Gianfranco Brambilla

Recent developments in the use and abuse of growth promoters

During the last few years, control within the European Union (EU) for illegal growth promoters in cattle and pigs revealed only a limited number of positives. Analysis of illegal preparations, however, showed that steroids, often (esters of) natural hormones, and -agonists are still used. Corticosteroids, controlled to a much lesser extent, seem to have become the most important group, while even thyreostats remain. Alarming information was obtained from specific investigations in which a large variety of products were found, some of which had never been reported to be misused in the field of growth promotion. For -agonists and quinoxaline compounds, analogues of known compounds are synthesised. Other compounds are readily available as they are registered as growth promoters in some countries outside Europe or are allowed for specific veterinary purposes. Some classes of veterinary drugs are misused for their secondary pharmacological effects, e.g. benzodiazepines as feed intake enhancers and non-steroidal anti-inflammatory drugs (NSAIDs) as pale meat-making agents. Several non-traditional substances are suspected to be used in the field of breeding animals. This is the case for growth hormones (GHs) and all substances acting over this anabolic compound, as for instance, orally GH secretagogue. Moreover, ecdysteroids, which according to old Russian studies, have anabolic activity, are actually very easy to purchase on the Internet. Recent findings in different classes of growth promoters are discussed in detail.

Clinical and pharmacological profile in a clenbuterol epidemic poisoning of contaminated beef meat in Italy

Long-acting beta adrenergic agonists, such as clenbuterol accumulate in the liver, but not meat of treated farm animals, and result in epidemic poisonings in consumers. We describe an outbreak of poisoning in 15 people, following the consumption of meat. Clinical symptoms (distal tremors, palpitations, headache, tachipnoea-dyspnoea, and also moderate hyperglycaemia, hypokalemia and leucocytosis) were seen in nine hospitalised patients, starting about 0.5-3 h after poisoning, and disappearing within 3-5 days later. Clenbuterol was found in the urine of all the symptomatic patients, at higher levels than pharmacokinetic computing (mean level 28 ng/ml, 36 h after ingestion), based on the levels found in the meat (1140-1480 ng/g edible tissue). Thus, epidemic poisoning can be produced following the consumption of contaminated meat. The need for a better definition of pharmaco- and toxico-kinetics, not only for drugs ingested as parent drug, but also when ingested as residues with animal tissues, is recommended.

Correlates of oxidative stress in wild kestrel nestlings (Falco tinnunculus)

The fitness of an organism can be affected by conditions experienced during early development. In light of the impact that oxidative stress can have on the health and ageing of a bird species, this study evaluated factors accounting for the variation in oxidative stress levels in nestlings of the Eurasian kestrel (Falco tinnunculus) by measuring the serum concentration of reactive oxygen metabolites and the serum antioxidant barrier against hypochlorite-induced oxidation. The ratio between these two variables was considered as an index of oxidative stress, with higher values meaning higher oxidative damage. Six-chick broods showed the highest level of oxidative stress, while no effect of sex was found. Age showed an inverse relationship with the oxidants and the levels of oxidative stress, with younger birds having higher levels. Hatching date, body condition, body mass and carotenoid concentration did not show any relationship with oxidants, antioxidants or degree of oxidative stress. These findings suggest that intrabrood sibling competition could play a role in determining oxidative stress, and that in carnivorous birds other antioxidant molecules could be more important than carotenoids to reduce oxidative stress.

Effect on plants of sulphadimethoxine used in intensive farming (Panicum miliaceum, Pisum sativum and Zea mays)

Abstract Animal wastes from intensive farming are generally collected for field fertilisation. They contain drugs that pollute soil. To evaluate the effect of sulphonamide contamination in terrestrial systems, sulphadimethoxine has been subjected to standardisable laboratory tests using seeds of Panicum miliaceum, Pisum sativum, and Zea mays. The drug alters normal post-germinative development and growth of roots, hypocotyls and leaves in the three species; this toxic effect depends on the bioaccumulation rate, and is higher for C4 plants (Panicum and Zea) than for a C3 plant (Pisum). The environmental risk of sludge application on soils and the possible contamination of food chains are discussed.

Use of molecularly imprinted polymers in the solid-phase extraction of clenbuterol from animal feeds and biological matrices

Clenbuterol molecularly imprinted polymers (MIPs) as chromatographic stationary phase for the solid-phase extraction (SPE) of the drug from biological samples have been prepared. Propylene columns filled with 500 mg of clenbuterol MIPs have been tested with respect to their loading capacity, memory effects, selectivity toward related drugs (mabuterol, clenproperol, clenisopenterol, ritodrine) and specificity toward interferences arising from heterogeneous matrices such as animal feeds, bovine urine and liver. Analytes were concentrated on Extrelut 20 columns and the residues resuspended in 70% acetonitrile. Application, washing and elution fractions were collected and analyzed by HPLC-diode array detection. Results indicate this MIP approach in SPE is extremely selective for clenbuterol, mabuterol, clenproperol and clenisopenterol (>95% found in the eluate), with a loading capacity of about 20 microg/100 mg of stationary phase. Ritodrine showed a recovery rate of 51%. The molecular recognition mechanism is so specific to allow clenbuterol detection and identification by conventional detectors at level of interest (ppb) also from complex matrices such as feeds, urine and liver.

Effect of sulphadimethoxine contamination on barley (Hordeum distichum L, Poaceae, Liliopsida)

Animal wastes from intensive farming are generally collected for field fertilisation. They may contain drugs that can become soil pollutants. To evaluate the possible effects of such contamination in terrestrial systems, sulphadimethoxine has been subjected to laboratory tests (in vitro, synthetic medium, and soil) using seeds of barley (Hordeum distichum L.). The drug suppressed normal post-germinative development and growth of roots and leaves in both test conditions; this effect was dependent on the bioaccumulation rate, which was higher on synthetic medium than in soil. Bioaccumulation was higher in roots than foliage and this was markedly evident in soil and, in particular, in soils with a low humus content. The environmental risk of sludge application on soils and the possible contamination of food chains are discussed.

Nitrite in feed: From Animal health to human health

Nitrite is widely consumed from the diet by animals and humans. However the largest contribution to exposure results from the in vivo conversion of exogenously derived nitrate to nitrite. Because of its potential to cause to methaemoglobin (MetHb) formation at excessive levels of intake, nitrite is regulated in feed and water as an undesirable substance. Forages and contaminated water have been shown to contain high levels of nitrate and represent the largest contributor to nitrite exposure for food-producing animals. Interspecies differences in sensitivity to nitrite intoxication principally result from physiological and anatomical differences in nitrite handling. In the case of livestock both pigs and cattle are relatively susceptible. With pigs this is due to a combination of low levels of bacterial nitrite reductase and hence potential to reduce nitrite to ammonia as well as reduced capacity to detoxify MetHb back to haemoglobin (Hb) due to intrinsically low levels of MetHb reductase. In cattle the sensitivity is due to the potential for high dietary intake and high levels of rumen conversion of nitrate to nitrite, and an adaptable gut flora which at normal loadings shunts nitrite to ammonia for biosynthesis. However when this escape mechanism gets overloaded, nitrite builds up and can enter the blood stream resulting in methemoglobinemia. Looking at livestock case histories reported in the literature no-observed-effect levels of 3.3mg/kg body weight (b.w.) per day for nitrite in pigs and cattle were estimated and related to the total daily nitrite intake that would result from complete feed at the EU maximum permissible level. This resulted in margins of safety of 9-fold and 5-fold for pigs and cattle, respectively. Recognising that the bulkiness of animal feed limits their consumption, these margins in conjunction with good agricultural practise were considered satisfactory for the protection of livestock health. A human health risk assessment was also carried out taking into account all direct and indirect sources of nitrite from the human diet, including carry-over of nitrite in animal-based products such as milk, eggs and meat products. Human exposure was then compared with the acceptable daily intake (ADI) for nitrite of 0-0.07 mg/kg b.w. per day. Overall, the low levels of nitrite in fresh animal products represented only 2.9% of the total daily dietary exposure and thus were not considered to raise concerns for human health. It is concluded that the potential health risk to animals from the consumption of feed or to man from eating fresh animal products containing nitrite, is very low.

Identification of main corticosteroids as illegal feed additives in milk replacers by liquid chromatography–atmospheric pressure chemical ionisation mass spectrometry

Corticosteroids were proposed as growth promoting agents to improve commercial quality of meat. We developed a liquid chromatography-atmospheric pressure chemical ionisation mass spectrometry (LC-APCI-MS) method able to identify the presence in milk replacers, when given by mouth, of dexamethasone, betamethasone, flumethasone, triamcinolone, predinisotone, prednisolone, methylprednisolone, fludrocortisone and beclomethasone, at levels in the range of 20-100 ppb. C18 solid-phase extraction, LC-RP C8 column separation, data acquisition (positive ions) in the scan range m/z 200-550 allowed us to differentiate and identify compounds by protonated molecules, their methanolic adducts and fragmentation patterns.

Laboratory models to evaluate phytotoxicity of sulphadimethoxine on terrestrial plants

Abstract Antimicrobial drugs soil contamination is a possible side effect of spreading animal wastes from intensive farming on arable land: waste often contain persistent drugs (like sulphonamides) that pollute soil. In laboratory tests different crop plants and weeds demonstrated, on both synthetic medium and soil, toxicity and sulphadimethoxine bioaccumulation. These data suggest potential adverse implications for organisms higher up the food net, including humans. The increasingly used technique ofsoil top dressing will impose the monitoring of antimicrobial contamination in arable lands and their crops, to protect natural ecosystems and consumer. A possible tool for this monitoring is the use of weeds that can constitute a “mesh” where antimicrobial contamination can be detected.

Hormetic effect(s) of tetracyclines as environmental contaminant on Zea mays.

Animal wastes from intensive pig farming as fertilizers may expose crops to antimicrobials. Zea mays cultivations were carried out on a virgin field, subjected to dressing with pig slurries contaminated at 15 mg L(-1) of Oxy- and 5 mg L(-1) of Chlor-tetracycline, and at 8 mg L(-1) of Oxy and 3 mg L(-1) of Chlor, respectively. Pot cultivation was performed outdoor (Oxy in the range 62.5-1000 ng g(-1) dry soil) and plants harvested after 45 days. Tetracyclines analyses on soils and on field plants (roots, stalks, and leaves) did not determine the appreciable presence of tetracyclines. Residues were found in the 45-day pot corn only, in the range of 1-50 ng g(-1) for Oxy in roots, accounting for a 5% carry-over rate, on average. Although no detectable residues in plants from on land cultivations, both experimental batches showed the same biphasic growth form corresponding to a dose/response hormetic curve.