PCL481H1 Lecture Notes - Lecture 3: Nfe2L2, Cytochrome P450, Cyp2E1
26 Feb 2017
School
Department
Course
Professor
Use of electric cell-substrate impedance sensing to assess in vitro cytotoxicity
• Senses the resistance of the cell membrane in cells that are unlabeled
• Resistance measurements of human umbilical vein epithelial cells (HUVEC)
o Exposed to increasing concentrations of cytochalasin B
o Shows a dose response curve where membrane resistance decreases
Intermediate markers of cellular toxicity
• Cellular levels of reactive oxygen species (ROS): looks at intracellular levels of ROS through assays
• Oxidative stress defense gene expression:
o Looks at how ROS affects gene transcription of defense mechanisms
• Damage to cellular macromolecules:
o Adducts, mass spectrometry and liquid chromatography for DNA, proteins, and lipids
o Looks at adducts to hemoglobin because they bind to SH groups found on them*
• Alterations in gene and protein expression:
o qPCR transcript quantitation
o Western blotting for protein expression, functional assays for enzyme activity
N-Hydroxylation of 4-AMinobiphenyl by CYP2E1 Produces Oxidative Stress in a Mouse model of chemically
induced liver Cancer.
• The DCF assay: Monitors ROS formation in live cells with dichlorogluorescein (DCF) dye.
• The N-hydroxylated metabolite of the mouse liver carcinogen *
• ABP produced oxidative stress in mouse hepatoma cells, which could be blocked by the antioxidant NAC
• The parent compound: ABP produced ROS in cells engineered to express cytochrome P450 CYP2E1
o Caused increased sensitivity to parent compound and increases ROS
o Meaning that CYP2E1 mediates ABP transformation to its ROS form
o Dichlorofluoroscene can enter cells
▪ High ROS in cells→ DCF will react with ROS
▪ Produces a product measured colourmetrically
o ABP is a pro-toxicant that gets converted to an ultimate toxicant:
▪ Super fucked up chemical that produces cancer→ very potent carcinogen
ABP exposure produces a greater Nrf2 antioxidant stress response in livers of female mice
• Nrf2: Nuclear factor (erythroid-derived 2)- like 2:
o A key transcription factor that orchestrates the antioxidant defense response in mouse liver
o Active in presence of ROS→ increases expression of proteins/factors (combat oxidative stress)
• Increase in liver Nrf2 protein is quantified by Western blotting
• Notice how antioxidant response is greater in females than in males
o Comparison of cells through ABP levels, the initial toxicant shows an increase of Nrf2
o This response is far greater in females
• Males are more likely to get cancer when given ABP
o Shows (maybe) females have stronger anti-oxidant response.
ABP exposure produces higher expression of antioxidant Nrf2 target genes in female mice
• Nrf2 target genes: this is a transcription factor, and activates the transcription of downstream genes:
o GGTT1, HMOX1
find more resources at oneclass.com
find more resources at oneclass.com
