1-amino-4-phenyl-1,2,3,6-tetrahydropyridine and 1-amino-4-phenylpyridinium salt, the 1-amino analogues of neurotoxins, MPTP and MPP+, induce apoptosis in PC12 cells: detection of apoptotic cells by Comet assay and flow cytometric analysis.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is known to induce parkinsonism in humans when it is oxidized to the 1-methyl-4-phenylpyridinium salt (MPP+). We previously reported the syntheses of 1-amino-4-phenyl-1,2,3,6-tetrahydropyridine (APTP) and 1-amino-4-phenyl-pyridinium salt (APP+), the 1-amino analogues of the dopaminergic neurotoxins, MPTP and MPP+, respectively, and demonstrated that both APTP and APP+ are cytotoxic to PC12 cells. In this study, we found that both APTP and APP+ induce apoptotic cell death in PC12 cells. Apoptosis was determined by the Comet assay and flow cytometric analysis. Prior to using the Comet assay for detection of apoptotic PC12 cells, Comet images of apoptotic and necrotic cells were first distinguished by using several standards. Comet images were classified into four groups (A to D) according to their shapes. Class D consisted of the apoptotic cells and was easily distinguished. We also demonstrated that apoptotic and necrotic PC12 cells can be easily differentiated and quantified using the convenient Comet assay.