Received: 28 January 2017 Revised: 7 March 2017 Accepted: 12 March 2017 DOI: 10.1002/jbt.21924 A reappraisal of the proposed metabolic and antioxidant actions of butylated hydroxytoluene (BHT) in the liver Lorena dos Santos Castro Lívia Bracht Jurandir Fernando Comar Rosane Marina Peralta Adelar Bracht Department of Biochemistry, University of Maringá, Maringá 87020900, Brazil Correspondence Adelar Bracht Abstract Butylated hydroxytoluene (BHT) was investigated for its metabolic actions in the perfused rat liver. Contrary to what is expected from an uncoupler, BHT up to 500 M did not stimulate oxy- gen uptake nor did it inhibit gluconeogenesis from lactate. Transformation of fructose into glucose was also not affected by BHT; only lactate production was slightly increased at the concentra- tion of 100 M. The uncoupling effect of BHT in isolated mitochondria was confirmed, but only at concentrations above 10 M; uncoupling at lower concentrations, 10 -9 to 10 -6 M, could not be confirmed. BHT, however, increased reactive oxygen species (ROS) production in isolated mito- chondria, starting at the concentration of 10 -8 M. This is the opposite of what can be expected from a compound with proven ex vivo antioxidant action. One cannot exclude the possibility that, in mitochondria, stimulation of ROS production rather than uncoupling could be the most signifi- cant effect of BHT. KEYWORDS energy metabolism, gluconeogenesis, oxidative stress, prooxidant activity, uncoupling 1 INTRODUCTION Butylated hydroxytoluene (BHT) is amply utilized as antioxidant in industrially processed foods, cosmetics, pharmaceutical, and petro- chemical products. [1] Its chemical structure is shown as an inset in Figure 1. It is a highly hydrophobic compound that presents high poten- tial of interacting with biological membranes, enzymes linked to mem- branes, and hydrophobic domains of proteins in general. As antioxi- dant, BHT inhibits lipid peroxidation, an effect that it also exerts in biological systems. [2] It was also shown, however, that BHT labilizes lisosomal membranes and disorganizes the mitochondrial structure. [3] This potential toxicity of BHT does not avoid its commercialization in the form of capsules as a food and health supplement. It has also been reported that the compound exerts antiviral effects, particularly against the herpes virus family. [4,5] There is also some popular litera- ture about the latter effect. [6] The most obvious interaction of BHT with the liver is biotransfor- mation, since this organ uses to be the most important site of the trans- formation of xenobiotics. The biotransformation of BHT in the liver is characterized by hydroxylations. [7] Hydroxylation of the methyl group in position 4 culminates with the formation of a carboxyl group, an event that facilitates renal excretion. The hydroxylated and carboxy- lated products are probably inert, but another biotransformation prod- uct, namely the BHT-quinone methide, is highly electrophilic and able to conjugate with reduced glutathione (GSH) as well as with macro- molecules such as proteins. [8,9] One of the most prominent toxic effects of BHT is tissue necrosis, which results from the reaction of the BHT- quinone methide with macromolecules. [10] With respect to energy metabolism there are at least two points of view on how the compound impairs the mitochondrial energy transduction. The first point of view regards BHT as a classical uncoupler or inhibitor of electron flow. In this view, BHT impairs mitochondrial energy transduction by a combination of uncoupling and inhibition of complex I at concentrations in the range between 50 and 750 M. [11,12] The pertinent experiments were done using phosphorylating mitochondria with respiratory rate measurements in the presence and absence of ADP or submitochondrial particles. [11] The second view was derived from experiments in which predomi- nantly non-phosphorylating mitochondria were used. [13] In the latter approach, the authors found oxygen uptake stimulation by BHT in the range from 10 -9 to 10 -6 M. If this represents uncoupling, BHT would possess a dynamic or ample uncoupling range. This could be a useful property in contrast to other uncouplers possessing a narrower range as it would diminish the risks of over dosage when using uncouplers for weight loss and other health promoting purposes. [14,15] Irrespective of the range in which BHT acts as an uncoupler or respiratory chain inhibitor, it has not yet been unequivocally demon- strated that this compound really acts as such in intact cell systems. In J Biochem Mol Toxicol. 2017;e21924. c  2017 Wiley Periodicals, Inc. 1 of 8 wileyonlinelibrary.com/journal/jbt https://doi.org/10.1002/jbt.21924