Anita Solhaug

Role of cell signalling involved in induction of apoptosis by benzo[a]pyrene and cyclopenta[c,d]pyrene in Hepa1c1c7 cells

The reactive metabolites of benzo[a]pyrene (B[a]P) and cyclopenta[c,d]pyrene (CPP) induced an accumulation/phosphorylation of p53 in Hepa1c1c7 cells, whereas inhibition of p53 reduced the apoptosis. Judged by the inhibiting effect of wortmannin, phosphatidyl‐inositol‐3 (PI‐3) kinases such as DNA‐dependent protein kinase (DNA‐PK), ATM (ataxia‐telangiectasia mutated), and/or ATR (ATM related kinase), appeared to be involved in the DNA damage recognition and the B[a]P‐/CPP‐induced accumulation of p53. B[a]P and CPP also induced phosphorylation of jun‐N‐terminal kinase (JNK) and p38 mitogen activated protein kinase (MAPK). While inhibition of JNK had no effects on the B[a]P‐/CPP‐induced apoptosis, inhibition of p38 MAPK activity reduced this effect. Interestingly, survival signals such as phosphorylation of Akt and Bad seemed to be induced by the B[a]P‐/CPP‐compounds. Furthermore, also extracellular signal‐regulated kinase (ERK)1/2 was activated and seemed to function as a survival signal in B[a]P‐/CPP‐induced apoptosis.

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. J. Cell. Physiol. 208: 527–537, 2006.

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-1β) 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-1β and possibly activation of the inflammasome through lysosomal damage and leakage of cathepsin B.

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.

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.

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.

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 β-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 of Action and Toxicity of the Mycotoxin Alternariol: A Review

The mycotoxin alternariol (AOH) is produced by Alternaria fungi. It occurs naturally in foodstuffs and is frequently found as contaminant in fruit and grain products. Most information regarding AOH toxicity and the potential mechanisms involved comes from in vitro studies, as only very limited in vivo studies have been performed. AOH forms reactive oxygen species (ROS) and interacts with DNA topoisomerase, thereby generating both single (SSB)- and double-strand DNA beaks (DSB). This triggers various DNA damage response pathways. AOH causes a marked reduction in proliferation in mammalian cells due to cell cycle arrest often in the G2 /M-phase. After an additional inhibition of cytokinesis, cells with abnormal nuclei as well as polyploidy are reported. In macrophages, AOH may increase autophagic activity and induce senescence. Furthermore, AOH is found to change the morphology and phenotype of various human macrophage cell models. Studies so far indicate that the AOH-induced effects are primarily a result of DSB via its effects on topoisomerase activity. Thus, most probably there will be a threshold for the AOH-induced effects, typically seen in the 5-10 μM range. These in vitro mechanistic studies further support the in vivo studies suggesting low acute toxicity. However, a decreased immune response to infections and/or a disturbed balance of the adaptive immune system when exposed together with other mycotoxins cannot be excluded. This hypothesis needs to be further explored with proper in vivo studies.

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).