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Recently, residues of 4-acetaminophen (paracetamol) have been found in urine of slaughter animals. To investigate its possible origin, a 7 week animal experiment was performed with 5 groups of 4 calves fed different amounts of roughage, and treated or not with 4-acetaminophen. Group 1 (control): new-born calves were fed, after the first days of colostrum, solely dairy based calf milk replacer (CMR). The animals from the other groups were 2–3 weeks of age at the start of the experiment and were fed the same CMR. Group 2 was administered oral Pracetam® 30 mg acetaminophen/kg Body Weight (BW) in the CMR, 5 days a week at alternate weeks, with maximum roughage. Group 3 was fed legal minimum roughage. Group 4 received the maximum roughage. Group 5 was administered human paracetamol pills 30 mg/kg BW once a week at weeks 2–6 with maximum roughage. The animals were housed in separate pens on slatted rubber floors without bedding. The controls were housed in a different room than the other animals. Urine was sampled once in the acclimatization period and for 5 days after treatment. At week 2, 4 and 6 at respectively 1, 2 and 3 days after treatment, one animal from groups 2 and 5 was sacrificed. An additional animal from group 4 was sacrificed at week 4, the rest of the animals at day 43. Urine, muscle, liver and kidney were analysed for residues of 4-acetaminophen and its metabolites. It appeared that controls and the low and high roughage animals had almost no 4-acetaminophen (

Livestock is an important and integral component of agriculture production in Yemen and contributes 28% of the total agricultural production income. Research in the field of Yemeni ethnoveterinary medicine is limited to a few studies. Therefore, our work aims to substantiate scientifically the ethnoveterinary use of some documented plant species based on a literature review of their bioactivities and toxicological properties. Searching the scientific literature has revealed various pharmacological activities that may support the claimed healing activities of 11 out of 14 plant species for some of their ethnoveterinary utilization. This comprises the use of Aloe spp. latex for constipation, worms, boils, and wounds; Boswellia sacra underbark for wounds and its oleo-gum resin for mastitis; Soqotraen Boswellia species as an insect repellent; Cissus rotundifolia for stomach pain; Cyphostemma digitatum as an appetite stimulant; Psiadia punctulate for bone fracture; Pulicaria undulata as an insect repellent; combinations of Aristolochia bracteolate with Sorghum bicolor grains for bloating; Rumex nervosus and salt for eye pimples; and Trigonella foenum-graecum seeds with Hordeum vulgare grains for constipation. Some plants were found to demonstrate various toxic effects in in vivo and in vitro experimental studies. The local administration of Calotropis procera latex was also reported to induce an intense inflammatory response. It can be concluded that our work has provided valuable scientific information on the biological and toxic activities of some Yemeni ethnoveterinary remedies that could be utilized for the benefit of farmers to ration the use of these remedies and avoiding their toxicity.

Thiouracil (2-thiouracil) is a thyreostatic compound that can be used as an illegal growth promoter. In bovine, porcine and other farm animals, low concentrations of thiouracil are detected in urine. There is much debate on which concentrations can be considered to originate from feed (���natural���) and which concentrations are caused by the illegal administration of thiouracil for growth-promoting purposes. Currently, a threshold value of 10 ��g/L in urine is applied. The threshold value is based on epidemiological data. Data on thiouracil from animals treated with thiouracil is scarce. We conducted a study whereby animals were fed with rapeseed, rapeseed with thiouracil, or regular feed with thiouracil (low and high concentration). It was determined that administration of thiouracil leads to concentrations higher than the current 10 ��g/L threshold of thiouracil and its metabolites in urine during treatment. Animals fed with rapeseed showed higher thiouracil concentrations than the control group, mostly above 10 ��g/L and in some cases above 30 ��g/L. In the discovery study, several biomarkers for thiouracil treatment were tentatively identified and confirmed with reference standards. One metabolite was identified as indicative for thiouracil abuse, namely 6-methyl-thiouracil. Another metabolite, 4-thiouracil, was indicative for endogenous formation and did not increase during 2-thiouracil treatment. 6-Methyl-thiouracil was not found in urine samples from the Dutch routine control programmes that contained (endogenous) 2-thiouracil above the threshold value. However, 4-thiouracil was found at high concentrations in the same samples when 2-thiouracil was present. This study���s overall conclusion is that the threshold value for thiouracil in bovine urine samples should be set at 10 ��g/L and for porcine urine samples at 30 ��g/L. Also, confirmation of 6-methyl-thiouracil and 4-thiouracil should be used as indicators for exogenous or endogenous origin in routine control monitoring programmes.

Antibiotics are used to control infectious diseases in both animals and humans. They can be life-saving compounds but excessive use in animal husbandry leads to the development of antibiotic resistance which can impact the public health. Since similar antibiotics are used in both animal and human healthcare, it is important to reduce the use of antibiotics in production animals. In the Netherlands policies have been developed aiming for a decrease of antibiotic usage in animals, and alternatives to antibiotics are investigated. Currently, a one-on-one relationship between farmer and veterinarian is successfully implemented and (national) registration of antibiotic usage is mandatory. Unfortunately, after a 70% decrease in antibiotic usage since 2009, this decrease is now stagnating in most sectors. Innovative strategies are required to facilitate a further reduction. One promising option is a focus on farm management and natural alternatives to antibiotics. The Dutch government has invested in the spread of knowledge of natural remedies and good animal management to support animal health via so called Barnbooks for farmers and veterinarians. Another option is the analysis of on-farm antibiotic use to prevent unregistered applications. New (bio)analytical strategies to monitor the correct and complete registration of antibiotic usage have been developed and trial-tested in the Netherlands. Such strategies support a risk-based monitoring and allow effective selection of high-risk (high antibiotic use or illegal antibiotic) users. Both effective monitoring and the availability and knowledge of alternatives is a prerequisite to achieve a further significant decrease in antibiotic veterinary usage.

Enteric methane (CH4) is the main source of greenhouse gas emissions from ruminants. The red seaweeds Asparagopsis taxiformis (AT) and Asparagopsis armata contain halogenated compounds, including bromoform (CHBr3), which may strongly decrease enteric CH4 emissions. Bromoform is known to have several toxicological effects in rats and mice and is quickly excreted by the animals. This study investigated the transfer of CHBr3 present in AT to milk, urine, feces, and animal tissue when incorporated in the diet of dairy cows. Twelve lactating Holstein-Friesian dairy cows were randomly assigned to three treatment groups, representing the target dose (low), 2× target dose (medium), and 5× target dose (high). The adaptation period lasted seven days, and subsequently cows were fed AT for 22 days maximally. The transfer of CHBr3 to the urine at days 1 and 10 (10–148 µg/L) was found with all treatments. On day 1, CHBr3 was detected in the milk of most cows in the low and medium treatment groups (9.1 and 11 µg/L, respectively), and detected in the milk of one cow in the high treatment group on day 9 (35 µg/L). Bromoform was not detected in milk and urine at day 17, nor at concentrations above the detection limit in feces and collected animal tissues. Two animals (low) were sacrificed, and their rumen wall showed abnormalities. Upon histological examination, signs of inflammation became visible. Animals regularly refused the feed or distinctively selected against AT. In conclusion, within the confines of the present experiment, CHBr3 does not accumulate in animal tissue, but can be excreted in urine and milk.

Microplastics (MPs) are considered an emerging issue as environmental pollutants and a potential health threat. This review will focus on recently published data on concentrations in food, possible effects, and monitoring methods. Some data are available on concentrations in seafood (fish, bivalves, and shrimps), water, sugar, salt, and honey, but are lacking for other foods. Bottled water is a considerable source with numbers varying between 2600 and 6300 MPs per liter. Particle size distributions have revealed an abundance of particles smaller than 25 µm, which are considered to have the highest probability to pass the intestinal border and to enter the systemic circulation of mammals. Some studies with mice and zebrafish with short- or medium-term exposure (up to 42 days) have revealed diverse results with respect to both the type and extent of effects. Most notable modifications have been observed in gut microbiota, lipid metabolism, and oxidative stress. The principal elements of MP monitoring in food are sample preparation, detection, and identification. Identified data gaps include a lack of occurrence data in plant- and animal-derived food, a need for more data on possible effects of different types of microplastics, a lack of in silico models, a lack of harmonized monitoring methods, and a further development of quality assurance.

A new method for detecting low levels of growth promoters in bovine meat was developed with the following goal: easy, fast and sensitive analysis of a wide range of compounds, with reduced consumption of chemicals and disposables. Several classes of growth promoters were included, i.e. resorcylic acid lactones (RALs) and steroids, the latter including corticosteroids and gestagens. For sample treatment, 0.5 g of homogenised bovine meat was simultaneously disrupted and extracted in a bead-ruptor machine. The organic extraction solvent was further processed by solid-phase extraction (SPE) clean-up using 96-Well Oasis® HLB Plates. Six SPE washing steps were applied to remove matrix compounds after which the growth promoters were eluted and analysed using UHPLC-MS/MS. To achieve lower detection levels and to reduce LC-solvent consumption, a narrow-bore column with an internal diameter of 1 mm was used, instead of the conventional 2.1 mm. During analysis, the mass spectrometer was operated in negative and positive ionisation mode (ion switching). The newly developed method was validated according to the Commission Decision 2002/657. The results demonstrate that the method meets the criteria as established in this Commission Decision. The precision of the method for exogenous steroids varies between 85 and 115%, the CCα for the compounds ranges from 0.1–0.9 μg kg−1 and the expanded measurement uncertainty was lower than 36%. Compared to our current in-house methods with analysis times of 2 days for a maximum of 24 samples, the new method offers improved sample throughput (96 samples in less than 24 h) and lower detection limits.

To investigate the potential transfer of pyrrolizidine alkaloids (PAs), laying hens were fed for 14 days with diets containing 0.5% of dried common ragwort, common groundsel, narrow-leaved ragwort or viper’s bugloss, or 0.1% of common heliotrope. This resulted in total PA levels in feed of respectively 5.5, 11.1, 53.1, 5.9 and 21.7 mg kg–1, with varying composition. PAs were transferred to eggs, in particular yolk, with steady-state levels of respectively 12, 21, 216, 2 and 36 µg kg–1. Overall transfer rates for the sum of PAs were estimated between 0.02% and 0.23%, depending on the type of PAs in the feed. In animals slaughtered shortly after the last exposure, levels in meat were slightly lower than those in eggs, levels in livers somewhat higher. When switched to clean feed, levels in eggs gradually decreased, but after 14 days were still above detection limits in the hens exposed to higher PA levels. Similar was the case for meat and especially kidneys and livers. It is concluded that the intake of PA containing herbs by laying hens may result in levels in eggs and meat that could be of concern for consumers, and as such should be avoided.

In agriforensics, time of administration is often debated when illegal drug residues, such as clenbuterol, are found in frequently traded cattle. In this proof-of-concept work, the feasibility of obtaining retrospective timeline information from segmented calf tail hair analyses has been studied. First, an ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) hair analysis method was adapted to accommodate smaller sample sizes and in-house validated. Then, longitudinal 1 cm segments of calf tail hair were analyzed to obtain clenbuterol concentration profiles. The profiles found were in good agreement with calculated, theoretical positions of the clenbuterol residues along the hair. Following assessment of the average growth rate of calf tail hair, time of clenbuterol administration could be retrospectively determined from segmented hair analysis data. The data from the initial animal treatment study (n = 2) suggest that time of treatment can be retrospectively estimated with an error of 3-17 days.

The current and expected growth of the world's population warrants an increased production of high-quality animal protein. Dairy farming is regarded as one of the important ways of satisfying this need to meet the growing demand for milk, especially in developing countries. The focus on crossbreeding and increasing the productivity of dairy cattle has, besides enhanced milk production, also resulted in an increased use of agro--chemicals, mainly antibiotics and anti-parasite drugs. The residues of these agro-chemicals, if not managed properly, could leak into the environment, affecting natural processes, biodiversity, and soil life. Public health can also be affected due to residues in milk and meat, especially in countries with insufficient food quality controls. These processes contribute to the growing global threat to human and animal health posed by multi-resistant microbes. This article discusses the differences and similarities of dairy farming, and the effect on public and environmental health, between the Netherlands, India, Ethiopia, and Uganda, emphasizing the strategies that have been developed during the E-Motive exchange project to reduce the use of antibiotics and other chemicals in dairy farming. Proposed solutions include raising consciousness about the risk of antibiotics and their effect on food quality, and implementing the Natural Livestock Farming five-layer approach for reducing the use of antibiotics and other chemicals. This approach is based on improving animal and farm management, revitalizing ethno veterinary knowledge and the use of medicinal plants, genetic improvement through strategic use of local breeds, establishing quality control systems in the dairy chain, and extra payment to farmers for residue-free milk.

The effect of 17ß-19-nortestosterone (17ßNT) treatment of barrows on residue levels and growth was evaluated. Five barrows were treated three times during the fattening period with 17ßNT phenylpropionate (Nandrosol, nandrolone phenylpropionate 50¿mg/ml,1¿mg/kg body weight). Another five barrows were untreated and five boars (untreated) were kept as positive control. Boars and treated barrows showed a 13 and 9% improvement in growth compared to untreated barrows, with mean final body weights of 121.6, 117.8 and 109.0¿kg, respectively. The bulbourethral glands of the treated barrows were three times heavier than untreated barrows. The histology of the prostate and bulbourethral gland of the treated barrows was comparable to the boars, whereas the control barrows showed atrophic glands. Levels of 17ßNT ester in hair from treated barrows were high, whereas boars and untreated barrows did not show levels above LLQ. It is concluded that analysis of hair can detect illegal treatment with 17ßNT ester in barrows. The size of the bulbourethral gland can also be used for screening in the slaughterhouse.

Background: Synthetic Amorphous Silica (SAS) is commonly used in food and drugs. Recently, a consumer intake of silica from food was estimated at 9.4 mg/kg bw/day, of which 1.8 mg/kg bw/day was estimated to be in the nano-size range. Food products containing SAS have been shown to contain silica in the nanometer size range (i.e. 5 - 200 nm) up to 43% of the total silica content. Concerns have been raised about the possible adverse effects of chronic exposure to nanostructured silica.Methods: Rats were orally exposed to 100, 1000 or 2500 mg/kg bw/day of SAS, or to 100, 500 or 1000 mg/kg bw/day of NM-202 (a representative nanostructured silica for OECD testing) for 28 days, or to the highest dose of SAS or NM-202 for 84 days.Results: SAS and NM-202 were extensively characterized as pristine materials, but also in the feed matrix and gut content of the animals, and after in vitro digestion. The latter indicated that the intestinal content of the mid/high-dose groups had stronger gel-like properties than the low-dose groups, implying low gelation and high bioaccessibility of silica in the human intestine at realistic consumer exposure levels. Exposure to SAS or NM-202 did not result in clearly elevated tissue silica levels after 28-days of exposure. However, after 84-days of exposure to SAS, but not to NM-202, silica accumulated in the spleen. Biochemical and immunological markers in blood and isolated cells did not indicate toxicity, but histopathological analysis, showed an increased incidence of liver fibrosis after 84-days of exposure, which only reached significance in the NM-202 treated animals. This observation was accompanied by a moderate, but significant increase in the expression of fibrosis-related genes in liver samples. Conclusions: although only few adverse effects were observed, additional studies are warranted to further evaluate the biological relevance of observed fibrosis in liver and possible accumulation of silica in the spleen in the NM-202 and SAS exposed animals respectively. In these studies, dose-effect relations should be studied at lower dosages, more representative of the current exposure of consumers, since only the highest dosages were used for the present 84-day exposure study. © 2014 van der Zande et al.; licensee BioMed Central Ltd.

To investigate the possibilities for screening and confirmation methods when the 'pour on' method of application is used for administration of growth promoters, an animal experiment was performed using a cocktail of a combination of growth promoters derived from (illegal) practice. Two cocktails were used, cocktail A consisting of stanozolol and estradiol benzoate and cocktail B consisting of stanozolol, estradiol benzoate and beclomethasone dipropionate. The intended dose per animal was 110 mg stanozolol, 25 mg estradiol and 10 mg beclomethasone. The experiment was performed on 20 male veal calves, 16 treated and 4 vehicle treated controls and 3 female veal calves, 2 treated and 1 vehicle treated control. Half of the animals were shaven prior to the application of the drugs. The cocktails were administered using two types of vehicles: vehicle A; Miglyol 840 with butylated hydroxytoluene and vehicle B; di(ethyleneglycol) monobutylether. During a 28 day treatment period, one group of animals was treated once a week, another group of animals was treated once every two weeks and slaughtered. Preliminary results showed that pour on application of anabolic steroids markedly increased growth performance of veal calves, the animals treated with cocktail A performed better than the animals treated with cocktail B. Macroscopically, the thymus was reduced in weight and size in the B animals. The bulbo-urethral glands were enlarged in all treated animals. Histologically all treated animals showed squamous metaplasia in the prostate, bulbo-urethral gland and Bartholins glands. Moreover, a changed secretion pattern was observed in both the prostate and the bulbo-urethral gland. Severe cortical atrophy was observed in the thymus and to a lesser extent the adrenals of the beclomethasone treated animals. The recently discovered 16 beta-hydroxy-metabolite of stanozolol was detected in urine, in relatively high concentrations. This is the first report of the excretion of this metabolite in urine after pour on administration showing the prospect for detection of dermal treatment. Estradiol levels were remarkably elevated (up to 200 micrograms l-1) exceeding the endogenous levels (< 1 microgram l-1).

To investigate whether clenbuterol-treated calves could contaminate untreated pen mates, three animal experiments were performed. (1) One calf of a pen of five was treated with clenbuterol by injection (Ventipulmin injection, REG NL 2532, 2.5 mL/100 kg) twice a day for 10 days. (2) In two pens, one animal was treated with clenbuterol via oral administration (Ventipulmin syrup, REG NL 2532, 4 mL/125 kg) for 4 weeks. (3) In two pens, one animal was treated with clenbuterol via the milk (Ventipulmin, REG NL 2532, 2.5 mL/100 kg body weight) twice a day for 10 days. Here, the animal was set apart during treatment, cleaned and put back into the group. Levels of clenbuterol were analysed in hair and urine with LC-MS/MS. Clenbuterol administered by injection could not be transferred from treated to untreated calves. In the second experiment, all pen mates were found positive for clenbuterol in the hair. This contamination was probably due to licking the mouth of the treated animal or saliva from the treated animal spoiling the floor. In the third experiment, no pen mates were found positive for clenbuterol in the hair. Clenbuterol was found in the urine and hair of only treated animals.

Precision cut liver slices (PCLSs) are widely used as a model to study hepatotoxicity. For culturing of PCLS diverse protocols are used which could affect slices viability and results. We aimed to identify the most optimal culture protocol for mouse PCLS. Slices were cultured for 24 h under different concentrations of serum, glucose, insulin, and oxygen. Thereafter, slices viability was assessed by biochemical methods. Transcriptome analysis was performed to identify changes introduced by culture at different oxygen concentrations (20%, 40%, 60%, and 80% of oxygen). Medium composition did not affect the slices viability. Although metabolic competence was unaffected by oxygen concentrations, culturing at 80% of oxygen yielded slices with the best biochemical characteristics. The comparison of uncultured vs. cultured slices revealed 2524 genes to be differentially expressed. Genes involved in drug metabolism, peroxisomal and mitochondrial functions were down-regulated while several adaptive/stress response processes were up-regulated. Moreover, 80% of oxygen was the most favorable condition with respect to maintenance of expression of genes involved in drug and energy metabolism. The outcome of this study indicates that mouse PCLS are a valuable tool in research on hepatic functions and toxicity, particularly if they are cultured under a controlled oxygen concentration of 80%.

Innovative techniques were developed in the Isafruit project in order to create a more ecological sustainable way of fruit growing. Before fruit growers will consider implementation of these innovations they need information concerning their economic sustainability. The economic model ProfitFruit is made to evaluate the profitability of innovative techniques. ProfitFruit includes a database with quantitative data concerning apple growing in three European fruit growing regions: North Germany (Jork), Switzerland and the Netherlands. Data include amounts of inputs and its costs, yield and prices, labour demand and costs for fixed assets. ProfitFruit calculates returns, gross margin, marginal gross margin, fixed costs as well as labour income of the entrepreneur. ProfitFruit is used for the economic evaluation of the Isafruit Casa sprayer, of mechanical thinning, and of hot water treatment to prevent storage rot. In general, the Isafruit Casa sprayer is the most profitable of these techniques. If the sprayer functions well, it will decrease costs and therefore increase returns when compared to a standard spraying machine. Therefore, fruit growers who can afford the investment might invest in this technique. Also mechanical thinning instead of chemical thinning may be profitable, especially if it results in better product quality and higher prices. Hot water treatment increases total costs, compared with the chemical way of protection. Therefore not many integrated fruit growers will decide to apply this technique, not even when it becomes more effective or if additional labour demand decreases. However, due to problematic storage rot and high prices for organic apples (no chemicals allowed), hot water treatment seems profitable in organic production. Also a desired reduction of the residues on apples might stimulate hot water treatment. In general, costs and labour demand will be critical success factors for implementation of innovative techniques which are meant to spare the environment.

Expert systems, or more precise decision support systems, are valuable tools for structuring the results of scientific research and to translate this to knowledge. The decision support sys‐ tem Determinator is now used for several years as a platform for models to identify subjects [1, 2]. The system is based on the two main different procedures for identification [3, 4]; a sin‐ gle access key (tree) and a free access key (matrix). The latter option provides the possibility to calculate the match between the subject as chosen by the user and the objects as included in the data model, based on a range of characteristics. In addition, a matrix allows to make selections, to filter the set of available objects and to compare two objects for their variability.

Isoxsuprine is a beta-agonist that can be used for growth promotion in cattle, but it is also used as registered veterinary medicine. To investigate if veterinary treatment of cows could lead to residues of isoxsuprine in the hair of their newborn calves, an animal experiment was performed. Four cows, treated on veterinary indication with isoxsuprine lactate (Duphaspasmin) before a caesarian section, were included in the experiment. Hair samples from cows and from their calves were analyzed. The animals were shaved every week for 16 weeks and levels of isoxsuprine were measured in hair. In the cows, the levels of isoxsuprine were highest (>15 µg/kg) just after administration of the isoxsuprine lactate. After two weeks in two cows, a sort of plateau was reached and then the levels decreased. After approximately 10-15 weeks the levels were around the CCa level of the method used (0.5 µg/kg). In calves, for the first two weeks after birth, no isoxsuprine was found above CCa level in three of the four animals. At about 20-30 days old, a maximum concentration of 4 µg/kg was found. Then the levels dropped again under the CCa level, after 60 days no levels above CCa level were found. In one animal, the levels never reached CCa level. We conclude that veterinary treatment of cows with isoxsuprine may temporarily lead to low levels of isoxsuprine in the hair of their newborn calves which can be measured for a maximum of 60 days after birth.

Traceability of food implies the ability to trace and follow a food, feed, or a food-producing animal or substance intended to be, or expected to be, incorporated into a food or feed, through all stages of production, processing, and distribution. The importance of traceability has grown due to the consumers' increasing attention to food safety and food quality, and due to the increasing complexity of food supply chains. There are several types of food traceability depending on how traceability is obtained and on what information it concerns, i.e., conventional traceability, genetic traceability, and geographic traceability. Optical, electronic, and biological identification methods are used to identify and transfer the information. The aim of this chapter is to explain the basic characteristics of traceability systems in the supply chain and to list the developments in this area. An extensive overview of analytical systems is described that may verify documentary information on the basis of analyses, i.e., DNA-based methods, chemical verification methods, visual markers for the determination of food and feed, and sensory analysis. It is concluded that in future new traceability systems will be developed that combine logistical data with analytical data that are derived from more informative multiplex approaches, increasingly comprising also data from different types of analytical approaches. In this way, the consumer may be even better informed.

Biotransformation of inactive prohormones like dehydroepiandrosterone (DHEA) can lead to the formation of potent androgens and subsequent androgenic responses in target tissues. In the present study, precision-cut bovine liver slices were used to study the effects of DHEA on the metabolite, transcript and androgenic activity level. Bovine liver slices were exposed for 6 h to various concentrations of DHEA. Changes in androgenic activity of the DHEA containing cell culture media were measured using a yeast androgen bioassay and metabolites were identified using ultra performance liquid chromatography time-of-flight mass spectrometry (UPLC–TOFMS), while gene expression in the DHEA-treated liver slices was examined using bovine microarrays and compared with the profile as obtained with 17s-testosterone (17s-T). An increase in androgenic activity was observed in the bioassay upon testing of samples from incubations of DHEA with liver slices and the formation of 4-androstenedione (4-AD), 5-androstene-3s,17s-diol, 17s-T, 7α-hydroxy-DHEA, 7-keto-DHEA and 17α-T could be confirmed by UPLC–TOFMS analysis. Exposure of liver slices to DHEA and the strong androgen 17s-T resulted in the identification of significantly up- and down-regulated genes and revealed similar gene expression profiles for both compounds. The results indicate that DHEA itself is biologically not very active, but is rapidly converted by the liver slices into the more androgen active compounds 4-AD and 17s-T. Moreover, the present data highlight the multi-functionality of bovine liver slices as an in vitro bioactivation model, allowing the assessment of androgen activity or gene expression as effect-based endpoints for prohormone exposure.

The aim of the present study was to demonstrate the applicability of a yeast androgen and estrogen bioassay in the detection of steroid esters in hair samples of animals treated with a hormone ester cocktail. The outcome of the activity screenings was critically compared with the results previously obtained with LC-MS/MS analysis. Hair samples of one pour-on treated animal, 10 ml DMSO containing 25 mg estradiol benzoate (EB), 60 mg testosterone decanoate (TD) and 60 mg testosterone cypionate (TC), were selected and analyzed with the androgen and estrogen yeast bioassay. Results showed that by the introduction of a hydrolysis step, bioassays can be used to screen for the presence of hormone esters in hair samples. Based on the difference in fluorescence responses between the nonhydrolyzed and the hydrolyzed hair samples, it was possible to detect the presence of EB up to at least 56 days after a single pour-on treatment and to detect the presence of TC and TD up to at least 14 days after the treatment. Although the LC-MS/MS analysis could detect TC and TD up to 49 days after treatment, bioassays have the advantage that they can also detect any (un)known steroid ester. Keywords Testosterone ester . Estradiol benzoate . Yeast bioassay . Untargeted analysis . Hair

Summary In this study the possibilities of combined indirect screening methods for the growth promotants clenbuterol and estradiol-17s benzoate and the combination of both these agents were investigated. Three groups of 5 male goat kids (two months of age) were treated for 4 weeks; clenbuterol per os, 10 μg/day/kg body weight via milkreplacer twice a day; 2 mg estradiol-17s benzoate weekly per i. m. injection; combined clenbuterol per os + estradiol-17s benzoate per injection. Five animals were not treated and served as controls. Nuclear Magnetic Resonance (NMR) was used as an initial tool to monitor the urine composition. The analysis of NMR spectra showed, that most treated animals had increased L-lactate/creatine ratios (L/C-ratio) in their urine, compared to control animals. For screening, L-lactate/creatine ratios could be determined directly in urine - in a simple, quick and inexpensive manner - by using enzymatic methods and an autoanalyzer. Histologically clenbuterol induced degenerative changes in the urethral and glandular epithelium of the prostate. Changes consisted of vacuolization of the epithelium and necrosis with nuclear pyknosis and fragmentation. Estradiol-17β benzoate induced metaplastic changes in the glandular tissue of the goat prostate. In combination with estradiol-17β benzoate, characteristics of both agents could be observed. The histological methods and observed effects can be used directly for screening. The combination of independent histological and L/C-ratio methods looks very promising with respect to screening of growth promotants. Since in both cases effects of growth promotants are detected, it may be expected that this approach is not limited to goats or clenbuterol and estradiol alone.

The application of anabolic steroids in food producing animals is forbidden in the EU since 1988, but the abuse of such drugs is a potential problem. The existing test systems are based on known compounds and can be eluded by newly emerging substances. The examination of physiological effects of anabolic hormones on different tissues to indirectly detect misuse might overcome this problem. Two studies were conducted with post-pubertal 24-months old Nguni heifers and pre-pubertal female 2–4 weeks old Holstein Friesian calves, respectively. The animals of the accordant treatment groups were administered combinations of estrogenic and androgenic compounds. The measurement of the gene expression pattern was undertaken with RT-qPCR. Target genes of different functional groups (receptors, angiogenesis, steroid synthesis, proliferation, apoptosis, nutrient metabolism and others) have been quantified. Several biochemical pathways were shown to be influenced by anabolic treatment. Both studies identified significant regulations in steroid and growth factor receptors (AR, ERβ, LHR, FSHR, Flt-1, PR, IGF-1R, Alk-6), angiogenic and tissue remodeling factors (VEGFs, FGFs, BMPs, ANGPT-2, MMPs, TIMP-2, CTSB), steroid synthesis (S5A1, HSD17, CYP19A1), proliferation (TNFα, IGF-1, IGFBPs, p53, c-fos; CEBPD, c-kit), apoptosis (CASP3, FasL, p53) and others (C7, INHA, STAR). Several genes were regulated to opposite directions in post-pubertal compared to pre-pubertal animals. PCA for Nguni heifers demonstrated a distinct separation between the control and the treatment group. In conclusion, anabolics modify hormone sensitivity and steroid synthesis, and they induce proliferative effects in the whole reproductive tract (uterus and ovary) as well as anti-angiogenic effects in the ovary. However, the extent will depend on the developmental stage of the animals.

Receptor binding transcription activation bioassays are valuable tools for the screening of steroid hormones in animal feed and supplements. However, steroid derivatives often lack affinity for their cognate receptor and do not show any direct hormonal activity by themselves. These compounds are thus not detected by these kinds of bioassays and need a bioactivation step in order to become active, both in vivo and in vitro. In this study a comparison was made between different in vitro activation methods for hormone esters and hormone glycosides. Testosterone acetate and testosterone decanoate were chosen as model compounds for the hormone esters, representing the broad range of steroid esters of varying polarities, while genistin was used as a substitute model for the steroid-glycosides. Concerning bioactivation of the steroids esters, the efficiency for alkaline hydrolysis was 90–100% and much better as compared to enzymatic deconjugation by esterase. As a result 1 µg testosterone ester per gram of animal feed could easily be detected by a yeast androgen bioassay. When comparing different enzyme fractions for deglycosilation, genistin was shown to be deconjugated most efficiently by ß-glucuronidase/aryl sulfatase from Helix pomatia, resulting in a significant increase of estrogenic activity as determined by a yeast estrogen bioassay. In conclusion, chemical and enzymatic deconjugation procedures for ester and glycoside conjugates respectively, resulted in a significant increase in hormonal activity as shown by the bioassay readouts and allowed effective screening of these derivatives in animal feed and feed supplements.

Background Within the European Union the use of growth promoting agents in animal production is prohibited. Illegal use of natural prohormones like dehydroepiandrosterone (DHEA) is hard to prove since prohormones are strongly metabolized in vivo. In the present study, we investigated the feasibility of a novel effect-based approach for monitoring abuse of DHEA. Changes in gene expression profiles were studied in livers of bull calves treated orally (PO) or intramuscularly (IM) with 1000 mg DHEA versus two control groups, using bovine 44K DNA microarrays. In contrast to controlled genomics studies, this work involved bovines purchased at the local market on three different occasions with ages ranging from 6 to 14 months, thereby reflecting the real life inter-animal variability due to differences in age, individual physiology, season and diet. Results As determined by principal component analysis (PCA), large differences in liver gene expression profiles were observed between treated and control animals as well as between the two control groups. When comparing the gene expression profiles of PO and IM treated animals to that of all control animals, the number of significantly regulated genes (p-value 1.5) was 23 and 37 respectively. For IM and PO treated calves, gene sets were generated of genes that were significantly regulated compared to one control group and validated versus the other control group using Gene Set Enrichment Analysis (GSEA). This cross validation, showed that 6 out of the 8 gene sets were significantly enriched in DHEA treated animals when compared to an 'independent' control group. Conclusions This study showed that identification and application of genomic biomarkers for screening of (pro)hormone abuse in livestock production is substantially hampered by biological variation. On the other hand, it is demonstrated that comparison of pre-defined gene sets versus the whole genome expression profile of an animal allows to distinguish DHEA treatment effects from variations in gene expression due to inherent biological variation. Therefore, DNA-microarray expression profiling together with statistical tools like GSEA represent a promising approach to screen for (pro)hormone abuse in livestock production. However, a better insight in the genomic variability of the control population is a prerequisite in order to define growth promoter specific gene sets that can be used as robust biomarkers in daily practice.

The abuse of synthetic esters of natural steroids such as testosterone and estradiol in cattle fattening and sports is hard to detect via routine urine testing. The esters are rapidly hydrolysed in vivo into substances which are also endogenously present in urine. An interesting alternative can be provided by the analysis of the administered synthetic steroids themselves, i.e., the analysis of intact steroid esters in hair by liquid chromatography tandem mass spectrometry (LC/MS/MS). However, retrospective estimation of the application date following a non-compliant finding is hindered by the complexity of the kinetics of the incorporation of steroid esters in hair. In this study, the incorporation of intact steroid esters in hair following pour-on treatment has been studied and critically compared with results from intramuscular treatment. To this end animals were pour-on treated with a hormone cocktail containing testosterone cypionate, testosterone decanoate and estradiol benzoate in different carriers. The animals were either treated using injection and pour-on application once or three times having 1 week between treatments using injection and pour-on application. Animals were slaughtered from 10-12 weeks after the last treatment. Both hair and blood plasma samples were collected and analysed by LC/MS/MS. From the results, it is concluded that after single treatment the levels of steroid esters in hair drop to CCbeta levels (5-20 microg/kg) after 5-7 weeks. When treatment is repeated two times, the CCbeta levels are reached after 9-11 weeks. Furthermore, in plasma, no steroid esters were detected; not even at the low microgramme per litre level but--in contrast with the pour-on application--after i.m. injection, significant increase of 17beta-testosterone and 17beta-estradiol were observed. These observations suggest that transport of steroid esters after pour-on application is not only performed by blood but also by alternative fluids in the animal so probably the steroid esters are already hydrolysed and epimerized before entering the blood.

A number of recent food safety incidents have involved chemical substances, while various activities aim at the early identification of emerging chemical risks. This review considers recent cases of chemical and biochemical risks, as a basis for recommendations for awareness and prevention of similar risks at an early stage. These cases include examples of unapproved genetically modified food crops, intoxications with botanical products containing unintentionally admixed toxic herbs, residues of unapproved antibiotics and contaminants in farmed aquaculture species such as shrimp and salmon; and adverse effects of chemical and biological pesticides of natural origin. Besides case-specific recommendations for mitigation of future incidents of the same nature, general inferences and recommendations are made. It is recommended, for example, to establish databases for contaminants potentially present within products. Pro-active reconnaissance can facilitate the identification of products potentially contaminated with hazardous substances. In international trade, prevention and early identification of hazards are aided by management systems for product quality and safety, rigorous legislation, and inspections of consignments destined for export. Cooperation with the private sector and foreign authorities may be required to achieve these goals. While food and feed safety are viewed from the European perspective, the outcomes also apply to other regions.

In livestock production, illegal use of natural steroids is hard to prove because metabolites are either unknown or not significantly above highly fluctuating endogenous levels. In this work we outlined for the first time a metabolomics based strategy for anabolic steroid urine profiling. Urine profiles of controls and bovines treated with the prohormones dehydroepiandrosterone (DHEA) and pregnenolone were analyzed with ultraperformance liquid chromatography in combination with time-of-flight accurate mass spectrometry (UPLC-TOFMS). The obtained full scan urinary profiles were compared using sophisticated preprocessing and alignment software (MetAlign) and multivariate statistics, revealing hundreds of mass signals which were differential between untreated control and prohormone-treated animals. Moreover, statistical testing of the individual accurate mass signals showed that several mass peak loadings could be used as biomarkers for DHEA and pregnenolone abuse. In addition, accurate mass derived elemental composition analysis and verification by standards or Orbitrap mass spectrometry demonstrated that the observed differential masses are most likely steroid phase I and glucuronide metabolites excreted as a direct result from the DHEA and pregnenolone administration, thus underlining the relevance of the findings from this untargeted metabolomics approach. It is envisaged that this approach can be used as a holistic screening tool for anabolic steroid abuse in bovines and possibly in sports doping as well.

Residues of illegally used hormones are regularly detected in animal products, feed, or cocktails recovered at farms. In order to better understand the effects of dietary exposure to ethinyl estradiol (EE2, 0.03-1 microg/kg body weight [bw]) and zearalenone (ZEA, 0.03-1 mg/kg bw), an immature rat uterotrophic assay was performed and effects were studied at morphological, histological, and gene expression levels. Ligand-mediated coregulator recruitment by estrogen receptor alpha (ERalpha) was studied in vitro. Uterine weight and epithelial cell height were increased dose dependently after a 3-day oral exposure of rats to the highest tested doses of EE2 or ZEA, respectively. At low doses 0.03 microg/kg EE2 and 0.1 mg/kg ZEA, edema, and vacuolization could already be observed in some animals. Exposure to 1 mg/kg ZEA resulted in severe damage of the uterine epithelial layer. Our study suggests similar coregulator recruitment and gene expression patterns for the two estrogenic compounds. Main regulated pathways were remodeling of extracellular matrix, alternative complement activation, cell proliferation, and estrogen-mediated calcium signaling. The level of regulation differed between EE2 and ZEA, attributing a much lower estrogenic potency to ZEA than to EE2. A major difference was their ability to recruit coregulator inhibitor of kappa B beta and induce expression of the matrix metalloproteinase 7 gene (381.4- and 6.9-fold upregulation by EE2 and ZEA, respectively), which plays an important role in the maintenance of the integrity of the epithelial layer of the uterus during proliferation and growth. This observation may explain the observed differences at the histological level.

Summary An animal experiment has been performed with 42 veal calves, 21 males and 21 females, which were fed and housed according to European regulations for veal calves. The animals were kept in six groups of seven animals and fed milk replacer supplemented with three different levels of lactulose (0%, 1% and 3%) and some roughage. At the start of the experiment the animals were 1–3 weeks of age and they were slaughtered at 26 weeks. From male animals prostate, bulbo-urethal gland and testes were sampled, from female animals Bartholin’s gland, uterus, cervix and ovaries were sampled. From all animals thyroid, thymus, adrenals, liver and kidneys were sampled. Histological investigation of the prostates and bulbourethral glands showed normal histology. This means that dilated tubules, strong secretion, increased mucinous glandular tissue and severe hyperplasia and squamous metaplasia, as is regularly observed in practice in the Netherlands, were not present in these animals. None of these prostates would be judged as positive in the screening for hormones as is performed by the Dutch Food and Consumer Product Safety Authority (VWA). The female calves also showed normal histology of Bartholin’s gland except for three animals that appeared to be in oestrus and showed some metaplasie of the ducts but with a normal gland to duct ratio. These animals would be judged as suspect. The liver and kidney showed minor alterations due to slight infections during the experimental period. This experiment showed that it is possible to raise veal calves according to the practice without getting positive histology in the prostate or Bartholin’s gland.

Within the European Union, the control for residues of illegal hormones in food-producing animals is based on urine analysis for a few target analytes using gas chromatography/mass spectrometry and/or liquid chromatography-tandem mass spectrometry. Recently, we developed a robust yeast bioassay screening tool for estrogens, which was validated as a qualitative screening method in accordance with EC decision 2002/657/EC. In this study, we present long-term performance data and a comparison of urine data obtained with this bioassay, and data from an established gas chromatography-tandem mass spectrometry (GC/MS/MS) confirmatory analysis method. More than 120 calf urine samples from a controlled reference experiment were analysed using both protocols. According to the GC/MS/MS method, only the natural estrogens 17alpha-estradiol and estrone were present in the non-compliant samples. The bioassay was less sensitive than GC/MS/MS for the relatively weak estrogenic compound 17alpha-estradiol, in accordance with expectations. Assuming that application of the mass spectrometric method is considered beyond reasonable doubt, the bioassay performed very well: only 5.6% of the calf urine samples found compliant in GC/MS/MS were screened false suspect in the bioassay screening method. The bioassay results of non-compliant urine samples under routine conditions were as predicted, taking into account the relative estrogenicity of the natural estrogens 17alpha-estradiol and estrone vs. 17beta-estradiol. Only one sample was screened false negative for 17alpha-estradiol and estrone. Application of this fast and simple estrogen bioassay in routine surveillance and control can significantly reduce GC/MS/MS sample workload and allow higher percentages of animals to be screened for potential hormone abuse.

A lifetime controlled reference experiment has been performed using 42 veal calves, 21 males and 21 females which were fed and housed according to European regulations and common veterinary practice. During the experiment feed, water, urine and hair were sampled and feed intake and growth were monitored. Thus for the first time residue analysis data were obtained from guaranteed lifetime-untreated animals. The analysis was focused on the natural hormones estradiol and testosterone and their metabolites, on 17 beta- and 17 alpha-nortestosterone, on 17 beta- and 17 alpha-boldenone and androsta-1,4-diene-3,17-dione (ADD), and carried out by gas chromatography tandem mass spectrometry (GC/MS/MS), an estrogen bioassay and liquid chromatography (LC) MS/MS. Feed, water and hair samples were negative for the residues tested. Female calf urines showed occasionally low levels of 17 alpha-estradiol and 17 alpha-testosterone. On one particular sampling day male veal calf urines showed very high levels of 17 alpha-testosterone (up to 1000 ng mL(-1)), accompanied by lower levels of estrone and 17 beta-testosterone. Despite these extreme levels of natural testosterone, 17 beta-boldenone was never detected in the same urine samples; even 17 alpha-boldenone and ADD were only occasionally beyond CC alpha (maximum levels 2.7 ng ng mL(-1)). The data from this unique experiment provide a set of reference values for steroid hormones in calf urine and demonstrate that 17 beta-boldenone is not a naturally occurring compound in urine samples. (c) 2006 Elsevier B.V. All rights reserved.

The abuse of esters of natural androgenic steroids in cattle fattening and sports is hard to control via routine urine testing. The esters are rapidly hydrolysed in vivo into substances which are also endogenously present in urine. In veterinary control strange findings of 17beta-testosterone and 17alpha-testosterone in urine are often ignored because of the lack of statistically sound reference data of naturally occurring levels. An interesting alternative for inconclusive urine analyses in veterinary control can be provided by the analysis of the administered steroids themselves, i.e. the analysis of intact steroid esters in hair. Unfortunately, the analysis of intact steroid esters is complicated not only by the vulnerability of the esters which precludes alkaline hydrolysis of the hair, but also by the wide polarity range of short and long-chain esters yielding very poor recoveries for either the one or the other. In this study, a multi-steroid esters LC/MS/MS screening method is presented for trace analysis of the synthetic intact esters of 17beta-testosterone and the undecylenate ester of 17beta-boldenone in bovine hair. The method, requiring only 200 mg of pulverised hair, features a mild digestion procedure using tris(2-carboxyethyl)phosphine hydrochloride (TCEP) and the use of four deuterium-labelled steroid esters as internal standards covering the wide polarity range of the analytes. In spiked hair samples for most of the analytes the limit of detection and the accuracy using isotope dilution were 2-5 ng/g and 97-105%, respectively. The applicability was demonstrated using hair samples from a controlled experiment in which six bovines were injected intramuscularly with two different doses of two commercial mixtures of testosterone esters, and with two different doses of boldenone undecylenate. Depending on the dose all administered testosterone- and boldenone esters were found to be incorporated in bovine hair following a single intramuscular injection, except testosterone propionate which dose might have been too low.

Due to the poor financial results of the fruit industry in the last decade, the changing trade structures and more consumer-driven fruit chains, Dutch fruit growers change their market behaviour. In these circumstances, the fruit industry itself and the applied fruit research are also changing. Applied Plant Research (PPO-Fruit) is involved in different projects related to consumer-driven fruit chain development and their side effects on fruit growers and firms. The purpose of this paper is to describe two projects about new Dutch fruit chains in which PPO-Fruit is involved and their impact on fruit growers and research. The projects are: a special variety chain, based on the Dutch apple variety Santana, which is scab resistant (Vf) and a regional crop chain ¿Betuwe framboos¿, (= Betuwe raspberry). It is clear that being part of a fruit chain means another way of working for the fruit growers and the other partners as well. They have to work according growing recipes and also recipes/procedures for other activities, like storage, packaging. This means a decrease of independence for the growers. PPO-Fruit works as a facilitator, a knowledge centre and executes experiments to solve new problems

Histopathology of male cattle previously found positive for beta-boldenone in urine in the Netherlands and in Italy was studied. The animals were derived from practice and several weeks had passed after the finding of beta-boldenone before the animals were examined. The animals consisted of 34 male veal calves and one finishing bull. In the prostate gland hypersecretion, cyst formation (45%) and hyperplasia of the urethral epithelium was observed, in the bulbo-urethral gland similar alterations were present. The testis showed reduced development and degeneration of the germinal epithelium (70%), leading to debris and syncytial cell formation in the lumina. Stromal proliferation was evident. In some animals the liver was sampled and showed periportal fibrosis, bile duct proliferation and sometimes necrosis. The bull also showed degeneration of the germinal epithelium of the testis and absence of sperm production, the prostate gland showed some secretion and had an atrophic appearance. It is concluded that beta-boldenone may lead to degeneration of the germinal epithelium of the testis and hypersecretion and cyst formation in the prostate and bulbo-urethral gland, which alterations may heal in time.

This study investigates the influence of low dosages of beta-agonists combined with other growth promoters on screening and confirmation methods in male veal calves. Five groups of four calves were treated for 6 weeks with combinations of low dosages of beta-agonists (beta AG, clenbuterol, mabuterol and mapenterol, 0.4 microgram/kg each twice daily) in combination with 17 beta-estradiol (E2, 5 mg per animal per 14 days), methylthiouracil (MTU, 2.857 mg/kg bw twice daily) and dexamethasone (DEX, 4 mg per animal per 10 days during the last 20 days of treatment). Another group of four untreated animals served as controls. The weight and size of the thymus was reduced in the DEX group, the MTU group showed enlarged thyroids. Histologically the prostates showed vacuolar degeneration in the beta AG animals and metaplasia in the E2 group. Some vacuolization was also observed in the controls. The testis showed impaired development in all treatment groups, E2 leading to the most severe changes. DEX led to cortical atrophy of the thymus. MTU induced hyperplastic changes in the thyroid. These results indicate that comedication does not markedly affect the histological changes induced in the hormonal target tissues. For screening purposes histology of the prostate can be used as a marker for oestrogens, whereas the weight of the thymus and thyroid can be used as an indication for the use of corticosteroids and thyreostatics, respectively. Vacuolization in the prostate appeared no reliable indication for beta-agonists. Samples of urine, faeces, liver and eye (retina/choroid) were analysed for beta-agonists. E2 significantly increased the levels of all beta-agonists in the liver and faeces, whereas DEX significantly reduced these levels. These observations show that additional medication with different groups of growth promoters can markedly alter the excretion of beta-agonists and thus influence (regulatory) control.

Prohormones such as dehydroepiandrosterone (DHEA) are steroid precursors that do not show hormonal activity by themselves. Abuse of these prohormones in cattle fattening is hard to prove because of strong in vivo metabolism and the difficulty to detect metabolites which are not significantly above endogenous levels. The aim of the present work was to develop an in vitro assay capable of detecting the indirect hormonal activity of prohormones that might be present in feed supplements and injection preparations. Sample extracts were incubated with a bovine liver S9 fraction in order to mimic the in vivo metabolic activation. Subsequently incubated extracts were exposed to a highly androgen-specific yeast bioassay to detect hormonal activity. Metabolic activation of DHEA, 4-androstene-3,17-dione (4-adione) and 5-androstene-3,17-diol (5-adiol) resulted in an increased androgenic activity caused by the formation of the active androgen 17beta-testosterone (17beta-T), as shown by ultra-performance liquid chromatography and time-of-flight mass spectrometry with accurate mass measurement. The developed in vitro system successfully mimics the hydroxysteroid dehydrogenase (HSD)- and cytochrome P450-mediated in vivo metabolic transitions, thus allowing assessment of both bioactivity and chemical identification without the use of animal experiments. Screening of unknown supplement samples claimed to contain DHEA resulted in successful bioactivation and positive screening results according to the androgen yeast biosensor.

Background Unexpected cholestasis substantially contributes to drug failure in clinical trials. Current models used for safety assessment in drug development do not accurately predict cholestasis in humans. Therefore, it is of relevance to develop new screening models that allow identifying drugs with cholestatic properties. Methods We employed mouse precision cut liver slices (PCLS), which were incubated 24 h with two model cholestatic compounds: cyclosporin A (CsA) and chlorpromazine (CPZ). Subsequently, transcriptome analysis using DNA microarrays and q-PCR were performed to identify relevant biological processes and biomarkers. Additionally, histology was carried out and levels of triglycerides (TG) and bile acids (BA) were measured. To verify the ex vivo mouse data, these were compared with publically available human data relevant for cholestasis. Results Whole genome gene expression analysis showed that CsA up-regulated pathways related to NF-¿B, ER stress and inflammation. Both CsA and CPZ down-regulated processes related to extracellular matrix (ECM) remodelling, BA homeostasis, Fxr signalling, and energy metabolism. The differential expression of a number of characteristic genes (e.g. Abcg5, Abcg8, Klf15, and Baat) could be confirmed by q-PCR. Histology revealed that CsA but not CPZ induced “ballooning” of hepatocytes. No effects on TG and BA levels were observed after incubation of PCLS with CsA and CPZ. A substantial number of processes altered in CsA- and CPZ-treated mouse PCLS ex vivo was also found to be affected in liver biopsies of cholestatic patients. Conclusion The present study demonstrated that mouse PCLS can be used as a tool to identify mechanisms of action of cholestatic model compounds. The induction of general stress responses and down-regulated Fxr signalling could play a role in the development of drug induced cholestasis. Importantly, comparative data analysis showed that the ex vivo mouse findings are also relevant for human pathology. Moreover, this work provides a set of genes that are potentially useful to assess drugs for cholestatic properties.

The EUBerry project is not only building on technical research but also studies berry production and consumption from a social sciences perspective. This paper aims to describe the approach used in the EUBerry project about business economics as well as marketing. In addition some preliminary results are presented. The social sciences part of EUBerry consists of four parts: economic viability, market situation, marketing strategies and evaluation. The economic viability of new production methods will be calculated beforehand as well as at the end of the project. The ex-ante analysis about critical success factors, with regard to expected costs and returns, gives the conditions for the improvement and implementation of new developing methods and can be seen as research guide. In this paper preliminary results of the ex-ante analysis for blueberries will be presented. With respect to market description, the initial market situation of fresh berries in selected European countries will be described. This description will be based on desk research as well as interviews. These results are taken as a basis for the development, testing and evaluation of marketing strategies, adapted from health benefits of berries in order to increase consumption. These marketing strategies will be tested together with SMEs. An overview of this marketing “plan of action method” will be given.