Effect of Rutaecarpine on the Benzo(a)pyrene-Induced DNA Damage In Vitro
Poster Number
2
Introduction/Abstract
Benzo[a]pyrene (BaP) is one of the most potent mutagens and carcinogens known. The metabolic pathway of BaP involves several steps initiated by cytochromeP450 (CYP) enzymes such as CYP1A1 to give the ultimate carcinogen benzo(a)pyrene-7,8-diol-9,10-epoxide (BPDE). BPDE is responsible for genotoxic effects by alkylating the DNA at the N2 position of guanine bases. Rutaecarpine, a pentacyclic indolopyridoquinazolinone alkaloid isolated from the unripe fruit of Evodia rutaecarpa, has been used in traditional medicine to treat various diseases. It has been reported as a CYP1 family inducer.
Purpose
Our objective is to determine if co-administered BaP and rutaecarpine would increase the formation of BPDE, which will ultimately increase the DNA damage.
Method
Cell Culture: Mucoepidermoid pulmonary carcinoma cells (H292) and hepatocellular carcinoma cells (Hep 3B) were seeded in 6 well plates and were incubated with benzo[a]pyrene (5 μM) and/or varying concentrations of rutaecarpine (1.25 μM, 2.5 μM, 5 μM, and 10 μM) for 24 hours.
EROD Assay: After the treaments, supernatant was discard, and cells were iucubated with fresh medium which mixed with 7-ethoxyresorufin. The activity of CYP1A1 in different treatment groups is measured as a rate of the O-deethylation of 7-ethoxyresorufin into resorufin (EROD assay). Resorufin can be detected using a fluorimetric assay. The fluorescence data was then normalized by BCA assay for protein.
MuseTM Multi-Color DNA Damage kit: The DNA damage levels of cells were determined by MuseTM Multi-Color DNA Damage kit. This kit provides a quick and easy way to detect the activation of ATM and H2A.X which reflects the extent of DNA damage using the Muse® Cell Analyzer.
Statistical analysis: Analysis of variance (ANOVA) with post hoc Tukey’s multiple comparisons test (compares means of each group with every other group) were performed on all the data sets. Data was analyzed using GraphPad Prism (Version 7.0). The acceptance level of statistical significance was P<0.05.
Results
The results showed that BaP (5 μM) and rutaecarpine (1.25-10 μM) significantly induced CYP1A1 enzyme activity (p
Significance
Our data showed that both rutaecarpine and benzo[a]pyrene are inducers of CYP1A1 enzyme, however, co-administration of rutaecarpine with BaP caused induction of CYP1A1 to a lesser extent. DNA damage results further supported the possibility that there is a protective effect of rutaecarpine against benzo[a]pyrene-induced DNA damage in both H292 and Hep 3B cells.
Location
DeRosa University Center
Format
Poster Presentation
Poster Session
Afternoon
Effect of Rutaecarpine on the Benzo(a)pyrene-Induced DNA Damage In Vitro
DeRosa University Center
Benzo[a]pyrene (BaP) is one of the most potent mutagens and carcinogens known. The metabolic pathway of BaP involves several steps initiated by cytochromeP450 (CYP) enzymes such as CYP1A1 to give the ultimate carcinogen benzo(a)pyrene-7,8-diol-9,10-epoxide (BPDE). BPDE is responsible for genotoxic effects by alkylating the DNA at the N2 position of guanine bases. Rutaecarpine, a pentacyclic indolopyridoquinazolinone alkaloid isolated from the unripe fruit of Evodia rutaecarpa, has been used in traditional medicine to treat various diseases. It has been reported as a CYP1 family inducer.
