G.D. Castro

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 liver microsomal and nuclear activation of methanol to hydroxymethyl free radicals

Recent studies from other laboratories reported that during methanol intoxication lipid peroxidation and protein oxidation in liver occurred. Further, they detected free radicals-PBN adducts in bile and urine of methanol poisoned rats. In this work, we report the presence in liver microsomes and nuclei of NADPH dependent processes of hydroxymethyl (HMet) radical formation. The detection of HMet radicals was performed by GC/MS of the trimethylsilyl derivatives of the PBN (N-tert-butyl-a-phenylnitrone)-radical adducts. The formation of HMet radicals was observed only under nitrogen, in these in vitro conditions. Formation of formaldehyde from methanol was observed in aerobic incubation mixtures containing either microsomes or nuclei but also under nitrogen using microsomes. The latter process was not inhibited by diphenyleneiodonium while the anaerobic microsomal one producing HMet was strongly inhibited by it. This shows that they are independent processes. Results suggest that both, liver nuclei and microsomes are able to generate free radicals during NADPH-mediated methanol biotransformation.

Further studies on the potential contribution of acetaldehyde accumulation and oxidative stress in rat mammary tissue in the alcohol drinking promotion of breast cancer

There is available evidence supporting a positive association between alcohol intake and risk of breast cancer. However, there is limited information regarding possible mechanisms for this effect. Past studies from our laboratory suggest that acetaldehyde accumulation in mammary tissue after alcohol intake may be of particular relevance and that cytosolic and microsomal in situ bioactivation of ethanol to acetaldehyde and free radicals and the resulting stimulation of oxidative stress could be a significant early event related to tumor promotion. In the present studies repetitive alcohol drinking for 28 days was found to produce significant decreases in the mammary tissue content of GSH and alpha tocopherol and in glutathione S‐transferase or glutathione reductase activities. In contrast, glutathione peroxidase activity was slightly increased. Malondialdehyde determinations did not show the occurrence of lipid peroxidation while the xylenol orange procedure gave positive results. The mammary microsomal metabolism of ethanol to acetaldehyde was not induced after an acute dose of ethanol or acetone able to induce the activity of its liver counterpart. The cytosolic pathway of alcohol metabolism instead was significantly enhanced by these two treatments. No increased generation of comet images was found either in mammary tissue or in liver under the experimental conditions tested. Results suggest that, while acetaldehyde accumulation in mammary tissue could be a critical event resulting from increasing production of acetaldehyde in situ plus an additional amount of it arriving via blood, other factors such as poor handling of the accumulated acetaldehyde could be also relevant. Copyright

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.

Cytochrome P450 reductase-mediated anaerobic biotransformation of ethanol to 1-hydroxyethyl-free radicals and acetaldehyde.

The ability of cytochrome P450 reductase to metabolize ethanol (EtOH) to acetaldehyde (AC) and 1-hydroxyethyl free radicals (1HEt) in anaerobic media was studied. Determination of AC was made by GC-FID analysis of the head space of incubation mixtures. The formation of 1HEt was established by GC-MS analysis of the adduct formed between the radical and the spin trap PBN. Results showed that pure human P450 reductase is able to biotransform EtOH to AC and 1HEt in a NADPH-dependent process under an oxygen-free nitrogen atmosphere. Pure FAD in the presence of NADPH was also able to generate AC and 1HEt from the alcohol. Anaerobic incubation mixtures containing either rat liver microsomes or pure nuclei were also able to biotransform EtOH to AC and 1HEt in the presence of NADPH. These processes were inhibited by antibody against rat liver microsomal P450 reductase. Results suggest that semiquinone forms of the flavin in P450 reductase may biotransform EtOH. These reactions might be of some significance in tissues where the P450 reductase is present in the absence of specific forms of cytochrome P450 known to be involved in EtOH metabolism (e.g. CYP2E1). However the toxicological significance of this enzymatic process remains to be established.

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.

Metabolism of ethanol to acetaldehyde by rat uterine horn subcellular fractions

Controversial studies from others suggested that alcohol intake could be associated with some deleterious effects in the uterus. Not all the effects of alcohol drinking on female reproductive organs can be explained in terms of endocrine disturbances. Deleterious effect of alcohol or its metabolites in situ could also play a role. Accordingly, we found a metabolism of alcohol to acetaldehyde in the rat uterine horn tissue cytosolic fraction mediated by xanthine oxidoreductase, requiring a purine cosubstrate and inhibited by allopurinol. This activity was detected by histochemistry in the epithelium and aldehyde dehydrogenase activity was detected in the muscular layer and in the serosa. There was a microsomal process, not requiring NADPH and of enzymatic nature, oxygen-dependent and inhibited by diethyldithiocarbamate, diphenyleneiodonium and partially sensitive to esculetin and nordihydroguaiaretic acid. The presence of metabolic pathways in the uterine horn able to generate acetaldehyde, accompanied by a low capacity to destroy it through aldehyde dehydrogenase, led to acetaldehyde accumulation in the uterus during ethanol exposure. Results suggest that any acetaldehyde produced in situ or arriving to the uterine horn via blood would remain in this organ sufficiently to have the opportunity to react with critical molecules to cause deleterious effects.

Inhibition of rat mammary microsomal oxidation of ethanol to acetaldehyde by plant polyphenols

We previously reported that the microsomal fraction from rat mammary tissue is able to oxidize ethanol to acetaldehyde, a mutagenic-carcinogenic metabolite, depending on the presence of NADPH and oxygen but not inhibited by carbon monoxide or other cytochrome P450 inhibitors. The process was strongly inhibited by diphenyleneiodonium, a known inhibitor of NADPH oxidase, and by nordihydroguaiaretic acid, an inhibitor of lipoxygenases. This led us to suggest that both enzymes could be involved. With the purpose of identifying natural compounds present in food with the ability to decrease the production of acetaldehyde in mammary tissue, in the present studies, several plant polyphenols having inhibitory effects on lipoxygenases and of antioxidant nature were tested as potential inhibitors of the rat mammary tissue microsomal pathway of ethanol oxidation. We included in the present screening study 32 polyphenols having ready availability and that were also tested against the rat mammary tissue cytosolic metabolism of ethanol to acetaldehyde. Several polyphenols were also able to inhibit the microsomal ethanol oxidation at concentrations as low was 10-50 μM. The results of these screening experiments suggest the potential of several plant polyphenols to prevent in vivo production and accumulation of acetaldehyde in mammary tissue.

Radioprotective effect of ethyl pyruvate alone or as a coadyuvant of amifostine

Entre los radioprotectores con uso clínico se destaca la amifostina (WR2721), eficaz pero con efectos secundarios que impiden su uso repetitivo. Es interés de los autores desarrollar radioprotectores menos tóxicos, por sí mismos o como coadyuvantes de amifostina. Ratas machos o hembras se expusieron a una dosis de rayos X de 2 Gy. Se ensayó el piruvato de etilo, solo o conjuntamente con amifostina. Cuarenta y ocho horas después de la exposición a la radiación, se realizó el recuento de eritrocitos, de leucocitos y la fórmula leucocitaria. Los efectos genotóxicos se evaluaron en leucocitos de sangre mediante el ensayo Cometa. Se realizaron también estudios de supervivencia a 60 días post-irradiación. En los animales irradiados disminuyeron los eritrocitos, y el recuento de leucocitos se redujo drásticamente respecto al control, presentando además una fórmula alterada. El tratamiento con piruvato de etilo resultó en una protección de los eritrocitos en ambos sexos. El daño genético disminuyó significativamente por el tratamiento con piruvato de etilo solo o combinado con amifostina, y en hembras se observó una mayor supervivencia solo con el tratamiento combinado. El piruvato de etilo mostró una acción radioprotectora significativa, que podría mejorarse aumentando la dosis o el tiempo de tratamiento, ya que tiene muy baja toxicidad.