Sara Divari

Corticosteroid Hormone Receptors and Prereceptors as New Biomarkers of the Illegal Use of Glucocorticoids in Meat Production

Despite the European ban on the use of growth promoters in cattle, veterinary surveillance reports indicate that the illicit use of corticosteroids persists both alone and in combination with anabolic hormones and β-agonists. Current control strategies should be informed by research into the effects of corticosteroids on bovine metabolism and improved through the development of specific, sensitive diagnostic methods that utilize potential molecular biomarkers of corticosteroid treatment. The actions of corticosteroids on target tissues are principally regulated by two receptors: the glucocorticoid receptor (GR) and the mineralocorticoid receptor (MR). The effects of these steroids are modulated by prereceptor enzyme-mediated metabolism: the two isoforms of the 11β-hydroxysteroid dehydrogenase (11β-HSDs) enzyme catalyze the interconversion between active glucocorticoids, such as cortisol, into inactive compounds, such as cortisone. This study aimed to determine whether the expression of the prereceptor system and of the corticosteroid receptors could be regulated in different target tissues by the administration of dexamethasone and prednisolone in cattle. It was observed that greater up-regulation of the GR and MR genes followed dexamethasone treatment in the muscle tissues than in the kidney, liver, and salivary glands; up-regulation of GR and MR expression following prednisolone treatment was higher in adipose tissue than in the other tissues. The thymus seemed to respond to dexamethasone treatment but not to prednisolone treatment. Both treatments significantly down-regulated 11β-HSD2 gene expression in the adrenal tissues, but only dexamethasone treatment down-regulated 11β-HSD2 expression in the bulbourethral and prostate glands. Together, these data indicate that the combination of GR, MR, and 11β-HSD2 could provide a useful biomarker system to detect the use of illicit glucocorticoid treatment in cattle.

Progesterone receptor gene expression in the accessory sex glands of veal calves

This study investigated progesterone receptor (PR) cDNA expression in the testes, prostate and bulbourethral glands of prepubertal calves treated experimentally with high and low doses of 17β-oestradiol and with testosterone. Tissue samples were examined histologically and immunohistochemically for PR. Western blot analysis and quantitative PCR against PR was performed on cDNA and protein extracted from the same tissues. Bulbourethral glands from animals treated with low and high dosages of 17β-oestradiol had 39- and 429-fold increases of PR transcript, respectively, compared with controls. In the prostate there were 7.5- and 16-fold increases, respectively. Animals treated with testosterone showed no increases in PR transcript. The results demonstrate that 17β-oestradiol specifically induces marked overexpression of the PR gene and protein, particularly in the bulbourethral gland.

Gene expression profiling of thymus in beef cattle treated with prednisolone

Glucocorticoids (GCs) are extensively used in livestock production, not only for their anti-inflammatory properties but also to improve the quality and quantity of meat in veal and beef production. In Italy, an increase in GC-positive cases has been observed in cattle since 2008, particularly prednisolone (PDN). Recent studies clearly demonstrate that both histopathological analysis and high-performance liquid chromatography tandem mass spectrometry (HPLC/MS-MS) were unable to detect PDN treatments. The aim of this study was to identify transcriptomic signatures of PDN administration in the thymus of experimentally treated animals by comparison with untreated controls, in order to identify gene expression changes or pathways alteration induced by the corticosteroid treatment. Microarray data analysis showed substantial modifications in thymus gene expression profiles after PDN treatment. Several of the 388 differentially expressed genes encoded pro-inflammatory and anti-inflammatory mediators or immune regulators which showed that PDN might have a role in the regulation of immunologic homeostasis, act on both innate and acquired components of the immunity and mainly induce the activation of immune tolerance and anti-inflammatory pathways. Thus, this study allowed to deepen the effects of PDN on the immune system and showed the potentiality of gene expression profiling by DNA-microarray as a powerful tool to complement the existing methods against the illegal use of growth promoting hormones, especially when working on samples collected after slaughtering.

17β-oestradiol-induced gene expression in cattle prostate: biomarkers to detect illegal use of growth promoters

The effects of 17β-oestradiol (E2) on gene expression in cultures of bovine primary prostate stromal cells (BPSCs) and prostate gland tissue were studied. In the first part of the study, BPSCs were grown in the presence of E2 from the first passage to the end of the experiment; a second group was treated in the same way but the treatment was suspended for 48 hours before the end of the experiment; a third group of BPSCs served as a control. In the second part of the study, five male veal calves, aged 130 days, were treated four times intramuscularly with 10 mg of E2 at intervals of two weeks and then euthanased two weeks after the last treatment. Quantitative PCR and immunohistochemistry were used to evaluate the expression of fibroblast growth factor (FGF) receptors (FGFRs), FGFs, progesterone receptor, androgen receptor and oestrogen receptor in BPSCs and prostate tissue. E2 induced a significant over-expression of progesterone receptor in both BPSCs and prostate tissue. There was also a marked up-regulation of FGFR types 1, 2 and 3 genes observed in the BPSCs.

Progesterone receptor up-regulation: a diagnostic tool for the illicit use of oestrogens in adult beef cattle

The monitoring of gene regulation via mRNA levels to detect anabolic sex steroid administration in cattle is a novel approach to detecting the illicit treatment of livestock in meat production. A previous study revealed that progesterone receptor (PR) gene expression levels were increased in the bulbourethral glands and prostates of 17β-oestradiol-treated prepubertal calves, suggesting that the PR can be used as a specific molecular biomarker for oestrogen treatment. The aim of this study was to verify the specificity and applicability of the PR to detect the illegal use of 17β-oestradiol in sexually mature beef cattle. Accessory sex glands were sampled from 42 male beef cattle that were divided into six experimental groups, including two control groups, K1 and K2. Group A cattle were treated with 17β-oestradiol (five weekly intramuscular doses of 20 mg), and group B cattle were treated with dexamethasone (40 daily doses of 0.7 mg per os). Group C cattle received an implant of Revalor-200 (200 mg of trenbolone acetate and 20 mg of 17β-oestradiol), and group D cattle received Revalor-200 plus dexamethasone (0.7 mg daily per os). 17β-Oestradiol, either alone or in combination with other steroids, up-regulated the PR gene and protein expression, even in the absence of detectable histological changes in the accessory sex glands, confirming the high sensitivity of PR gene expression as an indirect diagnostic screening tool to detect illicit oestrogen treatment in sexually mature male bovine.

A RIKILT yeast estrogen bioassay (REA) for estrogen residue detection in urine of calves experimentally treated with 17β-estradiol

17β-Estradiol is one of the most powerful sex steroids illegally used in bovine production. The objective of this study was to evaluate the application and the specificity of the RIKILT yeast estrogen bioassay (REA) for the detection of molecules with estrogenic activities in the urine of calves experimentally treated with anabolics. Four groups of six calves each received an injection of 17β-estradiol intramuscularly (group B), androsterone and gliburide (group A), and testosterone (group C) molecules at different dosage for 40 days. Group D was the control. The ability of the REA test to detect estrogenic activity in urine samples from all animals was assessed. All estrogen-treated animals (group B) showed as being positive up to 7 days after administration of the highest dosage of 17β-estradiol, while the other three groups showed as being negative. The identity of estrogenic molecules in the urine of group B (17β-estradiol, 17α-estradiol) was confirmed by gas chromatography-mass spectrometry (GC/MS). This is the first time the REA test has been applied to detect 17β-estradiol in the urine of calves treated with the hormone in vivo. The technique may offer an advantageous laboratory method for the veterinary surveillance of illegal steroid use.

Oxytocin precursor gene expression in bovine skeletal muscle is regulated by 17β-oestradiol and dexamethasone.

Growth promoter administration, in livestock, potentially poses a major threat to public health, due to the potential endocrine and carcinogenic activity of residues, accumulating in edible tissues, such as skeletal muscle. Therefore, development of new screening tests and methods for the detection of illicit treatments of food animals would be useful. In this study the serum concentrations of oxytocin peptide were measured in beef cattle receiving 17β oestradiol, dexamethasone or placebo over a period of 40 days. Changes in gene expression of oxytocin precursor in skeletal muscle were also examined in these animals. Serum analysis using an oxytocin EIA kit indicated a significant up-regulation of the biosynthesis of this nonapeptide only in cattle after 17β oestradiol, but not after dexamethasone or placebo treatment. Quantitative PCR (qPCR) analysis showed a significant overexpression of the oxytocin precursor gene by 33.5 and 13.3-fold in cattle treated with 17β oestradiol and dexamethasone, respectively, in comparison to placebo treated animals. Regulation of gene expression by some myogenic regulatory factors in skeletal muscle was also evaluated in these animal groups, confirming the activity of both growth promoters on this gene. To investigate the use of the oxytocin precursor gene as biomarker for 17β oestradiol and dexamethasone treatment in beef cattle, an absolute quantification of this gene by qPCR was developed. A standard curve was generated and developed with TaqMan® technology and optimal criterion value, sensitivity and specificity of this screening method were established through ROC analysis. This analysis suggested that the up-regulation of oxytocin precursor gene expression in skeletal muscle tissue is a valid marker for detection of illicit 17β oestradiol and/or dexamethasone use in beef cattle. This method may serve as a novel diagnostic tool in the screening phase, and, if introduced in routine testing, may significantly improve overall efficacy and success of the food screening process ordered by state authorities.

Regucalcin Expression in Bovine Tissues and Its Regulation by Sex Steroid Hormones in Accessory Sex Glands

Regucalcin (RGN) is a mammalian Ca2+-binding protein that plays an important role in intracellular Ca2+ homeostasis. Recently, RGN has been identified as a target gene for sex steroid hormones in the prostate glands and testis of rats and humans, but no studies have focused on RGN expression in bovine tissues. Thus, in the present study, we examined RGN mRNA and protein expression in the different tissues and organs of veal calves and beef cattle. Moreover, we investigated whether RGN expression is controlled through sex steroid hormones in bovine target tissues, namely the bulbo-urethral and prostate glands and the testis. Sex steroid hormones are still illegally used in bovine husbandry to increase muscle mass. The screening of the regulation and function of anabolic sex steroids via modified gene expression levels in various tissues represents a new approach for the detection of illicit drug treatments. Herein, we used quantitative PCR, western blot and immunohistochemistry analyses to demonstrate RGN mRNA and protein expression in bovine tissues. In addition, estrogen administration down-regulated RGN gene expression in the accessory sex glands of veal calves and beef cattle, while androgen treatment reduced RGN gene expression only in the testis. The confirmation of the regulation of RGN gene expression through sex steroid hormones might facilitate the potential detection of hormone abuse in bovine husbandry. Particularly, the specific response in the testis suggests that this tissue is ideal for the detection of illicit androgen administration in veal calves and beef cattle.

HPLC-ESI-MS/MS assessment of the tetrahydro-metabolites of cortisol and cortisone in bovine urine: promising markers of dexamethasone and prednisolone treatment

ABSTRACT The effects of long-term administration of low doses of dexamethasone (DX) and prednisolone (PL) on the metabolism of endogenous corticosteroids were investigated in veal calves. In addition to cortisol (F) and cortisone (E), whose interconversion is regulated by 11β-hydroxysteroid dehydrogenases (11βHSDs), special attention was paid to tetrahydrocortisol (THF), allo-tetrahydrocortisol (aTHF), tetrahydrocortisone (THE) and allo-tetrahydrocortisone (aTHE), which are produced from F and E by catalytic activity of 5α and 5β-reductases. A specifically developed HPLC-ESI-MS/MS method achieved the complete chromatographic separation of two pairs of diastereoisomers (THF/aTHF and THE/aTHE), which, with appropriate mass fragmentation patterns, provided an unambiguous conformation. The method was linear (r2 > 0.9905; 0.5–25 ng ml−1), with LOQQ of 0.5 ng ml−1. Recoveries were in range 75–114%, while matrix effects were minimal. The experimental study was carried out on three groups of male Friesian veal calves: group PL (n = 6, PL acetate 15 mg day–1 p.o. for 31 days); group DX (n = 5, 5 mg of estradiol (E2) i.m., weekly, and 0.4 mg day–1 of DX p.o. for 31 days) and a control group (n = 8). Urine was collected before, during (twice) and at the end of treatment. During PL administration, the tetrahydro-metabolite levels decreased gradually and remained low after the suspension of treatment. DX reduced urinary THF that persisted after the treatment, while THE levels decreased during the experiment, but rebounded substantially after the DX was withdrawn. Both DX and PL significantly interfered with the production of F and E, leading to their complete depletion. Taken together, the results demonstrate the influence of DX and PL administration on 11βHSD activity and their impact on dysfunction of the 5-reductase pathway. In conclusion, profiling tetrahydro-metabolites of F and E might serve as an alternative, indirect but reliable, non-invasive procedure for assessing the impact of synthetic glucocorticosteroids administration. GRAPHICAL ABSTRACT

17β-estradiol upregulates oxytocin and the oxytocin receptor in C2C12 myotubes

Background The endocrinology of skeletal muscle is highly complex and many issues about hormone action in skeletal muscle are still unresolved. Aim of the work is to improve our knowledge on the relationship between skeletal muscle and 17β-estradiol. Methods The skeletal muscle cell line C2C12 was treated with 17β-estradiol, the oxytocin peptide and a combination of the two hormones. The mRNA levels of myogenic regulatory factors, myosin heavy chain, oxytocin, oxytocin receptor and adipogenic factors were analysed in C2C12 myotubes. Results It was demonstrated that C2C12 myoblasts and myotubes express oxytocin and its receptor, in particular the receptor levels physiologically increase in differentiated myotubes. Myotubes treated with 17β-estradiol overexpressed oxytocin and oxytocin receptor genes by approximately 3- and 29-fold, respectively. A decrease in the expression of fatty acid binding protein 4 (0.62-fold), a fat metabolism-associated gene, was observed in oxytocin-treated myotubes. On the contrary, fatty acid binding protein 4 was upregulated (2.66-fold) after the administration of the combination of 17β-estradiol and oxytocin. 17β-estradiol regulates oxytocin and its receptor in skeletal muscle cells and they act in a synergic way on fatty acid metabolism. Discussion Oxytocin and its receptor are physiologically regulated along differentiation. 17β-estradiol regulates oxytocin and its receptor in skeletal muscle cells. 17β-estradiol and oxytocin act in a synergic way on fatty acid metabolism. A better understanding of the regulation of skeletal muscle homeostasis by estrogens and oxytocin peptide could contribute to increase our knowledge of muscle and its metabolism.