Diallyl Sulfide Attenuates Phip-Induced Cell Cycle Changes And Cell Death In Mcf-10a Cells

1549 Environmental factors, such as diet, have been shown to play a key role in cancer induction. One compound which has drawn much focus in this regard is 2-amino-1-methyl-6-phenylimidazo-[4,5b]pyridine (PhIP), the most abundant heterocyclic amine in the human diet. PhIP is bioactivated by cytochrome P450 enzymes to its N-hydroxy intermediate, N-OH PhIP, which is further metabolized by acetyl and sulfotransferases into DNA binding metabolites shown to induce tumors of the prostate, colon, & mammary glands. We propose that diallyl sulfide will attenuate PhIP-induced alterations in the cell cycle and cell viability. Seven million cells were plated in T-175 culture flasks and treated for 24, 48 and 72 hours with PhIP (100uM), DAS (100uM) and N-OH PhIP (5uM). DAS pretreated groups were dosed with 100uM of DAS for 6 hours prior to dosing with PhIP (100uM) or N-OH PhIP (5uM). All samples were fixed in absolute ethanol and stained with propidium iodide for cell cycle analysis on the FACSCalibur flow cytometer. In addition, cell viability was assessed using the CellTiter 96® AQueousOne Solution Cell Proliferation Assay. Fifty thousand cells were plated per well in 96 well plates and treated in the same manner as the flow treated samples for 24, 48 and 72 hours. A 20uL aliquot of AqueousOne Solution was added to each well and incubated at 37°C for four hours. The plates were read on the Bio-Tek Ex800 microplate reader, where the absorbance at 480 nm was determined for each well. PhIP at 24 and 48 hours reduced the percentage of cells in the S phase by 9% and 13%, respectively. At 48 hours, DAS further reduced the PhIP-induced alterations in S phase from 13% to 39%. However, DAS pretreatment increased the percentage of cells in S phase at 72 hours. Similar results were seen with N-OH PhIP. PhIP reduced cell viability at 24, 48 and 72 hours by 16%, 8% and 17%, respectively. Pre-treatment with DAS attenuated reduction in cell viability only at the 72 hour time point by 9%. Similar results were seen with N-OH PhIP. N-OH PhIP reduced cell viability at 24, 48 and 72 hours by 21%, 30% and 33%, respectively and DAS pretreatment attenuated this reduction at 72 hours by 11%. We conclude that PhIP inhibits DNA synthesis at 24 and 48 hours to allow cells to repair DNA damage. DAS enhances this inhibition in DNA synthesis. At 72 hours, DNA synthesis ensues after the damage has been repaired in the DAS/PhIP treated cells. The same is true with N-OH PhIP. Further studies are underway to investigate these phenomena. This research was supported by NIH/RCMI Grant #G12 RR03020.