carcinogenesis Flashcards
DNA damage and repair: explain how DNA can be damaged, recall the role of p53 in the detection of, and response to DNA damage, summarise the natural repair mechanisms for damaged DNA, explain how unrepaired DNA may become fixed as a mutation. Explain how the potential of a chemical / agent to damage DNA is assessed
2 things which can damage DNA (possibly leading to mutations and cancer, but also by damaging DNA can be used in chemotherapy)
chemicals (carcinogens), radiation
6 sources of chemicals
diet, lifestlye, environment, occupation, medical, endogenous
3 sources of radiation
ionising, solar, cosmic
4 ways DNA is damaged by carcinogens (base pairs are planar, and have hydrogen bonds, so easily activated)
DNA adducts and alkylation, base dimers and chemical cross-links, base hydroxylations (e.g. by reactive oxygen species) forming abasic site (no base present in nucleotide), double and single strand breaks
what is phase I metabolism
addition of functional groups
3 reactions in phase I metabolism
oxidation, reduction, hydrolysis
what mainly mediates phase I metabolism
cytochrome P450
what is phase II metabolism
conjugation of phase I functional groups
6 reactions in phase II metabolism
sulphation, glucoronidation, acetylation, methylation, amino acid conjugation, glutathione conjugation
what does phase II metabolism generate
polar (water soluble) metabolites
what are polycyclic aromatic hydrocarbons (common carcinogens)
common environmental pollutants formed from combustion of fossil fuels or tobacco
structure of polycyclic aromatic hydrocarbons
polycyclic carbon rings, double bonds
example of a polycyclic aromatic hydrocarbon carcinogen
benzo[a]pyrene (B[a]P)
what is the 2 step epoxidation of B[a]P in liver
benzo[a]pyrene -(P450)-> benzo[a]pyrene-7,8-oxide -(EH)-> benzo[a]pyrene-7,8-dihydrodiol -(P450)-> benzo[a]pyrene-7,8dihydrodiol-9,10-oxide -> epoxide -> DNA adducts
result of DNA adduction by B[a]P
binds to bases in DNA, causing mutational changes
what is aflatoxin B1 formed by, and where is this common
A. flavus mould (common on poorly stored grains and peanuts)
what is aflatoxin B1
potent human liver carcinogen
epoxidation of aflatoxin B1
aflatoxin B1 -(P450)-> aflatoxin B1, 2,3-epoxide -> DNA binding on N7 position (adduction of guanine)
what cancer does aflatoxin B1 epoxidation cause
liver
what is 2-naphthylamine and what cancer does it cause
past component of dye-stuffs which is a potent human bladder carcinogen
metabolism of 2-naphthylamine
2-naphthylamine -(CYP1A2)-> N-hyroxy-2-naphthylamine -(glucuronyl transferase)-> glucuronide binds to NOH -(urine pH)-> breaks down to nitrenium ion -> DNA-reactive electrophile causing bladder tumours
other carcinogen from solar (UV) radiation and cancer caused
pyrimidine (thymine) dimers, causing skin cancer
other carcinogen from ionising radiation
IC free radicals
what do IC free radicals include
oxygen free radicals (super oxide radical O2. and hydroxyl radical HO.)
what do oxygen free radicals possess, and what do they therefore seek out
possess unpaired electrons, so are electrophilic and seek out electron-rich DNA
what 3 things can happen during oxygen free radical attack on DNA
double and single strand breaks, apurinic and apyrimidinic sites (lose base), or base modifications
3 stage process of base modifications during oxygen free radical attack on DNA
ring-opened guanine and adenine -> thymine and cytosine glycols -> 8-hydroxyadenine and 8-hydroxyguanine (mutagenic)
what 2 factors influence rates of endogenous damage and repair
damage per hour, max repair rate (base pairs per hour)
enzyme system most frequently involved in activation of chemicals to metabolites that can damage DNA
cytochrome P450
what 3 things activate p53 during cellular stress (p53 lost in cancers as tumour suppressor gene)
mitotic apparatus dysfunction, DNA replication stress, double strand breaks
what can p53 (transcription factor) activate
DNA repair
4 types of DNA repair
direct reversal of DNA damage, base excision repair, nucleotide excision repair, during- or post-replication repair
what happens during direct reversal of DNA damage: thymine dimers and methyl/alkylation
photolyase splits cyclobutane thymine dimers -> excise dimers; methyltransferases (e.g. MGMT) and alkyltransferases remove alkyl groups from bases
what is base excision mainly for
apurinic/apyrimidinic damage (loss of bases), but no damage to phosphodiester bond and nucleotide (except base)
what happens during base excision repair
DNA glycosylases remove base -> AP endonucleases remove remaining nucleotide -> repair polymerase (e.g. Polb) fills gap -> DNA ligase completes repair
what is nucleotide excision repair mainly for
bulky DNA adducts
what happens during nucleotide excision repair
endonuclease breaks two phosphodiester bonds either side of affected base (plus others) -> stretch of nucleotides either side of the damage excised by helicase -> repair polymerases (e.g. Pold/b) fill gap -> DNA ligase completes repair
2 types of during- or post-replication repair
mismatch repair, recombinational repair
base excision repair pathway
mutagen exposure -> DNA glycosylase removes mutagen and base attached to -> AP-endonuclease removes full nucleotide of removed base -> DNA polymerase adds correct base -> DNA ligase attaches correct nucleotide
nucleotide excision repair pathway
mutagen exposure -> endonuclease breaks phosphodiester bonds either side of a stretch of nucleotides either side of damage -> helicase removes this stretch of nucleotides -> DNA polymerase adds correct bases -> DNA ligase attaches correct nucleotides
persistence of damage and chance of mutagenic event relationship
greater persistence of damage, the greater the chance of mutagenic event relationship
3 occasions when DNA double strand breaks are made
homologous recombination, physiological conditions during somatic recombination and transposition (variable region hypermutation in antibody), due to ionising radiation and oxidative stress
role of KU proteins during DNA double strand break repair
KU proteins hold DNA, forcing double stranded DNA back from DNA fragments
carcinogen-DNA damage 3 pathways
carcinogen damage leading to altered DNA -> [efficient repair and normal cell] OR [apoptosis and cell death] OR [incorrect repair/altered primary sequence -> DNA replication and cell division causes fixed mutations]
outcome if fixed mutations effect transcription and translation
aberrant proteins
outcome if fixed mutations are on critical targets
carcinogens
2 examples of critical targets
oncogenes, tumour suppressor genes
pathway of testing for DNA damage by potential carcinogen
structural alerts/SAR (predict if potential carcinogen) -> in vitro bacterial gene mutation assay -> in vitro mammalian cell assay -> in vivo mammalian assay -> investigative in vivo mammalian assays
describe the bacterial (Ames) test for mutagenicity of chemicals e.g. bacteria which can’t make histadine on histadine-free media
chemical to be tested (potential carcinogen) and rat liver enzyme preparation (S9) -> bacteria that do not synthesise histidine e.g. Salmonella strain -> conversion of chemical to reactive metabolite -> able to make histadine so form colonies
what is done to detect DNA damage in mammalian cells
treat mammalian cells with chemical in presence of liver S9 -> look for chromosomal damage
what happens in in vitro micronucleus assays
cells treated with chemical and allowed to divide -> binucleate cells assessed for presence of micronuclei (show presence of DNA damage)
in in vitro micronucleus assays, why might the kinetochore be stained
determine if chemical treatment caused clastgenicity (chromosomal breakage) or aneuploidy (chromosomal loss)
what happens in bone marrow micronucleus assay in mice or rats
treat animals with chemical -> examine bone marrow cells or peripheral erythrocytes for micronuclei (test so if fed by carcinogen, do you see effect of carcinogen)
normal spontaneous base modifications of DNA which must be corrected: deamination and examination
primary amino group unstable, so converted to ketogroups (e.g. cytosine to uracil)
base modifications of DNA which must be corrected: chemical modification
normally oxidation reactions e.g. thymine oxidised to thymine glycol; by hyper-reactive oxygen species, radiation and chemical agents; bases alkylated or covalently linked to larger molecules (“adducts”)
base modifications of DNA which must be corrected: photodamage
UV light absorbed by nucleic acid, inducing chemical change and forming thymine dimers (intra-DNA damage)
4 types of DNA damage
single base pair mismatch (causes bulge), thymine diamer (distortion of helix), gap (removal of many nucleotides), nick (removal of single nucleotide by single break of phosphodiester bond)
what is DNA mismatch repair
scrutinisation of DNA for bases that don’t pair properly, so proof-read by repliaction fork, with preference for newly synthesised strand
when does DNA mismatch repair only occur
only during replication
features of xeroderma pigmentosum caused by deficiencies of nucleotide excision repair
extreme sensitivity to UV light, causing severe pigmentation irregularities, melanomas and skin cancers, as well as other forms of cancer and neurological defects
2 other diseases caused by deficiencies of nucleotide excision repair
trichothiodystrophy, Cockayne’s syndrome
agents which overwhelm cells to cause DNA damage, so that they undergo apoptosis
alkylating, make bulky adducts, induce double strand breaks
