Impact of Alternaria toxins on CYP1A1 expression in different human tumor cells and relevance for genotoxicity.

The Alternaria toxins alternariol (AOH) and alternariol monomethyl ether (AME) have been reported previously to act as activators of the aryl hydrocarbon receptor (AhR) in murine hepatoma cells, thus enhancing the expression of cytochrome P450 (CYP) 1A monooxygenases. Concomitantly, both benzopyrones represent substrates of CYP1A, giving rise to catecholic metabolites. The impact of AOH and AME on CYP1A expression in human cells of different tissue origin colon (HT29), esophagus (KYSE510), liver (HepG2) and their effects on cell viability, generation of reactive oxygen species (ROS) and DNA integrity were investigated. ROS production was induced by both mycotoxins in all cell lines with AOH exhibiting the highest potency in esophageal cells concomitant with the most prominent CYP1A induction level. Of note, altertoxin-II (ATX-II), the more potent DNA-damaging mutagen formed by Alternaria alternata, induces CYP1A even at significant lower concentrations. AhR-siRNA knockdown in human esophageal cells supported the hypothesis of AhR-mediated CYP1A1 induction by AOH. However, DNA damage was minor at CYP1A1-inducing AOH concentrations. AhR-depletion did not affect the DNA-damaging properties of AOH indicating no substantial impact of AhR in this regard. However, in combination with xenobiotics prone to metabolic activation by CYP1A the induction of CYP1A by Alternaria toxins deserves further attention.

Bacterial quorum sensing molecule induces chemotaxis of human neutrophils via induction of p38 and leukocyte specific protein 1 (LSP1).

When bacteria colonize surfaces, they socialize and form biofilms. This process is well regulated and relies on the communication among the bacteria via so-called "quorum sensing molecules". Among those, N-(3-oxododecanoyl)-L-homoserine lactone (AHL-12), generated by Pseudomonas aeruginosa and other Gram-negative bacteria, activates not only bacteria but also interacts with mammalian cells. Among others, it activates phagocytic cells and - as we had shown previously - it is chemotactic for human polymorphonuclear neutrophils (PMN) in vitro. In the present study, we analyzed the signalling pathway of AHL-12 in PMN. We focused on the mitogen activated protein (MAP) kinase p38, because SB203580, an inhibitor of p38, prevented the AHL-12 induced chemotaxis. We found that in response to AHL-12, p38 was phosphorylated within minutes, as was its downstream target, the MAPKAP-Kinase-2 (MK2). In PMN, the major substrate of MK2 is the leukocyte specific protein 1 (LSP1), which binds to F-actin and participates directly in actin polymerization and cell migration. In response to AHL-12, LSP1 was phosphorylated and co-localized with F-actin in polarized PMN, suggesting that AHL-12-induced migration depended on p38 and LSP1 activation.

Furin cleavage of bacterial expressed glutathione-S-transferase-pro-transforming growth factor beta1 fusion protein in vitro.

To investigate the processing of transforming growth factor beta1 (TGFbeta1) pro-protein by furin protease we expressed a GST-pro-TGFbeta1 fusion protein in bacteria. Analysis of the furin digestion pattern revealed the liberation of 12.5 kDa TGFbeta1 monomers. There was no evidence for cleavage of an alternative furin site within the pro-protein.