Butylated Hydroxyanisole and Its Metabolitetert-Butylhydroquinone Differentially Regulate Mitogen-activated Protein Kinases

Abstract

Phenolic antioxidant butylated hydroxyanisole (BHA) is a commonly used food preservative with broad bio-logical activities, including protection against acute tox-icity of chemicals, modulation of macromolecule syn-thesis and immune response, induction of phase II detoxifying enzymes, and especially its potential tumor-promoting activities. Understanding the molecular ba-sis underlying these diverse biological actions of BHA is thus of great importance. Here we demonstrate that BHA is capable of activating distinct mitogen-activated protein kinases (MAPKs), extracellular signal-regulated protein kinase 2 (ERK2), and c-Jun N-terminal kinase 1 (JNK1). Activation of ERK2 by BHA was rapid and tran-sient, whereas the JNK1 activation was relatively de-layed and persistent. A major metabolite of BHA, tert-butylhydroquinone (tBHQ), also activated ERK2 but weakly stimulated JNK1 activity. Furthermore, tBHQ activation of ERK2 was late and prolonged, showing a kinetics different from that induced by BHA. ERK2 ac-tivation by both compounds required the involvement of an upstream signaling kinase MAPK/ERK kinase (MEK), as evidenced by the inhibitory effect of a MEK inhibitor, PD98059. Pretreatment with N-acetyl-L-cysteine, gluta-thione, or vitamin E attenuated ERK2 but not JNK1 activation by BHA and tBHQ. Modulation of intracellu-lar H 2 O 2 levels by direct addition of catalase or pretreat-ment with a catalase inhibitor, aminotriazole, also af-fected BHA-and tBHQ-stimulated ERK2 activity but not JNK1, indicating the involvement of oxidative stress in the ERK2 activation by these two compounds. However, we did not observe any generation of H 2 O 2 after expo-sure of cells to BHA or tBHQ using a H 2 O 2 -sensitive fluorescent probe, 2؅,7؅-dichlorofluorescein diacetate. Instead, BHA and tBHQ substantially reduced the amount of intracellular H 2 O 2 . Furthermore, BHA and tBHQ activation of ERK2 was strongly inhibited by ascorbic acid and a peroxidase inhibitor, sodium azide, suggesting the potential role of phenoxyl radicals and/or their derivatives. Taken together, our results indicate that (i) BHA and its metabolite tBHQ differentially reg-ulate MAPK pathways, and (ii) oxidative stress due to the generation of reactive intermediates, possibly phe-noxyl radicals but not H 2 O 2 , is responsible for the ERK2 activation by BHA and tBHQ, whereas the JNK1 activa-tion may require a distinct yet unknown mechanism.