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Dr Enouiu stayed as a long term IFCC Scinetific Exchange
scholar, September 1998 - August 1999, in the Centre du M�dicament
Nancy ( Head Prof G Siest). She worked under the responsability of
Dr Maria Wellman.
Oxidative damage of biomolecules (proteins, lipids, ADN) caused
by free radicals is involved in pathogenesis of different diseases
like cancer, atherosclerosis, inflamation, etc. Glutathione (GSH),
the major intracellular non protein thiol, is mainly known as an
important protector against free radical damage by providing
reducing equivalents for several key antioxidant enzymes and also
by scavenging hydroxyl radicals and singlet oxygen. However, it has
been reported that the GSH metabolism by
gamma-glutamyltranspeptidase (GGT) in the presence of iron leads to
reactive oxygen species (ROS) generation, by the autoxidation of
the metabolite of GSH, cysteinylglycine (CysGly) (Stark et al,
1993, Paolicchi et al, 1997). We proposed to study in vitro the
pro-oxidant role of GGT/GSH and the oxidative modifications that it
induces on proteins and lipids, known vulnerable targets of free
radical attack. The measurement of ROS generation in the presence
of different thiols and iron, by using dihydrorhodamine 123 (DHR
123), a leucodye nonspecifically oxidized by ROS to fluorescent
rhodamine 123, showed an antioxidant role of glutathione and a
pro-oxidant role of cysteine and CysGly. On the other hand,
cleavage of g-glutamyl moiety of GSH by GGT initiated the ROS
production in the presence of chelated iron. In analogy with GGT,
we hypothesized that GGT-rel, a distinct g-glutamyl cleavage enzyme
have also pro-oxidant properties. Using a HPLC method for thiols
dosage we showed that 3T3/GGT-rel transfected cell line metabolize
extracellular GSH to CysGly, and that induces a ROS production in
the presence of chelated iron. We studied the consequences of the
GGT/GSH generated oxidatif stres in the model of recombinant
apolipoprotein E (apo E) oxidation (Jolivalt et al, 1996). We found
a diminution of immunoaffinity towards specific antibody (by
Western Blotting technique) and a modification of HPLC profile of
apo E samples submitted to GGT/GSH/Fe3+ system. However, these
results do not allow to conclude an oxidative modification of
protein structure and identification of observed modifications by
analytical chemistry techniques remains quite difficult. We also
studied the lipid peroxidation (LPO) of polyunsaturated fatty acids
induced by GGT/GSH/Fe3+. LPO secondary aldehydic products, derived
as 2,4-dinitrophenylhydrazones were separated on TLC, HLPC and
analyzed in mass spectrometry. By these techniques we identified a
great complexity of carbonyl products (alkanals, alkenals,
alkadienals, hydroxyalkenals and dialdehydes), most of them being
similar to those produced in a known model for LPO
(Fe2+/ascorbate). We found a significant increase (1.3 to 5 fold)
for different carbonyl compounds in the samples containing GGT in
the oxidation mixture, as compared to the control, that clearly
proves the role of GGT in lipid peroxidation. In addition,
generation of highly reactive 4-hydroxyalkenals (such as
4-hydroxynonenal) with known toxic effects on cell membranes and
functions (Comporti, 1998) suggests that the GSH metabolism by GGT
in the presence of iron might represent a biological way for a LPO
toxic process. The present data justify a more detailed study of
GGT/GSH oxidant action on serum lipoproteins and membrane
phospholipids in order to correlate with mechanisms of
atherogenesis and carcinogenesis. In conclusion, our in vitro
studies demonstrate that the metabolism of GSH by GGT (or GGT-rel)
leads to ROS generation and oxidation of lipids and must be taken
into account as one of the physiopathological oxidation system.
Further studies will focus on the importance of GGT/GSH in the
pro-oxidant/antioxidant balance with particulary attention on its
involvement in the pathologies of cardiovascular system.
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