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Food Additives & Contaminants: Part A Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tfac20
Identification by a proteomic approach of a plasma protein as a possible biomarker of illicit dexamethasone treatment in veal calves a
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C. Guglielmetti , M. Mazza , M. Pagano , S. Carrella , S. Sciuto , S. Nodari , M. Pezzolato , a
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G.B. Richelmi , E. Baioni , M. Caramelli , P.L. Acutis & E. Bozzetta a
Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle d’Aosta, Turin, Italy Accepted author version posted online: 04 Mar 2014.Published online: 10 Apr 2014.
Click for updates To cite this article: C. Guglielmetti, M. Mazza, M. Pagano, S. Carrella, S. Sciuto, S. Nodari, M. Pezzolato, G.B. Richelmi, E. Baioni, M. Caramelli, P.L. Acutis & E. Bozzetta (2014) Identification by a proteomic approach of a plasma protein as a possible biomarker of illicit dexamethasone treatment in veal calves, Food Additives & Contaminants: Part A, 31:5, 833-838, DOI: 10.1080/19440049.2014.900191 To link to this article: http://dx.doi.org/10.1080/19440049.2014.900191
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Food Additives & Contaminants: Part A, 2014 Vol. 31, No. 5, 833–838, http://dx.doi.org/10.1080/19440049.2014.900191
Identification by a proteomic approach of a plasma protein as a possible biomarker of illicit dexamethasone treatment in veal calves C. Guglielmetti*, M. Mazza, M. Pagano, S. Carrella, S. Sciuto, S. Nodari, M. Pezzolato, G.B. Richelmi, E. Baioni, M. Caramelli, P.L. Acutis and E. Bozzetta Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle d’Aosta, Turin, Italy
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(Received 12 November 2013; accepted 21 February 2014) Corticosteroids have become the most widespread illegal growth promoters in veal calves and beef cattle. Testing for corticosteroids relies on either direct detection of compounds or their metabolites or indirect detection to identify changes in biological pathways. We used a comparative proteomic approach, based on two-dimensional electrophoresis (2DE), to identify plasma protein markers after short-term dexamethasone administration in veal calves. Twenty-three male Friesian veal calves were treated experimentally with dexamethasone sodium phosphate: 10 received low-dose administration of the drug (0.4 mg day–1 per os) for 20 consecutive days (treatment group); 10 received the drug at therapeutic dosage (2– 4 mg kg–1 i.m.) for 3 consecutive days (comparison group). Three animals were not treated (control group). Plasma samples were collected from each animal at six time points (T1–T6; treatment and control group) and at four time points (T1–T4; comparison group) and stored at –80°C until analysis. Plasma proteins were quantified and analysed in triplicate by 2DE. The images were analysed with Bionumerics® software. Comparison of 2DE maps obtained from blood samples at T1 (before treatment) and at T6 (final sampling) showed a significant disappearance (p < 0.001) of two protein spots at T6 in the treatment group. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis and immunoblotting identified these isoforms as serum paraoxonase/arylesterase 1 precursor (PON1). Synthesised in the liver and released into the blood, PON1 has an important role in lipid metabolism. The absence of variation of this protein in the comparison group suggests that the marker has good specificity for detecting illicit corticosteroid treatment. Keywords: dexamethasone; veal calves; meat; anabolic treatments; paraoxonase; PON1; 2D electrophoresis
Introduction Because chemical residues may constitute a potential risk for human health, the European Union has banned as a precautionary safety measure the use of growth-promoting hormones in food-producing animals since the 1990s. In spite of legislative regulation and intense inspection, the illicit use of these substances continues on European farms (Serratosa et al. 2006). There are, however, clear commercial benefits of increasing beef production yields through improved feed efficiency. In order to enhance carcasses and meat quality traits, corticosteroids, and dexamethasone in particular, are widely used as illicit growth promoters in veal calves and beef cattle, either alone or in combination with anabolic agents, especially at low dosages and primarily through oral administration (Gottardo et al. 2008). They are often used in association with sex steroids and βagonists to obtain a higher proportion of lean meat that consumers find more appealing (Meyer 2001). The strong pharmacological activity of synthetic corticosteroids renders their residues potentially dangerous for meat consumers. For example, glucocorticoids may be responsible for Cushing’s syndrome or other side-effects, including hypertension, hypokalemia, hypernatremia, metabolic alkalosis *Corresponding author. Email: [email protected] © 2014 Taylor & Francis
and connective tissue weakness. They may also lead to ulcer formation or cause permanent eye damage by inducing central serous retinopathy (Zhao et al. 2012). To date, the administration of synthetic hormones, such as dexamethasone, in livestock is approved by the European Union only for therapeutic indications; therefore, MRLs have been set for bovine edible tissues (0.75 μg kg–1 in kidney and muscle, 2 μg kg–1 in liver) and milk (0.3 μg kg–1) (Commission Regulation No. 37/2010). Depending on the particular glucocorticoid formulation, withdrawal periods of up to several weeks are recommended to prevent the accumulation of illegal residues in animal products. Dexamethasone has high bioavailability, is metabolised in the liver and excreted with the urine. In ruminants, dexamethasone, on entering the liver, undergoes reactions (hydroxylation at the six-position and reduction at the 3carbonyl group) catalysed by phase I drug-metabolising enzymes (DMEs); the resulting metabolite undergoes glucuronidation or sulfation by phase II DMEs. The conjugated metabolite leaves the liver and is eliminated via the urine. The glucocorticoid receptor (GR) is a steroid hormone-activated transcription factor. GR ligands are corticosteroid analogues, including dexamethasone.
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Dexamethasone interacts with several members of the nuclear receptor superfamily of transcription factors that contribute to the regulation of phase I and II DMEs involved in its biotransformation as well as the regulation of other target enzymes (Giantin et al. 2010). Testing for hormone abuse relies on the direct detection of illicit compounds or their metabolites by preliminary screening techniques such as immunoassay, then confirmatory analysis of positive samples by MS. The limiting factor is that the types of compounds being sought must be known beforehand and the analytes must be present at sufficiently high concentrations to enable detection at the method’s sensitivity, often expressed as its detection limit. Targeted GC/MS and LC/MS/MS may give unsatisfactory results when very low individual doses are administered, in which several products are combined to produce synergistic effects (“cocktails”), or with products known to be difficult to analyse because of their rapid metabolism (Courtheyn et al. 2002). Official feed and food controls by European Union member states require analytical screening for substances such as glucocorticoids in urine samples. Since 2008 Italy has carried out active epidemiological surveillance based on histological analysis of histopathological changes induced by anabolic substances in the thymus of regularly slaughtered calves (Bozzetta et al. 2011). To improve monitoring and detection of growth-promoter abuse, new analytical approaches have been developed for more effective alternative tests (Mooney, Le Bizec, et al. 2009; Pinel et al. 2010). By identifying alterations of physiological parameters in treated animals, such methods can provide faster, higher throughput and more sensitive forms of analysis that facilitate robust monitoring and improve detection rates of banned agents, with confirmatory residue analysis reserved for suspect samples. Screening with detection methods that allow identification and monitoring of changes in biological pathways and systems offers distinct advantages over existing testing methods because it does not require prior knowledge of the type of compounds or treatment regimes and because biomarker profiles remain altered for a longer period of time than the abused substance can be detected in circulation (Smits et al. 2012). For these reasons, there is an urgent need for screening methods that work by measuring indirect biomarkers and histological and physiological indicators. Numbering among the innovative methods that can help to identify animals treated with anabolic agents are the “omics” techniques (transcriptomic, proteomic and metabolomic) that permit simultaneous detection of biomarkers predictive of administration of specific substances (Gardini et al. 2006; Courant et al. 2009; Stella et al. 2011). The application of proteomic techniques to food safety has gained increasing interest (D’Alessandro & Zolla 2012; Ludwig et al. 2013; McGrath et al. 2013), as
previous studies on the responses of circulating protein components of the blood to growth-promoter administrations in cattle have identified several potential markers, including sex-hormone-binding globulin (Mooney, Bergwerff, et al. 2009), the aminoterminal propeptide of type III procollagen (Mooney et al. 2008), immunoreactive inhibin, osteocalcin (Cacciatore et al. 2009), and apolipoprotein A1 (Draisci et al. 2007). This study describes a comparative proteomic profiling approach, based on two-dimensional electrophoresis (2DE), to identify circulating protein markers in plasma indicative of dexamethasone administration in veal calves. In this study, which is part of a larger research project (data not reported), more calves were treated with several different compounds (17β-estradiol, testosterone, dexamethasone). Because a preliminary analysis of a small number of samples from each group showed the most interesting data related to the dexamethasone treatments, the present study reports the data pertaining only to these animals.
Materials and methods Animals and experimental design Twenty-three Freisian male veal calves bought between 15 and 35 days were farmed in boxes for 6 months. Each box had its own crib, multiple drinking troughs and a dedicated automated milk feeder system. To protect the animals against infections, upon arrival at the farm the animals were vaccinated against bovine respiratory syncytial virus (BRSV) and para-influenza type 3 (PI3) (Rispoval RS + PI3 IntraNasal; Pfizer Animal Health, New York, NY, USA). After 20 days, at about 1 month of age, the calves were vaccinated intramuscularly against bovine viral diarrhoea virus (BVDV) (Rispoval RS BVD; Pfizer Animal Health) and bovine rinotracheitis (IBR) (Bovilis IBR marker live; Intervet International, Boxmeer, the Netherlands). Daily clinical controls were carried out by a veterinarian and infections were treated without hormonal active substances. All animals had free access to fresh water and were fed standard milk replacer with an automatic milk feeder until 4 months of age, then 0.5 kg of corn were added to the diet twice a day. Before administration, all feeds, milk replacer and corn were analysed by ELISA to exclude the presence of corticosteroids (l’Screen Cortico, Tecna S.r.l., Trieste, Italy). During the sixth month, 10 randomly selected animals (treatment group) received dexamethasone 21 disodium phosphate (Desashock®) (0.4 mg day–1 per os) mixed with the milk, once a day for 20 days, and 10 other animals (comparison group) received the same drug (2–4 mg kg–1 i.m.), once a day for 3 days, to simulate therapeutic treatment. Three animals were not treated with dexamethasone (control
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Anabolic treatment Blood samples Therapeutic treatment Blood samples
Protein separation in the first dimension by isoelectric focusing (IEF) Before IEF, IPG strips (pH3-10NL, 18 cm) (Bio-Rad Laboratories) were rehydrated at RT overnight in a buffer containing 7 M urea, 2 M thiourea, 2% CHAPS, 10 mM DTT, 0.3% ampholytes pH 3–10, 0.3% ampholytes pH 5–8, 0.3% ampholytes pH 4–6 and 0.1% bromophenol blue. A volume of 150 μl of sample was loaded onto the IPG strip by the cup loading system (Bio-Rad Laboratories). IEF was performed on a Protean IEF System (Bio-Rad Laboratories): 350 V for 1 h, 450 V
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We analysed blood samples drawn at T1 and T6 from the 10 animals in the treatment group and at T1 and T4 from the 10 animals in the comparison group. Blood samples taken at T1 and T6 from the three animals in the control group were also analysed. Each sample was analysed in triplicate. Because the T1 samples were taken before treatment, they represent the control sample for each animal. Before beginning the preparation phase, plasma samples were quantified (Quick Start™ Bradford 1x Dye Reagent, Bio-Rad Laboratories, Hercules, CA, USA) according to the manufacturer’s instructions. For protein purification, solubilisation, denaturation and reduction, an aliquot of 360 µg ml–1 of total protein per sample was added to 150 μl of a buffer solution containing 7 M urea, 2 M thiourea, 4% CHAPS, 50 mM DTT, 250 mM EDTA and 0.1% bromophenol blue. The samples were incubated at RT overnight in end-over-end rotation, then centrifuged at 15 294g for 5 min at RT before loading on the strip for isoelectric focusing (IEF).
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Blood samples were taken from the anterior jugular vein using K2EDTA vacutainer tubes (Becton Dickinson, Sparks, MD, USA) throughout the study period from all 23 animals. Plasma was acquired by centrifugation at 4°C (1250g, 20 min) and stored at –80°C until analysis. Table 1 reports the sampling schedule for the two groups.
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group). The withdrawal period was respected before last blood sampling and slaughtering. Under the protocol of the experimental design, the samples from the animals treated for both anabolic and therapeutic purposes (treatment and comparison groups) were compared to ensure that the difference observed in terms of protein expression was attributable to simulated illegal treatment.
Table 1. Treatment and blood sampling schedule. Ten received dexamethasone for 20 consecutive days to simulate an anabolic treatment (treatment group); and 10 received the drug at therapeutic dosage for 3 consecutive days (comparison group).
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for 1 h, 2500 V for 3 h, 3500 V for 5 h, and 5000 V until reaching 80 000 Vh.
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Equilibration of the IPG Before transfer to the polyacrylamide gel for the second dimension, the IPG strips were washed twice, each time for 15 min, first with the reducing solution (6 M urea, 2% SDS, 50 mM Tris-HCl pH 8.8, 30% glycerol, 1% DTT and 0.1% of a solution of bromophenol blue) and then with the alkylating solution (6 M urea, 2% SDS, 50 mM Tris-HCl pH 6.8, 30% glycerol, 3% IAA and 0.1% bromophenol blue).
Protein separation in the second dimension by SDS-PAGE The second dimension was performed with homemade 8– 16% polyacrylamide gels on Protean Plus Dodeca Cell (Bio-Rad Laboratories) at 200 Vk for 6 h and 30 min with a Tris glycine buffer (25 mM Tris, 192 mM glycine, 0.1% SDS, pH 8.3), thermostated to 12°C.
Gel fixing and staining After electrophoresis, the gels were fixed in a solution containing 40% methanol and 10% acetic acid overnight at RT, washed four times for 15 min each in demineralised water, then stained with Blue Silver (Candiano et al. 2004).
Image analysis After destaining, images of the gels were acquired and subsequently analysed with BioNumerics® 2D Gel Types software (Applied Maths, Austin, TX, USA). The protein maps were then compared to detect qualitative and quantitative differences between the samples from the two groups.
Western blotting Pre- and post-treatment plasma samples (T1 and T6) from one animal in the treatment group were selected and analysed by 2DE as described above. Different IPG strips (pH 4–7, 17 cm) were used for IEF. After equilibration for both strips, the portion of interest was cut, loaded onto 15% minigel and subjected to electrophoresis at 200 Vk for 1 h. Proteins were transferred onto PVDF membrane using mini-trans-blot 150 Vk for 2 h. Non-specific binding was saturated in TBS-BSA 0.5%. Polyclonal Ab antiparaoxonase (PON1) (Aviva Systems Biology, San Diego, CA, USA) (0.2 μg ml–1) was incubated overnight at 4°C. Immunodetection was carried out with alkaline phosphatase-conjugated goat anti-rabbit IgG revealed by a chemiluminescent substrate. The image was captured on a photographic plate.
Statistical analysis The animals were randomly assigned to either the treatment or the comparison group, and the experimental design was planned to control for confounders such as sex, age and feed. Fisher’s exact test was used to calculate a p-value determining the probability that the observed differences in PON1 expression between the two groups was due to chance alone. Significance was set at p < 0–0.5.
Results and discussion Comparison of the protein maps obtained from the plasma samples taken at T6 showed the disappearance of two protein isoforms of about 43 kDa molecular weight and 4.8 isoelectric point (pI) in all the 10 animals in the treatment group (Figure 1). Analysis of the T4 samples, the last sampling time for the 10 animals in the pH3
LC-MS/MS analysis For protein identification, spots of interest were excised from the gel after staining with Blue Silver and bleached in 25 mM ammonium bicarbonate in 40% ethanol. After drying in acetonitrile, they were placed in 25 mM ammonium bicarbonate containing 0.6 g porcine trypsin overnight at 37°C. Peptides were extracted by sonication in 25 mM ammonium bicarbonate and analysed by LC-MS/ MS. Spectra were acquired in the range m/z (mass/charge) between 100 and 2200. Protein spots were identified by MASCOT® software (Matrix Science UK, London, UK).
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Figure 1. 2DE maps representative of plasma samples from a calf from the treatment group taken at the first (T1) and the last sampling time (T6). Identification of approximately 396 protein spots by Blue Silver staining. The box shows the disappearance of two protein spots of approximately 43 kDa and pI of about 4.8.
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Figure 2. 2DE maps representative of plasma samples from a calf from the comparison group taken at the first (T1) and the last sampling time (T4). Identification of approximately 394 protein spots by Blue Silver staining. The pair of spots of approximately 43 kDa and pI of about 4.8, as shown in the box, is present. The arrow in the inset indicates the weaker spot the T4.
antibody reactivity at T1 and the absence of reactivity at T6 (Figure 3). PON1, a protein synthesised in the liver and released into the blood, catalyses the hydrolysis of organophosphates. It also plays an important physiological role in lipid metabolism by hydrolysis of oxidised lipids, in the form of lipid hydroperoxides, generated on high-density lipoprotein (HDL) and low-density lipoprotein (LDL). By virtue of this ability it is considered an antioxidative/antiinflammatory component of HDL (Aviram et al. 1998). The decrease in PON1 following illicit dexamethasone treatment is correlated with its metabolism by the liver. Furthermore, PON1 activity is strongly inhibited by dexamethasone, as recently reported in a study in which this effect was measured in vitro (Arslan et al. 2012). Conclusion
comparison group, showed that the pair of spots of interest was present, albeit with a slightly weaker isoform characterised by a more acidic pI (Figure 2). Fisher’s exact test revealed a statistically significant difference in biomarker expression between the two groups (N = 20, p < 0.001). The expression of these markers in the comparison group remained unchanged after therapeutic treatment, allowing one to differentiate between the animals treated for anabolic or therapeutic purposes. Analysis of three samples taken at all six sampling times in the treatment group showed that the pair of spots disappeared between the fifth and sixth picks, confirming that prolonged treatment, for at least 20 days, is needed to reset the expression of these two protein markers. The three samples from the control group showed no variation of the two protein isoforms (data not shown). MS analysis of the two proteins identified them in two isoforms of the enzyme paraoxonase/arilesterase serum precursor (PON1). Using a polyclonal antibody anti-paroxonase 1, immunoblotting analysis of plasma samples taken at T1 and T6 from one animal in the treatment group confirmed identification by LC-MS/MS, showing
The data suggest that PON1 may be a useful marker for the detection of illicit dexamethasone treatment in veal calves. The absence of variation in PON1 seen in the animals treated with dexamethasone for therapeutic purposes suggests that this marker has good specificity, which seems to vary only after prolonged low-dose administration. Further studies on larger populations, and with other substances, are needed to improve the accuracy of results. When administered at low doses, dexamethasone is difficult to detect in matrices of biological origin. One way to improve the efficiency of residue testing programmes might be to apply screening methods that measure indirect parameters. Treatment-induced alterations in the proteomic profile of blood could be useful for identifying illegally treated animals already at the farm before they enter the human food chain. Positive or suspect animals could then be submitted to analysis with classical methods to detect any chemical residues, making the control of illicit treatment with growth-promoting agents more efficient and applicable on a larger scale. Funding
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This research was funded by the Italian Ministry of Health [grant number IZS PLV 18/08RC].
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Figure 3. 2DE coupled with Western blotting of a plasma sample from a calf from the treatment group taken at the first (T1) and the last sampling time (T6).
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Identification by a proteomic approach of a plasma protein as a possible biomarker of illicit dexamethasone treatment in veal calves a
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C. Guglielmetti , M. Mazza , M. Pagano , S. Carrella , S. Sciuto , S. Nodari , M. Pezzolato , a
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Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle d’Aosta, Turin, Italy Accepted author version posted online: 04 Mar 2014.Published online: 10 Apr 2014.
Click for updates To cite this article: C. Guglielmetti, M. Mazza, M. Pagano, S. Carrella, S. Sciuto, S. Nodari, M. Pezzolato, G.B. Richelmi, E. Baioni, M. Caramelli, P.L. Acutis & E. Bozzetta (2014) Identification by a proteomic approach of a plasma protein as a possible biomarker of illicit dexamethasone treatment in veal calves, Food Additives & Contaminants: Part A, 31:5, 833-838, DOI: 10.1080/19440049.2014.900191 To link to this article: http://dx.doi.org/10.1080/19440049.2014.900191
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Food Additives & Contaminants: Part A, 2014 Vol. 31, No. 5, 833–838, http://dx.doi.org/10.1080/19440049.2014.900191
Identification by a proteomic approach of a plasma protein as a possible biomarker of illicit dexamethasone treatment in veal calves C. Guglielmetti*, M. Mazza, M. Pagano, S. Carrella, S. Sciuto, S. Nodari, M. Pezzolato, G.B. Richelmi, E. Baioni, M. Caramelli, P.L. Acutis and E. Bozzetta Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle d’Aosta, Turin, Italy
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(Received 12 November 2013; accepted 21 February 2014) Corticosteroids have become the most widespread illegal growth promoters in veal calves and beef cattle. Testing for corticosteroids relies on either direct detection of compounds or their metabolites or indirect detection to identify changes in biological pathways. We used a comparative proteomic approach, based on two-dimensional electrophoresis (2DE), to identify plasma protein markers after short-term dexamethasone administration in veal calves. Twenty-three male Friesian veal calves were treated experimentally with dexamethasone sodium phosphate: 10 received low-dose administration of the drug (0.4 mg day–1 per os) for 20 consecutive days (treatment group); 10 received the drug at therapeutic dosage (2– 4 mg kg–1 i.m.) for 3 consecutive days (comparison group). Three animals were not treated (control group). Plasma samples were collected from each animal at six time points (T1–T6; treatment and control group) and at four time points (T1–T4; comparison group) and stored at –80°C until analysis. Plasma proteins were quantified and analysed in triplicate by 2DE. The images were analysed with Bionumerics® software. Comparison of 2DE maps obtained from blood samples at T1 (before treatment) and at T6 (final sampling) showed a significant disappearance (p < 0.001) of two protein spots at T6 in the treatment group. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis and immunoblotting identified these isoforms as serum paraoxonase/arylesterase 1 precursor (PON1). Synthesised in the liver and released into the blood, PON1 has an important role in lipid metabolism. The absence of variation of this protein in the comparison group suggests that the marker has good specificity for detecting illicit corticosteroid treatment. Keywords: dexamethasone; veal calves; meat; anabolic treatments; paraoxonase; PON1; 2D electrophoresis
Introduction Because chemical residues may constitute a potential risk for human health, the European Union has banned as a precautionary safety measure the use of growth-promoting hormones in food-producing animals since the 1990s. In spite of legislative regulation and intense inspection, the illicit use of these substances continues on European farms (Serratosa et al. 2006). There are, however, clear commercial benefits of increasing beef production yields through improved feed efficiency. In order to enhance carcasses and meat quality traits, corticosteroids, and dexamethasone in particular, are widely used as illicit growth promoters in veal calves and beef cattle, either alone or in combination with anabolic agents, especially at low dosages and primarily through oral administration (Gottardo et al. 2008). They are often used in association with sex steroids and βagonists to obtain a higher proportion of lean meat that consumers find more appealing (Meyer 2001). The strong pharmacological activity of synthetic corticosteroids renders their residues potentially dangerous for meat consumers. For example, glucocorticoids may be responsible for Cushing’s syndrome or other side-effects, including hypertension, hypokalemia, hypernatremia, metabolic alkalosis *Corresponding author. Email: [email protected] © 2014 Taylor & Francis
and connective tissue weakness. They may also lead to ulcer formation or cause permanent eye damage by inducing central serous retinopathy (Zhao et al. 2012). To date, the administration of synthetic hormones, such as dexamethasone, in livestock is approved by the European Union only for therapeutic indications; therefore, MRLs have been set for bovine edible tissues (0.75 μg kg–1 in kidney and muscle, 2 μg kg–1 in liver) and milk (0.3 μg kg–1) (Commission Regulation No. 37/2010). Depending on the particular glucocorticoid formulation, withdrawal periods of up to several weeks are recommended to prevent the accumulation of illegal residues in animal products. Dexamethasone has high bioavailability, is metabolised in the liver and excreted with the urine. In ruminants, dexamethasone, on entering the liver, undergoes reactions (hydroxylation at the six-position and reduction at the 3carbonyl group) catalysed by phase I drug-metabolising enzymes (DMEs); the resulting metabolite undergoes glucuronidation or sulfation by phase II DMEs. The conjugated metabolite leaves the liver and is eliminated via the urine. The glucocorticoid receptor (GR) is a steroid hormone-activated transcription factor. GR ligands are corticosteroid analogues, including dexamethasone.
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Dexamethasone interacts with several members of the nuclear receptor superfamily of transcription factors that contribute to the regulation of phase I and II DMEs involved in its biotransformation as well as the regulation of other target enzymes (Giantin et al. 2010). Testing for hormone abuse relies on the direct detection of illicit compounds or their metabolites by preliminary screening techniques such as immunoassay, then confirmatory analysis of positive samples by MS. The limiting factor is that the types of compounds being sought must be known beforehand and the analytes must be present at sufficiently high concentrations to enable detection at the method’s sensitivity, often expressed as its detection limit. Targeted GC/MS and LC/MS/MS may give unsatisfactory results when very low individual doses are administered, in which several products are combined to produce synergistic effects (“cocktails”), or with products known to be difficult to analyse because of their rapid metabolism (Courtheyn et al. 2002). Official feed and food controls by European Union member states require analytical screening for substances such as glucocorticoids in urine samples. Since 2008 Italy has carried out active epidemiological surveillance based on histological analysis of histopathological changes induced by anabolic substances in the thymus of regularly slaughtered calves (Bozzetta et al. 2011). To improve monitoring and detection of growth-promoter abuse, new analytical approaches have been developed for more effective alternative tests (Mooney, Le Bizec, et al. 2009; Pinel et al. 2010). By identifying alterations of physiological parameters in treated animals, such methods can provide faster, higher throughput and more sensitive forms of analysis that facilitate robust monitoring and improve detection rates of banned agents, with confirmatory residue analysis reserved for suspect samples. Screening with detection methods that allow identification and monitoring of changes in biological pathways and systems offers distinct advantages over existing testing methods because it does not require prior knowledge of the type of compounds or treatment regimes and because biomarker profiles remain altered for a longer period of time than the abused substance can be detected in circulation (Smits et al. 2012). For these reasons, there is an urgent need for screening methods that work by measuring indirect biomarkers and histological and physiological indicators. Numbering among the innovative methods that can help to identify animals treated with anabolic agents are the “omics” techniques (transcriptomic, proteomic and metabolomic) that permit simultaneous detection of biomarkers predictive of administration of specific substances (Gardini et al. 2006; Courant et al. 2009; Stella et al. 2011). The application of proteomic techniques to food safety has gained increasing interest (D’Alessandro & Zolla 2012; Ludwig et al. 2013; McGrath et al. 2013), as
previous studies on the responses of circulating protein components of the blood to growth-promoter administrations in cattle have identified several potential markers, including sex-hormone-binding globulin (Mooney, Bergwerff, et al. 2009), the aminoterminal propeptide of type III procollagen (Mooney et al. 2008), immunoreactive inhibin, osteocalcin (Cacciatore et al. 2009), and apolipoprotein A1 (Draisci et al. 2007). This study describes a comparative proteomic profiling approach, based on two-dimensional electrophoresis (2DE), to identify circulating protein markers in plasma indicative of dexamethasone administration in veal calves. In this study, which is part of a larger research project (data not reported), more calves were treated with several different compounds (17β-estradiol, testosterone, dexamethasone). Because a preliminary analysis of a small number of samples from each group showed the most interesting data related to the dexamethasone treatments, the present study reports the data pertaining only to these animals.
Materials and methods Animals and experimental design Twenty-three Freisian male veal calves bought between 15 and 35 days were farmed in boxes for 6 months. Each box had its own crib, multiple drinking troughs and a dedicated automated milk feeder system. To protect the animals against infections, upon arrival at the farm the animals were vaccinated against bovine respiratory syncytial virus (BRSV) and para-influenza type 3 (PI3) (Rispoval RS + PI3 IntraNasal; Pfizer Animal Health, New York, NY, USA). After 20 days, at about 1 month of age, the calves were vaccinated intramuscularly against bovine viral diarrhoea virus (BVDV) (Rispoval RS BVD; Pfizer Animal Health) and bovine rinotracheitis (IBR) (Bovilis IBR marker live; Intervet International, Boxmeer, the Netherlands). Daily clinical controls were carried out by a veterinarian and infections were treated without hormonal active substances. All animals had free access to fresh water and were fed standard milk replacer with an automatic milk feeder until 4 months of age, then 0.5 kg of corn were added to the diet twice a day. Before administration, all feeds, milk replacer and corn were analysed by ELISA to exclude the presence of corticosteroids (l’Screen Cortico, Tecna S.r.l., Trieste, Italy). During the sixth month, 10 randomly selected animals (treatment group) received dexamethasone 21 disodium phosphate (Desashock®) (0.4 mg day–1 per os) mixed with the milk, once a day for 20 days, and 10 other animals (comparison group) received the same drug (2–4 mg kg–1 i.m.), once a day for 3 days, to simulate therapeutic treatment. Three animals were not treated with dexamethasone (control
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Anabolic treatment Blood samples Therapeutic treatment Blood samples
Protein separation in the first dimension by isoelectric focusing (IEF) Before IEF, IPG strips (pH3-10NL, 18 cm) (Bio-Rad Laboratories) were rehydrated at RT overnight in a buffer containing 7 M urea, 2 M thiourea, 2% CHAPS, 10 mM DTT, 0.3% ampholytes pH 3–10, 0.3% ampholytes pH 5–8, 0.3% ampholytes pH 4–6 and 0.1% bromophenol blue. A volume of 150 μl of sample was loaded onto the IPG strip by the cup loading system (Bio-Rad Laboratories). IEF was performed on a Protean IEF System (Bio-Rad Laboratories): 350 V for 1 h, 450 V
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We analysed blood samples drawn at T1 and T6 from the 10 animals in the treatment group and at T1 and T4 from the 10 animals in the comparison group. Blood samples taken at T1 and T6 from the three animals in the control group were also analysed. Each sample was analysed in triplicate. Because the T1 samples were taken before treatment, they represent the control sample for each animal. Before beginning the preparation phase, plasma samples were quantified (Quick Start™ Bradford 1x Dye Reagent, Bio-Rad Laboratories, Hercules, CA, USA) according to the manufacturer’s instructions. For protein purification, solubilisation, denaturation and reduction, an aliquot of 360 µg ml–1 of total protein per sample was added to 150 μl of a buffer solution containing 7 M urea, 2 M thiourea, 4% CHAPS, 50 mM DTT, 250 mM EDTA and 0.1% bromophenol blue. The samples were incubated at RT overnight in end-over-end rotation, then centrifuged at 15 294g for 5 min at RT before loading on the strip for isoelectric focusing (IEF).
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Blood samples were taken from the anterior jugular vein using K2EDTA vacutainer tubes (Becton Dickinson, Sparks, MD, USA) throughout the study period from all 23 animals. Plasma was acquired by centrifugation at 4°C (1250g, 20 min) and stored at –80°C until analysis. Table 1 reports the sampling schedule for the two groups.
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group). The withdrawal period was respected before last blood sampling and slaughtering. Under the protocol of the experimental design, the samples from the animals treated for both anabolic and therapeutic purposes (treatment and comparison groups) were compared to ensure that the difference observed in terms of protein expression was attributable to simulated illegal treatment.
Table 1. Treatment and blood sampling schedule. Ten received dexamethasone for 20 consecutive days to simulate an anabolic treatment (treatment group); and 10 received the drug at therapeutic dosage for 3 consecutive days (comparison group).
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for 1 h, 2500 V for 3 h, 3500 V for 5 h, and 5000 V until reaching 80 000 Vh.
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Equilibration of the IPG Before transfer to the polyacrylamide gel for the second dimension, the IPG strips were washed twice, each time for 15 min, first with the reducing solution (6 M urea, 2% SDS, 50 mM Tris-HCl pH 8.8, 30% glycerol, 1% DTT and 0.1% of a solution of bromophenol blue) and then with the alkylating solution (6 M urea, 2% SDS, 50 mM Tris-HCl pH 6.8, 30% glycerol, 3% IAA and 0.1% bromophenol blue).
Protein separation in the second dimension by SDS-PAGE The second dimension was performed with homemade 8– 16% polyacrylamide gels on Protean Plus Dodeca Cell (Bio-Rad Laboratories) at 200 Vk for 6 h and 30 min with a Tris glycine buffer (25 mM Tris, 192 mM glycine, 0.1% SDS, pH 8.3), thermostated to 12°C.
Gel fixing and staining After electrophoresis, the gels were fixed in a solution containing 40% methanol and 10% acetic acid overnight at RT, washed four times for 15 min each in demineralised water, then stained with Blue Silver (Candiano et al. 2004).
Image analysis After destaining, images of the gels were acquired and subsequently analysed with BioNumerics® 2D Gel Types software (Applied Maths, Austin, TX, USA). The protein maps were then compared to detect qualitative and quantitative differences between the samples from the two groups.
Western blotting Pre- and post-treatment plasma samples (T1 and T6) from one animal in the treatment group were selected and analysed by 2DE as described above. Different IPG strips (pH 4–7, 17 cm) were used for IEF. After equilibration for both strips, the portion of interest was cut, loaded onto 15% minigel and subjected to electrophoresis at 200 Vk for 1 h. Proteins were transferred onto PVDF membrane using mini-trans-blot 150 Vk for 2 h. Non-specific binding was saturated in TBS-BSA 0.5%. Polyclonal Ab antiparaoxonase (PON1) (Aviva Systems Biology, San Diego, CA, USA) (0.2 μg ml–1) was incubated overnight at 4°C. Immunodetection was carried out with alkaline phosphatase-conjugated goat anti-rabbit IgG revealed by a chemiluminescent substrate. The image was captured on a photographic plate.
Statistical analysis The animals were randomly assigned to either the treatment or the comparison group, and the experimental design was planned to control for confounders such as sex, age and feed. Fisher’s exact test was used to calculate a p-value determining the probability that the observed differences in PON1 expression between the two groups was due to chance alone. Significance was set at p < 0–0.5.
Results and discussion Comparison of the protein maps obtained from the plasma samples taken at T6 showed the disappearance of two protein isoforms of about 43 kDa molecular weight and 4.8 isoelectric point (pI) in all the 10 animals in the treatment group (Figure 1). Analysis of the T4 samples, the last sampling time for the 10 animals in the pH3
LC-MS/MS analysis For protein identification, spots of interest were excised from the gel after staining with Blue Silver and bleached in 25 mM ammonium bicarbonate in 40% ethanol. After drying in acetonitrile, they were placed in 25 mM ammonium bicarbonate containing 0.6 g porcine trypsin overnight at 37°C. Peptides were extracted by sonication in 25 mM ammonium bicarbonate and analysed by LC-MS/ MS. Spectra were acquired in the range m/z (mass/charge) between 100 and 2200. Protein spots were identified by MASCOT® software (Matrix Science UK, London, UK).
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Figure 1. 2DE maps representative of plasma samples from a calf from the treatment group taken at the first (T1) and the last sampling time (T6). Identification of approximately 396 protein spots by Blue Silver staining. The box shows the disappearance of two protein spots of approximately 43 kDa and pI of about 4.8.
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Figure 2. 2DE maps representative of plasma samples from a calf from the comparison group taken at the first (T1) and the last sampling time (T4). Identification of approximately 394 protein spots by Blue Silver staining. The pair of spots of approximately 43 kDa and pI of about 4.8, as shown in the box, is present. The arrow in the inset indicates the weaker spot the T4.
antibody reactivity at T1 and the absence of reactivity at T6 (Figure 3). PON1, a protein synthesised in the liver and released into the blood, catalyses the hydrolysis of organophosphates. It also plays an important physiological role in lipid metabolism by hydrolysis of oxidised lipids, in the form of lipid hydroperoxides, generated on high-density lipoprotein (HDL) and low-density lipoprotein (LDL). By virtue of this ability it is considered an antioxidative/antiinflammatory component of HDL (Aviram et al. 1998). The decrease in PON1 following illicit dexamethasone treatment is correlated with its metabolism by the liver. Furthermore, PON1 activity is strongly inhibited by dexamethasone, as recently reported in a study in which this effect was measured in vitro (Arslan et al. 2012). Conclusion
comparison group, showed that the pair of spots of interest was present, albeit with a slightly weaker isoform characterised by a more acidic pI (Figure 2). Fisher’s exact test revealed a statistically significant difference in biomarker expression between the two groups (N = 20, p < 0.001). The expression of these markers in the comparison group remained unchanged after therapeutic treatment, allowing one to differentiate between the animals treated for anabolic or therapeutic purposes. Analysis of three samples taken at all six sampling times in the treatment group showed that the pair of spots disappeared between the fifth and sixth picks, confirming that prolonged treatment, for at least 20 days, is needed to reset the expression of these two protein markers. The three samples from the control group showed no variation of the two protein isoforms (data not shown). MS analysis of the two proteins identified them in two isoforms of the enzyme paraoxonase/arilesterase serum precursor (PON1). Using a polyclonal antibody anti-paroxonase 1, immunoblotting analysis of plasma samples taken at T1 and T6 from one animal in the treatment group confirmed identification by LC-MS/MS, showing
The data suggest that PON1 may be a useful marker for the detection of illicit dexamethasone treatment in veal calves. The absence of variation in PON1 seen in the animals treated with dexamethasone for therapeutic purposes suggests that this marker has good specificity, which seems to vary only after prolonged low-dose administration. Further studies on larger populations, and with other substances, are needed to improve the accuracy of results. When administered at low doses, dexamethasone is difficult to detect in matrices of biological origin. One way to improve the efficiency of residue testing programmes might be to apply screening methods that measure indirect parameters. Treatment-induced alterations in the proteomic profile of blood could be useful for identifying illegally treated animals already at the farm before they enter the human food chain. Positive or suspect animals could then be submitted to analysis with classical methods to detect any chemical residues, making the control of illicit treatment with growth-promoting agents more efficient and applicable on a larger scale. Funding
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This research was funded by the Italian Ministry of Health [grant number IZS PLV 18/08RC].
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Figure 3. 2DE coupled with Western blotting of a plasma sample from a calf from the treatment group taken at the first (T1) and the last sampling time (T6).
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