Interaction of phenolic uncouplers in binary mixtures: concentration-additive and synergistic effects.

The uncoupling activities of 14 binary mixtures of substituted phenols and of 4 binary mixtures of phenols and anisols were investigated at different pH values. Experiments were performed with time-resolved spectroscopy on membrane vesicles (chromatophores) of the photosynthetic bacteria Rhodobacter sphaeroides. Phenols are known to destroy the electrochemical proton gradient in energy-transducing membranes by a protonophoric mechanism. Anisols do not have protonophoric activity but disturb membrane structure and functioning as a nonspecific baseline toxicant. It was postulated in the literature that, for certain substituted phenols, the formation of a dimer between the phenoxide and the neutral phenol may contribute significantly to the overall protonophoric activity. In 13 of 14 mixtures of substituted phenols but in none of the mixtures of phenols with anisols, such a dimer appears to be formed between two different mixture partners. An extended shuttle mechanism of uncoupling, which includes a term for the contribution of such a mixed dimer, provided a good description of all experimental data. Opposite speciation favors interaction and ortho substituents abate interaction, which adds evidence for the dimerformation via a hydrogen bond between the phenol-OH and the phenoxide. These findings are significant not only regarding the mechanism of protonophoric action but also for the risk assessment process of chemical mixtures in the environment. When assessing the effect of mixtures, concentration addition is regarded as a reference X concept to estimate effects of similarly acting compounds. The substituted phenols in this work act according to the same action mechanism of uncoupling. Nevertheless, the overall effect of four of the investigated mixtures, which exhibit stronger dimer formation as compared to the single compounds or for which the resulting dimer is intrinsically more active, exceeded the effect calculated according to concentration addition considerably. In future work, this synergistic effect observed in-vitro has to be validated in-vivo to deduce its implications for the risk assessment process.