The notion that levodopa may be toxic to dopaminergic neurons leading to more rapid nigral degeneration has been a controversy for 25 years. It is based on a body of evidence that suggests that oxyradicals play an important role in the pathogenesis of cell death in PD (87). Evidence includes decreased glutathione, increased Fe2+, increased malondialdehyde, and decreased mitochondrial complex I activity in the substantia nigra (SN) (77). These changes appear to lead to apoptotic mechanisms of cell death (88). Dopamine, when metabolized by monoamine oxidase (MAO) or auto-oxidized, forms H2O2, a precursor to the toxic hydroxyl radical. In PD, after loss of a substantial number of nigral cells, those surviving neurons increase their dopamine metabolism, thus possibly increasing the risk of further free radical formation and neurodegeneration, especially in an environment where protective mechanisms such as glutathione are diminished and iron has accumulated. The use of levodopa may lead to an increase in dopamine formation and, in turn, an increase in dopamine metabolism with greater free radical formation (89,90). Although this theory has gained appeal, and although laboratory evidence supports this possibility, the theory remains controversial (90,91). However, detailed reviews (77,92-94) have indicated that there is no convincing evidence to suggest that levodopa is toxic.
The evaluations for levodopa toxicity have included both in vitro (cell culture) and in vivo studies in animals. In the cell culture studies, various cell types were used, including fetal mesencephalic cells, neuroblastoma, fetal fibroblasts, pheochromocytoma PC12 cells, chick sympathetic neurons, and others (95). Results of these studies were variable because of the levodopa concentrations used and culture conditions. High doses of levodopa are toxic to dopaminergic neurons in pure neuronal cultures. Mechanisms of toxicity include oxyradicals, mitochondrial toxic-ity, or apoptosis (96-98). However, as the conditions are set to more accurately reflect in vivo systems, the toxicity disappears and the neurons are able to resist injury. In fact, with exposure to previously toxic medium doses (20-100 |jm) and with glial cells and ascorbic acid present, levodopa actually has a trophic influence increasing cell survival and enhancing neurite outgrowth (77,99-101). The glial cells contain the protective enzymes, catalase and glutathione peroxidase, and provide a nutritive and protective environment. Levodopa exposure to these cultures actually increases cellular concentrations of reduced glutathione peroxidase and other anti-apoptotic molecules and may have other neurotrophic properties. This effect of levodopa may be due to the development of a low-level injury that activates protective mechanisms. Hence, both toxicity and protection may occur due to free radical development, the difference relating to the magnitude of that injury. At levels that are likely present in the extracellular fluid in the striatum of patients, as measured in animals by microdialysis (picomolar levels), it is unlikely that levodopa has any effect (92).
In vivo studies have included both unlesioned and lesioned animals. Several studies involved giving healthy animals levodopa for up to 18 months and they demonstrated no loss of dopaminergic neurons (102-106). Cotzias et al. (107) reported that mice that were given levodopa lived longer than the controls that were not given levodopa. Fahn (95) reviewed more than 15 studies of in vivo effects of levodopa and dopamine. Blunt et al. (108) lesioned rats with 6-OHDA, gave levodopa to some, and counted tyrosine hydroxylase (TH)-stained cells in the SN and ventral tegmental area (VTA). The unlesioned (healthy) side was unaffected by the levodopa, supporting the prior studies. The SN on the lesioned side lost 96% of its cells.
The VTA was less affected with 23% to 65% of the cells remaining. Levodopa further reduced surviving cell numbers to 10% to 35%. They concluded that either levodopa suppressed TH activity or caused increased cell death; however, this work has been criticized (109) and they were later unable to duplicate these findings.
Fukuda et al. (110) used MPTP-lesioned mice and examined the effect of levodopa and bromocriptine on total and TH+ cell counts. Levodopa further reduced cell counts in MPTP-treated mice, but it was of TH- cells; TH+ cells were unaffected. Bromocriptine had no effect and, interestingly enough, combined levodopa and bromocriptine actually resulted in a significant increase in surviving cells. Murer et al. (109) examined the effects of levodopa on nigrostriatal and VTA cells in rats with moderate and severe 6-OHDA lesions and sham-lesioned animals. Treatment was for six months, and the same experiment was performed at two separate times in two independent groups of rats. They measured three dopaminergic markers—TH, DAT, and vesicular monoamine transporter (VMAT2)—via radio-immunohistochemistry in the SN, VTA, and striatum. They also examined rotational behavior to assess pharmacologically relevant doses and postsynaptic receptor binding. The study failed to demonstrate any significant difference of effect on cell counts in SN and VTA in levodopa-treated animals compared to those treated with vehicle using all three markers. There was a trend toward increased TH staining in the SN of the moderately lesioned animals. At the level of the striatum, there was no effect of levodopa treatment in the sham-lesioned and severely lesioned animals; however, in the moderately lesioned animals, there was partial recovery of nerve terminals in the damaged area, suggesting a possible neurotrophic effect. The increased immunostaining in this region was significant compared to those rats treated with vehicle. It was suggested that this increased striatal activity with levodopa related to partial recovery via axonal sprouting by the remaining neurons. Levodopa also tended to reverse increased binding (upregulation) of dopamine receptors and diminished the development of behavioral supersensitivity, indicating that the doses of levodopa utilized were pharmacologically effective. These results indicate that levodopa was not toxic to neurons or their terminals in normal and moderately or severely lesioned animals. It may instead promote compensatory mechanisms at the terminals, and thus recovery of innervation of the striatum.
Datla et al. (111) demonstrated similar findings in rats with 6-OHDA and FeCl3 lesions; levodopa had no short-term or long-term effects on the number of TH+ cells. In contrast, in the 6-OHDA model, there may have been a protective effect since there was an increase in TH+ cells after 24 weeks. Although results of these animal studies appear to be conflicting, the latter studies appear to provide evidence that lev-odopa is not toxic.
Human studies have not supported the levodopa toxicity hypothesis. Quinn et al. (112) reported a non-PD patient who received high-dose levodopa for four years. Autopsy results demonstrated a normal SN. Rajput et al. (113,114) reported six patients with similar results. Three patients had essential tremor, two had dopa-responsive dystonia, and one had nonprogressive parkinsonism. Autopsies in two patients were normal. None of the essential tremor patients developed parkinsonism and the others showed no progression of disease clinically. This would indicate that levodopa is not detrimental to patients with normal or dysfunctional SN. Yahr et al.
(115) compared postmortem results in patients treated and never treated with lev-odopa and reported no difference in the pathology of the SNpc. Gwinn-Hardy et al.
(116) examined the effect of levodopa on a family of autosomal-dominant levodopa-responsive parkinsonism (PARK 4—a-synuclein triplication). There were 12 affected individuals, and survival duration and disease progression were compared in those treated and not treated. Survival was significantly different between the two groups, as was progression of disease, both in favor of levodopa therapy. These findings would indicate a neuroprotective effect of levodopa, not neurotoxicity.
Finally, the CALM-PD and REAL-PET studies utilized SPECT and PET imaging to compare progression of PD with an agonist versus levodopa therapy (49,51,53). The reduction in binding was less over several years for the agonists than for levodopa. This may be an indicator that levodopa is toxic or that the agonist is neuroprotective or it may simply reflect a differential pharmacological effect on the dopamine system measured by the imaging techniques. In the ELLDOPA study (59), the clinical data demonstrated less worsening of disease after levodopa washout in a dose-response fashion compared to placebo, suggesting neuroprotection. The SPECT data indicated that the reduction in B-CIT binding was greater with lev-odopa, suggesting neurotoxicity. Finally, if in fact the drug has a pharmacological effect on DATs, then the imaging results are invalid. If the clinical effect is due to a prolonged pharmacological effect of the drug, then the clinical data is invalid. At this point, the interpretation of these results remains elusive.
When one looks at the data from cell culture, animals, and humans, there is no convincing evidence that levodopa is toxic. This should not be a concern when considering therapy in PD patients.
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