Peter Goldman

Acetamide—a metabolite of metronidazole formed by the intestinal flora

Abstract Metronidazole is metabolized to acetamide in yields of between 8 and 15 per cent by cultures of rat cecal contents or Clostridium perfringens . The yield of acetamide is 6- to 9-fold greater than that of N -(2-hydroxyethyl)-oxamic acid which is also derived from metronidazole. When [2- 14 C] metronidazole is administered by gavage to conventional rats, 1.3 to 1.8 per cent of a 200 mg/kg dose is recovered as acetamide in the urine. An additional 0.9 to 2.4 per cent is recovered as acetamide in the feces. Acetamide is not detected, however, in either urine or feces when metronidazole is administered to germfree rats. The appearance of acetamide derived from metronidazole in conventional rats appears to be mediated by the intestinal microflora. The cleavage of the imidazole ring of metronidazole to yield both acetamide and N -(2-hydroxyethyl)-oxamic acid is consistent with nucleophilic attack at carbons 2 and 4 of a partially reduced nitroimidazole ring, which is then cleaved between positions 1 and 2 and between positions 3 and 4. Since acetamide has been shown previously to be a liver carcinogen for rats, its presence in the urine and the feces should be considered, together with other indirect evidence, when determining the possible risk of cancer to patients given metronidazole.

Role of the gastrointestinal microflora in amygdalin (laetrile)-induced cyanide toxicity☆

When conventional rats were given single oral doses of amygdalin (600 mg/kg), they sometimes experienced lethargy and convulsions, and usually died within 2 to 5 hr. Rats affected in this way had high concentrations of cyanide in their blood (2.6–4.5 μg/ml). Germfree rats receiving the same dose of amygdalin did not exhibit these symptoms and had blood cyanide concentrations (< 0.4 μg/ml) indistinguishable from those of conventional rats which did not receive amygdalin. After a non-toxic oral dose (50 mg), the recovery of amygdalin was higher in germfree than in conventional rats and only germfree rats had amygdalin in their feces. Furthermore, the intestinal contents of conventional rats, but not of germfree rats, catalyzed the release of benzaldehyde from amygdalin. It is concluded that the gastriointestinal flora are obligatory for the reactions which lead to the release of toxic amounts of cyanide from amygdalin (laetrile).

Role of the intestinal flora in the metabolism of misonidazole

Abstract The radiation sensitizer misonidazole is metabolized to its amino derivative [1-(2-aminoimidazol-1-yl)-3-methoxypropan-2-o1] in pure or mixed cultures of the intestinal microflora. This metabolite appears in the excreta of conventional rats but is not detectable in the excreta of germfree rats. Thus. its formation appears to be due to the activity of the intestinal flora in vivo as well as in vitro . CO 2 is liberated from 1ue5f8(2ue5f8aminoimidazolue5f81ue5f8yl)ue5f83ue5f8methoxypropanue5f82ue5f8o1 by pure and mixed cultures of the In cultures of Clostridium perfringens that lack urease, the release of CO 2 depends on added urease, suggesting that urea is an intermediate in this pathway.

Reduction of nitroheterocyclic compounds by mammalian tissues in vivo.

To determine whether nitro group reduction occurs in mammalian tissues, metronidazole (0.021, 0.064 and 10 mg/kg), misonidazole (0.015 mg/kg) and nitrofurazone (0.13 mg/kg), respectively, were administered to germfree rats. A reduced metabolite [1-(2-aminoimidazol-1-yl)-3-methoxypropan-2-ol] and two of its hydrolysis products, urea and (2-hydroxy-3-methoxypropyl)-guanidine, were found in the urines of germfree rats that received misonidazole. When nitrofurazone was administered, a reduced metabolite, 4-cyano-2-oxobutyraldehyde semicarbazone, was detected in the urines. However, acetamide and N-(2-hydroxyethyl)oxamic acid, fragmentation products from the reduction of metronidazole, were not found in significant concentrations in the urine when germfree rats received metronidazole. Apparently metronidazole is reduced so much more slowly than misonidazole and nitrofurazone in the tissues of germfree rats that its reductive metabolites are not detectable. This observation may be explained by the one-electron reduction potentials (E1 7) of these drugs, that of metronidazole (E1 7 = -486 mV) being lower than those of either misonidazole (E1 7 = -389 mV) or nitrofurazone (E1 7 = -257 mV). Under these circumstances, metronidazole reduction is not detected, either because its radical anion forms more slowly than that of the other nitroheterocyclic compounds or because its radical anion interacts more rapidly with oxygen to restore the parent compound.

Comparative misonidazole metabolism in anaerobic bacteria and hypoxic Chinese hamster lung fibroblast (V-79-473) cells.

The metabolism of the radiation sensitizer misonidazole was similar in anaerobic cecal contents and hypoxic Chinese hamster lung fibroblasts (V-79-473). Both systems formed the amino derivative of misonidazole, [1-(2-aminoimidazol-1-yl)-3-methoxypropan-2-ol] (AIM), and urea, as well as a metabolite, (2-hydroxy-3-methoxypropyl)-guanidine (G), which has not been described previously. It appears that the nitro group of misonidazole was reduced to form AIM and that this compound was then hydrolyzed to yield either urea or G, the latter in yields of 25% (tissue culture) to 55% (cecal contents). When tested with the Ames tester strain, both G and AIM were slightly mutagenic only for strain TA 98 and then only in the presence of the system for microsomal activation.

Association of salmonella mutants with germ-free rats: Characterization of the reverse mutational response to 2-nitrofluorene

Salmonella typhimurium strain TA1538 described by Ames, in association with otherwise germ-free rats, colonizes the gastrointestinal tract. The revertants enumerated in the feces of each of these rats varies in a coordinated manner in relation to the day on which the measurement is made. In response to the oral ingestion of a single dose of 2-nitrofluorene, the concentration of revertants in the feces increases and then returns essentially to control values within 6 days. When these rats are challenged repeatedly with a similar oral dose of 2-nitrofluorene the revertent concentration in the feces remains elevated for a progressively longer time. A change in phenotype of strain TA1538 during prolonged association rather than a change in the biology of the rat seems to explain this phenomenon. Firstly, germ-free rats exposed repeatedly to 2-nitrofluorene and then associated with strain TA1538 do not have the prolonged response characteristic of multiple challenges with 2-nitrofluorene. Secondly, strain TA1538 reisolated after several weeks of association with the otherwise germ-free rat shows other alterations such as decreased sensitivity to 2-nitrofluorene in the pour plate assay and a decreased sensitivity to crystal violet. In spite of limitations imposed by these alterations in strain TA1538, it is possible to demonstrate a dose-response relationship between the amount of 2-nitrofluorene ingested and the concentration of revertants in the feces of exposed rats.

Mutagenesis within the gastrointestinal tract determined by histidine auxotrophs of Salmonella typhimurium

The Ames Salmonella mutants can be maintained for months within the gastrointestinal tract of otherwise germfree rats. The ingestion of various carcinogens, but not structurally related non‐carcinogens, results in an increased concentration of his+ revertants in the feces. Rats, associated with strain TA1538, were also associated with either L. plantarum or B. vulgatus and with both of these strains. After 2‐nitrofluorene ingestion (3.4 mg) the concentration of fecal revertants increased except in rats associated only with strains TA1538 and B. vulgatus. The concentration of B. vulgatus in the stomachs of individual rats correlated negatively with their response to 2‐nitrofluorene. Since only B. vulgatus reduces nitrofluorene to the less mutagenic 2‐aminofluorene, it appears that B. vulgatus diminishes the revertant response by metabolic removal of the more potent direct acting mutagen from the gut. The ingestion of 1,2‐dimethylhydrazine (21 mg/kg) and azoxymethane (19 mg/kg) provokes increased fecal revertants only in some animals associated with strain TA1535 or TA100.

Role of sulfhydryl compounds in the Bactericidal effect of metronidazole

The bactericidal effect of metronidazole on Escherichia coli and Bacteroides fragilis can be partially reversed by cysteamine under conditions that lead to the formation of an adduct, the thioether, 4-(2-aminoethyl)thio-2-methylimidazole-1-ethanol (4-ATME). This adduct, which is not mutagenic for the Ames histidine auxotrophs of Salmonella typhimurium, forms at a rate that is independent of live bacterial cells and, therefore, can not be shown to relate to the biological effect of cysteamine. When treated with Raney nickel, this adduct yields 2-methylimidazole-1-ethanol. To determine whether a structurally related adduct forms with bacterial protein, a culture of B. fragilis was incubated with radiolabelled metronidazole and then treated with 5% trichloroacetic acid. That the radiolabel in the precipitate did not yield 2-methylimidazole-1-ethanol when treated with Raney nickel suggests that binding of metronidazole to cellular macromolecules does not involve thioether formation.