María I. Díaz Gómez

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.

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.

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.

Further studies on dimethylnitrosamine metabolism, activation and its ability to cause liver injury

Effects were studied of aminoacetonitrile (AAN), dibenamine (DB) diethyldithiocarbamate (DDTC) dimethylformamide (DMF), disulfiram (DS), and 2-mercapto-1-methylimidazole (MMI) on the in vitro dimethylnitrosamine (DMN) metabolism to CO2, covalent binding (CB) of DMN metabolites to nucleic acids in liver slices, DMN demethylase (DMNase) in male rat liver microsomes or 9,000 g supernatants and CB to microsome of 9,000 g supernatant proteins. Effects of those chemicals on DMN-induced rat liver necrosis were also studied, except for DS whose preventive effect was previously reported by our laboratory. All the chemicals significantly prevented DMN-induced liver necrosis, except for MMI that had no effect. All these compounds when added to incubation mixtures containing liver slices from Sprague-Dawley rats, significantly inhibited transformation of DMN to CO2 and CB to nucleic acids and when they were injected into animals and liver slices prepared afterwards, they did so except for MMI and DMF that had no effect. None of the chemicals tested except DDTC and MMI modified CB to microsome proteins whereas the CB to 9,000 g supernatant proteins was significantly decreased by all the chemicals except MMI. DMNase activity either in microsomes or 9,000 g supernatants was significantly inhibited by all the compounds except MMI.

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.

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.

Liver nuclear and microsomal CYP2E1-mediated metabolism of xenobiotics in rats chronically drinking an alcohol-containing liquid diet

In previous studies from our laboratory, the presence in highly purified liver nuclei of metabolic pathways for processing ethanol (EtOH), N-nitrosodimethylamine (NDMA), carbon tetrachloride and chloroform was reported. All these chemicals are known to be metabolized in liver microsomes, via cytochrome P450 2E1 (CYP2E1)-mediated processes. In the present work we checked whether rat liver nuclei from rats chronically drinking an alcohol-containing liquid diet exhibited an enhanced ability to metabolize chemicals known to require CYP2E1 participation for given metabolic transformations. The nicotinamide adenosine dinucleotide phosphate (NADPH)-requiring metabolism of p-nitrophenol to p-nitrocathecol; the activation of carbon tetrachloride to trichloromethyl radicals, covalently binding to proteins; and the ring hydroxylation of aniline and o-toluidine were studied. Comparison of the obtained nuclear activities against the one present in the microsomal counterpart, and their respective response to the EtOH inductive effect after repetitive exposure to it, was studied. The obtained results showed that rat liver nuclei exhibited less pnitrophenol hydroxylase activity than microsomes, but it was inducible by repetitive alcohol drinking to equivalent levels of those of microsomes from control animals. Nuclei exhibited the ability to activate CCl4, which was significantly enhanced by alcohol drinking. Aniline was ring hydroxylated in liver microsomes but not in nuclei from either control or EtOH-treated animals. In contrast, nuclei and microsomes metabolized o-toluidine to ring hydroxylated products. They are considered less toxic in nature but other authors reported a genotoxic effect for one of them. The production of the ring hydroxylated metabolites was enhanced by repetitive EtOH drinking. Results suggest that nuclear metabolism of xenobiotics might be relevant for either activations or detoxications mediated by CYP2E1 and that repetitive exposure to EtOH might significantly modulate those processes. Toxicology and Industrial Health 2006; 22: 367-374.

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.

Effect of inhibitors of drug metabolism on mitochondrial swelling and on carbon tetrachloride-induced lysosomal damage

Abstract Several inhibitors of drug metabolism were found to be very potent for causing mitochondrial swelling at concentrations in the range from 1 to 100 μ m . Moreover, they are also effective in increasing lysosomal loss of enzymes at concentrations from 1 to 100 μ m . The inhibitors of drug metabolism were not able to stabilize lysosomes against CCl 4 -induced lysosomal damage at concentrations from 0.001 to 100 μ m . 2-Diethylaminoethyl-2,2-diphenylvalerate hydrochloride (SKF 525A) administration to rats at a dose of 50 mg/kg ip increased the in vitro lysosomal lability to CCl 4 at 1 hr but not at longer periods of time after administration. Curiously some stabilizing effect was found 14 hr after SKF 525A administration if the injection was made at 8 pm instead of the usual time of 8 am . The administration of another inhibitor, ethyl N -(2-diethylaminoethyl) 2-phenyl-2-ethylmalonamate hydrobromide (Sch 5706) did not cause any effect on lysosomal stability.