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The goal of the present study was to develop an online web-based toolbox that contains generic physiologically based kinetic (PBK) models for rats and humans, including underlying calculation tools to predict plasma protein binding and tissue:plasma distribution, to be used for quantitative in-vitro-to-in-vivo extrapolations (QIVIVE). The PBK models within the toolbox allow first estimations of internal plasma and tissue concentrations of chemicals to be made, based on the logP and pKaof the chemicals and values for intestinal uptake and intrinsic hepatic clearance. As a case study, the toolbox was used to predict oral equivalent doses of in vitro ToxCast bioactivity data for the food additives methylparaben, propyl gallate, octyl gallate, and dodecyl gallate. These oral equivalent doses were subsequently compared with human exposure estimates, as a low tier assessment allowing prioritization for further assessment. The results revealed that daily intake levels of especially propyl gallate can lead to internal plasma concentrations that are close to in vitro biological effect concentrations, particularly with respect to the inhibition of human thyroid peroxidase (TPO). Estrogenic effects were not considered likely to be induced by the food additives, as daily exposure levels of the different compounds remained 2 orders of magnitude below the oral equivalent doses for in vitro estrogen receptor activation. Overall, the results of the study show how the toolbox, which is freely accessible through www.qivivetools.wur.nl, can be used to obtain initial internal dose estimates of chemicals and to prioritize chemicals for further assessment, based on the comparison of oral equivalent doses of in vitro biological activity data with human exposure levels.

Adverse outcome pathways (AOPs) describe causal relationships between molecular perturbation and adverse cellular effects and are being increasingly adopted for linking in vitromechanistic toxicology to in vivodata from regulatory toxicity studies. In this work, a case study was performed by developing a bioassay toolbox to assess key events in the recently proposed AOP for chemically induced liver steatosis. The toolbox is comprised of in vitroassays to measure nuclear receptor activation, gene and protein expression, lipid accumulation, mitochondrial respiration, and formation of fatty liver cells. Assay evaluation was performed in human HepaRG hepatocarcinoma cells exposed to the model compound cyproconazole, a fungicide inducing steatosis in rodents. Cyproconazole dose-dependently activated RARα and PXR, two molecular initiating events in the steatosis AOP. Moreover, cyproconazole provoked a disruption of mitochondrial functions and induced triglyceride accumulation and the formation of fatty liver cells as described in the AOP. Gene and protein expression analysis, however, showed expression changes different from those proposed in the AOP, thus suggesting that the current version of the AOP might not fully reflect the complex mechanisms linking nuclear receptor activation and liver steatosis. Our study shows that cyproconazole induces steatosis in human liver cells in vitroand demonstrates the utility of systems-based approaches in the mechanistic assessment of molecular and cellular key events in an AOP. AOP-driven in vitrotesting as demonstrated can further improve existing AOPs, provide insight regarding molecular mechanisms of toxicity, and inform predictive risk assessment.

Insecticidal Cry proteins from Bacillus thuringiensis(Bt) have been exploited in the development of genetically modified (GM) crops for pest control. However, several pests are still difficult to control such as the coleopteran boll weevil Anthonomus grandis. By applying in vitromolecular evolution to the cry8Ka1gene sequence, variants were generated with improved activity against A. grandis. Among them, Cry8Ka5 mutant protein showed coleoptericidal activity 3-fold higher (LC502.83 μg/mL) than that of the original protein (Cry8Ka1). Cry8Ka5 has been used in breeding programs in order to obtain coleopteran-resistant cotton plants. Nevertheless, there is some concern in relation to the food safety of transgenic crops, especially to the heterologously expressed proteins. In this context, our research group has performed risk assessment studies on Cry8Ka5, using the tests recommended by Codex as well as tests that we proposed as alternative and/or complementary approaches. Our results on the risk analysis of Cry8Ka5 taken together with those of other Cry proteins, point out that there is a high degree of certainty on their food safety. It is reasonable to emphasize that most safety studies on Cry proteins have essentially used the Codex approach. However, other methodologies would potentially provide additional information such as studies on the effects of Cry proteins and derived peptides on the indigenous gastrointestinal microbiota and on intestinal epithelial cells of humans. Additionally, emerging technologies such as toxicogenomics potentially will offer sensitive alternatives for some current approaches or methods.

•Extracts of Senecio jacobaeatest positive in the Ames test.•Extracts of Senecio jacobaeacontain substantial amounts of PAs.•Positive Ames test outcomes were not caused by PAs, but by free quercetin.

The main goal of this study was to assess the potential of the mouse thymoma EL-4 cell line in screening for chemical induced immunotoxicity. Therefore, EL-4 cells were exposed to two well-known immunotoxicants, organotin compound tributyltin oxide (TBTO, 0.5 and 1 μM for 3 or 6 h) and the mycotoxin deoxynivalenol (DON, 0.25, 0.5 and 1 μM for 3, 6 or 11 h). Previous studies in human Jurkat T cells and mouse thymus in vivoshowed that the primary mode of action of TBTO is induction of endoplasmic reticulum (ER) stress, T cell activation and apoptosis. DON induces ribotoxic stress and, similarly to TBTO, induces ER stress, T cell activation and apoptosis. In the present study, the effects of TBTO and DON on EL-4 mRNA expression were assessed by whole genome microarray analysis. The microarray data were then compared to those obtained with mouse thymuses in vivo, mouse thymocytes in vitro, and CTLL-2 cells and human Jurkat cells in vitroexposed to TBTO or DON. Analysis at the level of gene sets revealed that part of the previously detected modes of action of TBTO and DON were not observed in the EL-4 cell line. In EL-4 cells, TBTO induced genes involved in calcium signalling and ER stress but did not induce genes involved in T cell activation and apoptosis. DON induced RNA related processes and ribosome biogenesis. Furthermore, DON downregulated ER stress, T cell activation and apoptosis which is opposite to the mechanism of DON observed in the mouse thymus in vivoand in Jurkat T cells in vitro. Apparently, EL-4 cells lack factors that are necessary to link ribotoxic stress to ER stress. In addition, of the lack of T cell activation response of EL-4 cells to TBTO is likely due to the fact that these cells are in a constitutively activated state already. Based on the results obtained for TBTO and DON, it can be concluded that the EL-4 cell line has limited value for immunotoxicogenomics based screening.

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 AgNO3([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 AgNO3exposed rats, hereby demonstrating the formation of nanoparticles from Ag+in vivothat 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 vivoformation of silver nanoparticles, and of the long retention of silver in brain and testis should be considered in a risk assessment of silver nanoparticles.

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 vitromodel to mimic the human digestion. Food products subjected to in vitrodigestion 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 SiO2particles 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.

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 vitromodel 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 vitrointestine 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 AgNO3. Therefore, the observed effects of the silver nanoparticles are likely exerted by the silver ions that are released from the nanoparticles.

The potential effects of horizontal gene transfer on human health are an important item in the safety assessment of genetically modified organisms. Horizontal gene transfer from genetically modified crops to gut microflora most likely occurs with transgenes of microbial origin. The characteristics of microbial transgenes other than antibiotic-resistance genes in market-approved genetically modified crops are reviewed. These characteristics include the microbial source, natural function, function in genetically modified crops, natural prevalence, geographical distribution, similarity to other microbial genes, known horizontal transfer activity, selective conditions and environments for horizontally transferred genes, and potential contribution to pathogenicity and virulence in humans and animals. The assessment of this set of data for each of the microbial genes reviewed does not give rise to health concerns. We recommend including the above-mentioned items into the premarket safety assessment of genetically modified crops carrying transgenes other than those reviewed in the present study.

To study the effects of major histocompatibility complex (MHC) class II expression on T-cell development, we have investigated T-cell immune reconstitution in two MHC class II–deficiency patients after allogeneic bone marrow transplantation (allo-BMT). Our study showed that the induction of MHC class II antigen expression on BM graft-derived T cells in these allo-BMT recipients was hampered upon T-cell activation. This reduction was most striking in the CD8+ T-cell subset. Furthermore, the peripheral T-cell receptor (TCR) repertoire in these graft-derived MHC class II–expressing CD4+ and in the CD8+ T-cell fractions was found to be restricted on the basis of TCR complementarity determining region 3 (CDR3) size profiles. Interestingly, the T-cell immune response to tetanus toxoid (TT) was found to be comparable to that of the donor. However, when comparing recipient-derived TT-specific T cells with donor-derived T cells, differences were observed in TCR gene segment usage and in the hydropathicity index of the CDR3 regions. Together, these results reveal the impact of an environment lacking endogenous MHC class II on the development of the T-cell immune repertoire after allo-BMT.

To study the effects of major histocompatibility complex (MHC) class II expression on T-cell development, we have investigated T-cell immune reconstitution in two MHC class II–deficiency patients after allogeneic bone marrow transplantation (allo-BMT). Our study showed that the induction of MHC class II antigen expression on BM graft-derived T cells in these allo-BMT recipients was hampered upon T-cell activation. This reduction was most striking in the CD8+T-cell subset. Furthermore, the peripheral T-cell receptor (TCR) repertoire in these graft-derived MHC class II–expressing CD4+and in the CD8+T-cell fractions was found to be restricted on the basis of TCR complementarity determining region 3 (CDR3) size profiles. Interestingly, the T-cell immune response to tetanus toxoid (TT) was found to be comparable to that of the donor. However, when comparing recipient-derived TT-specific T cells with donor-derived T cells, differences were observed in TCR gene segment usage and in the hydropathicity index of the CDR3 regions. Together, these results reveal the impact of an environment lacking endogenous MHC class II on the development of the T-cell immune repertoire after allo-BMT.

Almost equal numbers ofDesulfovibrio, Veillonella, andAcetobacterium species were found in agar shake dilutions of anaerobic intertidal brackish sediments applying L-lactate as the only energy source and sulfate as electron acceptor. Pure cultures of these bacteria were studied in more detail in batch cultures as well as in L-lactate-limited chemostats. The maximal specific growth rates on L-lactate were determined in washout experiments and amounted to 0.16, 0.30, and 0.06 h−1 forDesulfovibrio baculatus H.L21,Veillonella alcalescens NS.L49, andAcetobacterium NS.L40, respectively. Competition for L-lactate was studied in energy-limited chemostats at a dilution rate of 0.02 h−1.D. baculatus H.L21 turned out to be the best competitor at low L-lactate concentrations provided that sufficient sulfate and iron were present.V. alcalescens NS.L49 was favored by the absence of sulfate and iron. Coexistence ofD. baculatus H.L21 andV. alcalescens NS.L49 was observed in a L-lactate-limited chemostat with additional sulfate and citrate. Syntrophic growth ofV. alcalescens NS.L49 andAcetobacterium NS.L40 occurred in a L-lactate-limited chemostat in the absence of sulfate. No coexistence betweenD. baculatus H.L21 andAcetobacterium NS.L40 was observed in a L-lactate-limited chemostat without sulfate. Addition of calcium-saturated illite to an energy-limited mixed culture ofV. alcalescens NS.L49 andAcetobacterium NS.L40 induced iron limitation and subsequent washout of theAcetobacterium species. Finally, the ecological niches of the 3 species in relation to the consumption of lactate were discussed.

In this study we analyzed fibroblasts derived from an MHC class II deficiency patient (type III bare lymphocyte syndrome). Northern blot analysis showed that upon induction with IFN-? these fibroblasts did not express HLA class II genes and displayed a strongly reduced level of HLA class I gene expression when compared with fibroblasts of a healthy individual. However, when analyzed by RT-polymerase chain reaction (PCR), residual expression could be detected for HLA-DRA, DPB, and DQA, but not for HLA-DRB, DPA, and DQB. The lack of HLA-DRB transcripts in the patient fibroblasts and the high degree of sequence polymorphism of HLA-DRB were exploited in the further analysis of these fibroblasts. Thus far, at least three, and probably four, complementation groups have been defined among patient-derived and experimentally-derived MHC class II-negative cell lines. Transient heterokaryons between the patient fibroblasts and representative B-lymphoblastoid cell lines from each of the complementation groups were analyzed by RT-PCR and Southern blotting, using HLA-DRB-specific primers and biotin-labeled sequence specific oligonucleotides, respectively. These analyses showed that the fibroblasts of this particular patient belonged to a novel complementation group in MHC class II deficiency.

An out-of-frame fusion between the penicillinase gene (penP) of Bacillus licheniformis and the ß-galactosidase gene (lacZ) of Escherichia coli was shown to direct the synthesis of an active ß-galactosidase with the same electrophoretic mobility as the wild-type protein, both in B. subtilis and E. coli. This synthesis was dependent on translation of the truncated penP gene and appeared to result from translational coupling. The fusion point between penP and lacZ contained the sequence AUAG, in which the UAG and AUA codons were in-frame with the penP and lacZ reading units, respectively. N-terminal amino acid sequence analysis of the ß-galactosidase protein suggested that, both in B. subtilis and E. coli, reinitiation of translation occurred at the AUA codon present at the gene fusion point.

The potential effects of horizontal gene transfer on human health are an important item in the safety assessment of genetically modified organisms. Horizontal gene transfer from genetically modified crops to gut microflora most likely occurs with transgenes of microbial origin. The characteristics of microbial transgenes other than antibiotic-resistance genes in market-approved genetically modified crops are reviewed. These characteristics include the microbial source, natural function, function in genetically modified crops, natural prevalence, geographical distribution, similarity to other microbial genes, known horizontal transfer activity, selective conditions and environments for horizontally transferred genes, and potential contribution to pathogenicity and virulence in humans and animals. The assessment of this set of data for each of the microbial genes reviewed does not give rise to health concerns. We recommend including the above-mentioned items into the premarket safety assessment of genetically modified crops carrying transgenes other than those reviewed in the present study.