Effects of Airborne Particulate Matter in Biomass Treatment Plants on the Expression of DNA Repair and IL-8 Genes

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Dear authors,

There are some concerns that need to be addressed as follows:

1. Please standardize all references to particulate matter (e.g., PM_0.49, PM_0.49-10, PM₁₀, and PM_2.5) using subscript notation consistently throughout the manuscript.
2. Line 43-44, please provide appropriate citations for the statements made in this section to support the claims.
3. Line 154, “0.25%” was missed.
4. The purpose of using genomic DNA in this study is unclear. Please clarify its role in the experimental workflow.
5. Table 1, XRCC1 is primarily involved in the Base Excision Repair (BER) pathway and also contributes to Single-Strand Break (SSB) Repair. While some studies have reported interactions between XRCC1 and components of the Nucleotide Excision Repair (NER) pathway, XRCC1 does not play a direct mechanistic role in NER. Please revise this classification accordingly and consider citing more directly relevant literature to reflect XRCC1’s functional role.
6. All figure captions should be placed beneath the corresponding figures.
7. Figure 2, “agro-zootechnic” and “agro-zoothenic” are used interchangeably, please choose one consistent label; “lignocellulosic” is misspelled as “lignocellolosic”; please unify x-axis formatting and legends across both panels; circles, star, and number appeared on the figure; consider showing sample sizes (n) in each group.
8. Figure 3, it’s unclear what exactly is being measured. Is it % cytotoxicity relative to control? Or a viability index? Please define the metric and include units. Additionally, some points are blue, some are white, a legend should be added to clarify their significance. Moreover, n-values should be stated somewhere.
9. Figure 4, Y axis, “mitochondrial”.
10. Figure 5, IL-8 is marked with a double asterisk indicating p < 0.01, yet the text states a p-value of 0.051, which is not statistically significant under the conventional threshold (p < 0.05). Please clarify whether the asterisk or the p-value is correct. For CRCC1, XPA, and XPF, the error bars look relatively large compared to the effect sizes.
11. Figure 6, it does not clearly indicate comparisons of IL-8 expression across different plants. Please revise to explicitly show these comparisons.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

In this manuscript by Zanchi et al., authors investigates the cytotoxic and gene regulatory effects of PM10 sub-fractions collected from biomass treatment plants, using human embryonic lung fibroblasts. The study provides important insight into the occupational risks associated with bioaerosol exposure in waste processing environments. This is interesting and well thought impactful study. However, following  concerns need to be addressed to strengthen this work.

1. The manuscript does not sufficiently justify the focus on PM10, especially given that PM2.5  are more strongly associated with DNA damage and cellular uptake in the literature. The rationale for excluding smaller particle sizes should be discussed.
2. The study does not identify specific chemical or microbial components within the PM samples. Without this information, it is difficult to determine which constituents are driving the observed biological effects.
3. Oxidative stress is a well-established mechanism underlying PM toxicity. However, the authors do not measure reactive oxygen species (ROS) or oxidative damage markers, which weakens the mechanistic interpretation.
4. While qPCR data are provided for selected genes, the study would benefit from broader transcriptomic analysis, such as RNA sequencing, to identify additional impacted pathways. Furthermore, western blot validation of key targets (e.g., ERCC1, IL-8) is recommended to confirm gene-level findings at the protein level.
5. In some experiments it is not clear whether untreated / mock controls were consistently included. negative controls is necessary for interpreting the results.
6. one of the major concern for me is use of only HELF cells. Validation in an additional cell line or model would strengthen the conclusions.
7. Although DNA repair gene expression is assessed, no direct evidence is shown to show DNA damage. Including a comet assay or γH2AX staining would provide more convincing evidence of genotoxicity.
8. The manuscript should clarify the duration of in vitro PM exposure in all assays. Additionally, it should specify the season if outside samples were collected and time frame over which environmental samples were collected.
9. The authors mention over 60 PM samples but do not clearly state whether they were analyzed independently or pooled. Pooling may obscure site-specific variability and statistics.
10. The authors should discuss limitations in discussion/conclusion section such as the using only one cell line, not measuring ROS or DNA damage, and potential variability in PM composition.
11. The study observes differential biological outcomes depending on the source of PM (e.g., composting vs. lignocellulosic), but does not systematically evaluate whether compositional differences between PM samples explain these effects. This question should be addressed more directly.
12. Manuscript contains some minor grammatical errors and needs proofreading.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

no comments for author.