Regucalcin Expression Profiles in Veal Calf Testis: Validation of Histological and Molecular Tests to Detect Sex Steroids Illicit Administration

Overview

Journal PeerJ

Specialties Biology
Environmental Health
General Medicine

Date 2021 Mar 1

PMID 33643712

Citations 1

Authors

Alessandro Benedetto

Elena Biasibetti

Chiara Beltramo

Valentina Audino

Simone Peletto

Elena Maria Bozzetta

Marzia Pezzolato

Affiliations

Soon will be listed here.

Abstract

Background: Sex steroids administration in meat producing animals is forbidden within the EU to preserve consumers' safety, but continuous monitoring to identify resurgence of their misuse is needed. Among biomarkers related to sex steroids abuse in veal calves the regucalcin (RGN) mRNA perturbations in testis have been described in RNA samples. To setup novel diagnostic method, to update current tests available in National Residue Control Plans (NRCPs) and in legal dispute when illicit practices on farm animals are suspected, the reliability of RGN profiling was assessed by histological and molecular techniques.

Methods: Formalin fixed paraffin embedded (FFPE) testis samples, chosen being the most effective preservation strategy adopted by histological NRCPs and allowing easier retrospective analysis if required by legal disputes, were analyzed from veal calves treated with nandrolone, 17β-estradiol and a cocktail of the two hormones. RGN levels were determined by quantitative Real Time PCR and Immunohistochemistry assays. Test performances were assessed and compared by multiple ROC curves.

Results: Both tests resulted sensitive and specific, allowing to enrich, in future field investigation, novel integrated diagnostic protocols needed to unveil sex steroid abuse.

Discussion: Developed RT-qPCR and IHC methods confirmed RGN as a useful and robust biomarker to detect illegal administration of sex steroid hormones in veal calves. The developed methods, successfully applied to ten years old FFPE blocks, could allow both retrospective analysis, when supplementary investigations are requested by authorities, and future implementation of current NRCPs.

Citing Articles

Transcriptional Biomarkers and Immunohistochemistry for Detection of Illicit Dexamethasone Administration in Veal Calves.

Benedetto A, Biasibetti E, Robotti E, Marengo E, Audino V, Bozzetta E Foods. 2022; 11(12).

PMID: 35742008 PMC: 9222442. DOI: 10.3390/foods11121810.

References

Casiraghi E, Huber V, Frasca M, Cossa M, Tozzi M, Rivoltini L . A novel computational method for automatic segmentation, quantification and comparative analysis of immunohistochemically labeled tissue sections. BMC Bioinformatics. 2018; 19(Suppl 10):357. PMC: 6191943. DOI: 10.1186/s12859-018-2302-3. View

Yamaguchi M, Yamamoto T . Purification of calcium binding substance from soluble fraction of normal rat liver. Chem Pharm Bull (Tokyo). 1978; 26(6):1915-8. DOI: 10.1248/cpb.26.1915. View

Maxim L, Niebo R, Utell M . Screening tests: a review with examples. Inhal Toxicol. 2014; 26(13):811-28. PMC: 4389712. DOI: 10.3109/08958378.2014.955932. View

Laurell H, Iacovoni J, Abot A, Svec D, Maoret J, Arnal J . Correction of RT-qPCR data for genomic DNA-derived signals with ValidPrime. Nucleic Acids Res. 2012; 40(7):e51. PMC: 3326333. DOI: 10.1093/nar/gkr1259. View

Ribeiro-Silva A, Zhang H, Jeffrey S . RNA extraction from ten year old formalin-fixed paraffin-embedded breast cancer samples: a comparison of column purification and magnetic bead-based technologies. BMC Mol Biol. 2007; 8:118. PMC: 2233637. DOI: 10.1186/1471-2199-8-118. View

Irwin R, Irwin T . A principled approach to setting optimal diagnostic thresholds: where ROC and indifference curves meet. Eur J Intern Med. 2011; 22(3):230-4. DOI: 10.1016/j.ejim.2010.12.012. View

Benedetto A, Pezzolato M, Beltramo C, Audino V, Ingravalle F, Pillitteri C . Real-time PCR assay for detecting illicit steroid administration in veal calves allows reliable biomarker profiling of formalin-fixed, paraffin-embedded (FFPE) archival tissue samples. Food Chem. 2020; 312:126061. DOI: 10.1016/j.foodchem.2019.126061. View

Bjorkman J, Svec D, Lott E, Kubista M, Sjoback R . Differential amplicons (ΔAmp)-a new molecular method to assess RNA integrity. Biomol Detect Quantif. 2016; 6:4-12. PMC: 4822209. DOI: 10.1016/j.bdq.2015.09.002. View

Cappallo-Obermann H, Schulze W, Jastrow H, Baukloh V, Spiess A . Highly purified spermatozoal RNA obtained by a novel method indicates an unusual 28S/18S rRNA ratio and suggests impaired ribosome assembly. Mol Hum Reprod. 2011; 17(11):669-78. DOI: 10.1093/molehr/gar037. View

10.

Wimmer I, Troscher A, Brunner F, Rubino S, Bien C, Weiner H . Systematic evaluation of RNA quality, microarray data reliability and pathway analysis in fresh, fresh frozen and formalin-fixed paraffin-embedded tissue samples. Sci Rep. 2018; 8(1):6351. PMC: 5910432. DOI: 10.1038/s41598-018-24781-6. View

11.

. Scientific Opinion on the public health hazards to be covered by inspection of meat from sheep and goats. EFSA J. 2020; 11(6):3265. PMC: 7163758. DOI: 10.2903/j.efsa.2013.3265. View

12.

Zeka F, Vanderheyden K, Smet E, Cuvelier C, Mestdagh P, Vandesompele J . Straightforward and sensitive RT-qPCR based gene expression analysis of FFPE samples. Sci Rep. 2016; 6:21418. PMC: 4761903. DOI: 10.1038/srep21418. View

13.

Pezzolato M, Botta M, Baioni E, Richelmi G, Pitardi D, Varello K . Confirmation of the progesterone receptor as an efficient marker of treatment with 17β-estradiol in veal calves. Food Addit Contam Part A Chem Anal Control Expo Risk Assess. 2015; 33(1):60-5. DOI: 10.1080/19440049.2015.1107918. View

14.

Kap M, Sieuwerts A, Kubista M, Oomen M, Arshad S, Riegman P . The influence of tissue procurement procedures on RNA integrity, gene expression, and morphology in porcine and human liver tissue. Biopreserv Biobank. 2015; 13(3):200-6. DOI: 10.1089/bio.2014.0076. View

15.

Zuker M . Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res. 2003; 31(13):3406-15. PMC: 169194. DOI: 10.1093/nar/gkg595. View

16.

Livak K, Schmittgen T . Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 2002; 25(4):402-8. DOI: 10.1006/meth.2001.1262. View

17.

Guglielmetti C, Mazza M, Pagano M, Carrella S, Sciuto S, Nodari S . Identification by a proteomic approach of a plasma protein as a possible biomarker of illicit dexamethasone treatment in veal calves. Food Addit Contam Part A Chem Anal Control Expo Risk Assess. 2014; 31(5):833-8. DOI: 10.1080/19440049.2014.900191. View

18.

Qin J, Zhang B . Best combination of multiple diagnostic tests for screening purposes. Stat Med. 2010; 29(28):2905-19. DOI: 10.1002/sim.4068. View

19.

Starvaggi Cucuzza L, Divari S, Mulasso C, Biolatti B, Cannizzo F . Regucalcin expression in bovine tissues and its regulation by sex steroid hormones in accessory sex glands. PLoS One. 2014; 9(11):e113950. PMC: 4240664. DOI: 10.1371/journal.pone.0113950. View

20.

Laurentino S, Correia S, Cavaco J, Oliveira P, de Sousa M, Barros A . Regucalcin, a calcium-binding protein with a role in male reproduction?. Mol Hum Reprod. 2011; 18(4):161-70. DOI: 10.1093/molehr/gar075. View