Sherell A. Morrison Claflin University and University of South Carolina

1. EFFECT OF RESERVATROL IN VITRO ON CYTOCROME P450 1A2, 2A AND 2B1/2 ACTIVITIES IN MOUSE LIVER MICROSOME Sherell A. Morrison Claflin University and University of South Carolina

2. Background • Lung cancer is the leading cause of death in the U.S. due to tobacco use. • A chemical which causes cancer is 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone also known as NNK (nicotine-derived nitrosaminoketone) • NNK is formed from nitrosation of nicotine during tobacco processing and cigarette smoking.

3. Background • Epidemiological studies suggest increased consumption of fruits and vegetables decrease risk for several cancers, including lung cancer. • Resveratrol is a compound found in grapes and red wines. • Resveratrol is synthesized in the leaf epidermis and the skin of grape berries.

4. NNK Pathway N=O N=O N=O O O OH N N N CH3 CH3 CH3 N N N NNK-N-OXIDE NNK NNAL O N=O N=O O O N N CH3 CH2OH OH N N N-OH O O O N + HCHO + H3C-N=NOH N N METHYLDIAZO- HYDROXIDE 4-(3-PYRIDYL)-4-OXOBUTYL- DIAZOHYDROXIDE KETO ALDEHYDE DNA and Protein Methylation DNA and Protein Pyridyloxobutylation Figure 1. Simplified metabolic scheme of NNK.

5. Our Goal • The overall goal of project is to determine the effect of resveratrol on cytochrome P450’s 1A2, 2A and 2B1/2 activities as a preliminary in vitro study to evaluate if resveratrol can potentially inhibit NNK activation.

6. METHODS • Livers and lung of female A/J mice were removed • Liver microsomes were prepared by differential centrifugation. • Different centrifuge speeds helped with separation of the supernatant and cytosol.

7. Cont. METHODS • Protein concentration of liver microsomes was determined by a spectrophotometer. • BCA (bicinchoninic acid) Protein Assay Reagent Kit was used to detect how much protein was present in each sample. • This assay consisted of a well known reduction on Cu+2 to Cu+1 . Results showed color development from light to dark purple. • Contained four samples: NNK, Control, Corn oil and Olive oil.

8. Protein Concentration Results Condition Average Concentration (ug/ul) 200/ug/Protein (ul) ________________________________________________________ Control 9.527 ug/ul 20.993ul _______________________________________________________ Corn oil 13.650 ug/ul 14.652ul

9. Stop-flow Assay for PROD and MROD • Activity assays using • methoxyresorufin (for P450 1A2) • Pentoxyresorufin (for P450 2B1/2) In order to measure resorufin formation we used a device called a flourometer. The first order of business is to obtain a standard curve which will help us to determine our sample readings.

10. Condition MROD PROD (pmol/mg/min) (pmol/mg/min) Control 422 30 Ethanol 395 31 *Ethanol is our vehicle control Stop-flow Assay Results

11. MROD Results

12. PROD Results

13. Conclusion • P450’s 1A2, 2B1/2 are present in the A/J mouse liver. • Resveratrol inhibits the o-dealkylation of methoxyresorufin and pentoxyresorufin in a dose dependent manner. • Since P450 1A2 is responsible for MROD and P450 2B1/2 is responsible for PROD, resveratrol is inhibiting these specific P450’s. • Since P450’s 1A2, 2B1/2 are involved in NNK activation it is possible that resveratrol may inhibit these P450’s therefore decreasing NNK activation and reducing the risk of lung tumor formation.

14. Experimental study dealing with P4502A which is responsible for metabolizing coumarin to 7-hydroxycoumarin. How is resveratrol inhibiting these specific P450’s? Competitive inhibition Noncompetitive inhibition Suicidal inhibition FUTURE RESEARCH