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Biomarkers
Biomarkers
- markers in biological systems with a sufficently
long half-life which allow location where in the
biological system change occur and to quantify the
change.
Toxicology ­ present status:
- identification of markers of long-term risks
: human toxicology ­ carcinogenesis
: ecotoxicology ­ early markers of toxic effects
Biomarkers - summary
Biomarker:
change which occurs as response to "stressors"
(xenobiotics, disease, temperature...) which extend
the adaptive response beyond the normal range
In vivo biomarkers:
changes measured in stressed animals
("classical biomarkers")
In vitro biomarkers
in vitro assessment for characterization of
xenobiotic potencies to induce specific biological
activity (genotoxicity, estrogenicity, dioxin-like
activity, tumor promotion ...)
Biomarkers
at different
levels
of biological
organisation
Biomarkers - classification
Categorization according to US Nat. Academy of Science
- Biomarkers of exposure
- Biomarkers of response or effect
- Biomarkers of susceptibility
Continuum exists
adducts with DNA ? response / ? exposure
Biomarkers & sampling
invasive / non-invasive
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Biomarkers
- Paracetamol
(1) paracetamol
(2) parent compound measurement - exposure
(3) activation to reactive metabolite (N-ac-p-benzoquinone, NAPQI) by CYPs; reaction with
GSH / measurement ­ levels of CYPs; levels of GSH ­ susceptibility)
(4) GSH-NAPQI conjugate ­ exposure, susceptibility
(5) NAPQI-protein adducts -> toxicity: exposure, effective dose
(6) adaptations: GSH depletion, inhibition of protein synthesis ­ biomarkers of response
(7) protein alkylation -> degeneration of hepatocytes: necrosis -> increase concentrations of
bile acids, bilirubin in plasma; start of inflamation in degraded tissue ­ response / effect
Human biomarkers ­ example
Human biomarkers ­ example
Specific (selective) in vivo biomarkers
- Biomarkers selectively reflecting specific types
(mechanisms) of toxicity
- E.g. inhibition of AcCholE :
exposure = organophosphates; effect = neurotoxicity
+ specific information
- multiple biomarkers must be measured
Non-specific (non-selective) in vivo biomarkers
- Biomarkers of general stress
- E.g. induction of Heat Shock Proteins (hsp)
+ general information about stress
- sensitive to many "stressors" (temperature, salinity ...)
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In vivo biomarkers
- Non-destructive
: blood / haemolymph collection & analyses
: skin, feather, hair ... contamination
- Destructive
: whole animal -> multiple biomarker evaluation
Non-destructive
biomarkers
What kind of biomarkers to measure ?
Do we know possible exposure (toxicant) ?
- specific biomarkers
? estrogenic effects in effluents
? dioxin-like effects, mutagenicity in urban areas
? neurotoxicity (AcChE) in rural areas
Do we expect varying exposure / contamination ?
- integrated approach
- non-specific biomarkers (hsp) as predictors of
stress level
Multiple biomarker evaluation
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Biomarkers
& Exposure
h: homeostatic conditions
c: reversible stage
r: irreversible effects of pollutants
Biomarkers:
- transitory
­ B5, B2; short period: B4
- continuous increase ­ B3
- repeated appearance (B5)
­ irreversible change
Biomarkers of Exposure
Biomarkers of
- internal dose (short / long term)
­ Cd in urine, DDE in fat tissues
- should be easy to sample (urine, breath)
- effective dose
- the chemical interacted with the target
= ADDUCTS
Biomarkers of Exposure - ADDUCTS
Selective aducts (chemical-specific)
- DNA aducts: styrene-oxide-O6-guanine; N7-guanyl-
aflatoxin B1; hemoglobin-pesticides
- chemical determination (HPLC/GC)
Aselective aducts
­ binding with DNA (proteins) but no info on structure of
aduct
- 32P-postlabelling assay
- identification of oxy-DNA (8-hydroxy-2´-deoxyguanosine)
- DNA-strand breaks ­ alkaline unwinding assay or comet
assay)
32P-postlabelling assay
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DNA-unwinding assessment Comet assay Genetic damage in fish exposed to BaP
PAH-DNA adducts
Occup. exposure
(Low / Intermed. / High)
Occupational
Non-exposed (NS)
vs.
Exposed (S)
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Biomarkers of susceptibility
Metabolism
- variability in specific enzymes
- susceptibility to modify toxicants: N-acetylation of
arylamines ­ NAT2
- null genotypes for conjugation enzymes (GSTM1)
Genotype
- familial cancers & susceptibility to genotoxins
Biomarkers of susceptibility
Biomarkers
of susceptibility
In vivo biomarkers of effects / response
Do we know the agent ? Do we expect the effect ?
: specific biomarkers / non-specific changes
Behaviour and Clinical biomarkers
Pathology
Clinical chemistry
Enzymatic changes
Protein synthesis
Oxidative stress markers
+ Human: Excretory products in urine
Tumor genes and tumor markers
cancer genes ras, myc, -fetoprotein (AFP)
suppressor genes p53, Rb
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Behaviour and clinical biomarkers
Parameters evaluated
- body weight
- food consumption
- fitness & welness
Interpretation
: ? biomarkers ? effects already demonstrated in vivo
- biomarkers of existing serious stress / intoxication
Behaviour and clinical biomarkers
Pathology
(-) Destructive methods, Time consuming, Professional requirements
(+) High relevance ­ organ/tissue changes
- microscopy of internal organs
: non-specific changes in internal organs
: specific changes in liver (dioxin-like POPs, cyanobacterial toxins)
: intersex / imposex formation (xenoestrogenicity)
- immunohistochemistry & microscopy
: determination of specific changes
: Fluorescein (FITC)- labeled antibodies (Ab) applications
- determination of vitellogenin in male organs (anti-Vtg Ab)
- autoimmunity (anti-nuclear Ab, ANA, in exposed organisms)
- chromosomal abnormalities & micronuclei evaluation
: karyotype biomarkers
: non-destructive (blood samples; plant tissues)
Pathology - Liver damage by
microcystins
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Pathology ­ Intersex microscopy
Oocytes
in testicular tissue
Immunohistochemical determination of
Vtg in male fish
Immunohistochemistry of
ANA in autoimmune serum
Chromosomal aberations
Micronuclei determinations
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Clinical chemistry
Non-destructive
Often specific interpretation
- determination of enzymatic activities in blood
- response to tissue/organ damage
- muscle damage: creatine kinase in serum
: isozymes - tissue specific (brain, muscle, heart);
- heart attack ­ isozymes of lactate dehydrogenase (LDH)
- liver damage ­ AST (....), ALT (....) in blood
: cyanotoxins, dioxin-like POPs
Example ­ changes in rat serum enzymes after CCL4 exposure
Enzymatic 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
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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)
Cholinesterase Inhibition in Bobwhite
0.0
20.0
40.0
60.0
80.0
100.0
0.0 1.0 2.0 3.0 4.0 5.0
Organophosphate Dose (g/L)
Cholinesterase
(%ofcontrol)
AcChE inhibition
mechanism & effects in birds
AcChE inhibition
mechanism & effects in birds
PPase 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
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MFO (CYP) activities MFO (CYP) activities
EROD assay
- endoplasmic reticulum (membrane bound) CYPs ­ mirosomal
vesicles (S9-fraction)
substrate: Ethoxyresorufin
-> Oxidation by CYP1A1 -> Fluorescence
EthoxyResorufin-O-Deethylase activity EROD
- other substrates: CYP isozymes: BROD, MROD, PROD ...
AHH (ArylHydrocarbon Hydroxylase) ~ similar method for MFO
- substrate: Benz[a]pyrene -> oxidation
Biomarker of organic pollution (exposure & effects)
: AhR-activating compounds (PCDD/Fs, PCBs, PAHs)
: often used in environmental studies
Locality:
Reference Exposed
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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
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
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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
Substrates reduced GSH + thiol selective probe (CDNB
GST
GSH + CDNB -> GS-CDNB
yellow product (A420), kinetic or endpoint determination
Kinetic assessment
stress -> Induction of GSTs
faster reaction -> slope of kinetic increase
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GST activity - example
Kinetic assessment of GSTs
stress -> Induction of GSTs
faster reaction -> slope of kinetic increase
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
GSH-related oxidative stress enzymes
Glutathion-reductase (GPx), Glutathion-peroxidase (GR)
Enzymatic reactions ­ differing in substrates (GSH +/- H202 ...)
- generally: NADPH consumption during reaction
- NADPH ­ easily determined (A340 nm)
Design ­ GPx:
Substrates (GSH, organic peroxide, NADPH)
Enzyme (biotic sample)
A340 kinetic record (slope of kinetics decrease with GPx activity)
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0 2 4 6 8
Blank
DMNQ (5 ug/L)
A 340nm
GPx activity induction by Dimethoxynaphthoquinone (DMNQ)
in Xenopus laevis embryos
-1.8
-1.6
-1.4
-1.2
-1
-0.8
-0.6
-0.4
-0.2
0
Blank 0.1 0.5 2.5 10
Slope
PROTEIN SYNTHESIS
Determination of specific proteins
amount quantification
- mRNA (in vitro assays)
- protein
- electrophoresis and Western-(immuno)blotting
- ELISA techniques
Complementary to enzymatic assays !!!
e.g. CYPs - mRNA -> protein amount -> activity
Examples
heat shock proteins (hsp90, hsp60, hsp 70, ubiquitin)
metalothioneins
Vitellogenin(-like) Vtg proteins in male
Superoxid dismutase (SOD)
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) ­ ,,CHAPERONES"
- hsp90, hsp60, hsp 70 ­ 60-90 kD molecular weight kD
- GENERAL STRESS biomarker, non-specific
- phylogenetically conserved (similar sequences in ,,all" organisms)
- structural similarity => easy determination:
electrophoresis + immunoblotting
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Heat Shock Proteins (hsp) HSP determination - example
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) Induction of SOD in plants
- protein electrophoresis + immunoblotting
SOD ­ superoxid dismutase;
induced by oxidative stress
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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
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 - ELISA
Vitelin-like proteins in mussels Oxidative stress markers
Several parameters respond to oxidative stress
: enzymes (GPx, GR, GSTs) - elsewhere
: antioxidants (GSH, vit E)
: markers of oxidative damage (MDA, 8OH-dG)
______________________
Determination of GSH (complex role in organism)
- 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
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Oxidative
stress
markers
Patologické
narušení
Metabolické
procesy
Xenobiotika
R
Volné radikály
R .
O2
O2 , OH , 1O 2
-.
Antioxidanty
Oxidované
aminokyseliny
ROS
Oxidované
nukleové kyseliny
Oxidované
nukleotidy
H2O2
CH 2
COOHH2N
OH
o-tyrozin
lipidní peroxidy
OOH
OH
OH
COOH
OH 8-izoprostan
N
NO
OH
tymidin glykol
O
N
N N
N
O
OH
H2N .
R
8-hydroxy-guanozin
R
R
konjugované dieny
alkany
aldehydické
produkty
R O
O O
R O
n-aldehydy
malondialdehyd
OH
R
O
trans-4-hydroxyalkeny
Malonyldialdehyde (MDA)
MDA ­ formed from oxidized membrane phospholipids
: determination: HPLC or TBARS method
TBARS ­ ThioBarbituric Acid Reactive Species
: less specific than HPLC (+/- aldehydes)
: easy determination (spectrophotometry)
Method:
1) sample extract (virtually containing MDA) + TBA
3) boiling (cca 30´ / 90°C)
=> formation of red/violet coloured product
4) determination by spectrophotometry (A 540 nm)
GSH & MDA - modulation / - example
patients control