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Identification of Na⁺/H⁺ exchange as a new target for toxic polycyclic aromatic hydrocarbons¹

INSERM U456, Détoxication et Réparation Tissulaire, Faculté de Pharmacie, Université Rennes 1, 35043 Rennes Cedex, France

²Correspondence: INSERM U456, Faculté de Pharmacie, Université Rennes 1, 2, avenue du Professeur Léon Bernard, 35043 Rennes cedex, France. E-mail: Dominique.Lagadic{at}rennes.inserm.fr

SPECIFIC AIMS

The aim of this study was to examine the effects of polycyclic aromatic hydrocarbons (PAHs), more specifically benzo(a)pyrene B(a)P, on intracellular pH (pH_i) in liver epithelial cells. Indeed, these ubiquitous environmental pollutants to which humans are commonly exposed elicit numerous important cellular effects, such as carcinogenic and apoptotic effects, also known to be affected by or to affect cellular H⁺ concentrations, notably through Na⁺/H⁺ exchanger 1 (NHE1) modulation.

PRINCIPAL FINDINGS

1. PAHs induce an early intracellular alkalinization due to NHE1 activation followed by a late acidification
The effects of B(a)P on pH_i were tested in rat liver F258 epithelial cell line using carboxy-SNARF-1 as pH-sensitive fluorophore and microspectrofluorimetry. B(a)P, tested at two concentrations (5 µM and 50 nM), induced an early transient alkalinization, followed by a late acidification. Such variations were detected in HEPES and in CO₂^-/HCO₃^- medium. However, at 5 µM, the time course of pH_i changes appeared to be faster than those at 50 nM. Determinations of pH_i recovery after an acid load showed an increase of acid efflux concomitantly to alkalinization. By using cariporide, we demonstrated that NHE1 was activated upon B(a)P treatment and was necessary for biphasic pH_i changes to occur (Fig. 1 ).

View larger version (20K): [in this window] [in a new window]   Figure 1. Effects of the NHE1-inhibitor cariporide on resting pHi and pHi recovery in B(a)P-treated cells. A) Resting pHi measurements recorded in F258 cells treated or not with B(a)P (50 nM) and/or cariporide (30 µM) in HEPES-buffered medium. Number in brackets next to each bar represents the number of coverslips used (i.e., number of performed measurements) from at least 3 independent experiments. Mean values correspond to the average of all pHi determinations. Basal pHi was 7.30 ± 0.04 (n=9) and 7.31 ± 0.03 (n=11) in control untreated and cariporide-treated cells, respectively. *P < 0.05 treated vs. control. B, C) Representative pHi recordings from 12 independent experiments in control + cariporide (B) and B(a)P + cariporide (C) -treated cells. Cells were superfused with HEPES-buffered medium with 30 µM cariporide and submitted to a NH4+ (20 mM) prepulse to activate acid extrusion.

2. CYP1A1-related metabolism of B(a)P is responsible for NHE1 activation through increased production of H₂O₂
The role for CYP1A1-related metabolism in B(a)P-induced pH_i changes was evaluated using -naphtoflavone (-NF), a known CYP1A1 inhibitor. This compound prevented any pH_i variation induced by B(a)P; dioxin, a specific ligand of the aryl hydrocarbon receptor, remained ineffective on pH_i. Dimethyl benzanthracene (DMBA), whose metabolism is known to produce reactive metabolites, was found to induce biphasic pH_i-changes. During the course of our experiments to evaluate the level of regulation of NHE1 by B(a)P, we found that the affinity of the exchanger toward H⁺ was increased with no change in either V_max or protein expression. A role for reactive oxygen species (ROS), which we showed to be produced during CYP1A1 metabolism of B(a)P, was next tested. We found that the antioxidant molecule thiourea prevented both H₂O₂ production and NHE1 activation.

3. NHE-1-related transient alkalinization is involved in B(a)P-induced apoptosis of liver epithelial cells
B(a)P-induced apoptosis, as estimated by Hoechst 33342 nuclei staining and caspase activity (estimated by cleavage of the substrate DEVD-AMC), was found to be prevented by -NF and bongkrekic acid (BgA), an inhibitor of mitochondria-dependent apoptosis. Concerning pH_i, BgA was shown to inhibit only the late acidification, indicating that mitochondria were involved in this pH_i change. Apoptosis was significantly reduced by cariporide added before the development of alkalinization (Fig. 2 ). Moreover, we found that DMBA induced a cariporide-sensitive apoptosis whereas dioxin and benzanthracene were without any toxic effects in F258 cells.

View larger version (17K): [in this window] [in a new window]   Figure 2. Role of NHE1 activation in PAH-induced toxicity. A) F258 cells were treated with 50 nM B(a)P alone or in the presence of 30 µM cariporide added at 0, 24, or 48 h after onset of B(a)P treatment. Treatments with B(a)P were performed during 72 h. Apoptotic nuclei were analyzed by Hoechst 33342 staining. *P < 0.05, **P < 0.01, cariporide- and PAH-treated cells vs. control PAH-treated cells (n=7 independent experiments). B) F258 cells were treated with 50 nM B(a)P alone or in the presence of 30 µM cariporide, 10 µM -NF, or 10 µM Z-Asp (a global caspase inhibitor). After 72 h treatment, cells were harvested and washed with PBS before being lysed. Caspase activities (as detected by cleavage of DEVD-AMC) were measured by spectrofluorometry and averaged from 5 independent experiments. *P < 0.05, B(a)P ± inhibitor vs. control ± inhibitor; #P < 0.05 B(a)P + inhibitor vs. B(a)P-treated cells.

CONCLUSIONS AND SIGNIFICANCE

This study is the first to identify intracellular H⁺ homeostasis as a new target for these compounds. Indeed, isoform 1 of the Na⁺/H⁺ exchanger was shown to be transiently activated by PAHs, likely via an increase of its affinity to H⁺. Whereas aryl hydrocarbon receptor was not directly involved, we found that NHE1 protein activation was triggered by the H₂O₂ production resulting from CYP1A1-dependent metabolism, illustrating the importance of ROS production in mediating some PAH effects. Another important finding was that this transient alkalinization was necessary for PAHs to induce apoptosis shown to be associated with a secondary, mitochondria-dependent intracellular acidification (Fig. 3 ). In support of this, we demonstrated that only PAHs (such as DMBA) capable of eliciting biphasic pH_i changes induced apoptotic effects. The precise targets of intracellular alkalinization in the apoptotic cascade remain to be elucidated. Our study points to NHE1 transient activation as a possible important early signal in the development of the apoptotic cascade induced by toxic xenobiotics such as PAHs.

View larger version (25K): [in this window] [in a new window]   Figure 3. Schematic diagram. Recapitulative model of NHE1-dependent, B(a)P-induced apoptosis. B(a)P, a lipophilic molecule, goes across the bilayer membrane. In the cytosol, B(a)P is metabolized by CYP1A1 into reactive metabolites that target NHE1 via H2O2 production, inducing alkalinization. This activation favors the apoptotic signaling via mitochondria, leading to a late acidification and caspase activity corresponding to the effector phase of apoptosis. Other pathways (such as the p53 pathway) are likely to be involved in this model and are represented by a dotted arrow.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0316fje

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