Keywords

1 Backgrounds

Parkinson’s disease (PD) is a neurodegenerative disorder characterized by a dramatic loss of dopaminergic neurons in the substantia nigra, and the subsequent deficiency of dopamine in the brain areas (Galvan and Wichmann 2008). Until now, very little is known about why and how the PD neurodegenerative process begins and progresses; however, an increasing body of evidence suggests that oxidative stress, mitochondrial dysfunction, and impairment of the ubiquitin-proteasome system may be involved in the pathogenesis of PD (Leroy et al. 1998; Schapira 2001; Balaban et al. 2005). Recent studies indicate that there are high levels of basal oxidative stress in the substantia nigra pars compacta (SNc) in the normal brain and this is increased in PD (Jenner 2003).

Oxidative stress in the brain easily leads to the lipid peroxidation reaction due to a high concentration of polyunsaturated fatty acids (PUFA), such as docosahexaenoic acid (DHA, C22:6/ω-3) and arachidonic acid (AA, C18:4/ω-6), which are present in the brain (Porter et al. 1995). The polyunsaturated fatty acids are located almost exclusively in the SN2-position of the phosphoglycerides found in the neural cell membranes. The beneficial physiological effects of DHA and AA have been frequently reported (Simopoulos 1999; Hadders-Algra 2008); however, the fatty acids are highly unsaturated, thus making them particularly susceptible to peroxidation. During the lipid peroxidation reaction, lipid hydroperoxides are generated as primary products. Subsequent decomposition leads to the formation of reactive mediates including aldehydes, which can covalently modify biomolecules. We have recently found that lipid hydroperoxides, the primary peroxidative products, can universally react with primary amino groups to form N-acyl-type (amide-linkage) adducts (Kato et al. 1997, 1999; Kato and Osawa 1998; Kawai et al. 2003, 2004, 2006). In our previous studies, the formation of linoleic acid-derived lysine modification adducts, N-(hexanoyl) lysine and N-(azelayl) lysine, and DHA-derived adducts, N-(succinyl) lysine and N-(propanoyl) lysine, have been identified in vitro or in vivo by LC-MS/MS or immunochemical analysis. In addition, the formation of N-(hexanoyl) lysine also was detected, as well as N-(glutaryl) lysine, during the reaction of oxidized arachidonic acid (AA) with the lysine residue. The N-acyl-type adducts are specific to the peroxidation of polyunsaturated fatty acids, therefore, their formations are the useful markers for the lipid peroxidation, protein modification and related dysfunction that occur in these fatty acids enriched tissues.

Dopamine is the endogenous neurotransmitter produced in nigral neurons. Dopaminergic neuronal loss can trigger not only prominent secondary morphological changes, such as density reduction of the dendritic spines, but also changes in the density and sensitivity of dopamine receptors (Galvan and Wichmann 2008); therefore, it is a sign of PD development. The reasons for dopamine loss are attributed to molecular instability of dopamine. Some possible causes of dopamine are characteristics of dopaminergic neurons (Bove et al. 2005), such as dopamine degradation by monoamine oxidase A (MAO-A) (Gotz et al. 1994) or auto-oxidation (Hald and Lotharius 2005) and the reaction with amino acid cysteine (LaVoie and Hastings 1999). Dopamine is a member of catecholamine family. The catechol structure of dopamine contributes to vulnerability to oxidative stress. Additionally, the NH2-teminals in dopamine’s structure may represent another reactive spot, however, little experimental evidence have been proven. Based on our previously described reaction between lipid hydroperoxides and NH2 residues, the possibility that reactive LOOH species derived from lipid peroxidation may modify dopamine to form amide-linkage dopamine adducts was investigated.

2 Chemical Formation of DHA- and AA-Derived Dopamine Adducts

Lipid hydroperoxides, the primary products of lipid peroxidation, could non-specifically react with primary amino groups to form N-acyl-type (amide-linkage) adducts, and also within the chemical structure of dopamine, an amino residue is present. The DHA and AA-derived four amide-linkage dopamine adducts, succinyl dopamine (SUD) and propanoyl dopamine (PRD), hexanoyl dopamine (HED) and glutaroyl dopamine (GLD), were chemically synthesized respectively. The chemical structures of the authentic adducts were identified by NMR. The formation of these dopamine adducts was further confirmed by HPLC-MS/MS analysis. Collision-induced dissociation (CID) of the authentic adducts, SUD (m/z 254), PRD (m/z 210), HED (m/z 252) and GLD (m/z 268), produced the same daughter ions at m/z 91, and 137. SUD, PRD and HED also produced daughter ions at m/z 154, whereas GLD did not. The ion at m/z 137 was detected with the highest peak intensity in the fragments, and this ion was also identified to be derived from the dopamine spectra (Fig. 4.1).

Fig. 4.1
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]+ ion m/z 254, 210, 252, and 268 of SUD, PRD, HED, and GLD, respectively, were subjected to CID, and the daughter ions were scanned (left, upper). The proposed structures of individual ions are shown (right, upper). The chemical structure composition of the dopamine adducts is proposed by fragmental analysis (lower)

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3 In Vitro and In Vivo Detection of Dopamine Adducts

In our previous studies, to determinate the in vitro formation of the dopamine adducts, the reaction of dopamine with DHA- or AA-hydroperoxides were carried out. The results showed that the four adducts including SUD, PRD, HED and GLD were successfully detected by HPLC-MS/MS among the reaction mixtures.

It has been reported that polyunsaturated fatty acids such as DHA and AA are significantly enriched in the brain (Tapiero et al. 2002), and that there are high levels of basal oxidative stress in the normal brain, which increases with aging (Lin and Flint 2006). To investigate whether the DHA- and AA- derived dopamine adducts can be formed in vivo, the brains of 7-week- and 27-week-old male F344/NSIc rats were removed and the homogenates were used. The detection of the dopamine adducts in the homogenates was carried out by HPLC-MS/MS. The whole adducts were detected in the 7- and 27-week rat brains in both the positive ion mode and negative ion mode of LC-MS/MS. The level of HED and PRD, which are derived from the CH3-teminous of AA and DHA, were more preferentially formed than that of SUD and PRD; however, no significant difference of adduct level was found between the 7- week and 27-week rats.

Dopamine is a natural neurotransmitter in the brain, and its deficiency is a sign of Parkinson’s disease (Ang 2006). Although the mechanism of neurodegeneration is not fully understood, some considerations include dopaminergic neuron abnormalities, dopamine degradation by monoamine oxidase A (MAO-A) or auto-oxidation and modification (Gotz et al. 1994; Hald and Lotharius 2005; LaVoie and Hastings 1999). The in vitro and in vivo detections of DHA- and AA-derived dopamine adducts established may indicate an additional clue to the causes of dopamine deficiency in PD. Although the level of the dopamine adducts was not obviously increased in the 27-week-old rat brain compared to the 7-week-old rat brain, 27 weeks represents only middle age for a rat and the level of basal oxidative stress is increased with age (Navarro and Boveris 2007; Forster et al. 1996; Boveris and Navarro 2008); therefore, further study should confirm these adduct formations in the brain using aging model rats such as 1 year age and more and also PD model animals.

4 Identification of HED as a Potent Inducer of Neuronal Apoptosis

In recent years, several dopamine oxidants and dopamine-modified adducts have been reported, such as neuromelanin (Wakamatsu et al. 2003), aminochrome (Graumann et al. 2002), 6-OHDA (Saner and Thoenen 1971) and 5-S-CDA (LaVoie and Hastings 1999), among them 6-OHDA has been generally known as a potent neurotoxin (Pezzella et al. 1997; Izumi et al. 2005; Maharaj et al. 2005). To test that some of these DHA- and AA-derived dopamine adducts could cause neuronal cell death. The effect of these dopamine adducts on the cell viability in SH-SY5Y cells was studied. The results showed that among the tested dopamine adducts, HED and PRD induced about 80 and 30 % of the cell death, respectively. On the other hand, SUD and GLD had almost no influence on the cell viability, suggesting the death of SH-SY5Y cells was induced only by the CH3-terminus-derived adducts, and not by the COOH-terminus-derived adducts. Of interest, two HED analogs, nonanoyl dopamine (NOD) and lauroyl dopamine (LAD), which were synthesized and characterized by more carbons than HED in the methyl terminus, also showed a significant toxicity to SH-SY5Y cells, suggesting that the number of carbon in the CH3-terminus-derived dopamine adducts might be associated with the adduct-induced cell death.

HED was a potent inducer of SH-SY5Y cell death compared to SUD, PRD and GLD. Because apoptosis is suggested to be involved in neurodegeneration, we then characterized whether HED-induced cell death in SH-SY5Y cells is apoptosis or not. The exposure to HED induces to a dose-dependent decrease in the viable cells. Moreover, the fragmented nuclei were found in cells exhibiting the typical morphological features of apoptosis. In addition, the gel electrophoresis of DNA from the SH-SY5Y cells exposed to HED also displayed nucleosomal DNA fragmentation. HED treatment also led to the time- and dose- dependent cleavage of PARP resulting in the accumulation of the 85-kDa fragment and decreasing in the 116-kDa protein, as well as in the accumulation of the active caspase-3, both which are hallmarks of apoptosis. Moreover, the pretreatment with the caspase-3 inhibitor significantly prevented SH-SY5Y cells from HED-induced DNA fragmentation, providing further evidence that HED induced a caspase-3-mediated apoptotic cell death.

Dopamine-derived metabolites have been reported to inflict damage on neuronal cells (Asanuma et al. 2003). For example, 6-hydroxydopamine (6-OHDA), a hydroxylated analogue of dopamine, has been demonstrated to induce apoptosis in several neuronal cell lines (Hanrott et al. 2006; Chalovich et al. 2006; Jia et al. 2008; Lee et al. 2008). In addition, dopamine autoxidation generating dopamine quinone can react with protein sulfhydryl groups leading to structural modifications of proteins and reduced levels of glutathione (GSH) (Berman and Hastings 1999). HED, an AA-derived dopamine adduct, caused significant cell death in SH-SY5Y cells. Furthermore, the events including DNA fragmentation, chromatin condensation, PARP cleavage and accumulation of active caspase-3 suggest that HED-induced cell death was apoptosis.

5 Regulation of HED-Induced Apoptosis in SH-SY5Y Cells

What might be the signaling mechanism underlying the HED-induced apoptosis is our interests. It is well accepted that reactive oxygen species (ROS) generation is a key contributor to neuronal apoptosis induced by neurotoxin compounds (Chinopoulos and Adam-Vizi 2006). Hence, experiments were first carried out to assess the ROS generation induced by the HED treatment and the possibility that the HED-induced apoptosis is mediated via ROS generation in SH-SY5Y cells. HED led to increased ROS generation in the cells compared to the DMSO-treated cells, whereas the other three dopamine adducts, SUD, PRD and GLD, had a much less effect on the cells. Furthermore, a dose-dependent increase in the ROS generation was found by dichlorofluorescein (DCF) fluorescence staining. The pretreatment with NAC, a potent antioxidant, clearly inhibited the PARP cleavage, indicating that the ROS generation might be critically involved in the HED-induced apoptosis. It is widely accepted that mitochondrial dysfunction may play very important roles in neuronal cell death (Kluck et al. 1997). The cytochrome c release from mitochondria was found in HED-treated cells.

The precise mechanisms regulating apoptotic events in neuronal cells remain largely unclear; however, high levels of ROS generation and the increases in the mitochondrial permeability appear to be common occurrences in many forms of apoptotic neuronal cell death. The finding that HED induced a significant ROS generation and that NAC pretreatment clearly blocked the apoptosis suggests that ROS generation is an essential trigger for HED-induced apoptosis in the SH-SY5Y cells. The source of ROS generation has not been identified, however, the catechol ring is kept in the structure of HED like dopamine and 6-OHDA, therefore, the catechol oxidation might be one of the important causes for the ROS generation in the HED-treated SH-SY5Y cells. The regulation of neuronal apoptosis is generally characterized by the several signaling mediators such as p-53, Bcl-2 family proteins and cytochrome c release (Gorman et al. 2000). A significant release of cytochrome c from mitochondrial fraction in HED-treated SH-SY5Y cells was found, suggesting that the apoptosis may be critically mediated via a mitochondrial abnormality.

6 Monoamine Transporters Are Important in HED-Induced Apoptosis and ROS Generation

Monoamine transporters including the dopamine transporter (DAT), norepinephrine transporter (NET) and 5-HT transporter (5-HTT), which are of fundamental importance for proper signaling between neurons, have been reported to associate with experimental neurotoxins-induced toxicity (Kita et al. 2003). HED possesses a dopamine-based chemical structure, suggesting that the above-described HED cytotoxicity that occurred in the SH-SY5Y cells might be mediated by uptake of HED by monoamine transporters. The pretreatment with both GBR12909 and Imipramine, the inhibitors of DAT and NET/5-HHT, respectively, clearly inhibited the occurrence of the HED-induced PARP cleavage and active caspase-3 expression in the SH-SY5Y cells. Furthermore, the ROS generation by HED was also found to be suppressed in these two inhibitor-pretreated cells. The result that both monoamine transporter inhibitors showed markedly inhibitive effect on the HED-induced apoptosis and ROS generation suggested that HED might be primarily transported into the SH-SY5Y cells by the monoamine transporters, and inflicted damage on the cells.

To characterize whether the HED-induced cytotoxicity is specific to neuronal cells, our study investigated the effect of HED on apoptotic cell death and ROS generation in mouse embryonic fibroblast NIH-3T3 cells in comparison to that of the SH-SY5Y cells. A dose-dependent analysis revealed that HED led to no apoptotic cell death in the NIH-3T3 cells estimated by Hoechst 33258 and Propidium Iodide (PI) nuclear staining. A further quantitative analysis of the apoptotic cells by flow cytometry also indicated apoptosis in SH-SY5Y cells, whereas not in the NIH-3T3 cells. Moreover, no ROS generation was found in the HED-treated NIH-3T3 cells; on the other hand, the HED analogs, NOD and LAD, also induced only a slight ROS generation in the NIH-3T3 cells. Monoamine transporter is known to be absent in NIH-3T3 cells, which may indicate that the HED-induced cytotoxicity might be specific to neuronal cells.

Monoamine transporters are of fundamental importance for proper signaling between neurons. Plasma membrane transporters, the major subclass of intracellular transporters (Gethe et al. 2006), include the dopamine transporter (DAT), norepinephrine transporter (NET), and 5-HT transporter (5-HTT). In this study, pretreatment with inhibitors of DAT, NET and 5-HTT significantly suppressed ROS generation and apoptosis events induced by HED. In the case of 6-OHDA, similar to HED, a high affinity for several catecholaminergic plasma membrane transporters, such as DAT and NET, is also essential for its entrance into the neuronal cells to inflict damage. The dependence of monamine transporter is considered to be due to a structural similarity between the HED, dopamine and norepinephrine. The necessity of the monoamine transporter in HED-induced cytotoxicity was further demonstrated by the result that HED could not induce apoptotic cell death and ROS generation in the monoamine transporter-absent NIH-3T3 cells, which also indicates that HED may selectively induce cytotoxicity in different cell lines.

7 Conclusion and Note

Four amide-linkage adducts of dopamine with DHA and AA were synthesized and the in vivo formation during the reaction of lipid hydroperoxides with dopamine were also revealed. HED, an AA-derived dopamine adduct, as a potent neurotoxin based on the significant induction of ROS generation and apoptosis in human neuroblastoma SH-SY5Y cells. The mechanism of HED-induced apoptosis has not been fully established in this study; however, it seems to be mediated by ROS generation, mitochondrial abnormalities, and monoamine transporter (Fig. 4.2).

Fig. 4.2
figure 2

Proposed mechanism of HED-induced apoptosis in SH-SY5Y cells

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In fact, either DHA or AA is located almost exclusively in the SN2-position of phosphoglycerides found in the neural cell membranes (Ma et al. 2007; Beermann et al. 2005); however, free fatty acid levels are reported to increase with aging due to an increasing degradation by PLA2 (Rosenberger et al. 2004; Qu et al. 2003; Rapoport 1999), a phospholipase A2, which selectively acts on phosphoglycerides (Diez et al. 1994). DHA is the most enriched polyunsaturated fatty acid in the brain, and it has been implicated that DHA concentration is decreased in AD brain (Bazan et al. 2002); hence, the DHA-derived dopamine adducts formed in this study may be useful biomarkers for not only PD but also AD.