extra Flashcards
What are three methods used to create transgenic animals?
Three methods for creating transgenic animals are:
DNA Microinjection: DNA is injected directly into the eggs of an organism using a fine glass pipette, with random incorporation of the transgene.
Retrovirus-Mediated Gene Transfer: Retroviral vectors are used to infect early embryos, integrating the transgene into the genome via reverse transcriptase.
Embryonic Stem Cell-Mediated Gene Transfer: Embryonic stem cells are cultured and inserted into blastocysts, allowing for germline gene insertion in the host organism.
What are some key animal models and considerations when selecting an experimental model for toxicological research?
A: Key animal models and considerations include:
Transgenic models to assess mutagenicity: Used to study mutations and genetic damage caused by toxicants.
Other transgenic reporter animals: Engineered to express reporter genes that can help monitor gene expression and toxicity.
Knock-out of endogenous genes: Animals with specific genes removed to study the role of those genes in toxicity or disease.
Knock-in models: Animals with specific genes inserted to examine the effects of new or modified genes in response to toxicants.
What are three techniques used to measure mRNA levels and assess changes in transcription due to toxicant exposure?
Three techniques used to measure mRNA levels are:
Quantitative Real-Time PCR (qRT-PCR): mRNA is reverse transcribed into DNA and then amplified to quantify relative mRNA levels.
Microarrays: A solid surface with attached oligo-probes that hybridize to specific DNA or RNA sequences, reporting their presence.
Next-Generation Sequencing (NGS): High-throughput sequencing technologies (e.g., Illumina, Roche 454) used to sequence entire genomes rapidly and measure transcript abundance.
What are some techniques used to quantify protein levels, and when would each be preferred?
Techniques to quantify protein levels include:
Western Blotting
- Use: Best for determining the relative amount of a specific protein in a sample.
- Preferred When: You need to confirm the presence and size of a protein, and compare expression across different samples or conditions.
Immunolocalization
- Use: Visualizes the cellular and subcellular location of a protein using antibodies and microscopy.
- Preferred When: You want to know where a protein is located within cells or tissues (e.g., cytoplasm vs. nucleus).
Immunoprecipitation
- Use: Detects interactions between two proteins by isolating one and probing for the second.
- Preferred When: You are interested in studying protein-protein interactions or complexes.
Enzyme-Linked Immunosorbent Assay (ELISA)
- Use: Quantifies protein levels in a sample by using antibodies or substrates attached to a plate, detected via spectrophotometry.
- Preferred When: You need to measure protein concentration in large sample sizes, or when high-throughput screening is needed.
Protein Microarrays
- Use: Allows large-scale detection of proteins by using hundreds of antibodies affixed to a plate.
- Preferred When: You need to analyze many proteins at once (e.g., proteomics or screening for multiple biomarkers).
What is being measured in assays that detect transcription factor activity, and what are some common methods used?
In assays that detect transcription factor activity, the interaction between the transcription factor and DNA is typically being measured, as transcription factors initiate transcription by binding to specific DNA regions.
Common methods to detect transcription factor activity include:
Electrophoretic Mobility Shift Assay (EMSA):
- Measures protein-DNA interactions by detecting slower migration of DNA-protein complexes on a non-denaturing gel.
- Use: Best for detecting whether a transcription factor is bound to its target DNA sequence.
ELISA-Based Assays:
- Detects transcription factor binding by using nuclear extracts, immobilized oligonucleotides, primary antibodies, and secondary antibodies with a detectable label.
- Use: Useful for quantifying the amount of transcription factor bound to DNA in cell or tissue extracts.
Chromatin Immunoprecipitation (ChIP) Assays:
- Determines if transcription factors are bound to specific DNA regions by isolating DNA-protein complexes, followed by DNA quantification.
- Use: Ideal for identifying specific DNA regions where transcription factors are active.
What are two common techniques used for measuring metabolites in vitro?
- Gas Chromatography (GC) or High-Performance Liquid Chromatography (HPLC):
- These techniques allow researchers to quantify metabolites in a sample and separate them based on characteristics like hydrophilicity or lipophilicity.
Mass Spectrometry (MS): - Mass spectrometry identifies metabolites by measuring their molecular weight, allowing researchers to distinguish different metabolites in the sample.
What is the common in vivo method for measuring metabolites, and how is it used?
Radioactive Labeling (Tracing):
- In vivo, radioactive isotopes are used to track and measure metabolites and parent compounds in biological samples like expired air, urine, blood, feces, or tissue.
Total Measurement: Radioactivity can be measured in a compartment to assess both parent compounds and metabolites together.
Separate Measurement: Parent compounds and metabolites can be measured separately by coupling radioactivity with techniques like GC, HPLC, or MS.
How can reactive oxygen species (ROS) and reactive nitrogen species (RNS) be measured directly in cells?
They can be measured directly using a substrate that reacts with ROS or RNS to produce a fluorescent or colored product, such as DCFDA dye used to measure total ROS production in human lung endothelial cells.
What are two indirect methods used to measure reactive oxygen and nitrogen species (ROS/RNS) in cells?
1) Measuring reaction products like lipid peroxidation, oxidative DNA damage, or depleted reduced glutathione. 2) Measuring the activity of detoxifying enzymes, such as catalase or superoxide dismutase, as indicators of cellular stress caused by free radicals.
Why are electrophiles and free radicals considered the most damaging metabolites of xenobiotics?
Electrophiles and free radicals are highly damaging because of their ability to react with crucial cellular biomolecules, like DNA and proteins.
- electrophiles can alkylate nucleophilic sites on DNA
- free radicals can abstract hydrogen atoms from C-H bonds in DNA.
These interactions often lead to toxic effects due to the structural and biochemical properties of the target molecules.
What is a toxicant vs a toxic metabolite vs a reactive molecule?
Toxicant
Reactive molecule is a ROS/RNS
Toxic metabolite is for example an electrophile
How does proximity influence the target of a xenobiotic in the body?
Proximity is crucial because a molecule often becomes a target simply due to its location relative to the toxicant and its suitable biochemistry for interaction. Reactive metabolites produced at a specific site are more likely to affect nearby cells or tissues, especially at the site of absorption or metabolism. If no appropriate target is nearby, the ultimate toxicant may circulate until it reaches a target that it can interact with. The reactivity of the toxicant also determines how far or long it can travel before causing an effect.
For example, the liver often becomes a target for alcohol toxicity since alcohol is extensively metabolized there, leading to significant liver damage. Although reactive molecules can interact with multiple targets, not all interactions necessarily produce toxic side effects.
What kind of a reaction is an electrophile-nucleophile
covalent
What kind of reaction results in the formation of free radicals?
Hydrogen abstraction (the removal of a H from an endogenous molecule)
provide the general equations for oxidation and reduction
reduction (oxidation number decreases):
oxidant + e –> product
oxidation (oxidation number increases):
reductant –> product + e
