JoséA. Castro

Prevention by cystamine of liver necrosis and early biochemical alterations induced by carbon tetrachloride

Abstract The effect of the previous administration of cystamine on the hepatotoxicity of orally and intraperitoneally administered carbon tetrachloride (CCl 4 ) was investigated. Cystamine significantly prevented the necrosis and fatty infiltration, as well as the depression of glucose 6-phosphatase and ethylmorphine N -demethylase activities caused by oral CCl 4 administration. These results are due to a delay in the gastrointestinal absorption of CCl 4 caused by cystamine. However, cystamine also prevented necrosis and fatty infiltration when CCl 4 was given intraperitoneally. Moreover, it also significantly blocked the irreversible binding of 14 C from 14 CCl 4 to liver microsomal lipids and the depression of glucose 6-phosphatase in spite of the fact that the microsomal lipid peroxidation process was not inhibited and that CCl 4 levels in the livers of cystamine-treated and untreated rats were similar. The destruction of ethylmorphine N -demethylase could not be prevented by cystamine when CCl 4 was given intraperitoneally. The results are discussed in relation to the postulated mechanistical similarity between CCl 4 liver injury and damage from ionizing radiation.

Studies on the mechanism of cystamine prevention of several liver structural and biochemical alterations caused by carbon tetrachloride

Cystamine and cysteamine give type II spectral changes by interaction with microsomal suspensions while CCl4 gives a type I spectral change. Neither cystamine nor cysteamine at saturating concentrations prevent the spectral binding of small amounts of CCl4. Cystamine and cysteamine decelerate the reduction of cytochrome P-450 by cytochrome P-450 reductase. Cystamine administration partially prevents the irreversible binding of 14CCl4 to microsomal lipids. This preventive effect does not significantly change when the 14CCl4 dose is increased about 4-fold. In spite of the fact that this irreversible binding is decreased by about 60% at 6 hr by the previous cystamine administration, lipid peroxidation is only slightly prevented at 6 and 10 hr after CCl4 and prevented to about 30% at 24 hr. Cystamine partially prevented the depression of glucose-6-phosphatase activity and the destruction of cytochrome P-450 caused by CCl4. Cystamine pronouncedly lowers body temperature of the rats, and it is as effective in preventing CCl4-induced necrosis and fatty liver at 24 hr as it is at 48 or 72 hr after CCl4 administration. Cystamine stabilizes lysosomes in vitro but labilizes them when administered to rats. Cysteamine labilizes lysosomes in vitro. These results may suggest that cystamine administration partially prevents several structural and biochemical alterations caused by CCl4 because it inhibits the CCl4-activation step to ·CCl3 free radical, which would take place during the reduction of the cytochrome P-450CCl4 complex by cytochrome P-450 reductase.

Reductive metabolism and activation of benznidazole

Benznidazole (Bz) (N-benzyl-2-nitro-1-imidazole-acetamide) is a drug used against Chagas disease. Rat liver microsomal and cytosolic fractions, but not mitochondria, exhibited Bz nitroreductase activity under anaerobic conditions in the presence of NADPH. Microsomal nitroreductase activity was enhanced by FAD and was inhibited totally by oxygen and partially by carbon monoxide. Liver cystosol fraction was able to reduce Bz nitrogroups in the presence of either N-methylnicotinamide or hypoxanthine as substrates. These enzyme activities were inhibited by menadione or allopurinol respectively. Under every experimental condition leading to enzymatic reduction of Bz nitrogroups and its inhibition or enhancement, reactive metabolites that bind covalently to proteins were also produced. This covalent binding was effectively prevented by reduced glutathione. Results suggest the participation of cytochrome P-450 and cytochrome c reductase in liver microsomal processes and of xanthine oxidase and aldehyde oxidase in liver cytosolic processes of Bz nitroreduction and activation to reactive metabolites that bind covalently to proteins. Possible pharmacological and toxicological implications of the described observations were discussed.

Therapeutic effectiveness of cystamine and cysteine to reduce liver cell necrosis induced by several hepatotoxins.

Abstract Cystamine or cysteine administration to rats receiving dimethylnitrosamine, thioacetamide, or bromobenzene 3, 6, or 12 hr before, partially prevented the liver necrosis produced by these substances at 24 hr. In contrast, both chemicals were unable to prevent the necrosis induced by allyl alcohol and aflatoxin B 1 . Since at either 6 hr (dimethylnitrosamine) or 12 hr (bromobenzene and thioacetamide), most of activation of the agents and the subsequent interaction with cell constituents has already occurred, these results suggest that cystamine or cysteine protect because they alter cellular response to the hepatotoxins. The results also suggest the possible application of these chemicals in therapeutics.

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.

Studies on pentane evolution by rats treated with nifurtimox or benznidazole

Sprague-Dawley male rats were treated with either 100 mg/kg Nifurtimox or Benznidazole p.o. and pentane evolution was measured at different periods of time. No significant increase in pentane evolution was observed in animals treated with Benznidazole during periods of time up to 10 h. In animals treated with Nifurtimox, a significant increase in pentane evolution was observed at 10 h but not at 3 or 6 h. The pentane evolution effect of Nifurtimox was compared to that of carbon tetrachloride. The latter was very intense up to 1 h and ceased thereafter. The possible participation of lipid peroxidation in the unwanted toxic side effects of Nifurtimox and Benznidazole is discussed.

Mechanism of chlorpromazine prevention of carbon tetrachloride-induced liver necrosis☆

Abstract Chlorpromazine (CPZ) is able to partially prevent the CCl 4 -induced liver necrosis in rats 24 hr but not 72 hr after its administration. Preventive effects at 24 hr were still observed when CPZ was given as late as 10 hr after CCl 4 . Prevention is not due to variation in CCl 4 concentration in liver. CPZ does not decrease either the covalent binding of CCl 4 reactive metabolites to cellular constituents or lipid peroxidation but causes a marked decrease in the body temperature of the rats. Our results indicate that CPZ only delays the onset of necrosis and suggest that this effect is mediated by the CPZ decreasing effects on body temperature, since the necrosis observed in the CPZ-treated animals injected with CCl 4 and maintained at 37°C is as intense as that observed in the group receiving CCl 4 alone.

Cholinesterase inhibition by phenothiazine and nonphenothiazine antihistaminics: analysis of its postulated role in synergizing organophosphate toxicity.

Abstract Several antihistaminic compounds inhibit in vitro pseudocholinesterase (ChE) from several sources (human, rat, or horse plasma), pI50 ranging from 5.0 to 6.0. This inhibition is competitive with acetylcholine (ACh) and is reversible. Acetylcholinesterase (AChE) from rat brain was nonsensitive to antihistaminics even at a concentration of 4 m m . Antihistaminics do not compete with either organophosphates or quaternary ammonium compounds in inhibiting ChE. Inhibition of ChE by antihistaminics is more pronounced at alkaline pH values than at acidic or neutral pH. Administration of promethazine ip to rats at a dose of 50 mg/kg resulted in a 90% inhibition of ChE 1 hr later, while the AChE was totally nonsensitive to the inhibitor even when the dose of the antihistamine was doubled. Results were analyzed in relation to the postulated role of ChE inhibition in antihistaminic potentiation of organophosphorus intoxication.

Carbon tetrachloride target lipids in rat liver microsomes. Effect of cystamine administration on their pattern of labeling by 14CCl4

Abstract 14C from 14CCl4 irreversibly binds to lipids from the smooth (SER) and rough (RER) endoplasmic reticulum. Most of the label is associated with the phospholipid fraction (> 95%). Prior cystamine administration decreased the extent of that binding but does not change its pattern of distribution. About the half of the label in phospholipids is in the phosphatidylcholine fraction; the other half is distributed similarly among lysophosphatidylcholine, sphingomyelin and phosphatidylethanolamine, while only a very minor fraction is associated with diphosphatidyl glycerol. No differences were found in the pattern of labeling of phospholipids in SER and RER.

Interaction of trichloromethyl free radicals with thymine in a model system: a mass spectrometric study

In previous studies from our laboratory we found that the CCl4 reactive metabolites produced during enzymatic in vitro or in vivo CCl4 biotransformation covalently bind to DNA. Further, chemically produced.CCl3 produce many adducts of unknown structure with the four DNA bases when the reaction proceeds in model systems. In the present work, we describe our attempt to elucidate by GLC/MS the structures of the adducts resulting when chemically generated.CCl3 interact with thymine. The following reaction products were identified: (i) 5-hydroxymethyl uracil; (ii) thymineglycol; (iii) 5-trichloroethyl uracil (tentative) and (iv) two isomeric 5,6-monochloro monohydroxy adducts of thymine (tentative). Reaction products found do not involve thymine positions directly participating in base-pairing processes. However, alterations in thymine structure reported if they occurred in DNA from livers of CCl4 poisoned animals, might potentially have biological significance that remains to be established.