Methods

     Part A: Liver Cell Isolation: An experimental laboratory rat was sacrificed humanely and the liver was salvaged for use in this project. The rats’ liver was surgically removed and perfused of blood using phosphate buffered saline solution. A single lobe of the liver was removed and was digested using a collagenase in William’s media for 30 minutes. The digested liver was then strained through a 530-micron filter to allow only single cells to be obtained. A small sample of the cells were tested for cell count and to assess cell viability with a microscope using Trypan Blue Dye.

     Part B: Dosing of Liver Cells with NDGA: The cells were diluted to the same cell concentration (about 1 x 105 cells/mL) and were cultured on 96-well micro-plates. A total volume of 100 mL was used where 5 mL of NDGA (at different amounts) was added to 95 mL of the cell suspension in media. After thorough mixing the micro-plates were covered and incubated under 37°C. Controls were then prepared with 95 mL cell suspension and 5 mL of NDGA. The incubated compounds were removed after 1-hour and 4-hour intervals and assessed for cell death. This assessment involved the addition of Trypan Blue to the cell mixtures. Following this, the cells were loaded onto a hematocytometer and counted under a microscope. Cells that were transparent and their membranes were clear silver in colour were regarded as live cells. Cells that had Trypan blue dye within the cell walls were considered dead.

     Part C: Investigation of the Potential Mechanism by which NDGA Causes Liver Cell Death: The following solutions were made: 2mM Salicylamide diluted in 10mL of double distilled water, 100mM MgCl₂ dissolved in 10mL of double distilled water, 50mM Na₂HPO₄ dissolved in double distilled water and changed to pH 7.4 using HCl, 10mM glutathione dissolved in the prepared 50mM Na₂HPO₄ at pH 7.4. Also an already prepared sample of 62.5mM of NDGA was taken.

The following reactions were done in open culture tubes and in duplicates.

     For all reactions everything except NAPDH was added and incubated in a shaking water bath for 5 minutes at 37°C (To bring incubation up to temperature and allow NDGA to reach active sites).

     After the 5 minutes were up the NAPDH was added to start the reactions and then it was left to incubate for 60 minutes.

     The reaction was then stopped by placing the open culture tubes in ice-cold water and then adding 50ml cold IS dissolved in CAN (50ml of 2mM salicylamide).

     The solution was then transferred to eppendorf tubes and centrifuged at 14,000 rpm for 10 minutes. The 150ml of the supernatant was transferred into HPLC vials for analysis. 1 tube was prepared with 4 ml of 62.5mM NDGA + 496 ml CAN to see the retention time of NDGA.

     The stock solutions were placed inside the HPLC machine in order to find the potential mechanism that metabolized NDGA kills hepatocytes with.

     The HPLC system will consist of Water’s Model 600 solvent delivery system, Model 486 variable UV-VIS detector, Model 717 Plus auto sampler and a Millennium data module. Absorbance was monitored at 280 nm. Chromatographic separation was carried out on a reversed phase C18 column. The analytes will be eluted under isocratic conditions using an appropriate mobile phase delivered at 1.0 mL/min. 20 µL of the supernatant from the liver microsomal system will be injected directly onto the analytical column for HPLC analysis.

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.: (C) Mithum Kularatne and Andrew Plishka - Canada Wide Virtual Science Fair 2007 :.