7. NAMs & Omics Revolution Flashcards
What are the classic measures of toxicity?
- Histopathology
- Clinical Chemistry
- Metabolism
- Physiology
- Enzymology
- Electron Microscopy
What are the new approach methodologies of toxicity?
- Information from the exposure of chemicals in the context of hazard assessment (seen with Dr. McKeague)
- in silico approaches
- in chemico approaches
- in vitro assays (seen with Dr. McKeague)
- high-throughput screening (seen with Dr. McKeague)
- high-content imaging (seen with Dr. McKeague)
All of the -omics: • genomics • epigenomics • transcriptomics • proteomics • lipidomics • metabolomics
What is the tiered protocol for toxicity testing recommended by the NIHS and Health Canada?
5 interacting levels in the tier:
- Start with the chemical structure
- What is happening in terms of structure activity relationship - in silico
- In vitro -cell lines
- Move to lower organisms like flies and worms. Fish are still debatable because they have backbones but a lot of work is still done on fish
- Assess toxicity in mammals
If there is toxicity along the way you stop. Furthermore, the steps are interconnected with loops where if there are problems at one level can go back and test a different approach/other chemicals.
What problems did we have in terms of proper toxicity testing ? What had to be done?
There were too many chemicals and not enough data. Therefore, compounds needed to be prioritized for more extensive toxicological evaluation, like high volume chemicals and the chemicals you think are most toxic. Also, since there are so many chemicals, we cannot test them all, so we want to be able to develop methods to predict their toxicity -> predictive toxicology.
Define “in silico” approaches & name them. What is the purpose?
In silico experiments are software-based. In silico means: by means of computer modelling or computer simulation.
In silico approaches include:
- Data management
- Bioinformatics
- *Quantitative structure activity relationship
- *Read across
- Modeling
- Reverse dosimetry
The purpose is: From what we know about a family of chemicals, can we predict if another chemical will be toxic or not.
What are “in chemico” approaches?
- Identify reactive compounds
- Use of analytical techniques
What are the different approaches to the read-across in silico method? Label the axes on the graph. (See graph on slide 9 of L7)
X-axis: carbon chain length
Y-axis: Toxicity value
There are points on the graph that are placed based on chemicals that we know (analogs?) (their toxicity and carbon chain length is known). Then, the chemical that we are interested in is added to the graph in order to find its toxicity.
-> Interpolation is when the chemical falls within the carbon chain length of the known chemicals and an estimated toxicity can be determined.
-> Extrapolation is when the chemical of interest is outside of the known chemicals and we extend the trend line to estimate the toxicity.
-> Chemicals can also be compared with a “category approach” where they are compared to other chemicals that are in the same family. They can be put into families based on structure (ex: carbon chain and R-groups) or function (ex: chemicals that have estrogenic activity). It’s also possible to have “outliers” from the categories/families (ex: has the same carbon chain structure but the R-group is different).
What is the purpose of the read-across method?
To be able to potentially find bad/toxic chemicals based on the computer analysis to identify whether or not we should stop the use of the chemicals.
What is the main goal of the “omics”? (Genomics - Transcriptomics - Proteomics – Lipidomics -Metabolomics)
To determine whether gene, RNA, protein or metabolite expression profiles or ”signatures” can serve as markers to predict toxicity. (see if the toxicant exposure is causing harm to any of the “omics” ex: RNA, proteins, etc.)
Define Genome.
All genes of an individual organism.
Define Genomics.
The study of all of the genes of a cell or tissue
at the DNA level.
Define Epigenomics.
The study of all epigenetic modifications, e.g., reversible modifications on a cell’s DNA or histones that affect gene expression without altering the DNA sequence
Define Transcriptomics.
The study of all of the gene transcripts of a cell or tissue (RNA level) -> ex: how does the mRNA respond to a given toxicant?
Define Proteomics.
The study of all of the proteins of a cell or tissue.
Define Lipidomics.
The study of all lipids in an organelle or a cell. Ex: how does the toxicant modify the lipid profile of the cell?
Define Metabolomics.
The study of all small chemicals in a cell.
Describe the Omics cascade from genome to phenotype. (slide 14 L7) Why is the cascade important?
This cascade describes how the “omics” funnel down and relate to each other.
- Genomics: At the DNA level we see what is possible in the cell (the potential).
- Transcriptomics: At the RNA level we see what appears to be happening in the cell (current direction).
- Proteomics: at the protein level we see the functional capabilities of the cell (proteins are what makes things happen).
- Metabolomics: at the metabolite level of the cell we see what is ACTUALLY happening in the cell because metabolites are the limiting currency of the cell. Therefore, what is happening in the cell is dependent on the metabolomics.
Define Toxicogenomics
Toxicogenomics: the effects of a toxicant on all of the genes of a cell or tissue at the DNA, RNA, protein and metabolite levels
Define Toxicoepigenomics
Toxicoepigenomics: The effects of a toxicant on the expression of genes in cells or tissue that is not mediated by altering the nucleotide sequence
What is the underlying hypothesis of toxicogenomics?
Hypothesis: The most sensitive marker of a toxicant on a cell is at the mRNA level. So… thats where you should study.
Gene expression PRECEDES protein changes and toxicity. If you see damage in a tissue that has occurred because proteins are effected, it’s most likely because transcripts of a protein are affected.
Furthermore, changes in gene expression can be measured at low doses of the toxicant.
What are the different ways to measure gene expression via mRNA transcription?
- Gene by gene analysis: Northern Blotting (look at one transcript at a time), RT- PCR (is it expressed or not expressed), qRT-PCR (quantify the amount of the transcript)
- DNA arrays and microarrays
- RNA sequencing
Give a classical example of a toxicogenomics flow scheme.
- Expose animal to a toxicant (multiple doses)
- Isolate a tissue or tissues you want to look at
- Isolate RNA and run your chips to find out which transcripts are being affected or not.
How is toxicogenomics being used? (what are the applications) What are the goals?
- Deciphering mechanism of action (pathway analysis) of toxicant
- Response at low doses (Could see an affect at transcript level before you see an affect in the actual tissue)
- Revealing potentially novel health effects (hypothesis generating science: Based on whats being affected you form your hypothesis, instead of the other way around.)
- Identification of perturbed pathways – targeted follow-up
Goals:
- Biomarker discovery
- Predictive toxicogenomics (so you don’t have to test on animals or people)
Describe array technology. Why was it exciting?
- You have nylon membranes and you spot DNA sequences on it (ex: 300-400 base pairs) which act as a probe to detect gene expression.
- Take RNA from tissue & reverse transcribe it to generate cDNA.
- Put cDNA with radioactive label on the membranes and look for radioactivity to see which genes are expressed.
It was exciting because you could look at 212 transcripts at once.