Overexpression of CYP2D6 attenuates the toxicity of MPP+ in actively dividing and differentiated PC12 cells.

Clonal pheochromocytoma cell lines overexpressing cytochrome P450 2D6 (CYP2D6) were established. CYP2D6 was localized in the endoplasmic reticulum, and its enzymatic activity in the microsomal fraction was confirmed by using high performance liquid chromatography analysis with [guanidine-14C]debrisoquine as a substrate. Overexpression of CYP2D6 protected both actively dividing and differentiated cells against the toxic effects of 1-methyl-4-phenylpyridinium ion at the concentration range of 20-40 microM, as assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The production of reactive oxygen species in the mitochondria was suppressed. The cytotoxicity of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine was unchanged in both actively dividing and differentiated cells overexpressing CYP2D6 versus mock-transfected controls at concentrations up to 500 microM. These results suggest that the lowered enzyme activity of CYP2D6 in individuals termed "poor metabolizers" may represent a risk factor from exposure to select neurotoxicants.

Generation of reactive oxygen species and activation of NF-kappaB by non-Abeta component of Alzheimer's disease amyloid.

Non-amyloid beta (Abeta) component of Alzheimer's disease (AD) amyloid (NAC) coexists with Abeta protein in senile plaques. After exposure to NAC fibrils, cortical neurons of rat brain primary culture became apoptotic, while astrocytes were activated with extension of their processes. NAC fibrils decreased the activity of reducing 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) in cortical neurons more markedly (IC(50) = 5.6 microm) than in astrocytes (IC(50) approximately 50 microm). The neuron-specific toxicity of NAC fibrils was indicated also by an increased release of lactate dehydrogenase from the cells. Neuronal apoptosis was suppressed by pre-treatment with the antioxidants, propyl gallate (PG) and N-t-butyl-phenylnitrone (BPN), or overexpression of human Bcl-2. Exposure to NAC fibrils enhanced generation of reactive oxygen species (ROS) in neurons and less efficiently in astrocytes, as demonstrated by oxidation of 2',7'-dichlorofluorescin. The site of ROS generation was shown to be mitochondria by oxidation of chloromethyl-tetramethyl rosamine. Exposure to NAC fibrils increased also the nuclear translocation of nuclear factor kappa B (NF-kappaB) and enhanced its DNA-binding activity, which was inhibited by PG and BPN more efficiently in neurons than in astrocytes. These results suggest that NAC fibrils increase mitochondrial ROS generation and activate NF-kappaB, thereby causing a differential change in gene expression between neurons and astrocytes in the AD brain.