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Redox homeostasis & oxidative stress
Redox homeostasis
- natural levels of oxidants (O2) and antioxidants in each cell
Disruption of redox homeostasis
-> depletion of oxygen: metabolism disruption, acidosis in
tissues, cell necrosis
-> overproduction of oxidants: depletion of antioxidants,
oxidation of biological molecules (membranes, proteins,
DNA ...)
-> disruption of signalls (GSH), carcinogenesis, health
problems, necrosis ...
= oxidative stress
Overproduction of oxidants
Oxygen ­ principal molecule in living organisms
Oxygen increase or reactive derivatives -> toxicity
ROS = Reactive Oxygen Species: Sources
- production in mitochondria (byproducts)
- redox-cycling (quinones of xenobiotics)
- Fenton-reaction (metals)
- oxidations mediated via MFO (CYP)
- depletion of antioxidants (reactive molecules)
Reactive Oxygen Species (ROS)
Druh Symbol Poločas života (s) při 37 °C
Superoxid O2
ˇ -
1 x 10 ­6
Hydroxylový radikál OHˇ
1 x 10 ­9
Alkoxylový radikál ROˇ
1 x 10 ­6
Peroxylový radikál ROOˇ
1 x 10 ­2
Singletový kyslík O2 1 x 10 ­6
Molekulární kyslík O2 > 10 2
ROS & mitochondria
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Fenton reaction Redox-cycling and ROS formation
O2 O2
O
O
e -
O -
O
.
. -
e -
O -
O -
2e -
Toxicity
of ROS
Patologické
narušení
Metabolické
procesy
Xenobiotika
R
Volné radikály
R .
O2
O2 , OH , 1O2
-.
Antioxidanty
Oxidované
aminokyseliny
ROS
Oxidované
nukleové kyseliny
Oxidované
nukleotidy
H2O2
CH 2
COOHH2N
OH
o-tyrozin
lipidní peroxidyOOH
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
Examples of chemical-induced
oxidative stress
- Metals: fenton reaction -> OH*
- Depletion of GSH:
reactive molecules, GST-conjugation,
metals: SH oxidation ...
- Redox-cycling chemicals: oxy-PAHs
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GSH
and its depletion Poškození Produkt Stanovení Citace
fosfolipidy
MDA
TBARS assay,
HPLC, HPLC s UV-
detekcí
Draper et al.
1993, Bird et
al. 1983, Selim
1977
DNA
8-OH-dG HPLC, metoda
s využitím
imunoafinitní izolace
Degan et al.
1991, Loft et al.
1992
proteiny
o-Tyr
spektrofotometricky,
HPLC, MS
Deneshvar et
al. 1997, De
Zwart et al.
1998
Vysvětlivky:
MDA malondyaldahyd
8-OH-dG 8-hydroxy-2
-deoxyguanozin
o-Tyr orthotyrozin
TBARS reaktivní látky s kyselinou thiobarbiturovou
HPLC vysokotlaková kapalinová chromatografie
MS hmotnostní spektrometrie
Biomarkers of oxidative damage
DNA damage
mutagenicity and genotoxicity
DNA:
- principal molecule for life of the cell
- structure and function carefully checked
- changes rapidly repaired
- irreversible changes -> cell death (apoptosis)
Mutagenesis
- changes in the sequences of deoxynucleotides
- deletions/insertions: changes in reading frame
- exchanges of nucleotides: changes in aminoacids
- natural mutations (billions of nucleotides/day)
: variability in genoms; reparations
- chemical-induced mutagenesis
Trosko and Ruch 1998, Frontiers in Bioscience 3:d208
AhR
ER
GJIC
Oxidační stres
IMPORTANT PROCESSES IN CANCEROGENESIS
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Chemical induced DNA damage
Bases analogs
- incorporation into DNA during replication
(5-Br-Uracil: AT -> GC)
HNO2, HSO3
-, Hydroxylamine, Methoxyamine-
- deamination of bases (GC -> AT)
Alkylsulphates, N-nitroso-alkyles, cis-platinum
- alkylation of bases; crosslinks of dsDNA
Polycyclic aromatic hydrocarbons (PAHs) & derivatives
(N-acetyl-2-aminofluorene (AAF), benzo[a]pyrene)
Mycotoxins (aflatoxins)
- require metabolic activation by CYPs
- aduct formation with DNA (biomarkers)
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Metabolic activation of PAH
and DNA-aduct formation
Metabolic activation
of aflatoxin
and formation
of DNA-aducts
Does chemically-induced genotoxicity has effects in vivo ?
- adducts from mitochondrial DNA ?
- distance between ,,source of radicals" and nuclear DNA ?
- protection mechanisms (mutation -> death)
Rubin (2002) Oncogene 21:7392
Thilly (2003) Nature Genetics 34(3):255
Mutations are not caused by chemicals
Chemicals only allow ,,unveil" previously existing mutations in
nuclear DNA (non-genotoxic events cause cancer !!!)
Physical factors & DNA damage
Ionizating radiation
- direct interaction with hydrogen atoms in water
(and bases)
-> OH* radicals; H2O2, O2-
- oxidation of bases; dimerization ...
UV radiation
- interaction with aromatic cycles (bases)
- base dimerization (T=T)
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Ionizing radiation effects on DNA DNA repair
Damage of DNA is carefully controlled
constitutively expressed proteins
Changes in DNA
induction of reparation enzymes ("SOS-repair")
= biomarker of DNA damage