Genotoxicity and gene expression modulation of silver and titanium dioxide nanoparticles in mice.

Recently, we showed that silver nanoparticles (AgNPs) caused apoptosis, necrosis and DNA strand breaks in different cell models in vitro. These findings warranted analyses of their relevance in vivo. We investigated the genotoxic potential and gene expression profiles of silver particles of nano- (Ag20, 20 nm) and submicron- (Ag200, 200 nm) size and titanium dioxide nanoparticles (TiO2-NPs, 21 nm) in selected tissues from exposed male mice including the gonades. A single dose of 5 mg/kg bw nanoparticles was administered intravenously to male mice derived from C57BL6 (WT) and 8-oxoguanine DNA glycosylase knock-out (Ogg1(-/-) KO). Testis, lung and liver were harvested one and seven days post-exposure and analyzed for DNA strand breaks and oxidized purines employing the Comet assay with Formamidopyrimidine DNA glycosylase (Fpg) treatment, and sperm DNA fragmentation by the sperm chromatin structure assay (SCSA). Based on an initial screening of a panel of 21 genes, seven genes were selected and their expression levels were analyzed in all lung and testis tissues sampled from all animals (n = 6 mice/treatment group) using qPCR. AgNPs, in particular Ag200, caused significantly increased levels of DNA strand breaks and alkali labile sites in lung, seven days post-exposure. Fpg-sensitive lesions were significantly induced in both testis and lung. The transcript level of some key genes; Atm, Rad51, Sod1, Fos and Mmp3, were significantly induced compared to controls, particularly in lung samples from Ag200-exposed KO mice. We conclude that the Ag200 causes genotoxicity and distinct gene expression patterns in selected DNA damage response and repair related genes.

SNX1 and SNX2 mediate retrograde transport of Shiga toxin.

The bacterial toxin Shiga toxin (Stx) is transported retrogradely from early endosomes to the Golgi apparatus on its way to the endoplasmic reticulum (ER) and the cytosol. In this study we explored the functions of the two phosphoinositide binding proteins Sorting nexin 1 (SNX1) and Sorting nexin 2 (SNX2) in endosomal sorting of the toxin. When Vero cells were depleted of either SNX1 or SNX2 by small interfering RNA (siRNA), Stx transport to the trans-Golgi network (TGN) was impaired by > or = 40%, whereas combined depletion of SNX1 and SNX2 gave a total inhibition of approximately 80%. Inhibition of PI(3)P formation by wortmannin resulted in a similar reduction. Thus, although being partly redundant, both SNX1 and SNX2 are required for efficient Stx trafficking to the Golgi apparatus.

ERK2 prohibits apoptosis-induced subcellular translocation of orphan nuclear receptor NGFI-B/TR3.

Transcription factor NGFI-B (neuronal growth factor-induced clone B), also called Nur77 or TR3, is an immediate early gene and an orphan member of the nuclear receptor family. The NGFI-B protein also has a function distinct from that of a transcription factor; it translocates to mitochondria to initiate apoptosis. Recently, it was demonstrated that NGFI-B interacts with Bcl-2 by inducing a conformational change in Bcl-2, converting it from protector to a killer. After exposing rat cerebellar granule neurons to glutamate (100 mum, 15 min), NGFI-B translocated to the mitochondria. Growth factors such as the epidermal growth factor activate the MAP kinase ERK, the activity of which may determine whether a cell survives or undergoes apoptosis. In the present study we found that the epidermal growth factor activated ERK2 in cerebellar granule neurons and that this activation prohibited glutamate-induced subcellular translocation of NGFI-B. Likewise, overexpressed active ERK2 resulted in a predominant nuclear localization of green fluorescent protein-tagged NGFI-B. Thus, activation of ERK2 may overcome apoptosis-induced subcellular translocation of NGFI-B. This finding represents a novel and rapid growth factor survival pathway that is independent of gene regulation.

Nuclear receptor and apoptosis initiator NGFI-B is a substrate for kinase ERK2.

NGFI-B is an inducible orphan nuclear receptor that initiates apoptosis. Growth factors such as EGF activate the MAP kinase ERK, whose activity may determine if a cell survives or undergoes apoptosis. EGF stimulation of cells leads to phosphorylation of threonine in NGFI-B. Thr-142 of NGFI-B is comprised in a consensus MAP kinase site and was identified as a preferred substrate for ERK2 (but not ERK1) in vitro. These results suggest that NGFI-B may be a molecular target for ERK2 signals and thereby a substrate for crosstalk between a growth factor survival pathway and an inducible regulator of apoptosis.