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Development of an in vitro mechanistic toxicity screening model using cultured hepatocytes
Thesis (PhD)--University of Pretoria, 2011.
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University of Pretoria
2013
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| _version_ | 1867613506664136704 |
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| access_status_str | Open Access |
| author2 | Cromarty, Allan Duncan |
| author_browse | Cromarty, Allan Duncan |
| author_facet | Cromarty, Allan Duncan |
| collection | Thesis |
| dc_rights_str_mv | © 2011 University of Pretoria. All rights reserved. The copyright in this work vests in the University of Pretoria. No part of this work may be reproduced or transmitted in any form or by any means, without the prior written permission of the University of Pretoria. |
| description | Thesis (PhD)--University of Pretoria, 2011. |
| format | Thesis |
| id | oai:repository.up.ac.za:2263/24162 |
| institution | University of Pretoria (South Africa) |
| last_indexed | 2026-06-10T12:37:14.131Z |
| license_str | Other — see source repository |
| provenance_str_mv | Harvested via OAI-PMH from UPSpace — University of Pretoria Institutional Repository |
| publishDate | 2013 |
| publishDateRange | 2013 |
| publishDateSort | 2013 |
| publisher | University of Pretoria |
| publisherStr | University of Pretoria |
| record_format | dspace |
| source_str | UPSpace — University of Pretoria Institutional Repository |
| spelling | oai:repository.up.ac.za:2263/24162 Development of an in vitro mechanistic toxicity screening model using cultured hepatocytes Cromarty, Allan Duncan Gulumian, Mary Steenkamp, Vanessa japie.vantonder@up.ac.za Van Tonder, Jacob John Apoptosis Cyp1a1 Ddd Dde Ddt Hepatotoxicity Mechanistic profiling UCTD Thesis (PhD)--University of Pretoria, 2011. In vitro testing includes both cell-based and cell-free systems that can be used to detect toxicity induced by xenobiotics. In vitro methods are especially useful in rapidly gathering intelligence regarding the toxicity of compounds for which none is available such as new chemical entities developed in the pharmaceutical industry. In addition to this, in vitro investigations are invaluable in providing information concerning mechanisms of toxicity of xenobiotics. This type of toxicity testing has gained popularity among the research and development community because of a number of advantages such as scalability to high throughput screening, cost-effectiveness and predictive power. Hepatotoxicity is one of the major causes of drug attrition and the high cost associated with drug development poses a heavy burden on the development of new chemical entities. Early detection of hepatotoxic agents by in vitro methods will improve lead optimisation and decrease the cost of drug development and reduce drug-induced liver injury. Literature highlights the need for a cellbased in vitro model that is capable of assessing multiple toxicity parameters, which assesses a wider scope of toxicity and would be able to detect subtle types of hepatotoxicity. The present study was aimed at developing an in vitro procedure capable of mechanistically profiling the effects of known hepatotoxin dichlorodiphenyl trichloroethane (DDT) and its metabolites, dichlorodiphenyl dichloroethylene (DDE) and dichlorodiphenyl dichloroethane (DDD) on an established liver-derived cell line, HepG2, by evaluating several different aspects of cellular function using a number of simultaneous in vitro assays on a single 96 well microplate. Examined parameters have been suggested by the European Medicines Agency and include: cell viability, phase I metabolism, oxidative stress, mitochondrial toxicity and mode of cell death (apoptosis vs. necrosis). To further assess whether the developed method was capable of detecting hepatoprotection, the effect of the known hepatoprotectant, N-acetylcysteine, was determined. Viability decreased in a dose-dependent manner yielding IC50 values of 54 μM, 64 μM and 44 μM for DDT, DDE and DDD, respectively. Evaluation of phase I metabolism showed that cytochrome P4501A1 activity was dose-dependently induced. Test compounds decreasedlevels of reactive oxygen species, and significantly hyperpolarised the mitochondrialmembrane potential. Assessment of the mode of cell death revealed a significant elevation of caspase-3 activity, with DDD proving to be most potent. DDT alone induced dosedependent loss of membrane integrity. These results suggest that the tested compounds produce apoptotic death likely due to mitochondrial toxicity with subsequent caspase-3 activation and apoptotic cell death. The developed in vitro assay method reduces the time it would take to assess the tested parameters separately, produces results from multiple endpoints that broadens the scope of toxicity compared to single-endpoint methods. In addition to this the method provides results that are truly comparable as all of the assays utilise the same batch of cells and are conducted on the same plate under the exact same conditions, which eliminates a considerable amount of variability that would be unavoidable otherwise. The present study laid a solid foundation for further development of this method by highlighting the unforeseen shortcomings that can be adjusted to improve scalability and predictive power. Pharmacology unrestricted 2013-09-06T16:48:35Z 2012-05-03 2013-09-06T16:48:35Z 2012-04-13 2011 2012-04-26 Thesis Van Tonder, JJ 2011, Development of an in vitro mechanistic toxicity screening model using cultured hepatocytes, PhD thesis, University of Pretoria, Pretoria, viewed yymmdd < http://hdl.handle.net/2263/24162 > D12/4/224/ag http://hdl.handle.net/2263/24162 http://upetd.up.ac.za/thesis/available/etd-04262012-112100/ © 2011 University of Pretoria. All rights reserved. The copyright in this work vests in the University of Pretoria. No part of this work may be reproduced or transmitted in any form or by any means, without the prior written permission of the University of Pretoria. application/pdf application/pdf application/pdf application/pdf application/pdf application/pdf application/pdf application/pdf application/pdf application/pdf application/pdf University of Pretoria |
| spellingShingle | Apoptosis Cyp1a1 Ddd Dde Ddt Hepatotoxicity Mechanistic profiling UCTD Development of an in vitro mechanistic toxicity screening model using cultured hepatocytes |
| title | Development of an in vitro mechanistic toxicity screening model using cultured hepatocytes |
| title_full | Development of an in vitro mechanistic toxicity screening model using cultured hepatocytes |
| title_fullStr | Development of an in vitro mechanistic toxicity screening model using cultured hepatocytes |
| title_full_unstemmed | Development of an in vitro mechanistic toxicity screening model using cultured hepatocytes |
| title_short | Development of an in vitro mechanistic toxicity screening model using cultured hepatocytes |
| title_sort | development of an in vitro mechanistic toxicity screening model using cultured hepatocytes |
| topic | Apoptosis Cyp1a1 Ddd Dde Ddt Hepatotoxicity Mechanistic profiling UCTD |
| url | http://hdl.handle.net/2263/24162 http://upetd.up.ac.za/thesis/available/etd-04262012-112100/ |