[Show abstract][Hide abstract] ABSTRACT: Real-time PCR has become an important tool for quantitative nucleic acids analysis. By now, a wide rang of real-time PCR instruments, PCR plastic consumables and sealing systems are available. In this study both transparent and white plates were taken to test possible position and plate effects on quantitative PCR results on different real-time platforms: ep realplex (Eppendorf, Hamburg, Germany) and the iQ5 (Bio-Rad, Palo Alto, USA). Heat and adhesive sealing was used to compare sealing systems. Heterogeneity in Ct values was calculated to show possible effects of plate type and sealing system. Heterogeneity in amplification efficiency should show possible positional effects of 96-well plates.
[Show abstract][Hide abstract] ABSTRACT: Deoxynivalenol (DON) is one of the most common Fusarium toxins in animal feed and poses a potential risk especially for monogastric animals like pigs. DON is known to modulate the immune system, dependent on dose and frequency of exposure. The aim of the study was to investigate the effects of chronic exposure to low levels of DON on the expression of immune relevant genes. In a feeding trial (84 days), 20 pigs were assigned equally to a control and a treatment group. The DON-content of the contaminated diet was 1.2 mg/kg from day 1 to 41, from day 42 it was elevated to 2.0 mg/kg. The control group (n = 10) was fed a diet with a DON concentration lower than 0.05 mg/kg. Blood samples were taken over the course of the study and ileum samples were taken at slaughter. Gene expression measurement was done using real-time RT-qPCR. For target genes, those cytokines were chosen, which were estimated to be implicated in the modulation of the immune system induced by DON ingestion. In ileum, significant down-regulations could be observed for IL-1β and IL-8 (p < 0.05). Most significant regulations in blood could be detected on day 45 after increasing the dietary DON content in the experimental diet. Herein, down-regulations of IL-1β, IL-8 and TNFα were demonstrated. In conclusion, the present study provides data concerning chronic application of DON in low doses, as little is known in this area. Down-regulations of immune-related transcription factors and pro-inflammatory immune factors could be demonstrated.
Full-text · Article · Nov 2011 · Mycotoxin Research
[Show abstract][Hide abstract] ABSTRACT: In quantitative single-cell studies, the critical part is the low amount of nucleic acids present and the resulting experimental
variations. In addition biological data obtained from heterogeneous tissue are not reflecting the expression behaviour of
every single-cell. These variations can be derived from natural biological variance or can be introduced externally. Both
have negative effects on the quantification result. The aim of this study is to make quantitative single-cell studies more
transparent and reliable in order to fulfil the MIQE guidelines at the single-cell level. The technical variability introduced
by RT, pre-amplification, evaporation, biological material and qPCR itself was evaluated by using RNA or DNA standards. Secondly,
the biological expression variances of GAPDH, TNFα, IL-1β, TLR4 were measured by mRNA profiling experiment in single lymphocytes.
The used quantification setup was sensitive enough to detect single standard copies and transcripts out of one solitary cell.
Most variability was introduced by RT, followed by evaporation, and pre-amplification. The qPCR analysis and the biological
matrix introduced only minor variability. Both conducted studies impressively demonstrate the heterogeneity of expression
patterns in individual cells and showed clearly today's limitation in quantitative single-cell expression analysis.
Full-text · Article · Jul 2011 · Nucleic Acids Research
[Show abstract][Hide abstract] ABSTRACT: The application of anabolic steroids in food producing animals is forbidden in the EU since 1988, but the abuse of such drugs is a potential problem. The existing test systems are based on known compounds and can be eluded by newly emerging substances. The examination of physiological effects of anabolic hormones on different tissues to indirectly detect misuse might overcome this problem. Two studies were conducted with post-pubertal 24-months old Nguni heifers and pre-pubertal female 2-4 weeks old Holstein Friesian calves, respectively. The animals of the accordant treatment groups were administered combinations of estrogenic and androgenic compounds. The measurement of the gene expression pattern was undertaken with RT-qPCR. Target genes of different functional groups (receptors, angiogenesis, steroid synthesis, proliferation, apoptosis, nutrient metabolism and others) have been quantified. Several biochemical pathways were shown to be influenced by anabolic treatment. Both studies identified significant regulations in steroid and growth factor receptors (AR, ERβ, LHR, FSHR, Flt-1, PR, IGF-1R, Alk-6), angiogenic and tissue remodeling factors (VEGFs, FGFs, BMPs, ANGPT-2, MMPs, TIMP-2, CTSB), steroid synthesis (S5A1, HSD17, CYP19A1), proliferation (TNFα, IGF-1, IGFBPs, p53, c-fos; CEBPD, c-kit), apoptosis (CASP3, FasL, p53) and others (C7, INHA, STAR). Several genes were regulated to opposite directions in post-pubertal compared to pre-pubertal animals. PCA for Nguni heifers demonstrated a distinct separation between the control and the treatment group. In conclusion, anabolics modify hormone sensitivity and steroid synthesis, and they induce proliferative effects in the whole reproductive tract (uterus and ovary) as well as anti-angiogenic effects in the ovary. However, the extent will depend on the developmental stage of the animals.
Full-text · Article · Jul 2011 · The Journal of steroid biochemistry and molecular biology
[Show abstract][Hide abstract] ABSTRACT: miRNAs are regulatory RNA molecules. The analytical interest rose over the past 10 years especially in clinical diagnostics as miRNAs show specific expression patterns in several human diseases like diabetes or cancer. Therefore, it is expected that miRNA profiles might be used as biomarkers in early diagnosis. The idea of establishing biomarkers is also present in veterinary drug analysis, e.g. in the surveillance of illegal use of anabolics. Transcriptomics is a promising approach in the detection of anabolics misuse. However, miRNA expression patterns have shown their superiority over mRNA patterns in clinical diagnostics. Thus, the influence of anabolic steroids on miRNA expression in bovine liver should be investigated and an expression pattern should be validated, which might be used as a treatment biomarker. An animal experiment was conducted with 18 heifers equally allocated to a control and a treatment group, which was implanted with TBA plus E2. Liver samples were screened for miRNA expression using PCR arrays. Expression of 11 prominent miRNAs was validated via single assay qPCR. Herein, the following expression pattern could be found with an up-regulation of miR-29c and miR-103 and a down-regulation of miR-34a, miR-181c, miR-20a and miR-15a (p<0.05 each). Using principal components analysis (PCA), the control group could clearly be distinguished from the treatment group, when integrating gene expression results from both miRNA and mRNA. So, the combination of different transcribed targets (mRNA plus miRNA) might be a promising approach to find a valid expression pattern to be used for anabolic treatment screening.
[Show abstract][Hide abstract] 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.
Full-text · Article · Oct 2010 · Experimental and Clinical Endocrinology & Diabetes
[Show abstract][Hide abstract] ABSTRACT: The use of anabolic steroids is forbidden for food producing animals in the EU. Owing to the advantages of anabolics for production profitability, illegal application is appealing. Anabolics are known to influence gene expression of several tissues. We focused on the liver because of its important role in nutrient and hormone metabolism. The aim of the present study was to find differentially regulated metabolic pathways, which might be used as treatment biomarkers.
A total of 18 Nguni heifers were allocated equally to a control group and a treatment group and were implanted with Revalor H. Expression of 34 target genes was measured using reverse transcriptase quantitative polymerase chain reaction (RT-qPCR).
Upregulation of androgen receptor and insulin-like growth factor 1 (IGF-1) and downregulation of IGF-2, insulin-like growth factor binding protein 2, steroid hormone binding globulin, insulin receptor α, insulin receptor β, tyrosine aminotransferase, 17β-hydroxy steroid dehydrogenase 2,3-hydroxy-methylglutaryl-coenzym-A-synthase, cathepsin B, hepatocyte growth factor, steroidogenic acute regulatory protein, apolipoprotein 2 and tumor necrosis factor α was demonstrated.
Several biochemical pathways showed different regulations on mRNA level under the influence of trenbolone acetate plus estradiol. The inhibition of nutrient metabolism and protein breakdown seems to support growth processes. IGF-1 plays an important role in growth and development and thus the upregulation of IGF-1 could be responsible for the stimulation of growth in treated animals. The upregulation of IGF-1 could also be revealed as a possible risk factor for the generation of artherosclerotic plaques, which are known as long-term side effects following the use of anabolic steroids. Principal components analysis of RT-qPCR results showed that both groups arrange together and can be clearly separated. Therefore, these might be used as possible biomarkers in bovine liver.
Full-text · Article · Aug 2010 · Hormone molecular biology and clinical investigation
[Show abstract][Hide abstract] ABSTRACT: The focus of this study was to evaluate data on the gene expression profiles induced by testosterone and a selective androgen receptor modulator (SARM, TAP Pharmaceutical Products Inc., Lake Forest, IL, USA) in androgen sensitive muscle tissue to obtain a better understanding on the molecular mechanisms of action and to identify biomarkers for SARM function in primate organs. A total of 24 male cyomolgus monkeys were divided into four groups: testosterone group, SARM1 group, SARM10 group, and control group, each consisting of six animals. The testosterone group was treated i.m. with 3.0 mg/kg Testostoviron
Full-text · Article · Jan 2010 · Hormone molecular biology and clinical investigation
[Show abstract][Hide abstract] ABSTRACT: Anabolic hormones, including testosterone, have been suggested as a therapy for aging-related conditions, such as osteoporosis and sarcopenia. These therapies are sometimes associated with severe androgenic side effects. A promising alternative to testosterone replacement therapy are selective androgen receptor modulators (SARMs). SARMs have the potential to mimic the desirable central and peripheral androgenic anabolic effects of testosterone without having its side effects. In this study we evaluated the effects of LGD2941, in comparison to testosterone, on mRNA expression of selected target genes in whole blood in an non-human model. The regulated genes can act as potential blood biomarker candidates in future studies with AR ligands. Cynomolgus monkeys (Macaca fascicularis) were treated either with testosterone or LGD2941 for 90 days in order to compare their effects on mRNA expression in blood. Blood samples were taken before SARM application, on day 16 and on day 90 of treatment. Gene expression of 37 candidate genes was measured using quantitative real-time RT-PCR (qRT-PCR) technology. Our study shows that both testosterone and LGD2941 influence mRNA expression of 6 selected genes out of 37 in whole blood. The apoptosis regulators CD30L, Fas, TNFR1 and TNFR2 and the interleukins IL-12B and IL-15 showed significant changes in gene expression between control and the treatment groups and represent potential biomarkers for androgen receptor ligands in whole blood.
Full-text · Article · May 2009 · The Journal of steroid biochemistry and molecular biology
[Show abstract][Hide abstract] ABSTRACT: In the EU, the use of anabolic steroids in food producing animals has been forbidden since 1988. The routine methods used in practice are based on the detection of hormonal residues. To overcome these routine methods, growth-promoting agents are sometimes administered at concentrations below the detection limit and new anabolic substances are designed. Therefore, new monitoring systems are needed to overcome the misuse of anabolic agents in meat production. In this study, a new monitoring system was applied: the quantification of mRNA gene expression changes by quantitative real time reverse transcription polymerase chain reaction (qRT-PCR). Blood was selected as ideal tissue for biomarker screening. From the literature, it is known that steroid hormones affect mRNA gene expression of the different blood cells, which can easily be taken from the living animal. In an animal trial, 18 Nguni heifers were separated to two groups of nine animals. One group served as untreated control and the other group was treated with a combination of trenbolone acetate plus estradiol for 39 days in order to allow the detection of the effect on mRNA expression in blood at three time points. Candidate genes used for developing a biomarker pattern were chosen by screening the actual literature for anabolic effects on blood cells. It could be demonstrated that the combination of trenbolone acetate plus estradiol significantly influences mRNA expression of the steroid receptors (ER-alpha and GR-alpha), the apoptosis regulator Fas, the proinflammatory interleukins IL-1alpha, IL-1beta and IL-6 and of MHCII, CK, MTPN, RBM5 and Actin-beta. Advanced statistical analysis by Principal Components Analysis (PCA) indicated that these genes represent potential biomarkers for this hormone combination in whole blood.
Full-text · Article · May 2009 · Analytica chimica acta
[Show abstract][Hide abstract] ABSTRACT: Doping with anabolic agents is a topic in sports where strength is crucial, e.g. sprinting, weight lifting and many more. Testosterone and its functional analogs are the drugs of choice taken as pills, creams, tape or injections to increase muscle mass and body performance, and to reduce body fat. Stanozolol (17β-hydroxy-17α-methyl-5α-androst-2-eno[3,2c]pyrazol) is a testosterone analogue with the same anabolic effect like testosterone but its ring structure makes it possible to take it orally. Therefore, stanozolol is one of the most frequently used anabolic steroids.
Common verification methods for anabolic drugs exist, identifying the chemicals in tissues, like hair or blood samples. The idea of this feasibility study was to search for specific gene expression regulations induced by stanozolol to identify the possible influence of the synthetically hormone on different metabolic pathways. Finding biomarkers for anabolic drugs could be supportive of the existing methods and an additional proof for illegal drug abuse.
In two separate cell cultures, human HFDPC (hair follicle dermal papilla cells) from a female and a male donor were treated with stanozolol. In the female cell culture treatment concentrations of 0 nM (control), 1 nM, 10 nM and 100 nM were chosen. Cells were taken 0 h, 6 h, 24 h and 48 h after stimulation and totalRNA was extracted. Learning from the results of the pilot experiment, the male cell culture was treated in 10 nM and 100 nM concentrations and taken after 0 h, 6 h, 24 h and 72 h. Using quantitative real-time RT-PCR expression of characteristics of different target genes were analysed.
Totally 13 genes were selected according to their functionality by screening the actual literature and composed to functional groups: factors of apoptosis regulation were Fas Ligand (FasL), its receptor (FasR), Caspase 8 and Bcl-2. Androgen receptor (AR) and both estrogen receptors (ERα, ERβ) were summarized in the steroid receptor group. The growth factor group included the insulin like growth factor receptor (IGF1R) and growth hormone receptor (GHR). Fibroblast growth factor 2 (FGF2) and keratinocyte growth factor (FGF7) were summarized in the hair cycle factor group. 5α-Steroidreductases (SRD5A1, SRD5A2) represented the enzyme group. Three reference genes were taken for relative quantification: ubiquitin (UBQ), glycerinaldehyde-3-phsophate-dehydrogenase (GAPDH), and β-actin (ACTB).
In cell culture 1 AR, FasR, FGF2 showed significant regulations within one treatment time, significant gene expressions over time were analysed for Caspase 8. In cell culture 2 AR, FasR and SRD5A2 were significantly regulated within one treatment time.
In this feasibility study first biomarker for a screening pattern of anabolic agents could be identified providing the rationality to investigate modified, metabolic pathways in the whole hair follicle.
Full-text · Article · Feb 2008 · Biomarker insights
[Show abstract][Hide abstract] ABSTRACT: With this feasibility study a first step towards a new monitoring system for hormonal treatments was done. Screening of regulation and function of anabolic sex steroids via modified gene expression of mRNA in various tissues could be a new approach to trace treatments with unknown drugs or newly combined cocktails. In the study, uterus, liver and muscle tissue from 24 cycling heifers were taken after the animals were treated either with Melengestrol Acetate (MGA), Finaplix-H (200 mg Trenbolone Acetate) or Ralgro (36 mg Zeranol) for 56 days. In every treatment group always two heifers were given 1-fold, 3-fold and 10-fold doses of the standard preparation, the control group without any treatment consisted of two animals. The different tissue gene expression profiles were investigated via the candidate gene approach. Totally 57 candidate genes were selected according to their functionality by screening the actual literature and composed to functional groups: angiogenesis, apoptosis, cell cycle, endocrine factors, energy metabolism, inflammatory factors, muscle function, oncogenes, protein metabolism and transcription factors. Gene expression was measured using quantitative real-time RT-PCR (qRT-PCR) technology. From 24 tested candidate genes in the liver, 17 showed a significant regulation. Eight genes were influenced by MGA, 9 by Finaplix-H, and 4 by Ralgro. For the muscle tissue 19 genes were tested with the result that in the neck muscle 11 genes were regulated and in the hind limb muscle 8 genes. In the neck 5 genes were affected by MGA, 6 by Finaplix-H and 3 by Ralgro. Only 2 genes were influenced by MGA in the hind limb muscle. Finaplix-H affected 6 and Ralgro 4 genes. In the uterus 29 target genes were tested and 13 were significantly influenced by the anabolic sex steroids. Under Finaplix-H treatment eight target genes were regulated and Ralgro and MGA showed a significant regulation in four target genes. The highest gene expression changes under anabolic treatment were observed in the uterus. The analyzed genes showed significant regulations but further studies, testing different animal husbandry conditions will be needed to identify meaningful expression patterns for the different tissues. With the investigation of the regulation and possible function of anabolic sex steroids via gene expression, a preparatory work for the development of an expression pattern for drug screening was made.