When less is more: shortening the Lpp protein leads to increased vancomycin resistance in Escherichia coli.

Vancomycin is a naturally occurring cell-wall-targeting glycopeptide antibiotic. Due to the low potency of this antibiotic against Gram-negative pathogens, such as Escherichia coli, there is a limited knowledge about interactions between vancomycin and this group of bacteria. Here, we show that an in-frame 63 bp deletion of the lpp gene caused a fourfold increase in vancomycin resistance in E. coli. The resulting protein, LppΔ21, is 21 amino acids shorter than the wild-type Lpp, a helical structural lipoprotein that controls the width of the periplasmic space through its length. The mutant remains susceptible to synergistic growth inhibition by combination of furazolidone and vancomycin; with furazolidone decreasing the vancomycin MIC by eightfold. These findings have clinical relevance, given that the vancomycin concentration required to select the lpp mutation is reachable during typical vancomycin oral administration for treating Clostridioides difficile infections. Combination therapy with furazolidone, however, is likely to prevent emergence and outgrowth of the lpp-mutated Gram-negative coliforms, avoiding exacerbation of the patient's condition during the treatment.

Furazolidone and Nitrofurazone Metabolic Studies in Crucian Carp by Ultra-Performance Liquid Chromatography Tandem Mass Spectrometry.

In this work, the detection of the furazolidone (FZD) and nitrofurazone (NFZ) metabolites residuals in crucian carp are focused. Crucian carps of identical size were exposed to the mixed nitrofuran antibiotics under optimized bath conditions at a concentration of 50 mg/L, 26 ± 0.5°C for 24 h. Then, liquid chromatography-electrospray ionization-triple quadrupole mass spectrometry (LC-ESI-MSMS) was performed after the drug exposure experiments when the nitrofuran metabolites were enriched in organisms. During the period of 0-144 h, residue levels of the 3-amino-2-oxazolidinone (AOZ) gradually decreased with a prolonged sampling time. The changing trend in semicarbazide (SEM) with the sample collection duration is divided into two stages, and its concentration showed a trend of rising first and then falling. The metabolite concentration-time curve demonstrates that 24 h was used as a sampling time, and fish muscle was selected as tissue samples in the further quantitative study. A novel crucian carp-enrichment procedure coupled to LC-ESI-MSMS quantitative method was further explored based on much metabolite data. According to the exponential curve of the SEM-to-AOZ concentration ratio at a precisely designed FZD-to-NFZ mass ratio, the final FZD content of the veterinary NFZ antibiotics was 0.069 ± 0.005% (in terms of mass).

Evaluation of Giardia lamblia thioredoxin reductase as drug activating enzyme and as drug target.

The antioxidative enzyme thioredoxin reductase (TrxR) has been suggested to be a drug target in several pathogens, including the protist parasite Giardia lamblia. TrxR is also believed to catalyse the reduction of nitro drugs, e.g. metronidazole and furazolidone, a reaction required to render these compounds toxic to G. lamblia and other microaerophiles/anaerobes. It was the objective of this study to assess the potential of TrxR as a drug target in G. lamblia and to find direct evidence for the role of this enzyme in the activation of metronidazole and other nitro drugs. TrxR was overexpressed approximately 10-fold in G. lamblia WB C6 cells by placing the trxR gene behind the arginine deiminase (ADI) promoter on a plasmid. Likewise, a mutant TrxR with a defective disulphide reductase catalytic site was strongly expressed in another G. lamblia WB C6 cell line. Susceptibilities to five antigiardial drugs, i.e. metronidazole, furazolidone, nitazoxanide, albendazole and auranofin were determined in both transfectant cell lines and compared to wildtype. Further, the impact of all five drugs on TrxR activity in vivo was measured. Overexpression of TrxR rendered G. lamblia WB C6 more susceptible to metronidazole and furazolidone but not to nitazoxanide, albendazole, and auranofin. Of all five drugs tested, only auranofin had an appreciably negative effect on TrxR activity in vivo, albeit to a much smaller extent than expected. Overexpression of TrxR and mutant TrxR had hardly any impact on growth of G. lamblia WB C6, although the enzyme also exerts a strong NADPH oxidase activity which is a source of oxidative stress. Our results constitute first direct evidence for the notion that TrxR is an activator of metronidazole and furazolidone but rather question that it is a relevant drug target of presently used antigiardial drugs.

Furazolidone in chicken: case study of an incident of widespread contamination.

1. Furazolidone, a nitrofuran antibiotic, was prohibited from the use in food-producing animals in the European Union (EU) in 1997. In 2002, the EU restricted the import of poultry meat and aquaculture species from countries where furazolidone residues had been detected. 2. By 2004, however, residues of the side-chain metabolite, 3-amino-2-oxazolidinone (AOZ) of furazolidone, were detected in chicken meat produced in Northern Ireland. 3. With the random spread of positive results across farms of a single integrated organisation, including organically reared flocks, it seemed unlikely that the source of residues was due to illegal use of the drug, but more likely caused by a source of contamination. 4. Potential sources investigated were as follows: furazolidone contamination of feedstuffs, a "hot spot" of furazolidone in poultry houses, contamination occurring within breeding stocks and transferred with the birds to broiler growing houses, and furazolidone contamination of the water supply. 5. Furazolidone contamination was associated with birds reared in houses more than 10 years old. 6. Contamination was traced to the water supply of poultry houses, where un-dissolved furazolidone, legally administered prior to 1997, had settled to the bottom of water storage tanks. It remained un-disturbed until 2004 when the integrator changed the procedure for cleaning water tanks between crops of birds. 7. The use of Proxitane, a hydrogen peroxide disinfectant, caused effervescence within the tank such that small quantities of furazolidone were dissolved, delivered to birds via drinkers and subsequently caused residues in the broiler meat. 8. The environmental impact of the contamination was investigated by testing soil and grass from land adjacent to an organic poultry house to which birds had access. 9. Mechanisms of contamination and how residues may be spread throughout a large integrated poultry system are not restricted to furazolidone. Incidents of contamination are even more likely when using licensed drugs where the drugs may be present on-farm in large quantities.

Application of a modified enzyme-linked immunosorbent assay for 3-amino-2-oxazolidinone residue in aquatic animals.

Furazolidone has been banned from use in food animals because of its carcinogenicity and mutagenicity, but its continued misuse is widespread in aquacultures. Therefore, there is an urgent need for a simple, reliable, and rapid method for the detection of its marker residue, 3-amino-2-oxazolidinone (AOZ), in aquatic products. In this regard, we modified a simplified indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) to address this need. A good linearity was achieved over a concentration range of 0.05-12.15 microg L(-1), and the IC(50) value was 0.96 microg L(-1). The sample preparation was simple and effective included water bath treatments, acid hydrolysis combined with overnight derivatization of AOZ by benzaldehyde. The limit of detection and the limit of quantification were 0.15 and 0.3 microg kg(-1). The recoveries of AOZ in all tissues were between 78.0-95.3% at the levels of 0.3, 1.0, and 2.0 microg kg(-1). The inter-assay variability was less than 19.1%. The modified ic-ELISA was applied in quantification of AOZ elimination in carp. The results showed that AOZ was quite difficult to eliminate. Good correlations of the results obtained by ELISA and LC-MS/MS were observed in incurred carp muscle (r=0.9923) and carp plasma (r=0.9915) at the levels of 2.5-571.8 microg kg(-1) (microg L(-1)). Better results were obtained by modified ic-ELISA when compared with commercial ELISA kit. Therefore, the present assay is considered a rapid, accurate, reliable, and inexpensive method for the detection of furazolidone-residues in the edible tissues of aquatic animals.

Trichomonas vaginalis: metronidazole and other nitroimidazole drugs are reduced by the flavin enzyme thioredoxin reductase and disrupt the cellular redox system. Implications for nitroimidazole toxicity and resistance.

Infections with the microaerophilic parasite Trichomonas vaginalis are treated with the 5-nitroimidazole drug metronidazole, which is also in use against Entamoeba histolytica, Giardia intestinalis and microaerophilic/anaerobic bacteria. Here we report that in T. vaginalis the flavin enzyme thioredoxin reductase displays nitroreductase activity with nitroimidazoles, including metronidazole, and with the nitrofuran drug furazolidone. Reactive metabolites of metronidazole and other nitroimidazoles form covalent adducts with several proteins that are known or assumed to be associated with thioredoxin-mediated redox regulation, including thioredoxin reductase itself, ribonucleotide reductase, thioredoxin peroxidase and cytosolic malate dehydrogenase. Disulphide reducing activity of thioredoxin reductase was greatly diminished in extracts of metronidazole-treated cells and intracellular non-protein thiol levels were sharply decreased. We generated a highly metronidazole-resistant cell line that displayed only minimal thioredoxin reductase activity, not due to diminished expression of the enzyme but due to the lack of its FAD cofactor. Reduction of free flavins, readily observed in metronidazole-susceptible cells, was also absent in the resistant cells. On the other hand, iron-depleted T. vaginalis cells, expressing only minimal amounts of PFOR and hydrogenosomal malate dehydrogenase, remained fully susceptible to metronidazole. Thus, taken together, our data suggest a flavin-based mechanism of metronidazole activation and thereby challenge the current model of hydrogenosomal activation of nitroimidazole drugs.

Multi-residue monitoring for the simultaneous determination of five nitrofurans (furazolidone, furaltadone, nitrofurazone, nitrofurantoine, nifursol) in poultry muscle tissue through the detection of their five major metabolites (AOZ, AMOZ, SEM, AHD, DNSAH) by liquid chromatography coupled to electrospray tandem mass spectrometry--in-house validation in line with Commission Decision 657/2002/EC.

Following the ban of four nitrofurans in the mid-90s (furazolidone, furaltadone, nitrofurantoine, nitrofurazone), the nifursol, a veterinary drug from the nitrofuran class of antibacterials which has been used prophylactically as feed additive for treating turkeys against histomoniasis (blackhead disease) was also declared in Annex IV of the European Union Directive no. 90/2377/EC in 2002 according to the Regulation no. 1756/2002/EC. As for the four other nitrofurans, nifursol disappears from tissues within a few days after treatment of food-producing animals. But toxic metabolites are still present for longer periods (several weeks or even months). The major metabolite that can readily be monitored in the tissues following nifursol abuse is the 3,5-dinitro-salicylic acid hydrazine (DNSAH). This article displays some improvements and the revalidation of the analytical method by liquid chromatography coupled to electrospray tandem mass spectrometry (LC-esiMS/MS) already in use in our laboratory for monitoring nitrofuran metabolites but also including the nifursol metabolite at the confirmatory minimum required performance level (MRPL) of 1 microg kg(-1). The validation is applied both to artificially and to naturally incurred turkey muscle.

Determinations of residual furazolidone and its metabolite, 3-amino-2-oxazolidinone (AOZ), in fish feeds by HPLC-UV and LC-MS/MS, respectively.

The antibacterial drug furazolidone belonging to the group of nitrofuran antibacterial agents has been widely used as an antibacterial and antiprotozoal feed additive for poultry, cattle, and farmed fish in China. During application a large proportion of the administered drug may reach the environment directly or via feces. Although the use of furazolidone is prohibited in numerous countries, there are indications of its illegal use. It is known that furazolidone can be rapidly metabolized to 3-amino-2-oxazolidinone (AOZ) in the body of the target organism. In this study, a total of 21 fish feed samples, including 17 commercial fish feeds from local markets in China (representing 15 different formulations) and 4 fish feeds obtained from Germany and Turkey, respectively, are analyzed to determine whether the drug is still illegally used or commercially available feeds are contaminated by this drug. High-performance liquid chromatography (HPLC) and liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) methods have been implemented to determine furazolidone and its metabolite AOZ in fish feeds containing animal protein, respectively. An efficient and convenient cleanup method for the determination of furazolidone in fish feeds is developed, and a simple cleanup method for the determination of AOZ is used. Method recoveries for samples used were determined as 87.7-98.3% for furazolidone at two spike levels of 2.0 and 5.0 ng g-1 and as 95.6-102.8% for AOZ at spike levels of 0.4 and 0.8 ng g-1. Limits of detections were 0.4 ng g-1 for furazolidone and 0.05 ng g-1 for AOZ. The established methods are therefore suitable for the determination of furazolidone and its metabolite AOZ in fish feeds at trace contamination levels. Using the established methods, all fish feed samples have been proved to be furazolidone negative; however, AOZ is tested in 16 of 17 fish feeds obtained from local markets in the Hubei province of China, with a positive rate as high as 94.1%.

Depletion of four nitrofuran antibiotics and their tissue-bound metabolites in porcine tissues and determination using LC-MS/MS and HPLC-UV.

Depletion of the nitrofuran antibiotics furazolidone, furaltadone, nitrofurantoin and nitrofurazone and their tissue-bound metabolites AOZ, AMOZ, AHD and SEM from pig muscle, liver and kidney tissues is described. Groups of pigs were given feed medicated with one of the nitrofuran drugs at a therapeutic concentration (400?mg?kg(-1)) for ten days. Animals were slaughtered at intervals and tissue samples collected for analysis for six weeks following withdrawal of medicated feed. These samples were analysed both for parent nitrofurans (using LC-MS/MS and HPLC-UV), and for tissue-bound metabolites (using LC-MS/MS). The parent drugs were detectable only sporadically and only in pigs subjected to no withdrawal period whatsoever. This confirms the instability of the four major nitrofuran antibiotics in edible tissues. In contrast, the metabolites accumulated to high concentrations in tissues (ppm levels) and had depletion half lives of between 5.5 and 15.5 days. The metabolites of all four drugs were still readily detectable in tissues six weeks after cessation of treatment. This emphasizes the benefits of monitoring for the stable metabolites of the nitrofurans.

Detection of 3-amino-2-oxazolidinone (AOZ), a tissue-bound metabolite of the nitrofuran furazolidone, in prawn tissue by enzyme immunoassay.

Furazolidone, a nitrofuran antibiotic, is banned from use in food animal production within the European Union. Increasingly, compliance with this ban is monitored by use of analytical methods to detect a stable tissue-bound metabolite, 3-amino-2-oxazolidinone (AOZ). Widespread use of furazolidone in poultry and prawns imported into Europe highlighted the urgent need for development of nitrofuran immunoassay screening tests. The first enzyme-linked immunoabsorbant assay for detection of AOZ residues in prawns (shrimps) is now described. Prawn samples were derivatized with o-nitrobenzaldehyde, extracted into ethyl acetate, washed with hexane and applied to a competitive enzyme immunoassay based on a rabbit polyclonal antiserum. Assay limit of detection (LOD) (mean + 3 s) calculated from the analysis of 20 known negative cold and warm water prawn samples was 0.1 microg kg(-1). Intra- and interassay relative standard deviations were determined as 18.8 and 38.2%, respectively, using a negative prawn fortified at 0.7 microg kg(-1). The detection capability (CCbeta), defined as the concentration of AOZ at which 20 different fortified samples yielded results above the LOD, was achieved at fortification between 0.4 and 0.7 microg kg(-1). Incurred prawn samples (n = 8) confirmed by liquid chromatography coupled with tandem mass spectrometry detection to contain AOZ concentrations between 0.4 and 12.7 microg kg(-1) were all screened positive by this enzyme-linked immunoabsorbant assay. Further data are presented and discussed with regard to calculating assay LOD based on accepting a 5% false-positive rate with representative negative prawn samples. Such an acceptance improves the sensitivity of an ELISA and in this case permitted an LOD of 0.05 microg kg(-1) and a CCbeta of below 0.4 microg kg(-1).

Evaluation of the carcinogenic effects of furazolidone and its metabolites in two fish species.

The major metabolite from the use of furazolidone (FZD) in mammals, birds and fish is 2,3-dihydro-3-cyanomethyl-2-hydroxy-5-nitro-1alpha, 2-di(2-oxo-oxazolidin-3-yl)iminomethyl-furo[2,3-beta]furan, also called 3-amine-2-oxazolidone (AOZ). A minor metabolite was identified as N-(5-amine-2-furfuryliden)-3-amine-2-oxazolidone (FOZ). To assess the potential carcinogenicity of FZD and the metabolic mixture of AOZ/FOZ, 11 mg FZD/kg feed/day was fed for 12 weeks to mollies (Poecilia formosa), an ornamental fish species prone to develop tumors. The rate of tumors was quantified and defined both in mollies and their offspring. Then, some fish was made into fishmeal and incorporated into fish food at 500 g of meal/kg of food and fed to other mollies for 12 weeks. The rate of tumors was assessed. A similar trial design was carried out in tilapia fish (Oreochromis niloticus) by adding 50 mg FZD/kg to the feed for 90 days. All animals were placed in glass fishponds under controlled laboratory conditions. Each week, a significant biomass was collected from both groups to assess the macroscopic and histopathological changes. All mollies developed melanohistiocytomic tumors in the liver and other organs. Offspring from surviving mollie females stimulated to breed showed no changes compared to control animals. None of the mollies fed with the mollie-meal food contaminated with AOZ/FOZ developed tumors. Neither tilapia medicated with FZD nor tilapia fed with tilapia-meal contaminated with AOZ/FOZ developed tumors. These results do not support the established viewpoint that FZD must be banned from trophic chains based on its potential carcinogenic properties.

Identification of a furazolidone metabolite responsible for the inhibition of amino oxidases.

1 Furazolidone, a drug widely used in human and veterinary medicine, exhibits inhibition of monoamine oxidase activity, as observed in the tissues of a number of different animal species, including man. The aim of the current study was to determine which of the two possible metabolites, 3-amino-2-oxazolidone (AOZ) or beta-hydroxyethylhydrazine (HEH), a well-known carcinogenic compound, is involved in the toxicological effects reported. 2 A new spectrometric method was set up to differentiate intracellular HEH from AOZ inside cells. This method works well at low pH where both AOZ and HEH are free in solution and available to react with the chemical chromophore (DAB). 3 The results confirm that furazolidone has to be metabolized in the intact cell in order to exhibit mitochondrial monoamine oxidase inhibition, whereas AOZ itself is able to exert a reversible monoamine oxidase inhibition. AOZ also inhibits bovine serum amino oxidase. On the contrary, HEH gives irreversible inhibition of both enzymes. However, the reversible nature of the AOZ inhibition with respect to HEH suggests that the two metabolites act by different mechanisms which do not require the biotransformation of AOZ to HEH. 4 Cell lysates, previously incubated with AOZ, were directly analysed and the formation of HEH from AOZ was not detected, supporting the conclusion that the amino oxidase inhibition observed on treatment with furazolidone was attributable to AOZ and not to HEH.

[Characterization of Vibrio cholerae eltor in the city of Kazan in 2001]. / Kharakteristika kholernykh vibrionov él'tor, vydelennykh v g. Kazan' v 2001 g.

Information on V. cholerae eltor isolated in the focus of cholera in Kazan in 2001 at different periods of the outbreak is presented. The identity of strains isolated from patients, vibriocarriers and environmental objects, including their antibioticograms (sensitivity to cyprofloxacin and resistance to trimethoprim--sulfamethoxazole, streptomycin, furazolidone and nalidixic acid, which may be regarded as markers), is shown. Variable tandem repetitions in the DNA of 30 isolates strains of different origin have been determined. The results of this determination make it possible to classify all these strains as one genotype, which confirms the suggestion on the circulation of one subclone of the infective agent of cholera in the focus. As revealed in this investigation, the isolated strains are labile with respect to diagnostic phage eltor, while ctx+ strains are resistant to phage eltor ctx+.

Metabolism of furazolidone: alternative pathways and modes of toxicity in different cell lines.

1. The metabolism and cytotoxicity of the antimicrobial nitrofuran drug furazolidone have been studied in Caco-2, HEp-2 and V79 cell lines. Free radical production, metabolite pattern, formation of bound residues, inhibition of cellular replication and protection by the antioxidant glutathione were compared for the three cell lines. 2. All three cell lines produced the same nitro-anion radical with similar kinetics. Little further metabolic breakdown was observed in V79 cells, whereas Caco-2 and HEp-2 cells showed extensive degradation of furazolidone, but with different end patterns. 3. Under hypoxic conditions, the colony-forming ability was extensively impaired in HEp-2 cells whereas the other two cell lines were less affected, suggesting that irreversible damage to DNA occurred prevalently in HEp-2 cells. In V79 cells the absence of oxygen caused a 25-fold increase in the formation of protein-bound residues. 4. Brief exposure to furazolidone caused a 50% loss of endogenous glutathione in Caco-2 cells, but no loss could be detected in V79 and HEp-2 cells. Consistently, when glutathione was depleted by buthionine-[S,R]-sulphoximine (BSO) and diethylmaleate (DEM) treatment, the viability of V79 and HEp-2 cells was minimally affected by furazolidone, whereas that of Caco-2 cells was substantially reduced. 5. It is concluded that the cytotoxicity of furazolidone in these cell lines can be exerted by a number of different mechanisms, possibly related to different metabolic pathways. The cytotoxicity of nitrofuran drugs, therefore, cannot be ascribed to a single toxic intermediate, but in Caco-2 cells furazolidone is extensively metabolized and detoxified by GSH, in V79 is only partially activated and then bound to proteins, whereas in HEp-2, once activated, may react with DNA.

A contribution to safety assessment of veterinary drug residues: in vitro/ex vivo studies on the intestinal toxicity and transport of covalently bound residues.

1. The gastrointestinal fate of protein-bound residues of the model compound furazolidone (FZD) was investigated in vitro and ex vivo. Protein-bound residues were generated in rat liver microsomes, isolated by solvent extraction and digested with 0.5% hydrochloric acid and Pronase E. 2. During digestion, 3-amino-2-oxazolidinone (AOZ), the side chain of furazolidone, was partly released from bound residues. 3. The absorption of free AOZ and digested protein-bound residues was tested in isolated perfused rat gut segments (IPGS) and in the intestinal cell line Caco-2. Free AOZ was transfered both in the IPGS model and in Caco-2 monolayer cultures, while no indications for passage of bound residues were obtained. 4. No acute toxicity of AOZ or digested food residues respectively was observed in gut segments and Caco-2 cells at concentrations that were substantially above maximum residue levels to be expected in food of animal origin after administration of therapeutic doses. 5. The results demonstrate that digestive processes can alter the chemical nature of drug residues and yield degradation products that may be bioavailable for the consumer. Thus, the covalent binding of xenobiotics to macromolecular tissue constituents cannot necessarily be regarded as an irreversible endpoint of residue bioavailability and toxicity.

Characterization of furazolidone apical-related effects to human polarized intestinal cells.

In studying the effects of furazolidone (FZ) on the human intestinal Caco-2 cell line grown on microporous membrane, we have previously demonstrated a higher toxicity when the compound was administered at the apical (AP) side than at the basolateral (BL) side. Moreover, we have also shown the production, in the intact cells, of a nitroanion radical from FZ by a cytochrome c P450 reductase. The aim of the present study was to investigate which specific cell structures and functions are involved in the observed domain-related toxicity. The relevance of alterations in integrity and selective properties of the intestinal barrier as first-pass site for ingested molecules is also discussed. We have confirmed that, as expected, the Caco-2 cells are protected from FZ injury by a specific inhibitor of the cytochrome c P450 reductase, and we have shown that this protection is more active on the apical side of the cells. In sublethal conditions, FZ causes increased permeability to 3H-mannitol and, to a different extent, to 3H-inulin. Again the effect is higher when the cells are apically exposed. We have thus examined the tight junctions morphology: a disruption of the apical perijunctional actin-bound cytoskeleton was detected by rhodamine-phalloidin staining and microtubule disorganization by antitubulin fluoresceinated antibodies. Again, the effect was more evident when the cells were apically treated with FZ. Preferential transport and accumulation of the compound by active transport mechanisms could be excluded, since transport of FZ was linear and no intracellular accumulation was detected either from the AP and or the BL sides. All together these results may suggest that the AP formation of the active metabolite and its possible reactivity with SH groups of perijunctional microfilaments could be responsible of the higher FZ apical toxicity. This study shows that polarized differentiated cells are very interesting in vitro models to investigate specific cellular domains as targets of toxic effects and to detect subtle changes that may be induced, in absence of cell death, in specialized epithelial layers.

Evidence of 14C-furazolidone metabolite binding to the hepatic DNA of trout.

Furazolidone (FZ) is a nitrofuran drug commonly used in aquaculture. In the present study, [methylidene-14C]-FZ or [oxazolone-4,5-14C]-FZ was offered to rainbow trout (Oncorhyncus mykiss) in medicated feed at a daily dose of 135 mg/kg b. wt. for 10 days. The trout were sacrificed at specific time points post-dosing and the liver removed for DNA-bound 14C characterization. Both forms of the 14C-labelled FZ were converted by trout to reactive metabolite(s) which bound irreversibly to the hepatic DNA. The amount of 14C bound to the hepatic DNA increased with post-dosing time and was higher in trout pretreated with [methylidene-14C]-FZ than in trout pretreated with [oxazolone-4,5-14C]-FZ. The identity of the FZ reactive metabolite(s) remained to be elucidated. However, a part of the FZ reactive metabolite(s) could be released as 3-amino-2-oxazolidone by acid hydrolysis. An appreciable amount of 14C was also found to bind irreversibly with the hepatic DNA of trout following an i.v. injection of [oxazolone-4,5-14C]-FZ. Results of these studies indicate that FZ is metabolized by trout to a reactive metabolite(s) which binds irreversibly to the DNA of trout liver.

The bioavailability of residues of the furazolidone metabolite 3-amino-2-oxazolidinone in porcine tissues and the effect of cooking upon residue concentrations.

Residues of furazolidone in pig tissues have previously been shown to be bioavailable in the rat. However, no specific furazolidone metabolite has been identified in the tissues of a second species. Tissues were taken from pigs that had been treated therapeutically with furazolidone, lyophilized and then fed to female Sprague Dawley rats for 3 days. Protein-bound and solvent-extractable residues containing the side chain metabolite 3-amino-2-oxazolidinone (AOZ) were detected in the liver, kidney and muscle of the rats using HPLC-thermospray mass spectrometry. Furazolidone-contaminated pig tissues which had undergone solvent extraction and thereby contained only bound residues, was fed to two rats. Bound and extractable AOZ was detected in liver, kidney and muscle. Since it is most likely that consumers would eat animal tissue which had been cooked, an experiment was carried out to determine the effects of cooking upon the concentrations of AOZ residues in pig tissues. Total AOZ concentrations were not significantly reduced in liver, kidney or muscle, following frying, grilling or microwaving.

The prevalence and possible causes of bound and extractable residues of the furazolidone metabolite 3-amino-2-oxazolidinone in porcine tissues.

Furazolidone is readily metabolized and rarely detectable in animal tissues. An alternative is to measure bound and extractable residues containing the 3-amino-2-oxazolidinone (AOZ) moiety. This compound was used to examine the incidence of furazolidone residues in Northern Ireland pigs. AOZ was found in 32/200 kidney samples. A depletion study showed that none of the samples contained AOZ residues at concentrations greater than that found in pigs fed medicated feed, and subjected to an obligatory 7-day withdrawal period. Furazolidone carry-over from medicated feed to subsequent unmedicated batches of feed was investigated in a local feed mill. Furazolidone could be detected only in the first two batches of ostensibly unmedicated feed. A series of feeds containing levels of furazolidone similar to those found in the feed mill carry-over study were prepared. These were fed to pigs, which were killed without withdrawal. The tissues contained AOZ residues, but at concentrations lower than those found in pigs fed medicated feed and properly withdrawn. The possible cross-contamination of unmedicated pigs following exposure to a cleaned house that had previously housed pigs undergoing furazolidone medication was investigated. Tissue AOZ residues were detected, even after comparatively short periods of exposure. However, the concentrations were again lower than those found in pigs fed medicated feed and properly withdrawn.