Antioxidant Modulation in Response to Metal-Induced Oxidative Stress in Algal Chloroplasts O. K. Okamoto, E. Pinto, L. R. Latorre, E. J. H. Bechara, P. Colepicolo Dept. Bioquı ´mica, Instituto de Quı ´mica, Universidade de Sa ˜o Paulo, C.P. 26077 05599-970, Sa ˜o Paulo, SP, Brazil Received: 4 April 2000/Accepted: 9 July 2000 Abstract. To investigate adaptive responses to metal stress at the subcellular level, the oxidative balance in isolated chloro- plasts was evaluated for the first time in the unicellular alga Gonyaulax polyedra exposed to the toxic metals Hg 2+ , Cd 2+ , Pb 2+ , and Cu 2+ . Different antioxidant responses were verified according to the metal and model of stress applied. Cells chronically exposed to metals exhibited high activity of the antioxidant enzymes superoxide dismutase and ascorbate per- oxidase, high glutathione content, and decrease of peridinin levels, whereas no significant changes were detected for -car- otene levels. In contrast, cells subjected to acute metal stress displayed twice as much -carotene but only a slight increase in superoxide dismutase and ascorbate peroxidase activities. The correlation of acute metal treatment and oxidative stress was inferred from the higher oxygen uptake and decreased reduced glutathione pool found in treated cells. In addition, increased oxidative damage to proteins and lipids occurred mainly in cells under acute stress. Pb 2+ was the most damaging toxicant, causing protein oxidation and lipid peroxidation even at chronic treatment. These results indicate that heavy metals are able to induce oxidative stress in chloroplasts of G. poly- edra, particularly under acute conditions. Nevertheless, the maintenance of a high antioxidant capacity within chloroplasts seems to be an important strategy during acclimation of G. polyedra to chronic metal stress. By acting at the subcellular site, where oxidative stress is triggered, induction of such chloroplast antioxidants might be crucial for cell survival dur- ing exposure to heavy metals. Oxygen activation to reactive species is a constant threat to photosynthetic organisms. Chloroplasts are cell compartments highly susceptible to oxidative stress due to elevated oxygen concentration, electron flux, and presence of metal ions in their microenvironment (Foyer 1996). For instance, excessive light energy may increase the levels of excited molecules, such as triplet chlorophyll and singlet oxygen (O 2 1 g). The latter is highly electrophilic and can oxidize other molecules. In addi- tion, superoxide anion (O 2 •- ) can be generated by oxygen reduction in photosystem I (Mehler reaction). Diffusion of O 2 •- into stroma is followed by its dismutation therein to oxygen and hydrogen peroxide (H 2 O 2 ). Reaction of H 2 O 2 with reduced metal ions produces hydroxyl radical (HO • ), a strong oxidant that can react with and damage several biomolecules (Takeda et al. 1995). Furthermore, chloroplasts have a complex system of membranes rich in polyunsaturated fatty acids, which are potential targets for peroxidation (Halliwell and Gutteridge 1999). Under normal conditions, these reactive ox- ygen species (ROS)– generating processes are slow, but they can be accelerated by xenobiotics and some environmental factors, such as high light or UV exposure (Rao et al. 1996; Mackerness et al. 1999). An important source of oxidative stress in biological systems is the intoxication with heavy metals, which involve different ROS-generating mechanisms (Stohs and Bagchi 1995). Tran- sition metals, such as iron (Fe 3+ ) and copper (Cu 2+ ), partici- pate in the well-known Haber-Weiss cycle, producing HO • (Winterbourn 1982). In contrast, metals without redox capac- ity, such as cadmium (Cd 2+ ), lead (Pb 2+ ), and mercury (Hg 2+ ), can enhance the pro-oxidant status by reducing the antioxidant glutathione (GSH) pool, activating calcium-dependent systems, and affecting iron-mediated processes. These heavy metals can also disrupt the photosynthetic electron chain, leading to O 2 •- and O 2 1 g production (Asada and Takahashi 1987). Thus, tolerance of photosynthetic organisms to environmental chal- lenge must be dependent on defense responses that prevent oxidative insult within chloroplasts. The antioxidant defense system of chloroplasts includes the enzymes superoxide dismutase (SOD), ascorbate peroxidase (Apx), and low molecular weight antioxidants, such as GSH, carotenoids, and phenolics (Rice-Evans et al. 1996). Chloro- plasts contain either CuZnSOD or FeSOD isoforms, which catalyze the dismutation of O 2 •- to O 2 and H 2 O 2 , whereas Apx reduces H 2 O 2 to H 2 O using ascorbate as electron donor (Fri- dovich 1997). The soluble antioxidant GSH acts directly as a reducing agent or indirectly as co-substrate of different enzy- mic reactions. Carotenoids also prevent photodamage mainly by acting as physical quenchers of electronically excited mol- ecules, besides functioning as photoreceptors (Krinsky 1989; Woodall et al. 1997). Modulation of these antioxidants is therefore an important adaptive response to withstand adverse Correspondence to: P. Colepicolo Arch. Environ. Contam. Toxicol. 40, 18 –24 (2001) DOI: 10.1007/s002440010144 ARCHIVES OF Environmental Contamination and T oxicology © 2001 Springer-Verlag New York Inc.