The Poisons Within: Application of Toxicity Mechanisms to

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PerspectiVe The Poisons Within: Application of Toxicity Mechanisms to Fundamental Disease Processes Daniel C. Liebler Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt UniVersity School of Medicine, NashVille, Tennessee 37232 ReceiVed February 10, 2006

Contents Introduction Toxicology and the Changing Research Enterprise Defining Toxicology and Redefining Toxicology Endogenous Toxicants, Reactive Intermediates, and Molecular Damage Oxidative Stress and Protein Damage in Environmentally-Related Diseases Conclusion

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Introduction Toxicology is the science of poisons. Paracelsus taught that any substance could be a poison in sufficiently high doses. The poisons of greatest interest to toxicologists have been environmental and workplace pollutants, drugs, personal care products, dietary constituents, and certain natural products. Indeed, the two dominant goals of toxicology over the past 50 years have been to assess adverse health effects of environmental pollutants and to manage the problem of toxicity in drug development and chemical safety (1). In both contexts, toxicology deals only with the adverse effects of exogenous agents. Accordingly, toxicology is what happens when living systems encounter exogenous chemicals in sufficiently high doses to cause injury. Toxicology is an essential element of the healthcare enterprise. Many lives have been saved and injuries prevented by the nowroutine detection of toxic drug candidates in early drug development and by the identification of adverse effects of potential household products and food additives through toxicity testing. Nevertheless, the focus on exogenous agents casts toxicology as an auxiliary discipline to the mainstream of biomedical research. What does toxicology have to offer beyond testing and safety assessment? First, toxicology brings a unique perspective to biology and medicine by addressing the adverse interplay of chemical and physical agents with living systems. This perspective is actually quite uncommon in most areas of biology, which are often uninformed by basic concepts of toxicity. Second, it offers a logical framework for probing the adverse impact of the environment with living systems. In this broad context, * To whom correspondence should be addressed. Tel: 615-322-3063. Fax: 615-343-8372. E-mail: [email protected].

“environment” encompasses diverse physical agents and chemical substances, ranging from chemical pollutants and drugs to dietary components and naturally occurring substances. Toxicology is both a practical discipline and a science, which, at its heart, is the study of mechanisms. Just as fundamental biology seems endlessly complex, so is the problem of how poisons interact with living systems. Mechanistic studies integrate chemistry and biology to understand how chemicals are absorbed, distributed, and metabolized and how chemicals and their metabolites interact with specific biomolecule targets to elicit their effects. From the late 1960s through the mid1990s, the confluence of toxicology and drug metabolism research yielded important fundamental discoveries in the chemistry and biochemistry of xenobiotic metabolism, which governs the activation of some toxicants and the detoxication of many others. This provided the driving force behind the discovery and characterization of the large CYP enzyme superfamily, as well as acetyltransferases, glucurosyltransferases, glutathione-S-transferases, and numerous conjugating enzymes, oxidases, reductases, and transporters. These discoveries were accompanied by studies of catalytic mechanisms that fundamentally informed understanding of how both xenobiotics and endobiotic molecules are processed in living systems. Historically strong research support for toxicology research and numerous high-quality research training programs were established throughout the United States, Europe, and Asia. These programs yielded three generations of toxicologists who have established an indispensable drug and chemical safety assessment infrastructure in the chemical and pharmaceutical industry, in regulatory agencies, and in the academic toxicology community. A stable, long-term need for toxicologists to support this regulatory and safety assessment infrastructure seems assured. Nevertheless, toxicology as a science faces an intellectual crisis and an uncertain future.

Toxicology and the Changing Research Enterprise The most fundamental challenge to toxicology is a gradual erosion of its perceived relevance within the biomedical research enterprise. The principal cause of this problem is an intellectual stasis in the toxicology field. Over 50 years of toxicology has taught us much about the mechanisms by which certain chemicals cause tissue-specific toxicities (1). However, essentially all of the key concepts in toxicity mechanisms have been around for at least 20 years. Increased sophistication in understanding of toxicity mechanisms is due mainly to advances in chemistry, biology, and biotechnology, rather than to advances

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in toxicology methods and concepts. This situation has contributed to the widespread perception that toxicology is an intellectual backwater in the postgenomic era. The linkage of toxicology and xenobiotic exposure separates the field from other areas of health and medicine. In principle, control of pollution, workplace hazards, and mitigation of adverse drug reactions could prevent most toxicities; yet, major diseases, such as heart disease, cancer, and diabetes, would still be with us. Toxicology has not yet established a clear relevance to diseases that have a major public health impactsdespite the fact that “environment” is widely viewed as a major contributor to many of these diseases. Toxicity mechanisms have been defined almost exclusively in the context of xenobiotic exposures, and it is not clear which of these exposures or mechanisms apply to diseases. This situation has had negative consequences for academic toxicologists studying mechanisms. The recent reorganization of the National Iinstitues of Health (NIH) peer review system essentially eliminated toxicology-oriented study sections, which leaves toxicology grant proposals to vie for favor among review groups with little familiarity with toxicology. Disease-based review panels routinely question the relevance of studying chemical toxicity based on the logic that avoiding the exposure solves the problem. The concept of using chemicals as probes of biology, although relevant, is poorly articulated by toxicologists and poorly received by reviewers. Despite some parallels, chemical toxicity models are generally poor models of complex human diseases. Thus, research funding for mechanistic studies of chemical toxicants has become increasingly difficult to obtain, despite continued growth of the overall biomedical research enterprise. Moreover, the increased emphasis on disease- and technology-based research in NIH funding has also marginalized toxicology. The National Institute of Environmental Health Sciences, which historically has funded much work in biochemical and molecular toxicology, is redirecting resources away from studies of chemical toxicity and toward studies that focus on human diseases with both environmental causes and major public health impacts (2).

Defining Toxicology and Redefining Toxicology There is no universally accepted definition of toxicology. The Society of Toxicology advances a broad view of the discipline and states that “Toxicology is the study of the adverse effects of chemical, physical, or biological agents on living organisms and the ecosystem, including the prevention and amelioration of such adverse effects” (www.toxicology.org). In the most widely read toxicology text, Casarett and Doull’s Toxicology: The Basic Science of Poisons, Michael Gallo writes, “Toxicology has been defined as the study of the adverse effects of xenobiotics and thus is a borrowing science that has evolved from ancient poisoners” (1). Both of these definitions implicitly or explicitly support the dominant view that toxicology is focused on exogenous agents. The time has come to redefine toxicology in two ways. First, the scope of toxicology must be expanded beyond exogenous agents to include endogenous toxicants that play key roles in important disease processes. Endogenous toxicants not only are key players in many disease processes but also are generated in response to exogenous toxicants. Second, toxicology should focus on mechanisms and processes that underlie the adverse biological consequences of endogenous toxicants. This focus is likely to identify mechanisms that contribute both to important disease processes and to the toxicities of exogenous poisons. This expanded scope for toxicology still accommodates work

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on exogenous poisons that has long defined toxicology. However, the most important consequence of this redefinition is to bring the strengths of toxicology squarely to bear on some of the most important problems in human disease. By doing so, toxicology secures an essential role in the mainstream of biomedical research.

Endogenous Toxicants, Reactive Intermediates, and Molecular Damage A key concept in a redefined toxicology is that xenobiotic toxicity and diseases involving oxidative stress and inflammation share common mechanisms and both involve similar types of molecular damage by reactive intermediates. The formation of oxidants is a hallmark of chemical toxicity, inflammation, and other types of environmental stresses. Although oxidative stress derives fundamentally from the excessive flux of reduced oxygen species, such as superoxide, hydrogen peroxide, and hydroxyl radicals, secondary products of lipid, DNA, and protein oxidation may play critical roles in oxidant-associated molecular pathologies. Lipid, DNA, protein, and carbohydrate oxidation all yield a variety of electrophiles (3-6). These products are well-known to form mutagenic DNA adducts, which are thought to contribute to oxidant-induced mutagenesis (7). However, reactive electrophiles and oxidants also readily react with proteins. The scope and significance of these modifications are just beginning to be explored with the aid of new analytical technologies. For years, relatively little was known about the target selectivity of endogenous electrophiles in modifying proteins. Since the discovery of covalent protein binding in the late 1940s, limitations in analytical methods precluded the identification of the molecular targets of damage on anything approaching a proteomic scale and the scope and selectivity of damage remained speculative. Nevertheless, the modification of protein targets by reactive metabolites of xenobiotic toxicants has been a central problem in toxicology (8, 9). A broader understanding of the chemistry of damage by endogenous reactive intermediates can provide a basis for understanding mechanisms of oxidant-induced stress that contributes to disease processes and mechanisms of xenobiotic toxicity. Recent advances in proteome analyses by mass spectrometry have enabled identification of the protein targets of electrophile probes (10) and model oxidants (11). These studies indicate that protein damage is highly selective and is dictated by both the properties of the protein targets and the chemistry of the electrophiles and oxidants. Indeed, the selectivity of protein modification by low concentrations of hydrogen peroxide allows it to participate in cell signaling through selective modification of thiols in protein tyrosine phosphatases (12). Small differences in structures of related intermediates can lead to very different effects. For example, the lipid oxidation product 4-oxononenal selectively activates p53-dependent stress responses, whereas the closely related 4-hydroxynonenal does not in the cell type studied (13). Similarly, the endogenous carbohydrate oxidation product methylglyoxal was recently shown to selectively modulate glucose-responsive transcriptional regulators (14) and stress response regulators (15). These few examples underscore the emergence of new opportunities to investigate mechanisms of toxicity and diseases by tracing the chemical biology of damage to biological molecules. Mechanistic investigation in toxicology has historically relied heavily on the application of pharmacological agents to perturb pathways, always based on the assumed specificity of the agents

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used. Of course, the lack of specificity of many of these agents, particularly in the context of injury, led to ambiguous and frequently erroneous interpretations of mechanism. The application of new genetic and targeted molecular manipulations makes possible the unambiguous evaluation of mechanisms and pathways related to injury (16-18). Moreover, the integration of gene expression profiling, targeted gene disruption strategies, and evaluation of changes in the context of networks and pathways reveals highly selective damage responses even to chemical and physical agents thought to act through widespread, nonspecific damage (19, 20).

Oxidative Stress and Protein Damage in Environmentally-Related Diseases Oxidants and oxidative stress play a central role in several human diseases with major health impacts. Three examples illustrate this point. The first is atherosclerosis, which is a major etiologic factor in cardiovascular disease. The oxidative modification of low-density lipoprotein leading to accumulation of oxidized lipid in macrophage-derived “foam cells” is considered an essential element of atherogenesis (21). Recent work suggests that oxidized phospholipids react covalently with lysine residues in apoB-100 to form adducts, which are recognized by scavenger receptors (CD36) and thus provide a means of uptake for the oxidized low-density lipoprotein (LDL) (22). The diversity of oxidizable lipids in LDL suggests that a number of lipid-derived electrophiles can contribute to disease pathogenesis (23). Nevertheless, the specific intracellular targets of these species and the mechanisms by which they contribute to the development of a disease phenotype remain unknown. Oxidative stress appears to contribute to the pathology of neurodegenerative diseases, including Parkinson’s and Alzheimer’s diseases, both of which appear to be caused in part by poorly understood gene-environment interactions (24). The etiology of these neurodegenerative diseases is characterized by toxic protein deposition. Parkinson’s disease is characterized by the formation of insoluble protein deposits, which contain aggregates of several proteins, including R-synuclein (25). Mutations in R-synuclein associated with rare familial Parkinson’s disease lead to misfolding of the synuclein protein and the generation of protofibrils, which are toxic to neurons in vitro (26). Quinone intermediates formed from autoxidation of dopamine and other catecholamines can stabilize toxic R-synuclein protofibrils (27). These observations raise the possibility that electrophiles associated with oxidative stress may modify R-synuclein or other proteins to induce misfolding and aggregation. Protein deposition and oxidative stress also are contributing factors to Alzheimer’s disease (24, 28). Recent work indicates that mutant forms of β-amyloid form neurotoxic protofibrils similar to those generated from mutant forms of R-synuclein (29). Covalent modification by lipid oxidation products was recently reported to stabilize misfolded amyloid forms (30). These observations clearly point to a key role for endogenous toxicants in neurodegenerative diseases. Oxidative stress is also a characteristic of “metabolic syndrome”, in which weight gain and abdominal obesity, insulin resistance, dyslipidemia, hypertension, and a systemic proinflammatory state occur together (31). Metabolic syndrome is associated with an increased risk of type 2 diabetes and cardiovascular disease, certain cancers, and other adverse clinical outcomes and is modulated by diet, lifestyle, and environmental factors (31). A persistent theme of current research in this area is the adverse interplay between obesity, inflammation, and oxidative stress. In obesity, fat cells are thought to contribute

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to insulin resistance in part through the release of free fatty acids, which interfere with insulin-mediated signal transduction (32, 33). Fat is subject to macrophage infiltration in obesity, which provides a mechanistic basis for the observed release of fatty acids and inflammatory mediators (34, 35). Mitochondrial loading with free fatty acids is postulated to lead to lipid oxidation and enhanced damage due both to enhanced mitochondrial production of oxygen radicals and to formation of reactive products of lipid oxidation (36). The association of obesity with oxidative stress was further supported by the analysis of urinary isoprostanes in 2828 subjects from the Framingham Heart Study (37). Isoprostanes were considered a “gold standard” for the assessment of systemic oxidative stress (38, 39), and isoprostane levels were positively associated with both body mass index and diabetes in the Framingham subjects. Oxidative stress and reactive lipid oxidation products are clearly correlated with the pathogenesis of type 2 diabetes and other diseases associated with metabolic syndrome. Of course, the key question is whether these agents play causative roles, and if so, how do they act?

Conclusion The historical focus of toxicology on exogenous agents has isolated the discipline from the mainstream of biomedical research. If toxicologists adhere to this status quo, further erosion of toxicology as a science seems inevitable. Given the unprecedented challenges and opportunities before the biomedical research enterprise, the estrangement of toxicology is a lost opportunity. Toxicology has evolved a unique perspective in biology by being the only discipline to embrace the study of mechanisms of chemical injury. These processes are common to cell and tissue damage both by endogenous toxicants in many important diseases and by xenobiotics. The time has come for toxicologists to recognize this opportunity, to broaden toxicology to address fundamental mechanisms shared by diseases and chemical toxicities, and to bring their unique strengths into the research mainstream. Comments on this perspective are invited. Please go to the CRT website (http://pubs.acs.org/journals/crtoec/index.html) and choose “Comments” to submit an opinion.

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