9. Genetic Basis Flashcards
What is genetic toxicology?
The effects of chemical and physical agents on DNA and on the genetic processes of living cells.
What are the different branches of genetic toxicology?
A toxicant will cause genetic alterations. These genetic alterations can be done to either:
- Germ cells–which can then have an effect on the offspring
- Somatic cells–which can lead to cell death, senescence (the process by which cells irreversibly stop dividing and enter a state of permanent growth arrest without undergoing cell death), aging, and cancer.
What can cause DNA damage?
DNA damage, due to environmental factors and normal processes inside the cell (ex: reactive oxygen species–ROS), occurs at a rate of 10,000 to 1,000,000 molecular lesions per cell per day.
What is an alkylating agent?
- Chemicals that have an electron deficiency so they are looking for something to bind (something that can give an electron) so that they can alkylate.
- Can cause DNA damage.
- Can be mono functional (bind to one base or one site that gives them an electron) or bifunctional/trifunctional like intra-or inter-strand crosslinks. ex: anti-cancer drugs they are multifunctional alkylating agents because they can cross link, have more than one spot.
What are the different types of DNA damage and what could cause them?
- Abasic sites (missing a base–either apurinic or apyridmidinic): caused by monofunctional alkylating agents)
- Abnormal bases (bases that are changed in a way that they are not the regular ATGC): can be due to x-rays
- Base adducts (a segment of DNA bound to a cancer-causing chemical): adducts such as benzo(a)pyrene and polycyclic aromatic hydrocarbons
- Single strand breaks: x-rays, UV, etc.
- Thymidine dimers or pyrimidine dimers (covalently bonded complex of two adjacent thymines on a single strand of DNA): UV
- Chemical mutagens
- Intercalations (insertion of molecules between the planar bases of DNA): acridines
- Radical formation: x-rays
- Interstrand crosslinks (covalent bond between 2 strands or within one strand): bifunctional or polyfunctional alkylating agents
- DNA-protein cross-links: x-rays, polyfunctional alkylating agents
- Double strand breaks: ionizing radiation
- Base insertions
- Base deletions
- A-G mismatch
- T-C mismatch
What is a mutation?
A small rearrangement of base pairs on the DNA strand, due to exposure to a physical or chemical agent or to errors in DNA replication. Causes altered transcripts and altered amino acids.
What is a mutagen?
A mutagen induces/increases the frequency of mutations; in some cases this occurs at DNA “hot spots”.
What is a DNA hot spot? Give an example.
Spot in DNA that is more susceptible to DNA damage. Ex: The DNA binding domain of the TP53 gene is a hot spot for DNA mutations which is frequently linked to different kinds of cancer including multiple myeloma (WBC cancer).
Explain how base damage may lead to mispairing.
An example of a base substitution mutation is:
If you don’t have the right hydrogen bonds in the right place, you don’t pair A to T or G to C. Get the wrong base so when you read the DNA you get the wrong message.
Ex: When a G-C base pair is exposed to Ethylmethane sulfonate (EMS), the G gets alkylated to make O6-ethylguanine. O6-ethylguanine pairs with T rather than C. Now, when the strands separate for the next replication, the T will base pair with an A. Therefore what was originally supposed to be a GC bond is now making TA.
What is the effect of an insertion or deletion?
They may change the reading frame due to a frameshift mutation.
What are the possible effects of mutations?
Mutated DNA can produce mutated RNA which can lead to:
- A stop in translation (so no protein)
- Too much protein
- Shortened protein
- A mutated protein
- or a normal protein
What are the different types of chromosomal aberrations (affects whole chromosome)? define them.
Aneugens: affect cell division and the mitotic spindle apparatus resulting in the loss or gain of whole chromosomes
Clastogens: induce breaks in chromosomes leading to sections of the chromosomes being added, deleted or rearranged
When does DNA repair lead to toxicity?
When the DNA damage exceeds the repair, there is an adverse outcome.
What are the different DNA repair pathways and which damage do they repair?
Mismatch repair: for base misspairs
Nucleotide excision repair: for base adducts, cross links, and base modifications
Base excision repair: for base modifications and single strand breaks
Recombination repair: for single strand breaks and double strand breaks (and for whole chromosomes)
Explain mismatch repair.
Enzymes cut the DNA, take away the wrong base and some bases around it (segment), synthesize the correct base sequence (segment), and replaces it. Base pair is now correct.
What is direct reversal mismatch repair?
An enzyme that specifically removes the group bound to the base that is causing it to bind to the wrong base.
Ex: O6 methylguanine transferase (MGMT) bound to a cysteine that removes the methyl group on Guanine (through its cycteine) so that G can properly bind Cytosine. Once the enzyme does its job, it commits suicide, therefore new enzymes must be synthesized for the next time this DNA damage occurs.
What is nucleotide excision repair?
Excision of a 12 nucleotide fragment, DNA synthesis by a DNA polymerase to fill in the missing gap, joining the strand by DNA ligase. Good for repair bulky adducts because it cuts out a large segment of DNA. A lot of machinery involved (including different XP enzymes).
What is Xeroderma Pigmentosum?
A nucleotide excision repair deficiency therefore person can’t be exposed to the sun. Is missing the repair process completely.
What is base excision repair?
Taking out a single damaged base and putting in the right one, then ligating the strand. Less machinery is involved.
What is recombination repair?
Used for double&single strand breaks and for whole chromosomes. When chromosomes are exposed to UV light they can break, and then be rejoined in the wrong way which is called chromosome aberrations. When repair systems try to repair broken chromosomes, sometimes mistakes in the repair can also cause aberrations.
What are the assays used for the detection of DNA damage? What gets tested?
- Structure based/ in silico analysis
Direct methods of detection:
- Looking at DNA strand breaks (use alkaline elution assay, comet assay–single cell gel electrophoresis)
- Looking at chemical adducts to DNA (use antibodies, chemical assays)
Detection of mutations:
- Ames test (bacteria)
- Mammalian cell gene mutation test – in vivo (LacZ MutaMouse or Pig-a gene mutation assay) or in vitro (mouse lymphoma cell assay)
Chromosome abberations:
- Micronucleus test
- Chromosome aberration test/metaphase analysis
Stress response:
- Gamma phosphorylated histone yH2Ax
- Gadd45 assays (GreenScreen)
Every human drug has to be tested for DNA damage and many of the environmental chemicals are tested as well. So these assays are widely used.
Explain how DNA damage is detected using Structure based/ in silico analysis.
Structural based: The interpretation of the chemical structure can give hints to the chemicals genotoxicity. For example, if we can see based off of its structure that the chemical is a reactive electrophile, then we know that it can form adducts with DNA or react with proteins.
In silico predictive models: Compare your potentially mutagenic chemical to other analogs that are similar in structure to see if your chemical reacts in a different way and how similar it is to the others. Uses computational programs and Quantitative structure-activity relationship (QSAR) modelling.
What is the alkaline elution assay? give an example.
The assay is a way of determining how fragmented the DNA is by running it through a filter after removing the bound proteins to see how big the pieces of DNA are. If the DNA is fragmented (small pieces) it will go through the filter faster. If it has long pieces (less fragmented), it will take longer to go through or will stay on the filter.
Example: alkaline elution of rat sperm treated with cyclophosphamide for 6 wks.
The cyclophosamide DNA was compared with the control DNA as well as DNA that had been exposed to radiation.
The control: Some DNA went through the filter
Cyclophosamide: an anti-cancer drug that binds to DNA and causes cross-links and base damage, so more DNA went through the filter than the control.
Radiation: causes many breaks and goes through the filter easily.
What is the comet assay?
Expose cells to radiation (UV or GAMMA) or a chemical, put them in agarose gel and then add a current to see what happens to cell. If there’s no DNA damage it will stay in the core. If the DNA is fragmented, it will run out of the core so it looks like a comet with a tail. Increased DNA breaks = increased comet.
How do you detect chemical adducts? Give an example.
Adducts can be detected using antibodies or chemical assays.
Ex: Feed mice with different doses of bezo(a)pyrene and then collect DNA from different areas such as bone marrow, small intestine, stomach, and liver, to see if different tissues covalently bind DNA to metabolites of bezo(a)pyrene in different amounts.
Results:
Liver has way more DNA adducts than the other tissues and it increases when exposed to BaP (Benzapyrene). Why does the liver have more? Because it is what is making the reactive metabolites of BaP and it’s actually the metabolites that bind to the DNA and cause damage.
What is the Ames test? How can the Ames test provide mutation specificity?
It is a bacterial mutagenicity assay.
Genetically engineered bacteria (salmonella) so that they can’t grow unless they had histidine. Then you add in the test chemical mutagen; add an extract of rat liver that has drug metabolizing enzymes (S9-supernatant); and adds enough histidine for the bacteria to go through some cell divisions, get exposed to the chemical, and potentially be mutated. Then looks for mutations.
There will be some spontaneous revertants in the control group which are bacteria that had had spontaneuous mutations even thought hey weren’t exposed tot he chemical mutagen. These mutations allow them to survive without histidine.
If the chemical being tested is a mutagen, there will be a large number of revertants due to mutations, therefore they will be able to grow without the histidine.
To determine how mutagenic the chemical is, you subtract the spontaneous revertants from the induced revertants.
There are different genotypes of the salmonella bacteria (strains) that have a different mutations (frame shift, transition/transversion, changes in DNA sequence, changes in bacterial coat). These different strains can be used to determine what kind of mutation the revertants must have done in order to undo their need for histidine.
What is the in vitro mammalian cell gene mutation test? What are other names for it?
Also called the mouse lymphoma cell assay. In vitro test.
There are 2 pathways that cells use to make nucleotides: the endogenous pathway (with dihydrofolate reductase- DFHR) and the salvage pathway (with tyrosine kinase–TK OECD 476). In this test, you engineer mammalian cells in order to delete the DFHR enzyme of the endogenous pathway. Then, the cells are exposed to the chemical of interest. If the chemical of interest is a mutagen, then it will mutate TK. As a selection process to test for mutagenicity, mutant cells and normal cells are exposed to trifluorothymidine (TFT), which causes the inhibition of cellular metabolism and halts further cell division. Cells deficient in TK are resistant to the cytotoxic effects of TFT and are able to proliferate to form colonies in the presence of TFT. Therefore, an increase in the number of colonies in response to a test substance correlates to the mutagenic potential of the test substance.
What are the 2 types of in vivo mammalian cell gene mutation tests?
- In vivo transgenic mutations: MutaMouse
2. In vivo erythrocyte Pig-a gene mutation assay
What is the in vivo transgenic mutations: MutaMouse test? Give an example of a chemical and the result.
An in vivo mammalian cell gene mutation tests where mice are engineered to have a LacZ reporter gene that is designed to detect induced mutations.
The reporter transgene (lacZ) is present in ALL cells (somatic and germ) and is easily recovered for analysis.
These transgenic mice are exposed to the chemical you are interested in testing. Then you take the DNA out of the mice’s cells (from wherever you want-germ or somatic) to see if they still have the LacZ gene or if the LacZ was mutated in any way. If it is untouched, then the chemical isn’t a mutagen, if it is not WT or if it is missing, then it is a mutagen.
Ex: MutaMice treated with Benzo(a)pyrene for 28 days . Took cell samples from Small intestine, bone marrow, stomach, and liver. They all had mutations but the small intestine had the most probably because it was exposed to the chemical the longest.
What is the in vivo erythrocyte Pig-a gene mutation assay?
This flow cytometry based assay can be done in rats, mice, or other animals such as monkeys.
The Pig-a protein is needed to synthesize glycosylphosphatidylinosital (GPI) which is a cell membrane anchor. The gene for GPI is on the X chromosome.
They administer fluorescent tagged antibodies against the GPI anchors. Then they draw blood to analyze the peripheral reticulocytes (RETs–immature RBCs that are released into the peripheral blood where they mature into RBCs) or RBCs by flow cytometry. If the chemical caused a mutation in Pig-a, then GPI wont be formed on RBCs, and there will be no fluorescence.
What is the micronucleus test? Give an example.
An assay used to test for chromosome aberrations (can be done in vivo or in vitro).
Mice are exposed to a chemical that blocks cell division (separation) which causes chromosomes to lag meaning big pieces of DNA won’t get included into the nucleus when the cell divides. To see this, their bone marrow or peripheral blood cells are examined for 72 hours for the presence of micronuclei (extra-nuclear bodies that contain damaged chromosome fragments and/or whole chromosomes that were not incorporated into the nucleus after cell division), such as broken pieces of chromosomes surrounded by a nuclear membrane. You can also end up with 2 nuclei in once cell because it wasn’t able to divide.
Ex: Mice were treated with different doses benzo(a)pyrene for 28 days and then the blood cells were scanned for micronuclei. As the dose of BaP increased, the number of micronuclei also increased.
What is the mammalian chromosome aberration test/metaphase analysis?
Metaphase chromosomes make the classical picture of chromosomes (karyotype). For classical cytogenetic analyses, cells are grown in short term culture and arrested in metaphase using a mitotic inhibitor. Visualize the chromosomes using fluorescence in order to see deletions, missing pieces, and to see if there is anything wrong with the chromosomes.
What is a histone stress response test? Give an example.
Gamma-H2Ax histone is phosphorylated when there is DNA damage due to a stress ex: double strand break. The histone is tagged with antibodies to measure if there is an increase.
Ex: there is more gamma-H2Ax phosphorylation in people who smoke cigarettes vs people who smoke tobacco because cigarettes cause more stress in the form of double strand breaks.
What is a Gadd45 stress response test?
When cells experience a stress, Gadd45 will increase. The assay is called GreenScreen: Link Gadd45 to GFP, so wherever Gadd45 is activated due to a stress, then the test tube will be green.
If test tube has cells and is not green, then the chemical is safe.
If test tube has less cells and no green, then the chemical is toxic because it killed the cells.
If the test tube has some cells and is also green, then the chemical is genotoxic (activation of stress response).
What is the ToxTracker assay?
Color coded assay where they can look at markers for DNA damage, p53 activation, oxidative stress, and protein damage.
On average, how many mutations will a child inherit from their parents?
around 70.
Why is it hard to find germ cell mutations?
You require 10000 offspring in order to find one mutation. So unless there is a clear phenotypic defect associated with the mutation, you won’t find it.
What are the different assays used to test for germ cell mutations?
TUNEL assay Comet Assay yH2Ax phosphorylation assay FISH analysis MutaMice Look for epigenetic inheritance via the germ line
Explain the assays used to test the DNA damage in human sperm of patients with testicular cancer, with Hodgkin lymphoma, or infertile patients.
The treatment of testis cancer, infertility, and Hodgkins lymphoma all cause DNA damage.
TUNEL assay: assay for DNA breaks.
Comet assay: DNA assay for breaks too that makes a tail
Results from both assays: Infertile men have the most DNA breaks, then testicular cancer, then Hodgkins, then the control.
Explain the yH2Ax phosphorylation assay for germ cells.
Mice were treated with a DNA damaging drug (Cisplatin) that caused double strand breaks in spermatogonial cells. Higher amounts of phosphorylated yH2Ax (histones) were found at higher doses of Cisplatin.
What is FISH analysis?
Treat rat with a chemical (ex: cyclophosamide) and observe their spermartozoa chromosomes. Can fluorescently tag rat sperm chromosomes (which are haploid) so there should only be one tag for each chromosome in the sperm cell. There should never be 2 of any chromosome in the haploid cell, but it is normal if there is no Y chromosome in the cell because it could mean that it is an X.
How are MutaMice used for germ cells?
Same as before. The LacZ transgene is in all cells including germ cells.
Ex: mice treated with Benzo(a)Pyrene will have many mutations detected in the LacZ reporter of spermatogonia.
Can also see if the DNA damage is transmitted to offspring.
What is the proposed strategy for genotoxicity testing for the following:
- Cosmetics
- Environmental chemicals, high production chemicals, veterinary drugs
- Pharmaceuticals (for human use)
- cosmetics: 3 in vitro tests covering 3 different end points. NO IN VIVO TESTS.
- Environmental chemicals, high production chemicals, veterinary drugs: 3 in vitro tests with 3 different endpoints. 1 appropriate in vivo test.
- Pharmaceuticals (for human use):
- Option 1: 2 in vitro tests and 1 in vivo test.
- Option 2: 1 in vitro test (ex: Ames) and in vivo test in 2 different tissues with 2 different endpoints.
What are the more pivotal assays used in genetic toxicology?
- Well characterized assay for gene mutation like the Ames test
- Mammalian assay for chromosome damage in vivo (metaphase analysis; micronucleus assay)
Why is genotoxicity testing more extensive for cancer?
Because many carcinogens are genotoxic.
What is CMR?
A carcinogenic Mutagenic Reprotoxicant. Chemical gets tested and can be tagged as CMR but there are different categories:
- Cat. 1A. KNOW its a CMR based on human evidence (hard to prove)
- Cat. 1B. presumed to have CMR potential for humans based on animal data
- Cat. 2. Suspected to have CMR potential for humans
- Cat. Evidence of adverse effects in milk or progeny is another category
