The aim of this work was to examine the effect produced by the suppression of NF-κ
B activity on the toxicity caused by BSO or AA in cells of hepatic origin, with a special interest in describing a possible role exerted by CYP2E1 activity (Jimenez-Lopez & Cederbaum, 2005
) in the developing toxicity.
The rationale for this study was to evaluate how NF-κ
B can modulate the toxicity of GSH depletion produced by BSO or addition of AA, whether CYP2E1 alters this modulation by NF-κ
B activity, whether BSO and AA toxicities show similar responses to this NF-κ
B modulation, and to evaluate possible mechanisms, e.g., oxidative stress, MAPK activation, caspase activation, in the overall pathway of cellular toxicity. Activation of NF-κ
B may occur as an adaptive mechanism to protect cells in response to the ensuing oxidative injury produced by GSH depletion, e.g., in human leukemic U937 cells (Filomeni et al., 2005
), or after a long-time AA treatment (Wu & Cederbaum, 2003
). In fact, synthesis of protective factors stimulated by NF-κ
B activation appears to be a critical survival mechanism to protect hepatocytes against apoptotic cell death caused by a variety of insults including TNF family members (Okano et al., 2003
; Pikarsky et al., 2004
; Uesugi et al., 2001
), and vice versa, the inhibition of NF-κ
B activity may reduce GSH synthesis by downregulating glutamate-cysteine ligase at the transcriptional level (Lu, 1999
; Yang et al., 2005
Depletion of GSH by BSO treatment was shown to enhance AA (Chen et al., 1997
) and CYP2E1 itself-derived ROS (Chen & Cederbaum, 1998
) toxicities in the E47 HepG2 cell subline that overexpresses CYP2E1. As the main antioxidant inside mammalian cells, GSH plays a pivotal role in preventing oxidative stress and damage to mitochondrial function produced by numerous toxins (Dickinson & Forman, 2002
). Results of the current study indicate that GSH depletion as a result of BSO treatment, but not AA-related lipid peroxidation, sensitizes both HepG2 cells and isolated rat hepatocytes to damage and cell death when inhibition of NF-κ
B activity occurs. The observed toxic effect depends on the concentration of BSO, the concentration of the specific NF-κ
B inhibitor and the coexposure time.
Treatment with AA increases lipid peroxidation in E47 cells as assessed by assaying for the production of thiobarbituric acid reactive substances (), and this increase in lipid peroxidation plays a central role in the developing AA toxicity (Caro & Cederbaum, 2004
). BAY11-7082 is a well-described selective and irreversible inhibitor of Iκ
(inhibitory subunit of the cytoplasmic NF-κ
B complex) phosphorylation, thereby preventing NF-κ
B nuclear translocation and activity (Hansson et al., 2005
). BAY11-7082 exposure in AA-treated cells does not further augment lipid peroxidation as compared to cells treated with AA alone, which would explain the absence of higher toxicity, i.e., incubation of cells in the presence of BAY11-7082 plus AA only produces additive but not synergistic toxic effects. Moreover, BAY11-7082 itself does not induce lipid peroxidation, suggesting that there is no causal relationship between this effect and the inhibition of NF-κ
Restoration of GSH with GSH ethyl ester completely blocked the BAY11-7082 plus BSO synergistic toxicity in E47 cells, confirming that GSH depletion is critically involved in the mechanism of toxicity leading to cell death. The protective effect produced by the broad-spectrum caspase inhibitor z-VAD-FMK against BAY11-7082 plus BSO toxicity indicates that cell death depends on caspase activation, at least partially, for the final execution; hence, apoptosis likely accounts for loss of viability after incubating with BAY11-7082 plus BSO, although future studies directly evaluating apoptotic indices are necessary.
ROS generated from CYP2E1 and other sources can be scavenged either by direct reaction with GSH or by the GSH plus glutathione peroxidase reaction (Dickinson & Forman, 2002
). To evaluate the possible role of ROS production as a signal involved in the mechanism of potentiated toxicity caused by GSH depletion plus NF-κ
B inhibition, the incubation medium was supplemented with a variety of antioxidants before initiating the toxicity phase. Surprisingly, neither trolox- nor catalase- nor NAC-exposed cells overcome the following loss of viability induced by BAY11-7082 plus BSO, indicating that an increased accumulation of ROS caused by these treatments is likely not critical in determining cell death. This is in agreement with results showing that although BSO alone increased ROS production (DCF fluorescence), no potentiation of this BSO-induced increase occurs in the presence of BAY11-7082.
Activation of p38 MAPK seems to be an important signal leading to loss of viability by BAY11-7082 plus BSO treatment, as the p38 MAPK inhibitor SB203580 strongly prevented the synergistic toxicity in E47 cells. Lack of protection by PD98059 or SP600125 suggests that neither extracellular signal-regulated kinase nor c-Jun N
-terminal kinase are involved in the mechanism underlying the synergistic toxicity. BSO treatment induced the phosphorylation and hence activation of p38 MAPK in CYP2E1-overexpressing liver cells, which could suppress the activation of NF-κ
B. BAY11-7082 treatment was also found to increase the p-p38/p38 MAPK ratio in E47 cells, which may contribute to inhibition of NF-κ
B activation by preventing phosphorylation and subsequent degradation of Iκ
(Schwenger et al., 1998
). It is noteworthy that the BAY11-7082 plus BSO combination induced a higher activation of p38 MAPK than BAY11-7082 alone or BSO alone at relatively early time periods prior to the developing toxicity, which might explain, at least in part, the appearance of the synergistic toxicity. In addition, the greater p-p38/p38 MAPK ratio after treatment with BAY11-7082 plus BSO was strongly prevented by SB203580, in accordance to the protection provided by SB203580 against the potentiated toxicity of BAY11-7082 plus BSO in these cells.
Despite the fact that CYP2E1-dependent oxidative stress plays an important role in potentiating loss of cell viability in the above models of toxicity, the current work shows that the expression of CYP2E1 does not appear to change the sensitivity threshold eliciting a toxic response after long-term BSO (but not AA) treatment when cells are coincubated with any of several NF-κB inhibitors. Together, these results suggest that redox changes caused by profound intracellular GSH depletion are critical enough to sensitize the liver cells to toxicity provoked by inhibition of NF-κB activity through a mechanism that does not depend on either the accumulation of ROS or lipid peroxidation, but is mediated by p38 MAPK.
Based on the protection by SB203580, pro-apoptotic actions of p38 MAPK can be presumably occurring and likely be responsible for the observed toxicity of BAY11-7082 plus BSO, as p38 MAPK activation has been shown to dysregulate mitochondrial function through distinct mechanisms (Cuenda & Rousseau, 2007
). In this respect, it is interesting to notice that provision of exogenous nitric oxide decreased the TNF-α
plus acute GSH depletion toxicity in murine hepatocytes (Matsumaru et al., 2003
), the BSO plus CYP2E1-dependent toxicity in liver cells (Wu & Cederbaum, 2004
), and the toxicity produced by BAY11-7082 plus BSO in E47 cells (). As previously discussed, the protective role of nitric oxide could be related to its ability to prevent apoptosis through the inhibition of caspases (Matsumaru et al., 2003
) as well as by inhibiting cytochrome P450 activity (Wu & Cederbaum, 2004
In summary, this report describes a synergistic toxicity induced by GSH depletion and additional suppression of NF-κB activity, hence loss of NF-κB-dependent protection, in liver cells. Activation of p38 MAPK is an important early signal leading to toxicity through, at least partially, a caspase-dependent pathway. Differences in response of BSO versus AA toxicity to inhibition of NF-κB suggests that not all prooxidants induce toxicity by NF-κB-modulated mechanisms in liver cells, which may complicate therapeutic interventions. Since a reduction of the intracellular GSH levels may occur under diverse prooxidant conditions, which could further interfere with the activation state of NF-κB, conclusions derived from this study may be of relevance in predicting possible enhanced toxic effects as a consequence of redox homeostasis unbalance in situations involving the inhibition of NF-κB activity, e.g., after exposure to a variety of drugs.