Exposure to Sublethal Concentrations of Benzalkonium Chloride Induces Antimicrobial Resistance and Cellular Changes in Klebsiellae pneumoniae Clinical Isolates.

Benzalkonium chloride (BAC) is widely used as a disinfectant and preservative. This study investigated the effect on antimicrobial susceptibility and the cellular changes that occurred after exposure of Klebsiella pneumoniae clinical isolates to sublethal concentrations of BAC. Minimum inhibitory concentration and minimum bactericidal concentration of BAC were determined for the collected 50 K. pneumoniae clinical isolates by broth microdilution method, and the tested isolates were adapted to increasing sublethal concentrations of BAC. The effect of adaptation on MICs of the tested 16 antimicrobial agents, the cell ultrastructure, efflux, and membrane depolarization of the tested isolates were examined. Interestingly, most K. pneumoniae isolates that adapted to BAC showed increased antimicrobial resistance, various morphological and structural changes, increased membrane depolarization, and enhanced efflux activity. The findings of this study suggest that the extensive use of BAC at sublethal concentrations could contribute to the emergence of antibiotic resistance in K. pneumoniae clinical isolates that might complicate the therapy of infections caused by this pathogen. In conclusion, the hazard associated with the prolonged exposure to sublethal concentrations of BAC represents a public health risk and therefore it should be a focus in both hospital and community sanitation practices.

Modulation of Staphylococcus aureus Response to Antimicrobials by the Candida albicans Quorum Sensing Molecule Farnesol.

In microbial biofilms, microorganisms utilize secreted signaling chemical molecules to coordinate their collective behavior. Farnesol is a quorum sensing molecule secreted by the fungal species Candida albicans and shown to play a central physiological role during fungal biofilm growth. Our pervious in vitro and in vivo studies characterized an intricate interaction between C. albicans and the bacterial pathogen Staphylococcus aureus, as these species coexist in biofilm. In this study, we aimed to investigate the impact of farnesol on S. aureus survival, biofilm formation, and response to antimicrobials. The results demonstrated that in the presence of exogenously supplemented farnesol or farnesol secreted by C. albicans in biofilm, S. aureus exhibited significantly enhanced tolerance to antimicrobials. By using gene expression studies, S. aureus mutant strains, and chemical inhibitors, the mechanism for the enhanced tolerance was attributed to upregulation of drug efflux pumps. Importantly, we showed that sequential exposure of S. aureus to farnesol generated a phenotype of high resistance to antimicrobials. Based on the presence of intracellular reactive oxygen species upon farnesol exposure, we hypothesize that antimicrobial tolerance in S. aureus may be mediated by farnesol-induced oxidative stress triggering the upregulation of efflux pumps, as part of a general stress response system. Hence, in mixed biofilms, C. albicans may influence the pathogenicity of S. aureus through acquisition of a drug-tolerant phenotype, with important therapeutic implications. Understanding interspecies signaling in polymicrobial biofilms and the specific drug resistance responses to secreted molecules may lead to the identification of novel targets for drug development.

Rifampicin-induced injury in HepG2 cells is alleviated by TUDCA via increasing bile acid transporters expression and enhancing the Nrf2-mediated adaptive response.

Bile acid transporters and the nuclear factor erythroid 2-related factor (Nrf-2)-mediated adaptive response play important roles in the development of drug-induced liver injury (DILI). However, little is known about the contribution of the adaptive response to rifampicin (RFP)-induced cell injury. In this study, we found RFP decreased the survival rate of HepG2 cells and increased the levels of lactate dehydrogenase (LDH), alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (AKP), γ-glutamyl-transferase (γ-GT), total bilirubin (TBIL), direct bilirubin (DBIL), indirect bilirubin (IBIL), total bile acid (TBA) and adenosine triphosphate (ATP) in the cell culture supernatants in both a concentration- and a time-dependent manner. RFP increased the expression levels of bile acid transporter proteins and mRNAs, such as bile salt export pump (BSEP), multidrug resistance protein 1 (MDR1), multidrug resistance-associated protein 2 (MRP2), Na+/taurocholate cotransporter (NTCP), organic anion transporting protein 2 (OATP2), organic solute transporter ß (OSTß) and Nrf2. Following the transient knockdown of Nrf2 and treatment with RFP, the expression levels of the BSEP, MDR1, MRP2, NTCP, OATP2 and OSTß proteins and mRNAs were decreased to different degrees. Moreover, the cell survival was decreased, whereas the LDH level in the cell culture supernatant was increased. Overexpression of the Nrf2 gene produced the opposite effects. Treatment with tauroursodeoxycholic acid (TUDCA) increased the expression levels of the bile acid transporters and Nrf2, decreased the expression levels of glucose-regulated protein 78 (GRP78), PKR-like ER kinase (PERK), activating transcription factor 4 (ATF4), and C/EBP-homologous protein (CHOP), and inhibited RFP-induced oxidative stress. Moreover, TUDCA reduced cell apoptosis, increased cell survival and decreased the levels of LDH, ALT, AST, AKP, γ-GT, TBIL, DBIL, IBIL, TBA and ATP in the cell culture supernatant. Therefore, TUDCA alleviates RFP-induced injury in HepG2 cells by enhancing bile acid transporters expression and the Nrf2-mediated adaptive response.

Pyrrolopyrimidine derivatives and purine analogs as novel activators of Multidrug Resistance-associated Protein 1 (MRP1, ABCC1).

Multidrug resistance (MDR) is the main cause of diminished success in cancer chemotherapy. ABC transport proteins are considered to be one important factor of MDR. Besides P-glycoprotein (P-gp, ABCB1) and Breast Cancer Resistance Protein (BCRP, ABCG2), Multidrug Resistance-associated Protein 1 (MRP1, ABCC1) is associated with non-response to chemotherapy in different cancers. While considerable effort was spent in overcoming MDR during the last two decades, almost nothing is known with respect to activators of transport proteins. In this work we present certain pyrrolo[3,2-d]pyrimidine derivatives with variations at positions 4 and 5 and purine analogs with variations at position 6 as novel activators of MRP1-mediated transport of the MRP1 substrate calcein AM and the anticancer drug daunorubicin in low nanomolar concentration range. Two different MRP1 overexpressing cell lines were used, the doxorubicin-selected human lung cancer cell line H69 AR and the transfected Madin-Darby Canine Kidney cell line MDCK II MRP1. No effect was observed in the sensitive counterparts H69 and MDCK II wild type (wt). Derivatives with higher molecular weight possessed also inhibitory properties at low micromolar concentrations, although most compounds were rather poor MRP1 inhibitors. Purine analogs derived from potent MRP1 inhibitors of the pyrrolopyrimidine class showed equal activating, but no inhibiting effects at all. All tested compounds were non-toxic and had only minor impact on P-gp or BCRP, showing no inhibition or activation.

Gibberellin derivative GA-13315 sensitizes multidrug-resistant cancer cells by antagonizing ABCB1 while agonizes ABCC1.

PURPOSE: GA-13315 is a gibberellin derivative that reveals antitumor and antineoplastic effects both in vitro and in vivo. In the present study, the chemosensitizing effects of GA-13315 in multidrug-resistant cell lines were examined and the underlying mechanisms were investigated. METHODS: Cytotoxicity and chemosensitizing effects of GA-13315 were determined by MTT assay. Function of ABC transporter was analyzed by measuring intracellular drug accumulation of doxorubicin and rhodamine 123 and by determining the ATPase activity of ABC transporter. Expression levels of apoptosis regulators were analyzed using real-time quantitative PCR and Western blot. RESULTS: GA-13315 selectively killed MCF-7/adr cells that overexpress P-glycoprotein (ABCB1) over the parent MCF-7 cells. In combination with conventional chemotherapeutic agents, GA-13315 at sub-toxic concentrations reversed the multidrug resistance mediated by ABCB1 but exacerbated the resistance conferred by multidrug resistance-associated protein 1 (ABCC1). GA-13315 increased intracellular accumulation of doxorubicin and rhodamine 123 in MCF-7/adr cells and in ABCB1-transfected HEK293 cells but facilitated drug flush-out from cells that overexpress ABCC1. GA-13315 inhibited the ATPase activity of ABCB1 while stimulated that of ABCC1. Moreover, the downregulated expression of Bax in MCF-7/adr cells was restored by GA-13315 markedly. CONCLUSION: These data suggest that GA-13315 sensitizes multidrug-resistant cells at least partially by impeding the efflux function of ABCB1. The upregulation of Bax by GA-13315 may also contribute to the sensitizing action. The opposite effects of GA-13315 on different ATP-binding cassette transporters and their implications in overcoming drug resistance require further investigation.

Oleanolic acid attenuates obstructive cholestasis in bile duct-ligated mice, possibly via activation of NRF2-MRPs and FXR antagonism.

Obstructive cholestasis is characterized by impairment of hepatic canalicular bile efflux and there are no clinically effective drugs to cure except surgeries. Previously we revealed that oleanolic acid (OA) protected against lithocholic acid (LCA)-induced intrahepatic cholestasis in mice. Cholestasis caused by LCA is characterized by segmental bile duct obstruction, whether OA possesses the beneficial effect on completed obstructive cholestasis induced by bile duct ligation (BDL) remains unknown. In this study, we demonstrated that BDL-induced mice liver pathological change, and increase in serum levels of ALT, AST and ALP were all significantly reduced by OA (20 mg/kg, i.p.). Meanwhile, OA also lowered total bilirubin and total bile acids levels in serum, as well as total bile acids level in liver, in contrast, urinary total bile acids output was remarkably up-regulated by OA. Gene expression analysis showed that OA caused significant increased mRNA expression of MRP3 and MRP4 located at hepatic basolateral membrane, and restoration of MRP2 and BSEP located at hepatic cannalicular membrane. Furthermore, significant NRF2 protein accumulation in nucleus was also observed in OA treated mice. In mice primary cultured hepatocytes, the effects of OA on MRP2, MRP3 and MRP4 expression were directly proved to be mediated via NRF2 activation, and BSEP downregulation induced by OA was in part due to FXR antagonism. Luciferase assay performed in Hep G2 cells also illustrated that OA was a partial FXR antagonist. Taken together, we conclude that OA attenuates obstructive cholestasis in BDL mice, possibly via activation of NRF2-MRPs and FXR antagonism.

Exploring the structure-activity relationships of ABCC2 modulators using a screening approach.

ABCC2 is a transporter with key influence on liver and kidney pharmacokinetics. In order to explore the structure-activity relationships of compounds that modulate ABCC2, and by doing so gain insights into drug-drug interactions, we screened a library of 432 compounds for modulators of radiolabeled ß-estradiol 17-(ß-d-glucuronide) (EG) and fluorescent 5(6)-carboxy-2',7'-dichlorofluorescein transport (CDCF) in membrane vesicles. Following the primary screen at 80µM, dose-response curves were used to investigate in detail 86 compounds, identifying 16 low µM inhibitors and providing data about the structure-activity relationships in four series containing 19, 24, 10, and eight analogues. Measurements with the CDCF probe were consistently more robust than for the EG probe. Only one compound was clearly probe-selective with a 50-fold difference in the IC50s obtained by the two assays. We built 24 classification models using the SVM and fused-XY Kohonen methods, revealing molecular descriptors related to number of rings, solubility and lipophilicity as important to distinguish inhibitors from inactive compounds. This study is to the best of our knowledge the first to provide details about structure-activity relationships in ABCC2 modulation.

Collateral sensitivity of resistant MRP1-overexpressing cells to flavonoids and derivatives through GSH efflux.

The multidrug resistance protein 1 (MRP1) is involved in multidrug resistance of cancer cells by mediating drug efflux out of cells, often in co-transport with glutathione (GSH). GSH efflux mediated by MRP1 can be stimulated by verapamil. In cells overexpressing MRP1, we have previously shown that verapamil induced a huge intracellular GSH depletion which triggered apoptosis of the cells. That phenomenon takes place in the more global anticancer strategy called "collateral sensitivity" and could be exploited to eradicate some chemoresistant cancer cells. Seeking alternative compounds to verapamil, we screened a library of natural flavonoids and synthetic derivatives. A large number of these compounds stimulate MRP1-mediated GSH efflux and the most active ones have been evaluated for their cytotoxic effect on MRP1-overexpressing cells versus parental cells. Interestingly, some are highly and selectively cytotoxic for MRP1-cells, leading them to apoptosis. However, some others do not exhibit any cytotoxicity while promoting a strong GSH efflux, indicating that GSH efflux is necessary but not sufficient for MRP1-cells apoptosis. In support to this hypothesis, structure activity relationships show that the absence of a hydroxyl group at position 3 of the flavonoid C ring is an absolute requirement for induction of MRP1-cells death, but is not for GSH efflux stimulation. Chrysin (compound 8) and its derivatives, compounds 11 and 22, exhibit a high selectivity toward MRP1-cells with a IC50 value of 4.1 µM for compound 11 and 4.9 µM for chrysin and compound 22, making them among the best described selective killer compounds of multidrug ABC transporter-overexpressing cells.

Impact of probe compound in MRP2 vesicular transport assays.

MRP2 is an efflux transporter that is expressed mainly in the canalicular membrane of hepatocytes, where it expels polar and ionic compounds into the bile. MRP2 is also present in the apical membrane of enterocytes and epithelial cells of proximal tubules of the kidney. Inhibition of MRP2 transport can lead to the accumulation of metabolites and other MRP2 substrates up to toxic levels in these cells. The transport properties of MRP2 are frequently studied with the vesicular transport assay. The assay identifies compounds that interact with MRP2 by measuring the effect of a compound on the transport of a radioactively labeled or fluorescent probe. We have compared the effect of eight selected test compounds (quercetin, disopyramide, paracetamol, indomethacin, diclofenac, estrone-3-sulfate, budesonide, and thioridazine) on the MRP2-mediated transport of three commonly used probes: 5(6)-carboxy-2,7-dichlorofluorescein, leukotriene C4 and estradiol-17-ß-d-glucuronide (E217ßG). Five of the test compounds had different probe-dependent effects on the MRP2-mediated transport, suggesting differences in the transport mechanism of the probes. Our results underline the complexity of substrate recognition by these efflux transporters and the difficulties in directly comparing results obtained with different assays, especially when different probes are used.

The role of turmerones on curcumin transportation and P-glycoprotein activities in intestinal Caco-2 cells.

The rhizome of Curcuma longa (turmeric) is often used in Asia as a spice and as a medicine. Its most well-studied component, curcumin, has been shown to exhibit poor bioavailability in animal studies and clinical trials. We hypothesized that the presence of lipophilic components (e.g., turmerones) in turmeric extract would affect the absorption of curcumin. The effects of turmerones on curcumin transport were evaluated in human intestinal epithelial Caco-2 cells. The roles of turmerones on P-glycoprotein (P-gp) activities and mRNA expression were also evaluated. Results showed that in the presence of α- and aromatic turmerones, the amount of curcumin transported into the Caco-2 cells in 2 hours was significantly increased. α-Turmerone and verapamil (a P-gp inhibitor) significantly inhibited the efflux of rhodamine-123 and digoxin (i.e., inhibited the activity of P-gp). It is interesting that aromatic turmerone significantly increased the rhodamine-123 efflux and P-gp (MDR1 gene) mRNA expression levels. The effects of α- and aromatic turmerones on curcumin transport as well as P-gp activities were shown here for the first time. The presence of turmerones did affect the absorption of curcumin in vitro. These findings suggest the potential use of turmeric extract (including curcumin and turmerones), rather than curcumin alone, for treating diseases.

Rapid, nongenomic stimulation of multidrug resistance protein 2 (Mrp2) activity by glucocorticoids in renal proximal tubule.

In renal proximal tubule, multidrug resistance protein 2 (Mrp2) actively transports many organic anions into urine, including drugs and metabolic wastes. Upon exposure to nephrotoxicants or during endotoxemia, both Mrp2 activity and expression are up-regulated. This may result from induced de novo synthesis of Mrp2 or post-transcriptional events involving specific signaling pathways. Here, we investigated glucocorticoid signaling to Mrp2 in killifish renal proximal tubules, a model system in which transport activity can be measured using a fluorescent substrate and confocal imaging. Exposure of tubules to dexamethasone rapidly increased Mrp2-mediated fluorescein methotrexate transport. Other glucocorticoid receptor (GR) ligands, cortisol and triamcinolone acetonide, also stimulated Mrp2-mediated transport. The GR antagonist, mifepristone 17ß-hydroxy-11ß-[4-dimethylamino phenyl]-17α-[1-propynyl]estra-4,9-dien-3-one (RU486), abolished stimulation by all three ligands, whereas the mineralocorticoid receptor antagonist, spironolactone, was ineffective. Consistent with action through a nongenomic mechanism, dexamethasone stimulation of Mrp2-mediated transport was insensitive to cycloheximide and actinomycin D, and immunohistochemistry revealed no alterations in Mrp2 expression at the luminal membrane. (9S-(9α,10ß,12α))-2,3,9,10,11,12-hexahydro-10-hydroxy-10-(methoxycarbonyl)-9-methyl-9,12-epoxy-1H-diindolo[1,2,3-fg:3',2',1'-kl]pyrrolo[3,4-i][1,6]benzodiazocin-1-one (K252a), an inhibitor of the tyrosine receptor kinase subfamily, reduced the dexamethasone effect, as did the specific hepatocyte growth factor receptor (c-Met) tyrosine kinase inhibitor, (2R)-1-[[5-[(Z)-[5-[[(2,6-dichlorophenyl)methyl]sulfonyl]-1,2-dihydro-2-oxo-3H-indol-3-ylidene]methyl]-2,4-dimethyl-1H-pyrrol-3-yl]carbonyl]-2-(1-pyrrolidinylmethyl)pyrrolidine (PHA-665752). Hepatocyte growth factor (HGF), an endogenous ligand for c-Met, stimulated Mrp2-mediated transport. This effect was reversed by PHA-665752 but not by RU486. Inhibition of mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK 1/2) also abolished the effects of dexamethasone and HGF. Our results disclose a novel mechanism by which glucocorticoids acting through GR, c-Met, and MEK1/2 cause rapid, nongenomic stimulation of Mrp2-mediated transport in renal proximal tubules. This up-regulation may be nephroprotective, enhancing efflux of metabolic wastes and toxicants during cell and tissue stress.

Influence of luminal monosaccharides on secretion of glutathione conjugates from rat small intestine in vitro.

Intestinal efflux transporters can significantly reduce the absorption of the drug after peroral application. In this work we studied secretion of glutathione conjugates triggered by glucose at the luminal side of the intestine. Glucose stimulated secretion of DNPSG, NEMSG and CDNB. We used some different monosaccharides and determined that glucose, galactose and alpha-methylglucopyranoside trigger the secretion, while mannitol and fructose do not. We concluded that interaction with SGLT transporter is the key process necessary for this triggering. To determine which of possible glutathione conjugate transporters (MRP2, MRP4, BCRP or RLIP76) is responsible for the secretion of glutathione conjugates, we used benzbromarone, a MRP inhibitor, and sulfanitran and furosemide, two allosteric MRP2 activators. Benzbromarone inhibited glucose stimulated DNPSG secretion, while allosteric activators additionally increased the secretion. We concluded that MRP2 transporter is related to glucose stimulated DNPSG secretion. Regarding the work of Kubitz et al. we tested the effect of changed medium osmolarity on DNPSG transport and determined that hypoosmolar conditions trigger secretion of DNPSG. These findings suggest that intestinal MRP2 activity has no basal level, but can be stimulated by hypoosmolarity and SGLT transport.