Effects of ozone exposure on human epithelial adenocarcinoma and normal fibroblasts cells

<div><p>Previous studies show variable ozone cytotoxicity and genotoxicity in cell cultures, laboratory animals and humans directly exposed to tropospheric ozone. The aim of this study was therefore to investigate and compare the cyto and genotoxic effects of ozone using adenocarcinoma human alveolar basal epithelial cells A549 and normal human fibroblasts Hs27. A cell culture chamber with controlled atmosphere (a simulation reactor) was built to inject a flow of 120 ppb of ozone, which is two times the threshold value for the protection of human health, fixed by the EU legislation. Cell proliferation was evaluated by a luminescent cell viability assay while we assessed the genotoxic potential of ozone by the induction of micronuclei as well as evaluating DNA strand breaks by the induction of micronuclei evaluated by means of the cytokinesis-block micronucleus (CBMN) assay as well as evaluating DNA strand breaks by Alkaline Comet Assay (CA) or Comet Assay. A549 cells viability decreases significantly at 24 hours treatment with 120 ppb of O₃ while at 48 hours and 72 hours O₃ treated cells viability doesn’t differ in respect to the control. However a significative decrease of A549 viability is shown at 72 hours vs. 48 hours in both treated and not-treated cells. The viability trend in the Hs27 cells did not show any significant changes in treated samples compared to the control in all conditions. The two genotoxicity biomarkers, the micronucleus and the comet tests, showed in both the cell types exposed to ozone, a significant increase in the number of micronuclei and in the tail DNA % in respect to the control even if at different times/cell type. Moreover, we found that O₃ provokes genotoxic effects more evident in A549 cancer cells than in normal fibroblasts Hs27 ones. We applied a cell growth simulation model referred to ozone treated or not cell lines to confirm that the ozone exposure causes a slackening in the cells replication.</p></div

Cell growth simulation models for the A549 cells.

<p>The model has been numerically solved for the time interval 0–72 hours in order to reproduce the measured curve of cell proliferation in both the conditions (control and treated cells).</p

Viability test in A549 and in Hs27 cells.

<p>The effects of 120 ppb O₃ on cell proliferation were evaluated following exposures for 0, 24, 48 and 72 hrs and compared to untreated cells (A549 Fig 2A and Hs27 Fig 2B). Triton-X-100 0.1% was used as positive control. Significance values were determined according to the t-Student: * = p<0,05; ** = p<0,005; *** = p<0,0005 error bars represent the standard error of the mean.</p

CBPI index in A549 and Hs27 cells 120 ppb O₃ treated.

<p>Cytokinesis Block Proliferation Index (CBPI) in human cells lines A549 and Hs27 treated with 120 ppb O₃ at 48, 72 hrs. The CBPI indicates the average number of nuclei per cell, and may be used to calculate cell proliferation. CBPI was calculated as follows: (1 × N1) + (2 × N2) + (3 × (N3 + N4))/N where N1–N4 represent the number of cells with 1–4 nuclei, respectively, and N is the total number of cells scored. The results are compared to the negative control and are the means ± ES. * <i>p</i> < 0.05.</p

RI Replication Index and micronucleus induction in A549 and Hs27.

<p>Induction of cytotoxicity according to RI and genotoxicity according to micronuclei test in A549 cells (panels A and C), and Hs27 cells (panels B and D) in both the condition (control and ozone exposure). Significance values determined according to the t-Student: * = p<0,05; ** = p<0,005; error bars represent the standard error of the mean.</p