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 me mbrane vesicles (chromatophores) of the photosynthetic bacteria Rhodobacter sphaeroides. Phenols are known to destroy the electrochemical proton gradi ent in energy-transducing membranes by a protonophoric mechanism. Anisols d o not have protonophoric activity but disturb membrane structure and functi oning as a nonspecific baseline toxicant. It was postulated in the literatu re that, for certain substituted phenols, the formation of a dimer between the phenoxide and the neutral phenol may contribute significantly to the ov erall protonophoric activity. In 13 of 14 mixtures of substituted phenols b ut in none of the mixtures of phenols with anisols, such a dimer appears to be formed between two different mixture partners. An extended shuttle mech anism of uncoupling, which includes a term for the contribution of such a m ixed dimer, provided a good description of all experimental data. Opposite speciation favors interaction and ortho substituents abate interaction, whi ch adds evidence for the dimer formation via a hydrogen bond between the ph enol-OH and the phenoxide. These findings are significant not only regardin g the mechanism of protonophoric action but also for the risk assessment pr ocess of chemical mixtures in the environment. When assessing the effect of mixtures, concentration addition is regarded as a reference concept to est imate effects of similarly acting compounds. The substituted phenols in thi s work act according to the same action mechanism of uncoupling. Neverthele ss, the overall effect of four of the investigated mixtures, which exhibit stronger dimer formation as compared to the single compounds or for which t he resulting dimer is intrinsically more active, exceeded the effect calcul ated 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.