Mod 3 Flashcards
(177 cards)
1
Q
What are the two groupings of models that are used experimentally to conduct toxicological experiments?
A
In vivo and in vitro
2
Q
What is in vivo?
A
Experiments that occur within a body, such as those with animals
3
Q
What is in vitro?
A
Experiments that occur outside a body, such as within a petri dish
4
Q
What is is a cell culture, and what three materials are usually involved in it?
A
Where cells from an organism are maintained and propagated under specified conditions.
Three materials:
- Vessel (ie. dish or flask)
- Nutrient media (“feeds” the cells)
- Incubator (house the cells at ideal temp, humidity, and oxygen/CO2 levels)
5
Q
What are the two types of cell cultures?
A
Primary cell culture and cell line
6
Q
What are cell lines?
A
Cell lines are subcultures of cells derived from a primary cell culture that are transferred to new dishes to continue growing and proliferating after reaching confluency in the original dish.
7
Q
What does confluency mean in the context of cell culture?
A
Confluency refers to the state when cells have grown to occupy all the available space in a culture dish, necessitating their transfer to a new dish to continue proliferating.
8
Q
How are primary cells obtained?
A
Primary cells are extracted directly from a tissue or organism and then transferred to a culture vessel for maintenance and proliferation under controlled conditions.
9
Q
What happens when primary cells reach confluency in a culture dish?
A
When primary cells reach confluency, they must be transferred to a new dish, becoming subcultures known as cell lines, to continue growing.
10
Q
What is the difference between a primary cell culture and a cell line?
A
A primary cell culture consists of cells directly extracted from a tissue or organism, while a cell line refers to subcultures derived from primary cells, which are transferred to new dishes to continue proliferating.
11
Q
What are continuous cell lines?
A
Continuous cell lines are derived from primary cell cultures but modified to prevent senescence, allowing them to replicate indefinitely.
These cells are often immortalized, sometimes through viruses or chemicals, and are frequently cancerous in nature.
12
Q
How do continuous cell lines become immortalized?
A
Continuous cell lines become immortalized either spontaneously or by intentional alteration using viruses or chemicals, resulting in traits that prevent senescence and allow indefinite replication.
13
Q
Why are many immortal cell lines cancerous?
A
Many immortal cell lines are cancerous because cancer cells inherently possess the ability to avoid senescence, enabling continuous replication.
14
Q
What are finite cell lines?
A
Finite cell lines are derived from primary cell cultures and have a limited number of cell divisions before they senesce, losing their ability to proliferate. Unless modified, these cells eventually stop growing.
15
Q
What is the key limitation of finite cell lines?
A
Finite cell lines are limited in the number of divisions they can undergo before senescence and can suffer from rapid dedifferentiation, making them short-lived in culture.
16
Q
What does senesce mean?
A
Senesce refers to the deterioration of cells with age, leading to a loss of their ability to proliferate.
17
Q
What is dedifferentiation?
A
Dedifferentiation occurs when cells regress from a specialized function to a more simplified, stem cell-like state.
18
Q
What is one key factor to consider when choosing a species for a cell line in toxicology research?
A
The species chosen should align with the experimental purpose. For example, if researching the toxicity of a chemical in human breast cancer cells, human breast cancer cell lines should be used instead of non-human cell lines.
19
Q
Why is the experimental purpose important when choosing a cell line?
A
The experimental purpose helps determine the type of cell line to use. For example, liver cells might be appropriate for studying liver toxicity, while cells derived from embryonic or fetal tissue could be used for developmental toxicity studies.
20
Q
What are the benefits of using continuous cell lines?
A
Continuous cell lines are easier to propagate, maintain, and provide increased consistency in results. They are also well-characterized, which helps with predictability in experiments.
21
Q
Why might a finite cell line be preferred in some experiments?
A
Finite cell lines may better express the correct cellular phenotypes and offer more options for cell types to culture, making them useful in studies requiring more in vivo-like characteristics.
22
Q
What is the difference between normal and transformed cell lines?
A
Normal cell lines have not undergone significant changes, while transformed cell lines have altered phenotypes and increased growth rates, which may affect their response to experimental conditions.
23
Q
What is a potential drawback of using transformed cell lines?
A
Transformed cell lines may have altered phenotypes that could affect how they respond to experimental toxicants, potentially impacting the validity of the results.
24
Q
Why is feasibility an important consideration when choosing a cell line?
A
Feasibility considerations include access to the cell line, ease of storage, availability of stocks, and whether the cell line is well-characterized. These factors influence the practical execution of experiments
25
How do cell characteristics influence the choice of a cell line?
Cell characteristics, such as protein expression, growth rate, and confluency, must align with the research question. For example, a cell line should express the proteins necessary for studying a specific signaling pathway.
26
Why is the length of study important in cell line selection?
The length of time cells are exposed to a chemical in an experiment may affect cell viability and replication, influencing the results. This should be considered when choosing an in vitro model.
27
What is the primary advantage of using cell culture in toxicology experiments?
The primary advantage of using cell culture in toxicology is the consistency and reproducibility of results that can be obtained.
28
What is the purpose of a cell death assay in toxicology research?
A cell death assay is used to assess whether exposure to a chemical affects the extent or rate of cell death in a particular cell type, with the total number of cells decreasing over time after exposure to a toxicant.
29
What does a proliferation assay measure?
A proliferation assay measures whether exposure to a chemical affects the ability of cells to proliferate or differentiate, typically observing a decrease in cell proliferation over time.
30
What type of experimental question would be addressed by studying phenotypic changes in cell culture?
An experiment studying phenotypic changes might ask: "Does exposure to a chemical alter the phenotype or morphology of the cell population?"
31
What is the focus of mechanistic studies in cell culture experiments?
Mechanistic studies aim to understand biological processes or the mechanism of action of a drug or toxicant. They may investigate whether chemical exposure causes mitochondrial dysfunction, cellular stress, or changes in gene/protein expression or signaling pathways.
32
What type of chemical is 5-AZA-CR, and what is its role in cell culture experiments? - probably dont need to know this
5-AZA-CR is a chemical that inhibits DNA methylation and is used in experiments to study the effects of DNA methylation changes on cell populations, such as in phenotypic alteration studies.
33
T or F:
In vitro experiments help detect whether a toxicant causes mutations in cellular DNA.
T
34
What is the Ames Test used for?
The Ames Test is used to detect mutations caused by chemical exposure, indicating whether a toxicant causes DNA mutations.
35
In the Ames Test, what kind of bacteria are used?
Bacterial cells that are unable to produce histidine (his-) are used in the Ames Test.
36
How does a positive Ames Test result occur?
A positive result occurs when a chemical exposure causes a mutation that allows the bacteria to produce histidine (his+), enabling growth in the absence of histidine.
37
What is a unique technique in cell culture that is not possible in other toxicology models?
Transfection is a unique technique used in cell culture.
38
What is transfection in the context of in vitro cell culture models?
Transfection is the process of inserting DNA that has been amplified into a suitable vector to introduce it into a mammalian or other cell.
39
What are two methods commonly used to amplify DNA for transfection?
Polymerase Chain Reaction (PCR) and molecular cloning.
40
Why is it important to know the type of cell culture used before attempting transfection?
Not all cell types are amenable to transfection, so it is important to ensure the chosen cell type can be transfected.
41
What can transfection allow a cell to do?
Transfection can enable a cell to stably or transiently express gene products such as proteins, reporters, or inhibitors that it wouldn’t normally express.
42
How can transfection help in toxicology research?
By transfecting cells with specific genes, researchers can induce the expression of proteins, reporters, or inhibitors. This allows them to observe how these gene products interact with the toxicant, revealing how the toxicant affects cellular processes and providing insights into its mechanism of action.
43
What is molecular cloning?
Molecular cloning is the isolation and generation of recombinant DNA molecules that are placed in organisms for replication and study.
44
What are the key steps in the process of transfection?
First, the plasmid of interest is collected and applied to the cells. Then, the cell membrane's permeability is increased chemically or electrically, allowing the plasmids to enter the cell and its nucleus. Once inside the nucleus, the cell expresses the gene product of the plasmid, often a protein.
45
What is one major limitation of cell culture experiments?
A key limitation is their lack of predictive ability. Since cells are cultured outside of their in vivo environment, the results from cell culture studies may not accurately predict outcomes in living organisms.
46
How does metabolism limit the predictive ability of cell culture experiments?
The metabolic capabilities of isolated cells may not reflect those of intact organisms. This is crucial in toxicology, as the lack of necessary enzymes in cell culture can lead to overlooked or misrepresented effects of toxicants. Research is focused on enhancing the metabolic relevance of cell culture assays to align better with in vivo data.
47
What are stem cells capable of building?
Stem cells are capable of building any tissue type that exists within the body.
48
What are the two unique features of stem cells?
1. They can differentiate into a number of different specialized cell types.
2. They can renew themselves indefinitely.
49
How can stem cells be defined?
Stem cells can be defined by the developmental stage of the animal from which they are derived and their biological characteristics based on their ability to differentiate.
50
What are the two types of stem cells based on their differentiation capabilities?
Pluripotent and multipotent stem cells.
51
What are pluripotent stem cells?
Pluripotent stem cells have the capacity to differentiate into all cell types within the body, across all germ lines (endoderm, mesoderm, and ectoderm).
52
What are multipotent stem cells?
Multipotent stem cells have the capacity to differentiate into any cell type within one of the germ lines (endoderm, mesoderm, or ectoderm).
53
How can the use of stem cells benefit the field of toxicology?
The unique properties of stem cells allow researchers to address questions that are not possible with conventional cell culture.
54
Why is stem cell toxicology considered more consistent with in vivo data?
Stem cell toxicology increases the predictive ability of the model over other types of cell culture, making it a useful in vitro model in toxicological research.
55
What are the three types of stem cells applicable in toxicological research?
1. Embryonic stem cells
2. Adult stem cells
3. Induced pluripotent stem cells
56
What are embryonic stem cells and their characteristics?
Embryonic stem cells are harvested from an embryo that is less than 5 days old and are pluripotent, meaning they can differentiate into any cell type.
57
Where are adult stem cells harvested from, and what is their limitation?
Adult stem cells are harvested from adult tissues such as bone marrow, skin, cord blood, and brain.
They are limited in the types of cells they can differentiate into compared to embryonic stem cells.
58
What are induced pluripotent stem cells, and how are they created?
These stem cells are harvested from somatic cells and are induced to a state in which they can give rise to a number of different cell types via genetic transformation.
Functionally, they are also pluripotent
59
Which type of stem cells is considered the most useful in research due to fewer ethical constraints?
Induced pluripotent stem cells (iPSCs) are considered the most useful because they do not have the ethical constraints associated with embryonic stem cells, while also possessing pluripotent capabilities that adult stem cells lack.
60
What are some advantages of using stem cells in toxicological studies?
1. Stem cells do not lose their replicative ability and do not require genetic manipulation.
2. They allow for assessment of developmental toxicity testing in vitro.
3. Toxicity tests in stem cells tend to have more consistency with in vivo data compared to conventional cell types.
61
What are some disadvantages of using stem cells in research?
1. Stem cells are technically difficult to culture, and there is a lack of established protocols, making their use intimidating.
2. A general lack of understanding means stem cells are often not well characterized.
62
What are co-culture systems used for in cell studies?
Co-culture systems are used to study interactions between different cell populations and are fundamental for investigating cell-cell interactions.
63
What defines a co-culture setup?
A co-culture setup involves two or more different populations of cells grown with some degree of contact between them.
64
How do recent advances in co-culture improve toxicity investigations?
Recent advances involve co-culturing different types of cells, making the models more predictive of human responses when investigating the toxicity of substances.
65
What are two promising models in co-culture systems?
The two promising models are the organotypic model and organ-on-a-chip.
66
What is the organotypic model in cell culture?
The organotypic model is a type of cell culture where two or more cell types from a complex tissue or organ are cultured together to mimic in vivo tissue, utilizing 3D culturing to allow for more natural cell interactions, including extracellular matrix formation.
67
How does the organotypic model improve the study of dermal toxicity?
For studying dermal toxicity, co-culturing keratinocytes and fibroblasts mimics the in vivo environment of the skin, potentially providing more accurate toxicity reflections compared to monocultures.
68
What is the organ-on-a-chip model?
The organ-on-a-chip model involves culturing cells on a specialized microchip designed to recapitulate the microenvironment of a human organ, providing a more accurate representation of the organ system of interest.
69
How does the organ-on-a-chip model differ from the organotypic model?
While both models aim to mimic in vivo conditions, the organ-on-a-chip model uses a microenvironment that better reflects the specific organ system being studied.
70
What physiological processes can be simulated in the organ-on-a-chip model for studying lung toxicity?
The organ-on-a-chip model allows for the culture of relevant lung cells and the simulation of physiological processes, such as air exposure and the stretch force of breathing, to better replicate the in vivo environment.
71
What is tissue culture in the context of cell and organ studies?
Tissue culture involves culturing entire tissues or organs, either as slices or whole organs, which has been used for longer than organotypic or organ-on-a-chip models and can produce similar experimental results.
Numerous organ types have been cultured, including the lung, liver, kidney, skin, and many more.
72
What is mouse or rat embryo culture, and why is it significant?
Mouse or rat embryo culture involves removing whole embryos, still in their yolk sac, from the uterus and culturing them for a short period. This method provides unique mechanistic insights beyond traditional in vitro or in vivo approaches.
73
What are two benefits of using embryonic culture to study toxicants?
1. The effects of a toxicant on the developing fetus can be assessed independently of the mother's influence.
2. Specific embryonic organs can be identified and cultured separately to isolate the effects of toxicant exposure on the development of particular embryonic structures.
74
What is the ectoplacental cone?
The ectoplacental cone is a structure responsible for interactions between the mother and the embryo.
75
Why might it be beneficial to use two different in vitro models?
Using two complementary in vitro models can provide a more comprehensive understanding of the research question being investigated.
76
In a study on the toxicity of an ingested xenobiotic that causes lung toxicity, what factors should a researcher consider when choosing an in vitro model?
The choice of in vitro model depends on several factors, including the specific mechanisms of action being studied, the relevance of the model to lung metabolism, and the experimental goals.
77
When investigating how a toxicant affects neuronal proteins, what in vitro model should be considered, and what are the reasons behind this choice?
Isolating neurons from animals, such as mice, is recommended because primary human neurons are not feasible for ethical reasons, and popular continuous cell lines like HeLa or human embryonic kidney cells may not accurately represent neuronal signaling pathways due to their different origins. HeLa cells raise ethical concerns, including lack of informed consent and racial disparities in medicine.
78
What are HeLa cells, and what ethical concerns are associated with their use?
HeLa cells are the oldest and most commonly used immortal cell line in research, derived from cervical cancer cells of Henrietta Lacks. Ethical concerns include the absence of informed consent, racial disparities, lack of protection of medical information, and commercialization profits without compensation to the Lacks family.
79
What are the four major types of experimental models used in toxicology?
1. Cell culture
2. Stem cell culture
3. Co-culture of cells
4. Tissue culture
80
What factors should be considered when choosing animal models for toxicological testing, especially in developmental toxicology?
Initially, it is advisable to use non-genetically manipulated animals to test the toxicity of new chemicals.
For subsequent studies, using disease models that represent the exposure population is appropriate.
In developmental toxicology, selecting an animal model with a placenta type similar to humans is important for accurately mimicking toxicant handling by the human placenta.
81
What are the reasons for the value of animal models in different fields of toxicology (developmental, cardiac, liver, and nervous system)?
Developmental Toxicology: Consideration of maternal and fetal contributions to a toxic response.
Cardiac Toxicology: Compensatory mechanisms that could activate following a toxic response.
Liver Toxicology: Changes in toxicant metabolism that accompany liver toxicity.
Nervous System Toxicology: Behavioral changes associated with toxicant exposure.
82
What are the 4 key research-level factors to consider when developing a toxicological research study?
1. Existing Knowledge: Evaluate the current body of research to identify knowledge gaps and suitable animal models. Mice are commonly used, providing an extensive knowledge base.
2. Statistics: Ensure adequate statistical power, often requiring large sample sizes for significant results. Mice and small mammals are advantageous for their availability and ability to provide large samples.
3. Generalizability: Assess whether findings in animal models can be applied to humans. Mice's genetic and physiological similarities to humans enhance their generalizability.
4. Logistics: Consider feasibility factors such as cost, availability, and housing of the animal models, with mice being widely accessible.
83
What should researchers consider regarding the genetic strain of their animal model?
Researchers should consider whether uniformity (using inbred strains) is beneficial for mechanistic work to reduce genetic variability or if a strain with genetic variation is more useful for their research.
84
Why is genetic manipulation of animal models significant in research?
Genetic manipulation allows for the study of specific genetic traits or diseases, enhancing the relevance and precision of research findings.
85
What is the difference between using natural and experimental animal models?
Natural models come from wild populations and may be necessary for certain ecological studies (particularly when evaluating the environmental toxicity of a chemical)
Experimental models are bred in controlled laboratories, providing consistency and reliability.
86
What biological properties should be considered when selecting an animal model?
Researchers should choose species that exhibit similar biological processes, particularly metabolism, to ensure relevance to human health and disease.
87
How do sex and lifespan of an animal influence research decisions?
Researchers need to consider sex differences in toxicant effects and select species with appropriate lifespans for the study's focus, such as aging or reproductive studies.
88
What is the average lifespan of laboratory mice, and why is it relevant to research?
Laboratory mice have an average lifespan of 3-4 years, making them suitable for studying aging and developmental processes.
89
Why are guidelines like those from the Organization of Economic Co-operation and Development (OECD) beneficial in toxicological research?
They offer a framework for experimental consistency across laboratories, helping to standardize methods and results.
90
What is acute oral dosing used for in toxicological studies?
It is the traditional OECD method used to determine the toxicity of a substance.
91
How many animals should be included in an acute oral testing experiment?
At least 5 animals of each sex per dose group.
92
What is the procedure for administering the toxicant in acute oral testing?
Animals are given a dose of the toxicant or a vehicle control (0 mg/kg) via oral gavage.
93
What is the waiting period after administering the toxicant in acute oral testing?
A wait time of 24 hours is typical to observe for a toxic response.
94
What aspects of toxicity can be assessed through acute oral testing?
Lethality, onset, nature, severity, and reversibility of the toxicity.
95
How is a dose-response curve created in acute oral testing?
By recording the percentage of the animal population that experiences toxicity at each dose, typically assessed 24 hours after exposure.
96
What is the time frame for acute toxicity tests? Name three examples.
Acute tests demonstrate the effects of a substance after one or multiple exposures in less than 24 hours.
- Eye irritation tests
- Dermal irritation/sensitization tests
- Safety pharmacology tests
97
What is the time frame for sub-chronic toxicity tests? Name two examples.
Sub-chronic tests demonstrate the effects of a substance after repeated doses over 5 days to 6 months.
- 90-day toxicity tests
- Repeated dose dermal tests
- Reproductive toxicity and teratogenicity tests
- Inhalation tests
98
What defines chronic toxicity tests in terms of exposure duration? Name two examples.
Chronic tests demonstrate the effects of a substance after long-term exposure over a period of more than 6 months.
- Chronic toxicity and carcinogenicity tests
- Single or multiple generation tests
99
What are special toxicity tests used for?
Special toxicity tests are used to investigate the toxicity of a toxicant on specific human organ systems, cell functions, or other specific criteria.
100
What is the focus of neurotoxicity tests?
Neurotoxicity tests identify selective toxicity to neurons or subtypes of neurons caused by a toxicant.
101
Why are behavior tests important in toxicity testing?
Behavior tests can identify changes in an animal's behavior after exposure to a toxicant, which may be difficult to detect with traditional biochemical or physiological assays.
102
What do immunotoxicity tests assess?
Immunotoxicity tests assess whether a toxicant interferes with proper immune function, increasing susceptibility to infection or carcinogenesis.
103
What are transgenic animals?
Transgenic animals are genetically modified organisms that have foreign DNA transplanted into their genome, which is transmitted through the germline so that every cell contains the transgene.
104
What is a knock-out animal model?
A knock-out animal model is one in which a specific gene has been deleted or knocked out, suppressing or removing the gene's product entirely.
105
What is a knock-in animal model?
A knock-in animal model is one in which a gene has been introduced or knocked in, allowing the animal to overexpress an existing gene or express a gene it does not normally express.
106
Which species are most commonly used as transgenic models?
The majority of transgenic animals are mice, as they were the first species in which transgenics were performed. Other species such as rats and pigs have also been developed as transgenic models.
107
What is the germline?
The genetic material that is passed down through generations to offspring
108
What is DNA microinjection in the context of creating transgenic animals?
DNA microinjection is a technique that involves using a fine glass pipette to manually inject DNA from one organism into the eggs of another, resulting in random incorporation (or not) of the transgene into the host cell’s DNA.
The high potential for random incorporation of the transgene can lead to inappropriate insertion and aberrant expression of the gene.
109
What are the potential issues with DNA microinjection?
The high potential for random incorporation of the transgene can lead to inappropriate insertion and aberrant expression of the gene.
110
What does retrovirus-mediated gene transfer involve?
This method uses retroviral vectors to infect early embryos and insert proviral DNA copies of their RNA genomes into the host DNA through reverse transcriptase.
111
What is embryonic stem cell-mediated gene transfer?
his approach involves using embryonic stem (ES) cells derived from the inner cell mass of the blastocyst, which are cultured and then reinserted into normal mouse blastocysts for germline gene insertion.
112
What are transgenic models used to assess mutagenicity?
These models are designed to evaluate the potential of substances to cause genetic mutations, which can lead to cancer.
113
What are other transgenic reporter animals?
These animals express a reporter gene that allows researchers to track the effects of toxicants and understand their mechanisms in real-time.
114
What are mutations in DNA, and why are they significant in toxicology?
Mutations are inheritable changes in the nucleotide sequence of a chromosome, and they can lead to cancer, birth defects, and other long-term diseases.
115
What is the process for creating a transgenic animal model for mutagenesis experiments?
A vector containing a reporter gene is inserted into an animal (typically a mouse) using transgenic methods to generate a line of transgenic animals.
116
What steps follow the exposure of a transgenic animal to a toxicant in mutagenesis experiments?
After exposure, genomic DNA is extracted from the transgenic animal, packaged into viral vectors, and then used to infect bacteria grown in selective in vitro conditions.
117
How do researchers determine the frequency of mutations in the transgenic model?
They count the number of colored bacterial colonies that contain the reporter gene, indicating the frequency of mutations.
118
How are transgenic animal models used to assess mutagenicity, and what is the process involved?
Transgenic animal models are created by inserting a vector containing a reporter gene (which produces a colored product) into an animal, typically a mouse.
After the transgenic animal is exposed to a toxicant, genomic DNA is extracted, packaged into viral vectors, and used to infect bacteria grown under selective in vitro conditions.
The frequency of mutations is then determined by counting the number of colored bacterial colonies that possess the reporter gene, indicating the mutation frequency.
119
What are transgenic reporter animals, and how are they used in toxicology studies?
Transgenic reporter animals express a reporter gene (like green fluorescent protein or luciferase) in response to specific stimuli, such as exposure to toxicants.
For example, a luciferase reporter mouse emits light only when estrogen receptor transcriptional activity occurs, allowing researchers to detect potential endocrine-disrupting compounds.
This method enables the real-time observation of toxic effects within the living organism.
120
What is the role of transgenic knock-out models of endogenous genes in toxicology research, and how do they function?
Transgenic knock-out models are used to evaluate mechanisms of toxicity by removing the expression of specific genes.
In some cases, double knock-outs are created, where two genes are knocked out to provide deeper insights into toxicological interactions.
For instance, researchers can engineer a knock-out model for a specific CYP (cytochrome P450) isoform involved in the metabolism of a toxicant. By exposing both wild-type and knock-out animals to the same dose of a toxicant, researchers can compare their responses, gaining valuable information on the role of the knocked-out gene product in the toxic response. This approach helps elucidate the mechanisms of toxicity linked to the toxicant.
121
How can researchers evaluate the role of specific genes in the metabolism of toxicants using knock-out models?
By engineering a knock-out model for a specific gene, researchers can expose both wild-type and knock-out animals to the same dose of a toxicant and compare their responses to understand the role of the gene in the toxic response.
122
What challenges do researchers face with traditional knock-out models?
Traditional knock-out models can lead to embryolethality, where the absence of a critical gene results in early developmental death.
123
What are conditional knock-out models, and why were they developed?
Conditional knock-out models allow for the selective deletion of a gene in specific cells, tissues, or at particular times, improving survival rates during embryonic development and providing more targeted insights into a gene's function.
124
What are the two types of conditional knock-out models?
Cell, Tissue, or Organ Specific: Gene deletion occurs only in specific tissues or organs.
Inducible: The gene can be silenced at a designated time through a chemical agent that initiates the knock-out.
125
What are knock-in models in transgenic animal research, and how are they used to study toxicants?
Knock-in models are transgenic animals created by introducing a human gene into a mouse to express a specific protein, often an enzyme, that is necessary for human-like metabolism of a toxicant.
This allows for more predictive results relevant to human scenarios. A specific type, known as "humanized mice," replaces a mouse gene with a homologous human gene, improving the generalizability of research findings.
Knock-in models can be developed for various genes, including those for transcription factors, signaling proteins, transporters, and receptors.
126
What types of biological macromolecules are analyzed in toxicology to determine the effects of a toxicant?
DNA, RNA, and proteins are analyzed using molecular, immunochemical, and 'omic' techniques. These analyses provide valuable information about the biological effects of toxicants on genes, proteins, and cellular functions.
127
How is Polymerase Chain Reaction (PCR) useful in toxicological research?
PCR is used to exponentially amplify specific segments of DNA, making it easier to detect and analyze genes or DNA sequences. This technique is valuable in toxicology for studying the genetic effects of toxicants.
128
What are some potential applications of PCR in toxicology?
PCR can be used to:
- Identify Genetic Polymorphisms: PCR helps detect polymorphisms in DNA that may make certain individuals more or less susceptible to toxicant effects, aiding in personalized risk assessments.
- Gene Expression Studies: PCR products can be processed and transfected into cells to express exogenous genes (genes from other sources) or overexpress endogenous genes (naturally occurring genes) to explore toxicant mechanisms.
- Mutagenesis Detection: PCR can amplify and identify mutations in gene sequences. Coupled with advanced genomics tools like whole-genome sequencing, this helps in the identification of toxicant-induced mutations.
129
What is epigenetics?
Epigenetics is the study of heritable changes in gene expression (active versus inactive genes) that do not involve changes to the underlying DNA sequence.
130
How does DNA methylation affect transcription?
DNA methylation can inhibit transcription by blocking the transcriptional machinery (like RNA polymerase) from binding to DNA, preventing the transcription of DNA to RNA.
131
How can toxicant exposure influence DNA methylation?
Toxicant exposure can lead to epigenetic changes, such as global or gene-specific DNA methylation, which may inhibit gene expression and result in health effects.
132
What is the difference between non-methylated and methylated DNA in terms of transcription?
Non-methylated DNA usually allows transcription machinery to bind and transcribe the gene, while methylated DNA usually blocks this process, preventing transcription.
133
Why is measuring mRNA levels important in toxicological research?
Measuring mRNA levels can indicate whether a toxicant exposure is producing changes in gene transcription. mRNA levels can serve as a proxy for protein levels, helping to assess the biological effects of toxicants.
134
What is qRT-PCR, and how is it used to measure mRNA levels?
qRT-PCR is a technique where mRNA transcripts are reverse transcribed into DNA by reverse transcriptase, followed by amplification and quantification to measure relative mRNA levels.
135
How do microarrays help in detecting mRNA or DNA sequences?
Microarrays use a solid surface with attached oligo-probes, which hybridize and report the presence of specific DNA or RNA sequences, making it possible to analyze gene expression.
136
What is Next-Generation Sequencing (NGS) and its role in toxicology?
NGS, also known as high-throughput or deep sequencing, encompasses modern sequencing technologies like Illumina and Roche 454.
It allows for the sequencing of entire genomes in a single day, providing detailed insights into genetic changes due to toxicants.
137
What are oligo-probes used for in molecular biology?
Oligo-probes are short nucleotide sequences synthesized to match a specific region of DNA or RNA, used as molecular probes to detect the presence of the target sequence in experiments like microarrays.
138
How can miRNA levels be useful in toxicological research?
miRNA levels can be measured to study epigenetic changes in toxicant-exposed vs. unexposed cells. miRNAs regulate gene expression by silencing mRNA at post-transcriptional and post-translational stages.
139
What is the main function of miRNA in gene regulation?
miRNA regulates gene expression by binding to complementary mRNA, leading to either degradation of the mRNA or inhibition of its translation, thus silencing the gene's expression.
140
How is miRNA produced in the cell?
miRNA is transcribed in the nucleus, modified, transported to the cytoplasm, and further processed into a single-stranded miRNA fragment.
141
Do miRNAs code for a gene product?
NO, but instead they have important regulatory roles in the form of post-transcriptional and post-translational silencing of gene products
142
What are the two mechanisms by which miRNA silences mRNA?
miRNA can silence mRNA by either inducing its degradation or preventing the translation machinery from translating the mRNA into protein.
143
How can exposure to toxicants affect miRNA levels?
Toxicants can alter miRNA levels, leading to changes in downstream mRNA expression, which can affect protein levels, activity, and cellular phenotype, potentially contributing to toxic responses.
144
What are the potential downstream effects of altered miRNA levels due to toxicant exposure?
Altered miRNA levels can lead to decreased mRNA expression, which may result in reduced protein levels and activity, potentially causing changes in cellular function and contributing to toxicity.
145
What are the primary techniques used to quantify protein levels in cells or tissues?
Common techniques include Western blotting, immunolocalization, immunoprecipitation, ELISAs (Enzyme-Linked Immunoabsorbant Assays), and protein microarrays.
146
What is Western blotting used for in protein analysis?
Western blotting is used to determine the relative amount of protein in a particular cell or tissue by creating whole cell lysates or nuclear/cytosolic fractions for analysis.
147
What does immunolocalization reveal about proteins?
A technique whereby tissue slices or cells can be prepared and exposed to different antibodies which will reveal the cellular and subcellular localization of proteins of interest when visualized under a microscope.
148
What is immunoprecipitation used for?
A technique that allows for the detection of interactions between two proteins, by first isolating one of the proteins and then probing for the second protein with another antibody.
Only if the proteins are associated, will the second protein be detected
149
What is an ELISA and how is it used to detect proteins?
In ELISAs, either the antibody or the substrate is first coated onto a plate, and the protein levels are measured using a spectrophotometer, with darker or more intense color indicating higher protein levels.
150
What is a protein microarray and its use?
A protein microarray uses hundreds of antibodies affixed to a single plate to allow large-scale detection of proteins in a sample, commonly used in proteomic analysis.
151
What is the difference between primary and secondary antibodies?
Primary antibodies bind directly to the target protein, while secondary antibodies bind to primary antibodies and are conjugated to a label for visualization or quantification.
152
Why are immunochemical techniques commonly used to quantify protein levels?
Immunochemical techniques are commonly used because proteins act as antigens, allowing them to be recognized by primary antibodies and secondary antibodies.
There are many different labels that can be used; one common group of labels is enzymes that react with a substrate to produce a detectable product
153
What is a kinase assay used for?
A kinase assay measures the formation of ADP, which is an indirect measure of the phosphotransferase activity of kinases.
154
What is a kinase?
A kinase is an enzyme that catalyzes the transfer of phosphate groups from high-energy, phosphate-donating molecules to specific substrates, a process known as phosphorylation.
155
What does phosphorylation entail?
Phosphorylation is when a substrate gains a phosphate group, donated by a high-energy ATP molecule.
156
Why is it important to detect transcription factor activity in toxicology?
Detecting transcription factor activity is important because toxicant exposures can alter the transcriptional activity of genes, affecting downstream gene and protein expression.
157
How can measuring transcription factor activity contribute to understanding toxicants?
By comparing transcription factor activity between exposed and unexposed samples, researchers can gain valuable insights into the mechanism of action of a toxicant.
158
What is typically being measured in assays that detect transcription factor activity?
The interaction between the transcription factor and the DNA
159
What is the role of transcription factors in gene expression?
Transcription factors interact with DNA to initiate the transcription of downstream genes.
160
What does EMSA stand for, and what is its purpose?
EMSA stands for Electrophoretic Mobility Shift Assay.
It is used to detect protein-DNA interactions by observing the migration of protein-DNA complexes through a non-denaturing gel.
161
How do protein-DNA complexes behave in an EMSA compared to free DNA?
Protein-DNA complexes migrate more slowly through the gel than free DNA fragments because they are larger.
162
How are ELISA-based assays used to detect transcription factor activity?
In ELISA-based assays, nuclear extracts containing transcription factors are hybridized with immobilized oligonucleotides on a plate.
Primary antibodies bind to the transcription factor, and a secondary antibody with a label allows detection of the bound transcription factor.
The amount of the bound transcription factor can be quantified based on the signal generated by the label on the secondary antibody. The more transcription factor that is present, the stronger the signal will be.
163
What is the purpose of Chromatin Immunoprecipitation (ChIP) assays?
ChIP assays determine whether proteins, such as transcription factors, are associated with specific regions of DNA.
164
What are the key steps involved in a ChIP assay?
1. Cross-link transcription factors to DNA.
2. Digest chromatin into small DNA fragments.
3. Use antibodies to isolate transcription factors while attached to DNA.
4. Dissociate DNA and protein to quantify DNA to determine the amount of active transcription factor.
165
What is the importance of determining metabolic pathways when studying a new toxicant?
Determining metabolic pathways is crucial for assessing:
- The contribution of metabolites to the overall toxicity of the toxicant.
- The role of metabolism in the excretion of the toxicant.
166
What factors influence the methods used to determine the metabolic pathways of a compound?
The methods depend on:
- Whether the study is conducted in vitro or in vivo.
- The species being studied.
- The specific research question being addressed.
167
What is the purpose of in vitro analysis in toxicology?
In vitro analysis is used to investigate the contribution of specific enzyme classes to the metabolism of a compound.
168
What are the different fractions of cells that can be used in in vitro analysis?
Cells can be separated into:
1. Cytoplasm only
2. Nucleus only
3. Mitochondria only
4. Endoplasmic reticulum only
5. Some combination of these components
169
Why is the endoplasmic reticulum of particular interest in metabolic studies?
The endoplasmic reticulum is important because it is where cytochrome P450 enzymes (CYP450s), which are crucial for drug metabolism, are located within the cell.
170
What is the purpose of measuring metabolite production in in vitro analysis?
The purpose is to determine the amounts of each metabolite produced after cells or cell fractions incubate with a toxicant under specific conditions.
171
What are two common methods used for measuring metabolites?
1. Gas Chromatography (GC) or High-Performance Liquid Chromatography (HPLC)
2. Mass Spectrometry (MS)
172
How do Gas Chromatography and HPLC function in measuring metabolites?
They allow researchers to determine the amounts of each metabolite in a sample and separate various metabolites based on factors like hydro/lipophilicity.
173
What is the role of Mass Spectrometry in metabolite analysis?
Mass Spectrometry is used to identify each metabolite based on its molecular weight.
174
What common technique is used in vivo for metabolic studies?
Radioactive labeling (or tracing).
175
What biological samples may researchers analyze to measure toxicants and respective metabolites?
Expired air, urine, blood, feces, and/or tissue.
176
How can parent compounds and metabolites of interest be measured together?
By measuring total radioactivity in the compartment of interest.
177
How can parent compounds and metabolites be measured separately?
By coupling radioactive measurement with techniques like gas chromatography, HPLC, mass spectrometry, or both.
