56, 255–261 (2000) Copyright © 2000 by the Society of Toxicology
TOXICOLOGICAL SCIENCES
FORUM Metals and Disorders of Cell Accumulation: Modulation of Apoptosis and Cell Proliferation Michael P. Waalkes,* ,1 Donald A. Fox,† J. Christopher States,‡ Steven R. Patierno,§ and Michael J. McCabe, Jr.¶ *Inorganic Carcinogenesis Section, Laboratory of Comparative Carcinogenesis, National Cancer Institute at National Institute of Environmental Health Sciences, 111 Alexander Drive, P.O. Box 12233, MD F0-09, Research Triangle Park, North Carolina; †College of Optometry and Department of Biology and Biochemistry, University of Houston, Houston, Texas; ‡Department of Pharmacology and Toxicology, University of Louisville School of Medicine, Louisville, Kentucky; §Department of Pharmacology, Molecular and Cellular Oncology Program, The George Washington University Medical Center, Washington, D.C.; and ¶Institute of Chemical Toxicology, Wayne State University, Detroit, Michigan Received March 7, 2000; accepted April 20, 2000
Key Words: arsenic; apoptosis; cell proliferation; chromium; lead; mercury.
Introduction As a class of agents, toxic metals are a concern of the highest priority for human exposure. The metals have a vast array of remarkably adverse effects, including those of carcinogenicity, neurotoxicity, and immunotoxicity. Metals are also non-biodegradable and persist in the environment. Anthropogenic use has led to global dispersion of metals in the environment. Because of their wide distribution and extensive use in modern society, some human exposure to toxic metals is inevitable. Defining the mechanisms of metal toxicity has been problematic because of the intricate nature of the interactions of metals with living systems. Additionally, many metals, like zinc, copper, calcium, trivalent chromium and iron, are essential to life and complex mechanisms have evolved for their safe transport and usage, including transport and storage proteins. Essential metals are involved in a variety of critical functions including control of gene transcription, nerve conductance, oxygen carrying and delivery, and as active centers in enzymes. Evidence is emerging that some essential metals, such as zinc, may also function in control of apoptotic cell death (Chai et al., 1999). Thus, the essential metals can impact a variety of critical molecular events within the cell, including events associated with regulation of cell accumulation such as gene expression, cell proliferation, and cell death by apoptosis. Some toxic metals may mimic the essential metals and thereby gain access to important molecular targets. Here they can markedly alter function. It is clear that toxic metals can both 1
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activate and inactivate cellular processes controlled by the essential metals. It is important to note that even essential metals can be toxic, and chromium is an excellent example of this dichotomy. The carcinogenic potential of many metals is a major issue in defining human health risk from exposure. For example, certain compounds of hexavalent chromium, a transition metal, are clearly human carcinogens, causing tumors in the respiratory system (IARC, 1990). Similarly, environmental or occupational arsenic exposures are definitively associated with human cancer (NRC, 1999). Arsenic, a metalloid, is a multi-site carcinogen and causes tumors in the lung, skin, urinary bladder, liver, and probably other sites (NRC, 1999). Defining mechanisms in metal carcinogenesis is critical to defining human risk. However, no clear mechanism has emerged for any of the metallic carcinogens. For many metals, aberrant cell proliferation, including alterations in apoptosis, is an attractive aspect of a hypothetical mechanism of cancer induction. Several metals also have the potential to be potent and debilitating neurotoxins. For instance, the neurotoxic effects of lead in humans are well established, and exposure can cause adverse effects ranging from IQ deficit to peripheral neuropathy (Verity, 1995). The neurotoxic effects of lead clearly are more pronounced in the earlier stages of development. Other metals, like mercury, are recognized as potential immunotoxins (Lawrence and McCabe, 1995). For instance, there appears to be a connection between experimental mercury exposure and autoimmunity. Both immunotoxicity and neurotoxicity potentially can have aberrant apoptosis as an important element. Apoptotic cell death should be considered as an ongoing, normal event in the control of cell populations. However, apoptosis can also be induced by a variety of toxicants, including many of the toxic inorganics, resulting in the loss of affected cell populations. Apoptosis essentially occurs when
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cellular damage, including damage to genetic material, has exceeded the capacity for repair. What has often received less attention is the concept that environmental agents, including metals, can impair apoptosis, and that suppression of the apoptotic response could facilitate aberrant cell accumulation, which may be a critical step in the pathogenesis of malignancy or autoimmunity. Thus, for many toxic metals, disorders of cell accumulation may be a crucial aspect of their toxicity, but these disorders have just recently begun to be recognized. For instance, there is now evidence in model systems that apoptosis is a key factor in the neurotoxicity of lead. With arsenic, cell cycle dysregulation and altered DNA repair and apoptosis may be important early events in transformation potentially leading to malignancy development. Similarly, the carcinogenic potential of chromium, which can be a potent DNA damaging agent, may be related to the emergence of resistance to apoptosis, while perturbation of normal apoptosis appears to be a key factor in mercury-induced autoimmunity. Understanding the mechanisms by which metals induce disorders of cell accumulation will be important in defining their toxic potentials in exposed populations. In an attempt to illuminate some of the most recent developments in metal-induced disorders of cell accumulation, a symposium on this topic was held at the 1999 Annual Meeting of the Society of Toxicology. The following are a synopsis of the presentations at this symposium. Mitochondria Coordinate the Upstream and Downstream Events in Lead-Induced Rod Photoreceptor-Selective Apoptosis (D. A. Fox, L. He, and A. T. Poblenz) Apoptosis is an active mode of cell death that is induced by a variety of physiological and pathological stimuli. Convergent evidence suggests that mitochondria and caspases play a central and fundamental role in the effector or executioner phase of apoptosis (Green and Kroemer, 1998; Susin et al., 1998). Early during the effector phase, the mitochondrial permeability transition pore (PTP) is opened by a variety of apoptotic 2⫹ inducers such as elevated matrix Ca pro-oxidants, and thiolreactive agents (Bernardi, 1999; Ichas and Mazat, 1998; Susin et al., 1998). This leads to mitochondrial depolarization and subsequently to the release of cytochrome c from mitochondria to cytosol (Green and Kroemer, 1998; Heiskanen et al., 1999; Susin et al., 1998). Following the formation of the apoptosome, upstream and downstream caspases are activated. The latter cleave downstream death substrates and activate endonucleases that cleave genomic DNA into high molecular weight (HMW) fragments, resulting in the apoptotic nuclear morphology. The opening of the mitochondrial PTP and the apoptotic process can be inhibited by a diverse group of agents such as antiapoptotic Bcl-2 family members and the drug cyclosporin A (Green and Kroemer, 1998; Susin et al., 1998). Sustained increases in intracellular [Ca 2⫹] trigger apoptosis in a diverse array of in vivo and in vitro systems (Nicotera et al., 1998; Susin et al., 1998; Trump and Berezesky, 1996).
Elevated rod photoreceptor [Ca 2⫹] appears to play a key role in the process of apoptotic rod cell death in humans and animals during inherited retinal degenerations, retinal diseases and injuries, and chemical exposure. These include patients with retinitis pigmentosa and cancer-associated retinopathy (Thirkill et al., 1987; van Soest et al., 1999), lead-exposed rats (Fox and Chu, 1998; Fox et al., 1997, 1999), retinal degeneration mice (Chang et al., 1993; Fox et al., 1999), rats injected with anti-recoverin monoclonal antibodies (Adamus et al., 1998), rats with hypoxic-ischemic injury (Crosson et al., 1990) and rats with light-induced damage (Edward et al., 1991). Low-to-moderate level lead exposure during development or adulthood produces rod and bipolar apoptotic cell death in rats (Fox and Chu, 1988; Fox et al., 1997) and apoptotic neuronal cell death in primary cultured cells (Oberto et al., 1996; Scortegagna et al., 1997). In addition, there is selective loss of cholinergic cells in the medial septum/vertical diagonal band complex, but not in the horizontal limb of the diagonal band or hippocampus of rats exposed to low-to-moderate levels of lead during development (Kawamoto et al., 1984; Tian et al., 1995). The mechanism of cell death was not reported. Moreover, developmental lead exposure results in elevated retinal and rod elemental [Ca 2⫹] and [Pb 2⫹] (Fox and Katz, 1992; Medrano and Fox, 1994). Although the molecular mechanism underlying the lead-induced apoptosis is unknown, there are two possible, though not mutually exclusive, triggering mechanisms: Ca 2⫹ overload and the generation of reactive oxygen species (ROS). To examine the mechanisms of developmental lead-induced selective rod and bipolar cell apoptosis (Fox and Chu, 1988; Fox et al., 1997), we established an ex vivo model where retinal Ca 2⫹ and/or Pb 2⫹ levels were elevated (He et al., 2000). Isolated whole adult rat retinas were incubated briefly in physiological buffers containing 0.1 mM–2 mM free Ca 2⫹ or 10 nM–10 mM free Pb 2⫹. The concentrations of Ca 2⫹ and Pb 2⫹ that produced an apparent 50% increase in the amount of HMW DNA fragments were 0.5 mM and 1 mM, respectively. In contrast to the developmental lead exposure model, the apoptotic cell death in the ex vivo model (detected by HMW DNA fragmentation and chromatin condensation) was localized only to the rods. There was no evidence of necrosis caused by Ca 2⫹ and/or Pb 2⫹. Ca 2⫹ and/or Pb 2⫹ depolarized rod mitochondria, caused the release of mitochondrial cytochrome c, increased the activity of caspase-9 and -3, and produced HMW DNA fragmentation. The effects of Ca 2⫹ and Pb 2⫹ were additive. The mitochondrial depolarization and cytochrome c release, activation of caspase-9 and -3, and rod cell apoptosis were completely blocked by cyclosporin A but not by FK506, indicating the site of action at the mitochondrial PTP. The activation of caspase-9 and -3, as well as the rod cell apoptosis, but not mitochondrial depolarization and cytochrome c release were blocked by DEVD-fmk (carbobenzoxy-Asp[OMe]-Glu[OMe]-Val-Asp[OMe]-fluromethylketone), indicating that caspase activation was downstream of the mitochondrial alter-
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ations (He et al., 2000). Decreases in GSH and NADH levels and the production of ROS occur in many forms of apoptosis, are triggered by Ca 2⫹ overload, and can initiate rod photoreceptor degeneration (Susin et al., 1998; Weigand et al., 1986). In addition, the probability of mitochondrial PTP opening is increased by the oxidation of glutathione and pyridine nucleotides (Bernardi, 1999). In rods isolated from retinas incubated in Ca 2⫹ and/or Pb 2⫹, the concentrations of GSH, GSSG, NADH 2 and NAD ⫹ as well as the phospholipid yield and conjugated diene content were not significantly different compared to controls. These results demonstrate that the Ca 2⫹ and/or Pb 2⫹-induced opening of the rod mitochondrial PTP was not due to oxidative stress (He et al., 2000). In summary, these results suggest that developmental leadinduced apoptosis in rod and bipolar cells is triggered by Pb 2⫹ and Ca 2⫹ overload, which is most likely due to the lead-induced inhibition of cGMP PDE activity in these two cell types (Fox and Farber, 1988; Fox et al., 1997; Srivastava et al., 1995a,b) ● results from direct mitochondrial alterations that are upstream of caspase activation ● is ultimately produced by the activation of downstream caspases, such as caspase-9 and -3. ●
Thus, mitochondrial alterations appear to play a central and coordinating role during the effector phase of lead-induced rod photoreceptor cell, and presumably bipolar cell, apoptosis. A similar apoptosis-signaling cascade may underlie rod-selective or neuronal apoptosis observed in humans and animals, with different retinal and neural degenerations resulting from Ca 2⫹ overload and/or Pb 2⫹ neurotoxicity. A thorough morphological analysis of selectively vulnerable target tissues in the brains of lead-exposed animals may reveal that similar apoptotic changes are occurring and thereby offer a mechanism for the irreversible behavioral and electrophysiological alterations observed in lead-exposed animals. The mechanistic knowledge gained from the delineation of the apoptotic cascade in rod photoreceptors will be useful for the development of specific neuroprotective strategies. (This work was supported by NIH Grant ES03183, a UH PEER Grant and a UHCO VRSG Grant.) Arsenic-Induced Dysregulation of Cell Cycle in Human Fibroblasts (J. C. States) Arsenic is a common contaminant of drinking water worldwide. Chronic ingestion of arsenic-contaminated drinking water can cause skin, bladder, and liver cancer. The mechanism of arsenic-induced carcinogenesis is unknown. Arsenite disturbs normal cell cycle progression in cells treated in vitro. Arsenite disrupts the mitotic spindle in normal diploid human fibroblasts (Yih, et al., 1997) and proliferating normal human peripheral lymphocytes (Ramirez, et al., 1997). The spindle disruption is associated with a delayed transit through M-phase in normal
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diploid fibroblasts. Arsenite-induced mitotic disruption is associated with aneuploidy in both normal diploid fibroblasts and proliferating normal peripheral lymphocytes. Arsenite-induced toxicity in proliferating lymphoblasts is reduced by expression of wild type p53 (Salazar, et al., 1997). Initial experiments compared the toxicity of NaAsO 2 in diploid human fibroblasts and SV40 transformed human fibroblasts. Confluent SV40 transformed human fibroblasts were very sensitive to arsenite and 10 mM NaAsO 2 was extremely toxic. In contrast, no toxicity was observed in non-dividing confluent diploid fibroblasts (G0/G1 arrested) treated with up to 100 mM NaAsO 2. T-antigen expressed in SV40 transformed cells binds to and inactivates p53. The differential sensitivity of SV40 transformed fibroblasts and non-transformed fibroblasts suggested that the sensitivity of SV40 transformed fibroblasts to arsenite was a consequence of p53 inactivation. This suggestion was supported by the observation that other cell lines with mutated p53 were also sensitive to arsenite treatment. Toxicity occurred sooner in faster growing cell lines and the kinetics of cell killing was inversely proportional to cell doubling time. The dependence on cell growth rate suggested that toxicity was related to the disruption of the cell cycle. The effects of arsenite on SV40 transformed fibroblasts were examined in detail. Cells were treated with 0 –10 mM NaAsO 2 containing [ 73As], and arsenic binding by, and toxicity to cells were determined. Arsenic uptake, measured by cell association of [ 73As], and arsenic-induced toxicity, measured by LDH release, were NaAsO 2 concentration-dependent in SV40 transformed human fibroblasts. The effect of NaAsO 2 on cell cycle was determined by flow cytometry. NaAsO 2 treatment induced the accumulation of cells with twice the normal DNA content in a concentrationdependent manner. Examination of arsenite-treated cells by light microscopy suggested the accumulation of mitotic cells. Electron microscopic examination indicated that the mitotic index was increased in arsenite-treated cells. These combined data indicate an M-phase arrest induced by arsenite treatment. Examination of arsenite-treated cells by light microscopy suggested the accumulation of cells with membrane blebs and/or cytoplasmic vacuoles. This observation suggested that arsenite induced apoptosis in the M-phase cells. Confirmation of apoptosis was obtained by both flow cytometric analysis and TUNEL assay. The results indicated that arsenite treatment induced an early transient accumulation of cells that excluded propidium iodide but bound annexin V, indicating an intact cellular membrane with flipping of membrane-bound phosphatidylserine as expected in early stage apoptotic cells. At later times, arsenite treatment induced the accumulation of TUNEL positive staining cells, indicating that DNA strand breaks accumulated in arsenite-treated cells as expected in late stage apoptotic cells. Arsenite-induced cell death, but not arseniteinduced accumulation of M-phase cells, was inhibited by pretreatment with a broad-spectrum caspase inhibitor, further supporting the hypothesis that arsenite induces apoptosis.
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SV40 transformation alters other cellular processes in addition to inactivating p53. Thus, it was of interest to determine if the sensitivity of SV40 transformed fibroblasts was caused by inactivation of p53 or some other alteration. Fibroblasts from Li-Fraumeni patients have only one functional p53 allele. Losing the active p53 allele can spontaneously immortalize these cells. Spontaneously immortalized Li-Fraumeni fibroblasts lacking active p53, transfected with a controllable p53 expression vector, were treated with arsenite. Induction of p53 expression arrests these cells in both G1 and G2 phases of the cell cycle (Agarwal et al., 1995). Non-cycling Li-Fraumeni syndrome fibroblasts expressing p53 from a transfected expression vector are resistant to arsenite-induced apoptosis compared with Li-Fraumeni fibroblasts not expressing p53. This result suggests that the sensitivity to arsenite of SV40 transformed fibroblasts is caused by the inactivation of p53. The results of these studies indicate that arsenite disrupts mitosis and, in cells that are phenotypically negative for p53 expression, induces apoptosis. The sensitivity of phenotypically p53-null cells to arsenite may have important implications for the mechanism of arsenic induced carcinogenesis. (This work was supported by ES06639, RR12242, ES06460, WSU OVPPR.) Mechanisms and Modulation of Chromium-Induced Apoptosis (S. R. Patierno) Certain chromium compounds are occupational respiratory carcinogens and are of growing environmental concern. These generally highly insoluble particulate forms of hexavalent chromium also cause respiratory tract irritation, ulceration, and perforation. They can cause tumor formation in animals and are genotoxic and mutagenic, and they transform cultured mammalian cells, but only at doses that also evoke some cell death. We have utilized cellular, molecular, pharmacological, and genetic approaches in studying chromium toxicity and carcinogenesis, and have used normal human respiratory fibroblasts and epithelial cells in culture as a toxicologically relevant experimental system (reviewed in Singh et al., 1998a). We have studied the interaction of particulate chromium compounds with these cells with respect to particle internalization and micro-environmental dissolution, using Cr-DNA adducts as an intracellular dosimeter of bioavailability (Singh et al., 1998b; 1999). We have found that structural DNA damage, especially in the form of DNA-Cr-DNA cross links, is mechanistically related to functional damage in the form of basespecific replication arrest and altered transcription (Bridgewater et al., 1994a,b, 1998; Manning et al., 1992; Xu et al., 1996). Depending on the rate, magnitude, and spectrum of genotoxicity and mitochondrial damage, and the nature of the affected signaling pathways, cells are fated to undergo either terminal growth arrest or p53-dependent apoptosis (Blankenship et al., 1997; Carlisle et al., 2000; Carlisle and Patierno, 2000).
We have found that the broad-spectrum protease inhibitor, Z-VAD-FMK, which blocks the executioner stage of chromium-induced apoptosis, does not increase survival of replication-competent cells. In contrast, cyclosporin A, which prevents the pre-apoptotic release of cytochrome c from the mitochondria, blocks chromium-induced apoptosis and increases the survival fraction of replication-competent but genetically damaged cells (Pritchard and Patierno, 2000, submitted). We are therefore exploring whether cells that manage to resist or escape apoptosis are predisposed to genomic instability and progression towards neoplasia. (This work was supported by NIEHS Grant ES05304.) Mechanisms Contributing to Systemic Autoimmune Disease: Mercury-Induced Tyrosine Phosphorylation and Disruption of the CD95/Fas Apoptotic Death Pathway (M. J. McCabe, Jr. and A. J. Rosenspire) Mercury (Hg) is a widespread environmental agent whose toxic potential to numerous organ systems, including the immune system, has been well recognized (reviewed in Lawrence and McCabe, 1995; Pollard and Hultman, 1997). The immunotoxicity of Hg is complex, involving on the one hand immunosuppression, yet on the other hand immunopotentiation. Several factors likely work in concert with mercury, leading to such disparate outcomes with respect to immune modulation. First, there is a clear gene/environment interaction, at least in rodents, where genetic susceptibility factors linked to the major histocompatibility complex as well as non-MHC–linked genes contribute to mercury-induced systemic autoimmune disease. Mercury-mediated immunosuppression also is genetically influenced. Second, the concentration of mercury (i.e., Paracelsus’ adage) together with the chemical form (i.e., organic vs. inorganic) greatly influences its immunotoxicity. Third, the cellular target (i.e., B cells more sensitive than T cells) and the activation state of the cells, influence the immunotoxicity of mercury at a given concentration. It is perhaps the immunostimulatory aspects of mercury that are most relevant and significant with respect to adverse health effects. Animal studies have established a connection between experimental exposure to mercury and the development of a lupuslike autoimmune disease. Similarly, case reports demonstrating a correlation between accidental Hg exposure and either the onset or the severity of autoimmune disease symptoms support a link between mercury intoxication and the etiology of human autoimmune disease. From this perspective, Hg is one of the few suspected environmental agents where a link between exposure to the agent and autoimmune disease has been indicated. Although genetic linkages to certain autoimmune diseases have been firmly established, few environmental substances have been rigorously associated with autoimmunity. Many systemic autoimmune diseases are associated with an abnormal accumulation of autoreactive lymphocytes resulting from defects in the termination of lymphocyte activation and
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growth via apoptosis (Hunig and Schiml, 1997; Mountz et al., 1994). The CD95 apoptotic death pathway is a major regulatory mechanism for controlling the life span of activated lymphocytes. Defects in this pathway have been linked to autoimmunity (Dianzani, et al., 1997; Watanabe-Fukunaga, et al., 1992). Inorganic mercury (Hg 2⫹) induces a systemic autoimmune disease in genetically susceptible strains of rodents, characterized by abnormal lymphoproliferation and accumulation of autoreactive lymphocytes (Kono et al., 1998; Pelletier et al., 1986). Additionally, epidemiological studies support a linkage between mercury exposure and autoimmunity (Dantas et al., 1997; Ro¨ger et al., 1992; Schrallhammer-Benkler et al., 1992). Inasmuch as many autoimmune diseases also are associated with abnormal lymphoproliferation and accumulation of autoreactive lymphocytes, we have studied the influences of Hg2⫹ on signaling pathways controlling cell growth and cell death. This talk presented evidence supporting the hypothesis that Hg2⫹modulates the CD95 apoptotic death pathway (Whitekus et al., 1999). Our experimental design employed human cell culture models (i.e., Jurkat cells and PHAactivated peripheral blood-derived CD4⫹ T cells) to study the effects of Hg 2⫹ on components of the CD95 death pathway and the epistasis of the effects of Hg2⫹ on this pathway. Non-toxic concentrations of Hg2⫹ significantly attenuate the concentrationand time-dependent response to anti-CD95 agonist, meaning that cells survive CD95 stimulation and accumulate in the presence of non-cytotoxic concentrations of inorganic mercury. However, other apoptosis-inducing stimuli such as ceramide and TNF are not attenuated by mercury, suggesting that the signaling component(s) targeted by Hg2⫹ is unique to the CD95 portion of the apoptotic death pathway. Inhibition of CD95-mediated apoptosis by Hg 2⫹ was confirmed using several criteria including DNA fragmentation, nuclear condensation, phosphatidylserine externalization, and PARP degradation. Hg2⫹ also attenuates induction of caspase-3 autoproteolysis. Mercury does not interfere with the anti-CD95 agonist binding, nor does it down-regulate the cell surface receptor density of CD95. Collectively, these data suggest that Hg 2⫹, either directly or indirectly, interferes with CD95mediated apoptosis by interacting with a signaling component downstream of the formation of the membrane proximal death inducing signaling complex (DISC) and upstream-of-caspase-3 activation. Additional studies are underway, aimed at linking Hg-mediated activation of protein tyrosine kinases and the CD95 death pathway. The plausibility of such altered signaling mechanisms in the etiology of Hg-induced systemic autoimmune disease and the integration of these findings with the published literature concerning Hg-induced immunopathogenesis were discussed. (This work was supported by NIH grant R21 ES10351.) Conclusions This symposium reviewed several major areas involving the role of aberrant cell accumulation, including enhanced or disrupted apoptosis and altered cell proliferation, in the toxicity of metallic agents. In this regard, apoptosis should be viewed as
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a normal process by which the correct functional cellular population dynamics are maintained. A variety of evidence indicates that caspases and mitochondria both play important roles in the initiation and execution of apoptosis. Target cell specific toxicity can certainly result in the loss of cell populations to apoptosis. Additionally, the apoptotic process, as in all biological processes, cannot be considered perfect, and proapoptotic stresses could result in the escape of cells that would otherwise be sufficiently damaged to require elimination. Theoretically, increasing the amount of apoptosis under the conditions of a constant percent of escape would increase the proportion of damaged, but replication-competent cells. Conversely, metals can also perturb or inhibit appropriate apoptosis. Thus, the disruption of the apoptotic process will, by nature, result in disorders of cell accumulation that can either reduce the number of normal cells or increase the number of abnormal cells. Therefore, defining the mechanism of metalinduced apoptosis and resistance to apoptosis will be important to defining the role of metals in selective cell toxicity and in proliferative diseases and may help in developing strategies for the prevention of metal-induced diseases. Clearly, many metals can activate the processes involved in apoptotic cell death. With regard to the adverse effects of metals, inappropriate activation of the apoptotic process could result in the death of selected sensitive cell populations and, in turn, result in loss of critical function. The selectivity of leadinduced apoptosis in rod cells is a relevant example of this specific cell loss that would clearly result in diminished tissue function. This could be a generalized mechanism of leadinduced neurotoxicity, and offers an attractive theory for the irreversible behavioral and neurophysiological damage seen in the brains of lead-exposed animals. Further definition of the role and mechanism of lead-induced apoptosis in neural cells may allow the development of preventative or amelioratory strategies in lead poisoning. Considering the important issues that still remaining with human lead exposure and the problematic nature of environmental lead abatement, effective preventative approaches would be highly desirable. There are also data suggesting that increased apoptosis can result in proliferative diseases like cancer. In this regard apoptosis that results from DNA damage, such as with certain chromium compounds, may, by increasing the apoptotic rate in a given tissue, increase the absolute number of cells which manage to avert apoptosis. Hexavalent chromium compounds are clearly potent genotoxins, although if this is due to direct DNA binding of chromium, the generation of radicals during cellular chromium reduction which attack DNA or some combination of these two events is still not well defined. Thus, the creation of a pool of apoptotically resistant, but genetically damaged cells, if still capable of replication, could be part of a sub-population that might be predisposed to mutagenesis and, subsequently, tumor formation. Chromium-induced apoptosis is p53 dependent, so mutations in the p53 gene might facilitate the development of resistance to apoptosis and increased survival of genetically damaged cells.
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Conversely, arsenic, which is generally considered not to be highly genotoxic, appears to cause apoptosis specifically in cells which are phenotypically negative for the expression of the tumor suppressor gene, p53. Such cells may already be predisposed for eventual tumor formation and arsenic may behave as a promotional agent in this regard. So both highly genotoxic and more minimally genotoxic inorganics may have a component of apoptosis as part of their carcinogenic mechanism. The definition of the role of apoptosis in metal carcinogenesis could provide important advances in an area of study in which mechanism has been very difficult to define. On the other hand, the perturbation of normal apoptosis can also be induced by metals, which could result in the development of immune disorders. For instance, many systemic autoimmune diseases are linked to excessive accumulation of autoreactive lymphocytes. These appear to result from defects in the termination of lymphocyte growth by way of apoptosis. Epidemiological and experimental data link mercury exposure and autoimmunity. This appears to occur selectively through the attenuation of the CD95 death pathway, probably by disruption of the signaling component prior to caspase-3 activation. These results may be important in defining the mechanisms of autoimmunity in general, as well as of autoimmunity resulting from metal exposures. Defining the specificity and mechanisms of apoptosis in metal toxicology is a difficult and complicated task. Clearly the causes of aberrant apoptosis induced by metals vary widely with the metallic agent and location of the cell populations in question. There is very likely no real unifying mechanism by which metals act to alter the apoptotic process. However, knowledge of the mechanisms can lead to important advances in our use of metals. For instance, arsenic salts now appear to be an effective chemotherapeutic for acute promyelocytic leukemia by causing apoptosis specifically within the tumor cell population (Look, 1998). Perhaps a better understanding of the mechanisms of metalinduced alterations in apoptosis will lead to further beneficial ways in which these effects can be utilized to kill specific cells or, on the other hand, blocked, to prevent metal toxicity. However, altered apoptosis must always be seen potentially as a doubleedged sword, in that reduced apoptosis, although it may reduce initial cytolethality, may allow damaged cells that should not survive to bypass this important control point and potentially form malignancies. In an analogous fashion, enhanced apoptosis may destroy specific critical cell populations, or, assuming a similar error rate, may also allow damaged cells to escape appropriate destruction. In summary, disorders of apoptosis and cell accumulation may play critical roles in some of the most severe and debilitating metal-induced afflictions, including neurotoxicity, autoimmunity, and carcinogenesis. Defining the mechanisms in aberrant apoptosis resulting from metal exposure may well allow for the development of preventative or ameliorative strategies in metal toxicology.
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