Transport and toxicity of ochratoxin A

Ochratoxin A (OTA) is a fungal metabolite produced by strains of Aspergillus and Penicillium. A known food contaminant, OTA has been associated with various renal diseases. Transport of OTA to affected tissues occurs through blood plasma. Native isoelectric focusing and native electrophoresis of human plasma indicates human serum albumin (HSA) is the primary plasma protein that binds OTA. Optical spectroscopy illustrates the OTA dianion occupies two binding sites on HSA with binding constants of 5.2 · 106 M−1 and 1.0 · 105 M−1. Preferential binding of the dianion results from a pK_a decrease of the phenolic group of OTA by more than three units.; Binding site locations are probed through fluorescence energy transfer between the lone Trp214 on HSA and OTA and the co-bound HSA ligand warfarin and OTA. Furthermore, competitive interactions between OTA and additional ligands provide information on OTA binding sites through fluorescence polarization. These experiments, in combination with binding of OTA to recombinant domains of HSA, allow the assignment of the high affinity dianion to Site I (domain II) and the secondary dianion to Site II (domain III).; The electrostatic interactions responsible for OTA binding are explored through derivatives of the toxin. Substitutions at the phenolic moiety or positions para to that moiety suggest stabilization of the derivative or OTA dianion enhances the corresponding binding constant. Isothermal calorimetry also shows the binding of the OTA dianion is accompanied by protonation of an amino acid in the binding site by proton uptake from the solvent.; In addition, delivery of OTA to proximal tubule cells by basolateral organic anion transporters (OATS) is investigated. [3H]-OTA uptake into Xenopus laevis oocytes and Madine Darby canine kidney (MDCK) cells transfected with two human OATS, hOAT1 and hOAT3, is examined in the presence of HSA. Inhibition of [3H]-OTA uptake by HSA is apparent under HSA concentrations far lower than in vitro concentrations. Thus, other pathways into kidney cells must occur.