429

2

1 READyITO-USE KIT PERMITTING STUDIES ON CELLS IN CULTURE

TOXICITY

P r o c e s s i n g and Analysis of I m a g e s f r o m O r g a n o t y p e Tissue Culture

PRELIMINARY RESULTS

M. BOUE-GRABOT, 0. BERNARDIN, J.F. PINON (BIOGIR SA, BP4 F 53611 GAZINET CEDEX)

a method previously described (M. BOUE-GRABOT et coll., ATLA, 1992, 20, 445-450) which makes it possible to study the cytotoxicity of non hydrosoluble substances without addition of any solubilisation adjuvant, a ready-to-use device was designed. Cell (SIRC) viability and sensitivity were controled : , Viability control with two colorimetric methods (MTT Emd Neutral Red) i Sensitivity control by comparison of the LC 50 of a preservative (methyl p-hydroxybenzoate) and a nonionic surfactant (octoxynol) after 24 hours incubation at 37~ in 96 well microplates and in the "KIT" after 3 or 6 days conservation in the laboratory, with or without previous transport by postal services. The results show that cells kept for at least 6 days a good viability and the same sensitivity to the toxic substances tested. This device may be useful for cytotoxicity studies, particulary as an alternative to the Draize test, though it was adapted from a method aimed to this purpose for several

E. Grapa, M. Sigot and M.F. Sigot Luizard Laboratoire de Biologie Cellulaire Exp6rimentale, Universit6 de Technologie de Compi6gne, BP 649, Compiegne, 60206 France

Based on

years.

A microcomputer-based image acquisition, analysis and processing system was developed for the study of biological specimens from organotype tissue culture. The objective of this research is to identify, classify and measure the different tissue zones in organotype tissue culture as one element of the evaluation of the cyto-compatibility of biomaterials and bioactive agents. Video images from a stereoscope were digitized, processed and analyzed. The regions corresponding to the tissue explant and the migration region were defined by segmentation and measured. The form parameters studied include the area, perimeter, minimum inclusion circle (Ferret's diameter), the shape length and the best-fit ellipse. These parameters describe the migration region, defined as the surface covered by cells after a period of time, which is one of the elements used in the cyto-compatibility evaluation by the organotype tissue culture method. The system was used to measure the cellular migration from chicken embryo tissue cultured on Thermanox | This method is adaptable and can be applied to other tissue types and/or biomaterials or active agents.

430

3

4

Titanium Perfusion Chamber

Incubated Hen's Eggs as an Alternative in Pharmacology and Toxicology

M. Dawson, School of Pharmacy, University of Strathclyde, Glasgow G1 1XW

@ E

A B C D E F

.... /

Body of Chamber Closing Ring Glass Coverslip Hypodermic Needle, closed with nonabsorbent cotton wool Water Jacket Gasket

Explanation Titanium and Tantalum are the only metals non-toxic to cells. The locking ring is better than 4 screws as it tightens evenly with less likelihood of breaking the cover-slip. There are 3 needles, for liquid flowing in, for liquid flowing out, and to equalise pressure changes in the chamber as it is moved from bench temperature to 37~ The non-absorbent cotton wool prevents bacterial contamination. The needles are bent upwards to prevent culture medium from wetting the wool. The gasket is of Dow Corning Silastic rubber, the only one nontoxic. The water jacket is as deep as the chamber to prevent convection currents as arise from a heated stage. It is better not to use continuous perfusion. This washes away enzymes and gives poorer cell growth.

N.-P. Luepke, G. Baron-Ruppert, S. Ibrahim, H. Meier, U. Wehking and Th. Wolf Department of Pharmacology and Toxicology University of Osnabrueck, Fed. Rep. Germany Pharmacodynamic as well toxicodynamic effects of chemicals and drugs must be evaluated by experimental studies, generally in animals, to meet regulatory requirements. Incubated hen's eggs offer a lot of possibilities to obtain required data in a rapid, sensitive, inexpensive, reproducible and reliable non-animal-system. This is true for tests on embryotoxicity and general/organ toxicity (HET), membrane irritation (HET-CAM), antiinflammation (modif. HET-CAM), vascular activity (HET-VASA), metabolism (HET-META) as well as for examination of haematologi-cal and clinical chemistry parameters (HET-HAEMA, HET-CC). Test substances (pure, dissolved, suspended) were applied on the chorionaUantoic membrane or in the egg albumen at various days of incubation. Test parameters are morphology (macroscopic, [electron]microscopic), vascular reactions (e.g. diameter, flux, thrombosis) and/or analysis of albumen, allantoic fluid and tissues (e.g. GC-MS, HPLC). The results obtained in our lab as well as in other labs during interlaboratory trials (coordinated by CEC, BGA, SDA) are reproducible and in good correlation to in vivo investigations. Incubated hen's eggs cannot yet replace completely the presently used animal tests in pharmacology and toxicology, but they provide especially in screening fast and manifold informations so that toxicological and priority classifications can be set.

431

6

5 IN

VITRO T E R A T O G E N I C POTENTIAL EMBRYONIC CELLS: OPTIMIZATION CULTURE CONDITIONS.

ON OF

T o x i c i t y a n d e f f i c a c y of c o s m e t i c s : pool

of

in

vitro

tests

on

a

Human

F. HEUTTE, O. BOUTHERIN-FALSON, M. FINET Laboratoires INNOTHERA, Pharmacologie cellulaire, Arcueil, France.

Immortalized Keratinocytes

The teratogenic potentiality can be studied in vitro on limb bud (LB) and central nervous system cells removed at an early stage of organogenesis (Flint et Orton, 1984). Our purpose was to study the interest of the parallel use of the two cellular types and to optimize the culture conditions to obtain teratogenic effects with a predictive way and without false-negative results. Nine reference compounds with different cellular m e c h a n i s m s were used (retinoic acid, 13aminopropionitrile, chlorcyclizine, cycloheximide, cyclophosphamide, EDTA, 5-fluoro uracil, hydroxyurea, N-nitroso N-m6thyl urea). With N-nitroso N-methyl urea (50 ~tg/ml), we determined a sensitive stage of organogenesis 12,5 days after conception for rats, corresponding to the appearance of anterior limb buds and to the teratogenic effects on neurogenesis, an event which no longer occurs 13 days after conception. On the other hand, the results indicate that CNS cells can become differentiated in monolayer at 105 cells/ cm 2, unlike LB cells whose different~iation requires hight cellular density (micromasses of 105 cells/ 5 ~tl). Cyclophosphamide (5 ~tg/ml), a proteratogen agent, was used to select a metabolic activation system. A coculture technique with porous membranes was preferred to the classic method of coculture. Excepted for EDTA, the eight reference agents have inhibited differentiation (0.01~IC50~570 ~tg/ml) in this double model but a difference in teratogenic activity was shown with retinoic acid (complete inhibition on LB ceils and without effect on CNS cells at 0,2 ~tg/ml). These results demonstrate the interest in the parallel use of two cellular types (limb bud and central nervous system) to achieve reliable predictive results and to apply to an in vitro early screening test with new compounds.

M.Lavazza + and S.Zava o ~ Biology and Genetics, University of

Flint O.P., Orton T.C. (1984),Tox. appL pharmacoL 76:383395.

E.DolfinP*, G.Conforti*, T.Dasdia o, M.Meloni-t-

Milan, *Mado Negri Institute, Milan, +Diana de Silva Cosmetiques, Rho, Milan, ITALY Our group presents a human keratinocyte cell line (NCTC-2544) for analysis of the

toxicity and efficacy of cosmetics. Different biological parameters were considered: the

cytoskeleton

(actin

and

vimentin

proteins analyzed by immunofluorecence and northern-blot - shape change - the mitotic index and doubling time

Dose-dependent differences were found in the actin filament structures. No difference was observed in intermediate filaments of vimentin after treatment with the cosmetic products. Cell shape changed drastically after treatments, the highest concentration (lmg/ml) causing a total block of cellular adhesiveness. "Welfare" was analyzed by treating cell monolayer with fetal serum at concentrations from 0% to 1%. The addition of the cosmetics (0.01mg/ml) to the serum free medium induced cell replication similar to the controls maintained with serum

432

7

8

Change in phenotype of human epidermal cells induced by specific p r o t e i n phosphatase

Effects of cytokines on the gamma interferon-induced tryptophanyl-tRNA synthetase expression by human cultured keratinocytes. R~ano A, Viac J, Schmitt D. INSERM U346, affili~e CNRS, HSpital Ed. Herriot, Pav. R, 69437 Lyon Cedex 03 FRANCE.

inhibitors.

Serres M., Haftek M., Smquet M.J. and Schmitt D. Inserm U346, Hopital E. Herriot, 69437 Lyon, France. Protein phosphorylation plays an essential role in regulating cellular mechanisnqs like proliferation, differentiation and oncogenesis. The. recent characterization of protein-tyrosine phosphatases and of proteinserine/threonine phosphatases suggests that dephosphorylation might play a crucial role in protein synthesis, gene transcription and numerous other processes. Functional adhesion molecules named "integrins" mediate cell adhesion to other cells and to the extracellular matrix. Tyr- and Ser-phosphorylation events can be triggered by integrins. In the present study, we investigated upon the mechanisms implicated in the spreading and adhesiveness of epidermal cells and particularly the role of protein phosphorylation on integrin expression, using specific protein-phosphatase inhibitors. This study was carried out on noriaal human keratinocytes and on a spontaneously immortalized human keratinocyte cell line HaCAT (J. Cell. Biol. (1988) 106, 761-771) which shows highly preserved phenotypic characteristics of normal keratinocytes, Preliminary results demonstrated a clear cut change in cell morphology (acantholysis) induced by pefabloc, okadaic acid and calyculin A, selective inhibitors for Ser-Thr phosphatases whereas orthovanadate, a potent inhibitor for Tyr-phosphatases was without effect. Other inhibitors of phosphatases such as N aF or Methylamine had no effect. Results from FACS analysis showed a significant decrease in E cadherin expression whereas 131 integrins slightly decreased and 134 remained unchanged. We checked the role of phosphatase inhibitors in interactions between integrins and the cytoskelcton structures by immunofluorescence stainings using specific antibodies. Further studies in laser confocal scanning microscopy and electron microscopy may give more reformation concerning the changes in cell morphology. This approach may be of interest for studies of epithelial wour:d healing in man.

Incubation of human keratinocytes with gamma interferon (~,-IFN) has been shown to potently induce the synthesis of a 53 kDa protein which was recently identified as tryptophanyl-tRNA synthetase (TRS). However, in spite of the high sensitivity of cultured keratinocytes to TRS induction by ~,-IFN, the study of inflammatory skin lesions selected for elevated levels of 7IFN within the epidermis, as assessed by HLADR expression on keratinocytes, has allowed the detection of the protein only in a few cases, suggesting regulatory mechanisms from soluble endogenous mediators with antagonistic activity on the induction of TRS by ~,-IFN. Among these mediators, we wondered whether " cytokines selected for a possible antiinflammatory activity and potentially derived from activated resident skin cells, such as IL4, IL-10, TNF-c~ and TGF-8 may be involved in the modulation of the keratinocyte TRS expression. To assess this possibility, we investigated the modulation of the synthesis of TRS by human cultured keratinocytes upon stimulation by various ~ , - I F N / c y t o k i n e combinations. The effects were evaluated by immunoblotting assay revealed by enhanced chemiluminescence, with ~he aid of a specific antibody to the TRS protein. Results failed to demonstrate any effect of the tested cytokines, whether on the basal level of the TRS, or on the ~,-IFN-induced enzyme expression in keratinocytes. It is thus unlikely that such cytokines can account for the infrequency of the TRS detection in inflammatory skin processes. Further investigations of alternative working hypotheses should help elucidate the regulation of TRS in human keratinocytes.

433

9

10

Effect of zinc on IFN-T or n i c k e l - i n d u c e d ICAM-1 expression of h u m a n keratinocytes.

MITOCHONDRIA ACTIVITY OF HUMAN S K I N F I B R O B L A S T S BY C O L D L I G H T MICROTITRATION FLUOR1METRY - E F F E C T S O F UVA -

A.Gu6niche 1, J . V i a c 1, G . L i z a r d 2, M . C h a r v e r o n 3, D . S c h m i t t l . I I N S E R M U346 Clinique Dermatologique, 2 I N S E R M U80 Centre de Cytom6trie en flux, H6pital Ed.Herriot, Lyon and 3Laboratoires Pierre Fabre, Castanet-Tolosan, Vigoulet Auzil; France.

C. KORWIN-ZMIJOWSKA*, P. RAT*, C. PHILIPPOT*,

Zinc therapies exert beneficial effects in several cutaneous pathologies through their antiinflammatory properties b u t the mechanisms of action are still uncertain. W e asked whether keratinocyte ICAM-1 expression, an important cell membrane antigen induced in inflammatory reactions, m a y be reduced by zinc. For this purpose, we used normal human keratinocytes d e r i v e d f r o m plastic skin surgery and cultured in low calcium m e d i u m (MCDB153). ZnSO4 ( 5 0 ~ t M ) w a s added to cultured cells either with IFN-~/ (10U/ml), a strong mediator of inflammation or nickel (5-10gg/ml), a sensitizing metal hapten, k n o w n to i n d u c e k e r a t i n o c y t e I C A M - 1 e x p r e s s i o n . U s i n g F A C S a n a l y s i s , the combination of zinc with nickel or the addition of ZnSO4 (24h) before IFN-~t or NiSO4 treatments reduced ICAM-1 induction of more than 52% (p<0.01). All these observations appem'ed already at 24h. Taken together, these data indicated that zinc can reduce keratinocyte ICAM-1 expression; this action m a y be i m p l i c a t e d in the antiinflammatory effect of Zn 2+ associated therapies in cutaneous inflammatory reactions.

L. PASCUAL LE TALLEC*, D. CASTELLI** and M. ADOLPHE* * Laboratoire de Pharmacologic Cellulaire de l'Ecole Pratique des Hautes Etudes, Institut Biomedical des Cordeliers, 15 rue de l'Ecole de M~decine 75006 Paris. France. ** Laboratoire d'Evaluation, RoC S.A., 48-50 rue de Seine 92703 Colombes. France.

Mitochondria have been proved to be the powerhouse of the aerobic ceil. Mitochondrial activity can be revealed, by a cationic fluorescent probe: Rhodamine 123. The incorporation of this dye is dependent on the maintenance of an transmembrane potentiel across the mitochondrial membrane. This is why Rhodamine 123 could be considered as a cellular activity energetic indicator. The objective of this work was to study: on the one hand, the possible differences in mitochondrial activity of human skin fibroblasts according to the age of the donor and, on the other hand the effects of UVA on these different cell populations. Cells were placed in Petri dishes and maintained in MEM supplemented with 10% fetal bovine serum and 5 gg/ml gentamicin. Cells were irradiated in PBS at a wavelengh of 365 nm and a UVA dose of 9 J/cm2. Rhodamine 123 (10 gg/ml) was incubated with the cells. The unfixed dye was washed out and the fluorescence of the Rhodamine 123 bounded to the mitochondrial membrane read with a cold light microtitration fluorimeter. Mitochondrial activity was studied on cells in exponential growth phase and at the plateau phase. Our first results seem to show that mitochondrial activity changes according to the age of the donor. Moreover, mitochondriai activity was increased after irradiation whatever the irradiated cellular populations. This enhancement, could be the sign of a defense mechanism. Cell functional energetic state appears observable and measurable by Rhodamine 123 incorporation in a quick and sensitive way. As the energetic state is one of the first parameters affected by UVA, this technique is very useful to evaluate early the cellular response to this irradiation.

434

11

12

ZINC OXIDE IN CUTANEOUS INFLAMMATORY REACTION

PRESENCE OF OPIOID RECEPTORS AT THE CELL SURFACE OF HUMAN M E L A N O M A (M4Beu) : A P P L I C A T I O N TO THE RESEARCH OF OPIOID TYPE PEPTIDES

M.F. ARIES, M. CHARVERON

a n d Y. G A L L

Centre de recherche dermo-cosm~tique PIERRE FABRE, CHU Rangueil, TOULOUSE.

I. R E N I M E L , J. F R A N C H I ,

S. C H A M B O N * , G. R E D Z I N I A K

Among the essential oli~r zinc plays an important role in cell metabolism, notably in the activation of numerous enzyme systems. In dermatology, zinc is frequently used in the treatment of inflammatory dermatoses, but also for wound healing.

Parfums Christian Dior P.O. B o x 58 45804 S a i n t J e a n de B r a y e FRANCE

The aim of this work was to demonstrate the potential of zinc oxide by evaluating its activity on the inflammatory response of human keratinocytes and on the proliferative response of human T lymphocytes.

* Laboratoire BIODEV Limoges - FRANCE

Using the ELISA technique, we investigated the arachidonic acid cascade, in particular prostaglandin 6KFIc~ production by SVKI4 and HaCat transformed human keratinoocytes which had been stimulated by the calcium ionophore A23187. For both cell types, zinc oxide at concentrations of 50ttM and 100 ~M, significantly inhibited the production of this prostaglandin. This inhibition was of the same order as that observed for similar concentrations of zinc gluconate, a well known anti-inflammatory agent. Human T lymphocyte proliferation was also studied by using the 5'BrdU incorporation ELISA technique. At concentrations of between 3 and 60 ltM, zinc oxide induced significant lymphocyte proliferation which was dosedependent. This study demonstrates the immunostimulatory properties of zinc oxide, probably relating to the known involvement of zinc in protein synthesis and cell division. Zinc oxide also shows certain anti-inflammatory capacities because it significantly inhibits prostaglandin 6KFlct production.

SUMMARY W e h a v e s h o w n t h a t the M4Beu, h u m a n m e l a n o m a c e l l s had on their surface opioYds r e c e p t o r s of m u (~) type. The b i n d i n g s i t e r e c o g n i z e d b y a s p e c i f i c l i g a n d of this t y p e of r e c e p t o r : the D A M G O has two c o n s t i t u e n t s : one w i t h high affinity . (Kd I = 1,89 10 -10M) a n d a n o t h e r one w i t h low a f f i n i t y (Kd 2 = 6,610-9M).

This r e c o g n i t i o n r e c e p t o r - l i g a n d going through a high affinity b i n d i n g site, a l l o w s us to use this c e l l u l a r m o d e l to d e t e c t the e x o g e n o p i o Y d p e p t i d e s from d i f f e r e n t o r i g i n s , w h i c h can be u s e d in c o s m e t i c s .

435

13 REORGANIZATION OF CYTOSKELETAL FILAMENTS AFTER CONTRACTION WITH ATP FROM HAIR FOLLICLE CELLS. LACHGAR S. (1,2,3), CHARVERON M. (1,2), CERUTI I. (2), BOUHADDIOUI N. (3), GALL Y. (2) and BONAFE J.L. (1) 1- Groupe de Recherche ClinJque et Bio-Clinique en Dermatologie, C.H.U. Rangueil, Toulouse. 2- Centre de Recherche Dermo-Cosm~tique Pierre FABRE, Vigoulet-Auzil, Castanet-Tolosan. 3- Laboratoire d~ndocrinologie, Facult6 des Sciences I, Marrakech. In order to study the metabolism of hair follicle cells in near physiological conditions, we were interested in investigating the three-dimensional interaction of hair follicle dermal papilla cells in culture, cells which are principally involved in hair growth. In a similar way to dermal fibroblasts, the dermal papilla cells (DPC) are also capable of producing a contraction of collagen gel. To be able to understand the dynamic and mechanical mechanisms involved in the contraction of lattices, we looked into the phenomenon of cell contraction, an indicator of the capacity of cellular deformability and movement. Using laser confocal microscopy, we decided to measure three morphometric parameters; cell surface area and the thickness and diameter of microfilaments. The arrangement and distribution of certain structural proteins (~-actin, ~x-actininand vimentin) have also been studied before and after exposure of DPC's to different concentrations of ATP (1 - 5 mM), after incubation times of 2, 5, 10, 20, 30 and 60 minutes. Our results showed that 1 mM ATP causes a reduction of approximately 50% in the surface area of the cell after 30 minutes of exposure. In contrast, the thickness of the DPC's had doubled after this contraction. With regard to microfilarnent diameter of DPC's, this was considerably reduced (-70%) after 30 minutes of contraction. Cultured cells not exposed to ATP then labelled with anti ct-actin antibodies showed an abundant microfilament network. After addition of ATP, the distribution of actin fibers became very heterogeneous. Peripheral filaments appeared irregular and diffuse. An aggregation of central actin filaments was observed in an advanced state of contraction. Concerning the labelling of a-actinin, which is normally present as bundles of fibers at the periphery of the cell, after contraction these appeared as discontinuous fibrous deposits in the immediate proximity of the plasma membrane. Apart from this, we observed no difference in the arrangement and distribution of vimentin filaments before and after contraction. These results demonstrate that, in a similar way to muscle cells, dermal papilla cells whose role in regulating hair growth has been well established, have contractile properties. The presence of a dynamic system of cytoskeletal filaments suggests a close link between the reorganization of these filaments after contraction with ATP and alterations in eell-substrate contacts already established before contraction. Whether the contraction of dermal papilla cells affects the regulation of blood flow at the hair follicle bulb, remains to be seen.

14 MEASUREMENT AND INDUCTION OF CYTOCHROME P450 - DEPENDENT ENZYME ACTIVITIES IN A RECONSITrLrl~D HUMAN EPIDERMIS. Roguet R., Cotovio J., Rougier A., Kremers P., Leclaire J. I'OREAL, Laboratoires de Recherche Fondamentale 93601 Aulnay sous Bois, FRANCE In recent years it has become increasingly apparent that the skin is an organ containing enzyme systems capable of metabolizing a wide range of xenobiotic agents and endogenous substrates. Skin metabolism can modify topically applied compounds changing their percutaneous absorption and increases or reduces their pharmacological and/ or toxicological activities. In vivo and in vitro data indicate that most of the enzyme activity of skin is localized in the epidermal layer. On the EPISKIN model, a reconstituted human epidermis in culture, the presence of cytochrome P450 dependent activities has been demonstrated. After topical admistration of various subtrates of monooxygenase, Ethoxycoumafin (ECOD) and Ethoxyreso~fin (EROD) deethylase activities have been measured (0.92 and 0.39 pmoles /hours /ttg DNA respectively), suggesting the presence of at least cytochrome P-450 IA family in reconstituted epidermis EPISKIN. The percentage of metabolized subtrates were 0.027 % (ECOD) and 1.27 % (EROD) of the total penetrated amount after 2 hours. Both activities were highly inducible after topical application of 3-methylcholanthrene (6.6 fold for ECOD and 5 fold for EROD). The phenobarbital, a well-known inducer of hepatic cytochrome P-450, has no effect on ECOD activity as reported in vivo in the skin. Topically applied testosterone revealed a high percentage of metabolization (17% of the quantity applied after 8 hours). In control and benzanthracene induced cultures, 5~-dihydrotestoterone, 5r androstene-dione (Sc~-reductase activities) and 7c~, 2o~ hydroxytestosterone (hydroxysteroid dehydrogenase activities) have been identified. These data suggest that the EPISKIN model simulates some aspects of the metabolic processes of endogenous or xenohiotic compounds. In addition, the possibility of testing interaction between nonhydrosohible drug or formulation and metabolic cutaneous capacities make EPISKIN an useful tool for testing xenobiotic absorption and skin biotransformation in vitro.

436

15

16

Determination of the cytotoxicity of s o d i u m l a u r y l s u l f a t e on reconstituted skin

RECONSTITUTED H U M A N EPIDERMIS FOR TESTING

R i v a l l a n d P.*, C o i f f a r d L.*, De RoeckH o l t z h a u e r Y.* * CAEC - U n i v e r s i t 6 d e N a n t e s 68 Bd E u g e n e O r i e u x 44000 Nantes The first matrix or skin equivalent w e r e u s e d b y Bell a n d I v e r s s o n , to c u l t i v a t e keratinocytes. W h e n u s i n ~ t h e k i n ~o f m o d e l d e s c r i b e d b y t h e s e authors, we were able to obtain s a m p l e s o f e q u i v a l e n t skin, i n w h i c h the keratinocytes, spread in several l a y e r s , a r e c o n f l u e n t o n t h e gel o f f i b r o b l a s t s o r g a n i z e d in s u b s t r a t e . On these samples, we tested the cytotoxicity of sodium laurylsulfate. We chose this surfactant because, in vitro, its c y t o t o x i c i t y o n c u l t u r e s o f f i b r o b l a s t s is s u p e r i o r to t h e o n e o b s e r v e d in vivo w h e n u s i n g t h e numerous cosmetics formulated with this primary matter. Two different c o n c e n t r a t i o n s of t h e toxic s u b s t a n c e w e r e s t u d i e d : 10 -4 a n d 10-5 g.1-1. T h e cells viability, a f t e r 30 m i n u t e s of c o n t a c t w i t h t h e toxic s u b s t a n c e , is d e t e r m i n e d u s i n g t h e MTT m e t h o d . The first results already raised the conclusion that the cytotoxicity of s o d i u m l a u r y l s u l f a t e is l o w e r o n s k i n equivalent than on culture of fibroblasts.

D. ROSDY (SKINETHIC, Nice)

A fully differentiated epithelium having the major features of human epidermis is produced routinely in vitro in our laboratory. This epidermis is reconstituted by growing normal human adult keratinocytes (NHK) in supplemented chemically defined medium MCDB 153, on inert polycarbonate filter substrates at the air-liquid interface for 14 days. Vertical sections stained for histology (HES) and indirect immunofluorescence studies show a correct stratification, a compact stratum corueum, and expression of the major differentiation markers. Number of cell-layers are directly controlled by the concentration of epidermal growth factor (EGF) in the medium. When well-known toxic compounds are applied topically on the stratum corneum, they induce important changes in histologic appearance of the epidermis, and, depending on the tested products, basal cells'viability m a y be lost in less than 24 hours. The effects range from massive induction of epidermal cornification to total destruction of the cell-layers, Inflammation mediators secreted by the keratinocytes into the defined medium can be quantified using ELISA kits. When cosmetic creams containing vitamin A are applied topically, the keratohyalin granules disappear from the granular layer, and the stratum comeum becomes smoother within 48 hours. When pharmaceutical creams containing retinoic acid are applied, expression of the simple epithelia's typical keratin 19 is observed, as well as hyperproliferation and loss of polarization of the basal cells. Moreover, a total inhibition of profilaggrin synthesis associated with the disappearance of keratohyalin granules are noticed. SKINETHIC (R) epidermis allow to detect these different effects depending on dosis and application time, with great accuracy and perfect reproducibility.

437

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18

Assessment of the cytotoxicity of compounds towards polymorphonuclear neutrophils. Application to the study of calcium inhibitors.

Techniques for separating polymorphonuclear neutrophils: comparison between density gradient technique and differential centrifugation.

A. C a b a n i s ,

~ , A. C a b a n i s , S. L e b ~ g u e , C. B r u n e t , M. Luyckx, T. Dine, M. C a z i n , J . C . C a z i n . Facultd de P harmacie, Laboratoire de Pharmacologie, Pharmacocindtique et Pharmacie Clinique, rue du Professeur Laguesse, B.P. 83, 59006 LILLE

]L..._C.ts

S. L e b ~ g u e ,

C. Brunet, T. Dine, J. B e h r a - M i e l l e t , M. L u y c k x . Facultd de Pharmacie, Laboratoire de P harmacologie, Pharmacocindtique et Pharrnacie Clinique, rue du Professeur Laguesse, B.P. 83, 59006 LILLE Three methods for measurment of cytotoxicity using Trypan blue, neutral red and lactate dehydrogenase (LDH) were adapted for polymorphonuclear neutrophils (PMNs) and then compared in presence of two calcium inhibitors, verapamil and diltiazem, after 30 min incubation time in fluid culture medium. LDH is a cytoplasmic enzyme which measured in the supernatant shows membrane damage. Total LDH is measured with lysed PMNs and results were expressed as LDH percentage. Neutral red dye is able to enter into viable cells. Dye is then extract with solvants and the amount of absorbed neutral red was measured with a spectrophotometer. Trypan blue dye is able to enter into death cells. The percentage of death PMNs is counted in a Thoma's chamber. Diltiazem appears to be twice less toxic than verapamil in Trypan blue and LDH methods (50% of toxicity for 250 gM of verapamil and for 500 laM of diltiazem). In neutral red determination, we observed less toxicity difference between both compounds (50% of toxicity for 220 ~ of verapamil and for 340 ~a2Vlof diltiazem). Trypan blue exclusion is generally used to determinate cellular viability, but this test is interesting for a first estimation of toxicity because of its simpleness and its steepness. The difference of results obtained between LDH test and neutral red test may be explained by the fact that this latter test seem to be more adapted to cells in solid culture medium. LDH determination may be advised in order to evaluate the cytotoxicity of compounds such as calcium inhibitors towards PNNs in fluid culture medium.

Three separation techniques of polymorphonuclear neutrophils (PMNs) from heparinized human blood have been compared in order to increase yield and purity of this preparation. A density gradient technique (Histopaque) followed by a haemolysis with ammonium chloride (method A) has been so compared to two other techniques: first, Boyum's technique (method B), a density gradient technique (Histopaque) followed by a 3% dextran sedimentation then a haemolyse by a hypotonic 0.2% NaCI solution (1) and secondary, Eggleton's technique (method C), a differential centrifugation with ammonium chloride (2). The method C is faster (35 min) than methods A (65 min) and B (95 rain). But the method C is more contaminated with lymphocytes (8%) and erythrocytes ( 1 . 6 x 1 0 6 / 1 0 6 PMNs) than methods A (2% lymphocytes and 0.3xi06 erythrocytes/106 PMNs) and B (2% lymphocytes and 0.2x106 erythrocytes/106 PMNs). The method A allows to obtain a high yield of PMNs (3.8x106 PMNs/ml of blood) identical to the method C (3.5x106 PMNs/ml of blood) and average to the method B (2.1x106 PMNs/ml of blood). So, the method A increases yield and purity of the PMNs preparation and is effective for in vitro studies. (1) Boyum A. Isolation of mononuclear cells and granulocytes from human blood. Scand J Clirl L~I~ Invest 1968; 21(suppl.):77-89. (2) Eggleton P, Gargan R, Fisher D. Rapid method for the isolation of neutrophils in high yield without the use of dextran or density gradient polymers. J Immuno| Methods 1989; 121:105-113.

438

19

20

In vitro inhibition by letosteine of polymorphonuclear neutrophils endogenous oxidant release.

Methotrexate increases the amount of hydrogen peroxyde released by polymorphonuclear neutrophils .

S. L e b ~ g u e , B. G r e s s i e r , C. B r u n e t , T. D i n e , M. L u y c k x . Facultd de Pharmacie, Laboratoire de Pharmacologie, Pharmacocindtique et Pharmacie Clinique, rue du Professeur Laguesse, B.P. 83, 59006 LILLE

S. L e b ~ g u e , ~ , C, Brunet, M, Luyckx, T. Dine, M. Cazin, J. K a b l a n , J . C . C a z i n . Facultd de Pharmacie, Laboratoire de Pharmacologie, Pharmacocindtique et Pharmacie Clinique, rue du Professeur Laguesse, B.P. 83, 59006 LILLE

Acute production of reactive oxygen species by polymorphonuclear neutrophils (PMNs) during the respiratory burst may induce lung damage and may promote the pathogenetic bases for the development of pulmonary emphysema. The present study was performed with the aim to evaluate the protective activity of letosteine, a two blocked-thiol compound endowed with potential reducing properties, like N-acetylcysteine and mesna free-thiol drugs known for their antioxidant properties (*), against hydrogen peroxide (I-I202)and hypochlorous acid (HOC1) in a free-ceUular system. In a first time, letosteine dissolved, in ranging concentrations (1 to 1000 I.tM), in phosphate buffer saline (PBS) and then incubated with H202 or HOCI didn't decrease the amount of these reactive oxygen species, this measured by a spectrophotometric technique. Secondly, when letosteine was previously hydrolysed with bicarbonate-carbonate buffer, the amount of H202 and HOCI was decreased, in a dose-dependant manner, with inhibitory concentrations 50% (IC50) of 200 IxM and 15 pM respectively. This study demonstrated that the letosteine SH groups which can become free, after in vivo metabolism, may have an important role in the mechanism of action by scavenging endogenous oxidants. In this case, a therapeutic intervention with letosteine may be envisaged during cellular cytotoxicity where oxidants are involved. * Gressier B., Cabanis A., Lebegue S., Brunet C., Dine T., Luyckx M., Cazin M., Cazin J.C. Decrease of hypochlorous acid and hydroxyl radical generated by stimulated human neutrophils: comparison in vitro of some thiol-containing drugs. M~h Finr EXp Clin Ph~rrnar 1994; 16(2) in press.

Polymorphonuclear neutrophils (PMNs) have the ability to liberate large amounts of reactive oxygen species like hydrogen peroxide (H202). These free radicals release may have beneficial effects in chemotherapy but also lead to cytotoxicity in case of prolongated inflammatory reaction. Some anticancer drugs, such as anthracyclines, are known to induce an increase production of reactive oxygen species. About methotrexate (MTX), results of previous studies have been a subject of some controversies. So, we have investigated whether MTX has antioxidant or pro-oxydant properties. This in vitro study demonstrated that MTX increased the amount of H202 released by stimulated PMNs in a dosedependant manner with a maximum increase of 45% (i.e. 22 la-Mof H202) for 500 ~VI of MTX, The mechanism which govern MTX reaction may be a result of an intraceUular pro-oxidant mechanism by intervention on the oxidative metabolism of PMNs rather than a chemical interaction because not any pro-oxidant effect has been observed in a free-cellular system. In order to prevent cellular toxicity of H202, particularly this leading to cell malignant transformations via DNA damage, MTX has been associated with a thiol containing compound, mesna, known for its antioxidant properties (*). When MTX and mesna were simultaneous incubated in increased concentrations, only the excess of H202 released by stimulated PMNs with MTX was suppressed. This association MTX-mesna might be used in anticancer therapy, during oxidative burst, particularly when methotrexate is used in high concentrations, in order to limite toxic effects induced by reactive oxygen species. * Gressier B, Cabanis A, Lebegue S, Brunet C, Dine T, Luyckx M, Cazin M, Cazin JC. Comparison of in vitro effects of two thiol-containing drugs on human neutrophils hydrogen peroxide production. Meth Find 1993; 15(2):101-105.

439

22

21 Evaluation of lipid pemmddaticn b~ labelling lymphocytes

with

Tc99m.

V. de BF/30 , A. PETIET , N. OOIAS-IZNHART Laboratoire de Biophysique, Fac. XavierBichat, Paris, Franoe. Labelling leukocytes with 99mTechnetium hexamethylpropylene amineoxime (99mTc-HMPAO) is used in nuclear medicin for the localization of infectious focus. Labelling is not specific to polynuclear cells. Some labelled lymphocytes present chromosomal aberrations, and would be able to form cluster which could be a risk for the patient if reinjected. Radiations induce lipid p e r o x i d a t i o n with m a l o n d i a l d e h y d e formation known for its mutagenicity. The aim of the study was to determine if the labelling of lymphocyte with technetium 99m (370MBq) induce a lipid peroxidation. We have used two t e c h n e t i u m complexes which differ in their cellular localization : 99mTcHMPAO (intracellular) or 99mTcp y r o p h o s p h a t e (membrane). Peroxidation was m e a s u r e d as the amount of M D A or T h i o b a r b i t u r i c A z i d Reactiv Substances (TBARS) present in the surnageant. It was quantified by fluorescence spectrometry. A rise of TBARS during incubation time is noted but no significant increase between labelling cells and control was observed. The kinetic formation of TBARS differ between patients. Labelling lymphocyte with 99mTc does not seem to induce a lipid peroxidation in vitro. We have to precise the absence of specificity of the M D A dosage and that M D A is a minor product of lipid peroxidation.

Flow-eytometric (FCM) analysis of proliferation, differentiation and apoptotie parameters in a model of hematopoiesis in vitro applicable to pharmaco-toxicologic studies M. Chantal LEglise*, Patrick Darodes de Tailly ~ Jean-Luc Vignot ~ B6n6dicte Sawicki**, Christian Rich6*.* Service de Pharmacologie, **Unit6 de M6decine Professionnelle, C.H.U.R., ~ d'Instruction des Arm6es, Brest, 29200 France. Grant ANC01 91 from INSERM -CNAM-B

Cultures in cell-suspension allow to study modifications of a number of parameters by FCM. We used two types of hematopoietic expansion from stem cell-enriched (CD34+) human umbilical cord blood mononuclear cells. The granulo-monocytic pathway was stimulated by recombinant human interleukine-3, and the erythroid lineage by rHIL-3 + erythropoietin. Three parameters were studied on a laser-argon flow cytometer (Epics-Profile| after incubation of cellular expansion in the continuous presence of drug and compared to control culture data. Proliferation activity was evaluated as a percent of cells in S-phase as measured by propidium iodide staining of DNA content or bromodeoxyuridine incorporation. Differentiation stages were evaluated as the expression of surface membrane antigens recognized by a panel of monoclonal antibodies. Apoptosis was characterised by hypostainability with propidium iodide.Three drugs illustrate applicability to cytotoxic studies: Fluorouracil in the granulo-monocytic pathway, cytosinarabinoside in the erythroid lineage, 3' azido 3'deoxythymidine for the differential toxicity between both lineages. FCM techniques enable us to complete the mechanistic approach of cytotoxicity.

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24

Inhibiting effect of DAS on granulo-monocytic progenitors development for concentrations inducing immunosuppressive effect. S. Lautraite, D. Parent-Massin, D. Thouvenot. Laboratoire de Microbiologie et Biochimie, Ecole sup6rieure de Microbiologie et S6curit6 Alimentaire, ISAMOR, Technop61e Brest-Iroise 29280 Plouzan6.FRANCE Diacetoxyscirpenol (DAS) is a mycotoxin which belongs to trichothecenes. Though DAS is not the most toxic trichothecene (LD50 3.4 mg.kg 1 per os for the rat ), it is more active than molecules without acetyl groups. This toxin is implicated in immunotoxicity inducing immunosuppressive effects such as phagocytosis and microbicidal activity reduction. The aim of this work was to determine wether the immunosuppressive effects due to DAS could not also be attributed to a reduced macrophages and granulocytes production after exposition to this molecule. Granulo-monocytic progenitors from human umbilical cord blood on one hand and granulomonocytic progenitors from rat bone marrow on the other hand, have been cultured in the presence of DAS (10 -8 M to 5.10 -1~ M) for 14 days. Cytotoxic concentrations and IC50 were determined on day 7, 10 and 14. Concentrations responsible for the granulomonocytic inhibition development (CI50 = 7.10 -9 M, on J7 for the rat) are quite similar to those known to be immunosuppressive (3.10 -9 M). Theses results show that immunosuppressive effects could be also due to the inhibition of the monocytes/macrophage production .

Comparison of the in vitro toxicity of T-2 toxin on human and rat hematopoietic progenitors (CFU-GM). S. Lautraite, D. Parent-Massin, D. Thouvenot. Laboratoire de Microbiologie et Biochimie, Ecole Sup&ieure de Microbiologie et S6curit6 Alimentaire, ISAMOR Technopfle Brest-Iroise 29280 Plouzan6. T-2 toxin is a mycotoxin produced by Fusarium which essentially contaminate cereals under cold and temperate climates, as well as farm produce stocked in bad conditions. This toxin belongs to the family of trichotecene. The stability of its epoxy group associated to the presence of an isovaleryl group makes it one of the most toxic trichothecene. It is implicated in a l i m e n t a r y intoxications that induce hematological troubles (neutropenia, thrombopenia, medullary aplasia) among which Alimentary Toxic Aleukia is the most well known. The in vitro evaluation of toxicity due to this molecule has been performed on human and rat cells using two granulo-monocytic progenitors (CFUGM) culture models. CFU-GM from human umbilical cord blood on one hand and CFU-GM isolated from rat bone marrow on the other hand, were cultured in the presence of T-2 toxin for 14 days. Seven T-2 toxin concentrations from 10.8 M to 10-1~M have been tested. The agregates number (colony, macrocluster and microcluster) on day 7, 10 and 14 allowed to evaluate T-2 toxin cytotoxicity level and IC50. This work showed a higher cytotoxicity of the toxin for rat than for human progenitors though IC50 were quite similar.

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26

TOXICOLOGIC RISK EVALUATION T O CAULERPENYNE, TOXIN ISOLATED FROM CA ULERPA TAXIFOL1A ON HUMAN CF.I .I .S .

Effects of cAMP on the megakarvoblastic Dami cells: yon W i l l e b r a n d factoG glycoprotein lib/Ilia and ce II oroliferation. A-M Dosne, V. Deguine, D. Lecoq, D. Kerbiriou-Nabias, D. Meyer. INSERM 143 affili~e CNRS, H~pital de Bic~tre, Le Kremlin Bic~tre. The Dami cells isolated from leukemic cells by S. Greenberg displays the properties of a promegakaryoblast. Proteins of the megakaryocyte lineage, such as membrane glycoproteins lib/Ilia and von Willebrand factor (vWF) are present and inducible by several hematopoietic factors suggesting the possible differentiation of these cells. We have used this model to study the effect of cAMP on the expression of these proteins since this nucleotide is known to promote the differentiation of several cell types. The cells were incubated with different compounds known to stimulate cAMP accumulation: dibutyryl cAMP (db-cAMP), two phosphodiesterases inhibitors, isobutylmethylxanthine (IBMX) or pentoxifylline (PTX) and an activator of adenylate cyclase, forskolin. Treatments with db-cAMP or IBMX (10-1000 gM) induced a dose-dependent increase in vWF synthesis reaching about 20 times the basal value. Associations of IBMX with forskolin produced a synergistic enhancement in vWF formation. PTX used alone did not enhance vWF production but a latent effect was revealed in the presence of forskolin or db-cAMP. The increase in vWF mRNA shown by Northern blot analysis demonstrates that the protein synthesis correlates with the transcript expression after db-cAMP or IBMX treatments, vWF production paralleled the accumulation of cAMP in the cells. Moreover vWF expression induced by combination of IBMX with forskolin was associated with an increase in the percentage of GPIIb/llla positive cells from 75% up to 96% and an important inhibition of cell growth. These data provide evidence that agents acting on cAMP metabolism induce vWF synthesis in the Dami megakaryoblastic cells. This suggests the potential interest of phosphodiesterase inhibitors for differentiation and cell proliferation control in megakaryoblastic cells.

V. Foumier 1, R. Lemde 2,3, C. Delescluze 4, p. Amade 2, D..___Parent-MassinI e t D. Pesando 2 1-Laboratoire de Microbiologie et Biochimie, Eeole Supdrieure de Microbiologie et Sdcuritd Alimentaire, ISAMOR, Technopole de Brest-Iroise, 29280 Plouzand.2UR 303 INSERM, Laboratoire de Physiologic Cellulaire et Compar~e, Facult6 des Sciences, Pare Valrose. BP 71, 06108 NICE CX 2.3- Laboratoire Environnement Marin Littoral, Facult6 des Sciences. 06108 NICE CX 2. 4- UR 303 INSERM, 1 Avenue Jean Lorrain, 06300 NICE.

Caulerpa taxifolia (Vahl) C. Agardh (Ulvophycea, Caulerpales) is an alga of tropical origin that was accidentally introduced into the Mediterranean sea. In 1994 this alga was observed from the south of Italy to Spain (Baleares). This species shows a marked development and can compete with the endemic flora and this invasion can be defined as a biological pollution. It is known that tiffs species as the other caulerpacean algae can develop an efficient strategy against grazers consisting of the synthesis of repulsive or toxic secondary metabolites. Among them, caulerpenyne, an acetylenic sesquiterpenoid, is the major sesquiterpene of Caulerpa taxifolia representing 0,2% of the wet weight in July whereas all the other terpenes represent less than 0,0004% The aim of this work is to evaluate the toxicity of purified caulerpenyne with two models of human cells in culture : hematopoietic progenitors (CFU-GM), and keratynocyte cell lines (HaCaT et HESV). Human CFU-GM, obtained from umbilical cord blood samples, were cultured in semi-solid agar (0,3%) with increasing concentrations of caulerpenyne ( from 0,I to 30 /~gtml). Aggregates growth were scored after 7, 10 and 14 days of culture. IC50 was equal to 1/~ghnl with tiffs model. ttmnan keratinocytes were obtained from cell lines (tlaCaT and HESV). 104 cells have been plated. After 24 hours of incubation, cells have been cultured in the presence o f caulerpenyne (from 0,1/~g/ml to 20 jug/ml). The viability cells was evaluated with MTF test. IC50 was equal to 12 ,ug/ml for HaCaT, and 16/~g/ml for HESV. The comparison of IC50 shows that these human cells, with a high mitosis ratio, are more sensitive to caulerpenyne than cells obtained from other organisms.

442

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28

EFFECTS OF LACTIC ACID ON IRON MOBILIZATION AND TBARS PRODUCTION ON SLICES FROM DIFFERENT RAT BRAIN REGIONS

Effects of ethanol on astrocytes in vitro.

B..Fauconneau, C. Tallineau, F. Huguet, R. Pontcharraud, A. Piriou

Laboratoire de Neurobiologie du D~veloppement, Institut de Biologie mol6culaire et cellulaire, CERMO,*Universit~ Joseph Fourier, Grenoble, FRANCE.

Institute of Xdnobiotic Studies, E.A. 1223, Faculty of Medicine and Pharmacy, F-86005 Poitiers

Various studies have shown the key role played by iron (mainly Fe 2+) in free radical production and lipid peroxidation. Works in the rat (Zaleska et al.) established a parallel between the concentration of iron in different brain regions and the quantity of thiobarbituric reactive substances (TBARS) measured in the absence of induced oxidative stress. Furthermore, we showed that lactic acid exerts a lipid peroxidant action by Fe 2+ release (Fauconneau et al. ; Huguet et al.). The aim of the present study was to compare the effect of lactic acid in vitro on slices from 3 brain regions. Cortex, hippocampus and striatum were chopped into 250 Ixm slices, incubated at 37~ for 180 min in Krebs-Ringer buffer in the presence of lactic acid 7 mM (final pH 5.5) and bubbled with 95%/5% O2/CO2. Fe 2+ release in supematant was evaluated, after addition of ferrozine, by measuring the absorbance of the resulting pink complex. TBARS was assayed after the slices were ground. The first results showed that TBARS production and Fe 2§ release induced by lactic acid were related to the quantity of total iron present in the brain region. Values for Fe 2+ release and TBARS were higher in striatum slices than in those from the other regions. These results indicate that slices from brain regions provide a good model for lipid peroxidation study and point to the important role played by tissular iron reserves in lactic acid-induced lipid peroxidation. R6ferences: M.M. Zaleska et al., Neurochem.Res., 1985. 10, 397-410. B. Faueonneau et al., Biochem. Mol. Biol. Int., 1993, 31, 421-427. F. Huguet et al., Presentationin 3rd Int. Conf. on CNS, Slices Preparation, Louisville,KY, juin 1994.

the

morphology

of

L. BARRET, A. SOUBEYRAN, Y. USSON*, R. SAXOD.

Recent studies demonstrate the susceptibility of astrocytes to ethanol neurotoxicity. The effects of ethanol on astrocytic metabolism have been examined in vitro (Davies et al., 1984, Dev. Brain Res.). In order to characterise the morphologic effects, we performed a morphometric study (using the Samba system) on astrocyte secondary cultures after exposure to ethanol at various concentrations (0.5, 1 and 2%). The cells were fixed, marked by a GFAP immuno-assay, and then stained by appropriate methods for microscopic examination. The following cell parameters were measured: perimeter, surface, index of shape and of convexity, and the ratio of the minimal diameter to the maximal diameter. Measurements were made at different times of exposure (24h, 48h, 72h, 96h) for the different ethanol concentrations and compared to controls. At least 120 cells were measured in each experimental condition. Two independent experiments were performed. Additional parameters were also taken into account such as the proportion of GFAP-positive cells and the percentage of cells viability. In such conditions, statistical analysis (2 factors ANOVA) revealed that the ethanol treatment caused large variations, mainly affecting the perimeter and the surface of the cells, but also the cellular shape which was particularly stable in controls and deeply affected on exposed cells. The mode of action of ethanol is presently under investigations in other conditions of exposure.

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30

Elaboration of a modified culture medium for astrocytes, compatible with Electron Spin Resonance Spectroscopy

PRIMARY CULTURE OF RABBIT KIDNEY PROXIMAL TUBULE CELLS ON COLLAGEN-IV COATED POROUS MEMBRANES : TRANSPORT PROPERTIES.

B. GONTHIER, A. SOUBEYRAN, H. EYSSERIC, and L. BARRET

Equipe de Neurobiologie du D6veloppement, Institut de Biologie Cellulaire et Mol6culaire. Universit6 Joseph Fourier, Grenoble, FRANCE Electron Spin Resonance (ESR) spectroscopy, using the spin-trapping technique, is a widely used method for the detection and identification of free radicals formed during the cellular metabolism of various xenobiotics. This spin-trapping technique has been successfully applied to the study of tissular and subcellular preparations. Unfortunately, for astrocyte culture, the reducing agents present in the medium (reductases, ascorbate, thiol functions...), lead to the spin-adduct degradation and thus, to the formation of non-detectable ESR products. In order to determine the cause of this degradation, we successively tested the effect of the different components of our medium, on radicalar species preservation. To this purpose we used a stable nitroxide, the 4-oxo TEMPO (4-OT), which is known to give, in aqueous solutions, a persistently hight ESR signal. Each component of our medium was tested in the presence of 4-OT and the stability of the signal was measured. In these conditions, we were able to identify the most agressive components for ESR signal (e.g. fetal calf serum, nutrient mixture F-12, and streptomycin-penicillin) and thus define the most suitable medium for free radical formation. Finally, we verified the cellular viability in the newly modified medium by determination of protein content.

I. Genestie, *J.P. Morin, B. Vannier et G. Lorenzon. D6pt de Toxicologie, Roussel Uclaf, Romainville. *lnserm U295, UER de M~decine-Pharmacie de Rouen, St Etienne du Rouvray. In vivo, nephrotoxic insults of many compounds on proximal tubule cell are often related to transepithelial transport and/or intracellular accumulation processes. In order to study these factors, a new model of kidney tubule cell culture has been developed in laboratory, allowing access to both apical and basolateral sides of the cells. Rabbit kidney proximal tubular cells are grown onto collagen-IV coated porous membranes. Under these conditions, the confluency is attained between 5 and 6 days after seeding. Monolayer integrity is maintained from this time and up to the fifteenth day of culture, as confirmed by inulin diffusion measurements. These cell monolayers are highly polarized, as shown by : 1) the development of pH and glucose, lactate and ammonia concentration gradients between the two compartments ; 2) the restoration during the first days of culture of a well developed brush border at the apical side ; 3) the detection of a specific probenecid sensitive transepithelial paminohippurate transport, only directed from the basolateral side to the apical one. In conclusion, this model seems highly suitable for the in vitro study of the nephrotoxic potential of compounds, in relation to transport and/or secretion processes.

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Mechanistic approach of metal dilution cytoprotective effect in cadmium induced intoxicated renal cell cultures, by Delbancut A., Barrouillet M.P., Maury-Brachet R., Boudou A., Dorfman P., Cambar J. Groupe d'Etude de Physiologie et Physiopathologie R~nales, Facultd de Pharmacie, 3, place de la Victoire 33000 Bordeaux.

MESANGIAL CELL CONTRACTION: LOW RESPONSIVENESS IN D I A B E T I C R A T . M . OUARDANI, J.P. GIROLAMI, and J. LEUNG-TACK. INSERM U388, Institut Louis Bugnard, CHU Rangueil, 31054 TOULOUSE F-actin disassembly is observed in diabetic mesangial cells.The aim of the experiments is to study the contractility of diabetic cells in response to vasoactive peptides. Mesangial contraction in response to bradykinin (BK) and mlgiotensin II (AII), is measured by using an image analysis system and expressed by the reduction in planar surface area (PCSA). Diabetic rats m'e injected with streptozotocin and sacrified 1 and 8 weeks after (D1, D8).When compared to normal cells (N), theese ceils show reduced responses at the level of the number of responding cells and intensity of the contraction. BK and AII induce a dose-response with a maximal PCSA at the concenu'ation of 106M (8% and 14%, for D8 and N). When D8 cells

Heavy metals as cadmium (Cd), an important environmental pollutant, induces a severe toxicity for renal tubules. Numerous protective agents against metal toxicity, as chelating agents or SH-rich compounds have been already purposed. The present study approaches the precise mechanisms of metal high dilutions protective effect in cultured renal tubular cell lines (LLCPK1). We have already shown the fair protective effect of 10-30 and 10-40 M Cd dilutions against Cd high cytotoxic concentrations. It is presently evidenced a significant large decrease in Cd cell penetration rate (6,68% vs 10,40% - p<0,05) and an increase in metallothionein synthesis (4,08 vs 6,03 gg MT/mg PT - p<0,05). In conclusion, metal high dilutions seem to protect tubular cells against Cd cytotoxicity by a decrease in Cd intracellular penetration and by a metallothionein biosynthesis increase. These in vitro studies open the way to further in vivo investigations to reduce this metal toxicity.

are pretreated 48h with insulin (Sgg/ml), cell contraction is completly restored. In diabetic cells, insulin can reverse low contractile activity, at a non-proliferating close (0,05p.g/ml). In normal cells, the activation of protein kinase C (PKC) by a phorbol ester (PMA), induces a slower response, but the intensity is similar to the responses to BK and AII. In contrast, with D8 cells, PKC activation only induces 70% of the responses to vasoactive peptides.When normal ceils are pretreated overnight with PMA 10-7M (inhibition of PKC), an reduction of PCSA by 50%, is observed. Contraction is not modified in diabetic cells. Similarly, the direct inhibition of PKC by calphostin (10-9M), lowers the contraction of normal cells by 50%, but has no effect in diabetic ceils. Our experiments demonstrate a loss of mesangial contractility during the induction of insulino-dependent diabetes, which can be due to a loss of PKC activity. However, the contractile alterations can be reverse by insulin at a nonproliferating dose. Theese results suggest a relationship between PKC activity and insulin.

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34

REORGANIZATION OF BILE CANALICULI IN RAT HEPATOCYTES CULTURED ON POROUS MEMBRANES.

IMMOBILIZED FROZEN HEPATOCYTES : A C E L L K I T F O R IN VITRO A N A L Y S I S OF HEPATIC DRUG METABOLISM AND CYTOTOXICITY TESTING

C. Gu&y, J. Secchi, B. Vannier and G. Lorenzon Centre de Recherches Roussel UCLAF Romainville, France.

Rat hepatocytes plated on collagen I coated porous membranes can be maintained in culture for at least 15 days in serum-free hormonally defined Waxman's medium, supplemented with "physiological" concentrations of insulin, glucagon and dexamethasone. The reorganization steps of bile duct structures were studied by scanning and trasmission electron microscopy. On the first day of culture, many invaginations were present on the intercellular membrane. After 4 days, those invaginations were replaced by a hemicanaliculus, probably resulting from a coalescence process. The juxtaposition of a corresponding hemicanaliculus on the intercellular membrane of the facing cells allowed the reorganization of a duct-like structure, in close similarity to bile canaliculi architecture in vivo. Under the culture conditions used for this study, these canalicular structures were well maintained until the 15 th day of culture. They were situated in the middle of the intercellular face of the hepatocyte membrane parallel to the cell monolayer and showed a morphology very close to that of the bile canaliculi as observed in vivo in the normal rat liver. As these canalicular structures reorganized, different signs typical of hepatocyte repolarization could be observed, such as canaliculus delimiting junctional complexes, the presence of Golgi apparatus as well as the appearance of microfilaments and microtubules in the cytoplasm close to the canaliculus.

Claire M.GUYOMARD, 1 Christophe G.CHESNE 1 and Andr6 J.GUILLOUZO 2 1 BIOPREDIC SA, 14-18, rue Jean Pecker, 35000 RENNES, FRANCE 2 INSERM U49, H6pital de Pontchaillou, 35033 RENNES, FRANCE

Isolated hepatocytes from various animal species either in suspension or in primary culture, are widely used for drug metabolism studies and cytotoxicity testing. However, availability of parenchymal cells from large mammals is limited. Moreover the cell isolation process is time-consuming, costly and frequently cell numbers are too large to be used immediately. Consequently, storage protocols are desirable. Our laboratory has been involved in the development of various protocols for short-term and long-term hepatocyte preservation. Short-term hepatocyle storage conditions include immobilisation in alginate gel beads and hypothermic preservation in Leibovitz medium. Long-term preservation is obtained by freezing and storage in liquid nitrogen. Hepatocytes entrapped in alginate beads function for a few days when placed in a culture medium. After 24 h in culture, ethoxTresomfin O-deethylase and pentoxyresomfin dealkylase were equal to 18 and 3 pmoles/min/mg protein respectively. Procainamide N-acetyl transferase, glucuroand sulpho-conjugates of paracetamol were equal to 1, 2 and 12 nmoles/h/mg protein respectively. These values were similar to those measured in 24 h conventional primary culture and cytotoxicity of several xenobiofics was similar both in vitro model systems. After cryopreservation, cell viability still represented 80 % of the initial viability as estimated by" oxygen consumption and trypan blue exclusion. Thawed cells expressed various liver-specific functions at levels close to those found in fresh cells. Recently we have devised a system "ready for use" for studying interspecies xenobiotic hepatic metabolism. This model, designed as "LIVERBEADS| allows to obtain drug metabolites in large amounts. LIVERBEADS| are now prepared reproducibly from various animal species. They can be stored and easily transferred to pharmaceutical companies.

446

35 The influence of picolines on the soluble glutathione transferases in cultured h u m a n hepatoma cells Paul J. Dierickx (1), Hilde. H. Bohets (1), Martine Duverger-van Bogaert (2) and Thaly Lakhanisky (I). Instituut voor Hygiene en Epidemiologie, Afdeling Toxikologie, Wytsmanstraat 14, 1050 Brussel, Belgium (1) and Universit~ Catholique de Louvain, TEMU, UCL 7237, 1200 Bruxelles, Belgium (2). Cultured cells usually lose a number of their in vivo properties, including metabolic capabilities. Hep G2 cells (an established cell line derived from a human hepatoma) have retained a number of the hepatocytic phase I and II reactions. The influence of picolines, related compounds and some classical enzyme inducers on the specific glutathione transferase (GST) activity and its subunit composition in cultured Hep G2 cells is reported here. All test chemicals were applied to the cells in DMEM, supplemented with 5 juM hydrocortisone (HC) and 10% fetal calf serum. An increased GST activity was observed for rifamycin, phenobarbital, pyrazine and the picolines, of which the 4-isomer was the strongest inducer. Pyridine reduced the GST activity, but no effect was observed with HC and benz[a]anthracene. The GST subunits were analyzed by HPLC. GST Jt, GST/t, GST B1 and B2 (class alpha) were found in control Hep G2 cells. GST ju disappeared or was strongly reduced under the influence of the test chemicals. Pyridine was the only chemical decreasing GST B1. All total GST increases were due to an augmented GST B1 concentration. Consequently, picolines stimulate GST expression in Hep G2 cells by influencing the class alpha GST B1.

36 CYTOPROTECTION IN VITRO IN A MODEL OF STREPTOZOTOCINTREATED PANCREATIC ISLETS. C. LUCAS-CLERC 1, C. GUYOMARD 2, C. CHESNE 2, C. MASSART 1. (1): GURIFA, Laboratoire de Biochimie M6dicale A,UFR M6dicale, 35043 RENNES CEDEX (2):BIOPREDIC,Technopole Atalante Villejean, 35000 RENNES. The present study demonstrates the outstanding properties of 003 FA, a new active synthetic molecule (SECMA, Pontrieux 22, FRANCE) which could consistently protect rat pancreatic islet cells against the cytotoxic damages induced by a w e l l - k n o w n diabetogenic agent, Streptozotocin or by the freezingthawing-process during a cryopreservation protocol. Material and methods: Rat pancreatic islets were isolated with collagenase, exposed for 30' to 0.5 mg/ml Streptozotocin (STZ) (Sigma Corp.) and cultured subsequently for 3 days with two different concentrations of 003 FA (5 or 10 mg/ml). Spontaneous Insulin Release and Response to an acute D-Glucose stimulation were evaluated. Results 1 - On control islets, low dose 003 FA (5 mg/l) seem to favor the spontaneous insulin release in normal culture conditions and also to increase the response of islets to an acute D-Glucose stimulation. 2 - The SZ-treated pancreatic islets showed markedly diminished insulin release after stimulation with the B cell nutrient glucose. However, low dose 003 FA (5 rag/l) was able to partially counteract the SZ-induced impairment of stimulated insulin release and also to preserve an active spontaneous basal insulin release all over the 3-days culture period. 3 - Finally, when 5 or 10 mg/'l 003 FA were added to the freezing medium, the stimulation index of treated cryopreserved islets was significantly higher than that of non-treated cryopreserved islets. In c o n c l u s i o n , the present observation suggests that the toxic injury induced by streptozotocin, a highly diabetogenic drug, could be counteracted by 003 FA, a new chemical able either to protect islet cells against toxic injury or to restore the metabolic functions of partially damaged B cells affording thus the possibility of clinical application in the field of Diabetologia.

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STUDY OF CARYOLYSINE T O X I C I T Y ON R A B B I T S ' R E S P I R A T O R Y E P I T H E L I U M IN PRIMARY CULTURE jr., GIULIANI, E.,BOIVIEUX-ULRICH, C; GUENNOU, and F.. MARANO. Laboratoire de Cytophysiologie et ToMcologie cellulaire, Universit~ de Paris VII, place Jussieu 75005 Paris.

I M P L I C A T I O N OF OXIDATIVE STRESS IN THE T O X I C I T Y OF M I N E R A L P A R T I C L E S CONTAINING IRON ON TRACHEAL EPITIIELIUM IN PRIMARY CULTURE C. GUIL1ANELLI, A. BAEZA, R. ZALMA *, H. PEZERAT* and F. MARANO. Laboratoire de Cytophysiologie et Toxicologie cellulaire, Universit( de Paris VII, place Jussieu 75005 Paris. *Laboratoire de rgactivit( de surface et structure, Universit( P. et M. Curie, CNRS, URA 1106 4, place Jussieu 75005 Paris.

Genotoxics and radiomimetics properties of sulfur and nitrogen mustards are well known since about fifty years. Nevertheless acute toxicity is still badly understood and its use often leads to appearance of irreversible vesicant type lesions on epitheliums with which they come into contact. Most of carried out researches deal with skin matters, however, very few things are known on respiratory toxicity, which constitutes however one of the major risks. Moreover, its use in chemotherapy induces secondary effects among which are found out pulmonary complications. Caryolysine (HN2), compared to Hyperite for its in vivo and in vitro cutaneous toxicity, causes serious lesions on rabbit trachea epithelial cells in primary culture. An important cell detachment is observed. It is dose dependent and the detached cells are often still alive. HN2 also causes a strong inhibition of the cellular growth from 0,05 raM. This growth inhibition is dose- and timedependent. Moreover, it is irreversible and 5 rain of treatment are enough to cause an important and irreversible inhibition, even after a 120 hrs recovering in a fresh medium. Whereas the studies carried out on the skin models indicate basal cells like being mustards privileged targets, ours microscopics observations disclose that ciliated cells are also affected by HN2. The scanning electron microscopy points out an important damage on the ciliated ceils: detachment, loss and blister formation of cilia, loss of mierovilli. The transmission electron microscopy confirms these results and shows altered mitochondria. We have also tested the HN2 effect on the ciliary beat. HN2 seems to act with an "everything or nothing" effect. Indeed, after a 24 hr treatment, some cells have a ciliary beat frequency comparable to control cells, whereas others have totally ceased to beat.

Mineral particles are responsible for numerous respiratory deseases. Some contain iron which reduction can lead to the production of oxygen activated species. The rote of iron in the toxicity of three iron containing mineral particles has been investigated on respiratory epithelium cells in primary culture: The mineral particles tested were canadian chrysotile, nemalite and hematite, each containing different percentages of iron II. These mineral particles cause severe lesions in the rabbit tracheal epithelial cells in culture. Studies of scanning and transmission electron microscopy show that the three particles are endocyted by the cells. The three types of particle induce an inhibition of cellular growth with nemalite being the most cytotoxic particle. This effect is strongly decreased by a pretreatment of the particles with desferoxamin, an iron chelator. Besides studies of acute toxicity, the capacity of the particles to induce in vitro squamous metaplasia, a preneoplastic lesion, has also been analysed. Two specific criteria for the transformation of the epithelium into its squamous form, have been chosen: the appearance of eytokeratin 13 and the formation of cells with cross-linked envelopes. For both criteria, nemalite appears as the most efficient to induce metaplasia, To conclude, nemalite seems to be the most active particle tested. The data obtained with desferoxamin suggests that its action could be attributed to the iron II available at its snrface, and thus to the oxygen activated species produced. This hypothesis is in agreement with the results of paramagnetic electronic resonance (RPE) measuring the production of OAS by particles,

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39 E F F E C T S O F B U T Y R I C ACID ON A R A T COLON CARCINOMA CELL LINE BOISTEAU O. ; MEFLAH K. ; BARBIEUX I. ; GREGOIRE M. INSERM CJF 90.11, NANTES. We have studied the effects of butyric acid on a rat colon carcinoma cell line, PROb, which has been described for its tumoral and metastasising potential. In rats with peritoneal carcinomatosis, we have observed that butyric acid, associated with IL-2 immunotherapy, results in tumor regression and cures 70% of the animals. Furthermore, we shown in vitro that butyric acid inhibits cell proliferation. Using cytometric analysis, we demonstrated that this inhibition is a result either of a block in the cell cycle in the G2+M phase or a polyploidy. Moreover, cells treated at confluence (0-5raM) were induced into apoptosis. Metabolism of treated cells is intense. Incorporation of 3H-Uridine and 35S-Methionine verified that RNA and protein synthesis increased with the dose of butyric acid used. These results confirm that butyric acid treatment induces cell differentiation. In vitro, we observed identical phenomenon : treated cells were polarized as shown by the presence of domes and an increase in alkaline phosphatase activity, which is characteristic of colon cell differentiation was observed. Since we have found that TGF production was increased in treated PROb cells, butyric acid effects could be mediated by the secretion o f autocrine growth factors.Present studies in our laboratory may help better understand the role of butyric acid in in vivo and in vitro cell behaviors, as well as the mechanisms responsible for the efficiency of experimental treatments of colon cancers.

40 IN VITRO QUINOLONES CYTOTOXIC1Y ON RABBIT TENOCYTES K.GERSANT, C.HECQUET & M.ADOLPHE Laboratoire de Pharmacologie Cellulaire 15, rue de rEcole de Mddecine 75006 PARIS Fluoroquinolones are antibacterial agents characterized by a remarkable bioavallability and a wide tissue diffusion. However, some adverse effects such as arthropathies and Achilles tendonitis have been reported. The study of the in vitro mechanism of action of these antibacterial agents has been undertaken on first passage rabbit tenocytes to understand these side effects. First, this model was characterized by the study of proteoglycans synthesis after Alcian blue coloration of the matrix and type I collagen mARNs transcripts was evaluated by Northern blotting. A study on the aging of the model, by serial passaging, was begun and has permitted to calculate the population doubling times at several passages. Our first results showed that first passage tenocytes have a doubling time of 20 hrs. The characterization of the matrix components showed a very low proteoglycans synthesis and Northern blotting analysis showed presence of type I collagen transcripts. Cytotoxicity of two fluoroquinolones (norfloxacin and pefloxacin) was evaluated by several methods such as incorporation of neutral red, MTr and Rhodamine 123 (respectively markers for lysosomes, mitochondrialsuccinate deshydrogenase and global mitochondrial activity) and compared to nalidixic acid cytotoxicity. Tenocytes viability was altered after treatmentby these antibacterial agents. The IC 50, respectively for nalidixic acid, pefioxacin and norfloxacin, were 304, 168 and 144 gg/ml when determined by Neutral red incorporation and were 262, 165 and 148 gg/ml when determinedby the MTT method. The study of oxidative agents production by flow cytometry and NO2- determination should permit to develop new hypothesis for the in vitro mechanism of action of fluoroquinolones.

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41 TRANSGLUTAMINASE ACTIVITY IN RABBIT ARTICULAR CHONDROCYTES L. BORGE, S.DEMIGNOT and M. ADOLPHE Laboratoire de pharmacologie cellulaire de I'EPHE 15 rue de l'Ecole de M6decine 75006 PARIS Transglutaminases (TGases) (EC 2.3.2.13) are a family of calcium-dependent acetyl transferases that catalyse the formation of an amide bond between the 7-carboxamide groups of peptidebound glutamine residues and the primary amino groups in a variety of compounds, including the eamino group of lysine in certain proteins. TGases have been shown to be involved in many cellular events such as growth, differenciafion, apoptosis, capacity to metastase_. Recently, it has been shown that phospholipases A2 (PLA2) are strongly activated by TGase. PLA2 being an enzyme involved in the inflamatory process of arthritis, we studied this enzyme activity in rabbit articular chondrocyte in culture. TGase activity was measured on confluent culture at different passages. In primary culture, TGase activity was high (8000 pmoles/mg of protein/30min) and rapidly decreased to a base level (1000 pmole/mg of protein/30min) from passage 3 until passage 7. After retinoic acid (RA) treatment of cell cultures, a chondrocyte dedifferentiating agent, TGase activity decreased (5000 pmole/mg of protein/30min) in primary culture compared to control cells whereas TGase activity increased of a factor two as soon as the second passage. This main result as well as results of other experiments (TGase activity localization, TGase sensitivity to trypsin) suggest that chondrocytes express two different TGase which are regulated in an opposite way by RA. The TGase down regulated by RA might be associated with the well differentiated phenotype of the chondrocyte in primary culture since, expressed as a fonction of passage number, the TGase activity correlates better with the dedifferentiation profile than with the growth declining capacity of the chondrocyte.

42 Effect of different immortalizing oncogenes expression on the regulatory sequences of type II collagene gene in rabbit articular chondrocyte. N. Steimberg, S. Viengchareun, S. Thenet-Gauci, M. Adolphe. Laboratoire de Phaxmacologie Cellulaire. 15 rue de rEcole de M6decine 75006 Paris. In order to develop new cellular models which could be of interest in toxicology research, Rabbit Articular Chondrocytes (RAC) have been immortalized with T+t of SV 40, T of SV 40 or m y c . The lines we obtained did not express in a stable manner the differentiated phenotype. Our aim was to establish whether this dedifferentiation was the result of the oncogene expression or was due to the subcultures in monolayer. Transient transfection of well-differentiated chondrocytes with these oncogenes would permit to be free from the effect of long time culture in monolayer. We have used a vector carrying a reporter gene (CAT) placed under the control of type II collagene promoter and enhancer (differentiation marker) to study more directly the effect of the different oncogenes on this gene transcription. RAC in primary culture have been co-transfected with this vector and with a plasmide carrying one given oncogene. CAT activity has been determined 72 hr after the transfection. The first results obtained with transient transfection with T + t, T of SV 40 or myc, seemed to show that the expression of these oncogenes did not decrease the activity of type II collagene regulatory sequences and was therefore not incompatible with chondrocytes differentiated state. Consequently, it should be possible to immortalize chondrocytes with a plasmide carrying one of these oncogenes placed under the control of the regulatory sequences of type II collagene gene. The chondrocytes clones selected after transfection with such vector will derive from cells able to activate these regions. This construction would permit the obtentionof clones able to express and maintain the chondrocyte differentiated phenotype. with higher rate