Mechanistic approaches for evaluating the toxicity of reactive organochlorines and epoxides in green algae.

Reactive electrophilic chemicals, such as reactive organochlorine compounds or epoxides, react specifically with a broad spectrum of nucleophilic biomolecules, including proteins and DNA. Conventional toxicity tests for algae, involving the observation of growth inhibition, i.e., the inhibition of cell multiplication, after several days, yield unreliable information for risk assessment because reactive compounds hydrolyze to different extents during the exposure period. The diversity of their modes of toxic action further complicates effect assessment and calls for methods yielding additional information on the mechanisms of toxicity. One of the primary targets of reactive chemicals in cells is the tripeptide glutathione (GSH), which is important for detoxification but can also be regarded as a toxicity sensor because changes in glutathione levels indicate stress. A vital system for algae is the photosynthetic system, which is indirectly affected by reactive chemicals. The test systems developed in this study for the assessment of reactive toxicity toward algae were therefore based not only on nonspecific toxicity indicators like growth inhibition but also on indicators for disturbance of photosynthesis (inhibition of photosystem II quantum yield) and glutathione metabolism. The application of the developed test systems on Scenedesmus vacuolatus after short-term exposure of 2 h showed that these tests can be used as fast screening tests for algal toxicity and in mode-of-action-based test batteries.

Applicability and limitation of OSARs for the toxicity of electrophilic chemicals.

The appropriate selection and application of quantitative structure-activity relationships (QSARs) for the prediction of toxicity is based on the prior assignment of a chemical to its mode of toxic action. This classification is often derived from structural characteristics with the underlying assumption that chemically similar compounds have similar mechanisms of action, which is often but not necessarily the case. Instead of using structural characteristics for classification toward a mode of toxic action, we used Escherichia coli based bioanalytical assays to classify electrophilic chemicals. Analyzing a series of reactive organochlorines, epoxides, and compounds with an activated double bond, three subclasses of reactive toxicity were distinguished: "glutathione depletion-related toxicity", "DNA damage", and "unspecific reactivity". For both subsets of specifically reacting compounds a direct correlation between effects and chemical reactivity was found. Reaction rate constants with either glutathione or 2'-deoxyguanosine, which was used as a model for complex DNA, served well to set up preliminary QSARs for either glutathione depletion-related toxicity or toxicity based on DNA damage in the model organism E. coli. The applicability of QSARs for electrophilic chemicals based on mechanistically relevant reaction rate constants is a priori limited to a small subset of compounds with strictly identical mechanism of toxic action and similar metabolic rates. In contrast, the proposed bioanalytical assays not only allowed the experimental identification of molecular mechanisms underlying the observable toxicity but also their toxicity values are applicable to quantitatively predict toxic effects in higher organisms by linear correlation models, independent of the assigned mode of toxic action.

Evaluation of bioanalytical assays for toxicity assessment and mode of toxic action classification of reactive chemicals.

The toxicity of electrqphiles, including reactive organochlorines, epoxides, and compounds with an activated double bond was investigated. A set of different bioanalytical assays based on genetically modified Escherichia coli strains was set up to quantify cytotoxicity and specific reactivity toward the important biological nucleophiles DNA and glutathione (GSH). The significance of GSH for detoxification was assessed by cellular GSH depletion as well as by growth inhibition of a GSH-deficient strain. Tests for DNA damage comprised the measurement of induction of DNA repair systems, DNA fragmentation, and growth inhibition of a strain deficient in major DNA repair mechanisms. The most suitable assays for detection of mechanisms that underlie the observable cytotoxicity of the tested electrophiles were two sets of strains either lacking GSH or DNA repair in combination with their corresponding parent strains. Comparison of toxicity observed in those strains suggests three clearly distinguishable modes of toxic action for electrophilic chemicals: "DNA damage", "GSH depletion-related toxicity", and "unspecific reactivity". The class of chemicals causing DNA damage includes the epoxides 1,2-epoxybutane, (2,3-epoxypropyl)benzene, and styrene oxide. The class of chemicals with GSH depletion-related toxicity includes compounds with an activated double bond, like acrylates and acrolein. All reactive organochlorines and some epoxides were classified as unspecifically reactive because their toxicity is initiated by reactions with both biological nucleophiles. The work presented here is a contribution for an alternative hazard and effect assessment of organic pollutants based on mode of toxic action classification.