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.

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 carcinogens and anticarcinogens. Oxygen radicals and degenerative diseases.

The human diet contains a great variety of natural mutagens and carcinogens, as well as many natural antimutagens and anticarcinogens. Many of these mutagens and carcinogens may act through the generation of oxygen radicals. Oxygen radicals may also play a major role as endogenous initiators of degenerative processes, such as DNA damage and mutation (and promotion), that may be related to cancer, heart disease, and aging. Dietary intake of natural antioxidants could be an important aspect of the body's defense mechanism against these agents. Many antioxidants are being identified as anticarcinogens. Characterizing and optimizing such defense systems may be an important part of a strategy of minimizing cancer and other age-related diseases.

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.

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.

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.

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.

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.

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.

Oxygen-free radicals and lipid peroxidation in adult respiratory distress syndrome.

Activated species of oxygen have been implicated as mediators of some acute lung injury. In adult respiratory distress syndrome (ARDS), polymorphonuclear leukocytes accumulate in the lung and release excessive amounts of O2-derived products into the extracellular environment. The effects of these O2 products on lung tissue are multiple. In particular, they can initiate lipid peroxydation in cellular membranes. Excessive lipid peroxydation in membranes destroys cells such as vascular endothelium. Lipid peroxides are also detrimental to cellular functions. Lipid peroxydation could then play a role in the pathogenesis of ARDS.

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.

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.

Antiinflammatory effects of free radical scavengers and antioxidants: further support for proinflammatory roles of endogenous hydrogen peroxide and lipid peroxides.

Scavengers of reactive oxygen species were tested by local administration during granulomatous inflammation in the rat, induced by the subdermal implantation of carrageenin-soaked sponges. Drugs were administered either in a single dose immediately after sponge implantation, or in daily doses on days 3-6 of inflammation. The effects of the injected drugs were assessed using the day 7 granuloma. When given at the moment of sponge implantation, catalase showed antiinflammatory effects, whereas superoxide dismutase did not. However, the addition of superoxide dismutase to catalase, prior to injection, markedly potentiated the inhibition of granuloma formation by catalase alone. Negative results obtained with scavengers of hydroxyl radicals and singlet molecular oxygen suggest that protection of superoxide dismutase by catalase from inactivation by hydrogen peroxide, is a likely explanation for the observed potentiation. When administered at the moment of the induction of inflammation, alpha-tocopherol and propyl gallate, both antioxidants, also inhibited granuloma formation. All drugs tested were either ineffective or even enhanced granuloma weight following administration into a preformed granuloma. An inhibitor of both pathways of arachidonate metabolism, phenidone, inhibited granuloma formation irrespective of the moment of administration. The results presented in this paper suggest proinflammatory roles for hydrogen peroxide and lipid peroxides and a possible involvement of hydroxyl radicals and singlet oxygen in the breakdown of collagen.

Occurrence of lipid oxidation products in foods.

Lipid oxidation products are ubiquitous in foods, although much variation exists in the levels present. Although these levels are generally low, the problem of lipid oxidation severely compromises the quality of some foods and limits the shelf-life of others. Lipid oxidation represents a key barrier in the development of new food products and processes, especially convenience items and processes required to manufacture them. Deleterious changes in foods caused by lipid oxidation include loss of flavour, development of off-flavours, loss of colour, nutrient value and functionally, and the accumulation of compounds which may be detrimental to the health of consumers. All foods that contain lipids are susceptible to oxidation but especially affected are foods which are dehydrated, subjected to high temperatures or cooked and subsequently stored, e.g. dehydrated eggs, cheeses and meats, foods fried in frying oils, and cooked (uncured) meats. Specific examples of compounds which are of health concern include lipid peroxides and the free radicals involved in their formation and propagation, malonaldehyde, and several cholesterol oxidation products. Coronary artery disease (CAD) may be in part caused by the consumption of lipid oxidation products.

Scurvy: old disease--new insight.

Scurvy was once the number one killer of seafarers on long voyages. One early theory with widespread acceptance was that rancid fat caused scurvy. I propose to show that rancid fat did, indeed, predispose seafarers to scurvy. I propose to show that Captain Cook's successful voyages in the latter half of the eighteenth century were in part due to removal of rancid fat from the diet of his crew and in part due to dietary anti-scorbutic factors.

The combined role of atheroma, cholesterol, platelets, the endothelium and fibrin in heart attacks and strokes.

In 1920 the typical American diet was rich in cholesterol and fat, especially saturated animal fat, with one-third the polyunsaturated vegetable fat as now; yet in that year, death from myocardial infarction (MI) in the United States was so rare that it had no name or medical recognition. In 1960, when MI deaths in the United States had soared to an alarming rate of 600,000, orthodox medicine concluded that cholesterol and saturated animal fat in food caused elevated cholesterol in blood, which caused cholesterol in atheroma, which in turn caused death from MI and strokes. It is suggested that human atheroma is made up mostly of fibers of either collagen or fibrin, smooth-muscle cells or dead smooth-muscle cells, that it contains but little cholesterol, and that it is present in both men and women and in populations having little or no MI as well as in those where MI is the greatest cause of death. It is suggested that MI is largely caused by coronary blood clots formed at the site of a break in the coronary artery endothelium; that the introduction of a new, unnatural dietary fatty acid--trans-trans linoleic acid--in margarine and refined vegetable oils in the 1920s, by inducing a deficiency of beneficial prostaglandin E1 (PGE1) while greatly increasing harmful thromboxane A2 (TXA2), caused vasoconstriction while the clumping of platelets was greatly increased, giving rise to the coronary blood clots that either cause or are part of the fatal process of MI. It is suggested that in fostering the increase of dietary trans-trans linoleic acid in polyunsaturated vegetable fats at the expense of saturated animal fat, orthodox medicine is fostering a principle cause of MI as the cure.

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.