Hemochromatosis: a model of metal-related human toxicosis.

Many environmental agents, such as excessive alcohol intake, xenobiotics, and virus, are able to damage the human body, targeting especially the liver. Metal excess may also assault the liver. Thus, chronic iron overload may cause, especially when associated with cofactors, diffuse organ damage that is a source of significant morbidity and mortality. Iron excess can be either of acquired (mostly transfusional) or of genetic origin. Hemochromatosis is the archetype of genetic iron overload diseases and represents a serious health problem. A better understanding of iron metabolism has deeply modified the hemochromatosis field which today benefits from much more efficient diagnostic and therapeutic approaches.

Recent advances in cancer treatment by iron chelators.

The development of new therapeutic alternatives for cancers is a major public health priority. Among the more promising approaches, the iron depletion strategy based on metal chelation in the tumoral environment has been particularly studied in recent decades. After a short description of the importance of iron for cancer cell proliferation, we will review the different iron chelators developed as potential chemotherapeutics. Finally, the recent efforts to vectorize the chelating agents specifically in the microtumoral environment will be discussed in detail.

Effect of Caseinophosphopeptides from αs- and ß-Casein on Iron Bioavailability in HuH7 Cells.

Two pools of caseinophosphopeptides (CPPs) obtained from αs- and ß-casein fractions (α-CPPs and ß-CPPs) were characterized. A total of 16 CPPs were identified in the α-CPPs pool, 9 of them derived from αs1-casein and 7 from αs2-casein. A total of 18 CPPs were identified in the ß-CPPs pool. Four of the identified CPPs contained the characteristic phosphoseryl-glutamic acid cluster SpSpSpEE. Calcein assay was used to compare the iron-binding capacity of the α- and ß-CPPs pools. At the concentration of 12.5 µM CPPs used in the iron bioavailability assays, ß-CPPs pools show greater iron-binding capacity than α-CPPs pools. HuH7 human hepatoma cells show many differentiated functions of liver cells in vivo and can be used to evaluate iron bioavailability (ferritin content and soluble transferrin receptor) from Fe-α-CPPs and Fe-ß-CPPs complexes. The α-CPPs and ß-CPPs pools did not improve ferritin content or soluble transferrin receptor in HuH7 cells.

Quilamine HQ1-44, an iron chelator vectorized toward tumor cells by the polyamine transport system, inhibits HCT116 tumor growth without adverse effect.

Tumor cell growth requires large iron quantities and the deprivation of this metal induced by synthetic metal chelators is therefore an attractive method for limiting the cancer cell proliferation. The antiproliferative effect of the Quilamine HQ1-44, a new iron chelator vectorized toward tumor cells by a polyamine chain, is related to its high selectivity for the Polyamine Transport System (PTS), allowing its preferential uptake by tumoral cells. The difference in PTS activation between healthy cells and tumor cells enables tumor cells to be targeted, whereas the strong dependence of these cells on iron ensures a secondary targeting. Here, we demonstrated in vitro that HQ1-44 inhibits DNA synthesis and cell proliferation of HCT116 cells by modulating the intracellular metabolism of both iron and polyamines. Moreover, in vivo, in xenografted athymic nude mice, we found that HQ1-44 was as effective as cis-platin in reducing HCT116 tumor growth, without its side effects. Furthermore, as suggested by in vitro data, the depletion in exogenous or endogenous polyamines, known to activate the PTS, dramatically enhanced the antitumor efficiency of HQ1-44. These data support the need for further studies to assess the value of HQ1-44 as an adjuvant treatment in cancer.

Synthesis and biological properties of Quilamines II, new iron chelators with antiproliferative activities.

To selectively target tumor cells expressing an overactive Polyamine Transport System (PTS), we designed, synthesized, and evaluated the biological activity of a new generation of iron chelators, derived from the lead compound HQ1-44, which we named Quilamines II. The structures of four new antiproliferative agents were developed. They differ in the size of the linker (HQ0-44 and HQ2-44) or in the nature of the linker (HQCO-44 and HQCS-44) between a hydroxyquinoline moiety (HQ) and a homospermidine (44) chain, the best polyamine vector. The Quilamines II were obtained after 6 to 9 steps by Michael addition, peptide linkage, and reductive amination or by using the Willgerodt-Kindler reaction. The biological evaluation of these second-generation Quilamines showed that modifying the size of the linker increased the selectivity of these compounds for the PTS. In addition, measurement of the toxicity of Quilamines HQ0-44 and HQ2-44 highlighted their marked antiproliferative nature on several cancerous cell lines as well as a differential activity on nontransformed cells (fibroblasts). In contrast, Quilamines HQCO-44 and HQCS-44 presented low selectivity for the PTS, probably due to a loss of electrostatic interaction. We also demonstrated that the HCT116 cell line, originating from a human colon adenocarcinoma, was the most responsive to the various Quilamines. As deduced from the calcein and HVA assays, the higher iron chelating capacity of HQ1-44 could explain its higher antiproliferative efficiency.

Iron-mediated effect of alcohol on hepatocyte differentiation in HepaRG cells.

The development of alcoholic liver diseases depends on the ability of hepatocyte to proliferate and differentiate in the case of alcohol-induced injury. Our previous work showed an inhibitory effect of alcohol on hepatocyte proliferation. However, the effect of alcohol on hepatocyte differentiation has not yet been precisely characterized. In the present study, we evaluated the effect of alcohol on hepatocyte differentiation in relationship with changes of iron metabolism in HepaRG cells. This unique bipotent human cell line can differentiate into hepatocytes and biliary epithelial cells, paralleling liver development. Results showed that alcohol reduced cell viability, total protein level and enhanced hepatic enzymes leakage in differentiated HepaRG cells. Moreover, it caused cell enlargement, decreased number of hepatocyte and expression of C/EBPα as well as bile canaliculi F-actin. Alcohol increased expression of hepatic cell-specific markers and alcohol-metabolizing enzymes (ADH2, CYP2E1). This was associated with a lipid peroxidation and an iron excess expressed by an increase in total iron content, ferritin level, iron uptake as well as an overexpression of genes involved in iron transport and storage. Alcohol-induced hepatoxicity was amplified by exogenous iron via exceeding iron overload. Taken together, our data demonstrate that in differentiated hepatocytes, alcohol reduces proliferation while increasing expression of hepatic cell-specific markers. Moreover, iron overload could be one of the underlying mechanisms of effect of alcohol on the whole differentiation process of hepatocytes.

Involvement of polyamines in iron(III) transport in human intestinal Caco-2 cell lines.

Natural polyamines such as putrescine (Put), spermidine (Spd), and spermine (Spm), which are present in the human diet in large amounts, associated with their active transporter, are assumed to play a role in non-heme iron uptake and iron bioavailability from nutrients. Enterocytes and hepatocytes play pivotal roles in the regulation of body iron homeostasis. In this study, we report the effects of natural polyamines on iron transport in the Caco-2 cell line. In enterocyte-like Caco-2 cells, polyamines did not significantly modulate the transepithelial iron flux across the cell monolayer cultured on permeable membranes. In contrast, Spd, Spm, and to a lesser extent, Put were shown to activate Caco-2 cell iron uptake and to induce an increase in the ferritin level. This iron co-transport in enterocytes, which involved an interaction between iron and polyamine then cell uptake of the polyamine-iron complexes by the polyamine transport system, was more pronounced in proliferating than in differentiated Caco-2 cells. Moreover, it was observed at physiological concentrations of both polyamines and iron. It could thus play a role in the rapid renewal of enterocytes. These data suggest the involvement of polyamines as components of the pool of transferrin-independent iron-chelating vectors. Further investigations are needed to demonstrate their biological relevance in physiological situations.

Polyaminoquinoline iron chelators for vectorization of antiproliferative agents: design, synthesis, and validation.

Iron chelation in tumoral cells has been reported as potentially useful during antitumoral treatment. Our aim was to develop new polyaminoquinoline iron chelators targeting tumoral cells. For this purpose, we designed, synthesized, and evaluated the biological activity of a new generation of iron chelators, which we named Quilamines, based on an 8-hydroxyquinoline (8-HQ) scaffold linked to linear polyamine vectors. These were designed to target tumor cells expressing an overactive polyamine transport system (PTS). A set of Quilamines bearing variable polyamine chains was designed and assessed for their ability to interact with iron. Quilamines were also screened for their cytostatic/cytotoxic effects and their selective uptake by the PTS in the CHO cell line. Our results show that both the 8-HQ moiety and the polyamine part participate in the iron coordination. HQ1-44, the most promising Quilamine identified, presents a homospermidine moiety and was shown to be highly taken up by the PTS and to display an efficient antiproliferative activity that occurred in the micromolar range. In addition, cytotoxicity was only observed at concentrations higher than 100 µM. We also demonstrated the high complexation capacity of HQ1-44 with iron while much weaker complexes were formed with other cations, indicative of a high selectivity. We applied the density functional theory to study the binding energy and the electronic structure of prototypical iron(III)-Quilamine complexes. On the basis of these calculations, Quilamine HQ1-44 is a strong tridentate ligand for iron(III) especially in the form of a 1:2 complex.

Glycosiderophores: synthesis of tris-hydroxamate siderophores based on a galactose or glycero central scaffold, Fe(III) complexation studies.

A series of five new hexadentate tris-hydroxamate ligands based on a d-galactose or a glycerol scaffold have been synthesized. Protonation and ferric complex formation constants have been determined from solution studies by potentiometric and spectrophotometric titrations. All ligands form 1:1 Fe:L complexes. The calculated pFe values at pH 7.4 span over the range 19.2-23.0 depending on the scaffold and on the length of the spacers between hydroxamate and central scaffold and on the N-methyl substitution. This new kind of artificial siderophores based on a glycoscaffold is of interest as it opens up an easy way to modulate the pFe.

Antiproliferative and iron chelating efficiency of the new bis-8-hydroxyquinoline benzylamine chelator S1 in hepatocyte cultures.

If a new generation of iron chelators specifically devoted for cancer chemotherapy emerged these last years, any of them has not yet been approved at this time. Accordingly, there is a need to optimize new chelating molecules for iron chelation therapy and cancer treatment. So, the objective of the present investigation was to characterize the antiproliferative activity and the iron chelating capacity of the iron chelator S1 [bis-N-(8-hydroxyquinoline-5-ylmethyl)benzylamine]. Its effects were compared to O-trensox which binds ferric iron with a very high affinity (pFe(3+)=29.5). For this purpose, primary rat hepatocyte stimulated by EGF and human hepatoma HepaRG cell cultures were used. In these models, the anti-proliferative effect, the inhibition of DNA synthesis and the iron-chelating efficiency of increasing concentrations of S1 and O-trensox (0 up to 200 µM) were investigated. In the two cell culture models, we observed that S1 was about 100 times more efficient than O-trensox and the antiproliferative effect of S1 in HepaRG cells appeared at concentrations as low as 0.1 µM without cytotoxicity. Moreover, the stoichiometry of S1 for iron seemed to be in the range S1/Fe(3+)=1. Using the calcein fluorescence assay, we demonstrated that the affinity of S1 for iron was better than that of O-trensox since it was at least two times more effective to restore the fluorescence of calcein previously quenched by iron. So, the iron chelating efficiency of S1 could explain at least partially its higher anti-proliferative effect compared to O-trensox. Finally, these results suggest that molecules such as S1 may constitute a promising starting point to improve cancer treatment.

Synthesis, Physicochemical Studies, Molecular Dynamics Simulations, and Metal-Ion-Dependent Antiproliferative and Antiangiogenic Properties of Cone ICL670-Substituted Calix[4]arenes.

Iron chelators, through their capacity to modulate the iron concentration in cells, are promising molecules for cancer chemotherapy. Chelators with high lipophilicity easily enter into cells and deplete the iron intracellular pool. Consequently, iron-dependent enzymes, such as ribonucleotide reductase, which is over-expressed in cancer cells, become nonfunctional. A series of calix[4]arene derivatives substituted at the lower rim by ICL670, a strong FeIII chelator, have been synthesized. Physicochemical properties and antiproliferative, angiogenesis, and tumorigenesis effects of two calix[4]arenes mono- (5a) or disubstituted (5b) with ICL670 have been studied. These compounds form metal complexes in a ratio of one to two ligands per FeIII atom as shown by combined analyses of the protometric titration curves and ESIMS spectra. The grafting of an ICL670 group on a calix[4]arene core does not significantly alter the acid-base properties, but improves the iron-chelating and lipophilicity properties. The best antiproliferative and anti-angiogenic results were obtained with calix[4]arene ligand 5a, which possesses the highest corresponding properties. Analyses of molecular dynamics simulations performed on the two calix[4]arenes provide three-dimensional structures of the complexes and proved 5a to be the most stable upon complexation.

Antiproliferative effect on HepaRG cell cultures of new calix[4]arenes. Part II.

Cell cycle progression is dependent on the intracellular iron level and chelators can lead to iron depletion and decrease cell proliferation. This antiproliferative effect can be inhibited by exogenous iron. In this work, we present the synthesis of some new synthetic calix[4]arene podands bearing diamino-tetraesters, diamino-tetraalcohols, diamino-tetraacid and tetraaryloxypentoxy groups at the lower rim, designed as potential iron chelators. We report their effect on cell proliferation, in comparison with the new oral chelator ICL670A (4-[3,5-bis-(2-hydroxyphenyl)-1,2,4-triazol-1-yl]-benzoic acid). The antiproliferative effect of these new compounds was studied in the human hepatocarcinoma HepaRG cell cultures using cell nuclei counting after staining with the DNA intercalating fluorescence dye, Hoechst 33342. Their cytotoxicity was evaluated by the extracellular LDH activity. Preliminary results indicated that their antiproliferative effect was mainly due to their cytotoxicity. The efficiency of these compounds, being comparable to that of ICL670, was independent of iron depletion. This effect remains to be further explored. Moreover, it also shows that the new substituted calix[4]arenes could open the way to valuable new approaches for medicinal chemistry scaffolding.

Ethanol effect on cell proliferation in the human hepatoma HepaRG cell line: relationship with iron metabolism.

BACKGROUND: Alcoholism increases the risk of cirrhosis and/or hepatocellular carcinoma development. Iron, like ethanol, modulates the cell growth. However, the relationship between alcohol and iron toward hepatocyte proliferation has not been clearly elucidated. The purpose of this study was to evaluate, in the human HepaRG cell line model, the impact of ethanol on hepatocyte proliferation in relation to modulations of iron metabolism and the protective effect of iron metabolism manipulation by chelators in alcohol liver diseases. METHODS: The human hepatoma HepaRG cell line model was used. Cell viability was determined by measuring succinate dehydrogenase activity, total protein level by the Bradford method. DNA synthesis was evaluated by [(3)H]-methyl thymidine incorporation. Cytotoxicity was studied by release of lactate dehydrogenase (LDH), aspartate aminotransferase (AST), alanine aminotransferase (ALT) in culture medium and apoptosis by measuring caspase 3/7 activity. Gene expression was analyzed by RT-qPCR. Total iron, soluble transferrin receptor, and ferritin levels were, respectively, measured by colorimetrical, immuno-nephelometrical, and immuno-turbidimetrical methods. Intracellular iron uptake and accumulation was examined by radionuclide (55)Fe (III) measurement and Perls staining. RESULTS: Results showed that ethanol decreased all the parameters associated with HepaRG cell proliferation (cell viability, total protein levels, and DNA synthesis) in a dose- and time-dependent manner. This effect was accompanied by cytotoxicity and apoptosis as evaluated by a significant increase in extracellular enzymes (LDH, AST, ALT) and caspase 3/7 activity, respectively. Ethanol exposure was accompanied by an increased cellular iron uptake, together with increased expression of genes involved in iron transport and storage such as l-ferritin, Divalent Metal transporter 1, transferrin, transferrin receptor 1, and ceruloplasmin. Ethanol impact was intensified by iron-citrate and decreased by iron chelators when added to the culture medium. CONCLUSIONS: The results indicated that (i) ethanol-induced iron metabolism dysfunction could be one of the underlying mechanisms of ethanol antiproliferative effect and (ii) exogenous iron may accentuate ethanol hepatoxicity. These data suggest that iron metabolism manipulation by chelators may be a useful therapeutic approach in alcohol-related liver diseases.

Increased Lipiodol uptake in hepatocellular carcinoma possibly due to increased membrane fluidity by dexamethasone and tamoxifen.

INTRODUCTION: Lipiodol is used as a vector for chemoembolization or internal radiotherapy in unresectable hepatocellular carcinomas (HCCs). The aim of this study is to improve the tumoral uptake of Lipiodol by modulating membrane fluidizing agents to optimize the effectiveness of Lipiodol vectorized therapy. METHODS: The effect of dexamethasone and tamoxifen on membrane fluidity was studied in vitro by electron paramagnetic resonance applied to rat hepatocarcinoma cell line N1S1. The tumoral uptake of Lipiodol was studied in vivo on rats with HCC, which had been previously treated by dexamethasone and/or tamoxifen, after intra-arterial administration of (99m)Tc-SSS-Lipiodol. RESULTS: The two molecules studied here exhibit a fluidizing effect in vitro which appears dependent on time and dose, with a maximum fluidity obtained after 1 hr at concentrations of 20 µM for dexamethasone and 200 nM for tamoxifen. In vivo, while the use of dexamethasone or tamoxifen alone tends to lead to increased tumoral uptake of Lipiodol, this effect does not reach levels of significance. On the other hand, there is a significant increase in the tumoral uptake of (99m)Tc-SSS-Lipiodol in rats pretreated by both dexamethasone and tamoxifen, with a tumoral uptake (expressed in % of injected activity per g of tumor) of 13.57 ± 3.65% after treatment, as against 9.45 ± 4.44% without treatment (P<.05). CONCLUSIONS: Dexamethasone and tamoxifen fluidify the N1S1 cells membrane, leading to an increase in the tumoral uptake of Lipiodol. These drugs could be combined with chemo-Lipiodol-embolization or radiolabeled Lipiodol, with a view to improving the effectiveness of HCCs therapy.

Antiproliferative effect on HepaRG cell cultures of new calix[4]arenes.

Cell cycle progression is dependent on the intracellular iron level, and chelators lead to iron depletion and decrease cell proliferation. This antiproliferative effect can be inhibited by exogenous iron. In this work, we present the synthesis of new synthetic calix[4]arene podands bearing alkyl acid and alkyl ester groups at the lower rim, designed as potential iron chelators. We report their effect on cell proliferation, in comparison with the new oral chelator ICL670 (4-[3,5-bis-(2-hydroxyphenyl)-1,2,4-triazol-1-yl]-benzoic acid). The antiproliferative effect of these new compounds was studied in human hepatocarcinoma HepaRG cell cultures using the MTT assay. Their cytotoxicity was evaluated by extracellular LDH activity. Preliminary results indicate that their antiproliferative effect is due to their cytotoxicity. The efficiency of these compounds, comparable to that of ICL670, was independent of iron depletion. This effect remains to be further explored. Moreover, it also shows that novel substituted calix[4]arenes could open the way to new valuable medicinal chemistry scaffolding.

Effects of deferasirox and deferiprone on cellular iron load in the human hepatoma cell line HepaRG.

Two oral chelators, CP20 (deferiprone) and ICL670 (deferasirox), have been synthesized for the purpose of treating iron overload diseases, especially thalassemias. Given their antiproliferative effects resulting from the essential role played by iron in cell processes, such compounds might also be useful as anticancer agents. In the present study, we tested the impact of these two iron chelators on iron metabolism, in the HepaRG cell line which allowed us to study proliferating and differentiated hepatocytes. ICL670 uptake was greater than the CP20 uptake. The iron depletion induced by ICL670 in differentiated cells increased soluble transferrin receptor expression, decreased intracellular ferritin expression, inhibited (55)Fe (III) uptake, and reduced the hepatocyte concentration of the labile iron pool. In contrast, CP20 induced an unexpected slight increase in intracellular ferritin, which was amplified by iron-treated chelator exposure. CP20 also promoted Fe(III) uptake in differentiated HepaRG cells, thus leading to an increase of both the labile pool and storage forms of iron evaluated by calcein fluorescence and Perls staining, respectively. In acellular conditions, compared to CP20, iron removing ability from the calcein-Fe(III) complex was 40 times higher for ICL670. On the whole, biological responses of HepaRG cells to ICL670 treatment were characteristic of expected iron depletion. In contrast, the effects of CP20 suggest the potential involvement of this compound in the iron uptake from the external medium into the hepatocytes from the HepaRG cell line, therefore acting like a siderophore in this cell model.

Synthesis and biological properties of iron chelators based on a bis-2-(2-hydroxy-phenyl)-thiazole-4-carboxamide or -thiocarboxamide (BHPTC) scaffold.

Bis-2-(2-hydroxy-phenyl)-thiazole-4-carboxamides and -thiocarboxamides (BHPTCs) form a family of gemini hexacoordinated bis-tridentate chelating scaffolds. Four molecules were synthesized and shown to chelate iron(III) efficiently with a 1:1 stoichiometry. A dithioamide BHPTC displayed promising antiproliferative activity in several cancerous cell lines, making this molecule an interesting lead compound for the design of new iron-chelating anticancer drugs. Conversely, diamide BHPTCs had significant cytoprotective activity against iron overload in HepaRG cells in vitro, and were as efficient as and less toxic than deferoxamine B (DFO).

Pleiotropic role of quorum-sensing autoinducer 2 in Photorhabdus luminescens.

Bacterial virulence is an integrative process that may involve quorum sensing. In this work, we compared by global expression profiling the wild-type entomopathogenic Photorhabdus luminescens subsp. laumondii TT01 to a luxS-deficient mutant unable to synthesize the type 2 quorum-sensing inducer AI-2. AI-2 was shown to regulate more than 300 targets involved in most compartments and metabolic pathways of the cell. AI-2 is located high in the hierarchy, as it controls the expression of several transcriptional regulators. The regulatory effect of AI-2 appeared to be dose dependent. The luxS-deficient strain exhibited decreased biofilm formation and increased type IV/V pilus-dependent twitching motility. AI-2 activated its own synthesis and transport. It also modulated bioluminescence by regulating the synthesis of spermidine. AI-2 was further shown to increase oxidative stress resistance, which is necessary to overcome part of the innate immune response of the host insect involving reactive oxygen species. Finally, we showed that the luxS-deficient strain had attenuated virulence against the lepidopteran Spodoptera littoralis. We concluded that AI-2 is involved mainly in early steps of insect invasion in P. luminescens.

Modulation of cell proliferation and polyamine metabolism in rat liver cell cultures by the iron chelator O-trensox.

The antiproliferative effects of the iron chelator O-trensox and the ornithine-decarboxylase (ODC) inhibitor alpha-difluoromethylornithine (DFMO) were characterized in the rat hepatoma cell line FAO, the rat liver epithelial cell line (RLEC) and the primary rat hepatocyte cultures stimulated by EGF. We observed that O-trensox and DFMO decreased cell viabilty and DNA replication in the three culture models. The cytostatic effect of O-trensox was correlated to a cytotoxicity, higher than for DFMO, and to a cell cycle arrest in G0/G1 or S phases. Moreover, O-trensox and DFMO decreased the intracellular concentration of spermidine in the three models without changing significantly the spermine level. We concluded that iron, but also polyamine depletion, decrease cell growth. However, the drop in cell proliferation obtained with O-trensox was stronger compared to DFMO effect. Altogether, our data provide insights that, in the three rat liver cell culture models, the cytostatic effect of the iron chelator O-trensox may be the addition of two mechanisms: iron and polyamine depletion.

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.