title: Mixture toxicity of reactive chemicals by using two bacterial growth assays as indicators of protein and DNA damage.
abstract: The mixture toxicity of reactive chemicals was investigated with a set of bioanalytical tests that quantify not only the toxic effects but also allow the identification of the preferred target of reactive chemicals in bacterial cells. Softer electrophiles such as acrylates react preferentially with thiol groups in proteins and peptides, and harder electrophiles such as epoxides preferentially attack DNA. In addition, some compounds, e.g., benzyl chloride, have no preference for a biological target and damage both DNA and proteins. A thiophenol was used as a model compound representing nucleophiles. We explored if the paradigms of mixture toxicity also hold true for reactive chemicals. Compounds with the same targets and the same modes of action should act concentration additive in mixtures, and compounds with different modes of action should act according to the concept of independent action. In addition, we investigated the potential for interaction of compounds of mixtures of electrophiles or electrophiles plus nucleophiles, which might lead to synergistic or antagonistic effects. The toxicity of mixtures of electrophiles with a single preferred target was consistent with the prediction for concentration addition. Unfortunately, the predictions for independent action did not differ much from those for concentration addition; therefore it was not possible to differentiate between these two models. Mixtures of two groups with different preferred target sites clearly showed concentration addition. In contrast, mixtures of compounds with multiple targets, i.e., compounds that show nonspecific reactivity toward any biological nucleophile, exhibited effects that lay distinctly between the predictions for concentration addition and independent action. We observed neither synergism (higher toxicity than predicted by concentration addition) nor antagonism (lower toxicity than predicted by independent action) for mixtures of electrophiles. Binary combinations of different electrophiles with the nucleophile 4-chlorothiophenol yielded smaller effects than those expected from the prediction for independent action. The degree of antagonism was correlated with the reaction rate constant of the electrophile with the thiol group of glutathione, which indicates that the interaction between the mixture components occur in the toxicokinetic phase and is purely a result of chemical reactivity between the mixture components. Overall, we conclude that the concepts of mixture toxicity apply not only for baseline toxicity and receptor-mediated mechanisms, as has been shown in a large number of studies, but also for reactive mechanisms of toxicity, provided that one has checked beforehand that no chemical reactions occur between the mixture components.
authors.affiliation: Department of Environmental Toxicology, Swiss Federal Institute for Aquatic Sciences and Technology Eawag, CH-8600 Dübendorf, Switzerland.
authors.forename: Manuela
authors.initials: M
authors.lastname: Richter
authors.forename: Beate I
authors.initials: BI
authors.lastname: Escher
mesh_terms: D000179:Acrylates / Q000633:toxicity; D001426:Bacterial Proteins / Q000096:biosynthesis; D001681:Biological Assay / Q000379:methods; D002623:Chemistry Techniques, Analytical; D004249:DNA Damage / Q000187:drug effects*; D004269:DNA, Bacterial / Q000235:genetics; D004336:Drug Antagonism; D004357:Drug Synergism; D004784:Environmental Monitoring / Q000379:methods*; D004852:Epoxy Compounds / Q000633:toxicity; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D005978:Glutathione / Q000096:biosynthesis; D007202:Indicators and Reagents / Q000633:toxicity*; D018675:Toxicity Tests / Q000379:methods*
chemical_list: D000179:Acrylates; D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004852:Epoxy Compounds; D007202:Indicators and Reagents; D005978:Glutathione