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)

Dicentric + difragment = typical aberration after ionizing radiation

Chromatid breaks – typical after chemicals

Chromatid exchange – typical after chemicals

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