1 In vivo biomarkers of effects / response Do we know the agent ? Do we expect the effect ? : specific biomarkers / non-specific changes Behavioural and Clinical biomarkers Pathology Clinical chemistry and hematology Enzymatic changes Protein synthesis biomarkers Oxidative stress markers Behavioural and clinical biomarkers Behavioural and clinical biomarkers Parameters evaluated - body weight - food consumption - fitness & welness Interpretation : are these ? biomarkers ? (effects already demonstrated in vivo) - biomarkers of existing serious stress / intoxication Behavioural and clinical biomarkers (Histo)pathology biomarkers Pathology (-) Destructive methods, Time consuming, Professional requirements (+) High relevance ­ organ/tissue changes 1) microscopy of internal organs : non-specific changes in internal organs : specific changes in liver (dioxin-like POPs, cyanobacterial toxins ..) : intersex / imposex formation (xenoestrogenicity) Example: Liver damage by cyanobacterial toxins microcystins 2 Endocrine disruption: Intersex microscopy Oocytes in testicular tissue Pathology 2) immunohistochemistry & microscopy : determination of specific changes : Fluorescein (FITC) - labeled antibodies (Ab) applications - toxicant induced autoimmunity: anti-nuclear Ab, ANA Pathology 3) Nuclear DNA characterization - micronuclei evaluation - chromosomal abnormalities : karyotype biomarkers (human genetic disorders) : non-destructive (blood samples; plant tissues) Clinical chemistry & hematology biomarkers Clinical chemistry & hematology Non-destructive (BLOOD, URINE sampling) Multipe parameters can be measured - responses to various types of stresses (including toxic stress) - ,,normal" value ranges known for humans, rats and few other species (limited use as biomarkers in other organisms) 3 Methods: - automatic biochemical and hematological analyzers - different ,,analytes" various principles of methods Clinical chemistry & hematology Often with specific interpretation: - determination of enzymatic activities in blood - tissue/organ-specific damage damage Examples (toxicological studies) - liver damage ­ AST (Aspartate aminotransferase), ALT (Alanine aminotransferase) in blood... : cyanotoxins, dioxin-like POPs - lactate dehydrogenase (LDH) - general cell damage - muscle damage: creatine kinase in serum : isozymes - tissue specific (brain, muscle, heart); Clinical chemistry & hematology Clinical chemistry & hematology Kinetic Spectrophotometry LDH assay - principle Example ­ changes in rat serum enzymes after CCL4 exposure + Human: Excretory products in urine Tumor genes and tumor markers - cancer genes ras, myc, - -fetoprotein (AFP) - suppressor genes p53, Rb Methods of determination: - ELISA (enzyme linked immunosorbent assays) Clinical chemistry & hematology 4 Changes in enzyme activities Enzymatic changes Toxicity mechanisms related to ,,enzyme changes": Inhibitions of AcChE (organo-phosphates) d-Aminolevulinic Acid Dehydratase (ALAD) (lead - Pb) Proteinphosphatases (microcystins) Inductions of detoxication & oxidative stress enzymes (hepatopancreas / liver / blood) MFO [CYP classes - EROD / MROD / BROD] Phase II enzymes (GSTs) Glutathion metabolism enzymes (GPx, GRs) (+) Rapid enzymatic assays, specific responses (-) Some ~ EXPOSURE biomarkers AcChE inhibition mechanism & effects in birds AcChE inhibition assay Model Substrate (butyryl-thio-choline, acetyl-thio-choline) - cleaved by AcChE -> formation of free ­SH groups - SH: thiol reactive probes: Ellman´s reagent (DTNB) - DTNB-S-choline: yellow colour (spectrophotometry A420) Spectrophotometry AcChE inhibition mechanism & effects in birds Proteinphosphatase inhibition assay Model substrates cleaved by PPase 32P-labelled protein -> free 32P radioactivity 6,8-difluoro-4-methylumbelliferyl phosphate -> fluorescence PP PP PPasa P P PP PP PPasa SUBSTRÁT SUBSTRÁT 5 MFO (CYP) activities MFO (CYP) activities EROD assay - Determination of CYP450 activity substrate: Ethoxyresorufin -> Oxidation by CYP1A1 -> Fluorescence EthoxyResorufin-O-Deethylase activity EROD (other substrates: CYP isozymes: BROD - butoxy..., MROD, PROD ...) Biomarker of organic pollution (exposure & effects) : AhR-activating compounds (PCDD/Fs, PCBs, PAHs) : often used in environmental studies Locality: Reference Exposed EROD variation on male and female carp from the Anoia and Cardener tributaries ­ seasonal variability & response at contaminated localities MFO-responses are SPECIES ­ SPECIFIC & not always related to clinical signs 6 MFO-responses are SPECIES ­ SPECIFIC & relative activity decreases with body size Potencies to induce CYPs (AhR) PCDD/Fs and co-planar PCBs - induction of MFO is structure-dependent; potencies & toxicities among compounds differ - international agreement on TEF/TEQ approach to characterize dioxin-toxicity in environmental samples (WHO) - each compound (only few selected in WHO agreement) relative potency (TEF) related to 2,3,7,8-TCDD 2,3,7,8-TCDD TEF = 1 Several other PCDD/Fs 0.1-1 PCBs 10-5 ­ 0.1 (No. 77, 126) - species-specific TEFs for humans / fish / birds - chemical analyses of samples => SUMA (concentrations x TEF) = TEQ (ng TCDD / sample) - EASY comparison of sample contamination TEFs for selected PCDDs TEFs for PCBs Congener Number IUPAC Chlorobiphenyl Prefix 1994 WHO TEFs(1) 1997 WHO TEFs(2) Humans/ Mammals Fish Birds PCB-77 3,3',4,4'-Tetra- 0.0005 0.0001 0.0001 0.05 PCB-81 3,4,4',5-Tetra- -- 0.0001 0.0005 0.1 PCB-105 2,3,3',4,4'-Penta- 0.0001 0.0001 <0.000005 0.0001 PCB-114 2,3,4,4',5-Penta- 0.0005 0.0005 <0.000005 0.0001 PCB-118 2,3',4,4',5-Penta- 0.0001 0.0001 <0.000005 0.00001 PCB-123 2,3',4,4',5'-Penta- 0.0001 0.0001 <0.000005 0.00001 PCB-126 3,3',4,4',5-Penta- 0.1 0.1 0.005 0.1 PCB-156 2,3,3',4,4',5-Hexa- 0.0005 0.0005 <0.000005 0.0001 PCB-157 2,3,3',4,4',5'-Hexa- 0.0005 0.0005 <0.000005 0.0001 PCB-167 2,3',4,4',5,5'-Hexa- 0.00001 0.00001 <0.000005 0.00001 PCB-169 3,3',4,4',5,5'-Hexa- 0.01 0.01 0.00005 0.001 PCB-170 2,2',3,3',4,4',5-Hepta- 0.0001 -- -- -- PCB-180 2,2',3,4,4',5,5'-Hepta- 0.00001 -- -- -- PCB-189 2,3,3',4,4',5,5'-Hepta- 0.0001 0.0001 <0.000005 0.00001 Phase II conjugation enzymes - GSTs GSTs - soluble and membrane (ER) variants - activities in cytoplasm or microsomes Methods Substrates: reduced GSH + thiol selective probe (CDNB) GST GSH + CDNB -> GS-CDNB yellow product, kinetic or endpoint determination Kinetic assessment stress -> Induction of GSTs faster reaction -> slope of kinetic increase GST activity - example Kinetic assessment of GSTs stress -> Induction of GSTs faster reaction -> kinetic slope increases 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0 2 4 6 8 Blank PHE - 3 ug/L A 420nm GST activity induction by 1,10-Phenanthroline in Xenopus laevis embryos 0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.16 0.18 Blank 0.1 0.3 1 3 Slope 7 Protein levels (synthesis) biomarkers PROTEIN SYNTHESIS Protein determination - amount (concentration) - activity (see enzymatic assays) Amount quantification - mRNA levels (in vitro assays) - protein - electrophoresis and Western-(immuno)blotting - ELISA techniques Examples heat shock proteins (hsp90, hsp60, hsp 70, ubiquitin) metalothioneins Vitellogenin(-like) Vtg proteins in male Heat Shock Proteins (hsp) Stress = synthesis of new proteins ~ equilibrium and homeostasis buffering - temperature (cold / heat) ­ cryo-preservation - salinity & metals ­ ion buffering - organic xenobiotics ­ detoxication New proteins must be folded (3D-structure) by ,,CHAPERONES" - hsp90, hsp60, hsp 70 (~ 60-90 kD molecular weight kD) HSP determination - example HSP = GENERAL STRESS biomarker, non-specific - phylogenetically conserved (similar sequences in ,,all" organisms) - structural similarity => easy determination: electrophoresis + immunoblotting (Western blotting) Low MW proteins (6-10 kD) rich of Cystein (-SH) - detected in numerous eukaryotic organisms - induced in the presence of metals or less specific stress (low O2, T) - long halflife (~ 25 days) - binding of divalent metals (Zn, Cd, Hg) => exposure elimination - natural function (?) ­ regulation of essencial metals in cells Metalothioneins (MTs, MT-like proteins) Vitellogenin Vtg - precursor of yolk proteins, phospho-protein -> egg formations (females) at oviparous animals - synthesised in liver and distributed via blood (haemolymph) : xenoestrogens & other endocrine disruptors -> increased levels or early production in FEMALES -> production in MALES 8 Vitellogenin VTG Determination 1) ELISA (exposed organisms - F/M, in vitro - in vivo - exposed organisms (biomarker in vivo) - in vitro production in hepatocytes exposed to effluents (marker of estrogen-like presence (-) specific Antibodies necessary for each species (low crossreactivity) 2) ,,Vitelin-like proteins" - total amount of ,,alkali-labile" phosphate in haemolymph (mussels) - alkaline extraction of P from sample & determination Vitellogenin in fish Kidd et al. (2007) PNAS Fig. 1. Mean SE (n = 4­7) VTG concentrations in whole-body homogenates of male (Lower) and female (Upper) fathead minnow captured in 1999­2003 from reference Lakes 114 and 442 and from Lake 260 before and during additions of 5­6 ngL-1 of EE2 (low catches of fish in Lake 260 in 2004 and 2005 did not allow for these analyses in the latter 2 years of the study). FEMALE MALE Vitelin-like proteins in mussels Biomarkers of oxidative stress Oxidative stress markers Several parameters respond to oxidative stress : enzymes (GPx, GR, GSTs) - enzymatic activities (see elsewhere) : antioxidants (GSH, vit E) : markers of oxidative damage - MDA, - 8OH-dG (see DNA damage) ______________________ Oxidative stress markers GSH determination - antioxidant (scavenger of ROS) & reactive molecules - conjugation molecules for detoxication - probable intracellular regulatory molecule (? apoptosis ?) Total glutathione = reduced GSH + oxidized GSSG GSH + Ellman´s reagent (DTNB) -> Reduced GSH GSH + Glut.Reductase + DTNB -> Total GSH Total ­ Reduced = Oxidized 9 Markers of oxidative DAMAGE Lipid peroxidation -> Malonyldialdehyde (MDA) MDA ­ malondialdehyde product of Lipid peroxidation Lipid peroxidation -> Malonyldialdehyde (MDA) MDA ­ formed from oxidized membrane phospholipids : determination: - HPLC - TBARS method TBARS ­ ThioBarbituric Acid Reactive Species : less specific than HPLC (+/- aldehydes) : easy determination (spectrophotometry) Method: 1) sample extract (with MDA) 2) add TBA 3) boil (cca 30´ / 90°C) => formation of red/violet coloured product 4) determination by spectrophotometry (A 540 nm) MDA modulation - examples Effects of antioxidants in young/old on oxidative damage (MDA)