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 biological activities, including protection against acute toxicity of chemicals, modulation of macromolecule synthesis and immune response, induction of phase II detoxifying enzymes, and especially its potential tumorpromoting activities. Understanding the molecular basis 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 transient, whereas the JNK1 activation was relatively delayed and persistent. A major metabolite of BHA, tertbutylhydroquinone (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 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. Pretreatment with N-acetyl-L-cysteine, glutathione, or vitamin E attenuated ERK2 but not JNK1 activation by BHA and tBHQ. Modulation of intracellular H 2 O 2 levels by direct addition of catalase or pretreatment with a catalase inhibitor, aminotriazole, also affected 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 exposure 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 regulate MAPK pathways, and (ii) oxidative stress due to the generation of reactive intermediates, possibly phenoxyl radicals but not H 2 O 2 , is responsible for the ERK2 activation by BHA and tBHQ, whereas the JNK1 activation may require a distinct yet unknown mechanism.

Antioxidants Differentially Regulate Activation of Nuclear Factor- κ B, Activator Protein-1, c-Jun Amino-Terminal Kinases, and Apoptosis Induced by Tumor Necrosis Factor: Evidence that JNK and NF- κ B Activation Are Not Linked to Apoptosis

“…On the other hand, the LPO inhibitors, BHA at a lower dose and BHT at a higher dose, could activate JNK on their own. These results agree with a recent report of JNK activation by BHA (Yu et al, 1997). These authors also showed that BHA-induced JNK activation is resistant to NAC and GSH.…”

Antioxidants Differentially Regulate Activation of Nuclear Factor- κ B, Activator Protein-1, c-Jun Amino-Terminal Kinases, and Apoptosis Induced by Tumor Necrosis Factor: Evidence that JNK and NF- κ B Activation Are Not Linked to Apoptosis

“…On the other hand, the LPO inhibitors, BHA at a lower dose and BHT at a higher dose, could activate JNK on their own. These results agree with a recent report of JNK activation by BHA (Yu et al, 1997). These authors also showed that BHA-induced JNK activation is resistant to NAC and GSH.…”

Role of a Mitogen-activated Protein Kinase Pathway in the Induction of Phase II Detoxifying Enzymes by Chemicals

“…2B). These results, together with our previous observation that tBHQ weakly stimulated JNK1 activity in HepG2 cells (37), suggest that the JNK pathway may not be involved or at least are not essential in tBHQ-or SUL-induced cell signaling that leads to the induction of gene expression.…”

“…The induced ERK2 activity appeared at 30 min, reached the maximum between 1 and 2 h after treatment with SUL, and then declined. Interestingly, the time course of ERK2 activation by SUL was very similar to that of ERK2 activation by tBHQ (37), suggesting that two inducers may regulate ERK2 activity through a common pathway. When Western blotting was performed, no change in the protein level of ERK2 was observed throughout the dose response as well as time course studies (Fig.…”

Specificity of endogenous fatty acid release during tumor necrosis factor-induced apoptosis in WEHI 164 fibrosarcoma cells

“…This is supported by the observation that BHA inhibits cell growth and is cytotoxic to WEHI and L929 cells at high concentrations (35). Furthermore, both BHA (56) and TNF (57) activate JNK-1, which is part of the SAPK cascade and participates in apoptosis (57,58). A shift in the coregulation of the ceramide-induced SAPK cascade involving JNK-1 and the cytoprotective MAPK cascade involving ERK-1 and ERK-2 has been proposed to regulate the balance between induction of apoptosis or cell protection (58).…”

Antioxidants Differentially Regulate Activation of Nuclear Factor- κ B, Activator Protein-1, c-Jun Amino-Terminal Kinases, and Apoptosis Induced by Tumor Necrosis Factor: Evidence that JNK and NF- κ B Activation Are Not Linked to Apoptosis

“…On the other hand, the LPO inhibitors, BHA at a lower dose and BHT at a higher dose, could activate JNK on their own. These results agree with a recent report of JNK activation by BHA (Yu et al, 1997). These authors also showed that BHA-induced JNK activation is resistant to NAC and GSH.…”

Role of a Mitogen-activated Protein Kinase Pathway in the Induction of Phase II Detoxifying Enzymes by Chemicals

“…2B). These results, together with our previous observation that tBHQ weakly stimulated JNK1 activity in HepG2 cells (37), suggest that the JNK pathway may not be involved or at least are not essential in tBHQ-or SUL-induced cell signaling that leads to the induction of gene expression.…”

“…The induced ERK2 activity appeared at 30 min, reached the maximum between 1 and 2 h after treatment with SUL, and then declined. Interestingly, the time course of ERK2 activation by SUL was very similar to that of ERK2 activation by tBHQ (37), suggesting that two inducers may regulate ERK2 activity through a common pathway. When Western blotting was performed, no change in the protein level of ERK2 was observed throughout the dose response as well as time course studies (Fig.…”

Specificity of endogenous fatty acid release during tumor necrosis factor-induced apoptosis in WEHI 164 fibrosarcoma cells

“…This is supported by the observation that BHA inhibits cell growth and is cytotoxic to WEHI and L929 cells at high concentrations (35). Furthermore, both BHA (56) and TNF (57) activate JNK-1, which is part of the SAPK cascade and participates in apoptosis (57,58). A shift in the coregulation of the ceramide-induced SAPK cascade involving JNK-1 and the cytoprotective MAPK cascade involving ERK-1 and ERK-2 has been proposed to regulate the balance between induction of apoptosis or cell protection (58).…”

Antioxidants Differentially Regulate Activation of Nuclear Factor- κ B, Activator Protein-1, c-Jun Amino-Terminal Kinases, and Apoptosis Induced by Tumor Necrosis Factor: Evidence that JNK and NF- κ B Activation Are Not Linked to Apoptosis

“…On the other hand, the LPO inhibitors, BHA at a lower dose and BHT at a higher dose, could activate JNK on their own. These results agree with a recent report of JNK activation by BHA (Yu et al, 1997). These authors also showed that BHA-induced JNK activation is resistant to NAC and GSH.…”

Role of a Mitogen-activated Protein Kinase Pathway in the Induction of Phase II Detoxifying Enzymes by Chemicals

“…2B). These results, together with our previous observation that tBHQ weakly stimulated JNK1 activity in HepG2 cells (37), suggest that the JNK pathway may not be involved or at least are not essential in tBHQ-or SUL-induced cell signaling that leads to the induction of gene expression.…”

“…The induced ERK2 activity appeared at 30 min, reached the maximum between 1 and 2 h after treatment with SUL, and then declined. Interestingly, the time course of ERK2 activation by SUL was very similar to that of ERK2 activation by tBHQ (37), suggesting that two inducers may regulate ERK2 activity through a common pathway. When Western blotting was performed, no change in the protein level of ERK2 was observed throughout the dose response as well as time course studies (Fig.…”

Specificity of endogenous fatty acid release during tumor necrosis factor-induced apoptosis in WEHI 164 fibrosarcoma cells

“…This is supported by the observation that BHA inhibits cell growth and is cytotoxic to WEHI and L929 cells at high concentrations (35). Furthermore, both BHA (56) and TNF (57) activate JNK-1, which is part of the SAPK cascade and participates in apoptosis (57,58). A shift in the coregulation of the ceramide-induced SAPK cascade involving JNK-1 and the cytoprotective MAPK cascade involving ERK-1 and ERK-2 has been proposed to regulate the balance between induction of apoptosis or cell protection (58).…”