Manganese inhibits mitochondrial aconitase: a mechanism of manganese neurotoxicity

Abstract

The symptoms of Mn-induced neurotoxicity resemble those of Parkinson's diseases. Since iron (Fe) appears to play a pivotal role in pathophysiology of Parkinson's disease, we set out to test the hypothesis that alterations in Fe-requiring enzymes such as aconitase contribute to Mn-induced neurotoxicity. Mitochondrial fractions prepared from rat brain were preincubated with MnCl2 in vitro, followed by the enzyme assay. Mn treatment significantly inhibited mitochondrial aconitase activity (24% inhibition at 625 microM to 81% at 2.5 mM, p<0.05). The inhibitory effect was reversible and Mn-concentration dependent, and was reversed by the addition of Fe (0.05-1 mM) to the reaction mixture. In an in vivo chronic Mn exposure model, rats received intraperitoneal injection of 6 mg/kg Mn as MnCl2 once daily for 30 consecutive days. Mn exposure led to a region-specific alteration in total aconitase (i.e. , mitochondrial+cytoplasmic): 48.5% reduction of the enzyme activity in frontal cortex (p<0.01), 33.7% in striatum (p<0.0963), and 20.6% in substantia nigra (p<0.139). Chronic Mn exposure increased Mn concentrations in serum, CSF, and brain tissues. The elevation of Mn in all selected brain regions (range between 3.1 and 3.9 fold) was similar in magnitude to that in CSF (3.1 fold) rather than serum (6. 1 fold). The present results suggest that Mn alters brain aconitase activity, which may lead to the disruption of mitochondrial energy production and cellular Fe metabolism in the brain.

Fig. 1

Inhibition by Mn of aconitase activity in mitochondrial fractions prepared from rat brain. Mitochondrial fractions (25 μg) were pretreated with various concentrations of Mn for 10 min followed by Fe (50 μM) activation for another 10 min. The rate of formation of aconitase from L-citrate (1.0 mM) was monitored at 240 nm. Values represent means ± S.D. (n = 3).

Fig. 2

Reversal of the Mn-induced inhibition of aconitase by Fe. Mitochondrial fractions (25 μg) were pretreated with or without Mn (2.5 mM) for 10 min followed by Fe activation for 5 min. The rate of formation of aconitase from L-citrate (1.0 mM) was monitored at 240 nm. Values represent means ± S.D. (n = 4).

Fig. 3

Reversal of the Mn-induced inhibition of aconitase by L-citrate. Purified enzyme (0.2 mg) was pretreated with 2.5 mM Mn followed by Fe (50 μM) activation for 10 min. The rate (V, nmol/mg/min) of formation of aconitase from L-citrate (S, mM) was monitored at 240 nm. Each point represents mean of three separate experiments.

Fig. 4

Effect of chronic exposure to Mn on total aconitase (ACO1 + ACO2) activities in selected brain regions. Rats received i.p. injections of 6 mg/kg Mn as MnCl₂ once daily for 30 days. The whole tissue homogenates (20–30 μg proteins) were used for assay of aconitase activity at day 31. Data represent mean ± S.E. (n = 4–6). * *: p < 0.01, †: p = 0.096.