Dose addition in mixtures of compounds with dissimilar endocrine modes of action in in vitro receptor activation assays and the zebrafish sexual development test.

BACKGROUND: Human exposure to pesticides is being associated with feminisation for which a decrease of the anogenital distance (AGD) is a sensitive endpoint. Dose addition for the cumulative risk assessment of pesticides in food is considered sufficiently conservative for combinations of compounds with both similar and dissimilar modes of action (MoA). OBJECTIVE: The present study was designed to test the dose addition hypothesis in a binary mixture of endocrine active compounds with a dissimilar mode of action for the endpoint feminisation. METHODS: Compounds were selected from a list of chemicals of which exposure is related to a decrease of the AGD in rats and completed with reference compounds. These chemicals were characterised using specific in vitro transcriptional activation (TA) assays for estrogenic and androgenic properties, leading to a final selection of dienestrol as an ER-agonist and flutamide, linuron, and deltamethrin as AR-antagonists. These compounds were then tested in an in vivo model, i.e. in zebrafish (Danio rerio), using sex ratio in the population as an endpoint in order to confirm their feminising effect and MoA. Ultimately, the fish model was used to test a binary mixture of flutamide and dienestrol. RESULTS: Statistical analysis of the binary mixture of flutamide and dienestrol in the fish sexual development tests (FSDT) with zebrafish supported dose addition.

Transferability and reproducibility of exposed air-liquid interface co-culture lung models.

BACKGROUND: The establishment of reliable and robust in vitro models for hazard assessment, a prerequisite for moving away from animal testing, requires the evaluation of model transferability and reproducibility. Lung models that can be exposed via the air, by means of an air-liquid interface (ALI) are promising in vitro models for evaluating the safety of nanomaterials (NMs) after inhalation exposure. We performed an inter-laboratory comparison study to evaluate the transferability and reproducibility of a lung model consisting of the human bronchial cell line Calu-3 as a monoculture and, to increase the physiologic relevance of the model, also as a co-culture with macrophages (either derived from the THP-1 monocyte cell line or from human blood monocytes). The lung model was exposed to NMs using the VITROCELL® Cloud12 system at physiologically relevant dose levels. RESULTS: Overall, the results of the 7 participating laboratories are quite similar. After exposing Calu-3 alone and Calu-3 co-cultures with macrophages, no effects of lipopolysaccharide (LPS), quartz (DQ12) or titanium dioxide (TiO2) NM-105 particles on the cell viability and barrier integrity were detected. LPS exposure induced moderate cytokine release in the Calu-3 monoculture, albeit not statistically significant in most labs. In the co-culture models, most laboratories showed that LPS can significantly induce cytokine release (IL-6, IL-8 and TNF-α). The exposure to quartz and TiO2 particles did not induce a statistically significant increase in cytokine release in both cell models probably due to our relatively low deposited doses, which were inspired by in vivo dose levels. The intra- and inter-laboratory comparison study indicated acceptable interlaboratory variation for cell viability/toxicity (WST-1, LDH) and transepithelial electrical resistance, and relatively high inter-laboratory variation for cytokine production. CONCLUSION: The transferability and reproducibility of a lung co-culture model and its exposure to aerosolized particles at the ALI were evaluated and recommendations were provided for performing inter-laboratory comparison studies. Although the results are promising, optimizations of the lung model (including more sensitive read-outs) and/or selection of higher deposited doses are needed to enhance its predictive value before it may be taken further towards a possible OECD guideline.

Smoking-Associated Exposure of Human Primary Bronchial Epithelial Cells to Aldehydes: Impact on Molecular Mechanisms Controlling Mitochondrial Content and Function.

Chronic obstructive pulmonary disease (COPD) is a devastating lung disease primarily caused by exposure to cigarette smoke (CS). During the pyrolysis and combustion of tobacco, reactive aldehydes such as acetaldehyde, acrolein, and formaldehyde are formed, which are known to be involved in respiratory toxicity. Although CS-induced mitochondrial dysfunction has been implicated in the pathophysiology of COPD, the role of aldehydes therein is incompletely understood. To investigate this, we used a physiologically relevant in vitro exposure model of differentiated human primary bronchial epithelial cells (PBEC) exposed to CS (one cigarette) or a mixture of acetaldehyde, acrolein, and formaldehyde (at relevant concentrations of one cigarette) or air, in a continuous flow system using a puff-like exposure protocol. Exposure of PBEC to CS resulted in elevated IL-8 cytokine and mRNA levels, increased abundance of constituents associated with autophagy, decreased protein levels of molecules associated with the mitophagy machinery, and alterations in the abundance of regulators of mitochondrial biogenesis. Furthermore, decreased transcript levels of basal epithelial cell marker KRT5 were reported after CS exposure. Only parts of these changes were replicated in PBEC upon exposure to a combination of acetaldehyde, acrolein, and formaldehyde. More specifically, aldehydes decreased MAP1LC3A mRNA (autophagy) and BNIP3 protein (mitophagy) and increased ESRRA protein (mitochondrial biogenesis). These data suggest that other compounds in addition to aldehydes in CS contribute to CS-induced dysregulation of constituents controlling mitochondrial content and function in airway epithelial cells.

An inter-laboratory effort to harmonize the cell-delivered in vitro dose of aerosolized materials.

Air-liquid interface (ALI) lung cell models cultured on permeable transwell inserts are increasingly used for respiratory hazard assessment requiring controlled aerosolization and deposition of any material on ALI cells. The approach presented herein aimed to assess the transwell insert-delivered dose of aerosolized materials using the VITROCELL® Cloud12 system, a commercially available aerosol-cell exposure system. An inter-laboratory comparison study was conducted with seven European partners having different levels of experience with the VITROCELL® Cloud12. A standard operating procedure (SOP) was developed and applied by all partners for aerosolized delivery of materials, i.e., a water-soluble molecular substance (fluorescence-spiked salt) and two poorly soluble particles, crystalline silica quartz (DQ12) and titanium dioxide nanoparticles (TiO2 NM-105). The material dose delivered to transwell inserts was quantified with spectrofluorometry (fluorescein) and with the quartz crystal microbalance (QCM) integrated in the VITROCELL® Cloud12 system. The shape and agglomeration state of the deposited particles were confirmed with transmission electron microscopy (TEM). Inter-laboratory comparison of the device-specific performance was conducted in two steps, first for molecular substances (fluorescein-spiked salt), and then for particles. Device- and/or handling-specific differences in aerosol deposition of VITROCELL® Cloud12 systems were characterized in terms of the so-called deposition factor (DF), which allows for prediction of the transwell insert-deposited particle dose from the particle concentration in the aerosolized suspension. Albeit DF varied between the different labs from 0.39 to 0.87 (mean (coefficient of variation (CV)): 0.64 (28%)), the QCM of each VITROCELL® Cloud 12 system accurately measured the respective transwell insert-deposited dose. Aerosolized delivery of DQ12 and TiO2 NM-105 particles showed good linearity (R2 > 0.95) between particle concentration of the aerosolized suspension and QCM-determined insert-delivered particle dose. The VITROCELL® Cloud 12 performance for DQ12 particles was identical to that for fluorescein-spiked salt, i.e., the ratio of measured and salt-predicted dose was 1.0 (29%). On the other hand, a ca. 2-fold reduced dose was observed for TiO2 NM-105 (0.54 (41%)), which was likely due to partial retention of TiO2 NM-105 agglomerates in the vibrating mesh nebulizer of the VITROCELL® Cloud12. This inter-laboratory comparison demonstrates that the QCM integrated in the VITROCELL® Cloud 12 is a reliable tool for dosimetry, which accounts for potential variations of the transwell insert-delivered dose due to device-, handling- and/or material-specific effects. With the detailed protocol presented herein, all seven partner laboratories were able to demonstrate dose-controlled aerosolization of material suspensions using the VITROCELL® Cloud12 exposure system at dose levels relevant for observing in vitro hazard responses. This is an important step towards regulatory approved implementation of ALI lung cell cultures for in vitro hazard assessment of aerosolized materials.

Dose Addition in the Induction of Craniofacial Malformations in Zebrafish Embryos Exposed to a Complex Mixture of Food-Relevant Chemicals with Dissimilar Modes of Action.

BACKGROUND: Humans are exposed to combinations of chemicals. In cumulative risk assessment (CRA), regulatory bodies such as the European Food Safety Authority consider dose addition as a default and sufficiently conservative approach. The principle of dose addition was confirmed previously for inducing craniofacial malformations in zebrafish embryos in binary mixtures of chemicals with either similar or dissimilar modes of action (MOAs). OBJECTIVES: In this study, we explored a workflow to select and experimentally test multiple compounds as a complex mixture with each of the compounds at or below its no observed adverse effect level (NOAEL), in the same zebrafish embryo model. METHODS: Selection of candidate compounds that potentially induce craniofacial malformations was done using in silico methods-structural similarity, molecular docking, and quantitative structure-activity relationships-applied to a database of chemicals relevant for oral exposure in humans via food (EuroMix inventory, n=1,598). A final subselection was made manually to represent different regulatory fields (e.g., food additives, industrial chemicals, plant protection products), different chemical families, and different MOAs. RESULTS: A final selection of eight compounds was examined in the zebrafish embryo model, and craniofacial malformations were observed in embryos exposed to each of the compounds, thus confirming the developmental toxicity as predicted by the in silico methods. When exposed to a mixture of the eight compounds, each at its NOAEL, substantial craniofacial malformations were observed; according to a dose-response analysis, even embryos exposed to a 7-fold dilution of this mixture still exhibited a slight abnormal phenotype. The cumulative effect of the compounds in the mixture was in accordance with dose addition (added doses of the individual compounds after adjustment for relative potencies), despite different MOAs of the compounds involved. DISCUSSION: This case study of a complex mixture inducing craniofacial malformations in zebrafish embryos shows that dose addition can adequately predicted the cumulative effect of a mixture of multiple substances at low doses, irrespective of the (expected) MOA. The applied workflow may be useful as an approach for CRA in general. https://doi.org/10.1289/EHP9888.

Optimization of an air-liquid interface in vitro cell co-culture model to estimate the hazard of aerosol exposures.

Inhalation exposure to environmental and occupational aerosol contaminants is associated with many respiratory health problems. To realistically mimic long-term inhalation exposure for toxicity testing, lung epithelial cells need to maintained and exposed under air-liquid interface (ALI) conditions for a prolonged period of time. In addition, to study cellular responses to aerosol particles, lung epithelial cells have to be co-cultured with macrophages. To that aim, we evaluated human bronchial epithelial Calu-3, 16HBE14o- (16HBE), H292, and BEAS-2B cell lines with respect to epithelial morphology, barrier function and cell viability under prolonged ALI culture conditions. Only the Calu-3 cells can retain the monolayer structure and maintain a strong tight junction under long-term ALI culture at least up to 2 weeks. As such, Calu-3 cells were applied as the structural barrier to create co-culture models with human monocyte-derived macrophages (MDMs) and THP-1 derived macrophages (TDMs). Adhesion of macrophages onto the epithelial monolayer was allowed for 4 h with a density of 5 × 104 macrophages/cm2. In comparison to the Calu-3 mono-culture model, Calu-3 + TDM and Calu-3 + MDM co-culture models showed an increased sensitivity in inflammatory responses to lipopolysaccharide (LPS) aerosol at Day 1 of co-culture, with the Calu-3 + MDM model giving a stronger response than Calu-3 + TDM. Therefore, the epithelial monolayer integrity and increased sensitivity make the Calu-3 + MDM co-culture model a preferred option for ALI exposure to inhaled aerosols for toxicity testing.

An Air-liquid Interface Bronchial Epithelial Model for Realistic, Repeated Inhalation Exposure to Airborne Particles for Toxicity Testing.

For toxicity testing of airborne particles, air-liquid interface (ALI) exposure systems have been developed for in vitro tests in order to mimic realistic exposure conditions. This puts specific demands on the cell culture models. Many cell types are negatively affected by exposure to air (e.g., drying out) and only remain viable for a few days. This limits the exposure conditions that can be used in these models: usually relatively high concentrations are applied as a cloud (i.e., droplets containing particles, which settle down rapidly) within a short period of time. Such experimental conditions do not reflect realistic long-term exposure to low concentrations of particles. To overcome these limitations the use of a human bronchial epithelial cell line, Calu-3 was investigated. These cells can be cultured at ALI conditions for several weeks while retaining a healthy morphology and a stable monolayer with tight junctions. In addition, this bronchial model is suitable for testing the effects of repeated exposures to low, realistic concentrations of airborne particles using an ALI exposure system. This system uses a continuous airflow in contrast to other ALI exposure systems that use a single nebulization producing a cloud. Therefore, the continuous flow system is suitable for repeated and prolonged exposure to airborne particles while continuously monitoring the particle characteristics, exposure concentration, and delivered dose. Taken together, this bronchial model, in combination with the continuous flow exposure system, is able to mimic realistic, repeated inhalation exposure conditions that can be used for toxicity testing.

Differences in cytotoxicity of lung epithelial cells exposed to titanium dioxide nanofibers and nanoparticles: Comparison of air-liquid interface and submerged cell cultures.

Air Liquid Interface (ALI) system has emerged as a useful tool for toxicity evaluation of nanomaterials related to inhalation since the system mimics the aerosol exposure. We compared the biological responses of lung epithelial cells exposed to titanium dioxide (TiO2) nanofibers and nanoparticles in ALI and submerged cell cultures systems. Cells were exposed to 2 and 10 µg/cm2 for 24 h, 48 h and 72 h and LDH release, TiO2 internalization, DNA-double strand breaks (DSBs) and ROS production were assessed. LDH release was similar in both systems and particles had higher cytoplasmic uptake in submerged systems. Both TiO2 types were located in the cytoplasm but nanofibers had nuclear uptake regardless to the system tested. Cells exposed to TiO2 nanofibers had higher DSBs in the ALI system than in submerged cell cultures but cells exposed to TiO2 nanoparticles had similar DSBs in both systems. ROS production was higher in cells exposed to TiO2 nanofibers compared to cells exposed to TiO2 nanoparticles. In conclusion, cytotoxicity of lung epithelial cells was similar in ALI or submerged cell cultures, however cells exposed to TiO2 nanofibers displayed higher toxicity than cells exposed to TiO2 nanoparticles.

Dose addition in chemical mixtures inducing craniofacial malformations in zebrafish (Danio rerio) embryos.

A challenge in cumulative risk assessment is to model hazard of mixtures. EFSA proposed to only combine chemicals linked to a defined endpoint, in so-called cumulative assessment groups, and use the dose-addition model as a default to predict combined effects. We investigated the effect of binary mixtures of compounds known to cause craniofacial malformations, by assessing the effect in the head skeleton (M-PQ angle) in 120hpf zebrafish embryos. We combined chemicals with similar mode of action (MOA), i.e. the triazoles cyproconazole, triadimefon and flusilazole; next, reference compounds cyproconazole or triadimefon were combined with dissimilar acting compounds, TCDD, thiram, VPA, prochloraz, fenpropimorph, PFOS, or endosulfan. These mixtures were designed as (near) equipotent combinations of the contributing compounds, in a range of cumulative concentrations. Dose-addition was assessed by evaluation of the overlap of responses of each of the 14 tested binary mixtures with those of the single compounds. All 10 test compounds induced an increase of the M-PQ angle, with varying potency and specificity. Mixture responses as predicted by dose-addition did not deviate from the observed responses, supporting dose-addition as a valid assumption for mixture risk assessment. Importantly, dose-addition was found irrespective of MOA of contributing chemicals.

Sensitive method for endotoxin determination in nanomedicinal product samples.

Aim: Nanomaterials and nanomedicinal products tend to interfere with various commonly used assays, including regulatory required endotoxin detection methods for medicines. We developed a method to quantify endotoxin levels that is compatible with nanomaterials and nanomedicinal products. Materials & methods: The method is based on measuring endotoxin indirectly via 3-hydroxylated fatty acids of lipid-A, using Ultra High Performance Liquid Chromatography coupled with mass spectrometry. The outcome was related to results of the commonly used Limulus Amebocyte Lysate method. Results: The ultra high performance liquid chromatography coupled with mass spectrometry method has clear advantages compared with other endotoxin determination assays; particularly the absence of nanospecific interference. Conclusion: The method is sensitive, straightforward and accurate in determining and quantifying endotoxin in nanomedicinal product samples.

Head skeleton malformations in zebrafish (Danio rerio) to assess adverse effects of mixtures of compounds.

The EU-EuroMix project adopted the strategy of the European Food Safety Authority (EFSA) for cumulative risk assessment, which limits the number of chemicals to consider in a mixture to those that induce a specific toxicological phenotype. These so-called cumulative assessment groups (CAGs) are refined at several levels, including the target organ and specific phenotype. Here, we explore the zebrafish embryo as a test model for quantitative evaluation in one such CAG, skeletal malformations, through exposure to test compounds 0-120 hpf and alcian blue cartilage staining at 120 hpf, focusing on the head skeleton. Reference compounds cyproconazole, flusilazole, metam, and thiram induced distinctive phenotypes in the head skeleton between the triazoles and dithiocarbamates. Of many evaluated parameters, the Meckel's-palatoquadrate (M-PQ) angle was selected for further assessment, based on the best combination of a small confidence interval, an intermediate maximal effect size and a gentle slope of the dose-response curve with cyproconazole and metam. Additional test compounds included in the CAG skeletal malformations database were tested for M-PQ effects, and this set was supplemented with compounds associated with craniofacial malformations or cleft palate to accommodate otherwise organized databases. This additional set included hexaconazole, all-trans-retinoic acid, AM580, CD3254, maneb, pyrimethanil, imidacloprid, pirimiphos-methyl, 2,4-dinitrophenol, 5-fluorouracil, 17alpha-ethynylestradiol (EE2), ethanol, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), PCB 126, methylmercury, boric acid, and MEHP. Most of these compounds produced a dose-response for M-PQ effects. Application of the assay in mixture testing was provided by combined exposure to cyproconazole and TCDD through the isobole method, supporting that in this case the combined effect can be modeled through concentration addition.

Is current risk assessment of non-genotoxic carcinogens protective?

Non-genotoxic carcinogens (NGTXCs) do not cause direct DNA damage but induce cancer via other mechanisms. In risk assessment of chemicals and pharmaceuticals, carcinogenic risks are determined using carcinogenicity studies in rodents. With the aim to reduce animal testing, REACH legislation states that carcinogenicity studies are only allowed when specific concerns are present; risk assessment of compounds that are potentially carcinogenic by a non-genotoxic mode of action is usually based on subchronic toxicity studies. Health-based guidance values (HBGVs) of NGTXCs may therefore be based on data from carcinogenicity or subchronic toxicity studies depending on the legal framework that applies. HBGVs are usually derived from No-Observed-Adverse-Effect-Levels (NOAELs). Here, we investigate whether current risk assessment of NGTXCs based on NOAELs is protective against cancer. To answer this question, we estimated Benchmark doses (BMDs) for carcinogenicity data of 44 known NGTXCs. These BMDs were compared to the NOAELs derived from the same carcinogenicity studies, as well as to the NOAELs derived from the associated subchronic studies. The results lead to two main conclusions. First, a NOAEL derived from a subchronic study is similar to a NOAEL based on cancer effects from a carcinogenicity study, supporting the current practice in REACH. Second, both the subchronic and cancer NOAELs are, on average, associated with a cancer risk of around 1% in rodents. This implies that for those chemicals that are potentially carcinogenic in humans, current risk assessment of NGTXCs may not be completely protective against cancer. Our results call for a broader discussion within the scientific community, followed by discussions among risk assessors, policy makers, and other stakeholders as to whether or not the potential cancer risk levels that appear to be associated with currently derived HBGVs of NGXTCs are acceptable.

A Dose-Response Modeling Approach Shows That Effects From Mixture Exposure to the Skin Sensitizers Isoeugenol and Cinnamal Are in Line With Dose Addition and Not With Synergism.

Currently, hazard characterization of skin sensitizers is based on data obtained from studies examining single chemicals. Many consumer products, however, contain mixtures of sensitizers that might interact in such a way that the response induced by a substance is higher than predicted in the hazard assessment. To assess interaction of skin sensitizers in a mixture, a dose-response modeling approach is applied. With this approach, it is possible to assess whether or not responses from mixtures of sensitizers can be predicted from the dose-response information obtained from individual chemicals using dose addition. We selected the skin sensitizers isoeugenol and cinnamal, frequently occurring together in consumer products, to be examined in an adjusted local lymph node assay (LLNA). Cell number and cytokine production (IL-10 and IFN-γ) of the auricular lymph nodes were measured as hallmarks of the skin sensitization response. We found that dose addition for these 2 skin sensitizers closely predicted the effects from mixtures of both chemicals across the broad dose range tested. Hence, isoeugenol and cinnamal show no synergistic effects in the LLNA. Therefore, hazard assessment and risk assessment of these substances can be performed without taking into account mixture exposure.

Developmental immunotoxicity testing of 4-methyl anisole.

The developmental immunotoxicity of 4-methyl anisole (4MA) was investigated in the rat. Four study designs were used, with either premating or post-weaning onset of exposure, continued to postnatal day 50, and with or without additional oral gavage of pups from postnatal day 10 onward. Reduced litter size (benchmark dose lower confidence limit (BMDL) 80mg/kg bw/day) was the most sensitive developmental parameter, with pup relative organ weight effects observed at similar BMDLs, in the absence of maternal toxicity. Eosinophil numbers were reduced at lower doses (BMDL 16mg/kg bw/day). KLH challenge resulted in increased IL-13 and TNF-α responses, and variably reduced IgG production (BMDL 27mg/kg bw/day). T4 levels were reduced by 11% at maximum with a BMDL of 73mg/kg bw/day. Differences between exposure cohorts were limited and were considered to be without biological significance. This study shows that 4MA induces developmental immunotoxicity at doses below those inducing developmental and general toxicity. These observations being independent of the study designs applied suggest that the post-weaning period, included in all designs, is the most relevant sensitive period for inducing 4MA mediated developmental immunotoxicity. Moreover, this study stresses the importance of including developmental immunotoxicity testing by default in regulatory toxicology.

Immunotoxicity of silver nanoparticles in an intravenous 28-day repeated-dose toxicity study in rats.

BACKGROUND: Nanosilver is used in a variety of medical and consumer products because of its antibacterial activity. This wide application results in an increased human exposure. Knowledge on the systemic toxicity of nanosilver is, however, relatively scarce. In a previous study, the systemic toxicity of 20 nm silver nanoparticles (Ag-NP) was studied in a 28-day repeated-dose toxicity study in rats. Ag-NP were intravenously administered with a maximum dose of 6 mg/kg body weight (bw)/day. Several immune parameters were affected: reduced thymus weight, increased spleen weight and spleen cell number, a strongly reduced NK cell activity, and reduced IFN-γ production were observed. METHODS: Prompted by these affected immune parameters, we wished to assess exposure effects on the functional immune system. Therefore, in the present study the T-cell dependent antibody response (TDAR) to keyhole limpet hemocyanin (KLH) was measured in a similar 28-day intravenous repeated-dose toxicity study. In addition, a range of immunological parameters was measured. Data obtained using the benchmark dose (BMD) approach were analyzed by fitting dose-response models to the parameters measured. RESULTS: A reduction in KLH-specific IgG was seen, with a lowest 5% lower confidence bound of the BMD (BMDL) of 0.40 mg/kg bw/day. This suggests that Ag-NP induce suppression of the functional immune system. Other parameters sensitive to Ag-NP exposure were in line with our previous study: a reduced thymus weight with a BMDL of 0.76 mg/kg bw/day, and an increased spleen weight, spleen cell number, and spleen cell subsets, with BMDLs between 0.36 and 1.11 mg/kg bw/day. Because the effects on the spleen are not reflected by increased KLH-specific IgG, they, however, do not suggest immune stimulation. CONCLUSIONS: Intravenous Ag-NP administration in a 28-day repeated-dose toxicity study induces suppression of the functional immune system. This finding underscores the importance to study the TDAR to evaluate immunotoxicity and not to rely solely on measuring immune cell subsets.

Sub-chronic toxicity study in rats orally exposed to nanostructured silica.

BACKGROUND: Synthetic Amorphous Silica (SAS) is commonly used in food and drugs. Recently, a consumer intake of silica from food was estimated at 9.4 mg/kg bw/day, of which 1.8 mg/kg bw/day was estimated to be in the nano-size range. Food products containing SAS have been shown to contain silica in the nanometer size range (i.e. 5-200 nm) up to 43% of the total silica content. Concerns have been raised about the possible adverse effects of chronic exposure to nanostructured silica. METHODS: Rats were orally exposed to 100, 1000 or 2500 mg/kg bw/day of SAS, or to 100, 500 or 1000 mg/kg bw/day of NM-202 (a representative nanostructured silica for OECD testing) for 28 days, or to the highest dose of SAS or NM-202 for 84 days. RESULTS: SAS and NM-202 were extensively characterized as pristine materials, but also in the feed matrix and gut content of the animals, and after in vitro digestion. The latter indicated that the intestinal content of the mid/high-dose groups had stronger gel-like properties than the low-dose groups, implying low gelation and high bioaccessibility of silica in the human intestine at realistic consumer exposure levels. Exposure to SAS or NM-202 did not result in clearly elevated tissue silica levels after 28-days of exposure. However, after 84-days of exposure to SAS, but not to NM-202, silica accumulated in the spleen. Biochemical and immunological markers in blood and isolated cells did not indicate toxicity, but histopathological analysis, showed an increased incidence of liver fibrosis after 84-days of exposure, which only reached significance in the NM-202 treated animals. This observation was accompanied by a moderate, but significant increase in the expression of fibrosis-related genes in liver samples. CONCLUSIONS: Although only few adverse effects were observed, additional studies are warranted to further evaluate the biological relevance of observed fibrosis in liver and possible accumulation of silica in the spleen in the NM-202 and SAS exposed animals respectively. In these studies, dose-effect relations should be studied at lower dosages, more representative of the current exposure of consumers, since only the highest dosages were used for the present 84-day exposure study.

Statin use and markers of immunity in the Doetinchem cohort study.

It has been suggested that statins can both stimulate and suppress the immune system, and thereby, may influence autoimmune diseases. Therefore, we studied effects of statins on innate and adaptive immunity, and self-tolerance by measuring serological levels of C-reactive protein (CRP), neopterin, immunoglobulin E (IgE) antibodies and the presence of autoantibodies (antinuclear antibodies (ANA) and IgM rheumatoid factor (RF)) in the general population. We conducted a nested case-control study within the population-based Doetinchem cohort. Data from health questionnaires, serological measurements and information on medication from linkage to pharmacy-dispensing records were available. We selected 332 statin users (cases) and 331 non-users (controls), matched by age, sex, date of serum collection, history of cardiovascular diseases, diabetes mellitus type II and stroke. Multivariate regression analyses were performed to estimate effect of statins on the immune system. The median level of CRP in statin users (1.28 mg/L, interquartile range (IQR): 0.59-2.79) was lower than in non-users (1.62 mg/L, IQR: 0.79-3.35), which after adjustment was estimated to be a 28% lower level. We observed an inverse association between duration of statin use and CRP levels. Elevated levels of IgE (>100 IU/mL) were more prevalent in statin users compared to non-users. A trend towards increased levels of IgE antibodies in statin users was observed, whereas no associations were found between statin use and levels of neopterin or the presence of autoantibodies. In this general population sub-sample, we observed an anti-inflammatory effect of statin use and a trend towards an increase of IgE levels, an surrogate marker for Th (helper) 2 responses without a decrease in neopterin levels, a surrogate marker for Th1 response and/or self-tolerance. We postulate that the observed decreased inflammatory response during statin therapy may be important but is insufficient to induce loss of self-tolerance.

Developmental immunotoxicity in male rats after juvenile exposure to ethanol.

The aim of the present study was to determine the sensitivity of the developing immune system to ethanol (EtOH) after exposure from postnatal day (PND) 10 onward. Adult Wistar dams and litters were exposed to EtOH via drinking water (0, 0.25, 1.5, 2.75, 4, 5.25, or 6.5% (w/v) EtOH ad libitum) and drinking water exposure of the F1 was continued from weaning until sacrifice. Immune assessments were performed at postnatal days (PNDs) 21, 42, and 70. Furthermore, Keyhole Limpet Hemocyanin (KLH) specific immune responses were evaluated following subcutaneous immunizations on PNDs 21 and 35. EtOH exposure affected innate immune responses, such as LPS-induced NO-production by adherent splenocytes, as well as adaptive immune responses as represented by KLH-specific parameters. The most sensitive developmental parameters included effects on maternal and pup bodyweight with calculated BMDs of 4.0% and 4.3% EtOH, respectively. The most sensitive immune parameters were affected at dose levels lower than those affecting developmental parameters and included KLH-specific immune responses, LPS-induced NO production by adherent splenocytes, and IL-10 production by ConA stimulated splenocytes. Calculated BMDs for these parameters were between 0.01% and 0.1% EtOH. A comparison of the results of this juvenile study with an extended one-generation reproductive toxicity study revealed that the juvenile study design may result in a higher sensitivity related to differences in the exposure design. These findings demonstrate the relative sensitivity of the developing immune system for EtOH exposure, the additional value of assessing functional immune parameters, and the importance of the juvenile window in developmental immunotoxicity testing.

Induction of skin sensitization is augmented in Nrf2-deficient mice.

Several in vitro DNA microarray studies have shown the importance of nuclear factor (erythroid-derived 2)-like 2 (Nrf2) in skin sensitization. Nevertheless, the exact in vivo role of the Nrf2-Keap1 pathway during the induction of skin sensitization remains unknown. To study the function of Nrf2, a local lymph node assay was performed in wild-type and Nrf2-deficient mice using 2,4-dinitrochlorobenzene. The Nrf2-deficient mice show a more pronounced response, indicating that Nrf2 is involved in dampening the induction of skin sensitization.