Modelli sperimentali cellulari e molecolari per la valutazione della tossicità di xenobiotici

Abstract

The aim of my thesis was to find two different experimental models to study cellular and molecular xenobiotics toxicity. In the first part of the present work we studied the interaction between two different plasma membrane transporters (OCTN2 and OCTN1), mercury reagents and heavy metals. Mercury and heavy metals in general cause toxic effects in many tissues interacting with protein cysteine (Cys) thiols. Transport systems represent critical targets of mercurials. Indeed, the majority of transport systems of higher eukaryotes containseveral Cys residues. One of the most up to date method of studying transport is the reconstitution of transportersin proteoliposomes. This method has been used as a useful approach to test the effect of HgCl2, methylmercury(MeHg) and Cadmium on the carnitine (OCTN2) transporter, extracted from rat kidney brush border membranes and reconstituted in liposomes by removing the detergent withhydrophobic chromatography columns, and the human organic cation transporter (OCTN1) overexpressed in E. coli, purified by Ni-chelating chromatography and reconstituted in liposomes by detergent removal with a batch-wise procedure.Transport was measured as [3H]carnitine uptake into proteoliposomescontaining carnitine (antiport reaction) in the case of rat OCTN2 and as [14C]tetraethylammonium uptake in the case of human OCTN1. Mercurials and heavy metals strongly inhibited the transport. Inhibition was reversed by1,4-dithioerythritol (DTE), L-cysteine (Cys), and N-acetyl-Lcysteine (NAC) indicating that it was caused by covalent reactionof mercurials and heavy metals with Cys residue(s) of OCTN2 and OCTN1.The presence of substrate prevented the inhibition in rat OCTN2 transporter indicating that the mercurial binding residue (Cys) is in the substrate binding site. No substrate protection was found in the case of the human OCTN1, so probably mercurial and heavy metal binding residue is away from substrate binding site. For the human OCTN1 we also tested the effect of chemical reagents which are known to form mixeddisulphides with proteins SH residues, MTS reagents. MTSEA exerted inhibition of transport very similar to those observed for the heavy metals and as the toxic compounds. To ascertain the involvement of Cys residues in the interaction of the human OCTN1 with the xenobiotics and to identify the possible target of the reagents, 7 mutants were prepared in which the seven cysteines present in the transmembrane domains or in the extracellular loop were mutated to alanine. An additional mutant lacking two Cys residues has also been constructed (C50A/C136A). We studies the dose-response curves of the transporter for each inhibitor, the mercury reagents showed similar behavior, both HgCl2 and MeHg strongly inhibited the WT and the mutants C81A, C113A, C236A, C270A and C374A. While a clear shift of the curves towards higher concentrations of the compounds was observed in the case of mutants C50A and C136A indicating a decrease of affinity of these mutants for the mercury reagents. The experiment on the double mutant C50A/C136A showed a nearly complete lack of inhibition by the two reagents demonstrating the two Cys residues were indeed the target of the mercury compounds. The homology model of the human OCTN1 confirms the experimental data obtained in this work, in fact the model shows that the two cysteine residues (Cys-50 and Cys-136) are exposed to the extracellular site of the plasma membrane and are accessible to sulfhydryl groups reagents. In the present work we studied, also, biomarkers expression and nephrotoxic effect induced by drugs in human kidney primary cells model. Kidney is a primary target of drug-induced toxicity. Toxic effects on the kidney related to drugs are both common and expected, given the kidney's roles in plasma filtration and maintenance of metabolic homeostasis. As such, glomerular, tubular and renal interstitial cells frequently are exposed to concentrations of drugs, which can induce changes in kidney function and structure. We tested three different drugs: 2-Bromoethanamine an analgesic, cisplatin a chemotherapeutic agent and cyclosporine a immunosuppressive agent. We found that after the addition of these drugs on human primary kidney cells there is an increase in the expression of two different biomarkers: Osteopontin (OPN) a distal tubule biomarker and KIM-1 a membrane glycoprotein expressed by proximal tubule cells after kidney injury. We also found the toxic concentrations after 24 hours of exposition to the three different drugs, we quantified the percentual of necrotic and apoptotic cells and we studied the toxicological effect of these drugs on cellular organelles like mitochondria. In conclusion proteoliposomes represent a suitable molecular model for studying the interaction of plasma membrane transport and toxic compounds, such as mercurials, and human primary cell culture is a valuable tool to study cell toxicity mechanisms of different drugs. Both the experimental models are a novel and potentially important tools in drug discovery and in the understanding of toxicity mechanism of xenobiotics compounds.