Induction of the multixenobiotic/multidrug resistance system in various cell lines in response to perfluorinated
Izabela Rusiecka1, Andrzej C Składanowski
1Department of Molecular Enzymology, Intercollegiate Faculty of Biotechnology UG-MUG, Medical University of Gdańsk, Gdańsk, Poland.
Abstract:
The multixenobiotic resistance (closely related to multidrug resistance) system controls transport across the plasma membrane as a defense against toxic molecules. Multixenobiotic resistance system consists of an efflux pump, ABCB1 (also named P-glycoprotein, P-gp), and/or a molecule of the ABCC family (also named multiple resistance associated protein, MRP). ABCB1 is able to increase efflux of many low-molecular foreign molecules. Measuring system induction may be used as a biomarker of cell/organism exposure to foreign substances. Various established cell lines were tested for constitutive and induced multixenobiotic resistance proteins by Western blotting immunodetection. The pumping function was indirectly assayed with Rhodamine B by visualization of cell fluorescence in the presence of verapamil. Changes in ABC proteins were measured by flow cytometry after exposition to various perfluorinated carboxylic acids. MCF7 and HeLa cells were found to contain the highest constitutive level of both ABCB1 and ABCC1. HEK293 exhibited much less ABCB1 and no activity of pumping out Rhodamine B. The pumping activity was found to be related to the amount of the cell-type specific 170 kDa ABCB1 protein. An 8-day exposure to 10(-4) M perfluorononanoic acid resulted in about 2-2.5-fold increase of ABCB1 level. That was confirmed also for short times by flow cytometry of cells exposed to perfluorinated acids and its natural congeners. Both ABCB1- and ABCC1-related fluorescence increased along with the carbon chain in acids from C(6) up to C(9) and decreased for C(10). Measuring of multixenobiotic resistance changes in vitro induced by chemicals may be a convenient test for screening for their potential toxicity.
Insights
The multixenobiotic resistance system, involving ABCB1 and ABCC proteins, can be induced by foreign substances. Measuring this induction serves as a biomarker for chemical exposure and potential toxicity screening.
Area of Science:
- Biochemistry and Molecular Biology
- Toxicology
- Cell Biology
Background:
- The multixenobiotic resistance (MXR) system, including ABCB1 (P-glycoprotein) and ABCC (MRP) proteins, is crucial for cellular defense against toxic molecules by controlling transport across the plasma membrane.
- Induction of MXR proteins can serve as a biomarker for detecting exposure to xenobiotics.
- ABCB1 efflux pumps are known to transport a wide range of low-molecular-weight foreign compounds.
Purpose of the Study:
- To investigate the constitutive and induced levels of MXR proteins (ABCB1 and ABCC1) in various cell lines.
- To assess the functional activity of the MXR system using Rhodamine B efflux assay.
- To evaluate the impact of perfluorinated carboxylic acids (PFCAs) on MXR protein expression and function as a potential toxicity screening method.
Main Methods:
- Western blotting for immunodetection of constitutive and induced MXR proteins.
- Rhodamine B efflux assay with verapamil to indirectly measure pumping function.
- Flow cytometry to quantify changes in ABCB1 and ABCC1 expression after PFCA exposure.
- Testing of various cell lines including MCF7, HeLa, and HEK293.
Main Results:
- MCF7 and HeLa cells showed the highest constitutive levels of both ABCB1 and ABCC1, with functional pumping activity correlated to ABCB1 levels.
- HEK293 cells had lower ABCB1 levels and no detectable Rhodamine B efflux activity.
- Exposure to perfluorononanoic acid (PFNA) significantly increased ABCB1 levels (2-2.5 fold) in a time-dependent manner.
- PFCA-induced fluorescence for both ABCB1 and ABCC1 increased with carbon chain length from C(6) to C(9) and decreased at C(10).
Conclusions:
- The study identifies specific cell lines (MCF7, HeLa) with high constitutive MXR protein levels and functional activity.
- Perfluorinated carboxylic acids can induce the expression of ABCB1 and ABCC1 proteins, suggesting their potential as toxic agents.
- Measuring MXR system changes in vitro offers a viable method for screening chemical-induced toxicity and exposure.
