José Alberto Castro

Studies on the destruction of liver microsomal cytochrome P-450 by carbon tetrachloride administration

Carbon tetrachloride impairs the oxidative enzymes in liver microsomes by decreasing the amount of cytochrome P-450. The destruction is probably not mediated by an increase in lipid peroxidation, since ethanol which promotes lipid peroxidation does not decrease cytochrome P-450 or the metabolism of ethylmorphine. The decrease in cytochrome P-450 is not due to destruction of liver cells or to altered permeability of endoplasmic reticulum because NADPH cytochrome c reductase was not detected in plasma and was not decreased in liver microsomes. The solvent effect of CCl4 probably does not account for the decrease in cytochrome P-450 because CHCl3 and CH2Cl2 did not decrease the cytochrome. Our results are in accord with the view that CCl4 acts through an active metabolite, but they do not necessarily imply that the active metabolite is formed by a cytochrome P-450 enzyme.

Studies on the irreversible binding of 14CCCl4 to microsomal lipids in rats under varying experimental conditions

Abstract In order to establish whether a relationship exists between the activity of hydroxylating drug metabolizing enzymes and the activation of CCl4, the study of the irreversible binding of the 14C from 14CCl4 to microsomal lipids was undertaken under differing conditions of drug metabolism. Liver and kidney microsomes have greater ability to activate CCl4 than their respective mitochondrial or 105,000 g supernatant fractions. In adrenals, an unexpectedly high CCl4 activating activity was found not only in microsomes but also in mitochondria. The CCl4 activating ability was significantly higher than in controls in the following experimental conditions: in SKF 525A treated rats; in adrenalectomized rats; and in 72 hr starved rats. Activation was not significantly changed in castrated, Sch 5706 treated or aminopyrine treated rats and was decreased in female, DPEA treated or nicotinamide treated rats. The results are discussed in relation to a possible activation of CCl4 occurring during the reduction of the CCl4-cytochrome P-450 complex mediated by cytochrome P-450 reductase or arising during the interaction of CCl4 with reduced cytochrome P-450.

Covalent binding of carbon tetrachloride metabolites to liver nuclear DNA, proteins, and lipids☆☆☆

Abstract 14 C from 14 CCl 4 irreversibly binds in vivo to liver DNA from strain A J mice and Sprague-Dawley rats. Binding of 14 CCl 4 to DNA was also observed in vitro in incubation mixtures containing microsomes and a NADPH-generating system as well as in tissue slices. Chemically induced ·CCl 3 (CCl 4 + benzoyl peroxide system) intensively binds to DNA. Liver nuclear proteins also irreversibly bind CCl 4 metabolites. Nuclear protein fractionation studies revealed that deoxyribonucleoproteins, acidic proteins, histones, and residual proteins are the favorite targets of metabolite interaction. Nuclear sap proteins are less intensively labeled. Nuclear lipids were markedly labeled by CCl 4 reactive metabolites. Most of the label is in the phospholipid fraction and diphosphatidylglycerol is most intensively labeled; phosphatidylethanolamine, phosphatidylcholine, lysophosphatidylcholine, and sphingomyeline are also labeled by metabolites. The interaction of ·CCl 3 with DNA and nuclear proteins could be relevant to CCl 4 -induced liver tumors and hepatotoxic effects.

Effect of adrenalectomy and cortisone administration on components of the liver microsomal mixed function oxygenase system of male rats which catalyzes ethylmorphine metabolism

Abstract The effects of adrenalectomy and cortisone administration on components of the mixed function oxygenase system of liver microsomes from adult male rats were studied. The N -demethylation of ethylmorphine (EM) as measured by formaldehyde production was used as an index of the overall activity of this system. EM demethylase activity in liver microsomes from adrenalectomized rats was 56 per cent of the value obtained with sham-operated controls, while cytochrome P-450 content fell by only 19 per cent. NADPH cytochrome c reductase and cytochrome P-450 reductase activities were decreased 68 and 69 per cent, respectively, in liver microsomes from adrenalectomized animals, while the apparent affinity constant ( K sp ) of EM for cytochrome P-450 spectral change was not altered. The decrease in maximal spectral change caused by adrenalectomy corresponded to the decrease in cytochrome P-450 content. Cortisone acetate administration (5 mg/kg/day for 8 days) to adrenalectomized rats prevented the decrease in EM demethylase activity. Accordingly, the steroid treatment increased the NADPH cytochrome c reductase and cytochrome P-450 reductase activities to 113 and 140 per cent, respectively, of the values obtained with the sham-operated controls, without significantly altering the cytochrome P-450 content or the kinetic constants for the spectral changes. These data suggest that the well known decrease in drug-metabolizing activity seen in liver microsomes of adrenalectomized animals is not related to changes in cytochrome P-450 content or to a decrease in the ability of cytochrome P-450 to bind drug substrates. However, there appears to be a relationship between the reductase activities and the adrenal function, because the changes in activities in NADPH cytochrome c reductase and cytochrome P-450 reductase in liver microsomes from adrenalectomized and cortisone-treated adrenalectomized rats paralleled the changes in EM demethylase activity.

Covalent binding of chloroform metabolites to nuclear proteins — No evidence for binding to nucleic acids

No evidence was found for covalent binding of CHCl3 metabolites to male Sprague-Dawley rats or strain A/J male mice liver DNA or RNA under different experimental conditions including phenobarbital or 3-methyl-cholanthrene or diethylmaleate treatments or multiple CHCl3 injections. In addition, no binding to DNA was observed when DNA was exposed to CHCl3 in incubation mixtures containing microsomes and NADPH or when tissue slices were incubated with CHCl3. In contrast, nuclear histone and non-histone proteins bound to CHCl3 metabolites.

Inhibition of the Rat Breast Cytosolic Bioactivation of Ethanol to Acetaldehyde by Some Plant Polyphenols and Folic Acid

Abstract: There is a well-established association between alcohol consumption and breast cancer risk. About 4% of the breast cancers in developed countries are estimated to be attributable to drinking alcohol. The mechanism of tumor promotion by alcohol remains unknown. Recent studies from our laboratory and others showed the ability of mammary tissue to bioactivate ethanol to mutagenic/carcinogenic acetaldehyde and free radicals. Xanthine oxidoreductase (XOR) is an enzyme involved in those biotransformation processes. In the present study, we provide evidence of the ability of different natural polyphenols and of folic acid derivatives to inhibit the biotransformation of alcohol to acetaldehyde by rat breast cytosolic XOR. Folic acid and dihydrofolic acid, at concentrations of 10 μM, inhibited 100% and 84%, respectively, of the cytosolic acetaldehyde formation. Thirty-five polyphenols were tested in these initial experiments: ellagic acid, myricetin, quercetin, luteolin, and apigenin inhibited 79-95% at 10 μM concentrations. The remaining polyphenols were either less potent or noninhibitory of acetaldehyde formation at similar concentrations in these screening tests. Results are relevant to the known preventive effects of folic acid against alcohol-induced breast cancer and to their potential preventive actions if added to foods or alcoholic beverages.

Metabolism of ethanol to acetaldehyde and increased susceptibility to oxidative stress could play a role in the ovarian tissue cell injury promoted by alcohol drinking

It is known that drinking alcohol can lead to reproductive problems in women. In this study, we analyzed the possibility that part of those effects were mediated through alterations of ovarian function related to ethanol oxidation to acetaldehyde occurring in situ. Biotransformation in the rat ovary cytosolic fraction was partially inhibited by allopurinol, suggesting the participation of xanthine oxidoreductase in the process. Microsomal pathway was of enzymatic nature, requiring nicotinamide adenine dinucleotide phosphate-oxidase (NADPH), sensitive to oxygen and significantly inhibited by sodium diethyldithiocarbamate, 4-methylpyrazole and diphenyleneiodonium. Aldehyde dehydrogenase activity was detected by histochemistry in the ovarian tissue, in the strome surrounding the follicle while no alcohol dehydrogenase was detected. However, biochemical determination of alcohol dehydrogenase and aldehyde dehydrogenase activities in rat ovarian tissue revealed the presence of some activity of both enzymes but significantly lower than those found in the liver. By repetitive exposure of animals to ethanol, the microsomal metabolism to acetaldehyde was increased but not in the case of the cytosolic fraction. In these animals, t-butylhydroperoxyde-promoted chemiluminiscence was increased in comparison to control, revealing an increased susceptibility to oxidative stress due to alcohol drinking. Ultrastructure of ovarian tissue from rats exposed chronically to alcohol revealed alterations at the level of the granulosa; theca interna and pellucida zones. In the secondary follicle, alterations consisted of marked condensation of chromatin attached to the nuclear inner membrane. Intense dilatation of the outer perinuclear space could be observed. There was a marked dilatation of the rough endoplasmic reticulum accompanied of significant detachment of ribosomes from their membranes. Mitochondria appeared swollen. In the zona pellucida, most of the cell processes from oocyte and corona radiata cells were absent or broken totally or in part. Results suggest that in the rat ovary, metabolism of ethanol to acetaldehyde may play a role in alcohol effects on female reproductive function.

Effects of alkylating agents of human plasma cholinesterase: The role of sulfhydryl groups in its active center

Abstract Several alkylating agents (bromobenzylcyanide, chloroacetophenon, and xylyl bromide) were found to inhibit cholisterase from human plasma. Less reactive alkylating compounds such as bromoacetone, chloroacetate, and iodoacetate did not inhibit the enzyme. Chloropicrin, which is a powerful oxidizing agent, was not found to inhibit either. Several mercaptide-forming agents, like p- chloromercuribenzoic acid, diphenylaminechloroarsine, and ethyl-dichloroarsine, were effective inhibitors. w- Chloroacetophenon was found to be an instantaneous and noncompetitive inhibitor. The inhibition was reversible by dilution or dialysis. The degree of inhibition was higher in an aqueous medium that when a less polar solvent was used. These findings suggest that a sulfhydryl group is not involved during the inhibition of cholinesterase by alkylating agents. The possibility of a reaction on the histidine or methionine groups of the enzyme is discussed. The inhibition also was dependent on the substrate employed during the activity assay. The order of percentage of inhibition was found to be acetylcholine > butyrylcholine > benzoylcholine. These data are used to offer an interpretation for some aspects of the lachrymatory action of these agents.

Studies on the interaction between inhibitors of drug metabolism and horse plasma cholinesterase.

Abstract Several inhibitors of drug metabolism were found to inhibit horse plasma cholinesterase activity at concentrations ranging from 0·3 μM to 20 μM. A detailed study of the inhibition was made using 2-diethylaminoethyl 2,2-diphenylvalerate (SKF 525A); 2,4 dichloro-6-phenylphenoxyethyldiethylamine (Lilly 18947) and N -(γ-dimethyl- aminopropyl-iminodibenzyl) (imipramine). The cholinesterase inhibition produced by these three compounds was very fast, competitive and reversible. Only SKF 525A was able to delay the irreversible inhibition of the enzyme by dimethyl-dichlorovinyl-phosphate, indicating that it is probably acting at the esteratic site. The inhibition by these compounds increases from pH 5·5 to pH 7·5–8·0, then decreases. Since the inhibitors are mainly in the basic form at pH 7·5, the results were interpreted as showing that the active portion of the inhibitor is located at the basic nitrogen atom and that this group interacts on a chemical group of the enzyme having a p K a about 8·6–9·0. In agreement with this assumption, changes in basicity of the nitrogen atom of the inhibitors explained very well the differences in inhibitory power between pairs of analog compounds. This hypothesis also explains why bovine erythrocyte acetylcholinesterase exhibited smaller affinity for inhibitors than the horse plasma enzyme, in spite of the similarities in the inhibition at different pH values. Changes in entropy for the inhibition of cholinesterase by Lilly 18947 were also in accordance with this hypothesis.

Alcohol drinking and mammary cancer: Pathogenesis and potential dietary preventive alternatives.

Alcohol consumption is associated with an increased risk of breast cancer, increasing linearly even with a moderate consumption and irrespectively of the type of alcoholic beverage. It shows no dependency from other risk factors like menopausal status, oral contraceptives, hormone replacement therapy, or genetic history of breast cancer. The precise mechanism for the effect of drinking alcohol in mammary cancer promotion is still far from being established. Studies by our laboratory suggest that acetaldehyde produced in situ and accumulated in mammary tissue because of poor detoxicating mechanisms might play a role in mutational and promotional events. Additional studies indicated the production of reactive oxygen species accompanied of decreases in vitamin E and GSH contents and of glutathione transferase activity. The resulting oxidative stress might also play a relevant role in several stages of the carcinogenic process. There are reported in literature studies showing that plasmatic levels of estrogens significantly increased after alcohol drinking and that the breast cancer risk is higher in receptor ER-positive individuals. Estrogens are known that they may produce breast cancer by actions on ER and also as chemical carcinogens, as a consequence of their oxidation leading to reactive metabolites. In this review we introduce our working hypothesis integrating the acetaldehyde and the oxidative stress effects with those involving increased estrogen levels. We also analyze potential preventive actions that might be accessible. There remains the fact that alcohol drinking is just one of the avoidable causes of breast cancer and that, at present, the suggested acceptable dose for prevention of this risk is of one drink per day.