These data suggest an important role for RNS in APAP-induced mitochondrial damage and toxicity. of Laboratory Animals as adopted by the U.S. National Institutes of Health. Mice were acclimatized one week prior to the experiments and fed until the time of sacrifice. Hepatocyte Isolation and Incubations Freshly isolated hepatocytes were obtained from 25 g IB-MECA male B6C3F1 mice by collagenase perfusion following a modification of the method of Grewal and Racz (12, 13, 23). Briefly, for each individual experiment, hepatocytes were isolated from a single IB-MECA mouse as previously described, followed by centrifugation at 140for 8 min in a 90% Percoll gradient to purify the cells, followed by a wash in media, and a 3 min centrifugation at 140to wash the Percoll from cells. Preparations yielding 40 million cells and cell viability 90% as determined by Trypan blue exclusion were used for the experiments. The hepatocytes were incubated at a concentration of 1 1,000,000 cells/mL in RPMI-1640 (supplemented with 25 mM HEPES, 10 IU heparin/mL, and 500 IU penicillin G/mL) in 125 mL Erlenmeyer flasks at 37 C under an atmosphere of 95% O2C5% CO2. APAP (1 mM), at a concentration similar to that occurring in animals treated with a toxic dose of APAP, was added to experimental hepatocytes, but no APAP was added to control flasks. At 2 h following drug addition, the hepatocytes were centrifuged for 2 min at 140for 2 min and the supernatants discarded. Cells were resuspended with 6.5 0.05 from the control. Results Protein Nitration in APAP Toxicity To determine the potential role of RNS in APAP toxicity, freshly isolated mouse hepatocytes were incubated with APAP (1 mM). At 2 h, the hepatocytes were washed and subsequently incubated with media alone. At 5 h, incubations were stopped and proteins assayed by Western blot analysis for 3-nitrotyrosine. Figure 1 shows the presence of nitrated proteins at5hin APAP-treated hepatocytes compared to those of the control. Control hepatocytes were not incubated with APAP but otherwise treated identically. Each lane contains hepatocyte proteins from a separate incubation obtained from a separate mouse. Open in a separate window Figure 1 Western blot analysis for nitrotyrosine in proteins of APAP-treated hepatocytes. Freshly isolated mouse hepatocytes were incubated with APAP (1 mM) as described in Experimental Procedures. Controls were incubated with media alone. At 5 h, incubations were terminated, and 3-nitrotyrosine levels (protein nitration) were determined using Western blot analysis as described in Experimental Procedures. Each lane contains protein from a separate incubation performed on hepatocytes obtained from a separate mouse. A time course for increasing levels of 3-nitrotyrosine in APAP-treated hepatocytes and control hepatocytes was performed by ELISA. There was a linear increase in protein nitration from 2 to5hin APAP-treated hepatocytes (Figure 2), and the relative increase in nitration correlated with the relative increase in APAP toxicity (Figure 3). Control incubations did not show significant protein nitration (Figure 2) or toxicity (Figure 3). IB-MECA Open in a separate window Figure 2 ELISA determination for nitrotyrosine in proteins of APAP-treated hepatocytes: time course and effect of various inhibitors of toxicity. Freshly isolated mouse hepatocytes were incubated with media alone or with APAP (1 mM) for 2 h. Subsequently, hepatocytes were washed to remove APAP (arrow) and incubated with media. To some incubations, cyclosporine A (10 = 3 from separate mice) which significantly increased from the 2 2 h wash are indicated by * ( 0.05). Open in a separate window Figure 3 Effect of MPT inhibitors on APAP-induced toxicity in freshly isolated hepatocytes. Hepatocytes were incubated with APAP (1 mM). Subsequently, hepatocytes were washed to remove APAP (arrow) and incubated in media alone (), media containing cyclosporine A (10 0.05). Samples (= 3 from separate KNTC2 antibody mice) which are significantly decreased from APAP alone at the same time point are designated by ?? (* ( 0.05). Further experiments (Supporting Information) were carried out to assess the extent of RNS using the oxidation of 2,7-dichlorodihydrofluorescein (DCFH2), which can occur by peroxynitrite as well as other oxidants (21, 22). Figure 1S (Supporting Information) shows that there was a IB-MECA significant increase in DCFH2 oxidation in APAP-treated hepatocytes when compared to that of the control hepatocytes. The increases in DCFH2 oxidation correlated with the relative increases in APAP-induced toxicity and with the relative protein nitration. Effect of MPT Inhibitors on APAP Toxicity and Protein Nitration We previously reported that the addition of the MPT inhibitors, cyclosporine A and trifluoperazine, inhibited toxicity (13). To determine whether the MPT inhibitor,.