Validation of putative biomarkers of furan exposure through quantitative analysis of furan metabolites in urine of F344 rats exposed to stable isotope labeled furan.

Humans are chronically exposed to furan, a potent liver toxicant and carcinogen that occurs in a variety of heat-processed foods. Assessment of human exposure based on the furan content in foods is, however, subject to some uncertainty due to the high volatility of furan. Biomarker monitoring is thus considered an alternative or complementary approach to furan exposure assessment. Previous work suggested that urinary furan metabolites derived from the reaction of cis-2-butene-1,4-dial (BDA), the reactive intermediate of furan, with glutathione (GSH) or amino acids may serve as potential biomarkers of furan exposure. However, some metabolites were also reported to occur in urine of untreated animals, indicating either background contamination via animal feed or endogenous sources, which may limit their suitability as biomarkers of exposure. The overall aim of the present study was to accurately establish the correlation between external dose and concentration of furan metabolites in urine over time and to discriminate against endogenous formation and furan intake via feed. To this end, the furan metabolites GSH-BDA (N-[4-carboxy-4-(3-mercapto-1H-pyrrol-1-yl)-1-oxobutyl]-L-cysteinylglycine), NAcLys-BDA (R-2-(acetylamino)-6-(2,5-dihydro-2-oxo-1H-pyrrol-1-yl)-1-hexanoic acid), NAcCys-BDA-NAcLys (N-acetyl-S-[1-[5-(acetylamino)-5-carboxypentyl]-1H-pyrrol-3-yl]-L-cysteine) and NAcCys-BDA-NAcLys sulfoxide (N-acetyl-S-[1-[5-(acetylamino)-5-carboxypentyl]-1H-pyrrol-3-yl]-L-cysteine sulfoxide) were simultaneously analyzed by stable isotope dilution ESI-LC-MS/MS as unlabeled and [13C4]-furan dependent metabolites following oral administration of a single oral dose of isotopically labelled [13C4]-furan (0.1, 1, 10, 100 and 1000 µg/kg bw) to male and female F344/DuCrl rats. Although a linear correlation between urinary excretion of [13C4]-furan-dependent metabolites was observed, analysis of unlabeled NAcLys-BDA, NAcCys-BDA-NAcLys and NAcCys-BDA-NAcLys sulfoxide revealed substantial, fairly constant urinary background levels throughout the course of the study. Analysis of furan in animal feed excluded feed as a source for these background levels. GSH-BDA was identified as the only furan metabolite without background occurrence, suggesting that it may present a specific biomarker to monitor external furan exposure. Studies in humans are now needed to establish if analysis of urinary GSH-BDA may provide reliable exposure estimates.

Buried free flaps in head and neck reconstruction: higher risk of free flap failure?

Thrombosis of the pedicle is central to free flap failure, and early revision of a compromised flap is the key to successfully salvage a flap. Therefore, the majority of free flaps in reconstructive head and neck surgery are used with the ability to visually examine the flap. Sometimes, due to intra-operative circumstances, it is necessary to use a flap that cannot be monitored externally. These flaps are called buried flaps and have the reputation of being put at risk. The current literature provides only limited data to support or disprove this position. A single institution retrospective review of patient charts between 2007 and 2015 was performed. Flap monitoring was carried out with hand-held Doppler of the pedicle hourly for the first 72 h in all cases. Additional duplex ultrasound was performed in the majority of buried flaps. A total of 437 flaps were included into the study. 37 flaps (7.8 %) were identified to fulfill the criteria of a buried free flap. In total, four patients had complications, three of which required operative reexploration. All interventions were successful, resulting in no flap loss in our series. An accurate operation technique combined with meticulous monitoring protocols supported by duplex ultrasound can result in satisfactory outcome of buried flaps. No enhanced risk of flap loss of buried flaps was found in our cohort.

Clinical impact of malnutrition on complication rate and length of stay in elective ENT patients: a prospective cohort study.

Malnutrition is considered as an independent risk factor for morbidity, mortality and a prolonged hospital stay for in-hospital patients. While most available data on the impact of malnutrition on health-related and financial implications refer to gastroenterologic or abdominal surgery patients, little is known about the impact of malnutrition on Ear Nose Throat (ENT)/head and neck surgery patients. The objective of this study was to investigate the impact of malnutrition on morbidity and length of hospital stay in an elective ENT/head and neck surgery patient cohort. The study was performed as a single-center, prospective cohort study at a tertiary referral centre. Nutritional risk at admission was assessed using the NRS-2002 screening tool. Multivariate regression models were used to determine independent risk factors for complications and a prolonged hospitalization. Three hundred fifty one participants were included in the study. A malignant disease was found in 62 participants (17.7 %). 62 patients (17.7 %) were at a moderate to severe risk of malnutrition. A bad general health condition and complications during hospital stay could be identified as independent risk factors for a prolonged hospitalization. Patients with a malignant tumor showed a more than fourfold higher risk of developing at least one complication. Malnutrition, however, was not statistically associated with a higher complication rate or a prolonged hospital stay. Our data suggests that malnutrition does not seem to play such an important role as a risk factor for complications and a prolonged hospital stay in ENT patients as it does in other disciplines like abdominal surgery or gastroenterology.

[Renal cell carcinoma: Drug therapy and prognostic models]. / Nierenzellkarzinom : Medikamentöse Therapie und prognostische Modelle.

Renal cell carcinoma (RCC) represents the sixth-leading cancer-specific cause of death worldwide. This is mainly caused by metastatic or locally advanced RCC. Approximately 25-30% of patients present with metastasis during the initial diagnosis. Furthermore, 20-30% of patients develop metastatic disease following initial curative surgery. Metastatic RCC is characterized by a poor prognosis with a median overall survival of less than 2 years. Today, targeted therapies such as VEGF receptor inhibitors and antagonists as well as mTOR inhibitors represent the standard of care in metastatic RCC. Conventional chemotherapies or cytokine-based medications have been abandoned due to inferior clinical efficacy compared with targeted therapies. In Germany, sunitinib, pazopanib, temsirolimus, and bevacizumab have been approved for first-line treatment and sorafenib, axitinib, and everolimus for second-line treatment. Prognostic models, assessing individual risk profiles, have been developed in the last 15 years, which are crucial for the design of trials, patient counseling, and initiation of goal-directed therapies.

Strong association of serum- and glucocorticoid-regulated kinase 1 with peripheral and adipose tissue inflammation in obesity.

OBJECTIVE: The serum- and glucocorticoid-regulated kinase 1 (SGK1) is an early transcriptional target of glucocorticoids and is activated via insulin. Here we investigate the regulation of SGK1 expression in human obesity, diet-induced murine obesity and human monocytic cell line THP-1 monocytes. SUBJECTS AND METHODS: SGK1 expression was studied in subcutaneous and omental adipose tissue (AT) of 20 morbidly obese and 20 age- and gender-matched non-obese controls in murine diet-induced obesity and the THP-1 cell line. The regulation of SGK1 by inflammatory signals was tested in THP-1 cells. RESULTS: Murine diet-induced obesity is associated with a significant upregulation of Sgk1 in gonadal AT. Sgk1 expression is highest in the macrophage-rich stromal vascular fraction and lower in adipocytes. In humans, AT SGK1 is predominantly expressed in CD14(+) macrophages and significantly upregulated in omental and subcutaneous AT of obese subjects. SGK1 mRNA expression in both omental and subcutaneous AT correlates with body mass index, circulating leptin and C-reactive protein, and the local expression of inflammatory markers including monocyte chemotactic protein-1 and macrophage inflammatory protein-1α. The expression of SGK1 in THP-1 cells is upregulated by inflammatory signals, such as lipopolysaccharide and tumour necrosis factor-α, as well as during the induction of monocyte-to-macrophage maturation. CONCLUSIONS: Our data present the first link between SGK1 and obesity-associated inflammation. SGK1 expression is stimulated in response to inflammatory signals and increased in AT macrophages. The characterisation of SGK1 functions in obesity and immunity may help identify potential therapeutic targets in the treatment of obesity.

[The supraclavicular flap for reconstruction of the head and neck]. / Der supraclaviculäre Lappen zur Rekonstruktion im Kopf-Hals-Bereich.

The complex anatomy of the head and neck region requires the ability to raise a wide spectrum of pedicled and free flaps, to ensure optimal reconstruction of various defects by the reconstructive surgeon. The supraclavicular (island) flap, which has almost been buried in oblivion, provides excellent potential to reconstruct even bigger defects of the head and neck region, while causing minimal donor site morbidity at the same time. Its benefits lie in the reliable skin island and its wide arc of rotation, resulting in excellent cosmetic and functional outcomes.

[Prevention of postprostatectomy incontinence: etiology and risk factors]. / Verhinderung der Postprostatektomieinkontinenz: Ätiologie und Risikofaktoren.

The prevention of postprostatectomy incontinence has always been a challenge for urologists. Improved understanding of male continence and changes in pelvic anatomy after radical prostatectomy and the implementation of new surgical techniques raised hope of reduced incontinence rates. Despite using the full potential of continence-sparing techniques and atraumatic robot-assisted surgery, postprostatectomy incontinence still occurs in 5-20 % of cases. Recently published data showed a trend of improved early return to continence using anterior suspension or posterior reconstruction in robot-assisted prostatectomy. Postprostatectomy incontinence has a negative impact on quality of life and causes high costs of treatment. Therefore, further research in the amelioration of postoperative continence is mandatory.

[Differential diagnosis of multilocular bladder tumors in young patients: nephrogenic adenoma of the bladder in a 25-year old male cystinuria patient]. / Differentialdiagnose multilokulärer Blasentumoren bei jungen Patienten : Nephrogene Adenome in der Harnblase eines 25-jährigen Patienten mit Zystinurie.

The occurrence of malignant neoplasms of the urinary tract in patients below the age of 40 years is rare. Nevertheless, finding masses with suspicious malignant appearance in young patients should alert the examiner particularly against the background of reported cases of urothelial or urachal carcinoma in childhood or adolescence. This report presents the case of a 25-year-old male patient with recurrent urolithiasis due to cystinuria. During elective stone therapy via ureteroscopy a tumor of distinct malignant appearance was conspicuous and was immediately resected. The histopathological findings revealed the diagnosis of a nephrogenic adenoma.

Resistance to the mTOR-inhibitor RAD001 elevates integrin α2- and ß1-triggered motility, migration and invasion of prostate cancer cells.

BACKGROUND: Inhibitors of the mammalian target of rapamycin (mTOR) might become a novel tool to treat advanced prostate cancer. However, chronic drug exposure may trigger resistance, limiting the utility of mTOR inhibitors. METHODS: Metastatic potential of PC3 prostate cancer cells, susceptible (PC3(par)) or resistant (PC3(res)) to the mTOR-inhibitor RAD001 was investigated. Adhesion to vascular endothelium or immobilised collagen, fibronectin and laminin was quantified. Motility, migration and invasion were explored by modified Boyden chamber assay. Integrin α and ß subtypes were analysed by flow cytometry, western blotting and real-time PCR. Integrin-related signalling, EGFr, Akt, p70S6kinase and ERK1/2 activation were determined. RESULTS: Adhesion was reduced, whereas motility, migration and invasion were enhanced in PC3(res). The α2 and ß1 integrin subtypes were dramatically elevated, integrins α1 and α6 were lowered, whereas α5 was nearly lost in PC3(res). Activation of the Akt signalling pathway was strongly upregulated in these cells. Treating PC3(par) cells with RAD001 reduced motility, migration and invasion and deactivated Akt signalling. Blocking studies revealed that α2 and ß1 integrins significantly trigger the motile behaviour of the tumour cells. CONCLUSION: Chronic RAD001 treatment caused resistance development characterised by distinct modification of the integrin-expression profile, driving prostate cancer cells towards high motility.

[A leak in the lung: endobronchial one-way valve placement as treatment for a persistent bronchopleural fistula]. / Das Leck in der Lunge: Bronchoskopische Ventilimplantation zur Behandlung einer persistierenden bronchopleuralen Fistel.

Patients with bronchopleural fistula are at an increased risk of morbidity and mortality. Treatment of the air leak includes intrathoracic drainage, antibiotic therapy and closure of the fistula, which conventionally has been performed via surgical means. In patients with limited respiratory capacity, less-invasive alternatives are required. Here we report on a 62-year-old patient with underlying severe COPD, who was admitted with a lung abscess and consecutively developed a persistent bronchopleural fistula. Treatment involved antibiotic therapy and endobronchial one-way valve placement, which resulted in termination of the air leak and full recovery.

Influence of anabolic combinations of an androgen plus an estrogen on biochemical pathways in bovine uterine endometrium and ovary.

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, ERß, 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.

Hsa-mir-145 is the top EWS-FLI1-repressed microRNA involved in a positive feedback loop in Ewing's sarcoma.

EWS-FLI1 is a chromosome translocation-derived chimeric transcription factor that has a central and rate-limiting role in the pathogenesis of Ewing's sarcoma. Although the EWS-FLI1 transcriptomic signature has been extensively characterized on the mRNA level, information on its impact on non-coding RNA expression is lacking. We have performed a genome-wide analysis of microRNAs affected by RNAi-mediated silencing of EWS-FLI1 in Ewing's sarcoma cell lines, and differentially expressed between primary Ewing's sarcoma and mesenchymal progenitor cells. Here, we report on the identification of hsa-mir-145 as the top EWS-FLI1-repressed microRNA. Upon knockdown of EWS-FLI1, hsa-mir-145 expression dramatically increases in all Ewing's sarcoma cell lines tested. Vice versa, ectopic expression of the microRNA in Ewing's sarcoma cell lines strongly reduced EWS-FLI1 protein, whereas transfection of an anti-mir to hsa-mir-145 increased the EWS-FLI1 levels. Reporter gene assays revealed that this modulation of EWS-FLI1 protein was mediated by the microRNA targeting the FLI1 3'-untranslated region. Mutual regulations of EWS-FLI1 and hsa-mir-145 were mirrored by an inverse correlation between their expression levels in four of the Ewing's sarcoma cell lines tested. Consistent with the role of EWS-FLI1 in Ewing's sarcoma growth regulation, forced hsa-mir-145 expression halted Ewing's sarcoma cell line growth. These results identify feedback regulation between EWS-FLI1 and hsa-mir-145 as an important component of the EWS-FLI1-mediated Ewing's sarcomagenesis that may open a new avenue to future microRNA-mediated therapy of this devastating malignant disease.

The potential of bovine vaginal smear for biomarker development to trace the misuse of anabolic agents.

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-1ß, the growth factors FGF7, EGF, EGFR, IGF-1R, TGFα and LTF, the oncogen c-jun and other factors like actinß 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.

Interleukin family member ST2 and mortality in acute dyspnoea.

OBJECTIVES: The study objective was to investigate the prognostic utility and patient-specific characteristics of ST2 (suppression of tumorigenicity 2), assessed with a novel sensitive assay. BACKGROUND: Suppression of tumorigenicity 2 signalling has been shown to be associated with death in cardiac and pulmonary diseases. DESIGN/SUBJECTS: In an international multicentre cohort design, we prospectively enrolled 1091 patients presenting with acute dyspnoea to the emergency department (ED). ST2 was measured in a blinded fashion using a novel assay and compared to B-type natriuretic peptide (BNP) and NT-proBNP. The primary end-point was mortality within 30 days and 1 year. The prognostic value of ST2 was evaluated in comparison and in addition to BNP and NT-proBNP. RESULTS: Suppression of tumorigenicity 2 concentrations was higher amongst decedents than among survivors (median 85 vs. 43 U mL⁻¹, P < 0.001) and also higher in patients with impaired left ventricular ejection fraction (LVEF) when compared with preserved LVEF (P < 0.001). In receiver operator characteristics analysis, the area under the curve (AUC) for ST2, BNP and NT-proBNP to predict 30-day and 1-year mortality were 0.76, 0.63 and 0.71, and 0.72, 0.71 and 0.73, respectively. The combinations of ST2 with BNP or NT-proBNP improved prediction of mortality provided by BNP or NT-proBNP alone. After multivariable adjustment, ST2 values above the median (50 U mL⁻¹) significantly predicted 1-year mortality (HR 2.3, P < 0.001). CONCLUSION: In patients presenting to the ED with acute dyspnoea, ST2 is a strong and independent predictor of 30-day and 1-year mortality and might improve risk stratification already provided by BNP or NT-proBNP.

[Prevention of postoperative urinary stress incontinence]. / Prävention der postoperativen Harninkontinenz.

Postoperative stress incontinence following operative treatment of prostate cancer represents a considerable percentage of overall male incontinence. Postoperative incontinence following radical prostatectomy ranges between 2.9 and 87% depending on author and patient characteristics. Especially patient-related factors such as body mass index, age, size of prostate, preoperative incontinence, and concomitant diseases as well as classification of incontinence and modality of data collection influence postoperative continence rates. However, recent publications demonstrate the important impact of different operative techniques with regard to postoperative continence.The preservation of the muscular urethral sphincter is of particular importance. Nevertheless, the preservation of further anatomical structures contributes to postoperative continence. Preservation of bladder neck, nerve sparing, and reconstruction of the vesicourethral junction are operative techniques to prevent postoperative incontinence. In the last decade different modifications of the operative technique have been investigated regarding specific effects on postoperative continence. The interpretation of these studies investigating these operative techniques showed improvement in early continence with shorter period of time to continence. Long-term follow-up revealed no significant advantages for these modifications with regard to continence compared to the standard procedure. To evaluate the long-term effects of certain modifications to prevent incontinence, randomized and well powered studies are necessary. It seems to be most likely that a combination of these preventive modifications will lead to improved postoperative continence rates.

[Immunomodulatory treatment approaches for prostate cancer]. / Immunmodulatorische Therapieansätze beim Prostatakarzinom.

During the last 10 years different strategies for immunotherapy of prostate cancer have been investigated. These included unspecific and specific strategies to modulate or stimulate the immune system. For unspecific immunotherapy of prostate cancer innate humoral or cellular immune mechanisms are being stimulated, which are not specific to malignant cells. The global stimulation of the innate immune system is supposed to augment the immune reaction to prostate cancer by initiating an inflammatory reaction or other existing immune mechanisms. The main mediators and effectors of the unspecific immune system include humoral factors such as cytokines, complement system, and acute phase proteins and cellular components such as neutrophils, macrophages/monocytes, mast cells, and natural killer cells.In contrast, specific immunotherapy aims at adaptive immunity. This portion of the immune system can be amplified and thus specifically target tumor cells. Generation of a tumor-specific T cell reaction by vaccination or application of antibodies are the most promising approaches of specific immunotherapy. In a PubMed-based search of the current literature, publications regarding immunotherapy of prostate cancer were identified. The present article focuses on publications presenting clinical studies which investigate immunomodulatory treatments of prostate cancer. The results of these publications are described and discussed.

No relevant difference in quality of life and functional outcome at 12 months' follow-up-a randomised controlled trial comparing robot-assisted versus conventional laparoscopic Nissen fundoplication.

PURPOSE: The present randomised pilot trial was designed to compare robot-assisted (RALF) and conventional laparoscopic fundoplication (CLF) focussing on post-operative quality of life (QOL) and functional outcome. Any long-lasting advantages for patients in this regard could be a justification for the use of RALF for the treatment of gastroesophageal reflux disease (GERD). METHODS: Forty patients with GERD were randomised to either RALF or to CLF. During a follow-up period of 12 months, patients' QOL and functional outcome were investigated using disease-specific questionnaires. RESULTS: There were no significant differences in the mean QOL (1.3 versus 1.1; P = 0.374) and functional outcome (1.27 versus 1.3; P = 0.913) between both groups. Minor side effects such as bloating and persistent diarrhoea were present in four patients of each group. CONCLUSION: The present study did not show any benefit for RALF over CLF regarding QOL and functional outcome at 12 months' follow-up.

Success and complication parameters for laparoscopic surgery: a benchmark for natural orifice transluminal endoscopic surgery.

BACKGROUND: Natural orifice transluminal endoscopic surgery (NOTES) currently represents an issue of particular interest among surgeons and gastroenterologists. The principle of NOTES is the reduction of the operative trauma by using natural orifices of the human body to access the abdominal cavity. Regarding the tendency to further minimization of the surgical trauma, NOTES may be considered as a logical step in the evolution of minimally invasive surgery. Pioneers of this technique regard NOTES as the successor to laparoscopic surgery in enabling surgeons and gastroenterologists to conduct scarless surgery. This might not only lead to better cosmetic results but also enhance the prospect of decreases in wound infections and incisional hernias, as well as reducing operative stress, postoperative immobility, and pain. MATERIAL AND METHODS: In this article the authors collect and review the existing literature concerning NOTES and establish a benchmark for the assessment of this new technique by stating results from conventional minimally invasive surgery as the gold standard. CONCLUSION: It is shown that publications investigating possible advantages or long-term results of NOTES are scarce. However, the investigation and verification of potential advantages and disadvantages represent the most important step in the development of a new technique. Only proven advantages would justify the broad implementation of a new technique in relation to its specific risks. Conventional laparoscopic surgery as the current standard of minimally invasive surgery will be the benchmark for NOTES with regard to most issues. Superiority of NOTES in at least several issues would be the best argument for its further implementation into clinical practice.

[DNA-protective potential of polyphenols in human mucosa cell cultures]. / DNA-protektives Potential von Polyphenolen in humanen Schleimhautzellkulturen.

BACKGROUND: The concept of field cancerization in the head and neck offers an excellent basis for chemopreventive interventions. Within the last few years, polyphenols, the most abundant phytochemicals in our diet, have been identified as interesting chemopreventive agents based on their multiple actions. This study was designed to add more experimental data regarding the chemopreventive features of epigallocatechin gallate (EGCG) and tannin (TA) in cultures of fresh biopsied tissue to epidemiologic studies and animal and cell line experiments. MATERIALS AND METHODS: Miniorgan cultures (MOC) were produced from oropharyngeal mucosa-cubes about 1 mm(3), epithelialized and with their tissue structure preserved. The MOC were incubated with EGCG (0.1 and 5 microM) and TA (1 and 5 microM) for 30 min on three consecutive days. On the 3rd day, DNA damage was introduced with metabolically activated tobacco carcinogen benzo[a]pyren-7,8-dihydrodiol-9,10-epoxid (BPDE) [9 microM] for 60 min. The resulting DNA damage was measured with alkaline single-cell microgel electrophoresis (comet assay) and quantified using the olive tail moment (OTM). RESULTS: By incubating MOC with the polyphenols, the DNA damage caused by BPDE was significantly decreased at all concentrations. CONCLUSION: To our knowledge, this is the first test using cell cultures produced from fresh biopsies that demonstrates ECGC and TA as promising chemopreventive agents and confirms nutritional studies.

Gene expression in hair follicle dermal papilla cells after treatment with stanozolol.

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 (17beta-hydroxy-17alpha-methyl-5alpha-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 (ERalpha, ERbeta) 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. 5alpha-Steroidreductases (SRD5A1, SRD5A2) represented the enzyme group. Three reference genes were taken for relative quantification: ubiquitin (UBQ), glycerinaldehyde-3-phsophate-dehydrogenase (GAPDH), and beta-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.