112 results on '"Ellinger-Ziegelbauer H"'
Search Results
2. Assessment of candidate biomarkers of drug-induced hepatobiliary injury in preclinical toxicity studies
- Author
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Adler, M., Hoffmann, D., Ellinger-Ziegelbauer, H., Hewitt, P., Matheis, K., Mulrane, L., Gallagher, W.M., Callanan, J.J., Suter, L., Fountoulakis, M.M., Dekant, W., and Mally, A.
- Published
- 2010
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3. Characterization of primary rat proximal tubular cells by gene expression analysis
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Weiland, C., Ahr, H.J., Vohr, H.W., and Ellinger-Ziegelbauer, H.
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- 2007
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4. Testosterone response of hepatic gene expression in female mice having acquired testosterone-unresponsive immunity to Plasmodium chabaudi malaria
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Delić, D., Ellinger-Ziegelbauer, H., Vohr, H. W., Dkhil, M., Al-Quraishy, S., and Wunderlich, F.
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- 2011
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5. Toxicogenomic analysis of N-nitrosomorpholine induced changes in rat liver: Comparison of genomic and proteomic responses and anchoring to histopathological parameters
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Oberemm, A., Ahr, H.-J., Bannasch, P., Ellinger-Ziegelbauer, H., Glückmann, M., Hellmann, J., Ittrich, C., Kopp-Schneider, A., Kramer, P.-J., Krause, E., Kröger, M., Kiss, E., Richter-Reichhelm, H.-B., Scholz, G., Seemann, K., Weimer, M., and Gundert-Remy, U.
- Published
- 2009
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6. Functional interaction of Filamin and TNF-Receptor Associated Factor 2 (TRAF2)
- Author
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LEONARDI, ANTONIO, ELLINGER ZIEGELBAUER H., FRANZOSO G., BROWN K., SIEBENLIST U., Leonardi, Antonio, ELLINGER ZIEGELBAUER, H., Franzoso, G., Brown, K., and Siebenlist, U.
- Published
- 2000
7. Mitochondrial complex I inhibitors: Mechanistic investigations in the context of safety evaluation
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Heinz, S., primary, Ellinger-Ziegelbauer, H., additional, Schladt, L., additional, Lawrenz, B., additional, Freyberger, A., additional, and Schmuck, G., additional
- Published
- 2015
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8. The HESI inter-laboratory miRNA project
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Thompson, K.L., primary, Chen, T., additional, Couttet, P., additional, Ellinger-Ziegelbauer, H., additional, Kanki, M., additional, Kelsall, J., additional, Boitier, E., additional, Nassirpour, R., additional, Searfoss, G., additional, Sharapova, T., additional, de la Moureyre-Spire, Catherine, additional, Yuen, P., additional, and O’Lone, R., additional
- Published
- 2013
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9. Multi-laboratory assessment of best practices for quantification of microRNAs associated with isoproterenol-induced myocardial injury in the urine and plasma of rats
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Thompson, KL, primary, Chen, T, additional, Couttet, P, additional, Ellinger-Ziegelbauer, H, additional, Kanki, M, additional, Kelsall, J, additional, Boitier, E, additional, Nassirpour, R, additional, Searfoss, G, additional, Sharapova, T, additional, de la Moureyre-Spire, C, additional, Yuen, P, additional, and O’Lone, R, additional
- Published
- 2013
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10. In Vitro - In Vivo Correlation of Gene Expression Alterations Induced by Liver Carcinogens
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Heise, T., primary, Schug, M., additional, Storm, D., additional, Ellinger-Ziegelbauer, H., additional, J. Ahr, H., additional, Hellwig, B., additional, Rahnenführer, J., additional, Ghallab, A., additional, Guenther, G., additional, Sisnaiske, J., additional, Reif, R., additional, Godoy, P., additional, Mielke, H., additional, Gundert-Remy, U., additional, Lampen, A., additional, Oberemm, A., additional, and G. Hengstler, J., additional
- Published
- 2012
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11. Evaluation of the rodent Hershberger bioassay: Testing of coded chemicals and supplementary molecular-biological and biochemical investigations
- Author
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Freyberger, A., primary, Ellinger-Ziegelbauer, H., additional, and Krötlinger, F., additional
- Published
- 2007
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12. BAY 68–5042, ein hoch-selektiver und potenter PPARδ-Agonist, reduziert Risikofaktoren des Metabolischen Syndroms
- Author
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Bischoff, H, primary, Dittrich-Wengenroth, E, additional, Ellinger-Ziegelbauer, H, additional, Ellinghaus, P, additional, Kretschmer, A, additional, and Thielemann, W, additional
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- 2007
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13. A naturally occurring short variant of the FTZ-F1-related nuclear orphan receptor xFF1rA and interactions between domains of xFF1rA.
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Ellinger-Ziegelbauer, H, primary, Gläser, B, additional, and Dreyer, C, additional
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- 1995
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14. FTZ-F1-related orphan receptors in Xenopus laevis: transcriptional regulators differentially expressed during early embryogenesis
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Ellinger-Ziegelbauer, H, primary, Hihi, A K, additional, Laudet, V, additional, Keller, H, additional, Wahli, W, additional, and Dreyer, C, additional
- Published
- 1994
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15. Beta 1-integrin is a maternal protein that is inserted into all newly formed plasma membranes during early Xenopus embryogenesis
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Gawantka, V., primary, Ellinger-Ziegelbauer, H., additional, and Hausen, P., additional
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- 1992
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16. A retinoic acid receptor expressed in the early development of Xenopus laevis.
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Ellinger-Ziegelbauer, H, primary and Dreyer, C, additional
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- 1991
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17. Physical and functional interaction of filamin (actin-binding protein-280) and tumor necrosis factor receptor-associated factor 2.
- Author
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Leonardi, A, Ellinger-Ziegelbauer, H, Franzoso, G, Brown, K, and Siebenlist, U
- Abstract
Tumor necrosis factor (TNF) receptor-associated factor 2 (TRAF2) is an intracellular protein involved in signal transduction from TNF receptor I and II and related receptors. TRAF2 is required for TNF-induced activation of c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK), and TRAF2 can also mediate activation of NF-kappaB. Here we have identified the actin-binding protein Filamin (actin-binding protein-280) as a TRAF2-interacting protein. Filamin binds to the Ring zinc finger domain of TRAF2. Overexpressed Filamin inhibits TRAF2-induced activation of JNK/SAPK and of NF-kappaB. Furthermore, ectopically expressed Filamin inhibits NF-kappaB activation induced via TNF, interleukin-1, Toll receptors, and TRAF6 but not activation induced via overexpression of NIK, a downstream effector in these pathways. Importantly, TNF fails to activate SAPK or NF-kappaB in a human melanoma cell line deficient in Filamin. Reintroduction of Filamin into these cells restores the TNF response. The data imply a role for Filamin in inflammatory signal transduction pathways.
- Published
- 2000
18. β-integrin is a maternal protein that is inserted into all newly formed plasma membranes during early Xenopus embryogenesis
- Author
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Gawantka, V., Ellinger-Ziegelbauer, H., and Hausen, P.
- Abstract
A monoclonal antibody (mAb 8C8) that recognizes the Xenopus β1-integrin chain was used to study the appearance, synthesis and distribution of this integrin subunit during the early development of Xenopus. Both the precursor and the mature form of β1-integrin are provided maternally. They do not increase significantly in amount until early gastrula when the level of both forms begins to rise gradually. Synthesis of β1-integrin from maternal mRNA is observed throughout the pregastrula phase, though it seems to add only little to the total β1-integrin of the embryo. Until late blastula only small amounts of precursor are processed into the mature form. Starting with the formation of the first cleavage membrane, mature β1-integrin is inserted into the newly formed plasma membranes of all cells. The membrane domains forming the outer surface of the embryo remain devoid of the antigen. The data suggest an as yet unknown function of β1-integrin during the cleavage phase.
- Published
- 1992
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19. Direct activation of the stress-activated protein kinase (SAPK) and extracellular signal-regulated protein kinase (ERK) pathways by an inducible mitogen-activated protein Kinase/ERK kinase kinase 3 (MEKK) derivative.
- Author
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Ellinger-Ziegelbauer, H, Brown, K, Kelly, K, and Siebenlist, U
- Abstract
The extracellular signal-regulated kinase (ERK) pathway, the stress-activated protein kinase (SAPK) pathway, and the p38 pathway are three major mitogen-activated protein kinase (MAPK) cascades known to participate in the regulation of cellular responses to a variety of extracellular signals. Upstream regulatory components of these kinase cascades, the MAPK/ERK kinase kinases (MEKK), have been described in several systems. We have isolated a cDNA encoding human MEKK3. Transfected MEKK3 has the ability to activate both SAPK and ERK pathways, but does not induce p38 activity, in agreement with a previous report on murine MEKK3 (Blank, J. L., Gerwins, P., Elliott, E. M., Sather, S., and Johnson, G. L. (1996) J. Biol. Chem. 271, 5361-5368). We now demonstrate that MEKK3 activates SEK and MEK, the known kinases targeting SAPK and ERK, respectively. Utilizing an estrogen ligand-activated MEKK3 derivative, we furthermore demonstrate that MEKK3 regulates the SAPK and the ERK pathway directly. Consistent with the fact that several SAPK-inducing agents activate the transcription factor NFkappaB, we now show that MEKK3 also enhances transcription from an NFkappaB-dependent reporter gene in cotransfection assays. The ability of MEKK3 to simultaneously activate the SAPK and ERK pathways is remarkable, given that they have divergent roles in cellular homeostasis.
- Published
- 1997
20. Data sharing on compound selection: a way to improve the outcome of research projects in the field of 3R-alternatives to animal testing’
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Mathieu Vinken, Yordanova Doktorova Tatyana, Ellinger-Ziegelbauer, H., J Ahr, H., Lock, E., Carmichael, P., Roggen, E., Delft, J., Kleinjans, J., Castell, J., Bort, R., Donato, T., Ryan, M., Corvi, R., Keun, H., Ebbels, T., Athersuch, T., A Sansone, S., Rocca-Serra, P., Stierum, R., Jennings, P., Pfaller, W., Gmuender, H., Tamara Vanhaecke, and Vera Rogiers
21. Characteristic expression profiles induced by carcinogens in rat liver
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Ellinger-Ziegelbauer, H., Ahr, H.-J., Gmuender, H., Pallez, C., and Koenig, J.
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- 2006
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22. Hershberger assay: Testing of coded chemicals and supplementary molecular and biochemical investigations
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Freyberger, A., Ellinger-Ziegelbauer, H., Brockes, A., and Krötlinger, F.
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- 2006
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- View/download PDF
23. The pattern of retinoic acid receptor gamma (RARgamma) expression in normal development of Xenopus laevis and after manipulation of the main body axis
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Ellinger-Ziegelbauer, H. and Dreyer, C.
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- 1993
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24. The sGC Activator Runcaciguat Has Kidney Protective Effects and Prevents a Decline of Kidney Function in ZSF1 Rats.
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Kraehling JR, Benardeau A, Schomber T, Popp L, Vienenkoetter J, Ellinger-Ziegelbauer H, Pavkovic M, Hartmann E, Siudak K, Freyberger A, Hagelschuer I, Mathar I, Hueser J, Hahn MG, Geiss V, Eitner F, and Sandner P
- Subjects
- Animals, Rats, Cyclic GMP, Glycated Hemoglobin, Heme, Obesity, Proteinuria, Clinical Trials, Phase II as Topic, Kidney, Renal Insufficiency, Chronic drug therapy
- Abstract
Chronic kidney disease (CKD) progression is associated with persisting oxidative stress, which impairs the NO-sGC-cGMP signaling cascade through the formation of oxidized and heme-free apo-sGC that cannot be activated by NO. Runcaciguat (BAY 1101042) is a novel, potent, and selective sGC activator that binds and activates oxidized and heme-free sGC and thereby restores NO-sGC-cGMP signaling under oxidative stress. Therefore, runcaciguat might represent a very effective treatment option for CKD/DKD. The potential kidney-protective effects of runcaciguat were investigated in ZSF1 rats as a model of CKD/DKD, characterized by hypertension, hyperglycemia, obesity, and insulin resistance. ZSF1 rats were treated daily orally for up to 12 weeks with runcaciguat (1, 3, 10 mg/kg/bid) or placebo. The study endpoints were proteinuria, kidney histopathology, plasma, urinary biomarkers of kidney damage, and gene expression profiling to gain information about relevant pathways affected by runcaciguat. Furthermore, oxidative stress was compared in the ZSF1 rat kidney with kidney samples from DKD patients. Within the duration of the 12-week treatment study, kidney function was significantly decreased in obese ZSF1 rats, indicated by a 20-fold increase in proteinuria, compared to lean ZSF1 rats. Runcaciguat dose-dependently and significantly attenuated the development of proteinuria in ZSF1 rats with reduced uPCR at the end of the study by -19%, -54%, and -70% at 1, 3, and 10 mg/kg/bid, respectively, compared to placebo treatment. Additionally, average blood glucose levels measured as HbA1C, triglycerides, and cholesterol were increased by five times, twenty times, and four times, respectively, in obese ZSF1 compared to lean rats. In obese ZSF1 rats, runcaciguat reduced HbA1c levels by -8%, -34%, and -76%, triglycerides by -42%, -55%, and -71%, and cholesterol by -16%, -17%, and -34%, at 1, 3, and 10 mg/kg/bid, respectively, compared to placebo. Concomitantly, runcaciguat also reduced kidney weights, morphological kidney damage, and urinary and plasma biomarkers of kidney damage. Beneficial effects were accompanied by changes in gene expression that indicate reduced fibrosis and inflammation and suggest improved endothelial stabilization. In summary, the sGC activator runcaciguat significantly prevented a decline in kidney function in a DKD rat model that mimics common comorbidities and conditions of oxidative stress of CKD patients. Thus, runcaciguat represents a promising treatment option for CKD patients, which is in line with recent phase 2 clinical study data, where runcaciguat showed promising efficacy in CKD patients (NCT04507061).
- Published
- 2023
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25. Integrated Genotoxicity Testing of three anti-infective drugs using the TGx-DDI transcriptomic biomarker and high-throughput CometChip ® assay in TK6 cells.
- Author
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Buick JK, Rowan-Carroll A, Gagné R, Williams A, Chen R, Li HH, Fornace AJ Jr, Chao C, Engelward BP, Frötschl R, Ellinger-Ziegelbauer H, Pettit SD, Aubrecht J, and Yauk CL
- Abstract
Genotoxicity testing relies on the detection of gene mutations and chromosome damage and has been used in the genetic safety assessment of drugs and chemicals for decades. However, the results of standard genotoxicity tests are often difficult to interpret due to lack of mode of action information. The TGx-DDI transcriptomic biomarker provides mechanistic information on the DNA damage-inducing (DDI) capability of chemicals to aid in the interpretation of positive in vitro genotoxicity data. The CometChip
® assay was developed to assess DNA strand breaks in a higher-throughput format. We paired the TGx-DDI biomarker with the CometChip® assay in TK6 cells to evaluate three model agents: nitrofurantoin (NIT), metronidazole (MTZ), and novobiocin (NOV). TGx-DDI was analyzed by two independent labs and technologies (nCounter® and TempO-Seq® ). Although these anti-infective drugs are, or have been, used in human and/or veterinary medicine, the standard genotoxicity testing battery showed significant genetic safety findings. Specifically, NIT is a mutagen and causes chromosome damage, and MTZ and NOV cause chromosome damage in conventional in vitro tests. Herein, the TGx-DDI biomarker classified NIT and MTZ as non-DDI at all concentrations tested, suggesting that NIT's mutagenic activity is bacterial specific and that the observed chromosome damage by MTZ might be a consequence of in vitro test conditions. In contrast, NOV was classified as DDI at the second highest concentration tested, which is in line with the fact that NOV is a bacterial DNA-gyrase inhibitor that also affects topoisomerase II at high concentrations. The lack of DNA damage for NIT and MTZ was confirmed by the CometChip® results, which were negative for all three drugs except at overtly cytotoxic concentrations. This case study demonstrates the utility of combining the TGx-DDI biomarker and CometChip® to resolve conflicting genotoxicity data and provides further validation to support the reproducibility of the biomarker., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Buick, Rowan-Carroll, Gagné, Williams, Chen, Li, Fornace, Chao, Engelward, Frötschl, Ellinger-Ziegelbauer, Pettit, Aubrecht and Yauk.)- Published
- 2022
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26. A Collaborative Initiative to Establish Genomic Biomarkers for Assessing Tumorigenic Potential to Reduce Reliance on Conventional Rodent Carcinogenicity Studies.
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Corton JC, Mitchell CA, Auerbach S, Bushel P, Ellinger-Ziegelbauer H, Escobar PA, Froetschl R, Harrill AH, Johnson K, Klaunig JE, Pandiri AR, Podtelezhnikov AA, Rager JE, Tanis KQ, van der Laan JW, Vespa A, Yauk CL, Pettit SD, and Sistare FD
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- Animals, Biomarkers, Tumor genetics, Carcinogenesis, Carcinogenicity Tests, Carcinogens toxicity, Genomics, Neoplasms chemically induced, Neoplasms genetics, Rodentia
- Abstract
There is growing recognition across broad sectors of the scientific community that use of genomic biomarkers has the potential to reduce the need for conventional rodent carcinogenicity studies of industrial chemicals, agrochemicals, and pharmaceuticals through a weight-of-evidence approach. These biomarkers fall into 2 major categories: (1) sets of gene transcripts that can identify distinct tumorigenic mechanisms of action; and (2) cancer driver gene mutations indicative of rapidly expanding growth-advantaged clonal cell populations. This call-to-action article describes a collaborative approach launched to develop and qualify biomarker gene expression panels that measure widely accepted molecular pathways linked to tumorigenesis and their activation levels to predict tumorigenic doses of chemicals from short-term exposures. Growing evidence suggests that application of such biomarker panels in short-term exposure rodent studies can identify both tumorigenic hazard and tumorigenic activation levels for chemical-induced carcinogenicity. In the future, this approach will be expanded to include methodologies examining mutations in key cancer driver gene mutation hotspots as biomarkers of both genotoxic and nongenotoxic chemical tumor risk. Analytical, technical, and biological validation studies of these complementary genomic tools are being undertaken by multisector and multidisciplinary collaborative teams within the Health and Environmental Sciences Institute. Success from these efforts will facilitate the transition from current heavy reliance on conventional 2-year rodent carcinogenicity studies to more rapid animal- and resource-sparing approaches for mechanism-based carcinogenicity evaluation supporting internal and regulatory decision-making., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Society of Toxicology.)
- Published
- 2022
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27. Urinary miRNA Profiles in Chronic Kidney Injury-Benefits of Extracellular Vesicle Enrichment and miRNAs as Potential Biomarkers for Renal Fibrosis, Glomerular Injury, and Endothelial Dysfunction.
- Author
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Petzuch B, Bénardeau A, Hofmeister L, Meyer J, Hartmann E, Pavkovic M, Mathar I, Sandner P, and Ellinger-Ziegelbauer H
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- Animals, Biomarkers metabolism, Disease Progression, Female, Fibrosis, Humans, Kidney metabolism, Male, Obesity metabolism, Rats, Renin metabolism, Extracellular Vesicles metabolism, Hypertension metabolism, MicroRNAs genetics, Renal Insufficiency, Chronic
- Abstract
Micro-RNAs (miRNAs) are regulators of gene expression and play an important role in physiological homeostasis and disease. In biofluids, miRNAs can be found in protein complexes or in extracellular vesicles (EVs). Altered urinary miRNAs are reported as potential biomarkers for chronic kidney disease (CKD). In this context, we compared established urinary protein biomarkers for kidney injury with urinary miRNA profiles in obese ZSF1 and hypertensive renin transgenic rats. Additionally, the benefit of urinary EV enrichment was investigated in vivo and the potential association of urinary miRNAs with renal fibrosis in vitro. Kidney damage in both rat models was confirmed by histopathology, proteinuria, and increased levels of urinary protein biomarkers. In total, 290 miRNAs were elevated in obese ZSF1 rats compared with lean controls, whereas 38 miRNAs were altered in obese ZSF1 rats during 14-26 weeks of age. These 38 miRNAs correlated better with disease progression than established urinary protein biomarkers. MiRNAs increased in obese ZSF1 rats were associated with renal inflammation, fibrosis, and glomerular injury. Eight miRNAs were also changed in urinary EVs of renin transgenic rats, including one which might play a role in endothelial dysfunction. EV enrichment increased the number and detection level of several miRNAs implicated in renal fibrosis in vitro and in vivo. Our results show the benefit of EV enrichment for miRNA detection and the potential of total urine and urinary EV-associated miRNAs as biomarkers of altered kidney physiology, renal fibrosis and glomerular injury, and disease progression in hypertension and obesity-induced CKD., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)
- Published
- 2022
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28. A cross-industry survey on photosafety evaluation of pharmaceuticals after implementation of ICH S10.
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Bauer D, Buckley LA, Delafoy L, Ellinger-Ziegelbauer H, Fellows MD, Gerets HHJ, Howe J, Kaijser G, Nicolette J, Pettersen BA, and Schimpf B
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- Dermatitis, Phototoxic pathology, Drug-Related Side Effects and Adverse Reactions pathology, Organisation for Economic Co-Operation and Development standards, Pharmaceutical Preparations standards, Sunlight adverse effects
- Abstract
A cross-industry survey was conducted by EFPIA/IQ DruSafe in 2018 to provide information on photosafety evaluation of pharmaceuticals after implementation of ICH S10. This survey focused on the strategy utilized for photosafety risk assessment, the design of nonclinical (in vitro and in vivo) and clinical evaluations, the use of exposure margins in risk assessment, and regulatory interactions. The survey results indicated that a staged approach for phototoxicity assessment has been widely accepted by regulatory authorities globally. The OECD-based 3T3 NRU Phototoxicity Test is the most frequently used in vitro approach. Modifications to this assay suggested by ICH S10 are commonly applied. For in-vitro-positives, substantial margins from in vitro IC
50 values under irradiation to Cmax (clinical) have enabled further development without the need for additional photosafety data. In vivo phototoxicity studies typically involve dosing rodents and exposing skin and eyes to simulated sunlight, and subsequently evaluating at least the skin for erythema and edema. However, no formal guidelines exist and protocols are less standardized across companies. A margin-of-safety approach (based on Cmax at NOAEL) has been successfully applied to support clinical development. Experience with dedicated clinical phototoxicity studies was limited, perhaps due to effective de-risking approaches employed based on ICH S10., (Copyright © 2021 Elsevier Inc. All rights reserved.)- Published
- 2021
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29. Methodological considerations for measuring biofluid-based microRNA biomarkers.
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Chorley BN, Atabakhsh E, Doran G, Gautier JC, Ellinger-Ziegelbauer H, Jackson D, Sharapova T, Yuen PST, Church RJ, Couttet P, Froetschl R, McDuffie J, Martinez V, Pande P, Peel L, Rafferty C, Simutis FJ, and Harrill AH
- Subjects
- Humans, Biomarkers, MicroRNAs, Toxicology
- Abstract
MicroRNAs (miRNAs) are small non-coding RNA that regulate the expression of messenger RNA and are implicated in almost all cellular processes. Importantly, miRNAs can be released extracellularly and are stable in these matrices where they may serve as indicators of organ or cell-specific toxicity, disease, and biological status. There has thus been great enthusiasm for developing miRNAs as biomarkers of adverse outcomes for scientific, regulatory, and clinical purposes. Despite advances in measurement capabilities for miRNAs, miRNAs are still not routinely employed as noninvasive biomarkers. This is in part due to the lack of standard approaches for sample preparation and miRNA measurement and uncertainty in their biological interpretation. Members of the microRNA Biomarkers Workgroup within the Health and Environmental Sciences Institute's (HESI) Committee on Emerging Systems Toxicology for the Assessment of Risk (eSTAR) are a consortium of private- and public-sector scientists dedicated to developing miRNAs as applied biomarkers. Here, we explore major impediments to routine acceptance and use of miRNA biomarkers and case examples of successes and deficiencies in development. Finally, we provide insight on miRNA measurement, collection, and analysis tools to provide solid footing for addressing knowledge gaps toward routine biomarker use.
- Published
- 2021
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30. Urinary miRNA Biomarkers of Drug-Induced Kidney Injury and Their Site Specificity Within the Nephron.
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Chorley BN, Ellinger-Ziegelbauer H, Tackett M, Simutis FJ, Harrill AH, McDuffie J, Atabakhsh E, Nassirpour R, Whiteley LO, Léonard JF, Carswell GK, Harpur E, Chen CL, and Gautier JC
- Subjects
- Animals, Biomarkers, Kidney, Nephrons, Rats, MicroRNAs genetics, Pharmaceutical Preparations
- Abstract
Drug-induced kidney injury (DIKI) is a major concern in both drug development and clinical practice. There is an unmet need for biomarkers of glomerular damage and more distal renal injury in the loop of Henle and the collecting duct (CD). A cross-laboratory program to identify and characterize urinary microRNA (miRNA) patterns reflecting tissue- or pathology-specific DIKI was conducted. The overall goal was to propose miRNA biomarker candidates for DIKI that could supplement information provided by protein kidney biomarkers in urine. Rats were treated with nephrotoxicants causing injury to distinct nephron segments: the glomerulus, proximal tubule, thick ascending limb (TAL) of the loop of Henle and CD. Meta-analysis identified miR-192-5p as a potential proximal tubule-specific urinary miRNA candidate. This result was supported by data obtained in laser capture microdissection nephron segments showing that miR-192-5p expression was enriched in the proximal tubule. Discriminative miRNAs including miR-221-3p and -222-3p were increased in urine from rats treated with TAL versus proximal tubule toxicants in accordance with their expression localization in the kidney. Urinary miR-210-3p increased up to 40-fold upon treatment with TAL toxicants and was also enriched in laser capture microdissection samples containing TAL and/or CD versus proximal tubule. miR-23a-3p was enriched in the glomerulus and was increased in urine from rats treated with doxorubicin, a glomerular toxicant, but not with toxicants affecting other nephron segments. Taken together these results suggest that urinary miRNA panels sourced from specific nephron regions may be useful to discriminate the pathology of toxicant-induced lesions in the kidney, thereby contributing to DIKI biomarker development needs for industry, clinical, and regulatory use., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Society of Toxicology.)
- Published
- 2021
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31. A cross-sector call to improve carcinogenicity risk assessment through use of genomic methodologies.
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Yauk CL, Harrill AH, Ellinger-Ziegelbauer H, van der Laan JW, Moggs J, Froetschl R, Sistare F, and Pettit S
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- Animals, Carcinogenicity Tests, Chemical Industry, Drug Industry, Humans, Carcinogens toxicity, Neoplasms chemically induced, Risk Assessment, Toxicogenetics
- Abstract
Robust genomic approaches are now available to realize improvements in efficiencies and translational relevance of cancer risk assessments for drugs and chemicals. Mechanistic and pathway data generated via genomics provide opportunities to advance beyond historical reliance on apical endpoints of uncertain human relevance. Published research and regulatory evaluations include many examples for which genomic data have been applied to address cancer risk assessment as a health protection endpoint. The alignment of mature, robust, reproducible, and affordable technologies with increasing demands for reduced animal testing sets the stage for this important transition. We present our shared vision for change from leading scientists from academic, government, nonprofit, and industrial sectors and chemical and pharmaceutical safety applications. This call to action builds upon a 2017 workshop on "Advances and Roadblocks for Use of Genomics in Cancer Risk Assessment." The authors propose a path for implementation of innovative cancer risk assessment including incorporating genomic signatures to assess mechanistic relevance of carcinogenicity and enhanced use of genomics in benchmark dose and point of departure evaluations. Novel opportunities for the chemical and pharmaceutical sectors to combine expertise, resources, and objectives to achieve a common goal of improved human health protection are identified., (Published by Elsevier Inc.)
- Published
- 2020
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32. TGx-DDI, a Transcriptomic Biomarker for Genotoxicity Hazard Assessment of Pharmaceuticals and Environmental Chemicals.
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Li HH, Yauk CL, Chen R, Hyduke DR, Williams A, Frötschl R, Ellinger-Ziegelbauer H, Pettit S, Aubrecht J, and Fornace AJ Jr
- Abstract
Genotoxicity testing is an essential component of the safety assessment paradigm required by regulatory agencies world-wide for analysis of drug candidates, and environmental and industrial chemicals. Current genotoxicity testing batteries feature a high incidence of irrelevant positive findings-particularly for in vitro chromosomal damage (CD) assays. The risk management of compounds with positive in vitro findings is a major challenge and requires complex, time consuming, and costly follow-up strategies including animal testing. Thus, regulators are urgently in need of new testing approaches to meet legislated mandates. Using machine learning, we identified a set of transcripts that responds predictably to DNA-damage in human cells that we refer to as the TGx-DDI biomarker, which was originally referred to as TGx-28.65. We proposed to use this biomarker in conjunction with current genotoxicity testing batteries to differentiate compounds with irrelevant "false" positive findings in the in vitro CD assays from true DNA damaging agents (i.e., for de-risking agents that are clastogenic in vitro but not in vivo ). We validated the performance of the TGx-DDI biomarker to identify true DNA damaging agents, assessed intra- and inter- laboratory reproducibility, and cross-platform performance. Recently, to augment the application of this biomarker, we developed a high-throughput cell-based genotoxicity testing system using the NanoString nCounter® technology. Here, we review the status of TGx-DDI development, its integration in the genotoxicity testing paradigm, and progress to date in its qualification at the US Food and Drug Administration (FDA) as a drug development tool. If successfully validated and implemented, the TGx-DDI biomarker assay is expected to significantly augment the current strategy for the assessment of genotoxic hazards for drugs and chemicals., (Copyright © 2019 Li, Yauk, Chen, Hyduke, Williams, Frötschl, Ellinger-Ziegelbauer, Pettit, Aubrecht and Fornace.)
- Published
- 2019
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33. Preclinical Combination Studies of an FGFR2 Targeted Thorium-227 Conjugate and the ATR Inhibitor BAY 1895344.
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Wickstroem K, Hagemann UB, Kristian A, Ellingsen C, Sommer A, Ellinger-Ziegelbauer H, Wirnitzer U, Hagelin EM, Larsen A, Smeets R, Bjerke RM, Karlsson J, Ryan OB, Wengner AM, Linden L, Mumberg D, and Cuthbertson AS
- Subjects
- Animals, Breast Neoplasms metabolism, Cell Line, Tumor, Cell Survival drug effects, Chelating Agents therapeutic use, DNA Damage, Drug Combinations, Drug Synergism, G2 Phase Cell Cycle Checkpoints radiation effects, Histones metabolism, Humans, Immunoconjugates chemistry, Immunoconjugates pharmacokinetics, Immunoconjugates therapeutic use, Mice, Mice, Nude, Molecular Targeted Therapy methods, Receptor, Fibroblast Growth Factor, Type 2 antagonists & inhibitors, Receptor, Fibroblast Growth Factor, Type 2 metabolism, Thorium pharmacokinetics, Thorium Compounds therapeutic use, Up-Regulation, Xenograft Model Antitumor Assays, Antibodies, Monoclonal, Humanized therapeutic use, Ataxia Telangiectasia Mutated Proteins antagonists & inhibitors, Breast Neoplasms radiotherapy, Protein Kinase Inhibitors therapeutic use, Radioimmunotherapy methods, Receptor, Fibroblast Growth Factor, Type 2 therapeutic use, Thorium therapeutic use
- Abstract
Purpose: Fibroblast growth factor receptor 2 (FGFR2) has been previously reported to be overexpressed in several types of cancer, whereas the expression in normal tissue is considered to be moderate to low. Thus, FGFR2 is regarded as an attractive tumor antigen for targeted alpha therapy. This study reports the evaluation of an FGFR2-targeted thorium-227 conjugate (FGFR2-TTC, BAY 2304058) comprising an anti-FGFR2 antibody, a chelator moiety covalently conjugated to the antibody, and the alpha particle-emitting radionuclide thorium-227. FGFR2-TTC was assessed as a monotherapy and in combination with the DNA damage response inhibitor ATRi BAY 1895344., Methods and Materials: The in vitro cytotoxicity and mechanism of action were evaluated by determining cell viability, the DNA damage response marker γH2A.X, and cell cycle analyses. The in vivo efficacy was determined using human tumor xenograft models in nude mice., Results: In vitro mechanistic assays demonstrated upregulation of γH2A.X and induction of cell cycle arrest in several FGFR2-expressing cancer cell lines after treatment with FGFR2-TTC. In vivo, FGFR2-TTC significantly inhibited tumor growth at a dose of 500 kBq/kg in the xenograft models NCI-H716, SNU-16, and MFM-223. By combining FGFR2-TTC with the ATR inhibitor BAY 1895344, an increased potency was observed in vitro, as were elevated levels of γH2A.X and inhibition of FGFR2-TTC-mediated cell cycle arrest. In the MFM-223 tumor xenograft model, combination of the ATRi BAY 1895344 with FGFR2-TTC resulted in significant tumor growth inhibition at doses at which the single agents had no effect., Conclusions: The data provide a mechanism-based rationale for combining the FGFR2-TTC with the ATRi BAY 1895344 as a new therapeutic approach for treatment of FGFR2-positive tumors from different cancer indications., (Copyright © 2019. Published by Elsevier Inc.)
- Published
- 2019
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34. Energy metabolism modulation by biguanides in comparison with rotenone in rat liver and heart.
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Heinz S, Freyberger A, Lawrenz B, Schladt L, Schmuck G, and Ellinger-Ziegelbauer H
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- Animals, Antineoplastic Agents toxicity, Dose-Response Relationship, Drug, Gluconeogenesis drug effects, Lactic Acid blood, Liver metabolism, Male, Metformin toxicity, Mitochondria drug effects, Mitochondria metabolism, Oxidative Phosphorylation, Phenformin toxicity, Rats, Wistar, Rotenone toxicity, Transcriptome drug effects, Antineoplastic Agents pharmacology, Energy Metabolism drug effects, Heart drug effects, Liver drug effects, Metformin pharmacology, Phenformin pharmacology, Rotenone pharmacology
- Abstract
The biguanide metformin, a widely used antidiabetic drug, has received great interest in oncology research in recent years after an epidemiological study showed a link between metformin treatment and a reduced cancer risk in diabetic patients. Since mitochondrial metabolism has become a target for possible cancer therapeutic approaches, especially for tumors relying on oxidative metabolism, mitochondrial complex I inhibition is under discussion to be responsible for the anti-cancer effect of metformin. Rotenone, a well-known strong mitochondrial complex I inhibitor, yet associated with toxic effects, has also shown anti-cancer activity. Thus, we compared metformin and phenformin, another biguanide previously on the market as antidiabetic, with rotenone, to elucidate potential mechanisms rendering biguanides apparently less toxic than rotenone. Therefore, we conducted in vivo rat studies with metformin and phenformin, based on an experimental design previously described for mechanistic investigations of the effects of rotenone, including blood and tissue analysis, histopathology and gene expression profiling. These investigations show that the mechanistic profile of phenformin appears similar to that of rotenone, yet at a quantitatively reduced level, whereas metformin displays only transient similarities after one day of treatment. A potential reason may be that metformin, but not rotenone or phenformin, self-limits its entry into mitochondria due to its molecular properties. Thus, our detailed molecular characterization of these compounds suggests that inhibition of mitochondrial functions can serve as target for an anti-cancer mode of action, but should be self-limited or balanced to some extent to avoid exhaustion of all energy stores.
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- 2019
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35. Prediction of human drug-induced liver injury (DILI) in relation to oral doses and blood concentrations.
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Albrecht W, Kappenberg F, Brecklinghaus T, Stoeber R, Marchan R, Zhang M, Ebbert K, Kirschner H, Grinberg M, Leist M, Moritz W, Cadenas C, Ghallab A, Reinders J, Vartak N, van Thriel C, Golka K, Tolosa L, Castell JV, Damm G, Seehofer D, Lampen A, Braeuning A, Buhrke T, Behr AC, Oberemm A, Gu X, Kittana N, van de Water B, Kreiling R, Fayyaz S, van Aerts L, Smedsrød B, Ellinger-Ziegelbauer H, Steger-Hartmann T, Gundert-Remy U, Zeigerer A, Ullrich A, Runge D, Lee SML, Schiergens TS, Kuepfer L, Aguayo-Orozco A, Sachinidis A, Edlund K, Gardner I, Rahnenführer J, and Hengstler JG
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- Administration, Oral, Algorithms, Animals, Cell Line, Cell Survival drug effects, Computer Simulation, Gene Expression drug effects, Hepatocytes drug effects, Humans, In Vitro Techniques, Maximum Tolerated Dose, Pharmaceutical Preparations administration & dosage, Pharmaceutical Preparations blood, Pharmacokinetics, Reproducibility of Results, Sensitivity and Specificity, Support Vector Machine, Chemical and Drug Induced Liver Injury diagnosis, Drug-Related Side Effects and Adverse Reactions diagnosis
- 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 (C
max ) 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.- Published
- 2019
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36. Toxicogenomics directory of rat hepatotoxicants in vivo and in cultivated hepatocytes.
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Grinberg M, Stöber RM, Albrecht W, Edlund K, Schug M, Godoy P, Cadenas C, Marchan R, Lampen A, Braeuning A, Buhrke T, Leist M, Oberemm A, Hellwig B, Kamp H, Gardner I, Escher S, Taboureau O, Aguayo-Orozco A, Sachinidis A, Ellinger-Ziegelbauer H, Rahnenführer J, and Hengstler JG
- Subjects
- Animals, Cells, Cultured, Chemical and Drug Induced Liver Injury genetics, Dose-Response Relationship, Drug, Down-Regulation genetics, Gene Expression, Gene Expression Profiling methods, Liver drug effects, Male, Oligonucleotide Array Sequence Analysis methods, Rats, Rats, Wistar, Up-Regulation genetics, Chemical and Drug Induced Liver Injury etiology, Hepatocytes drug effects, Toxicity Tests methods, Toxicogenetics methods
- Abstract
Transcriptomics is developing into an invaluable tool in toxicology. The aim of this study was, using a transcriptomics approach, to identify genes that respond similar to many different chemicals (including drugs and industrial compounds) in both rat liver in vivo and in cultivated hepatocytes. For this purpose, we analyzed Affymetrix microarray expression data from 162 compounds that were previously tested in a concentration-dependent manner in rat livers in vivo and in rat hepatocytes cultivated in sandwich culture. These data were obtained from the Japanese Toxicogenomics Project (TGP) and North Rhine-Westphalian (NRW) data sets, which represent 138 and 29 compounds, respectively, and have only 5 compounds in common between them. The in vitro gene expression data from the NRW data set were generated in the present study, while TGP is publicly available. For each of the data sets, the overlap between up- or down-regulated genes in vitro and in vivo was identified, and named in vitro-in vivo consensus genes. Interestingly, the in vivo-in vitro consensus genes overlapped to a remarkable extent between both data sets, and were 21-times (upregulated genes) or 12-times (down-regulated genes) enriched compared to random expectation. Finally, the genes in the TGP and NRW overlap were used to identify the upregulated genes with the highest compound coverage, resulting in a seven-gene set of Cyp1a1, Ugt2b1, Cdkn1a, Mdm2, Aldh1a1, Cyp4a3, and Ehhadh. This seven-gene set was then successfully tested with structural analogues of valproic acid that are not present in the TGP and NRW data sets. In conclusion, the seven-gene set identified in the present study responds similarly in vitro and in vivo to a wide range of different chemicals. Despite these promising results with the seven-gene set, transcriptomics with cultivated rat hepatocytes remains a challenge, because in general many genes are up- or downregulated by in vitro culture per se, respond differently to test compounds in vitro and in vivo, and/or show higher variability in the in vitro system compared to the corresponding in vivo data.
- Published
- 2018
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37. Two approaches for estimating the lower limit of quantitation (LLOQ) of microRNA levels assayed as exploratory biomarkers by RT-qPCR.
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Wolfinger RD, Beedanagari S, Boitier E, Chen T, Couttet P, Ellinger-Ziegelbauer H, Guillemain G, Mariet C, Mouritzen P, O'Lone R, Pine PS, Sharapova T, Yan J, Yuen PS, and Thompson KL
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- Animals, Calibration, Genetic Markers, Rats, Reverse Transcriptase Polymerase Chain Reaction statistics & numerical data, Workflow, Limit of Detection, MicroRNAs genetics, Reverse Transcriptase Polymerase Chain Reaction methods
- Abstract
Background: Circulating microRNAs are undergoing exploratory use as safety biomarkers in drug development. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) is one common approach used to quantitate levels of microRNAs in samples that includes the use of a standard curve of calibrators fit to a regression model. Guidelines are needed for setting assay quantitation thresholds that are appropriate for this method and to biomarker pre-validation., Results: In this report, we develop two workflows for determining a lower limit of quantitation (LLOQ) for RT-qPCR assays of microRNAs in exploratory studies. One workflow is based on an error threshold calculated by a logistic model of the calibration curve data. The second workflow is based on a threshold set by the sample blank, which is the no template control for RT-qPCR. The two workflows are used to set lower thresholds of reportable microRNA levels for an example dataset in which miR-208a levels in biofluids are quantitated in a cardiac injury model. LLOQ thresholds set by either workflow are effective in filtering out microRNA values with large uncertainty estimates., Conclusions: Two workflows for LLOQ determinations are presented in this report that provide methods that are easy to implement in investigational studies of microRNA safety biomarkers and offer choices in levels of conservatism in setting lower limits of acceptable values that facilitate interpretation of results.
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- 2018
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38. Advanced Good Cell Culture Practice for human primary, stem cell-derived and organoid models as well as microphysiological systems.
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Pamies D, Bal-Price A, Chesné C, Coecke S, Dinnyes A, Eskes C, Grillari R, Gstraunthaler G, Hartung T, Jennings P, Leist M, Martin U, Passier R, Schwamborn JC, Stacey GN, Ellinger-Ziegelbauer H, and Daneshian M
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- Animal Testing Alternatives methods, Animals, Cell Culture Techniques methods, Education, Germany, Humans, In Vitro Techniques, Quality Control, Cell Culture Techniques standards, Guidelines as Topic, Organoids, Pluripotent Stem Cells physiology
- Abstract
A major reason for the current reproducibility crisis in the life sciences is the poor implementation of quality control measures and reporting standards. Improvement is needed, especially regarding increasingly complex in vitro methods. Good Cell Culture Practice (GCCP) was an effort from 1996 to 2005 to develop such minimum quality standards also applicable in academia. This paper summarizes recent key developments in in vitro cell culture and addresses the issues resulting for GCCP, e.g. the development of induced pluripotent stem cells (iPSCs) and gene-edited cells. It further deals with human stem-cell-derived models and bioengineering of organo-typic cell cultures, including organoids, organ-on-chip and human-on-chip approaches. Commercial vendors and cell banks have made human primary cells more widely available over the last decade, increasing their use, but also requiring specific guidance as to GCCP. The characterization of cell culture systems including high-content imaging and high-throughput measurement technologies increasingly combined with more complex cell and tissue cultures represent a further challenge for GCCP. The increasing use of gene editing techniques to generate and modify in vitro culture models also requires discussion of its impact on GCCP. International (often varying) legislations and market forces originating from the commercialization of cell and tissue products and technologies are further impacting on the need for the use of GCCP. This report summarizes the recommendations of the second of two workshops, held in Germany in December 2015, aiming map the challenge and organize the process or developing a revised GCCP 2.0.
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- 2018
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39. Development and validation of a high-throughput transcriptomic biomarker to address 21st century genetic toxicology needs.
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Li HH, Chen R, Hyduke DR, Williams A, Frötschl R, Ellinger-Ziegelbauer H, O'Lone R, Yauk CL, Aubrecht J, and Fornace AJ Jr
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- Cell Culture Techniques, Cell Line, Tumor, Cells, Cultured, Chromosome Aberrations, DNA Damage, Genetic Markers, Humans, Reproducibility of Results, Risk Assessment, Biomarkers, Gene Expression Profiling, High-Throughput Screening Assays, Mutagenicity Tests, Transcriptome
- Abstract
Interpretation of positive genotoxicity findings using the current in vitro testing battery is a major challenge to industry and regulatory agencies. These tests, especially mammalian cell assays, have high sensitivity but suffer from low specificity, leading to high rates of irrelevant positive findings (i.e., positive results in vitro that are not relevant to human cancer hazard). We developed an in vitro transcriptomic biomarker-based approach that provides biological relevance to positive genotoxicity assay data, particularly for in vitro chromosome damage assays, and propose its application for assessing the relevance of the in vitro positive results to carcinogenic hazard. The transcriptomic biomarker TGx-DDI (previously known as TGx-28.65) readily distinguishes DNA damage-inducing (DDI) agents from non-DDI agents. In this study, we demonstrated the ability of the biomarker to classify 45 test agents across a broad set of chemical classes as DDI or non-DDI. Furthermore, we assessed the biomarker's utility in derisking known irrelevant positive agents and evaluated its performance across analytical platforms. We correctly classified 90% (9 of 10) of chemicals with irrelevant positive findings in in vitro chromosome damage assays as negative. We developed a standardized experimental and analytical protocol for our transcriptomics biomarker, as well as an enhanced application of TGx-DDI for high-throughput cell-based genotoxicity testing using nCounter technology. This biomarker can be integrated in genetic hazard assessment as a follow-up to positive chromosome damage findings. In addition, we propose how it might be used in chemical screening and assessment. This approach offers an opportunity to significantly improve risk assessment and reduce cost., Competing Interests: Conflict of interest statement: Georgetown University has filed a provisional patent application for the technology described in this paper, on which A.J.F. and H.-H.L. are inventors., (Copyright © 2017 the Author(s). Published by PNAS.)
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- 2017
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40. Time-matched analysis of DNA adduct formation and early gene expression as predictive tool for renal carcinogenesis in methylazoxymethanol acetate treated Eker rats.
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Klaus V, Bastek H, Damme K, Collins LB, Frötschl R, Benda N, Lutter D, Ellinger-Ziegelbauer H, Swenberg JA, Dietrich DR, and Stemmer K
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- Animals, Cell Proliferation drug effects, Cell Transformation, Neoplastic drug effects, Cell Transformation, Neoplastic pathology, DNA Damage drug effects, Dose-Response Relationship, Drug, Female, Gene Expression Regulation, Neoplastic, Guanine analogs & derivatives, Guanine metabolism, Kidney metabolism, Kidney pathology, Male, Methylazoxymethanol Acetate administration & dosage, Rats, Mutant Strains, Time Factors, DNA Adducts analysis, Kidney drug effects, Methylazoxymethanol Acetate toxicity
- Abstract
Genotoxic carcinogens pose great hazard to human health. Uncertainty of current risk assessment strategies and long latency periods between first carcinogen exposure and diagnosis of tumors have raised interest in predictive biomarkers. Initial DNA adduct formation is a necessary step for genotoxin induced carcinogenesis. However, as DNA adducts not always translate into tumorigenesis, their predictive value is limited. Here we hypothesize that the combined analysis of pro-mutagenic DNA adducts along with time-matched gene expression changes could serve as a superior prediction tool for genotoxic carcinogenesis. Eker rats, heterozygous for the tuberous sclerosis (Tsc2) tumor suppressor gene and thus highly susceptible towards genotoxic renal carcinogens, were continuously treated with the DNA alkylating carcinogen methylazoxymethanol acetate (MAMAc). Two weeks of MAMAc treatment resulted in a time-dependent increase of O
6 -methylguanine and N7-methylguanine adducts in the kidney cortex, which was however not reflected by significant expression changes of cyto-protective genes involved in DNA repair, cell cycle arrest or apoptosis. Instead, we found a transcriptional regulation of genes involved in the tumor-related MAPK, FoxO and TGF-beta pathways. Continuous MAMAc treatment for up to 6 months resulted in a mild but significant increase of cancerous lesions. In summary, the combined analysis of DNA adducts and early gene expression changes could serve as a suitable predictive tool for genotoxicant-induced carcinogenesis.- Published
- 2017
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41. Xenobiotic CAR Activators Induce Dlk1-Dio3 Locus Noncoding RNA Expression in Mouse Liver.
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Pouché L, Vitobello A, Römer M, Glogovac M, MacLeod AK, Ellinger-Ziegelbauer H, Westphal M, Dubost V, Stiehl DP, Dumotier B, Fekete A, Moulin P, Zell A, Schwarz M, Moreno R, Huang JTJ, Elcombe CR, Henderson CJ, Roland Wolf C, Moggs JG, and Terranova R
- Subjects
- Animals, Biomarkers metabolism, Calcium-Binding Proteins, Chlordan toxicity, Constitutive Androstane Receptor, Liver metabolism, Liver pathology, Male, Mice, Mice, Knockout, Phenobarbital toxicity, Up-Regulation drug effects, Gene Expression drug effects, Intercellular Signaling Peptides and Proteins genetics, Iodide Peroxidase genetics, Liver drug effects, RNA, Long Noncoding genetics, Receptors, Cytoplasmic and Nuclear agonists, Xenobiotics toxicity
- Abstract
Derisking xenobiotic-induced nongenotoxic carcinogenesis (NGC) represents a significant challenge during the safety assessment of chemicals and therapeutic drugs. The identification of robust mechanism-based NGC biomarkers has the potential to enhance cancer hazard identification. We previously demonstrated Constitutive Androstane Receptor (CAR) and WNT signaling-dependent up-regulation of the pluripotency associated Dlk1-Dio3 imprinted gene cluster noncoding RNAs (ncRNAs) in the liver of mice treated with tumor-promoting doses of phenobarbital (PB). Here, we have compared phenotypic, transcriptional ,and proteomic data from wild-type, CAR/PXR double knock-out and CAR/PXR double humanized mice treated with either PB or chlordane, and show that hepatic Dlk1-Dio3 locus long ncRNAs are upregulated in a CAR/PXR-dependent manner by two structurally distinct CAR activators. We further explored the specificity of Dlk1-Dio3 locus ncRNAs as hepatic NGC biomarkers in mice treated with additional compounds working through distinct NGC modes of action. We propose that up-regulation of Dlk1-Dio3 cluster ncRNAs can serve as an early biomarker for CAR activator-induced nongenotoxic hepatocarcinogenesis and thus may contribute to mechanism-based assessments of carcinogenicity risk for chemicals and novel therapeutics., (© The Author 2017. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)
- Published
- 2017
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42. Quantitative targeted bile acid profiling as new markers for DILI in a model of methapyrilene-induced liver injury in rats.
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Slopianka M, Herrmann A, Pavkovic M, Ellinger-Ziegelbauer H, Ernst R, Mally A, Keck M, and Riefke B
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- Animals, Chemical and Drug Induced Liver Injury pathology, Chromatography, Liquid, Dose-Response Relationship, Drug, Down-Regulation drug effects, Gene Expression Regulation drug effects, Liver pathology, Male, Methapyrilene administration & dosage, Rats, Rats, Wistar, Reproducibility of Results, Tandem Mass Spectrometry, Up-Regulation drug effects, Bile Acids and Salts metabolism, Biomarkers metabolism, Chemical and Drug Induced Liver Injury etiology, Liver drug effects, Methapyrilene toxicity
- Abstract
Recently, bile acids (BAs) were reported as promising markers for drug-induced liver injury (DILI). BAs have been suggested to correlate with hepatocellular and hepatobiliary damage; however a clear connection of BA patterns with different types of DILI remains to be established. To investigate if BAs can improve the assessment of liver injury, 20 specific BAs were quantitatively profiled via LC-MS/MS in plasma and liver tissue in a model of methapyrilene-induced liver injury in rats. Methapyrilene, a known hepatotoxin was dosed daily over 14-days at doses of 30 and 80mg/kg, followed by a recovery phase of 10days. Conventional preclinical safety endpoints were related to BA perturbations and to hepatic gene expression profiling for a mechanistic interpretation of effects. Histopathological signs of hepatocellular and hepatobiliary damage with significant changes of clinical chemistry markers were accompanied by significantly increased levels of indivdual BAs in plasma and liver tissue. BA perturbations were already evident at the earliest time point after 30mg/kg treatment, and thereby indicating better sensitivity than clinical chemistry parameters. Furthermore, the latter markers suggested recovery of liver injury, whereas BA levels in plasma and liver remained significantly elevated during the recovery phase, in line with persistent histopathological findings of bile duct hyperplasia (BDH) and bile pigment deposition. Gene expression profiling revealed downregulation of genes involved in BA synthesis (AMACR, BAAT, ACOX2) and hepatocellular uptake (NTCP, OATs), and upregulation for efflux transporters (MRP2, MRP4), suggesting an adaptive hepatocellular protection mechanism against cytotoxic bile acid accumulation. In summary, our data suggests that specific BAs with high reliability such as cholic acid (CA) and chenodeoxycholic acid (CDCA) followed by glycocholic acid (GCA), taurocholic acid (TCA) and deoxycholic acid (DCA) can serve as additional biomarkers for hepatocellular/hepatobiliary damage in the liver in rat toxicity studies., (Copyright © 2017 Elsevier B.V. All rights reserved.)
- Published
- 2017
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43. Mechanistic Investigations of the Mitochondrial Complex I Inhibitor Rotenone in the Context of Pharmacological and Safety Evaluation.
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Heinz S, Freyberger A, Lawrenz B, Schladt L, Schmuck G, and Ellinger-Ziegelbauer H
- Subjects
- Administration, Oral, Animals, Body Weight drug effects, Bone and Bones drug effects, Bone and Bones metabolism, Bone and Bones pathology, Brain drug effects, Brain metabolism, Brain pathology, Electron Transport Complex I antagonists & inhibitors, Gene Expression drug effects, Hematopoiesis drug effects, Liver drug effects, Liver metabolism, Liver pathology, Male, Mitochondria metabolism, Rats, Rats, Wistar, Electron Transport Complex I metabolism, Mitochondria drug effects, Rotenone pharmacology
- Abstract
Inhibitors of the mitochondrial respiratory chain complex I are suggested to exert anti-tumor activity on those tumors relying on oxidative metabolism and are therefore of interest to oncology research. Nevertheless, the safety profile of these inhibitors should be thoroughly assessed. Rotenone, a proven complex I inhibitor, has shown anti-carcinogenic activity in several studies. In this context rotenone was used in this study as a tool compound with the aim to identify suitable biomarker candidates and provide enhanced mechanistic insights into the molecular and cellular effects of complex I inhibitors. Rats were treated with 400 ppm rotenone daily for 1, 3 or 14 consecutive days followed by necropsy. Classical clinical endpoints, including hematology, clinical chemistry and histopathology with supporting investigations (FACS-analysis, enzymatic activity assays) were examined as well as gene expression analysis. Through these investigations, we identified liver, bone marrow and bone as target organs amongst approx. 40 organs evaluated at least histopathologically. Our results suggest blood analysis, bone marrow parameters, assessment of lactate in serum and glycogen in liver, and especially gene expression analysis in liver as useful parameters for an experimental model to help to characterize the profile of complex I inhibitors with respect to a tolerable risk-benefit balance.
- Published
- 2017
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44. Non-Lethal Endotoxin Injection: A Rat Model of Hypercoagulability.
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Brooks MB, Turk JR, Guerrero A, Narayanan PK, Nolan JP, Besteman EG, Wilson DW, Thomas RA, Fishman CE, Thompson KL, Ellinger-Ziegelbauer H, Pierson JB, Paulman A, Chiang AY, and Schultze AE
- Subjects
- Acute Disease, Animals, Biomarkers blood, Blood Coagulation drug effects, Blood Platelets metabolism, Disease Models, Animal, Endothelial Cells metabolism, Extracellular Vesicles metabolism, Fibrin Fibrinogen Degradation Products metabolism, Intercellular Adhesion Molecule-1 metabolism, Leukopenia chemically induced, Male, MicroRNAs blood, Neutrophils metabolism, Neutrophils pathology, Plasminogen Activator Inhibitor 1 metabolism, Rats, Rats, Wistar, Thrombophilia immunology, Time Factors, Lipopolysaccharides, Thrombophilia chemically induced, Thrombophilia physiopathology
- Abstract
Systemic inflammation co-activates coagulation, which unchecked culminates in a lethal syndrome of multi-organ microvascular thrombosis known as disseminated intravascular coagulation (DIC). We studied an endotoxin-induced inflammatory state in rats to identify biomarkers of hemostatic imbalance favoring hypercoagulability. Intraperitoneal injection of LPS at 15 mg/kg body weight resulted in peripheral leukopenia and widespread neutrophilic sequestration characteristic of an acute systemic inflammatory response. Early indicators of hemostatic pathway activation developed within 4 hours, including increased circulating concentrations of procoagulant extracellular vesicles (EVs), EVs expressing endothelial cell and platelet membrane markers, and high concentration of soluble intercellular adhesion molecule-1 (sICAM-1), plasminogen activator inhibitor-1 (PAI-1), and D-dimers. Inflammation persisted throughout the 48-hour observation period; however, increases were found in a subset of serum microRNA (miRNA) that coincided with gradual resolution of hemostatic protein abnormalities and reduction in EV counts. Dose-adjusted LPS treatment in rats provides a time-course model to develop biomarker profiles reflecting procoagulant imbalance and rebalance under inflammatory conditions., Competing Interests: Lilly supported the in-life phase of this study by contracting for laboratories services, animals and reagents at the Covance facility in Greenfield, IN. All other work was provided in-kind by the ILSI HESI Cardiac Safety Committee Cardiac Biomarkers Working Group, which is supported by sponsorships from member companies. HESI’s scientific initiatives are primarily supported by the in-kind contributions (from public and private sector participants) of time, expertise, and experimental effort. These contributions are supplemented by direct funding (that primarily supports program infrastructure and management) provided primarily by HESI’s corporate sponsors. All authors are members of the ILSI HESI Cardiac Safety Committee Cardiovascular Biomarkers Working Group, a global non-profit organization. James R. Turk, Abraham Guerrero and Padma K. Narayanan are employed by Amgen Inc. Elizabeth G. Besteman is employed by Merck Research Laboratories. Roberta A. Thomas and Cindy E. Fishman are employed by GlaxoSmithKline. Heidrun Ellinger-Ziegelbauer is employed by Bayer Pharma AG. April Paulman is employed by Covance Laboratories. Alan Y. Chiang and Albert E. Schultze are employed by Lilly Research Laboratories. There are no patents, products in development or marketed products to declare. This does not alter our adherence to all the PLOS ONE policies on sharing data and materials.
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- 2017
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45. Absolute Measurement of Cardiac Injury-Induced microRNAs in Biofluids across Multiple Test Sites.
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Thompson KL, Boitier E, Chen T, Couttet P, Ellinger-Ziegelbauer H, Goetschy M, Guillemain G, Kanki M, Kelsall J, Mariet C, de La Moureyre-Spire C, Mouritzen P, Nassirpour R, O'Lone R, Pine PS, Rosenzweig BA, Sharapova T, Smith A, Uchiyama H, Yan J, Yuen PS, and Wolfinger R
- Subjects
- Animals, Biomarkers blood, Biomarkers urine, Heart Injuries chemically induced, Isoproterenol toxicity, Male, Plasma chemistry, Rats, Rats, Wistar, Real-Time Polymerase Chain Reaction, Serum chemistry, Heart Injuries metabolism, MicroRNAs blood, MicroRNAs urine
- Abstract
Extracellular microRNAs (miRNAs) represent a promising new source of toxicity biomarkers that are sensitive indicators of site of tissue injury. In order to establish reliable approaches for use in biomarker validation studies, the HESI technical committee on genomics initiated a multi-site study to assess sources of variance associated with quantitating levels of cardiac injury induced miRNAs in biofluids using RT-qPCR. Samples were generated at a central site using a model of acute cardiac injury induced in male Wistar rats by 0.5 mg/kg isoproterenol. Biofluid samples were sent to 11 sites for measurement of 3 cardiac enriched miRNAs (miR-1-3p, miR-208a-3p, and miR-499-5p) and 1 miRNA abundant in blood (miR-16-5p) or urine (miR-192-5p) by absolute quantification using calibration curves of synthetic miRNAs. The samples included serum and plasma prepared from blood collected at 4 h, urine collected from 6 to 24 h, and plasma prepared from blood collected at 24 h post subcutaneous injection. A 3 parameter logistic model was utilized to fit the calibration curve data and estimate levels of miRNAs in biofluid samples by inverse prediction. Most sites observed increased circulating levels of miR-1-3p and miR-208a-3p at 4 and 24 h after isoproterenol treatment, with no difference seen between serum and plasma. The biological differences in miRNA levels and sample type dominated as sources of variance, along with outlying performance by a few sites. The standard protocol established in this study was successfully implemented across multiple sites and provides a benchmark method for further improvements in quantitative assays for circulating miRNAs., (Published by Oxford University Press on behalf of the Society of Toxicology 2016. This work is written by US Government employees and is in the public domain in the US.)
- Published
- 2016
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46. Beyond miR-122: Identification of MicroRNA Alterations in Blood During a Time Course of Hepatobiliary Injury and Biliary Hyperplasia in Rats.
- Author
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Church RJ, Otieno M, McDuffie JE, Singh B, Sonee M, Hall L, Watkins PB, Ellinger-Ziegelbauer H, and Harrill AH
- Subjects
- 1-Naphthylisothiocyanate pharmacology, Animals, Biomarkers blood, Chemical and Drug Induced Liver Injury pathology, Disease Models, Animal, Hyperplasia, Male, Rats, Sprague-Dawley, Biliary Tract pathology, Chemical and Drug Induced Liver Injury blood, Liver pathology, MicroRNAs blood
- Abstract
Identification of circulating microRNAs for the diagnosis of liver injury and as an indicator of underlying pathology has been the subject of recent investigations. While several studies have been conducted, with particular emphasis on miR-122, the timing of miRNA release into the circulation and anchoring to tissue pathology has not been systematically evaluated. In this study, miRNA profiling was conducted over a time course of hepatobiliary injury and repair using alpha-naphthylisothiocyanate (ANIT) and a proprietary compound, FP004BA. ANIT administration (50 mg/kg) to rats caused significant biliary epithelial cell and hepatocellular necrosis between 24 and 72 h, followed by resolution and progression to biliary hyperplasia by 120 h which was associated with miRNA release into the blood. FP004BA (100 mg/kg) was used to confirm associations of miRNA along a time course with similar hepatic pathology to ANIT. Treatment with ANIT or FP004BA resulted in significant alterations of overlapping miRNAs during the early and peak injury phases. In addition to well-characterized liver injury markers miR-122-5p and miR-192-5p, multiple members of the 200 family and the 101 family along with miR-802-5p and miR-30d-5p were consistently elevated during hepatobiliary injury caused by both toxicants, suggesting that these species may be potential biomarker candidates for hepatobiliary injury. After 14 days of dosing with 4BA, miR-182-5p remained elevated-while miR-122-5p and miR-192-5p had returned to baseline-suggesting that miR-182-5p may have added utility to monitor for hepatobiliary injury in the repair phases when there remains histological evidence of ongoing cellular injury., (© The Author 2015. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)
- Published
- 2016
- Full Text
- View/download PDF
47. Importance of investigating epigenetic alterations for industry and regulators: An appraisal of current efforts by the Health and Environmental Sciences Institute.
- Author
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Miousse IR, Currie R, Datta K, Ellinger-Ziegelbauer H, French JE, Harrill AH, Koturbash I, Lawton M, Mann D, Meehan RR, Moggs JG, O'Lone R, Rasoulpour RJ, Pera RA, and Thompson K
- Subjects
- Animals, Cellular Reprogramming drug effects, DNA Methylation drug effects, Dose-Response Relationship, Drug, Endpoint Determination, Environmental Monitoring standards, Gene Expression Regulation, Developmental drug effects, Genetic Markers, Humans, Risk Assessment, Stem Cells drug effects, Stem Cells pathology, Epigenesis, Genetic drug effects, Gene Expression Profiling standards, Toxicity Tests standards
- Abstract
Recent technological advances have led to rapid progress in the characterization of epigenetic modifications that control gene expression in a generally heritable way, and are likely involved in defining cellular phenotypes, developmental stages and disease status from one generation to the next. On November 18, 2013, the International Life Sciences Institute (ILSI) Health and Environmental Sciences Institute (HESI) held a symposium entitled "Advances in Assessing Adverse Epigenetic Effects of Drugs and Chemicals" in Washington, D.C. The goal of the symposium was to identify gaps in knowledge and highlight promising areas of progress that represent opportunities to utilize epigenomic profiling for risk assessment of drugs and chemicals. Epigenomic profiling has the potential to provide mechanistic information in toxicological safety assessments; this is especially relevant for the evaluation of carcinogenic or teratogenic potential and also for drugs that directly target epigenetic modifiers, like DNA methyltransferases or histone modifying enzymes. Furthermore, it can serve as an endpoint or marker for hazard characterization in chemical safety assessment. The assessment of epigenetic effects may also be approached with new model systems that could directly assess transgenerational effects or potentially sensitive stem cell populations. These would enhance the range of safety assessment tools for evaluating xenobiotics that perturb the epigenome. Here we provide a brief synopsis of the symposium, update findings since that time and then highlight potential directions for future collaborative efforts to incorporate epigenetic profiling into risk assessment., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)
- Published
- 2015
- Full Text
- View/download PDF
48. Glomerulonephritis-induced changes in kidney gene expression in rats.
- Author
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Pavkovic M, Riefke B, Frisk AL, Gröticke I, and Ellinger-Ziegelbauer H
- Abstract
We investigated a glomerulonephritis (GN) model in rats induced by nephrotoxic serum (NTS) which contains antibodies against the glomerular basement membrane (GBM). The anti-GBM GN model in rats is widely used since its biochemical and histopathological characteristics are similar to crescentic nephritis and Goodpasture's disease in humans (Pusey, 2003[2]). Male Wistar Kyoto (WKY) and Sprague-Dawley (SD) rats were dosed once with 1, 2.5 and 5 ml/kg nephrotoxic serum (NTS) or 1.5 and 5 ml/kg NTS, respectively. GN and tubular damage were observed histopathologically in all treated rats after 14 days. To obtain insight into molecular processes during GN pathogenesis, mRNA expression was investigated in WKY and SD kidneys using Affymetrix's GeneChip Rat genome 230_2.0 arrays (GSE64265). The immunopathological processes during GN are still not fully understood and likely involve both innate and adaptive immunity. In the present study, several hundred mRNAs were found deregulated, which functionally were mostly associated with inflammation and regeneration. The β-chain of the major histocompatibility complex class II RT1.B (Rt1-Bb) and complement component 6 (C6) were identified as two mRNAs differentially expressed between WKY and SD rat strains which could be related to known different susceptibilities to NTS of different rat strains; both were increased in WKY and decreased in SD rats (Pavkovic et al., 2015 [1]). Increased Rt1-Bb expression in WKY rats could indicate a stronger and more persistent cellular reaction of the adaptive immune system in this strain, in line with findings indicating adaptive immune reactions during GN. The complement cascade is also known to be essential for GN development, especially terminal cascade products like C6.
- Published
- 2015
- Full Text
- View/download PDF
49. Glomerulonephritis-Induced Changes in Urinary and Kidney MicroRNA Profiles in Rats.
- Author
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Pavkovic M, Riefke B, Frisk AL, Gröticke I, and Ellinger-Ziegelbauer H
- Subjects
- Acute Kidney Injury chemically induced, Acute Kidney Injury genetics, Acute Kidney Injury urine, Animals, Biopsy, Cisplatin, Disease Models, Animal, Gene Expression Regulation, Gene Regulatory Networks, Genetic Markers, Glomerulonephritis genetics, Glomerulonephritis immunology, Glomerulonephritis pathology, In Situ Hybridization, Kidney immunology, Kidney pathology, Male, MicroRNAs urine, Oligonucleotide Array Sequence Analysis, RNA, Messenger genetics, RNA, Messenger metabolism, Rats, Inbred WKY, Rats, Sprague-Dawley, Real-Time Polymerase Chain Reaction, Time Factors, Gene Expression Profiling methods, Glomerulonephritis urine, Kidney metabolism, RNA, Messenger urine
- Abstract
MicroRNAs (miRNAs) regulate gene expression post-transcriptionally and thus are involved in various physiological and pathological states. Due to their stability in biofluids miRNAs have also been proposed as biomarkers (BMs) for tissue injury. We investigated the usefulness of urinary miRNAs for detection of site-specific renal damage in an antiglomerular basement membrane glomerulonephritis (GN) model in rats by comparing GN-induced urinary miRNAs profiles to traditional and newer protein BMs, and to proximal tubular injury-induced urinary miRNA profiles observed previously after cisplatin (Cp) treatment. Male Wistar Kyoto and Sprague Dawley rats were dosed once with 1, 2.5, and 5 ml/kg nephrotoxic serum (NTS) or 1.5 and 5 ml/kg NTS, respectively. GN and tubular damage were observed histopathologically in all treated rats after 14 days. Although serum creatinine and BUN were not changed, several protein BMs and 74 urinary miRNAs were found to be increased 8 and 14 days after NTS administration. Of these 74 miRNAs, 5 were identified as increased after NTS but not after Cp treatment. Using in situ hybridization two of them, miR-10 b and -100, were found to be localized in distal segments of the nephron, potentially reflecting the tubular injury in those regions. Furthermore, evaluation of both miRNA and mRNA expression in the kidney revealed possible miRNA-mRNA interactions mostly associated with fibrotic and transforming growth factor β signaling. In conclusion, our investigations support the potential of urinary miRNAs as specific BMs for kidney injury, and suggest a role of miRNAs in pathological processes during GN in the kidney., (© The Author 2015. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)
- Published
- 2015
- Full Text
- View/download PDF
50. Urinary microRNA profiling for identification of biomarkers after cisplatin-induced kidney injury.
- Author
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Pavkovic M, Riefke B, and Ellinger-Ziegelbauer H
- Subjects
- Acute Kidney Injury chemically induced, Acute Kidney Injury pathology, Animals, Cluster Analysis, Disease Models, Animal, Genetic Markers, Male, Principal Component Analysis, Rats, Rats, Wistar, Time Factors, Urinalysis, Acute Kidney Injury genetics, Acute Kidney Injury urine, Cisplatin, Gene Expression Profiling methods, MicroRNAs urine, Oligonucleotide Array Sequence Analysis
- Abstract
Extracellular microRNAs (miRNAs) have emerged as novel biomarkers (BMs) for various pathological states. To evaluate whether urinary miRNAs could serve as biomarkers for drug-induced kidney injury, we performed a nephrotoxicity study in rats with cisplatin (Cp), which is known to induce renal proximal tubular lesions in several species. Male Wistar rats were treated with a single dose of Cp (0, 1 and 3mg/kg) and urine was collected on days 3, 5, 8, 15 and 26 for measurement of several biomarkers and for RNA isolation. MiRNA profiling experiments with urine samples derived from the 3mg/kg Cp dosed animals, identified 136 miRNAs significantly increased in urine 3 and 5 days after Cp administration. 18 miRNAs with distinct time-dependent profiles were further analyzed using specific miRNA assays and absolute quantification. We observed >20-fold changes for 11 of these 18 miRNAs measured in profiling experiments, and confirmed their direction of change and time course profile by absolute quantification. Furthermore we found mechanistic links between several miRNAs and simultaneously measured mRNAs in the kidney after Cp administration. These were associated with pathways suggested to be involved in Cp-induced nephrotoxicity including a DNA damage response, apoptosis, and cell cycle regulation. Overall our results indicate that miRNAs measured in urine may serve as BMs for nephrotoxicity in rats., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)
- Published
- 2014
- Full Text
- View/download PDF
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