Development of early elucidaion of chemical substance biotransformation with DMA chip

2002 Fiscal Year Final Research Report Summary

• Project/Area Number: 12307010
• Research Category: Grant-in-Aid for Scientific Research (A)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Hygiene
• Research Institution: Keio University
• Principal Investigator: OMAE Kazuyuki Keio University, School of Medicine, Professor, 医学部, 教授 (60118924)
• Co-Investigator(Kenkyū-buntansha): NAKASHIMA Hiroshi Keio University, School of Medicine, Instructor, 医学部, 助手 (80217710) ; NISHIWAKI Yuji Keio University, School of Medicine, Instructor, 医学部, 助手 (40237764) ; TAKEBAYASHI Toru Keio University, School of Medicine, Assistant Professor, 医学部, 専任講師 (30265780)
• Project Period (FY): 2000 – 2002
• Keywords: DNA chip / expression profile / species difference / trichloroethylene / dichloromethane

Research Abstract

In 2000 and 2001, trichloroethylene (TCE) or dichloromethane (DCM) was exposed to rats or mice, expression profiles of liver were obtained with DNA chip (Affymetrix), which contains ESTs as well as genes. TCE or DCM causes hepatoma to mice, but not to rats or human. Whereas intraperitoneal injection of TCE to rats caused activation of lipid metabolism, that to mice seemed to disturb cell proliferation system. Consecutive TCE oral administration up to 2 weeks revealed up-regulation of genes which are induced by peroxisome proliferator. We verified the results of DNA chip by real-time PCR, and sequenced EST clones which showed significant change. Inhalation exposure of DCM to rat up-regulated P450 2E1 gene.
A homologue search program was developed to compare expression profiles between species. Homologues were searched for probes that showed significant change on TCE exposure. New expression profile, in which mouse genes correspond with those of rat, was subjected to cluster analysis. Expression profiles of mouse and those of rat were almost separated.
Our goal is to know the plausibility of expression profile when we determine which animal is suit for extrapolation. We had planned comparison experiments between mouse, rat and human with flesh primary culture hepatocytes. We had established a culture system of which hepatocytes retain inducibility of P450. Mouse primary culture hepatocytes were exposed to TCE after a series of preliminary tests. The exposure condition was determined by real-time PCR, being based on the results of in vivo experiments. We are waiting for human hepatocytes to be delivered from United States.