Deleterious effects induced by oxidative stress in liver nuclei from rats receiving an alcohol-containing liquid diet.

Highly purified rat-liver nuclei were previously shown to have nuclear ethanol (EtOH) metabolizing system able to bioactivate alcohol to acetaldehyde and 1-hydroxyethyl radicals. These reactive metabolites were able to covalently bind to nuclear proteins and lipids potentially being able to provoke oxidative stress of nuclear components. In this study, the above-mentioned possibility was explored. Sprague Dawley male rats (125-150 g) were fed a standard Lieber and De Carli liquid diet for 28 days. Controls were pair-fed with a diet, in which EtOH was isocalorically replaced with carbohydrate. The presence of a chlorzoxazone hydroxylase activity inducible by the repetitive EtOH drinking further suggested the presence of CYP2E1 in the highly purified nuclei. Nuclei from EtOH-drinking rats evidenced significantly increased susceptibility to a t-butyl hydroperoxide challenge as detected by chemiluminescence emission, increased formation of protein carbonyls, and decreased content of protein sulfhydryls. In contrast, no significant changes in the nuclear lipid hydroperoxides formation or even decreases in the 8-oxo-7,8-dihydro-2-deoxyguanosine were observed. No significant differences were observed in different parameters of the alkaline Comet assay. In immunohistochemical studies performed, no expression of p53 was observed in the livers of the animals under the experimental conditions tested. Since nuclear proteins and lipids are known to play a role in cell growth, differentiation, repair and signaling, their alterations by either oxidative stress, or by covalent binding might be of relevance to liver tumor promotion.

Acetaldehyde accumulation in rat mammary tissue after an acute treatment with alcohol.

Previous studies reported the presence in rat mammary tissue of a cytosolic xanthine oxidoreductase pathway for the metabolism of alcohol to acetaldehyde and hydroxyl radicals and to the microsomal biotransformation of ethanol to acetaldehyde. It was also reported that after chronic ethanol drinking stressful oxidative conditions can be observed. The present work reports that even after single doses of ethanol, given at three different levels (6.3 g kg(-1); 3.8 g kg(-1) or 0.6 g kg(-1) p.o.), acetaldehyde accumulates for prolonged periods of time in the mammary tissue to reach concentrations higher than in blood (e.g. 5.1+/-1.2 nmol g(-1) versus 0.2+/-0.1 nmol ml(-1), for 6.3 g kg(-1) dose, 6 h after intoxication). The presence in rat mammary tissue of low activities of additional enzymes able to generate acetaldehyde was established (alcohol dehydrogenase: 0.97+/-0.84 mU mg(-1) protein; CYP2E1: 1.30+/-0.12 x 10(-2) pmol 4-nitrocatechol min(-1) mg(-1) protein) and a low activity of aldehyde dehydrogenase was observed in the cytosolic, mitochondrial and microsomal fractions (0.02+/-0.04; 0.35+/-0.09 and 0.72+/-0.19 mU mg(-1) protein, respectively). After a single high dose of ethanol, an increased susceptibility to oxidative stress was observed, as evidenced by changes in the shape of t-butylhydroperoxide induced emission of chemiluminescence in mammary tissue (6.3 g kg(-1) dose; at 3 and 6 h). In summary, the results show that even after single doses of ethanol, acetaldehyde, either formed in situ or arriving via blood, tends to accumulate in mammary tissue and that this condition might decrease cell defenses against injury.

Early nifurtimox-induced biochemical and ultrastructural alterations in rat heart.

Nifurtimox (Nfx) and Benznidazole (Bz) are being used for the treatment of the acute phase of Chagas' disease. Recently, they were also considered for use in the indeterminate phase. Both the nitroheterocyclic drugs have serious toxic side effects. The mechanism of Nfx toxicity is associated with the formation of reactive oxygen species (ROS) generated during nitroreduction. Potential effects on cardiac function have not been established yet, despite the well-known cardiopathy often produced by the disease itself. We describe experiments testing some acute effects of Nfx on the male Sprague Dawley rat heart. Nifurtimox was present in the heart at 1, 3 and 6 h after intragastric (i.g) treatment. In vitro studies on Nfx microsomal and cytosolic nitroreductase activities showed that only the microsomal fraction had the ability to nitroreduce it. Cytochrome P450 and cytochrome P450 reductase would be involved in the process as suggested by their response to specific inhibitors. Nifurtimox increased the cardiac protein carbonyl content at 1 and 3 h and decreased the protein sulfhydryl content at 3 h. In addition, 24 h after treatment ultrastructural alterations such as marked cytoplasmic vacuolization, separation and loss of myofibrils and mitochondrial swelling were observed. Results suggest that Nfx administration might aggravate pre-existing adverse cardiac conditions. Human & Experimental Toxicology (2007) 26, 781 -788.

Biochemical and ultrastructural alterations in the rat ventral prostate due to repetitive alcohol drinking.

Previous studies showed that cytosolic and microsomal fractions from rat ventral prostate are able to biotransform ethanol to acetaldehyde and 1-hydroxyethyl radicals via xanthine oxidase and a non P450 dependent pathway respectively. Sprague Dawley male rats were fed with a Lieber and De Carli diet containing ethanol for 28 days and compared against adequately pair-fed controls. Prostate microsomal fractions were found to exhibit CYP2E1-mediated hydroxylase activity significantly lower than in the liver and it was induced by repetitive ethanol drinking. Ethanol drinking led to an increased susceptibility of prostatic lipids to oxidation, as detected by t-butylhydroperoxide-promoted chemiluminiscence emission and increased levels of lipid hydroperoxides (xylenol orange method). Ultrastructural alterations in the epithelial cells were observed. They consisted of marked condensation of chromatin around the perinuclear membrane, moderate dilatation of the endoplasmic reticulum and an increased number of epithelial cells undergoing apoptosis. The prostatic alcohol dehydrogenase activity of the stock rats was 4.84 times lower than that in the liver and aldehyde dehydrogenase activity in their microsomal, cytosolic and mitochondrial fractions was either not detectable or significantly less intense than in the liver. A single dose of ethanol led to significant acetaldehyde accumulation in the prostate. The results suggest that acetaldehyde accumulation in prostate tissue might result from both acetaldehyde produced in situ but also because of its low aldehyde dehydrogenase activity and its poor ability to metabolize acetaldehyde arriving via the blood. Acetaldehyde, 1-hydroxyethyl radical and the oxidative stress produced may lead to epithelial cell injury.

Alcohol induction of liver nuclear ethanol and N-nitrosodimethylamine metabolism to reactive metabolites.

In previous studies from our laboratory we reported the presence in highly purified liver nuclei, free of contamination with other organelles, of an ethanol metabolizing system (NEMS) able to lead to acetaldehyde and 1-hydroxyethyl free radicals (1HEt). In the present study we tested whether this NEMS is inducible by chronic alcohol administration to rats and whether these nuclei also have increased ability to bioactivate N-nitrosodimethylamine (NDMA). Sprague Dawley male rats (125-150g) were fed with a nutritionally adequate liquid diet containing alcohol to provide 36% of total energy (standard Lieber-De Carli rat diet), for 28 days. Controls received an isocaloric diet without alcohol. Animals were sacrificed, livers were excised and microsomes and purified nuclear fractions were prepared. Both microsomes and nuclei from treated animals had significantly increased ability compared to controls, to biotransform ethanol to acetaldehyde using NADPH as cofactor under an air atmosphere. Both organelles also exhibited significantly increased capacity compared to controls, to bioactivate NDMA to formaldehyde and to reactive metabolites that bind covalently to proteins. Nuclear preparations from control animals were also able to metabolize NDMA to formaldehyde and reactive metabolites. Results indicate that liver nuclei may have a CYP2E1 able to bioactivate both NDMA and EtOH and that these processes are being induced by chronic alcohol drinking. The bioactivation of these xenobiotics to reactive metabolites in the neighborhood of nuclear proteins and DNA might have significant toxicological implications.

A liver nuclear ethanol metabolizing system. Formation of metabolites that bind covalently to macromolecules and lipids.

Recent studies from the laboratory reported the presence in highly purified liver nuclear preparations free of endoplasmic reticulum, mitochondria or cytosol, of an ethanol metabolizing group of enzymes (NEMS) leading to acetaldehyde and to hydroxyl and 1-hydroxyethyl (1HEt) free radicals. In the present study it is reported that when NEMS metabolize [14C]ethanol using NADPH as cofactor, its reactive metabolites bind covalently to nuclear proteins and lipids. No covalent binding to DNA was detected with presently used procedures. The covalent binding to nuclear proteins was acid labile and is mostly attributable to acetaldehyde. Additional evidence was attempted through studies where the acetaldehyde was identified as its 2,4-dinitrophenylhydrazone or as its pentafluorphenylhydrazone and gas chromatography (GC) analysis using electron capture detection. Values obtained were close to detection limit and of variable nature. The covalent binding to nuclear lipids involved phospholipids, fatty acids and esters and cholesterol free and esterified and it was only partially labile to acid treatment. Production of ethanol reactive metabolites such as acetaldehyde and free radicals, nearby liver nuclear DNA and nuclear proteins or lipids, might have significant toxicological consequences.

Mechanisms of the preventive properties of some garlic components in the carbon tetrachloride-promoted oxidative stress. Diallyl sulfide; diallyl disulfide; allyl mercaptan and allyl methyl sulfide.

Previous studies evidenced that garlic extracts and/or garlic components were able to prevent against chemically induced tumors or acute toxic effects of chemicals (e.g. CCl4 induced liver injury). The chemopreventive potential of garlic has been attributed to the presence in it of several bioactive organosulfur compounds. Those components might act as antioxidants able to scavenge free radicals. In the present work we describe initial studies on the antioxidative-stress properties of some garlic components such as: diallyl disulfide (DDS), diallyl sulfide (DAS), allyl mercaptan (AMT) and allyl methyl sulfide (AMS). We found that DAS, DDS and AMT but not AMS were able to trap trichloromethyl and trichloromethylperoxyl free radicals. Further, DDS but not DAS or AMT also inhibited CCl4 promoted liver microsomal lipid peroxidation. DAS, but not DDS, AMT or AMS was able to react with free radicals arised during UVC activation of hydrogen peroxide or terbutyl hydroperoxide but not with those produced during UVC activation of terbutyl peroxide. However, all garlic components tested absorbed energy from UVC and became partially destroyed in the process. AMT, but not DDS, AMS or DAS was able to destroy 4-hydroxynonenal, a key reactive aldehyde produced during lipid peroxidation. AMT and DDS were also able to prevent UVC plus CCl4 promoted oxidation of albumin in vitro, but DAS and AMS failed to do so. Results suggest that the antioxidative stress properties of garlic might result from the contributions of its sulfur component in different steps and not necessarily from the contribution of only one of them.

Cholesterol interaction with free radicals produced from carbon tetrachloride or bromotrichloromethane by either catalytic decomposition or via liver microsomal activation.

The reaction between cholesterol (Ch) and trichloromethyl or trichloromethyl peroxyl radicals was studied. The latter were generated from CCl4 either by benzoyl peroxide (BP) catalysis or via thermal activation or by liver microsomal NADPH-dependent biotransformation of CBrCl3. The structure of the products formed was elucidated by gas chromatography-mass spectrometry (GC/MS). Under aerobic conditions and using thermal activation of CCl4, the formation of 6 products was observed. Two (I and II) were dehydrated Ch derivatives (one also having a third double bond) (I). Another product was a delta(5)-3 ketone derivative of Ch (III). Two additional reaction products were determined as ketocholesterols (IV and V). One chloro Ch was also formed (VI). At low concentrations of BP, reaction was more extensive than under thermal activation, and the formation of peaks I to IV was also observed. When the reaction was conducted anaerobically and using thermal activation of CCl4 to generate radicals, only products I and II were formed in low yield. Under anaerobic conditions, but using catalyst, compounds I and III were produced plus two new isomeric ketocholesterol derivatives (VIII and IX) and also a compound having an extra hydroxyl group on the Ch structure (X). In order to check whether similar reactions are observable under biological experimental conditions, we used activation of CBrCl3 by liver microsomes. The incubation using only microsomes (without CBrCl3 or NADPH) showed two ketocholesterol peaks (A and B). In the presence of CBrCl3 we could detect peak B and hydroxycholesterol (C) and two others, ketocholesterols (D and E). D was the only peak showing close similarity (spectrum and retention time) to one of those observed in the chemical reaction system (V). The reaction of CBrCl3 in the presence of NADPH showed peaks B, C, D and E, in low abundance and a 7-ketocholesterol (F). If some of the reaction products reported here were formed during the intoxication with these haloalkanes, significant biological consequences might be expected.

Covalent binding of carbon tetrachloride reactive metabolites to liver microsomal and nuclear lipid and phospholipid classes from Sprague Dawley and osborne Mendel male rats.

The interaction between carbon tetrachloride (CCl4)-reactive metabolites and liver microsomal or nuclear lipids (covalent binding = CB) was studied in male rats from Osborne Mendel (OM) and Sprague Dawley (SD), strains with different cancer susceptibility. The in vitro or in vivo CB was more intense in OM than in SD. Most of the CB was with phospholipid (PL; SD > OM). The CB to cholesterol (CH) and cholesterol esters (CHE) was OM > SD. We also observed the presence of specific adducts only present in lipids from either OM or SD strains. The results were related to the well-known role of PL and CH derivatives in gene function control and cell growth.

Carbon tetrachloride promoted malondialdehyde formation in liver microsomal and nuclear preparations from Sprague Dawley or Osborne Mendel male rats.

CCl4 is a hepatic carcinogen in male Osborne-Mendel (OM) but not in Sprague Dawley (SD) male rats. We demonstrate the occurrence of NADPH-dependent CCl4-promoted lipid peroxidation processes (LP) leading to malondialdehyde (MDA) formation in liver microsomal and nuclear preparations from OM and SD rats which do not correlate with the cancer susceptibility of both strains. Our results suggest that MDA production might not be a rate determining step in the carcinogenic process. However, the formation of this reactive aldehyde proximal to DNA and nuclear proteins might play a role that remains to be elucidated.