Cellular Models and In Vitro Assays for the Screening of modulators of P-gp, MRP1 and BCRP.

Adenosine triphosphate (ATP)-binding cassette (ABC) transporters are highly expressed in tumor cells, as well as in organs involved in absorption and secretion processes, mediating the ATP-dependent efflux of compounds, both endogenous substances and xenobiotics, including drugs. Their expression and activity levels are modulated by the presence of inhibitors, inducers and/or activators. In vitro, ex vivo and in vivo studies with both known and newly synthesized P-glycoprotein (P-gp) inducers and/or activators have shown the usefulness of these transport mechanisms in reducing the systemic exposure and specific tissue access of potentially harmful compounds. This article focuses on the main ABC transporters involved in multidrug resistance [P-gp, multidrug resistance-associated protein 1 (MRP1) and breast cancer resistance protein (BCRP)] expressed in tissues of toxicological relevance, such as the blood-brain barrier, cardiovascular system, liver, kidney and intestine. Moreover, it provides a review of the available cellular models, in vitro and ex vivo assays for the screening and selection of safe and specific inducers and activators of these membrane transporters. The available cellular models and in vitro assays have been proposed as high throughput and low-cost alternatives to excessive animal testing, allowing the evaluation of a large number of compounds.

P-glycoprotein induction in Caco-2 cells by newly synthetized thioxanthones prevents paraquat cytotoxicity.

The induction of P-glycoprotein (P-gp), an ATP-dependent efflux pump, has been proposed as a strategy against the toxicity induced by P-gp substrates such as the herbicide paraquat (PQ). The aim of this study was to screen five newly synthetized thioxanthonic derivatives, a group known to interact with P-gp, as potential inducers of the pump's expression and/or activity and to evaluate whether they would afford protection against PQ-induced toxicity in Caco-2 cells. All five thioxanthones (20 µM) caused a significant increase in both P-gp expression and activity as evaluated by flow cytometry using the UIC2 antibody and rhodamine 123, respectively. Additionally, it was demonstrated that the tested compounds, when present only during the efflux of rhodamine 123, rapidly induced an activation of P-gp. The tested compounds also increased P-gp ATPase activity in MDR1-Sf9 membrane vesicles, indicating that all derivatives acted as P-gp substrates. PQ cytotoxicity was significantly reduced in the presence of four thioxanthone derivatives, and this protective effect was reversed upon incubation with a specific P-gp inhibitor. In silico studies showed that all the tested thioxanthones fitted onto a previously described three-feature P-gp induction pharmacophore. Moreover, in silico interactions between thioxanthones and P-gp in the presence of PQ suggested that a co-transport mechanism may be operating. Based on the in vitro activation results, a pharmacophore model for P-gp activation was built, which will be of further use in the screening for new P-gp activators. In conclusion, the study demonstrated the potential of the tested thioxanthonic compounds in protecting against toxic effects induced by P-gp substrates through P-gp induction and activation.

Induction and activation of P-glycoprotein by dihydroxylated xanthones protect against the cytotoxicity of the P-glycoprotein substrate paraquat.

Xanthones are a family of compounds with several known biological activities and therapeutic potential for which information on their interaction with membrane transporters is lacking. Knowing that P-glycoprotein (P-gp) acts as a cellular defense mechanism by effluxing its toxic substrates, the aim of this study was to investigate the potential of five dihydroxylated xanthones as inducers of P-gp expression and/or activity and to evaluate whether they could protect Caco-2 cells against the cytotoxicity induced by the toxic P-gp substrate paraquat (PQ). After 24 h of incubation, all tested xanthones caused a significant increase in both P-gp expression and activity, as evaluated by flow cytometry using the UIC2 antibody and rhodamine 123, respectively. Additionally, after a short 45-min incubation, all the tested xanthones induced a rapid increase in P-gp activity, indicating direct pump activation without increased P-gp protein expression. The tested compounds also increased P-gp ATPase activity in MDR1-Sf9 membrane vesicles, demonstrating to be P-gp substrates. Moreover, when simultaneously incubated with PQ, all xanthones significantly reduced the cytotoxicity of the herbicide, and these protective effects were completely reversed upon incubation with a specific P-gp inhibitor. In silico studies evaluating the interactions between xanthones and P-gp in the presence of PQ suggested that a co-transport mechanism may be operating. A quantitative structure-activity relationship model was developed and validated, and the maximal partial charge for an oxygen atom was the descriptor predicted as being implicated in P-gp activation by the dihydroxylated xanthones. These results disclose new perspectives in preventing PQ- and other P-gp substrates-induced poisonings.

Hb Plasencia [α125(H8)Leu→Arg (α2)] is a frequent cause of α+-thalassemia in the Portuguese population.

Hb Plasencia is a thalassemic hemoglobin (Hb) mutation caused by a leucine to arginine replacement at residue 125 of the α2-globin chain (HBA2:c.377T>G). This variant was first described in the heterozygous state in association with a very mild α-thalassemic phenotype in three members of a Spanish family from Plasencia, Western Spain. Reviewing the molecular characterization of 308 Portuguese individual suspected of having α-thalassemia (α-thal) we found Hb Plasencia to be the second most frequent mutation after the -α(3.7) deletion.

Hb Iberia [α104(G11)Cys → Arg,TGC>CGC (α2) (HBA2:c.313T>C)], a new α-thalassemic hemoglobin variant found in the Iberian Peninsula: report of six cases.

We report a new structural defect of the α2-globin chain presenting with moderate microcytic hypochromic anemia, in six individuals from three unrelated families, living in Portugal and Spain. α-Globin gene deletions were ruled out by gap-polymerase chain reaction (gap-PCR) and multiplex ligation-dependent probe amplification (MLPA). Direct sequencing of the α2-globin gene revealed a substitution of codon 104 [α104(G11)Cys→Arg, TGC>CGC (α2) (HBA2:c.313T>C)]. This new variant, not detectable by high performance liquid chromatography (HPLC) or electrophoresis, was called Hb Iberia, as it was observed for the first time in families from the Iberian Peninsula. Although the mutant allele is transcribed, as indicated by the balanced mRNA α/ß ratio, the abnormal α2 chain could not form a stable tetramer as the cysteine and arginine residues, located at the α1ß1 contact, differ in size, charge and hydrophobicity. Hb Iberia is the third mutation described at codon 104 on the α-globin genes, namely, Hb Sallanches (α2, TGC>TAC) and Hb Oegstgeest (α1, TGC>AGC), also characterized as unstable hemoglobins (Hbs), present on an α-thalassemic phenotype.