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TOXICOLOGY
Département des Sciences Biologiques, TOXEN, Université du Québec à Montréal, Montréal, Québec, Canada
Received October 8, 2006; accepted January 2, 2007.
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,
-unsaturated aldehyde to which humans are exposed in many situations. It is an environmental pollutant that is responsible for multiple respiratory diseases and has been implicated in neurodegenerative diseases such as Alzheimer's disease. The hypothesis of the study is that the antioxidant N-acetylcysteine (NAC), a precursor of glutathione, could protect cells against acrolein-induced apoptosis. Exposure of Chinese hamster ovary cells to a noncytotoxic dose of acrolein (4 fmol/cell) depleted intracellular glutathione to 45% of initial levels. NAC, which increased intracellular glutathione levels by 30%, afforded protection against acrolein-induced cytotoxicity (loss of cell proliferation) and apoptosis. NAC protected against apoptosis by diminishing acrolein-induced activation of the mitochondrial death pathway. NAC inhibited acrolein-induced Bad translocation from the cytosol to the mitochondria, as well as Bcl-2 translocation from mitochondria to the cytosol, as evaluated by Western blot analysis. However, NAC had no effect on acrolein-induced Bax translocation to mitochondria and cytochrome c liberation into the cytosol. Meanwhile, NAC inhibited depolarization of mitochondrial membrane potential, as evaluated by rhodamine fluorescence using flow cytometry. NAC also inhibited procaspase-9 processing, activation of enzymatic activity of caspase-9, -7, and -8, and poly(ADP-ribose) polymerase cleavage induced by acrolein. Inhibition of acrolein-induced apoptosis using NAC was confirmed morphologically by diminished condensation of nuclear chromatin, as evaluated by fluorescence microscopy. These findings suggest that NAC could be potentially useful as a protective agent for people exposed to acrolein.
,-unsaturated aldehyde pollutant to which humans are exposed in multiple situations (Kehrer and Biswal, 2000
). It is a major component of smoke from cigarettes and forest and house fires and is a constituent of automotive exhaust. It has been implicated in the development of atherosclerosis and various lung diseases, including chronic obstructive pulmonary disease (Borchers et al., 1999). In addition, acrolein is found in the vapors of overheated cooking oil from which severe human toxic exposures have occurred (Beauchamp et al., 1985). As one of the toxic products of endogenous lipid peroxidation (Adams and Klaidman, 1993), acrolein has been implicated in chronic neurodegenerative disorders such as Alzheimer's disease (Lovell et al., 2001; Pugazhenthi et al., 2006). Acrolein is a metabolic product of the anticancer drug cyclophosphamide, for which it may be involved in its therapeutic effect and its toxic side effects (Schwartz and Waxman, 2001).
Acrolein toxicity has been shown in many different cellular systems. It is known to induce apoptosis in several cell types such as keratinocytes (Takeuchi et al., 2001), neutrophils (Finkelstein et al., 2005), cultured neurons (Pugazhenthi et al., 2006), and Chinese hamster ovary (CHO) cells (Tanel and Averill-Bates, 2005), whereas necrosis occurs in others (Luo et al., 2005; Liu-Snyder et al., 2006).
Apoptosis is a physiological cell death process that plays an important role during development and maintenance of tissue homeostasis (Chandra et al., 2000). The mitochondrial pathway of apoptosis is triggered by different stresses such as growth factor deprivation, ionizing radiation, corticosteroids, and anticancer drugs. Antiapoptotic (Bcl-2 and Bcl-XL) and proapoptotic (Bax, Bad) members of the Bcl-2 family are thought to control apoptosis by modulating release of proapoptotic molecules such as cytochrome c and apoptosis-inducing factor from mitochondria. Bcl-2–Bax interactions at the outer mitochondrial membrane are known to prevent induction of the mitochondrial apoptotic pathway. Translocation of Bad to mitochondria is necessary for promoting formation of pores consisting of Bax-Bax dimers on the outer mitochondrial membrane, which may be responsible for the release of proapoptotic factors (Orrenius et al., 2003). Bad translocation to the mitochondria removes the inhibitory effect of Bcl-2 on Bax by interacting with Bcl-2 itself. The Bcl-2 family has two types of proapoptotic members: the Bax and Bak subfamily and the BH3-only members such as Bid, Bad, Bim/Bod, and p53-up-regulated modulator of apoptosis (PUMA) (Huang and Strasser, 2000). Studies with gene-targeted cells indicated that the presence of Bax or Bak is required for many forms of apoptosis (Lindsten et al., 2000), and each type of cell needs at least one of the antiapoptotic Bcl-2 family members to survive (Wei et al., 2000; Cory and Adams, 2002). The BH3-only proteins are activated in response to various apoptotic stimuli and are able to induce apoptosis through interacting directly with Bax and Bak (Wei et al., 2000) or binding to the hydrophobic groove of antiapoptotic members such as Bcl-2 or Bcl-xL, thus removing their inhibitory effect on proapoptotic molecules such as Bax and Bak.
Interaction of cytochrome c and dATP with apoptosis protease-activating factor in the cytosol leads to conversion of procaspase-9 to active caspase-9 through autoproteolytic cleavage (Chandra et al., 2000). Caspase-9 subsequently activates effector caspases such as caspase-3, -6, and -7 (Salvesen and Dixit, 1997). Effector caspases then cleave cellular protein substrates such as poly(ADP-ribose) polymerase (PARP), lamins, gelsolins, fodrins, and inhibitor of caspase activator DNase. The cell then exhibits the characteristic morphological features of apoptosis such as chromatin condensation, cytoskeletal changes, nuclear membrane breakage, cell blebbing, and formation of apoptotic bodies, which are then phagocytosed by macrophages. Elimination of these dying cells avoids an inflammatory response in surrounding tissues, such as occurs during necrosis.
Acrolein will rapidly bind to and deplete cellular nucleophiles such as glutathione (GSH) (Heck, 1997). The antioxidant GSH seems to have an important role in the detoxification of acrolein (Finkelstein et al., 2001). Cellular cytotoxic responses to acrolein are likely to depend on intracellular GSH levels. It is likely that increased GSH levels could attenuate toxicity of acrolein. To increase GSH levels, the thiol-containing compound N-acetyl-L-cysteine (NAC) can be used as a GSH precursor because GSH itself does not easily penetrate cells (Roberfroid and Calderon, 1995). NAC also increases cysteine pools inside cells and acts as a thiol-containing reducing agent (Cotgreave, 1997). Thiol compounds such as NAC could be potentially useful for protecting tissues and cells from acrolein-induced toxicity. Therefore, the objective of this study is to investigate whether NAC can protect cells against acrolein-induced cytotoxicity and activation of the mitochondrial pathway of apoptosis.
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(-MEM) (Gibco Canada, Burlington, ON, Canada) plus 10% fetal bovine serum (FBS) (Gibco Canada) and 1% penicillin (50 units/ml)-streptomycin (50 µg/ml) (Flow Laboratories, Mississauga, ON, Canada) in tissue culture flasks (Sarstedt, Montréal, QC, Canada) in a humidified atmosphere of 5% CO2 in a water-jacketed incubator at 37°C (Lord-Fontaine and Averill, 1999). The cells were grown to near confluence and were then incubated for 24 h with fresh culture medium. Confluent cells were then harvested using phosphate-buffered saline/citrate, washed by centrifugation (1000g, 3 min), and resuspended in -MEM for experimental studies.
Pretreatment with NAC. Pretreatment of cells with 1 mM NAC (Sigma-Aldrich Canada Ltd, Oakville, ON, Canada) was performed for 24 h on confluent cells in monolayer (Lord-Fontaine and Averill, 1999). Cells were subsequently washed three times to remove NAC and then harvested. Intact cells were tested for cell survival, total GSH levels, and analysis of apoptosis. Treatment with NAC did not cause any loss of cell viability as determined by the trypan blue exclusion assay. Plating efficiency did not decrease relative to control cells.
Dosage of GSH. Total GSH was determined as described previously (Lord-Fontaine and Averill, 1999). The rate of formation of 5-thio-2-nitrobenzoic acid, which is proportional to total GSH concentration, was followed at 412 nm by an enzymatic assay using GSH reductase. Sample values were calculated from a standard curve of nanomole of GSH versus rate and expressed as nmol/106 cells.
Clonogenic Cytotoxicity Assay. Cytotoxicity was evaluated as the ability of cells to proliferate to form macroscopic colonies after a toxic insult with acrolein using a clonogenic cell survival assay (Lord-Fontaine and Averill, 1999). Cells (105/ml) were incubated with acrolein (Aldrich Chemical Co, Milwaukee, WI) in -MEM containing 10% FBS for 1 h at 37°C. Cytotoxicity was expressed as the mean number of macroscopic colonies (>50 cells) obtained relative to the mean number of colonies obtained in the control.
Morphological Analysis of Apoptosis. To visualize nuclear morphology and chromatin condensation, cells were incubated with acrolein for 4 h, and then apoptotic cells were stained with Hoechst 33258 dye (0.06 mg/ml) (Tanel and Averill-Bates, 2005). Propidium iodide (PI) (50 µg/ml) was added to stain necrotic cells. Observations were made by fluorescence microscopy (Carl Zeiss Ltd, Toronto, ON, Canada), and photographs were taken by digital camera (camera 3CCD, Sony DXC-950P; Empix Imaging Inc., Mississauga, ON, Canada). Images were analyzed by Northern Eclipse software (Empix Imaging Inc.). The fractions of apoptotic and necrotic cells were determined relative to total cells (obtained using bright field illumination). A minimum of 600 cells was counted per dish.
Determination of Caspase Activity by Fluorescence Spectroscopy. Freshly harvested cells (0.5 x 106) were incubated with acrolein in -MEM at 37°C. Cells were lysed by freezing at –20°C for 20 min, and reaction buffer (20 mM piperazine-N,N'-bis(2-ethanesulfonic acid), 100 mM NaCl, 10 mM dithiothreitol, 1 mM EDTA, 0.1% 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonic acid, and 10% sucrose, pH 7.2) was added (Stennicke and Salvesen, 1997). The kinetic reaction was started after addition of the appropriate caspase substrate at 37°C using a spectrofluorimeter (Spectra Max Gemini; Molecular Devices, Sunnyvale, CA) (Tanel and Averill-Bates, 2005). Caspase-7 activity was measured by cleavage of the fluorogenic substrate I MCA-VDQVDG-WK(DNP)-NH2 (Calbiochem, La Jolla, CA) with 
max excitation at 325 nm and max emission at 395 nm. Caspase-8 activity was measured by cleavage of the substrate Z-IETD-AFC to produce trifluorocoumarin (AFC) with max excitation at 415 nm and max emission at 490 nm. Caspase-9 activity was measured by cleavage of Ac-LEHD-AFC to produce AFC.
Subcellular Fractionation and Immunodetection of Bad, Bax, Bcl-2, Cytochrome c, Caspase-9, and PARP. After treatment with acrolein, cells were washed in buffer A (100 mM sucrose, 1 mM EGTA, and 20 mM 3-(N-morpholino)-propane sulfonic acid, pH 7.4) and resuspended in buffer B (buffer A plus 5% Percoll, 0.01% digitonin, and a mixture of protease inhibitors: 10 µM aprotinin, 10 µM pepstatin A, 10 µM leupeptin, 25 µM calpain inhibitor I, and 1 mM phenylmethylsulfonyl fluoride). After 30-min incubation on ice, lysates were homogenized using a hand potter (Duall 22; Kontes Glass Co., Fisher Scientific, Toronto, ON, Canada). Unbroken cells and nuclei were pelleted by centrifugation at 2500g for 10 min. The supernatant was centrifuged at 15,000g for 20 min, and the resultant pellet was designated as the mitochondrial fraction. A further centrifugation of the supernatant fraction at 100,000g for 1 h resulted in a pellet fraction designated as the microsomal fraction, whereas the supernatant was designated as the cytosolic fraction (Samali et al., 1999). For immunodetection of caspase-9 and PARP, whole cell lysates were used.
Proteins (30 µg) were quantified according to Bradford (1976), separated using a 15% SDS-polyacrylamide gel, and then transferred to a polyvinylidene difluoride membrane, as described previously (Tanel and Averill-Bates, 2005). The blots were probed with the following primary antibodies: anti-Bad, anti-Bax, anti-Bcl-2, anti-caspase-9, anti-PARP (Santa Cruz Biotechnology, Santa Cruz, CA), or anti-cytochrome c (BD Biosciences, Mississauga, ON, Canada). Secondary antibodies (1:1000) consisted of horseradish peroxidase-conjugated goat anti-mouse, anti-rabbit, and anti-goat IgG (Biosource, Camarillo, CA). Proteins were detected using the enhanced chemiluminescence plus kit (PerkinElmer, Boston, MA), and protein expression was quantified using a scanning laser densitometer (GE Healthcare, Chalfont St. Giles, Buckinghamshire, UK). Purity of mitochondrial and cytosolic fractions was verified using antibodies to cytochrome c oxidase (Santa Cruz Biotechnology) and GST
1 (Calbiochem), respectively. Caspase-9 and PARP expression in whole cell lysates was quantified using IPGel software (BD Biosciences Bioimaging, Rockville, MD), relative to -tubulin loading controls.
Flow Cytometry Analysis of Mitochondrial Membrane Potential (
m). Freshly harvested cells (106) were incubated with acrolein or the positive control p-trifluoromethoxy-phenyl-hydrazone (FCCP) in -MEM for 1 h at 37°C. To measure m, cells were incubated with rhodamine 123 (800 ng/ml) for 5 min (Tanel and Averill-Bates, 2005). PI was added to identify dead cells. Cells were analyzed with a FACScan flow cytometer equipped with an argon laser emitting at 488 nm (Becton Dickinson, Oxford, UK). Data were acquired and analyzed using Lysis II software (Becton Dickinson). The mean fluorescence intensity of 20,000 cells was calculated for each sample. Rhodamine 123 fluorescence was detected on the FL1 detector, and PI fluorescence was detected on the FL2 detector. For each sample, only live cells were selected, and their mean fluorescence in FL1 was analyzed. Apoptotic cells, which undergo a decrease in mitochondrial membrane potential, incorporate less of the rhodamine 123 dye, therefore emitting less fluorescence on the FL1 detector. Control measurements of mitochondrial mass were carried using the fluorescent probe MitoTracker Green (Invitrogen, Carlsbad, CA).
Statistical Analysis. Statistical differences between control and treated groups without NAC for Figs. 1, 2, 3 (I and J), 7D, 9A, and 10 (A and B) were determined using a one-way analysis of variance with a Bonferroni-Holm (a stepwise method) post-test correction for multiple comparisons. An adjustment was made to limit the family-wise error rate to 5% by calculating an adjusted p value, which is a simulated based p value obtained from the multivariate t distribution (number of simulations = 1 million) (Westfall et al., 1999). Statistical differences between control and treated groups with or without NAC for Figs. 1, 2, 3 (I and J), 7D, 9A, and 10 (A and B) were determined using a two-tailed unpaired Student's t test. Data for Figs. 4 (B and C), 5 (B and C), 6 (B and C), 8 (B and C), 9 (C and D), and 11 (B and C) were analyzed using a bilateral t test. Values are expressed as mean ± S.E.M. Differences were considered statistically significant at p < 0.05.
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). Nevertheless, the possible protective effect of antioxidants against acrolein-induced apoptosis is not known. Therefore, we investigated whether NAC, a precursor of GSH, can protect cells against acrolein-induced cytotoxicity and apoptosis. Induction of cytotoxicity (loss of cellular proliferation) occurred after a 1-h exposure to acrolein concentrations of 50 fmol/cell and higher (Fig. 1). Exposure to 350 fmol/cell induced 5 logarithms (105) of cell killing. NAC afforded significant protection against cytotoxicity of acrolein. The protective effect was only partial because acrolein cytotoxicity was not completely prevented.
Based on the known reactivity of ,-unsaturated aldehydes such as acrolein with thiols (Heck, 1997), it is likely that acrolein could mediate its toxic effects primarily by depleting cellular GSH and/or by modifying protein cysteine residues. Indeed, exposure of cells to acrolein caused rapid and severe depletion of GSH (Fig. 2). GSH levels were reduced to 36% of initial levels, from 2.28 ± 0.08 to 0.82 ± 0.13 nmol/106 cells, by treatment with 4 fmol/cell of acrolein during 30 min. We determined whether NAC could prevent the loss of GSH following exposure to acrolein by increasing GSH levels. Pretreatment of cells with 1 mM NAC for 24 h effectively increased total intracellular GSH levels in untreated control cells by approximately 30%, from 2.28 ± 0.08 to 3.22 ± 0.17 nmol/106 cells (Fig. 2). However, NAC partially decreased the loss of intracellular GSH in cells exposed to a low dose of acrolein (4 fmol/cell) but not at higher concentrations (
10 fmol/cell) (Fig. 2). Even though initial levels of GSH were elevated, acrolein still caused a severe loss of GSH in cells pretreated with NAC (Fig. 2).
Given that NAC protected cells against acrolein-induced cytotoxicity (Fig. 1), it is possible that NAC could trigger a change in the type of cell death from necrosis to apoptosis. Morphological analysis shows that acrolein induces both apoptosis and necrosis in CHO cells (Fig. 3). Apoptosis was revealed by condensation of chromatin with the fluorescent probe Hoechst (blue), whereas necrosis was revealed by the fluorescent probe PI (red). Acrolein (50 fmol/cell) induced apoptosis and necrosis in 21 and 3.5% of cells, respectively (Fig. 3, C, I, and J). A higher concentration of acrolein (100 fmol/cell) switched the mode of cell death from apoptosis (8.5%) to necrosis (20%) (Fig. 3, D, I, and J). We evaluated whether the NAC-induced increase in levels of intracellular GSH could attenuate cell death induced by acrolein. When NAC-pretreated cells were exposed to 50 fmol/cell of acrolein (Fig. 3, G and I), the fraction of apoptotic cells decreased from 21% to 2%. NAC also diminished necrosis in cells exposed to 50 and 100 fmol/cell of acrolein (Fig. 3, G, H, and J). Therefore, NAC increased the threshold level of acrolein for induction of apoptosis from 50 to 100 fmol/cell, relative to cells with native levels of GSH (Fig. 3, C and G–I). Treatment with NAC alone did not affect control cells (Fig. 3, E versus A). Taken together, these results suggest that NAC is an effective compound to inhibit acrolein-induced cytotoxicity and acrolein-induced apoptosis.
NAC Inhibits Acrolein-Induced Translocation of Bad and Bcl-2. We previously reported that acrolein can induce apoptosis by the mitochondrial pathway in CHO cells (Tanel and Averill-Bates, 2005). However, the effect of acrolein on proteins in the Bcl-2 family is not known. Acrolein could alter the balance between proapoptotic and antiapoptotic Bcl-2 proteins at the mitochondrial membrane. Therefore, we examined whether acrolein could induce the translocation of Bad, Bax, and Bcl-2 between the cytosolic and mitochondrial subcellular compartments. Exposure to acrolein (20 fmol/cell) for 1 h resulted in translocation of the proapoptotic protein Bad from the cytosol to the mitochondria (Fig. 4). Translocation of Bad to mitochondria was partially inhibited by NAC (Fig. 4, A and B). Acrolein also induced translocation of the proapoptotic protein Bax from the cytosol to the mitochondria (Fig. 5). However, NAC did not inhibit Bax translocation, but rather NAC alone promoted Bax translocation to mitochondria from the cytosol (Fig. 5). In addition, acrolein induced translocation of the antiapoptotic protein Bcl-2 from mitochondria (Fig. 6, A and B) to the cytosol (Fig. 6, A and C). This was inhibited by NAC. Together, these findings indicate that acrolein has a proapoptotic effect at the mitochondrial membrane. NAC played an antiapoptotic role at the mitochondrial level by inhibiting acrolein-induced translocation of Bad to mitochondria and by maintaining Bcl-2 levels at the mitochondrial membrane.
NAC Inhibits Acrolein-Induced Depolarization of Mitochondrial Membrane Potential. The balance between Bcl-2 proteins at the mitochondrial membrane is often a determinant in the opening of the permeability transition pore (PTP) and loss of mitochondrial membrane potential (MMP) under apoptotic stimuli. Because NAC can exert protective, antiapoptotic effects at the level of the mitochondrial membrane, we examined whether it could prevent mitochondrial membrane depolarization in acrolein-treated cells. Acrolein (10–20 fmol/cell) caused depolarization of MMP (Fig. 7, C and D). FCCP was used as a positive control for MMP depolarization (Fig. 7, A and D). However, pretreatment with NAC afforded a significant level of protection against acrolein-induced mitochondrial depolarization (Fig. 7, C and D). NAC alone caused a small decrease in MMP (Fig. 7B). To verify that changes in rhodamine fluorescence signals were not caused by changes in the total mitochondrial mass, the effect of acrolein (20 fmol/cell), with and without NAC treatment, was determined on the fluorescence uptake of the redox-insensitive mitochondrial dye MitoTracker Green (Fig. 7E). There was no evidence of a shift in fluorescence intensity in cells treated with acrolein alone or with acrolein and NAC, confirming that changes in rhodamine fluorescence were indeed caused by changes in MMP, thus ruling out the possibility that acrolein and/or NAC affected the mitochondrial mass.
A decrease in MMP often leads to the release of cytochrome c from mitochondria. Indeed, acrolein (20 fmol/cell) caused release of cytochrome c into the cytosol (Fig. 8). However, NAC did not seem to inhibit this effect but instead induced a small increase in cytochrome c levels in both the mitochondrial and cytosolic fractions.
NAC Inhibits Acrolein-Induced Activation of Caspase-9. Because NAC inhibited acrolein-induced translocation of Bad and Bcl-2, as well as mitochondrial membrane depolarization, it is likely that NAC could inhibit postmitochondrial events such as caspase-9 activation. Here we show that exposure to acrolein (4–50 fmol/cell) for 1 h activated the initiator caspase-9 (Fig. 9A), the first downstream caspase induced via the mitochondrial death pathway. Caspase-9 activation by acrolein was totally prevented by pretreatment with NAC (Fig. 9A). In addition, pretreatment with NAC inhibited the cleavage of procaspase-9 (Fig. 9, B–D) and the generation of the p35 active cleavage fragment induced by acrolein (Fig. 9D). These findings show that NAC can inhibit acrolein-induced apoptosis at the postmitochondrial level.
NAC Inhibits Acrolein-Induced Activation of Caspase-7 and -8 and PARP Cleavage. We next determined whether NAC could inhibit other events in the apoptotic cascade. Acrolein induced activation of the effector caspase-7 (Fig. 10A) and initiator caspase-8 (Fig. 10B), and these events were significantly inhibited in cells that had been pretreated with NAC. Because caspase-7 was inhibited by NAC, we investigated whether NAC could prevent the cleavage of PARP, which is a downstream substrate of caspase-7. PARP is a key nuclear enzyme involved in DNA repair and has a complex role in cell death. Acrolein (20 fmol/cell) caused cleavage of PARP protein from the full-length 116 kDa form to generate the characteristic apoptosis-related 85-kDa cleavage fragment (Fig. 11, A–C). Indeed, NAC inhibited the cleavage of PARP induced by acrolein (Fig. 11, A–C).
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). This study provides new information on the implication of the mitochondrial pathway in acrolein-induced apoptosis, for which Bcl-2 family proteins are critical regulators. This is the first study to show that acrolein stimulates translocation of the proapoptotic Bcl-2 proteins, Bax and Bad, from the cytosol to mitochondria, as well as the translocation of antiapoptotic protein Bcl-2 from mitochondria to the cytosol. The translocation of proapoptotic proteins such as Bax and Bad to mitochondria is an important step in removing the inhibitory effect of Bcl-2 on apoptosis at the mitochondrial level, thus promoting release of cytochrome c.
Acrolein induced a decrease in MMP and liberation of cytochrome c from mitochondria into the cytosol. The implication of the PTP in acrolein-induced apoptosis was already shown through inhibition of nuclear changes by cyclosporine A (Tanel and Averill-Bates, 2005), a blocker of opening of the PTP. Although the mechanisms are not completely understood, it is considered that cytochrome c can be released from mitochondria by several mechanisms, including the PTP, as well as through channels on the membrane involving Bax-Bax interactions (Orrenius et al., 2003). At the postmitochondrial level, liberation of cytochrome c leads to activation of caspase-9 and -7. Caspase-7 is an effector caspase that cleaves many substrates such as PARP. Several studies have reported that acrolein can inhibit caspase-3 activity (Finkelstein et al., 2001; Tanel and Averill-Bates, 2005), which could occur by direct alkylation of its cysteine residue by acrolein.
The cellular effects of acrolein are believed to be caused primarily by its ability to react rapidly with reduced thiols such as GSH (Heck, 1997). GSH plays a key role in cellular reductive processes and detoxification of harmful oxidative species and various xenobiotics (Meister and Anderson, 1983). GSH redox status is critical for a variety of biological process, including transcriptional activation of various genes, regulation of cell proliferation, inflammation, and apoptosis. An important consequence of severe GSH depletion caused by toxic compounds such as acrolein is the resulting perturbation of the cellular redox balance between pro-oxidants and antioxidants in favor of a pro-oxidant state. This would leave the cell vulnerable to damage, and possibly death, induced by normal levels of cellular oxidants (
, H2O2) or mild exposure to other toxic compounds (Luo et al., 2005).
The present study shows that acrolein causes GSH depletion at much lower concentrations (4 fmol/cell) than those that induce cytotoxicity and apoptosis (30–50 fmol/cell). This indicates that GSH depletion is an earlier event than cytotoxicity, evaluated by loss of cellular proliferation. There was also a correlation between acrolein-induced changes in GSH levels and inhibition of cell proliferation in A549 lung carcinoma cells (Horton et al., 1997).
This is the first study that links GSH to induction of apoptosis by acrolein because pretreatment with NAC, a GSH precursor, inhibited acrolein-induced apoptosis. Acrolein concentrations (20 fmol/cell) that completely depleted GSH levels coincided with those required to induce early apoptotic events, such as translocation of Bax and Bad, mitochondrial membrane depolarization, liberation of cytochrome c, and activation of caspase-9. Furthermore, this is the first study to report protection against acrolein-induced apoptosis with a thiol antioxidant. However, it was reported that sulfur compounds (e.g., thiamine, NAC, and ascorbic acid) (Sprince, 1985), -tocopherol, and ascorbic acid (Nardini et al., 2002), as well as the nucleophilic drug hydralazine (Kaminskas et al., 2004), could afford protection against acrolein-induced cytotoxicity. Indeed, NAC inhibited acrolein-induced apoptosis at both the mitochondrial and postmitochondrial levels. Caspase-9 activation, which is considered an important indicator for activation of the mitochondrial pathway of apoptosis, was totally prevented by NAC. NAC inhibited acrolein-induced activation of caspase-7, PARP cleavage, and chromatin condensation, which are downstream events from both caspase-9 and -8. Total inhibition of caspase-9 activation and partial inhibition of caspase-8 activation suggest that the mitochondrial pathway is primarily involved in protective mechanisms involving NAC. Caspase-8 activation mediates apoptotic signaling via death receptor pathways (Saikumar et al., 1999).
At the mitochondrial level, NAC inhibited the translocation of Bad to mitochondria and the translocation of Bcl-2 to cytosol, which was stimulated by acrolein exposure. NAC maintained antiapoptotic Bcl-2 protein at the mitochondrial membrane. Therefore, NAC changed the balance between proapoptotic and antiapoptotic Bcl-2 proteins at the mitochondrial membrane in favor of an antiapoptotic state, as confirmed by protection against the acrolein-induced decrease in MMP. Surprisingly, NAC did not seem to protect cells against acrolein-induced Bax translocation to the mitochondria. However, it is likely that the antiapoptotic effect of NAC may predominate by increasing Bcl-2. Even though NAC does not prevent Bax translocation to mitochondria, NAC ensures increased levels of Bcl-2 at the mitochondrial membrane. Bcl-2 binds to Bax to prevent its proapoptotic effects.
Surprisingly, NAC did not protect cells against acrolein-induced cytochrome c release from mitochondria to the cytosol. However, it was reported that the liberation of cytochrome c persists in neutrophils exposed to acrolein even though apoptotic features such as caspase-9 and -8 activation and externalization of phosphatidylserine were inhibited by acrolein (Finkelstein et al., 2005). This suggests that cytochrome c release is not necessarily a hallmark in acrolein-induced apoptosis. Moreover, GSH depletion caused cytochrome c release even in the absence of apoptosis (Ghibelli et al., 1999). This again suggests that cytochrome c release is not necessarily a terminal event leading to apoptosis, but could be the consequence of a redox disequilibrium arising from increased mitochondrial generation of reactive oxygen species that, under some circumstances, may be associated with apoptosis (Ghibelli et al., 1999). The mechanisms involved in cytochrome c release during apoptosis are not completely understood and seem to involve multiple and distinct pathways, involving Bax-Bax dimers, t-Bid, the mitochondrial permeability transition (MPT), and oxidation of cardiolipin (Orrenius et al., 2007). Further work is required to characterize the mechanisms involved in the mitochondrial regulation of apoptosis.
Depletion of GSH and other cellular thiols has been correlated with apoptosis in a number of cell types (Sato et al., 1995; Aoshiba et al., 1999). It was suggested that the effects of acrolein might be the result of redox changes of critical protein cysteine residues secondary to GSH depletion or to direct oxidation/alkylation of protein thiol residues that may occur along with or subsequent to the loss of GSH (Finkelstein et al., 2001). However, the relationship between GSH depletion and apoptosis is not clear, and GSH depletion itself may not be sufficient to trigger apoptosis. In fact, it has been proposed that apoptosis could involve efflux of reduced GSH from the cell (Ghibelli et al., 1995). Thus, depletion of cellular GSH may be a consequence rather than a cause of apoptosis.
During recent years, many compounds have been tested as precursors of GSH or as inducers of the enzymes related to its synthesis (Anderson, 1997). Increasing GSH levels could be of great interest in designing new therapeutic strategies against environmentally toxic compounds, such as acrolein, and against the side effects of many therapeutic agents. NAC has proven to be a useful compound for increasing GSH synthesis. The protective role of NAC against acrolein-induced cytotoxicity and apoptosis could be explained by its ability to stimulate synthesis of GSH, to increase cellular thiol pools, or to act as a thiol-containing reducing agent. Besides increasing cellular thiols, NAC can act as an antioxidant by directly reducing reactive oxygen species such as OH·, H2O2, and HOC1. NAC reduced protein carbonyls induced by acrolein, markers for lipid peroxidation, in rats developing acute hepatic injury (Kitamura et al., 2005). An advantage of using NAC as a protective agent is that it already has approval for clinical use. In fact, NAC is used clinically to treat overdose of the hepatotoxic drug acetaminophen (Perry and Shannon, 1998). Administering the cysteine delivery compound NAC to human subjects seems to be safer than administering cysteine itself because cysteine has been reported to have toxic effects on the central nervous system (Dizdar et al., 2000).
Based on these results, we conclude that NAC seems to have a beneficial effect in protecting cells against acrolein-induced apoptosis and inhibition of cell proliferation. These findings also suggest that intracellular GSH levels may play an important role in cellular toxicity of acrolein and, in particular, the induction of apoptosis and cytotoxicity. These findings may also be relevant to our understanding of the toxicity of environmental exposures to low doses of acrolein and the normal tissue toxicity of cyclophosphamide, which generates acrolein as one of its metabolites.
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: CHO, Chinese hamster ovary; PARP, poly(ADP-ribose) polymerase; GSH, glutathione; NAC, N-acetylcysteine; MEM, minimum essential medium; FBS, fetal bovine serum; PI, propidium iodide; AFC, amino trifluorocoumarin; FCCP, p-trifluoromethoxy-phenyl-hydrazone; PTP, permeability transition pore; GST, glutathione S-transferase; MMP, mitochondrial membrane potential.
Address correspondence to: Diana A. Averill-Bates, Département des Sciences Biologiques, Université du Québec à Montréal, CP 8888, Succursale Centre Ville, Montréal, QC H3C 3P8, Canada. E-mail: averill.diana{at}uqam.ca
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, p < 0.05 and 
