L6 Molecular end points in toxicity testing in vitro Flashcards
Aims: To introduce the general principles of molecular end point determination in toxicity testing, discuss some of the key approaches used and their application in a tiered screening strategy. Outcomes: - Understanding of the basic principles of molecular end point measurements - Ability to discuss key molecular methods and how to combine them in an appropriate manner in a tiered strategy of toxicity testing
Calcium and cAMP levels can be molecular end points. How can calcium be measured?
Using fluorescenct dyes, with single wavelength intensity modulating and radiometric dyes.
How can cAMP be measured in molecular end point measuring?
Monitoring cAMP is one of the most common ways to screen for agonists and antagonists against which large family of receptors?
Using cAMP assays.
GPCR’s.
A direct competitive immunoassay exists with Protein G immobilised anti-cAMP to a 96-well plate which camptures cAMP in cell lysate. After washing, cAMP can be quantified using HRP activity using optical density 450nm (OD450nm). OD450nm intensity inversely proportional to concentration of cAMP in samples.
Specific molecular markers related to biological model in study can be used as end point measurements. What is a good way of measuring them?
a) Antibody based detection.
Immunoblotting, ELISA, ria, IHC/ICC staining.
b) proteomic analysis
proteolysis and ID of fragments by MS e.g. MALDI-TOF analysis following tryptic digest.
c) gene expression assays
RT-PCR
What is a good control for RT-PCR?
Control, amplyify using primers to household genes.
How can the quantification be improved on RT-PCR?
Using real time RT-PCR
What is the overall principle of RT-PCR?
Solubilise cells –> RNA is precipitated –> using reverse transcriptase, cDNA is synthesised form this –> Gene-specific primers are added –> then added to PCR amplification machinery
What is the effect of Cadmium in toxicity assays?
Cadmium toxicity affects gene expression at quantifiable amounts. Can be used as control/reference for other toxicity studies.
DNA microarray analysis can also be used as end point measuring. How can this be applied to end point?
Doing multiple arrays, control and after toxin exposure. Differences indicate changes due to this toxin.
How are in vitro toxicity testing strategies developed?
Using a battery of tests of basal toxicity and multiple molecular endpoints. Applied to tiered system of increasing complexity.
Early basic tests indicate toxicity (cheaper/easier) used to initially validate before moving onto complex more definitive systems.
I.e. mitotic cel lines used initially then progress to primary cell cultures and whole organ cultures.
