Macrophages in vitro as a predictive model in polymer toxicology
Daly, Paul Michael
Organic polymers S2218600, S2429901 and S2219200 (referred to as Polymer 1, Polymer 2 and Polymer 3, respectively) of varying toxic potential, designed for use in cosmetic aerosols, were used as model substances to predict inflammatory potential. In vivo inflammogenic potential was evaluated by assessment of inflammatory cell profile (alveolar macrophage (AM), polymorphonuclear neutrophil (PMN)) of broncho-alveolar lavage fluid (BAL) 24hrs after a single instillation of either 0.5 mg or 2 mg polymer in Sprague Dawley rats. Pro-inflammatory Minusil particles and non-inflammatory titanium dioxide (TiO2) particles were used as controls. For comparison, cultured rat NR8383 AM-like cells, human THP-1 monocyte cells or human monocyte derived macrophages were treated with polymer for 24 h and supernatants analysed for indicators of cytotoxicity and inflammatory mediator release. In addition, after 6 h treatment, gene changes in the rat lung tissue and also in the rat NR8383 alveolar macrophage cell line were assessed using microarray to analyse the entire rat genome. The in vivo studies showed that Polymer 1, Polymer 3 and Minusil caused significant PMN influx into BAL. Polymer 3 and Minusil caused a significant increase in AM number in BAL. Polymer 2 and TiO2 had no effect on BAL cell profile. BAL tumour necrosis factor-α (TNFα) and macrophage inflammatory protein-2 (MIP-2) levels were significantly increased following instillation of Polymer 3 and Minusil. Thus the polymers and particles were ranked for potential to cause pulmonary inflammation: Polymer 3 > Minusil > Polymer 1 > Polymer 2 > TiO2. In vitro studies using cultured rat NR8383 AM-like cells showed that the polymers and particles could be ranked similarly for cytotoxic potential and their ability to stimulate the release of both TNFα and MIP-2. Cultured human monocyte derived macrophages detected the pro-inflammatory abilities of Polymer 3, as measured by cytotoxic potential and ability to stimulate TNFα, interleukin-8 (IL-8) and macrophage inflammatory protein-1α (MIP-1α), however, did not detect the pro-inflammatory abilities of Polymer 1. Cultured human THP-1 cells predicted the pro-inflammatory effects of Polymer 3 in rat lungs using the cytotoxicity assay and by changes in IL-1β, MIP-1α and IL-10 levels. The human THP-1 cell line did not predict the pro-inflammatory effects of Polymer 1 that were observed the rat lungs. Electron spin resonance (ESR) detected free radicals produced by the pro-inflammatory polymers and particles which had the ability to break bonds in super-coiled DNA and deplete intracellular glutathione (GSH). Microarray analysis of the canonical pathways activated by the pro-inflammatory polymers, Polymer 1 and Polymer 3, showed that 3 similar pathways were significantly activated in the instilled rats and the rat NR8383 AM-like cells following treatment. ‘Xenobiotic metabolism’, ‘IL-10 signalling’ and ‘leukocyte extravasation signalling’ pathways were significantly changed by the pro-inflammatory polymers. Use of these cell model alternatives in an industrial setting will refine and reduce in vivo testing and as these models are further developed and used alongside future new alternatives they will provide a substantial contribution towards the replacement of animal testing.