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1 Farnesyltransferase Inhibitors Reduce Ras Activation and Ameliorate Acetaminophen-Induced Liver Injury in Mice Banishree Saha and Dipankar Nandi Hepatotoxicity due to overdose of the analgesic and antipyretic acetaminophen (APAP) is a major cause of liver failure in adults. To better understand the contributions of different signaling pathways, the expression and role of Ras activation was evaluated after oral dosing of mice with APAP ( mg/kg). Ras guanosine triphosphate (GTP) is induced early and in an oxidative stress-dependent manner. The functional role of Ras activation was studied by a single intraperitoneal injection of the neutral sphingomyelinase and farnesyltransferase inhibitor (FTI) manumycin A (1 mg/kg), which lowers induction of Ras-GTP and serum amounts of alanine aminotransferase (ALT). APAP dosing decreases hepatic glutathione amounts, which are not affected by manumycin A treatment. However, APAP-induced activation of c-jun N-terminal kinase, which plays an important role, is reduced by manumycin A. Also, APAP-induced mitochondrial reactive oxygen species are reduced by manumycin A at a later time point during liver injury. Importantly, the induction of genes involved in the inflammatory response (including inos, gp91phox, and Fasl) and serum amounts of proinflammatory cytokines interferon- (IFN ) and tumor necrosis factor, which increase greatly with APAP challenge, are suppressed with manumycin A. The FTI activity of manumycin A is most likely involved in reducing APAP-induced liver injury, because a specific neutral sphingomyelinase inhibitor, GW4869 (1 mg/kg), did not show any hepatoprotective effect. Notably, a structurally distinct FTI, gliotoxin (1 mg/kg), also inhibits Ras activation and reduces serum amounts of ALT and IFN- after APAP dosing. Finally, histological analysis confirmed the hepatoprotective effect of manumycin A and gliotoxin during APAP-induced liver damage. Conclusion: This study identifies a key role for Ras activation and demonstrates the therapeutic efficacy of FTIs during APAP-induced liver injury. (HEPATOLOGY 2009;50: ) Abbreviations: APAP, acetaminophen; ALT, alanine aminotransferase; DCFDA, 2,7 -dichloro-dihydrofluorescein-diacetate; DTNB 5,5 -dithiobis 2-nitrobenzoic acid; FTI, farnesyltransferanse inhibitor; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GSH, glutathione; GTP, guanosine triphosphate; IFN, interferon- ; inos, inducible nitric oxide synthase; JNK, c-jun N-terminal kinase; NAPQI, N-acetyl-p-benzoquinone imine; PCR, polymerase chain reaction; ROS, reactive oxygen species; RT-PCR, reverse-transcription polymerase chain reaction; TNF-, tumor necrosis factor. From the Department of Biochemistry, Indian Institute of Science, Bangalore, India. Received May 6, 2009; accepted July 8, Supported by grants from the Government of India (to D. N.). B. S. received a student research fellowship from the Council of Scientific and Industrial Research. Address reprints requests to Dipankar Nandi, Department of Biochemistry, Indian Institute of Science, Bangalore , India. nandi@biochem. iisc.ernet.in; fax: Copyright 2009 by the American Association for the Study of Liver Diseases. Published online in Wiley InterScience ( DOI /hep Potential conflict of interest: Nothing to report. Additional Supporting Information may be found in the online version of this article. The liver is important for cellular metabolism and is highly susceptible to drug toxicity. Acetaminophen (APAP) is an analgesic and antipyretic drug that is used extensively for therapeutic purposes. It is readily absorbed by the gastrointestinal tract and metabolized by the cytochrome P450 dependent mixed-function oxidative enzyme pathway to form a reactive intermediate metabolite, N-acetyl-p-benzoquinone imine (NAPQI), which conjugates with glutathione (GSH) and is further metabolized. However, overdose of this drug leads to saturation of the conjugation pathways, and depletion of GSH results in NAPQI forming covalent bonds with protein and nonprotein thiols. 1 3 The binding of reactive metabolites to macromolecules present in the cellular milieu leads to hepatocyte death and increased release of liver alanine aminotransferase (ALT). 3 5 In fact, APAP-induced toxicity is a leading cause of both accidental and intentional poisoning and is a significant burden on the health care system

2 1548 SAHA AND NANDI HEPATOLOGY, November 2009 APAP-induced liver injury is an inflammatory process that is accompanied by increased infiltration and activation of immune cells. 6 Inflammatory cytokines also play an important role during APAP-induced acute liver injury. Tumor necrosis factor (TNF- ) and IL-1 are released in response to APAP intoxication and are responsible for pathological manifestations of APAP-induced liver injury. 7 Also, Ifn / mice resist APAP-induced liver injury, thus demonstrating a pivotal role for this cytokine. 8 Interestingly, oxidative stress plays an important role in this inflammatory process, and inhibition of reactive oxygen species (ROS) lowers liver injury. Currently, N-acetyl cysteine, the GSH substitute, is used to treat patients with APAP-induced liver injury. 2 There is great interest in studying the cellular processes and signaling events during APAP-mediated liver injury. Initially, NAPQI is produced followed by the depletion of GSH, but the hepatocytes remain viable. Subsequently, cells die over the next few hours due to oxidative stress and the opening of the mitochondrial permeability transition pore. 5 These events lead to a dramatic decline in mitochondrial bioenergetics and, ultimately, cell death. 3 5 Several studies have shown involvement of c-jun N-terminal kinase (JNK), a member of the stress-induced signaling pathway, during APAP-mediated cell death, using specific inhibitors and Jnk1 / and Jnk2 / mice Also, activation and association of JNK with mitochondria results in impaired mitochondrial bioenergetics followed by hepatocyte death. 13 These studies lead to an important question: How does APAP lead to JNK activation? A recent report has shown that apoptosis signalregulating kinase 1 lowers JNK activation during APAPinduced liver injury. However, the hepatoprotective effects with inhibition or lack of apoptosis signal-regulating kinase 1 were less compared with those observed with JNK inhibition or depletion. 14 Therefore, the contributions of other signaling molecules need to be investigated. Ras is a key signaling molecule which activates multiple signaling pathways and modulates several biological processes, including growth, transformation, differentiation, stress responses etc. 15 Previously, we had shown that Ras activation is responsible for the increase in ROS during interferon- (IFN- ) mediated in vitro growth suppression of the mouse hepatoma cell line, H6. 16 It was, therefore, important to assess the functional role of Ras activation in an in vivo disease or stress model. The APAPinduced liver injury was selected because it is dependent upon oxidative stress and the inflammatory response. A better understanding of the signaling pathways involved in this model of liver injury may lead to novel insights and possible therapeutic targets. In this study, we show that Ras is activated and is functionally important during oral dosing of mice with excess APAP. Notably, we have integrated our results with Ras activation together with expression and activation of other molecules, such as JNK, inducible nitric oxide synthase (inos), TNF-, and IFN-. This is the first study to demonstrate the role of nontoxic FTIs as therapeutic agents to ameliorate APAPinduced liver injury. Materials and Methods Chemicals. L-methionine and APAP were obtained from Sigma (St. Louis, MO). Manumycin A, gliotoxin, and GW4869 were purchased from Calbiochem (San Diego, CA). Peroxidase-conjugated goat anti-mouse or anti-rabbit antibodies were obtained from Jackson Immunoresearch Laboratories (West Grove, PA). Ethanol, ethylacetate, and trichloroacetic acid were purchased from S.D. Fine Chemicals (Mumbai, India). Guanidine- HCl and 2,4-dinitrophenylhydrazine were obtained from Himedia (Mumbai, India). Mice. Six- to eight-week-old BALB/c-mice (18-25 g) were obtained from the Central Animal Facility of the Institute. All experiments involving mice were performed according to institutional guidelines. Mice were starved overnight, and mg/kg APAP in autoclaved milliq was administered orally. Different amounts of L-methionine, manumycin A, gliotoxin, and GW4869 were administered intraperitoneally 1 hour after APAP dosing. ALT Measurements and Liver Histological Analysis. Liver injury was assessed by measuring serum ALT activity using a kit from Coral Clinical Systems (Goa, India). Liver tissue was dissected from mice treated under different conditions. Samples were fixed in 10% neutral formalin buffer and embedded in paraffin wax, and sections were stained with haematoxylin-eosin. Tissue sections were examined under a light microscope, and photographs were taken using a Nikon camera fitted to the microscope. Total RNA Isolation and Reverse-Transcription Polymerase Chain Reaction. Total RNA was extracted from liver samples using TRI reagent (Sigma Aldrich). Total RNA (10 g) was reverse-transcribed in a 40- L reaction mixture containing 50 U of MMLV-Reverse Transcriptase (New England Biolabs, Beverly, MA) and oligo dt (12-18mer) primer (GE Healthcare Life Sciences, Piscataway, NJ). Complementary DNA (2 L) was amplified using Taq polymerase (Bangalore Genei, Bangalore, India) in the presence of gene-specific primers (Supporting Table 1) with an optimal number of cycles at 94 C for 1 minute, optimal annealing temperature for 1 minute, and 72 C for 1 minute, followed by incubation at 72 C for 10 minutes. The polymerase chain reaction

3 HEPATOLOGY, Vol. 50, No. 5, 2009 SAHA AND NANDI 1549 (PCR) products were fractionated on a 1.5% agarose gel and visualized by ethidium bromide staining. To calculate the relative abundance of transcripts, the intensity of each band was determined by measuring the ethidium bromide fluorescence using Image Gauge software (Science Lab 2003; Fujifilm, Tokyo, Japan). All the intensities were calculated as a ratio of the respective genes and Hprt as control. Preparation of Liver Homogenates. Liver pieces were homogenized in homogenization buffer (50 mm Tris-HCl [ph 7.4], 150 mm NaCl, 1% Nonidet P40, 0.25% Na-deoxycholate containing protease inhibitors; Sigma) using a dounce homogenizer on ice. After centrifugation at 20,000g for 40 minutes at 4 C, supernatants were collected, snap-frozen in liquid nitrogen, and stored at 80 C for further analysis. Isolation of Liver Mitochondria. Liver sections were washed in phosphate-buffered saline and homogenized in a glass-teflon homogenizer in a homogenization buffer. The homogenate was centrifuged at 600g for 10 minutes at 4 C, and supernatant was collected and centrifuged at 7,000g for 10 minutes. The pellet was washed and resuspended in the homogenization buffer. Enriched mitochondria were assessed by the ratio of mitochondrial succinic acid dehydrogenase versus cytosolic lactate dehydrogenase activities 17. GSH Quantification. Ellman s reagent was used which involves the reduction of 5,5 -dithiobis 2-nitrobenzoic acid (DTNB) to a yellow product by sulfhdryl groups present in GSH. 18 Briefly, liver samples were homogenized in 1 ml 5% trichloroacetic acid, and homogenates were centrifuged at 10,000g for 30 minutes at 4 C. The protein content of trichloroacetic acid supernatants was quantified using Bradford s reagent. The supernatant (1 ml) was treated with 400 L of 2.5 mm DTNB and 1 ml of buffer containing 0.39 M Tris and M ethylene diamine tetraacetic acid (ph 8.9). The absorbance of the solutions was estimated at 412 nm against a blank using a spectrophotometer (Perkin-Elmer). The GSH content in samples corresponding to the absorbance were calculated using the extinction coefficient of DTNB (13,600 M 1 cm 1 ) and normalized for total protein content. Measurement of ROS. ROS production in enriched mitochondria was measured using 2,7 -dichloro-dihydrofluorescein-diacetate (DCFDA). Enriched mitochondria were resuspended in phosphate-buffered saline containing 5 M DCFDA, and incubated in the dark for an hour. DCF fluorescence was measured using a Perkin- Elmer spectrofluorimeter (monitoring emission, 520 nm; excitation wavelength, 485 nm). 19 Ras Activation. Briefly, 0.5 to 1 ml liver homogenate was incubated with 5 to 10 g Raf1-Ras binding domain agarose (glutathione S-transferase Ras binding domain fusion protein, corresponding to the human Ras binding domain, residues of Raf1) that binds to Ras-GTP (Millipore, Billerica, MA). The reaction mixture was kept at 4 C for 45 minutes with constant agitation, and the agarose beads were pelleted by way of centrifugation (10 seconds, 14,000g, 4 C), washed three times in Ras assay buffer, and resuspended in Laemmli reducing sample buffer. After boiling and centrifugation, the samples were loaded on a 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis gel. The proteins were transferred to a nitrocellulose membrane and probed with 1:2,000 anti- Ras. Detection was performed using 1:5,000 goat antimouse horseradish peroxidase conjugate (Jackson Laboratories) using chemiluminescence (ECL Detection, Millipore) and visualized on LAS-3000 (Fujifilm). Anti glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Millipore) was used at 1:2,000 dilution to verify equal protein loading per lane. 16 JNK Activation and inos Western Analysis. Liver homogenates ( g protein) were separated on a 10% SDS-PAGE and transferred to nitrocellulose membranes (Millipore) and the blots were blocked using 5% nonfat milk dissolved in phosphate-buffered saline containing 0.05% Tween 20. Blots were incubated with the desired primary antibodies (phospho-jnk, JNK, or inos), washed and incubated with secondary antibodies. Finally, the proteins were detected using ECL (Millipore). The software used to quantify the Ras-GTP, JNK, inos, and GAPDH amounts was the Multigauge software from Fujifilm. Cytokine Enzyme-Linked Immunosorbent Assay. The serum samples collected from the mice from different experiments were tested for the presence of TNF- and IFN- using enzyme-linked immunosorbent assay kits from ebioscience. The manufacturer s protocol was followed for the assay, and the linear range of detection was ,000 pg/ml. Statistical Analyses. Proper evaluations were performed using the Mann-Whitney U test for comparison between two groups, whereas multiple groups were compared using Kruskal-Wallis one-way analysis of variance. Graphpad Prism software was used for these analyses, and P 0.05 was considered significant. Results Ras Is Activated During APAP-Induced Liver Injury. To address whether Ras was activated during liver injury, mice were given 400 mg/kg APAP orally. Serum ALT amounts were detected after 2 hours and peaked between 6 and 12 hours after APAP treatment (Fig. 1A).

4 1550 SAHA AND NANDI HEPATOLOGY, November 2009 Fig. 1. Ras is activated during APAP-induced liver damage. Mice were orally dosed with 400 mg/kg of APAP for different time intervals, and blood and liver samples were taken for analysis. (A) Analysis of serum ALT levels was performed after APAP challenge. Data are expressed as the mean standard error (n 6 mice). *P (B) Liver homogenates were prepared after APAP dosing, and Ras-GTP was detected. GAPDH was used as a control for the amount of protein loaded. Ras-GTP levels in the control liver were taken as one, and the fold increase was calculated using densitometric analysis. Data are expressed as the mean standard error (n 5 mice). *P The amounts of APAP in the serum were measured in a time-dependent manner (Supporting Fig. 1). APAP was quickly absorbed after dosing and its amounts in sera were maximal within 15 minutes but greatly reduced by 4 hours. ALT was also measured from the identical sera samples, and the amounts increased after 4 hours. A clear inverse correlation between serum amounts of APAP and ALT was observed. In addition, histological examination revealed that some liver injury was seen at 6 hours but maximum injury was observed after 12 hours (Supporting Fig. 2). Mice given APAP showed increased Ras-GTP amounts in the liver as early as 2 hours with a maximum at 6 hours after treatment (Fig. 1B). Ras Activation Is Mediated by Oxidative Stress. The maturation of Ras involves the conversion of pro-ras to Ras, which enables binding to the plasma membrane. This process includes posttranslational modifications of which farnesylation plays a key role. Consequently, farnesyl transferase inhibitors (FTIs) prevent Ras maturation and binding to the plasma membrane. 20,21 Manumycin A is one such FTI; however, it also exhibits neutral sphingomyelinase inhibitor activity. To address whether the sphingomyelinase inhibitory activity of manumycin A plays any role during liver injury, GW4869, which is a specific neutral sphingomyelinase inhibitor, was also used for experimentation (Supporting Fig. 3). 22 To compare the effects of FTIs, L-methionine administration (250 mg/kg) was used as a positive control. L-methionine s probable mode of action is through direct reaction with oxygen-free radicals and enhancing the synthesis of new GSH, which reduces oxidative stress leading to decreased toxicity. 23 Administration of manumycin A, but not GW4869, in APAP-dosed mice led to decreased Ras-GTP amounts (Fig. 2A). Also, the increased serum ALT amounts observed upon APAP administration were reduced and statistically significant by manumycin A, but not GW4869, treatment (Fig. 2B). These results demonstrate that the FTI activity of manumycin A was responsible for lowering Ras activation and ALT amounts during APAP-induced liver injury. As expected, L-methionine administration reduced serum ALT amounts during APAP-induced liver injury (Fig. 2B). Interestingly, it also reduced Ras-GTP amounts (Fig. 2A), suggesting that the ROS generated during APAP-liver injury activates Ras, which is attenuated with L-methionine. Hepatoprotective Effect of Manumycin A. Histological analysis was performed to examine the effect of manumycin A on liver injury. Microscopic analysis of hematoxylin-eosin stained liver sections revealed normal hepatic architecture in control mice, whereas severe destruction of cell and tissue damage was observed in APAPtreated livers. Administration of manumycin A or L-methionine greatly reduced cell death and tissue damage compared with APAP (Fig. 3). These observations demonstrate that manumycin A reduced the progression and development of the APAP-mediated liver injury. Manumycin A Does Not Affect GSH Depletion but Reduces Late Mitochondrial ROS Induction by APAP. An important point to address was whether manumycin A inhibited the generation of the NAPQI, because this would affect the entire pathway. 24 It is known that NAPQI rapidly depletes GSH, and the extent of GSH

5 HEPATOLOGY, Vol. 50, No. 5, 2009 SAHA AND NANDI 1551 Fig. 2. Manumycin A, an FTI, lowers Ras activation and APAP-induced ALT. Mice were given APAP and, after 1 hour, treated with GW4869 (1 mg/kg), manumycin A (1 mg/kg), or L-methionine (250 mg/kg). (A) Ras activation was determined in liver homogenates. Data are expressed as the mean standard error (n 5 mice). *P (B) ALT amounts were determined in sera after 6 hours (top) and 12 hours (bottom) after APAP dosing. Data are expressed as the mean standard error (n 3 to 4 mice). *P 0.05 for 6 hours is shown. depletion prior to the onset of toxicity is a good biomarker for APAP metabolism. 5,11,24 Therefore, reduction in hepatic GSH amounts is considered an indirect readout of NAPQI formation. Mice were given oral doses of APAP, liver samples were dissected, and GSH amounts were determined. Total hepatic GSH amounts decreased rapidly within 2 hours of dosing with APAP, but gradually recovered with time (Fig. 4A). Similarly, mitochondrial GSH amounts were also reduced, although there was a slight lag and maximal drop was observed after 4 hours of APAP dosing (Fig. 4B). Increased total amounts of GSH were detected upon injection of L-methionine after 1 hour of APAP dosing. This observation is consistent with the known role of L-methionine to increase cellular amounts of GSH. 23 However, L-methionine did not increase mitochondrial GSH levels. The reduction in total and mitochondrial hepatic GSH amounts in mice injected with manumycin A 1 hour after oral APAP dosing was similar to that observed in control mice. Thus, it is unlikely that manumycin A affected APAP biotransformation. Furthermore, these results demonstrated that the mechanism of action of L-methionine and manumcyin A (Fig. 4A) were clearly distinct. The reduction in mitochondrial GSH amounts is known to increase ROS. 13 The effect of manumycin A on APAP-induced mitochondrial ROS production was studied. Mitochondrial ROS amounts increased gradually with time after APAP dosing, 13 and the enhanced ROS was from mitochondria as it was inhibited by rotenone, the mitochondrial complex I inhibitor (data not shown). Interestingly, no differences in mitochondrial ROS amounts were observed during the first 4 hours in mice dosed with APAP and treated with either L-methionine or manumycin A. However, at a later time point (6 hours), mitochondrial ROS amounts were reduced with L-methionine or manumycin A treatment (Fig 4C). Manumycin A Inhibits APAP-Induced Activation of Jnk1 and Jnk2 in Liver. JNK activation is important during APAP-induced liver injury 9 13 ; however, the major upstream molecules that activate JNK are still not identified. Because Ras is a well-known mediator of different mitogen-activated protein kinase signaling pathways, 15 we investigated this relationship in this system. The JNK phosphorylation pattern was studied in a kinetic manner, and phosphorylated Jnk1 (46 kda) and Jnk2 (54 kda) were detected early (after 2 hours of APAP treat-

6 1552 SAHA AND NANDI HEPATOLOGY, November 2009 Fig. 3. Histological analysis reveals the hepatoprotective effect of manumycin A. Mice were dosed with APAP and, after 1 hour, treated with L-methionine (250 mg/kg) or manumycin A (1 mg/kg). Liver samples were dissected and hematoxylin-eosin staining was performed. A representative histopathological examination is shown (magnification 250). ment) and were sustained until 12 hours (Fig. 5A). Notably, reduced phosphorylated Jnk1 and Jnk2 were observed in liver samples treated with manumycin A or L-methionine (Fig. 5B). These observations suggest that Ras activation leads to JNK phosphorylation, because it was inhibited by manumycin A. Effect of Manumycin A on APAP-Induced Gene Expression of Molecules Involved in the Inflammatory Process in Liver. APAP treatment led to enhanced gene expression of inflammatory molecules, including inos, gp91phox, and Fasl (Fig. 6Ai). Interestingly, APAP-enhanced gene expression was observed at later time points (6-12 hours after APAP dosing). This observation suggests that the initiation of liver injury led to APAP-induced gene expression. Therefore, it should be possible to reduce APAP-induced gene expression by inhibiting processes that occur earlier (such as Ras activation). Indeed, the induction of inos, gp91phox, and Fasl was reduced with manumycin A or L-methionine treatment (Fig. 6Aii). These effects were confirmed at the protein level, and elevated inos was observed upon APAP dosing (Fig. 6Bi), which was reduced after manumycin A or L-methionine administration (Fig. 6Bii). Manumycin A Treatment Reduces the Induction of Inflammatory Cytokines During Liver Injury. APAPinduced liver injury is also associated with increase in the amounts of proinflammatory cytokines.7,8 Initially, the gene expression of two proinflammatory cytokines, Ifn and Tnf, was investigated. The messenger RNA expression of these cytokines was enhanced by 6 to 12 hours after APAP dosing (Fig. 7Ai). Also, manumycin A or L-methionine treatment reduced the induction of these genes by APAP (Fig. 7Aii). Next, cytokine amounts were measured in the sera, which confirmed that maximal amounts of IFN- and TNF- were observed after 12 hours of APAP dosing (Fig. 7B,C). Furthermore, manumycin A or L-methionine treatment reduced APAP-induced IFN- and TNF- amounts in the sera. Because a significant increase in these cytokines was observed after 12 hours of APAP treatment, it is possible that increased Ras activation led to an oxidative environment that induced gene expression and increased serum amounts of proinflammatory cytokines (Fig. 7B,C). Taken

7 HEPATOLOGY, Vol. 50, No. 5, 2009 SAHA AND NANDI 1553 Discussion The role of Ras activation during liver injury caused by an overdose of APAP was investigated in this study. There are several important aspects of this in vivo liver injury model. First, mice were given APAP orally, which is physiologically relevant. Second, the expression of important signaling molecules, cytokines, ALT, and so forth, was studied in a kinetic manner. Third, the ability of L-methionine or FTIs to ameliorate liver injury initiated 1 hour after APAP challenge (i.e., therapeutic efficacy) was evaluated. Increased Ras activation was observed concomitant with increased liver injury, as measured by the presence of serum amounts of ALT, which is released by damaged Fig. 4. Manumycin A does not affect APAP-induced GSH depletion but reduces mitochondrial ROS amounts at a later time point. Mice were dosed with APAP and after 1 hour treated with manumycin A (1 mg/kg) or L-methionine (250 mg/kg). Liver samples were dissected at the indicated time points and (A) GSH amounts were measured in the total liver homogenate (B) mitochondria. (C) Liver mitochondria were incubated with DCFDA and ROS amounts were measured. Data are expressed as the mean standard deviation (n 3-4 mice). *P 0.05 versus APAP treatment alone. together, manumycin A reduced multiple APAP-induced inflammatory responses, which play an important role in liver injury. Gliotoxin Ameliorates APAP-Mediated Liver Injury. The effect of gliotoxin, a structurally distinct FTI (Supporting Fig. 3), 20 was evaluated during APAP-induced liver injury. Giotoxin reduced the increased Ras activation upon APAP dosing (Supporting Fig. 4A). In addition, histological analysis confirmed the hepatoprotective effect of gliotoxin in response to APAP dosing (Supporting Fig. 4B). Both manumycin A and gliotoxin reduced serum amounts of ALT and IFN- in a dose-dependent manner after APAP dosing (Supporting Fig. 5). The IC 50 values, in terms of reducing ALT and IFN- for manumycin A and gliotoxin were calculated to be mg/kg, mg/kg, and mg/kg, mg/kg, respectively. These experiments demonstrate the therapeutic efficacy of FTIs during APAP-induced liver injury. Fig. 5. Phosphorylation of JNK during APAP-induced liver damage is reduced by manumycin A. (A) Phospho-JNK levels were determined after oral dosing of mice with APAP. Phospho-JNK levels in the control liver was treated as one and the fold increase was calculated using densitometric analysis, as depicted in the bar diagram. Data are expressed as the mean standard error (n 4 mice). *P (B) Mice were dosed with APAP and, after 1 hour, one group of mice were treated with manumycin A (1 mg/kg) or L-methionine (250 mg/kg). Liver samples were collected after 6 hours, and western analysis of phospho-jnk and total JNK was performed. Data are expressed as the mean standard error (n 4 mice). *P 0.05.

8 1554 SAHA AND NANDI HEPATOLOGY, November 2009 Fig. 6. Manumycin A reduces the induction of inos and FasL during APAP-induced liver injury. (A) (i) Mice were dosed with APAP for different time intervals, liver samples were collected, and total RNA was isolated followed by complementary DNA synthesis and PCR. The mean fold modulation of genes with respect to Hprt for inos, gp91phox, FasL, and Fas was , , , and , respectively (n 5 mice). *P (ii) Mice were dosed with APAP alone or treated with manumycin A (1 mg/kg) or L-methionine (250 mg/kg). Liver samples were collected and gene expression was studied using reverse-transcription PCR (RT-PCR) was performed 6 hours after APAP dosing. (B) The protein amounts of inos under indicated conditions were determined by way of western blotting. Data are representative of one of the five mice in each group. hepatocytes (Fig. 1). To distinguish the role of Ras as the cause or the effect of liver injury, its activation was studied in liver samples from mice treated with APAP with or without L-methionine, which greatly reduces liver injury. Interestingly, Ras-GTP levels induced by APAP were reduced by L-methionine treatment (Fig. 2), demonstrating the role of oxidative stress in induction of Ras-GTP. Some cellular responses (e.g., activation of Ras and JNK and serum ALT amounts) increased within 2 to 6 hours. Most likely, the initial depletion of GSH caused oxidative stress leading to the initiation of liver injury (Figs. 4 and 5). Subsequently, activation of signaling cascades lead to enhanced gene expression of inos, gp91 phox, FasL, Tnf- and Ifn- (Fig. 6). Analysis of serum samples confirmed that TNF- and IFN- increased greatly at later time points after APAP treatment (Fig. 7). These latter changes result in severe liver injury (12 hours) as shown by tissue histological analysis (Fig. 3). These data have been incorporated as a model (Fig. 8) in which the initial liver injury leads to changes in gene expression and the increased inflammatory response results in greater liver injury. To evaluate the functional role of Ras activation, the effect of FTIs in liver injury due to overdose of APAP was investigated. Although FTIs affect other farnesylated proteins, such as nuclear lamin A, they reduce Ras signaling by direct measurements. 20,25 The specificity of FTIs and the issue of structural controls was addressed in two ways. Manumycin A is extensively used as an FTI, but it is also an in vitro inhibitor of neutral sphingomyelinase activity. 22 Therefore, the functional role of GW4869, which is a specific neutral sphingomyelinase inhibitor, was investigated. GW4869, unlike manumycin A, does not inhibit Ras activation and prevent liver injury (Fig. 2), which demonstrates that the FTI activity of manumycin A is involved in ameliorating liver damage. To confirm this observation, the functional role of another FTI, gliotoxin, which is structurally distinct from manumycin A, was investigated. Gliotoxin also inhibited Ras activation (Supporting Fig. 4A) and reduced serum amounts of ALT and IFN- during APAP-induced liver injury (Supporting Fig. 5). The IC 50 of manumycin A and gliotoxin with respect to reducing ALT and IFN- amounts during APAP-induced liver damage was comparable (Supporting Fig. 5). Together, these studies clearly demonstrate the functional role of FTIs in ameliorating APAP-induced liver injury. The ability of the liver to regenerate after injury is important during the recovery phase. One possibility is that FTIs may reduce liver injury by increasing liver regeneration; however, this appears to be unlikely. First, the kinetics of liver regeneration is late compared with the damage inflicted by APAP challenge It takes time for the liver to recover after injury and optimal regeneration occurs 72 hours after APAP dosing of mice. 26,27 Sec-

9 HEPATOLOGY, Vol. 50, No. 5, 2009 SAHA AND NANDI 1555 Fig. 7. The serum amounts of two inflammatory cytokines are reduced by manumycin A during APAP-induced liver damage. (A) (i) APAP-dosed mice were sacrificed after different time intervals (2, 6, 12 hours) and the expression of Ifn and Tnf genes in the liver was studied using RT-PCR. The mean fold modulation after 12 hours of APAP treatment was calculated to be and for Ifn and Tnf, respectively (n 5 mice). (ii) Liver samples from mice treated with APAP alone or treated with manumycin A or L-methionine were dissected, and RT-PCR was performed 6 hours after APAP dosing. Data are representative of one of five mice from each of the treatment groups. (B) IFN- and (C) TNF- levels were determined in the sera of mice that underwent different treatments. Each of the dots represent the data from a single mouse (n 5-7 mice per group). *P ond, activated Ras is induced in proliferating hepatocytes during liver regeneration. 29 Consistent with the role of Ras during cellular proliferation, inhibition of Ras decreases proliferation of hepatocytes. 30 This aspect is important as it has been shown that inhibition of liver regeneration lowers the ability to recover from APAPinduced liver damage. 26,28 Finally, it is important to point out that the efficiency of FTIs, like L-methionine, to reduce liver injury after APAP treatment was restricted to early time points after a single injection (Supporting Fig. 6). Therefore, it is unlikely that FTIs would inhibit APAP-induced liver damage by rapidly increasing liver regeneration. It is more likely that inhibition of Ras activation inhibits a major signaling pathway that lowers inflammatory responses leading to lower liver damage. This aspect is clinically relevant, and increased chances of recovery are observed in cases where liver injury is detected early and treated with N-acetyl cysteine. 28 Although both L-methionine and manumycin A treatment reduced APAP-induced liver injury, they appear to function in distinct manners. L-methionine acts in two ways: (1) it increases total cellular pools of GSH, which reduces the damage caused by NAPQI, and (2) it prevents signaling events, such as Ras activation (Fig. 2A), which are required for sustaining high mitochondrial ROS amounts. On the other hand, manumycin A does not

10 1556 SAHA AND NANDI HEPATOLOGY, November 2009 inflammatory responses (Figs. 4-7). These findings demonstrate that targeting Ras-GTP may be an effective strategy to treat pathological inflammation in the liver during injury. The relationship between Ras and the inflammatory response is an emerging area of investigation. Recent studies have shown the role of Ras in different inflammatory processes. 25,33 The anti-inflammatory effects of FTIs are important in several diseases, including collagen-induced arthritis 34 and atherosclerosis. 35 In addition, FTIs have been shown to inhibit corneal inflammation by inhibiting macrophage function and decreasing their survival. 36 The present findings add APAP-induced liver injury to the growing list of inflammatory pathological conditions that can be ameliorated by inhibiting Ras signaling. Fig. 8. A model depicting the key events that occur during APAPmediated liver damage. The early events in this process include GSH depletion, Ras and JNK activation, and mitochondrial ROS production, which initiate liver injury. The later phase is characterized by enhanced gene expression of inflammatory molecules and cytokine production that cause extensive liver damage. affect cellular amounts of GSH and early mitochondrial ROS production. The reduction in mitochondrial ROS amounts by manumycin A at a later stage may be due to its ability to reduce signaling events either directly (by way of Ras activation) or indirectly (by way of JNK activation). The observation that L-methionine and manumycin A inhibited mitochondrial ROS at a later time point is important, because signaling pathways need to remain activated to sustain mitochondrial ROS production. This observation agrees well with the two stages required for the action of JNK during APAP-induced liver injury. 13 Ras has been shown to mediate its effect in terms of both its antiapoptotic and proapoptotic properties by regulating the amounts of ROS. 31,32 Most likely, the initial oxidative stress results in Ras activation which, subsequently, enhances ROS generation. 16 Previously, JNK activation was shown to lead to increased mitochondrial ROS. 13 This study clearly shows that Ras is upstream of JNK activation during this process as manumycin A treatment lowers JNK activation. Overall, it appears that manumycin A reduced APAP-induced liver injury by lowering multiple inflammatory responses by directly reducing Ras activation and indirectly reducing JNK activation and other signaling pathways, leading to lowered Acknowledgment: We thank the Central Animal Facility, IISc, for providing mice for experimentation. The generous help and support by A. Ahmed, Manikandan P., M. Deobagkar, S. Chacko, V. Nandi, A. Deshpande, S. Chatterjee, and other members of the DpN laboratory are greatly appreciated. References 1. Kaplowitz N. Idiosyncratic drug hepatotoxicity. Nat Rev Drug Discov 2005;4: Larrey D, Pageaux GP. Drug-induced acute liver failure. Eur J Gastroenterol Hepatol 2005;17: Jaeschke H, Bajt ML. Intracellular signaling mechanisms of acetaminophen-induced liver cell death. Toxicol Sci 2006;89: Gujral JS, Knight TR, Farhood A, Bajt ML, Jaeschke H. Mode of cell death after acetaminophen overdose in mice: apoptosis or oncotic necrosis? Toxicol Sci 2002;67: Kon K, Kim JS, Jaeschke H, Lemasters JJ. Mitochondrial permeability transition in acetaminophen-induced necrosis and apoptosis of cultured mouse hepatocytes. HEPATOLOGY 2004;40: Jaeschke H. Role of inflammation in the mechanism of acetaminopheninduced hepatotoxicity. Expert Opin Drug Metab Toxicol 2005;1: Blazka ME, Elwell MR, Holladay SD, Wilson RE, Luster MI. Histopathology of acetaminophen-induced liver changes: role of interleukin 1 alpha and tumor necrosis factor alpha. Toxicol Pathol 1996;24: Ishida Y, Kondo T, Ohshima T, Fujiwara H, Iwakura Y, Mukaida N. A pivotal involvement of IFN-gamma in the pathogenesis of acetaminopheninduced acute liver injury. FASEB J 2002;16: Gunawan BK, Liu ZX, Han D, Hanawa N, Gaarde WA, Kaplowitz N. c-jun N terminal kinase plays a major role in murine acetaminophen hepatotoxicity. Gastroenterology 2006;131: Henderson NC, Pollock KJ, Frew J, Mackinnon AC, Flavell RA, Davis RJ, et al. Critical role of c-jun (NH2) terminal kinase in paracetamol-induced acute liver failure. Gut 2007;56: Latchoumycandane C, Goh CW, Ong MM, Boelsterli UA. Mitochondrial protection by the JNK inhibitor leflunomide rescues mice from acetaminophen-induced liver injury. HEPATOLOGY 2007;45: Bourdi M, Korrapati MC, Chakraborty M, Yee SB, Pohl LR. Protective role of c-jun N-terminal kinase 2 in acetaminophen-induced liver injury. Biochem Biophys Res Commun 2008;374: Hanawa N, Shinohara M, Saberi B, Gaarde WA, Han D, Kaplowitz N. Role of JNK translocation to mitochondria leading to inhibition of mito-

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