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Toxicology > Genotoxicology > Flashcards

Genotoxicology Flashcards

1
Q

Genotoxicology is -

A

the identification of substance that may interfere with the genetic code

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2
Q

Genotoxic carcinogens -
give examples

A

=. directly alters genetic materials
- Microlisions = metagenic effects on DNA base
- Macrolesion = effect on chromosome = Clastogenesis & Aneugenesis

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3
Q

what is clastogenesis

A

induction of chromosomal aberration due to loss, addition or rearrangement of DNA

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4
Q

what is aneugenesis

A

loss or gain of complete chromosome

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5
Q

what is mutagenesis

A

permanent small DNA change (point mutation, frameshift)
carried on to daughter cells

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6
Q

T/F there is a close relationship between mutagenesis and carcinogenesis

A

True

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7
Q

All _ are _ but not all _ are _

A

mutagens, genotoxins
genotoxins, mutagens

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8
Q

what is the response mechanism after DNA damage caused by mutagens

A

DNA repair mechanisms = trying to repair by removing damage prior to replication.
If DNA replication with DNA damage = mutation in the daughter cell

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9
Q

DNA damage in somatic cells can lead to

A

metagenesis = carcinogenesis
Ageing
teratogenesis (birth defects)

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10
Q

Mutations in germinal cells can cause

A

Genetic disease in both the nucleus and the mitocondria
More mutations means decrease in genomic stability.
More mutations means tumour cells undergo tumour clone expansion (tumour grows slowly which gives way to increase clone expansion)

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11
Q

How do most genotoxic carcinogens act

A

interact/bind directly with DNA. Causing cancer by directly inducing various types DNA damage. Causing DNA damage the formation of covalent bonds with DNA, leading to the development of DNA adducts (complexes DNA carcinogen) by intercalation or oxidation

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12
Q

what are DNA adducts

A

epoxides, lactones, platinum and amines

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13
Q

how do non-genotoxic carcinogens work ?

A

leads to DNA instability, capabke of inducing cancer by some 2nd mechanism that does not directly induce DNA damage.
DNA damage disrupts cellular structures chanong the rate of either cell proliferation or processess that increase the risk of genetic errors

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14
Q

what is an examples of non-genotoxic carcinogens

A

immuno-suppressants = cyclosporine

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15
Q

metabolic transformation of gentoxic carcinogens is important for _

A

activation and detoxification

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16
Q

many chemicals such as _, which are genotoxic require metabolic activation to become _

A
  • polycyclic aromatic hydrocarbons, aromatic amines, nitrosamines, aflatoxin B
  • carcinogen
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17
Q

why are pro carcinogens problematic

A

while they are hopefully detoxified by phase II metabolism. However they stimulate their own development

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18
Q

how do we determine if a chemical is mutagenic

A

epidemiological data
- measure of DNA adducts
- other measures of damage in exposed humans

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19
Q

what is the probelms with using epidemiological data to determine mutagenisis

A
  • other chemical exposure
  • cancer development can occur many years after exposure
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20
Q

how is mutagenisis determined in labs and what are the problems

A

loads of test to detect DNA damge
problems = Metabolic activation of chemical may be needs, Extrapolation of animal data to human

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21
Q

what are the classification of carcinogens

A

Human carcinogen (Group 1,A)
Probable human carcinogen (2A,B1-2)
Possible human carcinogen (2B,C)
Not classifiable (3,D)
Not carcibogenic (4,E)

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22
Q

Chemical carcinogens are part of which classification
why ?

A

1/A
Sufficient epidemiological data: (Occurrence of tumours at high rate, unusual site, unusual type (e.g. aflatoxin, asbestos))
Tumours occur at much greater rate than spontaneous tumours occurring in the non-exposed control + Tumours occur at specific sites

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23
Q

outline the evidence of carcinogenicity of chemical carcinogens from animal studies

A

Presence of benign or malignant tumours in multiple species and strains
Occurrence of tumours at higher rate/ unusual sites/ unusual types
Note: carcinogens may not cause cancer in all individuals. Some may cause cancer by a specific route of exposure (e.g. inhalation, ingestion, physical contact)

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24
Q

how can a substance be a possible carcinogen ?

A

Positive results in only one species strain or study
Mechanistic pointers/anecdotal evidence
Inadequate evidence in humans (lack of studies

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25
Q

what are the 3 main mechanism of interaction between genotoxic carcinogens and DNA

A

Addition of an alky grouo
Addition of bulky adducts
Formation of crosslinks between DNA strand

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26
Q

what are alkylating agents

A

electrophilic compounds with the ability to attack the nucleophilic centres of DNA: most reactive sites N7 and O6 of guanine

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27
Q

DNA alkylating may lead to

A

mispairing during replication unless repaired

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28
Q

which nucleotide is more susceptible to these agents

A

guanine

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29
Q

what are the 2 types of alkylating agents

A

monofunctional and bifunctional

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30
Q

monofunctional alkylating agents act by =

A

single alkylation step, e.g. dimethylnitrosamine, methyl methane sulphonate, vinyl chloride

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31
Q

bifucntional alkylating agents act by =

A
  • lead to alkylation + crosslinking of 2 strands by binding 2 Guanines, e.g. anticancer drugs: cyclophosphamide (nitrogen mustards)
  • (electrophilic attack of N7 -> release of Cl -> Formation bridge between 2 strands or intra strand)
    Cytotoxic /chemotherapeutic agent
  • Phosphoramide mustard is a breakdown product of aldophosphamide, a metabolite of cyclophosphamid
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32
Q

guanine can be attacked by various carcinogens _ and _ attaches a _

A

adduct formation PAHs
tamoxifen
Nitrogen

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33
Q

what also attacks a nitrogen

A

aflatoxin

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34
Q

what attacks the carbon in the pentamer of guanine

A
  • aromatic amine
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35
Q

what are the other direct but not covalent interaction of carcinogens with DNA

A
  • Intercalation
  • DNA damage by Removal of purine or pyrimidine base to give “apurinic” or “apyrimidinic” site
  • Reactive oxygen species
  • strand breaks can be induced by other agents
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36
Q

how does intercalation between DNA and carcinogens work ?

A

Mostly planar molecules that intercalate between stacked base pairs of double stranded DNA e.g. anticancer drugs such as doxorubicin, actinomycin

37
Q

how does DNA repair mechanism after damage results

A

result in replacement after replication by incorrect base or frameshift may occur

38
Q

single strand breaks are normally induced by _
double strand breaks typically due to _

A

chemical
radiation exposure

39
Q

Base substitution may lead to

A

changes in AA (coding region), altered regulation (promoter,mRNA stability/processing, regulatory regions), truncated proteins (premature stop codon

40
Q

frameshift after mutations due to chemical exposure results in

A

Typically a single base pair deletion which can have a profound effect on gene product if in a coding sequence

41
Q

what are the two phases of activation of procarcinogens

A

Phase I (mostly) P450s: CYP1A1, 1A2, 2A6, 2E1, and 3A4 involved in activation of >90% of pro-carcinogens. Additional role for epoxide hydrolase
Phase II enzymes also involved in activation reactions E.g. sulfotransferases (carbocations formation), acetyltransferases

42
Q

how does benzo[a]pyrene become activated

A

CYP1A1 forms a 7,8 epoxide. Then epoxide hydrolase forms a compound with a hydroxylation side chain
CYP1A1 then forms diol 9,10 epoxide which then goes on to form benzo[a]pyrene diol epoxide
this forms adduct with guanine G->A mutation

43
Q

Aflatoxin B1 is activated by CYP_ and what is the risk factor associated with exposure

A

CYP3A4
liver cancer

44
Q

where is aflatoxin derived from

A

mycotoxin found in food (cereals, corn, groundnuts (peanuts), pistachios)

45
Q

what are the 3 stages of tumoregenisis and what are they all influenced by

A

Tumour initiation - DNA damge, mutagenesis
Tumour promotion - clonal expansion
Tumour progression - malignant transformation
Carcinogen

46
Q

Tumour promoters are not _ but increase the effects of _ when given simultaneously

A

Carcinogens

47
Q

T/F tumour promoters effect DNA

A

False

48
Q

how do tumour promoter effect cancer progession

A

increase expansion of cancer clones by altering gene expression and cell proliferation)
(e.g. phorbol esters combined with polycyclic aromatic hydrocarbons (e.g. BaP) on mouse skin)
Mechanisms may involve stimulation of cell proliferation

49
Q

What is consider to not be directly genotoxins but they can produce tumour of mesenchymal origin

A

solid state carcinogens

50
Q

how does asbestos cause mesothelioma

A

generate OH radicals from hydrogen peroxide - lead to cancer development 30-40 years after exposure (exacerbated by smoking)

51
Q

Hows is p53 effected by mutations caused by smoking,

A

Mutations at “hot spots“ codons 157, 248 and 273 in TP53 gene
Can also get mutations at these positions by treating cells in vitro with BaP Diol Epoxide
Carcinogen signatures = Smokers + passive smokers

52
Q

what are the types of DNA repair mechanisms

A

Base excision repair (BER)
Nucleotide excision repair (NER)
Methyl group removal
Repair of strand breaks = (Homologous recombination repair + Non homologous end joining)

53
Q

how does base excision repair occur

A

1 - DNA glycosylase recognises the damaged base and cleaves the glycosidic bond between the base and deoxyribose in DNA backbone
enzymes involved include 8-oxoguanosine glycosylases (OGG1 and 2)
2 - AP endonuclease cleaves the phosphodiester bond in the DNA backbone near the AP site (Apurinic or apyrimidinic (AP) site)
3 - Small section of DNA including AP site is then removed and replaced
4 - DNA polymerase initiates repair by removing part of the damaged strand and replacing it by an undamaged base
5 - DNA ligase ligates the break

54
Q

what mechanism repairs non-bulky damage to bases resulting from oxidation (8-oxo-dG) methylation, deamination, or spontaneous loss of the DNA base

A

base excision repair

55
Q

what does nucleotide excision repair do ?

A

Removal of bulky adducts (e.g. due to benzo[a]pyrene, aflatoxin, aromatic amine; intercalating agents..)

56
Q

how does nucleotide excision repair work ?

A

Multi-enzyme repair complex recognises damage and removes 25 to 30 nucleotides surrounding site
-> DNA polymerase and ligase carry out repair

57
Q

what is xeroderma pigmentosum

A

Genetic deficiency in NER
Unable to repair UV-damaged skin and this leads to premature death from cancer

58
Q

what does NER to when cells are exposure to UV radiation

A

repairs T-T dimers

59
Q

how does O6 methylguanine DNA methyltransferade (MGMT) repair work

A

simple system
MGMT enzyme removes methyl and ethyl groups from guanine at O6 position
Effective against damage caused by monofunctional alkylating agents such as methyl or ethylnitrosourea, but not against crosslinking alkylating agents (bifunctional- need NER)
No requirement for DNA polymerase or ligase (no DNA removal, unlike BER and NER)
SOMETIMES CALLED “DIRECT REPAIR”

60
Q

methods to detect DNA damage are =
direct reporter -
indirect reporter -
Direct visualisation -

A

DR = Genetic reversion (the damages reverses the mutant phenotype = Ames test, HRPT/TK mammalian cell test
IR = transgenic mouse models
DV = micronucleus test, LC-MS, Comet test

61
Q

what in vivo testing is used to suggest mutagenic potentical of a chemical

A

Cytogenetic test
Gene mutation test
Suitable alternative test depending on individual chemical

62
Q

If results from both in vitro and in vivo tests are equivocal (neither negative nor positive) what additional tests may be needed

A

comet assay
transgenic animal mutation test

63
Q

what is the future for determining mutagenic potencial

A

genomic/proteomic tech

64
Q

how does ames assay work

A

Aim to evaluate a chemical’s genotoxicity by measuring its ability to induce reverse mutations (mutations which restore the wild type phenotype in a mutated gene) as a selected locus in a given bacterial strain

65
Q

what are the advantages of the ames assay

A
  • Predictive, good correlation with rodent carcinogens
  • short-term (4-8 weeks compared with 2years for in vivo models)
  • Relatively cheap
  • High-throughput
66
Q

what are the disadvantages

A

non-mammalian system
not 100% predictive

67
Q

what is the bacterial strain used in the ames test

A

Salmonella typhimurium carrying a mutation in a gene involved in histidine biosynthesis (→ requires His to grow)

68
Q

how would a positive result from the ames assay present

A

= exposed bacteria grow on His-free/ minimal medium: chemical induced a mutation→ reversion his-phenotype → chemical is a potential mutagen and carcinogen
= + and - S9 fractionated rat liver homogenate to allow potentially chemical to be activated by xenobiotic metabolism

69
Q

what are the conditions of a ames test

A

1 - Presence or absence S9 fraction: activation mutagen
2 - Controls: +ve cont (known mutagen) & -ve cont (known non-mutagen)
3 - Dose response: need to test different doses of the chemical
4 - Different tester strains: to detect different types of mutations (frameshift mutations(e.g. TA-1537 and TA-1538), point mutations(e.g. TA-1531)
To identify mutagens acting via different mechanisms
WHO guidelines: recommends using 5 different strains routinely

70
Q

what are the limitation of AMES test

A

False +VE: e.g. nitroglycerin is not carcinogenic or mutagenic but its metabolism by bacterial enzymes result in the release nitric oxide during test which can cause mutation
False -VE: ~ only 70% of all human carcinogens are +ve by Ames tes

71
Q

what is the aim of a mammalian mutation cell assay

A

The aim is to evaluate in vitro a chemical’s genotoxicity by measuring its ability to induce reverse mutations at a selected locus in mammalian cell lines (e.g. CHO, mouse lymphoma cells

72
Q

what is thioguanine

A

guanine analogue, when converted by hypoxanthine phosphoribosyl transferase (HPRT), incorporates in DNA in S-phase →kills cells
- used in the mammalian mutation cell assay

73
Q

what are the conditions of the mammalian mutation cell assay

A

1 - Cell lines:usually Chinese Hamster Ovary (CHO) cells deficient for HPRT
2 - Presence or absence S9 fraction: CHO cells exposed to test compound with/without liver enzymes from S9 fraction for 3 to 6 hours before addition of thioguanine to culture cell medium
3 - Controls: Need +ve controls: 1 - e.g. 3-methylcholanthrene (+S9)/ 2 - e.g. ethylnitrosourea (mutagenic without -S9)
4 - Results: survival difference (mutagen: cells do not grow; non-mutagen: cells grow)

74
Q

what are the advantages of mammalian mutation cell assay

A

closer to human model… but not human, mammalian system

75
Q

what are the disadvantages of mammalian mutation cell assay

A

HPRT gene located on X chromosome so may not test mutagenicity on all regions of DNA
Other possible tests include TK (tyrosine kinase; autosomal chr.) in mouse lymphoma cells: L5178 which is TK+/- (heterozygous, only 1 allele needs to be mutated)

76
Q

what is the aim of the micronucleus test

A

evaluate in vitro a chemical’s ability to generate clastogenic or aneugenic damage thus detect chemcials that modify chromosome structure and segregation is such a way as to lead to induction of micronuclei in interphase cells

77
Q

what are micronucleus

A

small nucleus formed when a chromosome or a chromosome fragment is not incorporated into one of the daughter nuclei during metaphase/anaphase transition of mitosis

78
Q

how does the micronucleus test carried out

A

Use human peripheral blood lymphocytes or a cell line in culture exposed to mutagen. Stimulate cell division then add test compound +/- S9, add cytochalasin B & stain for centromere. Score appearance of MN +/- centromer

79
Q

how are micronucleus formed

A

During mitosis, a lagging chromosome or a fragment of chromosome is trapped into a micronucleus.
Lagging chromosome = aneugenic events
chromosome fragment from unrepaired/misrepaired DNA (clastogenic event)

80
Q

explain Micronucleus testing using erythrocytes from bone marrow of mice treated with test compound

A
  • animals normally killed at several time points, erythrocytes are then isolated from bone marrow.
  • count number of polychromatic (young) erythrocyte cells with micronuclei due to test compound
  • expensive and uses lots of compounds
81
Q

how are tansgenic mouse models used to test possible mutagens

A

a number of different mouse strains have had bacterial genes introduced (LacZ/Lacl)
Can treat with test compound, isolate DNA from mouse tissue, determine whether bacterial DNA has been mutated using bacterial genetic assays

82
Q

how is 8-hydroxy-2’-deoxyguanosine (8-OHdG) used to detect damage

A

early response biomarker of oxidative DNA damage.
LC-MS (sensitive)
limitation = only measures oxidative DNA damage

83
Q

what is the comet assay and what are its advantages

A

quantify overall damage
- low cost, relatively fast and simple test

84
Q

method of
COMET in vitro assay =
COMET in vivo assay =

A
  • cells exposred to mutagens in tissue cells
  • measure of DNA damage in peripheral blood lymphocytes and other primary cells
    = Cells are lysed cells then DNA strands are unwound and separated in alkali buffer followed by electrophoresis, analysed under microscope
85
Q

how does COMET assay work

A

= short term loss of cell integrity
causes a DNA damage visulaised by microscopy
= kinetic software counts ~100 cells and quantifies the damage
= DNA stained with fluorescent stain
Damaged DNA forms COMET - quantitative measure of damage

86
Q

what is COMET primarly used to detect and what else

A

= single strand breaks and AP (apurinic and apyrimidinic) sites
= can also distinguish oxidative damage from covalent adducts by adding specific endonucleases with / without repair inhibitors in vitro after cell lysis before running assay

87
Q

what are the limitation of looking a single cells

A

very sensitive
dependant on cell cycle stage
quiescent cells less likely to undergo damage
more complication in vivo than in vitro

88
Q

how is risk to offspring tested

A

Germ cell tests
Can use transgenic (bacterial DNA) mouse strains as source of germ cells and look for mutations in bacterial DNA isolated from germ cells
COMET analysis on sperm cells: determine damage

89
Q

looks at offspring =

A

measure the rate of pregnancy loss
measure rate of gene mutation or chromosome abnormality in preogency = a lot of animals needed

Toxicology (11 decks)

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