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Contrasting effects of nitrofurans on plasma corticosterone in chickens following administration as a bolus or diet additive.

Administration of furazolidone as a bolus dose (8-500 mg/kg), produced a decrease in plasma corticosterone in chickens. In contrast, addition of furazolidone or furaltadone to the diet (0.04% or above, 10 days), increased plasma corticosterone. Pre-treatment with a 200-mg/kg bolus of furazolidone or furaltadone did not affect pentobarbitone anaesthesia time in the birds. In chickens pre-treated with a nitrofuran in the diet, however, pentobarbitone anaesthesia time was significantly less than that in controls. Furaltadone in the diet, produced significant increases in the amount of cytochrome P-450 and the activity of aniline hydroxylase in the liver microsomes. It is suggested that nitrofurans given in the diet stimulated corticosterone biosynthesis in the adrenal glands and induced mixed-function oxidase activity in the liver. Nitrofurans given as a bolus did not produce these effects. Furazolidone (200 mg/kg) produced severe anorexia, which lasted 2 days in T-line birds. The anorexia seemed to be associated with tissue damage in the birds rather than the ensuing adrenal cortical insufficiency.

Administration, Oral↗

Mutagenic action of nitrofurans on Euglena gracilis and Mycobacterium phlei.

There is a pronounced difference between the action of antibiotics and nitrofurans on Euglena gracilis. Those antibiotics that induce hereditary loss of chloroplasts do so only when they affect dividing cells. On the other hand, nitrofurans induce a mass mutation in both dividing and nondividing cells (under conditions of continuous illumination of cultures). It was found that a breakdown product, 5-nitro-2-furaldehyde, is liberated from furadantin and furoxone. This intermediate is responsible for the observed specific mutagenicity of 5-nitrofuran drugs. The mutagenic action of 5-nitro-2-furaldehyde is very similar to that of nitrosoguanidine. Both compounds induce bleached mutants of E. gracilis when acting on growing or resting cells, regardless of the dark or light conditions. Similarly, both compounds induce reverse mutations in auxotrophic strains of Mycobacterium phlei.

Animals↗

Involvement of active oxygen species in protein and oligonucleotide degradation induced by nitrofurans.

It is of great interest to know how nitrofurans are mutagenic and clastogenic. In particular, the 3-amino-2-oxazolidone (AOZ) ring, deriving from a cleavage of furazolidone, is not further metabolized and has been found to be part of protein-bound residues in edible tissues of farm animals and these might be released in the stomach of the consumer. The data in this paper show that isoniazide as well as AOZ and 3-amino-5-morpholinomethyl-2-oxazolidinone (AMOZ), the latter deriving from furaltadone, cause irreversible damage to the prosthetic group of enzymes as well as degrade their polypeptide chain and cause fragmentation of the backbone chain of cellular or isolated DNA and RNA. Cellular DNA was degraded into small fragments of 2000 Mb, while rRNA was completely destroyed. Nitrofuran derivatives and hydrazides, in fact, share an N-N moiety, which is assumed to play an essential role in the irreversible damage observed. The key to the molecular mechanisms by which these compounds cause their irreversible effects may lie in oxygen consumption and electron spin resonance measurements, which reveal that both nitrofurans and isoniazide produce oxygen radicals at various degrees of efficiency. AOZ and AMOZ are not metabolized into more reactive metabolites, being themselves able to react with atmospheric oxygen and induce protein and oligonucleotide damage. The reaction does not require metal ions, although their presence will accelerate it.

Caco-2 Cells↗

Generation of radical anions of nifurtimox and related nitrofuran compounds by ascorbate.

Nifurtimox analogues bearing triazol-4-yl, benzimidazol-1-yl, triazin-4-yl or related groups as counterpart of the (5-nitro-2-furfurylidene) amino group were reduced to their nitro anion radicals by ascorbate in anaerobic solutions at high pH. The ESR spectra of the radical anions showed hyperfine spin couplings restricted to the nitrofuran moiety. With these compounds, the spin density at the nitro group was greater than with nifurtimox, nitrofurazone and nitrofurantoin. At neutral pH, solutions containing ascorbate and nitrofuran derivatives consumed oxygen, the compounds bearing unsaturated nitrogen heterocycles being the most effective. Superoxide dismutase and catalase decreased the rate of oxygen consumption, thus demonstrating the production of superoxide and hydrogen peroxide, respectively. NMR spectra of the triazol-4-yl and triazin-4-yl nitrofuran derivatives showed a deshielding effect for the azomethine proton, which was undetectable with nifurtimox and nitrofurazone.

Ascorbic Acid↗

[Joint action of nitrofuran preparations and bile acids on bacteria of the genus Proteus].

Sensitivity of 25 fresh isolates of Proteus to some nitrofuran drugs most widely used in the clinical practice, such as furacillin, furagin, furazolidone and nitrofurantoin was studied. When the drugs were used in combination with some bile acids, i.e. desoxycholic, dehydrocholic, cholic and glycocholic acids, significant in vitro potentiation of the antibacterial activity of the nitrofurans against the isolates was observed. The combinations of the drugs with desoxycholic acid proved to be most effective. In the presence of this acid the bacteriostatic dose of the drugs decreased several thousand times. Combination of the nitrofurans with the other acids resulted in an increase in the antimicrobial activity amounting to several hundred times. The combinations of the drugs with the bile acids had not only bacteriostatic but also bactericidal effect.

Bile Acids and Salts↗

[Determination of metabolites of nitrofuran antibiotics in royal jelly by high performance liquid chromatography-tandem mass spectrometry].

Nitrofurans are a group of widely used veterinary antibiotics that have been banned in many countries. This has generated a great deal of interests and demands for assay of nitrofurans in animal food products. To our knowledge, this is the first time that a high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method has been successfully developed for simultaneously analyzing the metabolites of four nitrofuran antibiotics (furazolidone, furaltadone, nitrofurazone and nitrofurantoin) in royal jelly. Trichloroacetic acid solution was used both to precipitate proteins and to provide acidic reaction condition. Four isotope internal standards were utilized to improve the quantitative precision. The limits of detection (LODs) were 0.03 microg/kg for the metabolite of furaltadone and 0.05 microg/kg for the other three metabolites. The limits of quantitation were 0.20 microg/kg for the metabolite of furaltadone and 0.25 microg/kg for the other three metabolites. The linear range was 0.4-20 ng/mL for all the target analytes. The recoveries calibrated by internal standard were in the range of 97.7%-104.8 with the relative standard deviations (RSDs) of 2.7-9.7. It showed that this method could meet the requirements of national monitoring plan in China and the Minimum Required Performance Limits (MRPL) set by the European Union.

Anti-Bacterial Agents↗

[Effect of nitrofuran derivatives on the oxygen consumption by rat liver mitochondria].

Utilization of oxygen in rat liver mitochondria was studied using two nitrofuran derivatives 5-nitro-2-furaldehyde semicarbazone (furacin) and soluble derivative of nitrofuran--M-106-P. The substances at 10(-3) M concentration increased O2 consumption in mitochondria with simultaneous decrease in the respiratory control. After blocking of respiration with KCN, antimycine, rhothenone, the nitrofurans stimulated O2 consumption in mitochondria up to 30% as compared with controls.

Animals↗

[Biochemical bases for the effect of combining kanamycin and nitrofuran resistance genes in Escherichia coli cells].

The fact of a significant increase in resistance to aminoglycosides when nfr genes with chromosomal or plasmid localization are combined with the plasmid genes coding for kanamycin-transferase in E. coli cells is confirmed. Gel-filtration of homogenates of the cells with and without pLD105 plasmid carrying nfr gene and of the cells with a chromosomal nfr gene revealed a 10 kD polypeptide when the plasmid is present. Relying on these results, it is concluded that the discovered polypeptide fulfils two roles: inhibiting of specific nitrofuran-reductase, which leads to nitrofurans resistance and a drop of transmembrane electric potential contributing to the increase of resistance to aminoglycosides (kanamycin) in strains with the plasmid nfr gene. Absence of the 10 kD polypeptide in the cells with a chromosomal nfr gene and other data are indicative of a possible existence of a different mechanism of resistance to nitrofurans and an increase of resistance to aminoglycosides in the strains with a chromosomal nfr mutation.

Drug Resistance↗

Planar chromatography for the multiclass, multiresidue screening of chloramphenicol, nitrofuran, and sulfonamide residues in pork and beef.

A method is described for multiclass and multiresidue qualitative detection of chloramphenicol, nitrofuran, and sulfonamide residues in animal muscle. The drugs are extracted from 1 g tissue with 2 mL ethyl acetate and purified by silica solid-phase extraction. After elution of the cartridge, the collected solution is evaporated, and the residue is dissolved in methanol and chromatographed on a Si60 high-performance thin-layer chromatography plate. After evaporation of solvent, nitrofurans are visualized first by their specific UV photochemical reaction with pyridine. Then chloramphenicol is reduced to its amino derivative, and this derivative and the sulfonamides are visualized by long-wave UV after reaction with fluorescamine. Chloramphenicol, nitrofurans, and sulfonamides are detected at residue level of 10, 5, and 100 micrograms/kg, respectively, or less in pork and beef.

Animals↗

Metabolism in vivo of furazolidone: evidence for formation of an open-chain carboxylic acid and alpha-ketoglutaric acid from the nitrofuran in rats.

The in vivo metabolism of an antibacterial nitrofuran, furazolidone [N-(5-nitro-2-furfurylidene)-3-amino-2-oxazolidone] was investigated. When the nitrofuran was administered orally to rats, two new-type nitrofuran metabolites, N-(4-carboxy-2-oxobutylideneamino)-2-oxazolidone and alpha-ketoglutaric acid, were isolated from the urine, together with 3-(4-cyano-2-oxobutylideneamino)-2-oxazolidone and N-(5-acetamido-2-furfurylidene)-3-amino-2-oxazolidone. In addition, the present study showed that the corresponding aminofuran was an intermediate in the conversion of furazolidone to these metabolites.

Administration, Oral↗

Role of renal metabolism and excretion in 5-nitrofuran-induced uroepithelial cancer in the rat.

5-Nitrofurans have been used in the study of chemical carcinogenesis. There is substantial evidence that N-[4-(5-nitro-2-furyl)-2-thiazolyl] formamide (FANFT) is deformylated to 2-amino-4-(5-nitro-2-furyl)thiazole (ANFT) in the process of FANFT-induced bladder cancer. Paradoxically, ANFT is less potent as a uroepithelial carcinogen than FANFT when fed to rats. Feeding aspirin with FANFT to rats decreases the incidence of bladder cancer. Isolated kidneys were perfused with 5-nitrofurans to determine renal clearances and whether aspirin acts to decrease urinary excretion of the carcinogen. In FANFT-perfused kidneys, FANFT was deformylated to ANFT and excreted (1.06 +/- 0.22 nmol/min) at a rate eightfold higher than excretion of FANFT. In kidneys perfused with equimolar ANFT, excretion of ANFT was 0.25 +/- 0.05 nmol/min, which suggests a coupling of renal deformylation of FANFT to excretion of ANFT in FANFT-perfused kidneys. Neither aspirin nor probenecid altered the urinary excretion or half-life of FANFT or ANFT. In rats fed 0.2% FANFT as part of their diet, coadministration of aspirin (0.5%) increased urinary excretion of ANFT during a 12-wk feeding study, which suggests decreased tissue binding or metabolism of ANFT. Kidney perfusion with acetylated ANFT (NFTA), a much less potent uroepithelial carcinogen, resulted in no ANFT excretion or accumulation, which indicates the specificity of renal deformylase. Renal deformylase activity was found in broken cell preparations of rat and human kidney. These data describe a unique renal metabolic/excretory coupling for these compounds that appears to explain the differential carcinogenic potential of the 5-nitrofurans tested. These results are consistent with the hypothesis that aspirin decreases activation of ANFT by inhibiting prostaglandin H synthase.

Administration, Oral↗

Simultaneous separation of nitrofuran antibiotics and their metabolites by using micellar electrokinetic capillary chromatography.

Mixtures comprising nitrofuran antibiotics (NFA) and nitrofuran metabolites (NFM) were resolved for the first time by using MEKC. Sodium deoxycholate (SDC) was chosen as the micelle-forming surfactant. Optimization of separation conditions was achieved by using a central composite experimental design (CCD) approach. Experimental parameters such as concentration ratio of borate to phosphate in the buffer, pH of the running electrolyte and voltage were investigated. The effect of concentration of the surfactant on resolution was significant. Under optimal conditions of 80 mM SDC, pH 9.0, (20 mM borate + 20 mM phosphate) and 16 kV, the resolution between eight consecutive peak pairs ranged from 1.9 to 11.8. Due to the absence of a UV-active chromophore in the metabolites, they were derivatized with 2-nitrobenzaldehyde (2-NBA). In order to mimic a proposed extraction procedure for the analysis of both NFA and/or derivatized NFM in a sample, aqueous samples (prederivatized with 2-NBA) were extracted by using C(18) SPE cartridges. After washing with H(2)O, the cartridges were eluted with a small portion of organic solvent with weak elution characteristics to remove excess 2-NBA (hexane was chosen). Target analytes were then recovered with ACN. Excellent reproducibility of migration time (t(mig)) was achieved for all analytes using the developed MECC approach, with absolute t(mig) <1% RSD and t(mig) ratio <0.2% RSD, and peak area ratio was 4% RSD. The LOD for each compound, calculated by extrapolating to an S/N of 3, were found to be 0.19-2.0 microg/mL.

Animals↗

Metabolic reduction of novel 3,4-dichloro-5-nitrofurans in Salmonella typhimurium.

To gain insight on biochemical mechanisms of mutagenesis and carcinogenesis by the experimental carcinogens, 5-nitrofurans, a new series of 3,4-dichloro-5-nitrofurans, comprised of 3,4-dichloro-5-nitro-2-acetylfuran (I), 3,4-dichloro-5-nitro-2-bromoacetylfuran (II), methyl 3,4-dichloro-5-nitro-2-furoate (III), were synthesized and tested for their activation to mutagenic forms in the standard plate assay using Salmonella typhimurium TA98, TA100, and TA100NR, a derivative of TA100 deficient in nitroreductase activity. The mutagenic responses in TA98 were 2- to 6-fold lower compared to TA100. Furthermore, I and II were less active in TA100NR, while compound III was about four times more mutagenic in TA100NR compared to the parent strain TA100. Incubation of III with NADPH and bacterial lysates showed that the extent of reduction was greater in TA100 compared to TA100NR. High-pressure liquid chromatography analysis of the ethyl acetate extract obtained from incubation of III with lysates of TA100 revealed the formation of four metabolites with retention times of about 4.0, 5.7, 10.0, and 14.3 minutes. The spectroscopic and chromatographic properties of the components with retention times of 10.0 and 14.3 minutes were identical to two derivatives obtained by chemical reduction of III, and thus represent nitroreduction products. These derivatives have been identified as cis- and trans-oxime isomers of methyl 3,4-dichloro-2-furoate, based on spectroscopic analyses. These oximes were not mutagenic for TA100. Furthermore, III was more mutagenic under anaerobic conditions, suggesting that secondary superoxide or nitroanion free radicals generated from nitroreduction are not responsible for the mutagenicity of III. In addition, the higher mutagenic response in TA100NR, and the lack of mutagenic activities of the amino and the oxime analogs of III suggest that the mutagenic activation of III might be due to the nitroso intermediate or involve mechanisms other than nitroreduction.

Chromatography, High Pressure Liquid↗

[Multi-method for determining residues of chemotherapeutics, antiparasitics and growth promotors in foods of animal origin. 2. Determination of nitrofurans and nicarbazin (dinitrocarbanilide components)].

Nitrofurans and dinitrocarbanilide are extracted from food according to the multiresidue method and detected by Reversed-phase-HPLC with UV-detection at 360 nm. Records of the UV-spectra confirm the results qualitatively. Possibilities for further confirmation are discussed. Substances in amounts below 0.01 mg/kg can only be determined with particular care. Statistical confirmation of this method and possible problems are mentioned. An interlaboratory collaborative study confirmed the method: furazolidone, furaltadone, nitrofurazone, nitrofurantoin, and nicarbazin (the dinitrocarbanilide containing component) were added to homogenized eggs in amounts of 0.005-0.150 mg/kg. The procedure can be standardized as an official analytical method at a detection limit of 0.005 mg/kg for nitrofurans and 0.01 mg/kg for nicarbazin.

Animals↗

Antibacterial activity of some alpha-substituted 2-methyl-5-nitrofurans.

The minimum inhibitory concentration values against Gram-negative and Gram-positive bacteria were determined and compared for a selected group of synthesized alpha-substituted 2-methyl-5-nitrofuran derivatives. In vitro oxidation of thiols to disulfides by 2-(iodomethyl)-5-nitrofuran indicated that oxidation of enzyme-thiol groups to disulfide bonds was a possible mode of action; but was discounted by noninhibition of thiol enzymes by these compounds. Electron-microscopic studies of the morphology of bacteria after treatment with these derivatives showed the formation of unusual elongation, branching and atypical rod shapes in E. coli, while S. aureus manifested multibud formation with some cytoplasmic protrusions. The possible mode of action of these compounds is discussed.

Anti-Bacterial Agents↗

Cis-trans isomerization of nitrofuran derivatives by xanthine oxidase.

Enzymatic cis-trans isomerization of nitrofuran derivatives was 3-(5-Nitro-2-furyl)-2-(2-furyl)-demonstrated with milk xanthine oxidase. acrylamide (AF-2) and 3-(5-nitro-2-furyl)-2-(5-bromo-2-furyl)acrylamide (NFBFA) were mainly converted from the cis to the trans form by this enzyme supplemented with an electron donor. This enzymatic reaction was further characterized with respect to its cofactor requirements. Finally, a new cis-trans isomerization mechanism, which is based on transfer of a single electron by a nitroreductase such as xanthine oxidase to a nitrofuran derivative to give the anion free radical, was proposed.

Air↗

Nitrofuran inhibition of yeast and rat tissue glutathione reductases. Structure-activity relationships.

Nitrofuran derivatives bearing unsaturated five- or six-membered nitrogen heterocycles or related substituents were more effective inhibitors of yeast and rat tissue glutathione reductases than those bearing other groups, such as nifurtimox, nitrofurazone and 5-nitro-2-furoic acid. The inhibitory action proved independent of electron withdrawal from the reduced enzyme, as a consequence of redoxcycling of the nitro group. Uncompetitive kinetics was obtained with nitrofurantoin and nifurtimox. Most of the assayed nitrofurans inhibited the yeast enzyme Coenzyme A glutathione disulfide reductase activity, though less than oxidized glutathione reduction. The transhydrogenase activity was not inhibited to a significant degree. Benznidazole (a 2-nitroimidazole derivative), 2-nitroimidazole, 5-nitroindole and chloramphenicol did not inhibit glutathione reductase. Under the same experimental conditions, liver glutathione peroxidase was not affected by the nitro compounds.

Animals↗

Formation and excision of nitrofuran-DNA adducts in Escherichia coli.

When Escherichia coli were incubated with the strong mutagen 2[14C]2-amino-4-(5-nitro-2 furyl)-thiazole (ANFT) radioactivity became tightly and presumably covalently bound to DNA. Hydrolysis of the DNA with nucleases yielded low molecular weight radioactive material. The bound radioactivity was associated with at least two functionally and chemically distinct adducts. One of these was rapidly removed in uvr+ E. coli while the other was more persistent. Analysis of enzymatic hydrolysates on a Dowex AG 50W-X4 column showed that the 'excisable adducts' were chromatographically different from most of the persistent ones. ANFT caused daughter-strand gaps when the DNA of treated cells was replicated, provided this DNA contained excisable adducts. In situations where removal of these adducts was complete no gaps were found in newly synthesized DNA. 3-[14C]2-(2-furyl)-3-(5-nitro-2-furyl) acrylamide (AF2) (another strongly mutagenic nitrofuran) became bound to the DNA of E. coli WP2 uvrA to a slightly greater extent than did [14C]ANFT. In contrast, [14C]nitrofurazone (the semicarbazide of 5-nitro-2-furaldehyde) a much weaker mutagen, gave considerably less binding. With AF2 and ANFT there was roughly the same relation between the amount of adduct formed and the subsequent yield of daughter strand gaps when the DNA replicated while with nitrofurazone the yield of gaps per adduct was somewhat lower. Incubation in vitro of [14C]ANFT with DNA in the presence of an E. coli nitrofuran reductase preparation also resulted in the binding of 14C to DNA.

DNA, Bacterial↗