Evaluating genotoxic risks of indoor air pollutants: Insights from the National institute of biology, Slovenia

Air pollution is one of the most significant global health challenges, contributing to around 8 million deaths worldwide each year.  Notably, in 2020 alone, approximately 3.2 million deaths were attributed to indoor air pollution. Considering people spend up to 90% of their time indoors on average, maintaining good indoor air quality is essential for protecting public health. 

As part of the EDIAQI project, a wide range of pollutants were identified in households and schools. Among them, benzo[g,h,i]perylene (BGP) and benzo[b]fluoranthene (BBF) were frequently detected. These compounds belong to the group of polycyclic aromatic hydrocarbons (PAHs), which are known for their high toxic potential, including cytotoxic, mutagenic, genotoxic and carcinogenic effects.

Genotoxic compounds cause DNA and chromosomal damage either directly or through secondary mechanisms, leading to mutations that may ultimately result in cancer and other chronic diseases. Exposure to genotoxic pollutants can have adverse health effects, which may appear with a delay and even at extremely low concentrations, especially with chronic exposure (Kisby et al., 2006). Therefore, exposure to air contaminated with genotoxic pollutants poses a long-term global health risk, with effects that may only become evident after years or even decades. However, there is still limited information on the potential genotoxic effects of BBF and BGP.

To address these data gaps, the Department of Genetic Toxicology and Cancer Biology in Slovenia, at the National Institute of Biology (NIB), evaluated the genotoxic activity of BBF and BGP at non-cytotoxic concentrations, both as single compounds and as binary mixtures (1:1 ratio), through in vitro analysis using the human hepatocellular carcinoma (HepG2) cell line.  This cell line retains the activities of various xenobiotic-metabolising enzymes (CYPs and phase II enzymes),  expresses  P53, and is a well-validated cell model for toxicological studies (Knasmüller et al., 2004). 

The potential genotoxicity of BBF and BGP was evaluated using the comet assay and the cytokinesis-block micronucleus (CBMN) assay. The comet assay (also known as single-cell gel electrophoresis) is a highly sensitive technique for detecting DNA damage at the single-cell level. It can detect DNA single- and double-strand breaks, alkali-labile sites such as apurinic/apyrimidinic sites, DNA-DNA and DNA-protein cross-links, and single-strand breaks associated with incomplete excision repair (Collins et al., 2023). 

Complementary to the comet assay, the CBMN assay provides valuable insights into chromosomal damage by detecting micronuclei (MN; a biomarker for chromosomal breaks and/or the loss of whole chromosomes), nucleoplasmic bridges (NPBs; a biomarker for DNA misrepair and/or telomere end fusions), and nuclear buds (NBUDs; a biomarker of amplified DNA elimination and/or DNA repair complexes) in exposed binucleated cells (Fenech et al., 2011). Evaluation of these parameters is time consuming, therefore NIB continue to use the automated Metafer System from MetaSystem, Germany   (https://ediaqi.eu/articles/robots-rescue-automated-analysis-cytogenetic-biomarkers-genotoxic-risk-assessment)

The findings indicate that BBF and BGP increased DNA strand breaks after prolonged exposure (24 hours) when tested as single compounds and in the binary mixture (1:1 ratio). However, after short exposure (4 hours), increased DNA damage was observed only in the binary exposure. Among the two pollutants, only BBF, when tested as a single compound, led to a significant increase in the formation of micronuclei. When tested in a binary mixture (1:1 ratio), an increase in both MN and NPB was observed; however, no combined effects were detected at non-cytotoxic concentrations. These results indicate that both PAHs and their binary mixture can affect DNA integrity, posing a potential risk to exposed population. Therefore, it would appear that further research is urgently needed to assess their impact on human health.

Considering that no safe exposure threshold can be established for carcinogenic compounds, it is crucial to minimise exposure to carcinogenic air pollutants. Although PAH exposure and indoor air pollution in general present significant health risks, public awareness is still lacking, with many people unaware of these potential hazards within their own homes. Raising awareness is therefore imperative. Only by understanding the risks associated with polluted indoor air can Europe take meaningful steps to improve air quality and protect citizens’ health. It is thus high-time to confront this often-overlooked issue and adopt measures that enhance indoor air quality for all. 

Note: This article has been published on behalf of Bojana Žegura, Matjaž Novak, Alja Štern & Martina Štampar, National Institute of Biology, Ljubljana, Slovenia

REFERENCES: 

Collins, A., Møller, P., Gajski, G., Vodenková, S., Abdulwahed, A., Anderson, D., Bankoglu, E.E., Bonassi, S., Boutet-Robinet, E., Brunborg, G., Chao, C., Cooke, M.S., Costa, C., Costa, S., Dhawan, A., de Lapuente, J., Bo’, C. Del, Dubus, J., Dusinska, M., Duthie, S.J., Yamani, N. El, Engelward, B., Gaivão, I., Giovannelli, L., Godschalk, R., Guilherme, S., Gutzkow, K.B., Habas, K., Hernández, A., Herrero, O., Isidori, M., Jha, A.N., Knasmüller, S., Kooter, I.M., Koppen, G., Kruszewski, M., Ladeira, C., Laffon, B., Larramendy, M., Hégarat, L. Le, Lewies, A., Lewinska, A., Liwszyc, G.E., de Cerain, A.L., Manjanatha, M., Marcos, R., Milić, M., de Andrade, V.M., Moretti, M., Muruzabal, D., Novak, M., Oliveira, R., Olsen, A.-K., Owiti, N., Pacheco, M., Pandey, A.K., Pfuhler, S., Pourrut, B., Reisinger, K., Rojas, E., Rundén-Pran, E., Sanz-Serrano, J., Shaposhnikov, S., Sipinen, V., Smeets, K., Stopper, H., Teixeira, J.P., Valdiglesias, V., Valverde, M., van Acker, F., van Schooten, F.-J., Vasquez, M., Wentzel, J.F., Wnuk, M., Wouters, A., Žegura, B., Zikmund, T., Langie, S.A.S., Azqueta, A., 2023. Measuring DNA modifications with the comet assay: a compendium of protocols. Nat. Protoc. 1–61. https://doi.org/10.1038/s41596-022-00754-y

Fenech, M., Kirsch-Volders, M., Natarajan, a. T., Surralles, J., Crott, J.W., Parry, J., Norppa, H., Eastmond, D. a., Tucker, J.D., Thomas, P., 2011. Molecular mechanisms of micronucleus, nucleoplasmic bridge and nuclear bud formation in mammalian and human cells. Mutagenesis 26, 125–132. https://doi.org/10.1093/mutage/geq052

Kisby, G.E., Olivas, A., Standley, M., Lu, X., Pattee, P., O’Malley, J., Li, X., Muniz, J., Nagalla, S.R., 2006. Genotoxicants target distinct molecular networks in neonatal neurons. Environ. Health Perspect. 114, 1703–1712. https://doi.org/10.1289/ehp.9073

Knasmüller, S., Mersch-Sundermann, V., Kevekordes, S., Darroudi, F., Huber, W.W., Hoelzl, C., Bichler, J., Majer, B.J., 2004. Use of human-derived liver cell lines for the detection of environmental and dietary genotoxicants; Current state of knowledge. Toxicology 198, 315–328. https://doi.org/10.1016/j.tox.2004.02.008