DDT.4 Flashcards
Toxicants
Any substance (poison) that causes harm to a living organism including the human body.
Toxicants when introduced in the organism:
- Distributed
- Metabolized
- Interact with cellular macromolecules
- Result in toxic endpoint
- Excretion and repair process can mitigate toxin effects
Know the fate of a toxicant
chart on slide 3
Toxicology
The study of toxicants, chemical or physical.
Analytical toxicology
Identification of toxicants and their metabolites.
Toxicity testing
Use of living systems to estimate toxicity effects. Simple cell culture techniques to use of animals to detemine acute and chronic exposure to toxicants.
Toxicologic pathology
Investigates the subcellular, cellular, tissue and organ changes due to toxicants.
Structure activity studies
Identifies relationships between structure and toxic effects and is used as predictor for toxicity.
Statistics and epidemiology
Determine significance and risk associated with toxicant exposure and within human populations.
Mutagens
A mutagen is a chemical or physical phenomenon (radiation) that can cause changes to the composition of DNA.
Mutagens increase the rate of mutations in DNA compared to the spontaneous mutation rate.
Mutations can lead to development of cancer. Many mutagens are also carcinogens.
Physical mutagens
Different forms of electromagnetic radiation:
UV light – ionising radiation. Causes dimerisation of pyrimidine, hydration of cytosine and can also indirectly damage DNA by production of reactive oxygen species.
X rays & Gamma rays – Shorter wave length, mostly travels through cell without collision. If collides with DNA, strand break takes place.
Particle radiation
Including alpha and beta particles- Upon impact with nuclear DNA, attract or repel charged areas on DNA, causing breaks in DNA.
Chemical mutagens
Many different compounds capable of causing mutations to DNA.
Tests available to measure the ability of compounds to cause damage to DNA.
How to test for potential chemical mutagens and carcinogens?
Ames test monitors a chemical’s ability to bring about a reverse mutation in Salmonella typhimurium strains that have defects in their histidine synthesis pathway. Strains do not grow in absence of histidine. Cells can mutate back to wild type and grow in the absence of histidine.
Explain the Ames test process.
- Chemical and suspension of cells are incubated with liver enzymes from rats. (Rat enzymes added to produce any activated electrophilic species from test chemical.)
- Bacterial test culture is plated onto agar plate with NO histidine.
Presence of colonies indicate some cells have reverted to wild type – positive result
A positive result in the Ames test shows that a compound is a confirmed mutagen.
Different strains can indicate particular types of mutations.
–> Base pair or frame shift
For validity + and – controls and varying concentration of chemical are used in test.
Carcinogenesis
The process through which cancer develops.
Chemical carcinogenesis
The study of chemical carcinogens and their modes of action. They change the composition of DNA and by various mechanisms cause cancer.
–> Proto-oncogenes, found in normal cells, which are involved in positive regulation of cell growth are often mutated in cancer cells.
–> Tumor suppressor genes, which are negative regulators of cell growth, are also commonly inactivated in cancer cells.
Carcinogenesis: Initiation to Progression
Conversion: transformation of a preneoplastic cell into one that expresses the malignant phenotype.
Progression: expression of the malignant phenotype and cells acquire aggressive characteristics such as genomic instability, uncontrolled growth and metastasis.
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Direct Carcinogens
Some carcinogens are intrinsically reactive and form adducts with DNA without metabolic modification.
Examples: Nitrogen mustards, Methyl nitrosourea.
Procarinogens
Procarinogens require metabolic activation before damaging DNA by covalent adduct formation or via reactive oxygen species.
(Procarcinogen –> Proximate carcinogen –> Ultimate carcinogen (forms adduct))
Examples: Benzo(a)pyrene, Aflatoxin, Dimethylnitrosamine.
Ultimate carcinogen
metabolite that reacts with DNA.
Proximate carcinogen
metabolite requiring further metabolism and results in the ultimate carcinogen.
How do mutagens and carcinogens cause damage to DNA?
Covalent adduct formation:
- Formation of bulky aromatic type adducts
- Alkylation (generally small adducts)
- Dimerization
- Deamination
- Other reactions
Oxidative damage:
Bulky Aromatic Adducts
The first chemically identified carcinogens were the polycyclic aromatic hydrocarbons. Very common environmental contaminants composed of variable numbers of fused benzene rings. Formed from incomplete combustion of fossil fuels and vegetable matter. Chemically inert so require metabolic activation via Cytochrome P450.
Ex: Benzo(a)pyrene.
Activation of Benzo(a)pyrene and adduct formation.
1 – BP is converted to Benzo(a)pyrene-7,8-diol by P450 and Epoxide hydrolase.
2 – P450 adds another epoxide group to positions 9,10.
3 – Diol epoxide can undergo cleavage of C-O bond of epoxide at position 10 and form carbocation intermediate (electrophile).
4 – Ultimate carcinogen targets N-2 deoxyguanine residues which can perform nucleophilic attack of carbocation.
Aflatoxin B1
Toxin produced by some Aspergillus species found in soil and areas with decaying vegetation. Can contaminate food sources.
Exposure to high levels can cause cirrhoses and liver cancer!!!! Chronic exposure is also associated with increase risk to liver cancer!!!!
Procarcinogen –> Aflatoxin B1 epoxide (ultimate carcinogen).
Targets N7 nitrogen in guanine when forming adduct.
Alkylating agents
Attach small alkyl groups to nucleophilic sites to form DNA adducts.
Dimethylnitrosamine and other nitrosamines:
- Industrial bi-products and also present in tobacco smoke and some foods like cured meats
- Procarcinogens that produce reactive diazonium and carbonium ions.
- Target various sites on DNA bases.
Adenine (N1, N3, and N7),
Cytosine (N3),
Guanine (N3, O6, and N7),
Thymine (O2, N3, and O4)
Some adducts lead to mutations while others are less likely to.
Can lead to loss of base due to instability –> Abasic site.
Deaminating agents
Nitrous acid HNO2 is a deaminating agent that converts:
Cytosine –> uracil
Adenine –> hypoxanthine
Guanine –> xanthine
The hydrogen-bonding potential of the modified base is altered, resulting in mismatch of bases.
Hypoxanthine can pair with cytosine causing a mismatch.
DNA repair mechanisms - Excision repair
Removal of errors due to adduct and oxidative damage.
DNA repair mechanisms - Mismatch repair
Removal of deamination and small deletion & insertion damage.
DNA repair mechanisms - Single strand break repair
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DNA repair mechanisms - Direct reversal repair
Removal of methyl groups from bases without removal of base.
Base excision repair of DNA damage
Removes a variety of base damages such as 8-oxo-guanine, deaminated bases and methylated bases.
8-oxo-guanine
Results from reactive oxygen species damage.
8-oxo-G primarily repaired by theDNA glycosylase
8-oxo-G base flipped out of the double helix and cleavage of the N-glycosidic bond creating an abasic site.
Abasic site can then be repaired via polymerase and other enzymes (endonuclease, ligase, etc..)
Nucleotide excision repair of DNA damage
Repairs DNA with bulky adducts.
Adducts result from UV damage or electrophilic aromatic mutagens such as Benzo(a)pyrene.
Recognition of the damage initiates removal of a short single-stranded DNA segment containing damage.
The undamaged complementary strand is left intact
DNA polymerasecomplementary strand used as a template to synthesize replacement sequence.
DNA ligase completes repair process.
