BEU, Alm.del - 2017-18 - Bilag 73: Invitation til teknisk gennemgang af resultaterne fra forskningsprojekterne om brandmænds udsættelse for partikler den 17. januar 2018, fra beskæftigelsesministeren

Beskæftigelsesudvalget 2017-18
BEU Alm.del Bilag 73
Offentligt

Mutagenesis,

2017, 00, 1–11

doi:10.1093/mutage/gex021

Original Manuscript

Association between polycyclic aromatic

hydrocarbon exposure and peripheral blood

mononuclear cell DNA damage in human

volunteers during ire extinction exercises

Maria Helena Guerra Andersen, Anne Thoustrup Saber,

Per Axel Clausen,

Julie Elbæk Pedersen,

Mille Løhr, Ali Kermanizadeh,

Steffen Loft, Niels Ebbehøj,

Åse Marie Hansen,

1,3

Peter Bøgh Pedersen,

Ismo Kalevi Koponen,

Eva-Carina Nørskov,

Peter Møller* and

Ulla Vogel

1,5,

Department of Public Health, Section of Environmental Health, University of Copenhagen, Øster Farimagsgade 5A,

DK-1014 Copenhagen K, Denmark,

The National Research Centre for the Working Environment, Lersø Parkalle 105,

DK-2100 Copenhagen Ø, Denmark,

Department of Occupational and Environmental Medicine, Bispebjerg Hospital,

Bispebjerg Bakke 23, DK-2400 Copenhagen NV, Denmark,

Department of Public Health, Section of Social Medicine,

University of Copenhagen, Øster Farimagsgade 5A, DK-1014 Copenhagen K, Denmark,

Danish Technological Institute,

Teknologiparken, Kongsvang Allé 29, DK-8000 Aarhus C, Denmark, and

Department of Micro- and Nanotechnology,

Technical University of Denmark, DK-2800 Kgs, Lyngby, Denmark

*To

whom correspondence should be addressed. Department of Public Health, Section of Environmental Health, University

of Copenhagen, Øster Farimagsgade 5A, DK-1014 Copenhagen K, Denmark. Tel: +35327654; Email:

[email protected].

The

National Research Centre for the Working Environment, Lersø Parkalle 105, DD-2100 Copenhagen Ø, Denmark. Tel: +45 39

16 52 01; Email:

[email protected]

Received 24 May 2017; Editorial decision 10 July 2017; Accepted 11 August 2017.

Abstract

This study investigated a number of biomarkers, associated with systemic inlammation as well

as genotoxicity, in 53 young and healthy subjects participating in a course to become ireighters,

while wearing personal protective equipment (PPE). The exposure period consisted of a 3-day

training course where the subjects participated in various live-ire training exercises. The subjects

were instructed to extinguish ires of either wood or wood with electrical cords and mattresses. The

personal exposure was measured as dermal polycyclic aromatic hydrocarbon (PAH) concentrations

and urinary excretion of 1-hydroxypyrene (1-OHP). The subjects were primarily exposed to

particulate matter (PM) in by-stander positions, since the self-contained breathing apparatus

effectively prevented pulmonary exposure. There was increased dermal exposure to pyrene

(68.1%, 95% CI: 52.5%, 83.8%) and sum of 16 polycyclic aromatic hydrocarbons (ƩPAH; 79.5%,

95% CI: 52.5%, 106.6%), and increased urinary excretion of 1-OHP (70.4%, 95% CI: 52.5%; 106.6%)

after the ireighting exercise compared with the mean of two control measurements performed

2 weeks before and 2 weeks after the ireighting course, respectively. The level of Fpg-sensitive

sites in peripheral blood mononuclear cells (PBMCs) was increased by 8.0% (95% CI: 0.02%, 15.9%)

compared with control measurements. The level of DNA strand breaks was positively associated

with dermal exposure to pyrene and

ƩPAHs,

and urinary excretion of 1-OHP Fpg-sensitive sites

reserved. For permissions, please e-mail: [email protected].

BEU, Alm.del - 2017-18 - Bilag 73: Invitation til teknisk gennemgang af resultaterne fra forskningsprojekterne om brandmænds udsættelse for partikler den 17. januar 2018, fra beskæftigelsesministeren

M. H. G. Andersen et al.

were only associated positively with PAHs. Biomarkers of inlammation and lung function showed

no consistent response. In summary, the study demonstrated that PAH exposure during ireighting

activity was associated with genotoxicity in PBMCs.

Introduction

Fireighters are potentially exposed to numerous types of carcinogens

during ireighting. The exposure depends on the nature and source of

the ire, which the International Agency for Research on Cancer (IARC)

has broadly categorised as municipal, wildland, industrial, aviation, mil-

itary and oil well ire scenarios. IARC evaluated occupational ireight-

ing activity as possibly carcinogenic to humans (Group 2B), based on

increased risk of testicular and prostate cancer as well as non-Hodgkin’s

lymphoma (1). Pulmonary exposure to particles causes inlammation,

which is considered to be an important mechanism for both lung cancer

and systemic effects. Acute and chronic respiratory inlammatory effects

have been described in ireighters and noted by IARC as a potential

mechanism of carcinogenesis (1). Nevertheless, the IARC evaluation did

not ind evidence for increased risk of lung cancer among ireighters

(1) and recently a pooled analysis of case-control studies encompassing

approximately 15,000 cases and 17,500 controls showed no associa-

tion between ireighting and lung cancer risk with or without adjust-

ment for smoking (2). The IARC expert committee noted an insuficient

number of studies to evaluate genotoxic effects in ireighters.

Polycyclic aromatic hydrocarbons (PAHs) are an important

group of genotoxic compounds that can form DNA adducts (3).

Exposure to PAHs during ireighting can occur both by skin expo-

sure to soot and by inhalation of combustion particles (1). The expo-

sure to PAHs can be assessed by measurement of compounds such as

pyrene and benzo[a]pyrene (BaP) on the skin or the urinary excre-

tion of PAH metabolites such as 1-hydroxypyrene (1-OHP). The lat-

ter is the excreted metabolite of pyrene and is commonly used as a

biomarker for complex PAH exposure (4). It has been shown that

ireighting activity was associated with dermal exposure to PAHs

and increased urinary excretion of 1-OHP (5), indicating systemic

exposure to PAHs by dermal and possibly also inhalation routes.

There is a large body of literature on the association between

inhalation of particulate matter (PM) and DNA damage in terms of

DNA adducts, oxidatively damaged DNA and clastogenic effects in

peripheral blood mononuclear cells (PBMCs) and leucocytes from

humans (6). Such effects of air pollution particles on oxidation

damage to DNA in PBMCs are typically observed within hours of

controlled inhalation exposure (7). Likewise, particle-induced pul-

monary inlammation has been shown to induce systemic inlamma-

tion and acute phase response (8,9).

The aim of the present study was to investigate the effect of

ireighting activities on lung function, systemic inlammation and

DNA damage in circulating PBMCs. It is possible that pulmonary

inlammation spills over to the circulation and causes low-grade sys-

temic inlammation with elevated levels of C-reactive protein (CRP),

serum amyloid A protein (SAA), interleukin-6 and -8 (IL-6 and

IL-8), intracellular cell adhesion molecule-1 (ICAM-1) and vascular

cell adhesion molecule-1 (VCAM-1) (8). We performed an exposure

study of recruited healthy, young non-smoking volunteers from a

rescue education course for ireighting. This 3-day course involved

various smoke-diving exercises in a burn house and stays in lasho-

ver containers. The latter is a training unit that is used to mimic a

lashover condition of ires where objects in the room are heated to

their ignition point by thermal radiation. Flashover is a potential

life-threatning condition for ireighters, even when wearing full per-

sonal protective equipment (PPE), and they are trained to recognise

the signs of a possible developing lashover ire. The fuels for ires

in the irehouse were standard European Union wood pallets with

or without the addition of electrical cords and mattresses to mimic

a ire situation in a municipal setting. The study is part of a larger

investigation on occupational exposure to combustion-derived com-

pounds, cardiovascular risk factors and genotoxicity in ireighters.

Methods

Study subjects and study design

The study design, study population, airborne PM concentrations

and cardiovascular risk factors have previously been described (10).

Briely, we recruited conscripts from a rescue specialist educational

course, a 9-month education programme under the Danish Emergency

Management Agency. All subjects had the same housing and supply

of foods as they were accommodated in the same training camp dur-

ing the 9-month education programme. The subjects were healthy,

self-reported non-smokers, not pregnant and with no history of drug

or alcohol abuse. We used non-smokers in order to have maximum

exposure contrast with respect to PAH because smokers have higher

urinary levels of 1-OHP (4). Fifty-four subjects were enrolled from

four different and consecutive training classes (campaigns) that took

place in summer, autumn and winter of 2015 and spring of 2016:

campaign 1 (9 conscripts), campaign 2 (11 conscripts), campaign 3

(17 conscripts) and campaign 4 (17 conscripts) (Table

1).

One subject

from campaign 1 dropped out of the training course, and hence was

excluded from the study. The age of the 53 subjects varied from 18 to

26 years. Thirty-nine subjects had a body mass index (BMI) between

18.5 and 24.9 kg/m

and 14 had BMIs between 25.0 and 29.5 kg/m

The design was a cross-over study, where the subjects served

as their own controls, in three exposure scenarios separated by

2-week periods with the ireighting-related exposure in the middle.

The subjects extinguished ires from either combustion of standard

European Union wood pallets in the absence (campaign 1 and 2) or

presence (campaign 3 and 4) of foam mattresses and electrical cords.

Fireighting exercises were carried out during the 3 days using PPE,

including ireighting suits, ire helmet, lash hood, gloves, boots and

self-contained breathing apparatus. The self-contained air supply was

taken off outside the burn house during instruction, feedback and

breaks in zones that were considered smoke-free by the instructors

of the training course. The assessment of the personal exposure to

ultraine particles for two conscripts indicated that PM inhalation

exposure occurred mainly in situations where the subjects were in the

vicinity of the ires, although they were not actively extinguishing ires.

The PPE including the self-contained breathing apparatus

effectively prevented particle exposure during smoke-diving as

documented by measurement of the personal particle exposure in

the inhalation zone of two conscripts during smoke-diving (10).

However, dermal exposure occurred as a consequence of deposition

of soot on the skin. The present study used skin concentrations of

PAHs and urinary 1-OHP excretion as personal exposure markers.

The 1-OHP measurements from 43 out of the 53 subjects in this

study have been published previously (10).

The Danish Committee on Health Research Ethics of the Capital

Region (H-15003862) approved the study, and the study subjects

signed a written informed consent before entering the study.

PAH exposure and DNA damage after ireighting activity

Table 1.

The characteristics of the subjects in the control measurements

Characteristics

Age (years)

Height (cm)

Weight (kg)

BMI (kg/m

)

Allergies (n)

1-OHP (µmol/mol creatinine)

Pyrene (µg/m

)

ƩPAH

(µg/m

)

FEV1 (l)

FVC (l)

FEV1/FVC

DNA strand breaks (lesions/10

bp)

Fpg-sensitive sites (lesions/10

bp)

ICAM-1 (ng/ml)

VCAM-1(ng/ml)

CRP (mg/l)

SAA (mg/l)

Females (n = 12)

21.9 (2.4)

172.2 (3.1)

67.3 (9.6)

22.7 (2.9)

0.4 (0.3)

26.2 (13.3)

176.1 (146.7)

3.6 (0.5)

4.2 (0.6)

0.9 (0.1)

0.2 (0.1)

0.3 (0.1)

29.5 (7.9)

126.2 (25.8)

1.4 (1.3)

16.1 (12.5)

Males (n = 41)

21.2 (1.6)

181.4 (6.5)

77.5 (11.3)

23.5 (2.6)

0.4 (0.3)

27.8 (12.9)

143.5 (81.1)

4.7 (0.6)

5.6 (0.8)

0.8 (0.1)

0.2 (0.1)

0.3 (0.1)

30.0 (6.3)

139.3 (33.8)

0.9 (0.9)

18.0 (19.1)

All (n = 53)

21.4 (1.8)

179.3 (7.0)

75.2 (11.7)

23.3 (2.7)

0.4 (0.3)

27.5 (12.9)

150.8 (99.0)

4.4 (0.7)

5.3 (0.9)

0.8 (0.1)

0.2 (0.1)

0.3 (0.1)

29.9 (6.6)

136.3 (32.4)

1.0 (1.0)

17.6 (17.8)

Values are number or mean and standard deviation.

Values are mean of the means from both control measurements (before and after).

Three males did not deliver urine in any of the control measurements (n = 38).

BMI, body mass index; 1-OHP, 1-hydroxypyrene;

ƩPAH,

sum of 16 polycyclic aromatic hydrocarbons; FEV1, forced expiratory volume after 1 s; FVC, forced

vital capacity; ICAM-1, intercellular cell adhesion molecule-1; VCAM-1, vascular cell adhesion molecule; CRP, C-reactive protein; SAA, serum amyloid A protein.

Sample collection

Biological samples were collected on three occasions, namely 14 days

before the smoke-diving course, immediately after the 3-day course

exercises and 14 days subsequent to the end of the training session.

Dermal wiping of the neck was carried out to assess the PAH expo-

sure on the skin. We performed a pilot study to test different skin loca-

tions for optimal collection of PAHs in the main study (described in

detail in Supplementary data). In the main study, we used the neck as

area for the dermal wipes because the pilot study indicated that high

levels of PAH were wiped off this area when compared with the palms,

wrists and forehead. This observation was in line with earlier results

showing PAH concentrations in the order as neck > face > hand ≈ arm

(11). A skin area of approximately 3 cm × 6 cm on the back of the

neck was wiped with a ‘Alkoholswab’ (70% isopropanol/water, Mediq

Danmark A/S). The skin was wiped twice with the same wipe, irst with

one side of the wipe and then the other side. The operator wore nitrile

gloves (TouchNTuff, 92–600, Ansell), which were changed for each

wipe. The wipes were placed in 15-ml screw cap glass vials with foil-

lined lid (Wheaton), which were placed in the dark and transported

to the laboratory on the same day. The samples were stored at −18°C

until extraction and analysis. In these experiments, a skin area (back

of the neck) of nominally 18 cm

was used in all calculations. During

the campaigns, extracts of two blank wipes in screw cap glasses were

analysed for each series of wipe samples. None of the individual PAHs

were above the limit of detection (LOD) in any of the blanks.

For the enzyme-linked immunosorbant assay (ELISA) analysis of

SAA, CRP, ICAM-1, VCAM-1, IL-6 and IL-8, plasma was prepared by

10 min centrifugation at 4000 rpm (1780 ×

of blood collected into

ethylenediaminetetraacetic acid (EDTA)-coated tubes. The plasma

samples were prepared in a room adjacent to the ire house, kept on

ice and were transported to the laboratory on ice on the same day. At

arrival, the plasma samples were stored at −80°C until ELISA analysis.

PBMCs were isolated using Vacutainer Cell Preparation Tubes

(Vacutainer® CPT Becton Dickinson A/S, Brøndby, Denmark).

PBMCs were separated by 20 min centrifugation at 1650 ×

The PBMCs were diluted in 3-ml ice-cold medium (Rosswell Park

Memorial Institute [RPMI] medium with 10% foetal bovine serum

and 1% Pen/Strep) and kept on ice. The samples were transported

to the laboratory on ice on the same day. At arrival, the PBMCs

were separated by 15 min centrifugation at 300 ×

at 5°C and re-

suspended in 3-ml RPMI medium with 10% foetal bovine serum and

1% Pen/Strep. The same centrifugation procedure was repeated and

the PBMCs were re-suspended in freezing medium (RPMI with 50%

foetal bovine serum and 10% DMSO). The PBMCs were stored at

−80°C until analysis of DNA damage by the comet assay.

For the determination of 1-OHP, urine samples were collected in

the morning. The urine collected 2 weeks before and after exposure

was used to assess the control background levels. For the exposure

scenario, the urine was collected on the day after the 3-day ire extin-

guishing exercises. The urine samples were kept in cooling boxes

until arrival at the laboratory and at −20°C until analysis. Urinary

creatinine was used to standardise the result.

1-OHP analysis

Reverse-phase high-performance liquid chromatography (HPLC)

was used for the quantitative measurement of 1-OHP in urine using

a previously published method (12). We standardised for diuresis

with the concentration of creatinine as used in other studies (13).

We analysed a low (9.23 nmol/l) and a high (29.14 nmol/l) reference

sample together with the samples to assess equivalence between dif-

ferent runs.

Determination of PAH from skin wipes by GC-MS

analysis

The extraction of PAH was carried out by covering the wipes with

6-ml cyclohexane in 10-ml glass vials and sonication for 30 min in

an ultra-sonic bath (Branson 5200, output power 120 W at extrac-

tion of 25 samples at the same time). One millilitre of the super-

natant was transferred into a small glass vial and 30

µl

of internal

standard solution (10 ng/µl) added. The extracts were stored at

−18°C until analysis. The extracts were analysed by gas chromatog-

raphy and mass spectrometry (GC-MS) using a Bruker SCION TQ

(Bruker Daltonics, Bremen, Germany). The analysis was carried out

by injection of 1

µl

of the sample extract with an Bruker CP-8400

autosampler to a programmable temperature vapourising injector

at 280°C into the column with a He low of 1 ml/min. The column

was 30 m × 0.25 mm with 0.25-µm ilm thickness (VF-5MS, Agilent

Technologies, USA). The GC oven programme was set at 70°C for 4

min, ramp 1, 10°C min

−1

to 30°C, ramp 2, 45°C min

−1

to 325°C hold

for 7 min, and the transfer line and the source were kept at 275°C.

The MS was operated in a scan mode (mass range

m/z

50–500) in

electron ionization (EI) and in selected ion monitoring (SIM) for

each PAH.

ƩPAH

is the sum of the concentrations of 16 PAH (ana-

lytical details are shown in Supplementary Tables S1 and S2).

M. H. G. Andersen et al.

slides had been prepared in duplicates on two different assay runs

(including different electrophoresis). The nuclei were scored by visual

classiication based on a ive-class scoring system (arbitrary score

range: 0–400) as previously described (17). We had assay control sam-

ples in each experiments (corresponding to each electrophoresis) that

consisted of KBrO

-exposed THP-1 cells (5 mM for 1 h at 37°C). The

number of Fpg-sensitive sites was obtained as the difference in scores

of parallel slides incubated with and without Fpg. These scores were

transformed to lesions per 10

bp by means of a calibration curve

based on induction of DNA strand breaks by ionising radiation,

which has a known yield. We used an investigator-speciic conversion

factor of 0.01989 lesions/10

bp per score in 0–100 range (16), based

on the assumption that an average molecular weight of a DNA bp is

650 Dalton and 1 Gy yields 0.29 breaks per 10

Dalton DNA (18).

Lung function measurement

The lung function was assessed using a Vitalograph S spirometer

(Buckingham, UK) measuring forced vital capacity (FVC) and forced

expiratory volume after 1 s (FEV1). A single spirometer was used

in all tests to ensure standardisation of the measurements, with the

equipment calibrated before each testing session. All measurements

were performed with the conscripts standing and using a nose clip.

Up to three measurements were taken to obtain reproducible trac-

ings with the two highest FVC, FEV1 and FEV1/FVC.

Statistical analysis

We used R statistical language and the package

lme4

(19) to perform a

linear-mixed effects analysis of the relationship between the biomarkers

and exposure. As a ixed effect, we used factorial variable of exposure

(before/exposure/after). The exposure term in the statistical analysis was

either exposure period (i.e. one exposure and two non-exposure periods

within each campaign) or type of ire (i.e. wood or wood with mat-

tresses and electrical cords). As random effects, we used by-subject inter-

cepts. We have not adjusted for inter-individual confounders because

the subjects were their own controls.

P-values

were obtained with the

function

glht

from

multcomp

(20). The results on urinary excretion of

1-OHP were cubic root transformed and DNA strand breaks, CRP and

SAA were logaritmically transformed because of a skewed distribution

of residuals for the normal data. A few outlier values were eliminated

from the dataset (1-OHP: 3.48-µmol/mol creatinine, control before;

VCAM-1: 13210.3 ng/ml, exposure; CRP: 11.0 and 35.5 mg/l, control

before and exposure, respectively; SAA: 572.4 and 596.2 mg/l, control

before and exposure, respectively). The percent changes were obtained

by dividing the estimate change with the intercept value from the mixed

model graph line and multiplying with 100, except for CRP, SAA and

DNA strand breaks where the percent changes were obtained directly

from the effect estimated using the expression: (exp

estimate

− 1)×100.

Welch

t-test

was used to compare the difference in means of effect

change between exposed and unexposed scenarios for the different

types of ire. A few subjects were eliminated from the dataset for the

t-test

analysis due to missing values in the control or exposure meas-

urements (1 subject for DNA strand breaks and Fpg-sensitive sites, 3

subjects for Pyrene and

ΣPAH

and 10 subjects for 1-OHP).

Inlammation markers analysis

The concentrations of sICAM-1 (Cat. No. 560269) and sVCAM-1

(Cat. No: 560427) were assessed in plasma with BD cytometric bead

array system, utilising Accuri CFlow®Plus software (BD Bioscience)

according to methods described previously (14). Plasma levels of SAA

and CRP were determined by ELISA kits from Invitrogen (CA, USA)

and IBL International GMBH (Hamburg, Germany), respectively, as

described previously (15). Plasma levels of IL-6 and IL-8 were deter-

mined by ELISA kit from BD Biosciences (Cat. No. 555244 and Cat.

No. 555220) according to the manufacturer’s speciications.

DNA damage analysis

The levels of DNA strand breaks and formamidopyrimidine DNA

glycosylase(Fpg)-sensitive sites were detected by the comet assay as

described previously (16). Briely, PBMCs were embedded in 0.75%

low-melting point agarose (Sigma-Aldrich A/S, Brøndby, Denmark) on

GelBond ilms (Lonza Copenhagen Aps, Vallenbæk Strand, Denmark)

and lysed (1% Triton X-100, 2.5-M NaCl, 100-mM Na

EDTA,

10-mM Tris, pH = 10) overnight at 4°C. The Gelbond ilms were

washed 3 times for 5 min in endonuclease buffer (40-mM HEPES,

0.1-M KCl, 0.5-mM Na

EDTA, 200-µg/ml bovine serum albumin, pH

= 8). Subsequently, the nuclei were incubated for 45 min with Fpg

at 37°C. The Fpg enzyme was a gift from Professor Andrew Collins

(University of Oslo, Norway). Thereafter, the Gelbond ilms were

immersed in an alkaline solution (300-mM NaOH, 1-mM Na

EDTA,

pH > 13) for 40 min and subsequently subjected to electrophoresis for

20 min at 0.83 V/cm (cathode to anode) and 300 mA. After electro-

phoresis, the Gelbonds were washed 3 times for 5 min in Tris buffer

(0.4-M Tris-HCl, pH = 7.5), rinsed with milliQ water and dried in

96% ethanol. The nuclei were scored using an Olympus luorescence

microscope at 40× magniication with visual inspection after staining

with YOYO-1 in phosphate buffered saline (PBS) (Molecular Probes,

Eugene, OR, USA). All samples from one subject were coded and

analysed simultaneously in order to minimise inter-assay variation.

In addition, each batch of comet assay experiments included samples

from the four campaigns in order to control for inter-day experimen-

tal variation. We analysed 100 comets per slide and there were 4 slides

for each sample, corresponding to a total number of 400 nuclei. The

Results

PAH exposure assessment

Skin exposure to soot occurred both by handling contaminated

equipment and during ireighting. In a pilot study, PAH content of

wipes of different skin areas was assessed immediately before (i.e.

after putting on the PPE, but before smoke diving) and after smoke

diving (Figure

1).

The PAH content on the skin wipes from the neck

was high both before and after smoke diving, whereas there were

substantial differences in the

ΣPAH

concentrations from different

parts of the body. The high PAH content on skin before smoke div-

ing may be due to transfer of soot from contaminated equipment

to the skin while taking on the PPE before smoke diving. Soot can

be transmitted to the head and neck from the palms by touching

by hands, perhaps to wipe out sweat or reaction to tickling. Our

observation from the pilot study was that all conscripts had patches

PAH exposure and DNA damage after ireighting activity

of soot on their head and neck both before and after smoke diving.

For the main study, we selected skin wipes from the back of the neck.

During the main study, ireighting increased skin exposure to PAH

by 79.5% (95% CI: 52.5, 106.6) and pyrene by 68.1% (95% CI:

52.5, 83.8), respectively, compared with control situations (Figure

Table 2,

supplementary table S3 and supplementary igure S1).

The internal dose of PAH was assessed using 1-OHP excretion in

urine. The levels of 1-OHP were not normally distributed. The mean

(SD) and median (25%–75% quartiles) were 0.41 (0.40)-µmol/mol

creatinine and 0.27 (0.19–0.43)-µmol/mol creatinine, respectively,

in the ‘before exposure’ samples. The 1-OHP excretion was 0.68

(0.53)-µmol/mol creatinine and 0.51 (0.28–0.98)-µmol/mol creati-

nine, respectively, after the smoke exposure after the smoke-diving

exercises. In the ‘after exposure’ samples, the values were 0.48

(0.23)-µmol/mol creatinine and 0.41 (0.23–0.60)-µmol/mol creati-

nine, respectively. Fireighting increased 1-OHP concentration in

urine by 70.4% (95% CI: 33.4, 113.5) compared with the mean of

the two control periods (Figure 3,

Table 2

and supplementary igure

S2). Urinary 1-OHP concentrations were positively associated with

ƩPAH

and pyrene content on the skin (Table 3).

A stratiication of the exposure markers according to the type

of fuel for the ires is outlined in

Table 4.

The ireighting exer-

cises using only wood pallets were associated with higher expo-

sure in terms of pyrene (P < 0.001) and

ƩPAH

(P < 0.001) on the

skin as well as 1-OHP (P < 0.001) in urine when compared

with the ire that was supplemented with electrical cords and

mattresses.

Systemic inlammation

SAA, CRP, ICAM-1 and VCAM-1 levels in plasma were unaffected

by exposure (Figure 4,

Table 2)

and type of ire (Table 4). The levels

of IL-6 and IL-8 were below the LOD (only 11 and 7 observations

were detectable, respectively, of a total of 158).

Lung function

The conscripts had unaltered lung function after ireighting when

compared with the control periods (Table 2). There were statistically

signiicant differences between the types of ire and lung function

tests; however, these biological responses are regarded as ambiguous

as no discerning pattern is recognised (Table 4).

Figure 1.

Mean levels of

ƩPAH

(16 compounds) on the skin before and after a single episode of ireighting measured by skin wipes. Error bars are standard error

of mean (n = 4–6). The subjects were wearing the personal protection equipment before the irst skin wiping (pilot study). The same skin area was wiped before

and after ireighting. The recovery of the wiping is unknown but non-exhaustive, thus values may be considered minimum levels.

Figure 2.

Levels of pyrene (a) and

ƩPAH

(b) on skin on the back of the neck. Each symbol represents one measurement. The black solid lines have been obtained

from mixed effects linear regression.

Table 2. Percent changes (95% conidence interval) in outcome levels estimated by mixed effects model

Exposure vs before

Pyrene

ƩPAH

1-OHP

ICAM-1

VCAM-1

CRP

SAA

FEV1

FVC

FEV1/FVC

DNA strand

breaks

Fpg-sensitive sites

M. H. G. Andersen et al.

Exposure vs after

117.4 (96.1, 138.7)***

84.6 (59.8, 109.5)***

49.7 (14.7, 91.1)**

5.1 (−1.6, 11.8)

−1.1 (−9.5, 7.3)

56.1 (11.3, 119.0)**

16.4 (−11.4, 52.9)

0.5 (−1.4, 2.4)

−0.4 (−2.0, 1.1)

0.9 (−0.4, 2.1)

21.2 (5.4, 39.4)**

5.4 (−3.0, 13.7)

After vs before

−37.1 (−50.4, −23.9)***

−5.5 (−28.7, 17.7)

50.4 (11.6, 97.3)**

−3.1 (−9.6, 3.5)

1.5 (−7.0, 10.1)

−14.3 (39.0, 20.6)

−4.0 (−27.1, 26.3)

−1.5 (−3.4, 0.5)

0.4 (−1.2, 1.2)

−1.7 (−2.9, −0.4)**

−8.1 (−20.1, 5.7)

4.6 (−4.1, 13.3)

Exposure vs unexposed

68.1 (52.5, 83.8)***

79.5 (52.5, 106.6)***

70.4 (33.4, 113.5)***

3.5 (−2.0, 9.0)

−0.4 (−6.5, 5.7)

24.4 (−7.7, 67.7)

2.5 (−19.9, 31.2)

−0.2 (−1.9, 1.5)

−0.2 (−1.7, 1.2)

0.1 (−0.8, 0.9)

15.6 (−0.6, 34.4)

8.0 (0.02, 15.9)*

36.7 (23.2, 50.2)***

74.6 (50.8, 98.2)***

124.8 (73.1, 185.7)***

1.9 (−4.6, 8.4)

0.4 (−8.1, 9.0)

33.8 (−4.8, 88.2)

11.7 (−15.1, 47.0)

−0.9 (−2.0, 1.0)

−0.03 (−1.6, 1.6)

−0.8 (−2.1, 0.4)

11.4 (−3.2, 28.2)

10.2 (1.4, 18.9)*

Unexposed corresponds to the mean between ‘Before’ and ‘After’ for each subject.

Three individuals with missing data.

ƩPAH,

sum of 16 polycyclic aromatic hydrocarbons; 1-OHP, urinary excretion of 1-hydroxypyrene adjusted for excreted creatinine concentration; ICAM-1,

intercellular adhesion molecule-1; VCAM-1, vascular cell adhesion molecule-1; CRP, C-reactive protein; SAA, serum amyloid A protein; FEV1, forced expiratory

volume after 1 s; FVC, forced vital capacity.

Results are percent change from the mixed effect model in

Figs. 2–5.

The data are based on 53 individuals. *,**,*** Signiicantly different (P < 0.05,

P < 0.01

and

P < 0.001

respectively).

(P < 0.001;

Table 4).

A similar pattern was observed for Fpg-

sensitive sites, although it did not reach statistical signiicance (P

= 0.09;

Table 4).

Discussion

This study showed that participation in a 3-day ireighting training

with smoke-diving exercises was associated with elevated levels of

ƩPAH

on the skin, 1-OHP in urine and Fpg-sensitive sites in PBMCs.

No effect of exposure on DNA strand breaks in PBMCs and lung

function (FEV1, FVC or FEV1/FVC) or markers of systemic inlam-

mation was observed.

The subjects were exposed to soot on the skin. Potential inhala-

tion exposure to PM occurred in situations when the subjects were

not using the self-contained breathing apparatus. The inhalation

exposure typically occurred at locations where the subjects were

gathered for instructions or waiting for their turn of smoke diving.

The skin exposure to

ƩPAH

and pyrene was considerably increased

after the smoke-diving exercises on wood ires. Previously, it has

been shown that ireighting exercises with wood ires are associ-

ated with markedly increased whole-body PAH exposure (5). We

used 1-OHP as biomarker of the internal PAH dose since 1-OHP is

the metabolite of pyrene (4). Urinary concentrations of 1-OHP were

increased after ireighting training with wood and mixed fuel ires,

indicating systemic PAH exposure. The 1-OHP concentrations in

control measurements were similar to previous reported background

levels (<0.5-µmol/mol creatinine) (4), although higher than previous

reports of values in the range of 0.1- to 0.2-µmol/mol creatinine in

Danish adults (21). This was not caused by drift in the analytical

method of the 1-OHP measurements as determined by Westgard

control charts of the reference samples in the method (22). However,

during the control periods, the subjects were educated in ireighting

by demonstration of techniques and equipment that may result in

low-level exposures to PAH. Nevertheless, it should be emphasised

that the subjects were their own controls in the present study and the

effect of exposure is not affected by the baseline level of 1-OHP. The

urinary excretion of 1-OHP increased, especially in the subjects who

participated in the ireighting exercises on wood pallets (0.6-µmol/

Figure 3.

Concentration of 1-OHP in urine after ireighting activity

(exposure) and control periods (before and after). Each symbol represents

one measurement. The black solid line has been obtained from mixed-effects

linear regression. The dashed line represents the ‘no observed genotoxicity

effect level for occupational exposure to PAH’ (4).

Levels of DNA damage in PBMCs

Fireighting was not statistically signiicantly associated with

increased levels of DNA strand breaks, whereas ireighting activ-

ity increased the levels of Fpg-sensitive sites by 8% (0.02, 15.9;

Figure 5, Table 2

and supplementary igures S3 and S4). DNA

strand break levels correlated positively with

ƩPAH

levels on the

skin (P < 0.001) and with 1-OHP levels in urine (P < 0.001;

Table

3).

The levels of Fpg-sensitive sites were positively correlated with

PAH levels on skin (P < 0.01) but not with 1-OHP levels in urine.

The induction of DNA strand breaks was stronger after the ire-

ighting exercise with wool pallet fuel as compared to mixed fuels

PAH exposure and DNA damage after ireighting activity

Table 3.

The associations between biomarkers of external PAH exposure, internal PAH dose (i.e. 1-OHP) and genotoxicity in PBMCs

assessed at three different time-points for 53 volunteers.

Dependent variable

1-OHP (urine)

DNA strand breaks (PBMCs)

Fpg-sensitive sites (PBMCs)

Pyrene (skin)

ƩPAH

(skin)

ƩPAH

(skin)

Predictor

Pyrene (skin)

ƩPAH

(skin)

1-OHP (urine)

ƩPAH

(skin)

Pyrene (skin)

1- OHP (urine)

ƩPAH

(skin)

Pyrene (skin)

1-OHP (urine)

Pyrene (skin)

Estimate ± SE

0.00295 ± 0.00116*

0.00061 ± 0.000148***

0.6492 ± 0.1679***

0.0015 ± 0.0003***

0.0033712 ± 0.00059***

−0.3656 ± 3.4941

0.02595 ± 0.01004**

0.0001584 ± 0.0003691

35.331 ± 6.685***

269.866 ± 51.441***

5.3257 ± 0.3961***

1-OHP, 1-hydroxypyrene;

ƩPAH,

sum of 16 polycyclic aromatic hydrocarbons; PBMCs, peripheral mononuclear blood cells. The estimates are based on

non-transformed (pyrene,

ƩPAH

and Fpg-sensitive sites), cubic root transformed (1-OHP) and logarithmically transformed (DNA strand breaks) data, using

linear-mixed effects analysis. The estimates cannot be compared across rows because different transformations of the biomarkers have been used to obtain normal

distribution of residuals. *,**,*** Signiicantly different (P < 0.05,

P < 0.01

and

P < 0.001,

respectively).

Table 4.

Average effect change between exposure and unexposed situations for two different types of ires: wood pallets and wood pallets

with mattresses and electrical cords (mixed fuel)

Net effect of exposure

Wood pallet fuel

Pyrene (µg/m

)

ƩPAH

(µg/m

)

1-OHP (µmol/mol creatinine)

ICAM-1 (ng/ml)

VCAM-1 (ng/ml)

CRP (mg/l)

SAA (mg/l)

FEV1 (l)

FVC (l)

FEV1/FVC

DNA strand breaks (lesions/10

bp)

Fpg-sensitive sites (lesions/106 bp)

Mixed fuel

12.3 (12.4)

35.1 (35.2)

0.1 (0.5)

1.59 (6.44)

2.21 (31.08)

0.25 (1.6)

4.04 (24.9)

−0.12 (0.25)

−0.13 (0.21)

−0.001 (0.03)

−0.021 (0.05)

0.008 (0.08)

Difference between wood pallet and mixed fuel

18.1 [9.6; 26.6]***

257.4 [179.6; 335.2]***

0.5 [0.1; 0.8]***

1.54 [−4.91;1.82]

7.55 [−25.45; 10.34]

0.14 [−0.92; 0.65]

1.81 [−11.47; 15.09]

0.30 [0.17; 0.43]***

0.34 [0.20; 0.48]***

0.004 [−0.01; 0.02]

0.23 [0.12; 0.35]***

0.05 [−0.008, 0.107]

30.4 (14.3)

292.5 (144.7)

0.6 (0.5)

0.05 (5.44)

−5.35 (30.47)

0.11 (1.2)

5.85 (21.3)

0.19 (0.21)

0.21 (0.25)

0.003 (0.02)

0.213 (0.23)

0.058 (0.11)

Average difference (and SD) between exposure and the mean of control measurements (i.e. periods before and after the exposure period).

Welch

t-test

(mean difference and 95% CI).

ƩPAH,

sum of 16 polycyclic aromatic hydrocarbons; 1-OHP, urinary excretion of 1-hydroxypyrene adjusted for excreted creatinine concentration; ICAM-1,

intercellular adhesion molecule-1; VCAM-1, vascular cell adhesion molecule-1; CRP, C-reactive protein; SAA, serum amyloid A protein; FEV1, forced expiratory

volume after 1 s; FVC, forced vital capacity.

*,**,*** Signiicantly different (P < 0.05,

P < 0.01

and

P < 0.001

respectively).

mol creatinine), although the levels did not exceed the level that has

been deined as a ‘no observed genotoxicity effect level for occu-

pational exposure’ (1.0-µmol/mol creatinine) (4). The amount of

wood for the ires was similar in all campaigns. Thus, the difference

in PAH exposure between the campaigns might be due to seasonal

changes in temperature and wind. The seasonal changes may also

have had an impact on the behaviour of the subjects. The subjects

might have been waiting in different locations in the four campaigns.

For instance, it is possible that the subjects were more outside during

the summer (e.g. in vicinity to the ire trucks that engines running)

and stayed inside the irehouse during the winter.

The ireighting training is a multifaceted situation with elevated

PAH exposure, heat and physical exercise. Each of these conditions

or their combinational effects may be genotoxic. Nevertheless, PM,

PAHs, certain volatile organic and semi-quinone compounds are

well-described genotoxic carcinogens (23). PAHs are able to pen-

etrate the skin, and it has been shown that a single application of

pharmacological coal tar on the skin of hairless mice induces PAH–

DNA adduct formation in skin and liver as well as increased levels

of DNA strand breaks and increased mutation frequency in epider-

mal cells (24). Previous observations did not indicate higher levels of

PAH–DNA adducts in leucocytes from municipal ireighters when

compared with matched controls (25). Likewise, there was no asso-

ciation between levels of PAH–DNA adducts in leucocytes and ire-

ighting activity in wildland ireighters in California, USA (26). A

study on volunteer ireighters demonstrated no difference in levels

of PAH–DNA adducts in lymphocytes between pre- and post-shift

samples, although the subjects stated that they had been exposed to

plumes of smoke from oil well ires (27).

Here, we assessed both DNA strand breaks and the Fpg-sensitive

sites in PBMCs. Previous studies on trafic-related air pollution

exposures or wood smoke are summarised in

Table 5.

Exposure

to air with mixed pollution from busy streets appears to increase

the levels of, in particular, Fpg-sensitive sites whereas diesel exhaust

M. H. G. Andersen et al.

Figure 4.

Concentrations of ICAM-1 (a), VCAM-1 (b), CRP (c) and SAA (d) in plasma after ireighting activity (exposure) and control periods (before and after).

Each symbol represents one measurement. The black solid lines have been obtained from mixed-effects linear regression.

Figure 5.

Levels of DNA strand breaks (a) and Fpg-sensitive sites (b) in peripheral blood mononuclear cells after ireighting activity (exposure) and control

periods (before and after). Each symbol represents one measurement. The black solid lines have been obtained from mixed-effects linear regression.

PAH exposure and DNA damage after ireighting activity

Table 5.

Percent difference in DNA strand breaks and Fpg-sensitive sites in PBMCs between particle-rich (E) and control (C) exposures

Description

Subjects in Cotonou, Rep

Benin (cross-sectional study)

Exposure contrast

>250,000 vs <10,000

UFP/cm

(during midday)

DNA strand breaks (E/C)

33%(18,49)

0.16 ± 0.10

0.12 ± 0.05

(P < 0.05)

0% (−56, 56)

0.06 ± 0.08 (IQR)

0.06 ± 0.10 (IQR)

(NS)

50% (20, 80)

0.24 ± 0.21 (IQR)

0.16 ± 0.16 (IQR)

(P < 0.05)

−51%

(−65, 31)

0.04 ± 0.03

0.08 ± 0.08

(NS)

50% (−2.0, 102)

0.06 ± 0.08

0.04 ± 0.05

(NS)

3% (−17, 15)

(NS)

4% (−8, 15)

0.59 ± 0.39

0.57 ± 0.31

(NS)

13% (−44, 132)

0.06 ± 0.01

0.05 ± 0.01

(NS)

15.6% (−0.6, 34.4)

0.30 ± 0.20

0.23 ± 0.10

(NS)

Fpg-sensitive sites (E/C)

145% (136, 155)

0.27 ± 0.05

0.11 ± 0.03

(P < 0.001)

300% (188, 412)

0.08 ± 0.08 (IQR)

0.02 ± 0.04 (IQR)

(P < 0.001)

39% (18, 61)

0.53 ± 0.28 (IQR)

0.38 ± 0.33 (IQR)

P < 0.05

−15%

(−31, 4.9)

0.23 ± 0.13

0.25 ± 0.12

(NS)

−17%

(−37, 4.0)

0.40 ± 0.32

0.48 ± 0.30

(NS)

−13%

(−69, 4.6)

(NS)

9% (−8.1, 27)

0.35 ± 0.31

0.32 ± 0.31

(NS)

−5%

(−34, 25)

0.11 ± 0.01

0.13 ± 0.02

(NS)

8.0% (0.02, 15.9)

0.34 ± 0.11

0.32 ± 0.08

(P < 0.05)

Ref

(42)

Cycling in Copenhagen

Denmark

32,400 vs 13,400 UFP/cm

(7)

Controlled exposure to

street air

10,067 vs 235 UFP/cm

(43)

Controlled wood smoke

exposure (3 h)

>95,000 vs <10,000

UFP/cm

(29)

Controlled wood smoke

exposure (3 h)

71,036 vs 222 UFP/cm

(30)

Controlled exposure to diesel

exhaust (3 h)

Controlled exposure to street

air (5 h)

(276 vs 2 µg/cm

of PM

)

23,000 vs 1800 UFP/cm

(44)

(45)

One-week stay in a recon-

structed Viking age house

(with indoor ire)

This study

657 µg/m

of PM

2.5

(14)

0.7 vs 0.4 µm/mol creatinine

og 1-OHP

Not applicable

The effect is reported as percent change (95% conidence) intervals per exposure unit in bold text. The exposure units differ between studies, indicating that it is

not a stoichiometric measure. The second and third line represents the particle-rich exposure and reference, respectively. The values are lesions/10

bp. The fourth

line reports the statistical outcome of the study as either a

P-value

or not statistically signiicant (NS).

UFP, ultraine particles; IQR, inter-quartile range; 1-OHP, 1-hydroxypyrene.

alone or wood burning for heating in a stove or a ire place showed

no apparent effects on these biomarkers. The levels of DNA strand

breaks (15.6%, 95% CI: −0.60%; 34.4%) and Fpg-sensitive sites

(8.0%, 95% CI: 0.02%; 15.9%) are not overtly different from levels

reported in other studies using the same protocol for assessment of

DNA damage in PBMCs. Another study has shown increased levels

of DNA strand breaks in lymphocytes from a municipal ireight-

ing episode at a chemical plant where the ireighters had not worn

PPE (28). Studies on controlled exposure to wood smoke indicated

a lack of genotoxicity in terms of DNA strand breaks and oxida-

tively damaged DNA in PBMCs (29,30). However, a recent study

showed increased levels of DNA damage in wideland ireighters

(31). Interestingly, we found positive associations between DNA

strand breaks levels and all measures of PAH exposure (pyrene,

ƩPAH

and 1-OHP), whereas the level of Fpg-sensitive sites was

associated with dermal

ƩPAH

levels. DNA strand breaks measured

by the alkaline comet assay represent strand breaks in the DNA,

alkaline labile sites and breaks that occur as a consequence of base

and nucleotide excision repair of DNA lesions. The Fpg-modiied

comet assay detects oxidatively damaged DNA and has widely been

used in studies on air pollution exposure in biomonitoring studies

(32,33). The association between heavy PAHs such as BaP and levels

of oxidatively damaged DNA is in keeping with observations that

metabolites of BaP can generate superoxide anion radicals via redox

cycling (34). The strong associations between PAHs including pyrene

on skin, 1-OHP and DNA strand breaks in PBMCs indicate that skin

exposure to PAH contributes to the increased DNA strand break

levels. Importantly, these associations were found in the same con-

centration range of urinary 1-OHP as the acceptable exposure limit

of 1.0-µmol/mol creatinine.

There have been numerous studies on associations between expo-

sure to physical exercise and levels of DNA damage in leucocytes or

lymphocytes. Physical activity typically entails a short-term increase

in the body temperature due to the increased energy expenditure.

Our previous studies demonstrated no effect on DNA strand breaks

or Fpg-sensitive sites in PBMCs after an exhaustive exercise test

(35). However, other studies have demonstrated elevated levels of

DNA strand breaks and oxidatively damaged DNA in PBMCs or

leucocytes after long-term and exhaustive exercise such as complet-

ing a marathon race, which is associated with tissue damage and

inlammation (36). Heat exposure is a well-known inducer of heat

shock genes and DNA repair pathways in cell cultures, whereas the

mechanisms of genotoxicity, including DNA strand breaks, are not

elucidated (37). The effect of physical hyperthermia or intermittent

heat exposures on DNA damage levels in PBMCs remains to be

investigated in controlled studies. Associations between fever-asso-

ciated diseases and genotoxicity are most likely confounded by the

direct effect of the infectious agent on the genome.

We did not observe any consistent effect on lung function,

plasma levels of cytokines and acute phase proteins, although CRP

levels were statistically signiicantly increased after ireighting

training when compared with the control samples collected 2 weeks

after the ireighting training (table

2).

In a controlled study, inhala-

tion exposure to wood combustion particles increased levels of SAA

and IL6 in human volunteers (38). However, low-level exposures

did not increase markers of systemic inlammation (39). A meta-

analysis indicated little association between controlled exposure to

combustion-derived PM and systemic low-grade inlammation in

humans (40). It should be emphasised that inlammation is not a

pre-requisite for generation of DNA strand breaks and oxidatively

damaged DNA in either lung tissue or the blood compartment

(8,41).

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Conclusion

In conclusion, exposure of young, healthy non-smoking subjects to a

3-day ireighting course resulted in increased skin exposure to PAH,

including pyrene, increased urinary 1-OHP levels and increased lev-

els of Fpg-sensitive sites in PBMCs.

ƩPAH

and pyrene levels on skin

were strongly associated with 1-OHP.

ƩPAH

on skin and 1-OHP in

urine both correlated with DNA strand break levels in PBMCs.

Supplementary Material

Supplementary data is available at

Mutagenesis

online.

Funding

This work was supported by The Danish Working Environment Research

Fund (BIOBRAND, grant 34-2014-09/20140072567, Danish Centre for

Nanosafety, grant 20110092173/3 and Danish Centre for Nanosafety II).

Acknowledgments

The technical assistance from Anne Abiltrup, Ulla Tegner, Inge Christiansen,

Vivi Kofoed-Sørensen and Lisbeth Carlsen is gratefully acknowledged. We

have established a reference group which includes stakeholders from, for

example, ire brigades, trade unions and The Danish Emergency Management

Agency. We thank the reference group for involvement in the overall study

design. A special thanks to the Danish Emergency Management Agency where

the measurements took place. We are also grateful to the study participants for

the considerable time and willingness put into this study.

Conlict of interest statement: None declared.

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