Regular articles

REQUIRED EFFORT TO EVOLVE TOXICITY TESTING USING IN VITRO METHODS

MASAMI WATANABE
Division of Radiation Biology, Department of Health Sciences, Faculty of Pharmaceutical Sciences, Nagasaki University, Nagasaki, 852, Japan

Opinion :AATEX 3(3):81-84

Abstract
The procedures used to conduct chemical safety evaluations have developed over the years and continue to evolve with our understanding of the science of toxicology. The main objective of toxicity testing is to generate a toxicological database which can be utilized in human safety evaluation. Historically, the database to be utilized in the safety evaluation process was developed using whole-animal testing, human epidemiological studies and, in some cases, accidental human exposure data. However, as a result of recent biotechnological advances in the areas of cell culture and bioanalytical methodologies, new possibilities for in vitro studies and their applications to toxicity testing have been created. In light of these developments, the traditional approach to toxicity testing should be reevaluated.

TOXICITY OF 10 MEIC CHEMICALS TO MITOCHONDRIA BY FLUORESCENCE IMAGING

XINHAI WANG¹, KEN-ICHI HIRANO², YOSHIRO KOBAYASHI³, KAORU SAIJO¹ and TADAO OHNO^1
1RIKEN Cell Bank, The Institute of Physical and Chemical Research (RIKEN), 3-1-1 Koyadai, Tsukaba Science City, Ibaraki 305, Japan; ²Tsukuba Research Laboratory, Hamamatsu Photonics Co., Ltd., 5-9-2 Tokodai, Tsukuba Science City, Ibaraki 300-26, Japan; ³Department of Biomolecular Science, Faculty of Sciences, Toho University, 2-2-1 Miyama, Funabashi City, Chiba 274, Japan

Regular Article :AATEX 3(3):85-93

Abstract
The in vitro toxicity of 10 MEIC chemicals was determined using a fluorescence microscope-video camera-image processor system. Human lung carcinoma cells were loaded with rhodamine 123 which accumulates specifically in mitochondria in living cells. Rhodamine 123 fluorescence in mitochondria relative to that in the cytoplasm of the cells was measured after 3 hour treatment with the MEIC chemicals. Results showed that the ED50 values of the chemicals determined by this method were highly correlated with the corresponding values determined by the MTT (r=0.949) and lactate dehydrogenase-release assays (r=0.948) after 48 hours. This fluorescence-image processing was faster for measuring changes in mitochondrial function than the MTT assay, and is useful for measuring the direct toxic effects of chemicals on organelle’s functions in situ.

N VITRO CYTOTOXICITY OF 20 MEIC CHMICALS WITH “QUANTUM-TYPE” RELEASE OF RHODAMINE 123 DETECTED BY FLOW CYTOMETRY

XINHAI WANG^1,2, SHU QIN LIU¹, SONGHUA CHEN^1,3, XI RUI GE³, KAORU SAIJO^l and TADAO OHNO^1
1RIKEN Cell Bank, The Institute of Physical and Chemical Research (RIKEN), 3-1-1, Koyadai, Tsukuba Science City, Ibaraki 305, Japan
²Institute of Laboratory Animals, Beijing agricultural University, 2 West Yuanmingyuan Road, Beijin 100094, China
³Shanghai Institute of Cell Biology, Chinese Academy of Science, 320 Yue Yang Road, Shanghai, China

Regular Article :AATEX 3(3):94-105

Abstract
We determined the in vitro cytotoxicity of 20 MEIC chemicals by flow cytometry. Suspension-cultured Daudi cells were pre-stained with rhodamine-123 and treated with MEIC chemicals for 3 hr. Rhodamine-123 localizing preferentially in mitochondria was detected quantitatively. Relative changes in cell size and intracellular structure were also determined simultaneously by monitoring the relative forward and side scattering fluorescence intensity, respectively, by flow cytometry. Different patterns of these 3 parameters were observed in cells treated with different chemicals. For example, acetylsalicylic acid decreased rhodamine-123 content at the 50% effective dose (ED50) Of 1.07 mM but did not affect the other 2 parameters. However, amitriptyline-HCl decreased all 3 parameters near ED50 Values, while digoxin increased the side scattering light intensity suggesting that a dramatic change may have occurred in the intracellular structure. Based on these patterns, we classified toxicity of the MEIC chemicals into 5 types. ED50 Values for the rhodamine-123 release caused by the test chemicals were highly correlated with those derived from MTT assay, LDH-release assay, and the image analysis assay which we developed previously. This simple flow cytometric assay provides a new sight in cytotoxicity assays of chemicals.

PROTECTIVE EFFECTS OF 2,3–DIMERCAPTO-1-PROPANOL ON BIS (TRIBUYULTIN) OXIDE-INDUCED CYTOTOXICITY IN ISOLATED RAT HEPATOCUTES

N. SUSA, S. UENO, and Y. FURUKAWA
Department of Veterinary Public Health, School of Veterinary Medicine and Animal Sciences, Kitasato University, Towada, Aomori 034, Japan

Regular Article :AATEX 3(3):106-110

Abstract
The effects of 2, 3-dimercapto-1-propanol (BAL) on bis (tributyltin) oxide (TBTO)-mediated cell injury were studied using isolated rat hepatocytes. The cells (1×106 cells/ml) were incubated with or without TBTO alone, or with both TBTO and BAL at 37C for 60 min. Simultaneous application of 100 FLM BAL and 100 FLM TBTO reduced the rate of cell injury as evaluated by lactate dehydrogenase leakage and cellular tin content in comparison to TBTO alone. BAL also prevented the stimulation of lipid peroxidation as measured by the production of malonedialdehyde and protected against the depletion of levels of antioxidants such as glutathione (GSH), vitamin C, and vitamin E, and antioxidant enzyme levels such as glutathione reductase and superoxide dismutase induced by TBTO. The results obtained in the present study revealed that BAL is able to prevent TBTO-induced cytotoxicity and suggest that this protective effect may be related to a reduction of the cellular tin content.

COMPARISON OF CYTOTOXICTY OF SURFACTANTS ON A RANGE OF MAMMALIAN CELLS CULTURED UNDER VARIOUS CONDITIONS

H. KOJIMA, A. HANAMURA, A. SATO and H. KONISHI
Biochemical Research Institute, Nippon Menard Cosmetic Co., Ltd. Ohgaki-shi, Gifu 503, Japan

Original paper :AATEX3(3):111-120

Abstract
To facilitate development of an alternative test to predict Draize rabbit eye irritation scores (Draize eye score), we investigated optimal conditions for cytotoxicity testing using cultured mammalian cells. Different cytotoxic markers, various cells, treatment periods and serum concentrations in culture medium were assayed using eight surfactants as test agents.
Good correlations with in vivo results were gained with exposure periods of 24 or 48 hours. Little difference between cytotoxic markers was found exception of the lactate dehydrogenase (LDH) assay. The serum concentration in the medium exerted an effect regardless of the primary cell or cell line, cytotoxicity being more pronounced in cells cultured in serum free medium. However, the correlation with Draize eye score was lower for serum.

Key words: eye irritation, cytotoxicity, in vitro.