Developmental neurotoxicity (DNT) and many forms of reproductive toxicity (RT) often manifest themselves in functional deficits that are not necessarily based on cell death, but rather on minor changes relating to cell differentiation or communication. The fields of DNT/RT would greatly benefit from in vitro tests that allow the identification of toxicant-induced changes of the cellular proteostasis, or of its underlying transcriptome network. Therefore, the ‘human embryonic stem cell (hESC)-derived novel alternative test systems (ESNATS)’ European commission research project established RT tests based on defined differentiation protocols of hESC and their progeny. Valproic acid (VPA) and methylmercury (MeHg) were used as positive control compounds to address the following fundamental questions: (1) Does transcriptome analysis allow discrimination of the two compounds? (2) How does analysis of enriched transcription factor binding sites (TFBS) and of individual probe sets (PS) distinguish between test systems? (3) Can batch effects be controlled? (4) How many DNA microarrays are needed? (5) Is the highest non-cytotoxic concentration optimal and relevant for the study of transcriptome changes? VPA triggered vast transcriptional changes, whereas MeHg altered fewer transcripts. To attenuate batch effects, analysis has been focused on the 500 PS with highest variability. The test systems differed significantly in their responses (<20 % overlap). Moreover, within one test system, little overlap between the PS changed by the two compounds has been observed. However, using TFBS enrichment, a relatively large ‘common response’ to VPA and MeHg could be distinguished from ‘compound-specific’ responses. In conclusion, the ESNATS assay battery allows classification of human DNT/RT toxicants on the basis of their transcriptome profiles.Electronic supplementary materialThe online version of this article (doi:10.1007/s00204-012-0967-3) contains supplementary material, which is available to authorized users.

Reduced glutathione (GSH) is critical for many cellular processes, and both its intracellular and extracellular concentrations are tightly regulated. Intracellular GSH levels are regulated by two main mechanisms: by adjusting the rates of synthesis and of export from cells. Some of the proteins responsible for GSH export from mammalian cells have recently been identified, and there is increasing evidence that these GSH exporters are multispecific and multifunctional, regulating a number of key biological processes. In particular, the multidrug resistance-associated proteins (Mrp/Abcc) appear to mediate GSH export and homeostasis. The Mrp proteins mediate not only GSH efflux, but they also export oxidized glutathione derivatives (e.g., glutathione disulfide (GSSG), S-nitrosoglutathione (GS-NO), and glutathione-metal complexes), as well as other glutathione S-conjugates. The ability to export both GSH and oxidized derivatives of GSH, endows these transporters with the capacity to directly regulate the cellular thiol-redox status, and therefore the ability to influence many key signaling and biochemical pathways. Among the many processes that are influenced by the GSH transporters are apoptosis, cell proliferation, and cell differentiation. This report summarizes the evidence that Mrps contribute to the regulation of cellular GSH levels and the thiol redox state, and thus to the many biochemical processes that are influenced by this tripeptide.

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Adverse outcome pathways (AOPs) are a recent toxicological construct that connects, in a formalized, transparent and quality-controlled way, mechanistic information to apical endpoints for regulatory purposes. AOP links a molecular initiating event (MIE) to the adverse outcome (AO) via key events (KE), in a way specified by key event relationships (KER). Although this approach to formalize mechanistic toxicological information only started in 2010, over 200 AOPs have already been established. At this stage, new requirements arise, such as the need for harmonization and re-assessment, for continuous updating, as well as for alerting about pitfalls, misuses and limits of applicability. In this review, the history of the AOP concept and its most prominent strengths are discussed, including the advantages of a formalized approach, the systematic collection of weight of evidence, the linkage of mechanisms to apical end points, the examination of the plausibility of epidemiological data, the identification of critical knowledge gaps and the design of mechanistic test methods. To prepare the ground for a broadened and appropriate use of AOPs, some widespread misconceptions are explained. Moreover, potential weaknesses and shortcomings of the current AOP rule set are addressed (1) to facilitate the discussion on its further evolution and (2) to better define appropriate vs. less suitable application areas. Exemplary toxicological studies are presented to discuss the linearity assumptions of AOP, the management of event modifiers and compensatory mechanisms, and whether a separation of toxicodynamics from toxicokinetics including metabolism is possible in the framework of pathway plasticity. Suggestions on how to compromise between different needs of AOP stakeholders have been added. A clear definition of open questions and limitations is provided to encourage further progress in the field.

Purpose: Although the central role of the immune system for tumor prognosis is generally accepted, a single robust marker is not yet available.Experimental Design: On the basis of receiver operating characteristic analyses, robust markers were identified from a 60-gene B cell-derived metagene and analyzed in gene expression profiles of 1,810 breast cancer; 1,056 non-small cell lung carcinoma (NSCLC); 513 colorectal; and 426 ovarian cancer patients. Protein and RNA levels were examined in paraffin-embedded tissue of 330 breast cancer patients. The cell types were identified with immunohistochemical costaining and confocal fluorescence microscopy.Results: We identified immunoglobulin k C (IGKC) which as a single marker is similarly predictive and prognostic as the entire B-cell metagene. IGKC was consistently associated with metastasis-free survival across different molecular subtypes in node-negative breast cancer (n ¼ 965) and predicted response to anthracycline-based neoadjuvant chemotherapy (n ¼ 845; P < 0.001). In addition, IGKC gene expression was prognostic in NSCLC and colorectal cancer. No association was observed in ovarian cancer. IGKC protein expression was significantly associated with survival in paraffin-embedded tissues of 330 breast cancer patients. Tumor-infiltrating plasma cells were identified as the source of IGKC expression.Conclusion: Our findings provide IGKC as a novel diagnostic marker for risk stratification in human cancer and support concepts to exploit the humoral immune response for anticancer therapy. It could be validated in several independent cohorts and carried out similarly well in RNA from fresh frozen as well as from paraffin tissue and on protein level by immunostaining. Clin Cancer Res; 18(9); 2695-703. Ó2012 AACR.

Several studies suggest a link between circadian rhythm disturbances and tumorigenesis. However, the association between circadian clock genes and prognosis in breast cancer has not been systematically studied. Therefore, we examined the expression of 17 clock components in tumors from 766 node-negative breast cancer patients that were untreated in both neoadjuvant and adjuvant settings. In addition, their association with metastasis-free survival (MFS) and correlation to clinicopathological parameters were investigated. Aiming to estimate functionality of the clockwork, we studied clock gene expression relationships by correlation analysis. Higher expression of several clock genes (e.g., CLOCK, PER1, PER2, PER3, CRY2, NPAS2 and RORC) was found to be associated with longer MFS in univariate Cox regression analyses (HR<1 and FDR-adjusted P < 0.05). Stratification according to molecular subtype revealed prognostic relevance for PER1, PER3, CRY2 and NFIL3 in the ER+/HER2- subgroup, CLOCK and NPAS2 in the ER-/HER2- subtype, and ARNTL2 in HER2+ breast cancer. In the multivariate Cox model, only PER3 (HR = 0.66; P = 0.016) and RORC (HR = 0.42; P = 0.003) were found to be associated with survival outcome independent of established clinicopathological parameters. Pairwise correlations between functionally-related clock genes (e.g., PER2-PER3 and CRY2-PER3) were stronger in ER+, HER2- and low-grade carcinomas; whereas, weaker correlation coefficients were observed in ER- and HER2+ tumors, high-grade tumors and tumors that progressed to metastatic disease. In conclusion, loss of clock genes is associated with worse prognosis in breast cancer. Coordinated co-expression of clock genes, indicative of a functional circadian clock, is maintained in ER+, HER2-, low grade and non-metastasizing tumors but is compromised in more aggressive carcinomas.

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Metastasis from primary tumors remains a major problem for tumor therapy. In the search for markers of metastasis and more effective therapies, the tumor metabolome is relevant because of its importance to the malignant phenotype and metastatic capacity of tumor cells. Altered choline metabolism is a hallmark of cancer. More specifically, a decreased glycerophosphocholine (GPC) to phosphocholine (PC) ratio was reported in breast, ovarian, and prostate cancers. Improved strategies to exploit this altered choline metabolism are therefore required. However, the critical enzyme cleaving GPC to produce choline, the initial step in the pathway controlling the GPC/PC ratio, remained unknown. In the present work, we have identified the enzyme, here named EDI3 (endometrial differential 3). Purified recombinant EDI3 protein cleaves GPC to form glycerol-3-phosphate and choline. Silencing EDI3 in MCF-7 cells decreased this enzymatic activity, increased the intracellular GPC/PC ratio, and decreased downstream lipid metabolites. Downregulating EDI3 activity inhibited cell migration via disruption of the PKCα signaling pathway, with stable overexpression of EDI3 showing the opposite effect. EDI3 was originally identified in our screening study comparing mRNA levels in metastasizing and nonmetastasizing endometrial carcinomas. Both Kaplan-Meier and multivariate analyses revealed a negative association between high EDI3 expression and relapse-free survival time in both endometrial (P < 0.001) and ovarian (P = 0.029) cancers. Overall, we have identified EDI3, a key enzyme controlling GPC and choline metabolism. Because inhibition of EDI3 activity corrects the GPC/PC ratio and decreases the migration capacity of tumor cells, it represents a possible target for therapeutic intervention.