C. Di Giacomo

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.

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.

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.

Expression of bone morphogenetic protein-6 and transforming growth factor-β1 in the rat brain after a mild and reversible ischemic damage

We have examined the distribution of transforming growth factor-beta1 (TGF-beta1) and bone morphogenetic protein-6 (BMP-6) in the brain of rats subjected to a mild and reversible ischemic damage produced by a 20-min occlusion of both carotid arteries without occlusion of the vertebral arteries. We have selected this model to study how the expression of trophic factor of the TGF-beta superfamily changes in neurons that recover from a transient insult. Immunocytochemical analysis showed a loss of TGF-beta1 in neurons of all hippocampal subfields immediately after the ischemic period, followed by a recovery of immunoreactivity in CA1 and CA3 neurons after reperfusion. BMP-6 immunoreactivity was also lost in most hippocampal neurons, but immunostaining became particularly intense in the interstitial space after both ischemia and reperfusion. An interstitial localization of BMP-6 was also observed in the cerebral cortex, particularly after reperfusion. Mild ischemia also induced substantial changes in the expression of TGF-beta1 and BMP-6 within the cerebellar cortex. In control animals, these factors appeared to be localized in granule cells (TGF-beta1) and Purkinje cells (both), whereas the molecular layer was not immunopositive. Both TGF-beta1 and BMP-6 were highly expressed in the interstitial spaces of the cerebellar cortex either 20 min after ischemia or 20 min after reperfusion. Taken collectively, these results suggest that a mild and reversible ischemia stimulates the release of BMP-6 from neurons into the interstitial space. We speculate that BMP-6, besides functioning during brain development, may also regulate neuronal resistance to insults of the adult brain.

MAP2, Synaptophysin Immunostaining in Rat Brain and Behavioral Modifications after Cerebral Postischemic Reperfusion

Plasticity in the central nervous system after cerebral ischemia is a controversial issue; focal cerebral ischemia produces an area of infarction that is surrounded by neurons that may respond to nearby damage by creating new synapses. In the present study the expression of the postsynaptic microtubule-associated protein 2 (MAP2) and the presynaptic marker protein, synaptophysin, was investigated by immunocytochemical techniques in the CA1 sector of hippocampus and in cerebellum of rats made ischemic by bilateral clamping of common carotid arteries and reperfused for 7 and 30 days. In addition, ischemia-induced behavioral alterations were also evaluated after 7 and 30 days of reperfusion. The present study demonstrates a decreased postsynaptic MAP2 immunoreactivity, representative of neuronal loss, particularly in CA1 sector of hippocampus and in cerebellum of ischemic rats reperfused for 7 days. After 30 days of reperfusion, MAP2 immunostaining was similar to control. In the same brain sections an increased presynaptic synaptophysin immunoreactivity has been observed only after 30 days of reperfusion. These data suggest compensatory regenerative changes associated with synaptic remodelling and are supported by behavioral recovery observed under the same experimental conditions.

Lipid peroxidation in rat cerebral cortex during post-ischemic reperfusion: Effect of exogenous antioxidants and Ca++-antagonist drugs

Although the role of oxidant‐antioxidant metabolism in total ischemia and reperfusion in the central nervous system and cardiac myocardium have been well studied, less is known about the consequences of partial ischemic episodes. Here we show that reperfusion contributes to free radical formation as judged by conjugated diene formation. Also, antioxidants and Ca++ antagonists were able to reduce free radical formation. These results would suggest that free radical generation following ischemia and reperfusion may result from more than one injury process in cerebral cortex.

Aliphatic α,γ-bis(Amides) of Methotrexate. Influence of Chain Length on In-vitro Activity Against Sensitive and Resistant Tumour Cells

The synthesis of short-chain alkyl bis(amides) (heptyl-, isoheptyl-, octyl-) and nonylamide derivatives as lipophilic derivatives of methotrexate is reported. Direct amine substitution on methotrexate diethyl ester and a carbodiimide-assisted coupling method were used. The compounds were screened for in-vitro inhibitory activity against bovine liver dihydrofolate reductase and growth inhibition of human lymphoblastoid methotrexate-sensitive and resistant CCRF-CEM cells and erythroleukaemic K562 cells. The lipophilic methotrexate derivatives, despite showing lower activity against methotrexate-sensitive tumour cell lines, maintained similar activity to methotrexate, even against a methotrexate-transport resistant cell subline, since they can penetrate tumour cells by a passive route, by-passing the deficient cellular carrier system for the folates. Increasing the lipophilicity of methotrexate is a possible strategy to overcome the clinical resistance to the drug by tumour cells.

Fibroblast growth factor-2 and transforming growth factor-?1 immunostaining in rat brain after cerebral postischemic reperfusion

Several trophic factors are known to regulate the survival and growth of neurons in brain and peripheral tissues. Several findings suggest that basic fibroblast growth factor‐2 (FGF‐2) plays an important role in the “self‐repair” responses that follow injuries such as trauma and brain ischemia and that FGF‐2 contributes to the repair of damaged tissue. Transforming growth factor‐β (TGF‐β) is a potent growth‐regulatory protein secreted by virtually all cells. In the present study, we used immunohistochemical techniques to investigate whether FGF‐2 and TGF‐β1 participate in the healing of damaged tissue following partial brain ischemia. The profile of the observed immunoreactivities indicated that TGF‐β1 and FGF‐2 release varies between the different cerebral areas subjected to ischemic insult. Moreover, the sectorial heterogeneity of immunocytochemical response suggests that, during postischemic reperfusion, neuronal recovery may be due not only to neuron–glia interaction but also to neurochemical conditions involving inhibitory interneurons. J. Neurosci. Res. 63:136–142, 2001.