Transfer of nitroimidazoles from contaminated beeswax to honey.

Nitroimidazoles are not authorised for the treatment of honey bees in the European Union. However, they can be found in honey largely because they are illegally used in apiculture for the treatment of Nosema. The aim of the study was to examine the possible transfer of nitroimidazoles (metronidazole, ronidazole, dimetridazole and ipronidazole) from contaminated beeswax to honey. The wax foundations fortified with a mixture of four nitroimidazoles at three concentration levels (1000, 10,000 and 100,000 µg kg-1) were placed in beehives to let the honeybees (Apis mellifera L.) draw out the contaminated wax foundations to honeycombs. At 1 month from the start, the frames filled with capped honey were removed from the hives for a first sampling of honey. Next, the honeycombs were further incubated for 5 months in the laboratory at 35°C and sampled monthly. In the sampled honey, the concentrations of nitroimidazoles and their main metabolites (hydroxymetronidazole, 2-hydroxymethyl-1-methyl-5-nitroimidazole, hydroxyipronidazole) were determined by LC-MS/MS and compared with those determined in the nitroimidazole-containing wax foundations. Each of the tested nitroimidazoles could migrate from beeswax to honey kept in the contaminated combs at each tested concentration level. Higher maximum concentrations of residues in honey sampled from contaminated combs at 1000, 10,000 and 100,000 µg kg-1 were observed for metronidazole (28.9, 368.5 and 2589.4 µg kg-1 respectively) and ronidazole (27.4, 232.9 and 2351.2 µg kg-1 respectively), while lower maximum concentrations were measured for dimetridazole (0.98, 8.4 and 67.7 µg kg-1) and ipronidazole (0.9, 7.9 and 35.7 µg kg-1 respectively). When we took into account that a frame completely filled with honey on both sides of the comb contained 110 g of beeswax and 2488 g of honey, and that this ratio was constant, then maximum amounts of initial metronidazole, ronidazole, dimetridazole and ipronidazole that migrated from contaminated wax foundations to honey could be calculated: 65-89%, 55-63%, 1.7-2.7% and 1.4-2.3%, respectively.

Metabolism of a 5-nitroimidazole in susceptible and resistant isogenic strains of Bacteroides fragilis.

We investigated the metabolism of dimetridazole (1,2-dimethyl-5-nitroimidazole) (DMZ) by the resting cell method in a susceptible strain of Bacteroides fragilis and in the same strain containing the nimA gene, which conferred resistance to 5-nitroimidazole drugs. In both cases, under strict anaerobic conditions DMZ was metabolized without major ring cleavage or nitrate formation. However, one of two distinct metabolic pathways is involved, depending on the susceptibility of the strain. In the susceptible strain, the classical reduction pathway of nitroaromatic compounds is followed at least as far as the nitroso-radical anion, with further formation of the azo-dimer: 5,5'-azobis-(1,2-dimethylimidazole). In the resistant strain, DMZ is reduced to the amine derivative, namely, 5-amino-1,2-dimethylimidazole, preventing the formation of the toxic form of the drug. The specificity of the six-electron reduction of the nitro group, which is restricted to 4- and 5-nitroimidazole, suggests an enzymatic reaction. We thus conclude that nimA and related genes may encode a 5-nitroimidazole reductase.

Residues of dimetridazole in eggs after treatment of laying hens.

Laying hens were dosed orally with dimetridazole (DMZ) (50 and 250 mg/kg) for 3 days or intramuscularly (50 mg/kg), also for 3 days, and the residues were determined by liquid chromatography in albumen and yolk. The sensitivity of the whole procedure was 2 ng/g. The drug was excreted preferentially into the yolk (about 57% of the total) and the elimination period lasted for 4-6 days after treatment.

The identification and assay of 2-methyl-5-nitroimidazole in pigs treated with dimetridazole.

An unidentified metabolite of dimetridazole (DMZ), found in pig plasma, muscle and kidney, was shown by chromatography and spectroscopy to be 2-methyl-5-nitroimidazole (2-MNI), resulting from N-demethylation of DMZ. This route of degradation competes with the oxidation pathway previously described. The concentration of 2-MNI in the plasma of pig fed medicated diet (DMZ 0.0125%) ranged from 29 to 83 ppb, 2 hours after the morning meal, similar to DMZ, but lower than that of the major metabolite, 2-hydroxymethyl-1-methyl-5-nitroimidazole (HMMNI). Its elimination profile in plasma was biphasic, similar to those of HMMNI and DMZ. Early and terminal half lives were 2.6 and 9.1 h respectively. None of the metabolites could be detected in any of the tissues studied 49 hours after withdrawal.