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
11 – BIOMARKERS
of EXPOSURE and SUSCEPTIBILITY
OPVK_MU_stred_2

1212569_21823227.jpg logo_mu_cerne.gif
Biomarkers of Exposure
•Biomarkers of internal and effective dose
depends on toxicokinetics
•
Biomarkers of internal dose (short / long term)
– examples: Cd in urine, DDE in fat tissues
– - should be easy to sample (urine, breath)
– - instrumental analytical methods (analyses of toxicant)
•
•
• Biomarkers of effective dose
- the chemical interacted with the biological target
à analyses of ADDUCTS
• Two types of adducts: selective and non-selective

1212569_21823227.jpg logo_mu_cerne.gif
SELECTIVE ADDUCTS OF TOXICANTS with BIOMOLECULES
•SELECTIVE = CHEMICAL-SPECIFIC
•
•Adducts with DNA
styrene-oxide-O6-guanine
N7-guanyl-aflatoxin B1
•
•Hemoglobin-pesticides adduct
•
•
•Methods of analyses:
• - analytical chemistry
• - extraction from biological sample
• - chemical determination by HPLC or GC
•

1212569_21823227.jpg logo_mu_cerne.gif 1880



1212569_21823227.jpg logo_mu_cerne.gif
PAH-DNA adducts
PAH (polycyclic aromatic hydrocarbons)
* often high variability
* may have difficult interpretation
1886
Occup. exposure
(Low / Intermed. / High)
Occupational
Non-exposed (NS)
vs.
Exposed (S)

1212569_21823227.jpg logo_mu_cerne.gif
•– binding with macromolecules (DNA, proteins) with no further information on the structure of
actual adduct
(i.e. causative agent not clear)
•
•Typical nonselective biomarker methods
• - 32P-postlabelling assay
• - oxidized DNA: 8-hydroxy-2´-deoxyguanosine
–
Non-selective adducts

1212569_21823227.jpg logo_mu_cerne.gif 1846
32P-postlabelling assay
principle
•Digestion of NA
•Enzymatic labelling with 32P (kinase)
•TLC or HPLC analyses of products
TLC result
(thin layer chromatography)
A - 2-5 = various adducts
B - controls

1212569_21823227.jpg logo_mu_cerne.gif
8-hydroxy-2´-deoxyguanosine analysis
Oxidative damage to DNA
- many causes à 8-OH-dG is the most common marker of DNA oxidation
Analysis: analytical chemistry methods
- HPLC
- immunochemistry (ELISA)

1212569_21823227.jpg logo_mu_cerne.gif
Biomarkers of susceptibility


1212569_21823227.jpg logo_mu_cerne.gif
Toxicokinetics
determines susceptibility of an individual at various levels
à Biomarkers
of susceptibility
Will the individual be sensitive?
Will patient respond to a drug?
1876

1212569_21823227.jpg logo_mu_cerne.gif
Importance of susceptibility biomarkers
1875

1212569_21823227.jpg logo_mu_cerne.gif
Biomarkers of susceptibility
•Susceptibility depends on genotype and metabolism
• - genetic polymorphism in detoxification enzymes
• - variability in specific isoenzymes
•
àsusceptibility to „activate“ toxicants:
example: N-acetylation of arylamines – NAT2
àsusceptibility to genotoxins
Ã
àfamily cancers
àsusceptibility to drugs (including anticancer drugs)

1212569_21823227.jpg logo_mu_cerne.gif
Example: genetic polymorphism
SNPs - single nucleotide polymorphism
•SNPs
à affects protein functions
• à in specific cases (see example) some SNPs identified
•
•à PERSONALIZED MEDICINE
•
•To identify SNP as a biomarker
• Many genotypes (from many individuals) must be sequenced and compared with phenotype (e.g.
responsiveness to certain drug)

1212569_21823227.jpg logo_mu_cerne.gif
Cyclophosphamide (anticancer drug) and its toxicity
Genetic
polymorphism

1212569_21823227.jpg logo_mu_cerne.gif
Example: genetic polymorphism
Alleles known
to be involved
in polymorphism
http://www.deenabio.com/wp-content/uploads/2011/04/SNPQuickReference.jpg

1212569_21823227.jpg logo_mu_cerne.gif
Personalized medicine
http://www.bayerpharma.com/html/images/upload/Oncology/personalisierte_medizin_en.jpg
SNP diagnostics:
1) DNA isolation
2) Multiplication of specific gene eg. CYP
3) SNP identification
... Molecular biology methods such as
* NA sequencing
* Probe pairing ... number of variants
•
http://upload.wikimedia.org/wikipedia/en/0/01/SNP-invader-1.jpg