Agata Campisi

Bioflavonoids as antiradicals, antioxidants and DNA cleavage protectors

Flavonoids have recently aroused considerable interest because of their broad pharmacological activity. In fact, flavonoids have been reported to have antiviral, antiallergic, antiplatelet, anti-inflammatory and antitumoral activities. The pharmacological properties of bioflavonoids have been ascribed both to the concomitant inhibition of enzymes involved in the production of free radicals and to their free-radical scavenging and iron chelating capacity. However the antioxidant capacity of bioflavonoids due to free-radical scavenging and/or to iron chelating is still controversial. In this study, we have investigated the free-radical scavenging capacity of bioflavonoids (rutin, catechin, and naringin). In addition, the effects of these polyphenols on xanthine oxidase activity, spontaneous lipid peroxidation, and DNA cleavage were investigated. The bioflavonoids under examination showed a dose-dependent free-radical scavenging effect, a significant inhibition of xanthine oxidase activity, and an antilipoperoxidative capacity. In addition, they showed a protective effect on DNA cleavage.

L-propionyl-carnitine as superoxide scavenger, antioxidant, and DNA cleavage protector

L-Propionylcarnitine, a propionyl ester of L-carnitine, increases the intracellular pool of L-carnitine. It exhibits a high affinity for the enzyme carnitine acetyltransferase (CAT) and, thus, is readily converted into propionyl-coenzyme A and free carnitine.It has been reported that L-propionylcarnitine possesses a protective action against heart ischemia–reperfusion injury; however, the antioxidant mechanism is not yet clear. L-Propionylcarnitine might reduce the hydroxyl radical production in the Fenton system, by chelating the iron required for the generation of hydroxyl radicals. To obtain a better insight into the antiradical mechanism of L-propionylcarnitine, the present research analyzed the superoxide scavenging capacity of L-propionylcarnitine and its effect on linoleic acid peroxidation. In addition, the effect of L-propionylcarnitine against DNA cleavage was estimated using pBR322 plasmid. We found that L-propionylcarnitine showed a dose-dependent free-radical scavenging activity. In fact, it was able to scavenge superoxide anion, to inhibit the lipoperoxidation of linoleic acid, and to protect pBR322 DNA from cleavage induced by H2O2 UV-photolysis.

Nitric oxide-related toxicity in cultured astrocytes: effect of Bacopa monniera.

There is growing evidence that high concentrations of nitric oxide (NO), generated by activated astrocytes, might be involved in a variety of neurodegenerative diseases, such as Alzheimers disease, ischemia and epilepsy. It has recently been suggested that glial cells may produce NO under superoxide radical stimulation by enzyme-independent mechanism. This suggests that also natural antioxidants may have therapeutical relevance in neurodegenerative diseases. Studies of Bhattacharya et al. have evidenced that Bacopa monniera (BM) (family Scrophulariaceae), an Ayurvedic medicinal plant clinically used for memory enhancing, epilepsy, insomnia and as a mild sedative, is able to reduce the memory-dysfunction in rat models of Alzheimers disease, but the molecular mechanisms of this action are yet to be determined. In the present study, we examined the effect of a methanolic extract of BM on toxicity induced by the nitric oxide donor, S-nitroso-N-acetyl-penicillamine (SNAP), in culture of purified rat astrocytes. Our results indicate that, after 18 h of treatment, SNAP induced an increase in the production of reactive species, but did not induce the rupture of cellular membrane. Conversely, this NO donor induced a fragmentation of genomic DNA compared to control astrocytes. The extract of BM inhibited the formation of reactive species and DNA damage in a dose dependent manner. This data supports the traditional use of BM and indicates that this medicinal plant has a therapeutic potential in treatment or prevention of neurological diseases.

Propofol attenuates peroxynitrite-mediated DNA damage and apoptosis in cultured astrocytes: an alternative protective mechanism.

Background:The concentration of peroxynitrite in the brain increases after central nervous system injuries. The authors hypothesized that propofol, because of its particular chemical structure, mitigates the effects of peroxynitrite-mediated oxidative stress and apoptosis by the induction of heme oxygenase (HO)-1 in primary cultured astroglial cells. Methods:Primary cultured astroglial cells were incubated for 18 h with a known peroxynitrite donor (3 mm SIN-1) in the presence or absence of propofol (40 &mgr;m, 80 &mgr;m, 160 &mgr;m, and 1 mm). The protective effects of propofol were evaluated by 3(4,5-dimethyl-thiazol-2-yl)2,5-diphenyl-tetrazolium bromide cytotoxicity assay, lactic dehydrogenase release, DNA ladderization by Comet assay, and caspase-3 activation by Western blot analysis. Results:Appropriate propofol concentrations (ranging from 40 &mgr;m to 1 mm) significantly increased HO-1 expression and attenuated SIN-1–mediated DNA ladderization and caspase-3 activation. The protective effects of propofol were mitigated by the addition of tin mesoporphyrin, a potent inhibitor of HO activity. The addition of a specific synthetic inhibitor of nuclear factor &kgr;B abolished propofol-mediated HO-1 induction, suggesting a possible role of this nuclear transcriptional factor in our experimental conditions. Conclusions:The antioxidant properties of propofol can be partially attributed to its scavenging effect on peroxynitrite as well as to its ability to increase HO-1 expression at higher concentrations, a property that might be relevant to neuroprotection during anesthesia.

Effect of acetyl-l-carnitine on lipid peroxidation and xanthine oxidase activity in rat skeletal muscle

It has been reported that acetyl-l-carnitine (AcCn) can reduce the degenerative processes in the central nervous system of rats, modify the fluidity of membranes and decrease the accumulation of lipofuscins in neurones. In light of these considerations we have assayed the in vitro effect of acetyl-l-carnitine on spontaneous and induced lipoperoxidation in rat skeletal muscle; in addition, the effect of AcCn on XD/XO ratio was evaluated. The presence of AcCn (10–40 mM) in incubation medium significantly reduced MDA and conjugated diene formation in rat skeletal muscle; moreover, a significant decrease in induced MDA levels was observed when microsomal preparation where incubated in the presence of 10–40 mM AcCn. Since a significant reduction of XO activity was detected in the presence of 10–80 mM AcCn, the reduced lipid peroxidation by AcCn seems to be due to an inhibition of XO activity.

Reactive oxygen species levels and DNA fragmentation on astrocytes in primary culture after acute exposure to low intensity microwave electromagnetic field

The exposure of primary rat neocortical astroglial cell cultures to acute electromagnetic fields (EMF) in the microwave range was studied. Differentiated astroglial cell cultures at 14 days in vitro were exposed for 5, 10, or 20min to either 900MHz continuous waves or 900MHz waves modulated in amplitude at 50Hz using a sinusoidal waveform and 100% modulation index. The strength of the electric field (rms value) at the sample position was 10V/m. No change in cellular viability evaluated by MTT test and lactate dehydrogenase release was observed. A significant increase in ROS levels and DNA fragmentation was found only after exposure of the astrocytes to modulated EMF for 20min. No evident effects were detected when shorter time intervals or continuous waves were used. The irradiation conditions allowed the exclusion of any possible thermal effect. Our data demonstrate, for the first time, that even acute exposure to low intensity EMF induces ROS production and DNA fragmentation in astrocytes in primary cultures, which also represent the principal target of modulated EMF. Our findings also suggest the hypothesis that the effects could be due to hyperstimulation of the glutamate receptors, which play a crucial role in acute and chronic brain damage. Furthermore, the results show the importance of the amplitude modulation in the interaction between EMF and neocortical astrocytes.

Lipid peroxidation and antioxidant enzymatic systems in rat retina as a function of age

In the present study, we have assayed the enzymatic activity of Cu,Zn−SOD, Mn−SOD, GSH−Px, GSH-Red, Cat, and G6PD in rat retina as a function of age. Conjugated diene levels and MDA formation were also determined. The conjugated diene levels in rat retina were found to increase significantly with age, accompanied by a marked decrease in GSH−Px and Cat activities. No agerelated change in MDA levels and in GSH-Red and G6PD activity was found, whereas a significant increase in SOD activity was observed between 1 and 4 months. Decreased GSH−Px and Cat activity is related to increased lipid peroxidation with age.

Free radical scavenger depletion in post-ischemic reperfusion brain damage

In the present study the influence of pretreatment with various GSH depletors such as buthionine sulfoximine (BSO) and diethylmaleate (DEM) was investigated in rats following cerebral postischemic reperfusion. Moreover, the effect of diethyldithiocarbamic acid (DDC), inhibitor of endogenous Cu,Zn-SOD, was evaluated. A significant depletion (40% of control value) of GSH levels was observed 24 h after DEM administration; after 48 h the value reached control levels. BSO showed maximal GSH depletion (59%) 24 h after administration and it was constant for almost 48 h. DDC administration caused a marked decrease (60%) of Cu,Zn-SOD activity 4 h after the injection and induced a marked decrease in percentage of survival with respect to control (untreated, ischemic) rats, when administered 4 h before ischemia. BSO and DEM prolonged the survival time of animals when administered 24 h before ischemia. This last paradoxical effect is unclear at present, but it might be due to an influence on glutamate cascade.

Antioxidant activity and protective effect on DNA cleavage of extracts from Cistus incanus L. and Cistus monspeliensis L.

The genus Cistus includes many typical species of Mediterranean flora; Cistus species are used as antidiarrheics, as general remedies for treatment of various skin diseases in folk medicine and as anti-inflammatory agents. These species contain flavonoids that are considered to be chain-breaking antioxidants. In this work, we have investigated the effects of crude aqueous extracts from Cistus incanus and Cistus monspeliensis on DNA cleavage and their free-radical scavenging capacity. In addition, their effect on lipid peroxidation in rat liver microsomes was evaluated. These extracts showed a protective effect on DNA cleavage and a dose-dependent free-radical scavenging capacity; Cistus monspeliensis was more active than Cistus incanus; these results were confirmed by a significant inhibition of lipid peroxidation in rat liver microsomes. The experimental evidence, therefore, suggests that because of their antioxidant activity these extracts may offer excellent photoprotection for skin and may be useful in the treatment of human diseases where oxidative stress plays a key role.

In vitro evaluation of idebenone-loaded solid lipid nanoparticles for drug delivery to the brain.

Context: Solid lipid nanoparticles (SLN) are regarded as interesting drug delivery systems and their preparation techniques have gained a great deal of attention. Objective: To evaluate the feasibility of preparing idebenone (IDE) loaded SLN from O/W microemulsions by the phase-inversion temperature (PIT) method. Since SLN have been proposed to improve drug delivery to the brain, IDE was chosen as model drug due to its activity in the treatment of neurodegenerative diseases. Materials and Methods: Cetyl palmitate was used as solid lipid to prepare SLN containing two surfactant/cosurfactant mixtures, isoceteth-20/glyceryl oleate (SLN A) and ceteth-20/glyceryl oleate (SLN B) by the PIT method. Results and discussion: All the formulations tested showed a mean particle diameter ranging from 30 to 95 nm and a single peak in size distribution. Stability tests showed that SLN B were more stable than SLN A. IDE release was dependent both on the type of primary surfactant used and the amount of loaded drug. IDE-loaded SLN were effective in inhibiting 2,2′-azobis-(2-amidinopropane)dihydrochloride (APPH)-induced lactic dehydrogenase (LDH) release and reactive oxygen species (ROS) production in primary cultures of astrocytes obtained from rat cerebral cortex. It is noteworthy that SLN B2 (containing ceteth-20 as primary surfactant and 0.7% w/w IDE) were able to prevent entirely both the LDH release and ROS production induced by APPH. Conclusion: The PIT method provided SLN with good technological properties. The tested SLN could be regarded as interesting carriers to overcome the blood brain barrier and increase the efficacy of the loaded drug.