Cellular dielectric spectroscopy: a powerful new approach to label-free cellular analysis.

The past decade has seen a number of significant changes in identifying higher quality lead compounds earlier in the drug discovery process. Cell-based assay technologies yielding high-content information have emerged to achieve this goal. Although most of these systems are based on fluorescence detection, this article describes the development and application of an innovative cellular assay technology based on radio frequency spectrometry and bioimpedance measurements. Using this technique, the authors have discovered a link between cellular bioimpedance changes and receptor-mediated signal transduction events. By performing dielectric spectroscopy of cells across as pectrum of frequencies (1 KHz to 110 MHz), a series of receptor-specific, frequency-dependent impedance patterns is collected. These raw data patterns are used to determine the identity of the cellular receptor-signaling pathway being tested and to quantify stimulation endpoints and kinetics. The authors describe the application of this technology to the analysis of ligand-induced cellular responses mediated by the 3 major classes of G-protein-coupled receptors (GPCRs) and protein tyrosine kinase receptors. This single assay platform can be used with ease to monitor G(s), G(i), and G(q) GPCRs without the need for chimeric or promiscuous G-proteins, fluorophors, or tagged proteins. In contrast to other methods of monitoring cellular signal transduction, this approach provides high information content in a simplified, noninvasive, and biologically relevant fashion.

Cytotoxicity of antiviral nucleotides adefovir and cidofovir is induced by the expression of human renal organic anion transporter 1.

The transport of organic anions in proximal convoluted tubules plays an essential role in the active secretion of a variety of small molecules by the kidney. In addition to other anionic substrates, the human renal organic anion transporter 1 (hOATI) is capable of transporting the nucleotide analogs adefovir and cidofovir. To investigate the involvement of hOATI in the mechanism of nephrotoxicity associated with these two clinically important antiviral agents, Chinese hamster ovary (CHO) cells were stably transfected with hOATI cDNA. The resulting CHOhOAT cells showed probenecid-sensitive and pH-dependent uptake of p-aminohippurate (Km = 15.4 FtM, V,,, ..ax = 20.6 pmol/106 cells min), a prototypical organic anion substrate. In addition, the stably expressed hOATI mediated efficient transport of adefovir (Km, = 23.8 tLM, V, a,, = 46.0 pmol/106 cells min) and cidofovir (K, = 58.0 /iM, Vt,ax = 103 pmol/106 cells * min) such that the levels of intracellular metabolites of both nucleotides were > 1 00-fold higher in CHOh OAT cells than in parental CHO. Consequently, adefovir and cidofovir were approximately 500-fold and 400-fold more cytotoxic, respectively, in CHOh OAT cells compared to CHO. The cytotoxicity of both drugs in CHOh OAT cells was markedly reduced in the presence of hOATI inhibitors. The cyclic prodrug of cidofovir, which exhibits reduced in vivo nephrotoxicity, was a poor substrate for hOATI and showed only marginally increased cytotoxicity in CHOh OAT cells. In conclusion, these studies demonstrate that hOATI plays a critical role in the organ-specific toxicity of adefovir and cidofovir, and indicates that CHOh OAT cells may represent a useful in vitro model to investigate the potential nephrotoxicity of clinically relevant organic anion agents.