Milfred Seccia

PROTECTIVE EFFECT OF DEHYDROEPIANDROSTERONE AGAINST COPPER-INDUCED LIPID PEROXIDATION IN THE RAT

This study investigates the effectiveness and multitargeted activity of dehydroepiandrosterone (DHEA) as antioxidant in vivo. A single dose of DHEA was given IP to male rats. Liver and brain microsomes, and plasma low density lipoprotein (LDL), were isolated from rats sacrificed 17 h later. Liver and brain microsomes were challenged with CuSO(4) and, as index of lipid peroxidation, the production of thiobarbituric acid reactive substances (TBARS) was measaured. Also, plasma low-density lipoprotein (LDL) were challenged with copper and the time course of lipid peroxidation was evaluated following the formation of conjugated dienes. The onset of TBARS generation induced by copper was marked delayed in both liver and brain microsomes from DHEA-treated animals. Also, the resistance of LDL to oxidation, expressed by the duration of the lag-phase of the kinetic curve, was significantly enhanced in DHEA-treated rats. Results indicate that in vivo DHEA supplementation makes subcellular fractions isolated from different tissues and plasma constituents (LDL) more resistant to lipid peroxidation triggered by copper. The antioxidant effect on plasma LDL might be of special relevance to the proposed antiatherogenic activity of DHEA. Moreover, multitargeted antioxidant activity of DHEA might protect tissues from oxygen radicals damage.

The dynamic reduction of Cu(II) to Cu(I) and not Cu(I) availability is a sufficient trigger for low density lipoprotein oxidation.

Copper (II) is one of the most widely employed experimental models to induce in vitro low density lipoprotein (LDL) oxidation. It is generally assumed that Cu(I), generated from active reduction of Cu(II), is the real trigger for the peroxidation of polyunsaturated fatty acids in LDL. We have employed isolated human LDL challenged with Cu(II) and the Cu(I) chelator bathocuproine disulfonic acid (BC) to test the validity of this hypothesis. At lower Cu(II)/LDL molar ratios, BC completely inhibited copper-mediated LDL oxidation evaluated either as thiobarbituric acid reactive substances (TBARS) production or changes in apo B100 electrophoretic mobility. On the contrary, at higher Cu(II)/LDL molar ratios, BC did not prevent LDL oxidation but rather markedly stimulated both the generation of TBARS and the increase of apo B100 electronegativity. These oxidative modifications were completely prevented by the Cu(II) chelator EDTA. Furthermore, the BC-Cu(I) complex alone was neither redox active nor active inducer of TBARS generation. These findings indicate that, under these experimental conditions, [1] Cu(I) is not necessary to promote LDL oxidation, [2] the availability of Cu(II) is a prerequisite and [3] some of the reaction(s) involved in Cu(II) reduction may play an essential role in initiating LDL oxidation.

Different mechanisms are progressively recruited to promote Cu(II) reduction by isolated human low-density lipoprotein undergoing oxidation.

The kinetics of Cu(II) reduction and its relationship to the process of low density lipoprotein (LDL) oxidation were investigated in isolated human LDL incubated with CuSO4 by using the Cu(I) chelator and indicator dye bathocuproine disulfonate (BC). The inclusion of BC in the incubation medium containing isolated LDL and different concentrations of CuSO4 revealed a biphasic kinetics of Cu(II) reduction consisting of an early phase followed by a plateau phase and a subsequent extensive reduction phase. The amount of Cu(I) formed during the early phase, as well as the rate of its generation, were strictly dependent on both the level of Cu(II) available (saturation was observed at 20 and 50 microM CuSO4) and the concentration of alpha-tocopherol within native LDL particles. Artificial enrichment of LDL with different concentrations of alpha-tocopherol led to a parallel increase of both the amount of Cu(II) reduced and the rate of reduction. The late phase of Cu(II) reduction was strictly related to the availability of copper but was largely independent from alpha-tocopherol. Neither the amount of Cu(I) generated nor the rate of generation were saturated at concentrations of copper up to 100 microM. Comparable results were obtained by adding BC at different time-points to the LDL-copper mixture, in order to measure at the same time-points both the true rate of Cu(II) reduction and the generation of TBARS during the dynamic process of LDL oxidation. The rate of Cu(II) reduction was already high during the lag-phase of the LDL oxidation profile and progressively decreased as alpha-tocopherol concentration decreased. The subsequent increase in the rate of Cu(II) reduction paralleled the formation of TBARS during the extensive LDL oxidation phase. These results suggest that different mechanisms of Cu(II) reduction, namely alpha-tocopherol-dependent and independent (likely lipid peroxide-dependent), are progressively recruited during copper-promoted LDL oxidation.

Increased susceptibility to transglutaminase of eye lens proteins exposed to activated oxygen species produced in the glucose-glucose oxidase reaction.

In order to test whether a mild oxidative stress could promote the transglutaminase damaging effect on eye lens proteins, total lens soluble proteins and purified beta L-crystallin have been exposed to H2O2 slowly produced by the glucose-glucose oxidase reaction. Soon after the pretreatment, the substrate capacity of the lens proteins for an exogenous transglutaminase has been evaluated. Exposure to the oxidative stress increased the susceptibility of the lens proteins to transglutaminase. When ferrous ions were added to the preincubation medium, in order to convert the H2O2 into the hydroxyl radical, the increase was more evident.