Food-derived peroxidized fatty acids may trigger hepatic inflammation: a novel hypothesis to explain steatohepatitis.

BACKGROUND & AIMS: Obesity and hepatic steatosis are frequently associated with the development of a non-alcoholic steatohepatitis (NASH). The mechanisms driving progression of a non-inflamed steatosis to NASH are largely unknown. Here, we investigated whether ingestion of peroxidized lipids, as being present in Western style diet, triggers the development of hepatic inflammation. METHODS: Corn oil containing peroxidized fatty acids was administered to rats by gavage for 6 days. In a separate approach, hepatocytes (HC), endothelial (EC) and Kupffer cells (KC) were isolated from untreated livers, cultured, and incubated with peroxidized linoleic acid (LOOH; linoleic acid (LH) being the main fatty acid in corn oil). Samples obtained from in vivo and in vitro studies were mainly investigated by qRT-PCR and biochemical determinations of lipid peroxidation products. RESULTS: Rat treatment with peroxidized corn oil resulted in increased hepatic lipid peroxidation, upregulation of nitric oxide synthetase-2 (NOS-2), cyclooxygenase-2 (COX-2), interleukin-1ß (IL-1ß), and tumor necrosis factor-α (TNFα), elevation of total nitric oxides, and increase in cd68-, cd163-, TNFα-, and/or COX-2 positive immune cells in the liver. When investigating liver cell types, LOOH elevated the secretion of TNFα, p38MAPK phosphorylation, and mRNA levels of NOS-2, COX-2, and TNFα, mainly in KC. The elevation of gene expression could be abrogated by inhibiting p38MAPK, which indicates that p38MAPK activation is involved in the pro-inflammatory effects of LOOH. CONCLUSIONS: These data show for the first time that ingestion of peroxidized fatty acids carries a considerable pro-inflammatory stimulus into the body which reaches the liver and may trigger the development of hepatic inflammation.

Oxidative stress status in patients with chronic idiopathic urticaria

Aim: The controversial data related to oxidative stress status in patients with chronic idiopathic urticaria (CIU) have been reported. Therefore, the present study was aimed to contribute to this debate by determining oxidative stress markers along with some trace element levels inpatients with CIU. Methods: Twenty-five patients with CIU (10 males, 15 females) and 36 healthy controls were enrolled into the study. Erythrocyte lipid peroxidation status, scavenger enzyme activities and trace element levels were determined. Results: While erythrocyte MDA levels, erythrocyte GSH- Px activities and erythrocyte Zn levels showed no differences between the patient and control groups, a statistically significant decrease and increase were observed in erythrocyte CuZn-SOD activities and Cu levels, respectively, in the CIU patients when compared to those of the controls (p < 0.001 for both of them). Conclusion: In conclusion, an oxidative burden which can be relieved by some preserved antioxidant mechanisms seems to be present in patients with CIU even if they are clinically stable and it may probably have a role in the pathogenesis (AU)

Multiple roles of microsomal glutathione transferase 1 in cellular protection: a mechanistic study.

The aim of this study was to investigate the involvement of membrane-bound microsomal glutathione transferase 1 (MGST1) in cellular resistance against oxidative stress as well as its mechanism of protection. MGST1 is ubiquitously expressed and predominantly located in the endoplasmic reticulum and outer mitochondrial membrane. Utilizing MCF7 cells overexpressing MGST1 we show significant protection against agents that are known to induce lipid peroxidation (e.g., cumene hydroperoxide and tert-butylhydroperoxide) and an end-product of lipid peroxidation (e.g., 4-hydroxy-2-nonenal). Furthermore, our results demonstrate that MGST1 protection can be enhanced by vitamin E when toxicity depends on oxidative stress, but not when direct alkylation is the dominant mechanism. Mitochondria in MGST1-overexpressing cells were shown to be protected from oxidative insult as measured by calcium loading capacity and respiration. MGST1 induces cellular resistance against cisplatin. Here we used vitamin E to elucidate whether oxidative stress caused by cisplatin is significant for cell toxicity. The results indicate that oxidative stress and induction of lipid peroxidation are not the most prominent toxic mechanism of cisplatin in our cell system. We thus conclude that MGST1 protects cells (and mitochondria) by both conjugation and glutathione peroxidase functions. A new protective mechanism against cisplatin is also indicated.

A rat model for choroidal neovascularization using subretinal lipid hydroperoxide injection.

The purpose of this study was to develop and characterize a rat model of choroidal neovascularization (CNV) as occurs in age-related macular degeneration. The lipid hydroperoxide 13(S)-hydroperoxy-9Z,11E-octadecadienoic acid (HpODE) is found in submacular Bruch's membrane in aged humans and has been reported to generate neovascularization in a rabbit model. Three weeks after a single subretinal injection of 30 microg of HpODE, eyes of Sprague-Dawley rats were harvested. Follow-up fluorescein angiography was done on other animals until 5 weeks postinjection. Histological studies, immunohistochemical staining, and flatmount choroids for CNV measurements were performed. In addition, we used murine neuronal, bovine endothelial, and human ARPE19 cells for testing the in vitro effects of HpODE. CNV developed in 85.7% of HpODE-injected eyes. The neovascular areas were significantly greater in HpODE-injected eyes compared with those in control eyes (P = 0.023). The CNV had maximum dye leakage at 3 weeks, which subsided by the 5th week. Histologically, CNV extended from the choriocapillaris into the subretinal space. ED1-positive macrophages were recruited to the site. In vitro assays demonstrated that only 30 ng/ml HpODE induced cell proliferation and migration of endothelial cells. HpODE-induced CNV was highly reproducible, and its natural course seems to be ideal for evaluating therapeutic modalities. Because HpODE has been isolated from aged humans, the HpODE-induced rat model seems to be a relevant experimental model for CNV in age-related macular degeneration.

Dietary ALEs are a risk to human health--NOT!

Advanced lipoxidation end-products (ALEs) are formed by reaction of protein with lipid-derived reactive peroxyl and carbonyl compounds produced during food processing and cooking. There is concern that ALEs may induce damage in the gastrointestinal tract, affecting gut health, or enter the body and promote vascular inflammation and tissue damage. However, there is no direct evidence that ALE-proteins are a source of damage in the intestines or that they are transported into the circulation and cause pathology. Modification of proteins by ALEs impedes their digestion, and reactive ALEs released by gastrointestinal proteases would react with proteins or peptides in the gut, limiting their absorption. There are also potent enzymatic mechanisms for detoxifying ALEs or their precursors prior to their entry into the circulation. If ALEs gain access to the circulation, a battery of protective enzymes in tissue provides a second level of defense. These enzymes may be induced in intestinal epithelia and liver by low doses of ALEs, and adaptive responses would provide enhanced protection against future exposure to ALEs. Overall, except in persons with compromised organ function, e. g., vascular, hepatic, or renal diseases, there is little evidence that food ALEs will have any significant pathological effects.

Protective role of zinc-metallothionein (Zn-MT) in iron nitrilotriacetate (Fe-NTA)-induced renal oxidative damage.

Several studies have shown the role of thiol-rich proteins especially metallothionein (MT) in the therapeutic interventions against oxidative damage. Previously, we have provided strong evidence for the involvement of ROS in iron nitrilotriacetate (Fe-NTA)-induced renal toxicity, which may have relevance to its carcinogenicity. The purpose of this study was to evaluate the role of zinc metallothionein (Zn-MT) on the protection against Fe-NTA-induced renal oxidative damage. The results demonstrate that Zn-MT pretreatment provided protection against Fe-NTA-induced mortality in mice (40% protection). Similarly, Zn-MT pretreatment also provided protection against Fe-NTA-induced lipid peroxidation (26% inhibition, P < 0.001). It is proposed that Zn-MT protects kidney tissue against the noxious effect of Fe-NTA primarily by interference with lipid peroxides. It is concluded that Zn-MT may serve as an excellent physiological antioxidant against Fe-NTA-mediated renal oxidative damage.

Selenium supplementation acting through the induction of thioredoxin reductase and glutathione peroxidase protects the human endothelial cell line EAhy926 from damage by lipid hydroperoxides.

The human endothelial cell line EAhy926 was used to determine the importance of selenium in preventing oxidative damage induced by tert-butyl hydroperoxide (tert-BuOOH) or oxidised low density lipoprotein (LDLox). In cells grown in a low selenium medium, tert-BuOOH and LDLox killed cells in a dose-dependent manner. At 555 mg/l LDLox or 300 microM tert-BuOOH, >80% of cells were killed after 20 h. No significant cell kill was achieved by these agents if cells were pre-incubated for 48 h with 40 nM sodium selenite, a concentration that maximally induced the activities of cytoplasmic glutathione peroxidase (cyGPX; 5.1-fold), phospholipid hydroperoxide glutathione peroxidase (PHGPX;1.9-fold) and thioredoxin reductase (TR; 3.1-fold). Selenium-deficient cells pre-treated with 1 microM gold thioglucose (GTG) (a concentration that inhibited 25% of TR activity but had no inhibitory effect on cyGPX or PHGPX activity) were significantly (P<0.05) more susceptible to tert-BuOOH toxicity (LC(50) 110 microM) than selenium-deficient cells (LC(50) 175 microM). This was also the case for LDLox. In contrast, cells pre-treated with 40 nM selenite prior to exposure to GTG were significantly more resistant to damage from tert-BuOOH and LDLox than Se-deficient cells. Treatment with GTG or selenite had no significant effect on intracellular total glutathione concentrations. These results suggest that selenium supplementation, acting through induction of TR and GPX, has the potential to protect the human endothelium from oxidative damage.

The role of free radicals in chronic rhinosinusitis.

OBJECTIVE: To determine whether there is an increased amount of free radical-mediated damage in diseased vs healthy tissue from patients with chronic rhinosinusitis. DESIGN: Pathophysiologic study. Samples of heathly and diseased tissue were taken from each patient. Lipid peroxides (LPOs) are a by-product of free radical-mediated damage; LPO levels and LPO/protein ratios were determined for each patient. SUBJECTS: Consecutive series of 13 human subjects undergoing functional endoscopic sinus surgery to treat chronic rhinosinusitis. RESULTS: The mean LPO/protein ratio for healthy tissue was 3.52 x 10(-5), while that for the diseased tissue was 3.49 x 10(-5). There was no statistically significant difference in the LPO/protein ratio between healthy and diseased tissue (95% confidence interval, -3.00 x 10(-5) to 2.94 x 10(-5)). CONCLUSION: Free radical-induced damage, if present, was the same in infected and control tissues in this pilot investigation into the pathophysiologic characteristics of human chronic rhinosinusitis.

Oxidative DNA damage and cardiovascular disease.

Reactive oxygen species can directly cause covalent modifications to DNA. Alternatively, they can initiate the formation of lipid hydroperoxides, which undergo homolytic decomposition to the alpha,beta-unsaturated aldehyde genotoxins, 4-oxo-2-nonenal, 4,5-epoxy-2(E)-decenal, and 4-hydroxy-2-nonenal through two quite separate pathways. One pathway involves a complex rearrangement of the alkoxy radical derived from the lipid hydroperoxide. The other pathway involves the intermediate formation of 4-hydroperoxy-2-nonenal. Lipid hydroperoxides can also be derived from the action of lipoxygenases and cyclooxygenases on polyunsaturated fatty acids. 4,5-Epoxy-2(E)-decenal forms etheno-2'-deoxyadenosine adduct with DNA, a mutagenic lesion observed in human tissue DNA samples. Several new ethano- and etheno-DNA adducts have been identified from the reaction of 4-oxo-2-nonenal with DNA. Malondialdehyde, another genotoxic bifunctional electrophile, forms a propano adduct with 2'-deoxyguanosine (M1G-dR) rather than an etheno adduct. Very little is known about the consequences of lipid hydroperoxide-mediated DNA damage in cardiovascular diseases. This should prove to be an important area for future research.

Fish oil and cardiovascular disease: lipids and arterial function.

n-3 Fatty acids have been shown to modify several key risk factors for cardiovascular disease. However, it is not clear whether the apparent protection against cardiovascular disease is directly related to antiatherogenic functions of these fatty acids or is mediated through their modification of the risk factors through mechanisms not directly related to lipids. A major question concerns the importance of lipid modification, which is a potent outcome of fish-oil supplementation. On balance, lipid modification is likely to represent a significant antiatherogenic factor. The benefits include increased HDL(2)-cholesterol concentrations, reduced triacylglycerol-rich lipoprotein concentrations, reduced postprandial lipemia, and reduced remnant concentrations. In contrast, LDL-cholesterol concentrations have often been noted to rise and the potential of increased oxidizability of LDLs is potentially adverse with lipid modification, but this potential can be overcome with vitamin E supplementation. The characteristic lipid changes and the underlying mechanisms are reviewed. Additional benefits of fish oils include improved endothelial function and better arterial compliance (elasticity). Future trials will be needed to determine minimum effective dosages of eicosapentaenoic and docosahexaenoic acids over lengthy periods and to show cardiovascular disease reduction through intervention.

Molecular and cellular responses to oxidative stress and changes in oxidation-reduction imbalance in the intestine.

Recently, it has become increasingly apparent that oxidants, in addition to being agents of cytotoxicity, can play an important role in mediating specific cell responses and expression of genes involved in degenerative pathophysiologic states, such as inflammation and cancer. In particular, nuclear transcription factor kappaB (NF-kappaB), a multisubunit transcription factor, has been implicated in the transcriptional up-regulation of inflammatory genes in response to oxidants or changes in cellular oxidation-reduction status. This paper provides an overview of the cellular responses to oxidative stress and oxidation-reduction imbalance and the role of NF-kappaB in these responses and summarizes the current strategies used to study NF-kappaB activation and nuclear translocation, particularly in relation to dietary oxidant-mediated pathophysiology of the intestine.

Lipid peroxyl radicals from oxidized oils and heme-iron: implication of a high-fat diet in colon carcinogenesis.

A diet high in fat and iron is known as a risk factor in cancer epidemiology. However, the details of the molecular mechanism remains to be elucidated. We examined the possible implication of lipid peroxyl radicals generated from fatty acids and heme-iron in DNA damage, and hence in the possibility of colon cancer. F344 female rats were given N-nitroso-N-methylurea six times during a 2-week period and then fed diets containing different amounts of safflower oil and hemoglobin (rich in iron) for 36 weeks; the occurrence of colon cancer was determined by H&E staining. In this animal model, simultaneous feeding of a fat diet and heme-iron produced a significant increase (P < 0.05) in the incidence of colon cancer compared with a diet without hemoglobin. Electron paramagnetic resonance and chemiluminescence studies revealed that oxidized refined vegetable oils, particularly safflower oil, readily generated lipid peroxyl radicals in the presence of various heme compounds, and the peroxyl radicals did effectively cleave DNA. Unpurified native vegetable oils contain a high amount of peroxyl radical scavengers, whereas conventional refining processes seem to reduce the levels of many valuable anti-peroxyl radical compounds abundant in plant seeds. In conclusion, lipid peroxides and heme components generate peroxyl radical species that exert DNA-cleaving activity. A plausible explanation is that lipid peroxyl radicals thus generated, which originated from routine dietary components such as fat and red meat, may contribute, at least in part, to the high incidence of colon cancer.

Activation of eicosanoid metabolism in human airway epithelial cells by ozonolysis products of membrane fatty acids.

Inhaled ozone can react with a variety of cellular macromolecules within the lung. Recent analyses of the chemistry of ozone reactions with unsaturated fatty acids, which are present in all membranes and in mucus in the airways, indicate that ozonolysis yields one aldehyde and one hydroxyhydroperoxide molecule for each molecule of ozone. The hydroxyhydroperoxide molecule is unstable in aqueous environments, and subsequently yields a second aldehyde and hydrogen peroxide. The structure of common unsaturated fatty acids is such that attack by ozone at the carbon-carbon double bonds will yield 3-, 6-, and 9-carbon saturated and unsaturated aldehydes and hydroxyhydroperoxide. This study examines the effects of ozonolysis products on eicosanoid metabolism in human airway epithelial cells. Eicosanoid biosynthesis is important in a wide array of pathophysiological responses in the airway, and the release of eicosanoids by the epithelial barrier is likely to be significant in diseases induced by environmental factors. Previously, we demonstrated that ozone can increase eicosanoid synthesis from airway epithelial cells exposed in vitro. Human exposures to concentrations of ozone below the current National Ambient Air Quality Standard (0.12 ppm, not to be exceeded for more than one hour once per year) also resulted in increased eicosanoids in bronchoalveolar lavage fluid. To determine whether ozonolysis products could activate eicosanoid release, we exposed human airway epithelial cells to 3-, 6-, and 9-carbon aldehydes, hydroxyhydroperoxides, and hydrogen peroxide. We measured (1) eicosanoid metabolism using high-performance liquid chromatography and radioimmunoassays, and (2) the effects of the aldehydes, hydroxyhydroperoxides, and hydrogen peroxide on cell lysis. Eicosanoid release increased after exposure to aldehyde; release induced by 9-carbon (nonanal) aldehyde was greater than that induced by the 6-carbon (hexanal) or 3-carbon (propanal) aldehydes. Hydroxyhydroperoxides induced greater eicosanoid release than the corresponding aldehydes of equivalent chain length. Again, the longer the aliphatic chain length of the hydroxyhydroperoxide the greater the effect. These effects were noted at concentrations of hydroxyhydroperoxide below those that produce cell lysis, and the time course of the two responses was dissimilar. Because hydroxyhydroperoxides can degrade into an aldehyde and hydrogen peroxide, it is conceivable that the effects observed were attributable to the formation of either hydrogen peroxide or hydrogen peroxide and aldehyde. This mechanism is unlikely, however, because the effects of hydroxyhydroperoxides on eicosanoid release were dependent on chain length, whereas each hydroxyhydroperoxide can produce only one hydrogen peroxide molecule. Although hydrogen peroxide alone also stimulated eicosanoid metabolism, this effect was not augmented when aldehyde and hydrogen peroxide were added together. In addition, the dose of hydroxyhydroperoxide needed to produce an effect (10 to 100 microM) was lower than that of hydrogen peroxide (300 microM). We could not fully evaluate the effects of the unsaturated aldehydes and hydroxyhydroperoxides. Although the 6-carbon and 9-carbon cis-3-aldehydes could be synthesized from the cis-3-alcohols, the resulting aldehydes were not chemically stable. The cis-3-aldehydes were useful for producing the corresponding 1-hydroxy-alkenyl-hydroperoxides of high purity. These results support the method selected for chemical synthesis, but further studies are required to establish proper storage and handling methods before these compounds can be tested in assays of eicosanoid metabolism.

Toxic hydroperoxides in intravenous lipid emulsions used in preterm infants.

The unsaturated fatty acids that make up a large component of the lipid emulsion Intralipid are highly susceptible to peroxidation, and the products of this reaction could explain the toxicity that has been associated with the administration of some emulsions. Lipid peroxidation produces hydroperoxides, which can alter arachidonic acid metabolism or react to form organic free radicals, which then stimulate a cascade of damage to endogenous lipids. The lipid hydroperoxides and their breakdown products are also mutagens and carcinogens. To determine the degree of lipid peroxidation in Intralipid, we measured the lipid hydroperoxide content of three lots of 20% Intralipid using high-performance liquid chromatography with chemiluminescence detection. The average concentration was 290 +/- 29 mumol/L (SEM) lipid hydroperoxides (n = 15), a large portion of which was made up of trilinoleate derivatives. Measurements made on Intralipid samples collected from the end of the intravenous tubing after a 20-hour infusion cycle were not significantly different from measurements made on newly opened bottles. The lipid hydroperoxide content of some lipid emulsions may represent a clinically significant risk to premature infants, particularly those with preexisting lung disease.

Effect of L-carnitine derivatives on heart mitochondrial damage induced by lipid peroxidation.

We have incubated heart mitochondria with ferrous ions as catalyst of lipid peroxidation. Ferrous ions induced an increase of malondialdehyde formation and a reduction of mitochondrial oxygen consuming and calcium transporting capacities. L-Carnitine and Acetyl-L-Carnitine failed to prevent mitochondrial damage. Propionyl-L-Carnitine significantly improved mitochondrial function, but failed to reduce malondialdehyde formation. This protective effect was specific for Propionyl-L-Carnitine as propionic acid and L-Carnitine did not modify mitochondrial damage.

Phospholipid peroxidation as a factor in gallstone pathogenesis.

Phospholipid peroxidation markedly reduces the stability of mixed micellar systems composed of cholate, phosphatidylcholine and supersaturating levels of cholesterol. This suggests that lipid peroxidation is likely to play a significant role in the precipitation of cholesterol from gallbladder bile, thus in the pathogenesis of cholesterol gallstones. This conclusion is supported by studies of the nucleation time of cholesterol in gallbladder biles, which was significantly reduced by exposure to a stream of oxygen. This effect of phospholipid peroxidation on cholesterol solubility may occur in other biological fluids as well. In view of the increased lipid peroxidation in the elderly, it may explain the effect of age on the frequency of various diseases related to cholesterol precipitation.