Mario U. Dianzani

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.

The role of lipid peroxidation in liver damage

The consequences of the peroxidative breakdown of membrane lipids have been considered in relation to both the subcellular and tissue aspects of liver injury. Mitochondrial functions can be impaired by lipid peroxidation probably through the oxidation of pyridine nucleotides and the consequent alteration in the uptake of calcium. Several enzymatic functions of the endoplasmic reticulum are also affected as a consequence of peroxidative events and among these are the activities of glucose 6-phosphatase, cytochrome P-450 and the calcium sequestration capacity. Moreover, a release of hydrolytic enzymes from lysosomes and a decrease in the fluidity of plasma membranes can contribute to the liver damage consequent to the stimulation of lipid peroxidation. Extensive studies carried out in vivo and integrated with the use of isolated hepatocytes have shown that lipid peroxidation impairs lipoprotein secretion mainly at the level of the dismission from the Golgi apparatus, rather than during their assembly. However, such an alteration appears to give a late and not essential contribution to the fat accumulation. A more critical role is played by peroxidative reactions in the pathogenesis of acute liver necrosis induced by several pro-oxidant compounds as indicated by the protective effects against hepatocyte damage exerted by antioxidants. In addition, even in the cases where lipid peroxidation has been shown not to be essential in causing cell death there is evidence that it can still act synergistically with other damaging mechanisms in the amplification of liver injury.

Lipid peroxidation and cancer

Lipid peroxidation, firstly described as a mechanism of lipid deterioration by chemists, is now considered as a major mechanism of elementary lesion in living cells, and in many cases the major cause for their death. In living animals, it was firstly described to increase after irradiation [23,32,252] and then in vitamin E deficiency [57,258,259]. The discovery that it increases strongly in the liver of CC&-poisoned rats [64,195,196] opened wider perspectives to the meaning of this change in pathology. The onset of lipid peroxidation in the presence of CC& requires the metabolism of this substance in the smooth endoplasmic reticulum of the hepatocyte by the local drug metabolizing system involving cytochrome P-450 [221,222]. This leads to the formation of the trichloromethyl (CCls ’ ) free radical [4,64,192, 223,240]. In the presence of oxygen, this is converted into the trichloroperoxy (CC1s02 * ) free radical [ 116,174]. By using promethazine or vitamin E in an in vitro system, Slater’s group succeeded in showing that covalent binding of CCLderived free radicals to proteins, lipids or nucleotides is effected by CC13 * , whereas lipid peroxidation is started by CClsO* * . In fact, both promethazine and vitamin E are able to block completely at the same time the reactions of the last free radicals and lipid peroxidation whereas they are practically devoid of any effect both on the reactions of ccl, and on covalent binding. The use of promethazine or vitamin E in vivo in rats treated with CC& allowed the demonstration that some of the damages provoked by this haloalkane are due to covalent binding, whereas other types of damage were mostly due to lipid peroxidation [86,87,186,188]. Acute cell death occurring after Ccl, is mostly related to this chemical derangement, whereas the block in lipoprotein secretion, that is the most important cause for fatty liver, is due to the haloalkylation of apolipoproteins [ 1851. As these results were extended to the poisons [86,87.186] it became clear that lipid peroxidation may be an important mechanism in cell death. The demonstration that lipid peroxidation is low in rapidly proliferating tissues is rather old. The first paper in this field is that by Kohn and Liversedge [141], who found very low lipid peroxidation in rapidly proliferating tumours, as glioblastoma and fibrosarcoma,

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.

Free radicals : from basic science to medicine

Free radicals: Generation and mechanisms of damage.- Trevor Slater, free radical redox chemistry and antioxidants: from NAD+ and vitamin C to CCl4 and vitamin E, to thiols, myoglobin and vitamins A and D.- Regulation of gene expression in adaptation to oxidative stress.- Radiation-induced free radical reactions.- Nitric oxide and related radicals.- Mechanisms of oxidative cell damage.- Lipid peroxidation. An overview.- Lipid peroxidation in dividing cells.- Formation and metabolism of the lipid peroxidation product 4-hydroxynonenal in liver and small intestine.- DNA damage by reactive oxygen species. The role of metals.- Inflammation and a mechanism of hydrogen peroxide cytotoxicity.- Aging.- Free radical theory of aging.- Oxidative stress state in aging and longevity mechanisms.- Maillard reaction and oxidative stress are interrelated stochastic mechanisms of aging.- The glycoxidation: a non enzymatic mechanism of protein aging.- Marker or mechanism: possible pro-oxidant reactions of radical-damaged proteins in aging and atherosclerosis, an age-related disease.- Cancer.- Carcinogenesis and free radicals.- Derangements of cellular metabolism in the pre-malignant syndrome.- Molecular mechanisms of oxidant carcinogenesis.- Involvement of oxy-radicals in cancer cell killing and growth.- Free radicals and active states of oxygen in human cancer due to environmental pollutants: public health optimism and scientific skepticism.- Metabolic disorders.- Hepatotoxicity of experimental hemochromatosis.- Role of inducible cytochrome P450 in the liver toxicity of poly-halogenated aromatic compounds.- CBrCl3 toxicity in isolated rat hepatocytes: survey on reasonable cytotoxic mechanisms.- Evidence for a possible role of lipid peroxidation in experimental liver fibrosis.- Ethanol-inducible cytochrome P450 2E1. regulation, radical formation and toxicological importance.- Free radical-induced impairment of liver glycosylation processes in ethanol intoxication.- Oxidative damage and human alcoholic disease. Experimental and clinical evidence.- Oxidised low density lipoproteins.- Protein peroxides: formation by superoxide-generating systems and during oxidation of low density lipoprotein.- An in vitro approach to the study of inflammatory reactions in atherosclerosis.- Antioxidants.- Antioxidant defenses in eukaryotic cells: an overview.- Electron paramagnetic resonance studies on flavonoid compounds.- Antioxidant mechanisms of vitamin E and beta-carotene.- A nuclear pool of glutathione in hepatocytes.- Phospholipid hydroperoxide glutathione peroxidase is the major selenoperoxidase in nuclei and mitochondria of rat testis.- Medical applications of antioxidants: an update of current problems.- Free radicals and antioxidants in muscular and neurological diseases and disorders.- Pharmaceutical intervention for the prevention of post-ischemic reperfusion injury.- Metal-catalyzed free radical injuries in chilhood: disorders and pharmaceutical intervention.- Effect of antioxidants on oxidative modification of human low density lipoproteins.- Approaches to the therapy of glutathione deficiency.- Utilization of oral glutathione.- Author Index.

Role of ethanol-inducible cytochrome P450 (P450IIE1) in catalysing the free radical activation of aliphatic alcohols

Incubation of rat liver microsomes with 1-propanol and 1-butanol in the presence of NADPH and of the spin trapping agent 4-pyridyl-1-oxide-t-butyl nitrone (4-POBN) allowed the detection of free radical intermediates tentatively identified as 1-hydroxypropyl and 1-hydroxybutyl radical, respectively. Microsomes isolated from rats treated chronically with ethanol (EtOH) or with the combination of starvation and acetone treatment (SA), exhibited a two-fold increase in the ESR signal intensity as compared to untreated controls, whereas no increase was observed in phenobarbital-induced (PB) microsomes. Consistently, in reconstituted membrane vesicles, ethanol-inducible cytochrome P450IIE1 was twice as active as phenobarbital-inducible P450IIB1 in producing 1-butanol free radicals. In the microsomal preparations from EtOH and SA pretreated rats the addition of antibodies against cytochrome P450IIE1, but not of preimmune IgGs, lowered the ESR signal of 1-butanol radicals by more than 50%. The same antibodies decreased the free radical production by untreated microsomes by 35-40%, but were ineffective on microsomes from PB-treated animals. This indicated that cytochrome P450IIE1 is the major enzyme responsible for the free radical activation of alcohols in control and ethanol-fed rats. The generation of 1-hydroxybutyl radicals by EtOH microsomes was inhibited by 40, 48 and 68%, respectively, by the addition of isoniazid, tryptamine and octylamine, compounds known to specifically affect the NADPH oxidase activity of this isoenzyme. This effect was not due to the scavenging of the alcohol radical since none of these compounds affected the ESR signals originated from 1-butanol in a xanthine-xanthine oxidase system. When added to reconstituted membrane vesicles isoniazid, tryptamine and octylamine also decreased 1-butanol radical formation by P450IIE1 by 54, 38 and 66%, respectively. Such an inhibition corresponded to the effect exerted by the same compounds on O2- release from P450IIE1 containing vesicles. These results indicate that the capacity of cytochrome P450IIE1 to reduce oxygen is related to its ability to generate alcohol free radicals and suggest that ferric cytochrome P450-oxygen complex might act as oxidizing species toward alcohols.

Studies on fatty liver with isolated hepatocytes: II. The action of carbon tetrachloride on lipid peroxidation, protein, and triglyceride synthesis and secretion

Abstract This report describes some effects of poisoning with carbon tetrachloride on hepatocytes in single cell suspension. In isolated liver cells as well as “ in vivo ” CCl 4 stimulates lipid peroxidation, inhibits both protein synthesis and protein and lipoprotein secretion and induces fat accumulation within the cells. As the action of CCl 4 on lipid peroxidation, our data confirm that its increase induced by CCl 4 depends on the metabolism of this drug by the NADPH-cytochrome P 450 enzyme system. Furthermore, data reported here suggest that the onset of the CCl 4 -induced decrease of lipoprotein secretion is due to a derangement of the secretory pathway.

Spin trapping of free radical species produced during the microsomal metabolism of ethanol

Liver microsomes incubated with a NADPH regenerating system, ethanol and the spin trapping agent 4-pyridyl-1-oxide-t-butyl nitrone (4-POBN) produced an electron spin resonance (ESR) signal which has been assigned to the hydroxyethyl free radical adduct of 4-POBN by using 13C-labelled ethanol. The free radical formation was dependent upon the activity of the microsomal monoxygenase system and increased following chronic feeding of the rats with ethanol. The production of hydroxyethyl free radicals was stimulated by the addition of azide, while catalase and OH. scavengers decreased it. This suggested that hydroxyl radicals (OH.) produced in a Fenton-type reaction from endogenously formed hydrogen peroxide were involved in the free radical activation of ethanol. Consistently, the supplementation of iron, under various forms, also increased the intensity of the ESR signal which, on the contrary, was inhibited by the iron-chelating agent desferrioxamine. Microsomes washed with a solution containing desferrioxamine and incubated in a medium treated with Chelex X-100 in order to remove contaminating iron still produced hydroxyethyl radicals, although at a reduced rate. Under these conditions the free radical formation was apparently independent from the generation of OH. radicals, whereas addition of cytochrome P-450 inhibitors decreased the hydroxyethyl radical formation, suggesting that a cytochrome P-450-mediated process might also be involved in the activation of ethanol. Reduced glutathione (GSH) was found to effectively scavenge the hydroxyethyl radical, preventing its trapping by 4-POBN. The data presented suggest that ethanol-derived radicals could be generated during the microsomal metabolism of alcohol probably through two different pathways. The detection of ethanol free radicals might be relevant in understanding the pathogenesis of the liver lesions which are a consequence of alcohol abuse.

Activation of chloroform and related trihalomethanes to free radical intermediates in isolated hepatocytes and in the rat in vivo as detected by the ESR-spin trapping technique

When hepatocytes isolated from phenobarbital-induced rats were incubated with chloroform and the spin trap phenyl-t-butyl nitrone (PBN) under anaerobic conditions, a free radical-spin trap adduct was detectable by ESR spectroscopy. A similar incubation of hepatocytes in the presence of air resulted in an ESR signal that was eight times less intense than that seen under anaerobic conditions; incubation mixtures exposed to pure oxygen had no detectable adduct signal. A significant reduction in the signal intensity was also produced by the addition of cytochrome P-450 inhibitors such as SKF-525A, metyrapone and carbon monoxide, indicating that free radical formation depended upon the reductive metabolism of chloroform mediated by the mixed oxidase system. The origin of the CHCl3-derived free radical has been confirmed by using [13C]CHCl3, while the comparison between the ESR spectra obtained in the presence of deuterated chloroform (CDCl3) and bromodichloro-methane (CHBrCl2) suggests that the free radical derived from CHCl3 may be CHCl2. Free radical intermediates were also detected during the aerobic and anaerobic incubation of isolated hepatocytes with bromoform (CHBr3), and iodoform (CHI3). The intensity of the ESR signal obtained with the various trihalomethanes increases in the order CHCl3 less than CHBrCl2 less than CHBr3 less than CHI3. The formation of PBN-free radical adducts has also been observed in phenobarbital-induced rats in vivo when intoxicated with chloroform, bromoform or iodoform, suggesting that the reductive metabolism of trihalomethanes might be of relevance to their established toxicity in the whole animal.

Induction of differentiation in human HL-60 cells by 4-hydroxynonenal, a product of lipid peroxidation☆

4-Hydroxynonenal (HNE) is the major diffusible toxic product generated by lipid peroxidation of cellular membranes. The level of lipid peroxidation and, consequently, the concentration of its products are inversely related to the rate of cell proliferation and directly related to the level of cell differentiation. In the present paper the effects of HNE on the proliferation and differentiation of the HL-60 human promyelocytic cell line have been investigated. Repeated treatment at 45-min intervals with HNE (1 microM) was performed to maintain the cells in the presence of the aldehyde for 7 1/2 or 9 h. The effect of HNE on cell proliferation and differentiation was compared with dimethyl sulfoxide (DMSO)-treated cells. HNE causes a strong inhibition of cell growth without affecting cell viability. Moreover, HL-60 cells acquire the capability to produce chemiluminescence after soluble (phorbol myristate acetate) or corpuscolate (zymosan) stimulation. The phagocytic ability has also been calculated by counting the number of cells that phagocytize opsonized zymosan. Values were 43 and 55% after 10 or 12 HNE treatments, respectively, and 88% in DMSO-treated cells. Myeloperoxidase activity, 5 days after treatment, decreased by 85% in either HNE- or DMSO-treated cells while acid phosphatase activity increased with respect to untreated cells. Results obtained indicate that HNE at concentrations close to those found in the normal tissues can induce inhibition of proliferation and induction of differentiation in the HL-60 cell line.