Vânia Vilas-Boas

P-glycoprotein activity in human Caucasian male lymphocytes does not follow its increased expression during aging.

P‐glycoprotein (P‐gp) is a transmembrane protein that mediates the efflux of innumerous structurally unrelated compounds. It was initially found over‐expressed in tumor cells, associated to a multidrug resistance phenotype (MDR). Then, P‐gp was found constitutively expressed in excretory cells/tissues and in circulating cells, such as lymphocytes. Considering the importance of this transporter in the establishment of therapeutic protocols and the existence of contradictory results, this study aimed at evaluating the influence of aging in the expression and function of P‐gp in human lymphocytes, comparing two different methodologies to assess both parameters. P‐gp activity and expression were evaluated in lymphocytes isolated from whole blood samples of 65 healthy caucasian male donors, divided into two groups according to age (group 1: under 30‐years old; group 2: above 60‐years old). P‐gp expression was assessed using the anti‐P‐gp monoclonal antibody, UIC2, in the presence and in absence of vinblastine (Vbl). P‐gp activity was evaluated measuring the efflux rate of the fluorescent P‐gp substrate rhodamine 123 (Rho 123) and also using UIC2 shift assay. Flow cytometric analysis was performed to assess all the proceedings. Furthermore, P‐gp expression and each of the P‐gp activity determination methods were compared, through correlation analysis and linear regression models. We observed a significant age‐dependent increase in mean P‐gp expression (p = 0.029), which was not reflected in the transporters activity (p > 0.050). Statistical analysis allowed selection of UIC2 shift assay over Rho 123 efflux assay as a more selective method to assess P‐gp activity. Despite the significant correlation between P‐gp expression and P‐gp activity found in lymphocytes (Gp1(group 1)—r = 0.609, p < 0.001; Gp2—r = 0.461, p = 0.012), using UIC2 shift assay, these data reinforce the need for P‐gp activity assessment, rather than P‐gp expression determination alone, when starting new therapeutic regimens with P‐gp substrates, especially in men older than 60 years of age.

The mixture of "ecstasy" and its metabolites is toxic to human SH-SY5Y differentiated cells at in vivo relevant concentrations.

The neurotoxicity of “ecstasy” (3,4-methylenedioxymethamphetamine, MDMA) is thought to involve hepatic metabolism, though its real contribution is not completely understood. Most in vitro neurotoxicity studies concern isolated exposures of MDMA or its metabolites, at high concentrations, not considering their mixture, as expected in vivo. Therefore, our postulate is that combined deleterious effects of MDMA and its metabolites, at low micromolar concentrations that may be attained into the brain, may elicit neurotoxicity. Using human SH-SY5Y differentiated cells as dopaminergic neuronal model, we studied the neurotoxicity of MDMA and its MDMA metabolites α-methyldopamine and N-methyl-α-methyldopamine and their correspondent glutathione and N-acetylcysteine monoconjugates, under isolated exposure and as a mixture, at normothermic or hyperthermic conditions. The results showed that the mixture of MDMA and its metabolites was toxic to SH-SY5Y differentiated cells, an effect potentiated by hyperthermia and prevented by N-acetylcysteine. As a mixture, MDMA and its metabolites presented a different toxicity profile, compared to each compound alone, even at equimolar concentrations. Caspase 3 activation, increased reactive oxygen species production, and intracellular Ca2+ raises were implicated in the toxic effect. The mixture increased intracellular glutathione levels by increasing its de novo synthesis. In conclusion, this study demonstrated, for the first time, that the mixture of MDMA and its metabolites, at low micromolar concentrations, which represents a more realistic approach of the in vivo scenario, elicited toxicity to human SH-SY5Y differentiated cells, thus constituting a new insight into the context of MDMA-related neurotoxicity.

Performance enhanced UV/vis spectroscopic microfluidic sensor for ascorbic acid quantification in human blood.

Quantitative analysis of antioxidants in a fast, simple and accurate manner is of great importance in the view of real-time monitoring the health of individuals. Recently, we have developed a UV/vis spectroscopic microfluidic sensor to specifically quantify ascorbic acid based on the immobilization of ascorbate oxidase, a relatively unstable enzyme. In this work, three different strategies for the immobilization of the unstable enzyme, including alumina sol-gel encapsulation, physisorption to PDMS channels with, and without alumina xerogel modification, were compared to build a microsensor. We found that the loading amount of the enzyme is not the determinative factor for the performance of the microfluidic biosensor but the retained activity of the enzyme and diffusion in the microfluidic channel. Taking into account of the two factors, the protocol of adsorbing enzymes to alumina (Al2O3) xerogel modified PDMS surface was demonstrated to be the best for preparing the microfluidic sensor among the utilized protocols. The microsensor prepared under the optimized protocol was further used to quantify ascorbic acid in human blood, where only dozens of microliters of blood (few drops) was required, demonstrating its potential application in clinical diagnosis. The developed strategy is featured with optimized enzymatic activity, simple process of microfluidic platform, low sample consumption, and straightforward spectrophotometry based detection.

Mechanisms of P-gp inhibition and effects on membrane fluidity of a new rifampicin derivative, 1,8-dibenzoyl-rifampicin.

PURPOSE To assess P-glycoprotein (P-gp)-modulation ability and the mechanisms of P-gp inhibition mediated by a new synthetic rifampicin derivative, 1,8-dibenzoyl-rifampicin (DiBenzRif), in an in vitro model of the blood-brain barrier (BBB), RBE4 cells, and in membrane mimetic models (liposomes). METHODS P-gp expression (western blot) and activity [rhodamine 123 accumulation studies] were assessed until 72h of exposure to DiBenzRif. The effects on intracellular ATP levels and on P-gp ATPase activity were studied using luciferin-luciferase bioluminescence assay. Membrane fluidity changes were tracked by steady-state anisotropy measurements. Non-P-gp-related rhodamine 123 accumulation was evaluated using liposomes prepared with the main lipids present in RBE4 cell membranes. RESULTS A significant increase in intracellular rhodamine 123 content was observed in DiBenzRif-treated cells at all tested time-points. This effect was associated with a significant reduction in ATP intracellular levels, the inhibition of P-gp ATPase activity and a significant increase in membrane fluidity. DiBenzRif also favoured rhodamine 123 accumulation in a liposomal model of RBE4 cells, suggesting that it may be useful in increasing intracellular levels of substances that passively diffuse into the cells. CONCLUSION DiBenzRif-induced inhibitory effect on P-gp increases xenobiotic accumulation in BBB cells, which may contribute to the development of therapeutic adjuvants to enhance brain penetration of drugs.

Doxorubicin decreases paraquat accumulation and toxicity in Caco-2 cells

P-glycoprotein (P-gp) is an efflux pump belonging to the ATP-binding cassette transporter superfamily expressed in several organs. Considering its potential protective effects, the induction of de novo synthesis of P-gp could be used therapeutically in the treatment of intoxications by its substrates. The herbicide paraquat (PQ) is a P-gp substrate responsible for thousands of fatal intoxications worldwide that still lacks an effective antidote. The aim of the present work was to evaluate the effectiveness of such an antidote by testing whether doxorubicin (DOX), a known P-gp inducer, could efficiently protect Caco-2 cells against PQ cytotoxicity, 6 h after the incubation with the herbicide, reflecting a real-life intoxication scenario. Cytotoxicity was evaluated by the MTT assay and PQ intracellular concentrations were measured by gas chromatography-ion trap-mass spectrometry (GC-IT-MS). Also, the DOX modulatory effect on choline uptake transport system was assessed by measuring the uptake of [³H]-choline. The results show that DOX exerts protective effects against PQ cytotoxicity, preventing the intracellular accumulation of the herbicide. These protective effects were not completely prevented by the incubation with the UIC2 antibody, a specific P-gp inhibitor, suggesting the involvement of alternative protection mechanisms. In fact, DOX also efficiently inhibited the choline transport system that influences PQ cellular uptake. In conclusion, in this cellular model, DOX effectively protects against PQ toxicity by inducing P-gp and through the interaction with the choline transporter, suggesting that compounds presenting this double feature of promoting the efflux and limiting the uptake of PQ could be used as effective antidotes to treat intoxications.

Colchicine effect on P-glycoprotein expression and activity: In silico and in vitro studies

Colchicine is a P-glycoprotein (P-gp) substrate that induces its expression, thus increasing the risk for unexpected pharmacokinetic interactions with this drug. Because increased P-gp expression does not always correlate with increased activity of this efflux pump, we evaluated the changes in both P-gp expression and activity induced by colchicine using an in vitro model. Caco-2 cells were incubated with 0.1-100 μM colchicine up to 96 h. Cytotoxicity was evaluated by the MTT and LDH leakage assays, P-gp expression and activity were evaluated by flow cytometry and P-gp ATPase activity was measured in MDR1-Sf9 membrane vesicles. Furthermore, colchicine fitting in P-gp induction and competitive inhibition pharmacophore hypothesis, and docking studies evaluating the interaction between colchicine and P-gp drug binding pocket were tested in silico. Significant cytotoxicity was noted after 48 h. At 24 h a significant increase in P-gp expression was observed, which was not accompanied by an increase in transport activity. Moreover, colchicine significantly increased P-gp ATPase activity, demonstrating to be actively transported by the pump. New pharmacophores were constructed to predict P-gp modulatory activity. Colchicine fitted both the P-gp induction and competitive inhibition models. In silico, colchicine was predicted to bind to the P-gp drug-binding pocket suggesting a competitive mechanism of transport. These results show that colchicine induced P-gp expression in Caco-2 cells but the activity of the protein remained unchanged, highlighting the need to simultaneously evaluate P-gp expression and activity. With the newly constructed pharmacophores, new drugs can be initially screened in silico to predict such potential pharmacokinetic interactions.

Development of novel rifampicin-derived P-glycoprotein activators/inducers. synthesis, in silico analysis and application in the RBE4 cell model, using paraquat as substrate.

P-glycoprotein (P-gp) is a 170 kDa transmembrane protein involved in the outward transport of many structurally unrelated substrates. P-gp activation/induction may function as an antidotal pathway to prevent the cytotoxicity of these substrates. In the present study we aimed at testing rifampicin (Rif) and three newly synthesized Rif derivatives (a mono-methoxylated derivative, MeORif, a peracetylated derivative, PerAcRif, and a reduced derivative, RedRif) to establish their ability to modulate P-gp expression and activity in a cellular model of the rat’s blood–brain barrier, the RBE4 cell line P-gp expression was assessed by western blot using C219 anti-P-gp antibody. P-gp function was evaluated by flow cytometry measuring the accumulation of rhodamine123. Whenever P-gp activation/induction ability was detected in a tested compound, its antidotal effect was further tested using paraquat as cytotoxicity model. Interactions between Rif or its derivatives and P-gp were also investigated by computational analysis. Rif led to a significant increase in P-gp expression at 72 h and RedRif significantly increased both P-gp expression and activity. No significant differences were observed for the other derivatives. Pre- or simultaneous treatment with RedRif protected cells against paraquat-induced cytotoxicity, an effect reverted by GF120918, a P-gp inhibitor, corroborating the observed P-gp activation ability. Interaction of RedRif with P-gp drug-binding pocket was consistent with an activation mechanism of action, which was confirmed with docking studies. Therefore, RedRif protection against paraquat-induced cytotoxicity in RBE4 cells, through P-gp activation/induction, suggests that it may be useful as an antidote for cytotoxic substrates of P-gp.

RBE4 cells are highly resistant to paraquat-induced cytotoxicity: studies on uptake and efflux mechanisms

Paraquat (PQ) is a widely used, highly toxic and non‐selective contact herbicide, which has been associated with central neurotoxic effects, namely the development of Parkinsons disease, but whose effects to the blood–brain barrier (BBB) itself have rarely been studied. This work studied the mechanisms of PQ uptake and efflux in a rats BBB cell model, the RBE4 cells. PQ is believed to enter cells using the basic or neutral amino acid or polyamine transport systems or through the choline‐uptake system. In contrast, PQ efflux from cells is reported to be mediated by P‐glycoprotein. Therefore, we evaluated PQ‐induced cytotoxicity and the effect of some substrates/blockers of these transporters (such as arginine, L‐valine, putrescine, hemicholinium‐3 and GF120918) on such cytotoxicity. RBE4 cells were shown to be extremely resistant to PQ after 24 h of exposure; even at concentrations as high as 50 mM approximately 45% of the cells remained viable. Prolonging exposure until 48 h elicited significant cytotoxicity only for PQ concentrations above 5 mM. Although hemicholinium‐3, a choline‐uptake system inhibitor, significantly protected cells against PQ‐induced toxicity, none of the effects were observed for arginine, L‐valine or putrescine. Meanwhile, inhibiting the efflux pump P‐glycoprotein using GF120918 significantly enhanced PQ‐induced cytotoxicity. In conclusion, PQ used the choline‐uptake system, instead of the transporters for the basic or neutral amino acids or for the polyamines, to enter RBE4 cells. P‐glycoprotein extrudes PQ back to the extracellular medium. However, this efflux mechanism only partially explains the observed RBE4 resistance to PQ. Copyright

Several transport systems contribute to the intestinal uptake of Paraquat, modulating its cytotoxic effects.

Paraquat (PQ) is an extremely toxic herbicide upon oral ingestion that lacks a specific antidote. In case of intoxication, treatment primarily relies on limiting its intestinal absorption. In this study, we elucidate the intestinal transport mechanisms of PQ uptake using Caco-2 cells as a model of the human intestinal epithelium. The cells were incubated with a wide range of PQ concentrations (0-5000μM) for 24h with or without simultaneous exposure to different transporters substrates/inhibitors including, choline or hemicolinium-3 (for choline carrier-mediated transport system inhibition) and putrescine, trifluoperazine, valine, lysine, arginine or N-ethylmaleimide (for basic amino acid transport systems inhibition). PQ cytotoxicity was evaluated by the MTT reduction assay and correlated with PQ intracellular levels quantified by gas chromatography-ion trap-mass spectrometry (GC-IT/MS). Potential interactions of PQ with the substrates/inhibitors of the transport systems were investigated and discarded by infrared spectroscopy. Our results showed a significant reduction in PQ intracellular accumulation and, consequently, in PQ cytotoxicity, in the presence of both choline and hemicolinium-3, demonstrating that the choline carrier-mediated transport system is partially involved in PQ intestinal uptake. Likewise, PQ cytotoxicity and intracellular accumulation were significantly attenuated by simultaneous exposure to putrescine, trifluoperazine, valine, lysine, arginine and N-ethylmaleimide. These data suggested the involvement of more than one of the basic amino acids transport systems, including the y(+), b(0,+) or y(+)L systems. In conclusion, this study demonstrated that several transport systems mediate PQ intestinal absorption and, therefore, their modulation may provide alternative efficient pathways for limiting PQ toxicity in intoxication scenarios.

Combining CXCR4-targeted and nontargeted nanoparticles for effective unassisted in vitro magnetic hyperthermia

The use of targeted nanoparticles for magnetic hyperthermia (MHT) increases MHT selectivity, but often at the expense of its effectiveness. Consequently, targeted MHT is typically used in combination with other treatment modalities. This work describes an implementation of a highly effective monotherapeutic in vitro MHT treatment based on two populations of magnetic particles. Cells were sequentially incubated with two populations of magnetic particles: nonfunctionalized superparamagnetic nanoparticles and anti-CXCR4-functionalized particles. After removing the excess of free particles, an alternating magnetic field (AMF) was applied to produce MHT. The induced cytotoxicity was assessed at different time-points after AMF application. Complete loss of cell viability was observed 72 h after MHT when the iron loading of the anti-CXCR4-functionalized particles was boosted by that of a nontargeted population. Additionally, induction of necrosis resulted in more efficient cell death than did induction of apoptosis. Achieving a uniquely high effectiveness in monotherapeutic MHT demonstrates the potential of this approach to achieve complete loss of viability of cancer cells while avoiding the side effects of dual-treatment strategies that use MHT only as a sensitizing therapy.