J.A. Castro

Interaction of benznidazole reactive metabolites with nuclear and kinetoplastic DNA, proteins and lipids from Trypanosoma cruzi

Epimastigotes ofTrypanosoma cruzi (Tulahuen strain Tul 0 stock) biotransform benznidazole (N-benzyl-2-nitro-1-imidazone acetamide) to reactive metabolites that bind covalently to DNA, proteins and lipids of the parasite. These effects might be related to the trypanocidal action of benznidazole, a chemotherapeutic agent against Chagas disease.

N-Acetyl cysteine is an early but also a late preventive agent against carbon tetrachloride-induced liver necrosis

N-Acetyl cysteine (NAC) treatment 30 min before or 6 or 10 h after carbon tetrachloride (CCl4) administration significantly prevented the liver necrosis produced by the hepatotoxin at 24 h. NAC pretreatment was able to partially decrease the covalent binding of CCl4 reactive metabolites at 1 and 3 h of poisoning and, to a small extent, the concentration of CCl4 reaching the liver at 3 h. NAC also diminished partially the CCl4-promoted increases in lipid peroxidation at 3 h, but had an enhancing effect of its own of small intensity. Results suggest that early and late protective effects of NAC might be attributable to its prior conversion to cysteine and glutathione.

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.

Oxidation of ethanol to acetaldehyde and free radicals by rat testicular microsomes

A large number of epidemiological studies evidencing that excessive alcohol consumption is associated with impaired testosterone production and testicular atrophy are available in the literature. One hypothesis to explain the deleterious action of alcohol involves the in situ biotransformation to acetaldehyde, but it strongly suggests the need to learn more about the enzymatic processes governing alcohol metabolism to acetaldehyde in different cellular fractions since limited information is available in the literature. In this article we report studies on the metabolic conversion of alcohol to acetaldehyde and to 1-hydroxyethyl radicals in rat testicular microsomal fractions. The oxidation of ethanol to acetaldehyde in rat testes microsomal fraction was mostly of enzymatic nature and strongly dependent on the presence of NADPH and oxygen. Several compounds were able to significantly decrease the production of acetaldehyde: SKF 525A; diethyldithiocarbamate; esculetin; gossypol; curcumin; quercetin; dapsone; and diphenyleneiodonium. Microsomal preparations in the presence of NADPH were also able to produce both hydroxyl and 1-hydroxyethyl free radicals. Their generation was modulated by the presence of diphenyleneiodonium, gossypol, and deferoxamine. Results show that rat microsomal fractions are able to metabolize alcohol to deleterious chemicals, such as acetaldehyde and free radicals, that may be involved in ethanol toxic effects. Enzymes involved could include CYP2E1, P450 reductase, and other enzymes having lipoxygenase- /peroxidase-like behavior.

Benznidazole biotransformation in rat heart microsomal fraction without observable ultrastructural alterations: comparison to Nifurtimox-induced cardiac effects

Benznidazole (Bz) and Nifurtimox (Nfx) have been used to treat Chagas disease. As recent studies have de-monstrated cardiotoxic effects of Nfx, we attempted to determine whether Bz behaves similarly. Bz reached the heart tissue of male rats after intragastric administration. No cytosolic Bz nitroreductases were detected, although microsomal NADPH-dependent Bz nitroreductase activity was observed, and appeared to be mediated by P450 reductase. No ultrastructurally observable deleterious effects of Bz were detected, in contrast to the overt cardiac effects previously reported for Nfx. In conclusion, when these drugs are used in chagasic patients, Bz may pose a lesser risk to heart function than Nfx when any cardiopathy is present.

Late preventive effects of trifluoperazine on carbon tetrachloride-induced hepatic necrosis

As a very preliminary test for a possible role of calmodulin in CCl4-induced hepatic injury, we studied the effects of the anticalmodulin drug trifluoperazine (TFP) on several deleterious actions of CCl4 on the liver. TFP administrated 30 min before or 6 or 10 hr after CCl4 significantly prevented hepatic necrosis induced by the hepatotoxin at 24 hr but not at 72 hr. TFP did not modify the CCl4 concentrations reaching the liver, or the intensity of the covalent binding of CCl4-reactive metabolites to hepatic microsomal proteins or lipids or the CCl4-induced cytochrome P-450 and glucose 6 phosphatase destruction. TFP administration decreased body temperature between 0 and 1 degree C in controls and between 1.2 and 3.5 degrees C in CCl4-treated animals during the 24-hr observation period. When TFP-treated CCl4-poisoned animals were kept normothermic, protective effects were eliminated. One possibility is that the protective effect of TFP might be due to a nonspecific action related to decreased body temperature. Alternatively, prevention might result from TFP inhibition of a late-occurring process critical for CCl4-induced cell necrosis requiring calmodulin participation. If this alternative were in operation, protective consequences of this inhibitory effect of TFP should be either canceled or counteracted in the normothermic TFP + CCl4-treated animal.

Mechanism of the drug-metabolizing enzymes' induction by 2-diethylaminoethyl-2-2-diphenylvalerate-HCl (SKF 525 A).

Abstract Repetitive administration of 2-diethylaminoethyl-2-2-diphenylvalerate-HCl (SKF 525 A) produces a biphasic effect on the pentobarbital sleeping time of rats. It causes a significant prolongation effect after one daily dose, but it significantly shortens it when more daily doses are given. Administration of three daily doses of SKF 525 A results in increased activity of ethylmorphine N -demethylase and cytochrome P -450 ( P -450) content in liver microsomes while aniline hydroxylase and cytochrome c reductase activity in these preparations were not significantly increased. Repetitive administration of SKF 525 A increased [ 14 ]leucine incorporation and decreased ([ 14 C]guanidino)arginine disappearance from microsomal proteins. Results suggest that effects on pentobarbital sleeping time and on drug metabolism resulting from repetitive SKF 525 A administration would result from increases in P -450 content which might derive from increased microsomal protein synthesis and from decreased microsomal protein degradation.

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.

Evidence for hydroxyl free radical formation during paraquat but not for nifurtimox liver microsomal biotransformation. A dimethyl-sulfoxide scavenging study

The effect of several experimental conditions on methane (CH4) production from dimethylsulfoxide (DMSO) in incubation mixtures containing liver microsomes and NADPH generating systems was studied. The process was heat sensitive in part but a significant fraction was non-enzymatic in nature. CH4 formation from DMSO was not significantly modified by 2-diethylaminoethyl-2,2-diphenylvalerate. HC1 (SKF 525 A) or EDTA 1 mM and significantly enhanced under an atmosphere of (CO 80%+O2 20%) rather than under air. A marked increase in CH4 production was observed when paraquat (PQ) was included in incubation mixtures but not when nifurtimox (Nfx) was added. Results support the hypothesis of hydroxyl free radical (·OH) formation during PQ biotransformation but cast doubts about its production for the case of Nfx. The low temperature gas chromatographic separation of d3-CH4 from CH4 described opens the future possibility for detecting trace formation of ·OH in vivo, without interference from fecal CH4 formation by administering d6-DMSO to animals and collecting exhaled gases produced, in chambers containing the entire animal.

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.