1. Cancer Care Engineering: Studies on Oxidative Damage and SNPs Lisa M. Kamendulis, PhD James E. Klaunig, PhD Indiana University 1

2. Question: Is oxidative stress status altered in individuals with polyps or colorectal cancer? Approach Whole blood collected and processed from 235 individuals for: – Comet Analysis (Direct and Oxidative DNA damage) – Total Antioxidant Capacity (TEAC) – Focused SNP analysis Samples: – 104 controls – 92 polyps – 27 colon cancer 11 with 1 follow-up; 4 with 2 follow-up – 12 rectal cancer 3 with 1 follow-up; 2 with 2 follow-up – SNPs evaluated only on samples from the initial visit What Modulates Oxidative Stress? Dietary and lifestyle influences high fat diet, alcohol, smoking, etc.  increased levels Antioxidants  decreased levels Inflammation Sources: Mitochondria Endothelial Cells Immune Cells (including microglia) Peroxisomes Metabolism (P450) (Xenobiotics) Antioxidants GSH, Vitamin E, VitC, SOD, Catalase, etc Pro-oxidants H 2 O 2, O 2 -, HO, 1 O 2 2

3. Comet data (235 samples analyzed): Total Antioxidant Capacity (TEAC) Data (235 samples analyzed): Question: Is oxidative stress status altered in individuals with polyps or colorectal cancer? 3

4. Question: Do differences exist in SNPs for oxidative stress and damage, and selective genes associated with colorectal cancer? Oxidative Stress – Catalase – SOD2 – NOS3 – GSTP – GSTM1 DNA Damage/Repair – APEX1 – XRCC – OGG1 – TP53 Inflammation – IL-6 – IL-8 – PTGS2 (COX2) Vitamin D Status – VDR – CASR – CYP24A1 Metabolism – CYP1A2 – CYP3A4 SNPs evaluated (129 samples analyzed) 4

5. 5 APEX1 (I64V) CASR (A986S) Catalase (-262 C/T) CYP1A2 (5') CYP24A1 (intron) GSTM1 (N173K) GSTP1 (I105V) IL6 (5') IIIVVVAAASSS -- NNNKKK IIIVVV -- AAAGGG GTTTCCCTTTCCCTTTAAAGGGCCCGGG --AAAGGGCCCGGG Control (57) 1000084160584022791932601103059375670793 Polyp (52) 10000673126233604964425414341 5240801288 Colon Ca (15) 1000080137802000 80272053140503633531303367 Rectal Ca (5) 100008020040 2000100060400033672040 00100 IL8 (5') NOS3 (E298D) OGG1 (S326C) PTGS2 PTGS2 (Ex10 +837) SOD (A16V) TP53 (P72R) VDR (intron) XRCC (Q399R) -- EEEDDDSSSCCC -- VVVAAA RRRPPP -- RRRQQQ AAATTTGGGTTTCCCGGGCCCTTTAAAGGGAAAGGG CCCGGGCCCTTTCCCTTT Control (57) 225127 4247115837573261394616195823 652511 354223 51445 Polyp (52) 225127 37541048 444452404613254629 464410 31636 44506 Colon Ca (15) 2040 53407877720334740 20 5327 533313 40 20 274727 Rectal Ca (5) 60040 6040080020 60208002006040 206020 6020 60400 Question: Do differences exist in SNPs for oxidative stress and damage, and selective genes associated with colorectal cancer?

6. GSTM1 null shown to be protective, % null population decreases in colorectal cancer Polymorphism in promoter region of COX2 reported to change transcriptional activity. Not previously reported in colon cancer I64V amino acid change, function unknown. No differences observed in this population. 6

7. Next Steps Complete sample and data analysis – 3 additional samples collected – SNPs completed on all 235 samples Data Analysis/Evaluation – SNP tools – Correlation analysis with other endpoints 7