Regina Stoeber

Bio: Regina Stoeber is an academic researcher from Technical University of Dortmund. The author has contributed to research in topics: Breast cancer & Incubation period. The author has an hindex of 4, co-authored 6 publications receiving 128 citations.

Prediction of human drug-induced liver injury (DILI) in relation to oral doses and blood concentrations

Abstract: Drug-induced liver injury (DILI) cannot be accurately predicted by animal models. In addition, currently available in vitro methods do not allow for the estimation of hepatotoxic doses or the determination of an acceptable daily intake (ADI). To overcome this limitation, an in vitro/in silico method was established that predicts the risk of human DILI in relation to oral doses and blood concentrations. This method can be used to estimate DILI risk if the maximal blood concentration (Cmax) of the test compound is known. Moreover, an ADI can be estimated even for compounds without information on blood concentrations. To systematically optimize the in vitro system, two novel test performance metrics were introduced, the toxicity separation index (TSI) which quantifies how well a test differentiates between hepatotoxic and non-hepatotoxic compounds, and the toxicity estimation index (TEI) which measures how well hepatotoxic blood concentrations in vivo can be estimated. In vitro test performance was optimized for a training set of 28 compounds, based on TSI and TEI, demonstrating that (1) concentrations where cytotoxicity first becomes evident in vitro (EC10) yielded better metrics than higher toxicity thresholds (EC50); (2) compound incubation for 48 h was better than 24 h, with no further improvement of TSI after 7 days incubation; (3) metrics were moderately improved by adding gene expression to the test battery; (4) evaluation of pharmacokinetic parameters demonstrated that total blood compound concentrations and the 95%-population-based percentile of Cmax were best suited to estimate human toxicity. With a support vector machine-based classifier, using EC10 and Cmax as variables, the cross-validated sensitivity, specificity and accuracy for hepatotoxicity prediction were 100, 88 and 93%, respectively. Concentrations in the culture medium allowed extrapolation to blood concentrations in vivo that are associated with a specific probability of hepatotoxicity and the corresponding oral doses were obtained by reverse modeling. Application of this in vitro/in silico method to the rat hepatotoxicant pulegone resulted in an ADI that was similar to values previously established based on animal experiments. In conclusion, the proposed method links oral doses and blood concentrations of test compounds to the probability of hepatotoxicity.

Definition of transcriptome-based indices for quantitative characterization of chemically disturbed stem cell development: introduction of the STOP-Tox ukn and STOP-Tox ukk tests

Abstract: Stem cell-based in vitro test systems can recapitulate specific phases of human development. In the UKK test system, human pluripotent stem cells (hPSCs) randomly differentiate into cells of the three germ layers and their derivatives. In the UKN1 test system, hPSCs differentiate into early neural precursor cells. During the normal differentiation period (14 days) of the UKK system, 570 genes [849 probe sets (PSs)] were regulated >fivefold; in the UKN1 system (6 days), 879 genes (1238 PSs) were regulated. We refer to these genes as ‘developmental genes’. In the present study, we used genome-wide expression data of 12 test substances in the UKK and UKN1 test systems to understand the basic principles of how chemicals interfere with the spontaneous transcriptional development in both test systems. The set of test compounds included six histone deacetylase inhibitors (HDACis), six mercury-containing compounds (‘mercurials’) and thalidomide. All compounds were tested at the maximum non-cytotoxic concentration, while valproic acid and thalidomide were additionally tested over a wide range of concentrations. In total, 242 genes (252 PSs) in the UKK test system and 793 genes (1092 PSs) in the UKN1 test system were deregulated by the 12 test compounds. We identified sets of ‘diagnostic genes’ appropriate for the identification of the influence of HDACis or mercurials. Test compounds that interfered with the expression of developmental genes usually antagonized their spontaneous development, meaning that up-regulated developmental genes were suppressed and developmental genes whose expression normally decreases were induced. The fraction of compromised developmental genes varied widely between the test compounds, and it reached up to 60 %. To quantitatively describe disturbed development on a genome-wide basis, we recommend a concept of two indices, ‘developmental potency’ (D p) and ‘developmental index’ (D i), whereby D p is the fraction of all developmental genes that are up- or down-regulated by a test compound, and D i is the ratio of overrepresentation of developmental genes among all genes deregulated by a test compound. The use of D i makes hazard identification more sensitive because some compounds compromise the expression of only a relatively small number of genes but have a high propensity to deregulate developmental genes specifically, resulting in a low D p but a high D i. In conclusion, the concept based on the indices D p and D i offers the possibility to quantitatively express the propensity of test compounds to interfere with normal development.

Relevance of the incubation period in cytotoxicity testing with primary human hepatocytes.

Abstract: Primary human hepatocytes (PHHs) remain the gold standard for in vitro testing in the field of pharmacology and toxicology. One crucial parameter influencing the results of in vitro tests is the incubation period with test compounds. It has been suggested that longer incubation periods may be critical for the prediction of repeated dose toxicity. However, a study that systematically analyzes the relationship between incubation period and cytotoxicity in PHHs is not available. To close this gap, 30 compounds were tested in a concentration-dependent manner for cytotoxicity in cultivated cryopreserved PHHs (three donors per compound) for 1, 2 and 7 days. The median of the EC50 values of all compounds decreased 1.78-fold on day 2 compared to day 1, and 1.89-fold on day 7 compared to day 1. Median values of EC50 ratios of all compounds at day 2 and day 7 were close to one but for individual compounds the ratio increased up to almost six. Strong correlations were obtained for EC50 on day 1 and day 7 (R = 0.985; 95% CI 0.960–0.994), day 1 and day 2 (R = 0.964; 95% CI 0.910–0.986), as well as day 2 and day 7 (R = 0.981; 95% CI 0.955–0.992). However, compound specific differences also occurred. Whereas, for example, busulfan showed a relatively strong increase on day 7 compared to day 1, cytotoxicity of acetaminophen did not increase during longer incubation periods. To validate the observed correlations, a publicly available data set, containing data on the cytotoxicity of human hepatocytes cultivated as spheroids for incubation periods of 5 and 14 days, was analyzed. A high correlation coefficient of EC50 values at day 5 and day 14 was obtained (R = 0.894; 95% CI 0.798–0.945). In conclusion, the median cytotoxicity of the test compounds increased between 1 and 2 days of incubation, with no or only a minimal further increase until day 7. It remains to be studied whether the different results obtained for some individual compounds after longer exposure periods would correspond better to human-repeated dose toxicity.

Highlight report: Intratumoral metabolomic heterogeneity of breast cancer.

Abstract: Recently, Mikheil Gogiashvili and colleagues from TU-Dortmund have published a study about the metabolomics heterogeneity of breast cancer (Gogiashvili et al., 2017). The background of this study is the practically relevant question, whether measurement of a single biopsy is sufficient when analyzing tumors from a cohort of patients. In recent years metabolic profiling by high-resolution magic angle spinning nuclear magnetic resonance spectroscopy has been increasingly used to characterize the metabolome of breast cancer (Sitter et al., 2010; Giskeodegard et al., 2012; Cao et al., 2012; Choi et al., 2012; 2013). However, so far only a single study has addressed the possible influence of metabolic heterogeneity within a single breast tumor (Park et al., 2016). Therefore, the authors performed multi-core sampling of six small specimens from individual tumors and quantified 32 metabolites. Not unexpectedly, the intertumoral differences were larger compared to intratumoral differences (Gogiashvili et al., 2017). More importantly, a random forestclassifier trained on a sample set of individual tumors correctly predicted tumor identity of an additional set of independent cores from the same tumors (Gogiashvili et al., 2017). Therefore, the study shows that despite the intratumoral heterogeneity the analysis of only one or few replicates per tumor can be justified. This is of high relevance, when large cohorts of patients have to be analyzed. Currently, the majority of prognostic studies with cancer patients has been performed based on mRNA (Grinberg et al., 2017; 2015; Marchan et al., 2017; Cadenas et al., 2014; Ghallab et al., 2015; Lohr et al., 2015; Hellwig et al., 2016; Stock et al., 2015; Hammad et al., 2016) or immunostaining (Heimes et al., 2017; Mattsson et al., 2015; Schmidt et al., 2012; Barone et al., 2016). Studies with metabolic profiling by HR MAS 1H NMR are still relatively rare in breast cancer. Therefore, the present study of Gogiashvili and colleagues represents an important milestone in this field of research.

Role of WDR5 in breast cancer prognosis.

Abstract: Dear Editor, Recently, Punzi and colleagues published a study about the role of WDR5 in breast cancer metastasis (Punzi et al., 2019). WDR5 is involved in epigenetic regulation complexes and has been reported to influence the expression of numerous genes, including N-cadherin, Snail1 and vimentin (Aho et al., 2019; Ford and Dingwall, 2015; Wu et al., 2011; Chen et al., 2017; Tan et al., 2017). Several studies suggested WDR5 as a therapeutic target (Ye et al., 2019a, b; Macdonald et al., 2019; Aho et al., 2019; Zhang et al., 2018; Lu et al., 2018). In their present work, Punzi and colleagues report that downregulation of WDR5 by shRNA in breast cancer cells antagonizes the epithelial-to-mesenchymal transition through re-differentiation and reduces metastasis in a mouse model (Punzi et al., 2019). Moreover, an association of high WDR5 expression with shorter metastasis-free survival was observed in a cohort of breast cancer patients (Punzi et al., 2019). A further important finding of this study is that WDR5 activates TGFβ in breast cancer and that targeting the WDR5-TGFβ axis by a small molecular inhibitor reduces the migratory potential of breast cancer cells. Metastasis of breast cancer is a complex process (Loi et al., 2019; von Minckwitz et al., 2019; Gogiashvili, 2018; Stoeber, 2017). Besides epithelial-to-mesenchymal transition, genes involved in proliferation (Schmidt et al., 2018), immune cell infiltration (Schmidt et al., 2012, 2018; Heimes et al., 2017a, b; Godoy et al., 2014), oxidative stress response (Cadenas et al., 2010, 2014, 2019; Hellwig et al., 2016) and inflammatory factors (Mattsson et al., 2015; Sicking et al., 2014) are of relevance; also key enzymes of phosphocholine metabolism have been shown to control breast and ovarian cancer metastasis (Marchan et al., 2017; Stewart et al., 2012). It will be interesting to learn in the next years, whether WDR5 targeting compounds can be identified that have a perspective to be tested in clinical studies.

Adverse outcome pathways: opportunities, limitations and open questions.

Abstract: 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.

Recommendation on test readiness criteria for new approach methods in toxicology: Exemplified for developmental neurotoxicity

Abstract: Multiple non-animal-based test methods have never been formally validated. In order to use such new approach methods (NAMs) in a regulatory context, criteria to define their readiness are necessary. The field of developmental neurotoxicity (DNT) testing is used to exemplify the application of readiness criteria. The costs and number of untested chemicals are overwhelming for in vivo DNT testing. Thus, there is a need for inexpensive, high-throughput NAMs, to obtain initial information on potential hazards, and to allow prioritization for further testing. A background on the regulatory and scientific status of DNT testing is provided showing different types of test readiness levels, depending on the intended use of data from NAMs. Readiness criteria, compiled during a stakeholder workshop, uniting scientists from academia, industry and regulatory authorities are presented. An important step beyond the listing of criteria, was the suggestion for a preliminary scoring scheme. On this basis a (semi)-quantitative analysis process was assembled on test readiness of 17 NAMs with respect to various uses (e.g. prioritization/screening, risk assessment). The scoring results suggest that several assays are currently at high readiness levels. Therefore, suggestions are made on how DNT NAMs may be assembled into an integrated approach to testing and assessment (IATA). In parallel, the testing state in these assays was compiled for more than 1000 compounds. Finally, a vision is presented on how further NAM development may be guided by knowledge of signaling pathways necessary for brain development, DNT pathophysiology, and relevant adverse outcome pathways (AOP).