The effect of iron overload on rat plasma and liver oxidant status in vivo.
ABSTRACT: There is ample evidence implicating reactive oxygen species in a number of human degenerative diseases such as atherosclerosis and haemochromatosis. Although lipid peroxidation underlies many of the toxic effects of oxidative stress, there is a lack of a sensitive and reliable method for its assessment in vivo. To understand the implications of oxidative stress in vivo, we have used dietary iron overload (IO) in the rat. Oxidant status in these animals was determined by assessing depletion of endogenous antioxidants and formation of various lipid peroxidation products, including acylated F2-isoprostanes, a novel class of free-radical-derived prostaglandin-F2-like compounds. IO led to a significant decrease in the concentration of the antioxidants alpha-tocopherol and ascorbic acid in plasma, and alpha-tocopherol, beta-carotene and ubiquinol-10 in liver. Whereas there was no significant lipid peroxidation in plasma, hepatic F2-isoprostane levels were moderately but significantly increased in IO. In addition, IO caused a significant increase in plasma total and high-density lipoprotein cholesterol levels, an effect that was correlated with depletion of plasma ascorbic acid but not alpha-tocopherol. The data demonstrate that IO causes lipid metabolism disturbances and oxidative stress which is associated with substantial depletion of endogenous antioxidants and moderate lipid peroxidative damage.
Project description:Cigarette smoke (CS) is known to contain a large number of oxidants. In order to assess the oxidative effects of CS on biological fluids, we exposed human blood plasma to filtered (gas phase) and unfiltered (whole) CS, and determined the rate of utilization of endogenous antioxidants in relation to the appearance of lipid hydroperoxides. Lipid peroxidation was measured with a specific and sensitive assay that can detect lipid hydroperoxides at plasma levels as low as 10 nM. We found that exposure of plasma to the gas phase of CS, but not to whole CS, induces lipid peroxidation once endogenous ascorbic acid has been oxidized completely. In addition, CS exposure caused oxidation of plasma protein thiols and albumin-bound bilirubin, whereas uric acid and alpha-tocopherol were not consumed at significant rates. In plasma exposed to the gas phase of CS, low-density lipoprotein exhibited slightly increased electrophoretic mobility, but there was no apparent degradation of apolipoprotein B. Our results support the concept of an increased vitamin C utilization in smokers, and suggest that lipid peroxidation induced by oxidants present in the gas phase of CS leads to potentially atherogenic changes in lipoproteins.
Project description:We previously reported that probucol, a lipid lowering agent, protected mice from malaria infection via depletion in plasma α-tocopherol. The antioxidant α-tocopherol in host circulation is necessary for the malaria parasites to protect themselves from oxidative stress in erythrocytes where high amounts of reactive oxygen species are generated. To assess the potential for the clinical application of probucol as an anti-malarial therapy, it was necessary to determine the effects of probucol by using primate experiments. Here we verified that probucol induces an α-tocopherol decrement in cynomolgus macaque erythrocytes and plasma. After 2 weeks of probucol administration at doses of 200 or 400 mg/kg/day, the α-tocopherol contents in erythrocytes tended to decrease. The contents of hydroxyoctadecadienoic acids and 7β-hydroxycholesterol, peroxidation products derived from linoleic acid and cholesterol, respectively, increased in erythrocytes. On the other hand, plasma α-tocopherol concentration showed a marginal decrement. Plasma lipid peroxidation products were transiently increased in the early stages of probucol administration. No adverse effects were observed throughout the experiment, although the dosage of probucol was higher than the clinical maximum dosage. Considering that malaria proliferates in erythrocytes, probucol-induced disruption of redox homeostasis in erythrocytes could be effective in the inhibition of parasite proliferation.
Project description:While oxidative damage owing to reactive oxygen species (ROS) often increases with advancing age and is associated with many age-related diseases, its causative role in ageing is controversial. In particular, studies that have attempted to modulate ROS-induced damage, either upwards or downwards, using antioxidant or genetic approaches, generally do not show a predictable effect on lifespan. Here, we investigated whether dietary supplementation with either vitamin E (?-tocopherol) or vitamin C (ascorbic acid) affected oxidative damage and lifespan in short-tailed field voles, Microtus agrestis. We predicted that antioxidant supplementation would reduce ROS-induced oxidative damage and increase lifespan relative to unsupplemented controls. Antioxidant supplementation for nine months reduced hepatic lipid peroxidation, but DNA oxidative damage to hepatocytes and lymphocytes was unaffected. Surprisingly, antioxidant supplementation significantly shortened lifespan in voles maintained under both cold (7 ± 2°C) and warm (22 ± 2°C) conditions. These data further question the predictions of free-radical theory of ageing and critically, given our previous research in mice, indicate that similar levels of antioxidants can induce widely different interspecific effects on lifespan.
Project description:Ferritin and haemosiderin were shown, by the measurement of malondialdehyde production and loss of polyunsaturated fatty acids, to stimulate lipid peroxidation in liposomes. At pH 7.4 ascorbate was additionally required to achieve peroxidation; however, peroxidation occurred at pH 4.5 in the presence of iron-proteins alone. The damage was completely inhibited by the incorporation of chain-breaking antioxidants (alpha-tocopherol and butylated hydroxytoluene) into the liposomes. Metal chelators (desferrioxamine and EDTA) also completely inhibited lipid peroxidation. These and further results indicate that, at pH 4.5, even in the absence of a reducing agent, iron is released from haemosiderin and can mediate oxidative damage to a lipid membrane.
Project description:Cowden syndrome (CS), a Mendelian autosomal-dominant disorder, predisposes to breast, thyroid, and other cancers. Germline variations in succinate dehydrogenase genes (SDHx) occur in approximately 10% PTEN mutation-negative CS and CS-like (CSL) individuals (SDH(var+)). We previously showed that SDHx variants result in elevated reactive oxygen species (ROS), disruption of nicotinamide adenine dinucleotide (NAD) equilibrium, and destabilization of p53 hence apoptosis resistance in CS/CSL patient-derived lymphoblastoid cells. In the present study, we sought to address the tumorigenic impacts of increased ROS and the potential of protecting SDH(var+) cells with antioxidants.We measured the lipid peroxidation levels in patient-derived SDH(var+) lymphoblastoid cells and sequenced 74 controls or SDH(var+) germline DNA samples for mitochondrial hypervariable region II (HVRII) polymorphisms. SDH(var+) lymphoblastoid cells were treated with various antioxidants to check p53 expression and sub-G(1) cell population with cell-cycle analysis.We showed that elevated ROS results in higher lipid peroxidation in SDH(var+) cells. Accumulation of polymorphisms in mitochondrial HVRII was observed in SDH(var+) samples. Interestingly, ?-tocopherol (vitamin E) treatment, but not other antioxidants, rescued SDH(var+) cells from apoptosis resistance and protected SDH(var+) cells from oxidative damage such as decreased lipid peroxidation as well as partially recovered p53 expression and NAD/NADH levels.We conclude that disruption of complex II because of SDHx variants leads to increased ROS generation, specifically accompanied by lipid peroxidation. The lipid soluble antioxidant ?-tocopherol can selectively protect SDH(var+) cells from oxidative damage, apoptosis resistance, and rebalance redox metabolites NAD/NADH.
Project description:Selenocysteine (Sec) insertion sequence-binding protein 2 (SBP2) is essential for the biosynthesis of Sec-containing proteins, termed selenoproteins. Subjects with mutations in the SBP2 gene have decreased levels of several selenoproteins, resulting in a complex phenotype. Selenoproteins play a significant role in antioxidative defense, and deficiencies in these proteins can lead to increased oxidative stress. However, lipid peroxidation and the effects of antioxidants in subjects with SBP2 gene mutations have not been studied. In the present study, we evaluated the lipid peroxidation products in the blood of a subject (the proband) with mutations in the SBP2 gene. We found that the proband had higher levels of free radical-mediated lipid peroxidation products, such as 7?-hydroxycholesterol, than the control subjects. Treatment of the proband with vitamin E (?-tocopherol acetate, 100 mg/day), a lipid-soluble antioxidant, for 2 years reduced lipid peroxidation product levels to those of control subjects. Withdrawal of vitamin E treatment for 7 months resulted in an increase in lipid peroxidation products. Collectively, these results clearly indicate that free radical-mediated oxidative stress is increased in the subject with SBP2 gene mutations and that vitamin E treatment effectively inhibits the generation of lipid peroxidation products.
Project description:The plasma membrane (PM) contains redox enzymes that provide electrons for energy metabolism and recycling of antioxidants such as coenzyme Q and alpha-tocopherol. Brain aging and neurodegenerative disorders involve impaired energy metabolism and oxidative damage, but the involvement of the PM redox system (PMRS) in these processes is unknown. Caloric restriction (CR), a manipulation that protects the brain against aging and disease, increased activities of PMRS enzymes (NADH-ascorbate free radical reductase, NADH-quinone oxidoreductase 1, NADH-ferrocyanide reductase, NADH-coenzyme Q10 reductase, and NADH-cytochrome c reductase) and antioxidant levels (alpha-tocopherol and coenzyme Q10) in brain PM during aging. Age-related increases in PM lipid peroxidation, protein carbonyls, and nitrotyrosine were attenuated by CR, levels of PMRS enzyme activities were higher, and markers of oxidative stress were lower in cultured neuronal cells treated with CR serum compared with those treated with ad libitum serum. These findings suggest important roles for the PMRS in protecting brain cells against age-related increases in oxidative and metabolic stress.
Project description:During aging, oxidative stress affects the normal function of satellite cells, with consequent regeneration defects that lead to sarcopenia. This study aimed to evaluate tocotrienol-rich fraction (TRF) modulation in reestablishing the oxidative status of myoblasts during replicative senescence and to compare the effects of TRF with other antioxidants (?-tocopherol (ATF) and N-acetyl-cysteine (NAC)). Primary human myoblasts were cultured to young, presenescent, and senescent phases. The cells were treated with antioxidants for 24?h, followed by the assessment of free radical generation, lipid peroxidation, antioxidant enzyme mRNA expression and activities, and the ratio of reduced to oxidized glutathione. Our data showed that replicative senescence increased reactive oxygen species (ROS) generation and lipid peroxidation in myoblasts. Treatment with TRF significantly diminished ROS production and decreased lipid peroxidation in senescent myoblasts. Moreover, the gene expression of superoxide dismutase (SOD2), catalase (CAT), and glutathione peroxidase (GPX1) was modulated by TRF treatment, with increased activity of superoxide dismutase and catalase and reduced glutathione peroxidase in senescent myoblasts. In comparison to ATF and NAC, TRF was more efficient in heightening the antioxidant capacity and reducing free radical insults. These results suggested that TRF is able to ameliorate antioxidant defense mechanisms and improves replicative senescence-associated oxidative stress in myoblasts.
Project description:Oxidative modification of low-density lipoproteins in the arterial wall is a key feature of atherogenesis and widely believed to cause and/or accelerate lesion development. Linked to this is the expectation that vascular antioxidants are depleted during oxidation in vivo. However, whether alpha-tocopherol (vitamin E), an important lipid-soluble antioxidant, is depleted early in atherogenesis and can prevent lipid peroxidation in vivo is unresolved. To address this we examined the content of specific configurational isomers (cis/trans) of lipid hydro(pero)xides in lesions, which represent the major non-enzymic oxidation products, as formation and accumulation of cis/trans isomers is influenced by alpha-tocopherol in studies in vitro. Concordant with our previous findings that large amounts of oxidized lipid co-exist with relatively normal alpha-tocopherol levels in human lesions, we now show that cis/trans isomers predominate over other products in human carotid and aortic lesions and in lesion lipoproteins. Further, dietary vitamin E supplementation of rabbits after arterial injury significantly increases both the aortic levels of alpha-tocopherol and the overall content of cis/trans isomers. These data are fully consistent with alpha-tocopherol acting as a hydrogen donor during lipid oxidation in vivo and suggest that alpha-tocopherol does not prevent lipoprotein lipid oxidation in the diseased vessel wall.
Project description:The biomarker 8-iso-prostaglandin F2? (8-iso-PGF2?) is regarded as the gold standard for detection of excessive chemical lipid peroxidation in humans. However, biosynthesis of 8-iso-PGF2? via enzymatic lipid peroxidation by prostaglandin-endoperoxide synthases (PGHSs), which are significantly induced in inflammation, could lead to incorrect biomarker interpretation. To resolve the ambiguity with this biomarker, the ratio of 8-iso-PGF2? to prostaglandin F2? (PGF2?) is established as a quantitative measure to distinguish enzymatic from chemical lipid peroxidation in vitro, in animal models, and in humans. Using this method, we find that chemical lipid peroxidation contributes only 3% to the total 8-iso-PGF2? in the plasma of rats. In contrast, the 8-iso-PGF2? levels in plasma of human males are generated >99% by chemical lipid peroxidation. This establishes the potential for an alternate pathway of biomarker synthesis, and draws into question the source of increases in 8-iso-PGF2? seen in many human diseases. In conclusion, increases in 8-iso-PGF2? do not necessarily reflect increases in oxidative stress; therefore, past studies using 8-iso-PGF2? as a marker of oxidative stress may have been misinterpreted. The 8-iso-PGF2?/PGF2? ratio can be used to distinguish biomarker synthesis pathways and thus confirm the potential change in oxidative stress in the myriad of disease and chemical exposures known to induce 8-iso-PGF2?.