Characterization of biomolecules modified by oxidative mechanisms, especially via singlet oxygen, in chemical and biological systems

Abstract

Aerobic metabolism and various toxicological and pharmacological reactions can generated reactive oxygen species capable of damaging biological molecules and triggering deleterious side effects. To counteract these effects, the cell is equipped with a set of enzymatic and chemical antioxidant defense lines. A balance between pro-oxidants and antioxidants constitutes the normal condition of aerobic life; an imbalance in favor of the pro-oxidants is termed "oxidative stress". Singlet molecular oxygen (1O2) has been shown to be generated in biological systems and can be implicated in defense mechanisms against viruses and bacteria by phagocytic cells. Dark reactions (chemiexcitation) such as reactions catalyzed by peroxidases (myeloperoxidase) or oxygenases (lipoxygenase or cyclooxygenase), or the reaction of hydrogen peroxide with hypochlorite or peroxynitrite, or thermodecomposition of dioxetanes can be responsible for singlet oxygen generation in biological systems. Singlet oxygen is one of the major species mediating cytotoxic effects of photodynamic treatment. In addition to these cytotoxic effects, 1O2 can be responsible for ultraviolet A-induced activation of gene expression. Biological targets for 1O2 include unsaturated fatty acids, proteins, and DNA. However, unraveling the role of 1O2 in biological systems has been hampered by the difficulties to obtain 1O2 free from further reactive species. Our studies focus on providing the mechanism by which singlet oxygen and other reactive oxygen species play their physiological and pathological roles. We have been devoted to develop suitable 1O2 generators based on the thermolysis of endoperoxides. These compounds are chemically inert and have been employed as versatile sources of 1O2. This approach has been used in our studies for the detection of 1O2 induced damage in cells (DNA, lipids, proteins) and for screening of biologically occuring compounds for quenching 1O2. The purpose of the present project is to extend our understanding of the reactions between reactive oxygen species and biomolecules in vitro and in vivo emphasizing the following aspects: 1) synthesis of the endoperoxide derivates; 2) identification and quantification of 1O2 generated by the thermal decomposition of endoperoxides; 3) DNA damage in vitro and in cells induced by 1O2 generated by endoperoxides and by photosensitization of tetraruthenated porphyrins; 4) screening of naturally occuring compounds such as carotenoids, tocopherols, thiols, flavonoids for their ability to quench 1O2 and protect DNA damage in vivo and in cells; 5) chemical characterization of biomolecule damage induced by oxidative stress involving pathology; 6) mutagenesis by 5-aminolevulinic acid (ALA). (AU)