Myeloperoxidase-catalyzed redox-cycling of phenol promotes lipid peroxidation and thiol oxidation in HL-60 cells

Various types of cancer occur in peroxidase-rich target tissues of animals exposed to aryl alcohols and amines. Unlike biotransformation by cytochrome P450 enzymes, peroxidases activate most substrates by one-electron oxidation via radical intermediates. This work analyzed the peroxidase-dependent formation of phenoxyl radicals in HL-60 cells and its contribution to cytotoxicity and genotoxicity. The results showed that myeloperoxidase-catalyzed redox cycling of phenol in HL-60 cells led to intracellular formation of glutathionyl radicals detected as GS-DMPO nitrone. Formation of thiyl radicals was accompanied by rapid oxidation of glutathione and protein-thiols. Analysis of protein sulfhydryls by SDS-PAGE revealed a significant oxidation of protein SH-groups in HL-60 cells incubated in the presence of phenol/H2O2 that was inhibited by cyanide and azide. Additionally, cyanide- and azide-sensitive generation of EPR-detectable ascorbate radicals was observed during incubation of HL-60 cell homogenates in the presence of ascorbate and H2O2. Oxidation of thiols required addition of H2O2 and was inhibited by pretreatment of cells with the inhibitor of heme synthesis, succinylacetone. Radical-driven oxidation of thiols was accompanied by a trend toward increased content of 8-oxo-7,8-dihydro-2'-deoxyguanosine in the DNA of HL-60 cells. Membrane phospholipids were also sensitive to radical-driven oxidation as evidenced by a sensitive fluorescence HPLC-assay based on metabolic labeling of phospholipids with oxidation-sensitive cis-parinaric acid. Phenol enhanced H2O2-dependent oxidation of all classes of phospholipids including cardiolipin, but did not oxidize parinaric acid-labeled lipids without addition of H2O2. Induction of a significant hypodiploid cell population, an indication of apoptosis, was detected after exposure to H2O2 and was slightly but consistently and significantly higher after exposure to H2O2/phenol. The clonogenicity of HL-60 cells decreased to the same extent after exposure to H2O2 or H2O2/phenol. Treatment of HL-60 cells with either H2O2 or H2O2/phenol at concentrations adequate for lipid peroxidation did not cause a detectable increase in chromosomal breaks. Detection of thiyl radicals as well as rapid oxidation of thiols and phospholipids in viable HL-60 cells provide strong evidence for redox cycling of phenol in this bone marrow-derived cell line.     

Oxidative stress following traumatic brain injury in rats: quantitation of biomarkers and detection of free radical intermediates

Oxidative stress may contribute to many pathophysiologic changes that occur after traumatic brain injury. In the current study, contemporary methods of detecting oxidative stress were used in a rodent model of traumatic brain injury. The level of the stable product derived from peroxidation of arachidonyl residues in phospholipids, 8-epi-prostaglandin F(2alpha), was increased at 6 and 24 h after traumatic brain injury. Furthermore, relative amounts of fluorescent end products of lipid peroxidation in brain extracts were increased at 6 and 24 h after trauma compared with sham-operated controls. The total antioxidant reserves of brain homogenates and water-soluble antioxidant reserves as well as tissue concentrations of ascorbate, GSH, and protein sulfhydryls were reduced after traumatic brain injury. A selective inhibitor of cyclooxygenase-2, SC 58125, prevented depletion of ascorbate and thiols, the two major water-soluble antioxidants in traumatized brain. Electron paramagnetic resonance (EPR) spectroscopy of rat cortex homogenates failed to detect any radical adducts with a spin trap, 5,5-dimethyl-1-pyrroline N:-oxide, but did detect ascorbate radical signals. The ascorbate radical EPR signals increased in brain homogenates derived from traumatized brain samples compared with sham-operated controls. These results along with detailed model experiments in vitro indicate that ascorbate is a major antioxidant in brain and that the EPR assay of ascorbate radicals may be used to monitor production of free radicals in brain tissue after traumatic brain injury.     

High-performance liquid chromatography-based methods of enzymatic analysis: electron transport chain activity in mitochondria from human skeletal muscle

This study addresses an application of pyridine nucleotide enzymatic analyses to evaluate the activity of the mitochondrial electron transport chain (reduced nicotinamide adenine dinucleotide (NADH) oxidase) and Complexes I and II in samples of human muscle as small as approximately 10 mg wet weight. Key aspects in this adaptation are the use of high-performance liquid chromatography with fluorescence detection of NADH and use of alamethicin, a channel-forming antibiotic that enables an unrestricted access of substrates into the mitochondrial matrix. The procedure includes disintegration of tissue by Polytron homogenizer, extraction of myosin from myofibrillar fragments by KCl/pyrophosphate to facilitate release of mitochondria, and preparation of fractions of subsarcolemmal and intermyofibrillar mitochondria. Oxidation of NADH or succinate is assayed in the presence of 40 microg/ml alamethicin and the reaction is terminated by H(2)SO(4), which also destroys the remaining NADH. Nicotinamide adenine dinucleotide (NAD) or fumarate concentrations are measured using alcohol dehydrogenase or fumarase plus malic dehydrogenase reactions, respectively. Generation of NADH, assessed in auxiliary reactions in the presence of hydrazine, is strictly proportional to NAD or fumarate content across a concentration range of 1-20 microM. NADH is quantitatively analyzed with a detection limit of 3-5 pmol by HPLC using a reverse-phase Hypersil ODS column connected to a fluorescence detector.     

The Accelerated Failure Time Model Under Biased Sampling

Summary Chen (2009, Biometrics) studies the semi‐parametric accelerated failure time model for data that are size biased. Chen considers only the uncensored case and uses hazard‐based estimation methods originally developed for censored observations. However, for uncensored data, a simple linear regression on the log scale is more natural and provides better estimators.     

Characteristics of skeletal muscle mitochondrial biogenesis induced by moderate-intensity exercise and weight loss in obesity.

There are fewer mitochondria and a reduced oxidative capacity in skeletal muscle in obesity. Moderate-intensity physical activity combined with weight loss increase oxidative enzyme activity in obese sedentary adults; however, this adaptation occurs without a significant increase in mitochondrial DNA (mtDNA), which is unlike the classic pattern of mitochondrial biogenesis induced by vigorous activity. The objective of this study was to examine the hypothesis that the mitochondrial adaptation to moderate-intensity exercise and weight loss in obesity induces increased mitochondrial cristae despite a lack of mtDNA proliferation. Content of cardiolipin and mtDNA and enzymatic activities of the electron transport chain (ETC) and tricarboxylic acid cycle were measured in biopsy samples of vastus lateralis muscle obtained from sedentary obese men and women before and following a 4-mo walking intervention combined with weight loss. Cardiolipin increased by 60% from 47 +/- 4 to 74 +/- 8 microg/mU CK (P < 0.01), but skeletal muscle mtDNA content did not change significantly (1,901 +/- 363 to 2,169 +/- 317 Rc, where Rc is relative copy number of mtDNA per diploid nuclear genome). Enzyme activity of the ETC increased (P < 0.01); that for rotenone-sensitive NADH-oxidase (96 +/- 1%) increased more than for ubiquinol-oxidase (48 +/- 6%). Activities for citrate synthase and succinate dehydrogenase increased by 29 +/- 9% and 40 +/- 6%, respectively. In conclusion, moderate-intensity physical activity combined with weight loss induces skeletal muscle mitochondrial biogenesis in previously sedentary obese men and women, but this response occurs without mtDNA proliferation and may be characterized by an increase in mitochondrial cristae.     

Analysis of cardiolipin in human muscle biopsy

Cardiolipin is a phospholipid that is specific to the inner mitochondrial membrane and essential for numerous mitochondrial functions. Accordingly, a quantitative assay for cardiolipin can be a valuable aspect of assessing mitochondrial content and functional capacity. The current study was undertaken to develop a simple and reliable method for direct analysis of the major molecular species of cardiolipin and with particular application for analysis of human skeletal muscle. The method that is presented is based on derivatization of cardiolipin in a total lipid extract with 1-pyrenyldiazomethane (PDAM), to form stable, fluorescent 1-pyrenylmethyl esters. The derivatization reaction takes 30 min on ice in a two-phase system (chloroform:methanol:H(2)O:H(2)SO(4)) containing 0.5-1.0mM PDAM and detergent. The contents of the major cardiolipin species in the derivatization mixture can be estimated by HPLC separation with fluorescent detection during a 20 min run on a reverse phase column and with HPLC grade ethanol/0.5mM H(3)PO(4) as the mobile phase. The recovery is about 80%. The method is specific and sensitive with quantitation limits of 0.5-1 pmol cardiolipin. The response of the fluorescence detector (peak area) is linear across a range 5-40 pmol. The assay is linear over the range between 0.3 and 3.0mg of tissue (R(2)=0.998). The assay provides good reproducibility and accuracy (within 5-10%).     

Activation of the caffeine center of the sarcoplasmic reticulum at a reduced concentration of magnesium ions

The action of caffeine and Mg2+ on the efficacy of Ca2+ transport by terminal cisterns and longitudinal tubules of rabbit skeletal muscle sarcoplasmic reticulum (SR) was studied and compared. Addition of 5 to 10 mM caffeine to the incubation medium or a decrease in Mg2+ concentration from 4 to 0.1 mM led to a 3-fold diminution of the Ca/ATP ratio for the terminal cistern fraction. In longitudinal tubules, that effect was far less pronounced. The effects of caffeine and decreases in Mg2+ concentration were blocked by ruthenium red, tetracaine and dimethylsulfoxide. It is assumed that the decrease in Mg2+ concentration is accompanied by activation of the caffeine site of the SR, induced by the intravesicular caffeine-like factor.     

Effect of caffeine on the Ca2+-transport function of sarcoplasmic reticulum vesicles in the rat myocardium

The effects of caffeine on active transport of Ca2 by heavy and light fractions of rat myocardial microsomes were investigated with the use of a Ca2+-selective electrode and nephelometry. It was found that under the effect of caffeine (5 mM) the rate of Ca2 transport in the presence of oxalate decreased by 30 to 40%. The caffeine-induced inhibition was prevented by ruthenium and tetracaine, thus suggesting the inhibitor specificity. Since caffeine is a specific blocker of Ca2 transport to the terminal cisterns of the skeletal muscle sarcoplasmic reticulum, it is assumed that the microsomal fraction of rat myocardium contains terminal cistern fragments.     

Genomic divergence of zebu and taurine cattle identified through high-density SNP genotyping

Background

Natural selection has molded evolution across all taxa. At an arguable date of around 330,000 years ago there were already at least two different types of cattle that became ancestors of nearly all modern cattle, the Bos taurus taurus more adapted to temperate climates and the tropically adapted Bos taurus indicus. After domestication, human selection exponentially intensified these differences. To better understand the genetic differences between these subspecies and detect genomic regions potentially under divergent selection, animals from the International Bovine HapMap Experiment were genotyped for over 770,000 SNP across the genome and compared using smoothed F_ST. The taurine sample was represented by ten breeds and the contrasting zebu cohort by three breeds.

Results

Each cattle group evidenced similar numbers of polymorphic markers well distributed across the genome. Principal components analyses and unsupervised clustering confirmed the well-characterized main division of domestic cattle. The top 1% smoothed F_ST, potentially associated to positive selection, contained 48 genomic regions across 17 chromosomes. Nearly half of the top F_ST signals (n = 22) were previously detected using a lower density SNP assay. Amongst the strongest signals were the BTA7:~50 Mb and BTA14:~25 Mb; both regions harboring candidate genes and different patterns of linkage disequilibrium that potentially represent intrinsic differences between cattle types. The bottom 1% of the smoothed F_ST values, potentially associated to balancing selection, included 24 regions across 13 chromosomes. These regions often overlap with copy number variants, including the highly variable region at BTA23:~24 Mb that harbors a large number of MHC genes. Under these regions, 318 unique Ensembl genes are annotated with a significant overrepresentation of immune related pathways.

Conclusions

Genomic regions that are potentially linked to purifying or balancing selection processes in domestic cattle were identified. These regions are of particular interest to understand the natural and human selective pressures to which these subspecies were exposed to and how the genetic background of these populations evolved in response to environmental challenges and human manipulation.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-14-876) contains supplementary material, which is available to authorized users.