Inhalation and prenatal exposure to phthalates: influence on airway immunology

Phthalates are a class of chemicals that leak out of the plastic products that surround us in our everyday life. They are present in most indoor environments, where we are exposed through inhalation and dermal uptake. We are also exposed to phthalates through ingestion of food and beverages and application of cosmetic products. Population studies suggest a link between exposure to phthalates and the development or exacerbation of asthma and other respiratory symptoms, but a causal relationship is not yet established since the underlying biological effects are not well understood. In this 3-year project we investigate whether phthalates that we inhale or are exposed to in utero can affect respiratory and systemic immune responses. We conduct (i) studies of phthalate-induced effects in the immune cells in cell culture, (ii) a human exposure study in which we study the effects in the airways and blood after exposure to phthalates (to be conducted in Vancouver, Canada) and (iii) an experimental / population study of the immune response to cells from the respiratory tract of 7-year-old children from the Swedish mother-child study SELMA II, with partially known exposure to phthalates in utero. The cell culture studies show that dibutlyftalat (DBP), which is found in relatively high concentrations in indoor air, affects the differentiation of immune cells and their function. DBP accelerates differentiation of monocytes into macrophages, and can alter the macrophage immune response to bacteria, as studied using the bacterial lipopolysaccharide component (LPS). In primary immune cells from human volunteers, low concentrations of DBP decreased the LPS-induced immune response in airway macrophages while the effects in blood cells were minor. Contrary to what has been reported in the literature, we do not find that DBP changes other immune responses, such as the uptake of particles (phagocytosis), either in cell lines or in blood immune cells. We have also started to investigate how DBP causes effects in immune cells, i.e. which molecules and signaling pathways that are activated by DBP in these cells. In the effects of DBP on the accelerated macrophage differentiation, a nuclear receptor called PPAR (peroxisome proliferator-activated receptor) appears to be activated and involved in the effects induced by DBP. However, PPAR does not appear to be involved in the decreased LPS-induced immune response. The human inhalation study has been started, but is delayed since it was necessary to replace the original phthalate exposure method prior to start-up. The original method led to exposure through inhalation only, while the new method provides exposure through both skin and inhalation, which is more relevant for indoor exposure in daily life. Two subjects have completed the study and another four subjects are ready to start the exposures. Further recruitment and screening of volunteers is done consecutively and the aim is to finalize exposures of the 20 subjects within July 2018. Thus, the study will be completed after the end-date of this NRC-funded Project. A doctoral fellow has been recruited by our partner at the University of British Columbia in Vancouver to ensure study progress and finalization. During the 7-year follow-up of the Swedish mother-child study SELMA II, cells from the children's nose were collected using a soft brush. They were then cultured and stimulated with factors representing bacteria (LPS) or viruses (R848) to determine the cellular immune response. The collection ran until December 2016 and resulted in approximately 200 samples. In parallel with collection and stimulation of nasal cells, blood samples were also collected and subjected to similar stimulation, allowing for comparison of immune responses in cells from the nose and blood in the same individual. The samples are currently analyzed to determine the levels of various inflammatory markers. These data will then be linked to (i) parameters characterizing the nasal brushings sample (e.g. the distribution of different cell types in the sample) and the children's immunological status during sample collection (e.g. allergies, infections, recent vaccination or use of medication) and (ii) phthalate levels from pregnancy to assess any correlations between prenatal phthalate exposure and childhood immune response.

The indoor environment is important for health and well-being of the general population, but the health impact of a number of indoor pollutants is poorly characterised. In the proposed project we focus on phthalates, a group of chemicals that leak out fro m plastic and are present in most indoor environments. Epidemiological studies concerning both indoor environment and occupational exposures have linked phthalates to development and exacerbation of asthma and allergy, but the involved mechanisms are larg ely unknown. Prenatal exposure to these compounds has also been suggested to be involved in the development of childhood asthma but so far this is not confirmed. To investigate the effects of phthalates on airway immunology we will perform (i) in vitro investigations of mechanisms of phthalate-induced effects on airway cells, focusing on the functionality of immune cells, (ii) a human inhalation study investigating airway effects due to inhalation of phthalates and (iii) an experimental/epidemiological study of airway immune responses in 6 year old children with known high or low prenatal exposure. The project will provide new knowledge about immunological biomarkers for early effects of phthalate inhalation, the mechanisms involved in phthalate-indu ced airway pathobiology, and the role of exposure route for phthalate effects. Important outcomes of the project will be its contribution to risk assessment, identification of potential biomarkers for human inhalation exposure to phthalates, and deeper un derstanding of the overall toxicology of phthalates.