76 Cell Biology International Reports, Vol. 14, Abstracts Supplement 1990

WI33 NRN AND EyTExn: VALlDA’llON OF IN WTRO ALTF,RNA’l’lVEl To IXE DRAIZN EYJC BtBll’ATlON TEST

Michael Balls, Karen A Alki~~~u, Sandra I Reader, Richard H Clothier. Dcparbncnt of Human Morphology, University of Nottingham Medical School, Nottingham, NG7 2UH, UK

Public and sciontic concern over ihe contbmiog use of animals iu the. Dralzc. eye irritation lest has led to an explosion in the number of in vitro Draize alternatives currently being &velopcd. Many such systems still involve the sac&co of animal& and some such as the Leighton CAM method are clas&icd as animal experiments in lhe UK, under the Animals (Scienti@c &IX&~) Act 1986. As a result, FRAME is evaluating Iwo truly ia vitro altomativcs, namely, the neutral red release @RR) assay (developed by FRAME), and the commercially available EYTBX”’ assay (ROPAK Laboratories, Irvine, California).

Briefly, ia the NRR lest, a confluent layer of non-dividing mouse. 3T3-Ll fib&last-lie ceils, pnloaded with tbc Vital dye (neutral red), arc. em to hi conccntradons of tcsl chemical for 1 minute. The degree of dye released caa be used as an indicator of cellular damage.

The EYTBX”’ method involves a synthetic protein matrix, developed following the observation that a major component of all acute ocular toxicity determination is opacification of the cornea, spccitically that caused by precipitation of corn4 proteins.

A number of test materials, including both pure chemicals and formulatioq have been tested for thcii short-term toxicities using both methods. The rcaulls oblaincd will be d&cussed in the light of currently available in viva eye initation and human experience data.

Reader, S.J., Blackwell, V., O’Hara, R., Clothier, R.H., Gmm, G. & Balls, M. (1989). A vital dye release method for assessing the short- term cytotoxic effects of chemicals and formulations. AZ4 17, 28- 37. Gordon, V.C. & Berlpnao, H.C. (1987). EYTBX: An in virro method for evaluation of ocular irritancy. In: In vine Tmicology (Alternative Methods in Toxicology, Vol. 3, cd. A.M. Goldberg), pp. 641-649. New York: Mary AM Lie&l Inc.

w135 CULTURED RENAL EPITHELIA: A MODEL FOR in vitro NEPRROTOXICITY

Gerhard Gstraunthaler, Dieter Steinmassl, Walter Pfaller. Institute of Physiology, University of Innsbruck, Austria.

Renal epithelial cells in tissue culture have gained a powerful tool for studying epithelial transport and its regulation by metabolism, hormones and drugs (Gstraunthaler G. Renal Physiol. Eiochem. 11: 1, 1988). Thus, cultured kidney epithelia may also be utilized as an in vitro approach to investigate mechanisms of drug- induced nephrotoxicity.

The LLC-PKl cell line, which in tissue culture retained many proximal tubular properties was used to study adverse effects of cephalosporines and aminoglycosides. Treatment of LLC-PKI cells grown in plastic tissue culture dishes with cephaloridine, ceftazidime, cefotaxine or gentamicin caused a dose-dependent deterioration of cell monolayer integrity, changes in cell morphology, and release of cellular marker enzymes. LLC-PKI epithelia grown on permeable supports ensured measurements of transepithelial ion and solute transport. The most relevant and sensitive parameter found was the anion-to-cation permeability ratio. Drastic decreases in LLC-PKI epithelial peraselectivity were obtained at drug concentrations well below those leading to enzyme release. Thus, LLC-PKI epithelia may provide a valuable in vitro model system to investigate nephrotoxic nechanisas caused by xenobiotics and drugs at the cellular and subcellular level.

w134 A CYTOTOXICITY TEST BASED ON IN VITRO CUL- INJNM KERATI?WCVTES AND

FIBROBWSPS Massimo Malcovati, Chiara Giordani, Cristina Ranza- ti and Maria Luisa Tenchini. Dept. of Biology and Genetics for Medical Sciences, University of Milan, Milan, Italy. In recent years, growing attention has been paid to in vitro toxicity tests as a partial alternative to in viva tests. In this respect, it is essential to take into account the in viva target tissue. We de- veloped an in vitro cytotoxicity test based on the use of the main cellular components of skin, i.e. keratinocytes and fibroblasts. As a test system, several biomaterials currently used in direct con- tact with skin were used. In the assay, in vitro cultured human keratinocytes and fibroblasts were grown in direct contact with the biomaterial. The test was performed on cells at different growth stages. The effect of the biomate- rial was evaluated both qualitatively, by scoring cell morphology, and quantitatively, by determining the percent inhibition of cell growth, after 7 days of culture. In order to assess whether an observed cytotoxicity was due to physical and/or chemical factors, biomaterial-conditioned media were used. The assay proved to be sensitive and reproducible. The use of biomaterial-conditioned media allowed to detect "in trans" toxicity. Moreover, differential sensitivity of keratinocytes and fibroblasts was useful in discriminating between physical and che- mical components of toxicity.

W136 AUGMENTATION OF FETAL LUNG CELL SUP~OXIDEDISMUI.ASE(?3Ol3)ANDCATAi,ASE(CAT) A~pRolBcIsAaAINsTlUELEIXAL.EFFECfS OFELEVATED~ BuTwBsNOTATlENUATJ3~ MEDIA'IED~OFGROWIHORBXREASEOF PROSTAGlANDlN SYNIWESLT Keith TansweIl. David Olson and Bruce Freeman. Neonatal Research Division, Hospital for Sick Children, Toronto, Canada MSG 1X8; Departments of Pacdiatrics and Physiology, Lawson Research Institute, London, Canada; Departments of Anesthesiology and Biochemistry, University of Alabama at Birmh@am, Aw5294, USA

Fetal rat lung pnemnoqtes and fibroblasts were exposed to moderate (M%) or severe (95%) Ot insult. Both insults resulted ia cytotoxicity, as asses& both by lactate dehydrogenase and [lc]adcniae release, increased synthesis of prostaglandins (PGF-, PGFM, 6KF, PGEJ, and growth inhibition. Pretreatment of pneumcqtes with liposome (1 nmoUcm9 entrapped antioxidant enzymes increased SOD activity 3-fold and catalase activity 18-fold. Shnilar pretreatment of lung fibroblssu with liposomes (2 nmol/cm~ infxeased SOD activity by 50% and increased CAT activity 2.5-fold. Both cell types were protcctcd against the qtotoxic effects of 50 or 95% 0, by liposomemcdiated augmentation of antioxidant enzymes. However, such tmatment did not prevent &mediated inhibition of c-4 growth or &eased prostagtandin synthesis. Similar results wore obtained using polyethylene&co1 conjugated SOD and CAT.

We conclude: 1. Measurements of ce.ll death during in vitro studies of O2 toxicity are too insensitive for adequate evaluation of antioxidant therapies. 2. Enhancement of cellular SOD and catalase activities is not sufficient to prevent some manifestations of O1 toxicity.