1212569_21823227.jpg logo_mu_cerne.gif 1212570_28446780.jpg logo_mu_cerne.gif Luděk Bláha, PřF MU, RECETOX www.recetox.cz BIOMARKERS AND TOXICITY MECHANISMS 13 – BIOMARKERS of EFFECTS OPVK_MU_stred_2 1212569_21823227.jpg logo_mu_cerne.gif In vivo biomarkers of effects / response • •Behavioral and Clinical biomarkers •Pathology •Clinical chemistry and hematology •Enzymatic changes •Gene and protein expression biomarkers •Detoxification and oxidative stress markers • 1212569_21823227.jpg logo_mu_cerne.gif Behavioral and clinical biomarkers 1212569_21823227.jpg logo_mu_cerne.gif Examples of behavioral biomarkers 164 Concentrations affecting behaviour: often lower than LD50 à early markers of (lethal) toxicity 1212569_21823227.jpg logo_mu_cerne.gif Behavioral and clinical “biomarkers” •Interpretation •: are these really biomarkers ? (effects already demonstrated in vivo?) •= biomarkers of existing serious stress / intoxication • •Parameters evaluated -body weight -food consumption -fitness & welness • 1212569_21823227.jpg logo_mu_cerne.gif (Histo)pathology biomarkers 1212569_21823227.jpg logo_mu_cerne.gif Pathology •(-) Destructive methods, Time consuming, Professional requirements •(+) High relevance – organ/tissue changes - •1) microscopy of internal organs • A) observations of non-specific changes in internal organs B) specific changes, e.g. • in liver (dioxin-like POPs, cyanobacterial toxins ..) intersex / imposex formation (xenoestrogenicity) - 1 1 Example: Liver damage by cyanobacterial toxins microcystins 1212569_21823227.jpg logo_mu_cerne.gif Endocrine disruption: Intersex microscopy Testicular tissue http://histology-world.com/photoalbum/albums/userpics/normal_testis20x.jpg Oocytes within testis 1212569_21823227.jpg logo_mu_cerne.gif •2) immunohistochemistry & microscopy • : determination of “specific” changes in tissues : Fluorescein (FITC) - labeled antibodies (Ab) applications • Example à toxicant induced autoimmunity: anti-nuclear Ab (ANA test) 1 1212569_21823227.jpg logo_mu_cerne.gif •2) immunohistochemistry & microscopy • anti-nuclear Ab (ANA test) Systemic lupus http://what-when-how.com/wp-content/uploads/2012/05/tmpa83a17.png 1 ANA test * Determination of antibodies in patient blood acting against “nuclei” proteins (ANA) : target: permeated liver cells on slide : application of blood (Ab) : visualization (secondary Ab) http://www.euroimmun.com/uploads/pics/IFT_PRIN_11_engl.gif 1212569_21823227.jpg logo_mu_cerne.gif Pathology •3) Nuclear DNA damage characterization 3.1. micronuclei (MN) evaluation by microscopy : example: MNs in blood lymphocytes of hospital workers (exposed to anticancer drugs – they are often carcinogenic) • • • • • • 3.2 chromosomal abnormalities karyotype biomarkers (human genetic disorders) • 1 1a 1212569_21823227.jpg logo_mu_cerne.gif 1 1 1 3) Nuclear DNA damage characterization 3.3.COMET ASSAY 1212569_21823227.jpg logo_mu_cerne.gif Example results - Comet assay vs. radiation 1212569_21823227.jpg logo_mu_cerne.gif Standard clinical chemistry & hematology biomarkers 1212569_21823227.jpg logo_mu_cerne.gif 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) • •Blood analyses • - chemistry and biochemistry • - cells (hemogram) •Urine analyses • - chemistry, cells, bacteria etc. • https://www.innocentive.com/projectImages/challenge/ic9701396_2.jpg http://www.hivandhepatitis.com/0_2010_images/blood-biomarker.jpg 1212569_21823227.jpg logo_mu_cerne.gif Example: intoxication & liver damage à change in biomarker profiles in blood chemistry and urine à Further assays possible: 1212569_21823227.jpg logo_mu_cerne.gif Methods in clinical chemistry •Methods: - automatic biochemical and hematological analyzers • - different „analytes”: various principles of methods (see example à) 1212569_21823227.jpg logo_mu_cerne.gif •Example • - determination of enzymatic activities in blood • - interpretation: tissue/organ-specific damage damage • Examples (toxicological studies) -Liver damage (toxicants, POPs, alcohol) -AST (Aspartate aminotransferase), -ALT (Alanine aminotransferase) in blood -General damage in cell (tissue non-specific) -LDH - lactate dehydrogenase -Muscle damage: -creatine kinase in serum (isozymes - tissue specific – muscle vs heart); • •Other enzyme biomarkersà see further Methods in clinical chemistry 1212569_21823227.jpg logo_mu_cerne.gif Coloured product: kinetic spectrophotometry Methods in clinical chemistry: example LDH analysis Done in automatic analyzer: •Blood sample + addition of (NAD+ tetrazolium salt + diaphorase enzyme) •Incubation and spectrophotometry determination •Automatic evaluation à final value (LDH activity) à comparison with “limits” à highlighting for a doctor • 1212569_21823227.jpg logo_mu_cerne.gif Example – changes in rat serum enzymes after CCl4 exposure 1858 1212569_21823227.jpg logo_mu_cerne.gif 145 Cell damage (Liver) enzyme activity (LDH) is also highly variable and species-specific 1212569_21823227.jpg logo_mu_cerne.gif Biomarkers: Changes in enzyme activities 1212569_21823227.jpg logo_mu_cerne.gif Enzymatic changes •Biomarkers reflecting „enzyme changes“: • –EXAMPLES - inhibitions of specific enzymes (as also discussed earlier during the class: MoA) •AcChE (organo-phosphates) •Proteinphosphatases (microcystins) – – •(+) Rapid enzymatic assays, specific responses •(-) Some ~ EXPOSURE biomarkers • 1212569_21823227.jpg logo_mu_cerne.gif Reminder: AcChE inhibition mechanism 21 1212569_21823227.jpg logo_mu_cerne.gif •Model Substrate (butyryl-thio-choline, acetyl-thio-choline) • - cleaved by AcChE à formation of free –SH groups • - reaction of SH with thiol reactive probe = Ellman´s reagent (DTNB) • à DTNB-S-choline: yellow colour (spectrophotometry A420) • Spectrophotometry AcChE assessment 1212569_21823227.jpg logo_mu_cerne.gif 29 Changes in AcChE in birds after exposure to organophosphates 1212569_21823227.jpg logo_mu_cerne.gif Proteinphosphatase (PPase) inhibition assay •Model substrates cleaved by PPase –32P-labelled protein à free 32P radioactivity –6,8-difluoro-4-methylumbelliferyl phosphate à fluorescence • 1212569_21823227.jpg logo_mu_cerne.gif Biomarkers – assessing gene and protein expressions / levels 1212569_21823227.jpg logo_mu_cerne.gif Protein modulation: toxic response at several levels • • • Nature (2015) vol 520, p. 37 Toxicants induce various changes in the cell … … many of these changes result in à activation / deactivation of specific genes à modulated gene expression à modulated protein levels ... and protein activities 1212569_21823227.jpg logo_mu_cerne.gif How to measure gene and protein modulations? • • • Traditional methods of QUANTIFICATION at different levels - mRNA levels - PCR / quantitative RT-PCR - protein levels - electrophoresis and Western-(immuno)blotting - ELISA techniques - induced protein enzymatic activities associated with elevated protein levels - eg. enzymatic activity New types of complex techniques: “omics” à also will be discussed later Examples of targeted protein biomarkers – discussed further specific protein markers of disease / e.g. cancer heat shock proteins (hsp90, hsp60, hsp 70, ubiquitin) metalothioneins endocrine disruption biomarkers - Vitellogenin(-like) Vtg proteins in male - Aromatase Induction of detoxification enzymes - CYP450 / EROD - GST 1212569_21823227.jpg logo_mu_cerne.gif •Tumor genes and tumor markers • - cancer genes ras, myc – e.g. metastasing bowel cancer • - a-fetoprotein (AFP) – elevated during fetus development AND e.g. liver cancers • - tumor suppressor genes (e.g. p53) – indicate better prognosis for certain cancers • - PSA – prostate-specific antigen: prostate cancer in males (over 50 years of age) • •Methods of determination in practice: • ELISA (enzyme linked immunosorbent assays) • ESTABLISHED PROTEIN MARKERS – determination in blood 1212569_21823227.jpg logo_mu_cerne.gif Heat Shock Proteins (hsp) • • • General stress = synthesis of new proteins ~ equilibrium and homeostasis buffering - temperature (cold / heat) à proteins assuring cryo-preservation - salinity & metals à ion buffering - organic xenobiotics à detoxication New proteins must be folded to their 3D stucture by activity of „CHAPERONES“ Chaperons = hsp90, hsp60, hsp 70 ~ 60-90 kD molecular weight kD 1212569_21823227.jpg logo_mu_cerne.gif HSP determination - example • • • HSP = GENERAL STRESS biomarker, non-specific - phylogenetically conserved (similar genes in most of the organisms) - structural similarity à easy determination: electrophoresis + immunoblotting (Western blotting) http://proteomics.case.edu/proteomics/westernblot-sm.jpg 1 1212569_21823227.jpg logo_mu_cerne.gif 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) à exposure elimination - natural function (?) – regulation of essencial metals in cells Metalothioneins (MTs, MT-like proteins) 1212569_21823227.jpg logo_mu_cerne.gif Protein biomarkers of estrogenicity / ED-like effects ER = transcription factor controling number of target genes Target genes of ER = biomarkers of estrogenicity Major examples •Vitellogenin •Aromatase - CYP19A 1212569_21823227.jpg logo_mu_cerne.gif Vitellogenin (Vtg) Precursor of yolk proteins, phospho-protein („energy“ rich) à egg formations (females) at oviparous vertebrates Synthesized in liver and distributed via blood / haemolymph Xenoestrogens & other endocrine disruptors à increased levels or early production in FEMALES à production de novo in MALES 1 1212569_21823227.jpg logo_mu_cerne.gif 1)ELISA in exposed organisms (F/M) or in vitro (-) specific antibodies are necessary for each species (low crossreactivity of Abs) 2) „Vitelin-like proteins“ - total amount of „alkali-labile“ phosphate in haemolymph (mussels) - alkaline extraction of P from sample à spectrophotometric determination - Vitellogenin (Vtg) assessment 1212569_21823227.jpg logo_mu_cerne.gif 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 1212569_21823227.jpg logo_mu_cerne.gif Vitelin-like proteins in mussels 1212569_21823227.jpg logo_mu_cerne.gif Aromatase (CYP19A) - Levels inducible by estrogens - Catalyzes single enzymatic step androgens à estrogens Experimental assessment - mRNA (in reseach and practice) - 1. PCR / Quantitative-Real-Time-PCR 2. GM-organisms (zebrafish): reporter gene with GFP Green Fluorescence Protein under the control of aromatase promoter 1212569_21823227.jpg logo_mu_cerne.gif PCR principle 1212569_21823227.jpg logo_mu_cerne.gif Visualization of PCR product 1)Electrophoresis (qualitative) Intercalation dyes – e.g. ethidium bromide 1) 1) 1) 1) 1) 1) 1212569_21823227.jpg logo_mu_cerne.gif 2a) Real-time (quantitative) SYBR GREEN dye àmore DNA synthesized, more fluorescent dye incorporated àHigher fluorescence - Visualization of PCR product 1212569_21823227.jpg logo_mu_cerne.gif 2b) Real-time (quantitative) TaqMan probes (more DNA replications more fluorescent dye released) Visualization of PCR product 1212569_21823227.jpg logo_mu_cerne.gif “Quantitative” determination of PCR product 1212569_21823227.jpg logo_mu_cerne.gif qPCR determination of the aromatase gene in Zebrafish http://dx.doi.org/10.1016/j.ygcen.2005.12.010, 1212569_21823227.jpg logo_mu_cerne.gif GFP-reporter for estrogens in zebrafish embryo http://endo.endojournals.org/content/152/7/2542.full 1212569_21823227.jpg logo_mu_cerne.gif –Inductions of detoxication & oxidative stress enzymes (hepatopancreas / liver / blood) • MFO - CYP classes - EROD / MROD / BROD • Phase II enzymes (GSTs) • Glutathion metabolism enzymes (GPx, GRs) • http://pubs.rsc.org/services/images/RSCpubs.ePlatform.Service.FreeContent.ImageService.svc/ImageSer vice/Articleimage/2008/CS/b801558a/b801558a-f3.gif DETOXIFICATION / ANTIOXIDANT DEFENCES 1212569_21823227.jpg logo_mu_cerne.gif MFO (CYPs) - reminder 88 90 1212569_21823227.jpg logo_mu_cerne.gif • • • Determination of CYP1A1 activity “EROD” - EthoxyResorufin-O-Deethylase activity Substrate: Ethoxyresorufin : Oxidation by CYP1A1 (MFOs) à Fluorescence (easy determination) EROD = sensitive biomarker of organic pollution (exposure & effects) : AhR-activating compounds (PCDD/Fs, PCBs, PAHs) : often used in environmental studies Use of other substrates: assessment of other CYPs BROD – butoxy-ROD (CYP3A), MROD, PROD … Assessment of CYPs (MFO) – “EROD” 1212569_21823227.jpg logo_mu_cerne.gif 105 1212569_21823227.jpg logo_mu_cerne.gif 103 Locality: Reference Exposed 1212569_21823227.jpg logo_mu_cerne.gif E3 EROD variation on male and female carp from the Anoia and Cardener tributaries – seasonal variability & response at contaminated localities 1212569_21823227.jpg logo_mu_cerne.gif MFO responses (EROD) are strongly species specific & not always related to clinical signs 52 1212569_21823227.jpg logo_mu_cerne.gif MFO-responses (EROD) depends on animal size and metabolism rate 93 1212569_21823227.jpg logo_mu_cerne.gif Phase II conjugation enzymes - GSTs GSTs soluble and membrane (endoplasmic reticulum) variants: activities can be measured in cytoplasm or ER microsomes Methods Chemical reaction of reduced GSH + thiol selective probe (CDNB) GST GSH + CDNB à S-CDNB (formation of coloured product) kinetic or endpoint determination 1212569_21823227.jpg logo_mu_cerne.gif GST activity determination: example • • • Kinetic assessment of GSTs stress à Induction of GSTs faster reaction = increasing slope of the kinetics 1212569_21823227.jpg logo_mu_cerne.gif Biomarkers of oxidative stress 1212569_21823227.jpg logo_mu_cerne.gif Oxidative stress markers Several parameters respond to oxidative stress : enzymes – detoxification, antioxidants: GPx, GR, GSTs) .. - enzymatic activities (see elsewhere) : antioxidants – e.g. GSH (discussed further), vitamin E : markers of oxidative damage - membranes: MDA (discussed further) - DNA: 8OH-dG (see at DNA damage / adducts-exposure biomarkers) - proteins: oxidized forms (carbonyls) 1212569_21823227.jpg logo_mu_cerne.gif Oxidative stress markers GSH - antioxidant (scavenger of ROS) & reactive molecules - conjugation molecules for detoxication - probable intracellular regulatory molecule (? apoptosis ?) Total glutathione = reduced GSH + oxidized GSSG Method of determination (thiol selective probe DTNB) GSH + Ellman´s reagent (DTNB) à Reduced GSH GSH + GSH-reductase + DTNB à Total GSH Total – Reduced = Oxidized 1212569_21823227.jpg logo_mu_cerne.gif Example - GSH modulation by toxic nanoparticles Bláhová et al. 2014 Anal Bional Chem 406:5867–5876 1212569_21823227.jpg logo_mu_cerne.gif Markers of oxidative DAMAGE 1212569_21823227.jpg logo_mu_cerne.gif Lipid peroxidation à Malondialdehyde (MDA) MDA – malondialdehyde product of lipid peroxidation 1212569_21823227.jpg logo_mu_cerne.gif MDA – formed from oxidized membrane phospholipids : determination: - HPLC (instrumental) - TBARS (spectrophotometric) method TBARS – ThioBarbituric Acid Reactive Species : less specific than HPLC : easy determination (spectrophotometry) Method: 1) sample extract (with MDA) 2) add TBA 3) boil (cca 30´ / 90°C) à formation of red/violet coloured product 4) determination by spectrophotometry (A 540 nm) Malondialdehyde (MDA) determination TBA 1212569_21823227.jpg logo_mu_cerne.gif MDA modulation - examples Effects of nanoFeOxide particles on MDA in fish https://encrypted-tbn0.gstatic.com/images?q=tbn:ANd9GcTy3e9k01WAAiapIc54bT4bXPiQQ5yA_K0u_u_fIBh8U_C N3EkjOA http://www.scielo.cl/fbpe/img/ijmorphol/v31n3/art49_f3.jpg Induction of MDA (TBARS) by carbamazepine (and protection by antioxidants)