Effects of Exposure to High Temperature and Combustion by Products on Firefighter Saliva: An Evaluation of PH, Flow, TNF-α, IL-6, IL-10, Lactoferrin and Lysozyme

SUMMARY

This is a longitudinal study of 40 firefighters, in which the effects of firefighting on saliva were investigated using firefighting simulators. Salivary concentrations of TNF-α, IL-6, IL-10, lactoferrin and lysozyme were measured using the ELISA method, and salivary volume and pH were measured before and after firefighting exposure. The concentrations of TNF-α, IL-6, lactoferrin and lysozyme showed no significant changes, in contrast there was a decrease in the concentrations of IL-10 in pH and saliva production of firefighters after acute exposure to firefighting. We conclude that firefighting alters the composition of saliva.

Introduction

In addition to its digestive function, saliva is an important element in the composition of the immunological barrier that protects the upper airways, together with Waldeyer’s lymphatic ring and the mucosa that covers them.^1,2 The immune activity of saliva depends on innate factors such as the production of lysozymes and lactoferrins, and acquired ones with the production of specific antibodies. Its composition, activity and production are influenced by age, immunization, environmental influences, physical activities and diseases.³ Working as a firefighter is known to be an extreme activity, associated with dehydration and exposure to combustion by products. Studies have shown that this occupation affects the upper respiratory tract and leads to an inflammatory process in the lungs and larynx, and even systemic such as cardiovascular, but little has been studied on the relationship between this occupation and its effects on saliva.^4-6

Saliva is a fluid secreted by the salivary glands into the oral cavity and is one of the first innate and adaptive barriers through which harmful substances come into contact with the human body.⁷ It consists of water, electrolytes and proteins, among other things, and plays an important role in the initial immune defence. Among the proteins present in saliva, lactoferrin (LF), lysozyme, cytokines and immunoglobulins are of fundamental importance as they form the first line of defence against the invasion of harmful substances.⁸ LF is a multifunctional globular glycoprotein naturally found in saliva, tears, semen and colostrum, with higher concentrations observed in the latter. In in vitro studies, LF has been shown to be effective against a variety of viruses, including SARS CoV-2.⁹ In addition, it has unique immunomodulatory and anti-inflammatory properties that may be particularly important in the pathophysiology of severe viral diseases. On the other hand, lysozyme (LYS), which is found in tears, mucus, breast milk and saliva, is an enzyme with the unique ability to break down bonds in the bacterial cell wall and thus contribute to the destruction of pathogens. This process is essential for protection against infection and is a critical component of the human body’s innate immune system.¹⁰

Cytokines, glycoproteins with the ability to modulate the host’s inflammatory response, play a crucial role in the immune system. Among them, interleukin 6 (IL-6) and tumor necrosis factor-alpha (TNF-α) stand out as two of the most important pro-inflammatory cytokines. IL-6, which is mainly released by macrophages and T cells, not only triggers fever and the acute inflammatory response, but also stimulates the production of other cytokines and the growth of B cells, thereby strengthening the immune response. Stress events in the environment, such as infections and tissue damage, cause an immediate and transient expression of IL-6.^11,12 TNF-α, which is mainly produced by macrophages, lymphocytes and monocytes, plays an important role in attracting neutrophils and monocytes to the site of inflammation and increases the effectiveness of the immune response against invaders.¹³ Interleukin 10 (IL-10) plays a role in the homeostasis of the immune response. It is an important anti-inflammatory and antipyretic agent that acts as a macrophage deactivating factor and exerts inhibitory effects on neutrophils. In fact, IL-10 synthesis is induced by some infectious agents or they encode their own IL-10 homologs, resulting in suppression of the host immune response.¹⁴

Patients exposed to high temperatures can be expected to become dehydrated, resulting in decreased salivary flow and possibly a change in pH. Mineral loss due to acid dissolution has been shown to be essential for the proteolytic destruction of collagen.¹⁵ In addition to the proteins contained in saliva that help protect against the invasion of harmful substances, the chemical pH balance also plays an important role in this defence by creating an environment that can prevent the overgrowth of harmful microorganisms by acting as a chemical barrier.¹⁶

The aim of this study is to investigate the effects of exposure to high temperatures and combustion byproducts on the concentration of inflammatory and antimicrobial markers in the saliva of firefighters.

Materials and methods

General characteristics of the study and inclusion criteria

A prospective study was conducted on firefighters attending a fire safety instructor course at the Escola Superior de Bombeiros in Franco da Rocha in 2020 and 2023, using container fire simulators (Fig. 1). The laboratory analyses were carried out at the ENT Research Lab of the Department of Otorhinolaryngology and Head and Neck Surgery of the Universidade Federal de São Paulo. The project was approved by the Ethics Committee of Plataforma Brasil (Project number 4.572.579) and the participants were volunteers who all signed a voluntary informed consent form.

Fig. 1.

Demonstration of firefighting exercises in which exposure to fire can be observed

The inclusion criteria were to be a firefighter, to be between 18 and 50 years old, to have passed an annual medical and dental examination, to be approved and registered for the firefighter instructor course, and to have not been exposed to fires for at least 2 weeks. Firefighters who did not volunteer to participate in the study, active dental or stomatologic disease, chronic or acute sinusitis, history of chronic disease of the larynx, oral cavity or pharynx, pregnant women, severe systemic or autoimmune disease, and chronic use of corticosteroids, antibiotics or anti-inflammatory medications up to one month prior to the start of the study were excluded.

The study was conducted in two phases: in 2020, the concentrations of TNF-α, IL-6, IL-10, lactoferrin and lysozyme were evaluated, and in 2023, the saliva production and pH value were evaluated. In 2021 and 2022, the study was not conducted because the firefighter training courses were interrupted due to the COVID-19 pandemic. Each phase consisted of 20 firefighters forming 2 groups (before and after firefighter exposure). The firefighters were familiarised with firefighting through the use of fire simulation containers. The course for firefighting instructors lasted 4 weeks and included a total of 24 firefighting simulation sessions (6 sessions per week), each lasting 20 to 30 minutes.

The study phase conducted in 2020 consisted of: a control group (pre) before exposure to the simulators; a group after the prolonged exposure (post-4w) consisting of the same firefighters immediately after the fourth week of the course. In the study phase conducted in 2023, we added a 3^rd group to the ones defined in 2020, which consisted of the same firefighters after they had been exposed to the firefighting training, conducted immediately after the first week of the course. Saliva was collected from the firefighters 12 hours prior to the start of the firefighting instructor course and immediately after the last exposure in the first and/or fourth week of the course to examine the ongoing effects of firefighting on saliva, depending on the year the assay was conducted. For the group studied in 2023, saliva was additionally collected after the last exposure to firefighting. The addition of an assay by the end of the 1^st week sought to assess the acute response to firefighting-induced stress.

Samples and laboratory tests

The saliva of all firefighters was analysed during a 5-minute saliva sample 12 hours before the first exposure and immediately after the last exposure (in the 1^st and/or the 4^th weeks).

The saliva of the firefighters who participated in the 2020 phase was stored at the ENT Research Laboratory of the Universidade Federal de São Paulo at -80°C, where the samples were analysed for concentrations of TNF-α, IL-6, IL-10, lactoferrin and lysozyme. The concentration of cytokines (IL-6, IL-10 and TNF-α) was measured in the saliva samples using an ELISA assay (Thermo Fisher Scientific, Invitrogen, Vienna, Austria), following the manufacturer’s instructions, and results were obtained in pg/mL (catalogue numbers 88-7066, 88-7106 and 88-7346, respectively). The antimicrobial peptides (lactoferrin and lysozyme) were also measured using the ELISA method (EZ Assays, Florida, United States).

In 2020 and 2023, the samples were all analysed the same day. The results were expressed in μg/mL for lactoferrin and ng/mL for lysozyme (catalogue number CK-bio-12222 and CK-bio-12360, respectively). The reaction was evaluated by adding a TMB solution to each well of the plate, and the reaction was then stopped by adding a stop buffer (4N H2SO4). The reaction was read at 450 nm on a microplate reader (Mulitskan Sky, Thermo). In this way, the effects of temperature and combustion by-products on antimicrobial peptides (lactoferrin and lysozyme), IL-6, IL-10 and TNF-α in saliva were analyzed by comparisons between groups of firefighters from the 2020 study. Salivary pH monitoring was performed using the Supelco Mquant kit (batch HC293086) and the volume of saliva produced was measured during the 5-minute collections.

Statistical analysis

The data were analyzed using JAMOVI statistical software. The results of the quantitative variables were described by mean, standard deviation, median and interquartile range. The normality of quantitative variables was tested using the Shapiro-Wilk test. The Wilcoxon test or paired Student’s T test were applied according to the samples distribution to evaluate lactoferrin, lysozyme, IL-6, IL-10, and TNF-α. The Friedman and Durbin-Conover tests were used to assess pH and saliva volume, with p-values <0.05 indicating statistical significance.

Results

Anthropometric data

Participant characteristics, including height, weight, BMI and age, were recorded during the initial phase of the study and are shown in Table I. All participants in the study were male and aged 20 to 35 years. Height ranged from 167 cm to 188 cm, and weight ranged from 59 kg to 98 kg. BMI varied between 18.7 and 29.6 and none of the patients were smokers.

Table I.

Anthropometric data: mean, median, standard deviation (SD), maximum and minimum (n=40)

	Minimum	Maximum	Mean	SD	Median
Age (years)	20	35	27.5	7.3	30
Height (cm)	167	188	177.5	6.0	173
Weight (kg)	59	98	78.5	9.8	72
BMI (kg/m2)	18.7	29.6	23.4	1.4	23.5

Evaluation of IL-6, IL-10, TNF-α, lactoferrin and lysozyme

Table II shows the descriptive statistics of IL-6, IL-10, TNF-α, lactoferrin and lysozyme before and after a 4-week exposure to fire. No changes were found between the interleukins before and after exposure to fire. There was a decrease in the concentration of IL-10 (from 11.1 pg/ml to 9.22 pg/ml, p=0.004) after fighting the fire, as shown in Fig. 2 and Table III. On the other hand, analysis of TNF-α, IL-6, lactoferrin and lysozyme showed no differences between the groups before and after exposure to firefighting.

Table II.

Descriptive statistics of IL-6, IL-10, TNF-α, lactoferrin (Lf) and lysozyme (lys) before (pre) and after 4 weeks (post) exposure to fire

	IL-10 pre	IL-10 past	IL-6 pre	IL-6 post	TNF-α past	TNF-α post	Lf pre	Lf post	Lys pre	Lys post
N	20	20	20	20	20	20	20	20	20	20
Mean	10.8	9.43	9.41	7.89	64.1	76.4	8.43	9.11	46.2	44.0
Med	11.1	9.22	8.00	7.28	52.5	53.0	6.12	3.81	37.5	37.0
SD	2.02	1.33	6.59	1.78	32.7	53.1	6.94	9.05	22,0	24.5
IQR	2.75	2.05	0.77	1.07	9.70	23.6	3.19	7.38	26.4	32.5
Min	7.62	7.33	6.49	5.81	42.7	41.8	1.86	1.82	17.9	17.9
Max	14.7	12.1	37.8	13.?	184	217	35.4	29.4	102	100

Fig. 2.

Concentrations of IL-10 before (pre) and after 4 weeks (post) exposure to fire

Table III.

Concentrations of IL-6, IL-10, TNF-α, lactoferrin and lysozyme before (pre) and after 4 weeks (post) exposure to fire

	Pre	Post	p
IL-10	11.1 pg/ml	9.22 pg/ml	0.004
IL-6	9,41 pg/ml	7.89 pg/ml	0.374
TNF	64.1 pg/ml	76.4 pg/ml	1.000
Lactoferrin	8.43 μg/ml	9,11 μg/ml	0838
Lysozyme	46.2 ng/ml	44.0 ng/ml	0.304

Analysis of saliva production and saliva pH

Saliva production decreased after the first week of fire exposure (p<0.001) and increased again after the fourth week of fire exposure (p=0.03). There was no difference in saliva production between baseline and after the 4-week exposure, p=0.167 (Fig. 3). Following exposure to fire, there was a decrease in pH after an acute exposure of one week (p<0.01), which was maintained after a prolonged exposure of four weeks (p=0.008), compared to pre-exposure pH (Fig. 4).

Fig. 3.

Saliva production before exposure (pre) and after 1 and 4 weeks exposure to firefighting

Fig. 4.

Salivary pH before exposure (pre) and after 1 and 4 weeks exposure to firefighting

Discussion

The role of environmental factors in the development of inflammatory and infectious diseases associated with work is gaining increasing attention in the scientific literature. In this context, our study of firefighters exposed to fire by-products and high temperatures provides additional insight into the extreme environment to which they are exposed and its effects on saliva. This study has shown that firefighting has a direct effect on the saliva characteristics’ immune barrier, which may contribute to the invasion, development and progression of infectious diseases and the exacerbation of chronic inflammatory diseases such as periodontitis.

This finding is consistent with the role of environmental factors, including stress, in modulating immune responses through cellular and molecular mechanisms.¹⁷ Therefore, our study adds to the current understanding of the effects of environmental conditions during firefighting on the immune system by altering the salivary barrier.

The activity of enzymes depends on pH. LF having its optimal activity at neutral pH, the acidic pH of saliva after firefighting influences its immunomodulatory activities.¹⁸ The interaction between the protective properties of lactoferrin on oral pathologies and the immunological changes induced by extreme occupational exposures, as shown in our study on firefighters, could influence the ability of LF to act against inflammatory processes and microbial infections, and protect from a wide spectrum of diseases ranging from halitosis and xerostomia to alveolar or maxillary bone damage, gingivitis and even peri-implantitis. LF has the ability to stimulate the production of IL-10, which can modulate the immune response under unfavourable conditions.^19,20 IL-10 inhibits the production of other cytokines such as IFN-γ, IL-2, IL-12 and TNF-β, and deactivates macrophages that act on inflammatory cells, leading to inhibitory effects on T and natural killer cells.²¹ Furthermore, B cells, granulocytes, neutrophils, dendritic cells, keratinocytes and endothelial cells are all regulated by IL-10 in terms of growth and/or differentiation.²²

Although inflammation is crucial for defence against bacterial infection, it can lead to alveolar bone destruction when accompanied by excessive production of proinflammatory cytokines that negatively affect osteoblastogenesis and stimulate osteoclastogenesis.^23,24 Saliva analysis in firefighters showed that the production of IL-10 decreased in the group of firefighters after exposure compared to the same group before exposure, and could impact chronic inflammatory diseases in the oral cavity in this profession.

Studies have shown that alterations in the salivary immune barrier are associated with SARS-CoV-2 infections in firefighters, highlighting the importance of saliva as an immunological barrier during the COVID-19 pandemic.²⁵ In this study, we show that firefighting has an acute effect on this barrier (through saliva production, salivary pH change and interleukin imbalance), which may contribute to these workers being more susceptible to infectious epidemics.

This study underscores the importance of exploring interventions that positively impact the immune-inflammatory system for firefighters to prevent and avoid exacerbation of pre-existing oral diseases. These include agents such as LF, more efficient hydration, basing saliva pH, keeping a safe distance from the rest area, avoiding heat and combustion by-products, and using environmental protection equipment not only during firefighting but also during post-operation, provided smoke is still present.

Conclusion

Firefighting can alter the salivary immune barrier, indirectly by enhancing the inflammatory process and directly by altering saliva production and pH.