Carcinogens Flashcards
Carcinogens
- Carcinogens: chemical or physical sources that initiate or promote carcinogenesis
- Combinations of different carcinogens can increase the risk of tumor formation
- Several risk factors (e.g. working place, smoking, medication, sun burns) are associated with cancer
- Carcinogenic: an exposure that can lead to cancer in humans or animals
- Genotoxin: a physical or chemical agent that can induce DNA damages
- DNA damage: is an abnormal chemical structure in DNA, which causes changes in the structure of genetic material and prevents the replication mechanism from functioning and performing properly
- Mutation: an alteration of the nucleotide sequence in the genome
- Mutagen: a physical or chemical agent that can induce mutations
Genotoxin
- Mutagens: DNA adducts
- Clastogens: chromosome breaks
- Aneugens: mitotic spindle
Carcinogenesis
- tumor formation is a complex multistep process
Depending on the mode of action, carcinogens induce or promote carcinogenesis
-> Genotoxic carcinogens: initiate tumor formation by DNA damage and
mutagenesis (e.g. by DNA adduct formation)
-> Non-genotoxic carcinogens: promote tumor formation by pathways other than DNA damage (e.g. cell proliferation, cytotoxicity, hormonal effects)
Genotoxic carcinogens
- Direct DNA damage
- can be mutagenic
- Not concentration-dependent
- DNA damage is reversible; mutation not
-> In theory: One DNA damage event can potentially lead to tumor formation
Human genotoxic carcinogens
- Aflatoxin B1: moldy food
- Aristolochic acid: traditional Chinese medicine
- benzo(alpha)pyrene: tobacco smoke
- formaldehyde: tobacco smoke
- nitrosamine: processed food, tobacco smoke
- vinyl chloride: PVC manufacture
Non-genotoxic carcinogens
- No direct DNA damage
- Non-mutagenic
- Concentration-dependent
- Reversible
-> Provides growth advantage over normal cells
Human non-genotoxic carcinogens
- Asbestos: Manufacture, fire protection
- Cyclosporine: immunosuppressant
- 1,2-Dichlorobenzene: Agrochemical production
- Dioxins: Herbicides
- Estradiol: Hormone therapy
- Ochratoxin A: moldy food
- Phenobarbital: seizure treatment
Common types of DNA damages -> oxidative damages
- Life in oxygen-rich environment is a constant struggle to minimize oxidative damages
- 8-oxo-2’-deoxyguanosine (8-oxo-dG) is the most common oxidative lesion in duplex DNA
-> guanine has a lower one-electron reduction potential than other nucleosides - Human cells consist of highly efficient DNA repair pathways for oxidative damages -> Low mutagenic potential
Natural endogenous DNA damages -> Abasic (AP) sites: Depurinations, depyrimidinations
- β-N-gycosidic bond between base and deoxyribose is hydrolyzed
- Depurination occurs at a higher frequency than depyrimidination (weaker basicity)
Natural endogenous DNA damages -> Single-strand breaks (SSBs) are the most common lesions arising in cells
-> directly by disintegration of an oxidized sugar
-> indirectly during the DNA base-excision repair (BER)
* DNA double strand breaks are the most severe damages in the cell
* Repair of double-strand breaks can result in loss and rearrangement of genomic sequence
Natural endogenous DNA damages -> DNA alkylation (O6-methylguanine)
- O6-MG (alkylation of the oxygen atom of guanine) is highly mutagenic
- 1 of 8 unrepaired O6-MG damages is leading to a mutation
- Repaired by O6-methylguanine-DNA methyltransferase (MGMT)
- MGMT is a suicide enzyme (1 MGMT protein can repair 1 damage)
Natural endogenous DNA damages -> Deamination: Cytosine deamination
Spontaneous: hydrolysis reaction of cytosine into uracil, releasing ammonia
Why is thymine and not uracil found in DNA?
-> very frequent deamination conversions of C to U would not be caught by error- checking for non-DNA bases
-> would tremendously increase the mutation rate (C-G base-pairs would change to U-A)
-> uracil in DNA is quickly recognized and removed by multiple uracil DNA glycosylases
Activation of genotoxic carcinogens
- Many genotoxins are not genotoxic by themselves, but can form genotoxic metabolites through cellular metabolism (e.g. by cytochrome P450 enzymes)
Direct genotoxic carcinogens
- active without metabolic activation
Indirect genotoxic carcinogens (Pro-carcinogens)
- carcinogens after metabolic activation
- often species-, sex- and organ-specific
- Ultimate carcinogens are often highly reactive
- Usually electrophiles (i.e., molecules deficient in electrons)
- Attack nucleophilic (electron-rich) groups in DNA, RNA and proteins
DNA damage is not random
- Modifications of DNA can be at one or more DNA bases (or sites) and can also be formed with the DNA phosphate backbone
-> Exocyclic: number is superscripted
-> Endocyclic: number has normal size - Genotoxic substances can have specific reaction patterns:
-> Aristolochic acid: N6 of adenosine, N2 of guanosine
-> Benz[a]pyrene: N2 of guanosine
-> Formaldehyde: N6 of adenosine, N2 of guanosine, N4 of cytidine - Efficiency of DNA repair mechanisms can vary between different DNA damage sites
-> Guanosin-methylation: Repair efficiency for N7 methylation is higher than for O6-methylation
How to detect DNA adducts
- 32P-Postlabeling
- Immunoassay
- GC/MS and LC/MS
From DNA damage to mutation
- Cell division is required to fix a DNA damage into a mutation
- Once a DNA damage is fixed in a mutation, it can not be repaired
- Due to the structure transition mutations are more likely than transversion mutations
- Transition mutations are less likely to result in amino acid substitutions -> wobble base pair
- Silent mutation: TAT/TAC -> Tyrosine, TAG/TAA -> Stop codon, AGC/AGT -> Serine, AGG/AGA -> Arginine
Mutational signatures
Chemical exposures and DNA repair defects can lead to specific mutational patterns: Tobacco smoke, Temozolomide, Aristolochic acid, Aflatoxin B1, Defects in Mismatch repair
Our protection against genetic damage
- Although tens of thousands of DNA damages occur per day per cell, tumor formation is rare event due to:
-> Efficient DNA repair systems
-> Many mutations are “silent”
DNA mutations does not result in a change to the amino acid sequence
-> Mutations in non-coding sequences (e.g. introns)
-> Mutations in the third base of some codons
(e.g.: Proline: CCT, CCC, CCA, CCG)
(e.g.: Arginine: CGT, CGC, CGA, CGG, AGA, AGG)
Exception: change in codon usage can result in change of translation rate
-> Possibility of neutral mutations
Amino acid exchange does not lead to an altered protein function
e.g. exchange with a chemically similar amino acid (leucine/isoleucine)
-> Most of our genes are not involved in cancer Mutations in these genes do not lead to cancer
Tumor Initiation vs Promotion
DNA damage is prerequisite, but not sufficient for carcinogenesis
Genotoxic vs non-genotoxic carcinogens
Genotoxic mechanisms:
- DNA adducts
- Chromosome breakage, fusion, deletion, mis-segregation- non disjunction
-> leads to Genomic damage
Non-genotoxic mechanisms:
- inflammation
- immunosuppression
- reactive oxygen species
- receptor activation
- epigenetic silencing
-> leads to altered signal transduction
Non-genotoxic carcinogens
Non-genotoxic carcinogens lead to increase in cell proliferation (inhibition of apoptosis)
-> Direct mitogens (mimic growth factors)
-> Cytotoxic compounds (regenerative cell proliferation)
-> Hormone or hormone mimetics (high concentrations or with similar receptor affinity)
-> Compounds affecting the immune system (pro- and anti-inflammatory factors)
Can non-genotoxic carcinogen exposure lead to cancer without genotoxic carcinogen exposure?
“Bad luck”- mutations:
- Mutations that occur during cell replication without the contribution of genotoxic carcinogens
- Fast dividing cells undergo proliferation pressure and therefore have less time to repair DNA damages
- Non-genotoxic carcinogens can induce proliferation pressure
- Consequence: accumulation of “Bad luck” - mutations
Identifications of chemical carcinogens
- e.g. as part of the drug safety testing procedure:
Target discovery (100 projects) -> preclinic (20 compounds) -> Phase I (10 compounds) -> Phase II (5 compounds) -> Phase III (2 compounds) -> FDA approval and phase 4 (1-2 compounds) -> Commercial (1 compound) - Duration : 8-10 years
- 99 % of potential drugs do not pass toxicity or clinical phase tests
- 4/5 fail in preclinical studies
- Total costs per FDA approved drug: 500-600 Million US$
- Approximately 2000 animals per approved drug
Definition REACH
Registration, Evaluation, Authorisation and Restriction of Chemicals in the EU -> data on the hazardous properties of all substances manufactured or imported into the EU in quantities above 1 tonne per year (ca. 100.000 chemicals in europe)
Safety testing (e.g. for drugs)
- carcinogenic properties are evaluated in mutagenicity and carcinogenicity studies
- Carcinogenicity studies are the most time- and cost-consuming studies (animal studies)
- acute toxicity (weeks)
- Pharmacokinetics (weeks to months)
- Mutagenicity (Genotoxicity) (months)
- reproduction toxicity ( months to years)
- chronic toxicity (years)
- carcinogenicity (years)
Regulation of carcinogens
- Principle established by Paracelsus: any poison can be non-toxic if the dose is below an appropriate threshold
- But: no threshold dose at any exposure level for genotoxic carcinogens
- Pharmaceutical industry: drug development is stopped when genotoxicity tests are positive (exception: cancer therapy)
- For non-genotoxic carcinogens: may be used, if the intake level is below a threshold level, evaluated in acute toxicity studies
Carcinogenicity testing
2-years Rodent Bioassay:
* Rats (Sprague Dawley or Wistar) and mice (B6C3F1)
* Males and Females
* One control and three dosis (MTD (Maximum tolerated does), MTD/2, MTD/4) -> appropriate dose level selection is critical
* 50 animals per group -> 400-600 animals / study
* Histopathological examination of 24 potential target organs for neoplastic lesions
* Selected route of administration in animals should mimic as closely as possible the intended or expected route of human exposure
-> There is no in vitro method available which is considered sufficient to serve as replacement for animal studies!
