The Genotoxicity of Organic Extracts from Particulate Emissions Produced by Neat Gasoline (E0) and a Gasoline–Ethanol Blend (E15) in BEAS-2B Cells

Round 1

Reviewer 1 Report

Thank you for the opportunity to review the interesting study. The authors subjected bronchial epithelial cells to a gasoline PM emissions' organic fraction extract. The authors rightly point out the limitations of the study and hopefully future studies will include not only extracts but also the effects of complete particulate matter in vitro. 

Remarks

1. Line 126. Were 3 concentrations of PM used for all other methods? Or were 5 concentrations used for the cytotoxicity control and 3 for the other methods? 

2. Line 126-127.  0.5% DMSO chosen to match the concentration of DMSO in the PM samples, as it is not stated what volume of samples was applied.

3. The subsequent tests also used the DMSO control, effectively a control of the solvent used, and did not use negative and positive controls, which slightly reduces the effect of the interesting results presented. However, for example in γH2AX a positive control was used. 

4. Figure 4: E0 and E15 square bars below the diagram shows only 2 of three concentrations does it mean something special?

5. Line 536. Double dots after AhR.

6. There are no specific statistical methods section common for this kind of research.

Author Response

1. Line 126. Were 3 concentrations of PM used for all other methods? Or were 5 concentrations used for the cytotoxicity control and 3 for the other methods? 

We used a wider concentration range for cytotoxicity (5 doses) to select effective non-cytotoxic doses. For methods where the dose-response relationship was expected (Comet assay, apoptosis, micronucleus test), we used three doses. For other methods (gene expression analysis and γH2AX detection), the highest effective dose (50 µM) was used. The highest dose 50 µM was also selected with respect to our previous study (Libalova, 2018), where the same PM extracts (E0 and E15) were analyzed. In this study, the higher dose 100 µM was already cytotoxic for some gasoline PM extracts, therefore, 50 µM was used for all the tested extracts.

Libalova H, Rossner P Jr, Vrbova K, Brzicova T, Sikorova J, Vojtisek-Lom M, Beranek V, Klema J, Ciganek M, Neca J, Machala M, Topinka J. Transcriptional response to organic compounds from diverse gasoline and biogasoline fuel emissions in human lung cells. Toxicol In Vitro. 2018 Apr;48:329-341. doi: 10.1016/j.tiv.2018.02.002. Epub 2018 Feb 9. PMID: 29432896.

2. Line 126-127.  0.5% DMSO chosen to match the concentration of DMSO in the PM samples, as it is not stated what volume of samples was applied.

Reply: The concentration of stock solutions of PM extracts (50 mg PM/mL) was added to the text, please see the revised section 2.2. Chemical analysis of PM extracts (line 113). The maximal volume of the extract in the cell culture was 0.5% (v/v).

3. The subsequent tests also used the DMSO control, effectively a control of the solvent used, and did not use negative and positive controls, which slightly reduces the effect of the interesting results presented. However, for example in γH2AX a positive control was used. 

We used following positive controls in the study: BEAS-2B cells treated with KBrO3 and bleomycin was used for Comet assay and H₂O₂ for apoptosis and cytotoxicity. These positive controls were added to the corresponding graphs (please see the corrected Figure 1 and Figure 4). For micronucleus test, no positive control was used in this study. The metabolic competence of BEAS-2B cells and the ability of the laboratory to identify clastogens and aneugens under the conditions of the test protocol used was demonstrated in our previous study (Cervena, 2017). This is in accordance with OECD guidelines for the testing of chemicals (OECD, 2023). The positive control for gene expression was not applied, however, reference genes were selected previously according to the stability of gene expression during identical experimental conditions (Libalova, 2018) and qPCR analysis contains controls (such as IPC) that ensure the correctness and accuracy of the procedure. Negative controls (cells treated with medium) were involved in all experiments excepting the qRT-PCR experiment. As expected, no response was observed and was similar as for DMSO samples, no statistical difference was found. Therefore, we show the results for DMSO samples only as the vehicle control is more relevant. For qPCR experiment, we used only DMSO treated cells since the calculation of the fold change of each gene is always related to the DMSO sample.

Libalova H, Rossner P Jr, Vrbova K, Brzicova T, Sikorova J, Vojtisek-Lom M, Beranek V, Klema J, Ciganek M, Neca J, Machala M, Topinka J. Transcriptional response to organic compounds from diverse gasoline and biogasoline fuel emissions in human lung cells. Toxicol In Vitro. 2018 Apr;48:329-341. doi: 10.1016/j.tiv.2018.02.002. Epub 2018 Feb 9. PMID: 29432896.

Cervena T, Rossnerova A, Sikorova J, Beranek V, Vojtisek-Lom M, Ciganek M, Topinka J, Rossner P Jr. DNA Damage Potential of Engine Emissions Measured In Vitro by Micronucleus Test in Human Bronchial Epithelial Cells. Basic Clin Pharmacol Toxicol. 2017 Sep;121 Suppl 3:102-108. doi: 10.1111/bcpt.12693. Epub 2017 Jan 6. PMID: 27782363.

OECD (2023), Test No. 487: In Vitro Mammalian Cell Micronucleus Test, OECD Guidelines for the Testing of Chemicals, Section 4, OECD Publishing, Paris, https://doi.org/10.1787/9789264264861-en.

4. Figure 4: E0 and E15 square bars below the diagram shows only 2 of three concentrations does it mean something special?

Reply: The graph was corrected, please see the new Figure 4.

5. Line 536. Double dots after AhR.

Reply: The text was corrected, please see the revised version (line 569)

6. There are no specific statistical methods section common for this kind of research.

Reply: The chapter 2.9. Statistical analysis was added to the Materials and methods section.

Reviewer 2 Report

In this work, the authors compare the effects of organic compound mixtures extracted from PM extracts produced by neat gasoline (E0) and a blend containing 15% of ethanol (E15) on human BEAS-2B lung cells. Endpoints of toxicity assessed in this work include oxidative DNA damage, single/double DNA strand-breaks, micronucleus formation, apoptosis and induction of ROS- and aryl hydrocarbon receptor-related genes. Based on their experimental findings, the authors conclude that the addition of ethanol reduces the oxidative DNA damage induced by gasoline, whereas the other parameters didn’t change dramatically. In general, the research topic is interesting and the manuscript is well written and structured. However, I have two major points of concern, which should be addressed by the author team before I can recommend this paper for publication in the Journal of Xenobiotics.

 1.       Lack of positive control in nearly all assays. All parameters were assessed only after one timepoint per treatment. In order to see whether the assay would principally work at the chosen experimental conditions, I strongly recommend to include established chemicals/stimuli as positive control(s), in particular when assaying oxidative DNA damage, strand breaks, micronucleus formation and apoptosis.

 2.       qPCR analysis (fig. 5). I assume that gasoline PM induces oxidative stress and thereby enhances the activity of antioxidant/NRF2 signaling pathways. Hence, looking at gene expression level only after 24 h of treatment might be a little bit too late. I would recommend to include an earlier time point (e.g. 6 or 8 h) in this analysis.

Optional: Additional experiments confirming ROS formation (DCF assay etc.) and AHR activation (nuclear translocation or XRE reporter assay) would further increase the importance and rigor of this study.

Author Response

1. Lack of positive control in nearly all assays. All parameters were assessed only after one timepoint per treatment. In order to see whether the assay would principally work at the chosen experimental conditions, I strongly recommend to include established chemicals/stimuli as positive control(s), in particular when assaying oxidative DNA damage, strand breaks, micronucleus formation and apoptosis.

Positive controls were added to corresponding graphs, please see the reply to the third comment of the Reviewer 1.

2. qPCR analysis (fig. 5). I assume that gasoline PM induces oxidative stress and thereby enhances the activity of antioxidant/NRF2 signaling pathways. Hence, looking at gene expression level only after 24 h of treatment might be a little bit too late. I would recommend to include an earlier time point (e.g. 6 or 8 h) in this analysis.

Reply: In our previous study (Libalova, 2018), we exposed BEAS-2B cells with various gasoline PM extracts including the same E0 and E15 extracts, which are also used in the current study, for two time points (4h and 24h) and determined whole-genome gene expression changes by microarray analysis. After 4h incubation, various deregulated genes and pathways were found following E0 and E15 exposure, however, neither antioxidant genes (such as HMOX1, SOD1, TXNRD1 and others) nor antioxidant pathway was significantly deregulated. It is well known that whole PM from various sources induce strong antioxidant response in cell lines. However, we used PM extracts in our study that contain a mixture of organic compounds such as PAHs that may induce rather different toxic response. In our study, we focused on PAHs as these compounds are extremely toxic and are present in both extracts in considerable concentration. PAHs require metabolic activation to exert their toxicity, therefore, we selected the exposure time 24h to best reflect the toxic response to PAHs in cells. We assume that oxidative DNA damage originates from radicals and intermediates that are formed during the activation.

Libalova H, Rossner P Jr, Vrbova K, Brzicova T, Sikorova J, Vojtisek-Lom M, Beranek V, Klema J, Ciganek M, Neca J, Machala M, Topinka J. Transcriptional response to organic compounds from diverse gasoline and biogasoline fuel emissions in human lung cells. Toxicol In Vitro. 2018 Apr;48:329-341. doi: 10.1016/j.tiv.2018.02.002. Epub 2018 Feb 9. PMID: 29432896.

Optional: Additional experiments confirming ROS formation (DCF assay etc.) and AHR activation (nuclear translocation or XRE reporter assay) would further increase the importance and rigor of this study.

Reply: We definitely agree with the reviewer that additional experiments would be beneficial. Although detection of ROS by DCF assay may not be indicative as shown in our study with diesel PM extracts (Libalova, 2016), the following experiments including activation of AhR determined by reporter gene assay and other AhR-mediated toxic effect would be of great importance. Nevertheless, this study focused primarily on genotoxicity of E0 and E15 organic extracts and other experiments could be involved in a new follow-up study.

Libalova H, Rossner, P Jr., Vrbova K, Brzicova T, Sikorova J, Vojtisek-Lom M, Beranek V, Klema J, Ciganek M, Neca J, et al. Comparative Analysis of Toxic Responses of Organic Extracts from Diesel and Selected Alternative Fuels Engine Emissions in Human Lung BEAS-2B Cells. International Journal of Molecular Sciences. 2016; 17(11):1833. https://doi.org/10.3390/ijms17111833

Round 2

Reviewer 2 Report

The authors have adequately adressed my previous points of concern. I have no further comments and recommed the publication of this work in its present form.