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Protective effects of cysteine, methionine and vitamin C on the stomach in chronically alcohol treated rats
Abstract
A chronic intake of high dose alcohol may cause oxidative stress and inflammation in the stomach. It is hypothesized that cysteine-methionine and vitamin C may neutralize harmful compounds while potentiating the antioxidant capacity of the cell or tissue. The experimental animals were fed regular diets and were maintained for 90 days in the control group, the alcoholic group, which was given 2.5 g of 50% ethanol kg(-1) body wt. administered intragastrically every other day, or the alcoholic with antioxidant supplement group, to whom 2.5 g of 50% ethanol kg(-1) body wt. + a solution that contained 200 mg vitamin C, 100 mg cysteine and 100 mg methionine was administered intragastrically every other day. After the treatments, the stomach was taken for pathological and biochemical analysis. The stomach of the alcoholic group rats had higher scores of pathological findings compared with the control group, whereas the scores of the antioxidant-supplemented group were lower than the alcoholic group. In addition, the oxidized protein and lipid content in the stomachs of the alcoholic group were significantly higher than the control, but antioxidant supplementation lowered the amount of oxidation in the antioxidant supplemented group. The amount of stomach glutathione in the alcoholic group was higher than that of the control and antioxidant-supplemented groups. Interestingly, the level of total thiol in the stomach tissue of rats with antioxidant supplement was statistically higher than that of the control and alcoholic groups. In conclusion, the scores of the pathological findings in the stomach of rats with the antioxidant supplement were lower than the chronic alcohol-treated rats, albeit the amount of total thiol was increased in this group. Moreover, chronic alcohol treatment led to an increase in the level of lipid and protein oxidation in the stomach tissue of rats. A simultaneous intake of ascorbate/l-cys/l-met along with ethanol attenuated the amount of oxidation which suggested that cysteine-methionine and vitamin C could play a protective role in the stomach against oxidative damage resulting from chronic alcohol ingestion.
... On the other hand, increased oxidative stress plays a key role in the pathogenesis of ethanol-induced gastric damage, so that as oral ethanol produces gastric mucosal lesions and erosions, it increases lipid peroxidation, raises hydroxyl radicals generation, and causes DNA damage, while it lowers the gastric content of reduced glutathione. Also, ethanolinduced ulceration is linked to reduced mucosa microcirculation and to increased apoptosis1213141516. Vitamin C (VC) (ascorbic acid) is a water-soluble antioxidant that directly scavenges ROS, like superoxide and hydroxyl radicals, hydrogen peroxide, singlet oxygen, and hypochlorous acid, and also guarantees the chain-breaking antioxidant action of vitamin E (VE) by reducing the VE radical to VE [17, 18]. ...
Effects of GSE and vitamins C and E on aspirin- and ethanol-induced gastric ulcer and associated increases of lipid peroxidation in rats were compared. Two experiments were conducted. Rats were randomized into eight groups: a negative control and seven groups that received aspirin or ethanol for ulcer induction: one positive control (vehicle) and six with VC, VE, or GSE (25 and 250 mg/kg). Ulcer indexes and gastric levels of malondialdehyde (MDA) were quantified. VC, VE, and GSE (25 and 250 mg/kg) decreased aspirin, and ethanol-induced ulcers and MDA values compared with positive control group. The magnitude of aspirin ulcer reduction was comparable for all treatments, and MDA decrease with GSE was higher than with VC and tended to be greater, albeit none significantly, than with VE. GSE was more effective than VC and VE for lowering the ethanol ulcers, while the decrease of MDA levels with GSE was greater than with VC, but comparable to that achieved with VE. GSE protected against ethanol-induced gastric ulcers more effectively than VC or VE, while its protection against aspirin ulcers was comparable for all treatments. GSE produced the greatest reductions of gastric MDA in both models.
... A plausible explanation for the latter is the adaptation of the gastric mucosa to CMSinduced ROS, where the presence of either decreased [6,8,31] or increased [31,32] levels of endogenous antioxidants, with a net unchanged concentration, was previously reported in ulcer models including acute stress. Meanwhile, we observed a decline in the level of the anti-inflammatory cytokine IL-10, which accounts for the overwhelming antagonistic effect of TNF-α as reported by Brossart et al [33] , which may additionally aggravate gastric lesion formation. Recently, genetic IL-10 polymorphism has been found to predispose individuals to peptic ulcer [34] , thus lending further support to our findings. ...
To evaluate the effect of chronic mild stress (CMS) on the emergence of gastric ulcers and possible modulation by octreotide, a synthetic somatostatin analogue.
Adult male Wistar rats were subjected to nine different unpredictable random stress procedures for 21 d, a multifactorial interactional animal model for CMS. Octreotide was administered daily for 21 d at two dose levels (50 and 90 μg/kg) before exposure to stress procedure. Macro- and microscopical assessments were made, in addition to quantification of plasma corticosterone and gastric mucosal inflammatory, oxidative stress, and apoptotic biomarkers.
Exposure to CMS elevated plasma corticosterone (28.3 ± 0.6 μg/dL, P = 0.002), an event that was accompanied by gastric lesions (6.4 ± 0.16 mm, P = 0.01) and confirmed histopathologically. Moreover, the insult elevated gastric mucosal lipid peroxides (13 ± 0.5 nmol/g tissue, P = 0.001), tumor necrosis factor-α (3008.6 ± 78.18 pg/g tissue, P < 0.001), prostaglandin E2 (117.1 ± 4.31 pg/g tissue, P = 0.002), and caspase-3 activity (2.4 ± 0.14 OD/mg protein, P = 0.002). Conversely, CMS mitigated interleukin-10 (627.9 ± 12.82 pg/g tissue, P = 0.001). Furthermore, in animals exposed to CMS, octreotide restored plasma corticosterone (61% and 71% from CMS, P = 0.002) at both dose levels. These beneficial effects were associated with a remarkable suppression of gastric lesions (38% and 9% from CMS, P = 0.01) and reversal of derangements in gastric mucosa.
The current investigation provides evidence that exposure to CMS induces gastric ulceration, which was alleviated by administration of octreotide possibly possessing antioxidant, anti-inflammatory, and anti-apoptotic actions.
... Induction of enzymes involved in GSH metabolism such as GSH reductase has been reported. 27,28 Similar findings to ours have been reported in a recent rat study in which chronic alcohol ingestion induces higher GSH level and lipid and protein peroxidation in the rat stomach. 24 The mechanism by which the jejunum responds to chronic ethanol ingestion by independently regulating these 2 thiol redox pairs, Cys/CySS and GSH/GSSG, is unclear and requires additional investigation. ...
Alcohol consumption is associated with oxidative stress in multiple tissues in vivo, yet the effect of chronic alcohol intake on intestinal redox state has received little attention. In this study, we investigated the redox status of 2 major intracellular redox regulating couples: glutathione (GSH)/glutathione disulfide (GSSG) and cysteine (Cys)/cystine (CySS) in a rat model of chronic alcohol ingestion.
Sprague-Dawley rats were fed the liquid Lieber-DeCarli diet consisting of 36% ethanol of total calories for 6 weeks. Control rats were pair-fed with an isocaloric, ethanol-free liquid diet. Defined mucosal samples from the jejunum, ileum, and colon were obtained and analyzed by high-performance liquid chromatography (HPLC) for GSH and Cys pool redox status. Mucosal free malondialdehyde (MDA) was measured as an indicator of lipid peroxidation.
In the ethanol-fed rats, Cys and mixed disulfide (GSH-Cys) were significantly decreased in all 3 segments of intestinal mucosa. Free MDA was increased in jejunal but not in ileal or colonic mucosa. Chronic ethanol ingestion significantly increased mucosal GSH concentration in association with a more reducing GSH/GSSG redox potential in the jejunum, but these indices were unchanged in the ileum. In the colon, chronic ethanol ingestion increased oxidant stress as suggested by decreased GSH and oxidized GSH/GSSG redox potential.
Chronic alcohol intake differentially alters the mucosal redox status in proximal to distal intestinal segments in rats. Such changes may reflect different adaptability of these intestinal segments to the oxidative stress challenge induced by chronic ethanol ingestion.
Inflammation has attracted widespread attention because of its broad involvement in medical conditions. Examples are cancer, bowel diseases, Crohn's disease, asthma, chronic obstructive pulmonary disease, sarcoidosis, multiple sclerosis, as well as aging. Substantial evidence indicates involvement of reactive oxygen species (ROS) and oxidative stress (OS). There is debate whether radicals are responsible for the condition, arise from the condition, or both. There are various types of anti-inflammatory agents, with this review being concentrated on the antioxidant category. Mechanisms are discussed. The various types include phenols, resveratrol, curcumin, flavonoids, N-acetylcysteine, and nanoparticles. © Springer-Verlag Berlin Heidelberg 2014. All rights are reserved.
Objectives: Neutrophil-mediated inflammation and oxidative stress may be the important events that lead to gastroduodenal mucosal damage induced by aspirin. It is hypothesized that GSH-vitamin C-E may protect to the upper gastrointestinal mucosa against low dose aspirin's (L-ASA) adverse effects. Material and methods: Male rats were fed regular diets and maintained for 40 days in the control group (n=8), the L-ASA group (n=8), which was given aspirin (1.44 mg/kg/ day) administered intragastrically by feeding tube to rats, or the L-ASA with antioxidant supplement group (n=8), to whom 1.44 mg of aspirin/kg/day + a solution that contained 50 mg vitamin C, 25 mg vitamin E and 25 mg GSH was administered intragastrically by feeding tube to rats every day. After the treatments, blood, stomach and duodenum were taken for pathological and myeloperoxidase (MPO) activity, heat shock protein (Hsp) 70, lipid and protein oxidation analyses. Results: The stomach and duodenum of the L-ASA group rats had higher scores of pathological findings compared with the control group, whereas the severity of the lesions was found low in the antioxidant-supplemented group according to the L-ASA group. In addition, the gastric mucosal MPO activity and Hsp 70 levels in the L-ASA group were significantly higher than control, but antioxidant supplementation lowered the values of MPO and Hsp 70 in the antioxidant supplemented group (P=0.009, P=0.04). Conclusion: A simultaneous intake of GSH-vitamin C-E along with aspirin attenuated the gastric injury. GSH-vitamin C and E could play a protective role in the stomach against gastric damage resulting from low dose daily long-term aspirin use.
Objective: To study effects of liver hydrolysate (LH) containing various functional components which promote antioxidant and bioregulational effects on acute alcohol intoxication in mice. Methods: To investigate the effects of LH on hepatic ethanol metabolism, LH (1 g/kg body weight) were orally administered to 9-week-old male C3H/HeNCrj (C3H/He) mice (LH group), followed by oral administration of ethanol (2 g/kg body weight). The changes in concentrations of ethanol, acetaldehyde and acetic acid in serum and activities of enzymes related to ethanol metabolism in the liver were measured from 0 to 5 hours after the ethanol administration. Results: The concentrations of ethanol, acetaldehyde and acetic acid in serum of the LH group at 0.5, 2 and 3 hours after the ethanol administration were significantly lower than those of the control group (mice administered distilled water instead of LH). The specific activities of alcohol dehydrogenase (ADH), high-Km, aldehyde dehydrogenase (ALDH) in the cytosolic fraction and low-Km, high-Km and total ALDH in the mitochondrial fraction of the LH group at 2 hours after the ethanol administration were significantly lower than those of the control group. Conclusion: The present study demonstrates that oral administration of LH suppresses the increase in the serum concentration of acetaldehyde in mice. As acetaldehyde is considered to be a major contributing factor in the development of acute alcohol intoxication, the observed effects of LH may be relevant for symptom improvement in acute alcohol intoxication.
Radical-mediated damage to proteins may be initiated by electron leakage, metal-ion-dependent reactions and autoxidation of lipids and sugars. The consequent protein oxidation is O2-dependent, and involves several propagating radicals, notably alkoxyl radicals. Its products include several categories of reactive species, and a range of stable products whose chemistry is currently being elucidated. Among the reactive products, protein hydroperoxides can generate further radical fluxes on reaction with transition-metal ions; protein-bound reductants (notably dopa) can reduce transition-metal ions and thereby facilitate their reaction with hydroperoxides; and aldehydes may participate in Schiff-base formation and other reactions. Cells can detoxify some of the reactive species, e.g. by reducing protein hydroperoxides to unreactive hydroxides. Oxidized proteins are often functionally inactive and their unfolding is associated with enhanced susceptibility to proteinases. Thus cells can generally remove oxidized proteins by proteolysis. However, certain oxidized proteins are poorly handled by cells, and together with possible alterations in the rate of production of oxidized proteins, this may contribute to the observed accumulation and damaging actions of oxidized proteins during aging and in pathologies such as diabetes, atherosclerosis and neurodegenerative diseases. Protein oxidation may also sometimes play controlling roles in cellular remodelling and cell growth. Proteins are also key targets in defensive cytolysis and in inflammatory self-damage. The possibility of selective protection against protein oxidation (antioxidation) is raised.
The effects of a mega dose of ascorbic acid (AA) on alcohol induced peroxidative damages were investigated in guinea pigs. In the present study, four groups of male guinea pigs were maintained for 30 days as follows. (1) Control group (1 mg AA/100 g body wt); (2) Ethanol group (1 mg AA/100 g body wt. + 9 g ethanol/kg body wt); (3) AA group (25 mg AA/100 g body wt); (4) AA + ethanol group (25 mg AA/100 g body wt. + 9 g ethanol/kg). Results revealed that alcohol induced significant lipid peroxidation, since the lipid peroxidation products malondialdehyde (MDA), hydroperoxides and conjugated dienes were elevated. The activities of scavenging enzymes superoxide dismutase (SOD), catalase were reduced. However, supplementation of AA along with alcohol reduced the lipid peroxidation products in the liver and enhanced the activities of scavenging enzymes. Activities of glutathione peroxidase and reductase were also greater in guinea pigs given alcohol + AA in comparison with those given alcohol alone. Administration of ascorbic acid also reduced the activity of gamma-glutamyl transpeptidase (GGT), the marker enzyme of alcohol induced toxicity. The vitamin E level, which was reduced by alcohol intake, was raised by the co-administration of AA and alcohol. These studies suggest that a mega dose of AA helps in the prevention of alcohol induced oxidative stress by enhancing the antioxidant capacity and also by reducing the lipid peroxidation products.
A number of systems that generate oxygen free radicals and reactive aldehydic species are activated by excessive ethanol consumption. Recent studies from human alcoholics and from experimental animals have indicated that acetaldehyde and aldehydic products of lipid peroxidation, which are generated in such processes, can bind to proteins forming stable adducts. Adduct formation may lead to several adverse consequences, such as interference with protein function, stimulation of fibrogenesis, and induction of immune responses. The presence of protein adducts in the centrilobular region of the liver in alcohol abusers with an early phase of histological liver damage indicates that adduct formation is one of the key events in the pathogenesis of alcoholic liver disease. Dietary supplementation with fat and/or iron strikingly increases the amount of aldehyde-derived epitopes in the liver together with promotion of fibrogenesis.
Muscle tissue serves as a protein reservoir which is mobilized to meet the specific metabolic needs associated with various catabolic conditions in human subjects, such as trauma and critical illness. Glutathione is one of the most abundant short-chain peptides and a major source of non-protein thiol in the body, and tissue glutathione concentration is related to its oxidative capacity. Skeletal muscle is relatively unique with respect to a variety of metabolic properties, such as oxidative potential, patterns of amino acid utilization, and antioxidant enzyme activity. The glutathione concentration is not influenced by food intake, or by food deprivation. Moreover, there is no diurnal variation on muscle glutathione levels. Following elective surgery the muscle concentration of GSH (the reduced form) decreases by 40% 24 h post-operatively, while the concentration of GSSG (the oxidized form) remains unaltered. During critical illness a similar decrease in the GSH concentration is seen, but in addition a change in the redox status indicative of an elevated GSSG level occurs. Furthermore, correlations between the concentrations of glutamine as well as glutamate and GSH exist in these patients. From available evidence accumulated it is clear that glutathione plays a pivotal role in the maintenance of the intracellular redox status, the antioxidant vitamin levels, and the antioxidant enzyme functions under various metabolic conditions. The effectiveness of glutathione protection in the individual tissue depends on the tissue concentration of glutathione as well as the capacity of the tissue to import GSH and to export GSSG. The mechanisms by which catabolism regulates tissue glutathione levels and the enzyme activities associated with the gamma-glutamyl cycle are not completely understood and further studies need to be conducted.
There is a wealth of epidemiological information on antioxidants and their possible prevention of disease progression but very little of the research on antioxidants has involved intervention studies. In this study, the potential protective effect of vitamin C or E supplementation in vivo against endogenous and H2O2-induced DNA damage levels in lymphocytes was assessed. The supplementation involved fourteen healthy male and female non-smokers mean age 25-53 (SD 1.82) years, who were asked to supplement an otherwise unchanged diet with 1000 mg vitamin C daily for 42 d or 800 mg vitamin E daily for 42 d. DNA damage in H2O2-treated peripheral blood lymphocytes (PBL) and untreated PBL before and after supplementation, and during a 6-week washout period was assessed using an ELISA. At each sampling time-point, the red cell concentrate activities of superoxide dismutase, catalase and glutathione peroxidase were also determined. Supplementation with vitamin C or vitamin E decreased significantly H2O2-induced DNA damage in PBL, but had no effect on endogenous levels of DNA damage. The activities of the antioxidant enzymes superoxide dismutase and glutathione peroxidase were suppressed during the supplementation period. These supplementation regimens may be used to limit the possible adverse effects of reactive oxygen species (including those produced during the course of an immune response) on lymphocytes in vivo, and so help to maintain their functional capacity.
We summarize here results of studies designed to elucidate basic mechanisms of reactive oxygen (ROS)-mediated oxidation of proteins and free amino acids. These studies have shown that oxidation of proteins can lead to hydroxylation of aromatic groups and aliphatic amino acid side chains, nitration of aromatic amino acid residues, nitrosylation of sulfhydryl groups, sulfoxidation of methionine residues, chlorination of aromatic groups and primary amino groups, and to conversion of some amino acid residues to carbonyl derivatives. Oxidation can lead also to cleavage of the polypeptide chain and to formation of cross-linked protein aggregates. Furthermore, functional groups of proteins can react with oxidation products of polyunsaturated fatty acids and with carbohydrate derivatives (glycation/glycoxidation) to produce inactive derivatives. Highly specific methods have been developed for the detection and assay of the various kinds of protein modifications. Because the generation of carbonyl derivatives occurs by many different mechanisms, the level of carbonyl groups in proteins is widely used as a marker of oxidative protein damage. The level of oxidized proteins increases with aging and in a number of age-related diseases. However, the accumulation of oxidized protein is a complex function of the rates of ROS formation, antioxidant levels, and the ability to proteolytically eliminate oxidized forms of proteins. Thus, the accumulation of oxidized proteins is also dependent upon genetic factors and individual life styles. It is noteworthy that surface-exposed methionine and cysteine residues of proteins are particularly sensitive to oxidation by almost all forms of ROS; however, unlike other kinds of oxidation the oxidation of these sulfur-containing amino acid residues is reversible. It is thus evident that the cyclic oxidation and reduction of the sulfur-containing amino acids may serve as an important antioxidant mechanism, and also that these reversible oxidations may provide an important mechanism for the regulation of some enzyme functions.
Although it has been speculated that active oxdants and mitochondrial membrane damages play roles in ethanol‐induced gastric mucosal damages, its detail remains unknown. The present study was designed to investigate whether ethanol induces oxidative stress and mitochondrial permeability transition (MPT) before cell death of gastric mucosal cells. Rat gastric mucosal cells (RGM‐1) were kept in serum‐free Dulbecco's modified Eagle's medium before addition of various concentrations of ethanol. Nuclear morphological alternations and membrane barrier dysfunction of RGM‐1 cells were assessed by staining with Hoechst 33342 and propidium iodide, respectively. To assess the contribution of oxygen‐derived free radicals and intracellular glutathione, scavenger of hydrogen peroxide and the hydroxyl radical, N,N‐dimethylthiourea, glutathione precursor, N‐acetyl‐l‐cysteine, and an inhibitor of alcohol dehydrogenase, 4‐methylpyrazole were added before treatment with ethanol. To investigate MPT, calcein and tetramethylrhodamine methyl ester were loaded before addition of ethanol, and the changes of fluorescence intensity were monitored using a laser scanning confocal microscope. Ethanol (>5% v/v) dose‐dependently increased the number of propidium iodide‐positive cells, suggesting a diminished barrier function of cell membrane. After addition of ethanol, mithochondria were filled quickly with calcein indicating MPT, which was accompanied by mitochondrial depolarization, as shown by loss of tetramethylrodamine methyl ester before cell death. Ethanol‐induced cell death was significantly attenuated by simultaneous incubation with either N,N‐dimethylthiourea or N‐acetyl‐l‐cysteine, suggesting the importance of intracellular redox states in inducing cellular damage, whereas such change was not attenuated by 4‐methylpyrazole. Present results suggest that ethanol treatment induces intracellular oxidative stress and produces MPT and mitochondrial depolarization, which are preceding cell death in gastric mucosal cells. Intracellular antioxidants, such as glutathione, may have a significant protective action against ethanol in gastric mucosal cells.
The involvement of oxygen free radicals in the development of the ethanol-induced gastric mucosal damage has been investigated. We found that oral administration of superoxide dismutase reduced the incidence of ethanol-induced mucosal lesions. Reduction of superoxide dismutase activity by diethyldithiocarbamate led to a pronounced aggravation of mucosal damage. Inhibition of the chloride-bicarbonate channel by a stilbene derivative also aggravated the ethanol-induced hemorrhagic lesions. Neither glutathione peroxidase, catalase, nor ceruloplasmin were capable of inhibiting the development of mucosal damage. Compounds with scavenging properties such as thiourea, 1-phenyl-3-(2-thiazolyl)-2-thiourea, dimethyl sulfoxide, various inorganic compounds (elements of the first and second subgroups and of the sixth group of the periodic table) and sulfhydrylcontaining substances protected the gastric mucosa against ethanol-induced injury in a dose-related manner. Naturally occurring antioxidants such as -tocopherol, -carotene, and coenzyme Q10 were ineffective. The present results suggest that superoxide free radicals are involved in the development of ethanol-induced gastric mucosal lesions, probably via an interaction with cellular membranes.
Between one- and two-thirds of all alcohol abusers have impairment of muscle function that may be accompanied by biochemical lesions and/or the presence of a defined myopathy characterised by selective atrophy of Type II fibres. Perturbations in protein metabolism are central to the effects on muscle and account for the reductions in muscle mass and fibre diameter. Ethanol abuse is also associated with abnormalities in carbohydrate (as well as lipid) metabolism in skeletal muscle. Ethanol-mediated insulin resistance is allied with the inhibitory effects of ethanol on insulin-stimulated carbohydrate metabolism. It acutely impairs insulin-stimulated glucose and lipid metabolism, although it is not known whether it has an analogous effect on insulin-stimulated protein synthesis. In alcoholic cirrhosis, insulin resistance occurs with respect to carbohydrate metabolism, although the actions of insulin to suppress protein degradation and stimulate amino acid uptake are unimpaired. In acute alcohol-dosing studies defective rates of protein synthesis occur, particularly in Type II fibre-predominant muscles. The relative amounts of mRNA-encoding contractile proteins do not appear to be adversely affected by chronic alcohol feeding, although subtle changes in muscle protein isoforms may occur. There are also rapid and sustained reductions in total (largely ribosomal) RNA in chronic studies. Loss of RNA appears to be related to increases in the activities of specific muscle RNases in these long-term studies. However, in acute dosing studies (less than 1 day), the reductions in muscle protein synthesis are not due to overt loss of total RNA. These data implicate a role for translational modifications in the initial stages of the myopathy, although changes in transcription and/or protein degradation may also be superimposed. These events have important implications for whole-body metabolism.
Ethanol was given to male Wistar rats as an acute dose (5 g/kg) or continuously at 5% (w/v) in a liquid diet to provide 36% of the caloric requirement. Free radicals generated in the liver were collected as a stable adduct in bile following the in vivo administration of the spin trapping agent alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone (POBN; 700 mg/kg). [1-(13)C]ethanol was used to confirm the formation of the 1-hydroxyethyl radical and to demonstrate that this was ethanol-derived in the case of the single-dose treatment. Free radical production increased up to 1h after the acute dose and then plateaued over the next 30 min. During chronic exposure to ethanol, free radical generation increased significantly after 1 week and then declined again to remain at a low level over the next 2 weeks; this transient increase corresponded closely with the induction of cytochrome P-450 2E1 (CYP 2E1) in response to ethanol feeding. Single-cell electrophoresis was used to investigate effects on DNA. After an acute dose of ethanol, the frequency of single-strand breaks increased from 1 h to peak at 6 h but then declined again to control values by 12 h. During the chronic exposure, an increase in the frequency of DNA breaks was seen at 3 days, reached a peak at 1 week and then decreased slowly over the next 5 weeks. The effects of antioxidants on these parameters was investigated after an acute dose of ethanol. Pre-treatment with vitamin C (400 mg/kg, i.p., daily for 5 days) or vitamin E (100 mg/kg, i.p., for 5 days) prior to the administration of ethanol (5 g/kg) inhibited generation of the 1-hydroxyethyl-POBN adduct by 30 and 50%, respectively, and both agents prevented the increased frequency of DNA single-strand breaks caused by ethanol. The significance of the temporal coincidence of changes in the above parameters in response to ethanol is discussed.
Oxygen free radicals are implicated in the pathogenesis of stress and food/alcohol-induced gastrointestinal injury. We have investigated the effects of restraint stress, spicy food diet, high-fat diet and 40% ethanol on the enhanced production of reactive oxygen species, including superoxide anion and hydroxyl radicals, and on DNA fragmentation, lipid peroxidation and membrane microviscosity (indices of oxidative tissue damage) in gastric and intestinal mucosa of Sprague-Dawley rats. Furthermore, the protective ability of bismuth subsalicylate (BSS; 15 mg kg(-1) was determined against the gastrointestinal mucosal injury induced by these stressors. Animals on the high-fat diet consumed 31% more food as compared to other animals. Animals on the spicy food diet consumed ca. 23% more water as compared to control animals, and the high-fat diet animals consumed 17% less water. Restraint stress provided greater injury to both gastric and intestinal mucosa as compared to other stressors. Restraint stress, spicy food diet, high-fat diet and ethanol increased superoxide anion production by 10.0-, 4.3-, 5.7- and 4.8-fold, respectively, in the gastric mucosa, and by 10.4-, 5.3-, 7.0- and 5.5-fold in the intestinal mucosa. Exposure to restraint stress, spicy food diet, high-fat diet and 40% ethanol also increased hydroxyl radical production by ca. 14.3-, 4.5-, 3.5- and 4.8-fold, respectively, in the gastric mucosa, and by 17.0-, 4.8-, 3.5- and 4.7-fold in the intestinal mucosa. Bismuth subsalicylate administration to the animals provided significant protection against superoxide anion and hydroxyl radical production. Restraint stress, spicy food diet, high-fat diet and ethanol increased lipid peroxidation by 3.6-, 2.4-, 2.6- and 2.0-fold, respectively, in the gastric mucosa, and by 4.1-, 3.5-, 3.6- and 2.7-fold in intestinal mucosa. Administration of BSS decreased restraint stress, spicy food diet, high-fat diet and ethanol-induced gastric mucosal lipid peroxidation by ca. 26%, 36%, 45% and 18%, and intestinal mucosa lipid peroxidation by 20%, 21%, 46% and 42%, respectively. Approximately 4.0-, 2.0-, 2.4- and 2.0-fold increases in DNA fragmentation were observed in the gastric mucosa of rats exposed to restraint stress, spicy food diet, high-fat diet and 40% ethanol, respectively, and similar increases in the intestinal mucosa. These same four stressors increased membrane microviscosity by 11.6-, 6.1-, 7.3- and 5.4-fold, respectively, in the gastric mucosa, and by 16.2-, 7.9-, 9.5- and 7.8-fold in the intestinal mucosa. Bismuth subsalicylate exerted significant protection against DNA damage and changes in membrane microviscosity induced by the four stressors. Excellent correlations existed between the production of reactive oxygen species and the tissue damaging effects in both gastric and intestinal mucosa. In summary, the results demonstrate that physical and chemical stressors can induce gastrointestinal oxidative stress and mucosal injury through enhanced production of reactive oxygen species, and that BSS can significantly attenuate gastrointestinal injury by scavenging these reactive oxygen species.
The active component of aurothioglucose (ATG) in effecting changes in plasma sulfhydryl (SH) levels and plasma SH reactivity with 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) was determined. These two measurements are applied clinically to rheumatoid arthritis patients undergoing chrysotherapy. Normal rats were injected intramuscularly daily for seven days with 30 mumol of either ATG or sodium thioglucose (STG)/kg body wt or with an equivalent volume of the carrier, 0.05% benzyl alcohol. ATG but not STG significantly increased total SH levels in plasma, liver, and kidney. The seven-day treatment with ATG significantly increased glutathione levels in kidney but not in liver or plasma. Thus, gold(I) rather than thioglucose was the active moiety that affected SH levels in ATG-injected rats. In vivo, gold(I) was also the active moiety that stimulated plasma SH reactions with DTNB at pH 7.4, since injection of ATG but not STG stimulated the SH reactivity in fresh plasma. In vitro, ATG increased the rate of plasma reaction with DTNB at pH 7.4, thus, gold(I) ions acted as a catalyst in the SH-disulfide exchange reaction. This study demonstrates that gold(I) but not its thiol ligand strongly interacts with protein SH groups in the rat tissues. Such an interaction may play an important role in the biological actions of gold.
Concentrated ethanol causes gastric lesions by a mechanism that is poorly understood. We have investigated this mechanism in the rat stomach via gross morphologic, videomicroscopic, histochemical, and pharmacologic approaches. Within 1 min of contact, ethanol caused diffuse mucosal hyperemia. By 5 min, hyperemia greatly intensified at some mucosal sites. Beneath sites where mucosal hyperemia developed, intramural venules strongly constricted at 3-13 s postethanol, whereas submucosal arterioles dilated more than two times in diameter by 25 s. Submucosal venular constriction began sooner than arteriolar dilation (9 vs. 16 s, p less than 0.05). One-third of the gastric mucosal mast cells degranulated by 15 s postethanol; 50% discharged by 30 s. Ethanol-induced hyperemia was markedly reduced by lipoxygenase-selective inhibitors BW755C or nordihydroguaiaretic acid, or by the H1-antihistamine pyrilamine, but not by indomethacin, cimetidine, phentolamine, or methysergide. Based on these results, a model for the pathogenesis of ethanol-induced gastric lesions is proposed.
Ethanol-induced gastric mucosal damage is characterized by microcirculatory changes such as stasis and plasma leakage. Sluggish blood flow and stasis have also been observed after administration of exogenous leukotriene (LT) C4. The effect of ethanol on the release of LTC4 from rat gastric mucosa was therefore investigated. It was found that intragastric instillation of ethanol increases gastric mucosal release of LTC4 in a dose- and time-dependent manner parallel to the production of gastric lesions. The lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA) and the anti-ulcer drug carbenoxolone (CX) inhibited mucosal release of LTC4 and simultaneously protected against gastric damage caused by ethanol. It is concluded that increased formation of LTC4 and/or other 5-lipoxygenase-derived products of arachidonate metabolism may be involved in ethanol-induced gastric damage. Furthermore, inhibition of the 5-lipoxygenase pathway may be an important mechanism of action of gastric protective drugs.
Lesion formation due to oral administration of absolute ethanol could be prevented by parenteral pretreatment with antiperoxidative drugs such as butylated hydroxytoluene (BHT), quercetin and quinacrine. Also effective were allopurinol and oxypurinol, inhibitors of xanthine oxidase, but not superoxide dismutase (SOD) and hydroxyl radical scavengers, such as sodium benzoate and dimethyl sulfoxide (DMSO). BHT, quercetin, quinacrine and sulfhydryl compounds such as reduced glutathione and cysteamine which offer gastroprotection in vivo against ethanol inhibited lipid peroxidation induced in vitro by ferrous ion in porcine gastric mucosal homogenate, but SOD, sodium benzoate, DMSO, allopurinol and oxypurinol did not. These results suggest the possibility that an active species, probably derived from free iron mobilized by the xanthine oxidase system, other than oxygen radicals such as hydroxyl radicals, contributes to lipid peroxidation and lesion formation in the gastric mucosa after absolute ethanol administration.
Both in men and rats, most of the ethanol ingested at a low dose is metabolized before it reaches the systemic circulation. Oxidation of ethanol (mainly in the stomach) accounts for the bulk of this effect. This "first pass" metabolism (FPM) may be viewed as a barrier which protects against the systemic toxicity of ethanol. This barrier can be overcome by large doses of ethanol. Its efficiency is also reduced by a decrease in gastric alcohol dehydrogenase (ADH) activity secondary to chronic alcohol consumption.
Lipid peroxidation has been incriminated in some types of drug-induced liver injury, but it is unclear whether it contributes to or is present in alcoholic liver injury. In order to study this question, hepatic lipid peroxidation (measured as formation of diene conjugates) and hepatic GSH were assessed in baboons and rats after short- and long-term ethanol administration. Compared to controls, baboons fed alcohol for 1 to 4 years (chronic administration) had increased hepatic diene conjugates (16.9 +/- 4.8 OD/gm of liver) and depressed GSH (3.8 +/- 0.6 VS. 6.3 +/- 0.8 mumol/gm of liver; p less than 0.01) after an overnight withdrawal from ethanol. Administration of 1.6 gm/kg ethanol over 6 hr (acute administration) increased diene conjugates (17.6 +/- 4.3) and decreased GSH (3.2 +/- 0.5; p less than 0.01) in control animals and had an even greater effect in animals chronically fed alcohol (diene conjugates 48.5 +/- 9.2; GSH 1.8 +/- 0.3; p less than 0.001). In six rats fed alcohol for 5 to 6 weeks (chronic administration), an increase in diene conjugates was detected in microsomes (0.343 +/- 0.210 OD/mg of lipid) and mitochondria (0.143 +/- 0.061), accompanied by decrease in arachidonic acid and C22 polyenes, after acute ethanol administration (3 gm/kg p.o.) but no significant change in GSH. Simultaneous administration of methionine attenuated diene conjugate formation (0.107 +/- 0.058 and 0.035 +/- 0.020 OD/mg of lipid, respectively) and fatty acid changes. Thus chronic alcohol feeding potentiates lipid peroxidation produced by an acute dose of ethanol; these changes are not dependent on GSH depression but may be potentiated by it.
Ethanol induces hemorrhagic gastric erosions and causes a dose-dependent decrease in the concentration of nonprotein sulfhydryl
compounds in rat gastric mucosa. Sulfhydryl-containing drugs protect rats from ethanol-induced gastric erosions, whereas sulfhydryl
blocking agents counteract the mucosal cytoprotective effect of prostaglandin F2 beta. These observations suggest that endogenous
nonprotein sulfhydryls may mediate prostaglandin-induced gastric cytoprotection and that sulfhydryl drugs may have potential
for preventing or treating hemorrhagic gastric erosions.
Disturbance of the balance between the production of reactive oxygen species such as superoxide; hydrogen peroxide; hypochlorous acid; hydroxyl, alkoxyl, and peroxyl radicals; and antioxidant defenses against them produces oxidative stress, which amplifies tissue damage by releasing prooxidative forms of reactive iron that are able to drive Fenton chemistry and lipid peroxidation and by eroding away protective sacrificial antioxidants. The body has a hierarchy of defense strategies to deal with oxidative stress within different cellular compartments, and superimposed on these are gene-regulated defenses involving the heat-shock and oxidant stress proteins.
Alcohol-induced tissue damage results from associated nutritional deficiencies as well as some direct toxic effects, which have now been linked to the metabolism of ethanol. The main pathway involves liver alcohol dehydrogenase which catalyzes the oxidation of ethanol to acetaldehyde, with a shift to a more reduced state, and results in metabolic disturbances, such as hyperlactacidemia, acidosis, hyperglycemia, hyperuricemia and fatty liver. More severe toxic manifestations are produced by an accessory pathway, the microsomal ethanol oxidizing system involving an ethanol-inducible cytochrome P450 (2E1). After chronic ethanol consumption, there is a 4- to 10-fold induction of 2E1, associated not only with increased acetaldehyde generation but also with production of oxygen radicals that promote lipid peroxidation. Most importantly, 2E1 activates many xenobiotics to toxic metabolites. These include solvents commonly used in industry, anaesthetic agents, medications such as isoniazid, over the counter analgesics (acetaminophen), illicit drugs (cocaine), chemical carcinogens, and even vitamin A and its precursor beta-carotene. Furthermore, enhanced microsomal degradation of retinoids (together with increased hepatic mobilization) promotes their depletion and associated pathology. Induction of 2E1 also yields increased acetaldehyde generation, with formation of protein adducts, resulting in antibody production, enzyme inactivation, decreased DNA repair, impaired utilization of oxygen, glutathione depletion, free radical-mediated toxicity, lipid peroxidation, and increased collagen synthesis. New therapies include adenosyl-L-methionine which, in baboons, replenishes glutathione, and attenuates mitochondrial lesions. In addition, polyenylphosphatidylcholine (PPC) fully prevents ethanol-induced septal fibrosis and cirrhosis, opposes ethanol-induced hepatic phospholipid depletion, decreased phosphatidylethanolamine methyltransferase activity and activation of hepatic lipocytes, whereas its dilinoleoyl species increases collagenase activity. Current clinical trials with PPC are targeted on susceptible populations, namely heavy drinkers at precirrhotic stages.
Acetaldehyde, the first product of ethanol metabolism, has previously been shown to form potentially harmful adducts with various proteins. The aim of this study was to investigate whether acetaldehyde--either exogenous or metabolically derived--binds to gastric mucosal proteins. Homogenized rat gastric mucosa was incubated with various concentrations of radiolabeled acetaldehyde or ethanol for different time periods. Acetaldehyde-protein adducts were determined by a liquid scintillation counter. In addition, mucosa was incubated with nonlabeled ethanol, and the acetaldehyde formed was measured by using headspace gas chromatography. Incubation of gastric mucosa with (14C)-acetaldehyde led to a concentration- and time-dependent radiolabeling of mucosal proteins. Formation of acetaldehyde adducts occurred relatively rapidly within 30 minutes and even at low acetaldehyde levels (5 micromol/L). Stable adducts represented 77% +/- 5% (mean +/- SEM) of the total adducts formed. In the presence of ethanol, acetaldehyde production and adduct formation took place in a concentration- and time-dependent manner. 4-Methylpyrazole and sodium azide inhibited acetaldehyde production to 7% +/- 1% of control and decreased the amount of acetaldehyde adducts to 55% +/- 8%. Enhanced acetaldehyde formation (to 420% +/- 50%) was clearly reflected in increased adduct formation (550% +/- 110%). In conclusion, both exogenous and endogenous acetaldehyde binds to gastric mucosal proteins in vitro. Gastric mucosal acetaldehyde production and the consequent adduct formation could be a pathogenetic factor behind ethanol-associated gastric injury.
Acetaldehyde (AcH) at a concentration of 593 mM lowers the natural fluorescence of commercial human serum by 12%. It also lowers the fluorescence of a beta-naphthylamine standard curve (recovery) in serum by 17%. These results contrast with earlier reports showing that 447 mM AcH had no effect upon fluorescence of serum or a beta-naphthylamine standard curve in serum. Because 447 mM AcH and 593 mM AcH represent 2.5% and 3.3% AcH, it is apparent that there is a narrow window between which AcH may affect fluorescence by adduct formation with blood components and exogenous fluorophores. Nonetheless, serum has the capacity to bind > 2.5% (> 447 mM) AcH without alteration in fluorescence, suggesting that serum has a great carrying capacity for AcH, undoubtedly in the form of adducts to nucleophiles. These results are discussed in the light of toxicity of AcH and ethanol, the probable significance of the approximately 30 microM free AcH that is reported in chronic alcoholics and the planning of in vitro and in vivo studies with AcH.
Although it has been speculated that active oxidants and mitochondrial membrane damages play roles in ethanol-induced gastric mucosal damages, its detail remains unknown. The present study was designed to investigate whether ethanol induces oxidative stress and mitochondrial permeability transition (MPT) before cell death of gastric mucosal cells. Rat gastric mucosal cells (RGM-1) were kept in serum-free Dulbecco's modified Eagle's medium before addition of various concentrations of ethanol. Nuclear morphological aftemations and membrane barrier dysfunction of RGM-1 cells were assessed by staining with Hoechst 33342 and propidium iodide, respectively. To assess the contribution of oxygen-derived free radicals and intracellular glutathione, scavenger of hydrogen peroxide and the hydroxyl radical, N,N-dimethylthiourea, glutathione precursor, N-acetyl-L-cysteine, and an inhibitor of alcohol dehydrogenase, 4-methylpyrazole were added before treatment with ethanol. To investigate MPT, calcein and tetramethylrhodamine methyl ester were loaded before addition of ethanol, and the changes of fluorescence intensity were monitored using a laser scanning confocal microscope. Ethanol (>5% v/v) dose-dependently increased the number of propidium iodide-positive cells, suggesting a diminished barrier function of cell membrane. After addition of ethanol, mitochondria were filled quickly with calcein indicating MPT, which was accompanied by mitochondrial depolarization, as shown by loss of tetramethylrodamine methyl ester before cell death. Ethanol-induced cell death was significantly attenuated by simultaneous incubation with either N,N-dimethylthiourea or N-acetyl-L-cysteine, suggesting the importance of intracellular redox states in inducing cellular damage, whereas such change was not attenuated by 4-methylpyrazole. Present results suggest that ethanol treatment induces intracellular oxidative stress and produces MPT and mitochondrial depolarization, which are preceding cell death in gastric mucosal cells. Intracellular antioxidants, such as glutathione, may have a significant protective action against ethanol in gastric mucosal cells.
In this review first we evaluate evidence on the role of the neurobiological alterations induced by chronic ethanol consumption in the development of ethanol tolerance, dependence and withdrawal. Secondly, we describe the neuropathological consequences of chronic ethanol on cognitive functions and on brain structures. Chronic alcohol consumption can induce alterations in the function and morphology of most if not all brain systems and structures. While tolerance mechanisms are unlikely to contribute to the neuroadaptive changes associated with ethanol dependence, it is otherwise clear that repeated high, intoxicating doses of ethanol trigger those neuroadaptive processes that lead to dependence and contribute to the manifestation of the abstinence syndrome upon withdrawal. An unbalance between inhibitory and excitatory neurotransmission is the most prominent neuroadaptive process induced by chronic ethanol consumption. Due to the diffuse glutamatergic innervation to all brain structures, the neuroadaptive alterations in excitatory neurotransmission can affect the function of most if not all of neurotransmitter systems. The expression of the withdrawal syndrome is the major causal factor for the onset and development of the neuropathological alterations. This suggests a link between the neuroadaptive mechanisms underlying the development of ethanol dependence and those underlying the functional and structural alterations induced by chronic ethanol. In animals and humans, specific alterations occur in the function and morphology of the diencephalon, medial temporal lobe structures, basal forebrain, frontal cortex and cerebellum, while other subcortical structures, such as the caudate nucleus, seem to be relatively spared. The neuropathological alterations in the function of mesencephalic and cortical structures are correlated with impairments in cognitive processes. In the brain of alcoholics, the prefrontal cortex and its subterritories seem particularly vulnerable to chronic ethanol, whether Korsakoff's syndrome is present or not. Due to the role of these cortical structures in cognitive functions and in the control of motivated behavior, functional alterations in this brain area may play an important role in the onset and development of alcoholism.
The carbonyl assay has been developed as a general assay of oxidative protein damage to assess steady-state protein damage in animal tissues and body fluids. The assay is based on the fact that several ROS can attack amino acid residues in proteins (particularly histidine, arginine, lysine, and proline) to produce carbonyl functions that can react with 2,4-dinitrophenylhydrazine (DNPH) to generate chromophoric dinitrophenylhydrazones. This reaction can, therefore, be used to estimate the carbonyl content of proteins in human tissues and body fluids. Western blotting assays based on the use of anti-dinitrophenol antibodies have also been developed to identify oxidatively damaged proteins in tissues and body fluids after their extraction and derivatization with DNPH. The carbonyl assay has become widely used and many laboratories have developed individual protocols for it. Sometimes, the assay procedures used are not precisely specified, and when they are, they may differ from those originally proposed by the group of Stadtman et al. This point is important, because there is considerable variation in the baseline levels of protein carbonyls in certain tissues, depending on how the assay is performed.
Reactive oxygen species appears to be involved in the pathogenesis of ethanol-induced gastric mucosal injury in vivo. Because ingested ethanol diffuses into the gastric mucosa, targeting both epithelium and endothelium, in the present study we examined the possible protective effect of antioxidants on ethanol damage in gastric epithelial cells and endothelial cells in vitro. Cytotoxicity by ethanol was quantified by measuring 51Cr release. The effects of impairment of the glutathione redox cycle and of inhibition of cellular catalase were examined. The generation of superoxide was assessed by the reduction in cytochrome c. Ethanol caused a time- and dose-dependent increase in 51Cr release from epithelial cells. Incubation of cells with DL-buthionine-(S,R)-sulfoximine, while reducing glutathione production, dose dependently enhanced ethanol-induced injury. 1,3-Bis(chloroethyl)-nitrosourea, while inhibiting glutathione reductase activity, also sensitized cells to ethanol. In contrast, the inhibition of catalase with 3-amino-1,2, 4-triazole did not alter the susceptibility of epithelial cells to ethanol. Ethanol induced damage to endothelial cells in a similar fashion. In endothelial cells, however, neither impairment of the glutathione cycle nor inhibition of catalase influenced ethanol-induced damage. Epithelial cells, when exposed to ethanol, increased superoxide production as a function of ethanol concentration, whereas endothelial cells did not. The glutathione redox cycle, but not cellular catalase, plays a critical role in protecting epithelial cells against ethanol damage, whereas neither antioxidant seems to play a role in protection of endothelial cells. The distinct difference in antioxidant protection against ethanol appears to depend on the capability of each cell to produce cytotoxic oxygen species in response to ethanol exposure.
As cytochrome P-450 2E1 (CYP2E1) induction was related to oxidative stress in experimental models, the aim of this study was to investigate the relationship between CYP2E1 activity and markers of oxidative stress in 40 alcoholic patients entering a rehabilitation programme. Plasma oxidized proteins, lipid peroxides (LPO) and antibodies against hydroxyethyl radical (HER) or malondialdehyde (MDA) adducts were assessed as markers of the production of free radicals, whereas vitamin E levels were evaluated as a marker of the antioxidant defence. CYP2E1 activity was determined by using the 6-hydroxychlorzoxazone:chlorzoxazone blood metabolic ratio, 2 h after drug intake. This ratio was increased by 4-fold in alcoholics, compared to non-alcoholic patients, and was correlated with daily intake of ethanol, carbohydrate-deficient transferrin, and blood alcohol level at the time of admission to hospital. Plasma levels of LPO and oxidized proteins were slightly increased (20%) in alcoholic patients when compared with the control group, whereas those of vitamin E were found to be slightly decreased (by 18%). Antibodies against HER or MDA adducts showed a very significant increase. However, when alcoholic patients were divided into two groups according to low or high CYP2E1 induction, no significant difference was observed in the variation of these parameters, except for anti-HER adducts antibodies. Therefore, our study confirms the main involvement of CYP2E1 in HER production. By contrast, CYP2E1 does not appear to be the main factor responsible for the oxidative stress occurring during human chronic alcoholism. Free radicals from other sources may therefore contribute significantly to the generation of this oxidative stress.
In this article we have reviewed recent evidence in support of the hypothesis that acute/chronic alcohol toxicity is mediated primarily via the generation of damaging free radical species in various tissues. Studies in man, animal model or in vitro experimental systems have shown: (1) the demonstration of alcohol-induced free radical species directly via esr spectroscopic analysis; (2) increases in indirect markers of ethanol-induced free radical damage in tissues, such as lipid peroxides and protein carbonyl; (3) ethanol-induced alterations in the levels of endogenous tissue antioxidants. These data show the induction of free radicals by ethanol to be a complex interactive process. The classical pathway for ethanol metabolism, catalysed by alcohol dehydrogenase to form acetaldehyde, results in the formation of free radicals, resulting from concomitant changes in NADH levels and NADH/NAD+ redox ratios, which in turn modulate the activity of the free radical generating enzyme xanthine oxidase. The induction of CYP 2E1 in the microsomes results in the generation of HER, another major route by which ethanol induces free radical formation. In addition to the above, ethanol may also induce free radical formation via the reaction of aldehyde oxidase with acetaldehyde or NADH to generate oxyradicals via disturbance in the metabolism of the pro-oxidant iron, or via increased efflux from mitochondria following altered mitochondrial oxidative metabolism.
This study was designed to determine the ulcer-protecting effects of rutin, a natural flavone, against gastric lesions induced by 50% ethanol, the experimental model related to lesion pathogenesis with production of reactive species. The possible involvement of sulphydryl compounds (SH), neutrophil infiltration, and the capacity of this flavone to restrain the oxidative process produced in the gastric tissue were also investigated. The levels of thiobarbituric acid (TBA, as index of lipid peroxidation), the myeloperoxidase activity (MPO, as a marker of neutrophil infiltration), the content of mucosal sulphydryls (SH) groups and the activity of glutathione peroxidase (GSH-Px, an important antioxidant enzyme) were determined. Pretreatment with the highest dose of rutin (200 mg/kg), 120 min before 50% ethanol, resulted in the most effective necrosis prevention. TBA reactive substances in the gastric mucosa, were increased by ethanol injury, and this increase was inhibited by the administration of 200 mg/kg of rutin. However, the flavonoid was not able to modify the ethanol-induced neutrophil infiltrate expressed as myeloperoxidase activity. Exposure of the gastric mucosa to 50% ethanol induced a significant diminution in gastric non-protein SH content; this parameter also was not modified by the treatment with rutin. GSH-Px activity decreased in the gastric mucosa after ethanol-treatment. In contrast, rutin at all tested doses induced a significant increase in this enzymatic activity, higher than in control group. These results suggest that the gastroprotective effect of rutin in this experimental model appears through an anti-lipoperoxidant effect, and also by enhancement of the anti-oxidant enzymatic (GSH-Px) activity.
Increased oxidative stress has been speculated to be one possible mechanism of ethanol toxicity. This study evaluates malondialdehyde (MDA) and protein carbonyl content in serum as markers of oxidative stress and DNA damage in lymphocytes in the same patients with chronic alcoholism. Patients with chronic alcoholism showed a significant increase in MDA levels and protein carbonyl content of their serum as compared with non-alcoholic control subjects. Increases in endogenous and H2O2-induced DNA damage were also observed in lymphocytes of patients with chronic alcoholism. In addition, there were significant correlations between endogenous and H2O2-induced DNA damage and serum MDA or protein carbonyl content in patients with chronic alcoholism. These results clearly indicate the presence of oxidative stress in patients with chronic alcoholism.
The effect of two different doses (50 and 100 mg/kg body wt/day for 14 days) of 80% ethanolic extract of the leaves of Adhatoda vesica were examined on drug metabolizing phase I and phase II enzymes, antioxidant enzymes, glutathione content, lactate dehydrogenase and lipid peroxidation in the liver of 8 weeks old Swiss albino mice. The modulatory effect of the extract was also examined on extra-hepatic organs viz. lung, kidney and forestomach for the activities of glutathione S-transferase, DT-diaphorase, superoxide dismutase and catalase. Significant increase in the activities of acid soluble sulfhydryl (-SH) content, cytochrome P450, NADPH-cytochrome P450 reductase, cytochrome b5, NADH-cytochrome b5 reductase, glutathione S-transferase (GST), DT-diaphorase (DTD), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and glutathione reductase (GR) were observed in the liver at both dose levels of treatments. Adhatoda vesica acted as bifunctional inducer since it induced both phase I and phase II enzyme systems. Both the treated groups showed significant decrease in malondialdehyde (MDA) formation in liver, suggesting its role in protection against prooxidant induced membrane damage. The cytosolic protein was significantly inhibited at both the dose levels of treatment indicating the possibility of its involvement in the inhibition of protein synthesis. BHA has significantly induced the activities of GR and GSH in the present study. The extract was effective in inducing GST and DTD in lung and forestomach, and SOD and CAT in kidney. Thus, besides liver, other organs viz., lung, kidney and forestomach were also stimulated by Adhatoda, to increase the potential of the machinery associated with the detoxification of xenobiotic compounds. But, liver and lung showed a more consistent induction. Since the study of induction of the phase I and phase II enzymes is considered to be a reliable marker for evaluating the chemopreventive efficacy of a particular compound, these findings are suggestive of the possible chemopreventive role played by Adhatoda leaf extract.
Ethanol metabolism causes oxidative stress and lipid peroxidation not only in liver but also in extra-hepatic tissues. Ethanol administration has been shown to cause oxidative degradation and depletion of hepatic mitochondrial DNA (mtDNA) in rodents, but its in vivo effects on the mtDNA of extra-hepatic tissues have not been assessed. We studied the effects of an acute intragastric ethanol administration (5 g/kg) on brain, heart, skeletal muscle, and liver mtDNA in mice. Ethanol administration caused mtDNA depletion and replacement of its supercoiled form by linearized forms in all tissues examined. Maximal mtDNA depletion was about similar (ca. 50%) in all organs studied. It occurred 2 h after ethanol administration in heart, skeletal muscle, and liver but after 10 h in brain. This mtDNA depletion was followed by increased mtDNA synthesis. A secondary, transient increase in mtDNA levels occurred 24 h after ethanol administration in all organs. In hepatic or extra-hepatic tissues, mtDNA degradation and depletion were prevented by 4-methylpyrazole, an inhibitor of ethanol metabolism, and attenuated by vitamin E, melatonin, or coenzyme Q, three antioxidants. In conclusion, our study shows for the first time that ethanol metabolism also causes oxidative degradation of the mitochondrial genome in brain, heart, and skeletal muscles. These effects could contribute to the development of (cardio)myopathy and brain injury in some alcoholic patients. Antioxidants prevent these effects in mice and could be useful in persevering drinkers.
Most antioxidants show contradictory behaviors because in the biological environment, for unpredictable reasons, they can become prooxidants. Recently, a new simple method to monitor oxidative stress in serum was developed. This test detects the derivatives of reactive oxygen metabolites (D-Roms). Hydroperoxides are converted into radicals that oxidize N,N-diethyl-para-phenylendiamine and that can be detected through spectrophotometric procedures as U.CARR. (Carratelli units). One U.CARR. corresponds to 0.8 mg/L hydrogen peroxide. In normal subjects U.CARR. values range from 250 to 300. Values outside this range indicate a modification of the prooxidant/antioxidant ratio. On the basis of this method, we tested three different formulas of antioxidants (F1, F2, F3) in 14 apparently healthy volunteers (11 men and 3 women). Formula 1 was composed of 5 mg zinc, 48 microg selenium, 400 microg vitamin A (as retinol acetate), 50 microg beta-carotene, 15 mg vitamin E (as dl-alpha-tocopheryl acetate) and 10 mg L-cysteine. Formula 2 was composed of 30 mg bioflavonoids from citrus, 30 mg vitamin C (as L-ascorbic acid), 10 mg coenzyme Q(10) and 1 mg vitamin B-6 (as pyridoxine hydrochloride). Formula 3 was composed of Formula 1 plus Formula 2. Each formula was prepared in dry capsules (formulation D1, D2, D3) or in a fluid form (formulation P1, P2, P3). Each formulation was administered for 1 wk in a crossover design. A 15% deviation of U.CARR. levels was chosen as the cut-off value for a significant change in oxidative stress. Formulas F1 and F3 reduced mean U.CARR. levels in most of the treated subjects (t test, P < 0.05), whereas F2 was not active. Fluid formulations were more active than dry formulations (chi(2) test, P < 0.05). In some cases, a slight increase in oxidative stress was detected. These minimal increases were not related to any particular antioxidant formula. In one subject only, the administration of the dry formulation (D1), increased oxidative stress to a level that reached the cut-off value. In conclusion, when antioxidants are taken in combination at low dosages they reduce oxidative stress, and little relevant prooxidant activity is detectable.
The activities of alcohol dehydrogenase (ADH), catalase, microsomal ethanol-oxidizing system (MEOS) and aldehyde dehydrogenase (ALDH) were measured in gastric, small intestinal, colonic and rectal mucosal samples of rats fed on a liquid alcohol diet for 1 month. In the rectum and large intestine of control animals, the activities of ADH, MEOS and catalase were maximal, whereas the activity of ALDH was minimal. After chronic alcohol intoxication, MEOS activity increased significantly in the stomach. An activation of catalase and MEOS and a decrease of the low-K(M) ALDH activity were observed in the rectum of experimental animals. In rats consuming the alcohol diet, hypertrophy of crypts and an increased number of mitoses were noticed in colonic and rectal mucosa. Acute alcohol intoxication (2 g/kg, intragastrically) produced significantly higher acetaldehyde concentrations in the contents of the large intestine and rectum of rats receiving alcohol chronically compared to controls. Thus, after chronic alcohol intoxication, the large intestine regions showed a greater imbalance between the activities of acetaldehyde-producing and -oxidizing enzymes, which resulted in accumulation of acetaldehyde. This mechanism can account for the local toxicity of ethanol after its chronic consumption, and relates the development of mucosal damage and compensatory hyper-regenerative processes, and possibly carcinogenesis, in the colonic and rectal mucosae of alcoholics to the effects of acetaldehyde.
Protein carbonyl content, a measure of oxidative damage to hepatocellular proteins, and the activities of some thiol-containing proteins were assayed in the liver and plasma, as thiol-containing protein, appear to be targets for free radicals. These may be important in the mechanism of ethanol-induced liver injury.
Tap water containing ethanol at the concentration of 25% (v/v) and phenobarbital (500 mg/l) was the only source of drinking water for the experimental rats for 24 months. Another group of rats were administered 25% (v/v) ethanol alone in drinking water for 24 months. Control rats were administered either phenobarbital alone in drinking water or tap water for 24 months. At the end of 24 months, the rats were sacrificed. The protein carbonyl content, activities of glutamine synthase and biotinidase-sulfhydryl group containing enzymes were assayed in the liver along with alkaline protease, an enzyme that degrades oxidized proteins. The total thiol, albumin and the activity of biotinidase were measured in the plasma.
The protein carbonyl content of the liver was increased in the ethanol/phenobarbital-treated rats as well as in the ethanol-treated rats as compared with the controls. The activities of glutamine synthase and biotinidase were decreased significantly in the livers of ethanol/phenobarbital-treated rats as well as the ethanol-treated rats as compared with the controls. The activity of alkaline protease was increased significantly in both the ethanol-treated groups. In the plasma of ethanol/phenobarbital-treated rats as well as the ethanol-treated rats total thiol, albumin and the activity of biotinidase were decreased significantly as compared with the controls. The ethanol/phenobarbital-treated rats as well as the ethanol-treated rats developed fatty liver.
Damage to proteins occurs upon chronic ethanol intake in the rat, and it may play a role in the pathogenesis of alcohol-induced fatty liver.
This article contains the proceedings of a symposium at the 2002 RSA Meeting in San Francisco, organized and co-chaired by Thomas M. Badger, Paul Shih-Jiun Yin, and Helmut Seitz. The presentations were (1) First-pass metabolism of ethanol: Basic and clinical aspects, by Charles Lieber; (2) Intracellular CYP2E1 transport, oxidative stress, cytokine release, and ALD, by Magnus Ingelman-Sundberg; (3) Pulsatile ethanol metabolism in intragastric infusion models: Potential role in toxic outcomes, by Thomas M. Badger and Martin J.J. Ronis; (4) Free radicals, adducts, and autoantibodies resulting from ethanol metabolism: Role in ethanol-associated toxicity, by Emanuele Albano; and (5) Gastrointestinal metabolism of ethanol and its possible role in carcinogenesis, by Helmut Seitz.
In the present study we tested the ability of different antioxidant agents, used alone or in combination, to reduce the reactive oxygen species (ROS) levels and to increase the glutathione peroxidase (GPx) activity. Moreover, we tested the ability of such antioxidant agents to reduce the serum levels of proinflammatory cytokines IL-6 and TNFalpha. Fifty-six advanced stage cancer patients with tumors at different sites were included in the study: they were mainly stage III (12.5%) and stage IV (82.1%). The study was divided into two phases. In the 1st phase 28 patients were divided into five groups and a single different antioxidant agent was administered to each group. The selected antioxidant agents were: alpha lipoic acid or carboxycysteine-lysine salt, amifostine, reduced glutathione, vitamin A plus vitamin E plus Vitamin C. In the 2nd phase of the study 28 patients were divided into five groups and a combination of two different antioxidant agents was administered to each group. The antioxidant treatment was administered for 10 consecutive days. The patients were studied at baseline and after antioxidant treatment. Our results show that all single antioxidants tested were effective in reducing the ROS levels and three of them in increasing GPx activity, too. Among the combinations of antioxidant agents, three were effective in reducing ROS, while three were effective in increasing GPx activity (arm 4 was effective in both instances). Comprehensively, the "antioxidant treatment" was found to be effective both on ROS levels and GPx activity. Moreover, the antioxidant treatment was able to reduce serum levels of IL-6 and TNFalpha. Furthermore, a correlation was shown between the Eastern Cooperative Oncology Group Performance Status of patients and blood levels of ROS, GPx activity, serum levels of proinflammatory cytokines.
Alcohol is a constituent of the diet that is generally taken in on a voluntary basis. The amount and type of alcohol consumed along with the frequency of alcohol consumption can vary tremendously and can have divergent effects on an organism. Animal models have been developed to investigate the mechanisms by which both acute alcohol administration and chronic alcohol consumption affect the various organ systems of the body. The deleterious effects of alcohol, at least partly involve alcohol induced oxidative injury that has been documented by measurement of oxidant radicals, alterations in oxidant/antioxidant balance and oxidant induced changes in cellular proteins and lipids. In addition, evidence for alcohol-induced oxidant injury comes from studies in which pretreatment with antioxidants such as vitamin E, vitamin C, and agents that enhance antioxidant capacity attenuate alcohol induced effects. The susceptibility of tissues to alcohol-induced injury is related to their function and the method by which they are exposed to alcohol. For example, the stomach and liver are exposed to the highest concentrations upon ingestion and absorption of alcohol. The liver is also the major organ for metabolism, and with chronic alcohol use, P450 2E1 is induced. This enzyme activity however, adds additional oxidative stress to the liver. Although antioxidants can attenuate alcohol-induced injury, there is no one antioxidant that protects all organs during all modes of exposure. In addition, more studies are needed to determine if administration of antioxidants after alcohol exposure can reverse alcohol induce tissue damage. This review will summarize results from experiments in which alcohol has been delivered for a short time (acute) or prolonged period (chronic); in vivo or in vitro; at physiologic doses or at supraphysiologic doses. The effects of alcohol on various tissues will be presented and finally, the contribution of oxidant injury to alcohol induced tissue damage will be discussed.
Since 1922 when Wu proposed the use of the Folin phenol reagent for
the measurement of proteins (l), a number of modified analytical pro-
cedures ut.ilizing this reagent have been reported for the determination
of proteins in serum (2-G), in antigen-antibody precipitates (7-9), and
in insulin (10).
Although the reagent would seem to be recommended by its great sen-
sitivity and the simplicity of procedure possible with its use, it has not
found great favor for general biochemical purposes.
In the belief that this reagent, nevertheless, has considerable merit for
certain application, but that its peculiarities and limitations need to be
understood for its fullest exploitation, it has been studied with regard t.o
effects of variations in pH, time of reaction, and concentration of react-
ants, permissible levels of reagents commonly used in handling proteins,
and interfering subst.ances. Procedures are described for measuring pro-
tein in solution or after precipitation wit,h acids or other agents, and for
the determination of as little as 0.2 y of protein.
In this study, the effect of ascorbic acid (vitamin C), DL-alpha-tocopherol acetate (vitamin E), and sodium selenate (selenium) on ethanol-induced gastric mucosal injury in rats was investigated morphologically and biochemically. The gastric mucosal injury was produced by administration of 1 mL of absolute ethanol to each rat. Animals received vitamin C (250 mg/kg), vitamin E (250 mg/kg), and selenium (0.5 mg/kg) for 3 d 1 h prior to the administration of absolute ethanol. In gastric mucosa of rats given ethanol according to control groups, neuronal nitric oxide expression decreased. This immunoreactivity was much lower in the group given ethanol+vitamin C+vitamin E+selenium than the control group and the ethanol-induced group. Scanning electron microscopic evaluation of the ethanol-induced group, when compared to control groups, revealed degenerative changes in gastric mucosa, whereas a good arrangement in surface topography of gastric mucosa in the group given ethanol + vitamin C+vitamin E + selenium was observed. In the group administered ethanol, a reduction of the stomach glutathione (GSH) and serum total protein levels and increases in serum sialic acid, triglycerides, and stomach lipid peroxidation (LPO) levels were observed. Vitamin C+vitamin E+Se administration to alcohol-treated rats significantly increased the serum total protein, triglyceride levels, and stomach GSH levels and significantly lowered the levels of serum sialic acid and stomach LPO compared to untreated alcohol-supplemented rats. As a result of these findings, we can say that the combination of vitamin C, vitamin E, and selenium has a protective effect on ethanol-induced gastric mucosal injury of rats.