Identification of Na⁺/H⁺exchange as a new target for toxic polycyclic aromatic hydrocarbons in liver cells

Abstract

The ubiquitous environmental pollutants polycyclic aromatic hydrocarbons are responsible for important carcinogenic and apoptotic effects, whose mechanisms are still poorly understood, owing to the multiplicity of possible cellular targets. Among these mechanisms, alterations of ionic homeostasis have been suggested. In this work, the effects of benzo(a)pyrene [B(a)P] on pHi were tested in the rat liver F258 epithelial cell line, using the fluoroprobe carboxy-SNARF-1. After a 48-h treatment, B(a)P (50 nM) induced an alkalinization, followed by an acidification after 72 h and the development of apoptosis. Determinations of pH(i) recovery following an acid load showed an increased acid efflux at 48 h. Cariporide inhibited both the early alkalinization and the increased acid efflux, thus suggesting the involvement of Na+/H+ exchanger 1 (NHE1). Besides, alpha-naphtoflavone (alpha-NF), an inhibitor of CYP1A1-mediated B(a)P metabolism, prevented all pH(i) changes, and NHE1 activation was blocked by the antioxidant thiourea, which inhibited CYP1A1 metabolism-dependent H2O2 production. Regarding B(a)P-induced apoptosis, this was prevented by alpha-NF and bongkrekic acid, an inhibitor of mitochondria-dependent apoptosis. Interestingly, apoptosis was significantly reduced by cariporide. Taken together, our results indicate that B(a)P, via H2O2 produced by CYP1A1-dependent metabolism, induces an early activation of NHE1, resulting in alkalinization; this appears to play a significant role in mitochondria-dependent B(a)P-induced apoptosis.

Alterations of intracellular pH homeostasis in apoptosis: origins and roles

“…The striking point in all these studies is that the intracellular alkalinization seems to be an early event in the apoptotic cascade, occurring prior to caspase activation and DNA fragmentation. Furthermore, when tested, this phenomenon appears to be only transient, followed 56,60 or not 54,59 by an intracellular acidification (i.e. reaching pH i values below control levels).…”

“…Among the transporters responsible for the apoptosis-related alkaline shift of pH i , isoform 1 of the NHE would seem to be preferentially targeted, being activated for example following cytokine deprivation, 61 or upon exposure to PAT (V Lecureur and D Lagadic-Gossmann, unpublished data) or toxic PAHs. 60 This activation has recently been shown to result from protein phosphorylation by the p38 MAPK pathway, 61 H 2 O 2 being possibly involved in such an effect. 60 Regarding the involvement of ROS, it is worth noting that a role for anion superoxide has also been proposed in the NHE activation by phorbol esters; 62 such a role should therefore be tested in the future.…”

“…60 This activation has recently been shown to result from protein phosphorylation by the p38 MAPK pathway, 61 H 2 O 2 being possibly involved in such an effect. 60 Regarding the involvement of ROS, it is worth noting that a role for anion superoxide has also been proposed in the NHE activation by phorbol esters; 62 such a role should therefore be tested in the future. It is now well recognized that NHE1 activity is controlled by several kinases (see Putney et al 11 for a recent review).…”

“…Such a pH i change has been observed in cells undergoing situations as various as cytokine deprivation, 54 gamma radiation, 55 ceramide application 56 and exposure to xenobiotics, such as drugs targeting proteasome, 57 staurosporine, 58 potassium antimonyl tartrate (PAT) 59 or polycyclic aromatic hydrocarbons (PAHs). 60 Therefore, it appears that such a pH i change might represent a more generalized apoptotic feature and should be looked for in a more systematic way. Besides, to our knowledge, it seems that this event is mostly encountered when the mitochondria-dependent apoptotic pathway is involved.…”

Chronoregulation by Asparagine Deamidation

“…Additionally, a small change in pH between pH 7.0 and 8.0 can substantially increase the rate of deamidation of a protein (3). These findings are notable because there is an increase in pH to as high as pH 8.0 early in several forms of apoptosis, including DNA damage-induced apoptosis (25)(26)(27)(28)(29)(30). The increase in pH is mediated by the protein Na + /H + exchanger 1 (NHE1), a ubiquitously expressed protein that regulates cytosolic pH (25)(26)(27)(28)(29).…”