Formation of dopamine adducts derived from brain polyunsaturated fatty acids: mechanism for Parkinson disease

Abstract

Oxidative stress appears to be directly involved in the pathogenesis of the neurodegeneration of dopaminergic systems in Parkinson disease. In this study, we formed four dopamine modification adducts derived from docosahexaenoic acid (C22:6/omega-3) and arachidonic acid (C18:4/omega-6), which are known as the major polyunsaturated fatty acids in the brain. Upon incubation of dopamine with fatty acid hydroperoxides and an in vivo experiment using rat brain tissue, all four dopamine adducts were detected. Furthermore, hexanoyl dopamine (HED), an arachidonic acid-derived adduct, caused severe cytotoxicity in human dopaminergic neuroblastoma SH-SY5Y cells, whereas the other adducts were only slightly affected. The HED-induced cell death was found to include apoptosis, which also seems to be mediated by reactive oxygen species generation and mitochondrial abnormality. Additionally, the experiments using monoamine transporter inhibitor and mouse embryonic fibroblast NIH-3T3 cells that lack the monoamine transporter indicate that the HED-induced cytotoxicity might specially occur in the neuronal cells. These data suggest that the formation of the docosahexaenoic acid- and arachidonic acid-derived dopamine adducts in vitro and in vivo, and HED, the arachidonic acid-derived dopamine modification adduct, which caused selective cytotoxicity of neuronal cells, may indicate a novel mechanism responsible for the pathogenesis in Parkinson disease.

Figures

FIGURE 1.

Proposed chemical formation scheme and HPLC-MS/MS analysis of DHA- and AA-derived dopamine adducts.A, proposed reaction scheme of DHA- and AA-derived dopamine adduct formation. B, the [MH]+ ionm/z 254, 210, 252, and 268 of SUD, PRD, HED, and GLD, respectively, were subjected to collision-induced dissociation, and the daughter ions were scanned (upper left panel). The proposed structures of individual ions are shown (upper right panel). The chemical structure composition of the dopamine adducts is proposed by fragmental analysis (lower panel).

FIGURE 2.

HPLC-MS/MS analysis of the dopamine adducts formed during the reaction of dopamine with oxidized DHA and AA hydroperoxides. Dopamine (2 mm) was incubated with lipid hydroperoxides (10 mm) in 0.1 m phosphate buffer (pH 7.4) at 37 °C. Shown is selected ion monitoring of the transitions from m/z 254 (A), 210 (B), 252 (C), and 268 (D) to m/z137 for SUD, PRD, HED, and GLD, respectively. Top panels, authentic dopamine adduct; bottom panels, reaction mixture of DHA- or AA hydroperoxides with dopamine.

FIGURE 3.

Formation of the dopamine adducts in vivo. The levels of dopamine adduct formed in rat brain were determined by HPLC-MS/MS (data are shown as the means ± S.D. (n = 5)).

FIGURE 4.

Identification of HED as a potent inducer of cell death in SH-SY5Y cells. The cells were exposed to 100 μm sample for 24 h. Cell viability was measured by the MTT assay. In the MTT assay, the data are expressed as percentages of control culture conditions. A, potential comparison of cell death induction by DHA- and AA-derived dopamine adducts.B, effect of carbon numbers in C terminus in the structure of dopamine-derived adducts to cell viability in the cells (data are shown as the means ± S.D. (n = 3); ***, indicates p< 0.001).

FIGURE 5.

Apoptosis induced by HED.A, dose- and time-dependent cytotoxicity of HED. SH-SY5Y cells were exposed to 0–100 μm HED for different retention times. Cell viability was measured by the MTT assay. B, chromatin condensation in SH-SY5Y cells exposed to 10 μm HED. The cells were fixed with paraformaldehyde, stained with Hoechst 33258, and examined by fluorescence microscopy. Upper left panel, control (Cont) cells staining.Upper right panel, HED-treated cells staining. Lower graph, statistical analysis of apoptotic cells. C, DNA fragmentation in SH-SY5Y cells exposed to 25 μm HED or other samples for 12 h. Nucleosomal DNA fragmentation was visualized by agarose gel electrophoresis.D, PARP cleavage and active caspase-3 expression in SH-SY5Y cells exposed to 0–10 μm HED for 4 h and 8 h. The cleavage of PARP and expression of active caspase-3 were tested by Western blotting and statistically analyzed. E, effect of caspase-3 inhibitors on HED-induced DNA fragmentation. The inhibitor used was AC-DEVD-CHO. The SH-SY5Y cells were treated with 25 μm HED for 12 h in the presence or absence of inhibitor for 30 min. DNA fragmentation was visualized by agarose gel electrophoresis. All of the data are shown as the means ± S.D. (n = 3) (significantly different from control: * indicatesp < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001).

FIGURE 6.

ROS generation and cytochrome c release during HED-induced apoptosis.A, comparison of ROS generation inducted by DHA- and AA-derived dopamine adducts. The SH-SY5Y cells were treated with 10 μm dopamine adducts for 30 min and exposed to H2DCF-DA for 30 min. The fluorescence of DCF was measured by flow cytometer. B, dose-dependent ROS generation induced by HED. DCF fluorescence imaging was determined by fluorescence microscope. C, effect of antioxidant N-acetyl-l-cysteine on HED-induced PARP cleavage and accumulation of active caspase-3. 50 mmN-acetyl-l-cysteine was administrated in SH-SY5Y cells for 30 min before HED treatment. D, cytochrome c release induced by HED. The SH-SY5Y cells were treated with different concentrations of HED for 0, 4, and 8 h. The expressions of cytochrome c and cytochrome c oxidase IV (COX IV) in the mitochondrial fraction of HED-treated cells were assessed by Western blot. All of the data are shown as the means ± S.D. (n = 3) (significantly different from control: * indicates p < 0.05, ** indicatesp < 0.01, and *** indicates p < 0.001.DMSO, dimethyl sulfoxide; Cont, control.

FIGURE 7.

Effect of monoamine transporter inhibitors on apoptosis and ROS generation. The inhibitors used were GBR 12909 and imipramine for DAT and NET/5-HTT, respectively. 1 μm inhibitors were administrated in SH-SY5Y cells for 30 min before drug treatments. A, effect of monoamine transporter inhibitors on HED-induced PARP cleavage and accumulation of active caspase-3. Cleaved PARP and the expression of active caspase-3 were statistical analyzed. B, effect of monoamine transporter inhibitors on HED-induced and HED analog-induced ROS generation. All of the data are shown as the means ± S.D. (n = 3) (significantly different from control: *** indicates p < 0.001; significantly different from HED alone: # indicates p < 0.05, and ## indicatesp < 0.01). Cont, control.

FIGURE 8.

No cytotoxicity was induced by HED in NIH3T3 cells compared with in SH-SY5Y cells.A, apoptotic cells imaging. NIH3T3 cells were treated with different concentrations of HED for 12 h. PI and Hoechst staining were performed by fluorescence microscope. B, numbers of apoptotic cells. The apoptotic cells were analyzed by PI staining by using a flow cytometer. C, ROS generation. The fluorescence of DCF was measured by flow cytometer. The data are shown in B and C as the means ± S.D. (n = 3). Cont, control.