Sulforaphane is an isothiocyanate found in high concentrations in cruciferous vegetables like broccoli. Sulforaphane has received much attention due to the evidence that it inhibits phase I carcinogen-bioactivating enzymes and/or induces phase II antioxidant enzymes as well as metallothioneins (MTs) (Perocco et al., 2006; Clarke et al., 2008; Yeh & Yen, 2009). Since MTs and antioxidant enzymes are involved in the scavenging of reactive oxygen species (ROS), the question was raised whether sulforaphane can provide protection against increased oxidative stress and if sulforaphane exposure of a human hepatocellular carcinoma cell line, like HepaRG cells, will have a negative impact on phase I and II biotransformation in these cells. Oxidative stress was exogenously induced in HepaRG cells with tert- Butyl hydroperoxide (t-BHP). Phase I and phase II biotransformation pathways were assessed with caffeine, paracetamol, aspirin, sodium benzoate, and paraaminobenzoic acid, respectively, as probe substances. Through the use of a liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS/MS) assay, the biotransformation of caffeine in phase I and the formation of paracetamol, aspirin, sodium benzoate and para-aminobenzoic acid conjugates in phase II were investigated. This involved elucidating the time it took for the whole probe to be completely biotransformed during phase I biotransformation and the unique conjugates formed during phase II biotransformation in HepaRG cells.
The optimal t-BHP concentration and exposure time in HepaRG cells were standardized with a 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. LC-ESI-MS/MS assays to monitor phase I and phase II biotransformation were optimized and validated. The optimal sulforaphane concentration and exposure time in HepaRG cells were standardized with a MTT assay. To evaluate the possible protective effect of sulforaphane against oxidative stress, HepaRG cells were pre-incubated with sulforaphane followed by the induction of oxidative stress with t-BHP and the quantification of the amount of viable cells with a MTT assay. To investigate the effect of sulforaphane on phase I and phase II biotransformation pathways, HepaRG cells were first pre-incubated with sulforaphane followed by the addition of a specific probe substance and the assessment of the biotransformation of the probe with a LC-ESI-MS/MS assay.
The results partially supported the hypothesis of the study that sulforaphane will protect HepaRG cells against oxidative stress without negatively influencing phase I and phase II biotransformation. The results indicated that sulforaphane provided partial protection against t-BHP induced oxidative stress and had no effect on phase II paracetamol biotransformation in HepaRG cells. / Thesis, MSc (Biochemistry), North-West University, Potchefstroom Campus, 2013.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:nwu/oai:dspace.nwu.ac.za:10394/9642 |
| Date | January 2013 |
| Creators | Crous, Ané |
| Publisher | North-West University |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis |
Page generated in 0.0018 seconds