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Emerging biological risks: endotoxins?
Prof. Dick Heederik, PhD
IRAS, Division of Environmental Epidemiology University UtrechtThe Netherlands
Endotoxin (Lipopolysaccharide (LPS))
• First described by Richard Pfeiffer in 1892 (Zf f Hygiene) as heat stable, cell associated material from Vibrio cholerae
• part of the outer membrane of all gram negative bacteria and some blue algae
Measurement of endotoxin (CEN 2002)
• As described by CEN (NEN-EN 14031)– Classical inhalable dust
sampling – Extraction filter with dust– Measurement based on (kinetic
version of the) LymulusAmoebocyte Lysate Assay
– Results expressed relative to external standard in EndotoxinUnits/m3 of air
New industry wide exposure studies (EU/m³): endotoxin exposure database
96 (9)
170 (7)
593 (8)
233 (9)
GM (GSD)
2-8119
2-191434
2-149064
2–191434
range
716102Animal production sector
3064291Horticulture
4824 194Grains, seeds and legumes sector
3238 587Overall
AMN
(Spaan et al., J Environ Monit 2006)
LargeLarge populationpopulation at risk: at risk: agriculturalagricultural industryindustry, , relatedrelated foodfood processing industries, processing industries, metalmetal workingworking fluidfluid exposedexposed …….. >5%.. >5%--10 10 workforceworkforce
Elevation relative to reference company (Spaan et al., J Environ Monit 2006)
>54.6
20.0-54.6
7.4-20.0
2.7-7.4
1- 2.7
Reference
Multiplying factor
Pig farm (pulp), dairy farm&breeding, grain transshipment, grain harvest, poultry farms (eggs, free-range), grinding industry, grass drying, malting, flax culture and processing, onion trade
Canning vegetable, flower bulbs nurseries, animal feed industry, dairy farm, poultry farm (meat), corn processing, rice hulling
Mushroom compost, coffee/tea, abattoir (poultry), cucumber nursery, meal/flour tillage/processing, potato selection, flower bulb trade
Industrial bakery, nurseries/trade (tulips, tomatoes, mushroom, trees, chicory), abattoir (calf), sugar production, gardening companies
meat processing, vegetable processing (slicing, freezing), abattoirs (pig, cow), nurseries (roses, pot-plants)
Dried subtropical fruit (19 EU/m3)
Endotoxin
Correlation dust and endotoxin exposure
correlation dust and endotoxin exposurer=0.69, β=1.06
1
10
100
1000
10000
100000
1000000
0.001 0.01 0.1 1 10 100
inhalable dust (mg/m3), logarithmic scale
end
oto
xin
(EU
/m3)
, lo
gar
ithm
ic s
cale
Grains, seeds and legumes sector r=0.67, β=0.89
1
10
100
1000
10000
100000
1000000
0.001 0.01 0.1 1 10 100
inhalable dust (mg/m3)
end
otox
in (E
U/m
3)
Horticulture r=0.59, β=1.13
1
100
10000
1000000
0.001 0.01 0.1 1 10 100
inhalable dust (mg/m3)
end
oto
xin
(E
U/m
3)
Animal production sectorr = 0.66, β=1.07
1
10
100
1000
10000
0.001 0.01 0.1 1 10 100
inhalable dust (mg/m3)
end
otox
in (E
U/m
3)
2
Early evidence on health effects
• Cotton production
• Neal et al. JAMA 1942 suggested Enterobacteragglomerans might play a role in the development of byssinosis
• Rylander et al. was among the first to mention endotoxin in relation to byssinosis (Chest 1986)
• Since than, exposure and effects have been described in a range of industries
Review of health effects (up to 1997 www.gr.nl)
• Experimental animal studies
• Experimental studies in humans (injection and inhalation pure endotoxin, inhalation of dust containing endotoxin (cotton, pig stables)
• A series of large scale observational epidemiologic studies in agricultural workers and other occupational groups:
– Grain processing– (pig) farming– Composting– Metal working fluids, glass fiber
production, etc.
Health effects
• Acute:– Systemic and respiratory symptoms (dry cough, shortness of
breath, fever, shivering, joint pain) ICOH: ODTS 1000 EU/m3
(Rylander, 1997)– (Acute) lung function changes ICOH 100 EU/m3
– Inflammatory response (neutrophile, cytokines)
• Chronic:– Accelerated lung function decline (COPD?)
• Protective effects:– Atopy and allergy?– Lung cancer?
Acute respiratory changes in cotton dust exposed individuals: card room studies
• Experimental study in the US in humans (Castellan et al., 1987)
• NOEL for acute lung function changes around 9 ng/m3
• Formed the basis of the proposed exposure standard in the Netherlands 50 EU/m3
standard of 200 EU/m3
adapted
Interindividual variability in response
Kline et al. 1999. ARRCCM.
Atopy, and hay fever in farmers’ children (Braun-Fahrländer et al., 2002)
Endotoxin exposure during first year and current endotoxin exposure were associated with reduced atopy prevelance in children from farming and non-farming families
3
occupational endotoxin exposure protects against respiratory sensitization ast adult age
Portengen et al., JACI 2005
Endotoxin, respiratory symptoms and BHR (Smit et al., 2007 in preparation)
Modeled endotoxin exposure (EU/m3)
10 100 1000 10000
Lo
wer
res
pir
ato
ry s
ymp
tom
s (%
)
0
10
20
30
40
50
60
All participants aged 18-65 (n = 878)95% SE bands
Modeled endotoxin exposure (EU/m3)
10 100 1000 10000
Bro
nch
ial r
espo
nsi
ven
ess
to m
etac
ho
line
(%)
0
20
40
60
80
100
Change in FEV1 > 15%
Change in FEV1 >20%
95% SE bands, change in FEV1 >15%
95% SE bands, change in FEV1 >20%
Endotoxin, atopy (with and without farm childhood)(Smit et al., in preparation)
Modeled endotoxin exposure (EU/m3)
10 100 1000 10000
Hay
fev
er (
%)
0
5
10
15
20
25
30
35
All participants aged 18-65 (n= 878)95% SE bands
Modeled endotoxin exposure (EU/m3)
10 100 1000 10000
Ato
pic
sen
siti
sati
on
(%
)
0
20
40
60
80
No farm childhood (n = 235)Farm childhood (n= 194)95% SE bands, no farm childhood95% SE bands, farm childhood
Association of lung cancer with cumulative exposure to endotoxin: 20 year latency
0.4-0.70.5338594043-138177
0.4-0.80.5309572083-4042
0.5-0.90.7347752041-2082
0.5-0.90.7408811275-2041
reference1.0543122>0-1274
0.6-1.00.810902080
95%CIRR*SubcohortCasesCum-exp(EU-yrs)
*Hazard ratio, adjusted for age, smokingSource: Astrakianakis G, et al. JNCI (2007 in press)
Variability issues• Variability within and between
laboratories LAL assessment
• Most information on laboratories with different protocols (Reynolds et al., 2002, 2005; Chun et al., 2002)
• Performance improves when protocols are harmonized (Chun et al., 2002 and Linselet al., 2003)
• Major steps in harmonization can now be made (Spaan et al., 2007; Wouters et al., 2007)
– Assay type: KLARE and other kinetic versions (Milton et al., 1992; Thorne et al., 1997)
– Storage stability of samples (Douwes et al., 1995; Milton et al., 1997)
– Filter media (Douwes et al., 1995; Thorne et al., 1997; Milton et al., 1997)
– “Inhibition” and “enhancement”(Hollander et al., 1993)
– Extraction medium (Tweenoptimal) (Douwes et al., 1995; Thorne et al., 2003; USA ASTM protocol)
Variability issues
• Changes over time seem limited (supplier, lot, etc.)
• Variability in endotoxinbetween workers and over time larger than for chemical agents!
• Need to allow for this in the exposure assessment strategy
endotoxin concentration with kabivitrum based assay
0 2 4 6 8 10 12
repe
ated
ana
lysi
s
-2
0
2
4
6
8
10
12
kabivitrum 1988 against whittaker 1991 kabivitrum 1988 against kabivitrum 1993 kabivitrum 1993 against whittaker 1993 x = y
4
Other issues
• Information from a limited number of industries
• Exposure to a complex mixture
• extrapolation from one industry to another?
• Evidence remarkably consistent across industries
• Endotoxin often most potent constituent (in vitro evidence)
Fig. 1a: TNF-a productie in WBA - exp.1
0
100
200
300
400
500
600
700
0.1 1 10 100
zaad extract (microliter/test)
cyto
kin
e (p
g/m
L)
Lolium-1Festuca-1Wheat-1CauliflowerRed beetLPS
In conclusion
• No doubt about the role of endotoxin as cause of respiratory disease
• Quantitative evidence has become stronger, as well as insight inmechanisms
• Potential protective effects with regard to atopic responses: two sides of the coin?
• Introduction of CEN protocol on EU level needed so reduce interlabvariability
• High variability in endotoxin levels requires specific measurement strategy for biological agents
• Standard setting? SCOEL?