The design of this study has been described in detail in our earlier publication (Zhu et al., 2024). In brief, pre- and post-exposure assessments were conducted over a period of six months (Dec. 2021–Jun. 2022) to assess the biological effects of exposure to PAH from fire smoke on miRNA expression in biospecimens. Orientation days were scheduled to visit fire departments and conduct pre-exposure sampling campaigns, explaining the purpose of the study and the general procedures to the potential participants. After their eligibility was confirmed, participants signed an informed consent form approved by the Institutional Review Board (IRB #11466) of the University of Oklahoma Health Sciences. Baseline demographic characteristics, turnout gear practices, and firefighting-related practices were recorded via self-administered questionnaires. Unlike a traditional pre-post study design, we began to conduct post-exposure sampling campaigns due to the unpredictability of fire events. For baseline pre-exposure sampling, we revisited each participating fire department no earlier than 24 h (1 day) after the fire activity. The interval between the post- and pre-sampling campaigns ranged from 1 to 90 days.

The recruitment of study participants was a collaborative effort between local and regional fire departments in Oklahoma because groups of career and volunteer firefighters from different departments make runs together to a fire scene. These fire departments are located within a 100-km radius of the institutional field team to facilitate timely responses, as longer distances would cause delays. To be eligible, a firefighter had to be 18 years of age or older, with at least one year of experience subsequent to probation, and possess their own turnout gear, as PAH on their gear and related information on their gear practices were collected. To maximize control of the background PAH levels, firefighters were excluded who 1) used any tobacco products; 2) had eaten grilled, barbecued, or smoked foods within two days of the sampling collection; 3) had a history of chronic/infectious diseases; or 4) used medications. As a further precaution, volunteer firefighters who had potentially been exposed to PAH in a primary occupation were excluded.

The procedure for collecting PAH samples from firefighters has been previously described in detail in (Zhu et al., 2024). In brief, PAH deposited on the surface of firefighters’ turnout gear and skin were collected using a 100 cm² polyester fabric wipe (Alpha Wipes, Texwipe, Kernersville, NC) saturated with 1 ml of 99 % isopropyl. The collected wipes from the turnout gear and skin were analyzed by gas chromatography/mass spectrometry using EPA Method 8270D. For quality control, at least one field blank, without wiping, was collected for every ten samples.

Skin specimens were collected from firefighters using a skin tape strip (STS, D-Squame discs, 1.4 cm diameter, CuDerm, Dallas, TX). The tape was applied to the skin 20 times all around the wrist and neck areas opposite the environmental PAH skin wipe spots, with pressure equivalent to 225 g/cm² (D500 Pressure Instrument, CuDerm, Dallas, TX) for 3 s each time (Dyjack et al., 2018). The STS specimens were stored in microfuge tubes at −80 °C until further analysis. Blood samples were collected by a certified phlebotomist from the Oklahoma Shared Clinical and Translational Resources (OSCTR) at the University of Oklahoma Health Sciences. At the field site, blood samples were drawn in EDTA-containing tubes to avoid coagulation and processed into serum by centrifuging per standard operating procedure (World Health Organization, 2010). All of the collected biospecimens were transported on ice to the Laboratory for Molecular Biology and Cytometry Research (LMBCR) at the University of Oklahoma Health Sciences on the same day and stored at −80 °C for further analysis.

Polycyclic aromatic hydrocarbons (PAHs) are major organic byproducts generated through incomplete combustion processes during fires. Fire smoke, produced as firefighters work to suppress fires, contains a range of toxic chemical compounds, among which PAHs are considered particularly hazardous due to their persistence and potential health risks (ASTDR, 2015; Hwang et al., 2021; U.S. EPA, 2022). As far as we know, our network is the first to reveal a distinct topology with seven PAH analytes from fire smoke that interacted with 106 miRNAs (Fig. 3). The PAHs in this network, specifically Naphthalene, represent a class of possibly carcinogenic to humans (Group 2B) toxins while the rest of the PAHs are not classifiable (Group 3) as the level of evidence of carcinogenicity is insufficient (IARC, 2024). The network visualization demonstrates two primary interaction clusters between Naphthalene and 1-Methyl-anthracene, with one exception – miR-520b. We also found that PAHs primarily upregulate miRNA expression. These positive associations suggest that PAHs may contribute to disease development through miRNA upregulation, potentially affecting downstream gene expression patterns. This positive pattern aligns with previous findings that PAHs such as benzo[a]anthracene and benzo[k]fluoranthene can significantly upregulate miRNA expression, particularly miR-181 family members in hepatocellular carcinoma cells (Song et al., 2013). Similarly, benzo[a]pyrene can upregulate p53-targeting miRNAs, including miR-25, miR-92, miR-141, and miR-200a in multiple myeloma cells, as PAHs activate the aryl hydrocarbon receptor (AhR)(Gordon et al., 2015; Rude et al., 2024). In contrast, Acenaphthylene showed a negative interaction with a specific miRNA (miR-450b–3b). These distinct regulatory signatures suggest analyte-specific mechanisms of action, supporting previous findings that different PAHs can activate distinct molecular pathways leading to varied toxicological outcomes(Mallah et al., 2022).

Among the exposure-expression network combinations evaluated, PAHs detected on firefighting turnout gear showed the strongest associations with blood miRNA expression. Significant correlations were observed specifically for low molecular weight (LMW) PAHs, including acenaphthene, anthracene, naphthalene, and 1-methyl-naphthalene. LMW PAHs generally are greater bioavailability due to their higher volatility and lower hydrophobicity that enable rapid skin and respiratory absorption(Patel et al., 2020). These results suggest that dermal contact or inhalation of LMW PAHs from contaminated gear may induce systemic molecular alterations reflected in circulating miRNA profiles. However, we were not able to distinguish between the exposure routes of dermal absorption and inhalation of the PAHs from this study. The marked downregulation in skin tissue suggests that significant differentially expressed miRNA may participate in blood-specific regulatory circuits or act as suppressors of skin-related pathways. Thus, deposited PAH toxins on gear mechanisms may differ across biospecimen interfaces, with particularly pronounced effects of miRNA circulating in blood. This finding correlates with our earlier finding that exposure to PAHs on gear is more sensitive than on skin, and primarily transported via the blood(Zhu et al., 2024). Yet, whether the levels of miRNA in biospecimens are related to occupational factors, including detailed turnout gear practices and PAH exposure assessments, is still largely undetermined. The rest of the PAHs display distinct associations within miRNAs, suggesting analyte-specific miRNA regulatory mechanisms.

Pyrene, Anthracene, and Acenaphthylene show fewer miRNA associations, suggesting more localized or restricted biological interactions. A negative Skin-Blood pathway association is found with Acenaphthene, suggesting potential miRNA-mediated suppression mechanisms in response to exposure to Acenaphthene.

The simultaneous enrichment of both necroptosis and Th17 differentiation pathways suggests a potential interplay between programmed cell death mechanisms and immune regulation in our study, which warrants further investigation. Necroptosis appears as the most significantly enriched pathway, with five miRNA-target genes contributing to its enrichment. This genetically regulated form of necrotic cell death is emerging as an important pathogenic mediator in various human diseases(Choi et al., 2019). The strong enrichment of necroptosis suggests that regulated cell death mechanisms play a crucial role in the biological processes under investigation, potentially indicating cellular stress, inflammatory responses, or tissue injury mechanisms. Th17 cell differentiation represents the second most enriched pathway. Th17 cells are a subset of CD4⁺ T cells that secrete IL-17 and play critical roles in immune regulation, host defense, and autoimmune diseases(Yasuda et al., 2019). The significant enrichment of this pathway suggests active immune regulatory processes in our study participants, potentially indicating inflammatory responses or immune system activation(Graeber and Olsen, 2012).

The Firefighter Cancer Registry Act of 2018, enacted by the U.S. Congress, requires the Centers for Disease Control and Prevention (CDC) to develop and maintain a voluntary cancer registry, called the National Firefighter Registry for Cancer (NFR), focused on cancer incidence among firefighters(U.S. Congress, 2018). This ongoing project implies not only that the federal government recognizes the significant scope of research needed but also that scientific efforts to address the unique challenges of firefighters should be continued. Despite being a pilot, our study indicates that miRNA-based biomarker research could strongly complement the NFR. First, we recommend conducting longitudinal cohort studies to monitor firefighters throughout their professional careers, assessing both occupational exposures and biomarkers – miRNA expression dynamics – over time. Integrating NFR data with these findings could enable the development of personalized cancer risk assessments, facilitating earlier interventions such as enhanced screening protocols for firefighters identified with high-risk miRNA profiles.

Second, an occupational epidemiological study could effectively reconstruct comprehensive exposures by studying biomarkers such as miRNA expression levels and pathways. miRNA research in the biomedical and clinical research areas has exponentially increased over the past decade due to the fast development and commercial accessibility of the needed technology. miRNAs are important mediators and post-transcriptional regulators of expression because of their ability to degrade and/or suppress the translation of multiple mRNA molecules (Guo et al., 2010). Genetically and epigenetically altered miRNA, which is involved in carcinogenesis and metastasis, is associated with either oncogenes or tumor suppressor genes in cancer(Jeong et al., 2018; Li et al., 2018). Thus, miRNA provide crucial information for fully understanding cancer pathways and cancer etiology. The potential interaction between miRNAs and the pathways involved in cancer development can be used to integrate miRNA expression profiles(Jeong et al., 2016).

Furthermore, incorporating individual miRNA profiling in biomarker patterns with occupational exposure measurements will significantly reduce misclassification errors, resulting in better estimates for constructing individual job exposure matrices (JEMs) specific to this unique cohort group. Traditionally, a JEM is created using the identified health risks associated with exposure data (e.g., either primary measurements or a secondary database) and surrogate information (e.g., work history, job title). Collectively, direct measuring a panel of carcinogens (e.g., PAHs)-responsive miRNAs in accessible biospecimens (e. g., blood, skin) could enable early detection of high-risk firefighters and monitoring of intervention efficacy(Sisto et al., 2019). Furthermore, some of these miRNAs might be explored as therapeutic targets – for example, restoring a PAH-suppressed miRNA (like miR-216a-5p) in tumors or inhibiting an oncogenic miRNA (like miR-421) might reverse certain molecular damage of PAHs.

This study is the first to systematically investigate and quantify the mechanism by which PAHs on turnout gear and skin can alter miRNA expression, modulating its potential impact on carcinogenesis and addressing a critical gap in the literature. Given the small window of opportunity at a fire activity, short-term effects were explored by comparing pre- and post-exposure levels with unique work characteristics, including respirator practices. A recent study on wildland firefighters found that 65 miRNAs significantly changed between pre- and post-wildfire season samples, suggesting miRNA expression is a responsive epigenetic marker of short-term smoke exposure (Goodrich et al., 2025). Thus, the significance of this study is based on the scientific premise that specific targeted exposure to PAHs at pre- and post-fire activities allowed us to more accurately and reliably measure the alteration of miRNA expression and identify early indicators of the carcinogenic process. In summary, miRNAs act as critical mediators linking environmental carcinogen exposure to human cancer development. Fireground PAH exposure induces specific miRNA expression changes in firefighters, with consistent patterns observed across skin and blood biospecimens. These miRNAs target gene networks implicated in occupational carcinogenesis and immune modulation, offering promising biomarkers for occupational health surveillance. Thus, identifying exposure-response biomarkers may enhance risk prediction and support the development of effective intervention and prevention strategies.

miRNAs are both abundant, as they are found in red blood cells and extracellular vesicles (Woeller et al., 2016). While their half-lives vary, miRNAs demonstrate remarkable stability in blood once released into circulation due to their small size and presence in protein complexes. The experimental results found that the half-lives of miRNA were approximately 16.42 ± 4.2 h in blood (Wang and Liu, 2022). Thus, we collected blood-circulating miRNAs for assessing the health effects. Also, the skin tape strip (STS) is a valid sampling method and an adequate alternative to a whole skin biopsy, having been used for transcriptome gene expression analysis in clinical and pathological diagnostics(Dyjack et al., 2018; Kim et al., 2019). To our knowledge, this is the first miRNA study to investigate both blood and skin in an occupational cohort of firefighters. Despite its contributions, this study has limitations that warrant consideration. We determined that restricting our study to PAHs of all the complex toxic compounds present in fire smoke was our best approach. Simultaneously, assessing biological responses using miRNA oversimplifies the complex exposure to fire smoke that firefighters encounter. Thus, the correlation between PAHs and associated microRNAs in firefighter biospecimens should not be interpreted as evidence of causation. Our study may represent preliminary evidence on the association of accumulated PAH and miRNA expression. Although we did not include experimental validation to support bioinformatic analyses, we acknowledge its absence as a limitation. Nonetheless, our findings provide an initial framework for identifying candidate miRNAs responsive to fire-related carcinogen exposure, and lay the foundation for future studies. Another key limitation of this study is the relatively small sample size, which may limit the generalizability of the findings. Additionally, the cross-sectional design precludes establishing causal relationships between exposures and miRNA expression changes, highlighting the need for longitudinal studies to confirm these associations. Specifically, carcinogenic exposures, including those from PAHs, should be examined using longitudinal studies because miRNA alterations from reversible to irreversible depend on both dosage and duration of exposure (Jeong et al., 2018; Li et al., 2018). Initially, the field-exposure assessment was designed to measure PAH levels and biospecimens via repeated measurements. Yet, the study period (Dec. 2021–Jun. 2022) was harshly affected by the pandemic, so the original approach was not feasible. Thus, there was no record of repeated samples from the same participants nor the exact timing of the sampling within the 24-h window in this study. While the baseline for pre-fire activities was established using pre-exposure sampling at the fire departments, the post-sampling campaigns were conducted within 24 h after the fire suppression, but before the gear was washed. Initially, the post-fire sampling at the fire scene was designed so that the firefighters immediately doffed the outer layers of their gear at the scene while leaving the inner layers on for the rest of the work shift or longer(Wingfors et al., 2018), but this approach was challenging during the pandemic. We also were not able to monitor physical activity and stress after each fire activity. Finally, the severe thermal conditions and moist environment during fire extinguishment, in conjunction with the up-to-35 kg of multi-layered turnout gear worn by firefighters, leads to acute physical activity with a higher core heat stress, which may trigger epigenetic regulation including changes in miRNA expression.