Results

Toxicity of Anonaceae for dopaminergic neurons in culture

We extracted the most abundant alkaloids (reticuline and coreximine) and the main acetogenin (annonacin) from A. muricata and tested their neurotoxicity in primary neuronal cultures. After a 24 hours treatment, 50% of the dopaminergic neurons degenerated with 13 mM coreximine and 304 mM reticuline. Annonacin was much more potent than the alkaloids, with an effective concentration [EC50] of 18 nM. Annonacin was approximately 50-fold more potent than the classic complex I inhibitor MPP+ and 2-fold

Table 1. Comparison of the toxic effects of annonacin, rotenone and MPP+ for TH+ and MAP-2+ neuronal cells in mesencephalic cultures

Toxins

TH+ cells

MAP-2+ cells

EC50 (mM)

EC50 (mM)

Annonacin

0.018 ± 0.004

0.027 ± 0.005

Rotenone

0.034 ± 0.009

0.041 ± 0.011

MPP+

1.9 ± 0.3

72 ± 11

The cultures were exposed to each toxin for 24 h and then processed for TH or MAP-2 immunocytochemistry more potent than rotenone in inducing neuronal death (Table 1). Unlike MPP+ but similar to rotenone, cell death induced by alkaloids or annonacin did not require toxin uptake by the dopamine transporter and, consequently, was not restricted to dopaminergic cells. Raising glucose concentrations largely restored intracellular ATP levels and prevented neuronal demise. Thus, alkaloids, but more so annonacin, extracted from A. muricata promote dopaminergic neuronal death by impairing energy production.

Annonacin, induces nigral and striatal neurodegeneration in rats

Because of its toxicity in vitro, we asked whether chronic systemic exposure of a living animal to annonacin would result in neurodegeneration similar to that observed in atypical parkinsonian patients. We therefore, infused annonacin intravenously (3.8 and 7.6 mg per kg per day for 28 days) in rats and demonstrated that annonacin can cross the blood brain barrier intact, since unmeta-bolized annonacin was detected in the brain

Control

Annonacin

Fig. 1. Histological alterations visualized by Bodian silver impregnation in the striatum of annonacin (7.6 mg/kg/day)-treated rats. Enlarged and dystrophic nuclei (A) and dystrophic fibres (B) can be observed in annonacin-treated animal but not in control animal. Scale bar: 10 mm

parenchyma, and decreased brain ATP levels by 44%. Annonacin induced morphological alterations and a dose-dependent loss of nigral dopaminergic neurons and striatal dopa-minergic fibers (Fig. 1). Neurodegeneration was not restricted to nigrostriatal neurons but was pronounced and widespread in basal ganglia and brainstem nuclei, whereas hippocampus, cerebellum, and cerebral cortex were only moderately affected. The distribution of the lesions, including nigral and striatal neuronal cell loss, was similar to that of patients with atypical parkinsonism, reinforcing the hypothesis that systemic exposure to complex I inhibitors in fruit and extracts of Annonaceae can induce atypical parkinsonism.

Quantification of acetogenins in A. muricata

To quantify the exposure to acetogenins through consumption of A. muricata, we determined the concentrations of annonacin in extracts and preparations of fruit and leaves by matrix-assisted laser desorption-ionization mass spectrometry. An average fruit was estimated to contain about 15 mg of annonacin, a can of commercial nectar 36 mg and a cup of infusion or decoction 140 mg. As an indication of its potential toxi-city, an adult who consumes one fruit or can of nectar a day is estimated to ingest, over one year, the amount of annonacin that induced brain lesions in rats receiving purified annonacin by intravenous infusion.

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