BUTYLATED HYDROXYANISOLE AND ITS METABOLITE TERT-BUTYLHYDROQUINONE DIFFERENTIALLY REGULATE MITOGEN-ACTIVATED PROTEIN-KINASES - THE ROLE OF OXIDATIVE STRESS IN THE ACTIVATION OF MITOGEN-ACTIVATED PROTEIN-KINASES BY PHENOLIC ANTIOXIDANTS

Phenolic antioxidant butylated hydroxyanisole (BRA) is a commonly used food preservative with broad biological activities, including protect ion against acute toxicity of chemicals, modulation of macromolecule s ynthesis and immune response, induction of phase II detoxifying enzyme s, and especially its potential tumor-promoting activities. Understand ing the molecular basis underlying these diverse biological actions of BRA is thus of great importance, Here we demonstrate that BRA is capa ble of activating distinct mitogen-activated protein kinases (MAPKs), extracellular signal-regulated protein kinase 2 (ERK2), and c-Jun N-te rminal kinase 1 (JNK1). Activation of ERK2 by BHA was rapid and transi ent, whereas the JNK1 activation was relatively delayed and persistent . A major metabolite of BHA, tert-butylhydroquinone (tBHQ), also activ ated ERK2 but weakly stimulated JNK1 activity, Furthermore, tBHQ activ ation of ERK2 was late and prolonged, showing a kinetics different fro m that induced by BHA. ERK2 activation 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, Pretr eatment with N-acetyl-L-cysteine, glutathione, or vitamin E attenuated ERK2 but not JNK1 activation by BHA and tBHQ. Modulation of intracell ular H2O2 levels by direct addition of catalase or pretreatment with a catalase inhibitor, aminotriazole, also affected BHA-and tBHQ-stimula ted ERK2 activity but not JNK1, indicating the involvement of oxidativ e stress in the ERK2 activation by these two compounds. However, we di d not observe any generation of H2O2 after exposure of cells to BHA or tBHQ using a H2O2-sensitive fluorescent probe, 2',7'-dichlorofluoresc ein diacetate. Instead, BRA and tBHQ substantially reduced the amount of intracellular H2O2, Furthermore, BHA and tBHQ activation of ERK2 wa s strongly inhibited by ascorbic acid and a peroxidase inhibitor, sodi um azide, suggesting the potential role of phenoxyl radicals and/or th eir derivatives, Taken together, our results indicate that (i) BHA and its metabolite tBHQ differentially regulate MAPK pathways, and (ii) o xidative stress due to the generation of reactive intermediates, possi bly phenoxyl radicals but not H2O2, is responsible for the ERK2 activa tion by BHA and tBHQ, whereas the JNK1 activation may require a distin ct yet unknown mechanism.