Oxidative stress (OS) is a frequent complication of various disease conditions such as Alzheimer’s and Parkinson’s disease, atherosclerosis, preeclampsia, rheumatoid arthritis, diabetes including gestational diabetes, etc. OS is defined as an imbalance between the production of reactive species and the ability of an organism to detoxify the reactive intermediates and repair the damage. As a result of OS, the excess of reactive species such as oxygen superoxide (O2-), hydroxyl radical (OH), peroxynitrite (ONOO−), 4-hydroxynonenal (4HNE), etc., have a tendency to react with nearby proteins/nucleic acids/lipids changing their functionality or inactivating them completely. The organism has many ways to protect itself from the harmful effects of oxidants. One strategy employs antioxidants introduced to the body with food. The purpose of this thesis was to investigate the effect of reactive species on the active transport mediated by ABC efflux transporters as well as exploring the possibility of using antioxidants not as interceptors of reactive species but rather as inhibitors of metabolic enzymes and transporters. The BCRP/ABCG2 efflux transporter was selected for the investigation of the effect of reactive anion, ONOO−, generated during OS and the product of OS, 4HNE, formed after a series of chain reactions involving ROS. Experiments conducted with Sf9 membrane vesicles overexpressing BCRP/ABCG2 revealed that both species are capable of inactivating this ABC transporter with IC50 being 31 ± 2.7 μM and 92 ± 1.4 μM for ONOO− and 4HNE, respectively. In presence of 4HNE, Vmax decreased 4-fold and Km remained unchanged, suggesting a noncompetitive inhibition mechanism. However, with addition of 4HNE, positive cooperativity was also observed. With ONOO−, the situation was different: both Vmax and Km changed consistent with mixed type inhibition. Overall, OS-mediated BCRP/ABCG2 inactivation occurred at biologically relevant concentrations of the reactive species. Antioxidants are substances that are known to reduce the amount of ROS/RNS accumulated during OS, but this research considered the use of antioxidants not only as interceptors of ROS/RNS but rather as inhibitors of metabolic enzymes. The effect of the dietary antioxidant, quercetin (Qc), on the metabolism of 2-methoxyestradiol (2Me-E2), a promising potential anticancer agent was investigated. Qc possesses five hydroxyl groups, several of which are targets for UDP-glucuronosyltransferases (UGTs). Thus, the simultaneous presence of Qc and 2Me-E2 could result in decreased glucuronidation of 2Me-E2. Using the LS180 intestinal human colon adenocarcinoma cell line, glucuronidation of 2Me-E2 resulted in formation of only one major glucuronide, 2-Methoxyestradiol-3-glucuronide (2Me-E2-3G). Qc effectively reduced its formation (IC50 = 7.8 ± 0.26 μM) to a minimum level. The decrease in the activity of UGTs increased the intracellular concentration of parent 2Me-E2. Additional increase in cellular concentration of 2Me-E2 was achieved when LS180 cells were pre-incubated with Qc prior the addition of 2Me-E2. Transwell experiments with MDCKII – BCRP cells revealed that BCRP/ABCG2 did not appear to transport 2Me-E2. All in all, the present study showed that OS has a negative impact on active transport mediated by ABC transporters. This, in turn, can affect drug disposition and protection of endogenous organs and tissues. Antioxidants are one of the mechanisms that can effectively reduce the negative impact caused by oxidative species. Nevertheless, this research revealed that they can also be an effective tool to reduce the excessive metabolism of therapeutic drugs. Thus, Qc was found to be a dietary antioxidant that could reduce metabolism of 2Me-E2 and increase it intracellular concentration.
| Identifer | oai:union.ndltd.org:vcu.edu/oai:scholarscompass.vcu.edu:etd-1095 |
| Date | 01 January 2010 |
| Creators | Verenich, Svetlana |
| Publisher | VCU Scholars Compass |
| Source Sets | Virginia Commonwealth University |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Theses and Dissertations |
| Rights | © The Author |
Page generated in 0.0019 seconds