Anabolic treatments in bovines: quantification of plasma protein markers of dexamethasone administration.

The aim of this study was to profile plasma proteome responses in bulls experimentally treated with dexamethasone at anabolic dosage. Illicit use of active substances in animal husbandry remains a matter of concern in Europe. Corticosteroids are probably one of the most widespread growth promoter family illegally used in beef cattle and veal calves. Testing for corticosteroids relies on detection of drug residues or their metabolites in biological fluids or tissues. Their indirect detection by mapping altered physiological parameters may overcome limits linked to route of administration, dosage, biotransformation and elimination kinetics that can lower residual drug concentration, hampering official controls. A set of 11 proteins proposed in literature as potential markers of anabolic treatments with dexamethasone, was quantified in bovine plasma by targeted proteomics based on liquid chromatography-high resolution tandem mass spectrometry. Among investigated proteins, sex hormone-binding globulin (SHBG), histidine-rich glycoprotein (HRG) and paraoxonase-1 (PON1) were found to be biomarkers of treatment. To investigate further such biomarkers, an additional group of veal calves was experimentally treated with dexamethasone at anabolic. These animals also demonstrated a significant alteration in SHBG, HRG and PON1 concentration, suggesting that quantification of plasma markers have the potential to detect animals illegally exposed to dexamethasone.

TMT-Based Proteomics Profiling of Bovine Liver Underscores Protein Markers of Anabolic Treatments.

Illegal use of growth promoter compounds in food production exposes consumers to health risk. Surveillance of such practices is based on direct detection of drugs or related metabolites by HPLC-MS/MS. Screening strategies focusing on indirect biological responses are considered promising tools to improve surveillance. In this study, an untargeted shotgun proteomics approach based on tandem mass tags (TMTs) is carried out to identify proteins altered in bovine liver after different anabolic treatments. Three controlled pharmacological treatments with dexamethasone, a combination of dexamethasone and clenbuterol, or a combination of sexual steroids (trenbolone and estradiol) are analyzed. Untargeted TMT analysis of liver digests by high resolution MS allowed for the relative quantification of proteins. Thanks to partial least squarediscriminant analysis, a set of proteins capable to classify animals treated with dexamethasone alone (11 proteins), or in combination with clenbuterol (13 proteins) are identified. No significant difference is found upon administration of sexual steroids. After relative quantification of candidate markers by parallel reaction monitoring (PRM), two predictive models are trained to validate protein markers. Finally, an independent animal set of control bulls and bulls treated with dexamethasone is analyzed by PRM to further validate a predictive model giving an accuracy of 100%.

Absolute quantification of myosin heavy chain isoforms by selected reaction monitoring can underscore skeletal muscle changes in a mouse model of amyotrophic lateral sclerosis.

Skeletal muscle fibers contain different isoforms of myosin heavy chain (MyHC) that define distinctive contractile properties. In light of the muscle capacity to adapt MyHC expression to pathophysiological conditions, a rapid and quantitative assessment of MyHC isoforms in small muscle tissue quantities would represent a valuable diagnostic tool for (neuro)muscular diseases. As past protocols did not meet these requirements, in the present study we applied a targeted proteomic approach based on selected reaction monitoring that allowed the absolute quantification of slow and fast MyHC isoforms in different mouse skeletal muscles with high reproducibility. This mass-spectrometry-based method was validated also in a pathological specimen, by comparison of the MyHC expression profiles in different muscles from healthy mice and a genetic mouse model of amyotrophic lateral sclerosis (ALS) expressing the SOD1(G93A) mutant. This analysis showed that terminally ill ALS mice have a fast-to-slow shift in the fiber type composition of the tibialis anterior and gastrocnemius muscles, as previously reported. These results will likely open the way to accurate and rapid diagnoses of human (neuro)muscular diseases by the proposed method. Graphical Abstract Methods for myosin heavy chain (MyHC) quantification: a comparison of classical methods and selected reaction monitoring (SRM)-based mass spectrometry approaches.

Targeted proteomics for the indirect detection of dexamethasone treatment in bovines.

The illegal use of pharmacologically active compounds for growth promotion in food-producing species poses risks for consumer health and animal welfare. Surveillance relies on the quantification of drug residues in animal fluids or tissues, but the efficacy can be negatively affected due to undetectable residual concentrations in biological matrices. Consequently, techniques focusing on the indirect biological effects of exogenous compound administration have been proposed as more sensitive detection methods. The purpose of the present study is to develop a tandem mass spectrometry analytical method based on low-energy collision-induced dissociation (CID-MS/MS) using multiple reaction monitoring (MRM) for the quantification of 12 potential protein markers of skeletal muscle to detect anabolic treatments with dexamethasone. Protein markers identified in a previous study applying a 2D-DIGE proteomics approach have been quantified using the signature peptide method. A group of proteins were confirmed as reliable markers. Quantitative results enabled a predictive model to be defined based on logistic regression for the detection of treated animals. The developed model was finally cross-validated in an independent animal set. Graphical abstract Analytical workflow used for the quantification of indirect protein markers of dexamethasone treatment.

Proteomics for the detection of indirect markers of steroids treatment in bovine muscle.

Despite the ban by the European Union, anabolic steroids might still be illicitly employed in bovine meat production. The surveillance of misuse of such potentially harmful molecules is necessary to guarantee consumers' health. Analytical methods for drug residue control are based on LC-MS/MS, but their efficacy can be hindered due to undetectable residual concentrations as a result of low-dosage treatments. Screening methods based on the recognition of indirect biological effects of growth promoters' administration, such as the alteration of protein expression, can improve the efficacy of surveillance. The present study was aimed at identifying modifications in the muscle protein expression pattern between bulls treated with an ear implant (Revalor-XS®) containing trenbolone acetate (200 mg) and estradiol (40 mg), and untreated animals. The analysis of skeletal muscle was carried out using a tandem mass tags shotgun proteomics approach. We defined 28 candidate protein markers with a significantly altered expression induced by steroids administration. A subset of 18 candidate markers was validated by SRM and allowed to build a predictive model based on partial least square discriminant analysis. Our findings confirm the effectiveness of the proteomics approach as potential tool to overcome analytical limitations of drug residue monitoring.

Clinical biochemical and hormonal profiling in plasma: a promising strategy to predict growth hormone abuse in cattle.

Recombinant bovine somatotrophin (rbST) is widely used in some countries to increase milk production. Since 1994, both marketing and use of this substance have been prohibited within the European Union. In this context, the targeted plasma biochemical and hormonal profiling was assessed as a potential screening strategy to highlight rbST (ab)use in cattle. Twenty-one routinely measured clinical blood parameters, representative of main biological profiles (energetic, proteic, etc.), were measured in the plasma of six lactating cows before and after rbST treatment throughout a 23-day study period. Appropriate multivariate statistical analyses [principal component analysis (PCA) and orthogonal partial least square (OPLS)] enabled discriminating animal samples before and after treatment (days 0 vs. 2 to 9, P = 2.10(-9)) and highlighted the five most relevant blood parameters in this discrimination. Based on each five-analyte contribution, a simple mathematically weighted equation was suggested to predict the status of samples. A suspicious threshold was proposed, and the model was further tested with the status prediction of the supplementary samples from untreated (n = 20) and treated cows (n = 22). The calculated false-positive (10%) and false-negative (4.5%) rates were in accordance with the EU requirements for screening methods. Although the model needs to be further validated with additional samples, such targeted plasma biochemical and hormonal profiling already appears as a potential promising screening strategy to highlight rbST (ab)use in cattle.

Confirmation of protein biomarkers of corticosteroids treatment in veal calves sampled under field conditions.

In veal calf production, growth promoters are still illicitly used. Surveillance of misuse of such molecules is necessary to preserve human health. Methods currently adopted for their analysis are based on liquid chromatography-tandem mass spectrometry, but their efficacy can be affected by undetectable residual concentrations in biological matrices due to treatments at low-dosage or based on unknown anabolic compounds. The development of screening methods to identify the indirect biological effects of administration of growth promoters can improve the efficiency of drug residue monitoring. To this purpose, an integrated approach has been used to further validate the set of protein biomarkers defined in a previous controlled study to detect the use of corticosteroids through the changes caused in muscle protein expression. The thymus morphology of 48 samples collected under field conditions was evaluated to assess the presence of potential corticosteroids treatment. Animals were divided on the basis of their thymus characteristics in negative or suspected for illegal corticosteroids treatment. Drug residue analyses were performed on the liver, giving a satisfactory correlation with the histological examination (∼85%). Finally, the proteomics analysis of muscle protein extracts was carried out by 2D differential in gel electrophoresis, and proteins that were differentially expressed between the two animal groups (p value <0.01) were selected for MALDI-MS/MS analysis. This approach allowed us to identify 29 different proteins, and our findings indicate that the altered protein expression pattern can be used as an indirect method for the detection of illicit corticosteroids administration. A subset of the identified proteins was already reported in a previous controlled study, proving that these biomarkers can be used to develop a screening assay to improve the tools currently available for the detection of corticosteroids abuse in bovine meat production.

Evaluation of thymus morphology and serum cortisol concentration as indirect biomarkers to detect low-dose dexamethasone illegal treatment in beef cattle.

BACKGROUND: Corticosteroids are illegally used in several countries as growth promoters in veal calves and beef cattle, either alone or in association with sex steroids and ß-agonists, especially at low dosages and primarily through oral administration, in order to enhance carcasses and meat quality traits. The aim of the present study is to evaluate the reliability of the histological evaluation of the thymus, as well as the serum cortisol determination, in identifying beef cattle, treated with two different dexamethasone-based growth-promoting protocols and the application of different withdrawal times before slaughter. RESULTS: Our findings demonstrate that low dosages of dexamethasone (DXM), administered alone or in association with clenbuterol as growth promoter in beef cattle, induce morphologic changes in the thymus, resulting in increase fat infiltration with concurrent cortical atrophy and reduction of the cortex/medulla ratio (C/M). In fact, the C/M value was significantly lower in treated animals than in control ones, with both the protocols applied. The cut off value of 0.93 for the cortex/medulla ratio resulted to be highly effective to distinguish control and treated animals. The animals treated with DXM showed inhibition of cortisol secretion during the treatment period, as well as at the slaughterhouse, 3 days after treatment suspension. The animals treated with lower doses of DXM in association with clenbuterol, showed inhibition of cortisol secretion during the treatment period, but serum cortisol concentration was restored to physiological levels at slaughterhouse, 8 days after treatment suspension. CONCLUSIONS: The histological evaluation of thymus morphology, and particularly of the C/M may represent a valuable and reproducible method applicable to large-scale screening programs, due to the easy sampling procedures at slaughterhouse, as well as time and cost-saving of the analysis. Serum cortisol determination could be considered as an useful in vivo biomarker of dexamethasone illegal treatment in beef cattle during the fattening period, whilst it does not appear to be a good biomarker at the slaughterhouse, since the protocol of DXM administration, as well as the withdrawal period could affect the reliability of the method.

LC-HRMS-Based Non-Targeted Metabolomics for the Assessment of Honey Adulteration with Sugar Syrups: A Preliminary Study.

Honey is a natural product that is in great demand and has a relatively high price, thus making it one of the most common targets of economically motivated adulteration. Its adulteration can be obtained by adding cheaper honey or sugar syrups or by overfeeding honeybees with sugar syrups. Adulteration techniques are constantly evolving and advanced techniques and instruments are required for its detection. We used non-targeted metabolomics to underscore potential markers of honey adulteration with sugar syrups. The metabolomic profiles of unadulterated honeys and sugar beet, corn and wheat syrups were obtained using hydrophilic interaction liquid chromatography high-resolution mass spectrometry (LC-HRMS). The potential markers have been selected after data processing. Fortified honey (5%, 10% and 20%), honey obtained from overfeeding, and 58 commercial honeys were analyzed. One potential marker appeared with a specific signal for syrups and not for honey. This targeted analysis showed a linear trend in fortified honeys with a calculated limit of quantification around 5% of fortification.

Serum Metabolomics and Proteomics to Study the Antihypertensive Effect of Protein Extracts from Tenebrio molitor.

Hypertension is the leading risk factor for premature death worldwide and significantly contributes to the development of all major cardiovascular disease events. The management of high blood pressure includes lifestyle changes and treatment with antihypertensive drugs. Recently, it was demonstrated that a diet supplemented with Tenebrio molitor (TM) extracts is useful in the management of numerous pathologies, including hypertension. This study is aimed at unveiling the underlying mechanism and the molecular targets of intervention of TM dietary supplementation in hypertension treatment by means of proteomics and metabolomics techniques based on liquid chromatography coupled with high-resolution mass spectrometry. We demonstrate that serum proteome and metabolome of spontaneously hypertensive rats are severely altered with respect to their normotensive counterparts. Additionally, our results reveal that a diet enriched with TM extracts restores the expression of 15 metabolites and 17 proteins mainly involved in biological pathways associated with blood pressure maintenance, such as the renin-angiotensin and kallikrein-kinin systems, serin protease inhibitors, reactive oxygen scavenging, and lipid peroxidation. This study provides novel insights into the molecular pathways that may underlie the beneficial effects of TM, thus corroborating that TM could be proposed as a helpful functional food supplement in the treatment of hypertension.

Protein expression changes in skeletal muscle in response to growth promoter abuse in beef cattle.

The fraudulent treatment of cattle with growth promoting agents (GPAs) is a matter of great concern for the European Union (EU) authorities and consumers. It has been estimated that 10% of animals are being illegally treated in the EU. In contrast, only a much lower percentage of animals (<0.5%) are actually found as being noncompliant by conventional analytical methods. Thus, it has been proposed that methods should be developed that can detect the use of the substances via the biological effects of these substances on target organs, such as the alteration of protein expression profiles. Here we present a study aimed at evaluating if a correlation exists between the treatment with GPAs and alterations in the two-dimensional electrophoresis (2DE) protein pattern obtained from the biceps brachii skeletal muscle from mixed-bred cattle. After image analysis and statistical evaluation, protein spots that differentiate between treated and control groups were selected for analysis by mass spectrometry. A set of proteins could be defined that accurately detect the use of glucocorticoids and ß(2)-agonists as growth promoters through the changes caused in muscle differentiation. As a further validation, we repeated the analysis using an independent set of samples from a strain of pure-bred cattle and verified these proteins by Western blot analysis.

A novel tool to screen for treatments with clenbuterol in bovine: Identification of two hepatic markers by metabolomics investigation.

Surveillance of illegal use of growth promoters such as ß2-agonists in food producing animals rely on the detection of drug residues by LC-MS/MS. Screening strategies focusing on indirect physiological responses following administration of active compounds are promising approaches to strengthen existing targeted methods and ensure food safety. A metabolomics analysis based on LC-HRMS was carried out on liver extracts from bulls experimentally treated with clenbuterol combined with dexamethasone (n = 8) to mimic a potential anabolic practice, and control animals (n = 8). Nicotinic acid and 5'-deoxy-5'-methylthioadenosine were identified as biomarkers of treatment. Ratio values of such markers to others of the same metabolic pathways (nicotinamide or methionine) were used to develop a classification model to assign animals as treated with clenbuterol or non-treated. The classification model was tested on an external validation set comprising 74 animals either treated with different anabolic compounds (ß2-agonists, sexual steroids, corticosteroid), or non-treated, showing 100% sensitivity and specificity.

Fast and simultaneous analysis of carbamate pesticides and anticoagulant rodenticides used in suspected cases of animal poisoning.

Carbamate pesticides (CBs) are reported as one of the main causes of intentional or accidental poisoning of animals. Anticoagulant rodenticides (ARs) form the main class of poisons implicated in analyzed poisoned baits. These two groups of pesticide compounds include multiple substances, and thus, the development of a simple and rapid multiclass/multiresidue analytical method for simultaneous identification of both toxicant classes should be a useful strategy for analytical laboratories to reduce analysis time and cost. The present study aimed to elaborate and validate a rapid method to simultaneously determine 11 CBs and 8 ARs in samples of real matrices (bait, stomach content, and liver) from suspected animal poisoning cases. QuEChERS sample treatment and liquid chromatography coupled to hybrid high resolution mass spectrometry were used. The method resulted in good linearity (R2 ≥ 0.98) for all compounds, recovery was between 70% and 120% for CBs and 40-90% for ARs, and precision was ≤ 20% for all compounds. The method was successfully applied to the analysis of 871 real samples originating from suspected cases of animal poisoning, collected from April 2019 to October 2020. Furthermore, full scan dependent data acquisition allowed qualitative retrospective data analysis of an additional 15 compounds outside the scope of the method to be performed; these compounds could potentially be involved in unresolved poisoning cases.

Bioaccumulation and in vivo formation of titanium dioxide nanoparticles in edible mussels.

The aim of this study was to evaluate the bioaccumulation of titanium dioxide nanoparticles (TiO2NPs) in edible mussels bred in polluted artificial seawater. An in vivo study was conducted by exposing mussels to different concentrations of TiO2NPs (0.25 mg/L and 2.5 mg/L) or ionic titanium (1.6 mg/L) for 4 days. Inductively coupled plasma mass spectrometry (ICP-MS) showed titanium presence in all groups proportional to exposure levels (concentration range: 209-1119 µg/kg). Single particle ICP-MS revealed NPs in both TiO2NP treated mussels (concentration range: 231-1778 µg/kg) and in ionic titanium treated mussels (concentration 1574 µg/kg), suggesting potential nanoparticle formation in vivo. These results were confirmed by transmission electron microscopy with energy dispersive X-ray detection. Nonetheless, mussels eliminated more than 70% of the TiO2NPs after 3 days' depuration. These results show the potential for consumer exposure to TiO2NPs when contaminated mussels are consumed without a proper depuration process.

LC-HRMS/MS for the simultaneous determination of four allergens in fish and swine food products.

The inclusion on the label of packed foods of any ingredient or technological adjuvant causing allergies is required by EU food legislation. In this study a targeted proteomics method for detecting four allergens in animal-derived food matrices was developed. Liquid chromatography coupled with high-resolution tandem mass spectrometry (LC-HRMS/MS) was used to select marker peptides from four allergens and develop a quantitative method able to simultaneously detect the presence of milk, egg, crustaceans and soy. The method was validated on fish or swine processed food products contaminated at 5 µg g-1 for milk and egg and 10 µg g-1 for soy and crustaceans. The method was tested by analyzing commercial food products with high protein content and was compared to the ELISA technique. Our results indicated the presence of soy not reported on the food label of some products, pointing out the need for efficient controls to protect allergic consumers.

Evaluation and quantification of antimicrobial residues and antimicrobial resistance genes in two Italian swine farms.

Antimicrobial resistance genes (ARGs) are considered emerging environmental pollutants, posing potential risks for human and animal health: the misuse of antimicrobials in food-producing animals could favour the maintenance and spread of resistances in bacteria. The occurrence of ARGs in Italian swine farming - which has specific characteristics - was investigated in order to explore resistance spread dynamics. Two farrow-to-finish pig farms were longitudinally monitored: faecal samples from animals and environmental samples were collected. DNA was extracted and tetA, ermB, qnrS and mcr1 ARGs were analysed by qPCR for their ability to confer resistance to highly or critically important antimicrobials (CIAs). Moreover, 16SrDNA gene was analysed to assess bacterial abundance. ermB and tetA genes were found in animal samples and manure samples. On the contrary, mcr1 was exclusively found in weaners, while qnrS occurred in all animal categories but sows and finishers. Among the analysed genes, ermB and tetA showed the highest absolute and relative abundances. Our results indicate that ermB and tetA ARGs are widely disseminated in the explored farms, suggesting efficient maintenance among bacteria and persistence in the environment. Interestingly, the presence of qnrS and mcr1, limited to just a few animal categories, highlights inefficient dissemination of these genes in the farm environment, in particular for mcr1, a stable plasmid gene conferring resistance to the last-resort antimicrobial, colistin. Paying close attention only to the finishing phase would have hampered the discovery of resistances to CIAs at farm level, which we instead identified thanks to an intensive longitudinal monitoring programme.

The Prion Protein Regulates Synaptic Transmission by Controlling the Expression of Proteins Key to Synaptic Vesicle Recycling and Exocytosis.

The cellular prion protein (PrPC), whose misfolded conformers are implicated in prion diseases, localizes to both the presynaptic membrane and postsynaptic density. To explore possible molecular contributions of PrPC to synaptic transmission, we utilized a mass spectrometry approach to quantify the release of glutamate from primary cerebellar granule neurons (CGN) expressing, or deprived of (PrP-KO), PrPC, following a depolarizing stimulus. Under the same conditions, we also tracked recycling of synaptic vesicles (SVs) in the two neuronal populations. We found that in PrP-KO CGN these processes decreased by 40 and 60%, respectively, compared to PrPC-expressing neurons. Unbiased quantitative mass spectrometry was then employed to compare the whole proteome of CGN with the two PrP genotypes. This approach allowed us to assess that, relative to the PrPC-expressing counterpart, the absence of PrPC modified the protein expression profile, including diminution of some components of SV recycling and fusion machinery. Subsequent quantitative RT-PCR closely reproduced proteomic data, indicating that PrPC is committed to ensuring optimal synaptic transmission by regulating genes involved in SV dynamics and neurotransmitter release. These novel molecular and cellular aspects of PrPC add insight into the underlying mechanisms for synaptic dysfunctions occurring in neurodegenerative disorders in which a compromised PrPC is likely to intervene.

Semiquantitative immunohistochemical detection of progesterone receptors in male accessory sex glands as a screening assay for anabolic steroid use in bulls.

We investigated the immunohistochemical expression of progesterone receptors (PRs) in the prostate and bulbourethral glands of thirty-two 10-14-mo-old Charolais bulls following treatment with a low dosage of estrogens. Animals were divided into 2 groups: 16 animals (group T) were treated for 71 d with a therapeutic dose of trenbolone acetate and estradiol by subcutaneous implant, 16 animals (group C) received no treatment. Urine samples were collected both at the beginning of the trial and 9 times during the study. A semiquantitative analysis of immunohistochemistry (IHC) was performed by counting the number of positive cells in 10 randomly selected high-power fields (hpf). Both groups showed no significant histologic lesions. IHC examination showed positive cells in the epithelium of both glands, with different patterns of distribution between groups. In group C, IHC-positive cells per hpf varied from 0 to 40 in the prostate and from 0 to 32 in the bulbourethral gland. In group T, positive cells varied from 0 to 85 per hpf in the prostate and from 0 to 75 in the bulbourethral gland. The treated group showed significantly higher median numbers of positively stained cells in both organs than the controls ( p < 0.001). Chemical analysis of the urine samples confirmed that the experimental treatment mimics continuous, low-dose administration of anabolic steroids. IHC quantification showed good sensitivity with a high predictive power to correctly classify treated animals and could be used as a preliminary screening test in bulls.

Abuse of anabolic agents in beef cattle: Could bile be a possible alternative matrix?

European Union prohibited the use of anabolic agents in food producing animals since 1988. An efficient control of abuses is guaranteed not only by highly performing analytical methods, but also by knowledge of metabolic pathways, kinetics of elimination and tissue distribution. To obtain data concerning metabolites production and accumulation in bile, two typical growth promoting treatments are carried out in cattle. In the first study, sixteen beef cattle were implanted with trenbolone acetate and estradiol. In the second one, three animals were implanted with zeranol and three were fed a diet containing zearalenone. Methods based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) were developed and validated to quantify the analytes of interest. The results evidenced that the biliary concentrations of the marker residues were always higher than those determined at the same time in urine and liver which are the matrices generally collected within the official monitoring programmes.

Development and validation of a QuEChERS method coupled to liquid chromatography and high resolution mass spectrometry to determine pyrrolizidine and tropane alkaloids in honey.

Awareness about pyrrolizidine alkaloids (PAs) and tropane alkaloids (TAs) in food was recently raised by the European Food Safety Authority stressing the lack of data and gaps of knowledge required to improve the risk assessment strategy. The present study aimed at the elaboration and validation of a method to determine PAs and TAs in honey. QuEChERS sample treatment and liquid chromatography coupled to hybrid high resolution mass spectrometry, were used. The method resulted in good linearity (R2>0.99) and low limits of detection and quantification, ranging from 0.04 to 0.2µgkg-1 and from 0.1 to 0.7µgkg-1 respectively. Recoveries ranged from 92.3 to 114.8% with repeatability lying between 0.9 and 15.1% and reproducibility between 1.1 and 15.6%. These performances demonstrate the selectivity and sensitivity of the method for simultaneous trace detection and quantification of PAs and TAs in honey, verified through the analysis of forty commercial samples.