A new lactoferrin- and iron-dependent lysosomal death pathway is induced by benzo[a]pyrene in hepatic epithelial cells.

While lysosomal disruption seems to be a late step of necrosis, a moderate lysosomal destabilization has been suggested to participate early in the apoptotic cascade. The origin of lysosomal dysfunction and its precise role in apoptosis or apoptosis-like process still needs to be clarified, especially upon carcinogen exposure. In this study, we focused on the implication of lysosomes in cell death induced by the prototype carcinogen benzo[a]pyrene (B[a]P; 50 nM) in rat hepatic epithelial F258 cells. We first demonstrated that B[a]P affected lysosomal morphology (increase in size) and pH (alkalinization), and that these changes were involved in caspase-3 activation and cell death. Subsequently, we showed that lysosomal modifications were partly dependent on mitochondrial dysfunction, and that lysosomes together with mitochondria participate in B[a]P-induced oxidative stress. Using two iron chelators (desferrioxamine and deferiprone) and siRNA targeting the lysosomal iron-binding protease lactoferrin, we further demonstrated that both lysosomal iron content and lactoferrin were required for caspase-3 activation and apoptosis-like cell death.

Cisplatin-induced apoptosis involves membrane fluidification via inhibition of NHE1 in human colon cancer cells.

We have previously shown that cisplatin triggers an early acid sphingomyelinase (aSMase)-dependent ceramide generation concomitantly with an increase in membrane fluidity and induces apoptosis in HT29 cells. The present study further explores the role and origin of membrane fluidification in cisplatin-induced apoptosis. The rapid increase in membrane fluidity following cisplatin treatment was inhibited by membrane-stabilizing agents such as cholesterol or monosialoganglioside-1. In HT29 cells, these compounds prevented the early aggregation of Fas death receptor and of membrane lipid rafts on cell surface and significantly inhibited cisplatin-induced apoptosis without altering drug intracellular uptake or cisplatin DNA adducts formation. Early after cisplatin treatment, Na+/H+ membrane exchanger-1 (NHE1) was inhibited leading to intracellular acidification, aSMase was activated, and ceramide was detected at the cell membrane. Treatment of HT29 cells with Staphylococcus aureus sphingomyelinase increased membrane fluidity. Moreover, pretreatment with cariporide, a specific inhibitor of NHE1, inhibited cisplatin-induced intracellular acidification, aSMase activation, ceramide membrane generation, membrane fluidification, and apoptosis. Finally, NHE1-expressing PS120 cells were more sensitive to cisplatin than NHE1-deficient PS120 cells. Altogether, these findings suggest that the apoptotic pathway triggered by cisplatin involves a very early NHE1-dependent intracellular acidification leading to aSMase activation and increase in membrane fluidity. These events are independent of cisplatin-induced DNA adducts formation. The membrane exchanger NHE1 may be another potential target of cisplatin, increasing cell sensitivity to this compound.

c-Jun NH2-terminal kinase-related Na+/H+ exchanger isoform 1 activation controls hexokinase II expression in benzo(a)pyrene-induced apoptosis.

Regulation of the balance between survival, proliferation, and apoptosis on carcinogenic polycyclic aromatic hydrocarbon (PAH) exposure is still poorly understood and more particularly the role of physiologic variables, including intracellular pH (pH(i)). Although the involvement of the ubiquitous pH(i) regulator Na(+)/H(+) exchanger isoform 1 (NHE1) in tumorigenesis is well documented, less is known about its role and regulation during apoptosis. Our previous works have shown the primordial role of NHE1 in carcinogenic PAH-induced apoptosis. This alkalinizing transporter was activated by an early CYP1-dependent H(2)O(2) production, subsequently promoting mitochondrial dysfunction leading to apoptosis. The aim of this study was to further elucidate how NHE1 was activated by benzo(a)pyrene (BaP) and what the downstream events were in the context of apoptosis. Our results indicate that the mitogen-activated protein kinase kinase 4/c-Jun NH(2)-terminal kinase (MKK4/JNK) pathway was a link between BaP-induced H(2)O(2) production and NHE1 activation. This activation, in combination with BaP-induced phosphorylated p53, promoted mitochondrial superoxide anion production, supporting the existence of a common target for NHE1 and p53. Furthermore, we showed that the mitochondrial expression of glycolytic enzyme hexokinase II (HKII) was decreased following a combined action of NHE1 and p53 pathways, thereby enhancing the BaP-induced apoptosis. Taken together, our findings suggest that, on BaP exposure, MKK4/JNK targets NHE1 with consequences on HKII protein, which might thus be a key protein during carcinogenic PAH apoptosis.

Different mechanisms involved in apoptosis following exposure to benzo[a]pyrene in F258 and Hepa1c1c7 cells.

The present study compares and elucidates possible mechanisms why B[a]P induces different cell signals and triggers apparently different apoptotic pathways in two rather similar cell lines (hepatic epithelial cells of rodents). The rate and maximal capacity of metabolic activation, as measured by the formation of B[a]P-tetrols and B[a]P-DNA adducts, was much higher in mouse hepatoma Hepa1c1c7 cells than in rat liver epithelial F258 cells due to a higher induced level of cyp1a1. B[a]P increased intracellular pH in both cell lines, but this change modulated the apoptotic process only in F258 cells. In Hepa1c1c7 cells reactive oxygen species (ROS) production appeared to be a consequence of toxicity, unlike F258 cells in which it was an initial event. The increased mitochondrial membrane potential found in F258 cells was not observed in Hepa1c1c7 cells. Surprisingly, F258 cells cultured at low cell density were somewhat more sensitive to low (50nM) B[a]P concentrations than Hepa1c1c7 cells. This could be explained partly by metabolic differences at low B[a]P concentrations. In contrast to the Hepa1c1c7 model, no activation of cell survival signals including p-Akt, p-ERK1/2 and no clear inactivation of pro-apoptotic Bad was observed in the F258 model following exposure to B[a]P. Another important difference between the two cell lines was related to the role of Bax and cytochrome c. In Hepa1c1c7 cells, B[a]P exposure resulted in a "classical" translocation of Bax to the mitochondria and release of cytochrome c, whereas in F258 cells no intracellular translocation of these two proteins was seen. These results suggest that the rate of metabolism of B[a]P and type of reactive metabolites formed influence the resulting balance of pro-apoptotic and anti-apoptotic cell signaling, and hence the mechanisms involved in cell death and the chances of more permanent genetic damage.

Effects of nitrated-polycyclic aromatic hydrocarbons and diesel exhaust particle extracts on cell signalling related to apoptosis: possible implications for their mutagenic and carcinogenic effects.

Nitrated-polycyclic aromatic hydrocarbons (nitro-PAHs) and diesel exhaust particle extracts (DEPE) induced apoptosis in Hepa1c1c7 cells with the following potency: 1,3-dinitropyrene (1,3-DNP)>1-nitropyrene (1-NP) >> DEPE >> 1,8-dinitropyrene (1,8-DNP). The compounds induced cyp1a1, and activated various intracellular signalling pathways related to apoptosis. The CYP inhibitor alpha-naphthoflavone strongly reduced 1,3-DNP-induced cell death, whereas cell death induced by 1-NP was rather increased. Toxic 1,3-DNP and 1-NP were found to induce a concentration-dependent lipid peroxidation. 1,3-DNP caused pro-apoptotic events, including increased phosphorylation and accumulation of p53 in the nucleus, cleavage of bid and of caspases 8 and 3, down-regulation of bcl-x(L) and phosphorylation of p38 and JNK MAPK. Furthermore, 1,3-DNP increased the activation of survival signals including phosphorylation of Akt and inactivation (phosphorylation) of pro-apoptotic bad. Although less potent, rather similar effects were observed following exposure to DEPE, compared to 1-NP. The most important finding was that the most mutagenic and carcinogenic compound tested, 1,8-DNP, induced little (if any) cell death, despite the fact that this compound seemed to give the most DNA damage as judged by DNA adduct formation, increased phosphorylation of p53 and accumulation of cells in S-phase. Immunocytochemical studies revealed that the p53 protein did not accumulate into the nucleus suggesting that 1,8-DNP inactivated the pro-apoptotic function of the p53 protein by a non-mutagenic event. These results suggest that after exposure to 1,8-DNP more cells may survive with DNA damage, thereby increasing its mutagenic and carcinogenic potential.

Protective effect of monosialoganglioside GM1 against chemically induced apoptosis through targeting of mitochondrial function and iron transport.

Exogenous treatment with monosialoganglioside GM1 has been described to afford protection against different apoptotic insults. However, the underlying mechanisms remain to be determined. In this study, we focused on the effect of GM1 on the apoptotic cascade induced by benzo[a]pyrene (B[a]P) in rat hepatic F258 epithelial cells. We first demonstrated that a co-treatment with GM1 (80 microM) reduced B[a]P (50 nM)-induced apoptosis as evidenced by a decrease of both cell population exhibiting nuclear fragmentation and caspase 3 cleavage and activity. We next showed that the p53 phosphorylation and nuclear translocation as well as the intracellular alkalinization related to Na+/H+ exchanger 1 (NHE1) activation, two early events of the apoptosis induced by B[a]P, were not inhibited by GM1. In contrast, the late mitochondria-dependent acidification elicited by B[a]P was inhibited by GM1 co-treatment, and an inhibition of the oxidative stress was also observed. Because GM1 has been shown to reduce the low-molecular weight iron content related to ethanol-induced oxidative stress, we finally investigated the involvement of iron under our conditions. Using the two iron chelators deferiprone and desferrioxamine, we clearly showed that iron played an important role in B[a]P-induced apoptosis in F258 cells, and that B[a]P-treatment resulted in a significant GM1-sensitive increase in (55)Fe uptake. In conclusion, our results indicate that exogenous GM1 partly prevents B[a]P-induced apoptosis by interfering with mitochondria-related intracellular acidification and iron transport.

Multiple apoptotic pathways induced by p53-dependent acidification in benzo[a]pyrene-exposed hepatic F258 cells.

Polycyclic aromatic hydrocarbons (PAH), such as benzo[a]pyrene (B[a]P), are ubiquitous genotoxic environmental pollutants. Their DNA-damaging effects lead to apoptosis induction, through similar pathways to those identified after exposure to other DNA-damaging stimuli with activation of p53-related genes and the involvement of the intrinsic apoptotic pathway. However, at a low concentration of B[a]P (50 nM), our previous results pointed to the involvement of intracellular pH (pHi) variations during B[a]P-induced apoptosis in a rat liver epithelial cell line (F258). In the present work, we identified the mitochondrial F0F1-ATPase activity reversal as possibly responsible for pHi decrease. This acidification not only promoted executive caspase activation, but also activated leucocyte elastase inhibitor/leucocyte-derived DNase II (LEI/L-DNase II) pathway. p53 appeared to regulate mitochondria homeostasis, by initiating F0F1-ATPase reversal and endonuclease G (Endo G) release. In conclusion, a low dose of B[a]P induced apoptosis by recruiting a large panel of executioners apparently depending on p53 phosphorylation and, for some of them, on acidification.

Membrane fluidity changes are associated with benzo[a]pyrene-induced apoptosis in F258 cells: protection by exogenous cholesterol.

Polycyclic aromatic hydrocarbons (PAHs) such as benzo[a]yrene (B[a]P) constitute a widely distributed class of environmental pollutants, responsible for highly toxic effects. Elucidating the intracellular mechanisms of this cytotoxicity thus remains a major challenge. Besides the activation of the p53 apoptotic pathway, we have previously found in F258 hepatic cells that the B[a]P (50 nM)-induced apoptosis was also dependent upon the transmembrane transporter NHE1, whose activation might result from membrane alterations in our model. We here demonstrate that: (1) B[a]P induces a membrane fluidization surprisingly linked to NHE1 activation; (2) membrane stabilization by exogenous cholesterol protects cells from B[a]P-induced apoptosis, via an effect on late acidification and iron uptake.