Nucleic acids, particularly antibiotic resistance genes, are commonly found on surfaces within healthcare environments, with levels not reducing following cleaning. Within the UK, there are no regulations for testing disinfectants against nucleic acids. A series of commonplace in vitro methods were used to determine disinfectant-induced physical and functional damage to various nucleic acids; RNA (10 μg), genomic DNA (2 μg), and plasmids (1 μg). Using these methods, the optimal residence time (10 minutes) and working concentration (10%) were determined for a new disinfectant. Furthermore, comparison of disinfectants with different active ingredients including lactic acid (LA), sodium hydroxide (NaOH), chloroxylenol (PCMX) and quaternary ammonium compounds (QAC) were compared to controls. All disinfectants showed greater degradation by gel electrophoresis of genomic DNA and RNA than of purified plasmids. Functional analysis using qPCR and PCR demonstrated that no disinfectant tested (apart from control) could damage DNA to the level where PCR amplification was not possible, and only the NaOH reagent could achieve this for RNA. The set of methods described herein provides a platform for future standardisation and potential regulation regarding monitoring cleaning solutions for their activity against nucleic acids.

The majority of in vitro studies focusing upon particle–lung cell interactions use static models at an air–liquid interface (ALI). Advancing the physiological characteristics of such systems allows for closer resemblance of the human lung, in turn promoting 3R strategies. PATROLS (EU Horizon 2020 No. 760813) aimed to use a well-characterised in vitro model of the human alveolar epithelial barrier to determine how fluid-flow dynamics would impact the outputs of the model following particle exposure. Using the QuasiVivo^TM (Kirkstall Ltd., York, UK) system, fluid-flow conditions were applied to an A549 + dTHP-1 cell co-culture model cultured at the ALI. DQ₁₂ and TiO₂ (JRCNM01005a) were used as model particles to assess the in vitro systems’ sensitivity. Using a quasi- and aerosol (VitroCell Cloud12, VitroCell Systems, Waldkirch, Germany) exposure approach, cell cultures were exposed over 24 h at IVIVE concentrations of 1 and 10 (DQ₁₂) and 1.4 and 10.4 (TiO₂) µg/cm², respectively. We compared static and fluid flow conditions after both these exposure methods. The co-culture was subsequently assessed for its viability, membrane integrity and (pro-)inflammatory response (IL-8 and IL-6 production). The results suggested that the addition of fluid flow to this alveolar co-culture model can influence the viability, membrane integrity and inflammatory responses dependent on the particle type and exposure.

Exposure to indoor air pollutants (IAP) has increased recently, with people spending more time indoors (i.e. homes, offices, schools and transportation). Increased exposures of IAP on a healthy population are poorly understood, and those with allergic respiratory conditions even less so. The objective of this study, therefore, was to implement a well-characterised in vitro model of the human alveolar epithelial barrier (A549 + PMA differentiated THP-1 incubated with and without IL-13, IL-5 and IL-4) to determine the effects of a standardised indoor particulate (NIST 2583) on both a healthy lung model and one modelling a type-II (stimulated with IL-13, IL-5 and IL-4) inflammatory response (such as asthma).Using concentrations from the literature, and an environmentally appropriate exposure we investigated 232, 464 and 608ng/cm² of NIST 2583 respectively. Membrane integrity (blue dextran), viability (trypan blue), genotoxicity (micronucleus (Mn) assay) and (pro-)/(anti-)inflammatory effects (IL-6, IL-8, IL-33, IL-10) were then assessed 24 h post exposure to both models. Models were exposed using a physiologically relevant aerosolisation method (VitroCell Cloud 12 exposure system).No changes in Mn frequency or membrane integrity in either model were noted when exposed to any of the tested concentrations of NIST 2583. A significant decrease (p < 0.05) in cell viability at the highest concentration was observed in the healthy model. Whilst cell viability in the “inflamed” model was decreased at the lower concentrations (significantly (p < 0.05) after 464ng/cm²). A significant reduction (p < 0.05) in IL-10 and a significant increase in IL-33 was seen after 24 h exposure to NIST 2583 (464, 608ng/cm²) in the “inflamed” model.Collectively, the results indicate the potential for IAP to cause the onset of a type II response as well as exacerbating pre-existing allergic conditions. Furthermore, the data imposes the importance of considering unhealthy individuals when investigating the potential health effects of IAP. It also highlights that even in a healthy population these particles have the potential to induce this type II response and initiate an immune response following exposure to IAP.

The exposure of human lung and skin to carbon black (CB) is continuous due to its widespread applications. Current toxicological testing uses ‘healthy’ cellular systems; however, questions remain whether this mimics the everyday stresses that human cells are exposed to, including infection. Staphylococcus aureus lung and skin infections remain prevalent in society, and include pneumonia and atopic dermatitis, respectively, but current in vitro toxicological testing does not consider infection stress. Therefore, investigating the effects of CB co-exposure in ‘stressed’ infected epithelial cells in vitro may better approximate true toxicity. This work aims to study the impact of CB exposure during Staphylococcus aureus infection stress in A549 (lung) and HaCaT (skin) epithelial cells. Physicochemical characterisation of CB confirmed its dramatic polydispersity and potential to aggregate. CB significantly inhibited S. aureus growth in cell culture media. CB did not induce cytokines or antimicrobial peptides from lung and skin epithelial cells, when given alone, but did reduce HaCaT and A549 cell viability to 55% and 77%, respectively. In contrast, S. aureus induced a robust interleukin (IL)-8 response in both lung and skin epithelial cells. IL-6 and human beta defensin (hβD)-2 could only be detected when cells were stimulated with S. aureus with no decreases in cell viability. However, co-exposure to CB (100 µg/mL) and S. aureus resulted in significant inhibition of IL-8 (compared to S. aureus alone) without further reduction in cell viability. Furthermore, the same co-exposure induced significantly more hβD-2 (compared to S. aureus alone). This work confirms that toxicological testing in healthy versus stressed cells gives significantly different responses. This has significant implications for toxicological testing and suggests that cell stresses (including infection) should be included in current models to better represent the diversity of cell viabilities found in lung and skin within a general population. This model will have significant application when estimating CB exposure in at-risk groups, such as factory workers, the elderly, and the immunocompromised.

The indoor environment changes based on a variety of different factors, therefore changing the genotoxic potential of that environment. Despite the associations of exposure to outdoor air pollution and links to health impacts, enough is not currently known about the impacts to human health indoor air may have. Due to the variable nature of the indoor environment (multiple things can change this environment, including but not limited to, the use, occupants, materials within the environment, animals, the outside environment and ventilation) this can prove difficult. Indoor particulate matter (PM) of various sizes (PM2.5–PM10 for example) has been shown to induce reactive oxygen species, increase markers in oxidative stress, double strand DNA breaks, and cause 8-oxoguanine lesions in both in vitro and in vivo models (including humans). The particle type and source influence these responses, highlighting heating and cooking fuel sources that may be more genotoxic than others. A similar pattern has also been observed with volatile organic compounds (VOCs) with various mixtures of VOCs and different VOC compositions inducing DNA damage differently, with diet also being a contributing factor to genotoxic potential. A significant knowledge gap in the potential genotoxicity of indoor air pollution is the exposure of various biologicals found indoors. Moulds, other fungi, and allergens are an emerging health risk and are of significant public interest. Little is known about the inhalation toxicology and mechanisms of action of these components, and this is becoming increasingly important to understand due to public deaths linked to their exposure. There is overlap with the health effects of outdoor air pollution, where there has been much more research focus. Therefore, the aim of this review is to establish what is already known, the potential limitations of the current work and where we should focus research in the future.