Early life exposure to bisphenol A investigated in mouse models of airway allergy, food allergy and oral tolerance.

The impact of early life exposure to bisphenol A (BPA) through drinking water was investigated in mouse models of respiratory allergy, food allergy and oral tolerance. Balb/c mice were exposed to BPA (0, 10 or 100 µg/ml), and the offspring were intranasally exposed to the allergen ovalbumin (OVA). C3H/HeJ offspring were sensitized with the food allergen lupin by intragastric gavage, after exposure to BPA (0, 1, 10 or 100 µg/ml). In separate offspring, oral tolerance was induced by gavage of 5 mg lupin one week before entering the protocol for the food allergy induction. In the airway allergy model, BPA (100 µg/ml) caused increased eosinophil numbers in bronchoalveolar lavage fluid (BALF) and a trend of increased OVA-specific IgE levels. In the food allergy and tolerance models, BPA did not alter the clinical anaphylaxis or antibody responses, but induced alterations in splenocyte cytokines and decreased mouse mast cell protease (MMCP)-1 serum levels. In conclusion, early life exposure to BPA through drinking water modestly augmented allergic responses in a mouse model of airway allergy only at high doses, and not in mouse models for food allergy and tolerance. Thus, our data do not support that BPA promotes allergy development at exposure levels relevant for humans.

Prenatal exposure to bisphenol A interferes with the development of cerebellar granule neurons in mice and chicken.

In mice, prenatal exposure to low doses of bisphenol A has been shown to affect neurogenesis and neuronal migration in cortex, resulting in disturbance of both neuronal positioning and the network formation between thalamus and cortex in the offspring brain. In the present study we investigated whether prenatal exposure to bisphenol A disturbs the neurodevelopment of the cerebellum. Two different model systems were used; offspring from two strains of mice from mothers receiving bisphenol A in the drinking water before mating, during gestation and lactation, and chicken embryos exposed to bisphenol A (in the egg) on embryonic day 16 for 24h before preparation of cerebellar granule cell cultures. In the cerebellum, tight regulation of the level of transcription factor Pax6 is critical for correct development of granule neurons. During the development, the Pax6 level in granule neurons is high when these cells are located in the external granule layer and during their migration to the internal granule layer, and it is then reduced. We report that bisphenol A induced an increase in the thickness of the external granule layer and also an increase in the total cerebellar Pax6 level in 11 days old mice offspring. In cultured chicken cerebellar granule neurons from bisphenol A injected eggs the Pax6 level was increased day 6 in vitro. Together, these findings indicate that bisphenol A may affect the granule neurons in the developing cerebellum and thereby may disturb the correct development of the cerebellum.

Carbon nanofibers have IgE adjuvant capacity but are less potent than nanotubes in promoting allergic airway responses.

There is a growing concern for the possible health impact of nanoparticles. The main objective of this study was to investigate the allergy-promoting capacity of four different carbon nanofiber (CNF) samples in an injection and an airway mouse model of allergy. Secondly, the potency of the CNF was compared to the previously reported allergy-promoting capacity of carbon nanotubes (CNT) in the airway model. Ultrafine carbon black particles (ufCBP) were used as a positive control. Particles were given together with the allergen ovalbumin (OVA) either by subcutaneous injection into the footpad or intranasally to BALB/cA mice. After allergen booster, OVA-specific IgE, IgG1, and IgG2a in serum were measured. In the airway model, inflammation was determined as influx of inflammatory cells (eosinophils, neutrophils, lymphocytes, and macrophages) and by mediators (MCP-1 and TNF-α present in bronchoalveolar fluid (BALF)). CNF and CNT both increased OVA-specific IgE levels in the two models, but in the airway model, the CNT gave a significantly stronger IgE response than the CNF. Furthermore, the CNT and not the CNF promoted eosinophil lung inflammation. Our data therefore suggest that nanotube-associated properties are particularly potent in promoting allergic responses.

Long-term bisphenol A exposure accelerates insulitis development in diabetes-prone NOD mice.

Exposure to the endocrine disruptor (ED) bisphenol A (BPA) used in polycarbonate plastic and epoxy resins appears ubiquitous since BPA can be found in over 90% of analyzed urine samples from all age groups. There is a parallel occurrence of increased prevalence in type 1 diabetes mellitus (T1DM) and an increased exposure to EDs the last decades. T1DM is caused by insulin deficiency due to autoimmune destruction of insulin producing pancreatic beta cells and has been suggested to be induced by various environmental factors acting together with a genetic predisposition. The objective of the present study was to investigate the effect of BPA (0, 1 and 100 mg/l BPA in the drinking water) on T1DM development in nonobese diabetic (NOD) mice, spontaneously developing T1DM. Histological evaluation of pancreas from 12-weeks-old female mice revealed significantly increased insulitis in mice exposed to 1 mg/l BPA, while the insulitis was less severe at the higher BPA exposure. Serum glucose levels in the 1 mg/ml BPA group tended to be hyperglycaemic, also indicating an accelerated onset of T1DM. The high BPA exposure seemed to counteract the diabetes development in females and also in male NOD mice for both BPA concentrations. Prior to insulitis, both BPA concentrations resulted in increased apoptosis and reduced numbers of tissue resident macrophages in pancreatic islets. In conclusion, long-term BPA exposure at a dose three times higher than the tolerable daily intake of 50 µg/kg, appeared to accelerate spontaneous insulitis and diabetes development in NOD mice.

Particles from wood smoke and road traffic differently affect the innate immune system of the lung.

The effect of particles from road traffic and wood smoke on the innate immune response in the lung was studied in a lung challenge model with the intracellular bacterium Listeria monocytogenes. Female Balb/cA mice were instilled intratracheally with wood smoke particles, particles from road traffic collected during winter (studded tires used; St+), and during autumn (no studded tires; St-), or diesel exhaust particles (DEP). Simultaneously with, and 1 or 7 days after particle instillation, 10(5) bacteria were inoculated intratracheally. Bacterial numbers in the lungs and spleen 1 day after Listeria challenge were determined, as an indicator of cellular activation. In separate experiments, bronchoalveolar lavage (BAL) fluid was collected 4 h and 24 h after particle instillation. All particles tested reduced the numbers of bacteria in the lung 24 h after bacterial inoculation. When particles were given simultaneously with Listeria, the reduction was greatest for DEP, followed by St+ and St-, and least for wood smoke particles. Particle effects were no longer apparent after 7 days. Neutrophil numbers in BAL fluid were increased for all particle exposed groups. St+ and St- induced the highest levels of IL-1beta, MIP-2, MCP-1, and TNF-alpha, followed by DEP, which induced no TNF-alpha. In contrast, wood smoke particles only increased lactate dehydrogenase (LDH) activity, indicating a cytotoxic effect of these particles. In conclusion, all particles tested activated the innate immune system as determined with Listeria. However, differences in kinetics of anti-Listeria activity and levels of proinflammatory mediators point to cellular activation by different mechanisms.

Single-walled and multi-walled carbon nanotubes promote allergic immune responses in mice.

The adjuvant effect of particles on allergic immune responses has been shown to increase with decreasing particle size and increasing particle surface area. Like ultrafine particles, carbon nanotubes (CNTs) have nano-sized dimensions and a large relative surface area and might thus increase allergic responses. Therefore, we examined whether single-walled (sw) and multi-walled (mw) CNTs have the capacity to promote allergic responses in mice, first in an sc injection model and thereafter in an intranasal model. Balb/cA mice were exposed to three doses of swCNT, mwCNT, as well as ultrafine carbon black particles (ufCBPs, Printex90) during sensitization with the allergen ovalbumin (OVA). Five days after an OVA booster, OVA-specific IgE, IgG1, and IgG2a antibodies in serum and the numbers of inflammatory cells and cytokine levels in bronchoalveolar lavage fluid (BALF) were determined. Furthermore, ex vivo OVA-induced cytokine release from mediastinal lymph node (MLN) cells was measured. In separate experiments, differential cell counts were determined in BALF 24 h after a single intranasal exposure to the particles in the absence of allergen. We demonstrate that both swCNT and mwCNT together with OVA strongly increased serum levels of OVA-specific IgE, the number of eosinophils in BALF, and the secretion of Th2-associated cytokines in the MLN. On the other hand, only mwCNT and ufCBP with OVA increased IgG2a levels, neutrophil cell numbers, and tumor necrosis factor-alpha and monocyte chemoattractant protein-1 levels in BALF, as well as the acute influx of neutrophils after exposure to the particles alone. This study demonstrates that CNTs promote allergic responses in mice.

Allergy adjuvant effect of particles from wood smoke and road traffic.

There is growing evidence that in addition to augmenting the severity of asthma and allergic diseases, particulate air pollution also increases the incidence of allergy and asthma. We studied the adjuvant effect of particles from wood smoke and road traffic on the immune response to the allergen ovalbumin (OVA). OVA with and without particles was injected into one hind footpad of Balb/cA mice. All particles together with OVA significantly increased the level of OVA-specific immunoglobulin E (IgE) in serum, compared to groups given OVA or particles alone. Reference diesel exhaust particles (DEP) with OVA induced the highest levels of IgE, whereas no clear difference was observed between particles from road traffic and wood smoke. Road traffic particles collected in the autumn induced higher IgE values with OVA than corresponding particles collected during the winter season when studded tires are used, suggesting that studded tire-generated road pavement particles have less allergy adjuvant activity than exhaust particles. Compared to OVA or particles alone, all particles with OVA increased popliteal lymph node cell numbers, cell proliferation, ex vivo secretion of IL-4 and IL-10 after ConA stimulation, and the expression of several cell surface molecules (CD19, MHC class II, CD86 and CD23). Wood smoke particles with OVA induced somewhat higher cellular responses than road traffic particles, but less than DEP with OVA which seemed to be the most potent particle in inducing cellular as well as antibody responses. Thus, wood smoke particles had about the same capacity to enhance allergic sensitization as road traffic particles, but less than diesel exhaust particles.

Popliteal lymph node (PLN) assay to study adjuvant effects on respiratory allergy.

Different variants of the popliteal lymph node (PLN) assay have been published. Here we describe the adjuvant popliteal lymph node assay, an immune response assay to study the adjuvant activity of soluble substances as well as particulate matter. The substance to be studied for adjuvant activity is injected into the hind footpad of mice or rats together with an antigen. Adjuvant activity is determined as the increase in PLN weight and cell numbers in animals receiving antigen together with the substance under study, compared with PLN weight and cell numbers in animals given the antigen without the substance in question, and animals given the putative adjuvant alone. Because lymph node weight and cell numbers are immunologically non-specific parameters, specific immune response assays like serum antibody responses or antibody-forming cell numbers should additionally be performed. Different antigens and immune response assays may be used, depending on the research question asked. In relation to respiratory (or food) allergy, the assays should as a minimum include determination of specific IgE in serum, and preferably also IgG1 (mouse). Serum specific IgG2a antibody determination may be added to get an indication of the Th1-Th2-balance of the response. The adjuvant PLN assay, with cellular response assays performed in the draining popliteal lymph node and antibody determinations in serum, requires small amounts of test material. The assay offers a practical, sensitive and reproducible method to determine the adjuvant activity of soluble substances as well as particulate material, with the possibility to also perform mechanistic studies.

The capacity of particles to increase allergic sensitization is predicted by particle number and surface area, not by particle mass.

Particle exposure has traditionally been monitored as mass concentration of PM10 (particles with an aerodynamic diameter less than 10 microm), more recently also as PM2.5. The mass concentration is strongly influenced by the large particles. Therefore, particle mass is a poor measure for characterizing the amount of the small, possibly more biologically potent particles. We used polystyrene particles (PSP) ranging in diameter from 0.0588 to 11.14 microm, carbon black (CB), and diesel exhaust particles (DEP), to study the adjuvant effect of particles on the immune response to the allergen ovalbumin (OVA) after sc injection into the footpad of BALB/cA mice. At a given mass dose, the small particles (0.0588 and 0.202 microm PSP, CB, and DEP) increased the allergen-specific IgE serum levels to a substantially higher degree than the larger particles (1.053, 4.64, and 11.14 microm PSP). Further, in the draining lymph node during the primary response, the fine particles (0.202 microm) with OVA increased cell numbers, expression of surface markers (CD19, MHC class II, CD86, and CD23) and ex vivo production of IL-4 and IL-10, whereas the largest (11.14 microm) particles did not. Linear regression analyses indicated that the IgE response was not predicted by particle mass (R2 = 0.06), but was predicted by the total particle surface area (R2 = 0.64), number of particles (R2 = 0.62), and particle diameter (R2 = 0.58). In conclusion, we found that fine particles exerted stronger adjuvant effects on allergic responses than larger particles at equal mass doses. Consequently, the dose described as total particle surface area or particle number predicts the adjuvant effect of particles better than the currently used particle mass.

Effects of the environmental oestrogens bisphenol A, tetrachlorobisphenol A, tetrabromobisphenol A, 4-hydroxybiphenyl and 4,4'-dihydroxybiphenyl on oestrogen receptor binding, cell proliferation and regulation of oestrogen sensitive proteins in the human breast cancer cell line MCF-7.

Bisphenol A is extensively used in the manufacturing of epoxy resins and polycarbonate plastics, whereas several brominated and chlorinated analogues are used as flame retardants and intermediates in the plastic industry. Due to the structural relationship between these chemicals and the high production volumes, we wanted to characterize and compare their potential oestrogen-like potency using several end-points in MCF-7 cells: induction of pS2 protein and progesterone receptor, reduction of oestrogen receptor level, and stimulation of cell growth. Bisphenol A, tetrachloro- and tetrabromo-bisphenol A, 4-hydroxybiphenyl and 4,4'-dihydroxybiphenyl all showed oestrogen-like properties in MCF-7 cells. The chemicals tested had affinity to the oestrogen receptor isolated from MCF-7 cells, although their EC50s were 1,000 to 80,000 times higher than the EC50 of 17beta-oestradiol. Bisphenol A and 4-hydroxybiphenyl induced cell growth in MCF-7 cells, and the highest test concentrations induced responses, apparently exceeding the cell growth induced by 17beta-oestradiol. The other chemicals tested induced less than 50% of the maximum 17beta-oestradiol-stimulated cell growth. Bisphenol A, 4-hydroxybiphenyl, tetrabromobisphenol A and tetrachlorobisphenol A all increased the level of the oestrogen-regulated proteins, progesterone receptor and pS2, whereas 4,4'-dihydroxybiphenyl showed no such effect. Bisphenol A was the only chemical tested that clearly mimicked 17beta-oestradiol in its ability to reduce the level of cytosolic oestrogen receptors in MCF-7 cells. By measuring several oestrogen-dependent endpoints it seems that some xeno-oestrogens cause an imbalanced oestrogen-response. Their ability and potency in mimicking 17beta-oestrogen in one parameter is not necessarily accompanied by a similar effect in another oestrogen-linked parameter.