The Fusarium mycotoxin, 2-Amino-14,16-dimethyloctadecan-3-ol (AOD) induces vacuolization in HepG2 cells.

The mycotoxin 2-Amino-14,16-dimethyloctadecan-3-ol (AOD) has been isolated from cultures of the fungus Fusarium avenaceum, one of the most prevalent Fusarium species. AOD is an analogue of sphinganine and 1-deoxysphinganine, important intermediates in the de novo biosynthesis of cellular sphingolipids. Here we studied cellular effects of AOD using the human liver cell line HepG2 as a model system. AOD (10 µM) induced a transient accumulation of vacuoles in the cells. The effect was observed at non-cytotoxic concentrations and was not linked to cell death processes. Proteomic analyses indicated that protein degradation and/or vesicular transport may be a target for AOD. Further studies revealed that AOD had only minor effects on the initiation rate of macropinocytosis and autophagy. However, the AOD-induced vacuoles were lysosomal-associated membrane protein-1 (LAMP-1) positive, suggesting that they most likely originate from lysosomes or late endosomes. Accordingly, both endosomal and autophagic protein degradation were inhibited. Further studies revealed that treatment with concanamycin A or chloroquine completely blocked the AOD-induced vacuolization, suggesting that the vacuolization is dependent of acidic lysosomes. Overall, the results strongly suggest that the increased vacuolization is due to an accumulation of AOD in lysosomes or late endosomes thereby disturbing the later stages of the endolysosomal process.

Hyphae fragments from A. fumigatus sensitize lung cells to silica particles (Min-U-Sil): Increased release of IL-1ß.

Exposure to particulate matter (PM), such as mineral particles and biological particles/components may be linked to aggravation of respiratory diseases, including asthma. Here we report that exposure to Aspergillus fumigatus hyphae fragments (AFH) and lipopolysaccharide (LPS) induced both mRNA synthesis and release of pro-inflammatory interleukin-1 beta (IL-1ß) in both human THP-1 monocytes (THP-1 Mo) and phorbol 12-myristate 13-acetate (PMA)-differentiated THP-1 monocytes (THP-1 macrophages; THP-1 Ma); while Min-U-Sil alone enhanced the release of IL-1ß only in THP-1 Ma. Co-exposure to LPS or AFH with Min-U-Sil caused a synergistic release of IL-1ß when compared to single exposures. In contrast, Min-U-Sil did not markedly change LPS- and AFH-induced release of tumor necrosis factor alpha (TNF-α). The combined exposures did not increase the LPS- and AFH-induced expression of IL-1ß mRNA. Notably, the AFH- and LPS-induced IL-1ß responses with and without co-exposure to Min-U-Sil in THP-1 Mo were found to be caspase-dependent as shown by inhibition with zYVAD-fmk. Furthermore, co-exposure with AFH and Min-U-Sil resulted in similar synergistic releases of IL-1ß in primary human airway macrophages (AM; sputum), peripheral blood monocyte-derived macrophages (MDM) and in the human bronchial epithelial cell line (BEAS-2B). In conclusion, AFH induce both the synthesis and release of IL-1ß. However, Min-U-Sil further enhanced the cleavage of the induced pro-IL-1ß.

Characterization and pro-inflammatory responses of spore and hyphae samples from various mold species.

Mold particles from Aspergillus fumigatus, Penicillium chrysogenum, Aspergillus versicolor, and Stachybotrys chartarum have been linked to respiratory-related diseases. We characterized X-ray-inactivated spores and hyphae fragments from these species by number of particles, morphology, and mycotoxin, ß-glucan and protease content/activity. The pro-inflammatory properties of mold particles were examined in human bronchial epithelial cells (BEAS-2B) and THP-1 monocytes and phorbol 12-myristate 13-acetate (PMA)-differentiated THP-1. Spores from P. chrysogenum and S. chartarum contained some hyphae fragments, whereas the other preparations contained either spores or hyphae. Each mold species produced mainly one gelatin-degrading protease that was either of the metallo- or serine type, while one remains unclassified. Mycotoxin levels were generally low. Detectable levels of ß-glucans were found mainly in hyphae particle preparations. PMA-differentiated THP-1 macrophages were by far the most sensitive model with effects in the order of 10 ng/cm2 . Hyphae preparations of A. fumigatus and P. chrysogenum were more potent than respective spore preparations, whereas the opposite seems to be true for A. versicolor and S. chartarum. Hyphae fragments of A. fumigatus, P. chrysogenum, and A. versicolor enhanced the release of metalloprotease (proMMP-9) most markedly. In conclusion, species, growth stage, and characteristics are all important factors for pro-inflammatory potential.

Air pollution-related metals induce differential cytokine responses in bronchial epithelial cells.

Different transition metals have been shown to induce inflammatory responses in lung. We have compared eight different metal ions with regard to cytokine responses, cytotoxicity and signalling mechanisms in a human lung epithelial cell model (BEAS-2B). Among the metal ions tested, there were large differences with respect to pro-inflammatory potential. Exposure to Cd(2+), Zn(2+) and As(3+) induced CXCL8 and IL-6 release at concentrations below 100µM, and Mn(2+) and Ni(2+) at concentrations above 200µM. In contrast, VO4(3-), Cu(2+) and Fe(2+) did not induce any significant increase of these cytokines. An expression array of 20 inflammatory relevant genes also showed a marked up-regulation of CXCL10, IL-10, IL-13 and CSF2 by one or more of the metal ions. The most potent metals, Cd(2+), Zn(2+) and As(3+) induced highest levels of oxidative activity, and ROS appeared to be central in their CXCL8 and IL-6 responses. Activation of the MAPK p38 seemed to be a critical mediator. However, the NF-κB pathway appeared predominately to be involved only in Zn(2+)- and As(3+)-induced CXCL8 and IL-6 responses. Thus, the most potent metals Cd(2+), Zn(2+) and As(3+) seemed to induce a similar pattern for the cytokine responses, and with some exceptions, via similar signalling mechanisms.

Immunomodulatory effects of individual and combined mycotoxins in the THP-1 cell line.

Mycotoxins commonly contaminate food and may pose a risk for disease in humans and animals. As they frequently co-occur, mixed exposures often take place. Monocyte function, including differentiation into active macrophages, is a central part of the immune response. Here we studied effects of naturally co-occurring mycotoxins in grain on monocyte function, and effects of individual and combined exposure on the differentiation process from monocytes into macrophages. The THP-1 cell line was used as a model system. The mycotoxins 2-amino-14,16-dimethyloctadecan-3-ol (AOD), alternariol (AOH), enniatin B (ENNB), deoxynivalenol (DON), sterigmatocystin (ST) and zearalenone (ZEA) differently affected cell viability in THP-1 monocytes, with DON as the most potent. AOH, ZEA and DON inhibited differentiation from monocytes into macrophages. Using this differentiation model, combined exposure of AOH, ZEA and DON were mainly found to be additive. However, the combination AOH+ZEA had somewhat synergistic effect at lower concentrations. Furthermore, alterations in macrophage functionality were found, as single exposure of AOH and ZEA inhibited lipopolysaccharide (LPS) induced TNF-α secretion, while DON increased this response. Overall, the mycotoxins affected monocyte viability and differentiation into macrophages differently. Combined exposures affected the differentiation process mainly additively.

The mycotoxin alternariol induces DNA damage and modify macrophage phenotype and inflammatory responses.

Alternariol (AOH), a mycotoxin produced by Alternaria fungi, is frequently found as a contaminant in fruit and grain products. Here we examined if AOH could modify macrophage phenotype and inflammatory responses. In RAW 264.7 mouse macrophages AOH changed the cell morphology of from round to star-shaped cells, with increased levels of CD83, CD86, CD11b, MHCII and endocytic activity. TNFα and IL-6 were enhanced at mRNA-level, but only TNFα showed increased secretion. No changes were found in IL-10 or IL-12p40 expression. Primary human macrophages changed the cell morphology from round into elongated shapes with dendrite-like protrusions in response to AOH. The levels of CD83 and CD86 were increased, HLA-DR and CD68 were down-regulated and CD80, CD200R and CD163 remained unchanged. Increased secretion of TNFα and IL-6 were found after AOH exposure, while IL-8, IL-10 and IL-12p70 were not changed. Furthermore, AOH reduced macrophage endocytic activity and autophagosomes. AOH was also found to induce DNA damage, which is suggested to be linked to the morphological and phenotypical changes. Thus, AOH was found to change the morphology and phenotype of the two cell models, but either of them could be characterized as typical M1/M2 macrophages or as dendritic cells (DC).

Autophagy and senescence, stress responses induced by the DNA-damaging mycotoxin alternariol.

The mycotoxin alternariol (AOH), a frequent contaminant in fruit and grain, is known to induce cellular stress responses such as reactive oxygen production, DNA damage and cell cycle arrest. Cellular stress is often connected to autophagy, and we employed the RAW264.7 macrophage model to test the hypothesis that AOH induces autophagy. Indeed, AOH treatment led to a massive increase in acidic vacuoles often observed upon autophagy induction. Moreover, expression of the autophagy marker LC3 was markedly increased and there was a strong accumulation of LC3-positive puncta. Increased autophagic activity was verified biochemically by measuring the degradation rate of long-lived proteins. Furthermore, AOH induced expression of Sestrin2 and phosphorylation of AMPK as well as reduced phosphorylation of mTOR and S6 kinase, common mediators of signaling pathways involved in autophagy. Transmission electron microscopy analyzes of AOH treated cells not only clearly displayed structures associated with autophagy such as autophagosomes and autolysosomes, but also the appearance of lamellar bodies. Prolonged AOH treatment resulted in changed cell morphology from round into more star-shaped as well as increased ß-galactosidase activity. This suggests that the cells eventually entered senescence. In conclusion, our data identify here AOH as an inducer of both autophagy and senescence. These effects are suggested to be to be linked to AOH-induced DSB (via a reported effect on topoisomerase activity), resulting in an activation of p53 and the Sestrin2-AMPK-mTOR-S6K signaling pathway.

Mechanisms linked to differences in the mutagenic potential of 1,3-dinitropyrene and 1,8-dinitropyrene.

This study explores and characterizes the toxicity of two closely related carcinogenic dinitro-pyrenes (DNPs), 1,3-DNP and 1,8-DNP, in human bronchial epithelial BEAS-2B cells and mouse hepatoma Hepa1c1c7 cells. Neither 1,3-DNP nor 1,8-DNP (3-30 µM) induced cell death in BEAS-2B cells. In Hepa1c1c7 cells only 1,3-DNP (10-30 µM) induced a mixture of apoptotic and necrotic cell death after 24 h. Both compounds increased the level of reactive oxygen species (ROS) in BEAS-2B as measured by CM-H2DCFDA-fluorescence. A corresponding increase in oxidative damage to DNA was revealed by the formamidopyrimidine-DNA glycosylase (fpg)-modified comet assay. Without fpg, DNP-induced DNA damage detected by the comet assay was only found in Hepa1c1c7 cells. Only 1,8-DNP formed DNA adduct measured by 32P-postlabelling. In Hepa1c1c cells, 1,8-DNP induced phosphorylation of H2AX (γH2AX) and p53 at a lower concentration than 1,3-DNP and there was no direct correlation between DNA damage/DNA damage response (DR) and induced cytotoxicity. On the other hand, 1,3-DNP-induced apoptosis was inhibited by pifithrin-α, an inhibitor of p53 transcriptional activity. Furthermore, 1,3-DNP triggered an unfolded protein response (UPR), as measured by an increased expression of CHOP, ATF4 and XBP1. Thus, other types of damage possibly linked to endoplasmic reticulum (ER)-stress and/or UPR could be involved in the induced apoptosis. Our results suggest that the stronger carcinogenic potency of 1,8-DNP compared to 1,3-DNP is linked to its higher genotoxic effects. This in combination with its lower potency to induce cell death may increase the probability of causing mutations.

Inflammation-related effects of diesel engine exhaust particles: studies on lung cells in vitro.

Diesel exhaust and its particles (DEP) have been under scrutiny for health effects in humans. In the development of these effects inflammation is regarded as a key process. Overall, in vitro studies report similar DEP-induced changes in markers of inflammation, including cytokines and chemokines, as studies in vivo. In vitro studies suggest that soluble extracts of DEP have the greatest impact on the expression and release of proinflammatory markers. Main DEP mediators of effects have still not been identified and are difficult to find, as fuel and engine technology developments lead to continuously altered characteristics of emissions. Involved mechanisms remain somewhat unclear. DEP extracts appear to comprise components that are able to activate various membrane and cytosolic receptors. Through interactions with receptors, ion channels, and phosphorylation enzymes, molecules in the particle extract will trigger various cell signaling pathways that may lead to the release of inflammatory markers directly or indirectly by causing cell death. In vitro studies represent a fast and convenient system which may have implications for technology development. Furthermore, knowledge regarding how particles elicit their effects may contribute to understanding of DEP-induced health effects in vivo, with possible implications for identifying susceptible groups of people and effect biomarkers.

Alternariol induces abnormal nuclear morphology and cell cycle arrest in murine RAW 264.7 macrophages.

The mycotoxin alternariol (AOH), a frequent contaminant in fruit and cereal products, is known to induce DNA damage with subsequent cell cycle arrest. Here we elucidated the effects of AOH on stages of cell cycle progression using the RAW 264.7 macrophage model. AOH resulted in an accumulation of cells in the G2/M-phase (4N). Most cells exhibited a large G2 nucleus whereas numbers of true mitotic cells were reduced relative to control. Both cyclin B1 and p-cdc2 levels increased, while cyclin B1 remained in the cytoplasm; suggesting arrest in the G2/M transition point. Remarkably, after exposure to AOH for 24h, most of the cells exhibited abnormally shaped nuclei, as evidenced by partly divided nuclei, nuclear blebs, polyploidy and micronuclei (MN). AOH treatment also induced abnormal Aurora B bridges, suggesting that cytokinesis was interfered within cells undergoing karyokinesis. A minor part of the resultant G1 tetraploid (4N) cells re-entered the S-phase and progressed to 8N cells.

Season linked responses to fine and quasi-ultrafine Milan PM in cultured cells.

Exposure to urbane airborne particulate matter (PM) is related to the onset and exacerbation of cardiovascular and respiratory diseases. The fine (PM1), and quasi-ultrafine (PM0.4) Milan particles collected during different seasons have been characterised and the biological effects on human epithelial lung A549, monocytes THP-1 cells and their co-culture, evaluated and compared with the results obtained on the PM10 and PM2.5 fractions. Chemical composition and transmission electron microscopy (TEM) analysis of PM0.4 showed that this fraction was very similar to PM1 for biological responses and dimension. All the winter fractions increased within 1h the level of reactive oxygen species (ROS), while only summer PM2.5 had this effect on A549 cells. The phosphorylation of H2AX (γH2AX), a marker of double strand DNA breaks (DSBs), was increased by all the winter fractions on A549 and THP-1 cells while summer PM samples did not induced this effect. PM0.4 and PM1 biological effects are partly similar and related to the season of sampling, with effects on ROS and DNA damage induced only by winter PM fractions. The winter PM damaging effect on DNA correlates with the presence of organic compounds.

Mechanisms involved in alternariol-induced cell cycle arrest.

Alternariol (AOH), a mycotoxin produced by Alternaria sp, is often found as a contaminant in fruit and cereal products. Here we employed the murine macrophage cell line RAW 264.7 to test the hypothesis that AOH causes toxicity as a response to DNA damage. AOH at concentrations of 15-30µM almost completely blocked cell proliferation. Within 30min treatment, AOH (30µM) significantly increased the level of reactive oxygen species (ROS). Furthermore, DNA base oxidations as well as DNA strand breaks and/or alkaline labile sites were detected by the comet assay after 2h exposure of AOH. Cell death (mostly necrosis) was observed after prolonged exposure to the highest concentration of AOH (60µM for 24 and 48h) in our study. The DNA damage response involved phosphorylation (activation) of histone H2AX and check point kinase-1- and 2 (Chk-1/2). Moreover, AOH activated p53 and increased the expression of p21, Cyclin B, MDM2, and Sestrin 2; likewise the level of several miRNA was affected. AOH-induced Sestrin 2 expression was regulated by p53 and could at least partly be inhibited by antioxidants, suggesting a role of ROS in the response. Interestingly, the addition of antioxidants did not inhibit cell cycle arrest. Although the formation of ROS by itself was not directly linked cell proliferation, AOH-induced DNA damage and resulting transcriptional changes in p21, MDM2, and Cyclin B likely contribute to the reduced cell proliferation; while Sestrin 2 would contribute to the oxidant defense.

Enniatin B-induced cell death and inflammatory responses in RAW 267.4 murine macrophages.

The mycotoxin enniatin B (EnnB) is predominantly produced by species of the Fusarium genera, and often found in grain. The cytotoxic effect of EnnB has been suggested to be related to its ability to form ionophores in cell membranes. The present study examines the effects of EnnB on cell death, differentiation, proliferation and pro-inflammatory responses in the murine monocyte-macrophage cell line RAW 264.7. Exposure to EnnB for 24 h caused an accumulation of cells in the G0/G1-phase with a corresponding decrease in cyclin D1. This cell cycle-arrest was possibly also linked to the reduced cellular ability to capture and internalize receptors as illustrated by the lipid marker ganglioside GM1. EnnB also increased the number of apoptotic, early apoptotic and necrotic cells, as well as cells with elongated spindle-like morphology. The Neutral Red assay indicated that EnnB induced lysosomal damage; supported by transmission electron microscopy (TEM) showing accumulation of lipids inside the lysosomes forming lamellar structures/myelin bodies. Enhanced levels of activated caspase-1 were observed after EnnB exposure and the caspase-1 specific inhibitor ZYVAD-FMK reduced EnnB-induced apoptosis. Moreover, EnnB increased the release of interleukin-1 beta (IL-1ß) in cells primed with lipopolysaccharide (LPS), and this response was reduced by both ZYVAD-FMK and the cathepsin B inhibitor CA-074Me. In conclusion, EnnB was found to induce cell cycle arrest, cell death and inflammation. Caspase-1 appeared to be involved in the apoptosis and release of IL-1ß and possibly activation of the inflammasome through lysosomal damage and leakage of cathepsin B.

Biotransformation enzymes and lung cell response to 2-hydroxyethyl-methacrylate.

The aim of this in vitro study was to investigate possible involvement of cytochrome P450 (CYP) enzymes in modifying the toxic potential of 2-hydroxyethyl-methacrylate (HEMA). Primary cultures of CYP expressing rat alveolar type 2 cells were exposed to varying concentrations of HEMA. Nuclear translocation of aryl hydrocarbon receptor (AhR) after HEMA exposure (100 µM) was demonstrated by immunocytochemical staining. Using reverse transcriptase PCR, increased mRNA level of AhR-regulated genes encoding enzymes associated with detoxification of xenobiotics were found. Exposure to 1 mM HEMA rapidly (6 h) resulted in cells with an apoptotic like morphology as suggested by marked nuclear condensation. Cotreatment of the HEMA exposed cells with a CYP inhibitor (disulfiram) or an antioxidant (vitamin C) effectively rescued the cells from this fate. Despite this effect of vitamin C, no increased level of reactive oxygen species was observed in the HEMA exposed cells. Our results suggest that HEMA activates AhR regulated gene transcription and that CYP is involved in the formation of a highly reactive HEMA metabolite.

Mechanisms involved in lipid accumulation and apoptosis induced by 1-nitropyrene in Hepa1c1c7 cells.

1-Nitropyrene (1-NP) is a nitro-polycyclic aromatic hydrocarbon (nitro-PAH) present in diesel exhaust and bound to particular matter in urban air. We show that 1-NP and the referent PAH benzo(a)pyrene (BP) induce apoptosis and a lipid accumulation dependent on cytochrome P450 1A1-metabolites in mouse hepatoma cells, whereas 1-amino-pyrene had no effect. The caspase inhibitor, N-benzyloxycarbonyl-Val-Ala-Asp(O-Me) fluoromethyl ketone (Z-VAD-fmk), inhibits 1-NP-induced apoptosis, but failed to alter 1-NP-triggered lipid accumulation determined by Nile red staining. We further show that cholesterol and fatty acid contents are modified after nitro-PAH exposure and that 1-NP-induced cholesterol level is partially involved in related apoptosis. In parallel, the activity of the stearoyl-CoA desaturase 1 (SCD1), determined by fatty acid analysis, and its expression are reduced by 1-NP. The role of SCD1 in 1-NP-induced apoptosis is demonstrated in cells down-expressing SCD1, in which an increased apoptosis is observed, whereas the SCD1 overexpression elicits the opposite effects. In contrast, changes in SCD1 gene expression have no effect on the induced lipid accumulation. Moreover, 1-NP increases the activity of the AMP-dependent protein kinase (AMPK) leading to a caspase-independent apoptosis. Overall, our study demonstrates that the 1-NP-induced apoptosis is caspase- and AMPK-dependent, and is associated to a decrease of SCD1 expression which results in an alteration of lipid homeostasis.

DNA-damage, cell-cycle arrest and apoptosis induced in BEAS-2B cells by 2-hydroxyethyl methacrylate (HEMA).

The methacrylate monomer 2-hydroxyethyl methacrylate (HEMA) is commonly used in resin-based dental restorative materials. These materials are cured in situ and HEMA and other monomers have been identified in ambient air during dental surgery. In vitro studies have demonstrated a toxic potential of methacrylates, and concerns have been raised regarding possible health effects due to inhalation. In this study we have investigated the mechanisms of HEMA-induced toxicity in the human lung epithelial cell line BEAS-2B. Depletion of cellular glutathione (GSH) and an increased level of reactive oxygen species (ROS) were seen after 2h of exposure, but the levels were restored to control levels after 12h. After 24h, inhibited cell proliferation and apoptotic cell death were found. The results of the Comet assay and the observed phosphorylation of DNA-damage-associated signalling proteins including Chk2, H2AX, and p53 suggest that the toxicity of HEMA is mediated by DNA damage. Further, the antioxidant trolox did not counteract the HEMA-induced cell-cycle arrest, which indicates that the DNA damage is of non-oxidative origin.

TACE/TGF-α/EGFR regulates CXCL8 in bronchial epithelial cells exposed to particulate matter components.

Airborne particulate matter (PM) may induce or exacerbate neutrophilic airway disease by triggering the release of inflammatory mediators, such as CXC chemokine ligand (CXCL)8, from the airway epithelium. It is still unclear which PM components are driving CXCL8 responses, as most candidates occur at low concentrations in the dusts. We therefore hypothesised that different PM constituents may contribute through common mechanisms to induce CXCL8. Human bronchial epithelial cells (BEAS-2B) were exposed to different PM components (Zn²âº/Fe²âº salts, 1-nitropyrene, lipopolysaccharide and diesel exhaust/mineral particles). Gene expression patterns were detected by real-time PCR array. CXCL8 responses were measured by real-time PCR and ELISA. CXCL8 regulation was assessed with a broad inhibitor panel and neutralising antibodies. Epidermal growth factor receptor (EGFR) phosphorylation was examined by immunoprecipitation and Western blotting. Component-induced gene expression was mainly linked to nuclear factor-κB, Ca²âº/protein kinase C, phospholipase C, low-density lipoprotein and mitogenic signalling. Many inhibitors attenuated CXCL8 release induced by all PM components, but to varying extents. However, EGFR inhibition strongly reduced CXCL8 release induced by all test compounds and selected compounds increased EGFR phosphorylation. Interference with transforming growth factor (TGF)-α or tumour necrosis factor-α-converting enzyme (TACE), which mediates TGF-α ectodomain shedding, also attenuated CXCL8 release. Different PM constituents induced CXCL8 partly through similar signalling pathways but the relative importance of the different pathways varied. However, TACE/TGF-α/EGFR signalling appears to be a convergent pathway regulating innate immune responses of airway epithelial cells upon exposure to multiple airborne pollutants.

Role of thiol-complex formation in 2-hydroxyethyl- methacrylate-induced toxicity in vitro.

Methacrylate monomers that are found to leach from cured resin-based dental materials induce biological effects in vitro. The underlying mechanisms have not been fully elucidated although involvement of increased cellular reactive oxygen species (ROS) and DNA-damage has been suggested. In this in vitro study we have elucidated the impact of a commonly used methacrylate monomer, HEMA, on the level and oxidation state of cellular glutathione, intracellular ROS level, as well as the formation of complex between HEMA and glutathione. HEMA exposure rapidly led to increased level of ROS and reduced level of GSH (reduced form of glutathione). Antioxidants effectively counteracted the ROS increase, but had no effect on the GSH depletion. No change in glutathione-disulphide (GSSG; oxidized form of glutathione) concentration was detected in the HEMA treated cells, showing that oxidation of glutathione was not responsible for the reduced GSH concentration. Further we demonstrated spontaneous formation of a complex between HEMA and GSH. In conclusion, we showed that exposure to HEMA led to drop in cellular glutathione level probably caused by complex formation with HEMA. A similar covalent binding of HEMA to macromolecules combined with increased level of cellular ROS due to lower levels of GSH is suggested to be important factors triggering the toxic response.

3-Nitrobenzanthrone and 3-aminobenzanthrone induce DNA damage and cell signalling in Hepa1c1c7 cells.

3-Nitrobenzanthrone (3-NBA) is a mutagenic and carcinogenic environmental pollutant found in diesel exhaust and urban air pollution. In the present work we have characterised the effects of 3-NBA and its metabolite 3-aminobenzanthrone (3-ABA) on cell death and cytokine release in mouse hepatoma Hepa1c1c7 cells. These effects were related to induced DNA damage and changes in cell signalling pathways. 3-NBA resulted in cell death and caused most DNA damage as judged by the amount of DNA adducts ((32)P-postlabelling assay), single strand (ss)DNA breaks and oxidative DNA lesions (comet assay) detected. An increased phosphorylation of H2AX, chk1, chk2 and partly ATM was observed using flow cytometry and/or Western blotting. Both compounds increased phosphorylation of p53 and MAPKs (ERK, p38 and JNK). However, only 3-NBA caused an accumulation of p53 in the nucleus and a translocation of Bax to the mitochondria. The p53 inhibitor pifithrin-alpha inhibited 3-NBA-induced apoptosis, indicating that cell death was a result of the triggering of DNA signalling pathways. The highest phosphorylation of Akt and degradation of IkappaB-alpha (suggesting activation of NF-kappaB) were also seen after treatment with 3-NBA. In contrast 3-ABA increased IL-6 release, but caused little or no toxicity. Cytokine release was inhibited by PD98059 and curcumin, suggesting that ERK and NF-kappaB play a role in this process. In conclusion, 3-NBA seems to have a higher potency to induce DNA damage compatible with its cytotoxic effects, while 3-ABA seems to have a greater effect on the immune system.

The B[a]P-increased intercellular communication via translocation of connexin-43 into gap junctions reduces apoptosis.

Gap junctions are channels in plasma membrane composed of proteins called connexins. These channels are organized in special domains between cells, and provide for direct gap junctional intercellular communication (GJIC), allowing diffusion of signalling molecules <1 kD. GJIC regulates cell homeostasis and notably the balance between proliferation, cell cycle arrest, cell survival and apoptosis. Here, we have investigated benzo[a]pyrene (B[a]P) effects on GJIC and on the subcellular localization of the major protein of gap junction: connexin-43 (Cx43). Our results showed that B[a]P increased GJIC between mouse hepatoma Hepa1c1c7 cells via translocation of Cx43 from Golgi apparatus and lipid rafts into gap junction plaques. Interestingly, inhibition of GJIC by chlordane or small interference RNA directed against Cx43 enhanced B[a]P-induced apoptosis in Hepa1c1c7 cells. The increased apoptosis caused by inhibition of GJIC appeared to be mediated by ERK/MAPK pathway. It is suggested that B[a]P could induce transfer of cell survival signal or dilute cell death signal via regulation of ERK/MAPK through GJIC.