Ethanol-Induced Cell Death by Lipid Peroxidation in PC12 Cells

Free radical generation is hypothesized to be the cause of alcohol-induced tissue injury. Using fluorescent cis-parinaric acid and TBARS, lipid peroxidation was shown to be increased in the presence of trace amounts of free ferrous ion in PC12 cells. This increase in lipid peroxidation was enhanced by ethanol in a dose dependent manner and also correlated with loss of cell viability, as measured by increased release of lactate dehydrogenase (LDH). Resveratrol, a potent antioxidant, had a protective effect against lipid peroxidation and cell death. These findings strongly suggest that ethanol-induced tissue injury and cell death is a free radical mediated process, and may be important in alcohol-related premature aging and other degenerative diseases.     

Melatonin exerts a more potent effect than S-adenosyl-l-methionine against iron metabolism disturbances, oxidative stress and tissue injury induced by obstructive jaundice in rats

Melatonin and S-adenosyl-l-methionine (SAMe) prevent oxidative stress and tissue dysfunction in obstructive jaundice (OJ). Lipid peroxidation is exacerbated in the presence of trace amounts of iron (Fe). The study investigated the regulation by melatonin and SAMe the induction of oxidative stress, iron metabolism disturbances and tissue injury in an experimental model of OJ. Different parameters of lipid peroxidation, antioxidant status, tissue injury and Fe metabolism were determined in liver and blood. OJ induced Fe accumulation in liver, and increased transferrin (Tf) saturation and loosely bound Fe content in blood. Melatonin, and SAMe at lesser extent, enhanced protein Tf content in liver and blood, that reduced loosely bound Fe content in blood. Melatonin and SAMe did not affect ferritin (FT) and Tf mRNA expression, but reduced Tf receptor (TfR) mRNA expression in liver. In conclusion, the effect of melatonin and SAMe on Fe metabolism may be included in the beneficial properties of these agents on lipid peroxidation and tissue injury induced by OJ.     

Asbestos-catalysed lipid peroxidation and its inhibition by desferroxamine.

In an effort to understand the properties of asbestos fibres that might contribute to their being toxic, we incubated three different varieties of asbestos with phospholipid emulsions and looked for evidence of lipid peroxidation. Although all three types of asbestos were able to catalyse lipid peroxidation in the native state, this catalytic activity was inhibited by pre-washing of the asbestos with the iron chelator desferroxamine. This suggests that: lipid peroxidation may be one of the mechanisms by which asbestos produces tissue injury, and treatment with iron chelators might diminish the potential to produce this injury.     

Usual and Unusual Methods for Detection of Lipid Peroxides as Indicators of Tissue Injury in Cerebral Ischemia: What Is Appropriate and Useful?

1. Free radical-dependent lipid peroxidation processes have long been thought to contribute to brain damage following stroke or cerebral ischemia/reperfusion.2. The preponderance of evidence for this belief has been derived indirectly, through diminution of tissue injury indices (e.g., brain infarct volume) facilitated by application of free radical scavenger substances.3. Direct, unequivocal evidence for lipid peroxidation in terms of classical assays (detection of conjugated diene absorbance or thiobarbituric acid-reactive substances) is considerably less common, and its validity can be questioned.4. Correlations of treatment-induced diminishment of brain injury indices with reductions in lipid peroxidation level are rarer still.5. Reasons underlying the disparity between the belief that lipid peroxidation contributes to ischemic brain injury and direct evidence for this contribution (at least acutely) are proposed, along with evidence that new methods are being developed which should provide the basis for obtaining a definitive answer.     

Lipid peroxidation: a mechanism for alcohol-induced testicular injury

That alcohol abuse may lead to testicular lipid peroxidation is suggested by the fact that ethanol is a known testicular toxin and its chronic use leads to both endocrine and reproductive failure. Because testicular membranes are rich in polyenoic fatty acids that are prone to undergo peroxidative decomposition, it is reasonable to consider that lipid peroxidation may contribute to the membrane injury and gonadal dysfunction that occurs as a result of alcohol abuse and/or chronic use. The present report reviews the studies supporting the concept that testicular lipid peroxidation is a metabolic consequence of chronic alcohol administration to animals and that its presence correlates with the gonadal injury present in animals ingesting ethanol for prolonged periods. Consistent with such a mechanism for putative alcohol-associated testicular toxicity are the observed reductions in the testicular content of polyenoic fatty acids and glutathione (GSH) content of the testes of alcohol-fed animals as compared to isocalorically fed controls. The later finding demonstrates that ethanol modifies the precarious antioxidant balance of testicular tissue such that enhanced peroxidation can occur. It is well known that peroxidation injury can be attenuated when it occurs in association with dietary vitamin A supplementation. Thus, it is of interest to note that vitamin A, acting as an antioxidant, stabilizes testicular membranes by reducing lipid peroxidation and prevents the alcohol-induced atrophy that occurs in animals not receiving vitamin-A-enriched diets. Taken together, these observations suggest that the enhanced peroxidation of testicular lipids that occurs following ethanol exposure may be an important factor in the pathogenesis of alcohol-associated gonadal injury.     

Protection from nonfreezing cold injury by quinacrine, a phospholipase inhibitor

A recent study from our laboratory indicated additional tissue injury during rewarming of a cooled rabbit leg. Oxygen-derived free radicals were believed to play a role in such "rewarming injury." Since free radicals may attack membrane phospholipids, we analyzed the phospholipid composition in the leg tissue during cooling and rewarming. Our results indicated significant breakdown of membrane phospholipids, particularly phosphatidylcholine and phosphatidylethanolamine, with a corresponding accumulation of lysophosphatidylcholine and nonesterified fatty acids. Quinacrine, a phospholipase inhibitor, was able to preserve membrane phospholipids during rewarming of the cooled leg. Rewarming of cooled tissue was also accompanied by additional tissue injury, as evidenced by the increased release of lactic acid dehydrogenase and creatine kinase, as well as enhanced lipid peroxidation, as evidenced by increased malonaldehyde formation. Quinacrine reduced the release of these intracellular enzymes and decreased lipid peroxidation, suggesting its efficacy as a therapeutic agent against hypothermic injury.     

L-carnitine protects gastric mucosa by decreasing ischemia-reperfusion induced lipid peroxidation.

Studies have shown that reactive oxygen metabolites and lipid peroxidation play important roles in ischemia-reperfusion injury in many organs such as heart, brain and stomach. The aim of this study is to evaluate the antioxidant effect of L-carnitine on gastric mucosal barrier, lipid peroxidation and the activities of antioxidant enzymes in rat gastric mucosa subjected to ischemia-reperfusion injury. Rats were subjected to 30 min of ischemia followed by 60 min of reperfusion. L-carnitine (100 mg/kg), was given to rats intravenously five minutes before the ischemia. In our experiment, lesion index, thiobarbituric acid reactive substances, prostaglandin E2 and mucus content in gastric tissue were measured. The results indicated that the lesion index and the formation of thiobarbituric acid reactive substances increased significantly with the ischemia-reperfusion injury in the gastric mucosa. L-carnitine treatment reduced these parameters to the values of sham operated rats. The tissue catalase and superoxide dismutase activities and prostaglandin E2 production decreased significantly in the gastric mucosa of rats exposed to ischemia-reperfusion. L-carnitine pretreatment increased the tissue catalase activity and prostaglandin E2 to the levels of sham-operated rats but did not change superoxide dismutase activity. There were no significant difference in glutathione peroxidase activity and mucus content between the groups in the gastric mucosa. In summary, L-carnitine pretreatment protected gastric mucosa from ischemia-reperfusion injury by its decreasing effect on lipid peroxidation and by preventing the decrease in prostaglandin E2 content of gastric mucosa.     

Ischemic myocardial damage in spontaneously hypertensive rats (SHR) is enhanced after long-term feeding of an alpha-linolenic acid enriched diet.

In this study the influence of long-term feeding of an alpha-linolenic acid (LNA) enriched diet on the sensitivity of SHR to catecholamine-induced myocardial injury was investigated. An enhanced ischemic vulnerability after LNA supplementation was observed as indicated both by a marked decrease of enzyme activities in the myocardium and by a pronounced elevation of plasma enzymes. Distinctly higher TBARS levels in heart tissue and plasma of LNA rich fed SHR suggest that an exaggerated lipid peroxidation might contribute to the increased ischemic vulnerability. Non-enzymatic lipid peroxidation is favoured by a feeding-provoked enrichment in highly unsaturated fatty acids in tissue phospholipids. Under such conditions of enhanced substrate availability for radical-induced lipid peroxidation an increased requirement for antioxidants can be assumed which might not sufficiently be met by tocopherol-supplementation in SHR because of their known defects in antioxidative defense mechanisms.     

Oxidants and the pathophysiology of burn and smoke inhalation injury.

A skin burn is a common traumatic injury that results in both local tissue damage and a systemic mediator-induced response. There is evidence of both local and systemic oxidant changes manifested by lipid peroxidation in animal burn models and also in burned man. Both increased xanthine oxidase and neutrophil activation appear to be the oxidant sources. Animal studies have also demonstrated decreased burn edema, and also decreased distant organ dysfunction with the use of antioxidants, suggesting a cause-and-effect relationship, which needs to be tested in man. Smoke inhalation injury, a chemical injury to the airways caused by incomplete products of combustion, is frequently seen in conjunction with a body burn. Lipid peroxidation, both in lung and in distant organs, is also seen with this injury. The combined body burn and smoke inhalation injury lead to a marked increase in mortality rate and also an increase in the degree of generalized oxidant release and lipid peroxidation. Although data in man are limited, the available information, along with that from animal research on burns and smoke inhalation, indicates oxidants may well play a key role, and antioxidants may be of clinical therapeutic use.     

Glyceryl trinitrate, a nitric oxide donor, suppresses renal oxidant damage caused by potassium bromate

Nitric oxide (NO) is a short-lived, readily diffusible intracellular messenger molecule associated with multiple organ-specific regulatory functions. Endogenous stimulation or exogenous administration of NO have been shown to inhibit production of reactive oxygen species (ROS) and expression of oxidant-mediated molecular or tissue injury. Potassium bromate (KBrO3) is one such potent renal oxidant that acts through generation of ROS-mediated lipid peroxidation, and causes increased ornithine decarboxylase activity, enhanced rate of DNA synthesis and depletion of the antioxidant armoury of the tissue. In this study, we elucidate the effect of exogenous NO administration, using the NO donor glyceryl trinitrate (GTN), on KBrO3-induced nephrotoxicity, oxidative stress and cell proliferation. KBrO3 administration at a dose of 125 mg/kg body weight results in significant (P < 0.001) depletion in renal glutathione (GSH) content, and glutathione reductase (GR) activity with a concomitant increase in microsomal lipid peroxidation, and blood urea nitrogen (BUN) and creatinine levels. Parallel to these changes, we found significant enhancement in ornithine decarboxylase (ODC) activity and rate of renal DNA synthesis. Subsequent administration of GTN resulted in dose-dependent amelioration of GSH content and GR activity with concomitant inhibition of lipid peroxidation, and BUN and creatinine levels. In addition, GTN administration to KBrO3-intoxicated rats resulted in significant dose-dependent down regulation of enhanced ODC activity and rate of [3H]-thymidine incorporation in renal DNA, providing support for the protective role of NO in attenuation of KBrO3-induced oxidative stress and cell proliferation. Enhancement of oxidative tissue injury and cell proliferation on administration of the NO inhibitor, L-NAME, further demonstrates the protective efficacy of endogenous NO. These data suggest that NO inhibits KBrO3-induced tissue injury, oxidative stress and proliferative response in the rat kidney.     

Endotoxemia causes increased lung tissue lipid peroxidation in unanesthetized sheep

Our purpose was to determine whether lipid peroxidation of lung tissue, a reflection of O2 radical injury, occurs with endotoxin, and whether the degree of tissue change corresponds with the degree of increased protein permeability. Unanesthetized adult sheep with lung lymph fistulas were given Escherichia coli endotoxin at a dose of 2 micrograms/kg (n = 34). Tissue lipid peroxidation was measured using the thiobarbituric acid assay for malondialdehyde (MDA). The MDA content of lung tissue in nanomoles per gram increased from a control value of 48 +/- 8 to 98 +/- 18 at 5 h postendotoxin (2 micrograms/kg), whereas lung lymph protein transport (Cp), was increased 3- to 4-fold. The MDA content returned to base line with Cp by 24 h postendotoxin. Six sheep given endotoxin were pretreated with 12.5 mg/kg of ibuprofen, and six were infused with dimethylthiourea (DMTU) 0.75 g/kg. With ibuprofen, Cp was only increased 2.5- to 3-fold and MDA was increased to 69 +/- 15 nmol/g. With DMTU, the increase in Cp was comparable to that with endotoxin alone, as was the MDA of lung tissue with a value of 92 +/- 12 nmol/g. The correlation of tissue MDA with Cp in all animals was 0.83. We conclude that lipid peroxidation occurs in lung tissue after a moderately severe endotoxin injury with the degree of change corresponding to the degree of increased Cp.     

Modulation of cyclophosphamide-induced early lung injury by curcumin,an anti-inflammatory antioxidant

Cyclophosphamide causes lung injury in rats through its ability to generate free radicals with subsequent endothelial and epithelial cell damage. In order to observe the protective effects of a potent anti-inflammatory antioxidant, curcumin (diferuloyl methane) on cyclophosphamide-induced early lung injury, healthy pathogen free male Wistar rats were exposed to 20 mg/100 g body weight of cyclophosphamide, intraperitoneally as a single injection. Prior to cyclophosphamide intoxication oral administration of curcumin was performed daily for 7 days. At various time intervals (2, 3, 5 and 7 days post insult) serum and lung samples were analyzed for angiotensin converting enzyme, lipid peroxidation, reduced glutathione and ascorbic acid. Bronchoalveolar lavage fluid was analyzed for biochemical constituents. The lavage cells were examined for lipid peroxidation and glutathione content. Excised lungs were analyzed for antioxidant enzyme levels. Biochemical analyses revealed time course increases in lavage fluid total protein, albumin, angiotensin converting enzyme (ACE), lactate dehydrogenase, N-acetyl--D-glucosaminidase, alkaline phosphatase, acid phosphatase, lipid peroxide levels and decreased levels of glutathione (GSH) and ascorbic acid 2, 3, 5 and 7 days after cyclophosphamide intoxication. Increased levels of lipid peroxidation and decreased levels of glutathione and ascorbic acid were seen in serum, lung tissue and lavage cells of cyclophosphamide groups. Serum angiotensin converting enzyme activity increased which coincided with the decrease in lung tissue levels. Activities of antioxidant enzymes were reduced with time in the lungs of cyclophosphamide groups. However, a significant reduction in lavage fluid biochemical constituents, lipid peroxidation products in serum, lung and lavage cells with concomitant increase in antioxidant defense mechanisms occurred in curcumin fed cyclophosphamide rats. Therefore, our results suggest that curcumin is effective in moderating the cyclophosphamide induced early lung injury and the oxidant-antioxidant imbalance was partly abolished by restoring the glutathione (GSH) with decreased levels of lipid peroxidation.     

Critical role of acrolein in secondary injury following ex vivo spinal cord trauma

The pathophysiology of spinal cord injury (SCI) is characterized by the initial, primary injury followed by secondary injury processes in which oxidative stress is a critical component. Secondary injury processes not only exacerbate pathology at the site of primary injury, but also result in spreading of injuries to the adjacent, otherwise healthy tissue. The lipid peroxidation byproduct acrolein has been implicated as one potential mediator of secondary injury. To further and rigorously elucidate the role of acrolein in secondary injury, a unique ex vivo model is utilized to isolate the detrimental effects of mechanical injury from toxins such as acrolein that are produced endogenously following SCI. We demonstrate that (i) acrolein-Lys adducts are capable of diffusing from compressed tissue to adjacent, otherwise uninjured tissue; (ii) secondary injury by itself produces significant membrane damage and increased superoxide production; and (iii) these injuries are significantly attenuated by the acrolein scavenger hydralazine. Furthermore, hydralazine treatment results in significantly less membrane damage 2 h following compression injury, but not immediately after. These findings support our hypothesis that, following SCI, acrolein is increased to pathologic concentrations, contributes significantly to secondary injury, and thus represents a novel target for scavenging to promote improved recovery.     

Lipid peroxidation and tissue injury by ferric citrate in paraquat-intoxicated mice

To determine whether iron toxicity is caused by iron-catalyzed radical production, the in vivo effect of ferric citrate was studied in paraquat-intoxicated mice. Intraperitoneally injected Fe3+-citrate complex was distributed mainly in the liver and kidney, and promoted lipid peroxidation, as measured by expiratory ethane in both normal and paraquat-intoxicated mice. Plasma glutamic-oxaloacetic transaminase (L-aspartate: 2-oxoglutarate aminotransferase, EC 2.6.1.1) activity increased significantly only in paraquat and Fe3+-citrate-injected mice (PFe group). The rate of ethane production increased prior to the elevation of plasma glutamic-oxaloacetic transaminase levels, and was greater in the PFe group than in the mice, that were injected Fe3+-citrate alone. Pretreatment of animals with desferrioxamine mesylate inhibited both ethane production and elevation of plasma glutamic-oxaloacetic transaminase levels in the PFe group. Administration of 100% oxygen or glucose, which is expected to increase cellular NADPH, to the PFe group further elevated the plasma glutamic-oxaloacetic transaminase level, but had little effect on ethane production, indicating that tissue injury occurs independently of lipid peroxidation. These results suggest that iron toxicity is due to radical production and that, although iron stimulated lipid peroxidation, it might not be the only cause of tissue injury.     

Effect of oxygen concentration on the formation of molondialdehyde-like material in a model of tissue ischemia and reoxygenation

This study was conducted to explore the functional relationship between oxygen concentration during tissue reoxygenation after ischemia and the extent of postischemic lipid peroxidation, an indicator of reoxygenation injury. Excised rat liver or kidney tissue was rendered ischemic for 1 h at 37°C, minced into 1 mm³ fragments, and then reoxygenated for 1 h in flasks of buffered salt solution containing various amounts of oxygen. Production of malondialdehyde-like material (MDA) was measured to indicate lipid peroxidation. MDA production was minimal at oxygen tensions less than 10 mmHg, increased sharply from 10 to 50 mmHg, and plateaued at approximately 100 mmHg. A similar functional relationship was produced by a simple mathematical model of free radical mediated lipid peroxidation in biological membranes, suggesting that MDA production is indeed caudes by free radical oxidation of membrane phospholipids and that the oxygen effect is governed by simple competition between chain propagation and chain termination reactions within the membrane. These experimental and analytical results confirm that relatively low concentrations of oxygen are sufficient to produce oxidative damage in post-ischemic tissues.     

NMN recruits GSH to enhance GPX4-mediated ferroptosis defense in UV irradiation induced skin injury

Oxidative stress and lipid peroxidation are major causes of skin injury induced by ultraviolet (UV) irradiation. Ferroptosis is a form of regulated necrosis driven by iron-dependent peroxidation of phospholipids and contributes to kinds of tissue injuries. However, it remains unclear whether the accumulation of lipid peroxides in UV irradiation-induced skin injury could lead to ferroptosis. We generated UV irradiation-induced skin injury mice model to examine the accumulation of the lipid peroxides and iron. Lipid peroxides 4-HNE, the oxidative enzyme COX2, the oxidative DNA damage biomarker 8-OHdG, and the iron level were increased in UV irradiation-induced skin. The accumulation of iron and lipid peroxidation was also observed in UVB-irradiated epidermal keratinocytes without actual ongoing ferroptotic cell death. Ferroptosis was triggered in UV-irradiated keratinocytes stimulated with ferric ammonium citrate (FAC) to mimic the iron overload. Although GPX4 protected UVB-injured keratinocytes against ferroptotic cell death resulted from dysregulation of iron metabolism and the subsequent increase of lipid ROS, keratinocytes enduring constant UVB treatment were markedly sensitized to ferroptosis. Nicotinamide mononucleotide (NMN) which is a direct and potent NAD⁺ precursor supplement, rescued the imbalanced NAD⁺/NADH ratio, recruited the production of GSH and promoted resistance to lipid peroxidation in a GPX4-dependent manner. Taken together, our data suggest that NMN recruits GSH to enhance GPX4-mediated ferroptosis defense in UV irradiation-induced skin injury and inhibits oxidative skin damage. NMN or ferroptosis inhibitor might become promising therapeutic approaches for treating oxidative stress-induced skin diseases or disorders.     

Free radicals, lipid peroxidation and muscular ischemia]

The role of oxygen free radicals in ischemia and reperfusion injury of skeletal muscle has not been well defined, partly because of the relative resistance of this tissue to normothermic ischemia. Under normal conditions small quantities of oxygen free radicals are produced but they are quenched by intracellular free radical scavenging enzymes (superoxide dismutase, catalase and glutathione peroxidase) or alpha-tocopherol. The increase in malondialdehyde suggests increased lipid peroxidation initiated by free radical reactions. Lipid peroxidation is potentially a very damaging process to the organized structure and function of membranes. The results of recent studies indicate that: a) oxygen free-radicals mediates, at least in part, the increased microvascular permeability produced by reoxygenation, b) free radical scavengers can reduce skeletal muscle necrosis occurring after prolonged ischemia. Additional evidence support the hypothesis of the interrelationship between ischemic tissue and inflammatory cells. So capillary plugging by granulocytes and oxygen free radical formation may contribute to the ischemic injury.     

Purified human chondroitin-4-sulfate reduced MMP/TIMP imbalance induced by iron plus ascorbate in human fibroblast cultures

Imbalance between matrix metalloproteinases (MMPs) and tissue inhibitor of matrix metalloproteinases (TIMPs) is an important control point in tissue remodelling. Several findings have reported a marked MMP/TIMP imbalance in a variety of in vitro models in which oxidative stress was induced. Since previous studies showed that commercial hyaluronan and chondroitin-4-sulphate are able to limit lipid peroxidation during oxidative stress, we investigated the antioxidant capacity of purified human plasma chondroitin-4-sulfate in reducing MMP and TIMP imbalance in a model of ROS-induced oxidative injury in fibroblast cultures. Purified human plasma chondroitin-4-sulfate was added to the fibroblast cultures exposed to FeSO4 plus ascorbate. We assayed cell death, MMP and TIMP mRNA expression and protein activities, DNA damage, membrane lipid peroxidation, and aconitase depletion. FeSO4 plus ascorbate produced severe death of cells and increased MMP-1, MMP-2 and MMP-9 expression and protein activities. It also caused DNA strand breaks, enhanced lipid peroxidation and decreased aconitase. TIMP-1 and TIMP-2 protein levels and mRNA expression remain unaltered. Purified human plasma C4S, at three different doses, restored the MMP/TIMP homeostasis, increased cell survival, reduced DNA damage, inhibited lipid peroxidation and limited impairment of aconitase. These results further support the hypothesis that these biomolecules possess antioxidant activity and by reducing ROS production C4S may limit cell injury produced by MMP/TIMP imbalance.     

Mercuric chloride induced membrane damage in tomato cultured cells

Suspension cultures of tomato cells were used to study the membrane injury by the toxic concentration of mercuric chloride. Assessment of electrolyte leakage, UV-absorbance of the tissue leachates, relative leakage ratio, injury index, membrane lipid peroxidation, lipoxygenase activity, α-amino nitrogen and total soluble carbohydrate contents showed the extent of membrane damage as a function of the increasing concentration of mercuric chloride. It is suggested that the selected parameters can be used as qualitative tests for determination of stress-induced membrane damage     

Magnesium deficiency augments myocardial response to reactive oxygen species

Magnesium (Mg) deficiency and oxidative stress are independently implicated in the etiopathogenesis of various cardiovascular disorders. This study was undertaken to examine the hypothesis that Mg deficiency augments the myocardial response to oxidative stress. Electrically stimulated rat papillary muscle was used for recording the contractile variation. Biochemical variables of energy metabolism (adenosine triphosphate (ATP) and creatine phosphate) and markers of tissue injury (lactate dehydrogenase (LDH) release and lipidperoxidation), which can affect myocardial contractility, were assayed in Langendorff-perfused rat hearts. Hydrogen peroxide (100 micromol/L) was used as the source of reactive oxygen species. The negative inotropic response to H2O2 was significantly higher in Mg deficiency (0.48 mmol Mg/L) than in Mg sufficiency (1.2 mmol Mg/L). Low Mg levels did not affect ATP levels or tissue lipid peroxidation. However, H2O2 induced a decrease in ATP; enhanced lipid peroxidation and the release of LDH were augmented by Mg deficiency. Increased lipid peroxidation associated with a decrease in available energy might be responsible for the augmentation of the negative inotropic response to H2O2 in Mg deficiency. The observations from this study validate the hypothesis that myocardial response to oxidative stress is augmented by Mg deficiency. This observation has significance in ischemia-reperfusion injury, where Mg deficiency can have an additive effect on the debilitating consequences.