57, 6 –15 (2000) Copyright © 2000 by the Society of Toxicology
TOXICOLOGICAL SCIENCES
FORUM The Molecular Effects of Acrolein James P. Kehrer 1 and Shyam S. Biswal Division of Pharmacology and Toxicology, College of Pharmacy, The University of Texas at Austin, Austin, Texas 78712 Received May 8, 2000; accepted June 9, 2000
types of smoke (including cigarette smoke), in the exhaust from internal combustion engines, and in the vapors of overheated cooking oil where severe human toxic exposures have occurred (Beauchamp et al., 1985). In vivo, acrolein is a metabolic product of the anticancer drug cyclophosphamide and has been found to be formed from threonine by neutrophil myeloperoxidase at sites of inflammation (Anderson et al., 1997) Acrolein is the most reactive of the ␣,-unsaturated aldehydes, and will rapidly bind to and deplete cellular nucleophiles such as glutathione. It can also react with cysteine, histidine, and lysine residues of proteins and with nucleophilic sites in DNA. This reactivity is the basis for the cytotoxicity evident in all cells exposed to high concentrations of acrolein and may explain its genotoxic and mutagenic activity in Drosophila (Sierra et al., 1991). At lower acrolein doses, other biologic effects become evident. For example, acrolein decreases the proliferation of human lung carcinoma cell lines (Horton et al., 1997; Krokan, 1999; Ramu et al., 1996; Rudra and Krokan, 1999) at sublethal doses. The mechanism(s) of this effect is unknown, but may be related to the ability of acrolein to deplete cellular thiols or other nucleophiles, and/or to effects on gene activation, either directly or subsequent to effects on transcription factors, particularly those that are redox-regulated (Fig. 1). Acrolein has been identified as both a product and initiator of lipid peroxidation (Adams and Klaidman, 1993; Uchida, 1999). Since this oxidation process occurs in all cells, and is enhanced in many toxic and pathophysiological situations, the potential exists for virtually continuous exposure to acrolein. For example, acrolein protein adducts have been demonstrated in diabetic nephropathy (Suzuki and Miyata, 1999) and Alzheimer’s disease (Calingasan et al., 1999), both of which are associated with increased lipid peroxidation. It seems unlikely, however, that the levels of acrolein associated with endogenous exposures generated via oxidation processes have any significance in terms of function. Rather, acrolein adducts produced as a consequence of such exposures may serve as a useful biologic marker.
Acrolein is a highly electrophilic ␣,-unsaturated aldehyde to which humans are exposed in a variety of environmental situations, particularly as a component of smoke. In addition, as a metabolite of cyclophosphamide, acrolein is a major factor in the toxicity and perhaps the therapeutic activity of this important anticancer agent. The exposures to acrolein that are attained in vivo in most situations are quite low and the effects may differ from those seen at acutely toxic doses. At low doses, acrolein inhibits cell proliferation without causing cell death and may enhance apoptosis from secondary toxins, while at higher doses oncosis ensues. Although the acute toxicology of acrolein has been extensively investigated, both in animals and cultured cells, little information exists on the molecular effects of this reactive aldehyde. It is possible that the acrolein-mediated decrease in cell proliferation is caused by effecting changes in the expression of one or more growth- or stress-related genes or transcription factors secondary to a reduction in glutathione (GSH), which is rapidly depleted following acrolein treatment. It is apparent that the activation of the transcription factors nuclear factor kappa B (NF-B) and activator protein 1 (AP-1) can be inhibited by acrolein. The purpose of this review is to assess the literature currently available on the molecular effects of acrolein, to discuss the relationship between effects on glutathione with those on various genes, and to present some new data showing that acrolein actively stimulates genes associated with the electrophile response element. Key Words: acrolein; nuclear factor kappa-B (NF-B); activator protein 1 (AP-1); glutathione GSH); transcription factors; electrophile response element (ERE); gene array.
Acrolein is a highly electrophilic, ␣,-unsaturated aldehyde to which humans are exposed in industrial, environmental, and therapeutic situations. Industrially, acrolein is used as a herbicide and slimicide as well as a starting material for acrylate polymers and in the production of acrylic acid. Environmentally, acrolein exists naturally in foods and is formed during the combustion of organic materials. Thus, acrolein is found in all 1
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FIG. 1. Mechanisms by which acrolein may affect cells molecularly. Acrolein is highly electrophilic and will react with cellular nucleophiles, particularly thiols. This may affect gene expression or acrolein may directly interact with various genes and transcription factors. The net effect of acrolein is to diminish cell proliferation and possibly to enhance the susceptibility of cells to death by apoptosis or oncosis.
The extensive toxicology literature on acrolein has focused largely on its electrophilic nature and on the acute toxicologic effects both in vivo and in vitro. In general, it appears that acrolein largely induces cell death by oncosis (Rudra and Krokan, 1999). The ability of non-lethal doses of acrolein to inhibit cell division may reflect more subtle molecular effects, yet there are few publications describing the molecular basis for acrolein toxicity. A large number of genes and transcription factors have been identified as having effects on cell division and apoptosis. Acrolein appears to affect some of these transcription factors, particularly NF-B (Horton et al., 1999) and AP-1 (unpublished data). In addition, acrolein is capable of inducing the expression of the glutathione S-transferases (Uchida, 1999), although a complete analysis of the effects of acrolein on various genes has not been done. Acrolein as a Toxicant As noted above, acrolein is a widespread environmental pollutant. It is also a metabolic product of cyclophosphamide and allylamine and is believed to be the main cause of their toxicity (Boor, 1983; Fraiser et al., 1991). Acrolein is found in all types of smoke and is formed in polluted air as a photochemical reaction product. It has been estimated to represent 5% of the total aldehyde in polluted air (formaldehyde is the greatest component), but is proportionally the greatest problem because it is highly irritating, and, as an ␣,-unsaturated compound, is very electrophilic (Witz, 1989), making it capable of rapidly reacting at many cellular sites. Because of its ubiquitous presence in the environment and its high reactivity, the overt toxicity of acrolein has been extensively studied. In vitro concentrations of 25 to 100 M are lethal to pulmonary artery endothelial cells (Kachel and Martin, 1994; Patel and Block, 1993), bronchiolar epithelial
cells (Grafstro¨m et al., 1988), and both bronchial (Krokan et al., 1985) and cardiac fibroblasts (Torasson et al., 1989). Levels of acrolein in ambient air are estimated at only 0.04 to 0.08 ppm, although it is found at up to 90 ppm in cigarette smoke (Costa and Amdur, 1996). Subchronic-inhalation animal studies have reported physiological effects at 0.4 ppm, but as in studies with cultured cells, few if any have looked at the molecular effects of these, or lower, doses. High-dose acute acrolein toxicity has been suggested as involving oxidative stress subsequent to the loss of glutathione (GSH), or to be due to alkylation reactions at various nucleophilic sites in a cell—particularly in the nucleus. This dichotomy is somewhat confusing, since the loss of GSH is itself due to an alkylation reaction with acrolein. While oxidative stress can result from both an increase in the production of oxidants and a decrease in levels of antioxidants such as GSH, this latter occurrence is more likely to increase a cell’s susceptibility to oxidants and may not, in and of itself, lead to any adverse effects in the absence of an oxidant. Nevertheless, thiol redox balance is critical for numerous cell functions (Arrigo, 1999) and changes induced by acrolein seem certain to affect various signaling pathways. Oxidative stress is defined as a disturbance in the prooxidant-antioxidant balance in favor of the former, leading to potential damage (Sies, 1991). While this definition has been adequate in acute toxicity situations, it is more difficult to apply at the molecular level, since the changes that constitute “potential damage” are not easily determined and clearly depend on one’s definition of damage. It is possible that cellular redox balance is disrupted by an acrolein-mediated loss of GSH, and that this causes alterations in gene expression or transcription factor activation that affect proliferation and overall cell well-being. Alternately, or in addition, a more direct action of acrolein on various factors is possible. The direct alkylation of DNA by acrolein, while possible, seems unlikely at low doses that would be expected to react with the abundant levels of cellular GSH or other nucleophiles prior to reaching the nucleus. A lack of direct DNA damage is supported by the failure to observe induction of gadd153 at low acrolein doses in human lung adenocarcinoma A549 cells (Horton et al., 1997). However, a reaction with nucleophilic sites on transcription factors such as NF-B, and AP-1 (even within the nucleus) does occur (Horton et al., 1999). Acrolein, GSH and Apoptosis Cyclophosphamide, which is bioactivated to phosphoramide mustard and acrolein (Sladek, 1972), can reportedly induce apoptosis (Dole et al., 1995). It has been generally accepted that phosphoramide mustard is responsible for the therapeutic activity of cyclophosphamide and thus it is likely that this metabolite is the primary cause of apoptosis. Although acrolein alone can induce this form of cell death in alveolar macrophages (Li et al., 1997), this aldehyde appears more likely to
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induce oncosis in other cell types (Rudra and Krokan, 1999; unpublished data), perhaps because of its mechanism of action in acute situations. The ability of more subtle molecular effects, particularly at the low, repeated doses expected following treatment with cyclophosphamide, to modulate apoptosis, has not been studied much. Our preliminary data support the ability of acrolein to enhance apoptosis from a second agent, although this effect appears to be rather modest (Kern and Kehrer, 2000). GSH, which is clearly a major target for acrolein and has been implicated as a major factor controlling apoptosis, represents a likely factor that could link acrolein and this form of cell death. There has been a correlation in some, but not all, cells with the levels of GSH and resistance to, or induction of, apoptosis. Interestingly, depleting GSH with diethyl maleate (DEM) (which alkylates GSH via glutathione S-transferase) but not buthionine sulfoximine (BSO) (a selective inhibitor of ␥-glutamylcysteine synthetase, the rate-limiting step in GSH synthesis) appears to increase susceptibility to apoptosis (Mirkovic et al., 1997). The explanation for this finding has been related to differences in the subcellular pools that are affected (e.g., BSO does not deplete mitochondrial GSH). However, it is also possible that differences in how genes or transcription factors are altered in conjunction with treatments that change GSH levels may be critical. For example, DEM can inhibit NF-B at doses that do not affect GSH (Horton et al., 1999). Since NF-B can modulate apoptosis (Lin et al., 1999), such an effect clearly has the potential to alter cell death independently of GSH. Another explanation for apparent discrepancies in the role of GSH and oxidants in apoptosis is the report that the oxidative tonus of cells exposed to anti-fas antibody to induce apoptosis is increased by the stimulation of GSH efflux, not by the formation of reactive oxygen species (ROS) (van den Dobblesteen et al., 1996). Supplementing these cells with GSH did not prevent apoptosis, possibly because of rapid efflux. On the other hand, antioxidants may provide some protection by slowing oxidative processes that begin in the absence of adequate GSH. A key permissive factor in apoptosis may, therefore, be GSH, and modification of this cellular reducing agent by alkylation, oxidation, or extrusion may be critical in how apoptosis and/or proliferation is regulated at the molecular level. This hypothesis is supported by work showing that thiol modification, particularly in the mitochondria, is a factor in the apoptosis induced by agents such as nordihydroguaiaretic acid (Biswal et al., 2000b) and ebselen (Yang et al., 2000). Although GSH has been implicated as an important cellular molecule that may play a role in apoptosis (Beaver and Waring, 1995; Slater et al., 1996), whether changes in cellular levels of this tripeptide are a direct effector or a secondary modulating influence remains unclear. Depletion of GSH in polymorphonuclear leukocytes by DEM results in a dosedependent increase in apoptosis (Watson et al., 1996). This effect could be reversed by N-acetylcysteine but not the anti-
oxidant pyrollidine dithiocarbamate (PDTC). These authors suggested that tyrosine phosphorylation was the pathway through which apoptosis was signaled. This coincides with other data suggesting that the effects of N-acetylcysteine on apoptosis depend on effects on gene expression, not on its actions as a GSH precursor and/or antioxidant (Yan et al., 1995). This concept is supported by the observation that N-acetylcysteine, while preventing apoptosis in many systems, can induce it in several transformed cell lines independent of any effects on GSH (Liu et al., 1998). In addition, we have found that N-acetylcysteine can both enhance (Jurkat, Raji and WSU chronic lymphocytic leukemia cells) and inhibit (FL5.12 cells) the apoptosis induced by a single xenobiotic (MK886), depending on the cell line used (unpublished data). Other researchers have reported that the treatment of fibroblasts with BSO caused efficient depletion of GSH and resulted in apoptosis (Zucker et al., 1997). They suggested that the extent of GSH depletion is the critical determinant of whether or not apoptosis occurs. However, acrolein can deplete GSH to very low levels without affecting viability (at least in A549 cells) (Horton et al., 1997, 1999) suggesting that the extent of depletion alone is insufficient to induce cell death. Following removal of acrolein, GSH levels are rapidly restored. Whether the duration of GSH depletion is the critical factor in determining whether cells die has not been investigated, but is a likely factor. In the murine hematopoietic progenitor cell line, FL5.12, we have found that GSH is lost prior to apoptosis and that this loss is blocked by bcl-2 or bcl-x L (Bojes et al., 1997). We have also found that prolonged depletion of GSH with BSO can induce apoptosis in these cells, although the extent of depletion required significantly exceeds that evident when apoptosis is induced by growth factor withdrawal (Bojes et al., 1999). Additional data have been published showing that one of the functions of the antiapoptotic protein bcl-2 is to promote sequestration of GSH into the nucleus, thereby altering nuclear redox status and blocking changes characteristic of apoptosis (Voehringer et al., 1998). This further supports the importance of cellular GSH, particularly the nuclear pool, in regulating apoptotic cell death. The relationship between GSH and apoptosis is not yet clear and may involve other factors and genes, despite some evidence of a direct effect as described above. For example, the level of c-myc expression in a human hepatoma cell line appears to correlate with GSH content (Xu et al., 1997) and this gene may also be important in apoptosis (Saini and Walker, 1998). When cultured in serum-free medium plus zinc, hepatoma cells generate peroxides and rapidly undergo apoptosis, an effect that could be reversed by glutamine (Xu et al., 1997). The glutamine effect may result from its ability to act as an energy source or as a precursor for GSH, although these researchers concluded that its function as a GSH precursor was more important. It has been suggested that c-myc normally provides a proliferative stimulus involving hydrogen peroxide
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that is aborted in serum-free medium leading to activation of the apoptotic machinery (Xu et al., 1997). The peroxide produced might then modify factors affecting apoptosis, including NF-B and AP-1. What effect acrolein might have on c-myc, either directly or subsequent to its effects on GSH, is not known. In some cell types BSO does not appear to induce apoptosis, although it does markedly enhance the toxicity of alkylating agents and change the mode of death from apoptosis to necrosis (Fernandes and Cotter, 1994). GSH levels do not appear to be critical for the induction of death in GT1-7 cells since depletion with DEM, which causes a 100% loss of viability in control cells, does not induce any death in bcl-2 overexpressing cells (Kane et al., 1993). However, apoptosis per se was not assessed in this study, and the massive death in control cells is suggestive of a gross disruption of function. DEM was also reported to induce apoptosis in L929 transformants stably over-expressing human Fas (Hug et al., 1994). However, once again, the assay for viability was not specific for apoptosis and the 100% loss of viability within 3 to 6 h after treatment with DEM is not consistent with effects solely on GSH. Cell Proliferation, Gene Expression and the Thiol Redox State GSH participates in redox reactions via reversible oxidation of its active thiol and is the major regulator of the intracellular redox state. Under normal conditions, approximately 99% of this thiol exists in the reduced form at concentrations of from 0.5 to 10 mM, depending on the cell type. The intracellular distribution of GSH, particularly in terms of relative amounts of GSH and its disulfide (GSSG), in various compartments, remains unclear. It is evident, however, that GSH is present in cytosolic, mitochondrial, and nuclear pools, can distribute to the nucleus under various conditions (Briviba et al., 1993; Voehringer et al., 1998), and is involved in the redox regulation of signal transduction. The fluctuation of GSH levels in concert with DNA synthesis supports the involvement of this tri-peptide in the process of cellular division. Studies have shown that the redox state of normal fibroblasts becomes less reducing at confluency, when proliferation ceases while fibrosarcoma cells maintain a high reduction state and continue to proliferate despite increasing density (Hutter et al., 1997). Similarly, human bronchiolar epithelial cells and bronchiolar fibroblasts exhibit a correlation between GSH and proliferation (Atzori et al., 1994; Mallery et al., 1993). The generally high levels of GSH found in tumor cells could be a factor in their enhanced proliferative capacity. In K562 leukemia cells (Frischer et al., 1993) and human lung adenocarcinoma A549 cells (Kang and Enger, 1990), GSH varies proportionally with the rate of proliferation. Moreover, proliferation of A549 cells is inhibited when GSH is lowered with BSO (Kang and Enger, 1990). This effect is attenuated by
exogenous GSH (Kang, 1994), suggesting a functional role for GSH in proliferation. However, the growth modulatory effect of BSO on A549 cells grown in glutamine-deficient media correlates with changes in cellular glutamate levels and not with GSH levels (Kang, 1995). These investigators concluded that BSO inhibits glutamate-stimulated cell proliferation. Since exogenous GSH supplements the cells with glutamate via the ␥-glutamyl pathway, it bypasses the inhibition of amino acid uptake and overcomes the BSO-antiproliferative effect. While a positive correlation is often observed between GSH levels and cell proliferation, others have noted either no correlation (Spyrou and Holmgren, 1996) or even a suppression of proliferation following the administration of exogenous GSH (Cantin et al., 1990). Cell-specific differences may account for some of these variations, since addition of GSH to rat splenocytes stimulates proliferation (Pieri et al., 1992). Overall, the correlation between cell proliferation and cellular redox status is strong, but the mechanism by which such regulation occurs is not clear. GSH may play a role in both gene expression and the binding of various transcription factors to DNA. Studies of glucocorticoid receptor binding with DNA in COS2 cells have shown that depletion of GSH with DEM or BSO, or treatment of cells with H 2O 2, significantly decreases glucocorticoid receptor-DNA binding (Esposito et al., 1995). These authors also found that DEM significantly impaired the activation of CAT gene expression induced by dexamethasone in HeLa cells transfected with a plasmid containing the CAT gene under control of the glucocorticoid responsive element. In human T lymphocytes, depletion of GSH with BSO leads to a marked inhibition of the proliferative response (Walsh et al., 1995). However, while DNA and RNA synthesis are both reduced greater than 60% after 3 days of culture with BSO, no specific effects on mRNA expression of a number of critical T-cell genes required for activation and/or proliferation were detected. Acrolein and Specific Genes Relating to Proliferation and Apoptosis There are a large number of redox-sensitive genes and regulatory factors (Allen and Tresini, 2000), and the mechanism(s) by which alterations in the intracellular redox state may modulate gene expression in eukaryotes is clearly complex (Arrigo, 1999). Regulation via redox-sensitive transcription factors has generated substantial interest in recent years. These proteins bind to the promoter region of target genes allowing transcription to proceed. Redox regulation of this process can occur by activating these factors in the cytosol to forms that redistribute to the nucleus, or by affecting their binding to the promoter region. This latter process appears to require crucial cysteine moieties localized to the DNA binding domain. By binding to such nucleophilic amino acids, acrolein could directly affect transcription as well as modulate this process through its ability to deplete GSH. Although acrolein
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has not been widely studied for its ability to modulate the many genes known to affect cell division and apoptosis, some data exist demonstrating that, at least in select conditions, acrolein affects transcription factors important for proliferation and survival (see following sections). In contrast to the inhibition of cell growth seen with single in vitro acrolein treatments, it is interesting that repeated in vivo treatments with allylamine, which is metabolized by semicarbazide-sensitive amine oxidase to acrolein and hydrogen peroxide, modifies vascular smooth muscle cells such that when cultured they exhibit a persistent enhancement in their proliferative capacity (Ramos et al., 1993). Although not fully understood, this effect appears to involve the activation of NF-B as well as extracellular matrix proteins (Parrish and Ramos, 1997; Ramos, 1999). In vitro exposure to acrolein inhibits NF-B and AP-1 activation (see following section for more details). It is possible that the activation of these factors in vivo, and the augmented proliferation seen in vascular smooth muscle cells from allylamine-treated animals, represents an adaptation response to the acute inhibitory effects. However, the persistence of these changes also suggest a more fundamental alteration in the genome has occurred. NF-B NF-B is a member of the Rel protein family and resides in the cytoplasm in an inactive form as a homo- (p50:p50) or heterodimer (p50:p65) bound to IB (May and Ghosh, 1997). Upon activation, NF-B is translocated to the nucleus where it is an important mediator of transcription events associated with a variety of stress conditions. A few reports indicate NF-B may have a pro-apoptotic role in some systems (Sonenshein, 1997; van Antwerp et al., 1998). However, the preponderance of evidence suggests NF-B has a survival role and may be involved in the regulation of signaling events that control the expression of growth-related genes (Hutter and Greene, 2000). The activation of NF-B by TNF (Beg and Baltimore, 1996; van Antwerp et al., 1996), ionizing radiation or daunorubicin protects cells from apoptosis (Wang et al., 1996). Conversely, the inhibition of NF-B nuclear translocation induces apoptosis in B cells (Wu et al., 1996) and hepatocytes (Bellas et al., 1997) and enhances apoptotic death induced by various agents (van Antwerp et al., 1996; Wang et al., 1996). It has been suggested that the enhanced therapeutic activity often seen with combination chemotherapy including glucocorticoids is a consequence of these latter compounds ability to inhibit NF-B (Beg and Baltmore, 1996; Wang et al., 1996). Acrolein may have a similar effect and therefore improve the efficacy of cyclophosphamide therapy or enhance the toxicity of other xenobiotics. More so than most other factors, there is an extensive and rapidly expanding base of information regarding the role of oxidants in NF-B activation. This process appears to require both the presence of ROS and phosphorylation of the inhibitory
kappa B (IB) subunit that results in its ubiquitination, dissociation from the inactive complex, and degradation via the proteasome. A role for ROS is based on the observations that oxidizing conditions, including GSH depletion (Garcia-Ruiz et al., 1995), induce NF-B in several cell types; antioxidants can block this activation, and ROS production is enhanced by various NF-B inducers such as tumor necrosis factor ␣. In contrast, at the nuclear level, NF-B must be in the reduced form in order to bind to DNA. Thus, the redox status of a specific subcellular site is crucial for determining the activation state of NF-B (Flohe´ et al., 1997; Gius et al., 1999). For example, while oxidation can activate NF-B, BSO (which depletes GSH) (Dro¨ge et al., 1994) and diamide (which oxidizes GSH to GSSG) inhibit its DNA binding activity (Toledano and Leonard, 1991). The inhibition of NF-B activation by the antioxidant PDTC can be reversed by the treatment of inhibited nuclear extracts with 2-mercaptoethanol, a reducing agent (Brennan and O’Neill, 1996). PDTC increased GSSG levels in treated cells suggesting its effects on NF-B are due to a prooxidant rather than antioxidant activity, somewhat confusing the issue. In general, GSH appears to be required for NF-B activation following an exposure to oxidizing agents (Ginn-Pease and Whisler, 1996), a finding consistent with the need to reduce this transcription factor prior to its binding to DNA. Thus, GSH itself, or the GSH/GSSG ratio, may be a critical factor in the regulation of NF-B (Renard et al., 1997). Acrolein has rapid and profound effects on cellular GSH content, and based on the purported redox regulation of NFB, might be expected to affect NF-B activation, either indirectly through changes in GSH or more directly such as by binding to the critical nucleophilic cysteine on the p50 subunit and/or p65 subunits (Kumar et al., 1992; Toledano et al., 1993). It is also possible that acrolein exposures lead to increased intracellular calcium which, at least in kidney epithelial cells, is able to promote NF-B activation independent of thiol redox status (Woods et al., 1999). NF-B activity after exposure to acrolein may vary depending on the extent and duration of GSH depletion and the specific subcellular location of this depletion (with intranuclear GSH being critical). Li et al. (1999) reported that acrolein induced IB in rat alveolar macrophage cells within 15 min of a 5 M exposure. This induction apparently led to the inhibition of NF-B activation seen in gel-shift assays. Whether or not GSH was depleted at the doses used in this study, or whether GSH is a mediator of the observed effects, was not reported. In A549 cells treated with acrolein, a similar increase in IB does not occur, suggesting that the inhibition of activation of NF-B by acrolein is IB-independent (Horton et al., 1999). The effect of acrolein on NF-B may, therefore, be cell-type specific and other regulatory mechanisms could be involved. For example, it is possible that decreased NF-B activation could occur through decreased nuclear binding, which requires a reductive environment, or through changes in
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specific subunit components—perhaps covalent modification by acrolein. AP-1, Fos and Jun Activator protein 1 (AP-1) is a ubiquitous collection of protein complexes known to regulate transcription in response to environmental stimuli. It is composed of various gene products from the fos and jun proto-oncogene families. The products of these genes form homodimeric (Jun-Jun) and heterodimeric (Fos-Jun) complexes that bind to DNA, suggesting a crucial role of AP-1 in the control of cell proliferation (Rupec and Baeurle, 1995). In addition to being stimulated by a wide range of xenobiotics or factors that promote cell proliferation, several studies support the hypothesis that cellular redox state plays an important role in the activation of AP-1 (Pinkus et al., 1996; Rupec and Baeurle, 1995). AP-1 appears to be part of a general mechanism of regulation of gene expression following an alteration in redox balance. However, the pathways that control this expression are not clear. Antioxidants strongly activate AP-1 (Meyer et al., 1993; Schenk et al., 1994) yet growth factor-induced AP-1 activation appears to be ROSdependent (Sen and Packer, 1996). While AP-1 and NF-B seem to respond oppositely to antioxidants (Rupec and Baeurle, 1995), oxidants may have a role in the activation process of both factors (Pinkus et al., 1996). Some similarity in response is supported by our data showing that, as with NF-B, acrolein decreases AP-1 activation, probably through direct binding to Fos or Jun proteins (Biswal et al., 2000a). Irrespective of the specific role for oxidants and antioxidants, AP-1 activation appears to have links with the control of cell proliferation by NF-B, fos, and jun. Data suggesting a decreased activation of these transcription factors following acrolein treatments that deplete GSH are consistent with the activation reported with N-acetylcysteine, a treatment that increases GSH levels. On the other hand, the ability of N-acetylcysteine to prevent apoptosis in PC12 cells caused by trophic factor withdrawal appears to be due to effects on gene expression, not GSH levels (Yan et al., 1995). This suggests that at least some of acrolein’s effects on gene expression could be unrelated to its ability to deplete GSH. p53 DNA damage induced by chemicals and ionizing radiation is associated with the expression of negative regulators of the cell cycle. One of the most important of these regulators is the p53 gene product. The arrest of cells in G1 and G2 phases of the cell cycle mediated by p53 provides time for DNA repair or, in the absence of adequate repair, allows apoptosis to proceed. Studies on p53 and related genes have been extensive in recent years; p53 DNA binding and transcriptional activities are dependent on cysteines present in the DNA-binding domain (Hainaut and Milner, 1993). In addition, increasing the relative reduction of cellular thiols with N-acetylcysteine can elevate
p53 expression by increasing the rate of p53 mRNA translation (Liu et al., 1998). Thus, p53 activity is controlled at several levels by the thiol redox state of the cell. Acrolein may modify this control by binding to the cysteine residues preventing p53 from binding with its specific DNA target sequence or by altering the overall content of reduced thiols. Since p53 has a B site in its promoter region (Wu and Lozano, 1994), effects of acrolein on NF-B suggest this aldehyde may also alter p53 through this pathway. c-myc A transient increase in c-myc expression is an important signal for cells to enter S-phase (Kato and Dang, 1992; Saini and Walker, 1998). The blockade of endothelial-cell proliferation by non-lethal doses of tumor necrosis factor prevents this increase (Lo´pez-Marure et al., 1997). While such a correlation does not prove cause-effect, similar interference with c-myc expression during growth arrest has been reported in other model systems (Zentella and Massague, 1992). It must be noted, however, that the function of c-myc in cell proliferation is complex, with significant amounts of conflicting data in the literature. This gene appears to play a role in both transcriptional repression and activation. Among various factors that may be involved, NF-B has been reported to activate the c-myc promoter (La Rosa et al., 1994; van Antwerp et al., 1998) creating a link between these two factors. Furthermore, despite its clear importance in cell-cycle progression, it also seems to have a role in apoptosis. For example, the upregulation of c-myc has been found in apoptotic cells (Pandey and Wang, 1995). Two alternative translation products, c-Myc 1 (which suppresses growth) and c-Myc 2 (which stimulates growth) may explain at least a portion of this controversy. It is obvious, however, that this gene could play an important role in the effects of acrolein on proliferation and apoptosis. Other Factors A large number of additional genes and factors appear to be redox-sensitive and/or to contain cysteine residues that are critical for their binding to DNA (Allen and Tresini, 2000). These include transcription factors such as Elk-1, Egr, HSF, TTF-1, PAX-8, nuclear factor 1, upstream stimulatory factor, E2F, IIIC, U, Egr-1, vETS, the bovine papillomavirus type 1 E2 protein, Sp1, PB2/CBF, and glucocorticoid and estrogen receptors (Arrigo, 1999; Sen, 1998). Other redox sensitive targets include selected antioxidant enzymes, kinases, cytokines, ion transporters, phosphatases, and a few other proteins (Sen, 1998). Although not yet studied, acrolein would appear to be capable of modulating the activity of most if not all of these factors. Gene Expression after Acrolein Treatments As a highly electrophilic agent, acrolein might be expected to stimulate the family of genes regulated by the electrophile
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response element (ERE) also referred to as the antioxidant response element (ARE). ERE is a cis-acting DNA element that was first characterized in the promoter of the gene encoding the rat and mouse glutathione S-transferase Ya subunits (Rushmore and Pickett, 1990; Friling et al., 1990), which consists of two AP-1-like binding motifs. Because of the large number of potential targets for acrolein, we have used gene array technology to evaluate the genes that are modulated following exposure of human lung adenocarcinoma A549 cells to acrolein. This cell line was chosen because of the extensive data available on it regarding thiols and gene expression, and because, as a lung cell line, it has some relevance to potential environmental exposures. A cDNA microarray from Research Genetics (GF211) with 4000 known genes was used to study the effect of acrolein on gene expression. P 33-labeled cDNA was made from total RNA isolated from cells exposed to vehicle or 45 fmol/cell of acrolein for 30 min, followed by 30 min of recovery and hybridized to the membrane as per the manufacturer’s protocol. This dose and time of acrolein exposure depletes GSH by ⬃ 80% and inhibits proliferation without any lethality (Horton et al., 1997). The images were captured using the Cyclone Imager (Packard) and analyzed using the Pathways 2.0 software (Research Genetics). We found that expression of 732 genes were significantly upregulated and 282 genes downregulated beyond 3-fold. As expected, ERE genes including glutathione S-transferase and ␥-glutamylcysteine synthetase were among those upregulated to the greatest extent. The reason why such a large percentage of the genes examined responded is not known, but it may be related to the general reactivity of acrolein with nucleophiles thereby disrupting numerous pathways. A detailed analysis of genes whose expression is differentially regulated in response to low dosage of acrolein is in progress and is expected to aid in understanding the mechanism of action of this reactive aldehyde. Summary Acrolein is a common environmental contaminant and a compound formed by the metabolism of several xenobiotics, including the important anticancer drug, cyclophosphamide, which readily reacts with cellular nucleophiles. While the acute toxicity of this aldehyde has been well studied, more subtle molecular effects with lower doses remain unknown. Our findings that low acrolein doses can inhibit cell proliferation without affecting viability (Horton et al., 1997) and can enhance apoptosis by a secondary agent (Kern et al., 2000) provide an intriguing model system in which to examine how an important environmental agent can modulate the function of growth factors and cellular signaling events. Preliminary gene array data indicate that the expressions of numerous genes are both up- and downregulated in response to acrolein. Future experiments to assess the mechanism and functional importance of these changes will provide data to improve our un-
derstanding of the molecular mechanisms by which reactive environmental molecules can alter cell division and may assist in developing therapeutic strategies to protect normal tissues from environmental toxicants. ACKNOWLEDGMENTS Thanks go to Philip Bowman at the U.S. Army Institute of Surgical Research, Fort Sam Houston, Texas for his expert assistance with the microarray work. This work was supported by NIH grant ES09791. Support from NIEHS Center grant ES07784 is also acknowledged. S.S.B. is supported by a National Research Service Award (F32 ES05896) from the NIEHS. J.P.K. is the Gustavus and Louise Pfeiffer Professor of Toxicology.
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