1 Biomarkers in ecotoxicology. 2 Biomarkers Classic definition: Biochemical, physiological or...

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Biomarkers in

ecotoxicology

2

BiomarkersClassic definition: Biochemical, physiological or histological indicators of either exposure to or effects of, xenobiotic chamicals at the suborganismal or organismal level

Nato workshop (1993): A biological response that can be related to an exposure to, or toxic effect of, an environmental chemical or chemicals

Depledge (1993): A biolochemical, cellular, physiological or behavioural variation that can be measured in tissue or body fluid samples at the level of the whole organism (either individuals or populations) that provides evidence of exposure (exposure biomarkers) to and/or effects (health biomarkers) of one or morechemical pollutants

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Health and stressHealth (Bayne et al., 1985): The residual capacity of an organism to withstand stress.

Stress (Brett 1958): A state produced by an environment or other factor which extends the adaptive response of an animal beyond the normal range, or which disturbs the normal functioning to such an extent that the chances of survival are significantly reduced

Stressor (Lugo, 1981): A stressor is any condition or situation that causes a system to mobilise its resources and increase its energy expenditure. Stress is the response of the system to the stressor via this increase in energy expenditure.

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The driving forces behind biomarker development

• The problems with chemical analysis

• What do we measure?

• Temporal fluctuations in exposure

• Sensitivity vs effect?

• Bioavalability?

• Proof of exposure

• Proof of effect

• Prediction of ecological effects

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Impaired fitness

Disturbed population andecosystem stability

Chemical pollution- speciation- bioavailable residues

Sensoryinterference

Absorption

Molecular responsesPhysiological responses

Structural damage

Exposure / effectbiomarkers

Effect / healthbiomarkers

Pre

dic

tiv

eR

eact

ive

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BiomarkersAn ideal Health biomarker is sensitive to chemical stress and is irrefutably linked to the Darwinian fitness of the organism.

Darwinian fitness is the combined relative probability of survival and rate of reproduction of the individual.

An ideal Exposure biomarker is both sensitive and specific to exposure by a single chemical or group of chemicals.

The ideal biomarker in ecotoxicology combines the properties of both types.

Depledge, 1993

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Homeostasis

Healthy

Compensation

Stressed

Reversible

Non-compensation

curable

Irreversible

Non-curable

Hea

lth

Sta

tus

Inte

nsi

ty o

f B

iom

arke

r re

spo

nse

Intensity of Exposure

Depledge’s biomarker christmass wish 1993

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Health BiomarkersExposure Biomarkers

High specificity Medium/low specificity

Immunological responses Alad MFO

Scope for growth Egg-shell thinning AChE inhibition

Adenelate energy charge Metalothioneins

AChE inhibition DNA alterations

Egg-shell thinning Stress proteins

Hæms and porphorins Vitellogenin?

Behavioural alterations

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Alad: a highly specific biomarker for lead

poisoning

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Lead poisoning

• A historically prominent environmental toxin

• Symptomer: Porphyria/anaemia

Abdominal pain

Constipation

Peripheral neuropathy, Madness

Weight loss • Requirement: early warning biomarker

• ALAD: 5-aminolaevulnic acid dehydratase

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ChlorophylVitamin B12 etc

Pb Pb

Pb

12

The ALAD monomer

Zn binding site

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Protoporphyria in ducks fed lead contaminated food

Heinz et al, 1999

15Time (weeks)

2 4 6 8 10 12 14 16

Bo

dy

Mas

s (g

)

700

800

900

1000

1100

1200

1300

1400

The effect of lead contaminated diet on duck body weight

24% Pb sediment in commercial diet

24% clean sediment in commercial diet

Heinz et al, 1999

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Hem

oglo

bin

(g/d

l)13,0

13,5

14,0

14,5

15,0

15,5

16,0

16,540

42

44

46

48

Hem

ato

crit

val

ues

as

%

Con

Con

24%

3 %

Pb

6 %

Pb

12 %

Pb

24 %

Pb

Ala

d (I

U)

0

100

200

300

The effect of Pb contaminated

diet on blood parameters

in ducks

*

Heinz et al, 1999

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Biomarker developmentAfter Hugget et al. (1989)

• Relative sensitivity

• Inherent variability

• Biological specificity

• Chemical specificity

• Time to manifestation

• Linkage to higher level effects

• Field applicability

• Field validation

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Esterase inhibition

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Esterase classification

A-esterases(hydrolyse OP’s)

Paraoxonases

DFPase

B-esterases(Inhibited by OP’s)

Acetylcholinesterase

Buturylcholinesterase

Neurotoxicesterase

Carboxylesterase

C-esterases: Do not interact with OP’s or Carbamates

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-+

H3C-N-CH2-CH2-O-C-CH3

OH

OCH3CH3

-

Acetylcholine-receptor complex

-+

H3C-N-CH2-CH2-OH

O

OCH3CH3

-CH3

+C

Acetylcholine hydrolysed but bound

Hyd

roly

sis

-OH

CH3COOH+

(CH3)NCH2CH2OH

Regenerated enzyme + choline +acetic acid

Pro

duc

t re

lea

se (

rapi

d)

-OH

RO

Organophosphate-receptor complex

O-P-S-R

OR

-OH

O=P-S-R

OR

Hyd

rolysis

-

O

O=P-S-R

OR

-

OH

Prod

uct release

V slow

AChE Inhibition

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Relative specificity

• Species differences

• Intraspecific differences

•Use of oximes to reactivate enzyme

•Brain AChE shows least variablilty

N

CH3

CH

NO

2PAM

H

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Strategy for distinguishing Carbamates and OP’s

(Rotenburg et al, 1995)

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• Diurnal changes (up to 150% in starling)

• Seasonal changes (Brain AChE lowest var.)

• Age

N

CH3

CH

NO

2PAM

H

25

Effect of age IP

erc

en

tag

e o

f ad

ult

activ

ity

100

80

60

40

20

04 18 365Age of starlings (days)

Grue et al., 1981

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Effect of age IIP

lasm

a B

Ch

E (

µm

ol/m

in/l

pla

sma

) 1200

1000

800

600

400

2001 2 12

Age of mallard (weeks)

Bennett and Benet., 1991

4 7

300

200

100

Plasm

a B

Ch

E (µ

mo

l/min

/l plasm

a)

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• Diurnal changes (up to 150% in starling)

• Seasonal changes (Brain AChE lowest var.)

• Age

• Temperature/diet

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Effects of temperature and dieton ChE activity in QuailP

lasm

a C

hE

(IE

/l p

lasm

a) 3

2

1

1 3 28

Days

Ratner, 1982

147

Controls

Cold

Underfed

Parathion(15 mg/kg)

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Chemical Effect Animal Reference

DDE, PCB’s Plasma ChE up Quail Dieter (1974)

Crude oil Eratic changes in ChE

Mullet Chambers et al. (1979)

Cd, Hg, Pb Brain AChE down Rat Hrdina et al. (1976)

Pb Brain AChE down Rat Modak et al. (1975)

Cd ChE down Crab Reddy and Venugopal (1990)

Chemical specificity?

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32

.

Starling serumChE activity, 6hrs and • 24hrs after OP ingestion

Thompson et al 1991

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.

Starling serum BChE activity, 6hrs and • 24hrs after OP ingestion

Thompson et al 1991

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35

%

Links to fitness-related behaviours

Activity budgets of captive male starlings dosed with dicrotophos to give a 50% inhibition of AChE.

Grue and Shipley, 1981

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0

20

40

60

80

Zone of normal variation

Zone of reversible effects

Zone of irreversible effects

1 3 10 mg/Kg

280 420 ?

Dose of Fenitrothiong/ha

The utility of AChE mesurements in environmental management

% in

hibi

tion

of A

ChE

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Date Event Development stage Reference

1958Abnormal breakages in Peregrine falcon eggs

Problem? Ratcliffe (1958)

1965Dramatic decline in raptors in the holarctic (Madison meeting)

Time sequence

Consistency of replication

Hickey (1969)

1967Demonstration of the 1st occurance in 1940’s in UK

Time sequence Ratcliffe (1967)

1968In US decline in many raptors associated to 20% thinning

Time sequence

Link between biomarker and higher levels

Hickey & Anderson (1968)

1970Link to DDE in Alaskan eggs

First captivity exp. With Kestrel

Specificity of association

Link between biomarker and cause

Cade et al. (1971)

Wiemeyer and Porter (1970)

1973Enzymatic changes in avian oviduct caused by DDE

Mechanistic links between levels

Peakall et al. (1973)

1974Demonstration of DDE in eggs collected in 40’s

Specificity of association Peakall (1974)

1975

Demonstration of similar effects in field and controlled exp.s

Correlation between residues and thinning

Specificity of assoc

Strength of assoc

consistency

Lincer (1975)

Peakall et al. (1975)

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Conclusions

• Depledge’s Christmass wish is unlikely to be fulfilled in near future

• It is important to understand toxic mechanistic when attempting to understand environmental dammage

• Attaching blame to an environmental sinner will also in the future involve the use of biomarkers

1 Biomarkers in ecotoxicology. 2 Biomarkers Classic definition: Biochemical, physiological or...

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