509 Environmental Toxicology and Chemistry, Vol. 27, No. 3, pp. 509–518, 2008  2008 SETAC Printed in the USA 0730-7268/08 $12.00 + .00 MIXTURE TOXICITY AND GENE INDUCTIONS: CAN WE PREDICT THE OUTCOME? FREDDY DARDENNE,* INGRID NOBELS,WIM DE COEN, and RONNY BLUST Ecophysiology, Biochemisty, and Toxicology Group, Department of Biology, University of Antwerp, Groenenborgerlaan 171/U7, B-2020 Antwerp, Belgium ( Received 15 May 2007; Accepted 21 August 2007) Abstract—As a consequence of the nature of most real-life exposure scenarios, the last decade of ecotoxicological research has seen increasing interest in the assessment of mixture ecotoxicology. Often, mixtures are considered to follow one of two models, concentration addition (CA) or response addition (RA), both of which have been described in the literature. Nevertheless, mixtures that deviate from either or both models exist; they typically exhibit phenomena like synergism, ratio or concentration dependency, or inhibition. Moreover, both CA and RA have been challenged and evaluated mainly for acute responses at relatively high levels of biological organization (e.g., whole-organism mortality), and applicability to genetic responses has not received much attention. Genetic responses are considered to be the primary reaction in case of toxicant exposure and carry valuable mechanistic information. Effects at the gene-expression level are at the heart of the mode of action by toxicants and mixtures. The ability to predict mixture responses at this primary response level is an important asset in predicting and understanding mixture effects at different levels of biological organization. The present study evaluated the applicability of mixture models to stress gene inductions in Escherichia coli employing model toxicants with known modes of action in binary combinations. The results showed that even if the maximum of the dose–response curve is not known, making a classical ECx (concentration causing x% effect) approach impossible, mixture models can predict responses to the binary mixtures based on the single-toxicant response curves. In most cases, the mode of action of the toxicants does not determine the optimal choice of model (i.e., CA, RA, or a deviation thereof ). Keywords—Mixture Escherichia coli Stress gene Concentration addition Response addition INTRODUCTION Real-life exposure scenarios of ecosystems and humans to toxicants almost exclusively involve mixtures of chemicals. Nonetheless, until recently, traditional ecotoxicology has con- centrated on obtaining a better understanding of the mecha- nisms underlying the bioavailability of single compounds and their toxic effects on organisms. Today, most studies still focus on the impact of pure chemicals on the different levels of biological organization [1], sometimes taking into account the presence of factors modulating bioavailability [2–6], or they assess the impact of the whole environmental exposure without examining the activity of the individual compounds [7]. The former approach clearly adds to our understanding about the environmental impact of pollution, but it is not able to fully resolve the invariable complexity of environmental exposure, in which a multitude of physicochemical factors act together. Mixture toxicity is a complex issue and remains poorly un- derstood. This is illustrated by the fact that most of today’s en- vironmental legislation is based on risk assessment of single com- pounds or on the concentration addition (CA) model, which has only limited validity. Mixture toxicity can be influenced by dif- ferent interactions of the compounds composing the mixture and the cellular or organismal system. Chemical reactions between different compounds can directly alter the toxicity of one or more constituents of the mixture, whereas interference with cellular systems, such as uptake, transport, and receptor binding, can cause toxicants in a mixture to react in, for example, an additive, syn- ergistic, potentiating, or inhibiting manner. It generally is accepted that two models, CA and independent action (IA; or response addition [RA]), have a broader application and are suited to predict mixture toxicity from the toxicities of * To whom correspondence may be addressed (gosia.freddy@scarlet.be). Published on the Web 11/5/2007. the single toxicants. First described in 1926 [8], CA is applied when the chemicals in the mixture act through the same cellular mechanism on the same target (i.e., have the same mode of action). In this case, the effect of the mixture can be predicted from the known toxic units (i.e., the concentration of a compound divided by its x% effect concentration [ECx]) of all compounds in the mixture. Hence, given two compounds with the same tox- icological mode of action, one can replace the other in the mixture without having an effect on the overall toxicity—that is, the sum of all toxic units in the mixture equals one (see Eqn. 1). Thus, the basis of the CA model is n c i = 1 (1)  ECx i=1 i where c i equals the concentration of compound i and ECx i represents the x% effect concentration for compound i. The alternative model of IA [9] is valid for mixtures of toxicants with different modes of action and different targets, assuming no overlap or influence between each other. In this case, the overall effect of the mixture can be calculated from the effects of the individual toxicants at their respective con- centrations (Eqn. 2). This, the basis of the IA model is n E (c ) = 1 - [1 - E (c )] (2)  mix i i=1 where E(c mix ) equals the total effect of the mixture and E(c i ) is the effect of compound i. Note that both models assume the mixture under study is fully described in its chemical composition and that the (com- plete) dose–response curves of all compounds in the mixture are known. Both models are extensively covered in the liter- ature both theoretically [10] and as applied to different cases studies [11–15]. The scientific community has discussed which of both models would be the most widely applicable and how the best model can be chosen based on the specific traits of