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.

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.

The effects of a low extracellular concentration of potassium on the activity and numbers of Na+/K+ pumps in an EB-virus transformed human lymphocyte cell line.

The BM1A EB-virus transformed human lymphocyte cell line contains approximately 950,000 Na+/K(+)-ATPase sites per cell. The turnover number of each site is approx. 2240 molecules of rubidium per min. When cells are exposed to a low extracellular concentration of potassium the intracellular concentration of sodium rises, and the cells respond in the short term by increasing the Vmax of 86Rb+ uptake. In the longer term the cells respond by increasing both the Vmax of 86Rb+ uptake and the Bmax of [3H]ouabain binding. The suggestion that increases in the intracellular concentration of sodium is responsible for these changes is supported by the finding that monensin, which increases intracellular sodium without affecting intracellular potassium, is capable of inducing both the short- and long-term changes associated with a low external concentration of potassium.

Confirmation of carbadox and olaquindox metabolites in porcine liver using liquid chromatography-electrospray, tandem mass spectrometry.

A method is described for the quantitative determination of quinoxaline-2-carboxylic acid (QCA) and methyl-3-quinoxaline-2-carboxylic acid (MQCA), the metabolites that have been designated as the marker residues for the veterinary drugs, carbadox and olaquindox, respectively, in swine tissue. The method is suitable for use as a confirmatory method under EU National Surveillance Schemes. Porcine liver samples were subjected to protease digestion followed by liquid-liquid extraction. Further clean-up was performed by automated solid phase extraction (SPE) and was followed by a final liquid-liquid extraction step. Analysis was performed using a narrow bore column HPLC coupled to electrospray MS/MS, operated in positive ion mode. MS/MS product ions were monitored at m/z 102 and 75 amu for QCA, m/z 145 and 102 amu for MQCA and at m/z 106 and 152 amu for the d(4)-QCA and d(7)-MQCA internal standards, respectively. The method has been validated at 3.0, 10, 50 and 150 microg kg(-1) for both metabolites. The method performance characteristics-the decision limit (CCalpha) and the detection capability (CCbeta) have been determined for QCA at 0.4 and 1.2 microg kg(-1), respectively, and for MQCA at 0.7 and 3.6 microg kg(-1), respectively.

The effect of aromatase inhibition on sex steroids, gonadotropins, and markers of bone turnover in older men.

There is evidence that estrogen decreases bone turnover in men as well as women. We therefore hypothesized that older men would show increased bone resorption in response to inhibition of the aromatase enzyme, which converts androgens to estrogen. Fifteen eugonadal men over 65 yr were treated for 9 weeks with 2.0 mg/day of anastrozole, an aromatase inhibitor. After 9 weeks of treatment, there were significant decreases in estradiol, estrone, and sex hormone-binding globulin levels by 29%, 73%, and 16%, respectively, and total testosterone increased significantly by 56%. Despite the limited decrease of estrogen and the increase in testosterone, C-telopeptide of type 1 collagen showed a progressive significant increase of 11%, 24%, and 33% (P for trend = 0.033) above baseline at 3, 6, and 9 weeks, respectively. N-telopeptide of type 1 collagen values were highly correlated with C-telopeptide of type 1 collagen, but the change in N-telopeptide of type 1 collagen was not statistically significant. Bone-specific alkaline phosphatase and N-terminal type I procollagen peptides showed significant decreases of 8% and 11% of baseline at 9 weeks. Osteocalcin decreased significantly by 30% at 18 weeks. We conclude that aromatase inhibition can reduce estrogen levels in older men, but this effect is limited, perhaps because of feedback stimulation of testosterone production, and that endogenous estrogen derived from aromatization of testosterone plays a role in bone metabolism of older men by limiting the rate of bone resorption.

Effects of the disruption of transmethylation in the central nervous system: an animal model.

INTRODUCTION: Central nervous system (CNS) methyltransferases methylate a wide range of substrates including proteins, lipids, nucleic acids and hormones. In every instance the methyl donor is S-adenosylmethionine (SAMe) and the demethylated product is S-adenosylhomocysteine (SAH). Methylation can be disrupted when there is an inadequate supply of methionine synthase (following vitamin B12 deficiency or folate deficiency), SAMe synthetase (due to ethanol), or SAH hydrolase (for unknown reasons). MATERIAL AND METHODS: 5-week-old pigs were maintained in an environment of either air or nitrous oxide, which inhibits methionine synthase, and were fed either a methionine-unsupplemented or methionine-enriched diet. After 3 to 10 weeks, pigs were killed by pentobarbitone injection and the levels of methionine and SAMe in the pigs' brain, spinal cord, plasma, liver, and kidney assessed. RESULTS: Pigs maintained in nitrous oxide displayed a dramatic fall in methionine levels in plasma and brain tissues but maintained relatively normal SAMe levels in these tissues. Brain and spinal cord cystathionine levels were markedly elevated, especially in those animals receiving oral methionine, as in the absence of methionine synthase homocysteine can be metabolized only through the catabolic pathway to cystathionine and cysteine. CONCLUSION: Disorders such as vitamin B12 deficiency or folate deficiency inhibit methylation by limiting the availability of SAMe or by elevating levels of the inhibitor SAH. In either case, the disruption of a wide range of methylation reactions can cause clinical sequelae ranging from structural abnormalities such as myelopathy to functional abnormalities such as depression.

Lipid peroxidation induced in vivo by hyperhomocysteinaemia in pigs.

Much attention has been focused recently on the relationship between homocysteinaemia and the development of premature atherosclerosis. Hyperhomocysteinaemia constitutes as strong a risk factor for the development of the disease as either hypercholesterolaemia or smoking. Although the mechanism involved is unclear homocysteine exhibits prooxidative activity in vitro. This finding suggests that it may be involved in the oxidative modification of low density lipoprotein (LDL). In the current study hyperhomocysteinaemia was induced in eight domestic pigs by intermittent exposure to nitrous oxide for 4 weeks. At necropsy, cardiac tissue was removed and malondialdehyde (MDA) and the unsaturated fatty acid content were measured and compared with values obtained from air-breathing control animals. Nitrous oxide treated animals had significantly higher tissue concentrations of MDA than the controls. There was also a reduction in the contribution of linoleic and linolenic acids to the total fatty acid content of heart. The hyperhomocysteinaemic animals also had a significantly higher iron concentration in the heart than controls. Hyperhomocysteinaemia was associated with elevations in tissue iron stores and increased in vivo lipid peroxidation.

Enhancement of methotrexate cytotoxicity towards the MDA.MB.436 human breast cancer cell line by dipyridamole. The role of methotrexate polyglutamates.

MTX and dipyridamole are synergistic in their toxicity towards the MDA.MB.436 human breast cancer cell line. Dipyridamole increases net MTX uptake into the cells and increases the intracellular levels of MTXG7 to G10, the highest molecular weight polyglutamyl derivatives of MTX detected. During a recovery period, after completion of exposure to MTX with and without dipyridamole, levels of MTXG7 to G10 remained elevated in dipyridamole treated cells by comparison with controls. Dipyridamole, which has no intrinsic effect on cell growth, transforms a cytostatic response of MDA.MB.436 cells towards MTX into a cytotoxic response. The effect of dipyridamole is not mediated through an increase in prostacyclin biosynthesis.

The relationship between the activity of methionine synthase and the ratio of S-adenosylmethionine to S-adenosylhomocysteine in the brain and other tissues of the pig.

Using nitrous oxide to inactivate methionine synthase in vivo, the relationship of the activity of methionine synthase to the S-adenosylmethionine (AdoMet)/S-adenosylhomocysteine (AdoHcy) ratio was examined in neural and other tissues of the pig. Pigs were exposed to 15% nitrous oxide for varying intervals of up to 7 days or studied at varying intervals of recovery in air after 7 days nitrous oxide inhalation, and the rate of inactivation or resynthesis of methionine synthase was related to the corresponding AdoMet/AdoHcy ratios. The rate of inactivation of enzyme during nitrous oxide exposure was considerably faster in the liver and kidney than in the brain and spinal cord with activity levelling off between 10% and 20% of control values. The AdoMet/AdoHcy ratio fell in all tissues during nitrous oxide treatment, the fall being most marked in the brain and spinal cord where a 10-fold change occurred. This change was attributed mainly to a rise in AdoHcy levels. The recovery pattern of methionine synthase was broadly linear but was slower in the spinal cord (0.10 +/- 0.03% per hr; mean +/- SEM) than in any other tissue examined including brain (0.35 +/- 0.04% per hr). Correspondingly, the recovery of the AdoMet/AdoHcy ratio was also significantly slower in the spinal cord. When values for exposure and recovery were combined there was a significant correlation between the activity of methionine synthase and the AdoMet/AdoHcy ratio in both the brain (r = 0.90; P < 0.001) and the spinal cord (r = 0.92; P < 0.001). These results support the concept that the AdoMet/AdoHcy ratio is closely related to the pathogenic process which produces the neurologic lesions associated with a reduction in methionine synthase activity.

The effect of the rate of cell proliferation on the synthesis of methotrexate poly-gamma-glutamates in two human breast cancer cell lines.

The synthesis of methotrexate poly-gamma-glutamates by the MDA-MB-436 and MCF-7 human breast cancer cell lines is highly dependent on the rate of cell growth. Slowly proliferating cells accumulate methotrexate to the same extent as rapidly proliferating cells but convert a lower percentage of the drug to polyglutamate forms. The longest polyglutamate-derivatives of methotrexate are generally only synthesized when the cells are doubling rapidly. The MDA-MB-436 cells exhibit a biphasic response of doubling time and polyglutamation to increasing initial cell number. Extremes of cell density are associated with long doubling times and reduced polyglutamate synthesis. MCF-7 cells show increasing doubling time and decreasing polyglutamate synthesis in response to increasing initial cell number.

The effect of leucovorin on the synthesis of methotrexate poly-gamma-glutamates in the MCF-7 human breast cancer cell line.

The modulating effects of leucovorin on the synthesis of methotrexate (MTX) polyglutamates in the MCF-7 human breast cancer cell line have been investigated using a paired-ion high performance liquid chromatography (HPLC) system. Leucovorin decreased the intracellular level of MTX and profoundly affected polyglutamate synthesis irrespective of whether it was administered with or after MTX. Inhibition of MTX polyglutamate synthesis was also observed when concentrations of leucovorin too low to affect intracellular levels of MTX were employed. Leucovorin did not promote efflux of MTX from the MCF-7 cells and did not affect the distribution of the retained drug amongst the various polyglutamate forms.

Rapid plasma clearance and reduced rate and extent of urinary elimination of parenterally administered methotrexate as a result of severe vomiting and diarrhoea.

A patient received 200 mg methotrexate IM as part of a treatment schedule for malignant melanoma. Severe vomiting and diarrhoea began shortly after treatment and persisted for 4 h. During this period the methotrexate renal clearance rate was 37 ml . min-1, increasing to 97 ml . min-1 when vomiting and diarrhoea ceased. Only 26% of the administered dose was recovered in the urine up to 48 h after treatment, whilst the plasma clearance of methotrexate assessed over the same period was 208 ml . min-1. We conclude that a considerable proportion of the dose was lost from the gastrointestinal tract during the period of vomiting and diarrhoea, and that consequently enterohepatic circulation of methotrexate plays an important role in the pharmacokinetics of the drug.

Regulatory problems caused by contamination, a frequently overlooked cause of veterinary drug residues.

The occurrence of violative residues of veterinary medicines and other, unauthorised, drugs in food of animal origin is an issue of popular concern within the European Union. Violations can occur as a result of improper use of a licensed product or through the illegal use of an unlicensed substance. However, a "violative" analytical result does not necessarily mean that abuse has occurred. Contamination of animal feedingstuffs, environmental contamination and animal-to-animal transfer of drugs can also cause residue violations. This paper reviews these inadvertent causes of residues violations in food, and includes data generated using chromatographic and non-chromatographic methods of analysis.

Use of liquid chromatography-mass spectrometry in the analysis of residues of antibiotics in meat and milk.

The advent of affordable LC-MS systems has led to a massive increase in a number of publications describing quantitative methods for the analysis and confirmation of veterinary drug residues. The lack of volatility and thermal instability of many antibiotics makes LC-MS the method of choice for their analysis. In the review, analytical methods for the determination of residues of each of the major classes of antibiotics are presented.

Iso- and epi-iso-chlortetracycline and the principal metabolites of chlortetracycline in the hen's egg.

Chlortetracycline (CTC) is a broad spectrum antibiotic, licensed for use without any withdrawal period, in chickens laying eggs intended for human consumption. In the European Union, a maximum residue limit (MRL) in eggs of 200 microgram/kg for the sum of the concentrations of CTC and its 4-epimer (4-epi-CTC) has been established. Two major CTC metabolites have been identified in eggs. These compounds, iso-CTC and 4-epi-iso-CTC, have never previously been shown to be significant products of CTC metabolism in poultry or in any other species. The total amount of CTC present in eggs, as all of the chemical forms measured, can exceed the MRL by anything up to a factor of four (170-820 micrograms/kg).

Development and validation of a method for the confirmation of halofuginone in chicken liver and eggs using electrospray tandem mass spectrometry.

A method is described for the quantitative confirmation of halofuginone (HFG) residues in chicken liver and eggs. This method is based on LC coupled to positive ion electrospray MS-MS of the tissue extracts, prepared by trypsin digestion of the tissues followed by liquid-liquid extraction and final clean-up using Solid Phase Extraction (SPE). The [M+H](+) ion at m/z 416 is monitored along with four transitions at m/z 398, 138, 120 and 100. The method has been validated according to the draft EU criteria for the analysis of veterinary drug residues at 15, 30 and 45 microg kg(-1) in liver and 5, 15 and 50 microg kg(-1) in eggs. The new analytical limits, CCalpha and CCbeta were calculated for liver and were 35.4 and 43.6 microg kg(-1), respectively.

Vitamin E and exercise in the Northern Ireland population.

Serum vitamin E, vitamin E/cholesterol and physical activity and fitness were examined in a representative cross section (n = 1600) of the Northern Ireland population as part of the Northern Ireland health and activity survey. Serum vitamin E levels were measured by high-performance liquid chromatography, cholesterol by an enzymatic method, physical activity profile was recorded by computer assisted interview and physical fitness was determined by estimation of VO2 max. The levels of serum vitamin E and vitamin E/cholesterol ratio in the Northern Irish population were similar or higher than in other populations with lower incidences of coronary heart disease. The assessment of activity showed that 75% of the population fell below recommended activity levels likely to confer a cardioprotective effect. A significant relationship (P = 0.01) was found in males between serum vitamin E levels and lifetime participation in physical activity. Otherwise no relationship was found between serum vitamin E or vitamin E/cholesterol ratio and physical activity or fitness in the population.

A thermospray mass spectrometric assay for Fe-induced 4-hydroxynonenal in tissues.

A method is presented for the determination of 4-hydroxynonenal (HNE) in tissue homogenates following in vitro lipid peroxidation induced by iron (Fe++). HNE is measured as the pentafluorobenzyl oxime derivative using liquid chromatography thermospray mass spectrometry. In vitro metabolism of HNE via the glutathione/glutathione-S-transferase pathway was inhibited using iodoacetic and iodobenzoic acids. The assay has been used as an indicator of the peroxidizability of tissue samples from animals both adequate in and depleted of alpha-tocopherol. The concentrations of HNE produced in tissues taken from animals depleted of alpha-tocopherol were found to be up to 8 times higher than those taken from animals supplemented with alpha-tocopherol.

Regional brain monoamine concentrations and their alterations in bovine hypomagnesaemic tetany experimentally induced by a magnesium-deficient diet.

Monoamines are important brain neurotransmitters. An investigation was carried out to determine if hypomagnesaemic tetany was associated with alterations in regional brain monoamine concentrations in bovines. The results, established in cows with normal magnesium status, demonstrated that regional differences existed in the distribution and concentration of brain monoamines in the adult bovine, which were similar to those in other species. In magnesium-deficient cows, severe hypomagnesaemia and lowered cerebrospinal fluid (CSF) magnesium concentrations were associated with significant alterations in monoamine concentrations in some brain regions. Alterations in 3,4-dihydroxyphenylalanine (DOPA) and dihydroxyphenylacetic acid (DOPAC) concentrations in the corpus striatum, and dopamine (DA) in the cerebral cortex and cerebellum were recorded. These regions play an important role in both voluntary and involuntary motor function, and therefore these alterations may play a role in the aetiology of hypomagnesaemic tetany. However, there was no significant change in DA concentrations in the corpus striatum (the main dopaminergic region in the brain) associated with hypomagnesaemia. In addition, a significantly lower norepinephrine (NE) concentration in the corpus striatum of hypomagnesaemic animals was also recorded. Norephinephrine is generally excitatory and therefore lowered NE concentrations would be expected to result in depression rather than stimulation of motor function.

An immunohistochemical study of the distribution of biotin in tissues of pigs and chickens.

An optimised indirect peroxidase-anti-peroxidase immunohistochemical technique was used to detect endogenous biotin in frozen tissue sections from biotin-supplemented and biotin-depleted pigs and chickens. A monoclonal anti-biotin antibody was used as primary antibody in this technique. Immunoreactive biotin was detected in many tissues of both species including liver, kidney, pancreas, adipose tissue, adrenal gland, testis, brain, choroid plexus, cardiac and skeletal muscle, epithelium of the respiratory and digestive systems, skin and lymphoid tissues. The specificity of immunostaining for biotin was confirmed by the finding of reduced staining intensities in tissues of biotin-depleted animals compared to those of biotin-supplemented animals. The results of this study suggest that biotin has metabolic functions in a wider range of tissues than previously known. They also indicate that endogenous tissue biotin should be considered as a source of false-positive staining when immunohistochemical or histochemical techniques which use avidin or streptavidin reagents or anti-biotin antibodies as components of the detection system, are applied to tissue sections.