Henk van Loveren

THE EFFECT OF PARTICLE SIZE ON THE CYTOTOXICITY, INFLAMMATION, DEVELOPMENTAL TOXICITY AND GENOTOXICITY OF SILVER NANOPARTICLES

Silver nanoparticles are of interest to be used as antimicrobial agents in wound dressings and coatings in medical devices, but potential adverse effects have been reported in the literature. The most pronounced effect of silver nanoparticles and the role of particle size in determining these effects, also in comparison to silver ions, are largely unknown. Effects of silver nanoparticles of different sizes (20, 80, 113 nm) were compared in in vitro assays for cytotoxicity, inflammation, genotoxicity and developmental toxicity. Silver nanoparticles induced effects in all endpoints studied, but effects on cellular metabolic activity and membrane damage were most pronounced. In all toxicity endpoints studied, silver nanoparticles of 20 nm were more toxic than the larger nanoparticles. In L929 fibroblasts, but not in RAW 264.7 macrophages, 20 nm silver nanoparticles were more cytotoxic than silver ions. Collectively, these results indicate that effects of silver nanoparticles on different toxic endpoints may be the consequence of their ability to inflict cell damage. In addition, the potency of silver in the form of nanoparticles to induce cell damage compared to silver ions is cell type and size-dependent.

Contaminant-induced immunotoxicity in harbour seals: wildlife at risk?

Persistent, lipophilic polyhalogenated aromatic hydrocarbons (PHAHs) accumulate readily in the aquatic food chain and are found in high concentrations in seals and other marine mammals. Recent mass mortalities among several marine mammal populations have been attributed to infection by morbilliviruses, but a contributing role for immunotoxic PHAHs, including the polychlorinated biphenyls (PCBs), polychlorinated dibenzo-p-dioxins (PCDDs), and polychlorinated dibenzofurans (PCDFs) was not ruled out. We addressed this issue by carrying out a semi-field study in which captive harbour seals were fed herring from either the relatively uncontaminated Atlantic Ocean or the contaminated Baltic Sea for 2 years. We present here an overview of results obtained during this study. An impairment of natural killer (NK) cell activity, in vitro T-lymphocyte function, antigen-specific in vitro lymphocyte proliferative responses, and in vivo delayed-type hypersensitivity and antibody responses to ovalbumin was observed in the seals fed the contaminated Baltic herring. Additional feeding studies in PVG rats using the same herring batches suggested that an effect at the level of the thymus may be responsible for changes in cellular immunity, that virus-specific immune responses may be impaired, and that perinatal exposure to environmental contaminants represents a greater immunotoxic threat than exposure as a juvenile or adult. Together with the pattern of TCDD toxic equivalents of different PHAHs in the herring, these data indicate that present levels of PCBs in the aquatic food chain are immunotoxic to mammals. A review of contaminant levels in free-ranging harbour seals inhabiting polluted areas of Europe and North America suggests that many populations may be at risk to immunotoxicity. This could result in diminished host resistance and an increased incidence and severity of infectious disease.

Immunoglobulin-free light chains elicit immediate hypersensitivity-like responses

Immunoglobulin (Ig)-free light chains IgLC are present in serum and their production is augmented under pathological conditions such as multiple sclerosis, rheumatoid arthritis and neurological disorders. Until now, no (patho)physiological function has been ascribed to circulating Ig light chains. Here we show that IgLCs can confer mast cell–dependent hypersensitivity in mice. Antigenic stimulation results in plasma extravasation, cutaneous swelling and mast-cell degranulation. We show that IgLCs have a crucial role in development of contact sensitivity, which could be completely prevented by a novel IgLC antagonist. Although IgE and IgG1 are central to the induction of immediate hypersensitivity reactions, our results show that IgLCs have similar activity. IgLCs may therefore be a novel factor in the humoral immune response to antigen exposure. Our findings open new avenues in investigating the pathogenesis of autoimmune diseases and their treatments.

Bronchoconstriction and airway hyperresponsiveness after ovalbumin inhalation in sensitized mice

To investigate the mechanisms underlying airway hyperresponsiveness a murine model was developed with several important characteristics of human allergic asthma. Mice were intraperitoneally sensitized with ovalbumin and after 4 weeks challenge via an ovalbumin aerosol. After aerosol, lung function was evaluated with a non-invasive forced oscillation technique. The amount of mucosal exudation into the airway lumen and the presence of mast cell degranulation was determined. Tracheal responsiveness was measured at several time points after challenge. At these time points also bronchoalveolar lavage and histology were performed. Sensitization induced high antigen-specific IgE levels in serum. Inhalation of ovalbumin in sensitized mice induced an immediate but no late bronchoconstrictive response. During this immediate phase, respiratory resistance was increased (54%). Within the first hour after ovalbumin inhalation increased mucosal exudation and mast cell degranulation were observed. At 12 and 24 h after ovalbumin challenge, mice showed tracheal hyperresponsiveness (29% and 34%, respectively). However, no apparent inflammation was found in the lungs or bronchoalveolar lavage. From these results it can be concluded that hyperresponsiveness can develop via mechanisms independent of an inflammatory infiltrate. Since mast cell degranulation occurred after ovalbumin exposure, we hypothesize that mast cells are involved in the induction of airway hyperresponsiveness in this model.

Systemic and immunotoxicity of silver nanoparticles in an intravenous 28 days repeated dose toxicity study in rats.

Because of its antibacterial activity nanosilver is one of the most commonly used nanomaterials. It is increasingly used in a variety of both medical and consumer products resulting in an increase in human exposure. However, the knowledge on the systemic toxicity of nanosilver is relatively limited. To determine the potential systemic toxicity of silver nanoparticles (Ag-NP) with different sizes (20 nm and 100 nm) a 28-days repeated dose toxicity study was performed in rats using intravenous administration. The toxic effect of the 20 nm Ag-NP was performed using the bench mark dose (BMD) approach. Treatment with a maximum dose of 6 mg/kg body weight was well tolerated by the animals. However, both for 20 nm and 100 nm Ag-NP growth retardation was observed during the treatment. A severe increase in spleen size and weight was present which was due to an increased cell number. Both T and B cell populations showed an increase in absolute cell number, whereas the relative cell numbers remained constant. At histopathological evaluation brown and black pigment indicating Ag-NP accumulation was noted in spleen, liver, and lymph nodes. Ag-NP was also detected incidentally in other organs. Clinical chemistry indicated liver damage (increased alkaline phosphatase, alanine transaminase, and aspartate transaminase) that could not be confirmed by histopathology. Hematology showed a decrease in several red blood cell parameters. The most striking toxic effect was the almost complete suppression of the natural killer (NK) cell activity in the spleen at high doses. Other immune parameters affected were: decreased interferon-γ and interleukin (IL)-10 production by concanavalin-A stimulated spleen cells, increased IL-1β and decreased IL-6, IL-10 and TNF-α production by lipopolysaccharide stimulated spleen cells, increase in serum IgM and IgE, and increase in blood neutrophilic granulocytes. For the spleen weight a critical effect dose of 0.37 mg/kg body weight (b.w.) could be established. The lowest critical effect dose (CED) for a 5% change compared to control animals was observed for thymus weight (CED05 0.01 mg/kg b.w.) and for functional immune parameters, i.e. decrease in NK cell activity (CED05 0.06 mg/kg b.w.) and LPS stimulation of spleen cells (CED05 0.04 mg/kg b.w.). These results show that for nanosilver the most sensitive parameters for potential adverse responses were effects on the immune system.

Evaluation of the skin sensitizing potency of chemicals by using the existing methods and considerations of relevance for elicitation

The Technical Committee of Classification and Labelling dealing with harmonized classification of substances and classification criteria under Directive 67/548/EEC on behalf of the European Commission nominated an expert group on skin sensitization in order to investigate further the possibility for potency consideration of skin sensitizers for future development of the classification criteria. All substances and preparations should be classified on the basis of their intrinsic properties and should be labelled accordingly with the rules set up in the Directive 67/548/EEC. The classification should be the same under their full life cycle and in the case that there is no harmonized classification the substance or preparation should be self‐classified by the manufacturer in accordance with the same criteria. The Directive does not apply to certain preparations in the finished state, such as medical products, cosmetics, food and feeding stuffs, which are subject to specific community legislation. The main questions that are answered in this report are whether it would be possible to give detailed guidance on how to grade allergen potency based on the existing methods, whether such grading could be translated into practical thresholds and whether these could be set for both induction and elicitation. Examples are given for substances falling into various potency groups for skin sensitization relating to results from the local lymph node assay, the guinea pig maximization test, the Buehler method and human experience.

Endocrine effects of hexabromocyclododecane (HBCD) in a one-generation reproduction study in Wistar rats.

The brominated flame retardant (BFR) hexabromocyclododecane was tested in a one-generation reproduction assay in Wistar rats, enhanced for endocrine parameters. A solution of the compound in corn oil was mixed in the feed, targeting at dietary exposure of 0-0.1-0.3-1-3-10-30-100 mg/kg body weight/day (mkd) in parental rats during 10 (males) or 2 (females) weeks premating, during gestation and lactation, and in their F1 offspring from weaning until final necropsy. Effects were assessed in F1 animals. Livers of these animals showed increased HBCD concentrations, in a dose-dependent way. The trabecular bone mineral density of the tibia was dose-dependently decreased in females (BenchMark Dose Lower confidence bound, BMDL=0.056 mkd). The IgG response after immunization with sheep red blood cells (SRBC) was increased in males (BMDL=0.46 mkd). Further sensitive effects were decreased weight of the testis (BMDL=1.5 mkd), increased fraction of neutrophilic granulocytes (BMDL=7.7 mkd), decreased concentration of apolar retinoids in female livers (BMDL=1.3 mkd), and decreased plasma alkaline phosphatase in females (BMDL=8.6 mkd). CYP19/aromatase activity in the ovary was correlated to the concentration of gamma-HBCD in the liver. A developmental origin of these effects is considered, and this is also true for sensitive effects observed in neurobehavioural testing in littermates from the same experiment, i.e. in the brainstem auditory evoked potentials and in a catalepsy test [Lilienthal, H., Van der Ven, L.T.M., Piersma, A.H., Vos, J.G. Neurobehavioral effects of the brominated flame retardant hexabromocyclododecane (HBCD) in rats after pre- and postnatal exposure, in press]. The low BMDLs of these effects may raise concern for human health, particularly when based on body burdens of HBCD, which leads to critical margins of exposure particularly for the occupational setting.

Status of nutrition and health claims in Europe

Functional foods are closely associated with claims on foods. There are two categories of claims on foods: nutrition claims and health claims. Health claims on (functional) foods must be scientifically substantiated. In December 2006, the European Union published its Regulation 1924/2006 on nutrition and health claims made on foods. As concerns scientific evaluation, the EU-project PASSCLAIM resulted in a set of criteria for the scientific substantiation of health claims on foods. The European Food Safety Authority provides the scientific advise to the European Commission for health claims submitted under Regulation 1924/2006 and has hitherto published several hundreds of opinions on health claims, part of which are positive, part which are negative and a few with insufficient evidence. Antioxidant claims have been approved for the general function of vitamins but not for direct health effects in humans. Another issue with claims is consumer understanding. Consumers can hardly distinguish between graded levels of evidence, and they do make only little or no distinction between nutrition and health claims. Consumers understand nutrition and health claims different from scientists and regulators. Therefore, innovation in industry can readily proceed via approved nutrition claims and approved health claims. The market and the shelves in the stores will not be empty; rather they will look different in the years to come.

Pre-natal exposure to perfluoroalkyl substances may be associated with altered vaccine antibody levels and immune-related health outcomes in early childhood

Abstract Perfluoroalkyl substances (PFAS) are suggested to have immunosuppressive effects; exposure in utero and in the first years of life is of special concern as fetuses and small children are highly vulnerable to toxicant exposure. The objective of this study was to investigate the effect of pre-natal exposure to PFAS on responses to pediatric vaccines and immune-related health outcomes in children up to 3 years of age. In the prospective birth-cohort BraMat, a sub-cohort of the Norwegian Mother and Child Cohort Study (MoBa), pregnant women from Oslo and Akershus, Norway, were recruited during 2007–2008. Three annual questionnaire-based follow-ups were performed. Blood samples were collected from the mothers at the time of delivery and from the children at the age of 3 years. As a measure of pre-natal exposure to PFAS, the concentrations of perfluorooctanoate (PFOA), perfluorononanoate (PFNA), perfluorohexane sulfonate (PFHxS), and perfluorooctane sulfonate (PFOS) were determined in maternal blood from 99 BraMat participants. Main outcome measures were anti-vaccine antibody levels, common infectious diseases and allergy- and asthma-related health outcomes in the children up to the age of 3 years. There was an inverse association between the level of anti-rubella antibodies in the children’s serum at age 3 years and the concentrations of the four PFAS. Furthermore, there was a positive association between the maternal concentrations of PFOA and PFNA and the number of episodes of common cold for the children, and between PFOA and PFHxS and the number of episodes of gastroenteritis. No associations were found between maternal PFAS concentrations and the allergy- and asthma-related health outcomes investigated. The results indicate that pre-natal exposure to PFAS may be associated with immunosuppression in early childhood.

Monitoring immune modulation by nutrition in the general population: identifying and substantiating effects on human health.

Optimal functioning of the immune system is crucial to human health, and nutrition is one of the major exogenous factors modulating different aspects of immune function. Currently, no single marker is available to predict the effect of a dietary intervention on different aspects of immune function. To provide further guidance on the assessment and interpretation of the modulation of immune functions due to nutrition in the general population, International Life Sciences Institute Europe commissioned a group of experts from academia, government and the food industry to prepare a guidance document. A draft of this paper was refined at a workshop involving additional experts. First, the expert group defined criteria to evaluate the usefulness of immune function markers. Over seventy-five markers were scored within the context of three distinct immune system functions: defence against pathogens; avoidance or mitigation of allergy; control of low-grade (metabolic) inflammation. The most useful markers were subsequently classified depending on whether they by themselves signify clinical relevance and/or involvement of immune function. Next, five theoretical scenarios were drafted describing potential changes in the values of markers compared with a relevant reference range. Finally, all elements were combined, providing a framework to aid the design and interpretation of studies assessing the effects of nutrition on immune function. This stepwise approach offers a clear rationale for selecting markers for future trials and provides a framework for the interpretation of outcomes. A similar stepwise approach may also be useful to rationalise the selection and interpretation of markers for other physiological processes critical to the maintenance of health and well-being.Optimal functioning of the immune system is crucial to human health, and nutrition is one of the major exogenous factors modulating different aspects of immune function. Currently, no single marker is available to predict the effect of a dietary intervention on different aspects of immune function. To provide further guidance on the assessment and interpretation of the modulation of immune functions due to nutrition in the general population, International Life Sciences Institute Europe commissioned a group of experts from academia, government and the food industry to prepare a guidance document. A draft of this paper was refined at a workshop involving additional experts. First, the expert group defined criteria to evaluate the usefulness of immune function markers. Over seventy-five markers were scored within the context of three distinct immune system functions: defence against pathogens; avoidance or mitigation of allergy; control of low-grade (metabolic) inflammation. The most useful markers were subsequently classified depending on whether they by themselves signify clinical relevance and/or involvement of immune function. Next, five theoretical scenarios were drafted describing potential changes in the values of markers compared with a relevant reference range. Finally, all elements were combined, providing a framework to aid the design and interpretation of studies assessing the effects of nutrition on immune function. This stepwise approach offers a clear rationale for selecting markers for future trials and provides a framework for the interpretation of outcomes. A similar stepwise approach may also be useful to rationalise the selection and interpretation of markers for other physiological processes critical to the maintenance of health and well-being.