Food Additives & Contaminants
ISSN: 0265-203X (Print) (Online) Journal homepage: http://www.tandfonline.com/loi/tfac19
Metabolism of chlorpropham by adult rat hepatocytes co‐cultured with a liver epithelial cell line G. Carrera , J. Alary , Y. Lamboeuf , F. Anglade , C. Escrieut & C. Pinchon To cite this article: G. Carrera , J. Alary , Y. Lamboeuf , F. Anglade , C. Escrieut & C. Pinchon (1990) Metabolism of chlorpropham by adult rat hepatocytes co‐cultured with a liver epithelial cell line, Food Additives & Contaminants, 7:S1, S152-S154, DOI: 10.1080/02652039009373870 To link to this article: http://dx.doi.org/10.1080/02652039009373870
Published online: 10 Jan 2009.
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Citing articles: 1 View citing articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=tfac19 Download by: [University of Cambridge]
Date: 06 November 2015, At: 22:43
FOOD ADDITIVES AND CONTAMINANTS, 1990, VOL. 7, SUPPLEMENT NO. 1, S152-S154
Metabolism of chlorpropham by adult rat hepatocytes co-cultured with a liver epithelial cell line G. CARRERA, J. ALARY, Y. LAMBOEUF, F. ANGLADE, C. ESCRIEUT and C. PINCHON
Downloaded by [University of Cambridge] at 22:43 06 November 2015
INSERM U-87, 2 rue François Magendie, 31400 Toulouse, France
Introduction
Recent studies have shown that adult rat hepatocytes co-cultured with liver epithelial cells can survive for a period longer than 2 months maintaining most of their specific functions better than in primary cultures (Guguen-Guillouzo et al. 1983, Foliot et al. 1985). The aim of this work was to compare the long-term ability of xenobiotic metabolization by rat hepatocytes in primary culture and in co-culture with liver epithelial cells in order to use this model for in vitro studies reproducing in vivo semi-chronic toxicity experiments. The metabolism of a pesticide, chlorpropham (CIPC) was studied, for 2 weeks, in this co-culture model and in primary cultures of hepatocytes or epithelial cells.
Experimental
An epithelial cell line was obtained by trypsination of 10-day-old rat livers and purified in our laboratory by the method of Williams and Gunn (1974). Hepatocytes were obtained by Seglen's method (1973). Hepatocytes and epithelial cells were seeded at a density of 1 • 5 X 106 cells/ml medium as described by Begue et al. (1984). When hepatocytes and epithelial cells were confluent, 48 h after the seeding, CIPC (12 HM) was added daily and determined by HPLC as previously described (Alary et al. 1986). Simultaneously CIPC toxicity was evaluated by LDH, GOT and GPT release.
Results
As shown by the amount of unmetabolized CIPC (figure 1) and taking into account the recovery and precision of the method, epithelial cells in primary culture did not show any detoxication enzyme activity. In primary hepatocyte cultures, unchanged CIPC increased with time while in co-cultures CIPC metabolization was slightly increased during the same period. Free 4-OH-CIPC always remained lower than 2 /tg in all cases. In hepatocyte cultures, sulphation (figure 2) of 4-OH-CIPC decreased faster than glucuronidation (figure 3). In co-cultured hepatocytes, sulphation (figure 2) and glucuronidation (figure 3) were maintained and the sulphate/glucuronide ratio remained constant during the experimental period. Based upon LDH, GPT and GOT determination, CIPC (12 f-U) does not show any cytotoxicity. 0265-203X/90 $3.00 © 1990 Taylor & Francis Ltd.
Metabolism of chlorpropham by rat hepatocytes
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Figure 1. Unmetabolized CIPC versus time. M3 30
20
(J
i o
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15 13 days Figure 2. Formation of 4-OH-CIPC sulphate versus time.
Discussion In primary culture, our results show that hepatocytes rapidly lose their detoxication enzyme activities. This confirms earlier results showing that cytochrome P-450 decreased rapidly in primary culture of hepatocytes (Begue et al. 1984). In contrast, the maintenance of several specific functions such as albumin secretion (Guguen-Guillouzo et al. 1983), taurocholate uptake (Foliot et al. 1985) and cytochrome P-450 level (Begue etal. 1984) were demonstrated in hepatocytes co-cultured with liver epithelial cells. Begue etal. (1983) have shown that Ndemethylation, N-oxidation, reduction and glucuronidation of Ketotifen were also
G. Carrera et al.
S154
Downloaded by [University of Cambridge] at 22:43 06 November 2015
co-culture
3
5
7
9
11
13
15
days
Figure 3. Formation of 4-OH-CIPC glucuronide versus time.
maintained in co-cultured hepatocytes. Our metabolism study confirms these results and shows for the first time that sulphation remains high in co-cultured hepatocytes. The sulphate/glucuronide ratio remained constant throughout the experimental period, at the same value as that observed in isolated hepatocyte suspension (Carrera et al. 1988). Over a 15 day period and probably longer, co-culture of hepatocytes allows the maintenance of specific liver functions and particularly detoxication, in contrast to primary cultures. Consequently, co-culture of hepatocytes could be used as a suitable experimental tool for in vitro semi-chronic toxicity studies. Acknowledgement This work was supported by the Ministere Charge de l'Environnement. References ALARY, J., CARRERA, G., BERGON, M., PERIQUET, A., and VANDAELE, J., 1986, High liquid chromat-
ography determination of chloropropham and its metabolites in isolated rat hepatocyte incubations. Journal of Liquid Chromatography, 9, 3597-3606. CARRERA, G., FORGUES, S., ALARY, J., and LAPONTARIQUE, H., 1988, Effects of an experimental
hepatitis induced by D-galactosamine on the metabolism and toxicity of chlorpropham: in vivo and in vitro studies. Cellular and Molecular Aspects of Glucuronidation, edited by G. SIEST, J. MAGDALOU and B. BURCHELL (Paris: John Libbey Eurotext), pp. 285-288. BEGUE, J. M., LEBIGOT, J. F . , GUGUEN-GUILLOUZO, C., KIECHEL, J. R., and GUILLOUZO, A., 1983,
Cultured human adult hepatocytes: a new model for drug metabolism studies. Biochemical Pharmacology, 32, 1643-1646. BEGUE, J. M., GUGUEN-GUILLOUZO, C., PASDELOUP, N . , and GUILLOUZO, A.,
1984, Prolonged
maintenance of active cytochrome P-450 in adult rat hepatocytes co-cultured with another liver cell type. Hepatology, 4, 839-842. FOLIOT, A., GLAISE, D., ERLINGER, S., and GUGUEN-GUILLOUZO, C., 1985, Long-term maintenance of
taurocholate uptake by adult rat hepatocytes co-cultured with a liver epithelial cell line. Hepatology, 5, 215-219. GUGUEN-GUILLOUZO, C., CLEMENT, B., BAFFET, G., BEAUMONT, C., MOREL-CHANY, E., GLAISE, D.,
and GUILLOUZO, A., 1983, Maintenance and reversibility of active albumin secretion by adult rat hepatocytes co-cultured with another liver epithelial cell type. Experimental Cell Research, 143, 47-54. SEGLEN, P . O. 1973, Preparation of rat liver cells. II-Effects of ions and chelators on tissue dispersion. Experimental Cell Research, 776, 25-30. WILLIAMS, G. M. and GUNN, J. M., 1974, Long-term cell culture of adult rat liver epithelial cells. Experimental Cell Research, 89, 139-142.
ISSN: 0265-203X (Print) (Online) Journal homepage: http://www.tandfonline.com/loi/tfac19
Metabolism of chlorpropham by adult rat hepatocytes co‐cultured with a liver epithelial cell line G. Carrera , J. Alary , Y. Lamboeuf , F. Anglade , C. Escrieut & C. Pinchon To cite this article: G. Carrera , J. Alary , Y. Lamboeuf , F. Anglade , C. Escrieut & C. Pinchon (1990) Metabolism of chlorpropham by adult rat hepatocytes co‐cultured with a liver epithelial cell line, Food Additives & Contaminants, 7:S1, S152-S154, DOI: 10.1080/02652039009373870 To link to this article: http://dx.doi.org/10.1080/02652039009373870
Published online: 10 Jan 2009.
Submit your article to this journal
Article views: 4
View related articles
Citing articles: 1 View citing articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=tfac19 Download by: [University of Cambridge]
Date: 06 November 2015, At: 22:43
FOOD ADDITIVES AND CONTAMINANTS, 1990, VOL. 7, SUPPLEMENT NO. 1, S152-S154
Metabolism of chlorpropham by adult rat hepatocytes co-cultured with a liver epithelial cell line G. CARRERA, J. ALARY, Y. LAMBOEUF, F. ANGLADE, C. ESCRIEUT and C. PINCHON
Downloaded by [University of Cambridge] at 22:43 06 November 2015
INSERM U-87, 2 rue François Magendie, 31400 Toulouse, France
Introduction
Recent studies have shown that adult rat hepatocytes co-cultured with liver epithelial cells can survive for a period longer than 2 months maintaining most of their specific functions better than in primary cultures (Guguen-Guillouzo et al. 1983, Foliot et al. 1985). The aim of this work was to compare the long-term ability of xenobiotic metabolization by rat hepatocytes in primary culture and in co-culture with liver epithelial cells in order to use this model for in vitro studies reproducing in vivo semi-chronic toxicity experiments. The metabolism of a pesticide, chlorpropham (CIPC) was studied, for 2 weeks, in this co-culture model and in primary cultures of hepatocytes or epithelial cells.
Experimental
An epithelial cell line was obtained by trypsination of 10-day-old rat livers and purified in our laboratory by the method of Williams and Gunn (1974). Hepatocytes were obtained by Seglen's method (1973). Hepatocytes and epithelial cells were seeded at a density of 1 • 5 X 106 cells/ml medium as described by Begue et al. (1984). When hepatocytes and epithelial cells were confluent, 48 h after the seeding, CIPC (12 HM) was added daily and determined by HPLC as previously described (Alary et al. 1986). Simultaneously CIPC toxicity was evaluated by LDH, GOT and GPT release.
Results
As shown by the amount of unmetabolized CIPC (figure 1) and taking into account the recovery and precision of the method, epithelial cells in primary culture did not show any detoxication enzyme activity. In primary hepatocyte cultures, unchanged CIPC increased with time while in co-cultures CIPC metabolization was slightly increased during the same period. Free 4-OH-CIPC always remained lower than 2 /tg in all cases. In hepatocyte cultures, sulphation (figure 2) of 4-OH-CIPC decreased faster than glucuronidation (figure 3). In co-cultured hepatocytes, sulphation (figure 2) and glucuronidation (figure 3) were maintained and the sulphate/glucuronide ratio remained constant during the experimental period. Based upon LDH, GPT and GOT determination, CIPC (12 f-U) does not show any cytotoxicity. 0265-203X/90 $3.00 © 1990 Taylor & Francis Ltd.
Metabolism of chlorpropham by rat hepatocytes
S153
u D.
Downloaded by [University of Cambridge] at 22:43 06 November 2015
u
10
3
5
7
9
11
13
IS
d a y s
Figure 1. Unmetabolized CIPC versus time. M3 30
20
(J
i o
11
15 13 days Figure 2. Formation of 4-OH-CIPC sulphate versus time.
Discussion In primary culture, our results show that hepatocytes rapidly lose their detoxication enzyme activities. This confirms earlier results showing that cytochrome P-450 decreased rapidly in primary culture of hepatocytes (Begue et al. 1984). In contrast, the maintenance of several specific functions such as albumin secretion (Guguen-Guillouzo et al. 1983), taurocholate uptake (Foliot et al. 1985) and cytochrome P-450 level (Begue etal. 1984) were demonstrated in hepatocytes co-cultured with liver epithelial cells. Begue etal. (1983) have shown that Ndemethylation, N-oxidation, reduction and glucuronidation of Ketotifen were also
G. Carrera et al.
S154
Downloaded by [University of Cambridge] at 22:43 06 November 2015
co-culture
3
5
7
9
11
13
15
days
Figure 3. Formation of 4-OH-CIPC glucuronide versus time.
maintained in co-cultured hepatocytes. Our metabolism study confirms these results and shows for the first time that sulphation remains high in co-cultured hepatocytes. The sulphate/glucuronide ratio remained constant throughout the experimental period, at the same value as that observed in isolated hepatocyte suspension (Carrera et al. 1988). Over a 15 day period and probably longer, co-culture of hepatocytes allows the maintenance of specific liver functions and particularly detoxication, in contrast to primary cultures. Consequently, co-culture of hepatocytes could be used as a suitable experimental tool for in vitro semi-chronic toxicity studies. Acknowledgement This work was supported by the Ministere Charge de l'Environnement. References ALARY, J., CARRERA, G., BERGON, M., PERIQUET, A., and VANDAELE, J., 1986, High liquid chromat-
ography determination of chloropropham and its metabolites in isolated rat hepatocyte incubations. Journal of Liquid Chromatography, 9, 3597-3606. CARRERA, G., FORGUES, S., ALARY, J., and LAPONTARIQUE, H., 1988, Effects of an experimental
hepatitis induced by D-galactosamine on the metabolism and toxicity of chlorpropham: in vivo and in vitro studies. Cellular and Molecular Aspects of Glucuronidation, edited by G. SIEST, J. MAGDALOU and B. BURCHELL (Paris: John Libbey Eurotext), pp. 285-288. BEGUE, J. M., LEBIGOT, J. F . , GUGUEN-GUILLOUZO, C., KIECHEL, J. R., and GUILLOUZO, A., 1983,
Cultured human adult hepatocytes: a new model for drug metabolism studies. Biochemical Pharmacology, 32, 1643-1646. BEGUE, J. M., GUGUEN-GUILLOUZO, C., PASDELOUP, N . , and GUILLOUZO, A.,
1984, Prolonged
maintenance of active cytochrome P-450 in adult rat hepatocytes co-cultured with another liver cell type. Hepatology, 4, 839-842. FOLIOT, A., GLAISE, D., ERLINGER, S., and GUGUEN-GUILLOUZO, C., 1985, Long-term maintenance of
taurocholate uptake by adult rat hepatocytes co-cultured with a liver epithelial cell line. Hepatology, 5, 215-219. GUGUEN-GUILLOUZO, C., CLEMENT, B., BAFFET, G., BEAUMONT, C., MOREL-CHANY, E., GLAISE, D.,
and GUILLOUZO, A., 1983, Maintenance and reversibility of active albumin secretion by adult rat hepatocytes co-cultured with another liver epithelial cell type. Experimental Cell Research, 143, 47-54. SEGLEN, P . O. 1973, Preparation of rat liver cells. II-Effects of ions and chelators on tissue dispersion. Experimental Cell Research, 776, 25-30. WILLIAMS, G. M. and GUNN, J. M., 1974, Long-term cell culture of adult rat liver epithelial cells. Experimental Cell Research, 89, 139-142.
