1

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?