Biomarkers in ecotoxicology

Transcription

1 Biomarkers in ecotoxicology 1

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 2

3 Health and stress Health (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. 3

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 4

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

6 Biomarkers An 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,

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

8 Health Biomarkers Immunological responses Scope for growth Adenelate energy charge AChE inhibition Egg-shell thinning Hæms and porphorins Behavioural alterations Exposure Biomarkers High specificity Medium/low specificity Alad MFO Egg-shell thinning AChE inhibition Metalothioneins DNA alterations Stress proteins Vitellogenin? 8

9 Alad: a highly specific biomarker for lead poisoning 9

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 10

11 Chlorophyl Vitamin B12 etc Pb Pb Pb 11

12 The ALAD monomer Zn binding site 12

13 13

14 Protoporphyria in ducks fed lead contaminated food 14

15 The effect of lead contaminated diet on duck body weight Body Mass (g) Time (weeks) 15

16 , ,0 15,5 15,0 14,5 14,0 13,5 13,0 16 Alad (IU) Hemoglobin (g/dl) Hematocrit values as % Con Con 24% 3 % Pb 6 % Pb 12 % Pb 24 % Pb The effect of Pb contaminated diet on blood parameters in ducks *

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 17

18 Esterase inhibition 18

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

20 AChE Inhibition - CH 3 CH 3 O H 3 C-N-CH 2 -CH2-O-C-CH 3 + OH - - RO OR O-P-S-R OH Hydrolysis Product release (rapid) Acetylcholine-receptor complex - CH 3 CH 3 O CH 3 H 3 C-N-CH 2 -CH 2 -OH C + O+ - Acetylcholine hydrolysed but bound CH 3 COOH + (CH 3 )NCH 2 CH 2 OH - OH Regenerated enzyme + choline +acetic acid Organophosphate-receptor complex OR O=P-S-R OH - OR O=P-S-R O - OH - 20 Hydrolysis Product release V slow

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 21

22 Relative specificity Species differences Intraspecific differences Use of oximes to reactivate enzyme Brain AChE shows least variablilty N CH 3 2PAM CH N O H 22

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

24 Relative specificity Species differences Intraspecific differences N CH 3 Use of oximes to reactivate enzyme Brain AChE shows least variablilty Diurnal changes (up to 150% in starling) 2PAM CH N O H Seasonal changes (Brain AChE lowest var.) Age 24

25 Effect of age I 100 Percentage of adult activity Age of starlings (days) Grue et al.,

26 Bennett and Benet., Effect of age II Plasma BChE (µmol/min/l plasma) Age of mallard (weeks) Plasma BChE (µmol/min/l plasma)

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 27

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

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 29

30 Chemical specificity? 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) 30

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 31

32 Starling serum ChE activity, 6hrs and 24hrs after OP ingestion. 32

33 Starling serum ChE activity, 6hrs and 24hrs after OP ingestion. 33

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 34

35 Links to fitnessrelated behaviours Activity budgets of captive male starlings dosed with dicrotophos to give a 50% inhibition of AChE. % 35

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 36

37 The utility of AChE mesurements in environmental management % inhibition of AChE Zone of normal variation Zone of reversible effects Zone of irreversible effects ? Dose of Fenitrothion mg/kg g/ha 37

38 Date Event Development stage Reference 1958 Abnormal breakages in Peregrine falcon eggs Problem? Ratcliffe (1958) 1965 Dramatic decline in raptors in the holarctic (Madison meeting) Time sequence Consistency of replication Hickey (1969) 1967 Demonstration of the 1 st occurance in 1940 s in UK Time sequence Ratcliffe (1967) 1968 In US decline in many raptors associated to 20% thinning Time sequence Link between biomarker and higher levels Hickey & Anderson (1968) 1970 Link 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) 1973 Enzymatic changes in avian oviduct caused by DDE Mechanistic links between levels Peakall et al. (1973) 1974 Demonstration 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 38al. (1975)

39 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 Depledge s Christmass wish is unlikely to be fulfilled in near future 39