Validation of a method for the detection and confirmation of nitroimidazoles and corresponding hydroxy metabolites in turkey and swine muscle by means of gas chromatography–negative ion chemical ionization mass spectrometry

The results of a validation study of a GC–NCI–MS method for the quantitative determination of 5-nitroimidazoles {1,2-Dimethyl-5-nitroimidazole (dimetridazole, DMZ), 1-methyl-2-[(carbamoyloxy)methyl]-5-nitroimidazole (ronidazole, RNZ), 1-(2-hydroxyethyl)-2-methyl-5-nitroimidazole (metronidazole, MNZ) and 2-isopropyl-1-methyl-5-nitroimidazole (ipronidazole, IPZ)} including the hydroxy metabolites of these agents {2-hydroxymethyl-1-methyl-5-nitroimidazole (HMMNI), 1-(2-hydroxyethyl)-2-hydroxymethyl-5-nitroimidazole (MNZOH), and 1-methyl-2-(2′-hydroxyisopropyl)-5-nitroimidazole (IPZOH)} in turkey and swine muscle are presented. The validation was carried out according to the requirements of the draft for the revision of Commission Decision 93/256/EC, which is expected to be adopted by the European Commission in due course. The determination of the method’s performance parameters revealed decision limits (CC_α) between 0.65 and 2.8 μg/kg for DMZ, RNZ/HMMNI, MNZ and MNZOH. Confirmatory analyses according to the requirements of the forthcoming EC decision are possible for all analytes except for IPZ and IPZOH where already the decision limits (CC_α) were higher (5.2 μg/kg) than for the above-mentioned nitroimidazoles. The within-laboratory reproducibility and the mean recovery were in an acceptable range for all analytes.     

Nitroimidazoles, Part XXIII--activity of satranidazole series against anaerobic infections.

A large number of nitroimidazoles have been examined for in vitro activity against three anaerobes - Bacteroides fragilis (Bf), a strain of Bf resistant to metronidazole (16a) and Clostridium perfringens and many found to be active. Among these may be mentioned 1-methyl-5-nitroimidazoles carrying N - bound hetetocycles at position 2, such as satranidazole 1a, 1b, 1c, 1k, 1n and 1v which are at least twice as active as metronidazole (16a), ornidazole (16b) and tinidazole (16c). Even more active are 5-nitroimidazolyl benzimidazole 5d, -thiazolidinone 6b and thiadiazolidine dioxide 8a. Many other types of compounds derived from 1-methyl-2-amino-5-nitroimidazole are feebly active. Among 5-nitroimidazoles with a carbon substituent at position 2, 16a, 16b and 16c are equiactive while dimetridazole 14f is more active than 16a against Bf. Some 2-vinyl derivatives are very potent, with 18f and 18i being outstanding. Activity better than that of metronidazole is seen for nitroimidazooxazepines, e.g. 29d. 5-Nitroimidazoles are more active against anaerobes than 4-nitro isomers. Antianaerobic and antiamoebic activities generally run parallel in these classes of compounds. The study has led to the elaboration of the antianaerobic profile of satranidazole 1a.     

Absence of genotoxic effects of metronidazole and two of its urinary metabolites on human lymphocytes in vitro.

The antiprotozoan agent metronidazole (1-(2-hydroxyethyl)-2-methyl-5-nitroimidazole) and two of its major human urinary excretion products, 2-methyl-5-nitromidazole-1-yl acetic acid and 1-(2-hydroxyethyl)-2-hydroxymethyl-5-nitroimidazole were tested for genotoxic activity in human lymphocytes in vitro by analysis of chromosome aberrations, sister-chromatid exchanges and DNA-repair synthesis. The positive control compounds methyl methanesulphonate (MMS) and nitrogen mustard (HN2) showed significant genotoxic activity in these tests. No such activity of metronidazole and its two metabolites was detected in concentrations up to 1000 microgram/ml (5.8 X 10(-3) M). Nor did these 3 compounds influence DNA-repair synthesis induced by MMS and HN2. These results suggest that metronidazole, 2-methyl-5-nitroimidazole-1-yl acetic acid and 1-(2-hydroxyethyl)-2-hydroxymethyl-5-nitroimidazole have no direct genotoxic effect on human lymphocytes in vitro.     

Novel nitroimidazoles with trypanocidal and cell growth inhibition activities

Chagas' disease, caused by the protozoan parasite Trypanosoma cruzi, affects 18 million people in Latin America and is an important cause of heart disease. Although transmission has been reduced, an effective therapy for the infected population is lacking. New nitroimidazoles were designed and synthesized aimed at the development of a trypanocidal drug. The coupling of nitroimidazoles with heterocyclic N-trifluoromethyltriazolyl and pyrazolyl groups, 5-[N-(3-(5-trifluoromethyl)-1H-1,2,4-triazolyl)]amino-1-methyl-4-nitroimidazole (compound 4) and 5-N-(1-pyrazolyl)-1-methyl-4-nitroimidazole (compound 5). The in vitro trypanocidal effects of compounds 4 and 5 were evaluated. The results demonstrated that compound 5 was the most active compound, killing about 100% and 64% of the parasites in 0.3 mg/ml and 0.003 mg/ml concentrations, respectively. Interestingly, compound 4 also inhibited myeloma cell growth in a dose-dependent manner.     

Oxidation of nitrotoluenes by toluene dioxygenase: evidence for a monooxygenase reaction.

Pseudomonas putida F1 and Pseudomonas sp. strain JS150 initiate toluene degradation by incorporating molecular oxygen into the aromatic nucleus to form cis-1,2-dihydroxy-3-methylcyclohexa-3,5-diene. When toluene-grown cells were incubated with 2- and 3-nitrotoluene, the major products identified were 2- and 3-nitrobenzyl alcohol, respectively. The same cells oxidized 4-nitrotoluene to 2-methyl-5-nitrophenol and 3-methyl-6-nitrocatechol. Escherichia coli JM109(pDTG601), which contains the toluene dioxygenase genes from P. putida F1 under the control of the tac promoter, oxidized the isomeric nitrotoluenes to the same metabolites as those formed by P. putida F1 and Pseudomonas sp. strain JS150. These results extend the range of substrates known to be oxidized by this versatile enzyme and demonstrate for the first time that toluene dioxygenase can oxidize an aromatic methyl substituent.     

Oxidation of nitrotoluenes by toluene dioxygenase: evidence for a monooxygenase reaction.

Pseudomonas putida F1 and Pseudomonas sp. strain JS150 initiate toluene degradation by incorporating molecular oxygen into the aromatic nucleus to form cis-1,2-dihydroxy-3-methylcyclohexa-3,5-diene. When toluene-grown cells were incubated with 2- and 3-nitrotoluene, the major products identified were 2- and 3-nitrobenzyl alcohol, respectively. The same cells oxidized 4-nitrotoluene to 2-methyl-5-nitrophenol and 3-methyl-6-nitrocatechol. Escherichia coli JM109(pDTG601), which contains the toluene dioxygenase genes from P. putida F1 under the control of the tac promoter, oxidized the isomeric nitrotoluenes to the same metabolites as those formed by P. putida F1 and Pseudomonas sp. strain JS150. These results extend the range of substrates known to be oxidized by this versatile enzyme and demonstrate for the first time that toluene dioxygenase can oxidize an aromatic methyl substituent.     

Synthesis, antibacterial activity, and quantitative structure-activity relationships of new (Z)-2-(nitroimidazolylmethylene)-3(2H)-benzofuranone derivatives

A new series of (Z)-2-(1-methyl-5-nitroimidazole-2-ylmethylene)-3(2H)-benzofuranones (11a-p) and (Z)-2-(1-methyl-4-nitroimidazole-5-ylmethylene)-3(2H)-benzofuranones (12a-m) were synthesized and assayed for their antibacterial activity against Gram-positive and Gram-negative bacteria. Most of the 5-nitroimidazole analogues (11a-p) showed a remarkable inhibition of a wide spectrum of Gram-positive bacteria (Staphylococcus aureus, Streptococcus epidermidis, MRSA, and Bacillus subtilis) and Gram-negative Klebsiella pneumoniae, whereas 4-nitroimidazole analogues (12a-m) were not effective against selected bacteria. The quantitative structure-activity relationship investigations were applied to find out the correlation between the experimentally evaluated activities with various parameters of the compounds studied. The QSAR models built in this work had reasonable predictive power and could be explained by the observed trends in activities.     

Reduction by dithionite ion of adducts of metmyoglobin with imidazole, pyridine, and derivatives

The rate and equilibrium constants for the information of a number of metmyoglobin species Mb+X (X = imidazole, imidazole-H-, 1-methylimidazole, 2-methylimidazole, 4-nitroimidazole, 2-methyl-5-nitroimidazole, pyridine, 2-, 3-, and 4-picoline) and the rates of their reduction by dithionite have been measured at 25 degrees. Several different kinds of kinetic behavior for the reduction were observed. In all cases, a rate constant for direct reaction of Mb+X with SO2- can be assessed. The data strongly support attack of SO2- on the ligand, followed by electron transfer through the pi system to the metal ion.     

2-nitroimidazol-5-ylmethyl as a potential bioreductively activated prodrug system: reductively triggered release of the PARP inhibitor 5-bromoisoquinolinone.

5-Chloromethyl-1-methyl-2-nitroimidazole reacted efficiently with the anion derived from 5-bromoisoquinolin-1-one to give 5-bromo-2-((1-methyl-2-nitroimidazol-5-yl)methyl)isoquinolin -1-one. Biomimetic reduction effected release of the 5-bromoisoquinolin-1-one. The 2-nitroimidazol-5-ylmethyl unit thus has potential for development as a general prodrug system for selective drug delivery to hypoxic tissues.     

Peroxynitrite-induced reactions of synthetic oligo 2'-deoxynucleotides and DNA containing guanine: formation and stability of a 5-guanidino-4-nitroimidazole lesion

Peroxynitrite is a strong oxidizing agent that is formed in the reaction of nitric oxide and superoxide anion. It is capable of oxidizing and nitrating a variety of biological targets including DNA, and these modifications may be responsible for a number of pathological conditions and diseases. A recent study showed that peroxynitrite reacts with 2',3',5'-tri-O-acetylguanosine to yield a novel compound, tri-O-acetyl-1-(beta-D-erythro-pentafuranosyl)-5-guanidino-4-nitroimidazole, and, unlike other peroxynitrite-mediated guanine oxidation products, it is a stable and significant component formed even at low peroxynitrite concentrations. In this work, we studied the in vitro formation of the guanine-derived product, 5-guanidino-4-nitroimidazole, in synthetic oligonucleotides and DNA treated with peroxynitrite. When calf thymus DNA or oligonucleotides were reacted with peroxynitrite at ambient temperature, the modified base 5-guanidino-4-nitroimidazole was generated along with several other products. The oligonucleotides containing the 5-guanidino-4-nitroimidazole modification were purified by reverse-phase and anion-exchange HPLC and characterized by matrix-assisted laser desorption mass spectrometry. 5-Guanidino-4-nitroimidazole formation in peroxynitrite-treated DNA was characterized after enzymatic digestion of the reacted DNA to the nucleoside level. HPLC purification and electrospray ionization mass spectrometry (with selected reaction monitoring) enabled the analysis of this modified nucleoside with high sensitivity. The yield of 5-guanidino-4-nitroimidazole formed in single-stranded DNA was approximately 10-fold higher than that found in duplex DNA. With calf thymus DNA, 5-guanidino-4-nitroimidazole was dose-dependently formed at low peroxynitrite concentrations. In stability tests, a synthetic oligonucleotide containing the 5-guanidino-4-nitroimidazole modification was only partially cleaved by hot piperidine and was a weak substrate for Fpg glycosylase repair enzyme; in addition, this site was not cleaved by endonuclease III. These results suggest that nuclear DNA containing 5-guanidino-4-nitroimidazole may not be quickly repaired by DNA repair enzyme systems. Finally, primer extension experiments revealed that this lesion is a potential DNA replication blocker when polymerization is catalyzed by polymerase alpha and polymerase I (Klenow fragment, lack of exonuclease activity) but not with human polymerase beta. Replication fidelity experiments further showed that 5-guanidino-4-nitroimidazole may cause G-->T and G-->C transversions in calf thymus polymerase alpha and E. coli polymerase I.     

In vivo bypass efficiencies and mutational signatures of the guanine oxidation products 2-aminoimidazolone and 5-guanidino-4-nitroimidazole

The in vivo mutagenic properties of 2-aminoimidazolone and 5-guanidino-4-nitroimidazole, two products of peroxynitrite oxidation of guanine, are reported. Two oligodeoxynucleotides of identical sequence, but containing either 2-aminoimidazolone or 5-guanidino-4-nitroimidazole at a specific site, were ligated into single-stranded M13mp7L2 bacteriophage genomes. Wild-type AB1157 Escherichia coli cells were transformed with the site-specific 2-aminoimidazolone- and 5-guanidino-4-nitroimidazole-containing genomes, and analysis of the resulting progeny phage allowed determination of the in vivo bypass efficiencies and mutational signatures of the DNA lesions. 2-Aminoimidazolone was efficiently bypassed and 91% mutagenic, producing almost exclusively G to C transversion mutations. In contrast, 5-guanidino-4-nitroimidazole was a strong block to replication and 50% mutagenic, generating G to A, G to T, and to a lesser extent, G to C mutations. The G to A mutation elicited by 5-guanidino-4-nitroimidazole implicates this lesion as a novel source of peroxynitrite-induced transition mutations in vivo. For comparison, the error-prone bypass DNA polymerases were overexpressed in the cells by irradiation with UV light (SOS induction) prior to transformation. SOS induction caused little change in the efficiency of DNA polymerase bypass of 2-aminoimidazolone; however, bypass of 5-guanidino-4-nitroimidazole increased nearly 10-fold. Importantly, the mutation frequencies of both lesions decreased during replication in SOS-induced cells. These data suggest that 2-aminoimidazolone and 5-guanidino-4-nitroimidazole in DNA are substrates for one or more of the SOS-induced Y-family DNA polymerases and demonstrate that 2-aminoimidazolone and 5-guanidino-4-nitroimidazole are potent sources of mutations in vivo.     

‘Berenil’ and nitroimidazole combinations in the treatment of Trypanosoma brucei infection with central nervous system involvement

Three nitroimidazole compounds were tested for trypanocidal activity against early (3-day) T. brucei TREU 667 infections. Compound 1 (1-methyl-2-carbamoyl-oxy-methyl-5-nitroimidazole) at both 5 and 20 mg/kg given as four daily doses was ineffective, while Compound 2 (3-(1-methyl-5-nitroimidazole-2-yl)-3α, 4,5,6,7, 7α-hexahydro-1, 2-benz-isoxazole) at 4 × 80 mg/kg and Compound 3 (3-(1-methyl-5-nitroimidazole-2-yl)-4, 5-hexamethylene-Δ²-isoxazoline) at 4 × 20 mg/kg both elicited a permanent cure. When tested against late (21-day) infections of T. brucei 667 neither Compound 2 nor Compound 3 given singly, or in various combinations was effective in that parasitaemias returned rapidly in nearly all mice.When the trypanocidal drug ‘Berenil’ was administered followed by the Compound 3, the majority of the mice with a 21-day infection of T. brucei TREU 667 or T. brucei LUMP 1001 were cured permanently. When ‘Berenil’ alone was used the mice usually relapsed within a few weeks of treatment. The isolate used affected the outcome of the treatment. Higher dosages of ‘Berenil’ followed by Compound 3 were required to cure infections with T. brucei LUMP 1001 than with T. brucei TREU 667.The importance of these findings in the treatment of human sleeping sickness with central nervous system involvement is discussed.     

Synthesis of N-alkylated derivatives of imidazole as antibacterial agents

N-Alkylation of imidazole, 2-methylimidazole and 2-methyl-4-nitroimidazole have been carried out to achieve effective antibacterial agents. The products were then investigated for antibacterial activity against Escherichia coil, Staphylococcus aureus and Pseudomonas aeruginosa. Antibacterial effects of 1-alkylimidazole derivatives increase as the number of carbons in alkyl chain increases up to nine carbons. Also substitution of 2-methyl and 2-methyl-4-nitro groups on imidazole ring increases the antibacterial activity.     

Microbial transformation of nitroaromatic compounds in sewage effluent

The transformation of mono- and dinitroaromatic compounds was measured in sewage effluent maintained under aerobic or anaerobic conditions. Most of the nitrobenzene, 3- and 4-nitrobenzoic acids, and 3- and 4-nitrotoluenes and much of the 1,2- and 1,3-dinitrobenzenes disappeared both in the presence and absence of oxygen. Under anaerobiosis, 2,6-dinitrotoluene and 3,5-dinitrobenzoic acid disappeared slowly, but no loss was evident in 28 days in aerated sewage. Aromatic amines did not accumulate during the aerobic decomposition of the mononitro compounds. They did appear in nonsterile, but not in sterile, sewage incubated aerobically with the dinitro compounds and anaerobically with all the chemicals. Analysis by gas chromatography and combined gas chromatography-mass spectrometry showed that aniline was formed from nitrobenzene, toluidine was formed from 3- and 4-nitrotoluenes, and aminobenzoic acid was formed from 3- and 4-nitrobenzoic acids under anaerobiosis, and that nitroaniline was formed from 1,2- and 1,3-dinitrobenzenes, aminonitrotoluene resulted from 2,6-dinitrotoluene, and aminonitrobenzoic acid was a product of 3,5-dinitrobenzoic acid under both conditions. The isomeric forms of the metabolites were not established. Aniline, 4-toluidine, and 4-aminobenzoic acid added to sewage disappeared from aerated nonsterile, but not from sterile, sewage or sewage in the absence of oxygen. 2-Nitroaniline, 2-amino-3-nitrotoluene, and 2-amino-5-nitrobenzoic acid added to sewage persisted for at least 60 days in aerobic or anaerobic conditions. Gas chromatographic and gas chromatographic-mass spectrometric analyses demonstrated that acetanilide and 2-methylquinoline were formed from aniline, 4-methylformanilide and 4-methylacetanilide were formed from 4-toluidine, 2-methylbenzimidazole was a product of 2-nitroaniline, and unidentified benzimidazoles were formed from 2-amino-3-nitrotoluene in the absence of oxygen, and that 2-nitroacetanilide and 2-methyl-6-nitroacetanilide were formed from 2-nitroaniline and 2-amino-3-nitrotoluene, respectively, in the presence or absence of oxygen. It is suggested that the transformations of widely used nitroaromatic compounds should be further studied because of the persistence and possible toxicity of products of their metabolism.     

Microbial Transformation of Nitroaromatic Compounds in Sewage Effluent

The transformation of mono- and dinitroaromatic compounds was measured in sewage effluent maintained under aerobic or anaerobic conditions. Most of the nitrobenzene, 3- and 4-nitrobenzoic acids, and 3- and 4-nitrotoluenes and much of the 1,2- and 1,3-dinitrobenzenes disappeared both in the presence and absence of oxygen. Under anaerobiosis, 2,6-dinitrotoluene and 3,5-dinitrobenzoic acid disappeared slowly, but no loss was evident in 28 days in aerated sewage. Aromatic amines did not accumulate during the aerobic decomposition of the mononitro compounds. They did appear in nonsterile, but not in sterile, sewage incubated aerobically with the dinitro compounds and anaerobically with all the chemicals. Analysis by gas chromatography and combined gas chromatography-mass spectrometry showed that aniline was formed from nitrobenzene, toluidine was formed from 3- and 4-nitrotoluenes, and aminobenzoic acid was formed from 3- and 4-nitrobenzoic acids under anaerobiosis, and that nitroaniline was formed from 1,2- and 1,3-dinitrobenzenes, aminonitrotoluene resulted from 2,6-dinitrotoluene, and aminonitrobenzoic acid was a product of 3,5-dinitrobenzoic acid under both conditions. The isomeric forms of the metabolites were not established. Aniline, 4-toluidine, and 4-aminobenzoic acid added to sewage disappeared from aerated nonsterile, but not from sterile, sewage or sewage in the absence of oxygen. 2-Nitroaniline, 2-amino-3-nitrotoluene, and 2-amino-5-nitrobenzoic acid added to sewage persisted for at least 60 days in aerobic or anaerobic conditions. Gas chromatographic and gas chromatographic-mass spectrometric analyses demonstrated that acetanilide and 2-methylquinoline were formed from aniline, 4-methylformanilide and 4-methylacetanilide were formed from 4-toluidine, 2-methylbenzimidazole was a product of 2-nitroaniline, and unidentified benzimidazoles were formed from 2-amino-3-nitrotoluene in the absence of oxygen, and that 2-nitroacetanilide and 2-methyl-6-nitroacetanilide were formed from 2-nitroaniline and 2-amino-3-nitrotoluene, respectively, in the presence or absence of oxygen. It is suggested that the transformations of widely used nitroaromatic compounds should be further studied because of the persistence and possible toxicity of products of their metabolism.     

Generation of radical anions from metronidazole, misonidazole and azathioprine by photoreduction in the presence of EDTA

Ultraviolet irradiation of the nitroimidazole derivatives metronidazole, misonidazole, azathioprine and 1-methyl-4-nitroimidazole in aqueous solution with various reductants produced the respective nitro radical anions, as detected by electron spin resonance spectroscopy. The most effective reductant, yielding high concentrations of the radical anions, was EDTA at pH 10. NADH, NADPH, formaldehyde glutathione and methanol were also tested but were less efficient as reductants.     

Practical stereoselective synthesis of (2,3,4, 5-tetrahydro-1H-benzo[b]azepin-5-yl)acetic acid

Highly enantioselective asymmetric hydrogenation of readily accessible olefins, (E)- and (Z)-[1-(toluene-4-sulfonyl)-1,2,3, 4-tetrahydro-1H-benzo[b]azepin-5-ylidene]acetic acid (4a and 4b, respectively) and [1-(toluene-4-sulfonyl)-2, 3-dihydro-1H-benzo[b]azepin-5-yl]acetic acid (4c), is presented as an efficient and straightforward route to (R)-[1-(toluene-4-sulfonyl)-2,3,4, 5-tetrahydro-1H-benzo[b]azepin-5-yl]acetic acid [(R)-1] which is a key intermediate for the synthesis of non-peptide AVP V2-agonist. Hydrogenation of carboxylic acid 4c gave (R)-1 in quantitative yield and 85% ee using Ru(OAc)2[(S)-H8-BINAP], a Ru(II) complex of a partially hydrogenated BINAP (H8-BINAP), as a catalyst. When (R)-1 of 76% ee was transformed into the corresponding isopropylamide 6, pure enantiomer (R)-6 was obtained in 75% yield by recrystallization from MeOH.     

Selection for Growth on 3-Nitrotoluene by 2-Nitrotoluene-Utilizing Acidovorax sp. Strain JS42 Identifies Nitroarene Dioxygenases with Altered Specificities

Acidovorax sp. strain JS42 uses 2-nitrotoluene as a sole source of carbon and energy. The first enzyme of the degradation pathway, 2-nitrotoluene 2,3-dioxygenase, adds both atoms of molecular oxygen to 2-nitrotoluene, forming nitrite and 3-methylcatechol. All three mononitrotoluene isomers serve as substrates for 2-nitrotoluene dioxygenase, but strain JS42 is unable to grow on 3- or 4-nitrotoluene. Using both long- and short-term selections, we obtained spontaneous mutants of strain JS42 that grew on 3-nitrotoluene. All of the strains obtained by short-term selection had mutations in the gene encoding the α subunit of 2-nitrotoluene dioxygenase that changed isoleucine 204 at the active site to valine. Those strains obtained by long-term selections had mutations that changed the same residue to valine, alanine, or threonine or changed the alanine at position 405, which is just outside the active site, to glycine. All of these changes altered the regiospecificity of the enzymes with 3-nitrotoluene such that 4-methylcatechol was the primary product rather than 3-methylcatechol. Kinetic analyses indicated that the evolved enzymes had enhanced affinities for 3-nitrotoluene and were more catalytically efficient with 3-nitrotoluene than the wild-type enzyme. In contrast, the corresponding amino acid substitutions in the closely related enzyme nitrobenzene 1,2-dioxygenase were detrimental to enzyme activity. When cloned genes encoding the evolved dioxygenases were introduced into a JS42 mutant lacking a functional dioxygenase, the strains acquired the ability to grow on 3-nitrotoluene but with significantly longer doubling times than the evolved strains, suggesting that additional beneficial mutations occurred elsewhere in the genome.     

Genetically different clonal isolates of Trichomonas gallinae, obtained from the same bird, can vary in their drug susceptibility, an in vitro evidence

Trichomonas gallinae is a flagellated protozoon which parasitizes in the upper digestive tract of different birds, especially columbiformes (doves and pigeons) and falconiformes. The parasite is also a common inhabitant of the crop of psittacine birds and is frequently detected in budgerigars. The lesions associated with T. gallinae infection of the upper digestive tract range from mild inflammation of the mucosa to large caseous lesions that block the lumen of the oesophagus. Nitroimidazoles are considered to be the drugs of choice for the treatment of trichomonosis. However, only a few studies report the existence of resistant strains of T. gallinae to these drugs. Thus, in the present investigation cloned cultures of T. gallinae obtained from budgerigars and pigeons were analysed for the first time for their in vitro susceptibilities against four 5′-nitroimidazole derivates, including metronidazole, dimetridazole, ronidazole and ornidazole. Significantly different minimal lethal concentrations (MLCs) were observed for them against all four drugs. The lowest MLCs revealed the Trichomonas isolates obtained from two budgerigars, ranging from 2.0 ± 0.3 to 3.0 ± 0.7 μg/ml for metronidazole and dimetridazole, and from 2.0 ± 0.6 to 6.7 ± 1.7 μg/ml for ornidazole and ronidazole. Contrary to this, the highest MLCs were recorded for one Trichomonas isolate obtained from a pigeon, ranging from 83.3 ± 6.7 (for dimetridazole and ronidazole) to 103.3 ± 3.3 μg/ml (for metronidazole and ornidazole). The data obtained for the resistance testing were further compared with already available genetic data of the small subunit rRNA gene sequences and ITS-1, 5.8S rRNA and ITS-2 sequences, indicating a certain correlation between in vitro results and strain relationships.     

Biodegradation of 4-nitrotoluene by Pseudomonas sp. strain 4NT.

A strain of Pseudomonas spp. was isolated from nitrobenzene-contaminated soil on 4-nitrotoluene as the sole source of carbon, nitrogen, and energy. The organism also grew on 4-nitrobenzaldehyde, and 4-nitrobenzoate. 4-Nitrobenzoate and ammonia were detected in the culture fluid of glucose-grown cells after induction with 4-nitrotoluene. Washed suspensions of 4-nitrotoluene- or 4-nitrobenzoate-grown cells oxidized 4-nitrotoluene, 4-nitrobenzaldehyde, 4-nitrobenzyl alcohol, and protocatechuate. Extracts from induced cells contained 4-nitrobenzaldehyde dehydrogenase, 4-nitrobenzyl alcohol dehydrogenase, and protocatechuate 4,5-dioxygenase activities. Under anaerobic conditions, cell extracts converted 4-nitrobenzoate or 4-hydroxylaminobenzoate to protocatechuate. Conversion of 4-nitrobenzoate to protocatechuate required NADPH. These results indicate that 4-nitrotoluene was degraded by an initial oxidation of the methyl group to form 4-nitrobenzyl alcohol, which was converted to 4-nitrobenzoate via 4-nitrobenzaldehyde. The 4-nitrobenzoate was reduced to 4-hydroxylaminobenzoate, which was converted to protocatechuate. A protocatechuate 4,5-dioxygenase catalyzed meta-ring fission of the protocatechuate. The detection of 4-nitrobenzaldehyde and 4-nitrobenzyl alcohol dehydrogenase and 4-nitrotoluene oxygenase activities in 4-nitrobenzoate-grown cells suggests that 4-nitrobenzoate is an inducer of the 4-nitrotoluene degradative pathway.