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TOXICITY OF ERYTHROMYCINE, PARACETAMOL AND ISONIAZIDE IN HUMAN HEPATOCYTES IN PRIMARY CULTURE AND IN THE HUMAN HEPATOMA CELL LINE HEPG2. COMPARISON WITH RAT AND MOUSE HEPATOCYTES IN PRIMARY CULTURE

Studies, in video-microscopy, of agonist-induced calcium oscillations in human hepatocytes.

Catherine VIOLLON, Laurence NICOD, Catherine BENAY, Any REGNIER and Lysiane R[CHERT Laboratoire de Biologie Cellulaire, Facult6 de M6decine et de Pharmacie, Place Saint-Jacques, 25030 BESANCON

We have shown that hepatocytes in primary culture constitute an adequate in vitro model for the determination of species differences in the hepatotoxicity potential of various xenobiotics. Thus, the toxicity profile of erythromycine was equivalent in rat, mouse and human hepatocyte cultures (IC50 : 0.1-0.5 mM). In contrast, paracetamol and isoniazide displayed a similar profile of eytotoxicity in mouse and human hepatocytes (Paracetamol IC50: 5-8 mM ; Isoniazide IC50 : 40 raM), while no toxicity was noticed in rat hepatocytes at concentrations up to 20 mM for paracetamol and up to 40 mM for isoniazide. In addition, the toxicity profiles of paracetamol, isoniazide and erythromycine in human hepatocytes in primary culture where equivalent to those obtained with the human hepatoma cell line HepG2. It appears thus that HepG2 cells constitute a valuable tool for studying in vitro the mechanisms of hepatotoxicity in humans.

Tordjmarm T, Combettes L, Berthon B, Vons C*, Franco D* and Claret M.

INSERM U.274, Ix$t. 443, LIPS, 91405 Orsay Cedex and *H6pital Antoine B&zl&e,Clamart, France. Signalling between cells is achieved by a number of hormones, neurotransmitters and growth factors via changes in the concentration of cytosolic Ca2+ ([Ca2+]i). In hepatocytes, calcium signals are thought to be involved, among others function, in the control of bile secretion and canalicular contraction. We have studied hormone-induced calcium signals in isolated single cells and multicellular systems of human hepatocytes. Isolated human hepatocytes were prepared by coUagenase peffusion, from patients undergoing partial hepatectomy for benign or malignant turnouts, as already described in Hepatology (1991; 13:1126) and set in primary culture onto glass coverslips coated with collagen. Change in [Ca2+]i was measured, by video-microscopy, with the fluorescent dye fura2 after loading with fura2/AM (5 glVf) in a William's E medium at 37~ Addition of InsP3-dependent agonist such as norepinephrine (0.1-5 laM), angiotensine II (1 riM) or ATP (50-100 gM), induced [Ca2+]i oscillations. However, in contrast with rat hepatocytes, perfusion of vasopressin, even at maximal concentration (100 riM), had no effect on the resting [Ca2+]i. When observed on doublet or triplet of hepatocytes, perfusion of cells with noradrenaline (NOR, 0.1 glVl)evoked oscillations as well in single cells as in associated cells. However, associated hepatocytes display coordinated [Ca2+]i rises, with a first, a second (in doublets) and a third (in triplets) responding cells. This sequential responses were retained in the same order during the [Ca2+]i oscillations of a whole multiplet. These results: 1) show that Ca2+mobilising hormones, except vasopressin, generate Ca2+ oscillations in human hepatocytes, 2) strongly suggest that there is a gradual heterogeneity, in terms of hormonal sensitivity, among hepatocytes in multicellular systems. These functional differences might be related to the well established heterogeneity of the hepatocytes in the porto-centrilobular axis.

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M E T A B O L I S M AND G E N O T O X I C I T Y OF OCHRATOXIN A: USE OF HUMAN BRONCHIAL E P I T H E L I A L I M M O - R T A L I Z E D CELLS EXPRESSING C Y T O C H R O M E P450S. Grosse Y. * Macd K.w Pfeifer A.w Meiller B, * and Pfohl-Leszkowicz A. * 9 ENSAT, 145 Avenue de Muret, F-31076 Toulouse ; w NESTEC, Vers chez l~s Blanc, CH-1000 Lausanne.

DETECTION OF DNA-ADDUCTS AS BIOMARKERS OF EXPOSITION TO C A R C I N O G E N I C POLYCHLOROBIPHENYLS

We have recently shown that the mycotoxin ochratoxin A (OTA) was genotoxic, using the DNA-adduet detection [32p]-postlabelling method on four different organs of mice treated with OTA. Kidney was the target organ and some of the adducts were specific of every analyzed tissue, stating that OTA metabolism was different from organ to organ. We studied the influence of OTA metabolism on its genotoxic effect. Human bronchial epithelial immortalized cells (BEAS-2B) expressing or not cytochrome P450s (CYP) (1A2, 2A6, 2D6, 2El, 3A4) were incubated with 0.5 BM OTA. DNA-adducts were detected in all cells. Total DNA-adduct level ranged from 4 to 60 adducts per 10 9 nucleotides. Some adducts were common to all cell types, including cells expressing only phase II enzymes or peroxydases (N6o cells). Some other DNA-adducts were only induced by specific CYPs. The highest adducts level was found in cells where CYP 1A2 was expressed but CYP2D6 and 3A4 also induced DNA adducts. Pretreatment of cells by ethacrynic acid and Lbuthionine sulfoximine, which lower glutathione conjugation, led to a DNA-adducts general reduction, showing that this detoxication pathway leads to reactive metabolites. Owing to HPLC analysis of the culture media, three unknown OTA metabolites were found. One of these (called I), which needs to be identified, seemed to be correlated with DNA-adducts formation. These results indicate that OTA need not to be biotransfonned by CYPs to be activated (though they can play, like CYP1A2, a role in this activation), but that it could be converted via glutathione conjugation. During these reactions, a metabolite appears and specifically marks DNA adducts presence.

DUBOIS M., PFOHL-LESZKOWlCZ A.', KREMERS P. Laboratoirede ChimieMedicale,Universit6 de Li6ge, Belgium *ENSAT, Toulouse, France Xenobiotics penetrating a living cells are either eliminated unchanged or biotransformed into hydrosoluble metabolites, easier to eliminate. Cytoehrome P450 dependent monooxygenases are usually responsible for these biotrausformatious. Unfortunately, these reactions may also lead to the production of activated metabolites able to bind covalentlyto nucleophilic entities, like nucleic acids. This has often mutagenic consequences and may initiate a cancerous process. Polyehlorobiphenyls (PCBs) are widespread environmental pollutants producing various toxic effects namely at the hepatic level and suspected to be genotoxic and carcinogenic in humans. Cultured hepatocytes were exposed to a commercial mixture ofPCBs, Aroclor 1254, and to tetrachlorobiphenyl (TCB) in order to evaluate the DNA-adduct formation. The cells used were: fetal rat hepatocytes, expressing mainly CYPIA1, fetal quail hepatocytes, expressing CYP2B1 and CYP2E and a human cell line, Hep 132, expressing essentially CYP3A enzymes. TCB was genotoxic for the three cell types, aroclorproduces DNA-adducts only in rat hepatocytes. The EROD enzymatic activity, specific of CYP1A1, correlates with the amount of DNA-adducts formed as a function of the concentration of PCB in the culture medium. The ~P postlabelling of DNA-adducts method is a useful tool that, combinedwith the culture of hepatocytes, may constitute an excellent biomarker able to evaluate the genotoxicity of environmental pollutants.

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CELLULAR MODELS F O R T H E STUDY OF T H E I N D U C T I B I L I T Y OF PER()XISOMAL B-OXIDATION BY PEROXISOME P R O L I F E R A T O R S IN MAN AND RAT

Effect of various xenobiotics on nicotine C-oxidation by human liver microsomes

S. Duclos, L. C. Ramirez, J. Bride, C. Causeret et P. Bournot, Laboratoire de Biologie Mol~culaire et Cellulaire, Facult~ des Sciences Mirande, B. P. 138, 21004 Dijon Cedex.

P.H. Villard, B. Lacarelle, A. Durand, J. Covo, H. Point-Scoma, J. Catalin, A. Viala

The inductibility of peroxisomal ~3-oxidation by ciprofibrate, a hypolipidemic drug, was studied in h u m a n h e p a t o m a HepG2 cells, h u m a n hepatocytes, and rat hepatoma Fao and MH1C1 cells, in culture. The use of cytochemical, u l t r a s t r u c t u r a l techniques shows an increase in the number and size of peroxisomes in the rat cells treated with 250 gM ciprofibrate for 72 h, accompanied by a modification in the number and shape of the mitochondria. A more marked effect is seen on Fao cells. The effect is much less marked in HepG2 cells. In parallel, the palmitoyl-CoA oxidase activity is increased by ciprofibrate in rat cells by a factor as large as 20 in Fao cells, while it is hardly changed in human cells. The stimulation of the activity in Fao cells is also obtained in chemically defined culture media. Palmitoyl-CoA oxidase and enoyl-CoA hydratase/13-hydroxyacyl-CoA dehydrogenase were characterized in cellular homogenates and light mitochondrial fractions (L-fractions) by Western blot. The immunodetection used rabbit anti-sera raised against the purified enzymes. These enzymes are induced by ciprofibrate in rat cells, the induction being stronger in Fao cells than in M H I C 1 cells. In the human models, ciprofibrate as well as other proliferators such as nafenopin and WY 14,643, do not or only weakly induce these enzymes. The response of the human and rat cellular models is similar to the response in vivo of the species. These models, of different sensitivities to ciprofibrate, can be used to elucidate the molecular mechanisms of p e r o x i s o m a l 13-oxidation inductibility by physiological or xenobiotic proliferators. Supported by Sanofi-Winthrop and GIS Toxicologie. In collaboration with Dr. P. Maurel et al., I N S E R M U 128, Montpellier.

EA 859: Laboratoire de Pharmacocin~tique et de Toxicologie, Facult~ de Pharmacie de Marseille, 27, Bd Jean Moulin 13385 Marseille Cedex 5, France Cotinine is the major metabolite of nicotine and is widely used as an index of tobacco smoke exposure. Using cDNA expressed human cytochromes P450, previous studies have reported that CYP2A6 is the major isoenzyme involved in this metabolic pathway while CYP2B6 has the highest affinity toward nicotine. These studies also suggested the possible role of other cytochrome P450 isoforms (i.e. CYP2C8, 2C9, 2D6, 2E1 and 4B1). In order to clarify the role of the various human cytochrome P450 isoforms and to assess possible inhibition of cotinine formation, the aim of the present work was to study the inhibitory effect of various compounds on nicotine C-oxidation by human liver microsomes in presence of cytosol. Among the different xenobiotics studied, none except coumarin significantly inhibited the rate of cotinine formation. This study therefore demonstrated that CYP2A6 has a major role in cotinine formation whereas other isoforms have a negligible role. In conclusion, inhibition of this metabolic pathway by drugs cannot hinder the interpretation o f cotinine measurement as an index of tobacco smoke exposure.

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INTER INDIVIDUAL VARIABILITY OF CYP2B SUBFAMILY INDUCTION IN RAT: METHODOLOGICAL CONSEQUENCES.

Inductive effect of cigarette smoke on liver, lung and kidney cytochromes P450 in mouse

E. SAMPOL, B. LACARELLE, P. AUQUIER, H. SCOMA, J. COVO, J. CATALIN, A. DURAND

P.H. Villard, E. S~r6e, B. Lacarelle, Y. Barra, L. Attolini, B. Bruguerolles, J. Catalin, A. Durand.

EA 859, Facult~ de Pharmacie, 13385 MarseiUe Cedex 5

EA 859: Laboratoire de Pharmacocin~tique et de Toxicologie, Facult~ de Pharmacie de Marseille, 27, Bd Jean Moulin 13385 Marseille Cedex 5, France

Induction phenomenon has been extensively studied. Most authors have used pooled livers microsomes from different (2 to 6) animals, while others have prepared individual microsomes from each treated animal~ If a wide inter individual variability exists, the first approach could lead to misinterpretation and hinder statistical analysis. In this context, we compared the two approaches in rats treated by Phenobarbital (PB). Three lots of six rats were randomly assigned to three treatments (control, PB= 2mg/kg, PB= 20 mg/kg ). Microsomes from each rat liver (n=18) were prepared individually. In addition three pools of microsomes corresponding to the three groups of treatments were also prepared. The PROD activity which is representative of CYP2B subfamily activity was measured (n= 6 measures) in all microsomal samples (n= 21). Our results showed that the CV (%) on PROD activity determination was <15%. In all groups of treatment, a factor of 2 to 3 was observed for the inter individual variability of PROD activity. In the 20mg/kg group, the induction factor for pooled microsomes was about 30, whereas this factor was only about 2.5 in the 2mg/kg group. Concerning these pooled microsomes it was therefore difficult to conclude for the 2mg/kg group. On the other hand, statistical analysis performed on individual data from each animal has shown a highly significant induction effect in the [wo groups. In conclusion we can consider that pooling is valid when induction effect are strong. However if we want to demonstrate small induction effect we have to separate livers from each animals and performed an adequate statistical analysis.

Renal, pulmonary and hepatic microsomes of male NMRI mice were used to study the inductive effect of cigarette smoke on cytochrome P450 expression. Animals were daily exposed to tobacco smoke for 2 ($2), 4 ($4), 8 ($8) and 30 ($30) days. Liver enzymatic activities catalyzed by CYP1A, 2B, 2C, 2D, 2E1 and 3A were induced at a maximum in $8 group. Immunoquantification in liver microsomes indicated that: 1) CYP1A and 2B were the most induced in the $2 group, 2) C~'P2E1 and 3A were the most induced in the $8 group. Immunoquant!fication in lung microsomes indicated that: 1) eYP3A was not detected, 2) CYP1A and 2E1 were the most induced in groups $2 and $8 respectively, 3) CYP2E1 was more inducible than CYP1A, 4) CYP2B was considerably induced in an increasing manner during the treatment. Immunoquantification in kidney microsomes indicated that: 1) CYP1A and 3A were not detected, 2) CYP2B seemed to be only slightly expressed in the $2 group, 3) CYP2E1 was dramatically induced in an increasing manner during the treatment. In conclusion, the high inductibility of CYP2E1 in lung and kidney suggested that it could actively participate in carcinogenesis induced by cigarette smoke in man.

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Comparative transport and metabolism of Docetaxel in cultured hepatocytes from rat, dog, monkey and man F. MARRE1, G. de SOUSA2,G. SANDERINK3, R. RAHMANI2 1Faculte de Pharmacie, 87025 Limoges cedex. 21NSERM/CENTRE INRA, 06606 Antibes cedex 3RHONE-POULENC RORER, 94403 Vitry/Seine

D E V E L O P M E N T OF AN IN VITRO EQUIVALENT DERMIS FOR E V A L U A T I O N OF CUTANEOUS

Docetaxel, is a semi-synthetic spindle poison TaxoM which has demonstrated an excellent antitumor activity against various types of solid tumors. The aim of our work was to compare the transport and metabolic processes of Docetaxel in rat, dog, monkey and human hepatocytes in primary-culturp, seeded in 6-well or 12-well plates (0.3 or, 0.8 x 106 cells/well). Drug uptake was analyzed between 0 and 90 min and metabolic studies during 24 hrs on cultures exposed to 1-50 pM [14C]Docetaxel. Extra- and intra-cellular metabolites were quantified by HPLC. Whatever the species, the drug influx was directly proportional to the initial Docetaxel concentration. The steady-state was reached after 30 to 45 min of incubation. The maximal intracelllar incorporation was 2% (monkey), 4% (dog) and 12% (rat). It ranged from 2% (sample HL50) to 5% (sample HL51) in human hepatocytes. In the four species, the amount of metabolites produced during 24 hrs was dose dependent. Indeed, at 1 IJM (respectively 50 pM) Docetaxel, they represented 12.4% (3%) (in rat), 60.6% (10%) (in monkey), 98% (12%) (in dog), 17-53% (2-8%) (in human: HL45 and HL46). Four metabolites, more polar than the parent drug, were produced in the extracellular medium, whereas mainly unchanged drug was detected at the intracellular level. Except for the dog, the same major metabolite was synthetized in all species including man, although its formation rate showed significant variations. Our study hence demonstrates some interspecies and interindividual variabilities in Docetaxel hepatocyte uptake and metabolism which could determine its pharmacotoxicological activity.

TOXICITY.

Corinne AUGUSTIN, Christian COLLOMBEL and Odile DAMOUR. Laboratoire des substituts cutan6s, CNRS URA 1341, H6pital Edouard Herriot, 3 place d'Arsonval, LYON, FRANCE. The legal proceeding to evaluate toxicity of cosmetic, household, chemical and pharmaceutical products is still skin irritancy DRAIZE test on rabbit. Various in vitro techniques are currently being developed as alternatives to in vivo animal testing. Our in vitro model system is composed of 24 equivalent dermis (ED) comprising a chitosan-cross linked collagen-glycosaminoglycan matrix populated by foreskin fibroblasts. A potential advantage of this system is to study the effects of tested samples topically applied to the air-exposed ED allowing the test of complexe and insoluble formulations. In evaluating this system for irritancy testing, three different measures of toxicity were used: MTT reduction, LDH and IL6 release. The purpose of this study is to establish the validity of ED as a model for investigating cutaneous toxicity. After the standardization of ED preparation, we study the reproductibility versus sterm and cell passage fibroblasts and culture time by comparison of cadmium chloride IC50 using a Student's t test. Preliminary results suggest that the ED may be an useful in vitro model for the prediction of dermal irritation and allow the development of a skin equivalent-kit realised by seeding human keratinocytes onto this equivalent dermis.

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Stimulation of collagen synthesis b~ cosmetic materials : modelling with SKIN, an in vitro reconstructed skin

Uses of the in vitro reconstructed epidermis EPISKIN, for pharmacological studies of cosmetic products

A.DEGUERCy(1),C.CHESNE(1),F.VUILLE(2), R.VALLEE(3) (1) BIOPREDIC, 14-18, rue Jean Pecker,35000 RENNES (2) ADF CHIMIE,Le Bierson, 86370 NARCAY (3) CODIFINTERNATIONAL,La Tuilerie, 35610 ROZ-SUR-COUESNON Collagen contributes to skin mechanic properties (skin "firmness"), which are an important target for cosmetic products. Increase of its synthesis by active ingredients included in formulations can be screened using in vitro models. Reconstructed skins are made of fibroblasts embedded in a matrix similar to the dermis and are covered by a differenciated epidermis forming a stratum corneum. These models help to go further compared to cell monolayers : assay of lipophilic or finished products is possible and passage through is reproduced. Three microalguae protein extracts were tested on the artificial skin SKIN 2 (ZK1301, ADF CHIMIE, France) using collagen neosynthesis as the endpoint. Vitamin C was used as a reference compound. Test compounds and vitamin C were applied on the epidermis side, one hour per day during three successive days. Effects were evaluated by counting H3-proline incorporation in neosynthetised proteins. Results showed that vitamin C at 1 mM increased by a factor 3 the incorporation of H 3proline in proteins. Two alguae extracts increased t h e parameter by a factor 2 to 3 while the third one had no effect. These increases were superior to those found in fibroblast monolayers (ranging between 1.6 to 1.8). The difference could be explained by the higher concentrations we were able to test on SKIN2 (thanks to a lower cytotoxicity of the test compounds in this model) or to the higher sensitivity of the model.

A.DEGUERCY,C.CHESNE BIOPREDIC, 14-18, rue Jean Pecker, 35000 RENNES Claims made for cosmetic products are to be sustained by objective data. Clinical methods are often long and costly and not appropriate for screening purposes. Reconstructed skin and epidermis models offer numerous assets in pharmacology compared to cell monolayers : test compounds even lipophilic ones are applied as such on the top of the model and a formulation is not modified, passage of active ingredients through the model can be studied. Some studies performed with EPISKIN (IMEDEX, France) will be shown : 1) the effect of a natural extract on protein neosynthesis studied by incorporation of labelled leucine. Protein neosynthesis was increased in a dosedependant manner,-2) the water retention activity of ingredients and formulations was compared by the measurement of tritiated water evaporation through the epidermis, treated topically. Experimental conditions and comparison of ten finished products are presented, 3) transepidermic passage of three aminoacids, one free fatty acid and a sugar were studied. Permeability coefficient of these compounds were measured. EPISKIN model is seen as a useful model for the set up of experimental protocols extrapolated from animal models or which can not be performed easily or following ethical considerations in volunteers.

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INTERACTIVE LASER CYTOMETRY FOR A N A L Y Z I N G GENE EXPRESSION IN THE i n vitro R E C O N S T I T U T E D SKIN.

A SCID-hu M 6 i i s e Xenograft Model to Investigate The I m m u n u e Function of Human Skin

D.S.F. Biard 1, M-C V o z e n i n 1, Maratrat 2, M. Martin 1, F. Daburon 1

M.

1 Laboratoire de Radiobiologie Appliqu6e, CEASaclay, 91191 Gif sur Yvette, cedex, France. 2 RhSne Poulenc-Rorer. DSM-CRVA, BP14. 94403 Vitry sur Seine, cedex Alfortville, France.

For studying genetic disruptions giving rise to various cutaneous pathologies, we have focused our attention on dermal and epidermal interactions. For this reason, we develop the " i n v i t r o reconstituted human (or pig) skin" culture model. This model is constituted of an epidermal sheet stretching on a dermal matrix (human fibroblasts embedded into a rat collagen type I gel). The maturating program (proliferation versus diff6rentiation) of keratinocytes is strictly dependent on stromal cells. Fibroblastpopulated gels with keratinocytes are lifted on a metal grid to create an air-medium interface. In this model, mesenchymal cells and keratinocytes are nourished from below by diffusion through the gel while the apical side is exposed to the air. Our culture model has been designed for providing small gels (I cm ~) easily handling. In those conditions, a well-differentiated epithelium is achieved after two weeks of culture. At different times after chemical or physical treatments, skin specimens can be snap-frozen and frozen sections made (8 mm). Immunohistochemical staining analysis for structural proteins or proteins which are transiently expressed after a genotoxic injury (early response genes), is performed using an interactive laser cytometer (ACAS 570, M6ridian Inc.). This approach allows us to rapidly localize and 'quantitate fluorescence inside each cell of interest. By mean of this new technology, we have improved the use of the "in vitro reconstituted skin" model for assessing molecular mechanisms o f radiofibrosis or for unravelling early genetic events induced by oxydative stress.

~ ( 1 ) ~ . B. AUTRAN(2); F, CARPENT~ER(3); F.HADIDA(2); H. AGUT(4); J.F. ANDREANI(S); J.Y. CESBRON(1) (1)INSERMU415, InstitutPasteur,rue du Professeur,BP245 59019 Lille CedexFrance.;(2)URA62S)CERVI)GroupeHospitalierPiti~-Sail~tri~re, France; (3)Serviced'Anatomopathologie,GroupeHospitalierLacordaire, 59300; RoubaixFrance.(4)CNRSEP57,CERVI)GroupeHospitalierPiti6SaIl~tri6re,France.(5)Servicede Ch rurgieMaxilIo-Facialeet Stoniato|ogie, Centre Hospitalierdes arm6es Scrive,59998 lilleArmies,France Despite a lack o f constituent lymphocytes, the skin plays a n important role in the induction o f the immune response, mainly due to the activity o f Langerhans cells. For this reason, skin is currently a c o m m o n route for the administration o f vaccines. The skin can also be a site o f hypersensitivity reactions or autoimmune processes which induce skin lesions. A study o f these immune responses in humans, whether benefical or not is complicated by ethical considerations, In an attempt to study the immune function oflluman skin, under more controlled experimemal cono~tions, we have established a mouse-xenograft model that has p r o v i d e d an o p p o r t u n i t y for l o n g - t e r m investigations. Here, we present the first results of SCID mice immune reconstitution produced by the e n g r a f t m e n t o f h u m a n skin ( o b t a i n e d from reconstructive surgery) and transfer of syngeneic NK-depleted peripheral blood lymphocytes (SCIDhu PBL/Skin). Successful engraftment was obtained in 24 out of 25 mice. Some o f these were observed over a period o f 6 months. Sequential biopsies o f the skin grafts were performed, which revealed complete xenograft vascularisation within 4 weeks following transplantation. Anatomical-pathological analysis showed an histological structure similar to normal skin. To evaluate the immune capacity of this model, the SCID-hu P B L / S k i n mice were intradermally immunized by a recombinant pox virus. This vector was chosen for its ability to induce cytotoxic cellular immune response in humans. Immunohistochemical examination o f engrafted human skin demonstrated a predominantly perivascular infiltration o f CD8 + l y m p h o c y t e s . The production o f T l y m p h o c y t e clones from these graft is currently underwa~ A study o f the inter-relationship o f the immune system ~ d the skin through such experimental m o d e l s has significant potential, not only for the evaluation of new vaccines (DNA) but also to study the pathophysiology o f autoimmune disease.

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15 E F F E C T O F C E T I R I Z I N E ON ICAM-1 E X P R E S S I O N BY

Three-dimensional cultures of human cutaneous cells : interest and

KERATINOCYTES IN CULTURE

limit of cell lines.

.~.AN-LOUIS F.*, MICHI~ L.*, Mtt.LAC M.0' RIHOUX J.P?' ~

*

Braut-Boucher, F., Boukhelifa, M., Paulin, Y., Muriel, M-P., Giner,

*INSERM U 312, Laboratohe de Derraatologie, HSlital Saint-Louis, 75010

M., Pichon, J., Aubery, M and Font, J.

PARIS.0UCB Phmam S.A., 21, me de Neuilly, B.P. 314, 92003 NANTERRE Cdx

INSERM U.180 U F R Biomrdicale 45 rue des Saint-Prres, Universit~ Paris V 75006 Paris, France:

Intercellular adhesion molecule-1 (ICAM-1) is a member of immunogiobulin superfamily. The integrins LFA-1 (CDlla/CDI8) and

Attemps to limit the number of experiments on animals are made

Mac-I (CDllb/CDI8) are its ligands. It is involved in the leucocyte

and there is increasing interest for in vitro models. We have

adhesion to the vascular endothelium, thus facilitating the cell

developed a system of cultured normal human keratinocytes (NHK)

extravasation towards the tissues. It seems to play an important role in

grown at the air - liquid interface of a non coated artificial

inflammation and more specifically in the IgE-dependeat allergic reactions.

human epidermis (Font, J e t al. 1994) Nevertheless, two points are

In a previous study, we showed that cetirizine, a non-sedative anti-

difficult to control in primary cultures : the variability due to the

membrane on Falcon insert culture which display the architecture of

H1, inhibits/n vivo the cutaneous recruitment of eosinophils induced by

donor and the source of cells (adult human or foreskin)

allergens in allergic patients sensitive to grass pollen [J. Allergy Clin.

To minimize these drawbacks we used a spontaneously transformed

Iramunol. 1988, 82:101-109]. In order to better understand the mechanisms of actigu of cetirizine, we studied the effect of cetifizine on the ICAM-1 expression by keratinocytes. The ICAM-1 molecule is not constitutively expressed by keratinocytes but it can be induced by several stimuli such as IFN~/, TNFct, IL-lct or IL-115 [Arch. Dermatol. Res. 1990, 282:238-245]. In our work, we studied the effect of different concentrations of cetifizine on the expression of IFN~/-iudtteed ICAM-1 at the surface of cultured keratinocytes. The experiments were performed using primary cultures of human keratinocytes established from breast plastic surgery. Cells were cultured in Green medium supplemented with epidermal growth factors and 10% Foetal Calf Seraro. Subconflueut cultures were stimulated for 24 and 48 hours by several IFN~/doses, ranging from 10 U/ml to 100 U/ml, in the presence of defined concentrations of eetirizine (10-10M to 10-5M). The ICAM-1 expression was evidenced by immunocytochemistry using the alkaline phosphatase anti-alkaline phosphatase method (APAAP). Our results show : a) an absence of constitutive ICAM-1 expression on the surface of monolayer cultured keratinocytes; b) an ICAM-1 expression at the surface of IFN~/-stimulated keratinocytes. This expression varies according to the time and dose of stimulation with IFNy u) an inhibition of IFNy-induced ICAM-1 expression by cetifizine. This inhibition was dependent on the concentrations of cetirizine and the inhibition level coald reach 35% with 10"5Mofcetirizine. In conclusion, cetifizine inhibits the ICAM-1 expression of /n vitro IFN~l-s~imulated

keratinocytes. These results suggest that the

therapeutical efficiency of cetirizine could be due, at least in part, to the

in vivo modulation of the expression of adhesion molecules. It could also account for the inhibitory effect of cetirizine during the late phase of the allergic reaction.

non tumorigenic human keratinocytea, the HaCaT cell line (kindly provided by Dr. Fusenig). We observed that H a C a T cells cultivated in DMEM form a monolayer and exhibit the phenotypic morphology, the pattern differentiation markers (cytokeratins K1/K10) and integrin subunits of NHIC The homogeneity of the monolayer was controlled using the fluorescent vital probe calcein AM. The fluorescence was measured in a microtiter-plate scanning fluorometer (Fluostar, Tecan). O u r interest has focused on analyzing the capacity of H a C a T cells to differentiate and to establish a stratified phenotype under the airliquid interface culture conditions. The ultrastructural morphology, the expression of integrins and cytoskeleton proteins were examined at Day 7, Day 14 and Day 21 of the culture period in two media DMEM and D M E M / H a m ' s F12 supplemented with 10% FCS and adjusted to 1.5 mM. Ca 2+. After 7 days of culture in DMEM, multiple islands of HaCaT cells grew on the insert and exhibited a layer of basal cells which adhere closely to the porous membrane. Just above the basal cells, the suprabasal cells grew irregularly on two or three layers revealing many intermediate filaments (positive labeling of suprabasal cytokeratins K1 - K10) extending to the desmosomes. No further stratification was observed during the three week culture period. Suprabasal markers of differentiation (filaggrin, involucrin) and integrin topography were examined in view of the known skin differentiating model on insert. The findings suggest that H a C a T cells do not differentiate as well as

NHK in wltro but may, however, serve as an attractive model for investigations which need proliferative keratinocytes in the absence of the complete differentiation until stratum corneum. The two systems provide complementary in formations. pharmacological

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17

18 OKADAIC A C I D T R E A T M E N T INDUCES DNA ADDUCTS F O R M A T I O N I N B H K 2 1 C13 FIBROBLASTS AND HESV KERATINOCYTES Fessard V. 1, Pfohl-Leszkowicz Aft, Puiseux-Dao S. 3

1 L a b o r a t o i r e de Toxicologie M a r i n e , F a e u l t d de M d d e c i n e , A v e n u e de V a l o m b r o s e , 06 107 Nice C e d e x 2, F R A N C E 2 E . N . S . A . T . , L a b o r a t o i r e de T o x i c o l o g i e e t S~curitd alime/ataire, 145 A v e n u e de M u r e t , 31 076 Toulouse, FR/XeN.CE 3 C . E . M . A . T . M . A . , U n i v e r s i t d de P a r i s 7, 2 p l a c e J u s s i e u , T o u r 53-54, 75 251 P a r i s C e d e x 05, FRANCE

Okadaic acid (OA), a toxin involved in diarrhetic shellfish poisoning (DSP), h a s been shown to be a p o t e n t t u m o r p r o m o t e r in m o u s e s k i n a n d g l a n d u l a r s t o m a c h . However, more r e c e n t studies t e n d e d to show t h a t OA can also act as a genotoxic. In this study, using the 32p-postlabelling m e t h o d , DNA a d d u c t f o r m a t i o n w a s o b t a i n e d in two cell lines (BHK21 C13 fibroblasts and HESV keratinocytes) after t r e a t m e n t by OA for 24 hours. 19 adducts were observed with BHK21 C13 cells a n d 14 w i t h H E S V ones. T h i s n u m b e r decreased with t h e dose. 9 a d d u c t s were similar in the two strains while 10 were specific of BHK21 C13 cell line a n d 5 of HESV one. The genotoxic effect of OA showed in this study should lead to a more carefull survey of DSP outbreaks.

PROTECTION OF HUMAN DERMAL FIBROBLASTS BY LOW DOSES OF CADMIUM : study of metallothloneln Induction. BASSET L., MICHEL L. and DUBERTRET L. INSERM U312. Laboratoire de De~rmatologie. HSpital SaintLouis, 75010 Paris, France. The metallothionein (MT) is one of the main intracellular compound involved in the detoxification of heavy metals such as cadmium. Detoxification is achieved by the numerous thiols of this low molecular weight protein (~ 6500 D), allowing the chelation and the storage of the metals. MT synthesis can be induced at the transcriptional level by several factors including heavy metals, and more specifically cadmium. The aim of this study was to analyse the mechanism of induction of MT and its protective effect in cutaneous cells. Human dermal fibroblasts were chosen because they constitutively express low levels of intracellular MT (Bartsch et coll., Arch. Toxicol., 1990, 6 4 : 177-180). We studied the protective effect of a pretreatment of fibroblasts by low, nontoxic doses, of cadmium (first exposure) on the cytotoxicity induced by high concentrations of cadmium (second exposure). The experiments were conducted using primary cultures of human fibroblasts coming from plastic surgery. Cells were cultured and further propagated in EMEM 2 % f0etal calf serum. The subconfluent fibroblast cultures were treated first with subtoxic doses of cadmium for 24 hours (doses _< IC50, variable depending on the cell line) before being exposed to toxic doses of cadmium, for an additional 24-hour-period ("double induction"). The protective effect was measured by cellular proliferation analysis (3H thymidine incorporation) and by two cellular viability tests (neutral red, MT'F). In order to determine whether or not, the protective effect obtained by the first incubation of the cells with cadmium was linked to an increase in the intracellular content of MT, an ELISA assay was developped to measure the MT level both in control fibroblasts and in fibroblasts treated with various doses of cadmium for 24 hours (0.0I, 0.1, 0.5 and 1 }.tM). Our results showed that : - an initial incubation of fibroblasts with cadmium doses ranging from 0.1 to 1 p.M protected the cells against a further exposure to toxic concentrations of cadmium (> 10 p.M). - the MT content was increased in the 24-hour-cadmium treated cells in comparison with the control cells (e.g. : 5.28 I.tg/mg protein for 0.5 ~M versus 0.5 I.tg/mg protein in control cells). In conclusion, exposure of fibroblasts to low doses of cadmium allowed them to be protected against high concentrations of cadmium. Such protective effect might be due to an increase in metallothionein synthesis in fibroblasts.

204

19 Importance of fibroblasts and some components of extraceilular matrix on epidermal differentiation Robert M. 1, Noel-Hudson M.S. 1, Dazard J.E. 1, Muriel M.P. 2, Font j.2, Aubery M.2, and Wepierre j.1. 1,Laboratoire de Pharmacologiel Unit~ de Dermopharmacologie, Facult6 de Pharmacie, 5 rue JB C16ment, 92296 Ch~tenay-MalabryCedex, France. 2.Laboratoire de Glycobiologie et Reconnaissance Cellulaire, Unit6 INSERM 180, UFR Biom6dicale des Saints-P&es, 45 rue des Saints-P&es, 75006 Paris, France. In the presence of a defined medium (DMEM:Ham's F12 supplemented with Ultroser G 2%), keratinocytes cultured on a porous synthetic membrane and maintained at the air-liquid interface, are able to differentiate : the reconstructed epidermis displays many of the morphological characteristics of the native tissue from which it is derived (foreskin of a young child). This epidermal morphogenesis occurs in the absence of any dermal constituants, which indicates that keratinocytes possess the intrinsic information to organize themselves into an epidermal tissue. This model was used to study the influence of fibroblasts and/or some components of extracellular matrix on differentiation. The methods used are the following : transmission electron microscopy, quantitative and qualitative expressions of integrins' and 13 subunits, expression of epidermal markers (involucrin, transglutaminase, keratins K1/K10, filaggrin). Our results show that keratinocytes differentiation is prevented by proliferative fibroblasts, delayed by laminin (~61~ ligand) which increases the proliferative compartment. Fibroblasts and laminin association results in increasing numbers of spinous and granular layers but prevents the cornified layer formation. Moreover, the expression profile of integrins is quantitatively and qualitatively modified.

20 EVALUATION OF ALGINATE GELS AS A CULTURE MODEL FOR THE STUDY OF THE EFFECTS OF UVA ON HUMAN DERMAL FIBROBLASTS L. PASCUAL LE TALLEC, C. KORWINZ M I J O W S K A , et M. ADOLPHE.

Laboratoire de Pharmacologie Cellulaire de l'Ecole Pratique des Hautes Etudes, Institut Biomedical des Cordeliers, 15 rue de l'Ecole de M~decine 75006 PARIS. Until now UVA effects have been studied in vivo and in vitro with classical cellular monolayer culture model. This is why the effect of UVA were tested on a three dimensional culture model of alginate gels. However a model which would allow to recreate a cellular distribution closed to the denn in vivo would be of interest for the development of altemative methods. Human dermal fibroblasts, were cultured in 1.25% alginate gels. The UVA irradiation was performed with a lamp reproducing the solar spectrum, with UVB filter added. The UVA dose used was 4.5 Joules per cm 2. This dose corresponds to a 50% of growth inhibition in monolayer, two days after irradiation. Two other parameters were tested: the alginate type (from Fluka, Sigma, Kelco), and the irradiation medium (Hank's, PBS, DMEM). Effects of UVA were assessed by determining the cellular viability (by Trypan Blue exclusion) and by performing Neutral Red fluorescence test, based on the new concept of cold light fluorimetry (CLF). These tests were performed 24 hours after irradiation. Results revealed that alginate seems to be responsible only after UVA irradiation of a significant cellular toxicity. This cytotoxicity could be due to the production of oxygen species or to free radicals that would have been generated by irradiated alginate gels. However, our first results have showed that the cellular death was not mediated by the production of O superoxyde anions 02 -.

205

22

21 Compared

cytotoxicity

surfactants

tested

fibroblasts the

of

cultures

cellular

on by.

viability

various human

determining with

neutral

red

Arechabala

B.*, R i v a l l a n d P.*, C o i f f a r d L.*,

De R o e c k - H o l t z h a u e r Y.*

* CAEC - Univei-sit4 de N a n t e s 68 Bd E u g e n e O r i e u x 44000 Nantes

UMBILICAL CORD BLOOD, AS A SOURCE OF HUMAN CELLS USEABLE IN CELLULAR TOXICOLOGY : HEMATOPOIETIC PROGENITORS. ADVANTAGES, DISADVANTAGES, ETI-IICS. l-2 D. PARENT-MASSIN , J. F. ABGRALL', S. LAUTRArrE 1, B. RIO~ , H. HOELLINGER1. 1-Laboratoire de Microbiologie et Biochimie, ESMISAB, Brest. 2-Service Trousseau, CHR Morvan, Brest. Hematopoietic progenitors'can be used in toxicology in order to evaluate myelotoxicity of xenobiotics such as drugs, mycotoxins, pesticides, polluants, ... It is ea~ " to obtain routine hematopoietic progenitors by dissection of femur and extraction of bone rllalTOW.

Human hematopoietic progenitors can de obtained from three sources :

Bone marrow sternal aspiration, Extraction from head femur of patients undergoing arthroplasty surgery, - Umbilical cord bloodThe first source need inform consent from patient. It is a painfull taking, which must be realized in order to perform a rayelogram. The second source need also inform consent from patient. Ethic aspects are less complex. But, mononuclear cell number obtained after extraction from head femur, is often small. Elsewhere, these cells have a low proliferative capacitybecause they are obtained from aging persons. Umbilical cord blood is used as a source of transplantable progenitors cells. Its utilization in toxicolo~" does not present major ethic questiofi. It is possible to perform toxicologic studies with human cells which are obtained easly. These cells have an important proliferative activity. However, they present some differences with hematopoietic progenitors from adult. Advantages and disad~antages of these difference source of human hematopoietic progenitors are compared. -

-

T h e n e u t r a l r e d test is b a s e d o n t h e i n c o r p o r a t i o n a n d s t o r a g e o f this v i t a l d y e into living cells lysosoms. A linear relationship can be shown between the number of viable cells and the concentration of the dye that can be extracted by an alcohol/acetic acid solution, We u s e d this t e c h n i q u e to c o m p a r e the potential cytotoxicity of four surfactants belonging to different classes, which are two anionic surfactants (Texapon K1298" - Sodium Lauryl Sulfate Henkel; Texapon N40* Sodium Ether Sulfate - Henkel), one cationic surfactant (Benzethonium chloride* - Siber Hegner) a n d o n e n o n - i o n i c s u r f a c t a n t ( T w e e n 60* Monostearate of polyoxyethyl sorbitane ICI Speciality Chemicals). According to the letal 50 concentration, LC50 (gg.ml-1), the tested surfactants can be classified in the following o r d e r o f i n c r e a s i n g c y t o t o x f c i t y : T e x a p o n N40* (LCs0 = 2 9 0 ~tg.m1-1) < T w e e n 60* '(LC50 = 2 1 0 gg.m1-1) < T e x a p o n K 1 2 9 8 " (LCs0 = 62 gg.m1-1) < B e n z e t h o n i u m c h l o r i d e (LC50 = 8 rg.ml-1).

206

23

24

Evaluation and comparison of in vitro toxicity induced by 4 trichothecene mycotoxins on human and rat hematopoietic progenitors.

THE USE OF HUNL~,N BFU-E/CFU-E CULTURES IN CELLULAR TOXICOLOGY. ABOUT TWO EXAMPLES APPLIED TO ALLMENTARY AND ENVIRONMENTAL TOXICOLOGY.

Lautraite S., Rio B., Parent-Massin D., Hoellinger H. Laboratoire de Microbiologie et Biochimie, Ecole Suprrieure de Microbiologie et Srcurit6 Alimentaire de Brest, Technoprle Brest-Iroise, 29280 Brest-Plouzanr.

RIO B., PARENT-MASSLN D., LAUT1LSdTE S., HOELLINGER H. Laboratoire de Microbiologie et Biochimie, Ecole Sup~rieure de Microbiologie et S6curit~ Alimentaire de Brest, Technop61e Brest-Iroise, 29280 Brest-Plouzan&

T-2, HT-2, DAS and DON are trichothecene mycotoxins produced by different species of Fusarium which essentialy contaminate cereals and their derivative products. The aim of this work is to evaluate and to compare the myelotoxicity of T-2, HT-2, DON and DAS with cultures of human and rat hematopoietic progenitors. Granulo-monocytic progenitors (CFU-GM) from human ombilical cord blood or rat bone marrow have been cultured in the presence of one of these 4 mycotoxins. The comparison of the results obtained in accordance with time exposure, shows different dose-effects depending on :

- the origin of the cells. - the structure of the molecules. These observations suggest that the mecanisms of cytotoxicity are different depending on the structure of the trichothecene molecules and the origin of the cells.

BFU-E/CFU-E (Burst Forming UnitErythroid/Colony Forming Unit-Erythroid) are hematopoietic progenitors. Under growth factors influence, those precursors proliferate and differentiate to form eD-throblasts colonies. BFU-E/CFU-E from umbilical cord blood, .cultured in semi-solid medium in the presence of erythropoietin, are exposed to xenobiotics. The evaluation criteria of toxicity are cellular proliferation and differentiation, cellular morphology and hemoglobin synthesis. From these data, cytotoxic, non-toxic doses and CIs0 are determined. Spectrophotometric analysis allows to evaluate the ability of cells to synthetize porphyric compounds and hemoglobin. The levels of metabolic intermediates accumulated in the cells exposed to toxics were determined by HPLC. Two examples of the use &this model are proposed: Effect of an herbicide inhibitor of photosynthesis on erythropoiesis and hemoglobin synthesis. Study of the effect of a trichothecene mycotoxin, on cellular proliferation and differentiation of BFU-E/CFU-E.

207

25

26

In vitro evaluation of direct hemato-toxicity

Biological activity of umbilical-cordblood derived eosinophils maturing in

from 9 herbicides on human progenitors. B. SawickiL, JD Dewitte 2, C. Rich6 t and MC Lrglise 3. 1 Dep. Pharmacology, 2: Unit of Professional Medicine,3 :lab. Hematology, University Hospital 29609 Brest Cedex France

Herbicides, widely used in Britanny, have properties of remanence and accumulate in water, leading to their survey: f.e.: Atrazine: 480 tons/yr, tolerated threshold in water <2gg/ml. Our aim was the study of direct interactions of such molecules on human hemopoietic progenitor-cells in vitro. We compared the effects of herbicides from two origins: pure molecules and commercial specialties for Dinoterb (Herbogil| Alachlor (Lasso| Linuron (Afalon| Atrazine (Atraphyt| pure molecules only for 2,4D, 2,4MCPA, Mecoprop, Ioxynil, and; specialty only for Simazine (Simaphyt| Cell targets were HL60 cell-line (MTT in microplates), normal CFU-GM and granulo-monocytic expansion in liquid medium (IL-3 and GM-CSF stimulated) obtained from progenitors derived from umbilical cord blood. Inhibitory concentrations were screened on HL60 after 24h exposure. Each product concentration was tested in triplicate at least in 3 experiments. No inhibitory activity was observed with pure molecules, while some specialties were significantly more toxic: IC5os Herbogil@: 3 10-4 M and Lasso| 0,23 10-4M. Molecules active on cell-line were further explored on day 14 CFU-GM Mear~+SEM % control growth (10-SM)

2,4D

2,4MCPA Atrazine Simazine

161+15 101+4

104+6

50+ 7

2,4D confirmed a stimulatory activity on liquid expansions, without modification of differential counts. IC observed were far above the tolerated thresholds for Atrazine and Simazine. Adjuvents might play a role for Dinoterb and Alachlor. 2,4D stimulated human hematopoietic cell proliferation as in plant cells, suggesting risks of genotoxicity.

Grants ANCO1-91 from INSERM-CRAM de Bretagne

vitro

D. M. Z a r d i n i , A. Gallois, J.-L. Bueb, P. Heuschling, E. J. Tschirhart. Neuroimmunologie & Inflammatior~,,Centre de Recherche Public-Santr, 120 Route d'Arlon. L- 1150 Luxembourg. Recent progress in the pathobiology of allergic reactions has strongly suggested that eosinophils play crucial roles in the pathogenesis of allergic diseases, especially in bronchial asthma. Various bioactive substances such as granular cationic proteins and active oxygen species (02 ~) released by activated eosinophils, have been assumed to be involved in the tissue injury at the site of allergic reactions. In spite of their crucial role, the study of eosinophils has been hindered by the difficulty to obtain large numbers of these cells for analysis. In the presence of interleukin-3 and -5 (10 ng/ml each) eosinophil precursors from human umbilical cord blood mononuclear cells were regularly differentiated into mature eosinophil-like cells expressing normal morphology and cyanideresistant peroxidase. Using the fluorescent oxidative burst indicator dihydrorhodamine- 123, we have assessed O2" production in these in vi}ro differentiated cells after chemotactic peptide formyl-Methionyl-Phenyl-Alanine(fMLP) stimulation. We report here that fMLP (1 n M - 3 gM) induced a concentration-dependent production of 0 2 ~ with maximum effect at 1 ttM, which was correlated with ml increase in [CaZ+]i as measured with Fura-2. These results suggest that cord-derived eosinophils show a phenotype which, for the studied parameters, is comparable to that described for human peripheral blood eosinophils.

208

27

28

EVALUATION OF T H E IN VITRO E F F E C T O F D I R I T H R O M Y C I N AND ITS A C T I V E METABOLITE ON THE OXIDATIVE M E T A B O L I S M OF HUMAN N E U T R O P H I L Moutard I., Gressier B., Brunet CI., Luyckx M., Dine T, Cazin M, Cazin J.C. With technical assistance of Battez-Lebegue S. Laboratoire de Pharmacologie, Pharmacocin~tique et Pharmacie Clinique. Facult6 des Sciences Pharmaceutiques et Biologiques, 3 rue du Professeur Laguesse, B.P. 83, 59006 Lille Codex, France.

INTRACELLULAR HSP 70 DETECTION USING FLOW CYTOMETRIC METHOD F. Batteux*, P.Jaffray**, N.Conan*, C.Mathiot* *Laboratoire d'h~matologie, Institut Curie, Paris **Laboratoire de biochimie A, H6pital Cochin, 27 rue du Fbg St. Jacques, 75014 Paris, France. A nearly universal cellular response to a variety of environmental stresses or unfavorable conditions is the expression of families of related and highly conserved proteins called heat shock proteins (HSP).Among these proteins, it is thought that HSP 70 play a role in protecting the stressed cells towards a wide variety of factors including temperatures, toxic chemicals, oxidative stress etc. HSP measurements are often carried out using SDSPAGE analysis and/or immunoblotting.These methods are inappropriate to study cellular HSP biosynthesis of a mixed cells population as, for instance, human mononuclear cells.We are herein describing an intracellular HSP 70 detection method by flow cytometry (FACscan-B. D.) using a new fixing agent (orthopermeafixortho diagnostic). Human plasma mononuclear cells obtained using Ficoll preparation, are treated using the orthopermeafix solution. The cells are characterized with anti-CD3 and anti-CD14 antibodies. Intracellular HSP 70 measurements are carried out on treated cells as follows : cells at + 37~ 1), cells treatment at 43~ for 1.30 h (case2), cells treatment at 43~ for 1.30 h min and 3.30 h at 37~ 3).The results are setout below :

Polymorphonuclear neutrophils (PMN) are able to generate reactive oxygen species and so represent an important defence mechanism against bacteria infectious. For the purpose, we studied the possible interaction of a macrolide, dirithromycin and of its reae~dve metabolite, erythromycylamine, with the PMN'~oxidative metabolism represented by superoxide (02-) generation, the first species produced by PMN during inflammatory disorders. PMNwere isolated by a gradient density technique and then stimulated by a chemoattractant agent, FMLP. Potential cytotoxicity by measuring the release of the cytosolic enzyme LDH showed that the macrolide and its metabolite had no effect on cell viability for the studied concentrations. Our study exhibited two effects: the low concentrations of macrolide (near plasma and tissue levels) strongly increased the FMLP-induced response with dirithromycin (+22% for 0,1mg/1; +35% for lmg/l; +45% for 10mg/l) and the FMLP-induced response with erythromycylamine (+14% for 0,1mg/1; +24% for lmg/l; +35% for 10mg/l). Whereas, the macrolide strongly depressed the PMN oxidative response only for the highest concentrations, with inhibitory concentration 50% (IC50) of 400mg/l for dirithromycin and 600mg/l for its metabolite. These concentrations (> 100mg/l) are not thinkable in therapeutic. In summary, in antibacterial defence, dirithromycin and its reactive metabolite may increase the PMN bactericidal activity by involving in oxidative metabolism of these cells without generate any cytotoxicity.

q"

~

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"

" z o o ' ~ l d O 5 0 0 s o 0 "fo'oo FSC-H'tFSC-Height-->

"JI~P 7p ~e I

2 areeo: =sel

-1

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II 1"

[,li, 2

70 P=le2 tl" .

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t Bee: isdy~: c=ird 2 ~:=,Jel 3 Beck:,=se 2

This fixing agent which allows in a single step a good fixation, an excellent conservation of the morphology and the structure of the mixed cells suspension studied enables to obtain : i) an accurate scan analysis of the different cell populations ii) a strong reactivity of the intracellular HSP 70

209

29 Cytotoxicity of intrathyroidal or peripheral blood lymphocytes in monolayer or in follicle coeultures with autologous thyrocytes in Graves' disease. J. Gibassier, C.Lucas, M.L. Raoul, B. Le Goff, B. Genetet and C. Massart. Groupe de Recherches enImmunologie (Gurifa), UER M~dicales, rue L. Bernard, 35043 Rennes, France. In order to investigate the cellular interactions among thyrocytes and lymphocytes in thyroid glands from patients with Graves' disease, we studied morphology and metabolism o f thyrocytes cocultured with autologous intrathyroidal (ITL) or peripheral blood (PBL) lymphocytes. Thyroid glands were obtained from 9 patients with Graves'disease. Thyrocytes were prepared by dispase digestion. ITL or PBL were isolated on Ficoll-gradient. Cells were cocultured in F-12 medium supplemented with 10% fetal calf serum, penicillin (100U/ml) and streptomycin (50 jag/ml). After 5 days in culture,glutathion, lacticodehydrogenase,thyroglobulin , triiodothyronine measurements and neutral red uptake were performed. Interferon (IFN) gamma and tumor necrosis factor (TNF) alpha were also measured in the cell supematants by Elisa method. After 5 days in culture, thyrocytes cultured in monolayers with autologous PBL retracted and formed narrow strands. On the contrary, ITL induced no change in the cell morphology. All the metabolic tests significantly decreased when thyrocytes were cocultured in monolayer with PBL whereas they remained unchanged in the thyrocytes-ITL cocultures. When the thyrocytes were cultured in follicles, the metabolisms were dramatically or slightly decreased with PBL or ITL, respectively.On the other hand, IFN gamma and TNF alpha were produced by the lymphocytes cultured alone or in coculture. These results show the cytotoxicity of lymphocytes cocultured with autologous Graves' thyrocytes. The thyroid follicles reconstituted into collagen gel are a good and sensitive model to assess ITL toxicity.This cytotoxicity may be induced , at least in part, by the TNF alpha secreted by the lymphodytes.

30 INTERACTIONS BETWEEN ENDOTHELIAL CELLS AND T H E M O N O C Y T I C T H P - 1 CELLS.

M-E. Roux, D. Lecoq A-M Dosne. U143 INSERM, Hrpital de Bicfitre, Le Kremlin Bicfitre Cedex, 95275 Cedex, France. Systemic endothelial cell activation may occur in response to circulating bacterial products and also cytokines derived from infected monocytes. In order to analyze a monocyte dependent activation we have used a coculture of endothelial cells derived from the umbilical vein (HUVEC) with the monocytic THP-1 cells. The THP-1 cells were prestimulated or not with LPS for 30 min, washed and added to HUVEC for 3H and 24 H. After a 3H coincubation 30% of the prestimulated THP-1 cells became adherent to HUVEC as compared to 8% for the untreated cells. This was reversible within 24H. Pretreatment of the THP- 1 cells led to the expression of adhesion molecules as estimated by flow cytometry: 64% and 67% of HUVEC became positive for E selectin and ICAM-1 respectively after addition of prestimulated THP-1 cells as compared to the values of 17% and 32% obtained using untreated monocytic cells. After 24H coculture, membrane E-selectin expression reversed to basal value whereas ICAM-1 remained overexpressed on 72% of HUVEC. In the supernatants of prestimulated THP-1 cocultures a soluble form of E selectin was detected and the level of plasminogen activator inhibitor- 1 (PAL 1) was greatly increased. These results show that prestimulation of THP-1 cells induces a transitory adherence on HUVEC associated with the expression of adhesion molecules and production of soluble markers of endothelial activation. TNF alpha, the level of which was markedly increased after 3H, may be one of the possible mediator involved in this process. This model might be useful to analyze and modulate the different steps of the interactions between monocytic and endothelial cells.

210

31 vitro use of mesothelial cells

In

32 human

peritoneal

Fougeray S.*, Slingeneyer A.**, Bastide J.M.*, Mion C.**, Bastide M.* Laboratory of Immunology, Faculty of Pharmacy* and Division of Nephrology, University Hospital**, MontpeUier University, Montpellier, France. The aim of our study is to characterize human peritoneal mesothelial cells (ultmstructure, cytokine secretion) of end stage renal disease (ESRD) and non ESRD patients, to compare the two types of culture and finally to test different dialysis solutions on cell growth. Mesothelial cells are isolated from omentum tissues of ESRD and non ESRD patients using a collagenase solution. Ultrastructural study by transmission electron microscopy revealed the presence of microvilli on cell surface, lamellar bodies, many mitochondria and pinocytotic vesicles in the cytoplasm. IL-6 and IL-8 spontaneous secretion and after stimulation by IL-1B by ESRD and non ESRD mesothelial cells have been detected by ELISA test. Ultrastructural analysis showed no difference between ESRD and non ESRD mesothelial cells. A significant difference has been demonstrated for IL-6 secretion, the ratio stimuled ceUs/unstimulatedcells being 50 + 13 and 26 + 18 for ESRD and non ESRD cultures, respectively (P< 0.05, Mann Whitney test). The effects of two dialysis fluids, diluted 1/5 in culture medium, were evaluated using 3H-thymidine incorporation on 7 ESRD cultures and 7 non ESRD cultures. Compared to a traditional heat sterilized lactate solution (pH 5.5), a solution buffered with bicarbonate, stabilized with glycylglycine, (pH 7.35) and sterilized by filtration, promoted mesothelial cell growth. A fn'st evaluation of peritoneal dialysis solution biocompatibility can be realised in vitro on mesothelial cells from ESRD and non ESRD patients.

Mixed culture of pericytes and endothelial cells from human placental microvessels. Kacemi A. & Challier J.C., Physiopathologie du D6veloppement, Universit6 P. & M. Curie, Paris, France.

The development of endothelial cell, pericytes and myocytes cultures has allowed to explore the role of perivascular cells in angiogenesis, vasomotricity and coagulation. In this objective, we developed methods to isolate microvessels from human term placentas and to perform mixed cultures of endothelial cells and pericytes. Placental villi were passed through 640, 250~tm sieves and those retained by 75~tm sieve were collected and digested by collagenasedispase. Microvessels were separated by a Percoll gradient and digested again with the same enzyme mix. After a new Percoll separation, microvessels were explanted. Eight days later, the cell colonies were harvested by trypsin-EDTA and seeded to obtain primocultures. Rapidly, the pericytes with irregular border and microfilaments reacting to anti-t~-actin staining predominated. Some endothelial cells showing positive von Willebrand factor reaction remained in the culture. The cells built nodules formed by retraction of pericytes. Some neighbouring endothelial cells incorporated into the nodule. The nodules reacted positively either to a-actin or yon Willebrand factor staining. The formation of nodule points out to an attempt of vessels rebuilding or cell ageing in these primocultures.

211

33 EFFECT OF OCHRATOXIN A ON ARACHIDONIC ACID PATHWAY : USE OF EPITHELIAL BRONCHIAL CELLS Pinelli E.*, Grosse Y.*, Meiller B.*, Pipy B.w and PfohlLeszkowicz A.* *ENSAT, 145 avenue de Muret 31076 Toulouse, France ; w INSERM, CHU Rangueil, Toulouse In a previous study, we have shown that one of oehratoxin A (OTA) biotransformation pathway involved in DNAadducts formation would be correlated to the prostaglandine-H-synthase (PGHS) activity. In this study, we have analyzed (i)' the effect of OTA on the arachidonic acid biotransformation pathway using pulmonary epithelial cells in ~ulture, in presence or in absence of the transforming growth factor (TGF~), which is a phospholipase A2 and PGHS inductor (ii) the OTA-DNAadducts production in presence or not of two inhibitors (indomethacin 1ND or Nordihydroguarefic acid NDGA) (rio the OTA metabolites in the cell culture medium, by HPLC. Our results shown that OTA alone had no effect on the lipooxygenases (LPOX) and PGHS activity and induced only few adduets. When cells were stimulated with TGF[3, OTA potentiates the arachidonic acid pathway. In particular, the leucotrienes production is significantly increased. Moreover, DNA-adduct formation was 4 fold increased in this condition (2 adducts per 109 nucleotides compared to 0:5). Indomethacin which inhibits PGHS activity, increases DNA adducts formation (14 adducts per 109 nucleotides). NDGA which inhibits LPOX also increases DNA-adducts level (21 adducts per 10 9 nucleotides). Prestimulation of cells by TGF[5 potentiated these increases. DNA-adduct levels reached 35 and 51 adducts per I0 ~ nucleotides respectively for IND and NDGA. Two unknown OTA metabolites are found in the culture media after induction of TGF~. One of these (called I, rf 0.43) seemed to be correlated with DNA-adduct formation, the other (called A, rf 0.36) in the same time decreased. Altogether, these results indicated that OTA could be biotransformed by peroxidases functions of PGHS and LPOX.

34 In vitro local tolerability of human nasal mucosa: histopathoiogical and scanning electron microscopic evaluation of nasal powder forms containing Sandostatin | A. de Fraissinette ~, M. Kolopp 2, H. Felix3, I. Schiller 4, G. Fricker ~, C. Gammert ~, A. Pospischil4,j. Vonderscher ~, F. Richter~.~TRDDDS,2DS-Toxicology,Sandoz Pharma,CH-4002 Basel; 3Morphology Laboratory, ENT Dept. ZQrich; 4Institute for Veterinary Pathology, ZQrich; SENT Dept. Luzern. Switzerland. An in vitro human nasal model was developed as a tool to study the local tolerability of nasal powder forms using excised nasal mucosa in a diffusion chamber. The suitability of this model was tested using Sandostatin | (SMS) (0.05mg) as a reference drug enhanced by Avicel (microcristalline cellulose) or Lactose. The vehicle nasal spray was taken as a harmless control and 1% Chenodeoxycholate (CDC) as a harmful control in terms of local tolerability. SMS absorption was measured in the supernatant of the receiving chamber by radio-immunoassay. Its transepithelial pathway was detected by immuno-peroxidase staining on cross sections. The local tolerability for all tested forms was assessed by histopathological examination and scanning electron microscopy. The apparent permeation coefficient allowed us to range the drug absorption of the tested forms as following: Avicel>Spray>Lactose>1%CDC. No damages o f the citiated or goblet cells could be observed either with Avicel, Lactose or nasal spray vehicle in presence or absence of SMS. 1%CDC with or without drug showed a very strong and immediate destruction of the nasal epithelium. The validation of this in vitro model using human nasal mucosa will be further discussed as a tool for assessing the local tolerability of nasally applied forms.

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LEUKOTRIENE RECEPTORS IN HUMAN LUNG Isabelle Gorenne, Jean Pierre Gascard, Xavier Norel, Carlos Labat and Charles Brink CNRS URA 1159, Hrpital Marie Lannelongue, 133 av de la Rrsistance, 92350 Le Plessis Robinson, France Leukotrienes activate specific membrane receptors. Two types of receptors exist, one stimulated by leukotriene B4 (responsible for the chemotactic effects on leukocyte8) and another receptor for the.cysteinyl-leukotrienes C4, D 4 and E4. This latter receptor is present in the human lung (cys-LT-1 receptor) and stimulation by the cysteinyl-leuk~trienes produces a bronchoconstriction. 'gpecific cys-LT-1 antagonists have been developped which are potent inhibitors of the airway contraction induced by antigen in vitro. The bronchoconstriction produced by antigen in patients with atopy is also blocked by these antagonists suggesting that these compounds may have potential therapeutic value in the treatment of asthma. Cysteinyl-leukotrienes are also vasoconstrictor agents in the pulmonary vascular bed. Recently, the presence of a second receptor (cys-LT-2) in human pulmonary veins has been reported and there is no known antagonist available. The cysteinyl-leukotienes also produce an endothelium-dependent relaxation in human pulmonary vascular preparations, an effect which is associated with the liberation of nitric oxide. The receptors present on the endothelium which are associated with NO release are resistent to the cys-LT-1 antagonists. The balance between these two pathways in the regulation of pulmonary vascular tone by the cysteinyl-leukotrienes may be altered to favor the vasoconstrictor component during the evolution of certain pathologies such as pulmonary hypertension.

QUALIFICATION OF A HUMAN CELLULAR MODEL ADAPTED TO THE PREDICTIVITY OF A MOLECULE ACTIVITY FRANCHI J., COUTADEUR MC., BIESSEJP., REDZINIAK G. Parfums Christian DIOR Laboratories 45800 Saint Jean de Braye - FRANCE In fields like pharmacology or in vitro toxicology, the cellular models obtained from human tissue are more and more frequently used. The research of the efficacy of an active ingredient comes up against two difficulties : - the choice of the screening model (cellular type, reference substances, measured criteria...) - the inter-individuals variability which is also observed at the normal human cells culture level. Depending of the study objective, the biologist brings the answers to the first point. The second difficulty raises an important problem : to limit two major risks of erroneous conclusions : - assert that a molecule is not active when in reality it is not (c~ risk) - assert that a molecule is active when in reality it has an activity (13 risk) To reduce this two risks a maximum, it is possible to multiply the tests (it's expensive !) or to select used human cells. In fact it is important to well qualify a cellular model with reference molecules, to avoid to conclude to the inefficacy of an effector, when it is the chosen cell which does not answer to the studied parameter (Ig risk). The qualification will also allow to reduce the c~ risk improving the reliability of the conclusion.

9

To illustrate this remark, we have evaluated the growing capacity of 80 cell lines (normal human fibroblasts from different donors) under the influence of 5 reference molecules from different chemical families (proteins, lipids, glycolipids and vitamin). The measured parameter is the total proteins rate by the Bradford's method. The multidimensional statistical analysis in major components (ACP) and the hierarchical classification (CAH) allow to take into consideration the data chart crossing 80 cell lines and 7 criteria. It is then possible to perceive the sensible cells versus the ones wich are insensible to the agents influencing their growth, but m featuring their specific sensibility to certain effectors. This type of analysis allows to improve the predicfivity of any effect of a molecule at cellular level ; error risks are minimized at the beginning of the study by cells selection.

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37 STRESS MARKER PROTEIN TECHNOLOGY APPLIED TO THE QUANTITATIVE MEASURE OF PRODUCT TOXICITY Peter BROMLEY 1 , Claude SOUVIGNET 2 1 TWS Corporate Associates S.A. B&timent Athena, International Business Park. F-74166 ARCHAMPS (33) 50 31 50 80 2 Laboratoire HEMERIS ZI de I'Argenti6re, 6 rue de Chamechaude. F -38360 SASSENAGE. (33) 76 53 15 05 In response to toxic assaults to ce~ls in culture and to organisms, the production of a discrete series of stress proteins increases dramatically. Stress proteins remain intracellular and their quantitation is technically complex. Th~ "I'WS "Stress Promoter Technology" places marker genes under the expression control of stress promoters, and such cells or organisms will produce secretable marker proteins in a quantitative fashion, depending on the degree of toxicity of the test compound. In order to optimize the predictability of this toxicity screen to the human condition, a human cell line carrying a human hsp-70 stress promoter and a conveniant marker gene have been employed. HeLa cells producing Chloramphenicol acetyl transferase (CAT) that can be measured either by an ELISA immunoassay or by a radioactive label transfer assay. Product toxicity was assessed in the following way: the product to be tested is added to growth medium in the HeLa-CAT at selected dilutions. Cells are incubated at 37~ for 5 hours after which the medium is removed, Cells washed twice and fresh medium applied. Cells are incubated for a further 18 hours after which cells are collected, lysed and assayed for CAT content. The level of CAT produced by each ceil sample provides a precise and quantitative measure of the toxicity of test sample used. The "Stress Promoter Technology" has been applied to the quantitative toxicity assessment of 31 metal salts, a number of arsenate derivatives and valency variants and some organic molecules. This cell system provides a fully human derived cell line and cellular response, and further provides a global measure of product toxicity. Tests using HeLa-CAT cells are designed for early toxicity screening of compounds. Further cell-reporter gene constructs are underway that will allow species-specific and organ-specific toxicity but also screening programs for HSP inducing drugs.

Expression of stress proteins in cultured cells as an index of cell aggression. F. Delmas, V. Trocheris, N. Benali, E. Conilh & J.C. Murat (GREPEA, UFR/SVT, Universit~ Paul Sabatier, Toulouse, France) The study of cell reactions to external aggression relies upon indices of damages at the molecular level. For instance, metallothioneins are specifically overexpressed in the case of heavy metals toxicity. Hence, we propose that expression of stress proteins in cultured cells may represent a convenient biodetector of cellular insults caused by a wide variety of chimical or physical agents. In the present study, HT29 and HEPG2 human cell lines were used. Expression of stress proteins was measured by autoradiography and densitometric analysis of 35S-met labelled and electrophoretically separated proteins. Effects of heat shock, ethanol, propanol, nickel and cadmium were tested in our models under sublethal experimental conditions. Heat shock (45~ 30 min.) was found to induce a strong overexpression of HSP70 and 90, without affecting cell growth. 8% ethanol, given for 15 min, induces both an overexpression of HSP and a decrease, of growth rate. 2.5% propanol-1 for 15 min caused HSP68 expression to increase with no effect on growth rate. Higher concentrations were found to be toxic. Exposure to cadmium acetate was followed by decrease in growth rate and overexpression of HSP68/70 and HSP90, at 10~tM for HEPG2 cells and 50pM for HT29 cells. Nickel sulfate appears less aggressive: either 6 hrs at 500pM or 24 hrs at 2501.tM are needed to decrease growth rate and to increase HSP68 formation. As a rule, overproduction of stress proteins appears to be more sensitive than changes in growth rate as an index of cell aggression. Observing mRNA productien could render the test even more sensitive. Therefore, we think that a highly sensitive biosensor to environmental toxicity or nuisance may be developed from our experimental model.

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39 TOXICOLOGICAL ASSESSMENT OF PACKAGED WATER M.P. SAUVANT. D. PEPIN - Lab. Hydrologie-Hygi~ne, UFR Pharmacie, BP38, 63001 CLERMONT-FERRAND

In Europe, "inertness" of the packaging and "purity" of the foodstuffs, water included, in contact with it, are required. The reglementation lays down approved lists of substances and defines the analytical limits for specific migration of undesirable substances. Analytical determinations allow only specific substances to be inquired. On the other hand, toxicological b i o - a s s a y s allow the overall evaluation of product and the health risks to be specified. For toxicological assessment, the first difficulty is the choice of biological system. For some years, cell lines included L-929 fibroblasts - have been selected for screening and cytotoxicity testing of biomaterials, which are partly composed of polymers, similar to those used for water packaging. So, these cells have been firstly selected as model and Neutral Red Incorporation, MTT reduction and RNA synthesis rate as assays, for the toxicological evaluation of a natural water packaged in 1.5 litre polychloride vinyl (PVC), polyethylene terephthalate (PET) and glass-bottles and which h a d a storage-time r a n k i n g from 0 to 36 months. Furthermore, the same samples of packaged water have been concurrently tested with a second original model, s u c h as the cfliated protozoa T e t r a h y m e n a pyriformis GL a n d a n a l y s e d t o w a r d s the d e t e c t i o n of undesirable organic and inorganic substances. This methodology allowed firstly the quality of bottled water to be evaluated, and secondly the efficiency of b o t h cellular models to be performed. Whatever the lenght of storage and the packaging may be, no undesirable chemical w a s d e t e c t e d on b o t t l e d w a t e r . S o m e abnormalities have been detected by a s s a y s p e r f o r m e d w i t h b o t h L - 9 2 9 cells a n d Tetrahymena pyriformis GL on some samples of water bottled for more 18 months. However, these cytotoxic effects could not be significantly correlated to the lenght of storage or the packaging (PVC-, PET- or glass-bottles). The in vitro a s s a y s , p e r f o r m e d for the toxicological evaluation of p a c k a g e d water, revealed a potential risk to health, b u t it would not be possible to extrapolate these in vitro results for the consumer. They m u s t only be considered as "alarm" device in food toxicology.

40 IN VITRO B I O A S S A Y S AND ANALYTICAL DETERMINATIONS FOR TOXICITY TESTING OF WATER PLASTIC BOTTLES M.P. SAUVANT. D. PEPIN - Lab. Hydrologie-Hygi~ne, UFR Pharmacie, BP38, 63001 CLERMONT-FERRAND For some years, various plastic materials have been proposed for water packaging. In France, polyvinyl chloride (PVC) a n d polyethylene terephthalate (PET) are the most frequently used materials. As for all food packaging, the PVC- a n d PET-bottles m u s t agree to the principles of "inertness" of the packaging and of "purity" of the foodstuffs, i.e. water, in contact with it, according to the European Community Council regulation. To date, control of plastic materials is based on the absence of migration from the plastic materials into foodstuffs, evaluated by analytical determinations p e r f o r m e d on food s i m u l a n t s . M o r e o v e r , toxicological investigations are required. This study proposed a strategy a s s e s s m e n t of p a c k a g e d water quality and c o m p a r e d the chemical analytical technics arld some in vitro bio-assays (Neutral Red Incorporation assay, MTI" reduction assay, Coomassie Blue a s s a y and RNA synthesiS, determination by 3H-uridine incorporation), performed on L-929 fibroblasts, as well for the study of PVC- and PET-materials at the main stages of the manufactoring process of bottles, as for the study of packaged water. The d e t e r m i n a t i o n s have been c o n d u c t e d simultaneously on samples of a food simulant (deionized water) and of a natural water in contact with PVC-compound, PET-resin, PVCand PET-intermediate forms of transformation and also, PVC- and PET-finished bottles. Although some analytical a n d toxicological abnormalities have been detected on the food simulant after contact with the PVC-compound, the PET-resin and the intermediate products of bottles processing, no significant abnormality h a s b e e n detected on the n a t u r a l w a t e r p a c k a g e d in PVC- and PET-bottles in real conditions of a 24-month storage period. The a n a l y t i c a l a n d in vitro toxicological a p p r o a c h e s complemented one a n o t h e r a n d allowed the simultaneous control of both the packaged water and the plastic material to be performed.

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41 EYE IRRITANCY TESTING SET UP OF A BA'I-I-ERY OF ALTERNATIVE METHODS LEADING TO THE BEST CONCORDANCE in vivo/in vitro I. Fabre, J. Vincent, S. Camps, J.L. Peiffer, J. Maurin & J. Giroux Agencedu M6dicament,Unitade Pharmaco-Toxicologie 14rue Ecolede Pharmacie,F- 34000Montpellier Five alternative methods were assessed to evaluate the ocular irritancy of surfactants (21 raw materials, 19 formulations). The aim of our study was to select an alternative battery appropriate to predict the eye irritancy of surfactant based formulations. These assays were : the hen's egg chorioallantoic membrane (HET-CAM) test, the Neutral Red Uptake (NRU / SIRC cells), the Neutral Red Release (NRR / SIRC cells), the Agarose Overlay (AO / L929 cells) and the haemolysis test (HT). For each method, products were classified as Irritant or Non irritant. The cut off between these two categories were determined according to the literature and on the basis of in-house data. Cut off points for formulations were : in vivo Maximum Average Score > 30, HET - CAM score _>9, EC so NRU_>O,1% (w/v), ECso NRR _> 10% (w/v), AO _>30 (% of lysis area), EC so HT _>0,1% (w/v). The concordance level between in vivo and in vitro data was evaluated by the Kappa test. This analysis performed for each individual in vitro method gave the following results, according to the Landis and Koch classification (1) : HET-CAM : K = 0,45 (mild) NRU : K = 0,43 (mild) NRR : K = 0,70 (good) AO : K = 0,49 (mild) HT : K = 0,59 (mild) Same analysis performed for each combination of two and three methods allowed us to conclude that the sequence of the following assays : NRR/Haernolysis/NRU (Kappa: 1, with no false negative and positive response) represents a suitable battery for estimating the surfactant based formulations eye irritancy testing. 1- LandisJ.R.& KochG.G.(1977). Biometrics33,159-174.

42 Cytotoxicity evaluation of hematite (Fe203), Benzo(a)Pyren and pyren and analysis of particles by Laser Microprobe Mass Analyser (in-vitro) 9~ H I ~ 1 ,GOSSET p.1 ,MAREZT.1 ,HACHIMI A.2, MULLER J.F.2, HAGUENOERJ.M.1 1CERESTE, Institut de Medecine du Travail, 1-Place du Vei'dun 59 045 Lille Cedex 2LSMCL, Universit~ de Metz, Technopole 2000 1-bd Arago, F-57078 Metz Cedex

This work was supported by Grants from the European Communities of Steel and Coal (n ~ 7280/03/055) and Usinor Sacilor In this study we looked for a toxic effect of Iron oxide (Hematite ; Fe203), Benzo(a)Pyren (BaP) and pyren, alone or in combination. The lethal concentration 50% (LC50) was appreciated by colony-forming cell culture method. Cells were observed by electron microscopy and the valence of particles was analysed by LAMMA-500. We traced the survival curves as a function of doses. With F e 2 0 3 we observed a significant decrease (20%) at higher concentrations (0.5 mmol/l), (r=0.307 p<0.0005). Smaller quantities of BaP were highly toxic (r=0.76 ; p<0.0001). The association of BaP at the LC10 dose (0.05 ~tmol/I) with growing doses of F e 2 0 3 (0.0125 ; 0.025 ; 0.05 ; 0.1 ; 0.2 mmol/I), appeared to increase the toxic effect of BaP 3 to 4 times. Spectral analysis by LAMMA-500 in ionizing resonance revealed a peak of reduced Iron. 1

These results suggest that F e 2 0 3 alone is not very toxic but the association of this compound with BaP increases the toxicity of the latter. On the other hand, LAMMA-500 revealed a metabolization of Iron oxide into a reduced form. This reduced form seems to favour its binding with protein.

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44 Endothelins and Human Prostate Cell Lines

In-vitro cytotoxicity study of Magnetite (Fe304), Benzo(a)Pyren, Pyren alone or in combination and analysis by Laser Microprobe Mass Analyser

Aubin, P.*, Le Brun, G.*, Moldovan, F.*, Cussenot, O. ~ , Soliman, H.*, and Fiet, J.*. *Hormone Biochemistry Laboratory; ~176 Service, H6pital Saint-Louis, Paris, France.

SHIRALI p.1, B ~ I , MAREZT.t, MAUNIT B2, MULLER J.F.2, HAGUENOERJ.M1

Endothelins (ET) are the most powerful vasoactive peptides ever described. They are mainly secreted by endothelial tissue, but the synthesis of these peptides and the presence of receptors specific for them have been demonstrated in other tissues. ET-1 receptors (ET-A and ET-B) have recently been identified in vitro in normal human prostate tissue by means of autoradiography. However, the distribution of ET-1 receptors in prostate tissue has not been clearly described (there seem to be receptors in both stromal and epithelial tissues). To date, no study has demonstrated the presence of endothelins and their receptors in human prostate cells. In order to attempt this, we studied ET-1 and sought its receptors in several human prostate cell culture lines: PNT1 (normal human prostate epithelial ceils), PF1SV1 (normal human prostate fibroblasts transfected with the PMK16 plasmid), and in LNCAP, PC3, and DU 145 (cancerous human prostate cell lines). The presence of ET-1 was detected by indirect immunofluorescence (employing monoclonal antibodies prepared in our laboratory). ET-1 secretion was measured in the supernatants of the cell lines studied by radioimmunoassay after chron~tographic extraction. Prostatic ET-1 receptors were sought in membrane preparation binding studies (PC3, PNT1A, PNT2, and PF1SV1) as well as by studying binding in intact PC3 cells (competition and saturation studies), usin~ 125I-ET-1 with BQ123 (a selective ET-A receptor competitor) and IRL1038 (a selective ET-B receptor competitor). We also synthesized ET-1 (by solidphase peptide synthesis). Disulfide bridges were formed by oxidation in an alcaline medium, followed by hlgh-performance liquid chromatography (HPLC) purification. ET-1 was marked with 125I by the lactoperoxidase method. During indirect immunofluorescence studies, the anti-ET-1 monoclonal antibodies specifically bound in all the prostate cell lines and all ceils were found to secrete ET-1. The levels of ET-1 secreted were: DU145:27.3+1.1 fmol/ml; PC3:13.5+0.7 fmol/ml; LNCAP: 10.1+2.7 fmol/ml; and PNTIA: 19.0+1.0 fmol/ml. The membrane binding studies enabled us to demonstrate the existence of specific endothelin receptors. The endothelin receptors in the PC3 cells were identified and characterized as follows: Kd=2.32x10-10M, Bmax=4.21xl0-11M. The Hill coefficient of 0.98 indicated the presence of a single class of receptor. There were 5.5x103 sites per cell. ET-1 and BQ123 (which specifically competed with the A-subtype ET-1 receptor) displaced 1251-ET-1, whereas IRLI038, the specific inhibitor of the B-~ubtype ET-1 receptor, did not. In conclusion, we demonstrated a spontaneous ET secretion in cultured normal and cancerous human prostate and the presence of ET-1 receptors. These two properties render such cell lines intertesting as models for use in investigating endothelins and their role(s) in the prostate.

1CERESTE, Institut de Mddecine du Travail, 1-Place du Verdun 59 045 Lille Cedex 2LSMCL, Universite de Metz, Technopole 2000 1-bd Arago, F-57078 Metz Cedex This work was .supported by Grants from the European Communities of Steel and Coal (n ~ 7280/03/055) and Usinor Sacilor The pur~)ose of the study was to investigate the toxic effects'r of magnetite Fe304 (Fe2+, Fe3+), Benzo(a)Pyren (BaP) and pyren. Moreover, we looked for a possible synergic effect between the different compounds. The lethal concentration 50% (LC50) was determined by colony-forming cell culture methods. By LAMMA-500 (Laser Microprobe Mass Analyser) we determined the valence of particles in situ. There is a linear relation between Fe304 and cellsurvival rate (r=0.613 ; p<0.0001). Smaller quantities of BaP were highly toxic (r=0.763 ; p<0.0001) Pyren doesn't show a toxic effect (r=0.136 ; p>0.05). The association of BaP at the LCl0 dose (0.05 ~tmol/1) with growing doses of Fe304 (0.0125 ; 0.025 ; 0.05 ; 0.1 ; 0.2 mmol/1),appears to increase the toxic effect of BaP. With LAMMA-500, we observed the presence of a spectral peak of iron in treated cells. This study shows a synergistic effect between Fe304 and BaP. With LAMMA-500 we observed a metabolization of Iron oxide in L132 cells. With this technique it is no longer necessary to digest the biological matrix in an oxidizing medium in order to isolate the particles from the biological liquid. According to these results, it appears that the ionizing resonance technique could be extended to identify the metabolites in situ.

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Use of HaCaT cell line to study the toxicity Cytochromes P450 !Al/2.

Improvement of chondrocyte transfection by the calcium phosphate procedure.

N. Ledirac, C. Delescluse, G. de Sousa, M. Pralavorio, A. Cuany, J B Berg6, R. Rahmani

S.Viengchareun, S.Thenet-Gauci, N.Steimberg, M. Adolphe.

Equipe INSERM / INRA, 41 Bd du Cap, Antibes.

Laboratoire de Pharmacologie Cellulaire de l'Ecole Pratique des Hautes Etudes. 15 rue de l'Ecole de M6decine, 75006 Paris. France.

of pesticides and their potential to induce

We investigated the potential epidermotoxicity and P450 inducing effects of various insecticides (chlorinated hydrocarbons, organophosphates, carbamates and pyrethr0ids) on the HaCaT cell line. These cells were selected because they are human keratinocytes spontaneously immortalized in culture which maintain their full epidermal differentiation capacity. The toxicity was estimated by the MTT or the Neutral Red tests according to two protocols: either a 72 hours treatment, or 3 treatments of 24 hours. The two protocols gave similar IC50s suggesting that the toxicity did not come from metabolites. The chlorinated hydrocarbon insecticides were the most toxic. We also studied the induction of cytochromes P450 1Al/2 by measuring EROD activity, with 3Methylcholanthrene as the positive control. Our results were compared with those obtained on hepatocytes and HepG2. They show that HaCaT cells could represent a useful model to study the toxicity and the induction' of CYP1AI/2 by epidermotoxic xenobiotics.

Most of the genes encoding cartilage matrix proteins have now been characterized and the study of the mechanisms regulating their expression is fundamental for the comprehension of cartilage pathologies. Such studies depend on the possibility of introducing efficiently and in a reproducible manner these genes or their regulatory sequences into chondrocytes. However, relatively low transfection rates are usually obtained with this cell type, possibly due to the important extra-cellular matrix surrounding chondrocytes. With the aim to improve the transfection efficiency of chondrocytes, we treated these cells before and during transfection with hyaluronidase (E.C 3.2.1.35). This enzyme offers precipitates greater access to cell membrane since it removes pericellular proteoglycans. Transfection efficiencies were assessed by an in situ staining (X-gal staining) and by an enzymatic assay ([3-galactosidase assay) thanks to the expression of a lac Z reporter gene encoding [3-galactosidase. Results revealed that addition of 4 U/ml of hyaluronidase before and during transfection increases 2-4-fold the transfection efficiency of rabbit articular chondrocytes. Furthermore, it was possible to show that much better reproductibility was obtained using a "giant" precipitate for all dishes to be transfected, rather than using an individual precipitate for each dish as is customary. This latter improvement of the method is of course likely to be applicable to all cell models when studying regulation of DNA elements by chemicals, hormones or other environmental signals in transfection experiments.

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Behaviour of a fetal human articular chondrocyte cell line in monolayer and in tri-dimensional culture.

Immortalization of rabbit articular chondrocytes by the SV40 large T antigen under the control of the collagen II promoter and enhancer.

P. PENFORNIS, B. Benoit, S. Thenet-Gauci and M. Adolphe. Laboratory of Cellular Pharmacology. Ecole Pratique des Hautes Etudes, 15 rue de l'Ecole de Mtdecine, 75006 Paris, France. The development of an immortalized human articular cell line would be a valuable tool for toxicopharmacological studies. For this purpose we transfected femoral head chondrocytes obtained from a 13-week fetus with SV40 large T antigen. Two months later, proliferating clones emerged and have been expanded. After 4-months period, transfected cells entered in a crisis period during four months. Only two clones emerged from crisis and to date are proliferating with a constant growth rate. This behaviour seems to be close to the two step immortalization mechanism described for human fibroblasts. We did not observe any difference in p53 mRNA synthesis between pre and post-crisis cells. The collagen phenotype of clones is altered : synthesis of type 111 and type I collagen and no synthesis type II collagen. Since we showed that 3-D culture in alginate beads was able to red i f f e r e n t i a t e normal human articular chondrocytes de-differentiated by subculture, immortalized clones have been cultured in alginate beads during 17 and 31 days. Type II collagen synthesis has not been detected by northern blot analysis after this period. As cell agregates developed inside the alginate beads after extented period of culture, the next step of this study will be to evaluate the collagen phenotype in these cell agregates.

N. Steimberg, S. Viengchareun, S. Thenet-Gauci, P. Casanova and M. Adolphe. Laboratoire de Pharmacologic Cellulaire. 15 rue de l'Ecole de Mtdecine 75006 Paris. France A cell line of articular chondrocytes expressing in a stable manner the differentiated phenotype (which main marker is the type II collagen) would provide a very useful tool for the study of pharmacological effects of xenobiotics for therapeutic use, such as antirheumatic agents. In a first time, we showed, in transitory transfection experiments, that SV40 large T antigen did not induce directly the inhibition of the type II collagen gene. We constructed then a vector in which the SV40 large T expression is directed by the regulatory sequences (promoter and enhancer) of the rat type 11 collagen gene. Rabbit articular chondrocytes were transfected with this construct. Immortalized cells were obtained thanks to their capacity to escape from senescence. In parallel, cotransfections with this vector and with a plasmid carrying the Neo r gene, permitted the selection of G418 resistant clones. Whereas some clones died after several passages, others continued to proliferate and reached a regular growth (about passage 20). All these latter clones expressed the SV40 large T antigen of the expected size, as shown by western blot. Moreover, indirect immunofluorescence showed that 100% of these cells expressed SV40 large T antigen whereas 10 % to 25 % cells expressed the collagen II (instead of 50 to 70 % for primary cultures of chondrocytes). Thus, in stably transfected articular chondrocytes, the expression of SV40 Large T is not incompatible with the expression of type 1I collagen. These cell lines constitute an original model to study the regulation of growth and differentiation in immortalized chondrocytes.

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49 An immortalized human bronchial epithelial cell line: interest and limits for the study of ionic transports.

INDUCTION OF BLOOD-BRAIN BARRIER DIFFERENTIATION IN A RAT BRAIN DERIVED ENDOTHELIAL C E L L LINE

Dazy AC, Durin N, Legrand M, Houcine O, ~ Marano F

D. Lechardeur, B. Schwartz, D. Paulin* and D. Scherman

B,

Laboratoire de Cytophysiologie et Toxicologie Cellulaire, Universitd Paris VII, Paris, France. *Centre Hospitalier Intercommunal de Pneumologie 12F, Cr~teil, France.

We have examined the possiblity of using a human bronchial epithelial cell line (clone 16HBE14o-, Gruenert et al 1988, PNAS USA 85: 5951) for studying ion transport properties of human airway* epithelium. Cells from passage 20 to 40 were grown on porous membranes coated with collagen, in medium supplemented with 10% fetal bovine serum (FBS). After confluency was reached, FBS was suppressed and cells were cultured with an air-liquid interface for up to 5 weeks. They show no contact inhibition whether or not vit. A (10-TM) was added. The cell sheet was heterogenous, with monolayered areas of cubic cells presenting tight junctions, and: multilayered areas of small and large cells, necrotic cells, and ill defined apical junctions. Immunocytology showed that keratin filaments reacted with antibody MS/16B4 which recognizes K5 and K6 (hyperproliferating epithelia) but not. with antibody KB37 which recognizes basal cells nor with 1C7 which recognizes K13 (squamous metaplasia). The transepithelial potential difference of confluent cultures was very low (0.1 to 0.5 mV). The addition of norepinephrine (106M) to the basal medium induced a small increase in the apical secretion of 36C1", and a sinall and transient hyperpoladsation of the culture. However the effect was very variable and could not be repeated with a second addition of the hormone. This cell line does not appear to be well adapted for the study of transepithelial ionic fluxes.

UMR 133 CNRS/Rh6ne Poulenc Rorer, CRVA, 13 quai Jules Guesde, B.P. 14, 94403 Vitry/Seine. *Institut Pasteur, SCME, 25 rue du Dr. Roux, 75724 Paris. An immortalised brain capillary endothelial cell line displaying blood-brain characteristics may represent a useful tool to study the blood-brain barrier endothelial cell differentiation and for the in vitro prediction of brain drug penetration. To prevent the inherent troubles of ceils in primary culture, we established cerebral endothelial cell lines from rat cerebral capillaries after nuclear microinjection with a linear DNA insert in which the Simian Virus 40 large T antigen coding sequence is linked to a regulatory element from the human vimentin promoter. The CR3 cell line displayed endothelial morphological and biochemical characteristics for up to 30 passages. The CR3 ceils express endothelial cell markers such as synthesis of Von Willebrand (Factor VIIIrelated) factor, uptake of acetylated LDL and binding of Bandeiraea simplicifolia lectin. However, the CR3 cell line did not spontaneously express the specific blood-brain barrier markers glutamyl transpeptidase and mdr P-glycoprotein. However when the cells were treated with the differentiating agent all-trans retinoic acid, these blood-brain barrier markers were induced and functional. Retinoic acid treated CR3 cells may thus represent a useful tool for biological and pharmacological research related to the bloodbrain barrier.

220

51 Are DMSO-differentiated HL-60 cells an in vitro model of neutrophiis ? A. Gallois, J.-L. Bueb, E. J. Tschirhart. Neuroimmunologie & Inflammation, Centre de Recherche Public - Sant6. 120, route d'Arlon. L- 1150 LUXEMBOURG Neutrophils are major effector cells in inflammatory responses. DMSO-differentiated (~) HL-60 cells are used as a neutrophil model, but with some controversies. We aimed at characterizing these cells pharmacologically, studying their responses to f-Met-Leu-Phe (fMLP), as measured by intracellular calcium concentration ([Ca2+]i) variations, phosphoinositides (PI) turnover and superoxide ions (02") release, as well as their fMLP binding. HL-60 cell~ were grown in Iscove's with FCS/NCS (15~o/5%). Differentiation was induced by DMSO (1.3%) for 4 days. [Ca2+]i and 02" production were measured simultaneously by a fluorescent assay. For PI turnover, cells were incubated 48 hours with [3H]-myo-inositol, and IP3 eluted on Dowex resin. Binding studies (4~ were performed with [3H]-fMLP (1-60 nM) and non-specific binding was defined with 2 pM fMLP. Quiescent HL-60 cells did not respond to fMLP, despite fMLP binding. Differentiation induced a 5-fold increase in fMLP binding without modification of the affinity. In these ~ cells, 1 pM fMLP induced a transient increase in the cellular IP3 content (142+4 % of basal level, at 30 s) as well as a 500 nM increase in [Ca2+]i at 40-50 s, followed immediately by O2" production (10 pmole/min/106 cells). Elevations in [Ca2+]i and 02" production were concentrationdependent, EC50s being respectively 1.1+0.6 nM and 2.1+1.1 nM. Extracellular calcium deprivation reduced O2" production. In :~ HL-60 cells: 1) fMLP receptors become functional and their number increase, including a large receptor reserve. 2) O2" production follows the calcium response. 3) both responses are largely dependent upon extracellular calcium influx. These results indicate that, for the studied events, ~ HL-60 cells represent a good in vitro model of fMLP-stimulated neutrophils.

52 MODELIZATION OF TAU MODIFICATIONS ( A L Z H E I M E R ' S DISEASE TYPE) IN NEURONAL CULTURES M. LESORT, P. COURATIER, F. ESCLAIRE, J. HUGON. Alzheimer's disease (AD) is a neurodegenerative disorder. One of the histopathological hallmarks of AD is the presence of neurofibrillary tangles (NFTs). Prominent components of NFTs are the paired helical filaments (PHFs) which are composed of the accumulation of the microtubule associated protein phosphorylated tau. The goal of this work is to study the immunocytochemical modifications of tan protein in primary neuronal cultures of fetal rat exposed to glutamate or its agonist NMDA. 8-10 days after initial plating neuronal cultures were exposed to glutamate or to NMDA. Overactivation of glutamate receptors induces a neuronal degeneration which is dependent on the presence of extracellular calcium and the duration of exposure. This neurodegeneration is associated with a significant increase in tau immunoreactivity independently of its phosphorylation (monoclonal antibody tau 2 - Sigma) or related to a phosphorylated site at serine 202 (monoclonal antibody AT8 -Innogenetics). These studies used an immunocytochemical approach PAP or immunofluorescence quantified by laser confocal microscopy or immunoblot analysis. These modifications seem to be mediated by Ca++ influx or neuronal depolarisation. We have shown by in situ hybridization and by northern-blot analysis, that glutamate induces a significant increase in tan mRNA expression. Tau m o d i f i c a t i o n s (accumulation and phosphorylation) observed in the experimental model of neurodegeneration are reminiscent of that seen in AD. This model could allow pharmacological studies of tau modifications.

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54

INTERACTION BETWEEN ENDOTHELIN-1 AND C H I C K E N N A T R I U R E T I C PEPTIDE ON T H E C O N T R A C T I L I T Y OF C U L T U R E D C H I C K CARDIAC MYOCYTE.

Is Myocardial Cell Culture a Convenient Model to Study Myocardial Protection?

BEZIE Y.., MESNARD L*, SAMSON F*, PERRET C, MERCADIER JJ* et LAURENT S. INSERM U 337 Paris, *CNRS URA 1159 Le plessis-Robinson, France.

L.Camilleri, N.Moins, J.Papon, JMaublant, Ch.de Riberolles, A.Veyre. Chirurgie CardioVasculaire, INSERM U71, Universit6 d'Auvergne, 63000 Clermont-Ferrand. FRANCE.

We have previously shown that rat ANP was able to decrease contractility of cultured, spontaneously beating, chick embryo ventricular cells, and that this effect was opposite to endothelin1 (Et-1). Et-1 has been implicated as a secretagogue for'natriuretic peptides in vitro and in vivo. Interestingly, natriuretic peptides can inhibit Et-1 secretion from cultured endothelial cells, suggesting a negative feed-back mecanism between endothelial cells and cardiomyocytes.

The purpose of this study was to check the lack of toxicity of a cardioplegic solution (Plegisol, Abbott) and its possible protective effect against chemical hypoxia. Three-day old cultured beating cardiac myocytes isolated from newborn rats, were incubated for 90, 180, 270 or 360 min under normothermic (37~ or hypothermic (4~ conditions : (i) with four different concentrations of cardioplegic solution (0, 50, 75 and 100%), (ii) with the 50% concentration solution and metabolic inhibitors (KCN 5mM and iodoacetic acid 0,1 mM). Myocardial viability was assessed using propidium iodide fluorescence at the end of the experimental procedure or after 24 h of recovery in culture medium. Results were the following : (i) Cell viability was unaffected by the cardioplegic solution whatever the concentration and the incubation or recovery times. (ii) 50% concentration cardioplegia failed to protect against the detrimental effect of metabolic inhibitors whereas hypothermia did. Viability (% 4- sd) of cells incubated with metabolic inhibitors in a 50% cardioplegJcsolution was ; Recovery time 0 . 24 h Incubation 37~ 4~ 37oc 4oc 90min 78• 80• 8• 51_+9 270mir} 28• 83• 0 27• So, with this culture model, the Plegisol solution does not show any toxicity but appears uneffective to protect against a chemical hypoxia whereas the known beneficial effect of hypothermia is evidenced.

The aim of the study was to determine wether Et-1 could attenuate is own inotropic effect through an ANP-mediated decrease in contractility. Therefore, we studied, using a video-microscopy system, the contractility of isolated cultured chick ventricular myocytes in response to Et-1, chicken natriuretic peptide (ChNP), or both. In the same model, we studied, by northern-blot analysis, the time-dependent expression of ChNP in response to Et-l. Et-1 (10-SM), increased the chick cardiomyocytes contractility by 20-25% between the 5th and the 15th minute (p<0.05). Although ChNP (10-TM) did not significantly change the amplitude of contraction under basal conditions, it prevented the Et-l-induced increase in contractility (p<0.05) or reversed it (p<0.05). Et-1 stimulated the expression of the ChNP mRNA as soon as the 30th minute, with a maximal effect at the second hour of stimulation. At the 4th hour Et-I had no more effect on the expression of ChNP. These results, obtained in vitro, suggest an interaction between Et-1 and natriuretic peptides as autocrine/paracrine factors in the regulation of cardiac inotropy.

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55 EVIDENCE FOR A PROTECTIVE EFFFECT OF THREE XANTHIC DERIVATES IN CICLOSPORINE INDUCED CONTRACTION IN TWO IN VITRO GLOMERULAR MODELS. M. P O T I E R , B. L'AZOU, J. CAMBAR Laboratoire de Bioiogie Cellulaire Facult~ de Pharmacie 3, Place de la Victoire 33000 Bordeaux (FRANCE) Ciclosporine A (CsA), a higly potent immunosuppressive agent, induces in vivo a severe nephrotoxicity with a large decrease in renal haemodynamics. The aim of this study is to show the ability of three xanthic derivates, theophylline, caffeine and pentoxifylline, to diminish the CsA-induced vasoconstrictive effects in two in vitro rat glomerular models, e.g. isolated ~glomeruli and cultured mesangial cells. Isolated glomeruli are obtained by sieving method from rat renal superficial cortex in HBSS at pH 7,4. Mesangial cells are cultured from glomerular explants in RPMI 1640 medium with 15% Fetal Calf Serum. Area of either isolated glomeruli or mesangial cells is assessed by an image analyzer. Each glomerulus or mesangial cell serves as its own control by photographiing them prior to any drug incubation and after incubation for 10, 20 and 30 minutes either in HBSS control solution or with CsA alone or with CsA and xanthic derivates (theophylline, caffeine or pentoxifylline). CsA alone (10 -6 M)induces a dramatic time-dependent decrease in glomerular area (-7,4% at 10 rain, -11,6% at 20 min and 13,4% at 30 rain); control buffer solution does not induce any decrease. This effect is dose-dependant; at 30 minutes incubation, the area decrease for mesangial cells is about -11,3% (10 -8 M), -13% (10 -7 M),17% (10 -6 M). CsA (10 -6 M), after a 10 minute pretreatment with xanthic derivates, induces only a slight decrease on mesangial cell area: about -3,9% (pentoxifylline 10 -8 M ) , - 3 , 8 % (theophylline 10 -8 M) and - 1,1% (caffeine 108 M) at 30 minutes. In conclusion, the marked constriction induced by CsA in isolated glomeruli and mesangial cells can be partially prevented by xanthic derivates.

56 C A D M I U M N E P H R O T O X I C I T Y ASSESSED IN I S O L A T E D H U M A N G L O M E R U L I AND C U L T U R E D MESANGIAL CELLS. Barrouillet M.P, Potier M., Cambar J. Laboratoire de Biologie Cellulaire- Facult~ de Pharmacie - 3, place de la Victoire - 33000Bordeaux (FRANCE) Cadmium (Cd), an important pollutant, causes severe damages at renal glomerular and tubular level. Most of previous studies treated largely about tubular nephrotoxicity of Cd. The aim of the present study was to show the direct vasoactive effect of Cd in isolated glomeruli and mesangial ceils, incubated with various concentrations (10 -3 to 10 "10 M). Each glomerulus or mesangial cell served as its own control. Indeed, the same isolated glomerulus or mesangial cell was observed under microscope, at different times of incubation (0, 10, 20, 30 mn) with HBSS alone or with the metal and photographied. Glomeruli were isolated by passing the pulp through calibrated sieves and suspended in Hanks' balanced salt solution at pH 7,4; the mesangial cells grew in RPMI 1640 medium supplemented with 10% FBS, in atmosphere of 5% CO2, in a humidified incubator. Cytotoxicity was determined with neutral red test (24 hrs of contact with the toxic); the IC 50 of Cd in cultured mesangial cells was estimated at 2,4 10-5; 10 -4 M of Cd induced. Moreover, we found that different concentrations' of Cd could induce a dose- and time-dependent vasoactive effect in isolated glomeruli, as measured by quantification of decrease in glomerular area. Surface area reduction was -9,8 % (10 -9 M), -9,0 % (10 -8 M), 7,0 % (10 -7 M) and -6,2 % (10 -6 M). At 10-10 M of Cd, the area decrease was the same than in HBBS. The decrease is time-dependant: T10 (-5,2%), T20 (6,4 %), T30 (-7,4%), T40 (-9,8%) with 10 -9 M of Cd. We have observed this area reduction only in 70% of isolated glomeruli. The same experimental conditions were used on mesangial cells and the area reduction is also time- and dose-dependant, as well as glomeruli: -7,8 % (10 -7 M), -9,8% (10 -8 M), 12,3 %~(10 -9 M). These results suggest that Cd could significantly largely contract glomerular structures that can explain severe damages occurred in glomerular functions, especially in glomerular filtration.

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58

Early cytotoxic effects of mechlorethamine on rabbit's respiratory epithelium in primary culture

Evaluation of the t r a n s f o r m i n g potential of the ethylene glycol monobutyl ether (EGBE) in Syrian hamster embryo (SHE) cell system

I. Giuliani, F. Tournier, O. Houcine and F. Marano Laboratoire de Cytophysiologie et Toxicologic Cellulaire, Universit~ Paris VII, Paris, France.

Z. Elias, O. Poirot, M.C. Dhni~re, F. Terzetti, A.M. Marande INRS, Avenue de Bourgogne, 54501 Vandoeuvre (France)

The respiratory tract has been shown to be the major target of mustard gas. The mostI accepted hypothesis is that of Papirmeister et al. ( Fund. Appl. Tox. 1985, 5:S135-S149) which considers DNA'to be the first target of the drug in ceils. We have tested other hypotheses of lipid peroxidation induced by mechlorethamine (HN2) on primary culture of tracheal epithelial cells which were shown to be a suitable model in a previous study of acute toxicity (Giuliani et al. Cell Biol. Toxicol. 1994, 10:231-246). Flow cytometry showed that there were no modifications in the cell cycle analysis after one hours treatment by HN2 at the sublethal dose of 5.10 -5 M. After 5 hours of the same treatment the cells were arrested in phase S. However, lipid peroxidation, membrane and mitochondria alterations were observed after only 1 hour of: treatment. This early lipid peroxidation occured simultaneously with enzyme induction of superoxyde dismutase, catalase and glutathion reductase, whereas glutathion S transferase and glutathion peroxydase were inhibited. To conclude, we have shown that a sublethal dose of HN2 induces an early lipid peroxidation after only 1 hour of treatment. This epithelial response seems to occur before DNA alteration. Therefore, these results imply that other molecular targets may be implicated in early cytotoxic mechanisms induced by mustard gas.

EGBE is widely used for many industrial and domestic purposes. It has a hemolytic activity in several animal species but there are not yet data on its carcinogenicity or not. We have therefore investigated its transforming effect in in vitro SHE cells. In the cells treated continously (7d) with EGBE (0.125-1 mg/ml) any transformed colony was observed. But a brief exposure to EGBE (24 h) followed by treatment with 12-O-tetradecanoylphorbol-13acetate (TPA, 0.1 gg/ml) for 6d induced transformed colonies, the transformation frequencies (TF) (between 1.2 % and 4 %) depending upon EGBE dose. TF were still higher if the cells were simultaneously treated with EGBE and TPA. Co-exposure of cells to EGBE and benzo(a)pyrene (BP, 1 gg/ml) for 7d inhibited transformation by BP. The results suggest that : (1) EGBE could be an incomplete transforming agent, the initiated cells being expressed only by the promoting effects-of TPA ; the initiation of transformation by EGBE could by attributed to its genotoxic metabolite butoxyacetaldehyde (our observations) ; (2) EGBE could induce the inhibition of enzymes involved in the metabolism of BP, of cellular replication and/or other changes which will result in the suppression of BP-induced trahsformation. The carcinogenicity of EGBE and its in vivo modulatory effects on chemical carcinogenesis should be studied.

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59 Ni3S2 Uptake by G u i n e a Pig A l v e o l a r M a c r o p h a g e s ( G P A M ) and c o m p a r a t i v e a n a l y s i s of particles by EDS and LAMMA-500

SHIRALI p.1 BOUTINA.C1 , MAREZT.1 , HACHIMI A2, MULLERJ.F2, ~ 1 1CERESTE, Institutde M~decine du Travail, 1-Place du Verdun59 045 Lille Cedex 2LSMCL, Universit(~de Metz, Technopole 2000 1-bd Arago, F-57078Metz Cedex The aim of the present study is to determine the different pathways of incorporation of Ni3S2 into GPAM by characterization of nickel valence in cell membranes with 2 differents analytical investigations : "Energy Dispersive Spectrometry" (EDS) and "Laser Microprobe Mass Analyser" (LAMMA-500). By electron microscopy we observed 3 different pathways of Ni3S2 incorporation : - phagocytosis of crystalline form - endocytosis - transmembranous passage Elementary analysis by EDS of particles generated from Ni3S2 in different compartments revealed a decrease of sulphur concomitant with the of appearance of an organic Ni-P complex, either with the phosphate groups of membrane phospholipids or with phospho-transferring proteins. Spectral analysis by LAMMA-500 of reference subsulfide standards in ionizing resonance yields a distribution of ionized aggregates (NixSy +) characteristic of (~Ni3S2. This study revealed the different ionic form of nickel (58, 60) corresponding to various stages of metabolization. This study showed that phagocytosis is not the only way of Ni3S2 uptake and therefore not the only important pathway responsable for the carcinogenic or toxic effects of Ni3S2. By another way, both techniques (SED, LAMMA-500) are interesting for the determination of the elementary structure and valency of particles after metabolization.

60 INTRACELLULAR TRANSFORMATION OF URANIUM INSIDE THE ALVEOLAR MACROPHAGE

M-H.HENGE-NAPOLI*, R.GIBERT*, M.C.CHEYNETw E.ANSOBORLO* *lnstitut de Protection et de SOret(~ Nucldaire, Ddpartement de Protection de la sant~ de I'Homme et de Dosimdtrie,Service de DOSim~trie, B.P.n~ 92265 Fontenay-aux Roses Cedex, France. w LTPCM/INP . CNRS URA 29 - ENSEEG - BP 75 38402 - Saint Martin d' H~res Cedex, France.

Abstract:

The metabolic behaviour of a compound after inhalation depends on its ability to transfer from the pulmonary compartment to the blood, that is to say from its solubility. The alveolar macrophage is one of the first cell which interacts with the airborn particles. Previous studies have shown that the alveolar macrophage could lower the solubility of some uranium oxides and form uranyl phosphate needles. The aim of this work was to study the entering of the soluble uranyl ion from the extracellular compartment into the cell. Alveolar macrophages have been cultured using a gas phase technic on a medium containing, increasing concentrations of uranyl nitrate. For a concentration of 2.5 IJg.ml1 of uranium, the ATP content of the cells was increased of 25 % compared to the controls, which indicates a metabolic activation. The entering of uranyl into the cells has been studied at this concentration using transmission electronic microscopy and electron energy loss spectroscopy technics. We have observed that soluble uranium could enter the cells and concentrate at the periphery of vacuoles (probably lipidic). This indicates that the alveolar macrophage is able to concentrate soluble uranyl and to turn it into insoluble form, different of the phosphate needles previously observed.

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62

61 Behaviour of cultured rat liver endothelial cells and Kupffer cells after treatment with killed Propionibacterium avidum Dhouib M, Heuss R.~ Gendrault J.L.* and Lugnier A.

Laboraloire de Toxicologie'Fondamenlale e/ d'Ecoloxicologfe, D R E D EA 1327, Facul/e de Pharmac/'e de b~rasbourg, B.P. 24, 67401111Mrch Cedex, France; *Labora/oire de Virologie, I N G E R M U74, Facul/~ de ME=decine, ~7000 Gtrasbourg, France

Both types of cell were obtained from livers of Wistar rats by collagenase dissociation of the tissue, lysis of the hepatocytes and centrifugal elutriation. The cells were cultured for 24fhours in Dulbecco's Minimal Essential Medium supplemented with either fetal calf serum or Ultroser. They were then treated for one to four days with 1/50, 1/100, 1/250, 11500 and 1/1,000 dilutions of a suspension of 1.75 mg/ml killed Prop/on/bacler/um av/dum bacteria (PPA). Bacteria were cultivated under anaerobiosis in brain-heart infusion for 72 hours. Then, opaque suspensr in 2 ml of 0.9% NaCI was submitted to potential bactericidal condition (heat treatment). The result of the treatment on both types of cell was studied under electron microscopy, the characterization of Kupffer cells (KC) being ascertained through the cytochemical demonstration of endogenous peroxidase. The uptake of killed bacteria by KC was clearly demonstrated. Treated KC displayed phagocytosis against sheep red blood cells opsonized with the Fc fragment of IgG. On the other hand, no C3-mediated phagocytosis was observed as it would be the case after lipopolyseccharide (LPS) treatment. It is noteworthy that differences were observed in the liver when we treated the rat with PPA instead of LPS. Accordingly, alter Lp or Lv., injection, PPA induces NO synthase activity in a chronic and lasting manner whereas LPS gives only a transient induction.

E F F E C T OF P H A R M A C O L O G I C A L AGENTS ON TYPE II TRANSGLUTAMINASE EXPRESSION IN RABBIT ARTICULAR CHONDROCYTES IN CULTURE L. BORGE, SX)EMIGNOTand M. ADOLPHE Laboratoire de pharmacologie cellulaire de rEPHE 15 rue de rEcole de M&tecine75006 PARIS. Transglutaminases (TGases) (EC 2.3.2.13) are a family of calcium-dependent acetyl lransferases that catalyse the formation of an amide bond between the 7-carboxamide groups of peptide-bound glutamine residues and the primary amino groups of a variety of compounds, including the eamino group of lysine in certain proteins. TGases have been shown to be involved in many cellular events : formation of cross linked envelopes during squamous cell differentiation (type I TGase), of fibrin clot (Factor XIII), of apoptotics bodies (type II TGase). Moreover, type II TGase, ubiquitous, has been shown to be involved in growth, differentiation and stabilization of extracellular matrix in many cell types. The main biological fonction of articular chondrocytes is to produce the cartilage matrix, consisting of specific proteoglycans and collagen types, mainly type II collagen. In culture, chondrocytes rapidly dedifferentiate and so produce type I and type III collagen. We studied the type II TGase expression by western blot : - in articular chondrocytes in culture at differents passages (P0, P2 et P4), - treated or not by retinoic acid (RA : dedifferenciating agent in chondrocytes), treated by dihydrocytochalasine B (DHCB) or staurosporine (Stauro) which induce redifferentiation of cells dedifferentiated by subculture.

~nxxaJtLre

Under electron microscopy, endothelial cells after treatment with PPA do not present any significative change as compared to the control, but the effect of a prolonged and repetitive treatment remains to be studied.

Sdxdare RA EI-EB

Slam

Cot~gea type Type 1I 'IUese ext~ssion +/I andTIT ~ I mdTIT

IT

+-~

Ahesion (ell ++ +H-

4+'-H-

Results, summarised in the table, suggest that type II TGase expression is compatible with expression of type II collagen, the main differentiation marker. However, it was observed, a good correlation between type II TGase expression and adhesion capacity of cell. Type II TGase could be play an important role in cell adhesion, therefore in cell-matrix interaction.

226

63 Characterization of Endogenous Substrates of Transglutaminases in Rabbit Articular Chondrocytes in Culture Lajemi. Malika, Demignot. Sylvie and Adolpbe. Monique Laboratoire de Pharmaco-toxicologie cellulaire de I'EPHE. 15 rue de rEcole de M&tecine 75006 Paris. Studies have shown implications of TGases during many biologicals processes such as stabilization of extracellular matrices, apoptosis, growth and differentiation or, more recently, in signal transduction. To determine the role of the Iransglutaminases, a family of enzyme expressed m rabbit articular chondrocytes in culture (Demignot. Sylvie and al, Biochem.Biophys.Acta, 1995 in press), we studied their endogenous substrates. Transglutaminases (E.C 2.3.2.13) are a class of enzymes which are able t~) catalyze an acyl transfer between the Tcarboxyl group df peptide-bound glutamine and an acceptor amine such as pulrescine and cadav6rine. Incorporation of dansylcadaverine in cell lysates, detected by Western blot with an monoclonal antibody against dansyl (generously given by J.C Kvedar, Massachusetts, Charlestown, USA), revealed an important number of substrates among which three major proteins of 45, 58 and 220 kDa. Chondrocytes in monolayer culture incubated in fluoresceinecadaverine-supplementedculture medium were used to localize enzyme activity and detect colocalized substrates by fluorescence microscopy and Western blot respectively. In primary culture, transglutaminase was shown to be physiologicallyactive and mainly intracellular. When chondrocytes were dedifferentiated by subculture (passage 2) or retino'ic acid treatment, or incubated with exogenous tissue transglutaminase, we observed an incorporation of fluoresceinecadaverine into cells as well as in lattice structures similar to matrix fibronectin fibers. These endogenous proteins which incorporated fluoresceinecadaverine in culture were characterized, by western-blot, as the three proteins of 45, 58 and 220 kDa. Immunofluorescence microscopy and western-blot studies tend to strongly suggest that the 220 kDa corresponds to fibronectin. Concerning the 45 and 58 kDa proteins, we suggest that they may be actin and vimentin respectively since they are also major proteins in cell lysates as can be seen b.y sylver staining of SDS-PAGE gels. These results suggest that in rabbit articular chondrocytes, transglutaminases, may play a role in cell adhesion or extracellular matrices stabilization through cytoskeleton and extracellular matrix proteins elaborated by the chondrocyte.

64 STUDY O F Q U I N O L O N E S C Y T O T O X I C I T Y ON C U L T U R E D T E N O C Y T E S BY N E U T R A L R E D , M T T A N D R H O D A M I N E 123 ASSAYS

K . B E R N A R D - B E A U B O I S 1, C.HECQUET 1, O . H O U C I N E 2 & M.ADOLPHE 1 1) Laboratoire de Pharmacologie Cellulaire, Ecole Pratique des Hautes Etudes 15, rue de rEcole de M~decine, 75006 PARIS 2) Laboratoire de Cytophysiologie et Toxicologic Cellulaire, Universit6 Paris VII 2, place Jussieu, 75005 PARIS Many new quinolones have been developed and used widely because of their excellent antibacterial activity. However, some clinical studies have shown that substained treatments can generate, in patients over 60-year old, tendinitis and tendon rupture. To study the in vitro mechanism of action of these antibacterial agents, first passage and late passages (50) tenocytes from rabbit Achilles tendon were isolated, cultured in monolayer and finally incubated with quinolones compounds. First, we reported observations of the morphological and biological characteristics of ce~s cultured in monolayer for primary culture and subsequent passages. Data obtained by transmission electron microscopy and growth curve were complementary. After 50 passages, the generation time of tenocytes did not change and no sign of senescence could be seen with transmission electron microscopy. The characterization of the matrix components by Northern blotting showed, after a few passages, a decrease in the expression of mRNA encoding type I collagen. The effects of one quinolone (nalidixic acid) and two fluoroquinolones (pefloxacin and norfloxacin) were evaluated on cell viability, mitochondrial deshydrogenase and global mitochondrial activity by spectrophotometric evaluation of neutral red and MTT uptake and by cold light microfluorimetry determination of rhodamine 123 uptake. The IC 50 of nalidixic acid, pefloxacin and norfloxacin were 5.8 10-3 M, 5.4 10.4 M and 6.6 10-3 M respectively when determined by the neutral red cytotoxicity test on first passage tenocytes and 6.8 10-3 M, 6.4 10-4 M and 1.4 10-4 M when determined on late passages cells. The IC 50 of the three quinolones were similar when determined by the MTT or neutral red method. No significant cytotoxic effect was found after rhodamine 123 incorporation. These first results showed that quinolones were toxic to tenocytes'and that the cytotoxic effect of fluoroquinolones was more important than the one of nalidixic acid. Moreover, the cytotoxicity of these antibacterial agents did not seem to differ when evaluated on first or late passages tenocytes.

227

65 Immuno-Toxicology Today. Bernard Dugas. CNRS URA 625, HOpital la Piti~ Salpdtri~re, 75013 Pads, France. Objectives: To develop in vitro tests to evaluate the immunotoxicological properties of therapeutic agents, xenobiotics, and nutriements. In addition, these in vitro provide useful tools to study the cellular and molecular basis of various phsyiopathological situations (type IV hypersensitivity, oxidative stress to l.P~rA and LP,'B irradiation, xenobiotic toxicity etc.). Brief description: The development of these in vitro tests utilise recents advances in cellular and molecular immunology and related disciplines. In the first phase, it is based on reference subtances whose effects have been studied and elucidated in humans and/or experimental animals. Techniques used' for assessing these immune and inflammatory responses include cell activation and differentiation markers by FACS analysis and evaluation of cytokine and mediator production by multiple approaches such as bioassays, immunoenzymatic assays and mRNA levels by northern blot or reverse polymerase chain reaction analysis. This approach is focussed on four major area: 1) in vitro tests to evaluate the immunotoxicity of the different compounds, 2) the use of established animal models to develop further in vitro predictive assays for drug-induced toxicity, followed by the assessement of the validity of these tests in human situations, 3) the development of tests systems for predictive drug-specific human T cell responses m allergic drug reactions and 5) preparation of drug and compounds metabolites followed by evaluation of their immunotoxic potential. Selected in vitro tests will be then standardized as much as possible. This final approach will lead then to the definition and establishment of in vitro tests compatible with prenormative requirements.