Development of a Simple Double-layered Cell Culture System Using Caco-2 and TIG-1 Cells as a New Cytotoxicity Test

Yasuyuki Sakai ¹, Tomonori Arai ^1,2, Akiyoshi Sakoda¹, and Motoyuki Suzuki^1,3
1Institute of Industrial Science, University of Tokyo, 7-22-1 Roppongi, Minato-ku, Tokyo 106-8558, Japan
²Current address; Mitsubishi Tokyo Pharmaceutical Co. Ltd., 1,000 Kamoshida, Aoba-ku, Yokohama 227-0033, Japan
³United Nations University, 5-53-70 Jingumae, Shibuya-ku, Tokyo 150-8558, Japan

Corresponding author:Yasuyuki Sakai, Ph. D.
Institute of Industrial Science, University of Tokyo
7-22-1 Roppongi, Minato-ku, Tokyo 106-8558, Japan
Telephone: +81-3-3402-6231 Ext. 2458
Fax: +81-3-3408-0593
E-mail: yasuyuki@iis.u-tokyo.ac.jp

Running Head: Coculture of Caco-2 cells with fibroblasts

Original paper :AATEX 7(2,3):47-58,2001

Abstract
We investigated the effect of a Caco-2 cell barrier on toxicity expression in cultured human diploid fibroblasts, TIG-1 cells, in a simple double-layered culture system. The experimental setup used a 12-well plate for the TIG-1 cells and a companion membrane culture insert for the Caco-2 cells. Four model chemicals, i.e., caffeine, diclofenac sodium, indomethacin, and paraquat, which have different levels of in vivo human absorbability, were employed to test the feasibility of the culture system. In transport studies using a Caco-2 cell layer, not only initial apparent permeation coefficients of the four chemicals but also in vitro absorption ratios in equilibrium phases were well correlated with previously reported in vivo human absorbability. This phenomenon was explained by the differences in the permeation coefficients between the two directions across the Caco-2 cell layer. Changes in the dose-response relationships in terms of TIG-1 cell growth on the ninth day in the double-layered culture system were largely predicted from the cell growth in the single-layered system (without a Caco-2 cell layer) and the in vitro absorbability. However, small but significant discrepancies between the observed and the predicted dose-response relationships were found in the loading of diclofenac sodium and paraquat. The first discrepancy suggests the involvement of possible enhanced detoxification of diclofenac sodium by the Caco-2 cells. The latter discrepancy reflects the specific permeation kinetic of paraquat across the Caco-2 cell layer. Final toxicity in terms of TIG-1 cell growth in the double-layered system reflected in vivo toxicity better than that of the single-layered system without the Caco-2 cell layer. These results demonstrate that a Caco-2-cells-based permeation test combined with a single-cell-population-based cytotoxicity test is more useful in estimating in vivo human oral toxicity.

Keywords: Cocultivation, Caco-2 cells, Permeation, Human diploid fibroblast, Growth inhibition.

Color Doppler Echocardiograms Can Measure Hemodynamic Changes of Cardiovascular Systems in Chick Embryos

Takashi Sugiyama¹, Kazuru Saito², Hideyo Shimada¹ and Kanji Tshuchimoto^1,2
1Division of Pathophysiology, Center for Clinical Pharmacy and Clinical Sciences, School of Pharmaceutical Sciences, Kitasato University, 9-1, Shirokane 5-chome, Minato-ku, Tokyo 108-8641, Japan
²Kitasato Institute Hospital, 9-1, Shirokane 5-chome, Minato-ku, Tokyo 108-8642, Japan

Correspondence: Takashi Sugiyama, Ph.D., Division of Pathophysiology, Center for Clinical Pharmacy and Clinical Sciences, School of Pharmaceutical Sciences, Kitasato University

9-1, Shirokane 5-chome, Minato-ku, Tokyo 108-8641, Japan.
Tel: +81-3-3444-6161 ext 3231
Fax:+81-3-3446-9036
E-mail: sugiyamat @ platinum.pharm.kitasato-u.ac.jp

Original paper :AATEX 7(2,3):59-66,2001

Abstract
The aim of the present study was to evaluate the hemodynamic changes of chick embryos by using color Doppler echocardiography, which is one of the essential tools used to determine cardiovascular abnormalities in humans. Fertylisin (FTS), which induces cardiovascular abnormalities in rodents, was injected into the air sac of fertile eggs of White Leghorn chickens on the 2nd day of incubation and the chick embryos were observed grossly and physiologically on the 18th day of incubation. A high frequency of cardiovascular abnormalities including double aortic arch etc. was observed in the FTS-treated chick embryos . Color Doppler flow imaging echocardiograms and first Fourier transformation (FFT) patterns of the heart and aorta of FTS- or calmelose (control)-treated chick embryos were recorded on the 18th day of incubation by using echocardiography with 5 MHz transducer. The values of maximum velocity (MV), flow integration (FI) and acceleration time (AT) obtained from the FFT patterns in FTS-treated chick embryos were decreased compared with those of the control embryos, but the heart rate was increased. These findings suggest that color Doppler echocardiography could be applicable to evaluate the anatomical and physiological abnormalities of chick embryonic cardiovascular systems.

Keywords: chick embryo, Doppler echocardiography, cardiovascular anomaly, fertylisinIntroduction

Selected Presentations from the Symposium entitled Potential and Problems of Alternative Research
-Alternatives,Biosafety and Ethics in Laboratories with Human Cells-“(November 13-14, 1999, Tokyo)

Framework is Indispensable to Promote the Use of Human Material in Pharmacogenetics——69
Tohru Masui

Ethical Considerations on the Use of Human Tissues and Cells in Non-clinical Science, Techhnology and Practice—–71
Toshiharu Matsumura

Bioethics of Human DNA Sampling—–75
Hiraku Takebe

Biosafety in Laboratories Working with Human Materials——77
Reiko Hirai