In vivo biomarkers of effects / response lDo we know the agent ? Do we expect the effect ? : specific biomarkers / non-specific changes l lBehavioral and Clinical biomarkers lPathology lClinical chemistry and hematology lEnzymatic changes lProtein synthesis biomarkers lOxidative stress markers l Behavioral and clinical biomarkers Behavioral and clinical biomarkers lParameters evaluated -body weight -food consumption -fitness & welness l lInterpretation l: are these ? biomarkers ? (effects already demonstrated in vivo) l- biomarkers of existing serious stress / intoxication Behavioral and clinical biomarkers 164 (Histo)pathology biomarkers Pathology l(-) Destructive methods, Time consuming, Professional requirements l(+) High relevance – organ/tissue changes - l1) microscopy of internal organs l : non-specific changes in internal organs : specific changes in liver (dioxin-like POPs, cyanobacterial toxins ..) : intersex / imposex formation (xenoestrogenicity) - 1 1 Example: Liver damage by cyanobacterial toxins microcystins Endocrine disruption: Intersex microscopy 1 Oocytes in testicular tissue 1 1 1 Pathology l2) immunohistochemistry & microscopy l : determination of specific changes : Fluorescein (FITC) - labeled antibodies (Ab) applications - toxicant induced autoimmunity: anti-nuclear Ab, ANA 1 1 Pathology l3) Nuclear DNA characterization - micronuclei evaluation l - chromosomal abnormalities : karyotype biomarkers (human genetic disorders) l : non-destructive (blood samples; plant tissues) 1 1 1a Clinical chemistry & hematology biomarkers Clinical chemistry & hematology lNon-destructive (BLOOD, URINE sampling) l lMultipe parameters can be measured l l - responses to various types of stresses (including toxic stress) l l - „normal“ value ranges known for humans, rats and few other species (limited use as biomarkers in other organisms) l lMethods: - automatic biochemical and hematological analyzers l - different „analytes“ various principles of methods Clinical chemistry & hematology lOften with specific interpretation: - determination of enzymatic activities in blood l - tissue/organ-specific damage damage l lExamples (toxicological studies) -liver damage – AST (Aspartate aminotransferase), ALT (Alanine aminotransferase) in blood… : cyanotoxins, dioxin-like POPs - l- lactate dehydrogenase (LDH) - general cell damage l -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 1858 145 l+ Human: Excretory products in urine l Tumor genes and tumor markers - cancer genes ras, myc, l - a-fetoprotein (AFP) l - suppressor genes p53, Rb l lMethods of determination in practice: l - ELISA (enzyme linked immunosorbent assays) l Clinical chemistry & hematology Changes in enzyme activities Enzymatic changes lToxicity mechanisms related to „enzyme changes“: l –Inhibitions of lAcChE (organo-phosphates) ld-Aminolevulinic Acid Dehydratase (ALAD) (lead - Pb) lProteinphosphatases (microcystins) – –Inductions of detoxication & oxidative stress enzymes (hepatopancreas / liver / blood) l MFO [CYP classes - EROD / MROD / BROD] l Phase II enzymes (GSTs) l Glutathion metabolism enzymes (GPx, GRs) – l(+) Rapid enzymatic assays, specific responses l(-) Some ~ EXPOSURE biomarkers l AcChE inhibition mechanism 21 AcChE inhibition assay lModel Substrate (butyryl-thio-choline, acetyl-thio-choline) l - cleaved by AcChE à formation of free –SH groups l - SH: thiol reactive probes: Ellman´s reagent (DTNB) l - DTNB-S-choline: yellow colour (spectrophotometry A420) l Spectrophotometry AcChE inhibition mechanism & effects in birds 207 29 Proteinphosphatase inhibition assay lModel substrates cleaved by PPase –32P-labelled protein -> free 32P radioactivity –6,8-difluoro-4-methylumbelliferyl phosphate -> fluorescence l MFO (CYP) activities 88 90 MFO (CYP) activities l l l 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 105 103 Locality: Reference Exposed E3 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 52 MFO-responses are SPECIES – SPECIFIC & relative activity decreases with body size Related to the general metabolism rate 93 Phase II conjugation enzymes - GSTs l l l 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 l l l Kinetic assessment of GSTs stress -> Induction of GSTs faster reaction -> kinetic slope increases Protein levels (synthesis) biomarkers PROTEIN SYNTHESIS l l l Protein determination - amount (concentration) - activity (see enzymatic assays) Amount quantification - mRNA levels (in vitro assays) - protein levels - electrophoresis and Western-(immuno)blotting - ELISA techniques Examples heat shock proteins (hsp90, hsp60, hsp 70, ubiquitin) metalothioneins Vitellogenin(-like) Vtg proteins in male Aromatase Heat Shock Proteins (hsp) l l l 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 l l l 1 1 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) Protein biomarkers of estrogenicity ERs (transcription factors) control number of target genes Target genes = biomarkers of estrogenicity •Vitellogenin •Aromatase - CYP19A 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 1 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 = 4–7) VTG concentrations in whole-body homogenates of male (Lower) and female (Upper) fathead minnow captured in 1999–2003 from reference Lakes 114 and 442 and from Lake 260 before and during additions of 5–6 ng·L−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 Aromatase (CYP19A) Aromatase - inducible by estrogens - single enzymatic step androgens à estrogens Experimental assessment (in reseach and practice) - - PCR / Quantitative-Real-Time-PCR - - GM-organisms (zebrafish) (reporter gene – GFP – under the control of aromatase promoter) PCR principle Visualization of the PCR product 1)Electrophoresis (qualitative) Dyes – e.g. ethidium bromide 1) 1) 1) 1) 1) 1) Visualization of the PCR product Real-time (quantitative) SYBR GREEN -(more DNA synthesized, more fluorescent dye incorporated) - Visualization of the PCR product Real-time (quantitative) -TaqMan probes (more DNA replications more fluorescent dye released) Visualization of the PCR product QPCR determination of the Aromatase gene expression in Zebrafish http://dx.doi.org/10.1016/j.ygcen.2005.12.010, GFP-reporter for estrogens (zebrafish embryo) http://endo.endojournals.org/content/152/7/2542.full 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 Markers of oxidative DAMAGE Lipid peroxidation -> Malondialdehyde (MDA) MDA – malondialdehyde product of Lipid peroxidation Lipid peroxidation -> Malondialdehyde (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 1 Effects of antioxidants in young/old on oxidative damage (MDA)