DETECTION OF PRIMARY DNA-DAMAGE IN CHLAMYDOMONAS-REINHARDTII BY MEANSOF MODIFIED MICROGEL ELECTROPHORESIS

The assessment of genotoxic potential in surface water requires test m ethods, among which are those that detect initial DNA damage in organi sms of aquatic biocenosis. The microgel electrophoresis (MGE) ''comet assay'' was applied to a ubiquitous unicellular green alga (Chlamydomo nas reinhardtii) to detect DNA damage caused by genotoxins, For this, the test protocol described by Singh NP et al. [Exp Cell Res 175:184-1 91, 1988] was modified. Major modifications were the use of alkaline l ysis buffer with ionic detergents and the reduction of preincubation a nd electrophoresis rimes. Short-time exposure of Chlamydomonas to the well-known genotoxicants 4-nitroquinoline-1-oxide (4-NQO), N-nitrosodi methylamine, and hydrogen peroxide led to dose-dependent DNA damage. C hlamydomonas responded very sensitively to treatment with increasing d oses of 4-NQO. At a concentration of 25 nM, significant DNA damage was observed. At higher 4-NQO doses (> 100 nM), DNA damage was visible as complete DNA fragmentation into fine granules. N-Nitrosodimethylamine caused genotoxic effects at a concentration range from 0.014 to 0.14 mM without producing complete DNA fragmentation at the concentrations tested (highest dose, 140 mM). To evaluate the influence of illuminati on conditions during exposure, cells were incubated with increasing do ses of H2O2 (0.25-1.0 mM) in darkness and in light. Our results indica te that incubation in light enables Chlamydomonas to cope with oxidati ve stress more efficiently than under dark conditions. To a certain ex tent, cytotoxic as well as genotoxic effects of H2O2 depend on the ill umination condition or repair and anti-oxidative protection mechanisms activated by light, respectively. (C) 1997 Wiley-Liss, Inc.