METAL-INDUCED REACTIVE OXYGEN SPECIES PRODUCTION IN CHLAMYDOMONAS REINHARDTII (CHLOROPHYCEAE)(1).

Toxic effects of metals appear to be partly related to the production of reactive oxygen species (ROS), which can cause oxidative damage to cells. The ability of several redox active metals [Fe(III), Cu(II), Ag(I), Cr(III), Cr(VI)], nonredox active metals [Pb(II), Cd(II), Zn(II)], and the metalloid As(III) and As(V) to produce ROS at environmentally relevant metal concentrations was assessed. Cells of the freshwater alga Chlamydomonas reinhardtii P. A. Dang. were exposed to various metal concentrations for 2.5 h. Intracellular ROS accumulation was detected using an oxidation-sensitive reporter dye, 5-(and-6)-carboxy-2',7'-dihydrodifluorofluorescein diacetate (H2 DFFDA), and changes in the fluorescence signal were quantified by flow cytometry (FCM). In almost all cases, low concentrations of both redox and nonredox active metals enhanced intracellular ROS levels. The hierarchy of maximal ROS induction indicated by the increased number of stained cells compared to the control sample was as follows: Pb(II) > Fe(III) > Cd(II) > Ag(I) > Cu(II) > As(V) > Cr(VI) > Zn(II). As(III) and Cr(III) had no detectable effect. The effective free metal ion concentrations ranged from 10(-6) to 10(-9)  M, except in the case of Fe(III), which was effective at 10(-18)  M. These metal concentrations did not affect algal photosynthesis. Therefore, a slightly enhanced ROS production is a general and early response to elevated, environmentally relevant metal concentrations.