To assess the impact of a mixture containing dioxin-like and non-dioxin-like polychlorinated biphenyls (PCBs), male mice were initiated with N-nitroso-diethylamine and subsequently treated with PCB126, an Ah-Receptor agonist, and PCB153, acting via activation of the constitutive androstane receptor. The two congeners were given at two dose levels: the low dose was adjusted to induce ~150-fold increases in cytochrome P450 (Cyp)1a1 (PCB126) and Cyp2b10 mRNAs (PCB153), and the high dose was chosen as twice the low dose. To keep the liver PCB levels constant, mice were given initial loading doses followed by weekly maintenance doses calculated on the basis of the PCBs' half-lives. Mice were treated with the individual congeners (low and high dose) or with a mixture consisting of the low doses of the 2 PCBs. The following results were obtained: (1) the 2 PCBs produced dose-dependent increases in Cyp1a1 and Cyp2b10 mRNA, protein, and activity when given individually; (2) combined treatment caused more than additive effects on Cyp1a1 mRNA expression, protein level, and ethoxyresurofin activity; (3) changes in the levels of several proteins were detected by proteome analysis in livers of PCB-treated mice; (4) besides these biological responses, the individual PCBs caused no significant increase in the number of glucose-6-phospatase (G6Pase)-deficient neoplastic lesions in liver, whereas a moderate significant effect occurred in the combination group. These results suggest weak but significant response-additive effects of the 2 PCBs when given in combination. They also suggest that the Cyp biomarkers tend to overestimate the carcinogenic response produced by the PCBs in mouse liver.

BACKGROUND:Successful surgical repair of a regurgitant mitral valve (MV) is dependent on a comprehensive assessment of its complex anatomy. Although there is limited evidence of the feasibility and accuracy of intraoperative real-time 3-dimensional transesophageal echocardiography (RT3DTEE) in MV surgery, its use is increasing worldwide. We designed this prospective observational study of patients with mitral regurgitation to test initial findings on the accuracy of RT3DTEE images in the diagnosis of MV prolapse and chordal rupture relative to 2D imaging and to assess the potential of RT3DTEE for visualizing leaflet clefts.METHODS:TEE-certified anesthesiologists examined 62 consecutive patients undergoing MV surgery by acquiring a full standard set of 2D TEE sections and 3D zoom recordings. Offline, 2D and 3D images were presented independently and in randomized order to 2 expert interpreters. Accuracy was determined using the surgical findings as the "gold standard."RESULTS:Surgical inspection identified 52 cases of MV prolapse (MVP). RT3DTEE correlated stronger with the surgical findings than 2D TEE for detection and localization of MVP (difference in proportions=33.9%, P<0.001) and chordal rupture (difference in proportions=25.8%, P<0.001). The superiority of RT3DTEE was significant for scallops A2, P1, P2 in MVP and A2, P2 in chordal rupture (all P<0.05). In 22 patients, leaflet clefts were also surgically repaired, and RT3DTEE was feasible in accessing them (κ=0.65, confidence interval [0.44, 0.81]).CONCLUSION:Although 2D TEE is currently the standard tool for intraoperative imaging in MV surgery, RT3DTEE improves the visualization of MV pathology and increases the accuracy of interpretation by facilitating spatial orientation. Further investigations, particularly those aimed at establishing its cost effectiveness, are indicated.

High-throughput screening approaches are carried out for the toxicity assessment of a large number of chemical compounds. In such large-scale in vitro toxicity studies several hundred or thousand concentration-response experiments are conducted. The automated evaluation of concentration-response data using statistical analysis scripts saves time and yields more consistent results in comparison to data analysis performed by the use of menu-driven statistical software. Automated statistical analysis requires that concentration-response data are available in a standardised data format across all compounds. To obtain consistent data formats, a standardised data management workflow must be established, including guidelines for data storage, data handling and data extraction. In this paper two procedures for data management within large-scale toxicological projects are proposed. Both procedures are based on Microsoft Excel files as the researcher’s primary data format and use a computer programme to automate the handling of data files. The first procedure assumes that data collection has not yet started whereas the second procedure can be used when data files already exist. Successful implementation of the two approaches into the European project ACuteTox is illustrated.

Concentration-response studies are performed to investigate the potency of the substance under investigation. Data are typically evaluated using non-linear regression. A common model is the log-logistic model which includes parameters for lower and upper boundary of mean response, EC50 and Hill slope. Often, response and/or concentration data are transformed before proceeding with the analysis of their relationship. This is motivated by practical reasons, including comparability of results across different assays. We prove mathematically that a linear transformation of data will not change the EC50 and Hill slope estimates and only results in an identical transformation of the estimated parameters for lower and upper boundary of mean response. However, fixing some of the parameters may lead to erroneous estimates. This is of practical relevance when data are corrected for background signal and normalized by background corrected solvent control and a reduced model is used in which the response range is fixed between 100% and 0%. Computer simulations and a real data example are used to illustrate the impact of data transformations on parameter estimation. We further shed light on some common problems arising in the analysis of concentration-response data. Recommendations for practical implementation in concentration-response analysis are provided. (C) 2012 Elsevier Ireland Ltd. All rights reserved.

ABSTRACT Adeno-associated virus type 2 (AAV2) has gained much interest as a gene delivery vector. A hallmark of AAV2-mediated gene transfer is an intracellular conformational change of the virus capsid, leading to the exposure of infection-relevant protein domains. These protein domains, which are located on the N-terminal portion of the structural proteins VP1 and VP2, include a catalytic phospholipase A2 domain and three clusters of basic amino acids. We have identified additional protein sequence motifs located on the VP1/2 N terminus that also proved to be obligatory for virus infectivity. These motifs include signals that are known to be involved in protein interaction, endosomal sorting and signal transduction in eukaryotic cells. Among different AAV serotypes they are highly conserved and mutation of critical amino acids of the respective motifs led to a severe infection-deficient phenotype. In particular, mutation of a YXXQ-sequence motif significantly reduced accumulation of virus capsids around the nucleus in comparison to wild-type AAV2. Interestingly, intracellular trafficking of AAV2 was shown to be independent of PLA2 activity. Moreover, mutation of three PDZ-binding motifs, which are located consecutively at the very tip of the VP1 N terminus, revealed a nuclear transport-defective phenotype, suggesting a role in nuclear uptake of the virus through an as-yet-unknown mechanism.

Objective: To evaluate whether dynamic susceptibility-weighted contrast-enhanced (DSC), dynamic contrast-enhanced (DCE), and proton spectroscopic imaging (H-1-MRSI) can identify progression and predict treatment failure during follow-up before tumor size changes, contrast agent uptake, or when new lesions become obvious. The aim was also to find out which of the aforementioned techniques had the best diagnostic performance compared with each other and standard magnetic resonance imaging (MRI).Materials and Methods: Thirty-seven patients with gliomas (21 women, 16 men; mean age at inclusion, 48 +/- 14 years [standard deviation]) were assessed prospectively by H-1-MRSI (point-resolved spectroscopy), DCE, and DSC perfusion MRI, each after a single dose of gadobenate dimeglumine during follow-up. Histology was available in all cases (resection, N = 18; biopsy, N = 19). All patients with low-grade gliomas (n = 20) did not receive any radio-or chemotherapy after partial resection (n = 7) or biopsy (n = 13), whereas 17 patients with high-grade gliomas had received adjuvant radiotherapy immediately after surgery. Tumor progression (progressive disease, PD) was defined as increase in longest glioma diameter by at least 20% (Response Evaluation Criteria in Solid Tumors), appearance of new lesions, or new contrast-enhancement. DSC, DCE, and MRSI image analyses comprised a detailed semiquantitative region of interest (ROI) analysis of the different parameters. Wilcoxon signed-rank test, Wilcoxon rank sum test, and Cox regression were used for statistical analysis.Results: The median follow-up time was 607 days. Twenty patients showed PD (54%), 8 of 20 with low-grade (40%) and 12 of 17 with high-grade gliomas (71%). In PD, significant positive differences between log2-transformed ROI ratios at the last measurement in comparison to the first measurement (baseline) could be detected for tumor blood flow (P < 0.006) and volume (P < 0.001) derived from DSC and for maximum choline within tumor tissue (P = 0.0029) and Cho/Cr (P = 0.032) but not choline/N-acetyl-aspartate (P = 0.37) derived from MRSI. In contrast, these parameters were not significantly higher at last measurement in stable disease. Also, the differences between last value and baseline were significantly different between PD and stable disease for tumor blood flow (P < 0.004) and volume (P < 0.002) as well as for maximum choline within tumor tissue (P = 0.0011). The best prognostic parameter for PD at Cox analysis was time-dependent difference to baseline of log2 of relative regional cerebral blood flow normalized on gray matter (hazard ratio, 2.67; 95% confidence interval, 1.25-6.08; P = 0.01), while a prognostic value of MRS parameters could not be demonstrated.Conclusion: DSC perfusion imaging can identify progression and can predict treatment failure during follow-up of gliomas with the best diagnostic performance.

Objective: To evaluate whether dynamic susceptibility-weighted contrast-enhanced (DSC), dynamic contrast-enhanced (DCE), and proton spectroscopic imaging (1H-MRSI) can identify progression and predict treatment failure during follow-up before tumor size changes, contrast agent uptake, or when new lesions become obvious. The aim was also to find out which of the aforementioned techniques had the best diagnostic performance compared with each other and standard magnetic resonance imaging (MRI). Materials and Methods: Thirty-seven patients with gliomas (21 women, 16 men; mean age at inclusion, 48 ± 14 years [standard deviation]) were assessed prospectively by 1H-MRSI (point-resolved spectroscopy), DCE, and DSC perfusion MRI, each after a single dose of gadobenate dimeglumine during follow-up. Histology was available in all cases (resection, N = 18; biopsy, N = 19). All patients with low-grade gliomas (n = 20) did not receive any radio- or chemotherapy after partial resection (n = 7) or biopsy (n = 13), whereas 17 patients with high-grade gliomas had received adjuvant radiotherapy immediately after surgery. Tumor progression (progressive disease, PD) was defined as increase in longest glioma diameter by at least 20% (Response Evaluation Criteria in Solid Tumors), appearance of new lesions, or new contrast-enhancement. DSC, DCE, and MRSI image analyses comprised a detailed semiquantitative region of interest (ROI) analysis of the different parameters. Wilcoxon signed-rank test, Wilcoxon rank sum test, and Cox regression were used for statistical analysis. Results: The median follow-up time was 607 days. Twenty patients showed PD (54%), 8 of 20 with low-grade (40%) and 12 of 17 with high-grade gliomas (71%). In PD, significant positive differences between log2-transformed ROI ratios at the last measurement in comparison to the first measurement (baseline) could be detected for tumor blood flow (P < 0.006) and volume (P < 0.001) derived from DSC and for maximum choline within tumor tissue (P = 0.0029) and Cho/Cr (P = 0.032) but not choline/N-acetyl-aspartate (P = 0.37) derived from MRSI. In contrast, these parameters were not significantly higher at last measurement in stable disease. Also, the differences between last value and baseline were significantly different between PD and stable disease for tumor blood flow (P < 0.004) and volume (P < 0.002) as well as for maximum choline within tumor tissue (P = 0.0011). The best prognostic parameter for PD at Cox analysis was time-dependent difference to baseline of log2 of relative regional cerebral blood flow normalized on gray matter (hazard ratio, 2.67; 95% confidence interval, 1.25–6.08; P = 0.01), while a prognostic value of MRS parameters could not be demonstrated. Conclusion: DSC perfusion imaging can identify progression and can predict treatment failure during follow-up of gliomas with the best diagnostic performance.

As a part of EU-project ReProTect, a comparison of the dual re-circulating human placental perfusion system was carried out, by two independent research groups. The detailed placental transfer data of model compounds [antipyrine, benzo(a)pyrene, PhIP (2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine) and IQ (2-amino-3-methylimidazo(4,5-f)quinoline] has been/will be published separately. For this project, a comparative re-analysis was done, by curve fitting the data and calculating two endpoints: AUC120, defined as the area under the curve between time 0 and time 120 min and as t0.5, defined as the time when the fetal to maternal concentration ratio is expected to be 0.5. The transport of the compounds from maternal to fetal circulation across the perfused placenta could be ranked in the order of antipyrine > IQ > PhIP in terms of both t0.5 and AUC120 by both partners. For benzo(a)pyrene the curve fitting failed. These prevalidation results give confidence for harmonization of the placental perfusion system to be used as one of the test methods in a panel for reproductive toxicology to model placental transfer in humans.

The final stages of male gametogenesis are sensitive targets of DNA-reactive chemicals, most of which form adducts. Comet assay is a widely applied genotoxicity test that reveals DNA adducts through breaks formed during repair processes. However, sperm cells are essentially devoid of repair enzymes and comet assay is poorly sensitive in detecting chemically induced DNA lesions in sperm. To overcome such limitation, in a previous paper we proposed a modified protocol for comet assay. In this work we further tested the method treating bull sperm with additional mutagens (diethylsulfate, mitomycin C, bleomycin and colchicine) in parallel with the standard comet assay. No treatment-related increase of DNA migration was ever detected with the standard protocol. A dose-dependent effect of diethylsulfate, was obtained with the modified assay. As expected, the mitotic poison colchicine resulted negative even by the modified assay. Results with the other two compounds were consistent with their mechanism of action.

ReProTect is a project within the 6th European Framework Program which has developed alternative methods aimed to reduce or replace animal experimentation in the field of reproductive toxicology. In its final year, a ring trial, named the "Feasibility Study", was conducted, in which 10 blinded chemicals with toxicologically well-documented profiles were analyzed by employing a test battery of 14 in vitro assays. EC50 (half maximal effective concentration) or equivalent endpoints were determined and the test compounds were ranked relative to chemicals previously assayed in the tests of the battery. This comparative analysis together with a weight of evidence approach allowed a robust prediction of adverse effects on fertility and embryonic development of the 10 test chemicals in vivo. In summary, the vast majority of the predictions made based on the in vitro results turned out to be correct when compared to the whole animal data. The procedure used here, a nearest neighbor analysis coupled with a weight of evidence approach, may guide future activities in the field of alternative toxicity testing.

Despite more than a decade of research in the field of endocrine active compounds with affinity for the androgen receptor (AR), still no validated recombinant AR binding assay is available, although recombinant AR can be obtained from several sources. With funding from the European Union (EU)-sponsored 6th framework project, ReProTect, we developed a model protocol for such an assay based on a simple AR binding assay recently developed at our institution. Important features of the protocol were the use of a rat recombinant fusion protein to thioredoxin containing both the hinge region and ligand binding domain (LBD) of the rat AR (which is identical to the human AR-LBD) and performance in a 96-well plate format. Besides two reference compounds [dihydrotestosterone (DHT), androstenedione] ten test compounds with different affinities for the AR [levonorgestrel, progesterone, prochloraz, 17alpha-methyltestosterone, flutamide, norethynodrel, o,p'-DDT, dibutylphthalate, vinclozolin, linuron] were used to explore the performance of the assay. At least three independent experiments per compound were performed. The AR binding properties of reference and test compounds were well detected, in terms of the relative ranking of binding affinities, there was good agreement with published data obtained from experiments using recombinant AR preparations. Irrespective of the chemical nature of the compound, individual IC(50)-values for a given compound varied by not more than a factor of 2.6. Our data demonstrate that the assay reliably ranked compounds with strong, weak, and no/marginal affinity for the AR with high accuracy. It avoids the manipulation and use of animals, as a recombinant protein is used and thus contributes to the 3R concept. On the whole, this assay is a promising candidate for further validation.

Despite about two decades of research in the field of endocrine active compounds, still no validated human recombinant (hr) estrogen receptor-alpha (ERalpha) binding assay is available, although hr-ERalpha is available from several sources. In a joint effort, US EPA and Bayer Schering Pharma with funding from the EU-sponsored 6th framework project, ReProTect, developed a model protocol for such a binding assay. Important features of this assay are the use of a full length hr-ERalpha and performance in a 96-well plate format. A full length hr-ERalpha was chosen, as it was considered to provide the most accurate and human-relevant results, whereas truncated receptors could perform differently. Besides three reference compounds [17beta-estradiol, norethynodrel, dibutylphthalate] nine test compounds with different affinities for the ERalpha [diethylstilbestrol (DES), ethynylestradiol, meso-hexestrol, equol, genistein, o,p'-DDT, nonylphenol, n-butylparaben, and corticosterone] were used to explore the performance of the assay. Three independent experiments per compound were performed on different days, and dilutions of test compounds from deep-frozen stocks, solutions of radiolabeled ligand and receptor preparation were freshly prepared for each experiment. The ERalpha binding properties of reference and test compounds were well detected. As expected dibutylphthalate and corticosterone were non-binders in this assay. In terms of the relative ranking of binding affinities, there was good agreement with published data obtained from experiments using a human recombinant ERalpha ligand binding domain. Irrespective of the chemical nature of the compound, individual IC(50)-values for a given compound varied by not more than a factor of 2.5. Our data demonstrate that the assay was robust and reliably ranked compounds with strong, weak, and no affinity for the ERalpha with high accuracy. It avoids the manipulation and use of animals, i.e., the preparation of uterine cytosol as receptor source from ovariectomized rats, as a recombinant protein is used and thus contributes to the 3R concept (reduce, replace, and refine). Furthermore, in contrast to other assays, this assay could be adjusted to an intermediate/high throughput format. On the whole, this assay is a promising candidate for further validation.

To date there are no validated methods available to test androgenicity or antiandrogenicity in vitro. A problem with testing androgenicity using reporter genes is the possibility by other steroid receptors than androgen receptors to activate the same reporter gene, thereby lowering selectivity. To avoid this we have established a robust and very selective method, the AR CALUX® reporter gene assay, to test androgenic and antiandrogenic activity of compounds in vitro. This assay uses a human U2-OS cell line stably transfected with the human androgen receptor and an androgen receptor responsive reporter gene. We optimized protocols to be used in combination with AR CALUX cells and carried out an in house prevalidation. In addition we successfully transferred this assay to another laboratory, leading to comparable test results with a panel of androgen receptor agonists and antagonists. The assay was able to readily rank a range of chemicals on the basis of their EC50 values. The CALUX assay was found to be selective for androgens and seemed not influenced by signaling through other steroid receptors.

The human endometrium is unique among adult tissues. Its functions are modulated by numerous hormones and mediators. The aim of this study was to evaluate the suitability of human endometrial explants for studying functional effects of chemicals and drugs on gene expression biomarkers. Endometrial tissues were obtained by aspiration curettage and cultivated for up to 24 h. Relative mRNA concentrations were determined by reverse transcription quantitative real-time PCR. Viability was assessed by light microscopy, lactate dehydrogenase assay and scanning electron microscopy. It was acceptable after 6 h of culture but reduced after 24 h. Culture-induced alterations of mRNA levels were found for progesterone receptor, estrogen receptor(α), leukemia inhibitory factor and cyclooxygenase-2 in tissues from all cycle stages. The suitability of the model to detect chemical effects was demonstrated by the down-regulation of cyclooxygenase-2 mRNA by chlormadinone acetate in proliferative and secretory endometrium. The model is mainly restricted by interindividual variations and varying tissue quality. An advantage is the preservation of tissue composition. We conclude that human endometrial explants are a complex model due to limited viability, difficult standardization and intrinsic alterations during culture. Experiments with this model should be performed over a limited time period under strictly controlled conditions.

Short-term dynamic culture of rat testicular fragments was evaluated as a model to assess effects on steroidogenesis. A total of 11 compounds differentially affecting testosterone synthesis (aminoglutethimide, ketoconazole, danazol, flutamide, diethylstilbestrol, genistein, butylparaben, nonoxynol-9, dimethoate, RU 486, and cadmium chloride) were used to explore the performance of the assay. Testosterone secretion into the medium and testosterone retained in tissue fragments was determined as a measure of steroidogenesis. Three independent experiments per compound were performed. The known in vitro inhibitory properties of most compounds could be detected. Whenever significant inhibition of testosterone synthesis was observed, low effect concentrations for a given compound differed frequently only by a factor of ≤3.3, maximally by a factor of 10 from each other across the three experiments and besides a decrease of testosterone concentrations in the medium, corresponding reduction of testosterone levels in tissue fragment was obtained. On the other hand, despite the use of high concentrations, danazol and genistein were unexpectedly tested negative, and considerable variability of testosterone production within control and treatment groups of individual experiments were frequently observed. In contrast to cellular systems, specific assessment of cytotoxicity to the testosterone producing Leydig cells was not possible, effects requiring prolonged incubation times could be overlooked, and the observed variability of hormone production necessitates a considerable number of individual incubations per treatment group. On the whole, short-term dynamic culture of rat testicular fragments offers limited potential to assess effects on steroidogenesis by compounds of moderate to strong potency that directly interfere with steroidogenic enzymes or rapidly induce cytotoxicity.

A stem cell-based reporter assay was developed to detect drug-induced alterations in the canonical Wnt/β-catenin signaling pathway, which is involved in the regulation of early embryonic development. The so-called ReProGlo assay allows simultaneous determination of cell viability and luciferase reporter activity in a high throughput 96-well microtiter format. A clone of mouse embryonic stem (mES) cells stably expressing the SuperTopFlash reporter was established. This allows Wnt pathway activity determinations in undifferentiated mES cells and their differentiated descendants. Several test chemicals were analyzed in the new assay system. Known embryotoxicants like retinoic acid or lithium chloride induced concentration-dependent increases in reporter activity. The potency of valproic acid and a series of structural analogs to activate the Wnt pathway correlated well with their reported teratogenic activity in the mouse. Cyclophosphamide was also active but only after metabolic activation by hepatocytes. The new test may help to predict embryotoxic potential of chemicals.

The new European chemicals policy for the Registration, Evaluation, Authorisation and Restriction of Chemicals (the REACH system), requires the routine toxicological testing of chemicals produced in volumes of more than one tonne per year, in order to enhance environmental and consumer safety (1). Under the REACH system policy, it will be most probably necessary to test several thousand chemicals for their toxicological properties.

The olfactory receptor multigene family is organized in clusters spread throughout the genome. In the present study, we have sequenced two subregions of the mOR37 gene cluster on mouse chromosome 4. The resulting 100 kb of sequence revealed seven odorant receptor coding regions and one gene fragment. Sequence analyses reveal that the mOR37 gene cluster may represent a rather ancient cluster. The mOR37 genes exhibit a complex intron/exon structure, and some appear to be differentially spliced. All genes in the cluster share conserved sequence motifs 5′ of their putative initial exons, which represent potential binding sites for transcription factors. The clustered organization and conserved sequence motifs suggest common expression control mechanisms for these genes.