Documenti di Didattica
Documenti di Professioni
Documenti di Cultura
Original article
Scand J Work Environ Health 2008;34(4):278-287
doi:10.5271/sjweh.1272
Inflammation but no DNA (deoxyribonucleic acid) damage in
mice exposed to airborne dust from a biofuel plant
by Madsen AM, Saber AT, Nordly P, Sharma AK, Wallin H, Vogel U
Affiliation: National Research Centre for the Working Environment,
Lers Parkall, 105 DK-2100 Copenhagen, Denmark. amm@nrcwe.dk
Key terms: actinomycetes; airborne dust; asthma; beta-glucan;
biofuel plant; cancer; combustion particle; deoxyribonucleic acid;
DNA; DNA damage; endotoxin; genotoxicity; inflammation; mold;
mouse; occupational exposure; respirable dust
Print ISSN: 0355-3140 Electronic ISSN: 1795-990X Copyright (c) Scandinavian Journal of Work, Environment & Health
Original article
Scand J Work Environ Health 2008;34(4):278287
Objectives Particles in ambient air are associated with such health effects as lung diseases and cancer of the
lung. Exposure to bioaerosols has been found to be associated with respiratory symptoms. The toxic properties
of exposure to combustion and bioaerosol particles from biofuel plants have not been studied in detail. This
study investigated whether exposure to dust from biofuel plants induces DNA (deoxyribonucleic acid) damage
and inflammation in exposed mice.
Methods DNA damage and inflammation were evaluated in mice exposed through the intratracheal installation
of airborne dust collected at a biofuel plant at the straw storage hall and in the boiler room. The mice were given
either a single dose of dust (18 or 54 g) or four doses of 54 g on each of four consecutive days. Control mice
were exposed to a 0.9% sodium chloride solution.
Results In the mice exposed to 454 g of dust, the lung tissue mRNA (messenger ribonucleic acid) levels
of interleukin-6 (IL-6), monocyte chemoattractant protein-1 (MCP-1), and macrophage inflammatory protein-2
(MIP-2) were increased more than 10-fold if the dust was from the boiler room and 30- to 60-fold if the dust
came from the straw storage hall. The levels of DNA strand breaks in broncheoalveolar lavage (BAL) cells from
the mice exposed to dust did not differ from those in the control samples.
Conclusions The results indicate that the instillation of dust from a biofuel plant, at doses corresponding to
2 weeks of exposure to human endotoxins, results in a strong inflammatory response without detectable DNA
damage in BAL cells. The dust from the straw storage hall induced the strongest inflammatory response and had
the highest concentration of most microbial components.
Key terms actinomycetes; asthma; cancer; combustion particles; endotoxin; genotoxicity; beta-glucan, mold;
occupational exposure; respirable dust.
Correspondence to: Dr AM Madsen, National Research Centre for the Working Environment, Lers Parkall, 105 DK-2100
Copenhagen, Denmark. [amm@nrcwe.dk]
278
Madsen et al
Quantification of microorganisms
279
Number
of
mice
First
10
5
5
5
5
Second
15
15
15
280
Exposure
Madsen et al
Results
Microbial and metal content of the dust samples
The dust sampled from the straw storage hall contained
more of most of the microbial components than the dust
from the boiler room (table 2). The lyophilization of the
dust greatly affected the cultivability of the collected
fungi, and, after the lyophilization, only a minor fraction
of the fungal colonies was obtained when compared with
the amount before the lyophilization.
The content of inorganic elements in the dust used
for the exposure of the mice is presented in table 3. For
most of the inorganic elements, the concentration was
greater in the dust from the straw storage hall than in
the dust from the boiler room. The concentrations of
the elements in the straw dust were considerably lower
than in the dust from the boiler room and the straw
storage hall.
Human exposure at the plant
The person working in the storage hall, who mainly
drove a truck and transported straw, was exposed to
the following air concentrations: 0.62 mg dust/m3,
167104fungal spores/m3, 62 104 cfu of fungi/m3,
31cfu of Aspergilus fumigatus/m3, 394 104 bacteria/m3,
1298EU/m3, and 626 and 2.1 104 cfu of thermophilic
and mesophilic actinomycetes/m3, respectively. Another
Table 2. Fungal and bacterial components and pH of the dust
samples used for the exposure of the mice. (cfu = colony-forming
units, BD = below the detection level, EU = endotoxin units)
Microbial component
Unit
Boiler
Straw
room
storage hall
(unit/mg dust) (unit/mg dust)
Total fungi
Number
Mesophilic fungi
cfu
Aspergillus fumigatus
cfu
Cladosporium herbarum
cfu
Eurotium species
cfu
beta-Glucan
ng
beta-Glucosidase
Pmol/s
Total bacteria
Number
Mesophilic bacteria
cfu
Endotoxin
EU b
Mesophilc actinomycetes
cfu
Thermophilic actinomycetes
cfu
pH
130 104
172
+ a
+ a
BD
3.1 104
0.128
58 104
1063
196
35
20 104
7.19
a
b
170 104
35
+a
+a
+a
20 104
0.129
84 104
5231
286
1.7 104
6.9 104
7.10
281
Element
Boiler room
Straw storage hall
dust (ppm) (N=1) dust (ppm) (N=1)
Calcium
Potassim
Sodium
Aluminium
Magnesium
Iron
Manganese
Phosphorus
Zinc
Nickel
Copper
Lead
Chromium
Cadmium
53061
303154
44266
6789
5892
16434
361
1890
1770
568
530
127
38
5
Straw dust
(ppm) (N=3)
Mean
120068
39166
29817
30275
8684
49147
1014
3230
3077
203
1062
455
108
6
SD
11893 1714
5114
473
246
72
1510
810
1801
209
1825
757
149
38
1198
98
60
8
6
4
10
2
6
2
10
4
Table 4. Cellular profile for the bronchoalveolar lavage fluid after the intratracheal instillation of dust from the boiler room or the straw
storage hall.
Exposure
Dose
(g)
Animals
(N)
Total cells
(104)
Mean a SD a
Macrophages
(104)
Mean a
SD a
Neutrophils
(104)
Mean a
SD a
Lymphocytes
(104)
Mean a
SD a
Eosinophils
(104)
Mean a
SD a
1 0
1 18
1 54
1 18
1 54
9
5
5
4
4
12
9.1
10
4.8
3.9
1.5
3.1
6.6
2.4
1.3
12
8.8
9.9
4.7
3.7
3.5
3.1
6.5
2.3
1.3
0.13
0.13
0.05
0.04
0.03
0.18
0.10
0.04
0.02
0.03
0.17
0.13
0.13
0.099
0.13
0.07
0.07
0.07
0.06
0.03
0.007 0.02
0
0
0.004 0.009
0
Repeated exposures
0.9% sodium chloride
Boiler room dust
Straw storage hall dust
4 0
4 54
4 54
13
14
14
11
29 b
35 b
18.4
11.1
16.4
282
5.3
6.7
6.8
3.5
3.0
2.8
4.7
18 c
21 c
14.2
11.1
10.6
0.40
2.6 d
5.0 c
0.97
1.7
3.2
0.02
1.2 c
1.9 b
0.02
2.2
2.4
Madsen et al
Control
70
60
**
50
40
30
20
10
0
0.9% NaCl
Figure 180
1.a
Control
18 g dust
54 g dust
160
140
120
100
80
60
40
20
0
0.9% NaCl
18 g dust
54 g dust
Figure 1b
9000
Control
8000
**
7000
6000
5000
4000
3000
2000
1000
0
0.9% NaCl
18 g dust
54 g dust
Figure 1c.
283
45
0.9% NaCl (x 4)
40
35
Mean
30
25
20
15
10
5
0
FigureFigure
3
2
IL-6
MCP-1
MIP-2
100000
***
***
10000
)7
***
***
***
***
1000
100
10
0.9% NaCl (x 4)
Dust from boiler room
Dust from straw storage
Mean
( 4 x 54 g )
{
( 4 x 54 g )
{
284
Discussion
Exposure of mice to multiple doses of airborne dust
from a biofuel plant converting straw induced substantial
Madsen et al
p ulmonary inflammation, including neutrophil infiltration and increased cytokine expression in the lungs
of the mice. The mice exposed to a single dose only
responded significantly after exposure to the highest
dose of dust from the straw storage hall. No increase in
DNA-damaging effects in BAL cells was detected at the
studied points in time after either single or multiple exposures to both types of dust. Exposure to dust from the
straw storage hall caused a more than 60-fold increase
in IL-6 mRNA expression. In a study with the inhalation
of carbon black, at doses corresponding to the multiple
doses in this study, only a threefold increase in IL-6
mRNA expression was found, even though increased
DNA damage was detected (16). It is interesting to note
from this study that a strong inflammatory response does
not necessarily induce genotoxic effects in BAL cells
because in vitro studies have shown that genotoxicity
can be mediated by a particle-induced inflammatory
response (21). In accordance with this study, a similar
in vitro study with wood dust showed that the dust was
genotoxic, without an induced inflammatory response
(22).
To our knowledge, the genotoxic and inflammatory
effects of dust from biofuel plants have not been evaluated previously. However, some studies have evaluated
the effects of exposure to, for example, endotoxins and
other types of organic dust (23, 24). The inflammatory
effect of the biofuel dust may be caused by the high
concentrations of microorganisms and endotoxins in the
dust. The exposure of mice to endotoxins in this study
corresponds in the single dose of 18 g of dust from the
straw storage hall to 0.021 g endotoxin/kg mouse (if
the mouse weighed 20 g). In the multiple doses with a
total dust amount of 216 mg, this corresponds to 0.257
mg endotoxin/kg mouse. In an inhalation study with
humans, in which inflammatory responses were found,
the participants were exposed to higher concentrations
of endotoxins than those used in this study. Thus the
participants were exposed to 0.1 ml inhalant/kg body
weight with 0.9 g or 6.0 g endotoxins/ml inhalant (25,
26), corresponding to 0.090.6 mg endotoxins/kg body
weight. The dust used in our study also contained spores
of fungi and actinomycetes and the strong inflammatory
response to the dust exposure may have been caused
by the composition of different microbial components.
Thus synergistic effects on the induction of inflammation are found for endotoxin and organic dust particles
(27), and the fungus Metarhizium anisopliae has been
found to have an adjuvant effect on the allergic response
to ovalbumin in mice (28).
In this study, we chose to evaluate genotoxic and
inflammatory effects 1 hour after instillations of dust
because our previous studies have shown that genotoxicity occurs before the recruitment of neutrophils. Thus
in vivo studies have shown genotoxicity 1 hour after
285
286
Acknowledgments
Special thanks go to Signe H Nielsen, Hediye Avci,
Pernille Salvarli, Tina T Olsen, Anne-Karin Jensen, and
Gitte B Kristiansen for their expert technical assistance.
Extra thanks go to Jrgen Kystol at GEUS for performing the ICP-MS analysis.
We are also grateful to PSO-Eltra (5786) for its
financial support.
References
1. Madsen AM. Exposure to airborne microbial components in
autumn and spring during work at Danish biofuel plants. Ann
Occup Hyg. 2006;50:82131.
2. Madsen AM, Sharma AK. Sampling of high amounts of bioaerosols using a high-volume electrostatic field sampler. Ann
Occup Hyg. 2008;52:16776.
3. Madsen AM, Mrtensson L, Schneider T, Larsson L. Microbial
dustiness and particle release of different biofuels. Ann Occup
Hyg. 2004;48:32738.
4. Sebastian A, Madsen AM, Mrtensson L, Pomorska D, Larsson L. Assessment of microbial exposure risks from handling
of biofuel wood chips and straweffect of outdoor storage.
Ann Agric Environ Med. 2006;13:13945.
5. Viet SM, Buchan R, Stallones L. Acute respiratory effects
and endotoxin exposure during wheat harvest in Northeastern
Colorado. Appl Occup Environ Hyg. 2001;16:68597.
6. Eduard W, Douwes J, Mehl R, Heederik D, Melbostad E.
Short term exposure to airborne microbial agents during farm
work: exposure-response relations with eye and respiratory
Madsen et al
2007;632:7888.
23. Mtt J, Lehto M, Leino M, Tillander S, Haapakoski R,
Majuri ML, et al. Machanisms of particle-induced pulmonary
inflammation in a mouse model: exposure to wood dust. Toxicol Sci. 2004;93:96104.
24. Isral-Assayag E, Cormier Y. Adaptation to organic dust exposure: a potential role of L-selectin shedding? Eur Respir J.
2002;19:8337.
25. Jagielo PJ, Thorne PS, Watt JL, Frees KL, Quinn TJ, Schwartz
DA. Grain dust and endotoxin inhalation challenges produce
similar inflammatory responses in normal subjects. Chest.
1996;110:26370.
26. Jagielo PJ, Thorne PS, Watt JL, Frees KL, Quinn TJ, Schwartz
DA. Grain dust and endotoxin inhalation challenges produce
similar inflammatory responses in normal subjects. Chest.
1996;110:26370.
27. Pirie RS, Collie PM, Dixon PM, McGorum BC. Inhaled endotoxin and organic dust particulates have synergistic proinflammatory effects in equine heaves (organic dust-induced asthma).
Clin Exp Allergy. 2003;33:67683.
28. Instanes C, Ward MD, Hetland G. The fungal biopesticide
Metarhizium anisopliae has an adjuvant effect on the allergic
response to ovalbumin in mice. Toxicol Lett. 2006;161:219
25.
29. Schins RP, Knaapen AM. Genotoxicity of poorly soluble particles. Inhal Toxicol. 2007;19:18998.
30. Devlin RB, McDonnell WF, Becker S, Madden MC, McGee
MP, Perez R, et al. Time-dependent changes of inflammatory
mediators in the lungs of humans exposed to 0.4 ppm ozone
for 2 hr: a comparison of mediators found in bronchoalveolar
lavage fluid 1 and 18 hr after exposure. Toxicol Appl Pharmacol. 1996;138:17685.
31. Lund K, Dunster C, Sandstrm T, Kelly FJ, Sstrand P,
Schwarze P, et al. Inflammatory markers in bronchoalveolar
lavage fluid from human volunteers 2 hours after hydrogen
fluoride exposure. Hum Exp Toxicol. 2005;24:1018.
32. Madsen AM, Sharma AK. Sampling of high amounts of bioaerosols using a high-volume electrostatic field sampler. Ann
Occup Hyg. 2007;
33. Nielsen BH, Wrtz H, Breum NO, Poulsen OM. Microorganisms and endotoxin in experimentally generated bioaerosols
from composting household waste. Ann Agric Environ Med.
1997;4:15968.
34. Madsen AM. Airborne endotoxin in different background environments and seasons. Ann Agric Environ Med. 2006;13:81
6.
35. Arbejdstilsynet. At-vejledning. Grnsevrdier for stoffer og
materialer [The Danish working authority. work place exposure limits]. Copenhagen: Arbejdstilsynet; 2007. p 185.
36. Whlin P, Berkowicz R, Palmgren F. Characterisation of
traffic-generated particulate matter in Copenhagen. Atmos
Environ. 2006;40:21519.
287