mutagenes and carcinogenes in environment rndr z.polívková lecture no 519 – course: development...
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Mutagenes and carcinogenes in environment
RNDr Z.Polívková
Lecture No 519 – course: Development of cells and tissues
History of mutagenesis:
1928: Müller: X-irradiation – mutations in Drosophila
1946: Auerbach and Robson: mutagenity of yperite
1945: Hirošima, Nagasaki
Genotoxic effect - due to DNA binding - adducts, chemical modification of bases, DNA breaks
DNA and chromosomal damage - associated with tumor origin (mutagen = potential carcinogen)
Individual sensitivity to genotoxicants is influenced by polymorphism of genes involved in xenobiotic metabolism or in DNA reparation
= individual risk to cancer
DNA reparation (of damage caused by mutagenic and carcinogenic compounds) maintains genome stability
Genotoxicity
Consequences od unrepaired DNA damage - mutations:
• aging
• apoptosis (cell suicide)
• unregulated cell division - tumors
Exogenous DNA damageExamples of mutagenes/carcinogenes
PesticidesDDT
Industrial compounds PCBs (polychlorinated biphenyles)
Air pollutants benzo(a)pyrene,
Mycotoxins aflatoxin B1, ochratoxin A
Heavy metalschromium, arsenic, cadmium
Physical factorsUV, ionizing radiation
Endogenous DNA damagee.g.reactive oxygen species, …..
e.g.errors in replication, reparation…..
Sources of DNA damage
UV radiation (200-300nm) – pyrimidine dimers = covalent bonds that crosslink adjacent pyrimidine bases (C,T)
- free radicals
Ionizing radiation - free radicals
- DNA single-strand breaks (SSB) and double-strand breaks (DSB)
Chemicals - base alkylation (methylation)
- adducts = chemical entities attached to DNA
(procarcinogenes are metabolically converted into reactive carcinogens = oxidized forms)
Chemically modified bases have different pairing properties in replication
Environmental factors:
Endogenous damage:
hydrolysis – cleaves base from DNA strand (depurination, depyrimidination)
deamination (e.g. deamination of C→U – unrepaired U is misread as T during replication)
methylation and deamination – deamination of methyl C →T = mutation C →T
oxidation – (reactive oxygene species originate during metabolism → base oxidation (e.g.oxo-G is paired with A, point mutation C →A)
base cleavage (depurination, depyrimidination), DNA strand breaks
DNA breaks originate during normal cell processes – e.g.intermediate step of exision repair,…
Replication errors not detected by „proofreading“ activity of DNA polymerase
Direct mutagens Indirect mutagens -metabolic activation
Ionizing radiation
epoxides
Free radicals
cyt P-450
deto
xifik
catio
n
DNA
Addutson DNA bases,
oxygenated bases, DNA
breaks
Spontaneous or enzymaticreparation
Repair BER, NER, DSB repair
DNA replicatio
n
Mutations
Chromosomal aberrations
apoptopsis cell death
no mutationsR. Štětina,2007
Cell response to DNA damage – DNA repair
Response Mechanisms
Direct reversal of DNA damage: enzymatic fotoreactivation (dimers splitting) direct ligation of DNA breaks
methylation reversed by methyltransferase
Lesions on single DNA strand :
Excision of DNA damage Base excision repair (BER) Nucleotide excision repair (NER)
“mismatch repair" (MMR) Double strand breaks repair : Nonhomologous end joining - NHEJ Homologous recombination - HR Single strand annealing- SSA
Base excision repair- repair of bases damaged by reactive oxygen species, deamination, hydroxylation, methylation – origin during metabolic processes – only one base is damaged
damaged (chemically modified) base is removed by glycosylaseMin. 10 proteins (glycosylases) specific for each type of lesionsglycosylase
AP endonuclease
polymerase, ligase
abasic site removed by specific endonuclease
gap filled by DNA polymerase β and sealed with ligase
endonuclease
exonuclease
polymerase
ligase
Nucleotide excision repair
Incision near to the damaged site (e.g. dimere) by endonuclease
The oligonucleotide with the damaged base is removed by exonuclease
DNA polymerase δ and ε
fill the gap
The process is completed by sealing of strands by the ligase
=repair of lesions caused by exogenous mutagens (adducts, dimers..)defect of minimally two nucleotides with distortion of DNA double helix
30 proteins involved in NER (XPA-G, CS genes)
NER present in chromatin (not naked DNA) supposes remodeling of nucleosomes before repair in global genomic repair GG-NER
Repair in actively transcribing DNA, slightly different, more quick = transcription coupled repair TC-NER
Syndroms associated with error in NER:Xeroderma pigmentosumTrichothiodystrophyCockayne syndrome (TC-NER)
NER:
„Mismatch repair“ (MMR)
= repair of errors in base pairing and insertion or deletions of bases (origin during replication) - not detected by„proof reading“ activity of DNA polymerase
= repair of normal but mismatched bases on newly replicated DNA strand
- recognition and nick by endonucleas
- removal of DNA by exonuclease
- resynthesis by DNA polymerase delta
- ligation
Several proteins are involved (MSH.., MLH.., PMS..)
Error of MMR (germline defect in MMR gene + somatic mutation of second allele) → hereditary non-polyposis colon cancer (HNPCC)
HNPCC is connected with microsatelite instability = change in length in microsatellite sequences (= short repetition of 1-5 nucleotides) caused by insertion/deletion of nucleotides
Origin of double strand breaks (DSB):: endogenous: oxidative metabolism
topoizomerases (single strand breaks-SSB,DSB)
errors in DNA replication or reparation
DNA recombination – crossing over in meiosis
V(D)J recombination, „class switching“ of immunoglobuline genes
exogenous: radiation (ionizing, ultraviolet), chemicals
restriction endonucleases
DSB are induced directly – by ionizig radiation
or indirectly – by UV radiation, chemicals + enzymatic repair → SSB(single-strand breaks) → DSB (double-strand breaks)
Repair of double strand breaks
Double strand breaks repair :
NHEJ = nonhomologous end joining – mainly in G0, G1
- without presence of homologous template – more prone to errors
HR = homologous recombination
- needs presence of sister chromatid (feasible in G2, S phases of cell cycle)
- or presence of homologous chromosome (meiotic recombination)
SSA - single strand annealing- needs homology on the same chromosome
2 main mechanisms: NHEJ and HR - error free elimination of DSB
- or mutation and chromosomal aberration – consequence of erroneous reparation
Errors in DSB repair: Ataxia teleangiectasia, Nijmegen breakage syndrome, Fanconi anemia, trichothiodystrophy, cancers
1) NHEJ = nonhomologous end joining works in all stages of cell
cycle, in G0,G1 only NHEJ
- without need for homologous template - more prone to errors
connection of broken ends without sequentional homology on adjacent ends
-it is also mechanism of V(D)J recombination of immunoglobuline genes, or isotype switch
Ligation of broken ends on different chromosomes → CHA (translocations,
dicentrics..)
Genes involved: XRCC4, heterodimer of proteins Ku 70/Ku80….
DSB repair:
DSB repair :
2) HR = homologous recombination
presence of sister chromatid (in G2 or S phases of cell cycle) or presence of homologous chromosome (meiotic recombination)
HR– in meiosis = crossing-over - in mitosis between sister chromatids
Many genes responsible for HR: e.g. BRCA1, BRCA2
XRCC1,XRCC2,NBS1,Rad 51 genes etc.
double strand break
digestion by nucleases →3´single-stranded tails
strand invasion→joint molecules of intact and damaged DNA moleculesIntact DNA strands= template for synthesis
According Sumner 2003
Rad51
Rad51
binding of Rad 51 protein-search for homology
DNA polymerasefills gaps
resolution of Holliday junction - break
Double strand breaks repair by homologous recombination
3) SSA-“single strand annealing“
– uses of sequentional homology on the same chromosome (or between different chromosomes )
exonuclease digests one strand of both broken ends to leave single stranded tails – these tails start to search for homology between themselves – after trimming to size - ligation
- more prone to errors – loss of DNA on either side of the break
DSB repair
Consequences of unrepaired or missrepaired DNA
damage (DSB)
= chromosomal aberrations (CHA) = structural changes of
chromosome – breaks and rearrangements= early genotoxic effect
tumors = consequences of late genotoxic effect
Aberrations in somatic cells caused by mutagenes:
Aberration type is dependent on type of clastogenic (chromosomes
breaking) agents
on phase of cell cycle (in time of mutagen action )
e.g. Ionizing radiation : irradiation of human lymphocytes in vitro
before cultivation (lymphocytes of peripheral blood are in G0 phase)
- after cultivation → chromosome type of aberrations
irradiation in G2 (during cultivation)→ chromatid type of aberrations
(ionizing radiation = S-phase independent)
chemicals – chromatid aberrations – originate during replication
(S-phase dependent)
Frequency of dicentrics detected after irradiation by
unknown dose is used for biological dosimetry of radiation
exposure
Dicentrics are not stable aberrations !!!
Frequency of translocations (stable aberrations) – used in
retrospective dosimetry (translocations are detected by
FISH method)
Cytogenetic method
= biomarker of exposure to genotoxic compounds
= biomarker of effects on humans (prediction of cancer risk)
Results of prospective studies:
CHA are predictive for risk of malignancies,
The strongest association for carcinoma of stomach
Cytogenetic method – suitable for determination of exposure to genotoxicants (in groups or in individuals)
Interindividual variability in sensitivity to mutagens/carcinogens
Genetic factors influencing level of CHA:
• metabolism: activity of enzymes metabolizing compound to
ultimative carcinogene, activity of detoxification enzymes
polymorfism of enzymes (different activity) is genetically
determined by polymorfism of genes
• chromatin configuration – structural relations – possibility of
aberrations origin (“hot spots”)
• activity of DNA repair enzymes – influenced by polymorphism
of repair genes
Factors influencing level of CHA:
Nongenetic – acquired sensitivity:
Life style, smoking, nutrition (mutagenes/carcinogenes, anticarcinogenes in nutrition, alcohol),
Quality of environment – previous exposure, chronic exposure
Age
+ others
Exposure to mutagens/carcinogens• Environment: pollutant emission from industrial production
agriculture – pesticides, fertilizers
combustion of fossile fuels, wastes combustion
emissions from combustion engines …
• Nutrition: mutagenes/carcinogenes in nutrition:
origin during high temperature treatment of meat,
during foodstuffs storage,
by foodstuffs contamination
• Profesional exposure
• Life-style: smoking, alcohol, sunbathing, automobilism
• Treatment: chemotherapy, diagnostic and therapeutic irradiation
• Endogenous mutagens/carcinogens: NO, free radicals, nitrosamines
Metabolism of genotoxic compounds – interindividual variability
Phase I : derivatization: oxidation, reduction, hydrolysis → increase of hydrophility of compounds
enzymes: monooxygenases CYP450
= complex of inducibile, polymorfic enzymes
Phase II : conjugation – conjugation with molecule as glutathione ….
enzymes: glutathion-S-transferase (GST)
glukuronyltransferase
sulfo-, acetyltransferases …
= inducibile, polymorfic enzymes
Aim: conversion of lipophilic substances to polar, hydrophilic substances and excretion from organism
During the process of detoxification- increase of mutagenity of metabolites (so called indirect mutagenes – oxidative reactions→increase reactivity of product)
v
Prokarcinogene
Metabolic.activationIst phase enzymes
Ultimative carcinogen
Normal cell
Iniciated cell
Preneoplasticcells
Tumor cells
DetoxicationIInd phase enzymes
Iniciation1-2 days
Promotion10 years
Progression 1 year
Iniciation/promotion theory of tumor origin
Mutagens/carcinogens in nutrition
• Compounds originating during heat processing of foodstuffs, by storage of foodstuffs
• natural coumpounds in nutrition
• food additives
Mutagenes/carcinogens in nutrition
• Derivatives of main nutritional factors:
From proteins: by inadequate heat processing – heterocyclic amines (e.g. IQ=imidazochinoline) - in burned meat
- nitrosamines, N-nitrosocompounds (MNU=methylnitrosourea), polyamines- also endogenous origin)
- PAH – polycyclic aromatic hydrocarbons
From lipids – oxidized forms of fatty acids…
increased lipids supply → increased level of bile acids→secondary bile acids = stimulation of proliferation of intestinal epithelium
Lipid pyrolysis → polycyclic aromatic hydrocarbons
From saccharides by caramelization → heterocyclic compounds
From foods containing starch by frying→ acrylamide
Minerales – nitrozation reactions
• Food contaminants:
polycyclic aromatic hydrocarbons (PAH), aromatic amines,
chlorinated aliphatic hydrocarbons, chlorhydrines,
polychlorinated dioxines (natural origin by incomplet combustion of
organic compounds, e.g. during forest fire → soil contamination
animal food contamination, contamination of animal products)
polychlorinated biphenyles (PCB – industrially produced),
phtalates (combustion of fossile fuels-presented in the air, from plastics
transferred to foodstuffs)
nitrosamines, nitrates, Hg, Pb, As, Cd, Ni…
mycotoxines (aflatoxin, trichotecene mycotoxins….)
Food contaminants:
PAH - 65% in foodstuffs as contaminants of cereals, vegetable oils, leaf vegetables, fruits – from environment air (combustion engines, incomplet combustion of organic compounds)
- 35% origin during food technology – meat smoking, barbecued meat
Nitrosamines and other N-nitrosocompounds
Origin: by reduction of nitrates to nitrites and by their nitrosation
Endogenous nitrosation - role of enteric bacteria
Exogenous during beer processing, meat smoking tobacco smoke, engine emission
Mycotoxines = secondary metabolites produced by moulds
hepatotoxic, neurotoxic, cardiotoxic, cytotoxic, immunotoxic, haemorrhagic, alergenic, immunosupresive, mutagenic, carcinogenic effects
Aflatoxin B1 – Aspergillus flavus, A.parasiticus
cereals, peanut, nuts, spice…
hepatotoxic, proven mutagenic and carcinogenic compound for humans-
hepatocelular carcinoma - hepatitis B increases risk of tumor
Ochratoxin - Aspergillus, Penicillium
cereals, legumes, milk…
hepatotoxic, possible carcinogen for humans
Patulin – Aspergillus, Penicillium
apples and other fruits with brown rot…
possible mutagene, teratogene
Natural mutagens/ carcinogens
Examples:
Plant phenols – flavonoids, tannins, antrachinones
in lower doses mostly protective effects (antioxidants, anticarcinogens), some of them (flavonoid quercetin) in a high doses carcinogenic
Flavonoids and tannins - fruit, vegetables, legumes, some medicinal herbs
Antrachinones – medicinal herbs, aloe-emodin,
colouring substances in food
Hydrazines– in some edible mushrooms – destroyed by cooking
Imunosupressives:
polycyclic aromatic hydrocarbons (PAH)
polychlorinated biphenyles (PCB)
chlorinated dioxines
chlorinated aliphatic hydrocarbons
organic compounds of stannum, Cd
asbestos
benzen
mycotoxines
Examples of nutrition factors involved in the process of carcinogenesis
Iniciation factors promotion factors inhibition factors
mycotoxines increased energy supply vitamines C,E,A
natural mutagens increased lipids supply carotenoids
protein pyrolyzates increased salt supply plant phenoles
PAH alcohol indoles ….
nitrosamines selen
Nutrition protective factors – prevention of tumours and other diseases
• Vitamins – C, E, A, follic acid …..
• Minerals - Se, Ca, Mg, Zn
• Fiber
• Natural anticarcinogens:
carotenes, carotenoids (ß-carotene, lycopene)
flavonoids – grapes, red wine, vegetables, fruits
polyphenols, polyphenolic acids - ellagic acid, resveratrol, genistein, epigallocatechin gallate, curcumin
thioles: allyl sulphides – garlic, onion