JUNE 2006 VOLUME 19, NUMBER 6 © Copyright 2006 by the American Chemical Society
PerspectiVe The Future of ToxicologysDoes It Matter How Cells Die? Sten Orrenius* and Boris Zhivotovsky Institute of EnVironmental Medicine, DiVision of Toxicology, Karolinska Institutet, Box 210, Stockholm, SE-171 77, Sweden ReceiVed March 23, 2006
Contents Introduction Background Role of Gene Expression Role of Signal Transduction Role of Proteases Role of ROS Role of Cell Metabolism and ATP Generation Concluding Remarks
729 729 730 730 731 732 732 732
Introduction It is generally recognized that studies of the mechanisms of action of various poisons and chemical toxicants have been of critical importance for our understanding of fundamental biological processes. Claude Bernard (1813-1878) argued that the physiological analysis of organic systems can be done with the aid of toxic agents, and he applied this principle using curare to identify its site of action within the neuromuscular junction and carbon monoxide to demonstrate its formation of a complex with hemoglobin. More recent examples of biochemical lesions induced by toxicants, which have helped our understanding of basic cell physiology, include Sir Rudolph Peters’ discovery of the inhibition of the citric acid cycle by fluoroacetate (“lethal synthesis”) and the use of rotenone in studies of the mitochondrial respiratory chain. There are now numerous examples of * To whom correspondence
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toxins that have provided highly specific tools for the dissection of complex biological systems. Although many of these studies have originated in fields other than toxicology, their toxicological implications have been obvious and have led to further mechanistic studies in this area. In fact, it is clear that cross-talk with basic science has been instrumental for the development of toxicology in addition to furthering our understanding of fundamental biological processes. A recent example of technology transfer is the application of genomics, proteomics, and metabonomics in toxicological studies. However, in this perspective, we shall discuss another rapidly expanding research area in toxicology, i.e., the possible implications of the mode of cell death for our understanding of the toxicity of chemicals. The fact that there are different types of cell death was first recognized by pathologists and radiobiologists, but recent advances in the molecular characterization of these processes have made the cell death research field grow almost exponentially and spread into multiple disciplines, including toxicology.
Background Toxicants can kill cells by blocking vital metabolic processes. Auroleus Phillipus Theostratus Bombastus von Hohenheim (1493-1541), also known as Paracelsus and widely regarded as the father of modern toxicology, formulated the expression, “All things are poison and nothing is without poison”, suggesting that “dose determines the poison”. He was the first to emphasize the dose-response relationship of toxic substances. Indeed, depending on the concentration of the toxic agent and the susceptibility of the particular cell type, distinct pathways
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Figure 1. Dose determines the poison.
leading to cell death might become activated (Figure 1). Although several modes of cell death are now known, e.g., apoptosis, necrosis, autophagy, and mitotic catastrophe, the molecular mechanisms involved in apoptosis and necrosis have best been characterized. In 1971, John Kerr introduced the term “shrinkage necrosis” to describe the morphology of a process controlling tissue homeostasis (1). Previously, necrosis, or “accidental” cell death, was the predominant model and was also thought to be the universal mode of cell death. Later on, shrinkage necrosis was renamed “apoptosis” to distinguish physiological from pathological cell death (2). These two types of cell death exhibit distinct biochemistry and morphology (3). Apoptotic morphology is characterized by chromatin compaction and segregation against the nuclear envelope, condensation of the cytoplasm, nuclear fragmentation, and development of cell surface protuberances followed by the formation of apoptotic bodies with integral plasma membrane that are phagocytosed by macrophages or adjacent cells. Necrosis, in turn, is characterized by the clumping of chromatin, swelling of organelles, appearance of flocculent densities in the matrix of mitochondria, breakdown of cellular membranes, and cell disintegration. The order of morphological changes during apoptosis depends on the ability of the dying cell to engage in ATP-dependent processes of self-degradation. In contrast, necrosis can be reproduced experimentally by impairing a cell’s ability to produce ATP. Therefore, apoptosis has long been regarded as an active or “programmed” form of cell death, while necrosis has been seen as a form of death that is uncontrolled or pathological. However, recent evidence suggests that necrotic cell death can also occur as a programmed event that might contribute to organ development and to the maintenance of tissue homeostasis (4). Following the recognition of apoptosis as a distinct form of cell death, in 1987, Andrew Wyllie put forward two hypotheses related to its role in toxic or pathological cell death (5). He suggested that apoptosis might be induced by injurious stimuli of lesser amplitude than those causing necrosis in the same cells and that this might occur more readily in cell populations primed for apoptosis. However, recent studies have demonstrated that there is a cross-talk between the two modes of cell death and that features of both apoptosis and necrosis may coexist in the same cell (Figure 2). Moreover, inhibition of the apoptotic process, after it has been initiated, might result in cell death with necrotic features. This process is called “secondary necrosis”.
Role of Gene Expression To answer the question whether apoptosis and necrosis represent fundamentally different death modes, it is important to consider whether both of them can be directly modulated by gene expression. Although the requirement of sequential gene activation was clearly documented in the pioneering studies of programmed cell death in Caenorhabditis elegans (6), the need for gene activation during inducible cell death is less clear and often missing. The main players in the cell death process are constitutively present in most cells and are under control of inhibitory factors. Hence, protein synthesis might function
Figure 2. Factors that favor either apoptosis or necrosis.
mainly to generate triggers for the suicide process. Interestingly, immediate early gene proteins, including Fos, Jun, and Myc, are expressed during both apoptosis and necrosis. However, expression of the proto-oncogene, myc, a transcriptional activator of p53, can provide a proapoptotic signal when associated with Max. Importantly, the decision to proliferate or die by cells overexpressing c-myc depends on the presence or absence of extracellular survival factors and might require the function of the bcl-2 gene (7). p53 is pivotal in the cellular response to DNA damaging agents and causes cell cycle arrest to allow for DNA repair or, if this is not possible, cell deletion by apoptosis. Being transcriptionally activated, p53 induces the synthesis of several proapoptotic Bcl-2 family proteins followed by activation of the mitochondrially mediated apoptotic pathway (8). The recent identification of two homologues, p63 and p73, revealed that p53 is a member of a family of related transcription factors (9). Given that they share amino acid sequence identity, all three proteins should have redundant functions in the regulation of gene expression. Indeed, p73 can activate p53-regulated genes and suppress cell growth or induce apoptosis. Moreover, p73 and p53 are both induced by DNA damage, albeit through distinct mechanisms. Although the increased expression of p53 was observed during liver regeneration after partial hepatectomy and after treatment with carbon tetrachloride (10), there is no direct evidence that p53 might trigger necrosis. In the search for universal death-related genes, most attention has been focused on bcl-2. Overexpression of Bax and other proapoptotic members of the Bcl-2 family kills cells via apoptosis; however, overexpression of Bcl-2, surprisingly, protects cells from both apoptosis and necrosis (Figure 2), at least neuronal cells exposed to toxicants, oxidative stress, or hypoxia (for a review, see ref 11). Whether this effect is cellspecific remains unclear. The mechanism of Bcl-2 protection from necrosis might simply involve its antioxidant activity. Another possibility is that Bcl-2 prevents the consequences of mitochondrial dysfunction (i.e., collapse of the mitochondrial transmembrane potential, uncoupling of the respiratory chain, overproduction of superoxide anions, outflow of matrix calcium and glutathione, and release of soluble intermembrane space proteins), which might otherwise lead to a bioenergetic catastrophe with necrosis as the ultimate result. However, more work is required to understand the precise mechanism of the antinecrotic function of Bcl-2 and its homologues.
Role of Signal Transduction Signal transduction is thought to play a key role in the onset of both apoptosis and necrosis, and death signaling is often mediated by an increase in the intracellular Ca2+ level. Historically, the role of Ca2+ as a death trigger dates back to
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Fleckenstein’s proposal that excess Ca2+ entry into myocytes might be the mechanism that underlies cardiac pathology after ischemia (12). Subsequent studies emphasized the general importance of this observation, as both receptor overstimulation and cytotoxic agents were found to cause lethal Ca2+ influx into cells. As we learned more about intracellular Ca2+ distribution and fluxes, it also became clear that nondisruptive changes in Ca2+ signaling could have adverse effects, leading to alterations in cell proliferation and differentiation, as well as modulation of cell death (for a review, see ref 13). It is now known that Ca2+-dependent processes are interwoven with the mainstream apoptosis execution system, the caspases, and recent findings indicate that interference with Ca2+ sequestration into intracellular pools (i.e., the endoplasmic reticulum; ER) can be sufficient to trigger cell death as part of a stress response. In addition, Ca2+-dependent processes can be recruited to warrant the final elimination of dead or dying cells by promoting either their phagocytosis or their lysis. Finally, Ca2+ overload and Ca2+-dependent processes have recently been shown to activate and modulate the execution of a necrotic death program in C. elegans (14). The resulting mode of cell death is, most likely, determined by the concentration of cytosolic Ca2+. Whereas low to moderate Ca2+ elevations (200-400 nM) trigger apoptosis, higher concentrations of Ca2+ (>1 µM) are associated with necrosis (Figure 2). This might explain why excessive Ca2+ release from the ER normally results in apoptosis, while influx of extracellular Ca2+ through the plasma membrane is often associated with necrosis. As mentioned above, Bcl-2 family proteins act at multiple sites in the death pathways and, therefore, confer protection that is largely independent of the nature of the lethal agent. Both the ER and the mitochondria represent such sites of action for Bcl-2 family proteins, and one might speculate that they can modulate apoptosis and necrosis by affecting Ca2+ fluxes from the ER to the cytosol and mitochondria, thereby preserving mitochondrial integrity.
Role of Proteases Various proteases have been implicated in the initiation and execution of both apoptosis and necrosis (15). Some of them are regulated by Ca2+, e.g., the calpains, a family of cysteine proteases, which includes several tissue-specific isoforms (ncalpains) and two ubiquitous isozymes (µ-calpain and mcalpain). For activation, µ- and m-calpains require micromolar and millimolar Ca2+ concentrations, respectively. Cleavage of particular proteins by calpain might be essential for the development of the cell death process. For example, it has recently been shown that calpain-mediated cleavage of the endogenous calcineurin inhibitor cain (also known as cabin 1) results in the activation of calcineurin and the promotion of Ca2+-triggered cell death (16). Furthermore, Ca2+ entry via voltage-sensitive L type channels can activate calpain in brain regions affected by simulated stroke to cleave the Na+/Ca2+ exchanger NCX, in particular the NCX3 isoform required for extrusion of Ca2+. This leads to the inability of the cells to extrude Ca2+ and, hence, to a sustained intracellular Ca2+ increase (17). Accordingly, overexpression of calpastatin in granule neurons was able to block glutamate-induced NCX3 cleavage and to reduce neuronal death. Importantly, calpains can cleave several proteins, which are also targets for another family of “death proteases”, the caspases. Although both calpains and caspases are cysteine proteases, their functions and cleavage specificities are different. While cleavage of some proteins by either calpains or caspases activates their function during cell death, the proteolysis of others abrogates their
proapoptotic activity. Thus, cleavage of fodrin, Bcl-2, Bid, and Bcl-XL, although at different sites, promotes cell death, whereas cleavage of caspase-3, -7, -8, and -9 by calpain inactivates caspase function. On the other hand, calpain-mediated cleavage of procaspase-12 results in its activation, although the precise mechanism for this is unknown (18). Interestingly, the NCX cleavage by calpain discussed above has an intriguing parallel in the caspase-mediated cleavage of another Ca2+-extrusion pump in the plasma membrane, the Ca2+-ATPase, PMCA (19). It is likely that both of these proteolytic events can promote cell death by impairing cellular defense mechanisms, although under different circumstances. Hence, accumulating data indicate that there is cross-talk between calpain and caspases in the regulation of cell death. However, during apoptosis, calpains might also operate independently of caspases. For example, it was recently shown that both the precursor and the mature forms of recombinant AIF were cleaved near the amino terminus by calpain in vitro (20). Mitochondrial permeabilization by truncated Bid induced AIF release only in the presence of active calpain. Importantly, inhibition of calpain by calpeptin precluded AIF release, demonstrating that proteolytic activity was required for release. Both calpeptin and cyclosporine A also inhibited calciuminduced AIF release from isolated liver mitochondria, implicating the involvement of endogenous mitochondrial calpain activity in the release of AIF during permeability transition. Finally, studies in transgenic mice showed that calpastatin deficiency augmented kainate-evoked excitotoxicity in the hippocampus and resulted in mitochondrial changes associated with AIF release. Consistently, calpastatin overexpression suppressed these effects. These results define a novel mechanism of calpain involvement in cell death through mobilization of proapoptotic factors in a caspase-independent manner. Importantly, the involvement of caspases was documented only in apoptosis (Figure 2). The importance of another family of proteases, lysosomal cathepsins, in cellular and tissue autolysis during uncontrolled necrosis is well-established. Recent observations have suggested the involvement of lysosomes in programmed cell necrosis as well (21). Moreover, there are also some indications supporting the involvement of lysosomal activities in apoptosis. Hence, cathepsins are engaged in the execution of cell death triggered by agents causing lysosomal membrane permeabilization (LMP). LMP can also be induced by a variety of apoptotic stimuli, including lysosomotropic toxins, although the precise mechanism of permeabilization of the lysosomal membrane is unknown. It was suggested that in response to cell death stimuli, the lysosomal sphingomyelinase is activated and converts sphingomyelin into ceramide, which can be further converted into sphingosine by ceramidase. Sphingosine was shown to act as a detergent that might cause LMP. Because both ceramide and sphingosine are known to be involved in the regulation of apoptosis, it was hypothesized that at low concentrations (20 µM) it causes extensive lysosomal rupture and necrosis (22). However, it is unclear how partial rupture of the lysosomal membrane might occur. In some experimental systems, LMP is followed by mitochondrial permeabilization and caspase activation, which are two hallmarks of apoptosis. A possible mechanism linking lysosomes with mitochondria is provided by the observation that several cathepsins can cleave Bid, which is an important trigger of the mitochondrially mediated apoptotic pathway.
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However, the lethal effect of LMP and cytosolic cathepsins is not limited to the activation of the mitochondrial pathway. In some cancer cells treated with microtubule-stabilizing poisons, LMP occurs relatively early and cytosolic cathepsins mediate micronucleation and cell death in a caspase-independent manner. The precise signals leading to rupture of the lysosomal membrane are still unknown, although it was suggested that in some systems generation of reactive oxygen species (ROS) might induce LMP (23). In others, caspase-dependent LMP was observed. However, inhibition of caspase activity offered no protection but resulted in caspase-independent LMP and necrotic cell death. Furthermore, a sustained increase in calpain activity has been found to lead to lysosomal rupture and cell death in postischemic CA1 neurons. In this case, activated calpain was responsible for the spillage of hydrolytic cathepsins as well as lysosome-associated membrane protein-1 (LAMP-1) from lysosomes, leading to cell death (24). Thus, it is clear that calpains, caspases, and cathepsins do interact with each other and that cleavage of the same proteins by two (or more) proteases allows either protease alone, or both in combination, to activate apoptotic or necrotic processes.
Role of ROS It is well-known that physiological levels of ROS modulate transcription and can act as a defense mechanism to toxicants. However, excessive ROS production leads to oxidative stress, damage to intracellular molecules and organelles, and, ultimately, cell death. ROS are mainly generated in mitochondria and are neutralized by enzymes, e.g., superoxide dismutases, glutathione peroxidase, catalase, and peroxiredoxins, or via interaction with endogenous antioxidants or glutathione. One of the most reproducible inducers of apoptosis is mild oxidative stress, whereas sustained high concentrations of ROS lead to necrosis. Early work in our group revealed that a pancreatic β-cell line progressively underwent discrete responses of proliferation, apoptosis, and necrosis as the concentration of a pro-oxidant quinone in the medium was increased (25). Oxidative modification of proteins and lipids has been observed in cells undergoing apoptosis also in response to nonoxidative stimuli, suggesting that a redox shift might be a general feature of the effector phase of apoptosis. The caspases themselves are cysteine-dependent enzymes and, as such, are redox sensitive. Accordingly, studies of hydrogen peroxideinduced apoptosis suggested that mild oxidative stress causes caspase activation, whereas prolonged, or excessive, oxidative stress results in caspase inactivation and necrotic cell death (26). A physiological example of this is the NADPH oxidase-derived oxidants generated by stimulated neutrophils that prevent caspase activation in these cells. In addition to effects on caspase activation in the cytosol, the generation of ROS also seems to be associated with apoptotic events in other intracellular compartments. Thus, ROS production has recently been shown to be involved in the oxidation of mitochondrial cardiolipin by a peroxidase function of the cardiolipin-cytochrome c complex (27). This might lead to detachment of the hemoprotein from its binding to the mitochondrial inner membrane and its extrusion into the soluble cytoplasm through pores in the outer membrane. The peroxidase function of the cardiolipin-cytochrome c complex is compatible with the previously proposed “two-step hypothesis” of cytochrome c release and provides a plausible explanation for the protective effects against apoptosis reported for multiple mitochondrial antioxidant enzymes (28). ROS-induced DNA strand breaks lead to activation of the nuclear enzyme poly(ADP-ribose)polymerase-1 (PARP-1) fol-
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lowed by consumption of NAD+ and ATP, which, if excessive, can result in cell death by either apoptosis or necrosis. Activation of PARP results in destabilization of the surrounding chromatin, which allows access of repair enzymes to the damaged DNA. During apoptosis, PARP is cleaved by caspases and abrogates DNA repair. On the other hand, excessive PARP activation might result in necrosis by depletion of the cellular NAD+ and ATP pools. Accordingly, blocking PARP activity using inhibitors or RNA interference technology confers resistance to necrosis induced by DNA-alkylating agents (29).
Role of Cell Metabolism and ATP Generation Manipulating cell metabolism and ATP generation can affect cell fate. The apoptotic process is energy-dependent, and the main generation of ATP occurs through oxidative phosphorylation and/or glycolysis. Hence, decreasing the ATP level by incubation of cells in glucose-free medium, or with mitochondrial inhibitors, such as rotenone or S-nitrosoglutathione, results in cells resistant to apoptosis. Instead, prolonged incubation under ATP-depleting conditions leads to necrotic cell death. However, modulation of the intracellular ATP level might reverse the cell death mode from necrosis to apoptosis as well (Figure 2). Interestingly, Bcl-2, but not Bcl-XL, is able to reduce overall cell death in ATP-depleted cells regardless of whether it occurs by apoptosis or necrosis (30, 31). The mitochondrial permeability transition is considered to be a key event in necrosis and to amplify apoptosis by mediating the release of several proapoptotic proteins located in the mitochondrial intermembrane space. One of the proteins involved in the formation of the permeability transition pore is adenine nucleotide translocase (ANT), which mediates ADPATP exchange across the mitochondrial inner membrane. However, in ANT-deficient mitochondria, pore opening can still occur in response to treatment with well-known inducers, such as calcium ionophores, tert-butylhydroperoxide, and diamide. Hence, treatment of ANT-deficient hepatocytes with calcium ionophores resulted in necrosis, which could be blocked by cyclosporine A (32). In cells, cyclosporine A interacts with cyclophilin D (CypD), a matrix protein, which is also involved in pore formation. Recently, to analyze the role of CypD in cell death, knockout mice lacking this protein were created. Mitochondria isolated from tissues of such CypD-deficient mice did not undergo cyclosporine-A-sensitive permeability transition. Furthermore, CypD-deficient cells died normally in response to various apoptotic stimuli but showed resistance to necrotic cell death induced by ROS and calcium overload. Moreover, CypD-deficient mice demonstrated increased resistance to ischemia/reperfusion-induced cardiac injury (33). These results indicate that the mechanisms of mitochondrial membrane permeabilization might differ between apoptosis and necrosis and that CypD-dependent permeability transition pore formation triggers some forms of necrotic, but probably not apoptotic, cell death.
Concluding Remarks From the discussion above, it is obvious that cells can die in various ways and that toxicants can trigger multiple death pathways. However, it is equally clear that there is considerable cross-talk between these pathways and that features of both apoptosis and necrosis can coexist within a dying cell. In other words, the same lethal insult can result in distinct modes of cell death depending on the circumstances, e.g., the apoptosis/ necrosis threshold of the particular cell type, the metabolic state of the cell, exposure to other stresses, preconditioning, etc.
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So, does it matter how cells die? Yes, for our understanding of chemical hazards and the mechanisms by which toxicants can damage our cells and tissues, it certainly does! Cell death is the ultimate result of toxicity, and elucidating the signaling pathways involved is highly relevant for our understanding of the cellular targets and mechanisms of action of chemical toxins. Such knowledge can also be important in attempts to design less toxic analogues. Furthermore, using modern methodological approaches, e.g., genomics, proteomics, and lipidomics, knowledge of cell death mechanisms will help us understand how chemicals might interfere with cellular regulation of gene transcription and protein and lipid turnover. Is this important for the future of toxicology? Again, the answer is yes! Cell death research is a typical example of an area of potential cross-fertilization between toxicology and basic science. As exemplified above, mechanistic studies using toxicants have often contributed to our understanding of fundamental biological processes in the past. Similar studies have also helped us foresee the toxicological implications and potential health hazards of chemicals. Many of us believe that there is a risk that toxicology is becoming “too applied” and that maintenance of close contacts with fundamental biomedical research is a “must” for a successful future of our discipline. In our opinion, the cell death research field can serve as an important link between basic science and toxicology and as an area of cross-fertilization between these disciplines.
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