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Title: Investigation of Nrf2 activity measures for assessing the toxicological and pharmacological effects of drugs
Author: Mutter, F. E. M.
ISNI:       0000 0004 7428 6961
Awarding Body: University of Liverpool
Current Institution: University of Liverpool
Date of Award: 2018
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The transcription factor Nuclear factor erythroid 2-related factor 2 (NRF2) orchestrates the expression of a battery of cytoprotective genes containing antioxidant response elements (AREs) within their promotersequences. Activation of the NRF2 pathway has been implicated in the early stages of drug-induced toxicity, a major cause of drug attrition and public risk, and therefore holds potential as a pre-clinical marker of chemical entities liable to toxicity. Conversely, the upregulation of NRF2 activity holds great therapeutic potential. Many NRF2 modulating agents are currently undergoing clinical trials for the treatment of pathologies with an underlying oxidative stress component. The establishment of sensitive, accurate methods to monitor induction of the transcription factor are therefore vital. This thesis focuses on the characterisation of novel methods to measure NRF2 pathway activation following both toxic and therapeutic stimulation. In chapter 2, I use the transgenic Nrf2-Luciferase (Nrf2-Luc) mouse model (Oikawa et al., 2012), to test the hypothesis that localised activation of NRF2 represents an early marker of chemical stress associated with organ-specific drug toxicity. Following a hepatotoxic dose of paracetamol (APAP) a bioluminescent signal was observed in the liver but not kidneys or lungs of the Nrf2-Luc mice. On a cellular level, luciferase and Haem oxygenase 1 (Hmox1) staining colocalised around the centrilobular region, consistent with the known pathology of APAP. In response to a nephrotoxic dose of cisplatin, kidney specific bioluminescence was detected and colocalised with activation of the endogenous NRF2 pathway in tubular epithelial cells. A key finding was that activation of organ-specific bioluminescence correlated well with established biomarkers for liver and kidney injury respectively, highlighting the ability of NRF2 to reflect localised cellular insults associated with overt organ toxicity. Despite the emerging role of NRF2 as a marker of the response to drug toxicity and a therapeutic target, there are currently no non-invasive methods for monitoring NRF2 pathway activation in patients. In chapter 3 of this thesis, I hypothesised that organ-specific chemical perturbation instigates changes in NRF2 pathway activity in murine whole blood, providing potential markers of organ-specific injury. Here I provide evidence for the induction of NRF2-regulated genes in whole blood following exposure of C57BL/6 mice to APAP or the therapeutic NRF2-activating compound, CDDO-Me. The induction of NRF2- regulated transcripts, including Hmox1, was muted in mice administered APAP and the clinically used antidote, N-acetylcysteine (NAC). Insight into blood-based activity of the NRF2 pathway may provide evidence for the role of NRF2 in response to drug toxicity in humans and will be an important pharmacodynamic marker with the emergence of NRF2 modulators as therapeutic drugs. Primary human hepatocytes (PHH) are one of the most physiologically relevant in vitro systems for modelling drug-induced hepatotoxicity. However, the NRF2 pathway is not well characterised in these cells. In chapter 4, based on previous, unpublished microarray data of NRF2 pathway modulation in PHH, a group of potentially novel NRF2-regulated genes were identified: Coagulation factor II receptor-like 2 (F2RL2), NmrA-like family domain containing 1 pseudogene (LOC344887) and Tripartite Motif Containing 16 Like (TRIM16L). Following identification of potentially active cis-ARE sites in the promoters of these genes, I assessed direct activation by NRF2 using luciferase-based promotor-reporter assays. Despite evidence in multiple microarray datasets from our bioinformatic analysis that these genes are indeed modulated by NRF2, wild-type and mutant reporter constructs failed to produce differential luminescent responses in transfected HepG2 cells exposed to CDDO-Me or following overexpression of NRF2. During consideration of the well-established Nrf2-regulated gene SRXN1 as a positive control, I noticed two consecutive ARE sites within the region identified by Singh et al. to which NRF2 directly binds (Singh et al., 2009). Promotor-reporter analysis implicated both sequences in the activation of SRXN1 transcription as only cells transfected with the wild-type SRXN1 construct produced luminescence in response to Nrf2-activation. These data indicate the necessity of extended AREs for the activation of NRF2-regulated genes. The emergence of NRF2-activating compounds in the clinic highlights the application of therapeutic induction of the NRF2-dependent oxidative stress responses. The isothiocyanate, sulforaphane is among the most well studied clinical activators of NRF2. However due to its limited shelf life, stabilised analogues are needed. In the final experimental chapter of this thesis, I summarise data generated in collaboration with Evgen Pharma to assess the potencies of novel analogues of sulforaphane. Utilizing the H4IIe-8AREL reporter cell line, I show analogues with variable methane bridge length and sulfoxide group exhibited reduced potency towards NRF2 compared to the parent compound sulforaphane. The potencies of a subgroup of analogues as inducers of other luminescent reporters: pGL4- NQO1 and pGL4-5xARE, in a human liver cell line were also recorded. A key finding was the similar rank order of potencies of this subset of compounds in both cell lines. Development of sensitive assays for the in vitro assessment of NRF2 activators will be of increasing value as the number of therapeutic NRF2-modulating compounds entering the clinic grow. The establishment of robust methods to monitor NRF2 activation will inform key events underlying toxicity and provide insight into the subtle changes resulting in upregulation of the oxidative stress response to provide either an adaptive phenotype or one that is overwhelmed in resulting toxicity. Documentation of these mechanisms at a tissue or cellular level will inform adverse outcome pathways (AOPs), improving risk assessment frameworks. The specific stimuli NRF2 responds to must be thoroughly characterised to define the role of NRF2 and other stress response pathways following exposure to chemical and/or oxidative stressors. Conversely, development of effective, novel NRF2 inducers for use in the clinic requires sensitive screening platforms to determine the pharmacodynamic effects of these compounds.
Supervisor: Copple, Ian ; Park, B. K. Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral