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In utero exposure to benzene, a known environmental contaminant, is associated with
increased risk of childhood leukemia. We have previously shown that in utero benzene exposure can alter the redox sensitive transcription factor NF-κB, which is up-regulated in leukemia. We hypothesize that this is through benzene-induced reactive oxygen species (ROS) production
interfering with the signalling pathway involving NF-κB, p38-MAPK and c-Myb. The objectives were to determine if ROS and p38-MAPK mediate benzoquinone (BQ) induced changes in NF-κB activity, and if downstream targets of NF-κB are altered after BQ exposure. HD3 chicken erythroblast cells were transfected with either a c-Myb or an NF-κB luciferase linked reporter plasmid and exposed to 25 μM BQ for 2-24 hours. C-Myb and NF-κB activities were determined using luciferase transcription factor activity assays, Western blotting was conducted to assess changes in protein levels in non-transfected cells, immunofluorescence was used to determine nuclear translocation of NF-κB and the presence of ROS was determined via DCFDA assays. A TaqMan qRT-PCR kit was used to assess mRNA changes of c-myc and bcl-2, two targets of NF-κB. NF-κB activity was significantly increased following 16 and 24 hours of BQ exposure in
HD3 cells. DCFDA assays and pre-treatment with antioxidants indicated that BQ-mediated ROS production was responsible for increased NF-κB activity. Immunofluorescence and Western blotting indicated that NF-κB translocates into the nucleus after BQ exposure. P38-MAPK was activated through a ROS dependent pathway after 8-24 hours of BQ exposure, and pre-treatment with the p38-MAPK inhibitor, SB203580, attenuated BQ-mediated increased NF-κB activity,
partially due to increased IκB-α protein expression. At this point, the chosen downstream targets were not significantly different compared to control. Future studies should continue to evaluate
the role of p38-MAPK in this pathway as well as look at epigenetic changes in key signalling proteins. Evaluating the effects of toxicant exposure on cell signalling pathways is vital for understanding mechanisms of xenobiotic-induced toxicity. / Thesis (Master, Pharmacology & Toxicology) -- Queen's University, 2013-08-28 12:07:26.393
Date29 August 2013
CreatorsStokes, SAMANTHA
ContributorsQueen's University (Kingston, Ont.). Theses (Queen's University (Kingston, Ont.))
Source SetsLibrary and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada
LanguageEnglish, English
Detected LanguageEnglish
RightsThis publication is made available by the authority of the copyright owner solely for the purpose of private study and research and may not be copied or reproduced except as permitted by the copyright laws without written authority from the copyright owner.
RelationCanadian theses

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