Evelien Kramer

Distribution, elimination, and toxicity of silver nanoparticles and silver ions in rats after 28-day oral exposure.

We report the results of a 28-day oral exposure study in rats, exposed to <20 nm noncoated, or <15 nm PVP-coated silver nanoparticles ([Ag] = 90 mg/kg body weight (bw)), or AgNO(3) ([Ag] = 9 mg/kg bw), or carrier solution only. Dissection was performed at day 29, and after a wash-out period of 1 or 8 weeks. Silver was present in all examined organs with the highest levels in the liver and spleen for all silver treatments. Silver concentrations in the organs were highly correlated to the amount of Ag(+) in the silver nanoparticle suspension, indicating that mainly Ag(+), and to a much lesser extent silver nanoparticles, passed the intestines in the silver nanoparticle exposed rats. In all groups silver was cleared from most organs after 8 weeks postdosing, but remarkably not from the brain and testis. Using single particle inductively coupled plasma mass spectrometry, silver nanoparticles were detected in silver nanoparticle exposed rats, but, remarkably also in AgNO(3) exposed rats, hereby demonstrating the formation of nanoparticles from Ag(+)in vivo that are probably composed of silver salts. Biochemical markers and antibody levels in blood, lymphocyte proliferation and cytokine release, and NK-cell activity did not reveal hepatotoxicity or immunotoxicity of the silver exposure. In conclusion, oral exposure to silver nanoparticles appears to be very similar to exposure to silver salts. However, the consequences of in vivo formation of silver nanoparticles, and of the long retention of silver in brain and testis should be considered in a risk assessment of silver nanoparticles.

Presence of Nano-Sized Silica during In Vitro Digestion of Foods Containing Silica as a Food Additive

The presence, dissolution, agglomeration state, and release of materials in the nano-size range from food containing engineered nanoparticles during human digestion is a key question for the safety assessment of these materials. We used an in vitro model to mimic the human digestion. Food products subjected to in vitro digestion included (i) hot water, (ii) coffee with powdered creamer, (iii) instant soup, and (iv) pancake which either contained silica as the food additive E551, or to which a form of synthetic amorphous silica or 32 nm SiO(2) particles were added. The results showed that, in the mouth stage of the digestion, nano-sized silica particles with a size range of 5-50 and 50-500 nm were present in food products containing E551 or added synthetic amorphous silica. However, during the successive gastric digestion stage, this nano-sized silica was no longer present for the food matrices coffee and instant soup, while low amounts were found for pancakes. Additional experiments showed that the absence of nano-sized silica in the gastric stage can be contributed to an effect of low pH combined with high electrolyte concentrations in the gastric digestion stage. Large silica agglomerates are formed under these conditions as determined by DLS and SEM experiments and explained theoretically by the extended DLVO theory. Importantly, in the subsequent intestinal digestion stage, the nano-sized silica particles reappeared again, even in amounts higher than in the saliva (mouth) digestion stage. These findings suggest that, upon consumption of foods containing E551, the gut epithelium is most likely exposed to nano-sized silica.

Characterization of Translocation of Silver Nanoparticles and Effects on Whole-Genome Gene Expression Using an In Vitro Intestinal Epithelium Coculture Model

Applications of nanoparticles in the food sector are eminent. Silver nanoparticles are among the most frequently used, making consumer exposure to silver nanoparticles inevitable. Information about uptake through the intestines and possible toxic effects of silver nanoparticles is therefore very important but still lacking. In the present study, we used an in vitro model for the human intestinal epithelium consisting of Caco-2 and M-cells to study the passage of silver nanoparticles and their ionic equivalents and to assess their effects on whole-genome mRNA expression. This in vitro intestine model was exposed to four sizes of silver nanoparticles for 4 h. Exposure to silver ions was included as a control since 6-17% of the silver nanoparticles were found to be dissociated into silver ions. The amount of silver ions that passed the Caco-2 cell barrier was equal for the silver ion and nanoparticle exposures. The nanoparticles induced clear changes in gene expression in a range of stress responses including oxidative stress, endoplasmatic stress response, and apoptosis. The gene expression response to silver nanoparticles, however, was very similar to that of AgNO(3). Therefore, the observed effects of the silver nanoparticles are likely exerted by the silver ions that are released from the nanoparticles.

Beta-Carotene Reduces Body Adiposity of Mice via BCMO1

Evidence from cell culture studies indicates that β-carotene-(BC)-derived apocarotenoid signaling molecules can modulate the activities of nuclear receptors that regulate many aspects of adipocyte physiology. Two BC metabolizing enzymes, the BC-15,15′-oxygenase (Bcmo1) and the BC-9′,10′-oxygenase (Bcdo2) are expressed in adipocytes. Bcmo1 catalyzes the conversion of BC into retinaldehyde and Bcdo2 into β-10′-apocarotenal and β-ionone. Here we analyzed the impact of BC on body adiposity of mice. To genetically dissect the roles of Bcmo1 and Bcdo2 in this process, we used wild-type and Bcmo1 -/- mice for this study. In wild-type mice, BC was converted into retinoids. In contrast, Bcmo1-/- mice showed increased expression of Bcdo2 in adipocytes and β-10′-apocarotenol accumulated as the major BC derivative. In wild-type mice, BC significantly reduced body adiposity (by 28%), leptinemia and adipocyte size. Genome wide microarray analysis of inguinal white adipose tissue revealed a generalized decrease of mRNA expression of peroxisome proliferator-activated receptor γ (PPARγ) target genes. Consistently, the expression of this key transcription factor for lipogenesis was significantly reduced both on the mRNA and protein levels. Despite β-10′-apocarotenoid production, this effect of BC was absent in Bcmo1-/- mice, demonstrating that it was dependent on the Bcmo1-mediated production of retinoids. Our study evidences an important role of BC for the control of body adiposity in mice and identifies Bcmo1 as critical molecular player for the regulation of PPARγ activity in adipocytes

Carboxypeptidase E and thrombospondin-1 are differently expressed in subcutaneous and visceral fat of obese subjects

Abstract. The aim of this study was to identify candidate genes for visceral obesity by screening for genes strongly differentially expressed between human subcutaneous and visceral adipose depots. A cDNA microarray with human adipose-derived cDNAs was used as an initial screening to identify genes that are potentially differentially expressed between human subcutaneous and visceral abdominal fat tissues. For the two best candidates, carboxypeptidase E (CPE) and thrombospondin-1 (THBS1) (EST N72406), real-time RT-PCR was performed to confirm their depot specific expression in extremely obese individuals. Both genes appeared to be strongly differentially expressed, having a higher expression in the visceral depot than in the subcutaneous one. For THBS1, the difference in expression between the depots was greater in women than in men. The involvement of CPE and THBS1 in obesity allows us to suggest that the physiological processes controlled by these genes contribute to depot and gender-related differences in the metabolic complications of obesity.

Supplemental Calcium Attenuates the Colitis-Related Increase in Diarrhea, Intestinal Permeability, and Extracellular Matrix Breakdown in HLA-B27 Transgenic Rats

We have shown in several controlled rat and human infection studies that dietary calcium improves intestinal resistance and strengthens the mucosal barrier. Reinforcement of gut barrier function may alleviate inflammatory bowel disease (IBD). Therefore, we investigated the effect of supplemental calcium on spontaneous colitis development in an experimental rat model of IBD. HLA-B27 transgenic rats were fed a purified high-fat diet containing either a low or high calcium concentration (30 and 120 mmol CaHPO4/kg diet, respectively) for almost 7 wk. Inert chromium EDTA (CrEDTA) was added to the diets to quantify intestinal permeability by measuring urinary CrEDTA excretion. Relative fecal wet weight was determined to quantify diarrhea. Colonic inflammation was determined histologically and by measuring mucosal interleukin (IL)-1beta. In addition, colonic mucosal gene expression of individual rats was analyzed using whole-genome microarrays. The calcium diet significantly inhibited the increase in intestinal permeability and diarrhea with time in HLA-B27 rats developing colitis compared with the control transgenic rats. Mucosal IL-1beta levels were lower in calcium-fed rats and histological colitis scores tended to be lower (P = 0.08). Supplemental calcium prevented the colitis-induced increase in the expression of extracellular matrix remodeling genes (e.g. matrix metalloproteinases, procollagens, and fibronectin), which was confirmed by quantitative real-time PCR and gelatin zymography. In conclusion, dietary calcium ameliorates several important aspects of colitis severity in HLA-B27 transgenic rats. Reduction of mucosal irritation by luminal components might be part of the mechanism. These results show promise for supplemental calcium as effective adjunct therapy for IBD.

Impaired barrier function by dietary fructo-oligosaccharides (FOS) in rats is accompanied by increased colonic mitochondrial gene expression.

BackgroundDietary non-digestible carbohydrates stimulate the gut microflora and are therefore presumed to improve host resistance to intestinal infections. However, several strictly controlled rat infection studies showed that non-digestible fructo-oligosaccharides (FOS) increase, rather than decrease, translocation of Salmonella towards extra-intestinal sites. In addition, it was shown that FOS increases intestinal permeability already before infection. The mechanism responsible for this adverse effect of FOS is unclear. Possible explanations are altered mucosal integrity due to changes in tight junctions or changes in expression of defense molecules such as antimicrobials and mucins. To examine the mechanisms underlying weakening of the intestinal barrier by FOS, a controlled dietary intervention study was performed. Two groups of 12 rats were adapted to a diet with or without FOS. mRNA was collected from colonic mucosa and changes in gene expression were assessed for each individual rat using Agilent rat whole genome microarrays.ResultsAmong the 997 FOS induced genes we observed less mucosal integrity related genes than expected with the clear permeability changes. FOS did not induce changes in tight junction genes and only 8 genes related to mucosal defense were induced by FOS. These small effects are unlikely the cause for the clear increase in intestinal permeability that is observed. FOS significantly increased expression of 177 mitochondria-related genes. More specifically, induced expression of genes involved in all five OXPHOS complexes and the TCA cycle was observed. These results indicate that dietary FOS influences intestinal mucosal energy metabolism. Furthermore, increased expression of 113 genes related to protein turnover, including proteasome genes, ribosomal genes and protein maturation related genes, was seen. FOS upregulated expression of the peptide hormone proglucagon gene, in agreement with previous studies, as well as three other peptide hormone genes; peptide YY, pancreatic polypeptide and cholecystokinin.ConclusionWe conclude that altered energy metabolism may underly colonic barrier function disruption due to FOS feeding in rats.

Translocation of differently sized and charged polystyrene nanoparticles in in vitro intestinal cell models of increasing complexity.

Abstract Intestinal translocation is a key factor for determining bioavailability of nanoparticles (NPs) after oral uptake. Therefore, we evaluated three in vitro intestinal cell models of increasing complexity which might affect the translocation of NPs: a mono-culture (Caco-2 cells), a co-culture with mucus secreting HT29-MTX cells and a tri-culture with M-cells. Cell models were exposed to well characterized differently sized (50 and 100 nm) and charged (neutral, positively and negatively) polystyrene NPs. In addition, two types of negatively charged NPs with different surface chemistries were used. Size strongly affected the translocation of NPs, ranging up to 7.8% for the 50 nm NPs and 0.8% for the 100 nm NPs. Surface charge of NPs affected the translocation, however, surface chemistry seems more important, as the two types of negatively charged 50 nm NPs had an over 30-fold difference in translocation. Compared with the Caco-2 mono-culture, presence of mucus significantly reduced the translocation of neutral 50 nm NPs, but significantly increased the translocation of one type of negatively charged NPs. Incorporation of M-cells shifted the translocation rates for both NPs closer to those in the mono-culture model. The relative pattern of NP translocation in all three models was similar, but the absolute amounts of translocated NPs differed per model. We conclude that for comparing the relative translocation of different NPs, using one intestinal model is sufficient. To choose the most representative model for risk assessment, in vivo experiments are now needed to determine the in vivo translocation rates of the used NPs.

Factors influencing cDNA microarray hybridization on silylated glass slides

cDNA microarray technology is becoming the technique of choice for studying gene expression and gene expression patterns. Although experimental protocols are available, only limited methodological information on microarray manufacture, hybridization, and signal interpretation has been published. The aim of this paper is to provide more insight into the practical aspects of microarray construction and hybridization. The influence of the size, composition, and concentration of the spotted DNA fragments on the final hybridization signal and the effect of hybridization volume, sample concentration, and sample depletion have been tested and are discussed.

Salmonella induces prominent gene expression in the rat colon

BackgroundSalmonella enteritidis is suggested to translocate in the small intestine. In vivo it induces gene expression changes in the ileal mucosa and Peyers patches. Stimulation of Salmonella translocation by dietary prebiotics fermented in colon suggests involvement of the colon as well. However, effects of Salmonella on colonic gene expression in vivo are largely unknown. We aimed to characterize time dependent Salmonella-induced changes of colonic mucosal gene expression in rats using whole genome microarrays. For this, rats were orally infected with Salmonella enteritidis to mimic a foodborne infection and colonic gene expression was determined at days 1, 3 and 6 post-infection (n = 8 rats per time-point). As fructo-oligosaccharides (FOS) affect colonic physiology, we analyzed colonic mucosal gene expression of FOS-fed versus cellulose-fed rats infected with Salmonella in a separate experiment. Colonic mucosal samples were isolated at day 2 post-infection.ResultsSalmonella affected transport (e.g. Chloride channel calcium activated 6, H+/K+ transporting Atp-ase), antimicrobial defense (e.g. Lipopolysaccharide binding protein, Defensin 5 and phospholipase A2), inflammation (e.g. calprotectin), oxidative stress related genes (e.g. Dual oxidase 2 and Glutathione peroxidase 2) and Proteolysis (e.g. Ubiquitin D and Proteosome subunit beta type 9). Furthermore, Salmonella translocation increased serum IFNγ and many interferon-related genes in colonic mucosa. The gene most strongly induced by Salmonella infection was Pancreatitis Associated Protein (Pap), showing >100-fold induction at day 6 after oral infection. Results were confirmed by Q-PCR in individual rats. Stimulation of Salmonella translocation by dietary FOS was accompanied by enhancement of the Salmonella-induced mucosal processes, not by induction of other processes.ConclusionWe conclude that the colon is a target tissue for Salmonella, considering the abundant changes in mucosal gene expression.