The Potential of Bovine Vaginal Smear for Biomarker Development to Trace the Misuse of Anabolic Agents

Abstract

In the European Union the use of anabolic hormones in meat production is forbidden since 1988 and this ban of anabolic agents in animal production is strictly controlled. New hormone cocktails passing the detection systems are attractive for the practice and so new approaches to discover their illegal use have to be developed steadily. Verifying physiological effects caused by anabolic steroids will be a new way to develop potential monitoring systems. One promising matrix in female animals will be vaginal smear containing vaginal epithelial cells, because the vaginal epithelium is a primary steroid hormone responsive organ. In this study we quantified the gene expression in vaginal smear of sexually mature cattle in order to observe physiological effects. Further we aimed to establish a new screening method by testing the effect of a combination of certain anabolic steroid hormones on physiological regulations of mRNA expression of selected genes. In an animal trial Nguni heifers were treated with the anabolic combination trenbolone acetate plus estradiol. Vaginal smear samples were taken at 4 different time points. Gene expression of 27 candidate genes, selected by screening the actual literature for steroidal effects on vaginal epithelial cells, were estimated using quantitative real-time RT-PCR. There were different expression changes observed at different time points. It could be shown that the applied anabolic combination significantly influenced the expression of the steroid receptor ERα, the keratinization factor CK8, the proinflammatory interleukins IL-1α and IL-1β, the growth factors FGF7, EGF, EGFR, IGF-1R, TGFα and LTF, the oncogen c-jun and other factors like actinβ and ubiquitin 3. Using biostatistical tools like principal components analysis or hierarchical cluster analysis, the potential to develop a gene expression pattern for targeting the illegal use of growth promoters could be demonstrated.

anabolic agents - trenbolone acetate - estradiol - gene expression biomarker - real-time RT-qPCR

References

• 1 Andersson AM, Skakkebaek NE. Exposure to exogenous estrogens in food: possible impact on human development and health.  European Journal of Endocrinology. 1999;  140 477-485
• 2 Becker C, Riedmaier I, Reiter M. et al . Effect of trenbolone acetate plus estradiol on transcriptional regulation of metabolism pathways in bovine liver.  Hormone Molecular Biology and Clinical Investigation. 2010;  2 257-265
• 3 Cantiello M, Carletti M, Cannizzo FT. et al . Effects of an illicit cocktail on serum immunoglobulins, lymphocyte proliferation and cytokine gene expression in the veal calf.  Toxicology. 2007;  242 39-51
• 4 Cohen MS, Britigan BE, French M. et al . Preliminary observations on lactoferrin secretion in human vaginal mucus: variation during the menstrual cycle, evidence of hormonal regulation, and implications for infection with Neisseria gonorrhoeae.  American Journal of Obstetrics and Gynecology. 1987;  157 1122-1125
• 5 Daxenberger A, Ibarreta D, Meyer HHD. Possible health impact of animal oestrogens in food.  Human Reproduction Update. 2001;  7 340-355
• 6 Edgren RA. Oestrogen antagonisms: the effects of testosterone propionate and 17-ethyl-19-nortestosterone on oestrone-induced changes in vaginal cytology.  Acta Endocrinology. 1957;  25 365-370
• 7 Edgren RA. The modification of estrogen-induced changes in rat vaginas with steroids and related agents.  Annals of the New York Academy of Sciences. 1959;  83 160-184
• 8 Henricks DM, Dickey JF, Hill JR. Plasma estrogen and progesterone levels in cows prior to and during estrus.  Endocrinology. 1971;  89 1350-1355
• 9 Hom YK, Young P, Thomson AA. et al . Keratinocyte growth factor injected into female mouse neonates stimulates uterine and vaginal epithelial growth.  Endocrinology. 1998a;  139 3772-3779
• 10 Hom YK, Young P, Wiesen JF. et al . Uterine and vaginal organ growth requires epidermal growth factor receptor signaling from stroma.  Endocrinology. 1998b;  139 913-921
• 11 Iguchi T, Uchima FD, Bern HA. Growth of mouse vaginal epithelial cells in culture: effect of sera and supplemented serum-free media.  In Vitro Cell Development Biology. 1987;  23 535-540
• 12 Iguchi T, Uchima FD, Ostrander PL. et al . Growth of normal mouse vaginal epithelial cells in and on collagen gels.  Proceedings of the National Academy of Sciences. 1983;  80 3743-3747
• 13 Ing NH. Steroid hormones regulate gene expression posttranscriptionally by altering the stabilities of messenger RNAs.  Biology of Reproduction. 2005;  72 1290-1296
• 14 Kang JS, Lee BJ, Ahn B. et al . Expression of estrogen receptor alpha and beta in the uterus and vagina of immature rats treated with 17-ethinyl estradiol.  Journal of Veterinary Medical Science. 2003;  65 1293-1297
• 15 Kelver ME, Kaul A, Nowicki B. et al . Estrogen regulation of lactoferrin expression in human endometrium.  American Journal of Reproductive Immunology. 1996;  36 243-247
• 16 Kronenberg MS, Clark JH. Changes in keratin expression during the estrogen-mediated differentiation of rat vaginal epithelium.  Endocrinology. 1985;  117 1480-1489
• 17 Kubista M, Andrade JM, Bengtsson M. et al . The real-time polymerase chain reaction.  Molecular Aspects of Medicine. 2006;  27 95-125
• 18 Lange IG, Daxenberger A, Meyer HH. Hormone contents in peripheral tissues after correct and off-label use of growth promoting hormones in cattle: effect of the implant preparations Filaplix-H, Raglo, Synovex-H and Synovex Plus.  APMIS. 2001;  109 53-65
• 19 Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method.  Methods. 2001;  25 402-408
• 20 Meyer HHD. Biochemistry and physiology of anabolic hormones used for improvement of meat production.  APMIS. 2001;  109 1-8
• 21 Meyer HHD, Hoffmann S. Development of a sensitive microtitration plate enzyme-immunoassay for the anabolic steroid trenbolone.  Food Additives & Contaminants. 1987;  4 149-160
• 22 Meyer HHD, Rinke L, Dursch I. Residue screening for the beta-agonists clenbuterol, salbutamol and cimaterol in urine using enzyme immunoassay and high-performance liquid chromatography.  Journal of Chromatography. 1991;  564 551-556
• 23 Miyagawa S, Suzuki A, Katsu Y. et al . Persistent gene expression in mouse vagina exposed neonatally to diethylstilbestrol.  Journal of Veterinary Medical Science. 2004;  32 663-677
• 24 Moran C, Quirke JF, Prendiville DJ. et al . The effect of estradiol, trenbolone acetate, or zeranol on growth rate, mammary development, carcass traits, and plasma estradiol concentrations of beef heifers.  Journal of Animal Science. 1991;  69 4249-4258
• 25 Nelson KG, Sakai Y, Eitzman B. et al . Exposure to diethylstilbestrol during a critical developmental period of the mouse reproductive tract leads to persistent induction of two estrogen-regulated genes.  Cell Growth Differentiation. 1994;  5 595-606
• 26 Pentecost BT, Teng CT. Lactotransferrin is the major estrogen inducible protein of mouse uterine secretions.  Journal of Biological Chemistry. 1987;  262 10134-10139
• 27 Pessina MA, Hoyt Jr RF, Goldstein I. et al . Differential regulation of the expression of estrogen, progesterone, and androgen receptors by sex steroid hormones in the vagina: immunohistochemical studies.  Journal of Sexual Medicine. 2006;  3 804-814
• 28 Peters AR. Effect of trenbolone acetate on ovarian function in culled dairy cows.  Vet Rec. 1987;  120 413-416
• 29 Pfaffl MW, Daxenberger A, Hageleit M. et al . Effects of synthetic progestagens on the mRNA expression of androgen receptor, progesterone receptor, oestrogen receptor alpha and beta, insulin-like growth factor-1 (IGF-1) and IGF-1 receptor in heifer tissues.  Journal of Veterinary Medicine Series A-Physiology Pathology Clinical Medicine. 2002;  49 57-64
• 30 Pfaffl MW, Lange IG, Meyer HHD. The gastrointestinal tract as target of steroid hormone action: quantification of steroid receptor mRNA expression (AR, ERalpha, ERbeta and PR) in 10 bovine gastrointestinal tract compartments by kinetic RT-PCR.  The Journal of Steroid Biochemistry and Molecular Biology. 2003;  84 159-166
• 31 Prakash BS, Meyer HH, Schallenberger E. et al . Development of a sensitive enzymeimmunoassay (EIA) for progesterone determination in unextracted bovine plasma using the second antibody technique.  J Steroid Biochem. 1987;  28 623-627
• 32 Riedmaier I, Becker C, Pfaffl MW. et al . The use of omic technologies for biomarker development to trace functions of anabolic agents.  Journal of Chromatography A. 2009a; 
• 33 Riedmaier I, Tichopad A, Reiter M. et al . Identification of potential gene expression biomarkers for the surveillance of anabolic agents in bovine blood cells.  Analytica Chimica Acta. 2009b;  638 106-113
• 34 Sato T, Fukazawa Y, Ohta Y. et al . Involvement of growth factors in induction of persistent proliferation of vaginal epithelium of mice exposed neonatally to diethylstilbestrol.  Reproductive Toxicology. 2004;  19 43-51
• 35 Schilt R, Groot MJ, Berende PL. et al . Pour on application of growth promoters in veal calves: analytical and histological results.  Analyst. 1998;  123 2665-2670
• 36 Schroeder A, Mueller O, Stocker S. et al . The RIN: an RNA integrity number for assigning integrity values to RNA measurements.  BMC Molecular Biology. 2006;  7 3
• 37 Scippo ML, Degand G, Duyckaerts A. et al . Control of the illegal administration of natural steroid hormones in the plasma of bulls and heifers.  Analyst. 1994;  119 2639-2644
• 38 Stephany RW. Hormones in meat: different approaches in the EU and in the USA.  APMIS Suppl. 2001;  S357-S363
• 39 Swan SH, Liu F, Overstreet JW. et al . Semen quality of fertile US males in relation to their mothers' beef consumption during pregnancy.  Human Reproduction. 2007;  22 1497-1502
• 40 Teng CT. Lactoferrin gene expression and regulation: an overview.  Biochemistry and Cell Biology. 2002;  80 7-16
• 41 Traish AM, Kim SW, Stankovic M. et al . Testosterone increases blood flow and expression of androgen and estrogen receptors in the rat vagina.  Journal of Sexual Medicine. 2007;  4 609-619
• 42 van Ginkel LA. Immunoaffinity chromatography, its applicability and limitations in multi-residue analysis of anabolizing and doping agents.  Journal of Chromatography. 1991;  564 363-384
• 43 Zarkawi M, Galbraith H, Hutchinson JS. The action of trenbolone acetate, a synthetic anabolic steroid, on ovarian function in the guinea pig.  Lab Anim. 1991;  25 117-121

Correspondence