Hydroxyl radical formation and lipid peroxidation enhancement by chromium

Chromium VI compounds have been shown to be carcinogenic in occupationally exposed humans, and to be genotoxic, mutagenic, and carcinogenic in a variety of experimental systems. In contrast, most chromium III compounds are relatively nontoxic, noncarcinogenic, and nonmutagenic. Reduction of Cr⁶⁺ leads to reactive intermediates, such as Cr⁵⁺, Cr⁴⁺, or other radical species. The molecular mechanism for the intracellular Cr⁶⁺ reduction has been the focus of recent studies, but the details are still not understood. Our study was initiated to compare the effect of Cr⁶⁺-hydroxyl radical formation and Cr⁶⁺-induced lipid peroxidation vs those of Cr³⁺. Electron spin responance measurements provide evidence for the formation of long-lived Cr⁵⁺ intermediates in the reduction of Cr⁶⁺ by glutathione reductase in the presence of NADPH and for the hydroxyl radical formation during the glutathione reductase catalyzed reduction of Cr⁶⁺. Hydrogen peroxide suppresses Cr⁵⁺ and enhances the formation of hydroxyl radical. Thus, Cr⁵⁺ intermediates catalyze generation of hydroxyl radicals from hydrogen peroxide through a Fenton-like reaction. Comparative effects of Cr⁶⁺ and Cr³⁺ on the development of lipid peroxidation were studied by using rat heart homogenate. Heart homogenate was incubated with different concentrations of Cr⁶⁺ compounds at 22°C for 60 min. Lipid peroxidation was determined as thiobarbituric acid reacting materiels (TBA-RM). The results confirm that Cr⁶⁺ induces lipid peroxidation in the rat heart homogenate. These observations might suggest a possible causative role of lipid peroxidation in Cr⁶⁺ toxicity. This enhancement of lipid peroxidation is modified by the addition of some metal chelators and antioxidants. Thus, strategies for combating Cr⁶⁺ toxicity should take into account the role of the hydroxy radicals, and hence, steps for blocking its chain propagation and preventing the formation of lipid peroxides.     

Increase in free radical generation and lipid peroxidation following chemotherapy in patients with cancer

Several anti-cancer drugs are known to bring about their tumoricidal actions by a free radical dependent mechanism. Majority of the studies reported that adriamycin, mitomycin C, bleomycin, etc., augment free radical generation and lipid peroxidation process in vitro. Our results reported here suggest that following chemotherapy both stimulated and unstimulated human polymorphonuclear leukocytes generate increased amounts of superoxide anion and hydrogen peroxide. This was accompanied by increased formation of lipid peroxidation products as measured by thiobarbituric acid assay. These results confirm that many anti-cancer drugs augment free radical generation and lipid peroxidation even in an vivo situation.     

Paraquat- and diquat-induced oxygen radical generation and lipid peroxidation in rat brain microsomes

NADPH-menadione reductase activity by rat brain microsomes (Ms) was decreased 40-50% by 10 microM dicumarol, a potent inhibitor of DT-diaphorase, whereas no change in NADPH-paraquat (PQ) and -diquat (DQ) reductase activity was observed. NADPH-DQ reductase activity in brain Ms was 2.5-fold higher than NADPH-PQ reductase activity. The formation of PQ and DQ radicals was verified optically and observed directly by ESR spectroscopy in the NADPH-PQ and -DQ reductase reactions by brain Ms under anaerobic conditions. PQ- and DQ-induced superoxide formation was confirmed by the detection of DMPO-OOH ESR signals and followed by chemiluminescence (CL) of a Cypridina luciferin analogue (CLA). The kinetics and intensity of the CL were consistent with the observations that the reduction in DQ is faster than that in PQ. Thiobarbituric acid reactive substances (TBARS) and phospholipid hydroperoxides in brain Ms increased in the presence of NADPH and Fe3+. The generation of both lipid peroxidation products derived from brain Ms decreased with increasing concentrations of PQ and DQ. The inhibitory effect of DQ is more pronounced than that of PQ. The formation of PQ- and DQ-induced reactive oxygen species was not associated with lipid peroxidation in rat brain Ms.     

Ferritin and haemosiderin in free radical generation, lipid peroxidation and protein damage

Iron storage proteins, ferritin and haemosiderin, release iron to a range of chelators and reducing agents, including citrate, acetate and ascorbate. Released iron promotes both hydroxyl radical formation in the presence of hydrogen peroxide and lipid peroxidation in liposomes. Ferritin protein is modified in such reactions, both by free radical cleavage and addition reactions with aldehyde products of lipid peroxidation.     

Free radical generation, lipid peroxidation and essential fatty acids in uncontrolled essential hypertension

Vascular endothelium produces prostacyclin (PG12) and endothelium-derived vascular relaxing factor (EDRF), which are potent vasodilators and hence, may have a role in the regulation of blood pressure. Both PG12 and EDRF are readily degraded by free radicals, especially superoxide anion. Hence, we studied free radical generation and lipid peroxidation in patients with uncontrolled essential hypertension. It was observed that superoxide anion and hydrogen peroxide production by polymorphonuclear leukocytes (PMN) and the levels of lipid peroxides (measured by thiobarbituric acid assay) were higher in uncontrolled hypertensives compared to controls. Both free radical generation and the levels of lipid peroxides reverted to normal values when assayed after the control of hypertension. The calcium antagonist, verapamil, and beta-1 blocker, metoprolol, at the doses used inhibited free radical generation by phorbolmyristate acetate-stimulated PMNs. On the other hand, angiotensin II augmented free radical generation in normal PMN. In addition, it was also observed that both linoleic acid and arachidonic acid levels are low in the plasma of patients with hypertension compared to controls. These results suggest that increase in free radical generation by PMN and alterations in the plasma concentrations of essential fatty acids are closely associated with uncontrolled hypertension.     

Nicotine decreases agrin signaling and acetylcholine receptor clustering in C2C12 myotube culture

The clustering of acetylcholine receptors (AChRs) in skeletal muscle fibers is a critical event in neuromuscular synaptogenesis. AChRs in concert with other molecules form postsynaptic scaffolds in response to agrin released from motor neurons as motor neurons near skeletal muscle fibers in development. Agrin drives an intracellular signaling pathway that precedes AChR clustering and includes the tyrosine phosphorylation of AChRs. In C2C12 myotube culture, agrin application stimulates the agrin signaling pathway and AChR clustering. Previous studies have determined that the frequency of spontaneous AChR clustering is decreased and AChRs are partially inactivated when bound by the acetylcholine agonist nicotine. We hypothesized that nicotine interferes with AChR clustering and consequent postsynaptic scaffold formation. In the present study, C2C12 myoblasts were cultured with growth medium to stimulate proliferation and then differentiation medium to stimulate fusion into myotubes. They were bathed in a physiologically relevant concentration of nicotine and then subject to agrin treatment after myotube formation. Our results demonstrate that nicotine decreases agrin-induced tyrosine phosphorylation of AChRs and decreases the frequency of spontaneous as well as agrin-induced AChR clustering. We conclude that nicotine interferes with postsynaptic scaffold formation by preventing the tyrosine phosphorylation of AChRs, an agrin signaling event that precedes AChR clustering.     

Contractile C2C12 myotube model for studying exercise-inducible responses in skeletal muscle

Adequate exercise leads to a vast variety of physiological changes in skeletal muscle as well as other tissues/organs and is also responsible for maintaining healthy muscle displaying enhanced insulin-responsive glucose uptake via GLUT4 translocation. We generated highly developed contractile C(2)C(12) myotubes by manipulating intracellular Ca(2+) transients with electric pulse stimulation (EPS) that is endowed with properties similar to those of in vivo skeletal muscle in terms of 1) excitation-induced contractile activity as a result of de novo sarcomere formation, 2) activation of both the AMP kinase and stress-activated MAP kinase cascades, and 3) improved insulin responsiveness as assessed by GLUT4 recycling. Tbc1d1, a Rab-GAP implicated in exercise-induced GLUT4 translocation in skeletal muscle, also appeared to be phosphorylated on Ser(231) after EPS-induced contraction. In addition, a switch in myosin heavy-chain (MHC) expression from "fast type" to "slow type" was observed in the C(2)C(12) myotubes endowed with EPS-induced repetitive contractility. Taking advantage of these highly developed contractile C(2)C(12) myotubes, we identified myotube-derived factors responsive to EPS-evoked contraction, including the CXC chemokines CXCL1/KC and CXCL5/LIX, as well as IL-6, previously reported to be upregulated in contracting muscles in vivo. Importantly, animal treadmill experiments revealed that exercise significantly increased systemic levels of CXCL1/KC, perhaps derived from contracting muscle. Taken together, these results confirm that we have established a specialized muscle cell culture model allowing contraction-inducible cellular responses to be explored. Utilizing this model, we identified contraction-inducible myokines potentially linked to the metabolic alterations, immune responses, and angiogenesis induced by exercise.     

Permeability of C2C12 myotube membranes is influenced by stretch velocity

Mechanical signals are critical to the growth and maintenance of skeletal muscle, but the mechanism by which these signals are transduced by the cell remains unknown. This work examined the hypothesis that stretch conditions influence membrane permeability consistent with a role for membrane permeability in mechanotransduction. C2C12 myotubes were grown in conditions that encourage uniform alignment and subjected to uniform mechanical deformation in the presence of fluorescein labeled dextran to evaluate membrane permeability as a function of stretch amplitude and velocity. Within a physiologically relevant range of conditions, a complex interaction between the two aspects of stretch was observed, with velocity contributing most strongly at large stretch amplitudes. This suggests that membrane viscosity could contribute to mechanotransduction.     

Hydroxyl radical generation by postischemic rat kidney slices in vitro

To quantitate the formation of hydroxyl radicals (HO.) in ischemia and reoxygenation, dimethyl sulfoxide (DMSO) was added to "trap" evolving HO. in normal, in ischemic, and in ischemic and reoxygenated rat kidney slices, incubated in short-term organ culture in vitro. Hydroxyl radical generation was measured as the accumulation of the specific product of DMSO oxidation by HO., methane sulfinic acid (MSA) in the kidney tissue and surrounding medium using a new colorimetric assay. A mean difference of 7 nmol cumulative HO./gram tissue was detected in rat kidney slices subjected to ischemia and reoxygenation. This amount of HO. generation was not significantly greater than that found in nonischemic or in ischemic but not reoxygenated control tissues, and does not appear to represent the highly toxic burst of HO. radicals implied in current theoretical discussions of reperfusion injury. However, the addition of EDTA chelated iron (1:1) to the incubation medium led to marked postischemic HO. generation. We conclude that clearly toxic numbers of HO. radicals are not formed during reoxygenation in rat kidney slices, either because there is insufficient iron, because only a small fraction of cells in the kidney tissue make oxygen radicals, or because cellular defenses against HO. formation are more powerful than currently appreciated.     

Hypomorphic Smn knockdown C2C12 myoblasts reveal intrinsic defects in myoblast fusion and myotube morphology

Dosage of the survival motor neuron (SMN) protein has been directly correlated with the severity of disease in patients diagnosed with spinal muscular atrophy (SMA). It is also clear that SMA is a neurodegenerative disorder characterized by the degeneration of the alpha-motor neurons in the anterior horn of the spinal cord and atrophy of the associated skeletal muscle. What is more controversial is whether it is neuronal and/or muscle-cell-autonomous defects that are responsible for the disease per se. Although motor neuron degeneration is generally accepted as the primary event in SMA, intrinsic muscle defects in this disease have not been ruled out. To gain a better understanding of the influence of SMN protein dosage in muscle, we have generated a hypomorphic series of myoblast (C2C12) stable cell lines with variable Smn knockdown. We show that depletion of Smn in these cells resulted in a decrease in the number of nuclear 'gems' (gemini of coiled bodies), reduced proliferation with no increase in cell death, defects in myoblast fusion, and malformed myotubes. Importantly, the severity of these abnormalities is directly correlated with the decrease in Smn dosage. Taken together, our work supports the view that there is an intrinsic defect in skeletal muscle cells of SMA patients and that this defect contributes to the overall pathogenesis in this devastating disease.     

Oxygen concentration dependence of lipid peroxidation and lipid-derived radical generation: application of profluorescent nitroxide switch

Lipid-derived radicals and peroxides are involved in the pathogenesis of oxidative stress diseases and, although lipid peroxide production is a required reaction between a lipid radical and molecular oxygen, a useful lipid radical detection method has remained tentative. Also, the effect of oxygen concentration on lipid peroxide production must be considered because of the hypoxic conditions in cancer and ischemic regions. In this study, the focus was on nitroxide reactivity, which allows spin trapping with carbon-centred radicals via radical-radical reactions and fluorophore quenching through interactions with nitroxide's unpaired electron. Thus, the aim here was to demonstrate a useful detection method for lipid-derived radicals as well as to clarify the effects of oxygen concentration on lipid peroxide production using profluorescent nitroxide. This latter compound reacted with lipid-derived radicals in a manner inversely dependent on oxygen concentration, resulting in fluorescence due to alkoxyamine formation and, conversely, lipid peroxide concentrations decreased with lower oxygen in the reaction system. Furthermore, nitroxide inhibited lipid peroxide production and stopped oxygen consumption in the same solution. These results suggested that the novel application of profluorescent nitroxide could directly and sensitively detect lipid-derived radicals and that radical and peroxide production were dependent on oxygen concentration.     

Oxygen concentration dependence of lipid peroxidation and lipid-derived radical generation: Application of profluorescent nitroxide switch

Abstract

Lipid-derived radicals and peroxides are involved in the pathogenesis of oxidative stress diseases and, although lipid peroxide production is a required reaction between a lipid radical and molecular oxygen, a useful lipid radical detection method has remained tentative. Also, the effect of oxygen concentration on lipid peroxide production must be considered because of the hypoxic conditions in cancer and ischemic regions. In this study, the focus was on nitroxide reactivity, which allows spin trapping with carbon-centred radicals via radical–radical reactions and fluorophore quenching through interactions with nitroxide's unpaired electron. Thus, the aim here was to demonstrate a useful detection method for lipid-derived radicals as well as to clarify the effects of oxygen concentration on lipid peroxide production using profluorescent nitroxide. This latter compound reacted with lipid-derived radicals in a manner inversely dependent on oxygen concentration, resulting in fluorescence due to alkoxyamine formation and, conversely, lipid peroxide concentrations decreased with lower oxygen in the reaction system. Furthermore, nitroxide inhibited lipid peroxide production and stopped oxygen consumption in the same solution. These results suggested that the novel application of profluorescent nitroxide could directly and sensitively detect lipid-derived radicals and that radical and peroxide production were dependent on oxygen concentration.     

Effects of dietary zinc on free radical generation, lipid peroxidation, and superoxide dismutase in trained mice.

The purpose of this study was to investigate the effects of dietary zinc on free radical generation, lipid peroxidation, and superoxide dismutase (SOD) in exercised mice. In the first part of the study, 48 male weanling mice were randomly divided into three groups. They were fed a zinc-deficient diet containing 1.6 mg/kg zinc or were pair-fed or fed ad libitum a zinc-adequate diet supplemented with 50 mg/kg zinc. Half of each group received an exercise training program that consisted of swimming for 60 min per day in deionized water. The diets and exercise program persisted for 6 weeks. In the second part of the study, 64 mice were fed zinc-deficient diets for 6 weeks, and then one group was fed the zinc-deficient diet for an additional 3 weeks, and the other three groups were fed diets supplemented with 5, 50, and 500 mg/kg zinc, respectively. Half of each group also received the exercise program. Both blood and liver samples were examined. Free radicals in liver were directly detected by electron spin resonance techniques and the extent of lipid peroxidation was indicated by malonic dialdehyde (MDA). Both CuZn-SOD and Mn-SOD were measured. The results showed that exercise training increased the metabolism of zinc, and zinc deficiency induced an increased free radical generation and lipid peroxidation and a decreased hepatic CuZn-SOD activity in exercised mice. Furthermore, although exercise training had no effect on the level of free radicals in zinc-adequate mice, it could increase the hepatic mitochondrial MDA formation further in zinc-deficient animals and zinc deficiency would eliminate the exercise-induced increase in SOD activities which existed in zinc-adequate mice. A total of 50 mg/kg zinc supplemented in the diet was adequate to correct the zinc-deficient status in exercised mice while 5 mg/kg zinc had a satisfactory effect on the recovery of only sedentary zinc-deficient mice. However, 500 mg/kg zinc had a harmful effect on both sedentary and exercised zinc-deficient animals.     

The role of iron in oxygen radical mediated lipid peroxidation

The role of iron in the peroxidation of polyunsaturated fatty acids is reviewed, especially with respect to the involvement of oxygen radicals. The hydroxyl radical can be generated by a superoxide-driven Haber-Weiss reaction or by Fenton's reaction; and the hydroxyl radical can initiate lipid peroxidation. However, lipid peroxidation is frequently insensitive to hydroxyl radical scavengers or superoxide dismutase. We propose that the hydroxyl radical may not be involved in the peroxidation of membrane lipids, but instead lipid peroxidation requires both Fe2+ and Fe3+. The inability of superoxide dismutase to affect lipid peroxidation can be explained by the fact that the direct reduction of iron can occur, exemplified by rat liver microsomal NADPH-dependent lipid peroxidation. Catalase can be stimulatory, inhibitory or without affect because H2O2 may oxidize some Fe2+ to form the required Fe3+, or, alternatively, excess H2O2 may inhibit by excessive oxidation of the Fe2+. In an analogous manner reductants can form the initiating complex by reduction of Fe3+, but complete reduction would inhibit lipid peroxidation. All of these redox reactions would be influenced by iron chelation.     

Vaccinium ashei leaves extract alleviates insulin resistance via AMPK independent pathway in C2C12 myotube model

Blueberry (Vaccinium ashei) leaf has recently attracted attention as an anti-obesity food component. In this study, we examined the effects of blueberry leaf extract (BLEx) on insulin signaling in C2C12 differentiated myoblasts. The results showed that BLEx promotes the intracellular uptake of 2-[N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)amino]?2-deoxy-D-glucose (2-NBDG) and phosphorylation of Akt under insulin stimulation. In addition, pretreatment with BLEx ameliorated TNF-α-nduced insulin resistance with regard to 2-NBDG uptake and Akt phosphorylation. Moreover, BLEx prevented the TNF-α?induced activation of JNK and NF-kB pathways and phosphorylation of IRS-1 at serine residue. BLEx failed to induce phosphorylation of AMPK as well as did not prevent the restoration of 2-NBDG uptake under TNF-α–induced insulin resistance. Overall, skeletal muscle is a putative target for the anti-diabetic effect of BLEx by amelioration of insulin resistance.     

Glucosamine induces lipid accumulation and adipogenic change in C2C12 myoblasts

Hyperglycemia-induced activation of hexosamine biosynthesis pathway (HBP) has been implicated in the development of insulin resistance in skeletal muscles. In the present study, the content of uridine-5'-diphospho-N-acetylglucosamine, the end product of the HBP, was elevated in skeletal muscle of obese diabetic KKA(y) mice, compared with control mice. To elucidate the effect of elevated HBP in the skeletal muscle, we treated C2C12 myoblasts with glucosamine, an intermediate metabolite of the HBP. Glucosamine induced lipid accumulation and significantly increased the mRNA expression levels of peroxisome proliferator-activated receptor gamma, adiponectin, and aP2 in C2C12 myoblasts. Similar mRNA changes were observed in skeletal muscles of Sprague-Dawley rats treated with glucosamine infusion. Our results provide a possible explanation of hyperglycemia-induced insulin resistance in skeletal muscle.     

Superoxide anion generation and lipid peroxidation processes during hemodialysis with reused cuprophan dialyzers

In 11 chronic uremic patients superoxide anion (O2-.) generation and lipid peroxidation processes were determined during hemodialyses with cuprophan dialyzers used three times. Intradialytic changes observed during the first 20 min of hemodialysis, that is the period of the most marked alterations, with consecutive uses of the same dialyzer included: decreased whole blood O2-. generation at rest and following stimulation with opsonized zymosan, decreased reductions in the erythrocyte superoxide dismutase (SOD-1) activity, increased reductions in the plasma malonyldialdehyde (MDA) concentrations, and unchanged erythrocyte MDA concentrations. However, immediately before hemodialysis with third-used dialyzer whole blood O2-. generation at rest and following opsonized-zymosan stimulation, and erythrocyte SOD-1 activity were slightly, but statistically significantly, lower (p less than 0.05), while plasma MDA concentrations were higher (p less than 0.05) than those before hemodialysis with first-used dialyzer; erythrocyte MDA concentrations remained unchanged. The results seem to indicate that dialyzer reuse may exert beneficial effects on whole blood O2-. generation and protect erythrocyte membrane lipids from peroxidation, but, however, it leads to slightly increasing predialysis plasma lipid peroxidation.     

Hydroxyl radical generation by oligonucleotide derivatives of anthracycline antibiotic and synthetic quinone.

For the first time the covalent binding of anticancer anthracycline drugs and their potential synthetic analogs to oligonucleotides of different sequences is proposed for obtaining site-specific DNA scission in systems in vitro and in vivo. New compounds such as daunomycin (Dm) and synthetic naphthoquinone (NQ), covalently bound to the heptadeoxynucleotide of pCCAAACA (Dm-pN7) and decadeoxythymidilate (pT10p-NQ), have been obtained. These oligonucleotide derivatives can form specific complexes with complementary oligonucleotide sequences; these compounds and their complementary complexes can be reduced by purified NADPH-cytochrome P-450 reductase. Using the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO), it has been shown that in aerobic conditions Dm-pN7 and pT10p-NQ are capable of generating OH radicals with and without complementary oligonucleotides. The chemical stability of the compounds in redox reactions has been studied. Oligonucleotide derivatives of natural and synthetic quinones capable of generating OH radicals seem to be a promising tool for site-specific scission of DNA in solution and in cells.