Allyl Alcohol Activation of Protein Kinase C δ Leads to Cytotoxicity of

Jane F. Maddox*, Robert A. Roth, and Patricia E. Ganey ... Akshata Moghe , Smita Ghare , Bryan Lamoreau , Mohammad Mohammad , Shirish Barve , Craig ...
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Chem. Res. Toxicol. 2003, 16, 609-615

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Allyl Alcohol Activation of Protein Kinase C δ Leads to Cytotoxicity of Rat Hepatocytes Jane F. Maddox,* Robert A. Roth, and Patricia E. Ganey Department of Pharmacology and Toxicology, Michigan State University, East Lansing, Michigan 48824 Received November 4, 2002

Hepatotoxicity of allyl alcohol involves its bioactivation to acrolein and subsequent protein sulfhydryl loss and lipid peroxidation. However, the links between these events and hepatocellular death are not known. The purpose of these studies was to examine whether specific signal transduction pathways are associated with allyl alcohol toxicity in hepatocytes. Inhibition or augmentation of cyclic AMP and/or protein kinase A (PKA) by Rp-Ado-3N,5N-cyclic monophosphorothioate triethylamine salt or 3-isobutyl-1-methylxanthine had no effect on allyl alcohol-induced cell death. H-7, an inhibitor of PKA, PKC, and PKG, partially inhibited cell killing by allyl alcohol, whereas chelerythrine chloride, a nonselective PKC inhibitor, almost completely abolished allyl alcohol cytotoxicity. Neither 2,2N,3,3N,4,4N-hexahydroxy-1,1N,biphenyl-6,6N-dimethanol-dimethyl ether, a selective PKC R and β inhibitor, nor bisindolylmaleimide I, an inhibitor of PKC R, β, and , had any effect on allyl alcohol cytotoxicity. In contrast, rottlerin, a selective PKCδ inhibitor, blocked hepatocellular killing by allyl alcohol. Cytoprotection by chelerythrine chloride and rottlerin was not the result of inhibition of bioactivation of allyl alcohol because each inhibitor also prevented cell death from acrolein. Western blotting and immunohistochemical techniques revealed that allyl alcohol stimulated phosphorylation and translocation of PKCδ to hepatocyte membranes (i.e., activation), and this activity was inhibited by rottlerin. Cell death appeared to occur via oncotic necrosis rather than apoptosis based on single-stranded DNA ELISA and propidium iodide staining. Together, these results indicate that activation of PKCδ is a critical, early event in initiating hepatocyte injury and death from allyl alcohol.

Introduction Allyl alcohol (CH2dCHCH2OH) is widely used in manufacturing processes in food flavoring and fire retardant industries and is well-known as a hepatotoxicant (reviewed in 1). Oxidation of allyl alcohol to the active metabolite acrolein by alcohol dehydrogenase is necessary for hepatotoxicity to occur. If bioactivation of allyl alcohol is suppressed by inhibition of alcohol dehydrogenase with pyrazole, hepatic necrosis is prevented in vivo (2) and cytotoxicity is prevented in hepatocytes in vitro (3). Humans can also be exposed directly to acrolein from cigarette smoke, vehicle exhaust, and cyclophosphamide chemotherapy. Although acrolein is a reactive electrophile, the mechanism(s) by which hepatocellular damage and death occur upon exposure to allyl alcohol remains in question. Previous studies have suggested lipid peroxidation, depletion of reduced glutathione, or loss of protein sulfhydryls as general processes that may lead to hepatocellular toxicity; however, few proteins or processes have been specifically identified as being altered in function by allyl alcohol. A few studies have emerged regarding mechanisms by which acrolein may induce cell death. At nonlethal concentrations, acrolein significantly reduced activation of transcription factors NF-κB1 and AP-1 and inhibited proliferation of adenocarcinoma cells (4, 5). At a larger concentration, acrolein * To whom correspondence should be addressed. Tel: 517-432-6324. Fax: 517-432-2310. E-mail: [email protected].

induced phosphorylation and activation of epidermal growth factor receptor (EGFR) in keratinocytes, which led to cell death (6). On the other hand, in neutrophils, acrolein inhibited phosphorylation of the p38 ERK MAPK pathway and prevented apoptosis in these cells by preventing the activation of caspase-3 (7). Thus, seemingly disparate effects of acrolein on a few divergent pathways have been identified in various cell types, but a pathway to cell death remains to be identified in hepatocytes. The following studies were designed to examine mechanisms by which allyl alcohol causes hepatocyte death. We hypothesized that allyl alcohol affects critical signal transduction pathways, such as protein kinases, leading to death of hepatocytes. PKA and cyclic AMP activities have been implicated in both cell death and cell survival (8, 9). In hepatocytes in particular, cAMP-mediated events protected cells from death in donor livers for transplantation (10). The PKC family of Ser/Thr kinases participates in intracellular signaling that regulates diverse pathways, from cell growth to differentiation and death (11-15). Isoforms of PKC have been divided into three categories: (i) conventional (R, β, γ), which are 1 Abbreviations: ALT, alanine aminotransferase; DAG, diacylglycerol; HBDDE, 2,2N,3,3N,4,4N-hexahydroxy-1,1N,-biphenyl-6,6Ndimethanol-dimethyl ether; ERK, extracellular signal-regulated kinase; IBMX, 3-isobutyl-1-methylxanthine; MAPK, mitogen-activated protein kinase; NF-κB, nuclear factor-κB; PKA, protein kinase A; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; Rp-cAMPs, Rp-Ado3N,5N-cyclic monophosphorothioate triethylamine salt.

10.1021/tx025655n CCC: $25.00 © 2003 American Chemical Society Published on Web 04/16/2003

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calcium-dependent and activated by DAG or phorbol esters; (ii) novel (δ, , η, θ), which are calcium-independent and DAG- or phorbol ester-activated; and (iii) atypical (ζ, λ), which are calcium-independent and are not activated by DAG or phorbol esters. Among PKC isoforms, PKCδ activation, in particular, has been reported to play a role in both apoptotic and oncotic cell death (13). We investigated the effects of signal transduction inhibitors on allyl alcohol- or acrolein-induced cell death in isolated hepatocytes to identify specific pathways involved in the cytotoxicity. Via this approach, PKCδ was identified as critical to the cytotoxicity; therefore, activation of this protein was analyzed more specifically, and the mode of cell death was examined.

Materials and Methods Materials. Bisindolylmaleimide I was from Calbiochem (La Jolla, CA); propidium iodide was purchased from Molecular Probes, Inc. (Eugene, OR); In Situ Cell Death Detection Kit POD was from Roche Diagnostics Corporation (Indianapolis, IN); monoclonal antibody to PKCδ (Clone 14) was from Pharmingen (San Diego, CA); and horseradish peroxidase-conjugated goat antimouse antibody was from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Chelerythrine chloride, 1-(5-isoquinolinesulfonyl)-2-methyl-piperazine‚2HCl (H-7), HBDDE, IBMX, rottlerin, and Rp-cAMPs were obtained from Biomol Research Laboratories, Inc. (Plymouth Meeting, PA). All other chemicals were obtained from Sigma-Aldrich Chemicals (St. Louis, MO). Animals and Hepatocyte Isolation. Male, Sprague-Dawley rats (Crl:CD (SD)IGS BR; Charles River, Portage, MI) weighing 125-175 g were used for hepatic parenchymal cell isolation. Rats received humane care according to the criteria outlined in the Guide for the Care and Use of Laboratory Animals prepared by the National Academy of Sciences and published by the National Institutes of Health (NIH publication 86-23, revised 1985). They were housed under conditions of controlled temperature, humidity, and light (12 h dark, 12 h light). Rats were allowed food (Harlan Teklad Rodent Diet 8640) and water ad libitum. Hepatocytes were isolated from the livers of rats by collagenase perfusion using a modification of the method of Seglen (16) as described previously (17). Hepatocyte preparations were >85% viable as determined by exclusion of trypan blue. Cells were placed in Williams’ medium E supplemented with 10% fetal calf serum and 0.5 mg/mL gentamycin and plated either in 96 well tissue culture plates at a density of 1.2 × 104 cells per well or in 100 mm tissue culture dishes or collagen-coated, chambered glass slides at a density of 2.5 × 106 cells per dish or chamber. After a 2-3 h adherence period at 37 °C, the medium was removed and replaced with Williams’ medium E supplemented only with 0.5 mg/mL gentamycin. Assessment of Cytotoxicity and Effects of Pharmacologic Agents. Cells were exposed to various compounds for 0-2 h at 37 °C (see figure legends for details) before addition of allyl alcohol (0-100 µM) or acrolein (0-125 µM). They were then incubated for 90 min at 37 °C. Hepatocellular injury was assessed by measuring the release of ALT into the medium. After 90 min exposure to allyl alcohol or acrolein (18), 50 µL of the medium was transferred to a clean 96 well plate. The remaining medium was aspirated, and the cells were lysed with a 1.0% solution of Triton X-100. The activity of ALT was determined in both the medium and the lysates using Sigma Diagnostics kit no. 59. The total cellular ALT activity was calculated from the activity in the medium plus the activity in the lysate, and hepatocellular injury is presented as the percent of total cellular ALT released into the medium. PKCδ Immunoblotting. Hepatocytes in 100 mm tissue culture dishes were exposed to allyl alcohol (75 µM) or vehicle (medium) for 30 min to 2 h at 37 °C. Cells were washed once

Maddox et al. with ice-cold tris/sucrose buffer (20 mM Tris, 250 mM sucrose, pH 7.3) and scraped from the plate in 1 mL of the same buffer with protease inhibitors (Complete, Roche) added. The cells were snap-frozen in liquid N2, thawed, and sonicated (20% power, 2 × 15 s). Cell membranes were separated by centrifuging the cell lysates at 100 000g for 40 min at 4 °C. Supernatants were removed and used as cytosolic fractions, and membrane pellets were resuspended in 250 µL of tris buffer containing protease inhibitors. Protein concentrations of the fractions were determined using the BCA protein assay reagent (Pierce, Rockford, IL). Samples were stored at -80 °C until use. Proteins (30 µg) from the membrane and cytosolic fractions were mixed with SDS loading buffer and subjected to SDS-PAGE on a 10% acrylamide gel. Proteins were electrotransferred onto PVDF membranes. Membranes were blocked with 5% dry milk in trisbuffered saline plus 0.1% Tween 20 (TBST) overnight at 4 °C and were then incubated with the primary antibody to PKCδ for 1 h in TBST plus 5% dry milk at room temperature. Membranes were washed five times in TBST and incubated with a horseradish peroxidase-conjugated secondary antibody for 1 h. Protein bands were visualized by enhanced chemiluminescence (ECL, Amersham, Piscataway, NJ). The bands were analyzed by densitometry using a Chemi Doc Documentation System scanner (BioRad, Hercules, CA) and Quantity One software. Immunohistochemistry. Hepatocytes plated on collagencoated glass slides were exposed to rottlerin or vehicle for 2 h and treated with PMA, allyl alcohol, or vehicle for 1.5 h and then fixed with methanol:acetone (1:1). Fixed and washed cells were blocked with 5% goat serum and incubated with anti-PKCδ for 1 h at room temperature. Slides were then washed with PBS and incubated with goat antimouse fluorescent antibody (Alexa Fluor 488 GAM, Molecular Probes, Eugene, OR) for 1 h at room temperature. Cells were visualized using a fluorescent microscope, and digital images were obtained. TUNEL assays were performed using the In Situ Cell Death Detection (POD) Kit (Roche), and cells were visualized using a light microscope. Propidium iodide staining was completed by exposing cells to 500 nM propidium iodide for 4 min, washing with 2 × SSC buffer (0.3 M NaCl, 30 mM sodium citrate, pH 7.0), and visualizing with a fluorescent microscope. Single-Stranded DNA ELISA. Hepatocytes were seeded in 96 well plates at a density of 1.4 × 104/well and allowed to adhere in serum-containing medium for 2-3 h. The medium was then changed to serum-free as described above. Cells were exposed to allyl alcohol (25, 50, 75, and 100 µM) and incubated for 1.5 or 3 h at 37 °C. In parallel, wells were prepared in triplicate for assay of ALT release and single-stranded DNA (ssDNA) ELISA. For analysis of apoptosis, cells were fixed with 80% methanol and dried at room temperature. The ssDNA ELISA assay was performed according to the manufacturer’s instructions (Chemicon International, Temecula, CA). Cells for ALT assay were treated as described above. Statistical Analysis. Results are presented as the mean ( SEM. Data presented as percentages were first transformed (arcsin transformation) before analysis by repeated measures ANOVA. In all cases, the criterion for significance was p < 0.05.

Results Allyl alcohol caused a concentration-dependent release of ALT from isolated hepatocytes (Figure 1). H-7, which inhibits Ser proteases PKA, PKC, PKG, and myosin light chain kinases, reduced the cytotoxic activity of allyl alcohol in hepatocytes (Figure 1A). At 50 µM, a concentration well above the Ki for PKA, PKC, and PKG (3, 6, and 5.8 µM, respectively), H-7 decreased ALT release caused by 75 and 100 µM allyl alcohol by 28-38%. No further reduction in cytotoxicity was observed when the concentration of H-7 was increased to 100 µM. To determine more specifically whether PKA or PKC inhibi-

Allyl Alcohol Activates PKCδ in Hepatocytes

Figure 1. Protein kinase inhibitors reduce allyl alcoholmediated cytotoxicity. Hepatocytes were plated in 96 well tissue culture plates and exposed to (A) 50 or 100 µM H-7, n ) 3; (B) 5 µM Rp-cAMPs, n ) 3; or (C) 6 µM chelerythrine chloride, n ) 3-5, for 2 h. Cells were then exposed to 0-100 µM allyl alcohol for 90 min. Leakage of ALT was measured as a determinant of cytotoxicity. a ) significantly different from 0 µM allyl alcohol; b ) significantly different from vehicle at the same concentration of allyl alcohol, p < 0.05.

tion was causing this decrease, cells were treated with more selective signal transduction inhibitors. Rp-cAMPs, a selective, competitive inhibitor of cAMP-dependent protein kinase (i.e., PKA), had no effect on allyl alcohol hepatocyte toxicity (Figure 1B). In addition, IBMX, which increases cAMP via inhibition of phosphodiesterases, also had no effect on allyl alcohol cytotoxicity (data not shown). These results suggested that PKA was not involved in allyl alcohol signal transduction and hepatocyte death. In contrast, chelerythrine chloride, a selective inhibitor of PKC, almost completely prevented allyl alcohol-induced hepatocyte death (Figure 1C), indicating that PKC activation is critical to this response. In an attempt to determine more precisely which of the PKC isoforms was involved in allyl alcohol cytotox-

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Figure 2. Inhibition of PKCδ decreases allyl alcohol-induced cytotoxicity in hepatocytes. Hepatocytes were plated in 96 well tissue culture plates and exposed to (A) 100 µM HBDDE, n ) 3; (B) 100 nM bisindolylmaleimide I, n ) 3; or (C) 10 µM rottlerin, n ) 8, for 2 h, followed by exposure to 0-75 µM allyl alcohol for an additional 90 min. Leakage of ALT was measured as a determinant of cytotoxicity. a ) significantly different from 0 µM allyl alcohol; b ) significantly different from vehicle at the same concentration of allyl alcohol, p < 0.05.

icity, isoform-selective inhibitors were added to hepatocytes prior to treatment with allyl alcohol. HBDDE, a selective inhibitor of PKCR and PKCγ, did not significantly inhibit allyl alcohol cytotoxicity when used at concentrations double the IC50 for both enzyme isoforms (Figure 2A). Similarly, bisindolylmaleimide I, a PKCR, β, and  isoform inhibitor, had no effect on hepatocyte cell death caused by allyl alcohol (Figure 2B). In contrast, rottlerin, a selective inhibitor of PKCδ, almost completely blocked allyl alcohol-induced cytotoxicity in hepatocytes at all concentrations of allyl alcohol tested (Figure 2C). Inhibition of cytotoxicity by rottlerin alone suggested that PKCδ was the PKC isoform that participates in allyl alcohol-induced hepatocyte death. It was necessary to determine whether chelerythrine chloride and/or rottlerin were acting via interfering with the conversion of allyl alcohol to its active metabolite,

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Figure 4. Allyl alcohol induces a change in PKCδ immunostaining in isolated hepatocytes. Hepatocytes were plated on collagen-coated glass slides and exposed to vehicle, PMA (1 µM), allyl alcohol (100 µM), or acrolein (100 µM) for 30 min. Some cells were preincubated with rottlerin (10 µM) for 2 h or with 4-methylpyrazole (1 mM) for 10 min. Cells were fixed in MeOH: acetone (1:1) and stained with PKCδ antibody and AlexaFluor 488 (Molecular Probes) secondary antibody. Photographs are representative of three experiments with similar results.

Figure 3. PKCδ selective inhibitor reduces acrolein-mediated cytotoxicity. Hepatocytes were plated in 96 well tissue culture plates and exposed to (A) 6 µM chelerythrine chloride, n ) 3; or (B) 10 µM rottlerin, n ) 3, for 2 h, followed by exposure to 0-125 µM acrolein for an additional 90 min. Leakage of ALT was measured as a marker of cytotoxicity. a ) significantly different from 0 µM acrolein; b ) significantly different from vehicle at the same concentration of acrolein, p < 0.05.

acrolein. Therefore, similar experiments were performed with preexposure of cells to chelerythrine chloride or rottlerin, followed by treatment with acrolein instead of allyl alcohol. As was observed with allyl alcohol, each of these inhibitors significantly blocked cytotoxicity caused by acrolein (Figure 3A,B). These results indicate that inhibition of PKCδ reduces the cytotoxic effect of the active metabolite of allyl alcohol and is consistent with the interpretation that chelerythrine chloride and rottlerin did not inhibit allyl alcohol-induced cell death by preventing the conversion of allyl alcohol to acrolein. Given that inhibition of PKCδ reduced cytotoxic effects of allyl alcohol, it was of interest to determine whether exposure to allyl alcohol was associated with activation of PKCδ. PKCδ exists in a quiescent state in the cytosol and upon activation translocates to cell membranes. In vehicle-treated cells stained with a monoclonal antibody to PKCδ, fluorescence was diffuse throughout the cells (Figure 4). PMA is known to activate PKCδ (15), and after treatment with PMA, fluorescent staining was punctate, suggesting association of PKCδ with intracellular membranes. In particular, previous studies have demonstrated that phorbol esters induce translocation of PKCδ to mitochondrial membranes (14). A similar pattern of punctate staining was observed in cells exposed to either allyl alcohol or acrolein. In contrast, treatment of cells with rottlerin markedly reduced punctate staining in response to either allyl alcohol or acrolein. Rottlerin alone had no effect on PKCδ staining.

To evaluate whether conversion to acrolein is a critical step in the activation and translocation of PKCδ by allyl alcohol, hepatocytes were treated with the alcohol dehydrogenase inhibitor, 4-methylpyrazole, which prevents metabolism of allyl alcohol to acrolein. Treatment of hepatocytes with 4-methylpyrazole prior to exposure to allyl alcohol prevented translocation of PKCδ and the accompanying punctate staining pattern. To confirm that the punctate staining seen immunohistochemically reflected movement of PKCδ to cell membranes and to quantify this protein movement, immunoblots were performed on separated proteins from cell cytosol and membranes using the same PKCδ monoclonal antibody to identify the protein bands. Immunoblots showed doublet bands at approximately the correct molecular size (78 kDa), likely indicating phosphorylation of the protein upon activation (Figure 5A). Band density was quantified and represented as the percent of total PKCδ in the cytosol or the membranes (Figure 5B). Approximately 35% of total PKCδ was in the membranes of vehicle-treated cells, whereas PMA-treated cells (positive control) had 85% of PKCδ in the membranes. In cells treated with allyl alcohol, approximately 73% of PKCδ was in the membrane fraction. Together with results of immunohistochemical staining (Figure 4) and previous reports on PKCδ activation (13, 19), these results indicate that allyl alcohol caused translocation of PKCδ from the cytosol to intracellular membranes. Activation of PKCδ is associated with phosphorylation of its Tyr residues; therefore, Tyr kinases were inhibited with genistein, and the effect on allyl alcohol cytotoxicity was examined. Genistein reduced hepatocyte death at a concentration of 10 µM and was completely cytoprotective at 40 µM (Figure 6). Because PKCδ activation has been associated with cell death via both apoptosis and necrosis, it was of interest to establish which process allyl alcohol-treated hepatocytes undergo. Allyl alcohol-treated cells stained strongly

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totic cells due to chromatin condensation and digestion of DNA-stabilizing proteins but is not able to denature DNA in cells with single- or double-stranded DNA breaks in the absence of apoptosis (20, 21). ALT release was increased after exposure to 50 µM allyl alcohol for 1.5 or 3 h. Maximal ALT release was observed with 100 µM allyl alcohol. Under no conditions was ssDNA antibody binding (manifest as increased optical density) increased relative to vehicle-treated hepatocytes (data not shown). Of interest, TUNEL assays were also performed to examine DNA strand breaks and essentially all cells treated for 1.5 h with allyl alcohol stained positively for TUNEL, whereas vehicle-treated cells did not stain (data not shown), suggesting a strong false positive result for this apoptosis assay. Positive TUNEL staining in the absence of apoptotic cell death has been reported previously (22, 23). Figure 5. Allyl alcohol stimulates translocation of PKCδ from the cytosol to membranes. Isolated hepatocytes were exposed to PMA (positive control) or allyl alcohol for 10 min. Membrane and cytosolic fractions were separated by centrifugation, and proteins in each fraction were separated via SDS-PAGE. PKCδ was detected via immunoblotting. (A) Representative reproduction of immunoblot showing PKCδ bands. (B) Graphic representation of the density of the PKCδ bands, expressed as % of the total for each treatment. The gel photograph and bar graph are representative of three experiments with similar results.

Figure 6. Tyr kinase inhibitor genistein reduces allyl alcoholmediated cytotoxicity. Hepatocytes were plated in 96 well tissue culture plates and exposed to 10 or 40 µM genistein for 20 min, n ) 3. Cells were then exposed to 0-75 µM allyl alcohol for 90 min. Leakage of ALT was measured as a marker of cytotoxicity. a ) significantly different from 0 µM allyl alcohol; b ) significantly different from vehicle at the same concentration of allyl alcohol, p < 0.05.

Figure 7. Allyl alcohol induces cell death via oncotic necrosis. Hepatocytes were plated on collagen-coated glass slides and exposed to allyl alcohol (100 µM) for 3 h. Cells were stained with 500 nM propidium iodide for 4 min.

with propidium iodide (Figure 7), indicating leaking plasma membranes and a necrotic process. In addition, ssDNA ELISA was performed on these cells. This assay is based on detection of ssDNA with a monoclonal antibody after selective denaturation of DNA with formamide. Formamide is able to denature DNA in apop-

Discussion The toxic metabolite of allyl alcohol, acrolein, is a reactive electrophilic compound that can bind to a number of cellular molecules and cause adverse outcomes. It is known to bind and deplete GSH, causing oxidative stress, and to deplete cellular thiols as well as cause peroxidation of membrane lipids (reviewed in 24). These general effects have the potential to affect specific proteins and/or signal transduction pathways in cells, leading to previously described outcomes such as inhibited proliferation, apoptosis, and necrosis (4-7). The oxidative environment created in the cell by acrolein has particular potential to affect Cys residues on proteins. Previously, acrolein exposure was shown to promote disulfide bonding of Cys residues, leading to autophosphorylation and activation of Tyr kinases in fibroblasts (25, 26). Specific proteins and/or pathways associated with allyl alcohol or acrolein treatment of hepatocytes remain to be identified. The data presented here provide evidence that PKCδ is critically involved in the signal transduction pathway of allyl alcohol/acrolein-mediated cell death in hepatocytes. Selective pharmacologic agents were used to inhibit several protein kinase pathways to initiate the investigation. Although there exists some lack of specificity among these inhibitors, use of several selective inhibitors that led to the same results strengthens the conclusion. Furthermore, follow up using a specific antibody to PKCδ, showing its discrete activation and translocation by allyl alcohol, reinforced the pharmacologic evidence. H-7, a compound that inhibits myosin light chain kinases, PKA, PKG, and PKC, partially inhibited allyl alcohol-induced hepatotoxicity. Pharmacologic inhibitors or activators of PKA or its associated pathways (cAMP) did not reduce cell death from allyl alcohol, however, suggesting that the activity of H-7 to inhibit the myosin light chain kinases, PKG, or PKC was responsible for the cytoprotective effect. Almost complete inhibition of allyl alcohol cytotoxicity by the nonselective PKC inhibitor chelerythrine chloride narrowed the search for a signal transduction target. Because H-7 only partially suppressed allyl alcohol-induced toxicity, it is possible that inhibition of PKG or myosin light chain kinases has other cell damaging effects that counter the almost complete cytoprotective actions of PKC inhibition observed with chelerythrine chloride. In fact, antiapoptotic, or cell survival, pathways that are dependent on the activation of myosin

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light chain kinases (27) and cGMP signaling (28, 29) have been identified in several cell types. In addition, because H-7 is a relatively nonselective protein kinase inhibitor, it may be acting on another, unidentified protein, affecting cell death, as has been shown in neuronal cells (30). In subsequent experiments with more specific PKC inhibitors, only the PKCδ selective inhibitor, rottlerin, showed similar inhibition of allyl alcohol toxicity to chelerythrine chloride. Rottlerin also reduced acroleinmediated cytotoxicity, suggesting that its inhibitory effect on cell death was not due to inhibition of allyl alcohol bioactivation. Protein immunoblots and fluorescent microscopy with specific antibodies to PKCδ confirmed that allyl alcohol and acrolein activated PKCδ, lending support to the interpretation that this isoform of PKC is important in cell death caused by allyl alcohol. These results are in contrast to an earlier report in which acrolein inhibited phorbol ester-induced phosphorylation of a 40 kDa protein substrate of PKC in platelets (31). The disparity may relate to differences in isoforms of PKC expressed among different cell types or in the kinetics of the response. Rapid activation of PKCδ was observed in hepatocytes whereas inhibition of protein phosphorylation in platelets was observed after 1 h (31). On the other hand, results presented here are consistent with the observation that acrolein induced a rapid increase in Tyr phosphorylation of multiple proteins in human keratinocytes (6). Cys residues present in PKCδ are critical for binding to phorbol esters and/or DAG and, thus, for activation (32). Therefore, acrolein produced in allyl alcohol-exposed hepatocytes may have modified the Cys residues of PKCδ leading to disulfide bond formation and directly to activation. Alternatively, acrolein may have activated an upstream Tyr kinase that in turn, phosphorylated and activated PKCδ. PKCδ activation and translocation to membranes are associated with both apoptotic and necrotic cell death pathways (13, 14, 33). In cells exposed to H2O2, PKCδ has been shown to phosphorylate and activate a Tyr kinase, c-Abl, that targets the mitochondrial membrane and mediates the loss of mitochondrial membrane potential leading to necrotic cell death (13). Activation of PKCδ also increased reactive oxygen species production in myocytes and was directly related to the induction of apoptosis by hyperglycemia (34). At these concentrations and relatively short exposure periods (1.5-3 h), allyl alcohol-induced activation of PKCδ causes hepatocyte death via oncotic necrosis. This is evidenced by positive staining with propidium iodide and an absence of DNA labeling by a ssDNA antibody after formamide denaturation. Previously, acrolein was reported to cause both apoptosis and necrosis in alveolar macrophages (35). Acrolein produces “atypical apoptosis” in human keratinocytes, described as having the morphologic features of apoptosis including shrinkage of the cellular volume and nuclei but without production of a typical DNA ladder on gel electrophoresis (6). Acrolein inhibited activation of caspase 3 in neutrophils within 2 h of exposure (7), although treatment times as short as those employed in studies presented here were not examined. Similarly, in pro B lymphoid cells, a longer duration (12-24 h) of exposure to acrolein resulted in decreased ATP production and reduced activity of caspases 3, 8, and 9 (36). These results are consistent with initiation of injury in allyl alcohol-treated cells, followed

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by loss of ATP and reduction in caspase activity that would favor a necrotic mode of cell death (37). Together, these results indicate that allyl alcohol stimulates PKCδ translocation and activation in hepatocytes in vitro and that PKCδ activity is critical in precipitating cell death via necrosis. Specific mechanisms leading from activation of PKCδ to hepatocyte death remain to be defined.

Acknowledgment. We greatly appreciate Natasha Tasheva for technical assistance with these studies. This work was supported by NIH Grant ES08789. J.F.M. was supported, in part, by an Arthritis Investigator Award.

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