Differential effects of carboxyfullerene on MPP+/MPTP-induced neurotoxicity.

The effects of carboxyfullerene on a well-known neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and its active metabolite 1-methyl-4-phenyl-pyridinium (MPP+) were investigated. In chloral hydrate-anesthetized rats, cytosolic cytochrome c was elevated in the infused substantia nigra 4 h after an intranigral infusion of MPP+. Five days after local application of MPP+, lipid peroxidation (LP) was elevated in the infused substantia nigra. Furthermore, dopamine content and tyrosine hydroxylase (TH)-positive axons were reduced in the ipsilateral striatum. Concomitant intranigral infusion of carboxyfullerene abolished the elevation in cytochrome c and oxidative injuries induced by MPP+. In contrast, systemic application of carboxyfullerene did not prevent neurotoxicity induced by intraperitoneal injection of MPTP. In mice, systemic administration of MPTP induced a dose-dependent depletion in striatal dopamine content. Simultaneous injection of carboxyfullerene (10 mg/kg) actually potentiated MPTP-induced reduction in striatal dopamine content. Furthermore, systemic administration of carboxyfullerene (30 mg/kg) caused death in the MPTP-treated mice. An increase in the striatal MPP+ level and reduction in hepatic P450 level were observed in the carboxyfullerene co-treated mice. These data showed that systemic application of carboxyfullerene appears to potentiate MPTP-induced neurotoxicity while local carboxyfullerene has been suggested as a neuroprotective agent. Furthermore, an increase in striatal MPP+ level may contribute to the potentiation by carboxyfullerene of MPTP-induced neurotoxicity.

Inhibition of group A streptococcus infection by carboxyfullerene.

The effect of a water-soluble trimalonic acid derivative of fullerene, carboxyfullerene, against Streptococcus pyogenes infection was tested. Pretreatment with carboxyfullerene was able to protect mice from S. pyogenes infection in an air pouch model. S. pyogenes-induced death and skin injury were inhibited dose dependently by carboxyfullerene. Administration of carboxyfullerene via the peritoneum and air pouch at 3 h post-S. pyogenes infection was able to protect 33% of mice from death. Surveys of exudates of the air pouch of carboxyfullerene-treated mice revealed that survival of infiltrating neutrophils was prolonged and that the bacteria were eliminated as a result of enhanced bactericidal activity of the neutrophils. Furthermore, carboxyfullerene was able to directly inhibit in vitro growth of S. pyogenes. These data suggest that carboxyfullerene can be considered an antimicrobial agent for group A streptococcus infection.

Fullerenes as a new class of radioprotectors.

PURPOSE: To evaluate the radioprotective activity of C3, a regioisomer of water-soluble carboxyfullerene and a potent free radical scavenger, on both normal and tumour cells. MATERIALS AND METHODS: The murine committed bone-marrow stem cells for both granulocytes and monocytes (GM-CFC) were used to represent normal cells. For tumour cells, murine Ehrlich ascites tumour cells grown in regular tissue culture (EAT-T) and in the peritoneal cavity of CD1 mice (EAT-PC) and human HeLa cells were used. Cells were preexposed to varying concentrations (1-100 microg/ml) of C3 at 37 degrees C for 30 min before they were irradiated. Clonogenic assays were used to determine survival. The protection factor (PF), defined as the ratio of survival with and without C3, was then determined. RESULTS: C3 protected GM-CFC in a concentration-dependent manner up to 50 microg/ml, and no additional protection was seen at 100 microg/ml. The PF was 1.77 when bone-marrow cells were pre-exposed to 50 microg/ml of C3 before they were irradiated with 2 Gy. The value of PF increased to 2.38 when 4 Gy was used. In sharp contrast, C3 exerted less radioprotective effect on tumour cells. The PF values were 1.07, 1.43 and 1.07 for EAT-T, EAT-PC, and HeLa cells, respectively, when 2 Gy was given in the presence of 50 microg/ml of C3. These values increased to 1.40, 1.75 and 1.27, respectively, when 4Gy was given. The dose-modifying factors at 10% survival were 1.37 and 1.15 for GM-CFC and EAT-PC, respectively. CONCLUSION: C3 exhibits a radioprotective effect on a class of normal haemopoietic progenitor cells. It also protects tumour cells, but to a lesser degree. It appears that C3 and other water-soluble fullerenes have a potential to be a new class of cytoprotectors.

Overexpression of bcl-2 enhances LIGHT- and interferon-gamma -mediated apoptosis in Hep3BT2 cells.

LIGHT is a member of the tumor necrosis factor superfamily and is the ligand for LT-betaR, HVEM, and decoy receptor 3. LIGHT has a cytotoxic effect, which is further enhanced by the presence of interferon-gamma (IFN-gamma). Although LIGHT/IFN-gamma can activate caspase activity, neither benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethylketone nor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone can completely inhibit LIGHT/IFN-gamma-mediated apoptosis. Moreover, overexpression of Bcl-2 further enhances LIGHT/IFN-gamma-mediated apoptosis. It appears that LIGHT and IFN-gamma act synergistically to activate caspase-3, with the resultant cleavage of Bcl-2, removal of the BH4 domain, leading to conversion of Bcl-2 from an antiapoptotic to a proapoptotic form in p53-deficient hepatocellular carcinoma Hep3BT2 cells. Thus, LIGHT seems to be able to override the protective effect of Bcl-2 and induce cell death. Although benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethylketone and benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone can prevent the cleavage of Bcl-2 by LIGHT/IFN-gamma, they only partially inhibit apoptosis in Hep3BT2 cells that are overexpressing Bcl-2. In contrast, both LIGHT/IFN-gamma-mediated apoptosis and Bcl-2 cleavage are inhibited by free radical scavengers, indicating that free radicals may play an essential role in LIGHT/IFN-gamma-mediated apoptosis at a step upstream of caspase-3 activation. These results suggest that LIGHT signaling may diverge into multiple, separate processes.

Light-independent inactivation of dengue-2 virus by carboxyfullerene C3 isomer.

Carboxyfullerene (C60) is known as a photosensitizer for virus inactivation. Its regioisomer with C3 symmetry, named the C3 isomer, could also inactivate the dengue-2 virus without light when the dose of C3 isomer was increased to 40 microM, indicating the possible involvement of a light-independent mechanism. Further analysis showed that the C3 isomer blocked viral replication at the attachment and penetration stages, suggesting that a direct interaction between the C3 isomer and the virion is required for inactivation. The C3 isomer with a bipolar structure showed better lipid interaction and dengue-2 virus suppression than D3, another isomer that contains evenly distributed hydrophilic side chains. Moreover, the C3 isomer selectively inactivated enveloped viruses (viz., dengue-2 virus and Japanese encephalitis virus) instead of nonenveloped viruses (viz., enterovirus 71 and coxsackievirus B3). Collectively, these findings support the hypothesis that C3 isomer suppression of enveloped viruses is effected through its hydrophobic interaction with the viral lipid envelope. Our report, which demonstrates the light-dependent and -independent mechanisms of C60 on viral inactivation, will aid in the development of novel anti-viral agents for use against enveloped viruses.

C(60) and water-soluble fullerene derivatives as antioxidants against radical-initiated lipid peroxidation.

C(60), vitamin E, and three C(60) derivatives (polar 1 and water-soluble C(3)/D(3)C(60)s) were examined for their antioxidant effects on prevention of lipid peroxidation induced by superoxide and hydroxyl radicals. The protection effect on lipid peroxidation was found to be in the sequence: C(60) >/= vitamin E > 1 > none, for liposoluble antioxidants, and C(3)C(60) >> D(3)C(60) > none, for water-soluble ones. Fluorescence quenching of PyCH(2)COOH (Py = pyrene) by both C(3)- and D(3)C(60)s shows that the Stern-Volmer constant, K(SV), is about the same for both quenchers in aqueous solution. Upon addition of liposomes, the fluorescence quenching becomes more efficient: 5-fold higher in K(SV) for C(3)C(60) than for D(3)C(60). When Py(CH(2))(n)()COOH (n = 1, 3, 5, 9, or 15) was incorporated in lipid membranes, the K(SV)s all were small and nearly equal for D(3)C(60) but were quite large and different for C(3)C(60) with the sequence: n = 1 < 3 < 5 < 9 < 15. The better protection effect of C(3)C(60) on lipid peroxidation than that of D(3)C(60) is attributed to its stronger interaction with membranes. Overall, the antioxidation abilities of the compounds examined were rationalized in terms of the number of reactive sites, the location of antioxidant in lipid membranes, and the strength of interactions between antioxidants and membranes.

Inhibition of Escherichia coli-induced meningitis by carboxyfullerence.

The effect of a water-soluble malonic acid derivative of carboxyfullerence (C60) against Escherichia coli-induced meningitis was tested. C60 can protect the mice from E. coli-induced death in a dose-dependent manner. C60 administered intraperitoneally as late as 9 h after E. coli injection was still protective. The C60-treated mice had less tumor necrosis factor alpha and interleukin-1beta production by staining of brain tissue compared to the levels of production for nontreated mice. The E. coli-induced increases in blood-brain barrier permeability and inflammatory neutrophilic infiltration were also inhibited. These data suggest that C60 is a potentially therapeutic agent for bacterial meningitis.

Carboxyfullerene prevents iron-induced oxidative stress in rat brain.

Carboxyfullerene, a water-soluble carboxylic acid derivative of a fullerene, was investigated as a protective agent against iron-induced oxidative stress in the nigrostriatal dopaminergic system of anesthetized rats. Intranigral infusion of exclusive carboxyfullerene did not increase lipid peroxidation in substantia nigra or deplete dopamine content in striatum. Infusion of ferrous citrate (iron II) induced degeneration of the nigrostriatal dopaminergic system. An increase in lipid peroxidation in substantia nigra as well as decreases in K+-evoked dopamine overflow and dopamine content in striatum were observed 7 days after the infusion. Co-infusion of carboxyfullerene prevented iron-induced oxidative injury. Furthermore, tyrosine hydroxylase-immunoreactive staining showed that carboxyfullerene inhibited the iron-induced loss of the dopaminergic nerve terminals in striatum. The antioxidative action of carboxyfullerene was verified by in vitro studies. Incubation of brain homogenates increased the formation of the Schiff base fluorescent products of malonaldehyde, an indicator of lipid peroxidation. Both autooxidation (without exogenous iron) and iron-induced elevation of lipid peroxidation of brain homogenates were suppressed by carboxyfullerene in a dose-dependent manner. Our results suggest that intranigral infusion of carboxyfullerene appears to be nontoxic to the nigrostriatal dopaminergic system. Furthermore, the potent antioxidative action of carboxyfullerene protects the nigrostriatal dopaminergic system from iron-induced oxidative injury.

The antioxidative effect of carboxyfullerenes (C3/D3) on iron-induced oxidative injury in CNS.

Carboxyfullerenes, including two regioisomers C3 and D3, were investigated as antioxidants against iron-induced oxidative stress in vivo and in vitro. Both C3 and D3 dose-dependently inhibited autoxidation and iron-elevated lipid peroxidation in cortical homogenates. The antioxidative property of C3 was compared to Trolox (a water-soluble analogue of vitamin E) and glutathione. C3 was more effective than glutathione but was less effective than Trolox in inhibiting iron-induced elevation in lipid peroxidation. In urethane-anesthetized rats, intranigral infusion of iron degenerated the nigrostriatal dopaminergic system, including as elevation in lipid peroxidation in the infused substantia nigra (SN) and reductions in K(+)-evoked dopamine overflow and dopamine content in the ipsilateral striatum 7 days after the infusion. Local application of iron with C3 or D3 prevented iron-induced oxidative injuries. Our data suggest that carboxyfullerenes have a neuroprotective effect in preventing iron-induced oxidative injury in CNS.

Blockage of apoptotic signaling of transforming growth factor-beta in human hepatoma cells by carboxyfullerene.

Transforming growth factor-beta (TGF-beta) has been shown to induce apoptosis in normal hepatocytes and hepatoma cells both in vivo and in vitro. However, the mechanism by which TGF-beta induces apoptosis is not clear. The antiapoptotic activity of antioxidants including N-acetyl-L-cysteine (Ac-Cys), ascorbic acid and a novel free radical scavenger, carboxyfullerene (C60) on TGF-beta-treated human hepatoma Hep3B cells was examined. Only the water-soluble hexacarboxylic acid derivative of C60 was found to prevent TGF-beta-induced apoptosis. Antiapoptotic activity of C60 correlated its ability to eliminate TGF-beta-generated reactive oxygen species (ROSs). However, C60 did not interfere with TGF-beta-activated PAI-1 promoter activity in the Hep3B cells. These results indicate that the signaling pathway of TGF-beta-induced apoptosis may be related to the generation of ROSs and may be uncoupled from the TGF-beta-activated gene promoter activity. Furthermore, the regioisomer of C60 with a C3 symmetry was more potent in protecting cells from apoptosis than that with a D3 symmetry, and the C3 isomer had stronger interactions with lipid bilayers than the D3 isomer. The spectroscopic analysis revealed that the C3 isomer had stronger interactions with artificial lipid bilayers than the D3 isomer. Therefore, our study indicates that C60 may interact with membrane to eliminate TGF-beta-induced ROSs and to prevent apoptosis occur in human hepatoma cells.

Apoptotic signal of Fas is not mediated by ceramide.

Ceramide has been suggested as the secondary messenger mediating the apoptotic signal for Fas engagement. By using different inhibitors, we demonstrated here that ceramide is unlikely a mediator of Fas-initiated apoptosis. First, cAMP prevented cell death induced by ceramide but not by Fas. Second, ceramide-triggered, but not Fas-triggered, apoptosis was antagonized by the free radical scavenger C60. Third, the metal chelator pyrrolidinedithiocarbamate suppressed ceramide-initiated DNA fragmentation but had no effect on the Fas-induced cell death. Fourth, the SAPK/ERK kinase dominant negative mutant, which attenuated ceramide-induced cell death, did not prevent Fas-induced apoptosis. Finally, activation of NF-kappaB inhibited ceramide-induced but not Fas-initiated apoptosis. The fact that many antagonists of ceramide-induced apoptosis could not suppress Fas-mediated cell death clearly indicates that ceramide is not the mediator for Fas-initiated apoptotic signal.

Carboxyfullerenes as neuroprotective agents.

Two regioisomers with C3 or D3 symmetry of water-soluble carboxylic acid C60 derivatives, containing three malonic acid groups per molecule, were synthesized and found to be equipotent free radical scavengers in solution as assessed by EPR analysis. Both compounds also inhibited the excitotoxic death of cultured cortical neurons induced by exposure to N-methyl-D-aspartate (NMDA), alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), or oxygen-glucose deprivation, but the C3 regioisomer was more effective than the D3 regioisomer, possibly reflecting its polar nature and attendant greater ability to enter lipid membranes. At 100 microM, the C3 derivative fully blocked even rapidly triggered, NMDA receptor-mediated toxicity, a form of toxicity with limited sensitivity to all other classes of free radical scavengers we have tested. The C3 derivative also reduced apoptotic neuronal death induced by either serum deprivation or exposure to Abeta1-42 protein. Furthermore, continuous infusion of the C3 derivative in a transgenic mouse carrying the human mutant (G93A) superoxide dismutase gene responsible for a form of familial amyotrophic lateral sclerosis, delayed both death and functional deterioration. These data suggest that polar carboxylic acid C60 derivatives may have attractive therapeutic properties in several acute or chronic neurodegenerative diseases.