Parkinsonism after chronic exposure to the fungicide maneb (manganese ethylene-bis-dithiocarbamate).

Permanent parkinsonism was observed in a man with chronic exposure to the fungicide maneb (manganese ethylene-bis-dithiocarbamate). Symptoms developed at 37 years of age, two years after exposure had ceased. To our knowledge, this is the second report on parkinsonism associated with exposure to maneb. Manganese is a well-known parkinsonigen toxin in humans. More recently, it has been shown that dithiocarbamates can also induce extrapyramidal syndromes. The biochemical effects of manganese and dithiocarbamates are reviewed and their possible neurotoxic mechanisms are discussed. Both of these components may have played a role in this case.

It has long been known that chronic manganese intoxication induces parkin sonism among miners and some categories of manufacturers exposed to manganese ores (1)(2)(3)(4). Symptoms appear after a period of exposure ranging from six months to some years. They include bradykinesia, gait disturbances and postural instab ility, hypomimia, and postural and (less frequently ) rest tremor. Dystonia is often seen in intoxicated miners. Treatments with levodopa (L-dopa) and chelating agents (eg, edetic acid) have sometimes yielded positive results, while they have been ineffective at other times (1)(2)(3)(4). The few necropsy studies carried out on mangane se-induced parkinsonism have mainly shown degenerative lesions of the globus pallidum and subthalamic nucleu s, caudate nucleus, and putamen, with less frequent or less severe lesions of the substantia nigra, a scenario very different from that of idiopathic Parkinson's disease, in which the substantia nigra is typically involved and the strio-pallidal complex is spared (I). Chronic administration of manganese to monkeys also induces an extrapyramidal syndrome with extensive lesions of the basal ganglia (1). At the neuro chemical level, most studies have reported a depletion of dopamine in the striatum of animals chronically treated with mangan ese (1).
More recently several cases of permanent extrapyramidal syndromes have been observed followin g disulfiram (Antabuse") poisoning (5)(6)(7)(8)(9). These cases have sometimes been characterized by addi-tional pyramidal signs, peripheral neuropath ies, or behavioral abnormalities, and the computed tomography (CT) scans have detected bilateral lesions at the level of the basal ganglia in some of the subjects (7)(8)(9).
Ethylene-bi s-dithiocarbamate (EBDTC) and carbon disulfide are the main metabolite s of disulfiram (10). Carbon disulfide is itself a well-known toxin causing parkinsonism ( 11), and it can react with endogenous amino acids and monoamines to produce dithiocarbamates (12).
A possible role of EBDTC (6,9) or carbon disulfide (7,8) has been mentioned in some cases of disulfiram-associated parkinsonism. Moreover, some authors have suggested that EBDTC might be the compound directly responsible for the neurochemical changes and toxicity of disulfiram and carbon disulfide on the extrapyramidal system (12,13).
Besides their role in secondary parkin sonism, it has been hypothesized that mangane se ( I) and, recently also, dithiocarbamates (6) may also play a role in the pathogenesis of idiopathic Parkinson' s disease.
We observed permanent parkin sonism in a man with chronic exposure to the fungicide maneb (manganese ethylene-bis-dithiocarbamate) (figure 1) (14). This compound comprises a wide fungicidal spectrum; it acts mainly through contact with foliage and is used for several crops, including apples, pears, grains, tobacco, and beets. During the last 20 years the consumption of maneb and mancozeb (manganese zinc ethylene-bis-dithiocarbamate) has increased continuously in Italy. In the various marketed preparations containing maneb, the mangane se and EBDTC weight ratio is about 1:4. Copper is often added to the marketed preparations (14).

Case
A 47-year-old man came to our center in October 1991. His medical histor y showed no significant events in the past and no metabolic or neur opsychiatric diseases among relatives. From 23 to 33 year s of age he worked in the management and maintenance of the mech anical plant of a malt-producing mill. Then, betwe en 33 and 35 years of age, he planned, con structed, and tested a device for treating barley seeds with fung icides. In parti cular, during these years, the patient treated about 70 sacks of barley seeds (30 kg each) daily with 5 g of maneb fungicide in each sac k. He was exposed on the average for 4 h a day, 4 d a week, 4 months a year. During this time he handled abou t 45 kg of fungicides in a closed environment, with a window and ventil ation sys tem but without any personal prote ction (gloves or mask). In 1981, at the age of 37 years, two year s after the exposure to maneb had ceased, a mild tremor associated with paresthesias appeared in his right leg and later spre ad to the ipsilateral arm . A neurologist diagnosed extrapyramidal syndrome and prescribed amantadine (100 mg) and trihexyphenidyl (2 mg), which had a benefi cial effect on the symptoms. However, during th e follo wing three years the tremor worsened, spreading to the contralater al limb s. The symptoms then remained stable for about seven years . Our fir st neurol ogical examination showed mild, generalized bradykinesia and rigidit y, postural tremor in the right limbs, mild tremor of the lips, mild slowness of gait with reduced swinging of the ar ms, seborrhea, mild hyp omimi a, and slurred speech . No other neurological signs appeared in the examination. The blood analyses, the electrocardiogram , and the visual evoked potenti als were norm al. Cerebro spinal fluid samples were not avail able due to the patient' s refu sal to undergo lumbar puncture. The serum copper and ceruloplasmin levels wer e normal (copper: 105 ug . dl: ', norm al range 65-165 ; cerul oplasmin: 35. 1 mg . dl: ' , normal range 15-60), Wilson ' s dis-302 ease thus being excluded as a cau se of the parkinsonism. The urinary activity of arylsulfatase was also in the normal range, a finding excluding another rare metabolic cause of sec ondary parkinsoni sm .
The CT scan of the skull was norm al, the only exception bein g a mild enlargement of the right lateral ventricle . The acute administration of apomorphine (1 mg) subcutaneously or L-dopa (200 mg) orally had no effects on the symptoms. Thu s the chr onic treatment with amantadine and trihexyphenidyl was maintained.
In July 1992 the patient returned for a check-up bec ause of a worsening of the symptoms during the last six months. Our examinati on confirmed the worse ning of his symptoms and revealed the presence of rest tremor in all four limbs and the lips, particularly in the upper limb s and on the right side. A magnetic resonance imaging (MRI) scan of the brain was performed with a Magnetorn" imager (Siemens) with a 1.5-T magneti c fie ld. The asymmetry of the ventricular sys tem, already seen with the CT, with a mild enlargement of the right lateral ventricle, was con firmed. In proton den sity and Tz-weighted scans some small hyperintense areas appe ared in the bil ateral frontal and left parietal white matter. The se appearances can be interpreted as nonspecific gliotic foci . Th e basal ganglia were norm al (figures 2-3) . Finally, a neurop sychological evaluation showed no relevant abnormali ties. However, some perseverative errors and some diffi culties on the attentive tests and in shifting between semantic and alphabetic categories were noted.
In May 1993, during the last check-up, a furth er slight worsening of the extrapyra midal symptoms was found . The L-dopa challenge (250 mg orall y) was repeated , but again it had no effect on the symptoms.

Discussion
Manganese affects biological systems in numerous ways, but its primary neurotoxic mechani sm is still far from cle ar (15). Th e metal impairs the functioning of some recept ors and ionic channels of the plasma memb rane, the sys tems for signal transducti on and second messenger synthesis, some cellular enzymes, and other metalloproteins ( 15). One particular target of the metal is the mitoch ondria , where the mangane se alters the calcium homeosta sis and provokes an oxidati ve stress . Other mech anisms that have been sugges ted to explain the neurotoxic ity of manganese include dopamine auto-oxidation, stimulation of free radicals and 6-0H-dopamine production , and the redu cti on of levels of reduced glut athione , glut athione peroxidase and catalase ( 15).
The extrapyramida l syndromes assoc iated with the disulfiram -EBDTC-carbon disul fide group of compounds are much less stud ied and much less known . Dithiocarbamates are chel ating compounds which form lipoph ilic compl exes with various metallic ions (13). Such complexes tend to accumulate in the central nervous sys tem (CNS) and in other tissues with high lipid concentrations. EBDTC is one of the most studied dithiocarbamat es. It has been proposed as a chelating agent for the treatment of cadmium and nickel intoxication and even for Wilson ' s disease (13,16). However it has been shown that the effect of this comp ound , like that of other chelat ing agent s, is not a simple depletion but rather a redi stribution of metal s in the body, with a reduction of levels in some organs and an increase in others, including the CNS (13 , 16).
In particular , the administration of EBDTC to rats, with or without the addition of copper to the diet , increa ses the levels of copper in the CNS ( 13). Once they have passed the blood-brain barrier, the EBDTC-metal complexes may dissociate, and the metal may accumulate in the brain. Toxicity then ensues, probabl y through oxidativ e reaction s and free radical produ ction ( 13). Th is mechanism probably explains why the pretreatment of rats with EBDTC enhances the toxic effe cts of l-rnethyl-a -ph enyl-1,2,3 ,6-tetrahydrop yridine (MPTP), a selective toxin for the dopaminergic pathways, responsible for cases of perm anent human parkinsonism (17 , 18). Moreover, EBDTC inhibits some enzyme s which need metal ions to function, for example, superoxide disrnutase, a scavenger enzyme for superoxide radicals (19) and dopamine-Bvhydroxylase (12).
As regards maneb, only one case of acute intoxication by a mixture of maneb and zineb (zinc ethylene-bi s-d itniocarbamate) has been reported, with transi ent loss of consciou snes s, con vulsions, and hemiparesis, all of which disappeared after a few days (20) .
In 1988 two cases of parkinsonism were first obser ved in young agricultural workers expo sed to various fungicides , though mainly to maneb , for ScandJ Work Environ Health 1994, vol 20, no 4 four to five years (21). After thi s observ ation , a group of 69 workers from the sa me regi on was screened. The results showed a significantly increased frequency of rigidity with cogwheel phenomenon , headache, fatigue, nervousness, memory complaints, and sleepiness in the people expo sed to maneb. Postural trem or, bradyk inesia, and cerebellar signs were also seen in the exp osed group but with out statistical significa nce. The authors stated that occupational exposure to pesti cides containing manganese may induce signs of CNS manganese intoxication, and thus the role played by EBDTC in fungicide toxicity may be less important than that of mangane se (21).
Thi s stud y repre sents the second report of park insonism associated with chronic exp osure to maneb. The early onset (age 37 years ) and the stabilization of symptoms after an initial worsening suggest that some environmental factor s might have played a role in this case. More over, the intense, chronic and "s elective" exposure to the fungicide maneb makes this case very interesting.
In light of the biochemical effects of mangane se and EBDTC, a toxic cooperative mechanism can be hypothesized that is probably based on the accumulation of heav y metal s in the CNS (13) .
The latency of two years observed in this case between the end of exposure to the fungicide and the onset of extrap yramidal symptoms must be stre ssed. To our knowledge, in mang anese intoxications, such a phenomenon has never been reported. Moreover, after the exp osure to manganese has cea sed , the symptoms may improve, but they usually become stable and occasionally continue to worsen (1)(2)(3)(4). There is only one rep orted case in which mild gait disturbances appeared durin g exposure to manganese with the main extrapyramidal symptoms developing three years after the exposure had ceased (2). Sca nd J Work Environ Health 1994, vo l 20, no 4 For our ca se we hypothesized that the accumulation , induced by maneb, of heavy metals like mangan ese and copper in the CNS could have primed a form of delayed to xicity responsible for the delayed on set of symptoms. Indeed, in light of the effects of EBDTC on the brain , it has been stated that EBDTC and disulfiram treatments could enhance the risk of neurological disease s man y years later ( 13) . Moreover , delayed effects have been claimed to explain other cases of dithi ocarbamate toxicity (6) . As an altern ati ve hyp othesis, for this patient, the chronic exposure to maneb might have interacted with and accelerated a preexisting degenerative process, leading to the early cl inical appearance of parkinsonism.
For our patient a determination of blood or hair manganese levels was not performed. It is known that a rel ationship between manganese body levels and neurological symptoms doe s not exist (I, 21). Besides, such a determination , as well as that for EBDTC, would be of little rele vance years afte r the expo sure had ceased.
The lack of effects of L-dopa or apomorphine and the beneficial effects of amantadine and trihexyphenidylon this patient sug gest the presence of lesion s distal to the nigrostriatal pathway, in accordanc e with the results of a positron emission tomographic study on manganese-induced parkinsoni sm (2 2) .
The CT and MRI scans showed no relevant findings in our case. Bilateral pallidoputaminal lesions have been detected by CT (7-9) and MRI (7) in some case s of extrapyramidal syndromes after severe disul firam intoxication. As regards man gane se-induced parkinsoni sm, the MRI was abnormal in only one of the four cases examined (2 2).
Last, the neuropsychological examination of our patient detected, in the context of good global perform ance, so me per severative error s and attentive difficulties, findings compatible with an initial involvement of the frontal lobe fun ctions and in accordance with the cognitive pattern of parkinsonian patients (23 ).
Besides the involvement of manganese in form s of secondary parkinsonism, which are different from idiop athic Parkinson ' s dise ase on a pathological level , the role of mang anese, as well as that of dithiocarbamates in the etiopathogenesis of Parkinson' s disea se, is still unclear. Barbeau (1) proposed that initi al damage to the nigro str iatal system, cau sed by a variety of factors (including manganese intoxication) but able to induce a critical increase in dop amine turnover, could trigger a neurotoxic selfperpetuating mechan ism leading, through the production of oxidative stre ss in the surviving cell s, to the pro gre ssi ve loss of the dop aminergic neu ron s and to the development of Parkinson 's di sea se.
After the discovery of MPTP-induced park insonism (17), numerous studies have been de voted to the search for environmental toxins in Parkinson's disease etiopathogenesis (24) . Some epid emiol ogic sur-304 veys have identified risk factors such as living in rural areas , the use of well water, and exposure to pesticide s for Parkinson' s disease (24) . Another ca serefer ent study found instead that working in orchards and planer mills, two acti vities invol ving e xpos ure to fungicides, increased the risk of Parkinson's di sease (25).
In conclusion, thi s is the second report of permanent parkinsonism ob ser ved after chronic ex pos ure to the fun gicide maneb. Both of its components (manganese and EBDTC) are potential toxins for the extrapyramidal system and might have had toxic effect s. A better comprehension of the way the se substances act on basal ganglia may help to clarify the mechanisms of dela yed neurotoxicity and contribute to the und erstanding of the patho gen esis of other extrapyramidal disorders like Parkinson ' s dise ase , dy stoni as, and Wilson' s disease.