A cell culture model of isolated porcine urinary bladder epithelial cells for genotoxicity studies

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Pergamon

Toxic. in Vitro Vol. 8, No. 4, pp. 763-765, 1994

0887-2333(94)00105-7

Copyright © 1994 ElsevierScienceLtd Printed in Great Britain. All rights reserved 0887-2333/94 $7.00 + 0.00

A CELL C U L T U R E MODEL OF ISOLATED PORCINE U R I N A R Y BLADDER EPITHELIAL CELLS FOR GENOTOXICITY STUDIES W. F•LLMANN and C. GUHE Institut f/Jr Arbeitsphysiologie an der Universitfit Dortmund, Ardeystrasse 67, 44139 Dortmund, Germany A~tract--Monolayer cultures of porcine urinary bladder epithelial cells were obtained by isolating cells from bladders of freshly slaughtered pigs. The cultures were investigated morphologically and characterized according to their growth characteristics and enzymatic functions. Ultrastructural investigations demonstrated that the cells regain their in vivo polarization with apically situated membrane vesicles, tight junctions and desmosomes between neighbouring cells when they have built up a confluent monolayer. Membrane integrity and high cell viability was indicated by low levels of lactate dehydrogenase activity released into the medium. The chromosome set of the cells was stable during the first 5 wk of culture. Under standard culture conditions activities of the enzymes alkaline phosphatase and acid phosphatase were stable over a period of 4 wk. 7-glutamyl transpeptidase activity, indicating a dedifferentiation process, did not increase. Activity of NADH-dehydrogenase dropped, indicating a decrease in ER as a consequence of adaptation to the culture conditions. By measuring concentration dependent increase of sister chromatid exchanges (SCEs) induced by N-methyl-N'-nitro-N-nitrosoguanidine and 2-acetylaminofluorine, the suitability of this model for genotoxicity tests was estimated. These results indicate that the model can be used as a target for bladder cancer inducing agents to investigate the effects of such agents on the level of the target organ in further genotoxicity studies.

INTRODUCTION

Studies with primary cultures of porcine urinary epithelial cells were performed to investigate whether this model system can be used as a short-term assay for studying the genotoxic effects of chemicals, especially of aromatic amines. For an estimation of these effects the frequency of sister Chromatid exchanges (SCEs) was chosen as the visible endpoint.

The urinary bladder epithelium is the target organ for genotoxic effects of aromatic amines (Kadlubar et al., 1981). Epidemiological studies have shown that an occupational exposure to these chemicals has resulted in an increased incidence of bladder cancer (Hueper, 1969). Although many studies have been performed to show a connection between such exposure and the occurrence of bladder cancer, little is known about the mechanisms on the cellular level in the target organ. Additionally, there are few data concerning the enzyme complement, metabolic capacities and detoxification properties of epithelial cells of the urinary bladder. Cultures of isolated urinary bladder epithelial cells can therefore serve as a model system to study the attributes of this cell type. Although urinary bladders from humans are not readily available, some laboratories have established methods for the isolation and culture of human urothelium (Kirk et al., 1985; Messing et al., 1982; R a h m a n et al., 1987; Reznikoff et al., 1983). For routine culture a model based on bladders from animals is more suitable. Methods for the isolation and culture of human urothelium can be adapted for the urothelium of the pig.

MATERIALS AND METHODS Epithelium from bladders of freshly slaughtered pigs was removed mechanically from the underlying muscle tissue and subsequently cut into pieces, each of 0.5 cm 2 in area. After 20 min the supernatant containing the isolated cells was withdrawn, stored at 37°C and fresh trypsin solution was added to the tissue pieces. The tissue pieces were incubated for another 20 min, the supernatant of the second incubation period was withdrawn also, and united with the supernatant of the first incubation period. The obtained cell suspension was then centrifuged for 5 min at 50 g. The cell pellet was washed twice with H a m ' s F12 medium and cell viability was determined by trypan blue exclusion. Viability was 90% or more and routinely 6-10 × 10 6 cells per bladder were isolated; 5 × l0 s cells were seeded per 75 cm 2 plastic flask. The culture medium was H a m ' s F12 supplemented with 100 U penicillin/ml, 100 p g streptomycin/ml, 1.25 pg amphotericin/ml, 5 p g transferrin/ml, l0 # g insulin/ml, 0. l mM non-essential amino acids, 2.7 mg

Abbreviations :

2-AAF = 2-acetylaminofluorene; DMSO = dimethyl sulfoxide; LDH = lactate dehydrogenase; MNNG methyl - N- nitro - N- nitrosoguanidine; SCE = sister chromatid exchange.

= N-

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glucose/ml, 1 #g hydrocortisone/ml and 20 ng epidermal growth factor/ml. A karyotype analysis was performed according to the method of Freshney (1987) and enzyme measurements according to Bergmeyer (1983). SCEs were determined by a modified method according to Perry and Wolff (1974). For each assay, isolated epithelial cells from three urinary bladders were seeded and cultured for 1 day. Subsequently, the cultures were incubated with the test chemicals for 4 hr at the concentrations indicated; dimethyl sulfoxide served as a solvent control. After incubation, the culture medium was changed, 10 #M 5-bromo-2'-deoxyuridine were added and incubation was continued for another 28-29 hr. Then colcemid (0.1 pg/ml) was supplemented to prevent further cell division and arrest the chromosomes in the metaphase. After 16 hr the cells were detached from the substrate with 0.05/0.02% trypsin/EDTA solution (5 g/2 g/litre Puck's saline A solution) and washed with culture medium supplemented with 20% foetal calf serum to stop trypsin activity. The cells were then incubated in 0.075 M KCI solution, for 4 m i n and fixed with methanol-acetic acid (4:1, v/v) for 20 min. The cells were additionally heat fixed on glass slides and the sister chromatids were selectively stained with Hoechst 33258 dye. The SCE frequency was determined with a light microscope (magnification × 1000). For each assay, cells from three bladders were used, and two slides per bladder and concentration were evaluated (30 mitoses per slide). RESULTS

Isolated porcine urinary bladder epithelial cells maintained in culture regain their in vivo polarization, as indicated by apical microvilli, a basal lamina and

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Fig. 2. SCE frequency in cultured porcine urinary bladder epithelial cells incubated with 2-acetylaminofluorene (2AAF). DMSO served as a solvent control. For each concentration, six slides with cells from three different bladders were evaluated (30 mitoses per slide). tight junctions between neighbouring cells. Morphological structures typical of the urothelium (Hicks, 1975) occurred in the cultured monolayer--apically situated fusiform vesicles and an apical membrane of alternating thickness. A karyotype analysis indicated that the genotype of the cells is stable over 5 wk and cell viability and membrane integrity were good, as measured by the release of lactate dehydrogenase (LDH) activity into the culture medium (~<5% of total LDH activity) (Guhe and F611mann, 1994). Activities of marker enzymes for differentiation status (~-glutamyltranspeptidase), for lysosomes (acid phosphatase) and for luminal membranes (alkaline phosphatase) did not change significantly compared with the activities in the original tissue (Guhe and F611mann, 1994). Incubation of the cultures with the direct alkylating nitrosamine N - m e t h y l - N ' - n i t r o - N - n i t r o s o g u a n i d i n e (MNNG) resulted in a concentration-dependent increase in SCE frequency (Fig. 1). Above a concentration of 1 #M the agent is toxic, as indicated by a drastic decrease of the scorable metaphases. The aromatic amine 2-acetylaminofluorene (2-AAF) is not toxic in concentrations up to 100~M. Again a significant increase of the SCE frequency, dependent on the concentration of 2-AAF, can be observed (Fig. 2). DISCUSSION

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Fig. I. Sister chromated exchange (SCE) frequency in cultured porcine urinary bladder epithelial cells incubated with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Dimethyl sulfoxide(DMSO) served as a solvent control. For each concentration, six slides with cells from three different bladders were evaluated (30 mitoses per slide).

Primary cultures of porcine urinary bladder epithelial cells were successfully maintained in culture for several weeks. The cells preserved many features of bladder epithelium in vivo under the conditions described. This makes the model useful as an in vitro system representing the urinary bladder epithelium. Simultaneously, material from slaughtered pigs avoids the use of laboratory animals which otherwise would be killed for this purpose.

In vitro model of urinary bladder epithelium

Further studies have shown that prostaglandin H synthetase activity is constant in the cultures compared with that in fresh tissue and inducible by arachidonic acid. Furthermore, glutathione S-transferase activity is preserved under these culture conditions, but decreased to approximately 20% of the value of the original tissue after 4 wk in culture. In the case of N-acetyltransferase preliminary results indicate activities in fresh tissue that are conserved in culture (Guhe et al., 1994). Cytochrome P-450IAI was detectable by 7-ethoxyresorufin-O-deethylation measurements in isolated original tissue (Guhe et al., 1994) and studies on cytochrome P-450 activities in the cultures are under way. An indication that metabolizing capacities are present in the urothelium came from the results with 2-AAF. This aromatic amine differs from the direct alkylating agent M N N G in that it is an indirect carcinogen that has to be metabolized in order to affect D N A (Beland and Kadlubar, 1990). This implies that the cells have metabolic properties, either activities of cytochrome P-450 or prostaglandin H synthetase, and it is known that 2 - A A F can be activated by prostaglandin synthetase (Eling et al., 1988). These results show that the urothelium itself can activate indirect carcinogens, since in this cell culture model neither the liver metabolism nor a metabolic component system such as the S-9 microsome mix is present. In summary, this in vitro model system is a useful tool to study the mechanisms of genotoxic effect of aromatic amines on the cellular level. A further study of the properties of the cells will give information on the physiological and metabolic competence of the urothelium.

Acknowledgements--The authors acknowledge the excellent technical assistance of Mrs B. Kullik and Mrs R. Oberschmidt-Janning. Special thanks to Dr E. Hallier for assistance in the preparation of the manuscript. This work was supported by grant Fo 2.1-1328-120 from the Bundesgesundheitsamt (Zebet).

765 REFERENCES

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