Nrf2-mediated heme oxygenase-1 induction confers adaptive survival response to tetrahydropapaveroline-induced oxidative PC12 cell death.

Tetrahydropapaveroline (THP), a dopaminergic isoquinoline neurotoxin, has been reported to contribute to neurodegeneration in parkinsonism. As THP bears two catechol moieties, it undergoes autooxidation or enzymatic oxidation to produce reactive oxygen species (ROS), which may contribute to the THP-induced cell death. Although ROS are cytotoxic, the initial accumulation of ROS may provoke a survival response. In this study, treatment of PC12 cells with THP increased expression of heme oxygenase-1 (HO-1) as an adaptive survival response. Furthermore, THP-induced cytotoxicity was attenuated by the HO-1 inducer (SnCl2) and exacerbated by the HO-1 inhibitor (ZnPP). To elucidate the molecular mechanisms underlying THP-mediated HO-1 expression, we examined the possible involvement of NF-E2-related factor 2 (Nrf2), which plays an important role in the transcriptional regulation of detoxifying/antioxidant genes. THP treatment elevated nuclear translocation of Nrf2 and subsequent binding to antioxidant response element (ARE). PC12 cells transfected with dominant-negative Nrf2 exhibited increased cytotoxicity and decreased HO-1 expression after THP treatment. Moreover, U0126 and LY294002, which are pharmacologic inhibitors of extracellular signal-regulated kinase1/2 and phosphoinositide 3-kinase, respectively, attenuated HO-1 expression as well as Nrf2-ARE binding activity. Taken together, these findings suggest that HO-1 induction via Nrf2 activation may confer a cellular adaptive response against THP-mediated cell death.

Mechanism of DNA damage and apoptosis induced by tetrahydropapaveroline, a metabolite of dopamine.

Tetrahydropapaveroline (THP), a metabolite of dopamine, has been suspected to be associated with dopaminergic neurotoxicity of L-DOPA. THP induced apoptosis in human leukemia cell line HL-60 cells, but did not in its hydrogen peroxide (H(2)O(2))-resistant clone HP100. THP-induced DNA ladder formation in HL-60 cells was inhibited by a metal chelator. THP induced damage to (32)P-labeled DNA fragments in the presence of metals. In the presence of Fe(III)EDTA, THP caused DNA damage at every nucleotide. The DNA damage was inhibited by free hydroxy radical ((.)OH) scavengers and catalase, suggesting that the Fe(III)EDTA-mediated DNA damage is mainly due to (.)OH generation. In the presence of Cu(II), THP caused DNA damage mainly at T and G of 5'-TG-3' sequence. The inhibitive effect of catalase and bathocuproine on Cu(II)-mediated DNA damage suggested that H(2)O(2) and Cu(I) participate in the DNA damage. This study demonstrated that THP-induced apoptosis via reactive oxygen species generated from reaction of H(2)O(2) and metals plays an important role in cytotoxicity of L-DOPA.

Inhibition of dopamine biosynthesis by tetrahydropapaveroline.

Tetrahydropapaveroline (THP) at 5-15 microM has been found to induce L-DOPA-induced oxidative apoptosis in PC12 cells. In this study, the inhibitory effects of THP on dopamine biosynthesis in PC12 cells and tyrosine hydroxylase (TH) activity in bovine adrenal were investigated. Treatment of PC12 cells with THP at 2.5-10 microM significantly decreased the intracellular dopamine content in a concentration-dependent manner (21.3% inhibition at THP 10 microM). The activity of TH was also inhibited by the treatment with THP at 2.5-10 microM (23.4% inhibition at 10 microM). In addition, THP had an inhibitory effect on bovine adrenal TH (IC50 value, 153.9 microM). THP exhibited uncompetitive inhibition on bovine adrenal TH with a substrate l-tyrosine with the Ki value with L-tyrosine of 0.30 mM. Treatment with L-DOPA at 20-50 microM increased the intracellular dopamine content in PC12 cells and the increase in dopamine content by L-DOPA was in part inhibited when L-DOPA (20 and 50 microM) was associated with THP at non-cytotoxic (5-10 microM) or cytotoxic (15 microM) concentration ranges. However, the reduction of dopamine content by THP (15 microM) or THP (15 microM) associated with L-DOPA (20 and 50 microM) in PC12 cells was inversed by the antioxidant N-acetyl-cysteine (0.1mM). These results indicate that THP at 5-10 microM decreases the basal dopamine content and reduces the increased dopamine content induced by L-DOPA in part by the inhibition of TH activity, and that THP at 15 microM does by oxidative stress in PC12 cells.

Oxidative DNA damage and glioma cell death induced by tetrahydropapaveroline.

A series of naturally occurring isoquinoline alkaloids, besides their distribution in the environment and presence in certain food stuffs, have been detected in human tissues including particular regions of brain. An example is salsolinol (1-methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline) that not only induces neuronal cell death, but also causes DNA damage and genotoxicity. Tetrahydropapaveroline [THP; 6,7-dihydroxy-1-(3',4'-dihydroxybenzyl)-1,2,3,4-tetrahydroisoquinoline], a dopamine-derived tetrahydroisoquinoline alkaloid, has been reported to inhibit mitochondrial respiration and is considered to contribute to neurodegeneration implicated in Parkinson's disease. Since THP bears two catechol moieties, the compound may readily undergo redox cycling to produce reactive oxygen species (ROS) as well as toxic quinoids. In the present study, we have examined the capability of THP to cause oxidative DNA damage and cell death. Incubation of THP with phiX174 supercoiled DNA or calf thymus DNA in the presence of cupric ion caused substantial DNA damage as determined by strand scission or formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo), respectively. THP plus copper-induced DNA damage was ameliorated by some ROS scavengers/antioxidants and catalase. Treatment of C6 glioma cells with THP led to a concentration-dependent reduction in cell viability, which was prevented by the antioxidant N-acetyl-L-cysteine. When these cells were treated with 10microM THP, c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) were rapidly activated via phosphorylation, whereas activation of extracellular signal-regulated protein kinase (ERK) was inhibited. Furthermore, pretreatment with inhibitors of JNK and p38 MAPK rescued the glioma cells from THP-induced cytotoxicity, suggestive of the involvement of these kinases in THP-induced C6 glioma cell damage.

Tyrosinase protects human melanocytes from ROS-generating compounds.

The effects of two tetrahydroisoquinolines (TIQs), tetrahydropapaveroline (THP) and salsolinol (SAL), on human primary melanocytes were studied. These compounds are naturally occurring alkaloids deriving from the condensation of dopamine with aldehydes. The effects on the viability were studied by treating the cells with variable concentration of THP or SAL; both TIQs were well tolerated up to roughly 30 micro M. At higher concentrations, THP became overtly toxic while SAL showed no cytotoxic effect up to 100 micro M. TIQs treatment determined an impairment of intracellular activity of antioxidant enzymes, like SOD, DT-diaphorase, and glutathione peroxidase. A decrease of alpha-ketoglutarate dehydrogenase activity was also evidenced following TIQs treatment; a very strong diminution was found in THP-treated cells, whose viability was highly decreased. Both TIQs increased tyrosinase-specific mRNA transcription followed, in the case of SAL, by an activation of tyrosinase. In the presence of tyrosinase inhibitors TIQs treatment resulted in a sharp cytotoxic effect even at concentrations normally well tolerated. Taken together these data suggest that tyrosinase represents an outstanding protective mechanism against ROS-generating compounds, once primary detoxifying mechanisms are impaired or not available.

Dopaminergic neurotoxins, 6,7-dihydroxy-1-(3', 4'-dihydroxybenzyl)-isoquinolines, cause different types of cell death in SH-SY5Y cells: apoptosis was induced by oxidized papaverolines and necrosis by reduced tetrahydropapaverolines.

Dopamine-derived 6,7-dihydroxy-1-(3', 4'-dihydroxybenzyl)-isoquinolines, papaverolines and tetrahydropapaverolines, have been proposed to be neurotoxin candidates related to the pathogenesis of Parkinson's disease. In this paper, the cytotoxicity of papaverolines and their N-methyl derivatives was examined using human dopaminergic neuroblastoma SH-SY5Y cells as a model of dopamine neurons. Apoptotic and necrotic cell death were assessed by morphological observation of cells after staining with propidium iodide and Hoechst 33342. Papaveroline and N-methyl-papaveroline induced apoptosis in almost all the cells with typical features of condensed and fragmented nuclei. On the other hand, (R)- and (S)-tetrahydropapaveroline caused necrosis in cells. Tetrahydropapaverolines markedly reduced adenosine triphosphate (ATP) level, whereas papaverolines did not, suggesting that the types of cell death induced by these isoquinolines, necrosis and apoptosis, depend on ATP concentrations in the cells.