Experimental benznidazole encephalopathy:

Journal of Neurological Sciences 150 (1997) 3–11

Experimental benznidazole encephalopathy: I. Clinical-neurological alterations Carmen Lucia Leite Flores-Vieira b

a,b

a, , Amilton Antunes Barreira *

a ˜ Preto, SP, Brazil Department of Neurology, Psychiatry and Medical Psychology, University of Sao Paulo, 14048 -900, Ribeirao ˜ Preto, University of Sao Paulo, 14048 -900, Ribeirao ˜ Preto, SP, Brazil Department of Physiology, School of Medicine of Ribeirao

Received 30 December 1995; revised 23 October 1996; accepted 28 November 1996

Abstract Benznidazole (N-benzyl-2-nitro-1-imidazoleacetamide) is an antiprotozoan agent of the nitroimidazole group used extensively in South America to treat Chagas’ disease. In humans, its most important side effect is peripheral polyneuropathy, the frequency of which is dose related. To evaluate this effect, we administered benznidazole to adult, male, mongrel dogs at doses ranging from 5 to 40 mg / kg / day (0.5 to 4 times the dose used to treat chagasic patients). Subsequent neurological examination revealed apathy, ataxia, spastic tetraplegia with hyperreflexia of stretching reflexes, balance disorders and asymmetrical gait. These alterations appeared earlier and were more intense at the higher doses. Drug withdrawal also left dose- and time-dependent sequelae like ataxia, hypertonia, hyperreflexia and alterations of balance. No peripheral neuropathy was detected. The present findings suggest that a careful reevaluation of the side effects of benznidazole in humans is necessary.  1997 Elsevier Science B.V. Keywords: Benznidazole; Chagas’ disease; Experimental encephalopathy; Clinical-neurological alterations

1. Introduction Important side effects of nitroimidazole drugs like misonidazole and metronidazole result from their neurotoxicity (Coxon and Pallis, 1976; Schipper et al., 1976; Dische et al., 1977; Bradley et al., 1977; Said et al., 1978; Mamoli et al., 1979; Kusumi et al., 1980). Alterations in the righting reflex, persistent head falling, and disorders of behavior and balance have been observed in rats treated with misonidazole and metronidazole (Rogulja et al., 1973; Griffin et al., 1980). Benznidazole is an antiprotozoan agent with marked activity against Trypanosoma cruzi, the parasite that causes Chagas’ disease. The chemotherapeutic efficacy of benznidazole has been confirmed in vitro on the epimastigote, trypomastigote and amastigote forms of T. cruzi (Roche Research Department, unpublished data sheet). Chagas‘ *Corresponding author. Tel.:155 016 6330866; fax: 155 016 6339606; e-mail: [email protected] 0022-510X / 97 / $17.00  1997 Elsevier Science B.V. All rights reserved PII S0022-510X( 97 )05361-6

disease is endemic to almost all Latin American countries, including Mexico and the Central American nations (Kirchhoff, 1993), and benznidazole has been used widely to eliminate the circulating forms of the parasite which affects millions of people (WHO, 1981). Clinical trials using doses ranging from 5 to 10 mg / kg / day for periods of 30 to 90 days have demonstrated the therapeutic efficacy of benznidazole against these circulating forms (Levi et al., 1975; Ferreira, 1976; Barclay et al., 1978; Arruda Jr., 1986). The most important side effect, observed in chagasic patients treated with the drug, is peripheral polyneuropathy, the incidence of which ranges from 18 to 81%; the frequency and intensity of incidence are apparently dose-dependent. This polyneuropathy may be axonal in nature (Faria et al., 1986). There are no consistent reports on the side effects involving the CNS in humans. A single study of neurotoxic effects in dogs treated with benznidazole described strong contraction of the back and thigh muscles with opisthotonos, tail elevation and the

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development of a sustained, contracted posture with asso¨ ciated nystagmus (Scharer, 1972). In view of the scarcity of reports on the effects of benznidazole, the objectives of the present study were to examine and describe the clinical-neurological alterations in dogs treated with this drug, and after the withdrawal of treatment. Neurological syndromes and the characteristics of their development are analyzed as are the relationships between dose, time and intensity of the phenomena.

2. Material and methods

2.1. Experimental groups The study was conducted on 17 adult, male, mongrel dogs. The animals were divided into 3 experimental groups: a control group and two treated groups which received benznidazole at different doses for different periods of time. The control group consisted of 6 dogs that were submitted to neurological and electroencephalographic evaluation. Two control dogs were then sacrificed for histological study of the brain and spinal cord (see accompanying paper II). Six dogs (Group I, acute treatment for 15 days) received 30 mg / kg / day from day 1 to 15 and 10 mg / kg / day from day 16 to 30 (see Fig. 1). Since mortality was very high (5 / 6 in 30 days), five dogs (Group II, short-term acute treatment for 7 days followed by chronic treatment) were divided into two subgroups: IIa (2 animals, short-term acute treatment for 7 days followed by chronic treatment) and IIb (3 animals, chronic treatment). Subgroup IIa received 40, 20 and 5 mg / kg / day from days 1 to 7, 8 to 60

and 61 to the day of sacrifice (on days 125 and 382), respectively. Subgroup IIb received 20 and 5 mg / kg / day from days 1 to 30, and 31 to sacrifice (on days 105, 176 and 185), respectively. All dogs in Groups I and II (a,b) were neurologically evaluated as described below. Benznidazole was administered orally, twice daily, at 12 hour intervals in the form of commercially available tablets. There was no dietary restriction. The accompanying paper II also presents data obtained by electroencephalographic evaluation before, during and after (two animals) treatment. Histological data on the brain and spinal cord tissue of dogs that died spontaneously or were sacrificed at the end of the benznidazole treatment are also presented.

2.2. Neurological evaluation All dogs were evaluated neurologically according to the standardized method of Braund (1994). Postural reflexes, locomotion, mental status (level of consciousness) and cranial nerve dysfunction were evaluated on the basis of the criteria and scores (0, 1, 1.5 and 2) outlined in Table 1. Perception of pain was evaluated using the subject‘s reactions when the flexor and panniculus reflexes were tested and when the joints were submitted to passive movements. These alterations were scored as shown in Table 1, i.e., 05normal, 15mild, 1.55moderate and 25 severe. A different scale was used to classify muscle tone and spinal cord reflexes: 05absence; 15severely decreased; 1.55mild or moderately decreased; 25normal; 2.55mild or moderately increased and 35severely increased. The data in the Figures presented as histograms are

Fig. 1. Alterations in postural reflexes (Group I). Effect of benznidazole on postural reflexes during acute treatment for 15 days (Group I). The height of the bars indicates the postural reflex score per dog when tested on the day shown on the abscissa. The number of surviving dogs is indicated above each bar. The daily dose of 30 or 10 mg / kg / day is indicated at the top of the figure. During treatment with 30 mg / kg / day from days 1 to 15, the dogs exhibited moderate alterations in postural reflexes on day 9. On day 12, these alterations became severe even though the dose was reduced from 30 to 10 mg / kg / day on day 16, and persisted in the surviving dogs. Five spontaneous deaths occurred on days 16, 22, 26 (two animals) and 29.

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Table 1 Scores and criteria for the evaluation of mental status, locomotion, postural reflexes and cranial nerve dysfunction Degree of alteration Normal50

Mild51.0

Moderate51.5

Severe52

Alert and bright. Mildly apathetic. Reacts to visual, auditory and tactile stimuli

Alert and bright. Moderately apathetic. Reacts to visual, auditory and tactile stimuli with slow movements

Alert and bright Totally apathetic. Does not react to visual, auditory and tacitle stimuli.

Asymmetrical gait, mild ‘en bloc’ hindlimb, and normal forelimb movements. Mild difficulty to ascend and descend steps. Normal extension of head during locomotion

Asymmetrical gait, moderate ‘en bloc’ hindlimb and mild / moderate alterations of forelimb movements. Does not ascend and descend steps. Extension of head normal during locomotion

Asymmetrical gait, severe ‘en bloc’ hindlimb movements, crosses forelimbs during locomotion, staggering, and walking in small circles with no defined direction, loss of balance, The head droops constantly.

No alterations in wheelbarrowing, hemistanding and hemiwalking, hopping, proprioception, extensor postural thrust, righting, placing and tonic neck reactions. Cranial nerve dysfunction

Mild postural reaction deficits

Moderate postural reaction deficits.

Severe postural reaction deficits.

No alterations in the cranial nevers

Mild deficits (Only 1 alteration).

Moderate deficits (up to 2 alterations).

Severe deficits (several alterations).

Mental status (level of consciousness) Alert and bright. Reacts to visual, auditory and tactile stimuli.

Locomotion Symmetrical gait, alternating movement. The subject runs, ascends and descends steps, and avoids objects. Maintains the head extended in a normal position during locomotion

Postural reflexes a

a

Postural reflexes: Wheelbarrowing (walking with the forelimbs); hemistanding and hemiwalking and hemiwalking (standing or walking with hind and forelimbs on one side only); hopping (supporting body weight on one limb); proprioception (recognizing the position of a flexed, extended, or abnormally positioned limb); extensor postural thrust (supporting itself against gravity); righting (maintaining a normal position in relation to gravity and during motion) and placing reactions (initiating contact with the dorsal surface of the distal extremities when stimulated.

reported as the sum of the scores for the animals in a given group divided by the number of animals in the group.

3. Results

3.1. Clinical-neurological findings The acute treatment of six normal dogs with benznidazole (30 mg / kg / day) (Group I) for 15 days triggered severe clinical-neurological alterations. The data in Fig. 1 show alterations in postural reflexes, reported as average scores for the degree of alteration per dog, which were first observed on day 9 and were moderate (average score51.4, see Methods). Thereafter, the alterations became progressively worse and reached scores of 1.8 to 2.0 which correspond to severe alteration even though the dose was reduced from 30 mg / kg / day to 10 mg / kg / day on day 16

of treatment. The other parameters for measuring clinicalneurological alterations such as locomotion, mental status, muscle tone, spinal cord reflexes, and cranial nerve dysfunctions, the scoring of which is described in Table 1, were similarly affected, i.e., moderate alterations on day 9 which deteriorated to ‘severe’ by days 23 to 30. Spontaneous deaths occurred on day 16, 21, 26 (two animals) and 29. Thus, the number of animals in this group was reduced from 6 at the beginning of treatment to 1 by day 30. A summary of the neurological alterations observed in the experiment described in Fig. 1 follows. The alterations in postural reflexes observed during acute treatment with high doses of benznidazole (30 mg / kg / day) for 15 days were serious. The animals (Group I) presented moderate oscillatory movements of the head and trunk after day 9. Equilibrium was unstable and the animals overcame obstacles with difficulty. On day 12, the

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alterations became severe, the animals could stand only if supported; when unaided they would take a step and fall, hitting the floor with the muzzle. They exhibited no control over the position of the head which remained laterally flexed; balance was completely unstable due to intense oscillatory movements of the head and trunk. On day 15 of treatment, two dogs exhibited vertical nystagmus with the slow component upward which persisted until death. After dose reduction to 10 mg / kg / day on day 16 of treatment, severe alterations persisted until 5 spontaneous deaths occurred in this group. One dog continued to exhibit intense psychomotor activity up to day 20 and presented changes similar to those observed in the other animals on the following day. This animal died on day 26 of treatment. The other dog presented intense hypermetria in all four legs until death on day 29. On day 9, the animals (Group I) exhibited moderate alterations in locomotion with asymmetrical gait, widened base and ‘en bloc’ movements of the hind- and forelimbs. The head was kept in a normal position. On day 12, the alterations in locomotion were severe. When aided to stand the animals would take a few steps; gait was asymmetrical and ‘en bloc’, with crossing of the forelimbs, staggering, and walking in small circles with no defined direction. One dog exhibited motor agitation, did not stand still, and rapidly explored the surroundings, with mild ‘en bloc’ gait of the hindlimbs. After reduction of the dose to 10 mg / kg / day on day 16, there was no improvement in the alterations; the animals remained lying down, the head resting on the forelimbs. When raised to a standing position, the subjects collapsed. The alterations in mental status observed in Group I animals were mild apathy and poor ability to explore the surroundings on day 9. The subjects responded poorly to auditory, tactile and visual stimuli, except for one dog that exhibited intense psychomotor activity, running around the room and exploring the surroundings in a very rapid manner. On day 12, the animals presented severe apathy and lack of interest in their surroundings. They did not react to auditory, tactile or visual stimuli but remained bright and alert until spontaneous death or sacrifice. With the reduction in dose to 10 mg / kg / day on day 16, the clinical picture remained unchanged, in the survivors during the subsequent two weeks. Severe hypertonia and hyperreflexia in the fore- and hindlimbs, as illustrated in Fig. 2A, Fig. 2B, were observed in Group I until spontaneous death or sacrifice, except for one dog that exhibited intense psychomotor activity and presented moderate and mild hypertonia and hyperreflexia in the fore- and hindlimbs, respectively. After day 20 this animal presented alterations similar to those of the other treated dogs. Cranial nerve dysfunctions such as trismus were observed in Group I animals until spontaneous death or sacrifice.

Fig. 2. In (A) on day 12 of treatment with 30 mg / kg / day, the animal exhibited severe hypertonia and hyperreflexia in the four limbs. When placed in a standing position (B), the dog could not support its weight.

The acute treatment with high doses (40 mg / kg / day) of benznidazole for 7 days followed by chronic treatment at lower doses (20 and 5 mg / kg / day) is represented by Group II (a,b). It was surprising to observe that benznidazole at a dose of 30 mg / kg / day for 15 days was lethal to 5 of the 6 dogs (Group I) that received 10 mg / kg / day from days 16 to 30. Patients routinely receive 5 to 10 mg / kg / day which is not lethal but does cause peripheral polyneuropathy, the frequency of which is dose related. In the second series of experiments, shown in Fig. 3A, Fig. 3B, two dogs (Subgroup IIa) were treated with 40 mg / kg / day for 7 days and the dose reduced to 20 mg / kg / day on days 8 to 60, and to 5 mg / kg / day on day 61 (Fig. 3A). None of these animals died. After 7 days of treatment with a dose of 40 mg / kg / day, the animals (Subgroup IIa) exhibited severe clinical-neurological alterations. These alterations were similar to those in Group I which received 30 mg / kg / day for 15 days (Fig. 1). With the reduction in dose to 20 mg / kg / day on day 8,

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Fig. 3. Alterations in locomotion in A (Subgroup IIa) and B (Subgroup IIb). Effect of benznidazole on locomotion during acute treatment followed by chronic treatment. The data are presented as indicated in the legend to Fig. 1, except that the data in Panel A are for 2 dogs and in Panel B are for 3 dogs, all of which survived the entire experimental period. In A, the dogs (Subgroup IIa) also treated with high doses (40 mg / kg / day) of benznidazole for 7 days exhibited the same neurological alterations as those observed in Group I on days 12 to 30. When the dose was reduced from 40 to 20 mg / kg / day on day 8, none of the animals died. On days 15, 22 and 35, the alterations in locomotion were moderate and became severe on day 45. With dose reduction to 5 mg / kg / day, the changes again became moderate and persisted until sacrifice on days 125 and 382, respectively. In B, the dogs (Subgroup IIb) receiving 20 mg / kg / day from the beginning of the treatment exhibited the first alterations in locomotion only on day 20. These alterations were moderate and became severe on day 30. With dose reduction to 5 mg / kg / day on day 31, the changes became mild, moderate and severe on days 45, 60 and 110, and persisting until sacrifice on days 105, 176 and 185, respectively.

these neurological alterations were reversed to a moderate degree. In contrast, three animals (Subgroup IIb) receiving 20 mg / kg / day from the beginning of treatment presented the first neurological alterations only around day 20 (Fig. 3B) although their clinical picture was similar to that of Group IIa which received 20 mg / kg / day from days 8 to 60. The clinical-neurological alterations observed during the acute and chronic treatments in Subgroups IIa and IIb, respectively, are described below. The alterations in postural reflexes observed in the animals that received 40 mg / kg / day for 7 days (Subgroup IIa) were similar to those seen in Group I on day 12. After reduction of the dose to 20 mg / kg / day on day 8, these changes became moderate; the severe clinical picture was

reversed by day 15 and no deaths occurred. This alteration became severe again between days 40 and 50 of treatment. When the dose was reduced to 5 mg / kg / day, the severe alterations became moderate on day 70 and remained so until the animals were sacrificed on days 125 and 382. After 25 days of treatment with 20 mg / kg / day, the animals (Subgroup IIb) exhibited postural reflex alterations similar to those observed in Subgroup IIa during treatment with the same dose from days 8 to 60. The alterations in locomotion observed during the first week of treatment with 40 mg / kg / day (Subgroup IIa) were similar to those in Group I from days 12 to 30. When the dose was reduced to 20 and then to 5 mg / kg / day, the gait remained asymmetrical and ‘en bloc’ in the hindlimbs. The intensity of alterations was mild on day 45, became

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moderate on day 60 and remained so until the end of treatment (Fig. 3A). The alterations in locomotion observed in Subgroup IIb on day 20 were similar to those seen in Subgroup IIa in which the animals were given the same dose of benznidazole, i.e., 20 mg / kg / day (Fig. 3B). Fig. 4 illustrates normal, symmetrical gait (Fig. 4A), asymmetrical gait (Fig. 4B), unstable balance (Fig. 4C) and the effect of blindfolding on equilibrium (Fig. 4D). The alterations in mental status observed during the 7 days after 40 mg / kg / day in Subgroup IIa were similar to those observed between days 12 and 30 in Group I which received 10 mg / kg / day. When the dose was reduced to 20 mg / kg / day, the intensity of symptoms became moderate, returning to severe levels by day 45. After reduction to 5 mg / kg / day, the alterations disappeared completely; they became moderately intense by day 110 and remained so until the animals were sacrificed. In Subgroup IIb, the alterations in mental status were similar to those in Subgroup IIa in which the animals were submitted to 20 and 5 mg / kg / day, respectively. Severe hypertonia and hyperreflexia were observed in Subgroup IIa on day 7. With the reduction in dose to 20

and 5 mg / kg / day, the animals were able to stand although moderate alterations in muscle tone and spinal cord reflexes persisted until the end of treatment. In Subgroup IIb, moderate hypertonia and hyperreflexia were observed on day 20. These alterations became severe on day 30 and moderate by day 45 after reduction of the dose to 5 mg / kg / day on day 31. In this subgroup, the alterations returned to the severe level of intensity on day 110. Two animals in Subgroup IIa presented trismus during treatment with 40 mg / kg / day and hyperalgesia of the distal joints of the hindlimbs. These alterations disappeared after dose reduction.

3.2. Clinical-neurological alterations after drug discontinuation One week after drug discontinuation, one dog from each of Subgroups IIa and IIb which received acute and chronic treatment (5 mg / kg / day from days 61 to 104 and 61 to 342, respectively), exhibited normal mental status during the first week. On day 15, the dogs showed moderate alterations in locomotion, postural reflexes, muscle tone

Fig. 4. In (A), the normal dog exhibited symmetrical gait with alternate movements. During locomotion, the animal maintains the head extended in a normal position. In (B), on day 30 with 20 mg / kg / day, the animal exhibited moderate apathy and an asymmetrical gait with crossing of the forelimbs and unstable balance. In (C) on day 7 after acute treatment with high drug doses (40 mg / kg / day), the dog exhibited unstable balance, crossing of the four limbs, staggering, and walking in small circles. When the dog was blindfolded (D), the base of the four limbs became widened and the trunk arched to maintain balance.

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Fig. 5. In (A), on day 342 of treatment with 5 mg / kg / day, the animal exhibited paraparesis of the hindlimbs. After drug withdrawal in (B) the dog showed a mildly widened limb base when standing and stationary, and ‘en bloc’ gait in the hindlimbs.

and spinal cord reflexes. The alterations in locomotion and postural reflexes alteration were mild on day 30 and persisted until sacrifice. This reduction in intensity was not observed in muscle tone or spinal cord reflexes which were moderate until the end of treatment. Fig. 5 shows the dog which received 5 mg / kg / day from days 61 to 342 (Fig. 5A), and 30 days after the drug was discontinued (Fig. 5B). These data demonstrate the apparent reversibility of the neurotoxic effects of the drug, when measured in terms of mental status, postural reflexes, locomotion, muscle tone and spinal cord reflexes.

4. Discussion The most important result of the present study is that the neurotoxic effect of benznidazole on dogs is manifested on the central nervous system. This contrasts with the peripheral effects reported for chagasic patients who receive similar doses ranging from 5 to 10 mg / kg / day for

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periods of 30 to 90 days. The clinical-neurological findings obtained in the present study clearly show that the toxic effects of benznidazole on the CNS are dose dependent and partially time dependent. A second important observation is that the administration of 30 mg / kg / day of benznidazole (3 times the dose used for chagasic patients) for 15 days is extremely toxic and lethal to dogs. The administration of 40, 30 and 20 mg / kg / day of benznidazole to normal dogs consistently triggered the following neurological alterations: apathy, hypertonia, hyperreflexia, ataxia, loss of balance (involuntary oscillatory movements of the trunk and head) and asymmetrical gait. These alterations became manifest after one week of treatment with 40 mg / kg / day, after 12 days of treatment with 30 mg / kg / day, and about 30 days after treatment with 20 mg / kg / day. When these doses were reduced to 5 mg / kg / day improvement was seen accompanied later by reduction in the intensity of the neurological alterations; however moderate or severe hypertonia and exaggerated hyperreflexia persisted. The continuous use of 5 mg / kg / day was associated with the return of neurological alterations approximately 30 days after dose reduction. The neurological disorders were not manifested with the same intensity seen during the use of higher doses such as 30 and 40 mg / kg / day. After the drug was discontinued, there was a significant reduction, but not complete reversal, of neurological alterations such as mild hypertonia, hyperreflexia, loss of balance, ataxia and ‘en bloc’ gait which persisted until sacrifice 40 and 135 days after withdrawal of the drug. ¨ Scharer (1972), in a study of 4 dogs receiving 50 mg / kg / day of benznidazole for periods of 3 to 20 days, reported strong contractions of the back and thigh muscles, with opisthotonos and upright elevation of the tail which gradually evolved to a posture of sustained contraction. Death occurred on average a little less than one week after the contractures began. Significant ataxia set in starting on day 4 after drug withdrawal, although the dogs became normal two to three weeks later. Three dogs which received 100 mg / kg / day for 11 days presented contractures on day 9 and died between days 12 and 13 after the beginning of treatment. In the present study, when the neurological signs appeared, the animals always showed a tendency to present head flexion rather than opisthotonos. In the later phase, flexion contracture of the paravertebral musculature was present and was always much less severe than the limb contractures. Two animals exhibited vertical nystagmus with the slow component upward. No preferential tail posture was observed. Even when food was placed in its mouth, the animal had great difficulty in chewing and later was completely unable to chew due to trismus. An anorectic component was present since the dogs showed little interest in meals after the onset of the neurological disorders, leading to marked weight loss. Doses close to

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the limit of the daily toxic dose administered to mice, guinea pigs, rabbits (40 mg / kg / day) and rats (1000 mg / kg / day) did not cause the damage to the Purkinje cells ¨ previously reported in dogs (Scharer, 1972). Thus, it is clear that the sensitivity of certain systems to benznidazole toxicity is species dependent, suggesting that even doses significantly lower than 50 mg / kg / day can produce neurotoxic effects on the CNS of dogs and may be lethal. The differences between our observations and those of ¨ Scharer (1972) may be due to the fact that we used lower doses and followed more dogs for longer periods of time. Another factor may be that we employed a more systematic approach to evaluate the neurological effects of benznidazole on our animals. Toxic effects have been reported with the use of benznidazole in male Wistar rats that received doses of 80 mg / kg / day for 30 days. These animals exhibited severe testicular atrophy, arrest of spermatogenesis (Vieira et al., 1989), elevation of plasma FSH levels (Favaretto et al., 1990), and a marked reduction in the spermatogenic process. The data also suggest impairment of Sertoli cell secretory activity during chronic treatment and after drug discontinuation (Lamano-Carvalho et al., 1990). No changes in motility, sensitivity or in behavior, or pathological changes of the peripheral nervous system, were observed in these rats (Flores-Vieira and Barreira, unpublished data). Stevenson et al. (1989), studying the effects of the nitroimidazole compound, misonidazole, on the neurons of rats and mice in vitro, reported interruption of the neurofilament network, and dendritic retraction when low drug concentrations were used. With increasing concentrations, the toxic effects became more marked within a shorter period of time. These investigators concluded that the drug triggered dose- and time-dependent neurotoxicity. Our experimental model also exhibits dose- and time dependence, although, considering that the experiments were carried out in vivo, many other factors were involved in addition to those studied by Stevenson et al. (1989). The alterations observed in vitro were triggered by the direct effect of the nitroimidazole compound on the nerve cells. In the present model, we do not know whether the lesions are triggered by the drug or by its metabolites, or whether interference by systemic factors takes place, resulting in the occurrence of neurological disorders. Further studies on the qualitative and quantitative measurement of drug metabolites and their detection in the cerebrospinal fluid and brain tissue, as well as the quantification of changes in the affected cells at the fine structural level, may reveal the mechanisms underlying the lesions in the experimental model studied here. In conclusion, the results obtained with dogs suggest that a careful review of the side effects of the drug in man is necessary. Since subclinical or undetected central effects may occur in chagasic patients treated with benznidazole, a complete neurological evaluation is recommended.

Acknowledgments ˆ We thank Dr Lewis Joel Greene and Dr Irvenia Luiza de Santis Prada for helpful comments and discussion during this study. We are also indebted to Mrs. Maria Cristina ˆ Lopes, Mr. Antonio Ferreira, and Mr. Welder Tambelini for technical assistance, Mrs Maria da Penha and Mrs. Rosemeire Narosny Ribeiro for photography, and Mr. Luis ´ Flavio Degani for the videotaping work. The study was supported by CNPq, FAPESP and FAEPA.

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