The use of anabolic-androgenic steroids (AASs) as growth promoters in farm animals is banned in the European Union, representing both an illicit practice and a risk for consumer health. However, these compounds are still illegally administered, often in the form of synthetic esters. This work aimed to characterize significant coding RNA perturbations related to the illicit administration of testosterone and nandrolone esters in fattening pigs. A total of 27 clinically healthy 90-day-old pigs were randomly assigned to test and control groups. Nine animals were treated with testosterone esters (Sustanon®) and other nine with nandrolone esters (Myodine®). At the end of the trial, liver samples were collected and analyzed using RNAseq, allowing the identification of 491 differentially expressed genes (DEGs). The transcriptional signature was further characterized by a smaller sub-cluster of 143 DEGs, from which a selection of 16 genes was made. The qPCR analysis confirmed that the identified cluster could still give good discrimination between untreated gilt and barrows compared to the relative testosterone-treated counterparts. A conclusive field survey on 67 liver samples collected from pigs of different breeds and weight categories confirmed, in agreement with testosterone residue profiles, the specificity of selected transcriptional biomarkers, showing their potential applications for screening purposes when AAS treatment is suspected, allowing to focus further investigations of competent authorities and confirmatory analysis where needed.

Department of Infectious Diseases and Preventive Medicine Veterinary Research Institute 621 00 Brno Czech Republic

Dipartimento di Biotecnologie e Scienze della Salute Core Lab di Bioinformatica e Genomica Università degli Studi di Torino 10124 Turin Italy

Dipartimento di Medicina Veterinaria Università degli Studi di Bari Aldo Moro 70121 Bari Italy

Istituto Zooprofilattico Sperimentale del Piemonte Liguria e Valle d'Aosta 10154 Turin Italy

Istituto Zooprofilattico Sperimentale della Sicilia 90129 Palermo Italy

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Nachman K.E., Smith T.J.S. Hormone Use in Food Animal Production: Assessing Potential Dietary Exposures and Breast Cancer Risk. Curr. Environ. Heal. Rep. 2015;2:1–14. doi: 10.1007/s40572-014-0042-8. PubMed DOI

Qaid M.M., Abdoun K.A. Safety and concerns of hormonal application in farm animal production: A review. J. Appl. Anim. Res. 2022;50:426–439. doi: 10.1080/09712119.2022.2089149. DOI

Passantino A. A Bird’s-Eye View of Veterinary Medicine. IntechOpen; London, UK: 2012. Steroid Hormones in Food Producing Animals.

European Food Safety Authority Report for 2018 on the results from the monitoring of veterinary medicinal product residues and other substances in live animals and animal products. EFSA Support. Publ. 2020;17:1775E. doi: 10.2903/sp.efsa.2020.en-1775. DOI

European Food Safety Authority Report for 2019 on the results from the monitoring of veterinary medicinal product residues and other substances in live animals and animal products. EFSA Support. Publ. 2021;18:1997E. doi: 10.2903/sp.efsa.2021.en-1997. DOI

Marclay F., Mangin P., Margot P., Saugy M. Perspectives for Forensic Intelligence in anti-doping: Thinking outside of the box. Forensic Sci. Int. 2013;229:133–144. doi: 10.1016/j.forsciint.2013.04.009. PubMed DOI

EUROPOL Keeping Sport Safe and Fair: 3.8 Million Doping Substances and Fake Medicines Seized Worldwide. [(accessed on 11 June 2023)]. Available online: https://www.europol.europa.eu/media-press/newsroom/news/keeping-sport-safe-and-fair-38-million-doping-substances-and-fake-medicines-seized-worldwide.

Gadaj A., Ventura E., Ripoche A., Mooney M.H. Monitoring of selective androgen receptor modulators in bovine muscle tissue by ultra-high performance liquid chromatography-tandem mass spectrometry. Food Chem. X. 2019;4:100056. doi: 10.1016/j.fochx.2019.100056. PubMed DOI PMC

Gheddar L., Ameline A., Raul J.S., Kintz P. Designer anabolic steroids: A challenge for toxicologists. Toxicol. Anal. Clin. 2019;31:293–297. doi: 10.1016/j.toxac.2019.07.001. DOI

Poelmans S., De Wasch K., Noppe H., Van Hoof N., Van Cruchten S., Le Bizec B., Deceuninck Y., Sterk S., Van Rossum H.J., Hoffman M.K., et al. Endogenous occurrence of some anabolic steroids in swine matrices. Food Addit. Contam. 2005;22:808–815. doi: 10.1080/02652030500197805. PubMed DOI

Hartmann S., Lacorn M., Steinhart H. Natural occurrence of steroid hormones in food. Food Chem. 1998;62:7–20. doi: 10.1016/S0308-8146(97)00150-7. DOI

Nielen M.W.F., Nijrolder A.W.J.M., Hooijerink H., Stolker A.A.M. Feasibility of desorption electrospray ionization mass spectrometry for rapid screening of anabolic steroid esters in hair. Anal. Chim. Acta. 2011;700:63–69. doi: 10.1016/j.aca.2010.08.009. PubMed DOI

Janssens G., Courtheyn D., Mangelinckx S., Prévost S., Bichon E., Monteau F., De Poorter G., De Kimpe N., Le Bizec B. Use of isotope ratio mass spectrometry to differentiate between endogenous steroids and synthetic homologues in cattle: A review. Anal. Chim. Acta. 2013;772:1–15. doi: 10.1016/j.aca.2012.12.035. PubMed DOI

Pezzolato M., Baioni E., Maurella C., Benedetto A., Biasibetti E., Bozzetta E. The Italian strategy to fight illegal treatment with growth promoters: Results of the 2017–2019 histological monitoring plan. Ital. J. Food Saf. 2022:10. doi: 10.4081/ijfs.2021.9775. PubMed DOI PMC

Benedetto A., Pezzolato M., Biasibetti E., Bozzetta E. Omics applications in the fight against abuse of anabolic substances in cattle: Challenges, perspectives and opportunities. Curr. Opin. Food Sci. 2021;40:112–120. doi: 10.1016/j.cofs.2021.03.001. DOI

Skoupá K., Šťastný K., Sládek Z. Anabolic Steroids in Fattening Food-Producing Animals—A Review. Animals. 2022;12:2115. doi: 10.3390/ani12162115. PubMed DOI PMC

Wu Y., Bi Y., Bingga G., Li X., Zhang S., Li J., Li H., Ding S., Xia X. Metabolomic analysis of swine urine treated with β2-agonists by ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry. J. Chromatogr. A. 2015;1400:74–81. doi: 10.1016/j.chroma.2015.04.050. PubMed DOI

Li G., Fu Y., Han X., Li X., Li C. Metabolomic investigation of porcine muscle and fatty tissue after Clenbuterol treatment using gas chromatography/mass spectrometry. J. Chromatogr. A. 2016;1456:242–248. doi: 10.1016/j.chroma.2016.06.017. PubMed DOI

Stastny K., Putecova K., Leva L., Franek M., Dvorak P., Faldyna M. Profiling of metabolomic changes in plasma and urine of pigs caused by illegal administration of testosterone esters. Metabolites. 2020;10:307. doi: 10.3390/metabo10080307. PubMed DOI PMC

Peng T., Royer A.L., Guitton Y., Le Bizec B., Dervilly-Pinel G. Serum-based metabolomics characterization of pigs treated with ractopamine. Metabolomics. 2017;13:77. doi: 10.1007/s11306-017-1212-0. DOI

Beccuti M., Cordero F., Arigoni M., Panero R., Amparore E.G., Donatelli S., Calogero R.A. SeqBox: RNAseq/ChIPseq reproducible analysis on a consumer game computer. Bioinformatics. 2018;34:871–872. doi: 10.1093/bioinformatics/btx674. PubMed DOI PMC

Love M.I., Huber W., Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 2014;15:550. doi: 10.1186/s13059-014-0550-8. PubMed DOI PMC

Raudvere U., Kolberg L., Kuzmin I., Arak T., Adler P., Peterson H., Vilo J. G:Profiler: A web server for functional enrichment analysis and conversions of gene lists (2019 update) Nucleic Acids Res. 2019;47:W191–W198. doi: 10.1093/nar/gkz369. PubMed DOI PMC

Subramanian A., Tamayo P., Mootha V.K., Mukherjee S., Ebert B.L., Gillette M.A., Paulovich A., Pomeroy S.L., Golub T.R., Lander E.S., et al. Gene set enrichment analysis: A knowledge-based approach for interpreting genome-wide expression profiles. Proc. Natl. Acad. Sci. USA. 2005;102:15545–15550. doi: 10.1073/pnas.0506580102. PubMed DOI PMC

Grätz C., Bui M.L.U., Thaqi G., Kirchner B., Loewe R.P., Pfaffl M.W. Obtaining Reliable RT-qPCR Results in Molecular Diagnostics—MIQE Goals and Pitfalls for Transcriptional Biomarker Discovery. Life. 2022;12:386. doi: 10.3390/life12030386. PubMed DOI PMC

Vandesompele J., De Preter K., Pattyn F., Poppe B., Van Roy N., De Paepe A., Speleman F. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol. 2002;3:1–12. doi: 10.1186/gb-2002-3-7-research0034. PubMed DOI PMC

Markham N.R. Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res. 2003;31:3406–3415. PubMed PMC

Bustin S.A., Benes V., Garson J.A., Hellemans J., Huggett J., Kubista M., Mueller R., Nolan T., Pfaffl M.W., Shipley G.L., et al. The MIQE guidelines: Minimum information for publication of quantitative real-time PCR experiments. Clin. Chem. 2009;55:611–622. doi: 10.1373/clinchem.2008.112797. PubMed DOI

Pezzolato M., Biasibetti E., Gili M., Maurella C., Benedetto A., Marturella M., Ostorero F., Bozzo G., Bellino C., D’angelo A., et al. Assessment of the Physiological Values and the Reference Histological Profile Related to Sex Steroids in Veal Calves. Agriculture. 2023;13:1145. doi: 10.3390/agriculture13061145. DOI

Tereszkiewicz K., Kulig Ł., Antos P., Kowalczyk K. Influence of the Level of Sex Hormones in the Blood of Gilts on Slaughter Characteristics and Meat Quality. Animals. 2023;13:267. doi: 10.3390/ani13020267. PubMed DOI PMC

Kress K., Weiler U., Schmucker S., Čandek-Potokar M., Vrecl M., Fazarinc G., Škrlep M., Batorek-Lukač N., Stefanski V. Influence of housing conditions on reliability of immunocastration and consequences for growth performance of male pigs. Animals. 2020;10:27. doi: 10.3390/ani10010027. PubMed DOI PMC

Arrizabalaga Larranaga A., Groot M.J., Blokland M.H., Barbu I.M., Smits N.G.E., Sterk S.S. EURL Reflection Paper 2.0: Natural Growth Promoting Substances in Biological Samples: Presence—and Formation—of Hormones and Other Growth Promoting Substances in Food Producing Animals. 2022. [(accessed on 11 June 2023)]. Available online: https://edepot.wur.nl/606740.

Stella R., Bovo D., Mastrorilli E., Manuali E., Pezzolato M., Bozzetta E., Lega F., Angeletti R., Biancotto G. A Novel Tool to Screen for Treatments with Clenbuterol in Bovine: Identification of two Hepatic Markers by Metabolomics Investigation. Food Chem. 2021;353:129366. doi: 10.1016/j.foodchem.2021.129366. PubMed DOI

De Wasch K., Le Bizec B., De Brabander H., Andr F., Impens S. Consequence of boar edible tissue consumption on urinary profiles of nandrolone metabolites. II. Identification and quantification of 19-norsteroids responsible for 19-norandrosterone and 19-noretiocholanolone excretion in human urine. Rapid Commun. Mass Spectrom. 2001;15:1442–1447. doi: 10.1002/rcm.391. PubMed DOI

Hülsemann F., Fußhöller G., Lehn C., Thevis M. Excretion of 19-norandrosterone after consumption of boar meat. Drug Test. Anal. 2020;12:1581–1586. doi: 10.1002/dta.2958. PubMed DOI

Elgendy R., Giantin M., Montesissa C., Dacasto M. The transcriptome of muscle and liver is responding differently to a combined trenbolone acetate and estradiol implant in cattle. Steroids. 2016;106:1–8. doi: 10.1016/j.steroids.2015.11.002. PubMed DOI

Riedmaier I., Pfaffl M.W., Meyer H.H.D. The physiological way: Monitoring RNA expression changes as new approach to combat illegal growth promoter application. Drug Test. Anal. 2012;4:70–74. doi: 10.1002/dta.1386. PubMed DOI

Benedetto A., Pezzolato M., Robotti E., Biasibetti E., Poirier A., Dervilly G., Le Bizec B., Marengo E., Bozzetta E. Profiling of transcriptional biomarkers in FFPE liver samples: PLS-DA applications for detection of illicit administration of sex steroids and clenbuterol in veal calves. Food Control. 2021;128:108149. doi: 10.1016/j.foodcont.2021.108149. DOI

Benedetto A., Biasibetti E., Robotti E., Marengo E., Audino V., Bozzetta E., Pezzolato M. Transcriptional Biomarkers and Immunohistochemistry for Detection of Illicit Dexamethasone Administration in Veal Calves. Foods. 2022;11:1810. doi: 10.3390/foods11121810. PubMed DOI PMC

Zhao L., Yang S., Cheng Y., Hou C., You X., Zhao J., Zhang Y., He W. Identification of transcriptional biomarkers by RNA-sequencing for improved detection of β2-agonists abuse in goat skeletal muscle. PLoS ONE. 2017;12:e0181695. doi: 10.1371/journal.pone.0181695. PubMed DOI PMC

Dervilly-Pinel G., Royer A.L., Bozzetta E., Pezzolato M., Herpin L., Prevost S., Le Bizec B. When LC-HRMS metabolomics gets ISO17025 accredited and ready for official controls–application to the screening of forbidden compounds in livestock. Food Addit. Contam.—Part A Chem. Anal. Control. Expo. Risk Assess. 2018;35:1948–1958. doi: 10.1080/19440049.2018.1496280. PubMed DOI

Directorate-General for Health and Food . Safety Guidelines on EU Requirements for Entry of Animals and Products of Animal Origin Control Plans for Residues of Veterinary Medicines, Pesticides and Contaminants. Directorate-General for Health and Food; Brussel, Belgium: 2023.

Farschtschi S., Riedmaier-Sprenzel I., Phomvisith O., Gotoh T., Pfaffl M.W. The successful use of -omic technologies to achieve the ‘One Health’ concept in meat producing animals. Meat Sci. 2022;193:108949. doi: 10.1016/j.meatsci.2022.108949. PubMed DOI