Neurotoxins, parkinsonism and Parkinson's disease

Pharmac. Ther. Vol. 32, pp. 19 to 49, 1987 Printed in Great Britain.

NEUROTOXINS,

0163-7258/87$0.00+0.50 Copyright© 1987PergamonJournals Ltd

PARKINSONISM

AND

PARKINSON'S

DISEASE

J. WILLIAMLANGSTON,*t IAN IRWINt and GEORGEA. RICAURTE~ tlnstitute for Medical Research, 2260 Clove Drive, San Jose, California 95128, U.S.A. and ~Stanford University School of Medicine, Stanford, California 94305, U.S.A.

1. I N T R O D U C T I O N Neurotoxins have intrigued scientists investigating the etiology of Parkinson's disease for years. One does not have to look far to see why. A surprising number of toxins produce a parkinsonian state in humans, and this has been known for some time. Such an association evokes a number of tantalizing questions. Could one or more of these toxins be the actual cause of Parkinson's disease? Might there be one among them which could be successfully employed to produce the long sought animal model of Parkinson's disease? A number of these toxins not only cause a parkinsonian syndrome in humans, but also clearly induce degeneration of certain basal ganglia structures, including the substantia nigra. As loss of nerve cells in the substantia nigra is the core neuropathological feature of Parkinson's disease, neurotoxins have repeatedly lured investigators with their promise of yielding clues to the mechanism of nigral cell death in this disease. The very fact that the basal ganglia are targeted by a number of neurotoxins has not escaped attention either. Could there be a unifying theme here, one which might provide insight into the selective vulnerability of these important structures in the brain? Put another way, might the study of a toxin, or group of toxins which differ greatly in their chemical and biological properties, reveal the 'achilles heel' of the basal ganglia? These questions have been with us for a long time and, as we shall see, they have played an important role in the history of research into Parkinson's disease. It is with a great sense of pleasure, then, that we re-examine what has been learned to date, a n d sift through this information in light of more recent discoveries relating to toxins, parkinsonism, and Parkinson's disease. Because a number of concepts provide an essential foundation for the issues raised in this chapter, a certain amount of background information will be presented to set the stage for the ensuing discussion. This is done so that anyone reading this chapter, regardless of whether their background is in neurology, pharmacology, chemistry, pathology, or any other discipline will begin on a relatively equal footing. We would also like to explain why we have chosen to use the term 'neurotoxin' in this chapter rather than 'neurotoxicants'. Properly speaking, toxins are proteins which are produced by plants, animals and bacteria (e.g. botulinus toxin). In an attempt to recognize this distinction, the term neurotoxicant has been suggested for chemicals which are poisonous to the nervous system. However, because the term neurotoxin is somewhat gentler on the tongue and continues to enjoy wide-spread usage, it will be employed in this chapter to refer to any substance which damages the nervous system, even though the purists in the field will surely point out that the actual title of the chapter should have been 'Neurotoxicants, parkinsonism, and Parkinson's Disease'.

*To whom correspondence should be addressed. 19

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J.W. LANGSTONet al. 2. P A R K I N S O N I S M VS P A R K I N S O N ' S DISEASE 2.1. PARKINSON'S DISEASE

It may also appear that differentiating between the terms parkinsonism and Parkinson's disease is simply a matter of semantics, but its actual importance goes much deeper. Parkinson's disease was first described by James Parkinson in 1817. Characterized clinically by rigidity, tremor, akinesia and loss of postural reflexes, the disease typically begins after the age of 40, and progresses slowly. However, a clinical diagnosis will be correct at best only 90% of the time, as neuropathological examination is required for an unequivocal diagnosis of the disease (Forno, 1966). Stated another way, there is currently no clinical diagnostic test which is specific for Parkinson's disease. The cardinal neuropathological feature of this disease is loss of cells in the substantia nigra (Forno, 1982). Degenerative changes are typically seen in other pigmented nuclei in the brainstem as well, particularly the noradrenergic locus ceruleus. Lewy bodies, named after the individual who first described them in 1912 (Lewy, 1912), represent another characteristic neuropathological feature of Parkinson's disease. These intraneuronal inclusions are round, eosinophilic, and typically exhibit a peripheral pale halo. They are usually found within cell bodies, but may also lie in nerve cell processes. Lewy bodies are described here in some detail because they are highly characteristic of Parkinson's disease, although they may occasionally be seen in other diseases as well (Forno, 1982). Many, though not all, neuropathologists require their presence to confirm a diagnosis of Parkinson's disease. It is of interest to note that approximately 10% of individuals coming to autopsy over the age of 60 will exhibit Lewy bodies in the central nervous system (CNS) (Forno, 1969). These 'incidental' Lewy bodies have often been interpreted as evidence of preclinical Parkinson's disease. It is generally accepted, and will be an essential thesis here, that these clinical and pathological features constitute a specific and well-defined disease entity. This is a critical assumption if we are to begin talking about etiology. Obviously the task of tracking down the cause of any disease becomes much more complex if that entity is ill-defined, and may be next to impossible if its very existence is in doubt. 2.2. PARKINSONISM

The term 'parkinsonism', on the other hand, does not refer to a specific disease, but rather to any clinical condition exhibiting physical signs similar to those which are seen in Parkinson's disease. To understand the mechanism by which disorders other than Parkinson's disease might cause parkinsonism, a brief history of the pathophysiology and pharmacology of the disease is in order. It has been known for over 60 years that Parkinson's disease is characterized pathologically by loss of pigmented nerve cells in the substantia nigra (Tretiakoff, 1919). The subsequent discovery that this cell loss was accompanied by a decline in striatal dopamine was not to come for an additional 40 years, when Ehringer and Hornykiewicz (1960) demonstrated a striatal dopamine deficiency in Parkinson's disease. We now know that dopamine is produced in nigral cells and transported via their cell processes to the striatum, where it is stored in vesicles, to be released in the synapse as a neurotransmitter. It has frequently been argued that most, if not all, of the motor features of the disease are due to the loss of dopaminergic cells in the substantia nigra (Hornykiewicz, 1966; Fahn, 1982). The recent observation that MPTP, which appears to be selectively toxic to this same group of nerve cells, produces virtually all of the features of Parkinson's disease in humans (Ballard e t al., 1985) has further strengthened this argument. The essential point here is that a clinical picture of parkinsonism will emerge as the final expression of any process which interferes with the integrity of the nigrostriatal system or its output. Since dopamine is the primary neurotransmitter of the nigrostriatal system, anything which interferes with the production, storage, release or receptor recognition of dopamine is likely to cause parkinsonism as well. Given this information, one would predict that a number of classes of compounds might cause parkinsonism, and this is

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indeed the case. In this review, however, we will be focusing primarily on those compounds which cause irreversible parkinsonism. 3. REVERSIBLE AND IRREVERSIBLE PARKINSONISM Another critical distinction which we will be making in this chapter relates to the differences between pharmacological and toxicological agents. While pharmacological agents tend to produce reversible effects on biological systems, the core of toxicology relates to the production of effects which, by and large, are irreversible and often lead to cellular death (Carlsson, 1984). As might be expected based on the preceding discussion, there are a large number of agents which interfere with the function of nigrostriatal dopamine in a reversible manner. For example, reserpine induces parkinsonism by blocking the storage of dopamine in the synaptic vesicles in the terminals of nigrostriatal neurons (Stitzel, 1976). This eventually leads to a hypodopaminergic state which is fully reversible after reserpine is discontinued. Alpha-methyltyrosine produces a hypodopaminergic state by inhibiting tyrosine hydroxylase activity, the rate-limiting step in dopamine synthesis, and by itself forming a false transmitter which replaces dopamine at its storage site (Weiner, 1985). By far the most common cause of drug-induced parkinsonism in clinical practice results from the use of dopamine receptor blockers for the treatment of major affective disorders (Stephen and Williamson, 1984). However, although drug-induced parkinsonism represents a significant clinical problem, this condition is usually reversible and actual neuronal degeneration from long-term use of these agents has yet to be proven. In summary, pharmacological agents which induce parkinsonism usually affect neurotransmitter synthesis, storage or receptor sites. The parkinsonomimetic action of these compounds is reversible, since they are eliminated by enzymatic degradation, or excretion. A neurotoxin, on the other hand (at least as defined in this chapter), acts to damage a nerve cell, and its effect remains despite the removal of the damaging agent. Pharmacological agents may offer a number of advantages to scientists wishing to study the pharmacology and physiology of neuronal systems. However, for the study of neurodegenerative disease, toxic agents, because they actually induce neuronal degeneration, must be considered the tools of choice. Not surprisingly, agents of both classes have made important contributions to our understanding of Parkinson's disease. Carlsson's first observation regarding L-dopa was that it reversed the reserpine-induced syndrome of dopamine depletion in rats (Carlsson et al., 1957). In fact it was the similarity between reserpine-induced akinesia in rats and parkinsonism that led him to make the original suggestion that a central nervous system dopamine deficiency might play a role in Parkinson's disease (1959). Much of the early work with L-dopa by Cotzias and colleagues (1967), who eventually made this form of therapy a reality for patients with Parkinson's disease, was inspired by the similarities between the neurotoxic effects of manganese and Parkinson's disease (Cotzias, 1958). The discovery that 6-hydroxydopamine can selectively destroy catecholaminergic systems (Tranzer and Thoenen, 1968) has given birth to the speculation that endogenous generation of this compound could be involved in the pathogenesis of Parkinson's disease. Finally, the recent discovery of the neurotoxin MPTP (Langston et al., 1983) has created a dramatic resurgence of interest in the mechanisms of selective neuronal degeneration in Parkinson's disease (Lewin, 1984). The primary focus of this review will be on neurotoxins because of their promise as tools for the study of the neurodegenerative process. An understanding of this process is critical to the development of strategies for altering or preventing neuronal degeneration in Parkinson's disease, Alzheimer's disease, or any other degenerative disease for that matter. Further, such studies could provide clues regarding the etiology of one or more of these neurodegenerative diseases of aging. The major compounds to be discussed include manganese, carbon disulfide, and MPTP. Because of their potential relevance to the process of neuronal degeneration, 6-hydroxydopamine and amphetamines will be briefly

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reviewed as well. In each instance we will discuss the relevance of the compound to the issues described above. Particular emphasis will be given to the mechanism of action of these compounds, and how such knowledge might shed light on the process of neuronal degeneration in human disease. 4. M E C H A N I S M S OF N E U R O N A L DEATH Many biological disciplines are more concerned with how cells live rather than why they die. For example, most of the rapidly evolving fields in the neurosciences are devoted to the elucidation of normal neuronal behavior and function. For most pharmacologists, particularly those searching for new therapeutic agents, the first sign of toxicity usually provides a reason to immediately abandon an experimental compound. One can only guess at how many valuable experimental toxins are gathering dust in the vaults of major pharmaceutical companies. Even physicians who deal with the consequences of cell death on an almost daily basis are often unfamiliar with the mechanisms thought to underlie cellular demise. For example, it is the rare physician who is intimately familiar with 'free radical' or 'covalent binding'. A discussion of these and related concepts will constitute the last group of mini-reviews before proceeding to specific neurotoxins which cause parkinsonism. A discussion of the molecular mechanisms of cell death may seem a bit intimidating to the non-chemist, but we cannot stress how important it is to understand these issues in order to tackle the problems of cell death and neuronal degeneration. In the next two sections, we will briefly review the primary models of cellular injury. Hopefully, this will leave the reader well prepared for the ensuing discussion of the neurotoxins, particularly in regard to their mechanism of action. 4.1. FREE RADICALS

What are free radicals? Simply put, they are highly reactive chemical species (elements or molecules) with an unpaired electron. In the chemical industry they are widely used because of their ability to initiate and rapidly propagate chemical reactions. Using a single simple precursor, polymers of several thousand subunits can be created, sometimes with explosive speed, simply by adding a trace of free radical. This technology lies at the heart of the awesome proliferation of plastics and other synthetics which surround us in the modern-day world. In regard to biological systems, on the other hand, the history of free radicals is somewhat les s glorious. Early on they were associated with rancidification of foodstuffs, the derangement of cellular processes which occur in inflammation, and the action of toxic chemicals, and thus appeared to have no place in the orderly biochemical processes of life. However, cells are awash with all of the ingredients for the initiation and propagation of free radical reactions (e.g. polyunsaturated lipids, oxygen and transition metals), and recently oxygen radicals have acquired a certain status as a requirement for many biological processes (Brunori and Rotilio, 1984). Still, the cell is 'playing with fire' because unrestrained free radicals are capable of wreaking havoc. These highly reactive species, particularly the dreaded hydroxyl radical, can cause rapid peroxidation of lipid membranes (McBrien and Slater, 1982), and destruction of protein and DNA (Fridovich, 1979; Halliwell and Gutteridge, 1985). In the following discussion we will briefly explain the nature of active oxygen species, how they are formed, how cells protect against them, and finally how they damage cells.

4.1.1. Reactive Oxygen Species First, it is important to understand that oxygen exists chemically as a diatomic gas (dioxygen) and, consistent with its observed magnetic properties, it must possess two unpaired electrons as shown diagrammatically below: :0:0:

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Thus, using the above definition, molecular oxygen can be thought of as a di-radical. Therefore it will be attracted to sites where electrons are available (in this sense 02 or dioxygen can be thought of as 'electron hungry'). The electrophilic nature of oxygen facilitates its reactivity with electron-rich species (e.g. unsaturated lipids), and its ability to interact with reduced transition metals (i.e. metals which can serve as electron donors by virtue of their ability to change valency states). The unpaired electrons of dioxygen'also account for its ability to interact with light and radiation to form reduced oxygen radicals. But what is a reduced oxygen radical? The family of active oxygen species is best illustrated by reviewing the two successive one-electron reductions of dioxygen which are known to occur in a variety of systems (by reduction we refer to the process of adding electrons). This is shown in the following equation: O2+ 1 e- ~O~- + 1 e----, DIOXYGEN

02-

SUPEROXIDE PEROXIDE

As can be seen, the single-electron reduction of dioxygen results in a molecule which now has a single unpaired electron (this is shown by the dot before the minus sign), meeting our definition of a true free radical (in this case superoxide). With the reduction of superoxide (a second single-electron process), peroxide is formed. At physiological pH, peroxide exists as the equilibrium mixture of its conjugate acids, hydrogen peroxide (H202) and hydroperoxyl anion (HO 1- ). Once superoxide and hydrogen peroxide are present, the stage is set for the generation of what is thought to be one of the most damaging free radical species of all, the hydroxyl radical (. OH). To date, a mechanism for cellular defense against this radical has yet to be discovered. The hydroxyl radical can be generated from peroxide and superoxide via the Haber-Weiss reaction (1932) as shown below: O I - q- H202 --~ 02 -I- OH- +

"OH HYDROXYLRADICAL

It is now known that this reaction is extremely slow in the absence of metal ions. In their presence (particularly iron or copper), the formation of an hydroxyl radical is greatly accelerated. This occurs via the reactions shown below: H202 -t- F e 2+ --*OH- + F e 3+ -t- " O H 0~.- + F e 3+ ---*02 + F e 2+ .

In this case metal ions serve as redox cycling agents to catalyze the Haber-Weiss reaction. The hydroxyl radical can also be generated by the reaction of peroxide with reduced transitional metal ions (Fenton reaction) as illustrated below for manganese (Mn): H202 -1- Mn 2+ --*OH- + Mn 3+ + "OH

These three species (superoxide, peroxide and hydroxyl radical) have been referred to as the 'toxic triad' because they are all potentially damaging to biologic systems. 4.1.2. Free Radicals in Biological Systems It is important to note, however, that oxygen radicals can be produced as part of normal cellular reactions. For example, white blood cells (granulocytes) actually generate the entire array of oxygen radicals, effectively using these to kill bacteria (in fact, this granulocyte model is the most widely used to study the biology of free radicals) (Badwey and Karnovsky, 1980; Nathan and Cohn, 1980; Beaman and Beaman, 1984). Monoamine oxidase (MAO), a ubiquitous mitochondrial enzyme, produces one molecule of hydrogen peroxide every time a molecule of substrate is acted upon. Many enzyme reactions also utilize oxygen radical intermediates, but intermediates are generally never released from the active site of the enzyme. Oxygen radicals can also be produced under certain conditions by the action of transition metal ions, light, or ionizing radiation on dioxygen and by certain chemicals including ascorbic acid, catechols and a variety of toxins (for reviews, see Fridovich, 1979; Halliwell and Gutteridge, 1985).

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4.1.3. Cellular Defense Mechanisms If cells are indeed exposed to a continual flux of active oxygen species, and these are so damaging, one might ask why destruction is not sudden and devastating. The answer to this question probably includes both the ability of cellular structures to isolate oxygen radicals (Dormandy, 1978; Willson, 1977), and the presence of biological systems which inactivate oxygen radicals once they are formed. Oxygen radicals which are utilized in cellular processes are generally restrained. The respiratory assemblies of the inner mitochondrial membrane, for example, consist of an ordered grouping of several enzymes which collectively transfer four electrons to 02 to produce two molecules of water. O 2 + 4 e- + 4 H + ~ 2 H20 These enzyme reactions most likely produce oxygen radicals as intermediates, but they are carefully arranged so that the leakage of radicals is highly restricted. We might think of these systems as designed to prevent a toxic spill. When a spill does occur, a second line of defense is available in the form of enzymes which inactivate oxygen radicals. Catalase and glutathione peroxidase are specific for peroxide; superoxide dismutases are specific for superoxide (Fridovich, 1975; McCord and Fridovich, 1977). Although present in the brain, catalase and glutathione peroxidase concentrations are quite low (Gaunt and deDuve, 1976; Savolainen, 1978). The regional distribution of these enzymes indicates that certain structures in the basal ganglia contain the highest concentrations, and that overall brain concentrations decline with age. Furthermore, these enzymes are significantly reduced in Parkinson's disease, though it is not known whether this represents a cause or effect of cell death (Ambani et al., 1975). It is interesting that MAO, which produces peroxide, increases in the brain with age (Robinson et al., 1971; Nies et al., 1973; Fowler et al., 1980), paralleling glial proliferation, and appears to be normal (Bernheimer et al., 1962) or slightly elevated (Riederer and Jellinger, 1983) in Parkinson's disease. Thus, with advancing age, the cellular production of peroxide appears to be continually increasing against a failing line of defense. Superoxide dismutase activity has also been reported to be present in moderate amounts in brain tissue (Peeters-Joris et al., 1975; Fried, 1979), although to the best of our knowledge detailed studies of this enzyme have not been performed in the aging or parkinsonian brain. A final line of cellular defense may reside in the ability of simple, small molecules to inactivate oxygen radicals. Vitamins E and C, along with many others, have been reported to protect cells from free radical damage (Halliwell and Gutteridge, 1985). Simple aliphatic alcohols, such as ethanol and various other chemicals, appear to act as scavengers and afford some protection against hydroxyl radicals in experimental systems by reacting directly with them (Cohen et al., 1975; Heikkila et al., 1976b). 4.1.4. How Free Radicals Cause Damage There is no doubt that oxygen radicals can act to damage cells and cellular elements. Experimental systems which generate O1 have a deleterious effect on enzymes, DNA, cell membranes, virus particles, prokaryotic and eukaryotic cells in culture and animal tissues which have been exposed to them (Fridovich, 1979). In many cases, the addition of superoxide dismutase, catalase or scavengers protects against these effects. The subcellular effects of superoxide and hydroxyl radical probably involve the peroxidation of polyunsaturated lipids in membranes (McBrien and Slater, 1982). Free radical chain reactions are then promoted throughout the entire membrane. In addition to this primary damage, the attack on membranes may result in even greater secondary damage. As noted earlier, in mitochondria, the family of enzymes which generate and utilize active oxygen species are positioned carefully in a chain-like physical arrangement on the mitochondriai membrane. If the membrane structure is disrupted, then not only is the enzymatic process disrupted and the cell deprived of energy, but this 'topographical' safety factor may be deranged, leading to the uncontrolled release of radicals, which attack the membrane further and precipitate a chain reaction, or 'cellular meltdown'. As we will see, the

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generation of these toxic free radicals may represent the final common pathway for a number of the toxins to be discussed. In fact, similar mechanisms are frequently invoked to explain the aging process itself (Dormandy, 1983). 4.2. COVALENT BINDING

Cellular survival is dependent on the simultaneous operation of a wide variety of processes at the molecular level. The production and utilization of energy, the transport of essential materials, and the construction of cells is only possible because of the existence of a host of enzyme, transport and structural proteins which are continually at work. These, in turn, ultimately depend on DNA and RNA for their production. DNA, RNA and protein are all huge molecules (as many as 3,000,000 subunits in the case of DNA) which are built up by chemically linking together surprisingly simple subunits. Although their size is staggering, it is the chemical nature, and possible combinations of the individual subunits, which allows the creation of the versatile group of macromolecules which can perform tasks as simple as carrying oxygen or as complex as reproducing an entire organism. The subunits (amino acids in the case of protein and nucleic acids in the case of DNA and RNA) interact with each other, with water, and with substrate and cofactor molecules. It is these small interactions, repeated and varied throughout the vast parent molecule, which make possible the control of cellular metabolism and function. To some extent, however, nature is tolerant and as evolution and genetics have shown us, wide variations in the sequence of chemical subunits of proteins and nucleic acids are possible. Certain changes, however, may be fatal. 4.2.1. Why are Covalent Bonds Damaging? Some toxins damage cells by producing highly reactive metabolic intermediates which combine irreversibly with cellular macromolecules, forming permanent covalent bonds with specific chemical sites. The key word here is 'covalent'. Covalent bonds are to be distinguished from ionic bonds. The latter depend on the charge of the two participants, and tend to be easily broken, whereas covalent bonds are much more stable and are not easily broken within the biological environment without the aid of specific enzymes. If a compound covalently binds to one or more of the subunits of a macromolecule, unless there is a specific enzyme to reverse this bonding, it may interfere with the function of that molecule; hence, this type of bonding may inactivate enzymes, derange structural proteins, and alter DNA, causing mutations in genetic material. While the cell has ongoing repair mechanisms which can cope with a certain level of damage, and some level of inactivated macromolecules can be compensated for by new synthesis, the capacity for repair and restoration can be exceeded, and cell death ensues. Covalent binding of reactive intermediates is common to many toxic pathways, although to date much of the work has been done with peripheral neurotoxicity. One interesting example is n-hexane, a known peripheral neurotoxin, which is metabolized to a 2,5-diketone. This intermediate is believed to react with free amine groups (lysyl residues) of proteins to form pyrroles (Graham et al., 1984). It has been proposed that autoxidation of the pyrrole rings leads to covalent crosslinking between neurofilaments, which results in impairment of axoplasmic flow, and a resulting peripheral neuropathy (Graham et al., 1984). 4.2.2. Acetaminophen, The Cytochrome P-450 System and Toxic Overload Acetaminophen (Tylenol), a widely-used analgesic, is probably one of the best-studied compounds which causes cellular damage (hepatic necrosis) as the result of covalent binding (Mitchell and Jollow, 1975) when taken in excessive doses. A discussion of this drug also presents an opportunity to introduce two new concepts, that of the cytochrome P-450 mixed function oxygenase system, and toxic overload. Both of these are directly relevant to issues discussed later in this chapter. Acetaminophen is oxidized by hepatic microsomal mixed-function oxygenases (or the cytochrome P-450 system), which

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represents the major detoxifying system in the body (Brodie et al., 1958). The P-450 system oxidizes acetaminophen to an N-acetylhydroxylamine, which can rearrange to a number of reactive species (Mitchell et al., 1973a). One or more of these intermediates appear to be capable of forming covalent adducts to hepatocyte macromolecules and can cause extensive cell death. Normal therapeutic doses of acetaminophen, of course, do not cause liver necrosis or produce evidence of any cell death. However, there appears to be a toxic threshold above which cell death occurs. The explanation for this lies in P-450 activity and the exhaustion of cellular protective mechanisms. Microsomal cytochrome P-450 mixed-function oxygenases, which are present in mammalian liver, function as detoxifying enzymes, metabolizing drugs and other xenobiotics to more water-soluble compounds (Brodie et al., 1958). The ultimate goal of these biotransformations is to convert lipophilic compounds, which persist in membranes, to more hydrophilic derivatives which can be easily cleared from the organism by urinary excretion. Since some rather sophisticated chemistry is involved in these conversions, compounds need to be activated. Certain active intermediates are then chemically combined with substrates present in the cell (e.g. sulfur-containing glutathione) which render them more water-soluble. In the case of acetaminophen, the reactive intermediate is normally combined with glutathione (Mitchell et al., 1973b). This process is normally in equilibrium so that the amount of reactive intermediate produced does not exceed the amount of glutathione available. In the case of large overdoses of acetaminophen, however, the cellular supply of glutathione becomes exhausted and the reactive intermediate begins to react with the sulfhydryl groups in the amino acids of cellular proteins. Cytochrome P-450 mixedfunction oxygenases are an inducible system of enzymes, and exposure to certain foreign chemicals (e.g. hydrocarbons, phenobarbital) causes cells to greatly amplify P-450 activity by synthesizing more enzyme (Conney, 1967). In animal experiments with acetaminophen, induction greatly exacerbates toxicity. In a similar manner, pretreatment of animals with agents which deplete glutathione (e.g. diethylmaleate) potentiate the toxicity of acetaminophen (Mitchell and Jallow, 1975). An overdose of acetaminophen, with glutathione depletion, then represents an example of toxic overload. In this instance the situation might be analogous to running out of water while fighting a fire. N-acetylcysteine (Mucomyst) is used in the treatment of acetaminophen overdose as it serves as a form of glutathione replacement (Prescott and Critchley, 1983). In summary, covalent binding and oxygen-radical damage of cells are the molecular events which are thought to mediate the irreversible damage caused by many toxins. In the case of neurotoxins which cause parkinsonism, these two themes will appear again and again. As we will see, certain structures in the basal ganglia appear to be the target of these neurotoxins, and it can be hoped that by looking at a variety of them, it will be possible to determine common features which could help explain the selective vulnerability of one or more of these areas of brain.

5. MANGANESE 5.1. CLINICAL AND NEUROPATHOLOGICAL FEATURES

Historically, manganese intoxication was first observed in brownstone millers and in workers involved in mining and processing manganese ores, who inhaled toxic amounts of manganese dust (Couper, 1837). The symptoms of acute manganese toxicity in man are characterized by psychological disturbances which include memory impairment, disorientation, anxiety, and hallucinations. In fact the syndrome is distinct enough to have its own name, 'Locura manganica', which is said to be prevalent in the mining villages of Chile (Ansola et al., 1944). Manganese exposure may also cause damage to the liver and lung. Chronic manganism produces an irreversible syndrome which bears a striking resemblance to Parkinson's disease (Cotzias, 1958). The symptoms include fixed gaze, bradykinesia, postural difficulties, rigidity, tremor and dystonia. It is the dystonia, in addition to the

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often prominent mental status changes, which separates parkinsonism due to chronic manganese intoxication from Parkinson's disease (Barbeau, 1984a). At the neuropathologic level the two conditions appear to diverge even further. As would be expected, the core feature of involvement of the basal ganglia is indeed found, accounting for the clinical features of parkinsonism. However, autopsy reports (Casamajor, 1913; Ashizawa, 1927; Canavan and Drinker, 1934; Stadler, 1936; Voss, 1939, 1941; Flinn et al., 1940; Kawamura et al., 1941; Parnitzke and Peiffer, 1954) have shown the most extensive changes to be in the striatum and pallidum, areas which are spared in Parkinson's disease. Shrinking of the basal ganglia, together with marked degeneration of nerve cells, and the presence of gliosis, are noted in these areas. Substantia nigra degeneration, although a less prominent feature of manganese toxicity, has been observed to a variable degree. Interestingly, cell loss in the substantia nigra was marked in the case of a woman who had worked for 10 years in a battery factory and suffered a rigid-akinetic parkinsonian syndrome (Bernheimer et al., 1973). Although the neurotransmitter effects of manganese have been documented primarily in animal studies, it is of note that the only neurochemical report in a case of human manganese toxicity did show a depletion of striatal dopamine (Bernheimer et al., 1973). Norepinephrine was also lowered in the hypothalamus, while serotonin levels were normal. To our knowledge, Lewy bodies have never been observed in manganese-induced parkinsonism. Manganese-induced parkinsonism was originally treated by using chelating agents such as EDTA to attempt to remove managanese. This approach appeared to provide some benefit if used early in the condition, prior to neuronal destruction (Wynter, 1962), but no improvement was noted after more severe neurological signs had appeared (Tepper, 1961). With advances in the understanding of the neurochemical deficits in Parkinson's disease, L-dopa treatment seemed appropriate in patients with manganese-induced parkinsonism, but results have been somewhat contradictory. Six patients treated with L-dopa by Mena and colleagues (1970) showed improvement in rigidity, hypokinesia, and postural orientation. Rosenstock and colleagues (1971) also reported a therapeutic response in one patient. These results have not been confirmed by others (Cook et al., 1974; Greenhouse, 1982), although the possibility that there are two clinical subgroups of patients was suggested as an explanation. 5.2. DISTRIBUTION

Because manganese does cause a form of parkinsonism, the question arises as to whether or not manganese itself could be an etiological agent in Parkinson's disease. According to the Minerals Yearbook (1977), manganese is widely distributed in nature in the form of oxides, sulfides, carbonates and silicates and comprises about 0.1% of the Earth's crust. It is very common in iron ores (5.0-35.0%) and in many other minerals, as well as in coal and crude oil. Manganese also occurs naturally in water and air. Further, oxides of manganese have been used in the manufacture of glass since ancient times. More recently, the world production of manganese has been rising sharply--from 18 million tons in 1969 to about 27 million tons in 1975. Over 90% of the manganese produced is used in the making of steel. It is also utilized in the production of machinery and electrical equipment (batteries), in fertilizers, animal feeds, dyes, wood preservatives, glass and ceramics. Methylcyclopentadienyl manganese tricarbonyl (MMT) is increasingly being used as an anti-knock additive for gasoline and in 1976 was present in about 40% of unleaded fuels. Its use is expected to increase. Much of the manganese used in industry contributes to manganese pollution in the environment. Drinking water represents another possible source of manganese intoxication. Although the principal ores of manganese are only slightly soluble in water, gradual weathering and conversion to soluble salts result in appreciable concentrations in river and sea water. In addition, much of the manganese in water is absorbed on particulates. The concentrations of manganese in fresh water vary tremendously from a range of 0.00002-0.083 mg/liter in various remote American lakes (Kleinkopf, 1960) to several mg/liter in waters draining

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mineralized and industrial areas (Kozuka et al., 1971). The concentration of manganese in ground water (0.55mg/liter--Kimura et al., 1969), deep well water (0.22 to 2.76mg/liter--Itoyama, 1971), and in large-city, treated drinking-water supplies (0.002-1.0mg/liter--Schroeder et al., 1966) also shows wide variation. Only one epidemiological report is available on the adverse effects of drinking water contaminated with manganese (Kawamura et al., 1941). In this study 16 cases, three of which were fatal, occurred in a small Japanese community where 400 dry-cell batteries were found buried next to a well used for drinking water. The manganese content of the well water was 14 rag/liter. The subjects all exhibited psychological and neurological symptoms associated with manganese poisoning and high manganese levels were found in organs at autopsy. Finally, foods also represent a potentially large source of manganese. For example, cereal grains tend to have a high manganese content (wheat 13.740 mg/kg wet weight, rice 32.5 mg/kg wet weight). Meats, fish, fruits, vegetables and dairy products all have much lower amounts. Tea is known to contain very high amounts of manganese (780-930 mg/kg in finished leaves--Nakamura and Osada, 1957; and 3.6 mg/liter in liquid tea--Nakagawa, 1968). There is considerable national variation in dietary manganese intake. 5.3. RELATIONSHIP TO PARKINSON'S DISEASE This wealth of epidemiological data regarding manganese in the environment makes it a most interesting etiologic candidate in regard to Parkinson's disease. We know it's out there. As we will see later, absence of such data represents a current flaw regarding the etiological candidacy of a relative newcomer to the field of possible environmental toxins, MPTP. What puts manganese out of contention, in our opinion at least, is the lack of compatibility of manganese-induced parkinsonism with Parkinson's disease at the neuropathologic level. Until there is an absolute diagnostic marker for Parkinson's disease, its neuropathologic substrate has to be considered as the gold standard against which other models must be judged. The neuropathologic features of the two conditions, at least based on everything which is known to date, differ diametrically, one affecting the input side of basal ganglia (i.e. the substantia nigra in Parkinson's disease) and the other its output side (i.e. the striatum and globus pallidus in manganese toxicity). As mentioned earlier, the clinical features of the two conditions differ somewhat as well, further distinguishing them from each other. However, even though we are discarding manganese as an etiological candidate for the disease because of these important differences, there is still much to be learned by studying the mechanism of action of this element. 5.4. MECHANISM OF ACTION

Manganese, by virtue of its ability to assume multiple oxidation states, serves a role as an enzyme cofactor in mitochondrial superoxide dismutase (Fridovich, 1975), and mitochondria have an active system to concentrate manganese (Maynard and Cotzias, 1955). We have already pointed out that oxidative metabolism produces a number of active oxygen species (H202, O1-, and "OH) which can be damaging to biological systems. In normal concentrations manganese (when complexed with the enzyme superoxide dismutase) participates in the removal of superoxide by cycling between the trivalent and divalent state as is shown below: Enzyme-Mn 3+ + O1- --* 02 + Enzyme-Mn 2+ Enzyme-Mn 2+ + O1- -~ H202 + Enzyme-Mn 3+ NET 2 O1- --* H202 + 02 As can be seen, each cycle results in the removal of two superoxide molecules, and the production of hydrogen peroxide and dioxygen. Further, it is known that divalent manganese, in the presence of pyrophosphate, can act as a non-enzymatic superoxide dismutase, and appears to function this way in certain micro-organisms which are devoid of this important enzyme (Archibald and Fridovich, 1981). Thus, with respect to its protective role against superoxide, manganese can act both in an enzyme, and in the 'free

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form'. Of the three transition metals (Cu, Fe and Mn) utilized in the various superoxide dismutase systems, Mn appears unique in this ability. Of these three metals, the oxidized form of manganese (Mn 3+) is exceptional in another respect. While it is difficult to quantitatively measure the ability of substances to act as oxidants in biological systems, such measurements are routinely made as standard reduction potentials. The magnitude of a reduction potential reflects the oxidizing power of a species. The standard reduction potential for the one electron reaction of trivalent manganese (Mn 3+ + le- ~ Mn 2+) is + 1.51 V, which is nearly twice the potential for the reduction of Fe 3+, and almost ten times the potential for Cu 2+. Thus, Mn 3+ is an extremely powerful oxidant, capable of oxidizing many cellular constituents. All of these attributes of manganese suggest it should protect cells against damage, and in fact it is possible that the normal physiological role of manganese in the basal ganglia involves the enzyme-mediated dismutation of superoxide. How then might exposure to toxic amounts of manganese damage the nervous system? One possibility is that after excessive exposure to manganese, substantial amounts of unstable 'free' Mn 3+ could be generated by superoxide as shown in the following equation (Halliwell, 1984): Mn 2+ + OU --*H202 ÷ Mn 3+ As we will see in the text section, Mn 3+ may precipitate several damaging oxidations. Thus, while manganese serves a protective role, too much of this metal could lead to cell death. Alternatively, high concentrations of Mn 2+ could interact directly (Fenton reaction) with peroxide to produce hydroxyl radical. However, for the moment at least, both of these possible mechanisms remain speculative. 5.5. MANGANESE, DOPAMINE AND NEUROMELANIN

A discussion of manganese provides the ideal opportunity to broach two topics which are long-time favorites of investigators attempting to unravel the mysteries of nigral cell degeneration in Parkinson's disease. The first of these is dopamine. Eighty percent of CNS dopamine is found in the striatum (Carlsson, 1959). Since dopamine has been shown to be toxic in certain experimental systems (Graham et al., 1978), the question has frequently been asked whether or not dopamine itself might be neurotoxic in vivo (Cohen, 1983; Barbeau, 1984b). The topics of dopamine and manganese dovetail when it comes to a discussion of toxicity, because manganese has been shown to greatly enhance the oxidation of dopamine to its semiquinone and quinone (Donaldson et al., 1981; Graham, 1984). This enhanced oxidation probably occurs via Mn 3+ as is shown in the following formula: Mn 3+ + DOPAMINE -- Mn 2+ + DOPAMINE-SEMIQUINONE (DA-SQ) + H + Mn 3+ + DA-SQ = Mn 2+ + DOPAMINE-QUINONE (DA-Q) + H +. Dopamine semiquinone and quinone (Fig. 1) have been shown to be toxic to neuroblastoma cells (Graham et al., 1978). Interestingly, both free radical generation and covalent bonding have been implicated in this process. First, dopamine semiquinone may react with dioxygen to form the superoxide radical as is shown here: DA-SQ + 02 ~ DA-Q + OU + H + In dopaminergic cells, this process would be occurring in a setting where dopamine is being oxidized via MAO, a process which generates hydrogen peroxide. If enough of each were produced to overwhelm the cells antioxidant capacity, it can be seen that the stage would

0

//

(a)

CH2CH2NH 2

(b)

FIG. I. (a) Quinone; (b) DA semiquinone.

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al.

be set for the previously described Haber-Weiss reaction, with its potentially lethal consequences. Oxidized dopamine, particularly dopamine-quinone (ortho-quinone) can also covalently bond with the sulfhydryl group of certain enzymes, as is shown below: DA-Q + SULFHYDRYL ENZYME = DA-Q-SULFHYDRYL ENZYME COMPLEX. This covalentty bonded quinone-enzyme would result in the loss of the enzyme to the cell, where it could easily prove to be a lethal event. There is some evidence that these mechanisms may be at work in the in vivo situation. For example, in the rat, where intraventricular injection of MnC12 produces a 70-80% depletion of striatal dopamine, pretreatment of animals with L-dopa results in a more marked depletion of striatal dopamine (Parenti et al., 1986). Further, there is the intriguing case of manganese toxicity reported by Canavan and Drinker (1934). They noted the appearance of a black pigment in the cells of the caudate nucleus, where neuromelanin is not normally present. Melanin can be formed from the oxidation of dopamine. The finding of melanin in the striatum of manganese-induced parkinsonism would certainly be compatible with the proposition that manganese is accelerating the conversion of dopamine into a pigmented substance, and it seems likely that DA-SQ and DA-Q would be formed as an intermediate in this process. Thus, one can begin to see how the study of these toxic mechanisms has intrigued those trying to understand the nature of nigral cell degeneration for years. In fact, the formation of dopamine quinones has been speculated by some to be a key to the pathogenesis of Parkinson's disease itself. These speculations have been sparked by the observation that dopamine quinones can also be derived from the slow autoxidation of dopamine (Graham, 1978), probably as an intermediate in the formation of neuromelanin, the dark pigment which is increasingly deposited in catecholaminergic neurons with advancing age. Neuromelanin, because it appears to accumulate with aging (it is not present at birth) in the substantia nigra, has frequently been implicated in the process of nigral cell degeneration in Parkinson's disease. The role of neuromelanin is still poorly understood, but it has been suggested that its accumulation within nigral neuronal cells eventually results in cell death (Mann and Yates, 1983). In summary, it must be said that while a study of the mechanisms of manganese toxicity may have taught us a great deal, manganese itself cannot be considered as a promising candidate in the etiology of Parkinson's disease, in spite of the many parallels which can be drawn. This is because the brunt of the pathology is born by the striatum and pallidum in manganese intoxication. In this sense, manganese toxicity more closely resembles another disease which causes parkinsonism, known as striatonigral degeneration. In fact, the observation that manganese levels are elevated in striatonigral degeneration (Borit et al., 1975), has led to the suggestion that manganese might actually play an etiologic role in this condition. Finally, although the interactions between manganese and dopamine are of great interest, they may not help us explain the major effects of manganese toxicity, which are to be found in the cells of the striatum and globus pallidus, as these cells are not dopaminergic.

6. CARBON DISULFIDE 6.1. HISTORY AND DISTRIBUTION

The observation that carbon disulfide (CS2) was neurotoxic arose from its use in the 'cold' vulcanization of rubber to manufacture prophylactics, which at the time was a small cottage industry in France. The hygiene and ventilation in these shops was poor, and psychiatric and neurological disease became associated with the 'craftsmen' (Delpech, 1856; Laudenhaimer, 1899). Around the turn of the century, the viscose rayon industry was established, and the production of rayon from wood pulp, a process involving the use of large amounts of carbon disulfide, brought with it the recognition that chronic poisoning with carbon disulfide produced parkinsonism. It was in this setting that Quarelli

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(1928) observed varying degrees of parkinsonism in 30% of viscose rayon workers in Torrino. In a more recent study of workers in the American rayon industry, 21.7% appeared to have experienced some type of adverse affect; 1 in 6 of these individuals evidenced an extrapyramidal syndrome (Lewey, 1941). In this study the criteria for parkinsonism included the appearance of rigidity, the absence of arm swinging when walking, loss of postural reflexes, and the appearance of increased tone with the Noica maneuver. Because CS2 has been reported to cause parkinsonism, one might ask whether or not this compound is to be found in the environment. In fact, naturally-occurring CS2 has been found to appear only in small amounts--in the plume ash of volcanoes (Rasmussen et al., 1982) and in certain micro-organisms (Beauchamp et al., 1983), although it can also be formed by the combustion of sulfur-containing organic material. If we turn to man-made products, however, the story is quite different. Carbon disulfide was first synthesised in 1796 by Lampadius, and in less than 50 years had attained a prominent place in industry. Its excellent solvent properties resulted in its use as a phosphorous solvent in the manufacture of matches, and as a solvent for fats and oils (it is still used in the production of olive, and palm oils). Industrial uses represent by far the greatest contribution of CS2 to the environment. For example, atmospheric concentrations of CS2 are approximately l0 times greater in urban than in remote areas (Graedel et al., 1981). The greatest use of CS2 is in the rayon industry, since approximately 200 kg of CS2 are required to produce 1 ton of fibre. CS2 is also used in agriculture as a fumigant and fungicide, and in the production of cellophane and sulfur (Claus process). Large amounts of CS2 are produced in the operation of coal gassification plants. A recognition of the toxicity of CS2 has resulted in regulation, and a great reduction in the exposure of viscose rayon workers has resulted by decreasing the concentrations in the work place. This has led to a reduction in the incidence of neuropathy and other health effects from chronic poisoning. Because the vapors of carbon disulfide are more than 2.5 times denser than air, it also finds use as a fumigant in stored grain and for fumigation of soil. A recent report has cited a high incidence of parkinsonism in agricultural workers involved in the storage of grain, and suggested that CS: may play a role in this syndrome (Peters et al., 1986). It will be important to see if other investigators can confirm these observations. 6.2. RELATIONSHIP TO PARKINSON'S DISEASE

Could CS2 be an etiological candidate for Parkinson's disease, as well as a cause of parkinsonism? The arguments against this possibility are even stronger for CS2 than for manganese. First, CS2 does not produce a pure parkinsonian syndrome, nor are its effects restricted to neurotoxicity. It is a severe skin irritant, produces atherosclerotic cardiovascular changes, and renal, ophthalmologic, endocrine, and reproductive effects in exposed workers (Seppalainen and Haltia, 1980). It is also reported to produce both ototoxicity and liver damage. In regard to the nervous system, the clinical symptoms of chronic CS2 poisoning are more diffuse and, in some respects, differ considerably from those of Parkinson's disease (Seppalainen and Haltia, 1980). Polyneuropathy (usually accentuated in the legs and characterized by diminished muscle strength, patellar and/or plantar reflexes) is almost always present (Vigliani, 1954; Doyle Graham, personal communication). EMG studies have revealed slowed nerve conduction velocities, and such electrophysiological studies are primary to the diagnosis (Seppalainen et al., 1972). Ataxia, spastic paresis, mental deterioration and episodes of transient hemiplegia have been observed with higher levels of CS2 exposure (Vigliani, 1954). At lower levels of exposure, a syndrome consisting of headache, sleep disturbances, fatigue and decreased libido is common, and the incidence of suicide has been reported to be increased among rayon workers. Hence, in view of the multisystem involvement of the nervous system, in addition to the systemic effects of CS2, this compound does not appear to be a promising candidate as an etiologic agent for Parkinson's disease, even though man has successfully introduced it into the environment. J.P.T.3 2 / I ~

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6.3. NEUROPATHOLOGICALAND NEUROCHEMICALFEATURES In regard to the neuropathological effects of CS2 poisoning in humans, surprisingly little is known. In the single study of CS2 exposure of non-human primates, four rhesus monkeys were exposed, by inhalation, to 50ml/6 hr daily for 12 to 21 months (Richter, 1945). Symptoms of plastic cogwheel rigidity, bradykinesia, postural freezing, and tremor said to closely resemble Parkinson's disease were observed. The most striking and consistent pathological finding in these animals was a pronounced pallidonigral degeneration. It could be argued that these lesions may have been the result of anoxic episodes suffered by the animals, but in three of the four animals the onset of a parkinsonian state followed the episodes of unconsciousness by six to nine months. Although degenerative changes within the basal ganglia have been reported in dogs (Alpers and Lewey, 1940) and mice (Kuljak et aL, 1974) exposed to CS2, consistent or uniform involvement of these structures have not been observed, for the most part, in other species (Beauchamp, 1983). To compare the condition to Parkinson's disease, it should by now be clear that, as was true with manganese, the output structures of the basal ganglia (i.e. the striatum and globus paUidus) rather than the substantia nigra are predominantly affected, a critical differentiating factor from Parkinson's disease. Further, lesions in the cerebral cortex have also been reported in the dog, mouse, rat and cat (Beauchamp, 1983). Finally, vascular lesions in the brain have been described as part of the pathology of CS2 poisoning in cats, dogs and man (see Seppalainen and Haltia, 1980), and this has been proposed as one potential mechanism of CNS damage. Neurochemical data in humans and primates are not available but biochemical studies in rats have not shown dopamine depletion as a consequence of CS: exposure, although a depletion of CNS norepinephrine (NA) has been reported (Magos and Jarvis, 1970). Interestingly, animals studied after only two days of exposure showed a 16% elevation of striatal dopamine. These findings can probably both be explained by the subsequent observation that metabolites of CS2 inhibit dopamine-beta-hydroxylase (DBH) (McKenna and DiStefano, 1977). Other biochemical alterations have been observed in CS2-treated animals, including a marked decrease in brain synaptosomal Na+-K + dependent ATPase activity (Maroni et al., 1979), uncoupling of oxidative phosphorylation in brain mitochondria (Tarkowski and Sobczsak, 1971), increased proteolysis in rat brain, increases in brain acetylcholinesterase (Beauchamp et al., 1983), and decreased spinal cord copper in the rabbit (Cohen et al., 1959). 6.4. MECHANISM OF ACTION

The mechanism by which CS2 produces its toxic effects is not well understood, but it may well be multifactorial. Proposed theories have focused on its chemical reactivity, and it will probably come as no surprise to the reader that it is capable of forming covalent bonds to biomolecules. Thiol (SH), amino (NHR2) and hydroxy (OH) groups all react with CS2 to form addition compounds (Beauchamp et al., 1983). Perhaps one of the more interesting of these addition compounds, with respect to the mechanism of action of CS2, is the formation of dithiocarbamates, which is depicted below: S

S

II

II

C + HNR2"-* - S - - C - - N R 2

+ H+

II

S

The nitrogen can come from an amino acid, or any of the biologically present amines. Dithiocarbamates compounds are powerful chelators of metal ions including copper and zinc (Beauchamp et al., 1983). For example, rabbits treated with CS2 have copper levels in brain and spinal cord which are less than one half the value of controls (Scheel, 1967). Since essential metals act as cofactors for enzymes, the removal of these can have damaging consequences. Enzymes involved in both the biogenesis of catecholamines (e.g. DBH), and the inactivation of 0 2 (SOD), require metal ions as cofactors. Of particular interest here

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is the fact that copper is a crucial cofactor for DBH (McKenna and DiStefano, 1975), as noted earlier. DBH activity is decreased in rats exposed to CS2 (McKenna and DiStefano, 1977). That this inhibition is the consequence of dithiocarbamate formation in vivo is supported by the observation that in vitro inhibition of DBH is dependent on the preincubation of CS2 with amines capable of forming dithiocarbamates (McKenna and DiStefano, 1977). Dithiocarbamate derivatives of CS2 were also able to inhibit DBH. One dithiocarbamate is a known inhibitor of superoxide dismutase (Heikkila et al., 1976a); hence, CS2 damage to basal ganglia structures may arise out of the inhibition of this important enzyme which could leave the cell exposed to the potentially damaging effects of superoxide. Another mechanism which has been proposed for the toxic effects of CS2 involves the formation of covalent adducts with pyridoxamine (Vasak and Kopecky, 1967). The consequent derangement of B6 metabolism could be responsible for the neurotoxicity of CS2, and supplementation of the diet with B 6 delayed the onset of some of the neurotoxic effects of CS2 in rats (Teisinger, 1974), although it is still not clear why this would predispose basal ganglia structures to damage. In summary, CS2, for a variety of reasons, does not appear to be a likely candidate as an etiologic agent for Parkinson's disease. Because of its myriad effects on both the brain and systemic organs, it has also failed to find acceptance as a model for this disease. However, studies of its mechanism of action have provided some interesting insight regarding noradrenergic systems. Without clear evidence that CS2 induces a hypodopaminergic state, however, the relationship of this neurotoxin to the process of basal ganglia degeneration in Parkinson's disease remains uncertain. 7. 6-HYDROXYDOPAMINE (6OHDA) This is the first of the compounds to be discussed which was not discovered as the result of clinical observation, and has never been known to cause parkinsonism in humans. Nevertheless, because of its effects on certain neuronal systems, it promises to teach us something about the selective vulnerability of catecholaminergic nerve cells and hence will be discussed briefly. One could say that the era of experimental catecholaminergic neurotoxins began with 6OHDA. In 1968, Tranzer and Thoenen published their now-classic monograph detailing selective destruction of peripheral noradrenergic nerve endings with 6OHDA. It was this first successful 'chemical sympathectomy' that opened the door for investigating the effects of 6OHDA in the CNS. Unfortunately for those interested in using the compound to study central catecholaminergic neurons, 6OHDA does not cross the blood-brain barrier. Therefore, it has to be administered intraventricularly, intracisternally or directly into brain parenchyma. Once beyond the blood-brain barrier, however, 6OHDA acts in much the same way as it does in the periphery. It selectively destroys central dopaminergic and noradrenergic neurons (Ungerstedt, 1968; Bloom, 1971; Breese and Traylor, 1970; Hedreen and Chalmers, 1972). This destruction has been documented both neurochemically and morphologically. The selectivity of 6OHDA is believed to be due to its accumulation by dopaminergic and noradrenergic neurons through their high-affinity uptake systems (Thoenen and Tranzer, 1973; Kostrzewa and Jacobowitz, 1974). Indeed, compounds which interfere with catecholamine uptake protect against the neurotoxic action of 6OHDA (Thoenen and Tranzer, 1973; Kostrzewa and Jacobowitz, 1974). Not surprisingly, since its discovery, 6OHDA has become an extremely popular tool in neurobiological research. Specifically, 6OHDA has been widely used to lesion central catecholaminergic neurons, and determine the rote played by these neurons in maintenance of both normal and abnormal behavior. Exogenous 6OHDA has never been postulated to be a cause of Parkinson's disease. The reasons for this are twofold. First, 6OHDA does not cross the blood-brain barrier. Therefore, even if it were present in the environment (and there is no evidence that it is), 6OHDA would not be capable of reaching target sites in the brain such as the substantia

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nigra and locus ceruleus. Secondly, as noted above, 6OHDA, when administered systemically, destroys sympathetic nerve endings in the periphery. Since profound destruction of peripheral noradrenergic neurons is not a classic feature of Parkinson's disease, it is difficult to implicate exogenous 6OHDA as a cause of Parkinson's disease. Although exogenous 6OHDA has not been implicated as an etiologic agent in Parkinson's disease, it has been used extensively to study this disorder. The reason for this is clear. 6OHDA selectively destroys the two most prominently affected cell groups in the brain of patients suffering from Parkinson's disease--the dopaminergic neurons in the substantia nigra and the noradrenergic neurons in the locus ceruleus. In primates, bilateral intranigral administration of 6OHDA has been shown to produce a behavioral syndrome reminiscent of parkinsonism (Redmond et al., 1973; Kraemer et al., 1981). However, the technical difficulty of producing discrete nigral lesions has greatly limited the number of investigators that have been able to adequately explore the potential of developing a primate model of Parkinson's disease with 6OHDA. As will be discussed below, this only became feasible with the advent of MPTP, which destroys some of the same neurons in the brain as 6OHDA, but which is non-toxic systemically and crosses the blood-brain barrier (see below). The mechanism of action of 6OHDA has never been fully elucidated. Although it is known that 6OHDA must first gain entry into dopaminergic and noradrenergic neurons and attain a critical toxic concentration (for review, see Kostrzewa and Jacobowitz, 1974), the exact molecular series of events by which 6OHDA produces cell death remains incompletely understood. Any reader of this chapter by now will not be in the least bit surprised to learn that the two most commonly offered explanations in this regard are the production of free radicals (superoxide, the hydroxyl radical and hydrogen peroxide) and covalent bonding of quinone oxidation products (Heikkila and Cohen, 1971; Saner and Thoenen, 1971; Graham et al., 1978). Which, if either, of these mechanisms actually underlies the potent neurodegenerative action of 6OHDA remains to be ascertained. We turn now to a discussion of amphetamines, as they may give us a clue as to how 6OHDA might participate in the process of nigral cell degeneration in Parkinson's disease, even though the compound itself appears to be out of the running as an environmental agent. 8. METHAMPHETAMINE AND RELATED DRUGS Although methamphetamine and related drugs have not yet been reported to produce parkinsonism, we have chosen to include this class of compounds because of the recently accumulating evidence that these drugs are toxic to dopaminergic nigrostriatal neurons. Hence, insights into the mechanism of these effects could shed light on one or more mechanisms of nigral cell degeneration, which is the central theme of this chapter on toxins and parkinsonism. Methamphetamine is but one of literally hundreds of synthetic amphetamine derivatives. Amphetamine-like compounds probably constitute the largest class of stimulant drugs available to man. As Biel (1970) has noted, the amphetamine molecule has been the target of extensive structural modification, largely in an effort to develop an anorexic agent lacking the potent euphoric effect of amphetamine (Gunne, 1977). Methamphetamine and its congeners exert their powerful psychomotor stimulant effects indirectly by activating catecholamine-containing neurons in the brain, thereby increasing the concentration of dopamine and norepinephrine in the synaptic cleft (Moore, 1978). As is already clear from the section on manganese toxicity, dopamine itself is not above suspicion as a potentially toxic agent; therefore, any compound which increases dopamine concentrations is potentially of interest here. Typically, central effects of methamphetamine result in anorexia, hypodipsia, hyperthermia, psychomotor activation and respiratory stimulation (Moore, 1978). Humans also report lessened fatigue and a euphoric sense of well-being (Gunne, 1977). Given this spectrum of action, it is not surprising that amphetamine has been tried medically in the treatment of morbid obesity, narcolepsy, depression and parkinsonism (Innes and

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Nickerson, 1975). At times, amphetamine and related compounds have also been rampantly abused. Following the epidemics of methamphetamine abuse that took place in the United States, Japan, Great Britain and Sweden, a number of laboratories began investigating the long-term consequences of chronic exposure to methamphetamine. As a result of these studies, experimental evidence has accumulated which clearly shows that repeated high doses of methamphetamine exert a selective toxic effect on dopaminecontaining neurons in the brain. Repeated administration of methamphetamine leads to long-lasting reductions in brain dopamine content, the number of dopamine uptake sites, the concentration of the dopamine metabolites dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), as well as the activity of tyrosine hydroxylase (Seiden et al., 1975; Wagner et aL, 1980; Hotchkiss and Gibb, 1980; Kogan et al., 1976; Ricaurte et al., 1980; Steranka, 1982; Steranka and Sanders-Bush, 1980). Morphological studies have shown that these long-lasting dopamine neurochemical deficits are due to dopamine nerve terminal destruction by methamphetamine (Ricaurte et al., 1982; Ricaurte et al., 1984a; Ricaurte et al., 1984b; Ellison et al., 1978). Methamphetamine is not the only amphetamine derivative capable of destroying dopamine nerve fibers. Others possessing similar activity include amphetamine itself and cathinone, which is the principal active ingredient in Khat leaves (these are chewed throughout the Arab peninsula for much the same ends as South American indians use the Coca leaf) (Kalix and Braenden, 1985). Of the various dopaminergic systems in the brain, the nigrostriatal system is the most vulnerable to the toxic effect of methamphetamine (Ricaurte et al., 1980). The so-called 'mesolimbic' and 'mesocortical' dopamine neuron systems are also affected, but to a much smaller extent. By contrast, dopamine neurons in the hypothalamus appear refractory to the toxic effect of methamphetamine. This interesting difference appears to be related to the reduced affinity of the hypothalamic uptake transport mechansim for dopamine (Demarest and Moore, 1979). Methamphetamine-induced dopamine nerve terminal destruction has been demonstrated in a wide variety of experimental animals including rats, mice, guinea pigs, cats and rhesus monkeys (Seiden et al., 1975; Wagner et al., 1980; Hotchkiss and Gibb, 1980; Kogan et al., 1976; Ricaurte et al., 1980; Steranka, 1982; Steranka and Sanders-Bush, 1980). As yet, there is no direct evidence that amphetamines produce similar neurotoxicity in humans. However, the species generalization noted thus far suggests it could well occur, especially in individuals exposed to chronic high doses. If methamphetamine destroys dopamine nerve terminals in the human brain, the question arises as to why parkinsonism has not been observed in individuals abusing methamphetamine. A speculative answer to this important question is suggested by recent studies in subhuman primates. Rhesus monkeys given high doses of methamphetamine are known to suffer partial (60-70%) destruction of their nigrostriatal dopamine system (Seiden et al., 1975). Yet, these animals show no evidence of a movement disorder reminiscent of parkinsonism. That this is not due to failure of subhuman primates to develop a parkinsonian syndrome is indicated by recent studies with MPTP showing that rhesus and squirrel monkeys treated with this drug display prominent parkinsonism (Burns et al., 1983; Langston et al., 1984a). Indeed, with MPTP it has been possible to develop the first primate model of Parkinson's disease. Why then do methamphetamine-treated primates not develop parkinsonism? The answer probably lies in the fact that methamphetamine does not produce a large enough lesion in the nigrostriatal dopamine system to cause a clinically apparent syndrome. Studies using drugs to probe the integrity of dopamine neurons in methamphetamine-treated monkeys clearly show that these animals are more sensitive to agents that interfere with dopaminergic neurotransmission (Finnegan et al., 1982), suggesting that these animals do in fact have a partly lesioned dopaminergic system, but that the magnitude of the lesion is not sufficiently large to cause motor dysfunction. In a similar fashion, it may be that humans exposed to high doses of methamphetamine also suffer only partial damage of their nigrostriatal dopamine system. The extent of this damage may not be sufficiently large to cause clinically apparent parkinsonism.

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Recent studies with a subset of MPTP patients lend support to this concept. Numerous patients have now been identified who are known to have been exposed to MPTP, but as yet have no signs of parkinsonism (Ruttenbur et al., 1986). When some of these patients were studied with positron emission tomography (PET) scanning, they were found to have a partial depletion of striatal dopamine (Calne et al., 1985). Apparently, the damage to their nigrostriatal system has not been sufficiently large to produce overt parkinsonism. Of note is that with passage of time, some asymptomatic patients exposed to MPTP have developed symptoms of early parkinsonism (Langston, 1986). A tantalizing question raised by these observations is whether or not the large number of individuals exposed to high doses of methamphetamine might not also be at risk for developing Parkinson's disease at some time in the future. Studies on how amphetamine destroys dopamine nerve terminals have provided some fascinating new information. Evidence based on chromatographic analysis has recently been presented that amphetamine destroys dopamine neurons by inducing the in vivo formation of 6OHDA (Seiden and Vosmer, 1984), the potent dopaminergic neurotoxin discussed in the immediately preceding section. We would caution, however, that this most important finding requires more detailed analytic study, using techniques such as mass spectroscopy to unequivocally identify the presence of 6OHD in amphetamine-treated animals. It has been postulated that the 6OHDA found in the brain of amphetaminetreated animals is formed from auto-oxidation of endogenous dopamine (Seiden and Vosmer, 1984). If this hypothesis is correct, it would suggest another way in which dopamine itself might be made a toxic agent. Could the endogenous formation of 6OHDA, induced by an exogenous toxin, be a cause of nigral cell degeneration in Parkinson's disease? Are there other amphetamine-like compounds in the environment that are capable of stimulating the formation of endogenous 6OHDA? If so, could such environmental compounds play a role in the etiology of Parkinson's disease? These questions will be of great interest to address in the future. 9. MPTP 9.1. THE DISCOVERY AND CLINICAL SYNDROME Compared to the other neurotoxins discussed in the chapter, 1-methyl-4-phenyl-l,2,3,6tetrahydropyridine or MPTP [Fig. 2(a)] is a relative newcomer. For this reason, one might expect little to be known about it. However, in the short span since its discovery just three years ago, over 300 publications on MPTP have already appeared. One does not have to look far, however, to understand why this explosion of interest has occurred. First, the discovery of MPTP took place in a rather novel way, as it was mistakenly sold to heroin addicts as a narcotic substitute in northern California in 1982 (Langston et al., 1983). A number of them rapidly developed a severe, life-threatening parkinsonian syndrome. In retrospect, at least one previously published case of MPTP-induced parkinsonism has been identified as well (Davis et al., 1979). This places MPTP squarely in the camp of those compounds first discovered in humans, and the fact that we've been able to compare its clinical effects point for point with human parkinsonism has added an air of authenticity to the model. The first and most striking feature of MPTP toxicity in humans is that it produces an unalloyed parkinsonian state (Ballard et al., 1985). In fact, it is the purity of the parkinsonian condition that distinguishes MPTP-induced parkinsonism from other toxins discussed in this chapter. Not only does MPTP produce all of the the major features of Parkinson's disease, but many of the more subtle features of the disease, such as kinesia paradoxica and seborrhea are present as well. In regard to response to treatment, the parallel continues, as humans with MPTPinduced parkinsonism respond to the full array of anti-parkinsonian agents in a manner quite analogous to that seen in Parkinson's disease (Langston and Ballard, 1984). This further distinguishes it from manganese-induced parkinsonism and that caused by carbon disulfide; in these conditions the results of therapy are much less clear. Finally, the full

Neurotoxins, parkinsonismand Parkinson's disease CH 3

37

CH 3

I

I

(a)

(b)

CH 3

CH

+ CH (c)

(d)

Fro. 2. (a) MPTP; (b) MPP+; (c) MPDP+; (d) paraquat.

array of complications typically seen in idiopathic Parkinson's disease with L-dopa therapy are also encountered in patients with MPTP-induced parkinsonism (Langston and Ballard, 1984). In summary then, the clinical analogy between Parkinson's disease and MPTPinduced parkinsonism is nearly complete, and it must be considered perhaps the best agent discovered to date in terms of its ability to produce consistent and unalloyed parkinsonism (Ballard et al., 1985). 9.2. NEUROPATHOLOGY What about the neuropathology of MPTP neurotoxicity? Once again, the similarities between Parkinson's disease and MPTP-induced parkinsonism are much closer than any of the previously discussed neurotoxins. As has been stressed repeatedly throughout this review, the primary neuropathological feature in Parkinson's disease is degeneration of dopaminergic neurons of the substantia nigra and this is precisely the area affected by MPTP (Davis et al., 1979; Burns et al., 1983; Langston et al., 1984a; Forno et al., 1986a). This distinguishes it from the other toxins known to cause parkinsonism in man which, as we have seen, have their major effects in the striatum and pallidum rather than in the substantia nigra. While at first this may seem a major strength of the MPTP model, it has also been interpreted as a weakness, at least in terms of reproducing all of the neuropathological features of Parkinson's disease. In the latter, other pigmented brainstem nuclei may be affected, particularly the locus ceruleus (Forno, 1982). However, the original impressions of near total selectivity are beginning to crumble as further investigations take place. We have recently found in a series of six squirrel monkeys (of middle to old age) that MPTP in fact does induce degeneration in the locus ceruleus; five of the six animals were so affected (Fomo et al., 1986b). Mitchell and colleagues have also noted ceruleus damage in one monkey given MPTP (Mitchell et al., 1985). Hence, these observations bring the MPTP-model closer to Parkinson's disease. It will be recalled that earlier in this chapter we highlighted the importance of Lewy bodies as a neuropathologic feature of Parkinson's disease. Are Lewy bodies seen in

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J . W . LANGSTON et al.

MPTP-induced parkinsonism? Until recently, the answer would have been no. However, in the same group of older monkeys given M P T P (Forno et al., 1986b), three animals (all over the age of 15 years, or the equivalent of 60-70 years of age in the human) developed eosinophilic inclusions in the CNS which were intraneuronal and typically demonstrated a peripheral halo (Forno et al., 1986b). Even more interesting is the fact that these structures have been seen only in areas where Lewy bodies occur in human parkinsonism (Forno, 1982). The fact that these were seen only in older animals represents another parallel with Lewy bodies in humans, as these are typically seen only in older populations. While it would be premature to say that these structures are actually Lewy bodies, the fact that such inclusions are seen at all must be considered a very provocative finding, and one which may be moving us even closer to the idiopathic disease. 9.3. M P T P

AND THE ENVIRONMENT

These observations lead directly to the question of whether or not MPTP, or a similar compound, could be an etiological agent in Parkinson's disease. M P T P would seem to have passed many of the criteria failed by other neurotoxins in terms of clinical syndrome and neuropathological features. But does it exist in the environment? The answer, for the moment at least, is that we do not know. This lack of an 'environmental connection' must be considered the missing link in our quest to find a neurotoxin which is both present in the environment and can closely replicate the features of Parkinson's disease. On the other hand, it should be pointed out that the search for such compounds is just beginning. Two analogues of M P T P have already been discovered which are even more potent in producing dopamine depletion in the rodent striatum (Youngster et aL, 1986; Wilkening et aL, 1986). It seems possible that eventually we may have at our disposal a whole new class of catecholaminergic toxins. Further, we have recently found that M P T P produces its dopamine-depleting effects after oral, as well as intraperitoneal and intravenous administration (DeLanney, Irwin, Ricaurte and Langston--unpublished observations). This opens up a vast array of dietary compounds which must be considered in our search for an MPTP-like substance in the environment. Perhaps the most provocative finding to date regarding the possibility of an MPTP-like substance in the environment is that of Barbeau and colleagues (1986). These investigators, struck by the similarity between the major metabolite of MPTP, 1-methyl-4-phenylpyridinium ion or MPP + [Fig. 2(b)], and paraquat (a widely-used herbicide) [Fig. 2(d)], carried out a preliminary study of the prevalence of Parkinson's disease in Quebec province, and found the highest concentration of the disease in hydrographic region 3. Region 3 is the major agricultural area in Quebec province, and therefore has the highest use of pesticides. It also has higher concentrations of related industries, including paper mills and other forestry products (Barbeau et al., 1986). While it is as yet too early to be certain of the implications of these observations, they must be considered a tantalizing lead in the search for MPTP-like substances in the environment. It is probably safe to predict that there will be other such studies in the years to come. 9.4. MECHANISM OF ACTION

As promising as M P T P is as an etiologic candidate, the interest in elucidating its mechanism of action has generated no less excitement. As there are a number of recent detailed reviews on this subject (see Langston, 1985a; Langston, 1985b; Snyder and D'Amato, 1986; Langston and Irwin, in press), only a summary will be presented here (see Fig. 3). It is now known that M P T P is rapidly converted to a quaternary amine, l-methyl-4-phenylpyridinium ion or MPP + [Fig. 2(b)] (Langston et al., 1984c; Markey et al., 1984). This conversion is ubiquitous throughout the body and brain (with the possible exception of the eye where it appears to be much slower) (Langston et al., 1984c; Irwin and Langston, 1985) and takes place via an intermediate dihydropyridine, 1-methyl-4-phenyl-2,3-dihydropyridine or M P D P + [Fig. 2(b)] (Chiba et al., 1985a). The first step of this reaction, M P T P to M P D P +, appears to be mediated by monoamine

Neurotoxins, parkinsonism and Parkinson's disease

~

t3

39

DA NERVE TERMINAL

EXTRANIGROSTRIATAL COMPARTMENT

P ~

MPDP+ ~

(MAO-B)

MPP+

DA UPTAKE SYSTEM

(enzyme?)

GLIA? SEROTONERGIC NERONS? OTHER?

FIG. 3. Diagram showing the events which are thought to be responsible for the neurotoxic effects of MPTP after systematic administration (from Langston, J. William, MPTP: The promise of a new neurotoxin. In: Movement Disorders 2, Marsden C. D., and Fahn, S. (eds) Butterworth Scientific, London, in press).

oxidase B (Chiba et al., 1984, Heikkila et al., 1985; Salach et al., 1985). MAO B inhibitors block this transition in vitro (Chiba et al., 1984) and in vivo (Langston et al., 1984b; Markey et al., 1984) and prevent parkinsonism and nigral cell death in primates (Langston et al., 1984b; Cohen et al., 1985) as well as dopamine depletion in rodents (Heikkila et al., 1984; Markey et al., 1984). The next major link in the chain of events which appear to lead to toxicity is related to the dopamine uptake system. Javitch and colleagues (1985) have shown, and this was subsequently confirmed by Chiba and colleagues (1985b), that MPP + (but not MPTP) is taken up by the dopamine uptake system with the same affinity as dopamine itself. Thus we have a mechanism by which dopaminergic systems might be singled out for selective accumulation and hence neurotoxicity. However, not all dopaminergic systems within the brain are affected by MPTP, although there is some recent evidence MPTP may affect the dopaminergic ventral tegmental area in addition to the substantia nigra (Mitchell et al., 1985). Interestingly, the same hierarchy has been noted with amphetamine (i.e. the substantia nigra is more affected than the ventral tegmental area and the hypothalamus) (Demarest and Moore, 1979). Dopamine uptake blockers, predictably, protect against the dopamine-depleting effects of MPTP in rodents (Ricaurte et al., 1985; Snyder and D'Amato, 1985). However, in spite of repeated attempts, we have not been able to duplicate these results in the primate (Langston, DeLanney, and Irwin, unpublished observations). In fact, two animals receiving dopamine uptake blockers and MPTP became parkinsonian at doses of MPTP which do not produce a parkinsonian behavioral syndrome when MPTP is given alone. Whether these observations are simply the result of technical problems or in fact are pointing to some basic difference between rodents and primates is as yet unclear. 9.5. THEORIES OF CYTOTOXICITY

While this interesting chain of events has greatly advanced our understanding of MPTP, and highlighted the role of uptake systems in neurotoxicity (something that was relevant to the 6-hydroxydopamine story as well), we still know little about how this compound or its metabolites actually kill neurons. There are several major theories, however, which will be presented in this section. The first, and perhaps most attractive in many ways, was that MPTP might cause an increase in the availability of dopamine, which then might become cytotoxic through one

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J.W. LANGSTONet al.

or more of the previously discussed mechanisms (e.g. autoxidation to 6OHDA, the product of peroxides during the enzymatic oxidation of dopamine, or the formation of semiquinones and quinones which could play a role in the production of free radicals or form covalent bonds with enzymes as described previously). Although there is evidence that M P T P does cause a release of dopamine in vitro (Markstein and Lahaye, 1985), experimental data to date has not substantiated this mechanism of action. Compounds which deplete dopamine do not ameliorate toxicity (Fuller and Hemrick-Luecke, 1985; Schmidt et al., 1985), nor does pre-loading with L-dopa (which produces elevated striatal dopamine) (Melamud et al., 1985) appear to exacerbate toxicity. A second major theory was that a reactive intermediate might covalently bond with one or more enzyme systems (Langston et al., 1984c; Chiba et al., 1985a). The logical intermediate here would be M P D P - [Fig. 2(c)], which would be expected to covalently bond based on experimental work showing that such a process does occur ( M P D P ÷ bonds covalently at the 2 position of the nitrogen-containing ring with cyanide when they are co-incubated) (Chiba et al., 1985a). This theory also continues to bear exploration, although the increasing evidence that M P T P is converted to M P D P ÷ outside nigral neurons (Snyder and D'Amato, 1985; Ransom et al., in press) (presumably in glia) raises questions as to how this intermediate may be killing nigral neurons; rather, one might expect the damage to be occurring primarily in glia. The chemical similarity between MPP ÷ and paraquat has led some to suggest (Kopin et al., 1986) that the generation of free radicals by paraquat might serve as a model for MPP ÷ toxicity (see Figs 4 and 5). As can be seen [compare Figs 2(b) and (d)], paraquat can be viewed as essentially two MPP ÷ molecules together, and therefore has a doublepositive charge. Paraquat is thought to participate in a redox-cycling pattern (Fig. 4), which could be the source of almost unlimited amounts of superoxide and hydrogen peroxide (Bus and Gibson, 1984). This, in turn, could lead to the production of the hydroxy radical via the Haber-Weiss reaction. There is a major problem in attempting to extrapolate this mechanism to MPP ÷, however, in that it has been found that the one-electron reduction potential for formation of the MPP ÷ radical is much higher than for paraquat ( - 1 . 1 V for MPP ÷ as opposed to - 0 . 4 4 V for paraquat) (Sayre et al., in press) and there is no known biological system in the brain which might be expected to drive the reduction of MPP ÷ back to its radical. Thus, the possibility that the redoxcycling hypothesis might explain M P T P toxicity continues to be somewhat controversial. Although three different groups have shown that antioxidants appear to partially protect against M P T P toxicity (Perry et al., 1985; Wagner et al., 1985; Sershen et al., 1985), other investigators have been unable to reproduce these findings (Martinovits et al., 1986; Langston, Irwin, Langston and DeLanney, unpublished observations). The recent finding that diethyldithiocarbamate (DDC) (a superoxide dismutase inhibitor) (Corsini et al., 1986) exacerbates MPTP-induced neurotoxicity appears to support some type of free NADPH

Paraquat

NADP +

Paraquat Radical

NADPH

MPP +

NADP +

MPP + Radical

FIG. 4. Paraquat is reduced by NADPH-cytochrome P-450 reductase to the paraquat radical. The paraquat radical transfers one electron to dioxygen to produce superoxide anion. The oneelectron reduction potential (E0) is within the range of biological systems. The reduction occurs in microsomalfractions in the presence of NADPH, and is the suspectedmechanism of paraquatinduced toxicity. FIG. 5. Proposed mechanism of MPP+ redox cycling based on the paraquat model. The high one-electron reduction potential (E0), and the failure of MPP+ to generate superoxideanion in the presence of NADPH-cytochrome P-450 reductase and dioxygen do not support this model.

Neurotoxins, parkinsonism and Parkinson's disease

41

radical generation in MPTP neurotoxicity. We have found that DDC actually enhances the rate of oxidation of MPTP in vitro (Irwin, Langston and DeLanney, unpublished observation), and thus the enhancement of toxicity can be explained simply by the increased rate of generation of MPP +. For the moment, the final chapter regarding the exact role of free radicals and redox-cycling remains to be written regarding MPTP and MPP ÷. 10. NEURONAL DEGENERATION: A FAILURE OF THE DETOXIFICATION PROCESS? In this chapter, we have taken a lengthy tour through the current theories regarding causes of cell death, mechanisms of detoxification, and the relationship of these to a variety of neurotoxins which induce parkinsonism. In the final section of this chapter, we will try to extract the common elements among these various aspects of neurotoxicology, and attempt to look, in a fresh way, at some of the possible mechanisms which might underlie the process of neuronal degeneration. We believe that a number of new ideas are to be found in this rapidly evolving field, and one or more of these could provide insights as to why cell death actually occurs in human neurodegenerative disease. The first general thesis, however, will be a familiar one, and it is that the generation of active oxygen species and covalent bonding represent final common pathways for the induction of cell death by neurotoxins, and these same processes may underlie neurodegenerative disease. We should pause for a moment and note that excitotoxins represent another mechanism by which cell death can be induced in the CNS. Excitotoxins are undergoing a great deal of investigation at present, but are not discussed in this chapter because they are not known to cause parkinsonism. As we have seen in this review, both covalent bonding and free radical generation have been implicated in virtually every one of the major neurotoxins discussed. However, for the moment it must be said that the free radical hypothesis maintains the more prominent position, particularly in relation to 6OHDA, and MPTP. These are not new, however, but quite traditional concepts. What have we seen emerging as new or different ideas? 10.1. PROTECTING THE CENTRAL NERVOUS SYSTEM FROM TOXINS One novel idea which could be important in regard to neurotoxins and neurodegenerative disease relates to the concept of 'protoxins'. Before discussing this idea further, however, some broader concepts regarding mechanisms of detoxification are in order. It does not take long to realize that the brain might be exceptionally vulnerable to neurotoxins. The reason is simple. The brain is literally full of uptake systems used for the re-uptake of transmitters at nerve terminals, putatively to conserve these and to terminate their action. There is also evidence that there are uptake systems on cell bodies and even dendritic processes of neurons (Hamberger, 1967). In a sense then, one could view the brain as a structure which is comprised of hundreds of millions of small vacuum pumps capable of taking up a wide variety of compounds. We might compare this rather simplistically to the problem one has with very small children who are inclined to pick up everything that comes along and put it in their mouths. In this situation parents usually try to keep small and dangerous things out of reach. It would appear that nature has done very much the same thing in regard to the human brain by creating the blood-brain barrier. This structure is impermeable to many substances. In essence, it keeps the brain tightly compartmentalized, insulating it from the many potential toxins which could be taken up by neuronal systems and wreak havoc on the function of the CNS. There appears to be a second major difference between the brain and systemic circulation which further suggests that nature recognized the potential hazards of flooding the CNS with small, low molecular-weight compounds, which might be mistakenly taken-up as neurotransmitters. Here we are referring to the external compartmentalization of the cytochrome P-450 mixed-function oxygenase system discussed in some detail earlier. At first glance this would seem surprising. On closer examination, however, such an arrangement may make a great deal of sense. The fact that the hepatic system has open

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access to the circulation (and subsequent renal excretion) results in a strategy which is geared toward creating more polar, water-soluble compounds and conjugates from lipophilic precursors. The presence of large amounts of this enzyme within the central nervous system would not only result in the accumulation of a large number of potentially toxic reactive intermediates for uptake sytems to choose from, but would result in the creation of a series of compounds which could not be rapidly cleared from the CNS. Lipophilic compounds which act reversibly in the CNS (by virtue of their equilibration with the circulation and eventual removal to the periphery) might be converted to derivatives which might well be fatal to neurons if they were produced and accumulated within the CNS. We conclude, therefore (as nature already appears to have done before us), that the brain is not the environment for this approach. However, the CNS is not without the need for a detoxifying enzyme. It is now increasingly apparent that one of the substitutes for this system is monoamine oxidase (Westlund et al., 1985). Monoamine oxidase is a system of more limited versatility, catalyzing the oxidative deamination of a variety of amines (including amphetamine and methamphetamine). Could the process of neuronal degeneration in human disease result from the subversion of one or more of these detoxification mechanisms? We believe that the answer to this question could be yes, and that this failure of one of the brain's 'fail-safe mechanisms' might occur in at least two different ways. First, the process could be fooled, or diverted, in such a way that new toxins are produced rather than removed. Here, we come to the concept of 'protoxins'. The second mechanism, and one which has been well known for a long time, is that the capacity of systems designed to detoxify compounds may simply be exceeded. In the final sections of this chapter, we will review each of these, giving examples as we go from the toxins that we have discussed in this chapter. 10.2. FOOLtNG THE SYSTEM M P T P represents the best example of the first of these concepts. M P T P itself appears to be non-toxic (Langston et al., 1984c; Markey et al., 1984). Therefore, we would suggest that it represents an example of a true 'protoxin'. By this we mean a compound which is non-toxic but is eventually converted to a toxin by the body's own enzymatic machinery. Taking M P T P as a case in point, this compound passes through the blood-brain barrier by virtue of its lipid solubility. Even though a major protective barrier has now been overcome, a 'double deception' still appears to be necessary for the toxic effects of M P T P to occur. Once inside, the compound is recognized by the detoxifying-enzyme MAO as a potential danger (which it in fact does not appear to be, hence deception number 1), and is converted into MPP +. Now the protoxin (MPTP) has been converted to a toxin (MPP+). Still it would not damage cells unless it were selectively concentrated. This is where the uptake system comes into play. Because the toxin is now within the CNS compartment, it gains access to the uptake system of dopaminergic neurons and, based on current theory at least, is concentrated to toxic levels. MPP + is apparently recognized as 'dopamine-like substance' by the dopamine uptake system (in this case mistakenly--deception number 2); we have referred to this phenomenon as the 'Trojan horse effect'. Hence, it may be the very efficiency of the cellular machinery which activates and concentrates an agent which is responsible for neuronal death. The fact that it was necessary to start with a protoxin, and trick the cellular machinery no less than twice, gives an indication of the general effectiveness of these protective systems, and how difficult it is to fool them. A second mechanism by which our protoxin might inflict cellular damage is that it could provoke the conversion of a non-toxic substance in the brain to a toxin. Although oxidation of dopamine to quinones by M P T P and Mn may represent examples of this process, these proposed mechanisms remain, at least to some degree, speculative. Perhaps the most interesting example here is amphetamine, which (as described in detail above) may induce the autoxidation of dopamine to 6OHDA, a catecholaminergic toxin, in vivo (Seiden and Vosmer, 1984). In this scheme, dopamine itself actually becomes the protoxin (which we will refer to as an 'autoprotoxin' since it is an endogenous substance).

Neurotoxins, parkinsonism and Parkinson's disease

43

Methamphetamine may induce the conversion of this autoprotoxin (dopamine) to a toxic substance (6OHDA), and hence might be considered an 'autoprotoxin-inducer'. While amphetamines have not yet been shown to cause parkinsonism, it is the mechanism which is of particular interest here. We believe this idea deserves further exploration as another way in which environmental protoxins might provoke neurodegenerative disease. 10.3. AGING AND CELLULAR DEFENSES As mentioned earlier, a second mechanism which may play an important role in causing neurodegeneration resorts less to subterfuge, and more to the process of gradually overwhelming the neuronal cells antioxidant capacity. Here aging may be a critical factor, as has been frequently speculated in the past. With age the antioxidant capacity of the cell may decrease slowly, such that it becomes much more vulnerable to the assault of free radicals. There is now direct evidence that such a phenomenon may be directly relevant to MPTP toxicity (Ricaurte et al., 1986). It may be that this concept can be applied to other neurodegenerative diseases of aging as well. MAO is known to increase with age, presumably by virtue of glial proliferation. This proliferation is so great that glial fibers extend to fill virtually all the extraneuronal space, and eventually enshroud nearly all areas of the neuron except the synapse. This brings us to another concept which has only been hinted at earlier, and that relates to a potential role for glia in the process of neuronal degeneration. 10.3.1. Do Glia Become Killers? Glia have long been thought to be the great nourishers of neurons, a role which has the glial cell as protector. We would like to suggest that this association may not be without risk and that neurons may have a pay a price for this protection. Once again, MPTP appears to provide a striking example, as in this case glia appear to be converting a non-toxic substance into a compound which selectively kills neurons. This is a remarkable precedent, and could provide the foundation for a new category of disease. The intimate contact between neurons and glial cells suggest that the latter may very tightly control extracellular space, ion flow, neurotransmitter and metabolite levels, and free radical fluxes on the surface of neuronal membranes. Hence, it seems possible that glia could damage neurons in a variety of ways, particularly as the aging process begins to tip the balance in favor of glia (which appear to proliferate) at a time when neurons begin to lose their protective mechanisms. 10.3.2. Overwhelming Defenses Depletion with age of substances which serve an important role in the detoxification process could be important as well. Acetaminophen overdose provided an example of what happens when such substances are depleted, but this principle has not been welldemonstrated in the CNS as yet. Stores of glutathione are much more limited in the CNS than in the periphery but it is still possible that this or other compounds serve a similar function also. Reserves of compounds which are used to bind with neurotoxic products may decline with age. We believe this represents another field for further exploration, particularly in trying to define the process of neurodegeneration and its relationship to aging. 11. CONCLUSIONS In summary, we have seen that virtually all of the neurotoxins discussed here could provide valuable clues and insights as to the process of neuronal degeneration. These insights may help us explain not only why some neurotoxins cause parkinsonism, but could lead to an understanding of the process of neuronal degeneration in Parkinson's disease itself. Of the toxins discussed, only MPTP clearly remains in the running as a possible etiological candidate for Parkinson's disease, primarily because it affects the substantia

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J.W. LANGSTONet al.

n i g r a ( r a t h e r t h a n o t h e r b a s a l g a n g l i a s t r u c t u r e s ) a n d c a n g a i n access to the C N S a f t e r systemic exposure. I n a d d i t i o n , t h e s e t o x i n s m a y p r o v i d e n o v e l m e c h a n i s m s w h i c h c o u l d be i m p o r t a n t in r e g a r d to h u m a n n e u r o d e g e n e r a t i v e disease. T h e c o n c e p t t h a t it is p o s s i b l e to i n d u c e selective n e u r o n a l d e g e n e r a t i o n b y f o o l i n g b o t h t h e b l o o d - b r a i n b a r r i e r a n d the b r a i n ' s o w n e n z y m a t i c m a c h i n e r y c o u l d p r o v i d e a w h o l e n e w basis f o r the s t u d y o f n e u r o d e g e n e r a t i v e diseases, p a r t i c u l a r l y t h o s e a s s o c i a t e d w i t h aging. W i l l t h e s e c o n c e p t s p r o v i d e the basis f o r o n e o r m o r e h u m a n diseases? T h e a n s w e r to this q u e s t i o n is n o t yet k n o w n , b u t w e b e l i e v e t h e s e studies a r e o p e n i n g a v a s t n e w field f o r e x p l o r a t i o n . P e r h a p s as the result o f d e l v i n g i n t o t h e s e issues, a n s w e r s will be f o u n d to one or more of these previously impenetrable questions. Acknowledgements--We wish to thank Drs A. J. Trevor, N. Castagnoli and C. D. Marsden for many stimulating

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