant change in electrophoretic mobility or impairment in enzyme activity was observed. On the contrary, KGDC activity increased onefold (por =0.01) in old rats, suggesting that the aging myocardium is not affected by HNE adduction or compensates for such damage. Further analysis revealed that heightened KGDC activity was not due to increased protein content or gene expression, but correlates with a lower Km for alpha-ketoglutarate. Thus, contrary to that observed in vitro, the measurement of HNE-KGDC adduct in rat heart is more relevant as a marker of age-related protein oxidation than a factor of mitochondrial dysfunction.

This study investigated whether the age-related decline in hepatic ascorbic acid (AA) levels in rats was due to altered AA uptake. AA concentrations were 68% lower in freshly isolated hepatocytes from old (24-26 months) versus young (3-5 months; p

Oxidative mitochondrial decay is a major contributor to aging and neural degeneration. In old rats (vs. young rats) mitochondrial membrane potential, cardiolipin level, respiratory control ratio, and cellular O2 uptake are lower; oxidants/O2, neuron RNA oxidation, and mutagenic aldehydes from lipid peroxidation are higher. Ambulatory activity and cognition declines with age. Feeding old rats for a few weeks with acetyl-l-carnitine (ALCAR), a mitochondrial metabolite, plus R-lipoic acid (LA), a mitochondrial coenzyme and antioxidant, restores mitochondrial function; lowers oxidants, neuronal RNA oxidation, and mutagenic aldehydes; and increases rat ambulatory activity and cognition (Skinner box/Morris water maze). The mechanism appears to be that with age increased oxidative damage to protein causes a deformation of structure of key enzymes, with a consequent lessening of affinity (Km) for the substrate. The loss with age of carnitine acetyl transferase binding affinity can be mimicked by reacting it with malondialdehyde (a lipid peroxidation product which increases with age). Feeding the substrate ALCAR with LA restores the velocity of the reaction, Km for ALCAR and CoA, and mitochondrial function. Heme biosynthesis is predominantly in the mitochondria. Interfering with heme synthesis causes specific loss of complex IV with consequent release of oxidants and neuronal degeneration. Iron deficiency (25% of menstruating women in the US ingest

Vitamin C and thiol agents improve vasomotor function. To determine whether these compounds directly affect endothelial function, nitric oxide (NO) synthesis was measured in human aortic endothelial cells treated with ascorbic acid or the thiol modulating agents lipoic acid or L-2-oxothiazolidine-4-carboxylic acid (OTC). A dose-dependent increase in A23187-stimulated NO synthesis and elevated cGMP levels were observed in all cases except for OTC. Cellular GSH levels were not significantly increased, and the GSH/GSSG ratio was not significantly affected by treatment of the cells with lipoic acid, OTC, or ascorbic acid. Thus, vitamin C and lipoic acid potentiate endothelial NO synthesis and bioactivity by mechanisms that appear to be independent of cellular GSH levels and redox environment.

We assayed levels of lipid peroxidation, protein carbonyl formation, glutamine synthetase (GS) activity and both oxidized and reduced glutathione to study the link between oxidative damage, aging and β-amyloid (Aβ) in the canine brain. The aged canine brain, a model of human brain aging, naturally develops extensive diffuse deposits of human-type Aβ. Aβ was measured in immunostained prefrontal cortex from 19 beagle dogs (4–15 years). Increased malondialdehyde (MDA), which indicates increased lipid peroxidation, was observed in the prefrontal cortex and serum but not in cerebrospinal fluid (CSF). Oxidative damage to proteins (carbonyl formation) also increased in brain. An age-dependent decline in GS activity, an enzyme vulnerable to oxidative damage, and in the level of glutathione (GSH) was observed in the prefrontal cortex. MDA level in serum correlated with MDA accumulation in the prefrontal cortex. Although 11/19 animals exhibited Aβ, the extent of deposition did not correlate with any of the oxidative damage measures, suggesting that each form of neuropathology accumulates in parallel with age. This evidence of widespread oxidative damage and Aβ deposition is further justification for using the canine model for studying human brain aging and neurodegenerative diseases.

Mitochondrial-supported bioenergetics decline and oxidative stress increases during aging. To address whether the dietary addition of acetyl- l -carnitine [ALCAR, 1.5% (wt/vol) in the drinking water] and/or ( R )-α-lipoic acid [LA, 0.5% (wt/wt) in the chow] improved these endpoints, young (2–4 mo) and old (24–28 mo) F344 rats were supplemented for up to 1 mo before death and hepatocyte isolation. ALCAR+LA partially reversed the age-related decline in average mitochondrial membrane potential and significantly increased ( P = 0.02) hepatocellular O 2 consumption, indicating that mitochondrial-supported cellular metabolism was markedly improved by this feeding regimen. ALCAR+LA also increased ambulatory activity in both young and old rats; moreover, the improvement was significantly greater ( P = 0.03) in old versus young animals and also greater when compared with old rats fed ALCAR or LA alone. To determine whether ALCAR+LA also affected indices of oxidative stress, ascorbic acid and markers of lipid peroxidation (malondialdehyde) were monitored. The hepatocellular ascorbate level markedly declined with age ( P = 0.003) but was restored to the level seen in young rats when ALCAR+LA was given. The level of malondialdehyde, which was significantly higher ( P = 0.0001) in old versus young rats, also declined after ALCAR+LA supplementation and was not significantly different from that of young unsupplemented rats. Feeding ALCAR in combination with LA increased metabolism and lowered oxidative stress more than either compound alone.

It is well established that lipid metabolism is controlled, in part, by circadian clocks. However, circadian clocks lose temporal precision with age and correlates with elevated incidence in dyslipidemia and metabolic syndrome in older adults. Because our lab has shown that lipoic acid (LA) improves lipid homeostasis in aged animals, we hypothesized that LA affects the circadian clock to achieve these results. We fed 24 month old male F344 rats a diet supplemented with 0.2% (w/w) LA for 2 weeks prior to sacrifice and quantified hepatic circadian clock protein levels and clock-controlled lipid metabolic enzymes. LA treatment caused a significant phase-shift in the expression patterns of the circadian clock proteins Period (Per) 2, Brain and Muscle Arnt-Like1 (BMAL1), and Reverse Erythroblastosis virus (Rev-erb) β without altering the amplitude of protein levels during the light phase of the day. LA also significantly altered the oscillatory patterns of clock-controlled proteins associated with lipid metabolism. The level of peroxisome proliferator-activated receptor (PPAR) α was significantly increased and acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS) were both significantly reduced, suggesting that the LA-supplemented aged animals are in a catabolic state. We conclude that LA remediates some of the dyslipidemic processes associated with advanced age, and this mechanism may be at least partially through entrainment of circadian clocks.

Objectives. To describe levels of risky sexual behaviour, HIV testing and HIV knowledge among men and women in Guatemala by ethnic group and to identify adjusted associations between ethnicity and these outcomes. Design. Data on 16,205 women aged 15–49 and 6822 men aged 15–59 from the 2008–2009 Encuesta Nacional de Salud Materno Infantil were used to describe ethnic group differences in sexual behaviour, HIV knowledge and testing. We then controlled for age, education, wealth and other socio-demographic factors in a multivariate logistic regression model to examine the effects of ethnicity on outcomes related to age at sexual debut, number of lifetime sex partners, comprehensive HIV knowledge, HIV testing and lifetime sex worker patronage (men only). Results. The data show low levels of risky sexual behaviour and low levels of HIV knowledge among indigenous women and men, compared to other respondents. Controlling for demographic factors, indigenous women were more likely than other women never to have been tested for HIV and to lack comprehensive HIV knowledge. They were less likely to report early sexual debut and three or more lifetime sexual partners. Indigenous men were more likely than other men to lack comprehensive HIV knowledge and demonstrated lower odds of early sexual debut, 10 or more lifetime sexual partners and sex worker patronage. Conclusions. The Mayan indigenous population in Guatemala, while broadly socially vulnerable, does not appear to be at elevated risk for HIV based on this analysis of selected risk factors. Nonetheless, low rates of HIV knowledge and testing may be cause for concern. Programmes working in indigenous communities should focus on HIV education and reducing barriers to testing. Further research into the factors that underlie ethnic self-identity and perceived ethnicity could help clarify the relative significance of these measures for HIV risk and other health outcomes.

Children and families affected by HIV are at considerable risk for psychological distress. Community-based home visiting is a common mechanism for providing basic counseling and other services to HIV-affected families. While programs emphasize home visitor training and compensation as means to promote high-quality service delivery, whether these efforts result in measurable gains in beneficiaries' well-being remains largely unanswered. This study employs a longitudinal quasi-experimental design to explore whether these kinds of investments yield concomitant gains in psychological outcomes among beneficiaries. Baseline and follow-up data were collected over a two-year period from children aged 10-17 at the time of program enrollment and their caregivers, with 80% retention. In this sample of 1487 children and 918 caregivers, the psychological health outcomes of those enrolled in programs with home visitors who receive intensive training, organizational support, and regular compensation (termed "paraprofessional") were compared to those enrolled in programs offering limited home visiting services from lay volunteers. Applying multilevel logistic regression, no measurable improvements were found among paraprofessional enrollees, and three outcomes were significantly worse at follow-up regardless of program model. Children's behavior problems became more prevalent even after adjusting for other factors, increasing from 29% to 35% in girls and from 28% to 43% in boys. Nearly one-quarter of girl and boys reported high levels of depression at follow-up, and this was a significant rise over time for boys. Rates of poor family functioning also significantly worsened over time, rising from 30% to 59%. About one-third of caregivers reported high levels of negative feelings at follow-up, with no improvements observed in the paraprofessional group. Results highlight that children's and caregivers' psychological outcomes may be relatively impervious to change even in paraprofessional home visiting models. Findings underscore the need for programs serving HIV-affected families to add focused evidence-based psychological interventions to supplement traditional home visiting.

A diet supplemented with (R)-lipoic acid, a mitochondrial coenzyme, was fed to old rats to determine its efficacy in reversing the decline in metabolism seen with age. Young (3 to 5 months) and old (24 to 26 months) rats were fed an AIN-93M diet with or without (R)-lipoic acid (0.5% w/w) for 2 wk, killed, and their liver parenchymal cells were isolated. Hepatocytes from untreated old rats vs. young controls had significantly lower oxygen consumption (P0. 03) and mitochondrial membrane potential. (R)-Lipoic acid supplementation reversed the age-related decline in O2 consumption and increased (P0.03) mitochondrial membrane potential. Ambulatory activity, a measure of general metabolic activity, was almost threefold lower in untreated old rats vs. controls, but this decline was reversed (P0.005) in old rats fed (R)-lipoic acid. The increase of oxidants with age, as measured by the fluorescence produced on oxidizing 2',7'-dichlorofluorescin, was significantly lowered in (R)-lipoic acid supplemented old rats (P0.01). Malondialdehyde (MDA) levels, an indicator of lipid peroxidation, were increased fivefold with age in cells from unsupplemented rats. Feeding rats the (R)-lipoic acid diet reduced MDA levels markedly (P0.01). Both glutathione and ascorbic acid levels declined in hepatocytes with age, but their loss was completely reversed with (R)-lipoic acid supplementation. Thus, (R)-lipoic acid supplementation improves indices of metabolic activity as well as lowers oxidative stress and damage evident in aging.

Ascorbic acid recycling from dehydroascorbic acid and biosynthesis from gulono-1,4-lactone were used as measures of cellular response capacity to increased oxidative stress induced by tert-butylhydroperoxide. The hepatic ascorbic acid concentration was 54% lower in cells from old rats when compared to cells isolated from young rats (P

Mitochondrial function during aging was assessed in isolated rat hepatocytes to avoid the problem of differential lysis when old, fragile mitochondria are isolated. Rhodamine 123, a fluorescent dye that accumulates in mitochondria on the basis of their membrane potential, was used as a probe to determine whether this key function is affected by aging. A marked fluorescent heterogeneity was observed in hepatocytes from old (20–28 months) but not young (3–5 months) rats, suggesting age-associated alterations in mitochondrial membrane potential, the driving force for ATP synthesis. Three distinct cell subpopulations were separated by centrifugal elutriation; each exhibited a unique rhodamine 123 fluorescence pattern, with the largest population from old rats having significantly lower fluorescence than that seen in young rats. This apparent age-associated alteration in mitochondrial membrane potential was confirmed by measurements with radioactive tetraphenylphosphonium bromide. Cells from young rats had a calculated membrane potential of −154 mV, in contrast to that of the three subpopulations from old rats of −70 mV (the largest population), −93 mV, and −154 mV. Production of oxidants was examined using 2′,7′dichlorofluorescin, a dye that forms a fluorescent product upon oxidation. The largest cell subpopulation and a minor one from old animals produced significantly more oxidants than cells from young rats. To investigate the molecular cause(s) for the heterogeneity, we determined the levels of an age-associated mtDNA deletion. No significant differences were seen in the three subpopulations, indicating that the mitochondrial decay is due to other mutations, epigenetic changes, or both.

New evidence for the regulation of vitamin C homeostasis has emerged from several studies of human genetic variation. Polymorphisms in the genes encoding sodium-dependent vitamin C transport proteins are strongly associated with plasma ascorbate levels and likely impact tissue cellular vitamin C status. Furthermore, genetic variants of proteins that suppress oxidative stress or detoxify oxidatively damaged biomolecules, i.e., haptoglobin, glutathione-S-transferases, and possibly manganese superoxide dismutase, affect ascorbate levels in the human body. There also is limited evidence for a role of glucose transport proteins. In this review, we examine the extent of the variation in these genes, their impact on vitamin C status, and their potential role in altering chronic disease risk. We conclude that future epidemiological studies should take into account genetic variation in order to successfully determine the role of vitamin C nutriture or supplementation in human vitamin C status and chronic disease risk.

Duchenne muscular dystrophy is a progressive and incurable neuromuscular disease caused by genetic and biochemical defects of the dystrophin-glycoprotein complex. Here we show the regenerative potential of myogenic progenitors derived from corrected dystrophic induced pluripotent stem cells generated from fibroblasts of mice lacking both dystrophin and utrophin. We correct the phenotype of dystrophic induced pluripotent stem cells using a Sleeping Beauty transposon system carrying the micro-utrophin gene, differentiate these cells into skeletal muscle progenitors and transplant them back into dystrophic mice. Engrafted muscles displayed large numbers of micro-utrophin-positive myofibers, with biochemically restored dystrophin-glycoprotein complex and improved contractile strength. The transplanted cells seed the satellite cell compartment, responded properly to injury and exhibit neuromuscular synapses. We also detect muscle engraftment after systemic delivery of these corrected progenitors. These results represent an important advance towards the future treatment of muscular dystrophies using genetically corrected autologous induced pluripotent stem cells.

Several mitochondrial functions decline with age. The contributing factors include, the intrinsic rate of proton leakage across the inner mitochondrial membrane (a correlate of oxidant formation), decreased membrane fluidity, and decreased levels and function of cardiolipin, which supports the function of many of the proteins of the inner mitochondrial membrane. Oxidants generated by mitochondria appear to be the major source of the oxidative lesions that accumulate with age. Evidence supports the suggestion that age-associated accumulation of mitochondrial deficits due to oxidative damage is likely to be a major contributor to cellular, tissue, and organismal aging.

We have developed a high-throughput Spectral Unmixing Plate Reader (SUPR) which can acquire a fluorescence emission spectrum across 384 wells, with short (< 4 min) read time for the entire plate. The entire spectrum is recorded at once with a compact spectrometer equipped with an uncooled one-dimensional array detector. For application to FRET in living cells, FRET protein biosensors, with genetically encoded GFP (donor) and RFP (acceptor) tags, are expressed in HEK cells, and single-component spectra are acquired from water (Raman), untransfected cells (autofluorescence), cells expressing donor only, and cells expressing acceptor only. Each observed spectrum is decomposed into a linear combination of component spectra to determine FRET efficiency. The method enables unprecedented speed, precision, and accuracy. It rigorously and painlessly accounts for phenomena such as bleed-through of the donor emission into the acceptor channel, cross-talk associated with direct excitation of the acceptor, and cell autofluorescence. Excitation and detection are both from the top, so low-cost polypropylene plates typically used in qPCR work very well in this application. This novel technology has been combined with our previously developed fluorescence lifetime technology (Petersen et al, Rev Sci Inst, 2014) allowing for high-throughput quantitation of FRET efficiency from independent measurements. We have screened small-molecule libraries with an intramolecular cardiac calcium pump FRET biosensor, expressed in living cells (Gruber et al., J. Biomol Screening, 2014) using this multiplexed fluorescence detection platform and identified several previously unknown small-molecule effectors. The information obtained from both fluorescence lifetime and spectrum measurements reduced the number of false-positives and increased confidence in our hit selection methods. The development of this technology and screening assay will prove useful for drug discovery but also answering basic biological questions with fluorescence based assays.

Tumor necrosis factor (TNF) receptor 1 (TNFR1) and Death Receptor 5 (DR5) are members of the TNF receptor superfamily that are activated by binding to their ligands, TNFα and TNF-related apoptosis-inducing ligand (TRAIL) respectively. The TNFα-TNFR1 signaling pathway culminates with NF-κB activation and inflammatory response. The TRAIL-DR5 signaling pathway culminates in apoptosis. Recently, we have shown that TRAIL-induced apoptosis is initiated through the formation of large and highly structured networks held together by receptor dimers. In our current work, we use fluorescence lifetime measurements to study TNFR1. As an initial control to validate our approach, we monitored receptor-receptor binding affinity as a function of temperature and detergent concentration. Here, we have extended these initial studies to include investigation of the role of cholesterol and cytochalasin B. We have recently shown that cholesterol depletion in cells does not inhibit the formation of DR5 networks but, interestingly, does fail to initiate apoptosis. Others have shown that disruption of actin cytoskeleton by cytochalasin B in death receptor CD95, another member of the TNF receptor superfamily, induces and enhances apoptosis. Our lifetime measurements are used to investigate any potential connection between these cell-level observations and the biophysics of TNF-Receptor oligomerization.

Nuclear factor erythroid 2 (NF-E2) related factor 2 (Nrf2) is a transcription factor that governs the expression of over a hundred so-called phase II detoxification and antioxidant genes that are regulated through the antioxidant response element (ARE). Loss of Nrf2 activity has been implicated in cardiovascular disease, inflammation, aging, and cancer. Nrf2 is induced to accumulate in the nucleus when the cell encounters an oxidative stress, a fact that has been exploited experimentally to test the conditions under which ARE-containing genes are expressed. The nuclear levels of Nrf2 give an indication of whether an experimental treatment results in Nrf2 localization and induction. mRNA levels of phase II genes may be measured as a follow-up, but in order to show a direct link between nuclear Nrf2 accumulation and increases in gene expression, it is useful to show that Nrf2 binds to AREs in the promoters of target genes. The simplest way to do this is to employ a chromatin immunoprecipitation (ChIP) assay along with an examination of cellular Nrf2 levels by immunoblotting. Curr. Protoc. Toxicol. 45:17.13.1-17.13.13. © 2010 by John Wiley & Sons, Inc. Keywords: Nrf2; immunoblot; chromatin immunoprecipitation

The purpose of this protocol is to establish a primary hepatocyte culture system as a suitable model to examine age-related changes in Phase II detoxication gene expression. Hepatocytes are isolated using a two-step collagenase perfusion technique from young (3 to 6 months) and old (24 to 28 months) rats and placed in primary culture using collagen (Type I)-coated plates as the extracellular matrix. A supplemented William’s E Medium is used as the medium. This culture system maintains hepatocyte viability from both young and old rats for ~60 hr, as measured by lactate dehydrogenase activity, while also maintaining their respective phenotypes relative to Phase II detoxification. We thus conclude that a collagen-based cell culture system is suitable to study age-associated deficits in Nrf2/ARE-mediated Phase II gene regulation provided that experiments can be conducted within 60 hr after cell isolation.

Purpose Lipoic acid (LA) shows promise as a beneficial micronutrient in improving health, particularly in the elderly. Clinical and in vitro reports show that LA induces endogenous antioxidants and acts as an anti-inflammatory agent. LA also increases nerve conductance, improves diabetes-induced polyneuropathies, and remediates the age-associated cognitive decline in canines. Furthermore, LA significantly improves hypertriglyceridemia and glucose handling. From our preclinical research we have found that LA primarily influences three areas of health: cognition, stress response, and lipids. This study examines the effects of LA on human subjects in components of each of the three aforementioned areas of health.

Lipoic acid (LA) shows promise as a beneficial micronutrient toward improving elder health. Studies using old rats show that (R)-α-LA (R-LA) significantly increases low molecular weight antioxidants that otherwise decline with age. Despite this rationale for benefiting human health, little is known about age-associated alterations in absorption characteristics of LA, or whether the commercially available racemic mixture of LA (R,S-LA) is equally as bioavailable as the naturally occurring R-enantiomer. To address these discrepancies, a pilot study was performed to establish which form of LA is most effectively absorbed in older subjects relative to young volunteers. Young adults (average age = 32 years) and older adults (average age = 79 years) each received 500 mg of either R- or R,S-LA. Blood samples were collected for 3 h after supplementation. After a washout period they were given the other chiral form of LA not originally ingested. Results showed that 2 out of 6 elder males exhibited greater maximal plasma LA and area under the curve for the R-form of LA versus the racemic mixture. The elder subjects also demonstrated a reduced time to reach maximal plasma LA concentration following R-LA supplementation than for the racemic mixture. In contrast, young males had a tendency for increased bioavailability of R,S-LA. Overall, bioavailability for either LA isoform was much more variable between older subjects compared to young adults. Plasma glutathione levels were not altered during the sampling period. Thus subject age, and potential for varied response, should be considered when determining an LA supplementation regimen.

NFE2-related factor 2 (Nrf2) transcriptionally governs the cellular response to harmful electrophiles, xenobiotics, and reactive oxygen species. Its nuclear levels decline with age (Suh et al., 2004), which in part explains the age-related loss of phase II detoxification. However, little work has yet characterized how age affects Nrf2 DNA binding, or the role that alterations to the Nrf2 transcriptional apparatus plays in modulating Nrf2-mediated gene expression. In the present study we used immunoprecipitation assays to show that Nrf2 bound to the active antioxidant response element (ARE) of the catalytic subunit of glutamate cysteine ligase (GCLC) is significantly lower in hepatic chromatin from aged versus young rats. Moreover, the activity at this ARE locus is diminished during aging because of the presence of Bach1 and the absence of CREB-binding protein (CBP), a transcriptional repressor and co-activator, respectively. Further analysis reveals that Nrf2 occupies an alternate ARE site located -2.2 kb downstream from the normally active ARE binding site in livers of old rats, indicating an age-specific adaptation to maintain gene expression. Our results thus show that the conversion of Nrf2 binding from an active ARE to an alternative ARE element is not adequate to maintain basal expression of hepatic Gclc in old rats, which provides a potential mechanism for the age-related loss of glutathione synthetic and other phase II enzymes.

A wearable, multi-modal sensor is presented that can non-invasively monitor a patient's activity level and heart function concurrently for more than a week. The 4 in(2) sensor incorporates both a non-contact heartrate sensor and a 5-axis inertial measurement unit (IMU), allowing simultaneous heart, respiration, and movement monitoring without requiring physical contact with the skin [1]. Hence, this Oregon State University Life and Activity Monitor (OLAM) provides the unique opportunity to combine motion data with heart-rate information, enabling assessment of actual physical activity beyond conventional movement sensors. OLAM also provides a unique platform for non-contact sensing, enabling the filtering of movement artifacts generated by the non-contact capacitive interface, using the IMU data as a movement noise channel. Intended to be used in clinical trials for weeks at a time with no physician intervention, the OLAM allows continuous non-invasive monitoring of patients, providing the opportunity for long-term observation into a patient's physical activity and subtle longitudinal changes.

A transgenic mouse strain that expresses the hepatitis B virus (HBV) large envelope protein in the liver was used to determine the extent of oxidative DNA damage that occurs during chronic HBV infection. This mouse strain develops a chronic necroinflammatory liver disease that mimics the inflammation, cellular hyperplasia, and increased risk for cancer that is evident in human chronic active hepatitis. When perfused in situ with nitroblue tetrazolium, an indicator for superoxide formation, the liver of transgenic mice displayed intense formazan deposition in Kupffer cells, indicating oxygen radical production, and S-phase hepatocytes were commonly seen adjacent to the stained Kupffer cells. Similar changes were not observed in nontransgenic control livers. To determine whether these events were associated with oxidative DNA damage, genomic DNA from the livers of transgenic mice and nontransgenic controls was isolated and examined for 8-oxo-2'-deoxyguanosine, an oxidatively modified adduct of deoxyguanosine. Results showed a significant, sustained accumulation in steady-state 8-oxo-2'-deoxyguanosine that started early in life exclusively in the transgenic mice and increased progressively with advancing disease. The most pronounced increase occurred in livers exhibiting microscopic nodular hyperplasia, adenomas, and hepatocellular carcinoma. Thus, HBV transgenic mice with chronic active hepatitis display greatly increased hepatic oxidative DNA damage. Moreover, the DNA damage occurs in the presence of heightened hepatocellular proliferation, increasing the probability of fixation of the attendant genetic and chromosomal abnormalities and the development of hepatocellular carcinoma.

To determine the effects of age and lipoic acid supplementation on hepatic gene expression, we fed young (3 mo) and old (24 mo) male Fischer 344 rats a diet with or without 0.2% (wt/wt) R-α-lipoic acid (LA) for 2 wk. Total RNA isolated from liver tissue was analyzed by Affymetrix microarray to examine changes in transcriptional profiles. Results showed elevated proinflammatory gene expression in the aging liver and evidence for increased immune cell activation and tissue remodeling, together representing 45% of the age-related transcriptome changes. In addition, age-related increases in transcripts of genes related to fatty acid, triglyceride, and cholesterol synthesis, including acetyl-CoA carboxylase-β (Acacb) and fatty acid synthase (Fasn), were observed. Supplementation of old animals with LA did not reverse the necroinflammatory phenotype but, intriguingly, altered the expression of genes governing circadian rhythm. Most notably, Arntl, Npas2, and Per changed in a coordinated manner with respect to rhythmic transcription. LA further caused a decrease in transcripts of several bile acid and lipid synthesis genes, including Acacb and Fasn, which are regulated by first-order clock transcription factors. Similar effects of LA supplementation on bile acid and lipid synthesis genes were observed in young animals. Transcript changes of lipid metabolism genes were corroborated by a decrease in FASN and ACC protein levels. We conclude that advanced age is associated with a necroinflammatory phenotype and increased lipid synthesis, while chronic LA supplementation influences hepatic genes associated with lipid and energy metabolism and circadian rhythm, regardless of age.

The use of antioxidants is now often used as a pharmacological adjunct to limit infertility. Indeed, the lay public rightly perceives oxidative stress and, thus, antioxidant treatment as important modulators of infertility. While the direct effects of antioxidant treatment on the quality of semen and oocytes are still under investigation, a significant body of evidence points to loss of vascular tone as a root-cause of erectile dysfunction and, possibly, alterations to female reproduction. In this article, we will critically review the often neglected link between vascular dysfunction and infertility. A particular emphasis will be on the potential use of antioxidants to increase fertility and promote conception.

Increased oxidative stress and inflammation causally contribute to cardiovascular diseases, for which advanced age is a major risk factor. We found that indicators of oxidative stress, including NADPH oxidase activity and superoxide levels, were significantly increased in aortas of old (22-24 months) versus young (3-4 months) male F344 rats, whereas superoxide dismutase (SOD) activity was decreased. Aortic mRNA and protein levels of NOX4, the principal catalytic subunit of NADPH oxidase in vascular cells, also were increased with age, but not NOX2 and p22(phox). Indicators of inflammation, including activation of NFkappaB and upregulation of vascular cell adhesion molecule-1 (VCAM-1) in aorta, and monocyte chemotactic protein-1 (MCP-1) in plasma, also were significantly increased in old rats. Supplementation with 0.2% (wt/wt) (R)-alpha-lipoic acid (LA) for 2 weeks caused a nonsignificant decrease in NADPH oxidase activity in aged aorta and a significant decrease in mRNA--but not protein--levels of NOX4 and VCAM-1. Furthermore, LA reversed the age-dependent changes in aortic SOD activity and plasma MCP-1 levels. Hence, vascular oxidative stress and inflammation increase with age and are ameliorated by LA supplementation.

Mitochondrial sphingolipids play a diverse role in normal cardiac function and diseases, yet a precise quantification of cardiac mitochondrial sphingolipids has never been performed. Therefore, rat heart interfibrillary mitochondria (IFM) and subsarcolemmal mitochondria (SSM) were isolated, lipids extracted, and sphingolipids quantified by LC-tandem mass spectrometry. Results showed that sphingomyelin (approximately 10,000 pmol/mg protein) was the predominant sphingolipid regardless of mitochondrial subpopulation, and measurable amounts of ceramide (approximately 70 pmol/mg protein) sphingosine, and sphinganine were also found in IFM and SSM. Both mitochondrial populations contained similar quantities of sphingolipids except for ceramide which was much higher in SSM. Analysis of sphingolipid isoforms revealed ten different sphingomyelins and six ceramides that differed from 16- to 24-carbon units in their acyl side chains. Sub-fractionation experiments further showed that sphingolipids are a constituent part of the inner mitochondrial membrane. Furthermore, inner membrane ceramide levels were 32% lower versus whole mitochondria (45 pmol/mg protein). Three ceramide isotypes (C20-, C22-, and C24-ceramide) accounted for the lower amounts. The concentrations of the ceramides present in the inner membranes of SSM and IFM differed greatly. Overall, mitochondrial sphingolipid content reflected levels seen in cardiac tissue, but the specific ceramide distribution distinguished IFM and SSM from each other.

Mitochondrial membrane hyperpolarization and morphologic changes are important in inflammatory cell activation. Despite the pathophysiologic relevance, no valid and reproducible method for measuring mitochondrial homeostasis in human inflammatory cells is available currently. The purpose of this study was to define and validate reproducible methods for measuring relevant mitochondrial perturbations and to determine whether these methods could discern mitochondrial perturbations in type 2 diabetes mellitus (T2DM), which is a condition associated with altered mitochondrial homeostasis. We employed 5,5′,6,6′-tetrachloro-1,1′3,3′-tetraethylbenzamidazol-carboncyanine (JC-1) to estimate mitochondrial membrane potential (Ψ m ) and acridine orange 10-nonyl bromide (NAO) to assess mitochondrial mass in human mononuclear cells isolated from blood. Both assays were reproducible. We validated our findings by electron microscopy and pharmacologic manipulation of Ψ m . We measured JC-1 and NAO fluorescence in the mononuclear cells of 27 T2DM patients and 32 controls. Mitochondria were more polarized ( P = 0.02) and mitochondrial mass was lower in T2DM ( P = 0.008). Electron microscopy demonstrated diabetic mitochondria were smaller, were more spherical, and occupied less cellular area in T2DM. Mitochondrial superoxide production was higher in T2DM ( P = 0.01). Valid and reproducible measurements of mitochondrial homeostasis can be made in human mononuclear cells using these fluorophores. Furthermore, potentially clinically relevant perturbations in mitochondrial homeostasis in T2DM human mononuclear cells can be detected.

Alpha-lipoic acid is touted as a powerful antioxidant and possibly a conditionally essential nutrient in older mammals. The safety and efficacy of dl-alpha-lipoic acid was evaluated in 30 adult beagles that were evenly randomized into five groups, each of which was fed one of five different foods with varying inclusion rates of dl-alpha-lipoic acid (0, 150, 1500, 3000, and 4500 ppm). All dogs were fed their respective portion of food daily as their sole source of nutrition for 6 months. Evaluations included general health, body weight, food intake, hematologic and serum biochemical parameters, and glutathione:oxidized glutathione (GSH:GSSG) ratios in lymphocytes. No signs of toxicity were observed at any except the highest level of dl-alpha-lipoic acid inclusion, and no consistent abnormalities were noted in hematologic or biochemical measures at any level. There was a significant overall effect (P.05) of food on the difference of GSH:GSSG ratio between Day 84 and Day 0. All inclusions of dl-alpha-lipoic acid increased the ratio of GSH:GSSG with the largest numeric improvement occurring at the lowest inclusion rate (150 ppm).

Transport and distribution of vitamin C is primarily regulated by the function of sodium-dependent vitamin C transporters (SVCTs). SVCT1 is expressed in the small intestine, liver, and kidney, organs that play a vital role in whole body vitamin C homeostasis. Despite the importance of this protein, little is known about regulation of the gene encoding SVCT1, Slc23a1. In this study, we present the first investigation of the transcriptional regulation of human Slc23a1, identifying transcription factors that may influence its expression. A 1,239-bp genomic DNA fragment corresponding to the 5′-flanking region of Slc23a1 was isolated from a human hepatocarcinoma cell line (HepG2) and sequenced. When cloned into a reporter gene construct, robust transcriptional activity was seen in this sequence, nearly 25-fold above the control vector. Deletion analysis of the SVCT1 reporter gene vector defined the minimal active promoter as a small 135-bp region upstream of the transcriptional start site. While several transcription factor binding sites were identified within this sequence, reporter constructs showed that basal transcription required the binding of hepatic nuclear factor 1 (HNF-1) to its cognate sequence. Furthermore, mutation of this HNF-1 binding site resulted in complete loss of luciferase expression, even in the context of the whole promoter. Additionally, small interfering RNA knockdown of both members of the HNF-1 family, HNF-1α and HNF-1β, resulted in a significant decline in SVCT1 transcription. Together, these data suggest that HNF-1α and/or HNF-1β binding is required for SVCT1 expression and may be involved in the coordinate regulation of whole body vitamin C status.

Despite it being a quintessential Phase II detoxification gene, the transcriptional regulation of the rat γ-glutamate cysteine ligase catalytic subunit (GCLC) is controversial. Computer-based sequence analysis identified three putative antioxidant response elements (AREs) at positions −889 to −865 (ARE1), −3170 to −3146 (ARE2) and −3901 to −3877 (ARE3) in the 5′-flanking region of the transcriptional start site. Transfections of individual ARE-luciferase reporter gene constructs into H4IIE cells, a rat hepatoma cell line, identified ARE3 as the functional promoter. Chromatin immunoprecipitation assays using primary rat hepatocytes showed that the transcription factor Nrf2, which is known to regulate ARE-mediated genes, is associated with ARE3. Co-transfection of H4IIE cells with luciferase reporter plasmids containing Gclc ARE3 and an Nrf2 expression plasmid resulted in a 3-fold activation of ARE3-mediated transcription relative to controls. “Loss-of-function” analysis for Nrf2 by small interfering RNA (siRNA) revealed that ARE3-mediated expression was significantly impaired while site-directed mutagenesis of the ARE3-luciferase reporter abolished Nrf2-mediated induction. Treatment with two known Nrf2 inducers, R -(α)-lipoic acid and anetholedithiolethione, showed that the inducible expression of the GCLC gene was also regulated by the ARE3 element. Taken together, these results show that Nrf2 regulates the constitutive expression of rat Gclc through a distal ARE present in its 5′-flanking region. This is the first report showing that rat Gclc is under the transcriptional control of the Nrf2-ARE pathway on a constitutive basis.

Elevated blood triacylglycerol (TG) is a significant contributing factor to the current epidemic of obesity-related health disorders, including type-2 diabetes, nonalcoholic fatty liver disease, and cardiovascular disease. The observation that mice lacking the enzyme sn-glycerol-3-phosphate acyltransferase are protected from insulin resistance suggests the possibility that the regulation of TG synthesis be a target for therapy. Five-week-old Zucker Diabetic Fatty (ZDF) rats were fed a diet containing (R)-alpha-lipoic acid (LA, approximately 200mg/kg body weight per day) for 5 weeks. LA offset the rise in blood and liver TG by inhibiting liver lipogenic gene expression (e.g. sn-glycerol-3-phosphate acyltransferase-1 and diacylglycerol O-acyltransferase-2), lowering hepatic TG secretion, and stimulating clearance of TG-rich lipoproteins. LA-induced TG lowering was not due to the anorectic properties of LA, as pair-fed rats developed hypertriglyceridemia. Livers from LA-treated rats exhibited elevated glycogen content, suggesting dietary carbohydrates were stored as glycogen rather than becoming lipogenic substrate. Although AMP-activated protein kinase (AMPK) reportedly mediates the metabolic effects of LA in rodents, no change in AMPK activity was observed, suggesting LA acted independently of this kinase. The hepatic expression of peroxisome proliferator activated receptor alpha (PPARalpha) target genes involved in fatty acid beta-oxidation was either unchanged or decreased with LA, indicating a different mode of action than for fibrate drugs. Given its strong safety record, LA may have potential clinical applications for the treatment or prevention of hypertriglyceridemia and diabetic dyslipidemia.

The alpha-adrenergic agonist, phenylephrine (1.6 microM), caused a threefold stimulation of glutathione (GSH) transport from the lumen into the vasculature in isolated, vascularly perfused rat small intestine. Stimulation of GSH transport by phenylephrine was blocked by the alpha-adrenergic antagonists, prazosin or phentolamine. Norepinephrine and epinephrine (both alpha and beta agonists) also stimulated GSH absorption but not to the same extent as phenylephrine. Isoproterenol, a strict beta-adrenergic agonist, had no effect on the rate of GSH absorption. Under physiological luminal GSH concentrations, phenylephrine stimulated GSH efflux from the lumen, accumulation in the intestinal mucosa, and transport into the mesenteric vasculature. Phenylephrine did not stimulate the transport of polyethylene glycol, a high molecular weight molecule, and stimulated uptake of cysteine and glycine by 30%. This suggests that the effect of phenylephrine on GSH transport is not due to enhanced bulk flow through paracellular pathways. Studies with isolated small intestinal epithelial cells showed that phenylephrine also stimulated the release of GSH from the cells. Oral administration of phenylephrine with GSH caused a two- to fivefold transient increase in plasma GSH concentrations in rats. Phenylephrine alone or with the amino acid constituents of GSH caused no increase in plasma GSH concentration. Thus, absorption of dietary GSH is under hormonal regulation. The physiological importance of this regulation is not known, although such regulation may function to control utilization of dietary GSH for detoxication and may have therapeutic benefits for individuals with deficient GSH or increased risk of oxidative or chemically induced injury.

Akt is a highly regulated serine/threonine kinase involved in stress response and cell survival. Stress response pathways must cope with increasing chronic stress susceptibility with age. We found an age-related lesion in Akt activity via loss of phosphorylation on Ser473. In hepatocytes from old rats, basal phospho-Ser473 Akt is 30% lower when compared to young, but basal phospho-Thr308 Akt is unchanged. (R)-alpha-lipoic acid (LA), a dithiol compound with antioxidant properties, is effective against age-related increases in oxidative stress and has been used to improve glucose utilization through insulin receptor (IR) pathway-mediated Akt phosphorylation. Treatment with physiologically relevant doses of LA (50 microM) provided a 30% increase in phospho-Ser473. Furthermore, two phosphatases that antagonize Akt, PTEN and PP2A, were both partially inhibited by LA. Thus, LA may be a nutritive agent that can remediate loss of function in the Akt pathway and aid in the survival of liver cells.

The chemical reduction and oxidation (redox) properties of α-lipoic acid (LA) suggest that it may have potent antioxidant potential. A significant number of studies now show that LA and its reduced form, dihydrolipoic acid (DHLA), directly scavenge reactive oxygen species (ROS) and reactive nitrogen species (RNS) species and protect cells against a host of insults where oxidative stress is part of the underlying etiology. However, owing to its limited and transient accumulation in tissues following oral intake, the efficacy of nonprotein-bound LA to function as a physiological antioxidant has been questioned. Herein, we review the evidence that the micronutrient functions of LA may be more as an effector of important cellular stress response pathways that ultimately influence endogenous cellular antioxidant levels and reduce proinflammatory mechanisms. This would promote a sustained improvement in cellular resistance to pathologies where oxidative stress is involved, which would not be forthcoming if LA solely acted as a transient ROS scavenger. © 2008 IUBMB IUBMB Life, 60(6): 362–367, 2008