Proteomic analysis of MCF-7 cells treated with benzo[a]pyrene, dibenzo[a,l]pyrene, coal tar extract, and diesel exhaust extract

Polycyclic aromatic hydrocarbon (PAH) DNA adducts have been associated with carcinogenesis, which is accompanied by multiple alterations in gene expression. We used two-dimensional electrophoresis to distinguish protein expression changes induced in MCF-7 cells by individual PAH (B[a]P and DB[a,l]P) and PAH mixtures (coal tar extract [SRM 1597] and diesel exhaust extract [SRM 1975]). Spots of interest were identified by MALDI-TOF-TOF. Our results have shown alterations in the expression of heat-shock proteins, cytoskeletal proteins, DNA associated proteins, and glycolytic and mitochondrial proteins. The proteins that were universally altered in expression were actin cytoplasmic 1, tubulin alpha and myosin light chain alkali, cyclophilin B, and heterogeneous ribonucleoprotein B1 (a protein involved in access to telomerase and mRNA maturation). Additional proteins with altered expression include histone H2A.1, heat-shock protein 70-2, galectin-3, nucleoside diphosphate kinase, ATP synthase, and electron transfer flavoprotein. While sharing similarities, each PAH treatment exhibited a unique proteomic fingerprint.     

Identification and expression profile of a new cytochrome P450 isoform (CYP414A1) in the hepatopancreas of Venerupis (Ruditapes) philippinarum exposed to benzo[a]pyrene, cadmium and copper

With the objective to identify promising molecular biomarkers for marine pollution monitoring, a new cytochrome P450 gene was identified from Venerupis (Ruditapes) philippinarum and classified as a member of a new subfamily, CYP414A1. Phylogenetic analysis showed that CYP414A1 was closely related to members of the CYP2 family. CYP414A1 mRNA expression was significantly induced by 50 μg/L B[a]P at 96 h, while no significant change was found in 5 μg/L B[a]P-exposed samples. For heavy metals exposure, the expression of CYP414A1 was significantly up-regulated by Cd but sharply depressed by Cu exposure. These results suggested that CYP414A1 responded to various xenobiotics stresses, and could be used as a candidate biomarker of heavy metals and B[a]P.     

Effect of a dioxin-like PCB (CB 126) on the biotransformation and genotoxicity of benzo

Two experiments were performed to study the interaction between benzo[a]pyrene (BaP) and the planar, dioxin-like PCB congener 3,3′,4,4′,5-pentachlorobiphenyl (CB 126) in the flatfish dab (Limanda limanda). The first experiment involved four groups. Group I was treated with 10 μg/kg CB 126, group II was treated with 2 mg/kg BaP, group III was first treated with 10 μg/kg CB 126 and exposed to 2 mg/kg BaP 6 days later, and group IV was a control group. The second experiment was similar, except that the BaP dosage level was increased to 50 mg/kg. Pre-treatment with 10 μg/kg of CB 126 always caused the induction of hepatic cytochrome P450 1A (CYP1A), as measured by significant increases of the model reaction 7-ethoxyresorufin-O-deethylase (EROD) in microsomal preparations. Treatment of dab with BaP caused a significant EROD induction at the 50 mg/kg, but not at the 2 mg/kg level. Concurrent with EROD induction by either CB 126 or 50 mg/kg BaP, was a significant change in the biliary metabolite pattern in favour of 1-hydroxybenzo[a]pyrene and 3-hydroxybenzo[a]pyrene and towards a lower fraction of the procarcinogen BaP-7,8-dihydrodiol (7,8-DIOL). Pre-treatment with CB 126 did not cause an increase of hepatic BaP DNA adducts formed after treatment with either 2 or 50 mg/kg BaP. Glutathione S-transferase (GST) activities remained also unaffected by any of the treatments. The results of this study suggest that the pattern of BaP metabolites in bile depends on the level of CYP1A induction. Moreover, the concurrence of a potent CYP1A inducer and BaP does not necessarily lead to an increase in DNA adduct levels in liver tissue. The observation that the level of 7,8-DIOL is decreased despite a higher (CYP1A mediated) EROD activity explains, at least in part, the lack of induction of DNA adducts.     

Multiphoton spectral analysis of benzo[a]pyrene uptake and metabolism in a rat liver cell line

Dynamic analysis of the uptake and metabolism of polycyclic aromatic hydrocarbons (PAHs) and their metabolites within live cells in real time has the potential to provide novel insights into genotoxic and non-genotoxic mechanisms of cellular injury caused by PAHs. The present work, combining the use of metabolite spectra generated from metabolite standards using multiphoton spectral analysis and an “advanced unmixing process”, identifies and quantifies the uptake, partitioning, and metabolite formation of one of the most important PAHs (benzo[a]pyrene, BaP) in viable cultured rat liver cells over a period of 24 h. The application of the advanced unmixing process resulted in the simultaneous identification of 8 metabolites in live cells at any single time. The accuracy of this unmixing process was verified using specific microsomal epoxide hydrolase inhibitors, glucuronidation and sulfation inhibitors as well as several mixtures of metabolite standards. Our findings prove that the two-photon microscopy imaging surpasses the conventional fluorescence imaging techniques and the unmixing process is a mathematical technique that seems applicable to the analysis of BaP metabolites in living cells especially for analysis of changes of the ultimate carcinogen benzo[a]pyrene-r-7,t-8-dihydrodiol-t-9,10-epoxide. Therefore, the combination of the two-photon acquisition with the unmixing process should provide important insights into the cellular and molecular mechanisms by which BaP and other PAHs alter cellular homeostasis.     

Fluoranthene enhances p53 expression and decreases mutagenesis induced by benzo[a]pyrene

Fluoranthene (Fla) is the most abundant polycyclic aromatic hydrocarbon (PAH) in diesel particulate extracts. Benzo[a]pyrene (BaP) is genotoxic and is a prototype of PAH carcinogens. Fla's toxicity and mutagenicity are minor relative to BaP's. Our data showed that Fla enhanced BaP-induced p53 expression and promoted BaP-induced cell death. In contrast, Fla decreased BaP-induced mutagenesis. Fla had almost no influence on the cell cycle. However, the effect of cotreatment with BaP (1 μM) and Fla (10 μM) in regulating the cell cycle was greater than that of BaP (2 μM) alone. It is known that BaP activates the aryl hydrocarbon receptor (AhR), and, in turn, the AhR induces cytochrome P450 (Cyp)1a1 expression. The expression of Cyp1a1 corresponds well with the induction of apoptosis and mutagenesis by BaP. Fla did not activate the AhR or antagonize BaP's induction of AhR activity and Cyp1a1 expression. Therefore, the actions of Fla on BaP's toxicity were independent of the AhR signal and Cyp1a1. In summary, results indicated that Fla directs BaP-treated cells into death rather than mutagenesis, consequently preventing cells from being transformed. The novel cooperation between Fla and BaP provides valuable information for how to increase expression of the p53 tumor suppressor.     

Effects of benzo[a]pyrene on mitochondrial and nuclear DNA damage in Atlantic killifish (Fundulus heteroclitus) from a creosote-contaminated and reference site

Benzo[a]pyrene (BaP) is a known genotoxicant that affects both mitochondrial and nuclear DNA (mtDNA, nDNA). Here, we examined mtDNA and nDNA damage in the Atlantic killifish (Fundulus heteroclitus) from a highly contaminated Superfund site (Elizabeth River, VA, USA) and from a reference site (King's Creek, VA, USA) that were dosed with 10 mg/kg BaP. Using the long amplicon quantitative PCR technique, we observed similar increases in mitochondrial and nuclear DNA damage in King's Creek fish treated with BaP. Killifish from the Elizabeth River showed high levels of basal nDNA and mtDNA damage compared to fish from the reference site, but the level of damage induced due to BaP treatment was much lower in Elizabeth River killifish compared to King's Creek fish. Laboratory-reared offspring from both populations showed increased BaP-induced damage in mtDNA, relative to nDNA. Similar to the adult experiment, the Elizabeth River larvae had higher levels of basal DNA damage than those from the reference site, but were less impacted by BaP exposure. Measurements of oxidative DNA damage (8-oxo-deoxyguanine by LC–MS/MS) showed no differences among treatment groups, suggesting that the majority of DNA damage is from covalent binding of BaP metabolites to DNA. This study shows for the first time that mitochondria can be an important target of BaP toxicity in fish, indicating that BaP exposures could have important energetic consequences. Results also suggest that multi-generational exposures in the wild may lead to adaptations that dampen DNA damage arising from BaP exposure.     

Benzo[a]pyrene induces complex H2AX phosphorylation patterns by multiple kinases including ATM, ATR, and DNA-PK

H2AX is phosphorylated (γH2AX) by members of the phosphatidylinositol 3-kinase (PI3K) family, including ataxia telangiectasia-mutated (ATM), ATM- and Rad3-related (ATR) and DNA-PK in response to DNA damage. While it has been reported that benzo[a]pyrene (BaP) cannot induce γH2AX alone in several cell lines, we have shown that BaP alone could induce γH2AX in human amnion FL cells. Thus, we further examined the ability of BaP to induce γH2AX in different cell systems. It was shown that BaP-induced γH2AX in HeLa cells in a time- and dose-dependent manner. BaP also induced γH2AX in ATM^−/− mouse fibroblasts, DNA-PKcs^−/− mouse fibroblasts, and a genetically modified human osteosarcoma U2OS cell line. PI3K inhibitors caffeine and wortmannin were then used in an effort to identify the kinase(s) responsible for BaP-induced γH2AX. Unexpectedly, in BaP-treated HeLa cells, caffeine pretreatment did not inhibit but rather increased γH2AX level. On the other hand, caffeine or wortmannin can inhibit BaP-induced γH2AX in either U2OS, DNA-PKcs^−/− or ATM^−/− cells. Taken together, these data suggest that BaP alone can induce H2AX phosphorylation in certain cell systems, and that members of the PI3K family, including ATM, ATR, and DNA-PK can participate in the phosphorylation of H2AX in the various cell types.     

Importance of CYP1A1 and CYP1B1 in bioactivation of benzo[a]pyrene in human lung cell lines

Polycyclic aromatic hydrocarbons are ubiquitous environmental pollutants classified as carcinogens in humans and rodents. The cytochromes P4501A1 and 1B1 have both shown capacity to carry out bioactivation of the prototype PAH, benzo[a]pyrene (B[a]P) to its ultimate carcinogenic B[a]P-diol-epoxide-I-1 form. The part played by each enzyme in human lung cells, however, has not been clarified. To get further insight into their individual role in the metabolic activation of B[a]P, RNA-interference was used to down-regulate CYP1A1 and/or CYP1B1 gene expression in the human lung cell lines BEP2D and NCIH2009. Fluorescence-HPLC analysis revealed that formation of B[a]P-tetrol-I-1 (hydrolyzed form of the corresponding diol-epoxide) was dependent primarily on CYP1A1. In cells without down-regulation of CYP1A1, the B[a]P-tetrol-I-1 was the major tested isomer formed. In contrast, the B[a]P-cis- and trans-7,8-dihydrodiol isomers were readily formed in cells expressing high levels of either CYP-gene. Simultaneous down-regulation of CYP1A1 and CYP1B1 mRNA resulted in low levels of metabolites overall. Residual unmetabolized B[a]P levels followed the expression of CYP1A1 in an inverse manner. In conclusion, these results indicate a major role of CYP1A1 in the bioactivation of B[a]P to carcinogenic B[a]P-diol-epoxides and in overall metabolism of B[a]P in human lung cell lines. In contrast, both CYP1A1 and CYP1B1 contribute significantly to the formation of the B[a]P-cis- and trans-7,8-dihydrodiol isomers.     

Rapid induction of colonic adenocarcinoma in mice exposed to benzo[a]pyrene and dextran sulfate sodium

Previously, we reported that the mutation frequency was markedly increased in the colon after the oral treatment of mice with an environmental mutagen/carcinogen, benzo[a]pyrene (BP); however this was not followed by tumor development. The reasons for this are as yet unresolved. The purpose of the present study is to explore the mechanisms why a high frequency of mutations induced by BP in the colon is not associated with subsequent tumor development. We show in this study that oral administration of BP to CD2F₁ mice at 125 mg/kg/day for 5 days can lead to adenocarcinomas in the mouse colon both at Weeks 4 (5/8 mice) and 11 (100% of mice), but only in the presence of inflammation induced by 4% dextran sulfate sodium (DSS) in the drinking water for up to 2 weeks. These data indicate that, in this DSS model, BP induced mutagenic events lead to tumors in the mouse colon, a tissue which is not a BP target organ. DSS-induced inflammation in a tissue primed with mutagenic risk is a key to the induction of tumors in this model. This study provides a novel, rapid and useful colon carcinogenesis model (BP/DSS model).     

Benzo[a]pyrene-induced immunotoxicity in Japanese medaka (Oryzias latipes): relationship between lymphoid CYP1A activity and humoral immune suppression

Exposure to the environmental contaminant benzo[a]pyrene (BaP) results in suppression of immune function in both mammalian and fish species. This laboratory has previously demonstrated that a single intraperitoneal (IP) injection of BaP reduced lymphocyte proliferation, phagocyte-mediated superoxide generation, and antibody-forming cell (AFC) numbers in Japanese medaka (Oryzias latipes). The objective of the current study was to determine the role of BaP metabolism in the observed immunosuppression. Results from rodent studies have suggested that BaP elicits its immunotoxic effects via upregulation of cytochrome P4501A1 (CYP1A1) and the subsequent production of immunosuppressive BaP metabolites. In this study, exposure of medaka to 200 microg BaP/g BW significantly induced CYP1A expression or activity within lymphoid tissue 48 h post-IP injection; induction was observed specifically within distinct subpopulations of kidney mononuclear cells. Concurrent injection of fish with BaP and the CYP1A1 inhibitors alpha-naphthoflavone (ANF) or dehydroepiandrosterone (DHEA) resulted in inhibition of renal EROD activity and amelioration of BaP-induced suppression of medaka AFC numbers. Results of this study suggest that (1) BaP-induced suppression of medaka humoral immunity relies upon the CYP1A-catalyzed production of immunotoxic BaP metabolites and (2) BaP metabolites may be created in situ, directly by specific cells within kidney lymphoid tissue. Thus, apparently, mechanisms involved in BaP-induced immunosuppression have been phylogenetically conserved from fish to mammals.     

Tissue-specific induction of cytochromes P450 1A1 and 1B1 by polycyclic aromatic hydrocarbons and polychlorinated biphenyls in engineered C57BL/6J mice of arylhydrocarbon receptor gene

Tissue-specific induction of mRNA of cytochrome P450 (P450 or CYP) 1A1 and 1B1 by polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs) was investigated in wild and arylhydrocarbon receptor (AhR)-deficient C57BL/6J mice. Ratios of mRNA expression of CYP1A1 or CYP1B1 over beta-actin were determined and used to compare levels of expression and induction of these P450s by PAHs and PCBs in various organs. CYP1A1 mRNA was detected in control mice at very low levels in liver, lung, heart, kidney, intestine, thymus, testis, uterus, ovary, and brain and was highly induced in these organs by benzo[a]pyrene and 3,4,3',4'-tetrachlorobiphenyl in AhR(+/+) mice. In AhR(+/+) and AhR(-/-) mice, CYP1B1 mRNA was found to be constitutively expressed at significant levels in heart (the ratio of mRNAs of CYP1B1 to beta-actin was approximately 0.6), kidney ( approximately 0.8), intestine ( approximately 0.3), testis ( approximately 0.9), thymus ( approximately 0.4), uterus ( approximately 0.3), ovary ( approximately 1.4), and brain ( approximately 0.4), whereas it was low in liver and lung (the mRNA ratio to beta-actin was <0.2 in these cases). CYP1B1 in the latter two organs was highly induced by PAHs and 3,4,3',4'-tetrachlorobiphenyl in AhR(+/+) mice. The induction of CYP1B1 by PAHs and PCBs was more extensive in organs in which the constitutive expression of CYP1B1 was low. For example, CYP1B1 was induced 9-fold and 10-fold by benzo[a]pyrene and 3,4,3',4'-tetrachlorobiphenyl in livers of male and female mice, respectively, whereas in testis and ovary, the fold induction of CYP1B1 by two inducers was only 1.1 and 1.4, respectively. Liver microsomal xenobiotic oxidation activities were induced by these PAHs and PCBs in male and female AhR(+/+) mice. These results suggest that CYP1A1 and CYP1B1 are differentially regulated in their expression in extrahepatic organs of mice and could be induced by PAHs and PCBs with different extents of induction depending on the inducers used and the organs examined in AhR(+/+) mice. The findings of significant levels of constitutive expression of CYP1B1 in AhR(-/-) mice as well as AhR(+/+) mice in several organs including heart, kidney, thymus, testis, ovary, and brain in AhR(-/-) mice as well as AhR(+/+) mice are of importance in understanding the basis of toxicity and carcinogenesis by chemicals that are metabolized by CYP1B1.     

All-trans retinoic acid enhances the transport of phase II metabolites of benzo[a]pyrene by inducing the Breast Cancer Resistance Protein expression in Caco-2 cells

All-trans retinoic acid (atRA) is the most active metabolite of vitamin A. It is a ligand of retinoic acid receptors (RAR) as well as of retinoid X receptors (RXR) and effectively stimulates the RAR/RXR signalling pathway.     

Synergistic enhancement of H2O2 production in human epidermoid carcinoma cells by Benzo[a]pyrene and ultraviolet A radiation

Benzo[a]pyrene (BaP) is an ubiquitous environmental pollutant with potential carcinogenecity. It was shown that BaP, upon irradiation by UV A, enhanced the formation of 8-hydroxy-2'-deoxyguanosine in purified DNA and in cultured cells. The purpose of this present study was to determine whether BaP and UV radiation synergistically generate reactive oxygen species (ROS) that consequently result in the oxidation of DNA bases. In this study, the levels of H(2)O(2) were measured as an indicator of ROS in A431 cells and primary human keratinocytes treated with BaP plus UV radiation. Production of H(2)O(2) significantly increased from cells treated with BaP plus UVB or UVA, with the latter having a much greater effect. The responses of A431 cells and primary human keratinocytes to BaP and UVA irradiation were similar in generation of extracellular H(2)O(2). Also, H(2)O(2) production proportionally correlated with UVA and UVB dose, but was independent of time or BaP concentration. Treatment with catalase and general ROS scavengers significantly decreased H(2)O(2) production from cells treated with BaP plus UVA, whereas scavengers of *O2-, *OH, and (1)O(2) had minimal effects. These results demonstrate that BaP synergistically enhances the production of H(2)O(2) from cultured cells by UVA and, to a lesser extent, by UVB, supporting the hypothesis that interaction of BaP and UVA can generate ROS and further substantiate oxidative DNA damage that may lead to carcinogenesis.     

Genotoxic activation of the environmental pollutant 3,6-dinitrobenzo[e]pyrene in Salmonella typhimuriumumu strains expressing human cytochrome P450 and N-acetyltransferase

3,6-Dinitrobenzo[e]pyrene (DNBeP) is a potent mutagen identified in surface soil in two metropolitan areas of Japan. We investigated whether DNBeP can cause genotoxicity through any metabolic activation pathway in bacteria using the parental strain Salmonella enterica serovar Typhimurium (S. typhimurium) TA1535/pSK1002, nitroreductase (NR)-deficient strain NM1000, the O-acetyltransferase (O-AT)-deficient strain NM2000, bacterial O-AT-overexpressing strain NM2009, and bacterial NR- and O-AT-overexpressing strain NM3009 established in our laboratory. To further clarify the role of human cytochrome P450 (P450 or CYP) and N-acetyltransferase (NAT) enzymes in the bioactivation of DNBeP to genotoxic metabolites, we determined the genotoxicity of DNBeP using a variety of umu tester strains expressing human P450 and NAT enzymes. The dose-dependent induction of umuC by DNBeP was observed at concentrations between 0.01 and 1 nM in the O-AT-expression strain, but not in the O-AT-deficient strain. In the CYP3A4-, CYP1A2-, CYP1A1-, and CYP1B1-expressing strains, DNBeP was found to be activated to reactive metabolites that cause the induction of umuC gene expression compared with the parent strain. The induction of DNBeP in the NAT2-expressing strain had a 10-fold lower concentration than that in the NAT1-expressing strain. Collectively, these results suggest that nitroreduction by human CYP1A2, CYP3A4, and CYP1A1 and O-acetylation by human NAT2 contributed to the genotoxic activation of DNBeP to its metabolites.     

Reduction of androgen receptor expression by benzo[alpha]pyrene and 7,8-dihydro-9,10-epoxy-7,8,9,10-tetrahydrobenzo[alpha]pyrene in human lung cells

5Alpha-dihydrotestosterone significantly increased cell growth of lung adenocarcinoma cell line H1355. Benzo[alpha]pyrene (BaP) was a pulmonary carcinogen found in cigarette smoke. Treatment with 1microM BaP tremendously reduced constitutive androgen receptor (AR) expression, as determined with Western immunoblotting and the real-time RT-PCR assay, as well as testosterone-induced AR protein levels in H1355 cells. Similarly, 1microM BaP significantly reduced AR mRNA levels in human bronchial epithelial cells BEAS-2B. Although BaP, 2,3,7,8-tetrachlorodibenzo-p-dixin and polychlorinated biphenyl 126 activated aryl hydrocarbon receptor (AhR), which subsequently induced cytochrome P4501A1 (CYP1A1) and P4501B1 (CYP1B1) expression in H1355 cells, unexpectedly, neither TCDD nor PCB126 reduced AR expression. Antagonizing AhR activation and cytochrome P4501 activity with alpha-naphthoflavone, or inhibiting CYP1B1 activity with 2,4,3',5'-tetramethoxystilbene, however, prevented BaP-induced AR reduction. Furthermore, 7,8-dihydro-9,10-epoxy-7,8,9,10-tetrahydrobenzo[alpha]pyrene, a BaP carcinogenic metabolite catalyzed by CYP1A1 and CYP1B1, significantly reduced AR expression in H1355 cells and human lung fibroblasts WI-38. This was the first study that reports that BaP and BPDE reduced endogenous AR expression. These data suggest that metabolically activated BaP may disrupt androgen function by reducing AR levels in androgen-responsive organs.     

Multidrug resistance protein (MRP) 4 attenuates benzo[a]pyrene-mediated DNA-adduct formation in human bronchoalveolar H358 cells

Multi-drug resistance protein (MRP) 4, an ATP-binding cassette (ABC) transporter, has broad substrate specificity. It facilitates the transport of bile salt conjugates, conjugated steroids, nucleoside analogs, eicosanoids, and cardiovascular drugs. Recent studies in liver carcinoma cells and hepatocytes showed that MRP4 expression is regulated by the aryl hydrocarbon receptor (AhR) and nuclear factor E2-related factor 2 (Nrf2). The AhR has particular importance in the lung and is most commonly associated with the up-regulation of cytochrome P-450 (CYP)-mediated metabolism of benzo[a]pyrene (B[a]P) to reactive intermediates. Treatment of H358, human bronchoalveolar, cells with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or (−)-benzo[a]pyrene-7,8-dihydro-7,8-diol (B[a]P-7,8-dihydrodiol), the proximate carcinogen of B[a]P, revealed that MRP4 expression was increased compared to control. This suggested that MRP4 expression might contribute to the paradoxical decrease in (+)-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene-2′-deoxyguanosine ((+)-anti-trans-B[a]PDE-dGuo) DNA-adducts observed in TCDD-treated H358 cells. We have now found that decreased MRP4 expression induced by a short hairpin RNA (shRNA), or chemical inhibition with probenecid, increased (+)-anti-trans-B[a]PDE-dGuo formation in cells treated with (−)-B[a]P-7,8-dihydrodiol, but not the ultimate carcinogen (+)-anti-trans-B[a]PDE. Thus, up-regulation of MRP4 increased cellular efflux of (−)-B[a]P-7,8-dihydrodiol, which attenuated DNA-adduct formation. This is the first report identifying a specific MRP efflux transporter that decreases DNA damage arising from an environmental carcinogen.     

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.