Effect on Markers of Cardiovascular, Reproductive and Cancer Risk From Firefighting Training (BIOBRAND3)

Effect on Markers of Cardiovascular, Reproductive and Cancer Risk From Firefighting Activities - a Study Under Different Training Methods

Epidemiological studies based on Danish registries have observed that Danish male firefighters have more cardiovascular disease, infertility diagnose and a trend to increased risk of cancer than other Danish employed males. Firefighting activities include a combination of stressors such as strenuous work under heat, smoke and soot known to be able to affect cardiovascular and reproductive health, with smoke and soot also being known to increase the risk of cancer. The training facilities of real-fire extinguishing exercises in Denmark operate using wood or natural gas fire, which will have differential gradients of smoke, soot and possibly heat. The investigators will use different training conditions to create gradients of the different stressors and investigate health effects thereof. With this approach, the investigators expect to be able to evaluate the individual contribution of the different stressors in markers of cardiovascular, cancer and reproductive health risk. The project will include approx. 35 young conscript participants on a firefighting course, followed in four sessions, three firefighting training sessions under different fire conditions (no fire, wood fire and gas fire) and one control scenario.

The study methodology is based on a crossover design on firefighting training under different conditions, with characterization of exposure and assessment of cardiovascular, cancer and reproductive effect biomarkers. The study will be performed in cooperation with the Danish Emergency Management Agency. The study will have one baseline session, while conscripts are in a classroom, and three sessions of firefighting-related exercises under different fire conditions, used currently in firefighting training programs in Denmark. The three firefighting training sessions will be controlled for equivalent work exercise using full protective gear and under different ambient conditions of firefighting training: Firefighting equivalent work (no fire), with exercises performed in a clean environment, without fire (no ambient temperature, soot or smoke). This type of exercise precedes or complements the training under real fire conditions. Firefighting under wood fire (wood fire), with exposure to ambient heat, smoke and soot. This is the most common training scenario used by Danish Emergency Management Agency training centres. Firefighting under gas fire (gas fire), with exposure to ambient heat, and expectably less smoke and soot than with wood fire. These conditions are used in some Danish training centres, with logistical advantages (ease of turning or putting out the fire and managing the fire fuel) and unknown effect relating to exposure prevention (smoke and soot). The order of the firefighting sessions will be as randomized as possible, and according to a selection of three sequence order options (no-wood-gas; wood-gas-no and gas-wood-no). Each campaign (corresponding to each rescue course) would desirably have one of these session sequences. It is not possible to blind the participants to the different scenarios, neither the field staff, but all the samples will be blinded for the subsequent laboratory analysis. The training sessions will have 1-3 weeks in between (accordingly with programmatic educational course schemes).

Reactive Hyperemia, Micro RNA, Heart Rate Variability, DNA Strand Breaks, Oxidative Stress, Heat Stress

Comet assay, 8-oxodG, PAH CALUX, hydroxyl-PAHs and nitro-PAH metabolites, microRNA, AhR activation, scrotal temperature, core body temperature, urine, serum, blood, follicle-stimulating hormone, inhibin B, EndoPAT

Participants will perform firefighting full day exercises under 3 different conditions (without fire, under wood fire and under gas fire), sessions which will occur with weeks in between, and biological markers will be assessed and compared in relation to a baseline session, where the participants are having theoretical classes.

It is not possible to blind the participants to the different scenarios, neither the field staff, but all the samples will be blinded for the subsequent laboratory analysis, namely blood analysis, urine analysis, wipe samples analysis will all be blinded.

Baseline measurements will be performed in the same schedule as measurements in the three other arms. In the baseline session, participants are in classroom before starting any firefighting exercise

Firefighting equivalent work, with exercises performed in a clean environment, without fire (no ambient temperature, soot or smoke). This type of exercise usually precedes or complements the training under real fire conditions.

Firefighting under wood fire, with exposure to ambient heat, smoke and soot. This is the most common training scenario used in the training centres. The participants will be in teams performing pre-defined tasks (knocking down the fire, moving heavy objects, and searching and rescuing metal stand in models

Firefighting under gas fire, with exposure to ambient heat, and expectably less smoke and soot than with wood fire. These conditions are used in some Danish training centres, with logistical advantages (ease of turning or putting out the fire and managing the fire fuel) and unknown effect relating to exposure prevention (smoke and soot). The participants will be in teams performing pre-defined tasks (knocking down the fire, moving heavy objects, and searching and rescuing metal stand in models

The participants will be performing firefighting equivalent work in a clean environment, without fire (no ambient temperature, soot or smoke).

The participants will be in teams performing pre-defined tasks (knocking down the fire, moving heavy objects, and searching and rescuing metal stand in models), under wood fire conditions.

The participants will be in teams performing pre-defined tasks (knocking down the fire, moving heavy objects, and searching and rescuing metal stand in models), under gas fire conditions.

Reactive hyperemia index (RHI) measured with the device EndoPAT 2000. A reactive hyperemia is induced by a blood cuff on the upper arm and the peripheral vasodilation response is assessed in the small digital vessels of a fingertip with a portable device connected to a computer, with RHI determined by an algorithm from the device, with lower index values corresponding to a worsen situation.

Baseline afternoon measurement, afternoon measurement immediately after firefighting without fire, afternoon measurement immediately after firefighting under wood fire and afternoon measurements immediately after firefighting under gas fire

Reactive hyperemia index (RHI) measured with the device EndoPAT 2000. A reactive hyperemia is induced by a blood cuff on the upper arm and the peripheral vasodilation response is assessed in the small digital vessels of a fingertip with a portable device connected to a computer, with RHI determined by an algorithm from the device, with lower index values corresponding to a worsen situation.

Baseline morning measurement, morning measurement in subsequent day after firefighting without fire, morning measurement in subsequent day after firefighting under wood fire and morning measurement in subsequent day after firefighting under gas fire

Heart rate variability (HRV) measured with the device EndoPAT 2000. The HRV is calculated using the initial 5.5 complete minutes before the cuff is applied. pNN50 is the proportion of successive NN intervals differing by more than 50 milliseconds divided by the total number of N intervals (given in percentage).

Baseline afternoon measurement, afternoon measurement immediately after firefighting without fire, afternoon measurement immediately after firefighting under wood fire and afternoon measurements immediately after firefighting under gas fire

Heart rate variability (HRV) measured with the device EndoPAT 2000. The HRV is calculated using the initial 5.5 complete minutes before the cuff is applied. pNN50 is the proportion of successive NN intervals differing by more than 50 milliseconds divided by the total number of N intervals (given in percentage).

Baseline morning measurement, morning measurement in subsequent day after firefighting without fire, morning measurement in subsequent day after firefighting under wood fire and morning measurement in subsequent day after firefighting under gas fire

Heart rate variability (HRV) measured with the device EndoPAT 2000. The HRV is calculated using the initial 5.5 complete minutes before the cuff is applied. RMSSD is the square root of the mean squared differences of successive NN intervals (given in milliseconds)

Baseline afternoon measurement, afternoon measurement immediately after firefighting without fire, afternoon measurement immediately after firefighting under wood fire and afternoon measurements immediately after firefighting under gas fire

Heart rate variability (HRV) measured with the device EndoPAT 2000. The HRV is calculated using the initial 5.5 complete minutes before the cuff is applied. RMSSD is the square root of the mean squared differences of successive NN intervals (given in milliseconds)

Baseline morning measurement, morning measurement in subsequent day after firefighting without fire, morning measurement in subsequent day after firefighting under wood fire and morning measurement in subsequent day after firefighting under gas fire

Heart rate variability (HRV) measured with the device EndoPAT 2000. The HRV is calculated using the initial 5.5 complete minutes before the cuff is applied. Ratio of low frequency and high frequency bands

Baseline afternoon measurement, afternoon measurement immediately after firefighting without fire, afternoon measurement immediately after firefighting under wood fire and afternoon measurements immediately after firefighting under gas fire

Heart rate variability (HRV) measured with the device EndoPAT 2000. The HRV is calculated using the initial 5.5 complete minutes before the cuff is applied. Ratio of low frequency and high frequency bands

Baseline morning measurement, morning measurement in subsequent day after firefighting without fire, morning measurement in subsequent day after firefighting under wood fire and morning measurement in subsequent day after firefighting under gas fire

Oxidized nucleobase 8-oxodG will be measured in urine samples by High-performance liquid chromatography (HPLC) as marker of oxidative stress, together with creatinine, for adjusting for urine concentration. Data will be reported as nanomol 8-oxodG per millimol creatinine.

Baseline, before firefighting without fire, day after firefighting without fire, before firefighting under wood fire, day after firefighting under wood fire, before firefighting under gas fire, day after firefighting under gas fire

DNA strand breaks will be measured by comet assay, and reported as number of lesions per 10^6 base pairs, transformed from percentage of DNA in tail using the calibration curve from the well-establish relationship between ionizing radiation dose and yield of strand breaks in DNA.

Baseline, before firefighting without fire, day after firefighting without fire, before firefighting under wood fire, day after firefighting under wood fire, before firefighting under gas fire, day after firefighting under gas fire

Core body temperature will be assessed by an ingestible pill thermometer with data recorded and reported as time series during the period in transit.

Baseline day, during the day of firefighting without fire, during the day of firefighting under wood fire and during the day of firefighting under gas fire.

Scrotal temperature will be assessed by skin sensor placed in the scrotum of male participants and reported as scrotal skin temperature time series.

Baseline day, during the day of firefighting without fire, during the day of firefighting under wood fire and during the day of firefighting under gas fire.

Core body temperature will be assessed by an ingestible pill thermometer and scrotal temperature will be assessed by skin sensor placed in the scrotum of male participants, to assess the thermoregulation of the scrotum during firefighting exercises. Time series of core body temperature and scrotal skin temperature will be analysed for eventual thermoregulation disruption.

Baseline day, during the day of firefighting without fire, during the day of firefighting under wood fire and during the day of firefighting under gas fire.

Circulating micro RNA candidates will be measured by RNA extraction from serum samples, reverse transcribed into complementary DNA (cDNA) and analysed with quantitative polymerase chain reaction (qPCR).

Baseline, before firefighting without fire, day after firefighting without fire, before firefighting under wood fire, day after firefighting under wood fire, before firefighting under gas fire, day after firefighting under gas fire

The aryl hydrocarbon receptor (AhR) activation will be assessed in vitro using urine samples on the PAH CALUX (Chemical Activated LUciferase gene eXpression bioassay) reporter assay. The smoke and soot exposures are complex mixtures of compounds with potential toxic effect. Routine measurements of PAHs are usually quantified for a target list of 16 common soot elements and even less chemical species for urinary metabolites, but many other compounds are present in both soot and metabolites mixtures. The toxicity of PAHs is primarily caused through the binding to AhR, and induction of AhR related genes and subsequent toxic pathways. The outcome will be measured in the form of benzo[a]pyrene equivalence.

Baseline, before firefighting without fire, day after firefighting without fire, before firefighting under wood fire, day after firefighting under wood fire, before firefighting under gas fire, day after firefighting under gas fire

The aryl hydrocarbon receptor (AhR) activation will be assessed in vitro using wipe samples on the PAH CALUX (Chemical Activated LUciferase gene eXpression bioassay) reporter assay. The smoke and soot exposures are complex mixtures of compounds with potential toxic effect. Routine measurements of PAHs are usually quantified for a target list of 16 common soot elements, but many other compounds are present in soot mixtures. The toxicity of PAHs is primarily caused through the binding to AhR, and induction of AhR related genes and subsequent toxic pathways. The outcome will be measured in the form of benzo[a]pyrene equivalence.

Baseline, before firefighting without fire, immediately after firefighting without fire, before firefighting under wood fire, immediately after firefighting under wood fire, before firefighting under gas fire, immediately after firefighting under gas

Baseline, before firefighting without fire, day after firefighting without fire, before firefighting under wood fire, day after firefighting under wood fire, before firefighting under gas fire, day after firefighting under gas fire

Baseline, before firefighting without fire, day after firefighting without fire, before firefighting under wood fire, day after firefighting under wood fire, before firefighting under gas fire, day after firefighting under gas fire

The internal dose of polycyclic aromatic hydrocarbons (PAHs), that would have the contribution from different exposure routes, will be assessed in first morning urine samples and measured for 7 isomer hydroxyl-PAH compounds and 5 nitro-PAH compounds, measured by high-performance liquid chromatography (HPLC)

Baseline, before firefighting without fire, day after firefighting without fire, before firefighting under wood fire, day after firefighting under wood fire, before firefighting under gas fire, day after firefighting under gas fire

Skin wipes will be sampled to determine the PAH composition of deposited soot on the neck area. The wipes will be analysed for the 16 US Environmental Protection Agency priority list of PAH compounds by HPLC.

Baseline, before firefighting without fire, immediately after firefighting without fire, before firefighting under wood fire, immediately after firefighting under wood fire, before firefighting under gas fire, immediately after firefighting under gas

Lung function will be measured by spirometry using the Spirometer device EasyOne Air. Forced Expiratory Volume at 1 second (FEV1).

Baseline, immediately after firefighting without fire, immediately after firefighting under wood fire and immediately after firefighting under gas fire

Lung function will be measured by spirometry using the Spirometer device EasyOne Air. Forced Vital capacity (FVC).

Baseline, immediately after firefighting without fire, immediately after firefighting under wood fire and immediately after firefighting under gas fire

Lung function will be measured by spirometry using the Spirometer device EasyOne Air. Peak Expiratory Flow (PEF).

Baseline, immediately after firefighting without fire, immediately after firefighting under wood fire and immediately after firefighting under gas fire

Lung function will be measured by spirometry using the Spirometer device EasyOne Air. Forced Expiratory Volume at 1 second (FEV1) and Forced Vital Capacity (FVC) ratio is calculated from device output.

Baseline, immediately after firefighting without fire, immediately after firefighting under wood fire and immediately after firefighting under gas fire

Baseline, before firefighting without fire, day after firefighting without fire, before firefighting under wood fire, day after firefighting under wood fire, before firefighting under gas fire, day after firefighting under gas fire

Muscle activity will be assessed to control for body workload through electromyography (EMG) using the portable device Nexus10. Bipolar surface EMG electrodes are applied to the skin over the muscles in 3 relevant body regions (shoulder, leg and back). The signals are collected with a data logger and reported as work load during a working day.

Baseline day, firefighting without fire day, firefighting under wood fire day and firefighting under gas fire day

The participants will be volunteers (both males and females) recruited among conscripts in training in a desirable involved group up to 35 individuals. Inclusion Criteria: legally competent, conscript subjects following a Rescue Specialist Educational course Exclusion Criteria: current smoking status, pregnancy, on prescribed medication, body mass index (BMI) bellow 19 or over 30, alcohol or drug abuse.

Supportive information and fully anonymized information will be shared and whenever possible (in supplementary material) published along with the results, but following European Union General Data Protection Regulation (GDPR) and Danish rules, no non or pseudo anonymized data might be shared.

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