Biomarkers in ecotoxicology

1. Biomarkers in ecotoxicology

2. Biomarkers Classic 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

3. Health and stress Health (Bayne et al., 1985):The residual capacity of an organism to withstand stress. Stress (Brett1958):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.

4. 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

5. Chemicalpollution - speciation - bioavailable residues Sensory interference Absorption Exposure / effect biomarkers Molecular responses Physiological responses Structural damage Predictive Effect / health biomarkers Impaired fitness Disturbed population and ecosystem stability Reactive

6. Biomarkers An ideal Healthbiomarkeris sensitive to chemical stress and is irrefutably linked to the Darwinian fitness of the organism. Darwinian fitnessis the combined relative probability of survival and rate of reproduction of the individual. An ideal Exposure biomarkeris both sensitive and specific to exposure by a single chemical or group of chemicals. The ideal biomarkerin ecotoxicology combines the properties of both types. Depledge, 1993

7. Healthy Stressed Health Status curable Non-curable Reversible Irreversible Homeostasis Compensation Non-compensation Intensity of Biomarker response Intensity of Exposure Depledge’s biomarker christmass wish 1993

9. Alad: a highly specific biomarker for lead poisoning

10. 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

11. Chlorophyl Vitamin B12 etc Pb Pb Pb

12. The ALAD monomer Zn binding site

14. Protoporphyria in ducks fed lead contaminated food Heinz et al, 1999

15. The effect of lead contaminated diet on duck body weight 24% clean sediment in commercial diet 24% Pb sediment in commercial diet Heinz et al, 1999

16. Heinz et al, 1999

17. Biomarker development • After Hugget et al. (1989) • Relative sensitivity • Inherent variability • Biological specificity • Chemical specificity • Time to manifestation • Linkage to higher level effects • Field applicability • Field validation

18. Esterase inhibition

19. Paraoxonases A-esterases (hydrolyse OP’s) DFPase Acetylcholinesterase B-esterases (Inhibited by OP’s) Buturylcholinesterase Neurotoxicesterase Carboxylesterase Esterase classification C-esterases: Do not interact with OP’s or Carbamates

20. RO OR - CH3 CH3 O O-P-S-R H3C-N-CH2-CH2-O-C-CH3 OH + OH - - Acetylcholine-receptor complex Organophosphate-receptor complex Hydrolysis Hydrolysis OR - O CH3 CH3 CH3 O=P-S-R H3C-N-CH2-CH2-OH C OH + O + - Acetylcholine hydrolysed but bound - CH3COOH + (CH3)NCH2CH2OH OR Product release (rapid) O=P-S-R Product release V slow O - OH - OH - Regenerated enzyme + choline +acetic acid AChE Inhibition

21. Biomarker development • After Hugget et al. (1989) • Relative sensitivity • Inherent variability • Biological specificity • Chemical specificity • Time to manifestation • Linkage to higher level effects • Field applicability • Field validation

22. 2PAM N CH CH3 N O H Relative specificity • Species differences • Intraspecific differences • Use of oximes to reactivate enzyme • Brain AChE shows least variablilty

23. Strategy for distinguishing Carbamates and OP’s (Rotenburg et al, 1995)

24. 2PAM N CH CH3 N O H Relative specificity • Species differences • Intraspecific differences • Use of oximes to reactivate enzyme • Brain AChE shows least variablilty • Diurnal changes (up to 150% in starling) • Seasonal changes (Brain AChE lowest var.) • Age

25. Effect of age I 100 80 60 Percentage of adult activity 40 20 0 4 365 18 Age of starlings (days) Grue et al., 1981

26. Effect of age II 1200 1000 300 800 Plasma BChE (µmol/min/l plasma) Plasma BChE (µmol/min/l plasma) 600 200 400 200 100 1 2 4 7 12 Age of mallard (weeks) Bennett and Benet., 1991

27. Relative specificity • Species differences • Intraspecific differences • Use of oximes to reactivate enzyme • Brain AChE shows least variablilty • Diurnal changes (up to 150% in starling) • Seasonal changes (Brain AChE lowest var.) • Age • Temperature/diet

28. Controls Cold Underfed Parathion (15 mg/kg) Effects of temperature and diet on ChE activity in Quail 3 Plasma ChE (IE/l plasma) 2 1 1 3 7 14 28 Days Ratner, 1982

29. Biomarker development • After Hugget et al. (1989) • Relative sensitivity • Inherent variability • Biological specificity • Chemical specificity • Time to manifestation • Linkage to higher level effects • Field applicability • Field validation

30. Chemical specificity?

31. Biomarker development • After Hugget et al. (1989) • Relative sensitivity • Inherent variability • Biological specificity • Chemical specificity • Time to manifestation • Linkage to higher level effects • Field applicability • Field validation

32. Starling serum ChE activity, 6hrs and • 24hrs after OP ingestion Thompson et al 1991 .

33. Starling serum BChE activity, 6hrs and • 24hrs after OP ingestion . Thompson et al 1991

34. Biomarker development • After Hugget et al. (1989) • Relative sensitivity • Inherent variability • Biological specificity • Chemical specificity • Time to manifestation • Linkage to higher level effects • Field applicability • Field validation

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

36. Biomarker development • After Hugget et al. (1989) • Relative sensitivity • Inherent variability • Biological specificity • Chemical specificity • Time to manifestation • Linkage to higher level effects • Field applicability • Field validation

37. Zone of normal variation Zone of reversible effects Zone of irreversible effects g/ha 280 420 ? Dose of Fenitrothion The utility of AChE mesurements in environmental management 0 20 40 % inhibition of AChE 60 80 mg/Kg 1 3 10

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