Pirlindole and dehydropirlindole protect rat cultured neuronal cells against oxidative stress-induced cell death through a mechanism unrelated to MAO-A inhibition

Abstract

It has been shown that the MAO (monoamine oxidase)-B inhibitor deprenyl (DPR, selegiline) protects some cell types against oxidative stress. By decreasing H(2)O(2) production, MAO-A inhibitors could also reduce oxidative stress. This study reports the effect of the MAO-A inhibitors, pirlindole (PIR), dehydropirlindole (DHP), brofaromine (BRO) and moclobemide (MCL) on primary-cultured brain cells exposed to iron-mediated toxicity. A comparison with trolox (TRO), a hydrosoluble vitamin-E analogue that protects against such an induced stress, was performed. Rat hippocampal or cortical cultured cells were exposed either to 2 microM FeSO(4) alone or in the presence of PIR, DHP, BRO, DPR, MCL or TRO. Cell survival (lactate-dehydrogenase measurements, 16 h incubation), intracellular peroxide production (DCF-fluorescence, 1 h incubation), lipoperoxidation (TBARS-fluorescence, 6 h incubation) and mitochondrial function (MTT-test, 16 h incubation) were assessed. PIR, DHP and TRO significantly protected cultures (P<0.05) against Fe(2+)-induced toxicity in a concentration-dependent manner. The EC(50s) of these compounds were 6, 12 and 19 microM, respectively, in hippocampal cells. For cortical cell cultures incubated in the presence of iron and PIR or DHP, EC(50s) were 5 and 6 microM respectively. All Hill coefficients were close to unity. BRO, MCL and DPR were not protective in any type of culture. The IC(50s) for the inhibition of MAO-A were 2, 2 and 0.2 microM for PIR, DHP and BRO, respectively. PIR, DHP and TRO, but not DPR, induced a significant decrease in both intracellular peroxide production and lipoperoxidation. They also improved mitochondrial function. These experiments show that PIR and DHP can protect hippocampal and cortical neurons against oxidative stress at pharmacologically relevant concentrations. This protective effect seems unrelated to inhibition of MAO-A, but possibly involves free radical scavenging.

Figure 1

(a) Determination of the protective effect of pirlindole, dehydropirlindole, brofaromine and trolox. Hippocampal cells were exposed to various concentrations of drug during 16 h in the presence of 2 μM FeSO₄. Cell death was then assessed by the LDH assay. Statistical analysis: ANOVA analysis and Dunn's post hoc tests, ^*P<0.05 compared to iron-treated cells, n=10 – 51. (b) Determination of the toxicity of pirlindole and dehydropirlindole. Hippocampal cells were exposed to pirlindole or dehydropirlindole during 16 h. Cell death was then estimated. Statistical analysis: ANOVA test, ^*P<0.05 compared to control cells (0 μM), n=4 – 18. (c) Determination of the protective effect of pirlindole, dehydropirlindole and brofaromine on cortical cells. Cortical cells were treated as in a. Statistical analysis: ANOVA analysis and Dunn's post hoc tests, ^*P<0.05 compared to iron-treated cells, n=8 – 30.

Figure 2

Measurement of the inhibition of MAO-A activity by pirlindole, dehydropirlindole or brofaromine. Cultured cortical cells were exposed during 3 h to various concentrations of MAO-A inhibitors. MAO activity was then measured. n⩾3 except for 10 and 50 μM PIR and 10 μM DHP (n=2).

Figure 3

Structural formulas of pirlindole, dehydropirlindole and trolox.