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P-GP efflux pump inhibition potential of common environmental contaminants determined in vitro
Georgantzopoulou, A. ; Skoczynska, E.M. ; Berg, J.H.J. van den; Brand, W. ; Legay, S. ; Klein, S.G. ; Rietjens, I. ; Murk, A.J. - \ 2014
Environmental Toxicology and Chemistry 33 (2014)4. - ISSN 0730-7268 - p. 804 - 813.
multidrug-resistance transporters - flavonoid-mediated inhibition - caco-2 cell-line - aquatic organisms - multixenobiotic resistance - perfluorinated compounds - drug transport - mytilus-californianus - screening assay - great-lakes
Across different species, cellular efflux pumps such as P-glycoprotein (P-gp; also termed multidrug resistance protein 1 [MDR1]) serve as a first line of defense by transporting toxic xenobiotics out of the cell. This mechanism is also active in aquatic organisms such as mussels, fish, and their larvae. Modulation of this resistance mechanism by chemical agents occurring in the environment could result in either higher or lower internal concentrations of toxic or endogenous compounds in cells. The aim of the present study was to explore and quantify the inhibition of the P-gp efflux pumps by several ubiquitous aquatic contaminants. The calcein-acetoxymethyl ester (calcein-AM) assay commonly used in pharmacological research was established with P-gp–overexpressing Madin–Darby canine kidney cells (MDCKII–MDR1) in a 96-well plate, avoiding extra washing, centrifugation, and lysis steps. This calcein-AM–based P-gp cellular efflux pump inhibition assay (CEPIA) was used to study the inhibition by commonly occurring environmental contaminants. Among others, the compounds pentachlorophenol, perfluorooctane sulfonate, and perfluorooctanoate strongly inhibited the P-gp–mediated efflux of calcein-AM while the chloninated alkanes did not seem to interact with the transporter. The fact that common pollutants can be potent modulators of the efflux transporters is a motive to further study whether this increases the toxicity of other contaminants present in the same matrices.
Adaptation of a Madin-Darby canine kidney cell line to suspension growth in serum-free media and comparison of its ability to produce avian influenza virus to Vero and BHK21 cell lines
Wielink, R. van; Kant-Eenbergen, H.C.M. ; Harmsen, M.M. ; Martens, D.E. ; Wijffels, R.H. ; Coco-Martin, J.M. - \ 2011
Journal of Virological Methods 171 (2011)1. - ISSN 0166-0934 - p. 53 - 60.
scale microcarrier culture - mdck cells - vaccine production - cultivation - strains - chickens - receptor - anoikis - system - steps
Madin–Darby canine kidney (MDCK) cells are currently considered for influenza vaccine manufacturing. A drawback of these cells is their anchorage dependent growth, which greatly complicates process scale-up. In this paper a novel MDCK cell line (MDCK-SFS) is described that grows efficiently in suspension and retained high expression levels of both a-2,6 and a-2,3 sialic acid receptors, which bind preferably to human and avian influenza viruses, respectively. The production of avian influenza virus by BHK21, Vero and MDCK-SFS cell lines was compared. Although BHK21 cells consisted of two populations, one of which lacks the a-2,3 receptor, they supported the replication of two influenza strains to high titres. However, BHK21 cells are generally not applicable for influenza production since they supported the replication of six further strains poorly. MDCK-SFS cells yielded the highest infectious virus titres and virus genome equivalent concentration for five of the eight influenza strains analyzed and the highest hemagglutination activity for all eight virus strains. Taken together with their suitability for suspension growth this makes the MDCK-SFS cell line potentially useful for large scale influenza virus production.
Glutathione-dependent interaction of heavy metal compounds with multidrug resistance proteins MRP1 and MRP2
Wortelboer, H.M. ; Balvers, M.G.J. ; Usta, M. ; Bladeren, P.J. van; Cnubben, N.H.P. - \ 2008
Environmental Toxicology and Pharmacology 26 (2008)1. - ISSN 1382-6689 - p. 102 - 108.
reduced glutathione - arsenic-glutathione - in-vitro - vincristine transport - complex-formation - drug-resistance - leukotriene c-4 - cells - cisplatin - conjugate
The interactions of three heavy metal-containing compounds, cisplatin (CDDP), arsenic trioxide (AS(2)O(3)), and mercury dichloride (HgCl2), with the multidrug resistance transporters MRP1 and MRP2 and the involvement of glutathione (GSH)-related processes herein were investigated. In Madin-Darby canine kidney cells stably expressing MRP1 or MRP2, viability, GSH content, calcein efflux and polarized CSH efflux were measured as a function of exposure to CDDP, As2O3 and HgCl2. In isolated S integral]9-MRP1 and S integral 9-MRP2 membrane vesicles, the interaction with MRP-associated ATPase activity was measured. In the latter model system adduct formation with GSH is not an issue. The data show that (1) CDDP interacts with both MRP1 and MRP2, and GSH appears to play no major role in this process, (2) As2O3 interacts with MRP1 and MRP2 in which process GSH seems to be essential, and (3) HgCl2 interacts with MRP1 and MRP2, either alone and/or as a metal-GSH complex.
Breast cancer resistance protein (Bcrp1/Abcg2) limits net intestinal uptake of quercetin in rats by facilitating apical efflux of glucuronides
Sesink, A.L.A. ; Arts, I.C.W. ; Boer, V.C.J. de; Breedveld, P. ; Schellens, J.H.M. ; Hollman, P.C.H. ; Russel, F.G.M. - \ 2005
Molecular pharmacology 67 (2005)6. - ISSN 0026-895X - p. 1999 - 2006.
lactase-phlorhizin hydrolase - multidrug-resistance - oral bioavailability - dietary quercetin - biliary-excretion - fumitremorgin-c - heart-disease - in-vitro - transporter - cells
The intestinal absorption of the flavonoid quercetin in rats is limited by the secretion of glucuronidated metabolites back into the gut lumen. The objective of this study was to determine the role of the intestinal efflux transporters breast cancer resistance protein (Bcrp1)/Abcg2 and multidrug resistance-associated protein 2 (Mrp2)/Abcc2. To study the possible involvement of Mrp2, we compared intestinal uptake of quercetin-3-glucoside between control and Mrp2-deficient rats, using an in situ intestinal perfusion system. The contribution of Bcrp1 was determined using the specific inhibitor fumitremorgin C (FTC) in Mrp2-deficient rats. Furthermore, vectorial transport of quercetin was studied in Madin-Darby canine kidney (MDCK)II cells transfected with either human MRP2 or murine Bcrp1. In these MDCKII cells, we showed an efficient efflux-directed transport of quercetin by mouse Bcrp1, whereas in control and MRP2-transfected cells no vectorial transport of quercetin was observed. In Mrp2-deficient rats, intestinal uptake of quercetin from quercetin-3-glucoside, efflux of quercetin glucuronides to the gut lumen, and plasma concentration of quercetin were similar to that in control rats. When intestinal Bcrp1 was inhibited by FTC in Mrp2-deficient rats, total plasma concentrations of quercetin and its methylated metabolite isorhamnetin after 30 min of perfusion were more than twice that of controls (12.3 ± 1.5 versus 5.6 ± 1.3 µM; p <0.01), whereas uptake of free quercetin from the intestinal lumen was not affected. Instead, inhibition of Bcrp1 lowered the efflux of quercetin glucuronides into the perfusion fluid by approximately 4-fold. In conclusion, Bcrp1 limits net intestinal absorption of quercetin by pumping quercetin glucuronides back into the lumen.
Inhibition of multidrug resistance proteins MRP1 and MRP2 by a series of a,ß-unsaturated carbonyl compounds
Wortelboer, H.M. ; Ustafa, M. ; Zanden, J.J. van; Bladeren, P.J. van; Rietjens, I.M.C.M. ; Cnubben, N.H.P. - \ 2005
Biochemical Pharmacology 69 (2005)12. - ISSN 0006-2952 - p. 1879 - 1890.
acid phenethyl ester - organic anion transporter - s-transferase p1-1 - p-glycoprotein - ethacrynic-acid - glutathione-conjugate - cells - curcumin - agents - rat
To study the possible interplay between glutathione metabolism of and MRP inhibition by thiol reactive compounds, the interactions of a series of ¿,ß-unsaturated carbonyl compounds with multidrug resistance proteins 1 and 2 (MRP1/ABCC1 and MRP2/ABCC2) were studied. ¿,ß-Unsaturated carbonyl compounds react with glutathione, and therefore either their parent compound or their intracellularly formed glutathione metabolite(s) can modulate MRP-activity. Inhibition was studied in Madin-Darby canine kidney cells stably expressing MRP1 or MRP2, and isolated Sf9-MRP1 or Sf9-MRP2 membrane vesicles. In the latter model system metabolism is not an issue. Of the series tested, three distinct groups could be discriminated based on differences in interplay of glutathione metabolism with MRP1 inhibition. Curcumin inhibited MRP1 transport only in the vesicle model pointing at inhibition by the parent compound. The glutathione conjugates of curcumin also inhibit MRP1 mediated transport, but to a much lesser extent than the parent compound curcumin. In the cellular model system, it was demonstrated that glutathione conjugation of curcumin leads to inactivation of its inhibitory potential. Demethoxycurcumin and bisdemethoxycurcumin inhibited MRP1 in both the vesicle and cellular model pointing at inhibitory potency of at least the parent compound and possibly their metabolites. A second group, including caffeic acid phenethyl ester inhibited MRP1-mediated calcein transport only in the MDCKII-MRP1 cells, and not in the vesicle model indicating that metabolism appeared a prerequisite to generate the active inhibitor. Finally cinnamaldehyde, crotonaldehyde, trans-2-hexanal, citral, and acrolein did not inhibit MRP1. For MRP2, inhibition was much less in both model systems, with the three curcuminoids being the most effective. The results of this study show the importance to study the complex interplay between MRP-inhibitors and their cellular metabolism, the latter affecting the ultimate potential of a compound for cellular MRP-inhibition.
Interplay between MRP-inhibition and metabolism of MRP-inhibitors: the case of curcumin
Wortelboer, H.M. ; Usta, M. ; Velde, A.E. van der; Boersma, M.G. ; Spenkelink, A. ; Zanden, J.J. van; Rietjens, I.M.C.M. ; Bladeren, P.J. van; Cnubben, N.H.P. - \ 2003
Chemical Research in Toxicology 16 (2003)12. - ISSN 0893-228X - p. 1642 - 1651.
multidrug-resistance protein - glutathione-s-transferase - chemopreventive agent curcumin - organic anion transporter - p-glycoprotein - drug-resistance - reduced glutathione - rat hepatocytes - ethacrynic-acid - leukotriene c-4
The multidrug resistance proteins MRP1 and MRP2 are efflux transporters with broad substrate specificity, including glutathione, glucuronide, and sulfate conjugates. In the present study, the interaction of the dietary polyphenol curcumin with MRP1 and MRP2 and the interplay between curcumin-dependent MRP inhibition and its glutathione-dependent metabolism were investigated using two transport model systems. In isolated membrane vesicles of MRP1- and MRP2-expressing Sf9 cells, curcumin clearly inhibited both MRP1- and MRP2-mediated transport with IC50 values of 15 and 5 muM, respectively. In intact monolayers of MRP1 overexpressing Madin-Darby canine kidney (MDCKII-MRP1) cells, curcumin also inhibited MRP1-mediated activity, although with a 3-fold higher IC50 value than the one observed in the vesicle model. Interestingly, MRP2-mediated activity was hardly inhibited in intact monolayers of MRP2-overexpressing MDCKII (MDCKII-MRP2) cells upon exposure to curcumin, whereas the IC50 value in the vesicle incubations was 5 muM. The difference in extent of inhibition of the MRPs by curcumin in isolated vesicles as compared to intact cells, observed especially for MRP2, was shown to be due to a swift metabolism of curcumin to two glutathione conjugates in the MDCKII cells. Formation of both glutathione conjugates was about six times higher in the MDCKII-MRP2 cells as compared with the MDCKII-MRP1 cells, a phenomenon that could be ascribed to the significantly lower glutathione levels in the cell line. The efflux of both conjugates, identified in the present study as monoglutathionyl curcumin conjugates, was demonstrated to be mediated by both MRP1 and MRP2. From dose-response curves with Sf9 membrane vesicles, glutathionylcurcumin conjugates appeared to be less potent inhibitors of MRP1 and MRP2 than their parent compound curcumin. In conclusion, curcumin clearly inhibits both MRP1- and MRP2-mediated transport, but the glutathione-dependent metabolism of curcumin plays a crucial role in the ultimate level of inhibition of MRP-mediated transport that can be achieved in a cellular system. This complex interplay between MRP inhibition and metabolism of MRP inhibitors, the latter affecting the ultimate potential of a compound for cellular MRP inhibition, may exist not only for a compound like curcumin but also for many other MRP inhibitors presently or previously developed on the basis of vesicle studies.
Investigating the Use of Proxies for Fecundity to Improve the Management of Western Horse Mackerel
Oliveira, J.A.A. de; Roel, B.A. ; Dickey-Collas, M. ; Darby, C.D. - \ 2003
- p. 1 - 16.
|Susceptibility of bovine umbilical cord endothelial cells to bovine herpesviruses and pseudocowpox virus.
Wellenberg, G.J. ; Verstraten, E.R.A.M. ; Jongejan, F. ; Oirschot, J.T. van - \ 2002
Veterinary Research Communications 5 (2002). - ISSN 0165-7380 - p. 407 - 417.
polymerase-chain-reaction - herpes-simplex virus - infection - replication - rabbits - type-4
The purpose of the study was to determine the susceptibility of bovine umbilical cord endothelial (BUE) cells to bovine herpesvirus (BHV) 1, BHV2, BHV4 and BHV5, and to pseudocowpox virus. the detection limits and growth curves of these viruses in BUE cells were compared with those in Vero, Madin-Darby bovine kidney (MDBK), or bovine fetal diploid lung (BFDL) cells. Detection limits were determined by inoculating cell cultures with serial 10-fold dilutions of these viruses, and growth curves by titration of virus, harvested at various times after infecting cells at a multiplicity of infection of 0.1. The detection limits of BHV2 and BHV4 were lower in BUE cells than in Vero or MDBK cells, and cytopathic effects were observed earlier in BUE cells. In addition, BHV2 and BHV4 grew to higher titres in BUE cells than in Vero or MDBK cells. BUE cells appeared to be equally susceptible to BHV5, but less susceptible to BHV1.1 and BHV1.1 than MDBK cells. The study showed that BUE cells are highly susceptible to BHV2 and BHV4, and that the use of BUE cells can improve the laboratory diagnosis of these viruses. The use of BUE cells could also improve the isolation and growth of pseudocowpox virus.
1H NMR analysis of the partly-folded non-native two-disulphide intermediates (30-51,5-14) and (30-51,5-38) in the folding pathway of bovine pancreatic trypsin inhibitor.
Mierlo, C.P.M. van; Kemmink, J. ; Neuhaus, D. ; Darby, N.J. ; Creighton, T.E. - \ 1994
Journal of Molecular Biology 235 (1994). - ISSN 0022-2836 - p. 1044 - 1061.
Partially folded conformation of the (30-51) intermediate in the disulphide folding pathway of bovine pancreatic trypsin inhibitor : 1H and 15N resonance assignments and determination of backbone dynamics from 15N relaxation measurements
Mierlo, Carlo P.M. van; Darby, Nigel J. ; Keeler, James ; Neuhaus, David ; Creighton, Thomas E. - \ 1993
Journal of Molecular Biology 229 (1993)4. - ISSN 0022-2836 - p. 1125 - 1146.
Bovine pancreatic trypsin inhibitor (BPTI) - Disulphide bonds - Folding intermediate - NMR - Protein folding
An analogue of the important folding intermediate of BPTI with only the disulphide bond between Cys30 and Cys51 has been characterized by 1H and 15N NMR techniques. In particular, the dynamics of the polypeptide backbone were characterized using (1H)-15N NOE and 15N T1 and T2 relaxation data. The intermediate is partially folded, with part of the polypeptide chain stably folded and the remainder flexible or unfolded. The folded portion consists of the major elements of native-like secondary structure interacting through the hydrophobic core of the molecule. The 15N relaxation data show that the N-terminal 15 residues are very flexible, and the (1H,1H) NOESY data show that these residues have no NOE interactions with the remainder of the molecule. The segment of residues 37 to 41 is also flexible. These observations explain why during folding this intermediate most readily forms any of the possible disulphide bonds between Cys5, Cys14 and Cys38, including the non-native 5-14 and 5-38 bonds. The native-like folded portion of the molecule limits the possible disulphide bonds that can be formed to those in the remainder of the polypeptide chain. Also, forming the non-native disulphide bonds need not involve any disruption of that folded structure, as the Cys residues involved are in flexible regions of the molecule.
|BPTI as a model protein for understanding protein folding pathways.
Darby, N.J. ; Mierlo, C.P.M. van; Creighton, T.E. - \ 1993
In: Innovations on proteases and inhibitors / Avilés, F.X., - p. 391 - 406.
|The partially folded conformation of the Cys30-Cys51 intermediate in the disulphide folding pathway of bovine pancreatic trypsin inhibitor.
Mierlo, C.P.M. van; Darby, N.J. ; Creighton, T.E. - \ 1992
Proceedings of the National Academy of Sciences of the United States of America 89 (1992). - ISSN 0027-8424 - p. 6775 - 6779.
Kinetic roles and conformational properties of the non-native two-disulphide intermediates in the refolding of bovine pancreatic trypsin inhibitor.
Darby, N.J. ; Mierlo, C.P.M. van; Scott, G.H.E. ; Neuhaus, D. ; Creighton, T.E. - \ 1992
Journal of Molecular Biology 224 (1992). - ISSN 0022-2836 - p. 905 - 911.
The 5-55 single-disulphide intermediate in folding of bovine pancreatic trypsin inhibitor
Darby, N.J. ; Mierlo, C.P.M. van; Creighton, T.E. - \ 1991
FEBS Letters 279 (1991)1. - ISSN 0014-5793 - p. 61 - 64.
Bovine pancreatic trypsin inhibitor - Nuclear magnetic resonance, Circular dichroism - Protein folding: Disulphide intermediate
An analogue of the BPTI folding intermediate that contains only the disulphide bond between Cys-5 and Cys-55 has been prepared by mutation or the other four Cys residues to Ser. On the basis of its circular dichroism and 1H-nuclear magnetic resonance spectra and its electrophoretic mobility, this intermediate is shown to be at least partially folded at low temperatures. This probably accounts for several of the unique properties of this intermediate observed during folding.
(14-38, 30-51) Double-disulphide intermediate in folding of bovine pancreatic trypsin inhibitor : A two-dimensional 1H nuclear magnetic resonance study
Mierlo, Carlo P.M. van; Darby, Nigel J. ; Neuhaus, David ; Creighton, Thomas E. - \ 1991
Journal of Molecular Biology 222 (1991)2. - ISSN 0022-2836 - p. 353 - 371.
bovine pancreatic trypsin inhibitor (BPTI) - disulphide bonds - folding intermediate - n.m.r. - protein folding
An analogue of the BPTI folding intermediate that contains only the disulphide bonds between Cys14 and Cys38 and between Cys30 and Cys51 has been prepared in Escherichia coli by protein engineering methods. The other two Cys residues of native BPTI (at positions 5 and 55) have been replaced by Ser. Essentially complete proton resonance assignments of the analogue were obtained by employing two-dimensional 1H nuclear magnetic resonance techniques. The intermediate has a more extended conformation in the N-terminal (residues 1 to 7) region and there are other differences in the C-terminal (residues 55 to 58) region. The remainder of the protein is substantially identical to native BPTI. The conformational properties of the analogue can explain several aspects of the kinetic role that the normal (14-38, 30-51) intermediate plays in the folding of BPTI.
Two-dimensional 1H nuclear magnetic resonance study of the (5-55) single-disulphide folding intermediate of bovine pancreatic trypsin inhibitor
Mierlo, Carlo P.M. van; Darby, Nigel J. ; Neuhaus, David ; Creighton, Thomas E. - \ 1991
Journal of Molecular Biology 222 (1991)2. - ISSN 0022-2836 - p. 373 - 390.
bovine pancreatic trypsin inhibitor (BPTI) - disulphide bonds - folding intermediate - n.m.r. - protein folding
An analogue of the bovine pancreatic trypsin inhibitor (BPTI) folding intermediate that contains only the disulphide bond between Cys5 and Cys55 has been prepared in Escherichia coli by protein engineering methods, with the other four Cys residues replaced by Ser. Two-dimensional 1H nuclear magnetic resonance studies of the analogue have resulted in essentially complete resonance assignments of the folded form of the protein. The folded protein has a compact conformation that is structurally very similar to that of native BPTI, although there are subtle differences and the folded conformation is not very stable. Approximately half of the protein molecules are unfolded at 3 °C, and this proportion increases at higher temperatures. The folded and unfolded conformations are in slow exchange. The conformational properties of the analogue can explain many aspects of the kinetic role that the normal (5-55) intermediate plays in the folding of BPTI.