* Liver Research Institute, University of Arizona, College of Medicine, Tucson, Arizona 85724; Department of Pharmacology and Toxicology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205;
Department of Cell and Developmental Biology, University of North Carolina, Chapel Hill, North Carolina 27599
Received January 13, 2004; accepted April 7, 2004
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ABSTRACT |
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Key Words: acetaminophen; hepatotoxicity; oxidant stress; N-acteylcysteine; cultured hepatocytes.
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INTRODUCTION |
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During the recovery phase of the cellular glutathione content, substantial increases in the cellular and especially mitochondrial levels of glutathione disulfide (GSSG) are found (Jaeschke, 1990, Knight et al., 2001
). These data have been interpreted as evidence for a mitochondrial oxidant stress during acetaminophen hepatotoxicity. However, these conclusions are not without criticism. Although GSSG formation is generally considered a reliable and specific indicator for cellular oxidant stress (Jaeschke et al., 1988
; Lauterburg et al., 1984
; Smith, 1991
), an interfering factor is the severe initial depletion of cytosolic and mitochondrial GSH after AAP overdose. Because of these low GSH levels, only a small shift of the GSSG-to-GSH ratio to a more oxidized state can be observed (Knight et al., 2001
). It takes at least 4 h or longer until enough GSH is re-synthesized to measure GSSG levels higher than baseline, which would be considered solid evidence for an intracellular oxidant stress (Jaeschke, 1990
; Knight et al., 2001
). However, at that time, the release of cytosolic liver enzymes indicates cell injury. Thus, Smith and coworkers argue that this oxidant stress is most likely a consequence of cell injury rather than an early event, which might be relevant for the mechanism of cell death (Rogers et al., 2000
; Smith et al., 1985
;). Since the depletion of GSH is a prerequisite for the injury (Mitchell et al., 1973
), measurement of GSSG formation cannot unequivocally answer the question whether the oxidant stress precedes cell injury or is only a late event of the injury. Therefore, we used 2',7'-dichlorofluorescein (DCF) fluorescence as a marker of oxidant stress in a cell culture model to evaluate the time course of reactive oxygen formation in relationship to cell viability.
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MATERIALS AND METHODS |
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Mouse hepatocyte isolation. Primary hepatocytes were isolated from mice anesthetized with pentobarbital sodium solution (Nembutal, Abbott Laboratories, North Chicago; 50 mg/kg ip) as previously described (Hatano et al., 2000; Shen et al., 1991
) with some modifications. Briefly, the inferior vena cava was cannulated and the liver was first perfused in situ with an oxygenated Hanks' buffer salt solution (HBSS) containing 100 U/ml penicillin/streptomycin (Gibco, Grand Island, New York), pH 7.4 (8 ml/min, 37°C for 10 min), followed by perfusion with oxygenated HBSS containing 1 mM Ca2+ and Mg2+, penicillin/streptomycin (100 U/ml), and 0.04% collagenase D (Roche Molecular Biochemicals, Mannheim, Germany), pH 7.4 for 10 min. The liver was removed and then gently minced in HBSS containing 1 mM Ca2+ and Mg2+, penicillin/streptomycin (100 U/ml), and 1 x 107 M insulin (Sigma), pH 7.4. The liver cell suspension was then filtered with Falcon cell strainers (40, 70, and 100 mm; Becton Dickinson, Bedford, MA) and centrifuged at 50 x g for 2 min. From the isolation of one mouse liver, a typical yield was about 5060 x 106 hepatocytes. Cell viability, as determined by trypan blue exclusion, was generally >90%, and cell purity was >95% hepatocytes. Cells were plated on 6-well plates (6 x 105 cells/well) or 24-well plates (8 x 104) (Biocoat collagen I cellware plates; Becton Dickinson) in Williams's Medium E (Gibco) containing 10% fetal bovine serum (Gibco), 100 U/ml penicillin/streptomycin, and 1 x 107 M insulin and cultured at 37°C with 5% CO2. After an initial 4-h attachment period, cultures were washed with phosphate-buffered saline (PBS) and then plain culture medium (controls) or media containing various concentrations of AAP were added. Preliminary experiments indicated that concentrations of 510 mM AAP caused a dose-dependent cell injury in cultured mouse hepatocytes within 612 h. We selected 5 mM AAP for all further experiments. In some experiments, cells were treated with 20 mM N-acetylcysteine (dissolved in 10X PBS, pH 7.4) (Harman and Self, 1986
) either 1 h before or 2 h after AAP administration.
Cell viability. Cell viability was assessed by trypan blue uptake and LDH release. After removal of the cell medium, hepatocytes were incubated with 0.8% trypan blue solution for 3 min at room temperature. Trypan blue-positive cells were counted in 4 different fields (x10; a total of approximately 1500 cells). For LDH release measurements, medium was removed from cells and lysis buffer containing 25 mM HEPES, 5 mM EDTA, 0.1% CHAPS, and 1 mg/ml each of pepstatin, leupeptin, and aprotinin, pH 7.5, was added to the hepatocytes for 5 min. Cells were removed from wells with a cell scraper and placed into a test tube. After sonication, cells were centrifuged for 20 min at 15,000 rpm at 4°C. Aliquots of the cell lysate or medium added to a reaction mixture in potassium phosphate buffer (60 mM, pH 7.5) containing 0.72 mM pyruvate and 216 mM NADH. The kinetics of absorbance decrease at 340 nm was measured with a spectrophotometer (UV-1601PC, Shimadzu Scientific Instruments, Columbia, MD).
Glutathione. For cell glutathione measurements, media was removed and 0.5 ml of 3% sulfosalicylic acid was added to the cells. Each well was scraped with a cell scraper and the precipitated proteins centrifuged for 5 min. The acidic supernatant was diluted in 100 mM potassium phosphate buffer (KPP), pH 7.4, and assayed with a modified Tietze assay as described (Jaeschke and Mitchell, 1990). A 10% SDS solution was added to the pellet to solubilize the proteins. Protein concentrations were assayed using the bicinchoninic acid kit (Pierce, Rockford, IL). All chemicals were purchased from Sigma Chemical Co. (St. Louis, MO) unless stated otherwise.
Detection of reactive oxygen intermediates. Cultured hepatocytes were treated with 5 mM APP for 1.5, 3, 6, 9, or 12 h. At the indicated time points, hepatocytes were rinsed in phosphate-buffered saline (PBS). Cells were loaded with 1 mM 5- and 6-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate (CM-H2DCFDA; Molecular Probes, Inc., Eugene, OR) for 30 min. CM-H2DCFDA, a cell-permeable indicator for reactive oxygen species, is nonfluorescent until hydrolyzed by intracellular esterases and oxidized by intracellular reactive oxygen species. After loading the cells, the linear increase in fluorescence intensity was monitored in a thermostated fluorescence microplate reader (Spectra Max Gemini EM, Molecular Devices, Corp., Sunnyvale, CA) at 37°C for 1 h, using an excitation wavelength of 490 nm and an emission wavelength of 530 nm. The slope of the curve was used to calculate the change in fluorescence intensity per min.
XTT assay. Cell viability was also determined using the 2,3-bis[2- Methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxyanilide inner salt (XTT) system according to the manufacturer's instructions (Sigma). The XTT assay measures the activity of mitochondrial and extramitochondrial dehydrogenases (Bernas and Dobrucki, 2002; Huet et al., 1992
) and therefore provides an indicator of overall functional cell viability. The tetrazolium ring of XTT is cleaved by dehydrogenases of viable cells to produce soluble orange formazan, which can be detected spectrophotometrically. After adding XTT, the cells were incubated for 2 h and the increase in formazan absorbance was read at a wavelength of 450 nM on a microplate reader (SpectraMax 190, Molecular Devices, Corp., Sunnyvale, CA).
Statistics. All results were expressed as mean ± SE. Comparisons between multiple groups were performed with one-way ANOVA or, where appropriate, by two-way ANOVA, followed by a post hoc Bonferroni test. If the data were not normally distributed, we used the Kruskal-Wallis Test (nonparametric ANOVA) followed by Dunn's Multiple Comparisons Test; p < 0.05 was considered significant.
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RESULTS |
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DISCUSSION |
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A critical question remains: do this early oxidant stress and mitochondrial dysfunction actually contribute to cell death in these hepatocytes? To address this question, we enhanced cellular GSH levels before AAP or stimulated GSH synthesis, after exposure to AAP as previously shown in vivo (Bajt et al., 2003; Knight et al., 2002
). In contrast to the in vivo situation, where intravenously supplied GSH is degraded in the kidney and the amino acids can be taken up by hepatocytes for de novo glutathione synthesis (Wendel and Jaeschke, 1982
), cells in culture have to be supplied with precursor cysteine in the form of the nontoxic N-acetylcysteine (NAC). Treatment with NAC before exposure to AAP has been shown previously to attenuate covalent binding and cell injury in vivo (Corcoran et al., 1985
) and in isolated hepatocytes (Harman and Self, 1986
). Our data demonstrate the enhanced baseline GSH levels after NAC pretreatment, which resulted in reduced loss of functional viability (XTT assay), less oxidant stress, and attenuated cell necrosis after AAP treatment. However, when NAC was supplied at 2 h after AAP exposure, the initial metabolic dysfunction had already occurred, as indicated by the XTT assay. Under these conditions, the rapid restoration of cellular glutathione levels within the next hour prevented the further deterioration of the cellular respiration and reduced the overall cell injury at 9, 12, and 24 h as indicated by less trypan blue uptake. These findings support the conclusion that NAC treatment 2 h after AAP exposure strengthened the detoxification potential of the GSH/ glutathione peroxidase system as previously shown in vivo (Knight et al., 2002
). Consequently, these data suggest that the oxidant stress contributed to the progression of cell injury in this model.
A number of investigators concluded previously that reactive oxygen species might play a role in AAP-induced cell injury of cultured hepatocytes. Several studies showed a beneficial effect of treatment with vitamin E (Nagai et al., 2002), the iron-chelator deferoxamine (Gerson et al., 1985
; Adamson and Harman, 1993
) and catalase/superoxide dismutase (Kyle et al., 1987
). On the other hand, pretreatment with iron or inhibitors of glutathione peroxidase or glutathione reductase enhanced AAP toxicity in hepatocytes (Adamson and Harman, 1993
; Gerson et al., 1985
; Kyle et al., 1987
). However, many of these findings obtained with cultured cells could not be reproduced in vivo. For example, neither the beneficial effect of iron chelation nor the enhanced injury with inhibition of glutathione reductase was observed after AAP overdose in vivo (Smith et al., 1986
; Smith and Mitchell, 1985
). In addition, glutathione peroxidase gene-deficient mice were as susceptible to AAP as wild-type animals (Knight et al., 2002
). Moreover, despite positive effects against iron/allyl alcohol-induced lipid peroxidation and liver injury, treatment with a- or g-tocopherol did not protect against AAP-induced liver injury in mice (Knight et al., 2003
). These discrepancies between the effects of antioxidants against liver cell damage in vivo versus cultured cells suggest a more prominent role of reactive oxygen species and lipid peroxidation in cell culture. As recently reviewed by Halliwell (2003)
, some of the higher oxidant stress in cultured cells may be due to the generally higher oxygen concentrations in incubators, compared to the oxygen levels hepatocytes are exposed to in vivo. However, while these experimental design issues may affect the exact mechanism of injury propagation, the answer to the most critical question addressed in this study, i.e., does the oxidant stress precede injury, should not have been affected.
In summary, our data demonstrate that AAP causes a rapid depletion of glutathione, a functional deterioration, and reactive oxygen formation in cultured hepatocytes during the first 3.5 h after exposure. Substantial cell necrosis indicated by the increase of cell membrane permeability occurs several h later. Treatment with NAC before or after AAP attenuates the functional deterioration of the cells and reduces cell necrosis. We conclude that AAP-induced oxidant stress precedes cell necrosis and that the oxidant stress is involved in the propagation of the cell injury in cultured primary mouse hepatocytes.
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ACKNOWLEDGMENTS |
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NOTES |
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1 To whom correspondence should be addressed at the Liver Research Institute, University of Arizona College of Medicine, 1501 N. Campbell Ave, Room 6309, Tucson, AZ 85724. Fax: (520) 626-5975. E-mail: jaeschke{at}email.arizona.edu.
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