The identification of potential alternative biomarkers of nitrofurazone abuse in animal derived food products

Semicarbazide (SEM) was considered to be a characteristic protein-bound side-chain metabolite of the banned veterinary drug nitrofurazone and used as a marker of nitrofurazone abuse. It was recently discovered that SEM can arise in food from sources other than nitrofurazone. This uncertainty over the source of SEM may be overcome if alternative markers specific to tissue-bound nitrofurazone residues can be determined. The structure of nitrofurazone metabolites in vivo and particular proteins to which they are bound are not known. These proteins with altered structure due to the presence of the drug metabolites can be considered as potential alternative biomarkers of nitrofurazone abuse. The proteins implicated in the in vivo binding of nitrofurazone were separated and identified. A crude mixture of proteins extracted from the liver of a rat treated with the drug was separated using a series of different techniques such as preparative isoelectric focusing and size exclusion HPLC. Multiple fractions were assayed by LC-MS/MS to detect the presence of SEM. The proteins containing SEM residues were identified by peptide mass mapping using trypsin digestion and MALDI-TOF. The first protein identified as containing high concentration of SEM was albumin. It was also shown that low molecular weight species within a protein mixture whose main constituent was glutathione S-transferase contained a high concentration of SEM. The chemical composition of these components is under investigation. Preliminary data suggest the SEM forms part of a nitrofurazone metabolite conjugated to glutathione. (C) 2008 Elsevier Ltd. All rights reserved.

Nitrofurazone accumulates in avian eyes - a replacement for semicarbazide as a marker of abuse

Intact nitrofurazone is present in whole eyes of chickens fed varying levels of this banned antibiotic and may therefore be used as an alternative to the controversial marker residue, semicarbazide, to monitor for abuse of this drug in primary production.

Kinetics of semicarbazide and nitrofurazone in chicken eggs and egg powders

The accumulation, depletion and partitioning of semicarbazide (SEM) and its parent compound nitrofurazone (NFZ) in eggs were studied using hens fed NFZ at therapeutic and sub-therapeutic levels. Dietary NFZ correlated strongly with NFZ and total SEM in eggs, while 28% of observed SEM was present in the form of parent NFZ. Depletion half-life in eggs was 2.4 days for SEM and 1.1 days for NFZ. NFZ accumulated preferentially in yolk (57-63%) as opposed to albumen, while 71-80% of SEM was found in yolk. In whole egg, 29% of SEM was present as tissue-bound residues compared with 80% in breast muscle. Whilst NFZ and SEM were partly degraded by pasteurization and spray drying, sufficient NFZ remained to suggest it might be detectable in egg powders when SEM is observed at low µg kg -1 concentrations. NFZ was detectable in whole eggs during ingestion of only 0.1% of the therapeutic NFZ dose, making detection of intact NFZ in eggs a feasible means to prove conclusively the administration of this banned compound.

Synthesis and in vitro evaluation of potential antichagasic hydroxymethyinitrofurazone (NFOH-121): A new nitrofurazone prodrug

The synthesis of mutual prodrugs of nitrofurazone with primaquine, using specific and nonspecific spacer groups, has been previously attempted seeking selective antichagasic agents. The intermediate reaction product, hydroxymethylnitrofurazone (NFOH-121), was isolated and tested in LLC-MK2 culture cells infected with trypomastigotes forms of Trypanosoma cruzi showing higher trypanocidal activity than nitrofurazone and benznidazol in all stages. The mutagenicity tests showed that the prodrug was less toxic than the parent drug. Degradation assays were carried out in pH 1.2 and 7.4. (C) 2003 Elsevier Ltd. All rights reserved.

Cruzain inhibition by hydroxymethylnitrofurazone and nitrofurazone: investigation of a new target in Trypanosoma cruzi

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Voltammetric behavior of nitrofurazone and its hydroxymethyl prodrug with potential anti-chagas activity

Chagas' disease is a serious health problem for Latin America. The situation is worsened by the lack of efficient chemotherapy. The two available commercial drugs, benznidazole and nifurtimox, are more effective in the acute phase of the disease. Nitrofurazone is active against Trypanosoma cruzi, however its high toxicity precludes its current use in parasitosis. Hydroxymethylnitrofurazone is a prodrug of nitrofurazone. It is more active against Trypanosoma cruzi than nitrofurazone, besides being less toxic. This work shows the voltammetric behavior of nitrofurazone and a comparison with those of metronidazole and chloramphenicol using cyclic, linear sweep and differential pulse voltammetries. For these drugs also the prediction of the diffusion coefficients using Wilke-Chang equation was performed. The reduction of nitrofurazone is pH-dependent and in acidic medium the hydroxylamine derivative, involving four electrons, is the principal product formed. In aqueous-alkaline medium and with a glassy carbon electrode pre-treatment the reduction of nitrofurazone occurs in two steps, the first involving one electron to form the nitro-radical anion and the second corresponding to the hydroxylamine derivative formation. Hydroxymethylnitrofurazone presented the same voltammetric behavior and electroactivity, indicating that the molecular modification performed in nitrofurazone did not change its capacity to be reduced. A brief discussion regarding the differences in biological activity between the two compounds is also presented. ©2005 Sociedade Brasileira de Química.

Pharmacokinetics of hydroxymethylnitrofurazone and its parent drug nitrofurazone in rabbits

The prodrug hydroximethylnitrofurazone (NFOH) presents antichagasic activity with greatly reduced toxicity compared to its drug matrix nitrofurazone (NF). Besides these new characteristics, the prodrug was more active against the parasite T. cruzi amastigotes. These advantages make the prodrug a possible therapeutic alternative for the treatment of both acute and the chronic phase of Chagas disease. However, the knowledge of pharmacokinetic profile is crucial to evaluate the feasibility of a new drug. In this study, our objective was to evaluate the in vivo formation of NF from the NFOH single administration and to evaluate its pharmacokinetic profile and compared it to NF administration. A bioanalytical method to determine the NF and NFOH by LCMS/MS was developed and validated to perform these investigations. Male albino rabbits (n=15) received NF intravenously and orally in doses of 6.35 and 63.5 mg / kg respectively, and NFOH, 80.5 mg / kg orally. The serial blood samples were processed and analyzed by mass spectrometry. The system operated in positive and negative modes for the analites determination, under elution of the mobile phase 50:50 water: methanol. The administration of NFOH allowed the calculation of pharmacokinetic parameters for the prodrug, and the NF obtained from NFOH administration. Using the pharmacokinetic profile obtained from the NF i.v. administration, the oral bioavailability of NF from the administered prodrug was obtained (60.1%) and, as a key parameter in a prodrug administration, should be considered in future studies. The i.v. and oral administrations of NF differ in the constant of elimination (0.04 vs 0.002) and elimination half-life (17.32 min vs 276.09 min) due to the low solubility of the drug that hinders the formation of molecular dispersions in the digestory tract. Still, there was observed no statistical differences were observed between the pharmacokinetic parameters of orally administered NF and NF obtained from NFOH. The calculated area under the curve (AUC 0-∞) showed that the exposure to the parental drug was fairly the same (844.79 vs 566.44) for NF and NF obtained from the prodrug administration. The tendency to higher NF's mean residence time (MRT) as observed in the prodrug administration (956.1 min vs 496.3 min) guarantees longer time for the action of the drug and it allows the expansion of the administration intervals. These findings, added with the beneficial characteristics of the prodrug encourage new efficacy tests towards the clinical use of NFOH.

Enzyme immunoassay for semicarbazide - The nitrofuran metabolite and food contaminant

Semicarbazide (SEM), the marker residue for the banned nitrofuran veterinary antibiotic nitrofurazone (NFZ), has been detected regularly in foods (47% of recent nitrofuran EU Rapid Alerts involve SEM). However, the validity of SEM as a definitive marker for NFZ has been undermined by SEM arising from other sources including azodicarbonamide, a plastics blowing agent and flour treatment additive. An inexpensive screening test for SEM in food matrices is needed-all SEM testing currently uses expensive LC-MS/MS instrumentation. We now report the first production of antibodies against derivatised SEM. A novel carboxyphenyl SEM derivative was used to raise a polyclonal antibody that has been incorporated into a semi-quantitative microtitre plate ELISA, validated according to the criteria set out in Commission Decision 2002/657/EC, for use with chicken muscle. The antibody is highly specific for derivatised SEM, cross-reactivity being 1.7% with NFZ and negligible with a wide range of other nitrofurans and poultry drugs. Samples are derivatised with o-nitrobenzaldehyde and simultaneously protease digested before extraction by cation exchange SPE. The ELISA has a SEM detection capability (CC beta) of 0.25 mu g kg(-1) when a threshold of 0.21 mu g kg(-1) is applied to the selection of samples for confirmation (lowest observed 0.25 mu g kg(-1) fortified sample, n = 20), thus satisfying the EU nitrofurans' minimum required performance limit of 1 mu g kg(-1). N-FZ-incurred muscles (12) containing SEM at 0.5-5.0 mu g kg(-1) by LC-MS/MS, all screened positive by this ELISA protocol which is also applicable to egg and chicken liver. (C) 2007 Elsevier BN. All rights reserved.

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.

Residues of nitrofuran antibiotic parent compounds and metabolites in eyes of broiler chickens

Nitrofuran antibiotic residues in food continue to be of international concern. The finding of sources of semicarbazide (SEM), other than through the misuse of nitrofurazone, present a challenge to the use of SEM as a definitive marker residue for this drug. Detection of intact (parent) nitrofurazone would avoid confusion over the source of SEM residues. Broiler chickens were fed sub-therapeutic nitrofuran-containing diets and their tissues were analysed for parent compounds and metabolites by liquid chromatography coupled with tandem mass spectrometry detection (LC-MS/MS). Depletion half-lives in muscle were longer for tissue-bound metabolite residues, 3.4 days - 3-amino-2-oxazolidinone (AOZ), 3-amino-5-morpholinomethyl-2-oxazolidone (AMOZ) - to 4.5 days (SEM), than total metabolite residues, 2.0 days (AOZ) to 3.2 days (SEM). Metabolite concentrations were higher in eyes than in muscle. Metabolite half-lives in eyes ranged from 8.5 days (1-aminohydantoin (AHD)) to 20.3 days (SEM). Nitrofuran parent compounds were also detected in eyes. Furaltadone was detected in single eyes after 21 days' withdrawal of a 6 mg kg -1 furaltadone diet. When 50 eyes from broilers containing metabolites in muscle close to the 1 µg kg -1 minimum required performance level (MRPL) were pooled into single samples, 1.2 ng of furazolidone and 31.1 ng of furaltadone were detected, but nitrofurazone was not detected due to the long depletion half-life of SEM in muscle. Further studies are required to improve LC-MS/MS nitrofurazone sensitivity and refine the sample size necessary to use nitrofurazone detection in pooled eyes as a complement to SEM detection in muscle.

A Prodrug Approach to Improve the Physico-Chemical Properties and Decrease the Genotoxicity of Nitro Compounds

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Trypanosoma cruzi: establishment of permeable cells for RNA processing analysis with drugs

Pre-mRNA maturation in trypanosomatids occurs through a process called trans-splicing which involves excision of introns and union of exons in two independent transcripts. For the first time, we present the standardization of Trypanosoma cruzi permeable cells (Y strain) as a model for trans-splicing study of mRNAs in trypanosomes, following by RNase protection reaction, which localizes the SL exon and intron. This trans-splicing reaction in vitro was also used to analyze the influence of NFOH-121, a nitrofurazone-derivative, on this mechanism. The results suggested that the prodrug affects the RNA processing in these parasites, but the trans-splicing reaction still occurred.

Rapid test for the evaluation of the activity of the prodrug hydroxymethylnitrofurazone in the processing of Trypanosoma cruzi messenger RNAs

No fully effective treatment has been developed since the discovery of Chagas' disease by Carlos Chagas in 1909. Since drug-resistant Trypanosoma cruzi strains are occurring and the current therapy is effectiveness in the acute phase but with various adverse side effects, more studies are needed to characterize the susceptibility of T. cruzi to new drugs. Many natural and/or synthetic substances showing trypanocidal activity have been used, even though they are not likely to be turned into clinically approved drugs. Originally, drug screening was performed using natural products, with only limited knowledge of the molecular mechanism involved in the development of diseases. Trans-splicing, which is unusual RNA processing reaction and occurs in nematodes and trypanosomes, implies the processing of polycistronic transcription units into individual mRNAs; a short transcript spliced leader (SL RNA) is trans-spliced to the acceptor pre-mRNA, giving origin to the mature mRNA. In the present study, permeable cells of T. cruzi epimastigote forms (Y, BOL and NCS strains) were treated to evaluate the interference of two drugs (hydroxymethylnitrofurazone - NFOH-121 and nitrofurazone) in the trans-splicing reaction using silver-stained PAGE analysis. Both drugs induced a significant reduction in RNA processing at concentrations from 5 to 12.5 µM. These data agreed with the biological findings, since the number of parasites decreased, especially with NFOH-121. This proposed methodology allows a rapid and cost-effective screening strategy for detecting drug interference in the trans-splicing mechanism of T. cruzi.