9. Genetic Basis Flashcards

Question

How do you detect chemical adducts? Give an example.

Answer 1

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.

Answer 2

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.

Answer 3

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.

Answer 4

1. In vivo transgenic mutations: MutaMouse | 2. In vivo erythrocyte Pig-a gene mutation assay

Answer 5

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.

Answer 6

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.

Answer 7

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.

Answer 8

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.

Answer 9

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.

Answer 10

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).

Answer 11

Color coded assay where they can look at markers for DNA damage, p53 activation, oxidative stress, and protein damage.

Answer 12

around 70.

Answer 13

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.

Answer 14

``` TUNEL assay Comet Assay yH2Ax phosphorylation assay FISH analysis MutaMice Look for epigenetic inheritance via the germ line ```

Answer 15

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.

Answer 16

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.

Answer 17

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.

Answer 18

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.

Answer 19

1. cosmetics: 3 in vitro tests covering 3 different end points. NO IN VIVO TESTS. 2. Environmental chemicals, high production chemicals, veterinary drugs: 3 in vitro tests with 3 different endpoints. 1 appropriate in vivo test. 3. 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.

Answer 20

- Well characterized assay for gene mutation like the Ames test - Mammalian assay for chromosome damage in vivo (metaphase analysis; micronucleus assay)

Answer 21

Because many carcinogens are genotoxic.

Answer 22

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