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Evaluating the genotoxicity of urban PM2.5 using PCR-based methods in human lung cells and the Salmonella TA98 reverse test

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Abstract

A number of compounds found in particulate matter with an aerodynamic diameter <2.5 (PM2.5) can interact with DNA either directly or after enzymatic transformation to induce DNA modifications. These particulate matter (PM)-induced alterations in DNA may be associated with increased frequencies of pollution-associated diseases, such as lung cancer. In the present study, we applied different methods to assess the mutagenicity and genotoxicity of monthly PM2.5 organic extracts collected over a full year. We used the Salmonella assay, exposed cultured human embryonic lung fibroblasts and applied extracellular lactate dehydrogenase (LDH) and 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxyanilide inner salt (XTT) assays to assess the cytotoxicity of PM2.5 on the cells. We assessed both the expression levels of a number of DNA repair genes (using qRT-qPCR) and the genetic profile of the treated cells compared to the control. The expression levels of XRCC1 and APE1, which are involved in the first steps of base excision repair, as well as ERCC1, XPA and XPF, which encode nucleotide excision repair subunits, were analysed. The monthly mean of the PM2.5 collected was 35.16 ± 22.06 μg/m3. The mutagenicity of PM2.5 to TA98 was 46 ± 50 net revertants/m3, while the mutagenicity to TA98 + S9 was 17 ± 19 net revertants/m3. The mean IC50 values were 2.741 ± 1.414 and 3.219 ± 2.764 m3 of equivalent air in the XTT and LDH assays, respectively. A marked and significant increase in APE1 expression levels was observed in the exposed cells. This effect was also significantly correlated with mutagenicity (p < 0.01). No induced AFLP fragment profile alterations were detected. The proposed approach seems to be useful for integrated evaluation and for highlighting the mechanisms inducing DNA damage.

Mutagenic, cytotoxic and genotoxic effects that were recorded in vitro over a full year of investigation into PM2.5 organic extracts.

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Abbreviations

AFLP:

Amplified fragment length polymorphism

BER:

Base excision repair

LDH:

Extracellular lactate dehydrogenase

NER:

Nucleotide excision repair

PAHs:

Polycyclic aromatic hydrocarbons

PCR:

Polymerase chain reaction

PM:

Particulate matter

PM10:

Particulate matter with an aerodynamic diameter <10 μm

PM2.5:

Particulate matter with an aerodynamic diameter <2.5 μm

RT-qPCR:

Real-time quantitative polymerase chain reaction

XTT:

2,3-Bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxyanilide inner salt

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Acknowledgments

This study was funded by the University of Turin as a local project (ex. 60 %, 2012). The authors thank their colleagues (Prof. Giorgio Gribaudo and Dr. Anna Luganini) for supplying the HELF cells and the Environmental Protection Agency of Piedmont (particularly Dr. F. Lollobrigida) for collecting the PM2.5 samples. Moreover, the authors thank Dr. Roberto Arbore for his collaboration during his training period.

Authors’ contributions

Dr. Traversi designed the study and performed the PM2.5 sample extractions, Salmonella, cytotoxicity and gene expression assays. Dr. Cervella performed the AFLP assay. Dr. Gilli provided the PM2.5 samples and contacted the local Environmental Protection Agency. Dr. Gilli and Dr. Traversi analysed the data. Dr. Traversi prepared the manuscript. All authors approved the final manuscript.

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Authors and Affiliations

  1. Department of Public Health and Paediatrics, University of Torino, piazza Polonia 94, 10126, Torino, Italy

    Deborah Traversi & Giorgio Gilli

  2. Department of Life Sciences and System Biology, University of Torino, via Accademia Albertina 13, 10123, Torino, Italy

    Piero Cervella

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  1. Deborah Traversi
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  2. Piero Cervella
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  3. Giorgio Gilli
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Correspondence to Deborah Traversi.

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Responsible editor: Philippe Garrigues

Highlights

Fine particles are crucial urban pollution indicators.

PM2.5 produces marked mutagenic and cytotoxic effects in vitro.

APE1 gene overexpression provided an effect evidence, but no AFLP profile alterations were induced.

These effects displayed a significant seasonal trend.

Multi-assay approach displayed an advantage in conducting genotoxic assessments.

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Traversi, D., Cervella, P. & Gilli, G. Evaluating the genotoxicity of urban PM2.5 using PCR-based methods in human lung cells and the Salmonella TA98 reverse test. Environ Sci Pollut Res 22, 1279–1289 (2015). https://doi.org/10.1007/s11356-014-3435-1

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