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The Hepatocyte Suspension Assay Is Superior to the Cultured Hepatocyte Assay for Determining Mechanisms of Acetaminophen Hepatotoxicity Relevant to in ViWo Toxicity Received November 4, 2010
To the Editor: We thank Dr. Jaeschke and coauthors for submitting their letter to the Editor concerning our recent acetaminophen (APAP) toxicity studies in freshly isolated mouse hepatocytes. This gives us the opportunity to discuss the importance of our recent Chemical Research in Toxicology article (1) and to discuss the strengths of the freshly isolated hepatocyte suspension assay over the cultured hepatocyte assay in studying APAP toxicity.
Background There are a large number of manuscripts in the literature, which have utilized freshly isolated hepatocytes to study APAP toxicity (2-10). Freshly isolated hepatocytes are ideal for shortterm studies (up to six hours) of toxicity. A review of liver cell models in in Vitro toxicology was published by Guillouzo (11). We utilized the approach previously described by Boobis and co-workers (2-5) and by Racz and co-workers (6, 7). In this approach, APAP is incubated with freshly isolated mouse hepatocytes for 2 h under a 95% oxygen/5% CO2 atmosphere. During this time, GSH depletion and covalent binding of APAP to the protein of treated cells occur, but toxicity is minimal. Subsequently, the hepatocytes are washed to remove APAP and reincubated with media alone. Toxicity occurs in the reincubation phase (2-5 h); 60% of the cells lose viability after incubation with APAP. One key advantage of this approach is that the cytochrome P450 (CYP) enzyme activities needed for metabolism are still high compared to those of cultured hepatocytes (11). Another advantage is that by washing the hepatocytes to remove APAP, specific inhibitors can be added at 2 h without affecting the metabolism of APAP to the toxic metabolite since the toxic mechanism is already initiated. Thus, downstream events in APAP toxicity can be studied with this model. In a previous manuscript, we utilized confocal microscopy to show that APAP toxicity occurs with mitochondrial permeability transition (MPT). We showed that toxicity occurred with increased oxidative stress (increased fluorescence of dichlorodihydrofluorescein; DCFH2) and loss of mitochondrial membrane potential (altered fluorescence of JC-1). The toxicity, MPT, oxidative stress, and loss of mitochondrial membrane potential occurred in the reincubation phase (2-5 h). These events were blocked by adding the MPT inhibitors cyclosporine A or trifluoperazine, or N-acetylcysteine (NAC), at 2 h to the reincubation phase (12). These data indicated a role for MPT and increased oxidative stress in APAP toxicity. In our recent article (1), we utilized a similar approach to investigate the role of reactive nitrogen species as the mechanism of the increased oxidative stress in APAP toxicity by assaying for nitrotyrosine in proteins. Previously, we reported 3-nitrotyrosine protein adducts colocalized with APAP protein adducts and necrosis in the livers of APAP treated mice (13). We compared the formation of nitrated
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protein to increased oxidative stress as determined by increased DCFH2 oxidation (fluorescence). We showed that APAP toxicity in the freshly isolated hepatocytes occurred with nitration of proteins. MPT inhibitors and NAC added in the reincubation phase blocked nitration, toxicity, and DCFH2 oxidation. Inducible nitric oxide synthase (iNOS) inhibitors had no effect on nitration, toxicity, and DCFH2 oxidation. However, 7-nitroindazole, a neuronal NOS (nNOS) inhibitor, and LNMMA, a general NOS inhibitor, blocked nitration, toxicity, and DCFH2 oxidation. nNOS is believed to be in mitochondria as mtNOS (14, 15). Importantly, the nNOS inhibitor as well as the MPT inhibitor cyclosporine A, inhibited the loss of mitochondrial membrane potential, indicative of MPT. We interpreted these data to indicate that MPT occurred with activation of the mtNOS. The formation of increased reactive nitrogen species resulted in protein nitration and collapse of mitochondrial membrane potential (MPT). Specific Point 1: Why Do We Believe the Hepatocyte Suspension Assay Is Superior for APAP Toxicity Studies? In their first point, Dr. Jaeschke and co-workers believe that cultured hepatocyte assays are superior to freshly isolated hepatocyte suspension assays because the cells in suspension are more sensitive than the adhered cells. They state that the reason the cells are more sensitive is due to severe stress induced by keeping the cells in suspension and that the initial mitochondrial dysfunction triggered by APAP is further exaggerated by the high oxygen concentration. We respectfully disagree with this theory but do agree that the cultured cells are less sensitive. However, we believe that the reduced sensitivity of the cultured cells to the toxic effects of APAP is a result of the established fact that cultured hepatocytes lose cytochrome P450 enzyme activity (CYP) (11, 16-18). Guillouzo has suggested the loss of activity may be related to the loss of heme from CYP (11); in addition, cultured hepatocytes very rapidly lose CYP mRNAs, especially the critical CYP2E1 and CYP1A2 (19) important in APAP toxicity. Also, other changes occur such as the loss of mitochondria (18). CYP enzymes are necessary for the metabolism of APAP to the toxic metabolite N-acetyl-pbenzoquinone imine (NAPQI) which depletes GSH and covalently binds to proteins. These events initiate subsequent MPT, oxidant stress, and toxicity (20) (21). In Jaeschke and co-workers’ experiments, it is likely that the adherent cells have decreased CYP activity. With less CYP activity, the rate of metabolism of APAP to NAPQI would be greatly decreased. In APAP toxicity studies with cultured hepatocytes, it is necessary to incubate for long periods of time with APAP to obtain toxicity (12 h). A slower rate of formation of the reactive metabolite would result in a slower rate of GSH depletion and less covalent binding. In the freshly isolated hepatocytes, GSH was depleted by 0.5 h (22); however, in the cultured hepatocytes maximal GSH depletion was not observed until 4 h (23). The importance of these mechanisms in APAP toxicity is well characterized by multiple investigators (20-22). Also, it should be noted that the time course for the development of toxicity (ALT release) in our mouse hepatocytes is very similar to that reported for LDH release by Burcham and Harman (9). We believe that the reason that high APAP concentrations are necessary in the cultured hepatocyte assay is a result of
10.1021/tx1003744 2010 American Chemical Society Published on Web 11/17/2010
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loss of the CYP activities which have low Km values for APAP metabolism. APAP is metabolized to the toxic metabolite NAPQI primarily by three CYP enzymes: CYP2E1, CYP1A2, and CYP3A4 (24). Of these, CYP 2E1 is the major one. It has the lowest Km for the oxidation of APAP to NAPQI (0.18 mM) (25, 26). This enzyme is present in the centrilobular areas of the liver and not in the periportal areas of the liver (27, 28). The centrilobular areas are where toxicity, covalent binding, and protein nitration occur (20, 29, 13). The fact that we and others (6) obtained significant toxicity utilizing 1 mM APAP (five times the Km of CYP2E1) in hepatocyte suspensions, while 5-25 mM APAP is needed to obtain significant toxicity in cultured hepatocytes, suggests that these cultured cells have a decreased level of CYP2E1 activity. Thus, high concentrations of APAP are necessary to saturate the high Km forms of CYP that metabolize APAP. A decrease in CYP2E1 may occur as a result of the culturing process or by selective adherence of periportal hepatocytes instead of the centrilobular hepatocytes in the culturing process. Additional data support our belief that the hepatocyte suspension assay is more relevant to the in ViVo toxicity compared to the cultured hepatocyte assay for APAP toxicity studies. For example, following the administration of 300 mg/kg APAP to the mouse we observe significant increases in serum ALT (toxicity) by 4 h and peak ALT by 6 h. In the suspension assay, we found significant increases in ALT release at 3, 4, and 5 h with approximately 50-60% loss of cell viability at 5 h. In the cultured hepatocyte assay, toxicity occurs more slowly, and the APAP concentrations are much higher (5-25 mM). Using cultured hepatocytes, Bajt et al. (30) did not observe a significant LDH release until 6 h and observed 60% LDH release at 12 h. The APAP concentrations in the liver of mice following a hepatotoxic dose (300 mg/ kg) are approximately 1 µmol/g liver at 2 h and 0.5 µmol/g of liver at 4 h (Hinson, J. A., unpublished data). In our suspension assays, we used 1 mM APAP, which is five times over the CYP2E1 Km, and a concentration expected to saturate the CYP2E1 substrate binding site. Thus, the time for the development of toxicity in ViVo and in the hepatocyte suspension assay are very similar. Also, the concentration that we are using in the in Vitro assay is comparable to the APAP concentration in the liver of APAP treated mice at 2 h. Cultured hepatocytes utilize much longer incubation times (12 h) and much higher concentrations of APAP. Thus, the time course of toxicity and the concentration of APAP required to produce toxicity when adherent hepatocytes are used are much higher than what is observed in ViVo. Jaeschke et al. made the point that we utilized 95% oxygen/ 5% CO2 and that this high concentration of oxygen in their opinion contributed to the toxicity. We do not observe evidence of metabolic stress in these cells nor have problems been reported by others using the same oxygen concentration (2, 6, 9, 12). Our control cells had ALT release of approximately 20 IU/L into the media, and this level was maintained for the entire 5 h incubation. Thus, our control hepatocytes are very stable and showed little toxicity or loss of viability, which argues against a direct toxicity due to 95% oxygen. Our peak ALT level in the APAP treated cells at 5 h is approximately 400 IU/L (50-60% loss of viability). In our previous manuscript, we incubated the hepatocyte suspension with APAP for 1 h followed by washing and subsequently incubating in media alone. ALT release into media was not significantly different from that in control cells
(12). Thus, APAP exposure per se is not the mechanism leading to toxicity. Also, in the present article, we added the MPT inhibitors cyclosporine A and trifluoperazine to the reincubation media at 2 h, after first incubating with APAP and washing to remove APAP at 2 h, and there was no subsequent increase in toxicity (Figure 3) (1). Likewise, addition of the nNOS inhibitor 7-nitroindazole at 2 h blocked any further increase in toxicity (Figure 4) and protein nitration (Figure 2). Thus, the blockade of toxicity by specific inhibitors indicated that there was no inherent problem with the fragility of the hepatocytes due to keeping them in suspension with a high concentration of oxygen as suggested by Jaeschke et al. Moreover, toxicity occurred only with increased protein nitration (reactive nitrogen formation) and increased oxidative stress (1). It should also be noted that Burcham and Harman (9) reported toxicity data very similar to our data incubating APAP in a mouse hepatocyte suspension assay, They showed, as we did, no significant increase in cell death in the control cells over the course of the incubation. These control cells were treated in the same fashion as the APAP treated cells and undergo the same 95% oxygen/5% carbon dioxide atmosphere and suspension shaking methods. Also, the rate of the development of the toxicity in APAP treated cells was similar. An important factor in the hepatocyte suspension assay is maintaining adequate oxygen supply to the hepatocytes since decreased oxygen itself may cause toxicity. Jaeschke and co-workers recently published that higher oxygen concentrations (21%) in APAP cultured hepatocytes lead to accelerated cell death and mitochondrial oxidant stress compared to those with 10% oxygen (23). They believe hyperoxia occurred, and this increased the toxicity. We interpret their data to indicate that oxygen concentration was rate limiting for CYP metabolism of APAP to the toxic metabolite NAPQI at 10% oxygen. At the higher oxygen concentration (21%), there was greater GSH depletion than at the lower oxygen concentration (10%) at 1 h (see their Figure 4) (23). Oxygen is needed for CYP metabolism of APAP to the toxic metabolite NAPQI, which depletes GSH. Their data suggest that there was insufficient oxygen at 10% compared to 21% for optimal CYP metabolism of APAP to NAPQI (23). Thus, GSH is depleted more rapidly, and there would be more covalent binding in the incubation under the 21% oxygen atmosphere when compared with that under the 10% oxygen atmosphere because there is more toxic NAPQI metabolite produced. More covalent binding as a result of increased NAPQI formation would lead to greater oxidant stress and increased toxicity in the cells under 21% oxygen compared to those of the cells under 10% oxygen. Unfortunately, APAP covalent binding was not assayed by Jaeschke et al. However, the importance of GSH depletion and covalent binding in APAP toxicity are well described (31, 32). No studies were performed at 95% oxygen. Also, Kidambi et al. (33) reported that hepatocytes cocultured with endothelial cells in 95% oxygen maintained high levels of CYP activity. Specific Point 2: ROS or MPT, Which Comes First? The second point made by Jaeschke and co-workers is that previous data support the conclusion that the formation of ROS and peroxynitrite occurs prior to the MPT pore opening and is at least in part involved in triggering the MPT. They interpret the data in Table 1 to indicate that cyclosporine A completely inhibits DCFH2 oxidation and nitration of proteins. All of the data presented in Table 1 of our article (1) are calculated for between 2 and 5 h following APAP (see Table
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1, legend). Data were presented showing that oxidative stress did occur before toxicity (2 h). This included an increase in DCFH2 oxidation and tyrosine nitration (Supporting Information and Figure 2). Also, in Figure 6 we show that there is approximately a 25% decrease in mitochondrial membrane potential by 2 h. Consistent with the literature, we state in the article that MPT is caused by oxidative stress and leads to a large increase in oxidative stress. In the legend to the summary Figure 4, we state, “It is postulated that mitochondrial damage is a result of RNS and leads to additional RNS which causes additional mitochondrial damage” (1). Thus, we agree with Dr. Jaeschke and co-workers that indeed a low level of oxidant stress occurred before the induction of MPT and toxicity. Specific Point 3: What Is the Role of iNOS in APAP Toxicity? The third point made by Jaeschke and co-workers regards our conclusion that nNOS and not iNOS was the source of nitric oxide (NO) for peroxynitrite in the APAP treated hepatocytes. Our data in our article (1) show that two iNOS inhibitors (L-NIL and SAIT) did not inhibit APAP toxicity, protein nitration, or DCFH2 oxidation. However, the nNOS inhibitor (7-nitroindazole) inhibited APAP toxicity, protein nitration, DCFH2 oxidation, and loss of mitochondrial membrane potential. These data indicate that iNOS is not a contributor to the formation of reactive nitrogen species leading to toxicity in the hepatocyte suspension assay (1). We previously examined APAP induced hepatotoxicity in iNOS knockout mice. Even though there was a difference in serum ALT levels, histopathological examination of liver injury failed to demonstrate any significant differences between the amount of necrosis in the APAP treated iNOS knockout mice and the wild type mice. Since histopathology is considered to be the gold standard for toxicity studies, we believe that iNOS is not important in the development of APAP toxicity (34). In addition, Pohl’s laboratory found that iNOS knockout mice were equally sensitive to the hepatotoxicity of APAP as wild type mice (35). We also previously reported that iNOS inhibitors did not inhibit APAP toxicity in mice (36). Thus, our finding in our article that two iNOS inhibitors did not inhibit APAP toxicity in the hepatocyte suspension assay was consistent with our in ViVo data that iNOS is not an important player in APAP toxicity (1). However, iNOS, as does eNOS, may play a role in blood flow in the liver.
Conclusions Clearly, there are no perfect in Vitro models of APAP hepatotoxicity; therefore, it would be shortsighted to discount either of these models. We and others believe that suspension assays are appropriate for short-term toxicity studies because they retain the enzymatic ability to generate reactive metabolites clearly shown to be required for toxicity in ViVo. Adherent cells are clearly superior for longer-term toxicity studies and to study regeneration, but the loss of CYP activities in the cultured hepatocyte assay is a significant problem for studying the mechanisms of APAP toxicity relevant to in ViVo toxicity.
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Angela S. Burke WIL Research Laboratories, LLC, 1407 George Road, Ashland, Ohio 44805, United States Lee Ann MacMillan-Crow Department of Pharmacology and Toxicology, University of Arkansas for Medical Sciences, 4301 West Markham Street, Little Rock, Arkansas 72205, United States Jack A. Hinson* Department of Pharmacology and Toxicology, University of Arkansas for Medical Sciences, 4301 West Markham Street, Little Rock, Arkansas 72205, United States TX1003744
