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.

SummaryIn a recent study in rats, alanine am inotransferase (ALT ), the preferred plasma biomarker of hepatocellular injury in rats, was ineffective at detecting marked hepatic necrosis produced by acetam inophen (Hum a n a nd Expe rim e nta l To xic o lo gy 19, 277±83, 2000). In contrast, glutam at e dehydrogenase (GLDH) was markedly elevated. Accordingly, these enzymes were comprehensively evaluated as plasma biomarkers of hepatocellular injury in rats using several other models of hepatic injury, including partial hepatectom y and exposure to methapyrilene, dexamethasone, cyproterone, isoniazid, lead nitrate, and Wyeth-14643. Other enzymes also evaluated were aspartate aminotransferase (AST ), sorbitol dehydrogenase (SDH), and the hepatobiliary marker alk aline phosphatase (ALP). Compared to plasma ALT increases, plasm a GLDH increases were up to 10-fold greater, up to 3-fold more persistent, and occurred at tim es following hepatocellular injury when plasma ALT was not increased. Plasma GLDH act ivity was not inhibited by the test compounds, whereas ALT was substantially inhibited by both isoniazid and lead nitrate. While plasma GLDH activity was unaffected by induction, ALT was induced by cyproterone and dexamethasone, and ALP was induced by Wyeth-146 43 and partial hepatectomy. GLDH was concluded to be a more effective biom ark er of acute hepatic injury than ALT, AST, SDH or ALP in the rat, based primarily on the large increase following hepatocellular injury, prolonged persistence in the blood following injury, high sensitivity for detection of injury (including pre-necrotic injury), high tissue speci®city, and lower susceptibilit y to inhibition or induction.Keywords Glutamate dehydrogenase; rat; liver; plasm a; hepatotoxicity Drug-induced hepatocellular injury is most commonly evaluated in preclinical and clinical studies using alanine am inotransferase (ALT ) (Travlos e t a l. 1996, Fujii 1997 ). However, in the rat this enzym e has theoretical disadvantages as a hepatic biomark er since it has a relatively low intra-hepat ic activit y and short half-life in blood compared with that in the dog, monkey, and man (Evans 1996, Davy e t a l. 1988). Furthermore, literature review (Fujii 1997 ) and a recent study (O'Brien e t a l. 2000) indicate that in the rat , plasma ALT may fail to indicate hepatic necrosis.Glutamate dehydrogenase (GLDH; EC 1.4.1.3 ), a key enzyme in amino acid oxidation and urea production, has several features which mak e it at tract ive as a potential biomarker of drug-induced hepatocellular toxicity. It is highly conserved in structure, tissue-distribution, and function across a wide range of species (Schmidt & Schmidt 1988 ). In common with ALT, GLDH is a relatively liver-speci®c enzym e (Clam pitt 1978, Lindena e t a l. 1986 ); however, intracellular distribution is different. ALT is cytosolic in origin, whereas GLDH is located in the mitochondrial matrix (Zim merman 1974, Schmidt & Schmidt 1988

Repeated dosing of acetaminophen (paracetamol) to rats is reported to decrease their sensitivity to its hepatotoxic effects, which are associated with oxidative stress and glutathione depletion. We determined if repeated acetaminophen dosing produced adaptive response of key antioxidant system enzymes. Male rats (Sprague-Dawley, 10 weeks) were given 800, 1200, or 1600 mg/kg/day acetaminophen by oral gavage for 4 days. Liver was assayed for oxidative stress and antioxidant markers: malondialdehyde (MDA), thiobar-bituric acid reactive substance (TBARS), total antioxidant status (TAS), glutathione (GSH), glutathione reductase (GR), glutathione peroxidase (GPx), glucose-6-phosphate dehydrogenase (G6PD), catalase (CAT), and superoxide dismutase (SOD), and alanine transaminase (ALT) as a marker of hepatocellular injury. Acetaminophen at 1200/1600 mg/kg decreased GSH 26/47%, GPx 21/26%, CAT 35/28%, SOD 21/12%; and TAS 28/18% (correlated with CAT, r=0.91; SOD, r=0.66; GPx, r = 0.45). Despite antioxidant deficiencies, and no TBARS change, MDA decreased 26%/33%/37% at 800/1200/1600 mg/kg, which correlated with increased GR (61%/62%/76%, r = 0.77) and G6PD (130%/110%/190%, r = 0.78). Both MDA (r = 0.68) and G6PD (r = 0.71) correlated with hepatic ALT, which decreased 27%o/43%/48%, respectively. Resistance to acetaminophen hepatotoxicity produced by repeated exposure is partially attributable to upregulation of hepatic G6PD and GR activity as an adaptive and protective response to oxidative stress and glutathione depletion.

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