ILSI HESI Genotoxic Mechanisms Cytotoxicity Profiles Realtime PCR

ILSI / HESI - Genotoxic Mechanisms Cytotoxicity Profiles & Real-time PCR data from Tanabe (Taxol, Cisplatin in TK 6) Chinami ARUGA Exploratory Toxicology and DMPK Research Laboratories, Tanabe Seiyaku Co. , Ltd. 22 -Jan-2007

Study design Human TK 6 cells Chemicals: low, mid and high doses + time-matched controls Cisplatin Taxol (0. 1, 1 and 10 ug/m. L) (0. 01, 0. 03 and 0. 1 ug/m. L) Treatment 4 hr-treatment (4 hr) 4 hr-treatment + 3 hr-recovery (7 hr) 4 hr-treatment + 20 hr-recovery (24 hr) 2 independent biological replicates x 2 technical replicates Micronucleus observation in 24 hr RT-PCR (47+1 genes) ABI 7900 Low density arrays (cards) Housekeeping gene: 18 S Automatic threshold used on SDS v 2. 2 software partly reanalyzed using a manually fixed threshold DDCt method using the respective control at each time point

Cisplatin: Cyto- & Genotoxicity Profiles * MN(‰）: Permillage of micronucleated cells Fisher’s exact test: *; p<0. 05, **; p<0. 01

Taxol: Cyto- & Genotoxicity Profiles ** ** ** MN(‰）: Permillage of micronucleated cells Fisher’s exact test: *; p<0. 05, **; p<0. 01 **

Cisplatin: PCR data x -8 x -4 x – 1. 5 x 4 x 8

Taxol: PCR data x -8 x -4 x – 1. 5 x 4 x 8

Up-regulation Ten genes were up-regulated by both direct and indirect genotoxicant. Four genes were up-regulated only by direct genotoxicant. Two genes were up-regulated only by indirect genotoxicant. x -8 x -4 x – 1. 5 x 4 x 8

Down-regulation Twelve genes were down-regulated by both direct and indirect genotoxicant. Seven genes were down-regulated only by direct genotoxicant. Two genes were down-regulated only by indirect genotoxicant. x -8 x -4 x – 1. 5 x 4 x 8

Data comparison with the other laboratory Good concordance with data from Novartis except a few genes (Fos, Mdm 2)

Data comparison with the other laboratory -2 Same data with previous page

Outlines Genes not detected GSTA 2, GZMA, GZMB, LEF 1, CDKN 1 C, GADD 45 C. Dose & Time dependency Taxol: Change of gene expression was moderate even in severe genotoxicity concentration. 22 genes were up- or down-regulated by both chemicals. GDF 15, TP 53 I 3 were prominently up-regulated by both chemicals. 15 genes were specifically up- or down-regulated by each chemical. Good concordance with data from the other laboratory.

Next Step Etoposide, Na. Cl study in TK 6 Search for the discriminating genes Comparison with data from the other companies

Acknowledgment Tanabe, Exploratory Toxicology Group Hideko Yamazaki: Cell Culture and Cyto-&Genotox , RT-PCR and Analysis Naoko Murakami: RT-PCR Hisako Fujimura Wataru Toriumi