Eric R. Gremmer

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.

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

BackgroundSynthetic Amorphous Silica (SAS) is commonly used in food and drugs. Recently, a consumer intake of silica from food was estimated at 9.4xa0mg/kg bw/day, of which 1.8xa0mg/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 – 200xa0nm) up to 43% of the total silica content. Concerns have been raised about the possible adverse effects of chronic exposure to nanostructured silica.MethodsRats were orally exposed to 100, 1000 or 2500xa0mg/kg bw/day of SAS, or to 100, 500 or 1000xa0mg/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.ResultsSAS 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.ConclusionsAlthough 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.

Infection with the roundworm Toxocara canis leads to exacerbation of experimental allergic airway inflammation.

Background Epidemiological studies performed in developing as well as in western countries suggest that infection with Toxocara canis contributes to the development of atopic diseases.

Effects of Bifidobacterium animalis administered during lactation on allergic and autoimmune responses in rodents.

Probiotics are promoted as being beneficial to health and positive effects on the immune system have been reported. Beneficial immune effects have been attributed to several mechanisms, including stimulating T helper 1 (Th1) immunity. To explore the effects of the probiotic Bifidobacterium animalis on Th1‐ and Th2‐mediated immune responses, two different animal models representing either Th1‐ or Th2‐mediated immune responses were used: a rat model for experimental autoimmune encephalomyelitis (EAE) (Th1) and a mouse model for respiratory allergy induced by ovalbumin (OVA) (Th2). B. animalis administration started when the mice or rats were 2 weeks old. Respiratory allergy or EAE were induced when the animals were 6–7 weeks old. In the allergy model, B. animalis modestly reduced the number of infiltrating eosinophils and lymphocytes in the lungs, but no effects on allergen‐specific serum immunoglobulin E levels were found. Cytokine profiles assessed after culturing spleen cells with the mitogen concanvalin A (ConA) showed that B. animalis skewed the Th1/Th2 balance towards Th1 in females. However, allergen‐induced cytokine production in females was not affected by B. animalis. In males, B. animalis significantly decreased ConA‐induced interleukin‐13 and a trend towards lower levels of OVA‐induced Th2 cytokines. In the EAE model, B. animalis significantly reduced the duration of clinical symptoms by almost 2 days in males and improved the body weight gain during the experimental period compared with the control group. Our data show that B. animalis reduced several immune parameters in the allergy as well as in the autoimmunity model.

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

BackgroundNanosilver 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 20xa0nm 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 6xa0mg/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.MethodsPrompted 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.ResultsA reduction in KLH-specific IgG was seen, with a lowest 5% lower confidence bound of the BMD (BMDL) of 0.40xa0mg/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 0.76xa0mg/kg bw/day, and an increased spleen weight, spleen cell number, and spleen cell subsets, with BMDLs between 0.36 and 1.11xa0mg/kg bw/day. Because the effects on the spleen are not reflected by increased KLH-specific IgG, they, however, do not suggest immune stimulation.ConclusionsIntravenous 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.

Transfer of a two-tiered keratinocyte assay: IL-18 production by NCTC2544 to determine the skin sensitizing capacity and epidermal equivalent assay to determine sensitizer potency

At present, the identification of potentially sensitizing chemicals is carried out using animal models. However, it is very important from ethical, safety and economic point of view to have biological markers to discriminate allergy and irritation events, and to be able to classify sensitizers according to their potency, without the use of animals. Within the Sens-it-iv EU Frame Programme 6 funded Integrated Project (LSHB-CT-2005-018681), a number of in vitro, human cell based assays were developed which, when optimized and used in an integrated testing strategy, may be able to distinguish sensitizers from non-sensitizers. This study describes two of these assays, which when used in a tiered strategy, may be able to identify contact sensitizers and also to quantify sensitizer potency. Tier 1 is the human keratinocyte NCTC2544 IL-18 assay and tier 2 is the Epidermal Equivalent potency assay. The aim of this study is to show the transferability of the two-tiered approach with training chemicals: 3 sensitizers (DNCB, resorcinol, pPD) and 1 non sensitizer (lactic acid) in tier 1 and 2 sensitizers with different potency in tier 2 (DNCB; extreme and resorcinol; moderate). The chemicals were tested in a non-coded fashion. Here we describe the transferability to naïve laboratories, the establishment of the standard operating procedure, critical points, acceptance criteria and project management. Both assays were successfully transferred to laboratories that had not performed the assays previously. The two tiered approach may offer an unique opportunity to provide an alternative method to the Local Lymph Node Assay (LLNA). These assays are both based on the use of human keratinocytes, which have been shown over the last two decades, to play a key role in all phases of skin sensitization.

The role of Toll-like receptor-4 in pertussis vaccine-induced immunity

BackgroundThe gram-negative bacterium Bordetella pertussis is an important causative agent of pertussis, an infectious disease of the respiratory tract. After introduction of whole-cell vaccines (wP) in the 1950s, pertussis incidence has decreased significantly. Because wP were found to be reactogenic, in most developed countries they have been replaced by acellular vaccines (aP). We have previously shown a role for Toll-like receptor 4 (Tlr4) in pertussis-infected mice and the pertussis toxin (Ptx)-IgG response in wP-vaccinated children, raising the issue of the relative importance of Tlr4 in wP vaccination of mice. Here we analyze the effects of wP and aP vaccination and B. pertussis challenge, in Tlr4-deficient C3H/HeJ and wild-type C3H/HeOuJ mice. aP consists of Ptx, filamentous hemagglutinin (FHA), and pertactin (Prn).ResultsWe show an important role of Tlr4 in wP and (to a lesser extent) aP vaccination, induction of Th1 and Th17 cells by wP but not aP vaccination, and induction of Th17 cells by infection, confirming data by Higgins et al. (J Immunol 2006, 177: 7980–9). Furthermore, in Tlr4-deficient mice, compared to wild-type controls (i) after vaccination only, Ptx-IgG (that was induced by aP but not wP vaccination), FHA-IgG, and Prn-IgG levels were similar, (ii) after infection (only), lung IL-1α and IL-1β expression were lower, (iii) after wP vaccination and challenge, Prn-IgG level and lung IL-5 expression were higher, while lung IL-1β, TNF-α, IFN-γ, IL-17, and IL-23 expression were lower, and lung pathology was absent, and (iv) after aP vaccination and challenge, Prn-IgG level and lung IL-5 expression were higher, while Ptx-IgG level was lower.ConclusionTlr4 does not influence the humoral response to vaccination (without challenge), plays an important role in natural immunity, wP and aP efficacy, and induction of Th1 and Th17 responses, is critical for lung pathology and enhances pro-inflammatory cytokine production after wP vaccination and challenge, and diminishes Th2 responses after both wP and aP vaccination and challenge. wP vaccination does not induce Ptx-IgG. A role for LPS in the efficacy of wP underlines the usefulness of LPS analogs to improve bacterial subunit vaccines such as aP.

Lipopolysaccharide Analogs Improve Efficacy of Acellular Pertussis Vaccine and Reduce Type I Hypersensitivity in Mice

ABSTRACT Pertussis is an infectious disease of the respiratory tract that is caused by the gram-negative bacterium Bordetella pertussis. Although acellular pertussis (aP) vaccines are safe, they are not fully effective and thus require improvement. In contrast to whole-cell pertussis (wP) vaccines, aP vaccines do not contain lipopolysaccharide (LPS). Monophosphoryl lipid A (MPL) and Neisseria meningitidis LpxL2 LPS have been shown to display immune-stimulating activity while exerting little endotoxin activity. Therefore, we evaluated whether these LPS analogs could increase the efficacy of the aP vaccine. Mice were vaccinated with diphtheria-tetanus-aP vaccine with aluminum, MPL, or LpxL2 LPS adjuvant before intranasal challenge with B. pertussis. Compared to vaccination with the aluminum adjuvant, vaccination with either LPS analog resulted in lower colonization and a higher pertussis toxin-specific serum immunoglobulin G level, indicating increased efficacy. Vaccination with either LPS analog resulted in reduced lung eosinophilia, reduced eosinophil numbers in the bronchoalveolar lavage fluid, and the ex vivo production of interleukin-4 (IL-4) by bronchial lymph node cells and IL-5 by spleen cells, suggesting reduced type I hypersensitivity. Vaccination with either LPS analog increased serum IL-6 levels, although these levels remained well below the level induced by wP, suggesting that supplementation with LPS analogs may induce some reactogenicity but reactogenicity considerably less than that induced by the wP vaccine. In conclusion, these results indicate that supplementation with LPS analogs forms a promising strategy that can be used to improve aP vaccines.

Relative sensitivity of developmental and immune parameters in juvenile versus adult male rats after exposure to di(2-ethylhexyl) phthalate

The developing immune system displays a relatively high sensitivity as compared to both general toxicity parameters and to the adult immune system. In this study we have performed such comparisons using di(2-ethylhexyl) phthalate (DEHP) as a model compound. DEHP is the most abundant phthalate in the environment and perinatal exposure to DEHP has been shown to disrupt male sexual differentiation. In addition, phthalate exposure has been associated with immune dysfunction as evidenced by effects on the expression of allergy. Male wistar rats were dosed with corn oil or DEHP by gavage from postnatal day (PND) 10-50 or PND 50-90 at doses between 1 and 1000 mg/kg/day. Androgen-dependent organ weights showed effects at lower dose levels in juvenile versus adult animals. Immune parameters affected included TDAR parameters in both age groups, NK activity in juvenile animals and TNF-α production by adherent splenocytes in adult animals. Immune parameters were affected at lower dose levels compared to developmental parameters. Overall, more immune parameters were affected in juvenile animals compared to adult animals and effects were observed at lower dose levels. The results of this study show a relatively higher sensitivity of juvenile versus adult rats. Furthermore, they illustrate the relative sensitivity of the developing immune system in juvenile animals as compared to general toxicity and developmental parameters. This study therefore provides further argumentation for performing dedicated developmental immune toxicity testing as a default in regulatory toxicology.

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.