Time Dependence in Mixture Toxicity with Soft Electrophiles: 1. Combined Effects of Selected SN2- and SNAr-Reactive Agents with a Nonpolar Narcotic

Abstract

Frequently the toxicity of an organic chemical mixture is close to dose-additive, even when the agents are thought to induce toxicity at different molecular sites of action. These findings appear to conflict with the hypothesis that a strictly dose-additive combined effect will be observed for agents sharing a single molecular site of toxic action within the organism. In this study, several SN2-reactive (alpha-halogen) or S(N)Ar-reactive (halogenated dinitrobenzene) soft electrophiles were tested with a model nonpolar narcotic (NPN) to determine the toxicity of the combinations. A sham combination of the model NPN (3-methyl-2-butanone) was also tested as a positive control. The study design incorporated time-dependent toxicity (TDT) determinations at 15, 30, and 45 minutes using a Microtox (Vibrio fischeri) protocol that included testing seven duplicated concentrations for each single agent and mixture per combination. Additionally, in chemico reactivity was determined for each compound using thiol in glutathione as a model nucleophile. The model NPN alone lacked reactivity and TDT. The SN2-reactive agents individually showed varying levels of both reactivity and TDT alone, while the SNAr-reactive chemicals alone were reactive and had toxicity that was fully time-dependent between 15 and 45 minutes of exposure. Data analyses indicated that the sham combination was dose additive, as expected, whereas three of four SN2:NPN combinations showed effects close to that predicted for dose addition but with some differences. The fourth SN2:NPN combination, which included an alpha-halogen with full TDT, showed a less-than-dose-additive combined effect as did both of the SNAr:NPN pairings. By incorporating TDT values, shapes of the dose-response curves, chemical reactivity data with thiol, reactive mechanisms for the soft electrophiles, and quantitative structure activity relationship information on whether the toxicity of the individual soft electrophiles did or did not exceeded that predicted for baseline narcosis, the results suggested that the alpha-halogens elicited two toxic effects at the concentrations tested (reactivity and narcotizing effects), whereas toxicity induced by the halogenated dinitrobenzenes was essentially limited to reactive effects. Collectively, these results provide experimental evidence consistent with previous explanations as to why binary mixtures of industrial organic chemicals often show combined effects that are close to dose additive, even when the chemicals are thought to induce toxicity at different molecular sites of action.