Evaluation of cytotoxic and DNA damaging effects of catechol in HepG2-cells
Independent thesis Advanced level (degree of Master (Two Years)), 20 credits / 30 HE creditsStudent thesis
When cells are treated with a toxicant, cytotoxic and DNA damaging effects might appear. In most cases cytotoxic effects and DNA damage are linked, but for some compounds there is no cytotoxic effect but only DNA damage and these agents are of particular interest in risk assessments. Therefore, when evaluating cytotoxicity and genotoxicity, it is important to determine the relationship between those two effects regarding the concentration in which they are detected. The aim of the present study was to use HepG2 cells to evaluate catechol’s cytotoxicity and genotoxicity. Cytotoxicity was determined using ATP assay and DNA damage was assessed using comet assay, employing a protocol used by for example AstraZeneca safety assessment. Cytotoxicity can be caused by oxidative stress due to inhibition of mitochondrial activity, depletion of glutathione and production of ROS. DNA damage can be caused by oxidative damage and formation of DNA adducts. Catechol also induces apoptosis.
Cells were cultured for 48 hours before exposure to catechol. For the ATP assay, cells were exposed to 1 mM, 2 mM, 3 mM and 6 mM catechol for 24h and luminescence was recorded. Concentrations from 1 mM catechol showed significant cytotoxicity after 24 hours of exposure (P < 0.001). From 3 mM catechol, almost no viability was detected from the cells. To measure DNA damage, cells were exposed to 0.5 mM, 1 mM, 2 mM, 3 mM and 6 mM catechol for 3 hours. Only cells treated with concentrations from 3 mM showed significant DNA damage, compared to untreated cells (P<0,01).
HepG2 cells were found to be more sensitive to catechol induced cytotoxicity than to DNA damage because higher concentrations of catechol were needed to induce significant DNA damage. HepG2 cells are known to have low enzymatic metabolism, and since o-benzoquinone, an active metabolite from catechol also induces DNA damage, it can explain why higher concentrations are needed to obtain DNA damage. However, the observed concentration differences are most likely explained by the different exposure times used in the two different assays (3 hours for comet assay and 24 hours for ATP assay).
Place, publisher, year, edition, pages
2014. , 30 p.
catechol, HeG2, cytotoxicity, DNA damage, genotoxicity
Pharmacology and Toxicology
IdentifiersURN: urn:nbn:se:uu:diva-218118OAI: oai:DiVA.org:uu-218118DiVA: diva2:694909
Subject / course
Bachelor of Science Programme in Pharmacy
2014-01-16, 13:22 (English)
Hellman, Björn, FarmD
Brittebo, Eva, FarmD