To develop and validate a practical, in vitro, cell-based model to assess human hepatotoxicity potential of drugs, we used the new technology of high content screening (HCS) and a novel combination of critical model features, including (1) use of live, human hepatocytes with drug metabolism capability, (2) preincubation of cells for 3 days with drugs at a range of concentrations up to at least 30 times the efficacious concentration or 100 microM, (3) measurement of multiple parameters that were (4) morphological and biochemical, (5) indicative of prelethal cytotoxic effects, (6) representative of different mechanisms of toxicity, (7) at the single cell level and (8) amenable to rapid throughput. HCS is based on automated epifluorescence microscopy and image analysis of cells in a microtiter plate format. The assay was applied to HepG2 human hepatocytes cultured in 96-well plates and loaded with four fluorescent dyes for: calcium (Fluo-4 AM), mitochondrial membrane potential (TMRM), DNA content (Hoechst 33,342) to determine nuclear area and cell number and plasma membrane permeability (TOTO-3). Assay results were compared with those from 7 conventional, in vitro cytotoxicity assays that were applied to 611 compounds and shown to have low sensitivity (<25%), although high specificity ( approximately 90%) for detection of toxic drugs. For 243 drugs with varying degrees of toxicity, the HCS, sublethal, cytotoxicity assay had a sensitivity of 93% and specificity of 98%. Drugs testing positive that did not cause hepatotoxicity produced other serious, human organ toxicities. For 201 positive assay results, 86% drugs affected cell number, 70% affected nuclear area and mitochondrial membrane potential and 45% affected membrane permeability and 41% intracellular calcium concentration. Cell number was the first parameter affected for 56% of these drugs, nuclear area for 34% and mitochondrial membrane potential for 29% and membrane permeability for 7% and intracellular calcium for 10%. Hormesis occurred for 48% of all drugs with positive response, for 26% of mitochondrial and 34% nuclear area changes and 12% of cell number changes. Pattern of change was dependent on the class of drug and mechanism of toxicity. The ratio of concentrations for in vitro cytotoxicity to maximal efficaciousness in humans was not different across groups (12+/-22). Human toxicity potential was detected with 80% sensitivity and 90% specificity at a concentration of 30x the maximal efficacious concentration or 100 microM when efficaciousness was not considered. We conclude that human hepatotoxicity is highly concordant with in vitro cytotoxicity in this novel model and as detected by HCS.

Glutamate-cysteine ligase (GCL; also known as gamma-glutamylcysteine synthetase) is the rate-limiting enzyme in glutathione (GSH) synthesis. Traditional assays for the activity of this enzyme are based either on coupled reactions with other enzymes or on high-performance liquid chromatography (HPLC) assessment of gamma-glutamylcysteine (gamma-GC) product formation. We took advantage of the reaction of naphthalene dicarboxaldehyde (NDA) with GSH or gamma-GC to form cyclized products that are highly fluorescent. Hepa-1 cells which were designed to overexpress mouse GCL and mouse liver homogenates were used to evaluate and compare the utility of the NDA method with an assay based on monobromobimane derivatization and HPLC analysis with fluorescence detection. Excellent agreement was found between GCL activities measured by HPLC and NDA-microtiter plate analyses. This assay should be useful for high-throughput GCL activity analyses.

The role of intracellular oxidative stress in the mechanism of action of phosphotyrosine phosphatase (PTP) inhibitors was studied using three vanadiumbased compounds. Sodium orthovanadate (Na 3 VO 4 ), sodium oxodiperoxo(1,10-phenanthroline)vanadate(V) (pV(phen), and bis(maltolato)-oxovanadium(IV) (BMOV) differentially induced oxidative stress in lymphocytes. Treatment with pV(phen), which caused intracellular oxidation, induced strong protein tyrosine phosphorylation compared with Na 3 VO 4 and BMOV. Syk family kinases and the mitogen-activated protein kinase erk2 were rapidly activated by pV(phen) but not by BMOV or Na 3 VO 4 . In contrast, both BMOV and pV(phen) strongly activated NF-B. The antioxidant pyrrolidine dithiocarbamate (PDTC) greatly diminished the intracellular oxidation and protein phosphotyrosine accumulation induced by pV(phen). Pretreatment of cells with PDTC reduced and delayed the activation of Syk kinases and erk2. However, NF-B activation by pV(phen) was markedly enhanced in lymphocytes pretreated with PDTC, and another antioxidant, N-acetylcysteine, did not prevent the activation of NF-B by BMOV. These results indicate a role for oxidative stress in the biological effects of some PTP inhibitors, whereas NF-B activation by PTP inhibitors is mediated by mechanisms independent of intracellular redox status.Lymphocyte signal transduction requires the activation of protein tyrosine kinases (PTKs), 1 with subsequent assembly of signaling complexes, generation of second messengers, activation of transcription factors, and gene expression (1, 2). The balance of protein tyrosine phosphorylation within the cell is controlled by the relative activities of the PTKs and PTPs in the signaling network (3). Besides dephosphorylating a variety of PTK substrates, PTPs have been shown to directly modulate the activities of PTKs (4, 5). Thus PTPs serve a crucial function in lymphocytes by controlling both the initiation and termination of receptor-based signals.The inhibition of PTPs reveals PTK substrates on which phosphotyrosine accumulates in the absence of receptor engagement (6). Some of these substrates are key phosphoproteins in lymphocyte signal transduction pathways, suggesting that PTKs involved in transmission of receptor signals are activated by the absence of PTP regulation (6 -9). However, many of the PTP inhibitors used thus far to explore lymphocyte signal transduction pathways are redox-active compounds. For example, phenylarsine oxide, a thiol-reactive compound, and H 2 O 2 , which generates hydroxyl radicals, both act as potent PTP inhibitors (6, 10). The role of intracellular oxidation in the mechanism of action of PTP inhibitors is unknown, a question this study addresses.Vanadium-based PTP inhibitors, which have been extensively studied as insulin mimetic agents, stimulate glucose uptake and fatty acid synthesis in adipocytes and mimic receptor-based signals in lymphocytes (11-15). The widely used PTP inhibitor pervanadate is a peroxovanadium compound generated by reaction of H 2 O 2 wi...