Effects of di-n-butyl phthalate (DBP) on THP-1-derived macrophages

Phthalates are chemicals used as plasticizers in various plastics and consumer products, including PVC products, personal care products and pharmaceutical applicants. These chemicals may migrate into the environment, food and water and enter the human body via ingestion and inhalation, but also through the skin. Epidemiological studies suggest that phthalates may give asthmatic and allergic effects, and several other health effects. However, a strong causal link between phthalate exposure and airway effects at relevant exposure concentrations has not yet been established, and mechanisms possibly involved are not clearly defined. The objectives of this study were to evaluate effects of di-n-butyl phthalate (DBP) exposure on airway immune cells in vitro in terms of macrophage differentiation, as well as synthesis and release of the pro-inflammatory cytokines tumor necrosis factor alpha (TNFα) and interleukin-1 beta (IL-1β), during priming followed by challenge or not. The human monocytic cell line THP-1 was exposed to DBP for 24 hours and subsequently differentiated by phorbol myristate acetate (PMA) for an additional 24h or 48 hours. Effects on the macrophage differentiation process were examined by morphological observation using light microscopy and analysis of specific CD markers by flow cytometry. In addition, DBP-induced release of the pro-inflammatory cytokines TNFα and IL-1β from cells differentiated with PMA for 30 hours before an 18 hours rest was measured by enzyme-linked immunosorbent assay (ELISA). These differentiated cells were exposed to DBP for 24 hours and either primed (3 hours pre-exposure) or primed and challenged (last 3 hours of exposure) with lipopolysaccharide (LPS) or Zymosan bioparticles. In addition, the TNFα and IL-1β mRNA levels were measured by RT-PCR, after priming cells with LPS and challenging with Zymosan. Exposure to 20-80 µM of DBP for 24h accelerated the PMA-induced morphological macrophage differentiation and increased the expression of CD36. No major changes in expression of CD11b, CD71, CD14 and CD80 were seen. Exposure to 80 µM of the DBP metabolite mono-n-butyl phthalate (MBP), did not have the same impact on the differentiation process. In differentiated cells, 24h DBP exposure increased the release of TNFα and IL-1β in cells primed with low concentrations of LPS or Zymosan, known to be toll-like receptor 4 (TLR4) and TLR2 ligands respectively. These receptors are known to play key roles in the innate immune system. After a TLR challenge with LPS and/or Zymosan, DBP reduced the TNFα release, while the IL-1β release was not altered. The mRNA levels of TNFα support the observed changes in protein levels, suggesting involvement of pre-transcriptional events. In conclusion, DBP exposure accelerated macrophage differentiation of THP-1 monocytes and increased release of pro-inflammatory cytokines, but appeared to impair the functionality of macrophages following in vitro TLR challenges.