Evaluation of a cellular model for assessing cytoprotection by natural chemicals

Hashim, Maha Jalal (2013) Evaluation of a cellular model for assessing cytoprotection by natural chemicals. PhD thesis, University of Nottingham.

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There is considerable interest in the ability of plant-derived antioxidants to protect against oxidative damage associated with disease or exposure to toxic agents. In this study, the cytoprotection effect of the direct antioxidants quercetin (Q) and epigallocatechin-3-gallate (EGCG) and the indirect antioxidants, sulforaphane (SFN) and indole-3-carbinol (I3C) was assessed in a cellular protection assay. This assay involved two cytoprotection patterns: (a) 20-hour exposure to phytochemical followed by 5-hour exposure to t-BHP; (b) simultaneous exposure to phytochemicals and t-BHP for 5 hours. HepG2 cells were cultured to a confluent monolayer and exposed to phytochemical +/- t-BHP in serum-containing or serum-free medium, after which cell damage mediated by oxidant stress was assessed by neutral red uptake. Results showed that Q, EGCG and I3C were effective while SF was inactive and toxic to the cells by itself at high concentration during long incubation. On the other hand, short time of incubation with Q, EGCG and SF displayed identical results to prolonged exposure. However, I3C was devoid of protection activity. Moreover, results showed that serum has a major impact on antioxidant activity.

The toxicity effects of t-BHP on HepG2 cells in the presence and/or absence of phytochemicals was evaluated. This assay included exposure of HepG2 cells to phytochemicals for 20 hours prior exposure to different concentrations of t-BHP for 5 hours. Results indicated that full cytoprotection was provided by Q and EGCG while partial protection was displayed by I3C and SFN. On the other hand, results showed complete cell death to have occurred at 0.4 and 0.8 mM t-BHP after 5 hours of incubation with oxidant/ in the absence of phytochemicals. Additionally, presence of serum reduces the toxic effect of t-BHP effects. The possible mechanism of the toxicity of t-BHP on HepG2 was studied to ascertain if t-BHP initiated apoptosis in HepG2 cells, through determination of activated caspase by different techniques. Basically, HepG2 cells were exposed to +/- 0.4 and 0.8 mM t-BHP for specific times. Results showed no strong evidence for apoptosis although caspase-3 activity increased significantly (p≤0.05) in treated HpG2 cells with 0.8 mM t-BHP at 150 minutes. Similarly, significant increases in ROS and lipid peroxidation in treated HepG2 cells with 0.8 mM t-BHP (p≤0.05 and 0.01 respectively) at 150 minutes were obtained. Moreover, a (non-significant) decline in GSH amount was reported.

Treatment of the cells with Q and I3C under the conditions used in the cytoprotection study prevented the weak activation of caspase-3 identified by western blot.

The possible mechanism (s) of protective properties of the phytochemicals in induction of phase ll detoxifying enzymes was studied by use of an intact-cell assay of quinone reductase based upon duroquinone-mediated reduction of ferricyanide. However, studies demonstrated that Q and EGCG themselves acted as intermediate electron donor in the assay, and so interfered with the assay; I3C and SFN did not share this property.

In conclusion, the work presented in the thesis has indicated that the four selected compounds possess cytoprotection effect against oxidative stress induced by t-BHP under particular conditions, and has provided further insights into mechanism of toxicity of t-BHP.

Item Type: Thesis (University of Nottingham only) (PhD)
Supervisors: Fry, J.R.
Pratten, M.K.
Subjects: QS-QZ Preclinical sciences (NLM Classification) > QV Pharmacology
Faculties/Schools: UK Campuses > Faculty of Medicine and Health Sciences > School of Biomedical Sciences
UK Campuses > Faculty of Medicine and Health Sciences > School of Life Sciences
Item ID: 13398
Depositing User: EP, Services
Date Deposited: 21 Nov 2013 12:53
Last Modified: 13 Oct 2017 19:07
URI: http://eprints.nottingham.ac.uk/id/eprint/13398

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