Vol.1 No.3
Contents
Validation of in vitro tests for general toxicity
Bjorn Ekwall
Dept. Toxicology, Univ. of Uppsala Biomedical Center
AATEX 1(3):127-141
INTERLABORATORY VALIDATION STUDY OF THE ADVANCED TISSUE SCIENCES’ SKIN2TM DERMAL MODEL AND MTT CYTOTOXICITY ASSAY KITS
DENNIS TRIGLIA1, INGER KIDD1, BART DE WEVER2 and RAOUL ROOMAN2
1Advanced Tissue Science, Inc. (formerly Marrow-Tech, Inc), LaJolla, CA, USA; 2Dept. of Cell Biology, Life Science, Janssen Research Foundation, Beerse, Belgium
AATEX1(3):142-147
MEASUREMENT AND ANALYSIS OF INTRACELLULAR ION DISTRIBUTIONS IN SINGLE LIVING CELLS WITH FLUORESCENT INDICATOR DYES
ATSUO MIYAKAWA
Deportment of Medical Photonics, Hamamarsu University School of Medicine Handa-cho 3600, Hamamutsu-shi, 431-31 JAPAN
AATEX 1(3):148-153
Abstract
The distributions of intracellular pH, Na+, K+, Mg2+ and Ca2+ concentration is measured using various type fluorescent indicator dyes. The measurement of these intracellular ion distributions performed by a ratio imaging method. The equipment constructed with a fluorescence microscope and digital image processor.
Many type indicator dyes had the property of collection in nucleus, but only Na+ dye had a collection in cytoplasm. The intracellular pH, K+ and Ca2+ distribution was observed homogeneously distribution. However, the intracellular Na+ and Mg2+ shows the concentration in cytoplasm and nucleus were clearly different.
Abstract
Two factors of critical importance to the adoption of in vitro alternative assays are the predictive potential of the assay system and its intra- and interlaboratory reproducibility. Many in vitro alternatives to animal testing have been proposed, but only a few of them have been subjected to interlaboratory validation studies in order to assess their reproducibility and relevance to in vivo data. The study described in this paper was designed to assess the shippability, reproducibility and relevance to in vivo data of the Advanced Tissue Sciences Skin2TM Dermal Model substrate in concert with Advanced Tissue Sciences’ MTT Cytotoxicity Assay Kits. This 3-dimensional Dermal Model, which consists of several layers of human, foreskin-derived, metabolically active fibroblasts grown to form a tissue equivalent on nylon mesh, was used as the substrate to test the toxicity of 12 chemicals proposed by the Commission of the European Communities (i.e., toluene, n-hexane, 1-butanol, chloroform, sodium dodecyl sulfate, benzalkonium chloride, silver nitrate, tributyltin chloride, dibutyltin dichloride, 2-butoxyethyl acetate, acetaldehyde, and 2-methoxyethanol). Dermal Model kits and MTT Assay Kits were manufactured by Advanced tissue sciences (formerly Marrow-Tech) in the U.S. and shipped to Janssen Pharmaceutica in Belgium. Two runs of each of the twelve chemicals were performed in each lab one week apart and MTT-50 values were determined for each compound. The in vitro data were also compared with existing in vivo data and found to be highly correlative. Interlaboratory reproducibility was also excellent, demonstrating that the skin2 human Dermal Model product remains stable during intercontinental shipment and that the MTT Assay Kits, also supplied by Advanced Tissue Sciences, provide a standardized method for assessing the toxicity of test agents.
MEASUREMENT OF CELLULAR RESPONSES TO TOXIC AGENTS USING A SILICON MICROPHYSIOMETER
H.G. WADAI1, J.C. OWICKI1, L.H. BRUNER2, K.R. MILLERS3, K.M. RALEY-SUSMAN4, P.R. PANFILI1, G.M.K. HUMPHRIES1 & J.W. PARCE1
1Molecular Devices Corp, Menlo Park, CA 94025; 2Procter & Gamble Co., Miami Valley Lab., Cincinnati, OH 45239-8707; 3Microbiological Associates, Rockville, MD 20850; 4Vassar College, Poughkeepsie, NY 12601
AATEX 1(3):154-164
Abstract
The silicon microphysiometer monitors cellular metabolism in vitro and has been used to detect and study biological responses to xenobiotics. This instrument uses a light addressable potentiometric sensor to measure millipH changes in micro-flow chambers maintained at 37‹C. Cells are immobilized in the flow chambers. Cell proton excretion can be measured as the acidification of the medium when medium flow through the chamber is stopped. The rate of acidification is a measure of catabolism, which produces lactic and carbonic acids. Through continual cycling of on and off periods of flow, nondestructive metabolic measurements may be made every few minutes. When a cell affecting agent is introduced into the fluid stream a change in acidification rate indicates either the stimulatory or toxic effect of the agent on the cells. Thus, rapid cellular responses to chemical agents can be detected within minutes and quantitated by the magnitude of acidification rate change.
Receptor-mediated cell activation by specific ligands, such as hormones, neurotransmitters, and growth factors, causes increases in acidification rate within minutes of receptor-ligand binding. This provides a receptor-specific response that can be monitored during exposure to materials as a possible indicator of cell-type specific toxicity. Receptor specific toxicity in hippocampal neurons has also been tested using extracellular acidification as the measurement of the toxicity of glutamate receptor overstimulation.
In tests for chemotherapeutic efficacy, antiviral drug activity and toxicity, acidification rates have been used as the cells activity and viability indicator. Agents, such as detergents, that produce irritancy or other non-specific toxicity have been evaluated for their effects on extracellular acidification rate and found to decrease acidification rate. Concentrations at which acidification rates are reduced by 50% were determined for test substances and correlated with animal ocular irritation test results. Recovery after insult is readily measured and may be an important index of irritancy or toxicity. These examples illustrate the broad range of cell affecting agents that cause cellular responses detectable in the microphysiometer using extracellular acidification rate.
METHODS FOR EVALUATING TERATOGENIC ACTIVITIES OF CHEMICALS AND SERUM FLUIDS USING MICROMASS CULTURE SYSTEM
TOSHIE TSUCHIYA, YOHIAKI IKARASHI AND AKITADA NAKAMURA
Division of Medical Devices, National Institute of Hygienic Sciences, 1-18-1, Kamiyoga, Setagaya-ku, Tokyo 158, Japan
AATEX 1(3):165-171
Abstract
Mouse or rat embryonic midbrain (MB) and limb bud (LB) cells were prepared from day 10-13 embryos and cultured as micromass cell islands for 5-7 days. Differentiation was determined by the number of stainable foci of differentiated ceils, and the concentration at which each compound inhibited the formation of differentiated foci by 50% of the control value (IC50) was estimated for evaluating the teratogenic potentials of compounds.
We apply the micromass teratogen test to the studies on the causes of TBZ-induced teratogenesis and species differences in the ETU-induced teratogenesis.
AN ASSAY SYSTEM FOR DEVELOPMENTAL TOXICITY USING EMBRYOS AND LARVAE OF EXNOPUS LAEVIS MICHIKO K. SAKAMOTO, SHIN MIMA AND TAKASHI TANIMURA
MICHIKO SAKAMOTO, SHIN MIMA and TAKASHI TANIMURA
Department of Anatomy, Kinki University School of Medicine, Osakasayama City, Osaka 589, Japan
AATEX 1(3):172-177
Abstract
Xenopus larvis embryos and larvae were exposed to seventeen chemicals for the evaluation of Xenopus laevis embryo and larva system as one of alternatives of devclopmental toxicity of chemicals. All of the chemicals induced some developmental toxicity in a dose-dependent manner. Based on our data and those of other investigators, it is considered that Xenopus embryo and larva system is a good candidate for a simple and effective test system to evaluate developmental toxicants. Furthermore, advantages and disadvantages of this system are discussed.
The aims and activities of FRAME
Michael Balls
Chairman of the Trustees FRAME