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Influence of medium composition and culture conditions on glutathione S-transferase activity in adult rat hepatocytes during culture

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Summary

Glutathione S-transferase (GST) activity was measured in adult rat hepatocytes during either pure culture or coculture with another rat liver cell type in various media. Addition of nicotinamide, selenium, or dimethylsulfoxide, deprivation of cyst(e)ie and the use of two complex media were tested. Whatever the conditions used, after a constant decrease during the first 24 h, GST remained active over the whole culture period (1–2 wk). However, various patterns were observed: GST activity either remained relatively stable to approximately 50% of the initial value or showed a moderate or strong increase. The highest values were found in pure hepatocyte cultures maintained in the presence of nicotinamide or dimethylsulfoxide. Similar changes were observed using 1-chloro-2,4-dinitrobenzene or 1,2-dichloro-4-nitrobenzene as substrates for GST. Addition of 10−4 M indomethacin resulted in 37 to 60% inhibition of enzyme activity. Thus, these results demonstrate that GST remained expressed during culture but its levels markedly varied depending on the medium composition and type and age of culture.

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References

  1. Alin, P.; Jensson, H.; Guthenberg, C., et al. Purification of major basic glutathione transferase isoenzymes from rat liver by use of affinity chromatography and fast protein liquid chromatography. Anal. Biochem. 146:313–320; 1985.

    Article  PubMed  CAS  Google Scholar 

  2. Bégué, J. M.; Guguen-Guillouzo, C.; Pasdeloup, N., et al. Prolonged maintenance of active cytochrome P-450 in adult rat hepatocytes co-cultured with a rat liver epithelial cell line. Hepatology 4:839–842; 1984.

    PubMed  Google Scholar 

  3. Bissell, D. M.; Guzelian, P. S. Ascorbic acid deficiency and cytochrome P-450 in adult rat hepatocytes in primary monolayer culture. Arch. Biochem. Biophys. 192:569–576; 1979.

    Article  PubMed  CAS  Google Scholar 

  4. Croci, T.; Williams, G. M. Activities of several phase I and phase II xenobiotic biotransformation enzymes in cultured hepatocytes from male and female rats. Biochem. Pharmacol. 34:3029–3035; 1985.

    Article  PubMed  CAS  Google Scholar 

  5. Fahl, W. E.; Michalopoulos, G.; Sattler, G. L., et al. Characteristics of microsomal enzyme controls in primary cultures of rat hepatocytes. Arch. Biochem. Biophys. 192:61–72; 1979.

    Article  PubMed  CAS  Google Scholar 

  6. Fraslin, J. M.; Kneip, B.; Vaulont, S., et al. Dependence of hepatocyte-specific gene expression on cell-cell interactions in primary culture. EMBO J. 4:2487–2491; 1985.

    PubMed  CAS  Google Scholar 

  7. Guguen, C.; Guillouzo, A.; Boisnard, M., et al. Etude ultrastructurale de monocouches d'hépatocytes de rat adulte cultivés en presence d'hémisuccinate d'hydrocortisone. Biol. Gastroenterol. 8:223–231; 1975.

    CAS  Google Scholar 

  8. Guguen-Guillouzo, C.; Guillouzo, A. Modulation of functional activities in cultured rat hepatocytes. Mol. Cell. Biochem. 53/54:35–56; 1983.

    Article  Google Scholar 

  9. Guguen-Guillouzo, C.; Clément, B.; Baffet, G., et al. Maintenance and reversibility of active albumin secretion by adult rat hepatocytes co-cultured with another liver epithelial cell type. Exp. Cell Res. 143:47–54; 1983.

    Article  PubMed  CAS  Google Scholar 

  10. Guzelian, P. S.; Bissell, D. M.; Meyer, U. A. Drug metabolism in adult rat hepatocytes in primary monolayer culture. Gastroenterology 72:1232–1239; 1977.

    PubMed  CAS  Google Scholar 

  11. Habig, W. G.; Pabst, M. J.; Jakoby, W. B. Glutathione S-transferases. The first enzymatic step in mercapturic acid formation. J. Biol. Chem. 249:7130–7139; 1974.

    PubMed  CAS  Google Scholar 

  12. Holme, J. A.; Soderlund, E.; Dybring, E. Drug, metabolism activities of isolated rat hepatocytes in monolayer culture. Acta Pharmacol. Toxicol. 52:348–356; 1983.

    Article  CAS  Google Scholar 

  13. Isom, H. C.; Secott, T.; Georgoff, I., et al. Maintenance of differentiated rat hepatocytes in primary culture. Proc. Natl. Acad. Sci. USA 82:3252–3256; 1985.

    Article  PubMed  CAS  Google Scholar 

  14. Jefferson, D. M.; Clayton, D. F.; Darnell, J. E., et al. Post-transcriptional modulation of gene expression in cultured rat hepatocytes. Mol. Cell. Biochem. 4:1929–1934; 1984.

    CAS  Google Scholar 

  15. Ketterer, B.; Beale, D.; Meyer, D. J. The structure and multiple functions of glutathione transferases. Biochem. Soc. Trans. 10:82–84; 1982.

    PubMed  CAS  Google Scholar 

  16. Ketterer, B.; Beale, D.; Taylor, J. B., et al. The genetic relationships and inducibility of soluble glutathione transferases of the rat liver. Biochem. Soc. Trans. 11:466–467; 1983.

    PubMed  CAS  Google Scholar 

  17. Lake, B. G.; Paine, A. J. The effect of hepatocyte culture conditions on cytochrome P-450 linked drug metabolizing enzymes. Biochemistry 31:2141–2144; 1982.

    CAS  Google Scholar 

  18. Mannervik, B.; Guthenberg, C. In: Jakoby, W. B., ed. Methods in Enzymology. Glutathione transferase (human placenta). New York: Academic Press; 1981:231–235.

    Google Scholar 

  19. Maslansky, C. J.; Williams, G. M. Primary cultures and the levels of cytochrome P-450 in hepatocytes from mouse, rat hamster, and rabbit liver. In Vitro 18:683–693; 1982.

    Article  PubMed  CAS  Google Scholar 

  20. Michalopoulos, G.; Sattler, G.; Sattler, C., et al. Interaction of chemical carcinogens and drug metabolizing enzymes in primary cultures of hepatic cells from the rat. Am. J. Pathol. 85:755–772; 1976.

    PubMed  CAS  Google Scholar 

  21. Morel-Chaney, E.; Gullouzo, C.; Trincal, G., et al. “Spontaneous” neoplastic transformation in vitro of epithelial cell strains of rat liver: cytology, growth and enzymatic activities. Eur. J. Cancer 14:1341–1352; 1978.

    Google Scholar 

  22. Morgenstern, R.; DePierre, J. W. Microsomal glutathione transferase. Purification in inactivated form and further characterization of the activation process, substrate specificity and amino acid composition. Eur. J. Biochem. 134:591–597; 1983.

    Article  PubMed  CAS  Google Scholar 

  23. Paine, A. J.; Hockin, L. J. Nutrient imbalance causes the loss of cytochrome P-450 in liver cell culture: formulation of culture media which maintain cytochrome P-450 at in vivo concentrations. Biochem. Pharmacol. 29:3215–3218; 1980.

    Article  PubMed  CAS  Google Scholar 

  24. Paine, A. J.; Hockin, L. J.; Legg, R. F. Relationship between the ability of nicotinamide to maintain nicotinamide adenine dinucleotide in rat liver cell culture and its effect on cytochrome P-450. Biochem. J. 184:461–463; 1979.

    PubMed  CAS  Google Scholar 

  25. Paine, A. J.; Legg, R. F. Apparent lack of correlation between the loss of cytochrome P-450 in hepatic parenchymal cell culture and the stimulation of haem oxygenase activity. Biochem. Biophys. Res. Commun. 81:672–679; 1978.

    Article  PubMed  CAS  Google Scholar 

  26. Paine, A. J.; Williams, L. J.; Legg, R. F. Apparent maintenance of cytochrome P-450 by nicotinamide in primary cultures of rat hepatocytes. Life Sci. 24:2185–2192; 1979.

    Article  PubMed  CAS  Google Scholar 

  27. Reid, L. M.; Narita, M.; Fujita, M., et al. In: Guillouzo, A.; Guguen-Guillouzo, C. eds. Isolated and cultured hepatocytes. London: John Libbey, Paris: INSERM 1986:225–258.

    Google Scholar 

  28. Sells, M. A.; Chernoff, J.; Cerda, A., et al. Long-term culture and passage of human fetal liver cells that synthesize albumin. In Vitro 21:216–220; 1985.

    CAS  Google Scholar 

  29. Tipping, E.; Ketterer, B. The influence of soluble binding proteins on lipophile transport and metabolism in hepatocytes. Biochem. J. 195:441–452; 1981.

    PubMed  CAS  Google Scholar 

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Authors and Affiliations

  1. INSERM, Unité de Recherches Hépatologiques U 49, Hôpital de Pontchaillou, 35033, Rennes Cédex, France

    Y. Vandenberghe, D. Ratanasavanh, D. Glaise & A. Guillouzo

  2. Dienst Toxicologie, Vrije Universiteit Brussel, Brussels, Belgium

    Y. Vandenberghe, D. Ratanasavanh, D. Glaise & A. Guillouzo

Authors
  1. Y. Vandenberghe
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  2. D. Ratanasavanh
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  3. D. Glaise
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  4. A. Guillouzo
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Additional information

Y. V. was supported by Instituut voor Wetenschappelÿk Onderzoek in Landbouw en Nijverheid. This work was supported by INSERM.

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Vandenberghe, Y., Ratanasavanh, D., Glaise, D. et al. Influence of medium composition and culture conditions on glutathione S-transferase activity in adult rat hepatocytes during culture. In Vitro Cell Dev Biol 24, 281–288 (1988). https://doi.org/10.1007/BF02628828

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