Drug residue formation from ronidazole, a 5-nitroimidazole. V. Cysteine adducts formed upon reduction of ronidazole by dithionite or rat liver enzymes in the presence of cysteine.

When ronidazole (1-methyl-5-nitroimidazole-2-methanol carbamate) is reduced by either dithionite or rat liver microsomal enzymes in the presence of cysteine, ronidazole-cysteine adducts can be isolated. Upon reduction with dithionite ronidazole can react with either one or two molecules of cysteine to yield either a monosubstituted ronidazole-cysteine adduct substituted at the 4-position or a disubstituted ronidazole-cysteine adduct substituted at both the 4-position and the 2-methylene position. In both products the carbamoyl group of ronidazole has been lost. The use of rat liver microsomes to reduce ronidazole led to the formation of the disubstituted ronidazole-cysteine adduct. These data indicate that upon the reduction of ronidazole one or more reactive species can be formed which can bind covalently to cysteine. The proposed reactive intermediates formed under these conditions may account for the observed binding of ronidazole to microsomal protein and the presence of intractable drug residues in the tissues of animals treated with this compound. They may also account for the mutagenicity of this compound in bacteria.

Drug residue formation from ronidazole, a 5-nitroimidazole. VI. Lack of mutagenic activity of reduced metabolites and derivatives of ronidazole.

The potential toxicity of ronidazole residues present in the tissues of food-producing animals was assessed using the Ames mutagenicity test. Since ronidazole is activated by reduction, reduced derivatives of ronidazole and metabolites formed by enzymatic reduction of ronidazole were tested for mutagenicity. When tested at levels several orders of magnitude higher than that at which ronidazole was mutagenic, 5-amino-4-S-cysteinyl-1,2- dimethylimidazole , a product of the dithionite reduction of ronidazole in the presence of cysteine, the 5-N-acetylamino derivative of ronidazole and 5-amino-1,2- dimethylimidazole all lacked mutagenic activity in Ames strain TA100. The metabolites of ronidazole formed by the incubation of ronidazole with microsomes under anaerobic conditions were also not mutagenic. These data demonstrate that although ronidazole is a potent mutagen, residues from it which may be present in the tissues of food-producing animals lack any mutagenic activity.

Effect of liver enzymes on the mutagenicity of nitroheterocyclic compounds: activation of 3a,4,5,6,7,7a-hexahydro-3-(1-methyl-5-nitro-1H-imidazol-2-yl)- 1,2-benzisoxazole and deactivation of nitrofurans and nitroimidazoles in the Ames test.

The effect of liver enzymes (S9) on the mutagenic response of nitroimidazoles and nitrofurans in the Ames test was evaluated with strain TA100. A diminished response was observed with a 5-nitroimidazole and 5-nitrofurans when the S9 preparation was incorporated in the agar layer. Preincubation with S9 under anaerobic conditions prior to adding the bacteria resulted in a greater and sometimes complete loss of the mutagenic effect. The loss of mutagenic potency was dependent on both incubation time and quantity of the S9 preparation. These results suggest that metabolites formed after reductive metabolism are neither mutagenic (presumably due to the loss of the nitro group) nor capable of activation to mutagenic metabolites. One 5-nitroimidazole, 3a,4,5,6,7,7a-hexahydro-3-(1-methyl-5-nitro -1H-imidazol-2-yl)-1,2-benzisoxazole (MK-0436), gave an increased response in the presence of S9 in both the plate test and when preincubated under aerobic conditions. 7 metabolites were produced by the incubation. 4 monooxygenated metabolites were isolated and found to possess significant mutagenic activity. 2 synthetic dihydroxy analogs were more mutagenic than MK-0436. Similar results were obtained with S9 preparations from human liver and the livers of control, phenobarbital and Aroclor-1254 pretreated rats.

Drug residue formation from ronidazole, a 5-nitro-imidazole. IV. The role of the microflora.

When radioactive 1-methyl-5-nitroimidazole-2-methanol carbamate, ronidazole, labeled at the 4,5-ring positions was administered orally to germ-free and conventional rats, a much larger fraction of the radioactivity was excreted in the feces of the conventional animals. Determination of the total radioactive residues present in the carcass, blood, plasma, liver, fat and kidney 5 days after dosing indicated that the carcass of the germ-free animals contained a greater quantity of residue than that of conventional rats. On the other hand, the blood of the conventional animals contained a much higher level of radioactivity than that of the germ-free animals. These results show that while the microflora influence the distribution of the drug their presence is not obligating for the formation of persistent tissue residues in rats dosed with ronidazole.

Drug residue formation from ronidazole, a 5-nitroimidazole. I. Characterization of in vitro protein alkylation.

The metabolic activation of [14C]ronidazole by rat liver enzymes to metabolite(s) bound to macromolecules was investigated. The alkylation of protein by [14C]ronidazole metabolite(s) was catalyzed most efficiently by rat liver microsomes, in the absence of oxygen utilizing NADPH as a source of reducing equivalents. Based on a comparison of total ronidazole metabolized versus the amount bound to microsomal protein, approximately one molecule alkylates microsomal protein for every 20 molecules of ronidazole metabolized. Protein alkylation was strongly inhibited by sulfhydryl-containing compounds such as cysteine and glutathione whereas methionine had no effect. Based on HPLC analysis of ronidazole, cysteine was found not to inhibit microsomal metabolism of ronidazole ruling out a decrease in the rate of production of the reactive metabolite(s) as the mechanism of cysteine inhibition.

Drug residue formation from ronidazole, a 5-nitroimidazole. II. Involvement of microsomal NADPH-cytochrome P-450 reductase in protein alkylation in vitro.

Purified liver microsomal NADPH-cytochrome P-450 reductase is able to catalyze the activation of [14C]ronidazole to metabolite(s) which bind covalently to protein. Like the reaction catalyzed by microsomes, protein alkylation catalyzed by the reductase is (1) sensitive to oxygen, (2) requires reducing equivalents, (3) is inhibited by sulfhydryl-containing compounds and (4) is stimulated several fold by either flavin mononucleotide (FMN) or methytlviologen. A cytochrome P-450 dependent pathway of ronidazole activation can be demonstrated as judged by the inhibition of the reaction by carbon monoxide, metyrapone and 2,4-dichloro-6-phenylphenoxyethylamine but the involvement of specific microsomal cytochrome P-450 isozymes has not been definitively established. Milk xanthine oxidase is also capable of catalyzing ronidazole activation. Polyacrylamide sodium dodecyl sulfate (SDS)-gel electrophoresis reveals that the reactive intermediate(s) of ronidazole does not alkylate proteins selectively.

Drug residue formation from ronidazole, a 5-nitroimidazole. III. Studies on the mechanism of protein alkylation in vitro.

Ronidazole (1-methyl-5-nitroimidazole-2-methanol carbamate) is reductively metabolized by liver microsomal and purified NADPH-cytochrome P-450 reductase preparations to reactive metabolites that covalently bind to tissue proteins. Kinetic experiments and studies employing immobilized cysteine or blocked cysteine thiols have shown that the principal targets of protein alkylation ara cysteine thiols. Furthermore, ronidazole specifically radiolabelled with 14C in the 4,5-ring, N-methyl or 2-methylene positions give rise to equivalent apparent covalent binding suggesting that the imidazole nucleus is retained in the bound residue. In contrast, the carbonyl-14C-labeled ronidazole gives approx. 6--15-fold less apparent covalent binding indicating that the carbamoyl group is lost during the reaction leading to the covalently bound metabolite. The conversion of ronidazole to reactive metabolite(s) is quantitative and reflects the amazing efficiency by which this compound is activated by microsomal enzymes. However, only about 5% of this metabolite can be accounted for as protein-bound products under the conditions employed in these studies. Consequently, approx. 95% of the reactive ronidazole metabolite(s) can react with other constituents in the reaction media such as other thiols or water. Based on these results, a mechanism is proposed for the metabolic activation of ronidazole.

Problems associated with the use of cycloheximide to distinguish between animal drug residues bound to protein and those incorporated into protein.

The user of cycloheximide to distinguish between covalently-bound drug residues in animals and residues due to the incorporation of drug fragments into endogenous molecules was explored. The results indicated that cycloheximide prevented the absorption of both glycine and ronidazole from the gastro-intestinal tract, an effect that complicates its use in the characterization of drug residues in animals.

Effect of overloading pathways on toxicity.

Overloading of metabolic pathways results in a disproportionate relationship between the concentration of drug and/or metabolites and dosage. As a result, both the degree and type of toxicity may be affected. Metabolic pathways which may be overloaded include transport, drug or metabolite binding, enzymatic transformations of drug or metabolites and systems utilizing conjugating cofactors. Examples in which the degree of toxicity is affected by overloading the transport and distribution system or by overloading conjugation mechanisms or by overloading plasma and tissue binding capacities will be discussed. These include the acute toxicity of streptomycin, chronic toxicity of some nitrofurans and toxic effects of bromobenzene and warfarin. The effect of route of administration on the degree of toxicity of Cambendazole for laboratory and target animals will be discussed. Overloading enzymatic transformation of drug and/or metabolites results in disproportionate changes in the blood and tissue levels and relative quantities of drug and metabolites. This may affect the type of toxicity observed when the drug is administered by differing routes or methods. An example of this phenomenon may be the chronic toxicity in rats given FANFT in the diet or dosed by gavage. The relevance of these toxicity effects in considering the human hazard of tissue residues of animal health products will be discussed.

Identification of a glutathione conjugate of cambendazole formed in the presence of liver microsomes.

The incubation of multiply labeled (2H, 3H, 13C, 14C) cambendazole and glutathione with hepatic microsomes from phenobarbital-dosed hamsters results in the formation of polar metabolites. The major metabolite has been characterized by a variety of isotopic, spectrometric, chromatographic, and degradative/synthetic techniques as a glutathione conjugate of cambendazole in which substitution is on the 4-position of the benzimidazole nucleus. The same metabolite is produced by hepatic microsomes from the rat.

Cambendazole and nondrug macromolecules in tissue residues.

The anthelmintic cambendazole is rapidly metabolized to at least 13 urinary metabolites. Radioactivity was found in liver for weeks after a single dose in cattle, but even at 3 days' withdrawal, cambendazole and metabolites previously identified in urine accounted for only a small fraction of liver radioactivity. The radioactivity was ubiquitously distributed in protein and nucleic acid fractions, and [14C] glutamic acid was identified, indicating incorporation of 14C into the endogenous pool. Part of the residual liver radioactivity at 7 days was convertible chemically to 5-nitrobenzimidazole, indicating a drug-related macromolecular residue. However, data from rats fed radiolabeled steer liver indicate that the residue is minimally bioavailable and therefore of substantially less toxicological concern than cambendazole itself.