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.

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.

Rat breast microsomal biotransformation of ethanol to acetaldehyde but not to free radicals: its potential role in the association between alcohol drinking and breast tumor promotion.

We recently showed that mammary cytosolic xanthineoxidoreductase had the ability to bioactivate ethanol (EtOH) to acetaldehyde (AC) and free radicals. In the present study, we report that the microsomal fraction also biotransforms EtOH to AC. One pathway requires NADPH and the others do not. Both need oxygen. The NADPH-dependent pathway is not inhibited by CO:O(2) (80:20) or SKF 525A and that excludes the participation of cytochrome P450. It is inhibited by diethyldithiocarbamate (DDTC), sodium azide, and diphenyleneiodonium (DPI) but not by desferrioxamine, which suggests a possible role of a non-iron copper-requiring flavoenzyme. The process was partially inhibited by thiobenzamide (TBA), methylmercaptoimidazole (MMI), and nordihydroguaiaretic acid (NDG) but not by dapsone, aminotriazole, or indomethacin. These results suggest the potential participation of flavine monooxygenase and of lipooxygenase or of peroxidases/oxidases having similar characteristics but not of lactoperoxidase or cyclooxygenase. The pathway not requiring NADPH could also be partially inhibited by DDTC, NDG, azide, DPI, and TBA or MMI but not by the other chemicals. Little activity proceeds under nitrogen. Oxidases or peroxidases might be involved. No formation of 1-hydroxyethyl radicals was detected either in the presence or absence of NADPH. The nature of the EtOH bioactivating enzymes involved remains to be established. However, the fact remains that an activation of EtOH to AC was found in mammary tissue and could have a significant effect in some stages of the process of breast tumor promotion by EtOH.

Rat ventral prostate microsomal biotransformation of ethanol to acetaldehyde and 1-hydroxyethyl radicals: its potential contribution to prostate tumor promotion.

Rat ventral prostate microsomal fraction was able to biotransform ethanol to acetaldehyde and 1-hydroxyethyl radicals (1HEt) in the presence of NADPH and oxygen. The enzymatic processes involved were not inhibited by desferrioxamine, CO, SKF 525A, 4-methylpyrazole, or polyclonal antibody against P450 reductase but they were significantly inhibited by diethyldithiocarbamate, 2-mercapto-1-methylimidazol, thiobenzamide, or diphenyleneiodonium chloride. Results would suggest the partial participation in these ethanol bioactivation processes of flavin containing monooxygenase (FMO) and/or other flavin dependent oxidases/peroxidases and of a non-iron metal-containing enzymes. Acetaldehyde and free radicals production by prostate microsomal fraction might potentially contribute to tumor promotion in heavy alcohol drinkers.

Cytosolic xanthine oxidoreductase mediated bioactivation of ethanol to acetaldehyde and free radicals in rat breast tissue. Its potential role in alcohol-promoted mammary cancer.

Epidemiological evidence links alcohol intake with increased risk in breast cancer. Not all the characteristics of the correlation can be explained in terms of changes in hormonal factors. In this work, we explore the possibility that alcohol were activated to acetaldehyde and free radicals in situ by xanthine dehydrogenase (XDh) and xanthine oxidase (XO) and/or aldehyde oxidase (AO). Incubation of cytosolic fraction with xanthine oxidoreductase (XDh+XO) (XOR) cosubstrates (e.g. NAD+, hypoxanthine, xanthine, caffeine, theobromine, theophylline or 1,7-dimethylxanthine) significantly enhanced the biotransformation of ethanol to acetaldehyde. The process was inhibited by allopurinol and not by pyrazole or benzoate or desferrioxamine and was not accompanied by detectable formation of 1HEt. However, hydroxylated aromatic derivatives of PBN were detected, suggesting either that hydroxyl free radicals might be formed or that XOR might catalyze aromatic hydroxylation of PBN. No bioactivation of ethanol to acetaldehyde was detectable when a cosubstrate of AO such as N-methylnicotinamide was included in cytosolic incubation mixtures. Results suggest that bioactivation of ethanol in situ to a carcinogen, such as acetaldehyde, and potentially to free radicals, might be involved in alcohol breast cancer induction. This might be the case, particularly also in cases of a high consumption of purine-rich food (e.g. meat) or beverages or soft drinks containing caffeine.

Rat ventral prostate xanthine oxidase bioactivation of ethanol to acetaldehyde and 1-hydroxyethyl free radicals: analysis of its potential role in heavy alcohol drinking tumor-promoting effects.

The ability of the ventral prostate cytosolic fractions to biotransform ethanol to acetaldehyde and 1-hydroxyethyl (1HEt) radicals was tested. Acetaldehyde formation was determined by GC-FID analysis in the head space of incubation mixtures. 1HEt was determined by spin trapping with PBN followed by extraction, silylation of the adduct and GC-MS of the product. Prostate cytosol was able to biotransform ethanol to acetaldehyde in the presence of NADH, hypoxanthine, xanthine, caffeine, theobromine, theophylline, and 1,7-dimethylxanthine but not in the presence of N-methylnicotinamide. All these biotransformations were inhibited by allopurinol and were sensitive to heating for 5 min at 100 degrees C. The biotransformation of ethanol to acetaldehyde in the presence of purines as cosubstrates was accompanied by the formation of hydroxyl and 1HEt radicals as detected by GC-MS, and the process was inhibited by allopurinol. Results suggest that prostate cytosolic xanthine oxidase is able to bioactivate ethanol to acetaldehyde and free radicals. The potential of these processes to be involved in tumor-promoting effects of heavy alcohol drinking in conjunction with high meat and/or purines consumption is analyzed. Multifactorial epidemiological studies considering that possibility might be convenient. Teratogenesis Carcinog. Mutagen. 21:109-119, 2001.

Liver nuclear ethanol metabolizing systems (NEMS) producing acetaldehyde and 1-hydroxyethyl free radicals.

Biotransformation of ethanol by liver nuclei was studied. The formation of acetaldehyde was determined by GC/FID. The 1-hydroxyethyl (1HEt) formation was established by spin trapping of the radical with N-t-butyl-alpha-phenylnitrone (PBN) followed by GC/MS. Liver nuclei, free of endoplasmic reticulum, cytosol or mitochondria, were able to biotransform ethanol to acetaldehyde in the presence of NADPH under air. Only 22% activity was observed in the absence of the cofactor. Twenty-six percent of the NADPH-dependent activity and 47% of the NADPH-independent activity were observable under nitrogen. Aerobic biotransformation was inhibited by CO, SKF 525A, 4-methylpyrazole and by diethyldithiocarbamate. This suggests that CYP2E1 is involved in the process. However, the formation of acetaldehyde was able to proceed under a pure CO atmosphere. The lack of inhibitory effects of 2-mercapto-1-methylimidazol and thiobenzamide excludes the potential participation of the NADPH flavin monooxigenase system. The formation of hydroxyl radicals in the process is suggested by the partial inhibitory effect of 5 mM mannitol and 5 mM sodium benzoate and by the fact that the 1HEt was detected. The NADPH-dependent anaerobic ethanol biotransformation pathway was stimulated by FAD and inhibited to some extent by iron chelators. The relevance of a liver nuclear ethanol biotransformation, generating reactive metabolites, such as acetaldehyde and free radicals, nearby DNA, nuclear proteins and lipids is discussed.

Hydroxyl and 1-hydroxyethyl free radical detection using spin traps followed by derivatization and gas chromatography-mass spectrometry.

Detection of hydroxyl free radicals is frequently performed by electron spin resonance (ESR) following spin trapping of the radical using 5,5-dimethylpyrroline N-oxide (DMPO) to generate a stable free radical having a characteristic ESR spectrum. The necessary ESR equipment is expensive and not readily available to many laboratories. In the present study, a specific and sensitive gas chromatography-mass spectrometry (GC/MS) method for detection of hydroxyl and hydroxyethyl free radicals is described. The DMPO or N-t-butyl-alpha-phenylnitrone (PBN) radical adducts are extracted and derivatized by trimethylsylilation and analyzed by GC/MS. To standardize the method, .OH and 1-hydroxyethyl radicals were generated in two different systems: 1) a Fenton reaction in a pure chemical system in the absence or presence of ethanol and 2) in liver microsomal suspensions where ethanol is metabolized in the presence of NADPH. In the Fenton system both radicals were easily detected and specifically identified using DMPO or PBN. In microsomal suspensions DMPO proved better for detection of .OH radicals and PBN more suitable for detection of 1-hydroxyethyl radicals. The procedure is specific, sensitive and potentially as useful as ESR.