HNE interacts directly with JNK isoforms in human hepatic stellate cells.

4-Hydroxy-2,3-nonenal (HNE) is an aldehydic end product of lipid peroxidation which has been detected in vivo in clinical and experimental conditions of chronic liver damage. HNE has been shown to stimulate procollagen type I gene expression and synthesis in human hepatic stellate cells (hHSC) which are known to play a key role in liver fibrosis. In this study we investigated the molecular mechanisms underlying HNE actions in cultured hHSC. HNE, at doses compatible with those detected in vivo, lead to an early generation of nuclear HNE-protein adducts of 46, 54, and 66 kD, respectively, as revealed by using a monoclonal antibody specific for HNE-histidine adducts. This observation is related to the lack of crucial HNE-metabolizing enzymatic activities in hHSC. Kinetics of appearance of these nuclear adducts suggested translocation of cytosolic proteins. The p46 and p54 isoforms of c-Jun amino-terminal kinase (JNKs) were identified as HNE targets and were activated by this aldehyde. A biphasic increase in AP-1 DNA binding activity, associated with increased mRNA levels of c-jun, was also observed in response to HNE. HNE did not affect the Ras/ERK pathway, c-fos expression, DNA synthesis, or NF-kappaB binding. This study identifies a novel mechanism linking oxidative stress to nuclear signaling in hHSC. This mechanism is not based on redox sensors and is stimulated by concentrations of HNE compatible with those detected in vivo, and thus may be relevant during chronic liver diseases.

Lipid peroxidation in rat AH-130 hepatoma cells enriched in vitro with arachidonic acid.

Tumor cells generally display low lipid peroxidation. A low content of polyunsaturated fatty acids in membrane phospholipids is a possible cause of their decreased susceptibility to lipid peroxidation. To investigate the importance of substrate availability in eliciting lipid peroxidation and to study cell viability in conditions of stimulated lipid peroxidation, AH-130 hepatoma cells were enriched with arachidonic acid. The enriched hepatoma cells showed increased mortality correlated with the increased incorporation of arachidonic acid in membrane phospholipids. When 0.5 mM arachidonic acid was added to hepatoma cells, this fatty acid reached a percentage content similar to that found in hepatocytes. Hepatoma cells enriched with this concentration were further incubated to determine their susceptibility to lipid peroxidation; mortality increased in parallel with increased thiobarbituric acid-reactive substance production. The highest mortality was in hepatoma cells treated with ascorbate/FeSO4. Mortality in normal cells was low, although they had a high production of thiobarbituric acid-reactive substances. The high capability of normal cells to metabolize the products of lipid peroxidation might explain the different viabilities of normal cells and hepatoma cells. It may therefore be possible to modify the composition of fatty acids of hepatoma cells in order to sensitize them to the toxic effect of prooxidant agents.

Electron spin resonance studies on normal human uterus and cervix and on benign and malignant uterine tumors.

Electron spin resonance (ESR) studies at -130 degrees have been made on frozen samples of normal human cervix and uterus and on frozen samples of various pathological conditions of the cervix and uterus including fibroleiomyoma and carcinoma. Fifty-five samples of normal cervix and endometrium, 40 samples of nonmalignant disturbances, 15 benign tumor samples, and 20 malignant samples were studied. Very strong ESR signals were seen in frozen powders and frozen intact samples of normal cervix and endometrium and in nonmalignant gynecological conditions. In many cases, the ESR signal was greatly decreased or even undetectable in cancer samples. The substance(s) responsible for the ESR signal in frozen intact tissue (g = 2.11 to 2.15) is decreased in concentration when the sample is ground to powder under liquid nitrogen, and an anisotropic signal (g = 2.002 to 2.035) then becomes much more evident. The ESR signals in intact and in powder samples are sensitive to temperature variations; the signals disappear around 0 degrees, and only the intact samples show significant recovery of signal on recooling. The anisotropic g values and temperature sensitivity in the powders may result from an organic peroxy radical that is more strongly associated with a metal ion in intact samples.

Calmodulin antagonism and growth-inhibiting activity of triphenylethylene antiestrogens in MCF-7 human breast cancer cells.

The triphenylethylene antiestrogen tamoxifen has been shown previously to inhibit both calmodulin and protein kinase C activities, which are involved in the control of cell proliferation. We have studied the effect of several derivatives of the triphenylethylene antiestrogen family on the inhibition of both calmodulin-dependent cyclic adenosine 3':5'-monophosphate-phosphodiesterase activity and proliferation of breast cancer cells cultured with 0.5 microM estradiol in order to prevent interaction of these drugs with the estrogen receptor. We have observed that hydroxylation of the triphenylethylene molecule significantly decreases its ability to inhibit the calmodulin-dependent phosphodiesterase activity in vitro. Furthermore, the growth-inhibiting activity of several antiestrogens and other calmodulin antagonists [R24571, trifluoperazine, N-(6-aminohexyl)-5-chloronaphthalene-1-sulfonamide, and N-(6-aminohexyl)-1-naphthalenesulfonamide] correlated with their antagonistic effects on calmodulin activity. The level of activity was determined as follows: R24571 greater than tamoxifen = N-demethyltamoxifen = nafoxidine greater than 4-hydroxytamoxifen greater than 3,4-dihydroxytamoxifen = trifluoperazine greater than N-(6-aminohexyl)-5-chloronaphthalene-1-sulfononamide greater than metabolite A greater than N-(6-aminohexyl)-1-naphthalenesulfonamide. On the other hand both protein kinase C-activating and -inhibiting drugs (phorboltetradecanoate-13-acetate and tamoxifen, respectively) have a synergistic inhibitory effect on the growth of MCF-7 cells. Our data suggest that antiestrogen interactions with calmodulin and not protein kinase C may play a role in mediating the drug-induced estrogen-independent inhibition of breast cancer cell growth.

4-Hydroxynonenal, a product of cellular lipid peroxidation, which modulates c-myc and globin gene expression in K562 erythroleukemic cells.

Several studies point to the existence of an inverse correlation between cellular lipid peroxidation and both cell proliferation and neoplastic transformation. Here, we show that 4-hydroxynonenal (HNE) concentrations close to the level found in normal cells (in the range of 1 and 3 microM) can specifically induce changes in the expression of c-myc and gamma-globin mRNA in K562 cells, without inducing any toxic effects or affecting cell viability. Since we have determined that K562 cells have undetectable levels of endogenous lipid peroxidation, all these effects can be assigned to the exogenous HNE treatment. After a 1-h treatment with 1 microM HNE, c-myc mRNA levels decrease transiently during the first 4 h, rebounding later to higher levels, and normalizing to basal expression after 4 days. Run-on experiments show a transient transcriptional block 20 min after HNE treatment and subsequent posttranscriptional regulation. According to S1 mapping, mRNA changes are exerted on c-myc transcripts initiated from both the principal constitutive start sites (P1 and P2). gamma-Globin mRNA levels concomitantly increase 3- to 4-fold, but no significant changes of housekeeping gene expression are observed. On the basis of these results it appears that the restoration in human erythroleukemic K562 cells of HNE concentrations closer to the level in normal cells can modulate the expression of specific genes.

Stimulation of lipid peroxidation increases the intracellular calcium content of isolated hepatocytes.

Lipid peroxidation induced in isolated rat hepatocytes by FeCl3 (0.1 mM) was associated with an increase in the cytosolic free Ca2+ and in the ionophore-mobilizable Ca2+ content of both mitochondrial and extramitochondrial (endoplasmic reticular) pools. Ca2+ accumulation was completely prevented by the antioxidants promethazine and vitamin E succinate and was not linked to the inhibition of plasma membrane (Ca2+ + Mg2+)-ATPase and Ca2+ transport or to the depletion of intracellular ATP content. Moreover, preincubation of the hepatocytes with the Ca2+ channel blockers verapamil and nifedipine inhibited the elevation of cytosolic Ca2+, as well as the ion accumulation without interfering with the stimulation of lipid peroxidation by iron. These results suggest that peroxidative alterations of the hepatocyte plasma membranes might perturb the functions of verapamil- and nifedipine-sensitive Ca2+ channels resulting in a net influx of Ca2+, which is subsequently sequestrated in the intracellular compartments.

Resistance to oxidative stress by hyperplastic and neoplastic rat liver tissue monitored in terms of production of unpolar and medium polar carbonyls.

The susceptibility of rat liver tissue to oxidative stress during its neoplastic transformation was analyzed by both qualitative and quantitative measurements of the carbonyl products of lipid peroxidation. Diethylnitrosamine was used as initiating agent of hepatocarcinogenesis and lipid peroxidation levels were monitored in the homogenates from normal liver, hyperplastic nodules and tumour, incubated in the presence or in the absence of ascorbate or adenosine diphosphate-iron complex. While the basal levels of lipid peroxidation in the three experimental conditions were found to be quite similar, in the presence of the pro-oxidant stimulus a remarkable reduction in aldehyde production was shown not only by the hepatoma tissue but also by the preneoplastic nodules.

Action of lipid peroxidation products on phosphoinositide specific phospholipase C.

4-Hydroxynonenal (HNE), a major lipid peroxidation product, displays several biological actions. Among them, the differentiation of human HL-60 cells and the stimulation of neutrophil oriented migration occur at concentrations which can be actually found in normal tissues and in body fluids. In spite of its chemotactic activity, HNE fails to increase neutrophil oxidative metabolism. The action of the aldehyde on cell migration appears to be mediated by a phosphoinositide specific phospholipase C. The acceleration of phosphatidylinositol turnover induced by 10 pM 4-hydroxyoctenal, another lipid peroxidation product, is prevented by the pretreatment of neutrophils with pertussis toxin. The mechanism of action of these 4-hydroxyalkenals appears to follow pathways common to other chemoattractants, but some differences can be found too. In particular HNE seems unable to stimulate phospholipase D activity. The action of 4-hydroxyalkenals and other lipid peroxidation products on transmembrane signalling systems and on phospholipid metabolism might regulate several cell functions, such as motility, proliferation and differentiation.

In vitro evidence for CCl4 metabolites covalently bound to lipoprotein micelles.

CCl4-induced impairment of the lipoprotein secretion pathway of intact rat hepatocytes was carried out using 14CCl4 to check the possibility of binding to lipoproteins by CCl4 metabolites. After separation of different cell suspension fractions by means of ultracentrifugation and chemical precipitation procedures, a significant amount of the radioisotope was found covalently bound to the lipid and protein components of low density lipoproteins. Suitable experiments demonstrated that the bound radioisotope was represented by CCl4 metabolites and not by unactivated CCl4.

Free radical activation of acetaldehyde and its role in protein alkylation.

The formation of carbon centered free radicals, identified as methylcarbonyl species, was observed using ESR spectroscopy and the spin trapping agent 4-pyridyl-1-oxide-N-t-butyl nitrone (4-POBN) during the oxidation of acetaldehyde by xanthine oxidase. The reaction was dependent upon the presence of OH. radicals and was inhibited by the addition of superoxide dismutase, catalase or OH. radical scavengers. The generation of methylcarbonyl radicals was associated with a doubling of stable acetaldehyde adducts with serum albumin, and 4-POBN or superoxide dismutase and catalase, completely blocked this effect. Thus, methylcarbonyl radicals contributed to acetaldehyde-mediated protein alkylation which is involved in causing toxic as well as immunological reactions ascribed to acetaldehyde.

Inhibition of D1, D2, and A-cyclin expression in HL-60 cells by the lipid peroxydation product 4-hydroxynonenal.

4-Hydroxynonenal (HNE), a product of lipid peroxidation, is an highly reactive aldehyde that, at concentration similar to those found in normal cells, blocks proliferation and induces a granulocytic-like differentiation in HL-60 cells. These effects are accompained by a marked increase in the proportion G0/G1 cells. The mechanisms of HNE action were investigated by analyzing the expression of the cyclins and cyclin-dependent protein kinases (CDKs), controlling the cell cycle progression. Data obtained by exposing cells to dimethyl sulfoxide (DMSO) were used for comparison. 4-Hydroxynonenal downregulated both mRNA and protein contents of cyclins D1, D2, and A until 24 h from the treatments, whereas DMSO inhibited cyclin D1 and D2 expression until the end of experiment (2 days) and induces an increase of cyclin A until 1 day. Cyclins B and E, and protein kinase CDK2 and CDK4 expressions were not affected by HNE, whereas DMSO induced an increase of cyclin E, B, and CDK2 from 8 h to 1 day. These data are in agreement with previous results indicating a different time-course of accumulation in G0/G1 phases of cells treated with HNE and DMSO and suggest that the HNE inhibitory effect on proliferation and cell cycle progression may depend by the downregulation of D1, D2, and A cyclin expression.

Lipid peroxidation in human diseases: evidence of red cell oxidative stress after circulatory shock.

Erythrocytes obtained from human patients with circulatory shock of different aetiology consistently showed a strong increase in lipid peroxidation-derived aldehydes in comparison with red cells of normal adults. The highly toxic compound 4-hydroxynonenal has been recovered exclusively in the erythrocytes of the patients.

On the role of lipid peroxidation in the pathogenesis of liver damage induced by long-standing cholestasis.

Previous studies have suggested a possible involvement of free radical reactions in the pathogenesis of cholestatic liver injury as well as in the modulation of hepatic fibrogenesis. In this study we investigated whether lipid peroxidation is involved in the development of chronic liver damage induced by long-standing cholestasis. For this purpose we have used the rat model of bile duct ligation (BDL), which leads to liver fibrosis and cirrhosis. Using this model we observed that the development of chronic liver damage was associated with the onset of lipid peroxidation, as pointed out by detection of carbonyl compounds, 4-hydroxynonenal (HNE) and malondialdehyde (MDA), in BDL livers and of fluorescent adducts between MDA and serum proteins. Lipid peroxidation was a relatively late event (starting after 1-2 weeks of BDL) and was unrelated to the early development of liver necrosis and cholestasis (already evident after 72 h after BDL). A positive significant linear correlation between the kinetic of infiltration of neutrophils and of a monocyte/macrophage population in BDL livers and MDA and HNE generation in the same organs is presented, indicating a close link between lipid peroxidation and the activation of inflammatory cells. We also observed that a positive linear correlation exists between collagen deposition in these livers and hepatic production of MDA and HNE. This event, which is accompanied by an increase in the number of fat storing cells (FSC, the cells that produce collagen in fibrotic liver), suggests that lipid peroxidation in this model may contribute to stimulate collagen synthesis by proliferating FSC.

Mechanisms responsible for carbon tetrachloride-induced perturbation of mitochondrial calcium homeostasis.

Incubation of isolated hepatocytes with CCl4 results in early reduction of the intracellular calcium content, mostly due to loss from the mitochondrial compartment. CCl4 treatment directly affects mitochondrial functions as indicated by the inhibition of Ca2+ uptake in cells permeabilized to the ion by digitonin exposure and by the reduction of intracellular ATP content in hepatocytes incubated in a glucose-free medium. Such mitochondrial damage is not caused by CCl4-induced stimulation of lipid peroxidation since it is not prevented by alpha-tocopherol, used at a concentration able to inhibit completely peroxidative reactions without interfering with CCl4 activation. All data together are in favour of a direct action of CCl4-reactive metabolites on liver cell calcium homeostasis.

Metabolic activation of 1,2-dibromoethane to a free radical intermediate by rat liver microsomes and isolated hepatocytes.

A one-electron reductive metabolism of 1,2-dibromoethane (DBE) is described that gives rise to a free radical intermediate, which can be stabilized by a spin trapping agent and detected by electron spin resonance spectroscopy. Using rat liver microsomes or isolated hepatocytes from phenobarbitone pretreated animals, under hypoxic conditions, it has been possible to trap a free radical intermediate and identify it by using 13C-DBE. Inhibition experiments have demonstrated that the site of activation is the microsomal drug metabolizing system.

Effect of 4-Hydroxynonenal on cell cycle progression and expression of differentiation-associated antigens in HL-60 cells.

4-Hydroxynonenal (HNE) is a highly reactive aldehyde produced by lipid peroxidation of cellular membranes that inhibits growth and induces differentiation in HL-60 cells. Its mechanisms of action were investigated by analyzing the cell cycle distribution and the appearance of differentiated phenotypes in HL-60 cells. Data obtained by exposing cells to DMSO for 7.5 h (same time as for HNE treatment) or for the whole length of the experiments (5 d) were used for comparison. HNE induced a marked increase in the proportion of G0/G1 cells after 1 and 2 d. The brief DMSO treatment did not affect the distribution, whereas continuous exposure led to a progressive accumulation of cells in G0/G1 (maximal at day 5). The proportion of phagocytic cells gradually increased in HNE-treated and DMSO long-exposed cultures from day 2 and peaked at day 5 (35 and 63%, respectively), whereas the effect of the brief DMSO treatment was negligible. The expression of CD11b and CD67 increased in cells treated with HNE or continuously exposed to DMSO, whereas CD36 was expressed at low levels on both treatments. These results indicate that the pathway of the granulocytic differentiation induced by HNE in HL-60 cells differs from that of DMSO: with HNE, growth inhibition precedes the onset of differentiation, whereas in DMSO-treated cells the two processes are chronologically associated.

Protein thiols in normal and neoplastic human uterine cervix.

Protein-thiol groups that react with dihydroxydinaphthyl disulphide during a 7 h incubation (so-called reactive protein thiols, PSHr) have been quantitatively measured on sections of human uterine cervix by microcytospectrophotometry. Measurements were made on areas (1 micron 2) of epithelium and adjoining stroma in samples of normal cervix, and in samples obtained from patients with dysplasia, carcinoma-in-situ and invasive cancer. The ratio of PSHr in epithelium to stroma is substantially reduced in the pathological conditions compared with normal and in apparently normal adjacent areas. Such changes in PSHr are discussed in relation to the redox balance of the tissue, and free radical disturbances previously described.

Enrichment with arachidonic acid increases the sensitivity of hepatoma cells to the cytotoxic effects of oxidative stress.

Hepatoma cells are, at most, moderately sensitive to oxidative stress. An important cause of this lack of sensitivity is the decreased content of polyunsaturated fatty acids in comparison with normal cells. These fatty acids are one cellular target of oxygen radicals, by which they are broken down into several toxic carbonyl compounds. If the membrane phospholipids of tumor cells are enriched with polyunsaturated fatty acids, such as arachidonic acid, they become able to undergo lipid peroxidation in the presence of prooxidants. This effect is studied in the highly deviated Yoshida AH-130 ascites hepatoma and in two rat hepatoma cell lines. In parallel to their increased lipid peroxidation, cells enriched with arachidonic acid and exposed to ascorbic acid/FeSO4 showed lower viability and growth than unenriched ones.

Inhibition of class-3 aldehyde dehydrogenase and cell growth by restored lipid peroxidation in hepatoma cell lines.

Hepatoma cells have a below-normal content of polyunsaturated fatty acids; this reduces lipid peroxidation and the production of cytotoxic and cytostatic aldehydes within the cells. In proportion to the degree of deviation, hepatoma cells also show an increase in the activity of Class-3 aldehyde dehydrogenase, an enzyme important in the metabolism of lipid peroxidation products and also in that of several drugs. When hepatoma cells with different degrees of deviation were enriched with arachidonic acid and stimulated to peroxidize by ascorbate/iron sulphate, their growth rate was reduced in proportion to the quantity of aldehydes produced and to the activity of aldehyde dehydrogenase. Therefore, 7777 cells, less deviated and with low Class-3 aldehyde dehydrogenase activity, were more susceptible to lipid peroxidation products than JM2 cells. It is noteworthy that repeated treatments with prooxidant also caused a decrease in mRNA and activity of Class-3 aldehyde dehydrogenase, contributing to the decreased growth and viability. Thus, Class-3 aldehyde dehydrogenase could be considered relevant for the growth of hepatoma cells, since it defends them against cell growth inhibiting aldehydes derived from lipid peroxidation.

4-Hydroxynonenal as a biological signal: molecular basis and pathophysiological implications.

Reactive oxygen intermediates (ROI) and other pro-oxidant agents are known to elicit, in vivo and in vitro, oxidative decomposition of omega-3 and omega-6 polyunsaturated fatty acids of membrane phospholipids (i.e, lipid peroxidation). This leads to the formation of a complex mixture of aldehydic end-products, including malonyldialdehyde (MDA), 4-hydroxy-2,3-nonenal (HNE), and other 4-hydroxy-2,3-alkenals (HAKs) of different chain length. These aldehydic molecules have been considered originally as ultimate mediators of toxic effects elicited by oxidative stress occurring in biological material. Experimental and clinical evidence coming from different laboratories now suggests that HNE and HAKs can also act as bioactive molecules in either physiological and pathological conditions. These aldehydic compounds can affect and modulate, at very low and nontoxic concentrations, several cell functions, including signal transduction, gene expression, cell proliferation, and, more generally, the response of the target cell(s). In this review article, we would like to offer an up-to-date review on this particular aspect of oxidative stress--dependent modulation of cellular functions-as well as to offer comments on the related pathophysiological implications, with special reference to human conditions of disease.