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.

Organophosphorous insecticides as herbicide synergists on the green algae Pseudokirchneriella subcapitata and the aquatic plant Lemna minor

“…The second test is according to Jonker et al (2005) where positive a-values indicate antagonism while negative a-values indicate synergism. For all tests the corresponding P-value indicates whether the response surface deviates significantly from the reference model Insecticides as herbicide synergists 33 compound at the target site (Wehtje et al 1991;Richter and Escher 2005). The result of a mixture study based on growth represents a ''sum'' of all these possible interactions.…”

Combination effects of herbicides on plants and algae: do species and test systems matter?

“…Antagonism is often observed for herbicide mixtures and can have various causes. It can be caused by chemical interactions, which reduce the activity of the active compounds 36, 37. The uptake rate of one herbicide can be affected by the presence of another herbicide,38 as can the metabolisation rate, since some herbicides are known to affect the activity of enzymes active in the metabolism of other herbicides 39.…”

Tissue residue approach for chemical mixtures

“…However, there are also limits to implementing the TRA in mixture toxicity assessment. Although the mixture toxicity of reactive electrophilic chemicals can be described well by mixture toxicity concepts of CA and IA (Chen and Yeh 1996; Richter and Escher 2005), it will not be possible to use internal concentrations for these fast‐reacting chemicals as is discussed in more detail in the accompanying paper, which reviews the mechanisms and modes of toxic action in relation to the TRA (Escher et al 2011).…”