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.

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.

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.

DNA bases attack by reactive metabolites produced during carbon tetrachloride biotransformation and promotion of liver microsomal lipid peroxidation.

Free radicals produced during carbon tetrachloride biotransformation and the promoted lipid peroxidation process of liver microsomal lipids are able to attack the DNA bases guanine, cytosine and thymine to give at least three altered bases. They were identified as 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua); 5-hydroxycytosine (5OHCyt) and 5-hydroxymethyluracil (5OHMeU). No adenine adducts were detected. If these altered bases were formed during carbon tetrachloride poisoning and were not adequately repaired before cell replication, serious permanent consequences for liver cell DNA could be expected and this might be somehow involved in the liver carcinogenic effects of the haloalkane.

5-Methylcytosine attack by hydroxyl free radicals and during carbon tetrachloride promoted liver microsomal lipid peroxidation: structure of reaction products.

We recently reported that trichloromethyl and trichloromethylperoxyl radicals attack 5-methylcytosine (5MC) to give several products derived from hydroxylation, deamination or halogenation reactions. Hydroxyl radicals and lipid peroxidation (LP) are more frequently involved in deleterious pathological or toxicological processes than those CCl4 derived radicals and thus we considered it of interest to test whether they also alter 5MC. We observed that OH radicals generated by 0.1 mM Fe2+/2.5 mM H202 at 25 degrees C for 1 h led to the production of 5-hydroxymethylcytosine (5MHC). When OH generation was performed with UV light (254 nm, 3400 muWatt/cm2) and 2mM H202 during 4 min at 25 degrees C the following products were observed: 5-hydroxy-5-methylhydantoin, 5-hydroxyhydantoin, 5MHC, thymine glycol (two isomers) and 5-hydroxymethyl-6-hydroxycytosine. When 5MC was exposed to liver microsomal suspensions in the presence of NADPH generating system and carbon tetrachloride during 1 h at 37 degrees C and under air, the formation of only 5HMC was observed. Detection and identification of all reaction products was done by GC/MS analysis of trimethylsilyl derivatives of the bases. If similar reactions occurred in DNA, these results might be of relevance to gene control, differentiation and carcinogenesis.

Dithiothreitol inhibitory effects on carbon tetrachloride-promoted NADPH-dependent lipid peroxidation in liver microsomal suspensions.

In previous studies from our laboratory evidence was provided that generation in vivo of dithiothreitol (DTT) from DTT tetraacetate (DTTAC) was accompanied with preventive effects against CCl4-induced necrogenic effects on the liver. In that study, we reported the ability of treatment to decrease the intensity of covalent binding (CB) of the CCl4 reactive metabolites to cellular components but no evidence of preventive effects on CCl4-induced lipid peroxidation (LP) was obtained by the diene hyperconjugation technique. Now, we report that DTT at concentrations 1 or 3 mM inhibit at steps of the process after diene conjugation and prior to malondialdehyde formation. One of those steps might involve peroxides since we observed that DTT is able to significantly react with benzoyl peroxide in a model system. Others might also involve free radicals for in the present study we observed the reaction of DTT with trichloromethyl or trichloromethylperoxy free radicals generated from CBrCl3 in a model system. Reactions of DTT with free radicals and peroxides resulting in inhibition of CB and LP might be critical components in the preventive effects of DTTAC against CCl4-induced liver damage.

Decreased incorporation of 14C-leucine in different liver nuclear protein fractions at early stages of carbon tetrachloride poisoning in the rat.

Administration of CCl4 to 12-14 h starved Sprague Dawley male rats (220-240 g) at a dose of 5 ml of a 20% (v/v) olive oil solution/kg i.p. decreased the incorporation of 14C-leucine into microsomal and nuclear proteins at 1 or 2 h after the hepatotoxin. Decreased amino acid incorporation capacity in nuclear but not in microsomal proteins returned to control values at 4 h after poisoning. Cycloheximide given i.p. at a dose of 1 mg/kg in saline either alone or before CCl4 also decreased to a similar or greater extent the incorporation of 14C-leucine into both, microsomal and nuclear proteins at 1 h. The CCl4-induced decrease in amino acid incorporation was observable and significant even 1 h after CCl4 and involved histone and different non-histone nuclear protein fractions. Results suggest that decreased protein synthesis at the endoplasmic reticulum occurring in the very early stages of CCl4 poisoning might provoke temporary deficiencies in protein supplies to nuclei with as yet unknown consequences. However, some of the proteins involved have major regulatory properties in DNA expression.

Non-genotoxic carcinogens. Approaches to their risk assessment.

Epidemiological studies support the idea that most human cancers are related to chemicals present in the human environment. In turn, chemicals are believed to cause cancer via either genotoxic or non-genotoxic mechanisms. There were described in literature several simple, rapid and inexpensive short term tests to reasonably predict the genotoxic nature of chemicals but in contrast, there is no reliable test or battery of tests available to predict the carcinogenicity of non-genotoxic compounds and this poses a major problem to their risk assessment. In addition, there are conflictive opinions about risk assessment needs for both classes of carcinogens. Some workers believe that for non-genotoxic carcinogens, thresholds for exposure can be drawn while others do not. In this review, the reasons behind both of these opinions and the present hypotheses about the mechanism of action of non-genotoxic carcinogens are described and analyzed in relation to future needs.

Efecto del carcinogeno dimetilnitrosamina sobre el rinon del vison / Efect of carcinogen dimethylnitrasamine on the kidney of mink

Visones de un criadero que recibian alimentos, sobre la base de restos de pescado, evidenciaron un significativo aumento en su mortalidad, presencia de canceres hepaticos y alteraciones renales revelables histologicamente. Esos efectos fueron atribuibles a presencia, en el alimento, de dimetilnitrosamina (NDMA), en concentraciones 1,8 ug/g. En este trabajo se estudia en detalle el efecto de la NDMA sobre el rinon del vison. Visones que fueron tratados ip con NDMA(7 mg/kg en sol. fis.), mostraron dano evidenciable ultraestructuralmente en la corteza renal. El dano fue mayor en los tubulos proximales, que en los distales, pero era de naturaleza similar. Las celulas epiteliales tubulares de los animales intoxicados mostraron: a)Condensacion de la cromatina nuclear y dilatacion de la membrana perinuclear. b)Marcada hinchazon mitocondrial y ruptura de sus crestas con perdida de contenida de la matriz mitocondrial. c)Despegue de ribosomas y dilatacion del reticulo endoplasmico. d)Aumento del numero y tamano de las vacuolas autofagicas. e)Aparicion de gotas lipidicas en el citiplasma. En contraste con lo previamente establecido, para el caso de cancer hepatico del vison, el mecanismo del dano renal por NDMA no se pudo correlacionar directamente con la union de metabolitos reactivos de esta a proteinas o acidos nucleicos o la biotransformacion microsomal o mitocondrial de la NDMA o formaldehido. No obstante, el rinon biotransforma la NDMA a CO2, pero lo hace 3-4 veces menos intensamente que el rinon de rata. Los resultados sugeririan la presencia, en el caso del dano renal por NDMA, de mecanismos distintos de accion, a los habitualmente aceptados como responsables del dano hepatico o el renal en otras especies. Alternativamente, el dano renal puede deberse a dano hepatico concomitante

Efecto del carcinogeno dimetilnitrosamina sobre el rinon del vison / Efect of carcinogen dimethylnitrasamine on the kidney of mink

Visones de un criadero que recibian alimentos, sobre la base de restos de pescado, evidenciaron un significativo aumento en su mortalidad, presencia de canceres hepaticos y alteraciones renales revelables histologicamente. Esos efectos fueron atribuibles a presencia, en el alimento, de dimetilnitrosamina (NDMA), en concentraciones 1,8 ug/g. En este trabajo se estudia en detalle el efecto de la NDMA sobre el rinon del vison. Visones que fueron tratados ip con NDMA(7 mg/kg en sol. fis.), mostraron dano evidenciable ultraestructuralmente en la corteza renal. El dano fue mayor en los tubulos proximales, que en los distales, pero era de naturaleza similar. Las celulas epiteliales tubulares de los animales intoxicados mostraron: a)Condensacion de la cromatina nuclear y dilatacion de la membrana perinuclear. b)Marcada hinchazon mitocondrial y ruptura de sus crestas con perdida de contenida de la matriz mitocondrial. c)Despegue de ribosomas y dilatacion del reticulo endoplasmico. d)Aumento del numero y tamano de las vacuolas autofagicas. e)Aparicion de gotas lipidicas en el citiplasma. En contraste con lo previamente establecido, para el caso de cancer hepatico del vison, el mecanismo del dano renal por NDMA no se pudo correlacionar directamente con la union de metabolitos reactivos de esta a proteinas o acidos nucleicos o la biotransformacion microsomal o mitocondrial de la NDMA o formaldehido. No obstante, el rinon biotransforma la NDMA a CO2, pero lo hace 3-4 veces menos intensamente que el rinon de rata. Los resultados sugeririan la presencia, en el caso del dano renal por NDMA, de mecanismos distintos de accion, a los habitualmente aceptados como responsables del dano hepatico o el renal en otras especies. Alternativamente, el dano renal puede deberse a dano hepatico concomitante

Interaction of trichloromethyl radicals with phenylalanine.

Chemically or enzymatically generated trichloromethyl free radicals interact with liposoluble derivatives of phenylalanine. In vitro, in a chemical system to produce .CCl3 (benzoyl peroxide catalysis), this radical attacked N-acetyl-d,l-phenylalanine methyl ester (PheMeAc) to give a monochlorinated derivative (I) and an unsaturated imine type derivative of PheMeAc (II). Using a liver microsomal system to produce .CCl3 (microsomes + NADPH + CCl4 under nitrogen), the attack of PheMeAc did not result in I or II formation, but in production of benzene. The phenylalanine content in liver microsomal proteins from rats treated with CCl4 6 h before, was not significantly decreased. The results suggest that phenylalanine is a potential target of .CCl3.

Biotransformation of carbon tetrachloride and lipid peroxidation promotion by liver nuclear preparations from different animal species.

Liver nuclear preparations from male Syrian Golden hamster (SG); C3H mice and Sprague-Dawley (SD) rats were able to biotransform CCl4 to CHCl3. That ability was not NADPH dependent and proceeded to an equal extent under N2 or air. Studies in more detail with C3H mice preparations revealed that only one of the processes was of an enzymatic nature and that it was inhibited by 1 mM EDTA. There was a correlation between liver nuclear ability to biotransform CCl4 to CHCl3 in the species tested and their liver carcinogenic response to CCl4. That correlation was not observed when biotransformation was studied using liver slices instead of liver nuclei. Liver nuclear preparations from the 3 species were able to promote a lipid peroxidation (LP) process in the presence of CCl4. The process was fully NADPH dependent in the case of SG and SD preparations but not in C3H mice. Study of the process in detail in the case of C3H mice shows that in that case LP was heat and EDTA sensitive, particularly in the absence of NADPH. There was no correlation between the intensity of CCl4 promoted LP either in liver nuclear or liver slices preparations in the 3 species tested and their carcino genic response to CCl4. Results might suggest that LP does not determine or rate limit the process of cancer development by CCl4 but do not exclude its participation in a given stage of the overall process.

Species differences in the interaction between CCl4 reactive metabolites and liver DNA or nuclear protein fractions.

CCl4 has been reported to be a liver carcinogen for several mice strains, for Syrian Golden hamsters, but not for Sprague-Dawley rats. CCl4 is an experimental carcinogen for which no convincing evidence of mutagenicity is available despite the fact that CCl4 reactive metabolites bind covalently to liver DNA. Here we describe studies on the relationship between the intensities of the covalent binding (CB) of CCl4 reactive metabolites to liver DNA and nuclear proteins either in vivo or in vitro after activation to reactive metabolites by nuclear preparations, considering the known susceptibility of the C3H mice, Syrian Golden hamsters and Sprague-Dawley rats to CCl4. There was no correlation between the intensity of CCl4 carcinogenic effects on the liver and CB of CCl4 reactive metabolites to total DNA either in vitro or in vivo. A good correlation between carcinogenicity and CB to total nuclear proteins (in vivo or in vitro was found. Nuclear protein fractionation studies revealed CB of CCl4 reactive metabolites to both histone and non-histone proteins when nuclear preparations activated CCl4 either in the presence or absence of NADPH. Acidic and residual nuclear proteins were the favorite targets of the interaction with CCl4 reactive metabolites. A good correlation between CB to these nuclear protein fractions and CCl4 carcinogenicity in the three species was found.

Nitrosodimethylamine metabolism in rat ovaries. Interactions of its metabolites with nucleic acids and proteins.

N-Nitrosodimethylamine (NDMA) was metabolized by ovarian slices of noninbred Sprague-Dawley rats (200-250 g body wt, to CO2 and to reactive metabolites that bind covalently to nucleic acids. That ability was about 10 or 5 times smaller than the one observed in liver slices, respectively. Both ovarian microsomes and mitochondria were able to biotransform NDMA to formaldehyde and to reactive metabolites that bind covalently to proteins. Formaldehyde formation by microsomes was significantly higher than that by ovarian mitochondria but of the same order of magnitude. Ability to lead to covalent binding to proteins in microsomes was not significantly different from that in the respective mitochondrial fraction. DNA isolated from ovarian slices activating NDMA revealed the presence of the altered bases 7-methylguanine (7-MeGua) and O6-methylguanine (O6-MeGua) resulting from NDMA reactive metabolites' attack. Results suggest potential, mutagenic, carcinogenic and reproductive risks derived from women's exposure to NDMA present in tobacco smoke, food, beverages, workplace or other environmental sources.

[Slow-release theophyllines: serum and saliva levels in asthmatic children]. / Teofilinas retardadas: niveles séricos y salivales en niños asmáticos.

A total of 42 children, aged from 5 to 13, all having been diagnosed as extrinsic asthma cases, were observed with the aim of establishing the average dose needed to maintain serum concentration by means of the administration of a slow-release theophylline preparation. The resultant average dose was of 20.14 +/- 3.2 mg/kg/day for the under-9s and of 19.00 +/- +/- 4.67 mg/kg/day for the older ones. The overall average was 20.08 +/- 3.31 mg/kg/day (range: 14.28 mg/kg/day-28 mg/kg/day). Statistical analysis of salivary and serum theophylline levels simultaneously obtained was carried out, reading a lineal correlation coefficient of 0.89 (p less than 0.001), while the equation for the regression-line relationship was: Se = 1.52.Sa + 0.75. The preparation, with doses every 12 hours, proved to be efficient in maintaining therapeutic levels in most cases. The use of respective salivary levels could prove useful in later observations carried out on these children.

Studies on the mechanism of the acute and carcinogenic effects of N-nitrosodimethylamine on mink liver.

Outbreaks of liver necrosis and liver hemangiosarcoma were detected in a mink breeding colony in Argentina. Analysis of the Minks' food revealed the presence of 2.6 ppm dimethylnitrosamine (NDMA) in it, apparently as a result of the addition of nitrite as preservative. Previous studies gave evidence of the particular susceptibility of minks to NDMA and other hepatic insults. We have determined several biochemical parameters known to correlate with NDMA hepatotoxic effects and compared them with those in rat liver. NDMA administration to both species resulted in the formation of reactive metabolites able to interact with liver DNA to give N7-methylguanine and O6-methylguanine adducts. Biotransformation of NDMA by liver slices to CO2 was significantly lower in the mink than in the rat, whereas the covalent binding (CB) to nucleic acids was slightly lower than in in the rat. Aminopyrine N-demethylase activity was also significantly less in mink than in rat liver. The CB of NDMA reactive metabolites to microsomal proteins was not significantly lower in mink as compared to the rat, and the same holds true for the biotransformation of NDMA to formaldehyde by microsomal preparations. Results suggest that the high susceptibility of minks to NDMA might be partially due to a decreased ability to detoxicate NDMA but also to a higher intrinsic susceptibility of their liver cells to a given chemical insult.