Radical-mediated Mechanism Research Articles

The underlying mechanisms for self-healing of poly(disulfide)s.

Recently, self-healing polymers based on disulfide compounds have gained attention due to the versatile chemistry of disulfide bonds and easy implementation into polymeric materials. However, the underlying mechanisms of disulfide exchange which induce the self-healing effect in poly(disulfide)s remain unclear. In this work, we elucidate the process of disulfide exchange using a variety of spectroscopic techniques. Comparing a model exchange reaction of 4-aminophenyl disulfide and diphenyl disulfide with modified reactions in the presence of additional radical traps or radical sources confirmed that the exchange reaction between disulfide compounds occurred via a radical-mediated mechanism. Furthermore, when investigating the effect of catalysts on the model exchange reaction, it could be concluded that catalysts enhance the disulfide exchange reaction through the formation of S-based anions in addition to the radical-mediated mechanism.

Manganese superoxide dismutase deficiency triggers mitochondrial uncoupling and the Warburg effect.

Manganese superoxide dismutase (MnSOD) is a mitochondrially localized primary antioxidant enzyme, known to be essential for the survival of aerobic life and to play important roles in tumorigenesis. Here, we show that MnSOD deficiency in skin tissues of MnSOD-heterozygous knockout (Sod2+/−) mice leads to increased expresson of uncoupling proteins (UCPs). When MnSOD is deficient, superoxide radical and its resulting reactive oxygen species (ROS) activate ligand binding to peroxisome proliferator-activated receptor alpha (PPARα), suggesting that the activation of PPARα signaling is a major mechanism underlying MnSOD-dependent UCPs expression that consequently triggers the PI3K/Akt/mTOR pathway, leading to increased aerobic glycolysis. Knockdown of UCPs and mTOR suppresses lactate production and increases ATP levels, suggesting that UCPs contribute to increased glycolysis. These results highlight the existence of a free radical-mediated mechanism that activates mitochondria uncoupling to reduce ROS production, which precedes the glycolytic adaptation described as the Warburg Effect.

Peroxynitrite modified DNA presents better epitopes for anti-DNA autoantibodies in diabetes type 1 patients

Peroxynitrite (ONOO−), formed by the reaction between nitric oxide (NO) and superoxide (O2−), has been implicated in the etiology of numerous disease processes. Peroxynitrite interacts with DNA via direct oxidative reactions or via indirect radical-mediated mechanism. It can inflict both oxidative and nitrosative damages on DNA bases, generating abasic sites, resulting in the single strand breaks. Plasmid pUC 18 isolated from Escherichiacoli was modified with peroxynitrite, generated by quenched flow process. Modifications incurred in plasmid DNA were characterized by ultraviolet and fluorescence spectroscopy, circular dichroism, HPLC and melting temperature studies. Binding characteristics and specificity of antibodies from diabetes patients were analyzed by direct binding and inhibition ELISA. Peroxynitrite modification of pUC 18 plasmid resulted in the formation of strand breaks and base modification. The major compound formed when peroxynitrite reacted with DNA was 8-nitroguanine, a specific marker for peroxynitrite induced DNA damage in inflamed tissues. The concentration of 8-nitroguanine was found to be 3.8μM. Sera from diabetes type 1 patients from different age groups were studied for their binding to native and peroxynitrite modified plasmid. Direct binding and competitive-inhibition ELISA results showed higher recognition of peroxynitrite modified plasmid, as compared to the native form, by auto-antibodies present in diabetes patients. The preferential recognition of modified plasmid by diabetes autoantibodies was further reiterated by gel shift assay. Experimentally induced anti-peroxynitrite-modified plasmid IgG was used as a probe to detect nitrosative lesions in the DNA isolated from diabetes patients.

Mutual effects between aromatic amino acids and guanosine upon vitamin B2 photosensitization in the presence of visible light

Considering the importance of the visible-light-induced photodynamic effect in complex bioenvironments, mutual effects between the individual aromatic amino acids (AAs) tyrosine (Tyr), tryptophan (Trp), and histidine (His) and the nucleoside guanosine (GUO) were investigated in pH 7 aqueous solution with vitamin B2 (riboflavin (Rf)) as a dye sensitizer. The quantum yields of oxygen uptake (Φ–O2) for most of the AA−GUO mixtures studied, taken as a measure of overall photooxidation susceptibility, are not straightforwardly predictable from the individual behaviour of the components of the mixture. The final result depends on several connected factors, such as the respective abilities of the substrates as quenchers of the long-lived Rf triplet excited state and the generated reactive oxygen species singlet molecular oxygen (O2(1Δg)) and superoxide radical anion ([Formula: see text]). A mechanistic interpretation of the Rf-sensitized results can be roughly resumed as follows: Tyr at pH 7 exerts a protective effect on the photooxidation of the mixture Tyr−GUO due to the O2(1Δg) physical quenching by the AA. The same effect was observed for Trp−GUO and His−GUO at pH 7. In these cases, it is attributed to the quenching of3Rf* by GUO in detriment of the Type II route. For the system Tyr−GUO at pH 9, a marked decrease in the Φ–O2occurred for the mixture as compared with the respective Φ–O2for the individual components. It was ascribed to the participation of a radical-mediated mechanism without oxygen consumption in a competitive pathway with the [Formula: see text]-mediated route.

Glutathiolated Ras: Characterization and implications for Ras activation

Ras GTPases cycle between active GTP-bound and inactive GDP-bound forms to regulate a multitude of cellular processes, including cell growth, differentiation, and apoptosis. The activation state of Ras is regulated by protein modulatory agents that accelerate the slow intrinsic rates of GDP dissociation and GTP hydrolysis. Similar to the action of guanine-nucleotide exchange factors, the rate of GDP dissociation can be greatly enhanced by the reaction of Ras with small-molecule redox agents, such as nitrogen dioxide, which can promote Ras activation. Nitrogen dioxide is an autoxidation product of nitric oxide and can react with an accessible cysteine of Ras to cause oxidation of the bound guanine nucleotide to facilitate Ras guanine nucleotide dissociation. Glutathione has also been reported to modify Ras and alter its activity. To elucidate the mechanism by which glutathione alters Ras guanine nucleotide binding properties, we performed NMR, top-down and bottom-up mass spectrometry, and biochemical analyses of glutathiolated Ras. We determined that treatment of H-Ras, lacking the nonconserved hypervariable region, with oxidized glutathione results in glutathiolation specifically at cysteine 118. However, glutathiolation does not alter Ras structure or biochemical properties. Rather, changes in guanine nucleotide binding properties and Ras activity occur upon exposure of Ras to free radicals, presumably through the generation of a cysteine 118 thiyl radical. Interestingly, Ras glutathiolation protects Ras from further free radical-mediated activation events. Therefore, glutathiolation does not affect Ras activity unless Ras is modified by glutathione through a radical-mediated mechanism.

Covalent intermediate in the catalytic mechanism of the radical S-adenosyl-L-methionine methyl synthase RlmN trapped by mutagenesis.

The posttranscriptional modification of ribosomal RNA (rRNA) modulates ribosomal function and confers resistance to antibiotics targeted to the ribosome. The radical S-adenosyl-L-methionine (SAM) methyl synthases, RlmN and Cfr, both methylate A2503 within the peptidyl transferase center of prokaryotic ribosomes, yielding 2-methyl- and 8-methyl-adenosine, respectively. The C2 and C8 positions of adenosine are unusual methylation substrates due to their electrophilicity. To accomplish this reaction, RlmN and Cfr use a shared radical-mediated mechanism. In addition to the radical SAM CX(3)CX(2)C motif, both RlmN and Cfr contain two conserved cysteine residues required for in vivo function, putatively to form (cysteine 355 in RlmN) and resolve (cysteine 118 in RlmN) a covalent intermediate needed to achieve this challenging transformation. Currently, there is no direct evidence for this proposed covalent intermediate. We have further investigated the roles of these conserved cysteines in the mechanism of RlmN. Cysteine 118 mutants of RlmN are unable to resolve the covalent intermediate, either in vivo or in vitro, enabling us to isolate and characterize this intermediate. Additionally, tandem mass spectrometric analyses of mutant RlmN reveal a methylene-linked adenosine modification at cysteine 355. Employing deuterium-labeled SAM and RNA substrates in vitro has allowed us to further clarify the mechanism of formation of this intermediate. Together, these experiments provide compelling evidence for the formation of a covalent intermediate species between RlmN and its rRNA substrate and well as the roles of the conserved cysteine residues in catalysis.

Trifluoromethylation of arenes and heteroarenes by means of photoredox catalysis

Modern drug discovery relies on the continual development of synthetic methodology to address the many challenges associated with the design of new pharmaceutical agents. One such challenge arises from the enzymatic metabolism of drugs in vivo by cytochrome P450 oxidases, which use single-electron oxidative mechanisms to rapidly modify small molecules to facilitate their excretion. A commonly used synthetic strategy to protect against in vivo metabolism involves the incorporation of electron-withdrawing functionality, such as the trifluoromethyl (CF(3)) group, into drug candidates. The CF(3) group enjoys a privileged role in the realm of medicinal chemistry because its incorporation into small molecules often enhances efficacy by promoting electrostatic interactions with targets, improving cellular membrane permeability, and increasing robustness towards oxidative metabolism of the drug. Although common pharmacophores often bear CF(3) motifs in an aromatic system, access to such analogues typically requires the incorporation of the CF(3) group, or a surrogate moiety, at the start of a multi-step synthetic sequence. Here we report a mild, operationally simple strategy for the direct trifluoromethylation of unactivated arenes and heteroarenes through a radical-mediated mechanism using commercial photocatalysts and a household light bulb. We demonstrate the broad utility of this transformation through addition of CF(3) to a number of heteroaromatic and aromatic systems. The benefit to medicinal chemistry and applicability to late-stage drug development is also shown through examples of the direct trifluoromethylation of widely prescribed pharmaceutical agents.

A radical approach to diversity

Compounds containing the trifluoromethyl group have many uses, but their isomers must often be made using different multi-step routes. Two studies now show how several isomers can be made by the same route. See Letter p.224 Premature metabolic decomposition can stop potentially promising drugs from reaching their intended targets. One way of blocking this metabolism involves the addition of a trifluoromethyl (CF3) group to aromatic or heteroaromatic moieties in a candidate drug molecule. David Nagib and David MacMillan report a new method for this synthesis that is more economical than current methods in terms of both raw materials and energy input. They use an energy-saving compact fluorescent light bulb to excite a variety of commercially available photocatalysts, which promote the addition of CF3 to unactivated arenes and heteroarenes through a radical-mediated mechanism. The potential of the method is demonstrated by the trifluoromethylation of three molecules: a uracil analogue, a precursor of the acetylcholinesterase inhibitor donepezil and a vitamin (flavone).

The Effect of β-Carotene Against Adriamycin Toxicity on the Embryo Formation

Adriamycin is an anthracycline antibiotic widely used for the treatment of many types of cancer. The cytotoxic effect of Adriamycin occurs by a free radical-mediated mechanism. Thus, to prevent or reduce the toxic effect of Adriamycin, it is possible to use it in combination with antioxidants. The aim of this study was to evaluate a potential effect of β-carotene against Adriamycin-induced toxicity on the embryo formation. Pregnant rats were treated with Adriamycin, β-carotene, and their combination during the critical stages of embryogenesis. The first group was control group. Adriamycin was administered on day 9 (group 2a) and day 12 (group 2b) of gestation by a single intraperitoneal injection at a dose of 5 mg/kg. β-Carotene was given at a dosage of 0.6 mg/(kg·day) from day 6 to 10 or from day 9 (group 3a) to 13 (group 3b) of gestation 5 times per os; in the case of their combination, β-carotene was given per os 3 times before Adriamycin injection, one time simultaneously with Adriamycin and one time after its injection (groups 4a and 4b). Animals were euthanized on day 21 of gestation. Embryo resorptions and alive fetuses were counted, weighed, and measured. The embryos of each litter were examined macroscopically after the Buen solution fixation for the embryo defects. In order to render the skeleton visible, the soft tissues were macerated using caustic soda, stained with alizarin red, and cleared with glycerin. Adriamycin induced embryotoxicity; the combination of Adriamycin and β-carotene decreased the number of Adriamycin-induced embryo resorptions about two times. A gavage with Adriamycin alone decreased fetal body weights (P<0.05), while giving it in combination, the fetal body weight was similar to that in the control group. Adriamycin induced the retardation of skeletogenesis and external fetal malformations (microphthalmia, hydrocephaly, anencephaly, and others). After an exposure to β-carotene, external malformations (diaphragmatic hernia) of embryos were found only occasionally. β-Carotene in combination with Adriamycin produced no positive effect on Adriamycin-induced skeletodysgenesis or external malformations. Antioxidant β-carotene in combination with Adriamycin slightly reduced the Adriamycin-induced embryotoxicity, but produced no positive effect on Adriamycin-induced skeleto-dysgenesis or external malformations.

Photosynthesis-inspired design approach of a liquid phase heterogeneous photoreactor

A method of photocatalytic process intensification, which can be easily used for efficient photoreactor design, inspired by the mechanism of natural photosynthesis was developed and the possible mechanism was proposed. Four model compounds (phenol, Cr(VI), acid orange 7 and p-nitrophenol) with different photocatalytic reaction mechanisms, and using a Kenics static mixer, were selected to perform experiments for validation. Experimental results showed that the static mixer can intensify the indirect radical-mediated mechanism reaction, but had no effect on the direct surface reaction mechanism reaction. Meanwhile, both the illuminated static mixer and dark static mixer had similar intensification effects. Herein, we propose that the role the static mixer played in heterogeneous photocatalysis is the creation of intense mixing and increasing the interfacial mass transfer, which resulted in the enhanced mobility of reactive radicals from the catalyst surface or boundary layer to solution, thus intensifying the whole photocatalytic efficiency. Furthermore, the proposed separation of the illumination part and the mixing part for photoreactor design allows for easier scale-up and engineering applications.

Cholesterol oxidation in the retina: implications of 7KCh formation in chronic inflammation and age-related macular degeneration

This review will discuss the formation and potential implications of 7-ketocholesterol (7KCh) in the retina. 7KCh is a proinflammatory oxysterol known to be present in high amounts in oxidized LDL deposits associated with atheromatous plaques. 7KCh is generated in situ in these lipoprotein deposits where it can accumulate and reach very high concentrations. In normal primate retina, 7KCh has been found associated with lipoprotein deposits in the choriocapillaris, Bruch's membrane, and the retinal pigment epithelium (RPE). In photodamaged rats, 7KCh has been found in the neural retina in areas of high mitochondrial content, ganglion cells, photoreceptor inner segments and synapses, and the RPE. Intermediates found by LCMS indicate 7KCh is formed via a free radical-mediated mechanism catalyzed by iron. 7KCh seems to activate several kinase signaling pathways that work via nuclear factor κB and cause the induction of vascular endothelial growth factor, interleukin (IL)-6, and IL-8. There seems to be little evidence of 7KCh metabolism in the retina, although some form of efflux mechanism may be active. The chronic mode of formation and the potent inflammatory properties of 7KCh indicate it may be an "age-related" risk factor in aging diseases such as atherosclerosis, Alzheimer's, and age-related macular degeneration.

Determination of catalytic properties of TiO 2 coatings using aqueous solution of coumarin: Standardization efforts

Many types of supported photocatalytic TiO 2 continue to be the subject of extensive development worldwide. Besides industrial production and practical use of the new photocatalytic materials, there is an increasing need for a simple and reliable procedure for characterization of photocatalytic activities of newly developed materials. The aim of our work was to develop a method for the determination of quantum yields of supported photocatalysts by employing an aqueous solution of a model organic compound and different thin TiO 2 films. Additionally, also a newly defined parameter, the so-called mass efficiency, was introduced as an advantageous way of defining the photocatalytic activities. Coumarin (CM) was found to be an appropriate candidate for being a probe molecule when the photocatalytic degradation mainly occurs via the HO radical-mediated mechanism. An advantage of using CM is easy determination of highly fluorescent 7-hydroxycoumarin (7OHC). Different TiO 2 films were deposited via sol–gel methods on soda-lime glass slides that were precovered with a thin SiO 2 film, and via the sedimentation process from aqueous suspensions. Aqueous solutions of CM were irradiated using 365 nm radiation in the presence of titania films. Although Degussa P25 films showed approximately four times higher degradation rates compared to the sol–gel processed, its quantum yield was not more than 2.5 times higher. This could be explained by higher absorbance of Degussa P25 films per amount of the catalyst compared to sol–gel films. Since no information on the absorption characteristics of the material per unit of mass of the catalyst is included within the calculation of quantum yield, mass efficiency is suggested for the evaluation of photocatalytic properties of the coatings. It is evident from the present study that it is obligatory to evaluate the photocatalytic efficiencies of different area densities of the same photocatalyst in order to properly characterize the material's efficiency.

Mechanistic aspects of 4-amino-2,6-dichlorophenol-induced in vitro nephrotoxicity

4-Amino-2,6-dichlorophenol (ADCP) is a potent acute nephrotoxicant in vivo inducing prominent renal corticomedullary necrosis. In vitro, ADCP exposure increases lactate dehydrogenase (LDH) release from rat renal cortical slices at 0.05 mM or greater. The purpose of this study was to examine the ability of antioxidants, cytochrome P450 (CYP) and flavin adenine dinucleotide monooxygenase (FMO) activity modulators, indomethacin, glutathione and inhibitors of glutathione conjugate metabolism to attenuate ADCP cytotoxicity in vitro. Renal cortical slices prepared from untreated male Fischer 344 rats ( N = 4/group) were preincubated at 37 °C under a 100% oxygen atmosphere with an inhibitor or vehicle for 5–30 min. ADCP (0.05–0.5 mM) or vehicle was added and incubations continued for 120 min. At the end of the incubation period, LDH release was measured as an index of nephrotoxicity. ADCP cytotoxicity was partially attenuated by ascorbate (1.0 or 2.0 mM), but not by N, N′-diphenyl- p-phenylenediamine (DPPD), α-tocopherol or deferoxamine. Inhibitors of CYP (metyrapone, piperonyl butoxide and isoniazid) and FMO activity modulators (methimazole, N-octylamine) had no effect on ADCP cytotoxicity. Indomethacin or glutathione 1.0 mM completely and partially blocked ADCP 0.1 and 0.5 mM cytotoxicity, respectively. N-acetylcysteine, AOAA (an inhibitor of cysteine conjugate β-lyase) and probenecid (an organic anion transport inhibitor), but not AT-125 (an inhibitor of γ-glutamyl transferase), partially attenuated ADCP 0.1 mM cytotoxicity. Overall, these results suggest that reactive metabolites may be produced from ADCP primarily via a co-oxidation-mediated mechanism. The difference in the ability of ascorbate and glutathione to attenuate ADCP-induced cytotoxicity in vitro in kidney cells could indicate that alkylation via the reactive benzoquinoneimine metabolite might be responsible for cytotoxicity rather than a free radical-mediated mechanism.

Enhanced photocatalytic activity of TiO 2 by surface fluorination in degradation of organic cationic compound

Experiments were carried out to investigate the influence of TiO 2 surface fluorination on the photodegradation of a representative organic cationic compound, Methylene Blue (MB). The electropositive MB shows poor adsorption on TiO 2 surface; its degradation performs a HO. radical-mediated mechanism. In the F-modified system, the kinetic reaction rate enlarged more than 2.5 fold that was attributed mainly to the accumulating adsorption of MB and the increased photogenerated hole available on the F-modified TiO 2 surface.

Cytotoxic effects of B lymphocytes mediated by reactive oxygen species.

Reactive oxygen species (ROS) are produced in all mammalian cells as a result of norman cellular metabolism and due to the activation of oxidant-producing enzymes in response to exogenous stimuli. The balance between ROS production and antioxidant defenses determines the degree of oxidative stress. Generation of ROS has been associated with cell signaling, stress responses, cell proliferation, aging and cancer development. The ability of ROS to induce cellular damage and to cause cell death opens the possibility to exploit this property in the treatment of cancer through a fee radical-mediated mechanism.

Glutathione is involved in the antimalarial action of chloroquine and its modulation affects drug sensitivity of human and murine species of Plasmodium

Ferriprotoporphyrin IX (FP) is released inside the food vacuole of the malaria parasite during the digestion of host cell hemoglobin. FP is detoxified by its biomineralization to hemozoin. This process is effectively inhibited by chloroquine (CQ) and amodiaquine (AQ). Undegraded FP accumulates in the membrane fraction and inhibits enzymes of infected cells in parallel with parasite killing. FP is demonstrably degraded by reduced glutathione (GSH) in a radical-mediated mechanism. This degradation is inhibited by CQ and AQ in a competitive manner, thus explaining the ability of increased GSH levels in Plasmodium falciparum-infected cells to increase resistance to CQ and vice versa, and to render Plasmodium berghei that were selected for CQ resistance in vivo sensitive to the CQ when glutathione synthesis is inhibited. Some over-the-counter drugs that are known to reduce GSH in body tissues when used in excess were found to enhance the antimalarial action of CQ and AQ in mice infected either with P. berghei or Plasmodium vinckei. In contrast, N-acetyl-cysteine which is expected to increase the cellular levels of GSH, antagonized the action of CQ. These results suggest that some over-the-counter drugs can be used in combination with some antimalarials to which the parasite has become resistant.

A new approach for the detection of carbon-centered radicals in enzymatic processes using prefluorescent probes.

In this communication we propose a novel application for prefluorescent probes in the detection of free carbon-centered radicals in enzymatic processes. Prefluorescent probes combine a fluorescent moiety tethered to a paramagnetic nitroxide that acts as a fluorescence quencher. Trapping of a radical by the nitroxide group restores the fluorescence properties. The increase in fluorescence intensity with time reflects the formation and quenching of carbon-centered radicals and can be used for the quantitative evaluation of yields and kinetics. As a test system we used horseradish peroxidase, an oxidoreductase that is widely accepted to operate by a radical-mediated mechanism. We used the prefluorescent probe (quinoline-TEMPO), where a quinoline moiety has been tethered to 2,2,6,6-tetramethylpiperidin-1-oxyl.

Toxic effects of MPP + and MPTP in PC12 cells independent of reactive oxygen species formation

MPTP is a toxin presumed to damage dopamine-secreting neurons by an oxygen free radical-mediated mechanism. Two steps in MPTP metabolism are the primary candidates for oxygen free radical generation: (a) MPTP oxidation to MPP + by a monoamine oxidase and (b) NADH dehydrogenase inhibition by MPP +. In order to test the idea that MPTP toxicity is mediated by oxygen free radicals, we assessed lipid peroxidation and the effects of antioxidants in dopaminergic PC12 cells treated with MPTP or MPP +. For comparison purposes, we also examined the effects of the pro-oxidant tert-butyl-hydroperoxide (TBHP) and of the dopaminergic toxin 6-hydroxydopamine (6-OHDA) in PC12 cells. MPTP and MPP +, unlike TBHP, failed to induce lipid peroxidation in PC12 cells after a 4-h exposure. All toxins tested (MPTP, MPP +, TBHP and 6-OHDA) caused a dose-dependent decrease in [ 3H]dopamine ( 3H-DA) uptake in PC12 cultures. The hydroperoxide scavengers glutathione and N-acetyl-cysteine and the superoxide and peroxide scavenger EUK-134 protected PC12 cells from TBHP- and 6-OHDA-induced decrease in 3H-DA uptake. However, no protection by these antioxidants at various concentrations and time regimens was observed against MPTP- or MPP +-induced decreases in 3H-DA uptake in PC12 cells. In addition, incubation of PC12 cells with the energy-rich substrate, NADH, attenuated MPP +-induced decrease in 3H-DA uptake. These results suggest that MPTP-induced toxicity in dopaminergic PC12 cell cultures, does not involve oxygen free radical production, but rather may be caused by impairment in energy metabolism.

Ethanol-Related Gastric Mucosal Damage: Evidence of a Free Radical-Mediated Mechanism and Beneficial Effect of Oral Supplementation with Bionormalizer, a Novel Natural Antioxidant

Twenty-two healthy teetotal volunteers underwent gastroscopy during which biopsy samples from the antrum and body were taken for chemiluminescence assay, routine histology, and for malonyldialdehyde, xanthine oxidase and glutathione determination. Subjects were divided into 2 groups which, in a double-blind fashion, were randomly and orally given either (a) Bionormalizer 9 g at bedtime and 3 h prior examination, or (b) flavored sugar 9 g as placebo. During the second gastroscopy 40 ml of 80% ethanol were sprayed perendoscopically. Gastroscopy with biopsy was repeated 60 min later. As compared to the placebo group, subjects given Bionormalizer showed significantly reduced gastric mucosal damage at endoscopy and the histological level. When considering the placebo group, ethanol administration brought about a significant increase in the luminol-amplified chemiluminescence response in gastric mucosa as compared to the baseline value which was correlated with the histological score. The mean chemiluminescence value in the Bionormalizer group was significantly lower than in the placebo group. Ethanol ingestion brought about a significant increase in xanthine oxidase and malonyldialdehyde together with a decreased glutathione concentration. Bionormalizer significantly prevented such changes. The present data suggest that the natural antioxidant Bionormalizer when given orally promotes an effective protection against ethanol-induced gastric mucosal damage.

Distinct Mechanisms Underlie Neurotoxin-Mediated Cell Death in Cultured Dopaminergic Neurons

Oxidative stress is thought to contribute to dopaminergic cell death in Parkinson's disease (PD). The neurotoxin 6-hydroxydopamine (6-OHDA), which is easily oxidized to reactive oxygen species (ROS), appears to induce neuronal death by a free radical-mediated mechanism, whereas the involvement of free radicals in N-methyl-4-phenylpyridinium (MPP+) toxicity is less clear. Using free radical-sensitive fluorophores and vital dyes with post hoc identification of tyrosine hydroxylase-positive neurons, we monitored markers of apoptosis and the production of ROS in dopaminergic neurons treated with either 6-OHDA or MPP+. Annexin-V staining suggested that 6-OHDA but not MPP+-mediated cell death was apoptotic. In accordance with this assignment, the general caspase inhibitor Boc-(Asp)-fluoromethylketone only blocked 6-OHDA neurotoxicity. Both toxins exhibited an early, sustained rise in ROS, although only 6-OHDA induced a collapse in mitochondrial membrane potential temporally related to the increase in ROS. Recently, derivatives of buckminsterfullerene (C60) molecules have been shown to act as potent antioxidants in several models of oxidative stress (Dugan et al., 1997). Significant, dose-dependent levels of protection were also seen in these in vitro models of PD using the C3 carboxyfullerene derivative. Specifically, C3 was fully protective in the 6-OHDA paradigm, whereas it only partially rescued dopaminergic neurons from MPP+-induced cell death. In either model, it was more effective than glial-derived neurotrophic factor. These data suggest that cell death in response to 6-OHDA and MPP+ may progress through different mechanisms, which can be partially or entirely saved by carboxyfullerenes.