Characterization of catechol-thioether-induced apoptosis in human SH-SY5Y neuroblastoma cells.

Recent work has highlighted the involvement of a dopamine derivative, 5-S-cysteinyl-dopamine (CysDA), in neurodegeneration and apoptotic cell death. In this paper we study in further detail the apoptotic process activated by this catechol-thioether derivative of dopamine in SH-SY5Y neuroblastoma cells. CysDA activates a cascade of events by an initial perturbation of Calcium homeostasis in the cell. Cell treatment with the catechol-thioether induces an immediate rise in intracellular Ca(2+) concentration, as demonstrated by a shift in the indo-1 dye emission spectrum, and a sustained high calcium concentration at long times of incubation. Fluorescence microscopy data show that the treatment of cells induces mitochondrial transmembrane potential depolarization, a clear evidence of the onset of apoptotic process. Programmed cell death activation is also demonstrated by cytochrome c release from the mitochondria, by an increased activity of both caspase-8 and -9 and by the poly(ADP-ribose)polymerase (PARP-1) cleavage, yielding the typical 86 kDa fragment due to caspase-3 activity. Overall, our data support the hypothesis that CysDA may induce apoptotic death in neuronal cells, via an initial perturbation of calcium homeostasis in the cytosol.

UVA-induced modification of catalase charge properties in the epidermis is correlated with the skin phototype.

The harmful effects of UVA radiation (320-400 nm) on the skin have been related to the generation of reactive oxygen species. Pheomelanin, the pigment characteristic of fair-skinned individuals, amplifies these effects. In vitro, in the presence of photosensitizing agents, UVA light produces singlet oxygen, which reacts with several targets. We have investigated a possible correlation between melanin-type and the antioxidant defense system after UV, focusing on the activities of superoxide dismutase and catalase, which correlated with the phototype of epidermal reconstructs. UVA was more effective than UVB in damaging these enzymatic activities, especially catalase. Furthermore, UVA irradiation induced a free-radical-mediated damage in the cells, leading to an oxidation of cell proteins. On catalase, synthetic pheomelanin amplified this effect on specific targets, such as residues of tryptophan and methionine. UVA irradiation of low phototype reconstructed epidermis and of U937 through synthetic pheomelanin induced a modification in the electrophoretic properties of native catalase, which was counteracted by histidine, a quencher of singlet oxygen. These results demonstrate that pheomelanin could act as a photosensitizing agent, following UVA irradiation, inducing charge modifications of native catalase, by a mechanism involving singlet oxygen or its downstream products.

The pheomelanin precursor 5-S-cysteinyldopa protects melanocytes from membrane damage induced by ultraviolet A radiation.

Pheomelanin and pheomelanin precursors have been implicated as risk factors for induction of melanoma by ultraviolet radiation. The pheomelanin precursor, 5-S-cysteinyldopa, has been shown to sensitise DNA to oxidative damage by ultraviolet radiation. We here show that 5-S-cysteinyldopa significantly protects melanocytes from membrane damage (permeability) induced by ultraviolet A radiation. Thus, 5-S-cysteinyldopa, may at the same time sensitise DNA and protect membranes from damage induced by ultraviolet radiation.

5-s-Cysteinyl-conjugates of catecholamines induce cell damage, extensive DNA base modification and increases in caspase-3 activity in neurons.

A decrease in reduced glutathione levels in dopamine containing nigral cells in Parkinson's disease may result from the formation of cysteinyl-adducts of catecholamines, which in turn exert toxicity on nigral cells. We show that exposure of neurons (CSM 14.1) to 5-S-cysteinyl conjugates of dopamine, L-DOPA, DOPAC or DHMA causes neuronal damage, increases in oxidative DNA base modification and an elevation of caspase-3 activity in cells. Damage to neurons was apparent 12-48 h of post-exposure and there were increases in caspase-3 activity in neurons after 6 h. These changes were paralleled by large increases in pyrimidine and purine base oxidation products, such as 8-OH-guanine suggesting that 5-S-cysteinyl conjugates of catecholamines are capable of diffusing into cells and stimulating the formation of reactive oxygen species (ROS), which may then lead to a mechanism of cell damage involving caspase-3. Indeed, intracellular ROS were observed to rise sharply on exposure to the conjugates. These results suggest one mechanism by which oxidative stress may occur in the substantia nigra in Parkinson's disease.

Diffusible melanin-related metabolites are potent inhibitors of lipid peroxidation.

Although it has long been known that epidermal melanocytes produce and excrete a number of melanin-related metabolites, including 5.6-dihydroxyindole (DHI), 5,6-dihydroxyindole-2-carboxylic acid (DHICA), and 5-S-cysteinyldopa (CD), the possible functional significance of these compounds has been so far largely overlooked. We report now evidence that DHI, DHICA and CD exert potent inhibitory effects in different in vitro models of lipid peroxidation. The compounds, at 100 microM concentration, substantially decreased malondialdehyde (MDA) formation by lipid peroxidation in rat brain cortex homogenates. At 1.2 microM concentration, DHI proved as effective as alpha-tocopherol (alpha-T), one of the most potent endogenous antioxidants, in suppressing azo-induced peroxidation of linoleic acid in phosphate buffer (pH 7.4), containing 0.10 M SDS, whereas CD and DHICA at the same concentration were less active. DHI, CD and DHICA (all in the range 25 microM-0.5 mM) were also found to inhibit Fe (II)/EDTA-induced oxidation of 0.5 mM arachidonic acid at pH 7.4, as well as MDA formation by iron-promoted degradation of 0.5 mM 15-hydroperoxy-5,8,11, 13-eicosatetraenoic acid (15-HPETE). In both cases the inhibitory effects were much greater than those of ascorbic acid and glutathione. These results point to melanin precursors as a novel class of biological antioxidants which may contribute to defense mechanisms against oxidative injury in human skin.

5-S-cysteinyldopa, a diffusible product of melanocyte activity, is an efficient inhibitor of hydroxylation/oxidation reactions induced by the Fenton system.

Interest in 5-S-cysteinyldopa (5-S-CD), a major excretion product of normal and malignant melanocytes, has traditionally concentrated on its significance as a biosynthetic precursor of pheomelanins, the characteristic pigments of red hair, and as a specific biochemical marker for monitoring melanoma progression. The present study shows that 5-S-CD is a potent inhibitor of hydroxylation/oxidation reactions mediated by hydrogen peroxide and the Fe2+/EDTA complex under both aerobic and anaerobic conditions. The inhibitory effect of 5-S-CD, as determined by the deoxyribose and salicylic acid assays in phosphate buffer (pH 7.4), is much stronger than that of dopa, acetylsalicylic acid and mannitol, increases with increasing ligand-to-metal ratio, and is inversely proportional to the concentration of EDTA present in the Fenton system. Spectrophotometric evidence and competition experiments indicate that 5-S-CD forms a chelate complex with ferric ions (lambda max = 500 nm at pH 7.4), which may account for both an altered production of hydroxyl radicals by the Fenton reagent and a site-specific localization of oxidative damage on the chelate complex itself.

Dual role of melanins and melanin precursors as photoprotective and phototoxic agents: inhibition of ultraviolet radiation-induced lipid peroxidation.

Ultraviolet radiation (UVR) is one of the risk factors for skin cancer and the main inducer of melanin pigmentation, the major protective mechanism of mammalian skin against radiation damage. The melanin pigments, eumelanin and pheomelanin, are likely to be important in protection against UVR, but their precursors are generally considered as phototoxic. The available data suggest DNA damage as the mechanism of phototoxicity. However, the effect of melanin precursors on membrane damage through lipid peroxidation, another important and probably more relevant (from the point-of-view of the melanosomal confinement of these molecules) mechanism of phototoxicity, not known. As a model system for UVR-melanin-membrane interactions, we irradiated liposomes in the presence of eumelanin, pheomelanin and two of their major precursors, 5,6-dihydroxyindole (DHI) and 5-S-cysteinyldopa (SCD). The presence of the two melanin precursors substantially reduced the formation of lipid peroxidation products resulting from UVR exposure. The antioxidant activity of the melanin precursors was diminished under strong prooxidant conditions (presence of Fe3+). These results suggest that melanin precursors may have an important role in the protection of skin against the harmful effects of UVR including photocarcinogenesis.

Inhibition of human glutathione S-transferases by dopamine, alpha-methyldopa and their 5-S-glutathionyl conjugates.

The reversible and irreversible inhibition of human glutathione S-transferases (GST) by dopamine, alpha-methyldopa and their 5-S-glutathionyl conjugates (termed 5-GSDA and 5-GSMDOPA, respectively) was studied using purified isoenzymes. The reversible inhibition, using CDNB as substrate and expressed as I50, ranged from 0.18-0.24 (GST M1a-1a), 0.19-0.24 (GST M1b-1b) to 0.5-0.54 mM (GST A1-1) for 5-GSDA and 5-GSMDOPA, respectively. About 20% inhibition was observed for GST A2-2 and P1-1, using 0.5 mM of both 5-GSDA and 5-GSMDOPA. No significant reversible inhibition was observed with dopamine and alpha-methyldopa. Tyrosinase was used to generate ortho-quinones from dopamine and alpha-methyldopa which may bind covalently to GST and thereupon irreversibly inhibit GST. In this respect, GST P1-1 was by far the most sensitive enzyme. The inhibition (expressed as a % of control) after incubating 0.5 microM GST in the presence of 100 units/ml tyrosinase with 5 microM of the catecholamines for 10 min at 25 degrees, was 99% and 67% for dopamine and alpha-methyldopa, respectively. Moderate irreversible inhibition of GST A1-1 by both dopamine and alpha-methyldopa (33% and 25%, respectively), and of GST M1b-1b by dopamine (45%) was also observed. GST P1-1 is also the only isoenzyme susceptible to irreversible inhibition by 5-GSDA (33% inhibition), while no significant inhibition was observed with 5-GSMDOPA. A minor part of the inhibition by dopamine (23%), and the complete inhibition by 5-GSDA was restored by reduction with dithiotreitol. This suggests that GST P1-1 is inhibited by disulfide formation in the case of 5-GSDA, while this oxidative pathway also substantially contributes to the inactivation by dopamine. This was supported by the HPLC-profile of the GST P1-1 subunit which was strongly affected by dopamine, while for 5-GSDA after reduction with dithiotreitol the original elution profile of the subunit returned.

Enhancement of pheomelanogenesis by L-dopa in the mouse melanocyte cell line, TM10, in vitro.

Cells of TM10, an established cell line, are melanocytes that contain equal amounts of eumelanin (black pigment) and pheomelanin (yellow pigment). The content of pheomelanin drastically increased when the cells were cultured in growth medium containing 0.2mM-L-dopa (L-dihydroxyphenylalanine), which is the common precursor for both eumelanogenesis and pheomelanogenesis. After this treatment, the amount of pheomelanin was 3.7-fold greater than that of control in TM10, whereas the amount of eumelanin changed very little. In contrast, 5-S-cysteinyl-dopa, which is the specific precursor for pheomelanogenesis downstream of L-dopa, did not cause preferential increase in pheomelanogenesis. Ultrastructural observations also confirmed these results; in 0.2mM-L-dopa, an increase in the number of pheomelanosomes was observed in the cytoplasm of TM10 cells. Our results also suggest that the L-dopa treatment results in a decrease in tyrosinase activity per melanosome.

The mechanism of toxicity of 5-S-cysteinyldopa to tumour cells. Hydrogen peroxide as a mediator of cytotoxicity.

5-S-Cysteinyldopa, a melanin precursor, has been shown to possess selective toxicity to tumour cells in vitro and in vivo. The mechanism of cytotoxicity of the catechol was studied in comparison with L-dopa and 5-S-cysteaminyldopamine. Growth inhibition of human neuroblastoma cell line of YT-nu by 5-S-cysteinyldopa was completely depressed by addition of catalase. Superoxide dismutase and five drugs thought to scavenge hydroxyl radicals or quench singlet oxygen had little effect on the cytotoxicity. Hydrogen peroxide itself was also cytotoxic at low concns. These results indicated that hydrogen peroxide was a mediator of the cytotoxicity of 5-S-cysteinyldopa. It is suggested that reaction of the catechol with cellular superoxide radicals contributes to the production of hydrogen peroxide in addition to autoxidation. Catalase reduced the cytotoxicity of L-dopa by half, while it had no inhibitory effect on the strong cytotoxicity of 5-S-cysteaminyldopamine.

The effect of cysteine on oxidation of tyrosine, dopa, and cysteinyldopas.

The influence of cysteine on the oxidation of tyrosine, dopa, and monocysteinyldopas by mushroom tyrosinase was reexamined. During oxidation of tyrosine in the presence of cysteine the concentration of dopa increased slowly, whereas the concentration of cysteinyldopas increased more rapidly. When the concentration of cysteine decreased the cysteinyldopas were rapidly consumed and dopa concentrations increased sharply. Experiments on the oxidation of dopa by tyrosinase in the presence of cysteine showed that this thiol does not inhibit the oxidation. Dopa concentrations decreased more rapidly in the presence of cysteine because cysteine addition to dopaquinone prevented reformation of dopa from dopaquinone. Both 2-S-cysteinyldopa and 5-S-cysteinyldopa are substrates for tyrosinase. The oxidation of cysteinyldopas was inhibited at high cysteine concentrations. The greater part of 2,5-S,S-dicysteinyldopa formed during the oxidation of monocysteinyldopas in the presence of cysteine is derived from 5-S-cysteinyldopa, which is a better substrate for tyrosinase than 2-S-cysteinyldopa. The fact that cysteine binds more rapidly to 5-S-cysteinyldopaquinone than to 2-S-cysteinyldopaquinone further stresses the importance of 5-S-cysteinyldopa in the formation of 2,5-S,S-dicysteinyldopa. Oxidation of dopa in the presence of cysteine and glutathione or methionine showed that glutathione is added to dopaquinone but less rapidly than cysteine. Methionine showed insignificant addition to dopaquinone. When dopa or 5-OH-dopa is added to an incubate of cysteinyldopa and tyrosinase the oxidation of cysteinyldopa is accelerated owing to oxidation of cysteinyldopa by dopaquinone or 5-OH-dopaquinone.

Synthesis and antitumor activity of cysteinyl-3,4-dihydroxyphenylalanines and related compounds.

The natural catecholic amino acid 5-S-cysteinyl-3,4-dihydroxyphenylalanine (1) was selectively toxic to a variety of human tumor cell lines in culture and exhibited antitumor activity against L1210 leukemia and B-16 melanoma in mice at doses which were not toxic to the host. Structural analogues of 5-S-cysteinyl-3,4-dihydroxyphenylalanine including several new compounds, were synthesized and tested for growth inhibition of cultured cells of human neuroblastoma YT-nu and Chinese hamster fibroblasts Don-6. Some were also examined for antitumor activity against L1210 and B-16 in vivo. 4-S-Cysteinylcatechols and 2- and 4-S-cyteinylphenols, which cannot be prepared by conventional methods, were synthesized by the reaction of catechols and phenols with cystine and boiling aqueous HBr. 5-S-Cysteinyl- and 2-S-Cysteinyl-3,4-dihyroxyphenylalanine (1 and 2), L-3,4-dihydroxyphenylalanine (L-Dopa), and 2- and 4-S-cysteinylphenol (14 and 15) were toxic to the YT-nu cell line only, while 4-S-cysteinylcatechol (6), 3-S-cysteinyl-5-methylcatechol (8), 5-S-cysteaminyldopamine (9), and 4-methylcatechol were strongly toxic to both cell lines. Compound I (1000 mg/kg), 6 (500 mg/kg), and 8 (400 mg/kg) increased the life span of L1210-bearing mice by 50, 50, and 43%, respectively, and compounds 1 and 8 were marginally effective against B-16 melanoma as well. Compound 9 was too toxic to show any activity. There was a good correlation between the cytotoxicity and the in vivo activity.

Selective toxicity of 5-S-cysteinyldopa, a melanin precursor, to tumor cells in vitro and in vivo.

The effect of 5-S-cysteinyl-L-3,4-dihydroxyphenylalanine (cys-dopa), an intermediate in the pathway from L-3,4-dihydroxyphenylalanine (L-dopa) to pheomelanin, on the growth of eight human tumor cell lines in culture was compared to that of L-dopa. The tumor cell lines tested comprise two neuroblastomas (NB-1 and YT-nu), two amelanotic melanomas (HMV and SEKI), a gastric carcinoma (MKN-28), and three squamous cell carcinomas (HeLa-S3, KB, and a salivary gland carcinoma). Cys-dopa at a concentration of 1 mM inhibited growth of NB-1 (66%), YT-nu (67%), HMV (44%), SEKI (60%), MKN-28 (47%), HeLa-S3 (24%), KB (64%), and salivary gland carcinoma (33%), while L-dopa exhibited similar or even lower degree of inhibition at a concentration of 6 mM. On the other hand, both catechols had little effect on the growth of two fibroblasts derived originally from normal tissues (mouse fibroblast L929 and Chinese hamster fibroblast Don-6). Cys-dopa and L-dopa inhibited DNA and protein synthesis in YT-nu cells, but RNA synthesis was less affected. Treatment with cys-dopa at a dose of 1000 mg/kg i.p. for 7 days prolonged by 50% the life span of mice inoculated with L1210 leukemia. Normal mice given cys-dopa at a dose of 1000 mg/kg for 12 days showed no signs of toxicity. These results suggest the potential of cys-dopa as an antitumor agent.