Skip to main content
SpringerLink
Log in

Properties of peroxisomes and their induction by clofibrate in normal adult rat hepatocytes in primary culture

  • Published:
In Vitro Aims and scope Submit manuscript

Summary

Alterations in peroxisomes and catalase activity and their responsiveness to clofibrate in adult rat hepatocytes in primary culture were investigated.

The numbers of peroxisomes with and without crystalloid nucleoids per unit cytoplasmic area were preserved in cultured hepatocytes for 2 d after seeding at a level comparable to that of freshly isolated hepatocytes. At Day 3 in culture, the number of anucleoid peroxisomes was reduced in untreated hepatocytes, accompanied by more significant reduction in the number of nucleoid-containing peroxisomes, which decreased until Day 5. Peroxisome diameters were reduced in untreated hepatocytes at Day 2 and this decrease in the diameter was continued until Day 7. Catalase activity in untreated hepatocytes decreased markedly with culture age.

The number of anucleoid peroxisomes was significantly greater in hepatocytes treated with 2mM clofibrate in culture than in freshly isolated hepatocytes for 2 d or in untreated hepatocytes of the same culture age through 7 d. The number of nucleoid-containing peroxisomes in the treated cells began to decrease in 3 d, but was greater than that of untreated cells at Days 3 and 5. Peroxisomes with well-developed nucleoids were observed frequently in the treated cells even at Day 7. Peroxisome diameters were greater in the treated cells than in untreated cells at Days 3, 5, and 7. Catalase activity was always higher in the treated cells than in untreated cells. These results suggest that clofibrate is effective in inducing peroxisome proliferation as well as in maintaining the organelles in cultured hepatocytes.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Similar content being viewed by others

References

  1. Böck, P.; Kramar, R.; Pavelka, M. Peroxisomes and related particles in animal tissues. In: Alfert, M.; Beerman, W.; Franke, W. W.; Rudkin, G.; Sitte, P. eds. Cell biology monographs, vol. 7. New York: Springer-Verlag; 1980.

    Google Scholar 

  2. Tsukada, H.; Mochizuki, Y.; Fujiwara, S. The nucleoids of rat liver cell microbodies. Fine structure and enzymes. J. Cell Biol. 28: 449–460; 1966.

    Article  PubMed  CAS  Google Scholar 

  3. Hruban, Z.; Rechcigl, M., Jr. Microbodies and related particles: morphology, biochemistry and physiology. Int. Rev. Cytol. [Suppl.] 1: 1–296; 1966.

    Google Scholar 

  4. Tsukada, H.; Mochizuki, Y.; Konishi, T. Morphogenesis and development of microbodies of hepatocytes of rats during pre- and postnatal growth. J. Cell Biol. 37: 231–243; 1968.

    Article  PubMed  CAS  Google Scholar 

  5. Tsukada, H.; Mochizuki, Y.; Gotoh, M. Matrical inclusions induced by clofibrate in hepatic microbodies of rats fed 2-acetylaminofluorene. J. Natl. Cancer Inst. 54: 519–523; 1975.

    PubMed  CAS  Google Scholar 

  6. Tsukada, H.; Mochizuki, Y.; Itabashi, M.; Gotoh, M.; Morris, H. P. Response of microbodies in Morris hepatoma 9618A to clofibrate. J. Natl. Cancer Inst. 55: 153–158; 1975.

    PubMed  CAS  Google Scholar 

  7. Itabashi, M.; Mochizuki, Y.; Tsukada, H. Peroxisomes in liver tumors of rats induced by 3′-methyl-4-(dimethylamino)-azobenzene. Gann 66: 463–472; 1975.

    PubMed  CAS  Google Scholar 

  8. Itabashi, M.; Mochizuki, Y.; Tsukada, H. Changes in peroxisomes in preneoplastic liver of rats induced by 3′-methyl-4-dimethylamino-azobenzene. Cancer Res. 37: 1035–1043; 1977.

    CAS  Google Scholar 

  9. Tsukada, H.; Gotoh, M.; Mochizuki, Y.; Furukawa, K. Changes in peroxisomes in preneoplastic liver and hepatoma of mice induced by α-benzene hexacloride. Cancer Res. 39: 1628–1634; 1979.

    PubMed  CAS  Google Scholar 

  10. Sawada, N.; Furukawa, K.; Gotoh, M.; Mochizuki, Y.; Tsukada, H. Primary culture of preneoplastic hepatocytes from rats treated with 2-acetylaminofluorene and clofibrate: relationship between resistance to dimethylnitrosamine and responsiveness of peroxisomes. Gann 73: 1–6; 1982.

    PubMed  CAS  Google Scholar 

  11. Gotoh, M.; Mochizuki, Y.; Sawada, N.; Tsukada, H. Matrical inclusions of peroxisomes induced by clofibrate in preneoplastic hepatocytes of rats fed 3′ methyl-4-dimethylaminoazobenzene. Gann 73: 7–13; 1982.

    PubMed  CAS  Google Scholar 

  12. Alexander, R. W.; Grisham, J. M. Explantculture of rat liver. I. Method, morphology, and cytogenesis. Lab. Invest. 22: 56–62; 1970.

    Google Scholar 

  13. Gershensen, L. E.; Okigaki, T.; Andersson, M.; Molson, J.; Davidson, M. B. Fine structure and growth characteristics of cultured rat liver cells. Insulin effects. Exp. Cell Res. 71: 49–58; 1972.

    Article  Google Scholar 

  14. Sattler, C. A.; Michalopoulos, G.; Sattler, G. L.; Pitot, H. C. Ultrastructure of adult rat hepatocytes cultured on floating collagen membranes. Cancer Res. 39: 1539–1549; 1978.

    Google Scholar 

  15. Watanabe, H. A fine structural study of liver cultures. Exp. Cell Res. 42: 685–699; 1966.

    Article  PubMed  CAS  Google Scholar 

  16. Watanabe, H.; Essner, E. A comparative cytologic study of cultivation of hepatomas of different growth rates. Cancer Res. 29: 631–644; 1968.

    Google Scholar 

  17. Rose, G. G.; Kumegawa, M.; Cattoni, M. The circumfusion system for multipurpose culture chambers. II. The protracted maintenance of differentiation of fetal and newborn mouse liver in vitro. J. Cell Biol. 39: 430–450; 1968.

    Article  PubMed  CAS  Google Scholar 

  18. Williams, G. M.; Bermudez, E.; San, R. H. C.; Goldblatt, P. J.; Laspia, M. Rat hepatocyte primary cultures. IV. Maintenance in defined medium and the role of production of plasminogen activator and other proteases. In Vitro 14: 824–837; 1978.

    Article  PubMed  CAS  Google Scholar 

  19. Williams, G. M.; Stromberg, K.; Kroes, R. Cytochemical and ultrastructural alterations associated with confluent growth in cell cultures of epithelial-like cells from rat liver. Lab. Invest. 29: 293–303; 1973.

    PubMed  CAS  Google Scholar 

  20. Furukawa, K.; Mochizuki, Y.; Gotoh, M.; Sawada, N.; Tsukada, H. Changes in peroxisomes and catalase activity of Morris hepatoma 7316A in culture. Tumor Res. 15: 24–34; 1981.

    Google Scholar 

  21. Karasaki, S.; Simard, A.; De Lamirande, G. Ultrastructural analysis of malignant conversion in vitro of epithelial cells derived from normal adult rat liver. Eur. J. Cancer 12: 527–534; 1976.

    PubMed  CAS  Google Scholar 

  22. Bieri-Bonnit, F.; Bentley, P.; Waechter, F.; Suter, J.; Waldmeiner, U.; Stäubli, W. Effect of nafenopin on peroxisomal enzymes and DNA synthesis in primary cultures of rat hepatocytes. Ann. NY Acad. Sci. 386: 473–474; 1982.

    Article  Google Scholar 

  23. Gray, J. T. B.; Beamand, J. A.; Lake, B. G.; Foster, J. R.; Gangolli, S. D. Peroxisome proliferation in cultured rat hepatocytes produced by clofibrate and phthalate ester metabolites. Toxicol. Lett. 10: 273–279; 1982.

    Article  PubMed  CAS  Google Scholar 

  24. Gray, J. T. B.; Lake, B. G.; Foster, J. R.; Gangolli, S. D. Peroxisome proliferation in primary cultures of hepatocytes. Toxicol. Appl. Pharmacol. 67: 15–25; 1983.

    Article  PubMed  CAS  Google Scholar 

  25. Krisans, S. K.; Schlote, E. B.; Ruby, S. Induction of peroxisomal enzymes and lipogenesis in cultured rat hepatoma cells. Ann. NY Acad. Sci. 386: 468–472; 1982.

    Article  CAS  Google Scholar 

  26. Furukawa, K.; Gotoh, M.; Mochizuki, Y.; Tsukada, H. Primary cultures of adult rat hepatocytes: Induction and maintenance of peroxisomes (abstr.). Trans. Soc. Pathol. Jpn. 67: 108–109; 1978.

    Google Scholar 

  27. Berry, M. M.; Friend, D. S. High yield of preparation of isolated rat liver parenchymal cells. A biochemical and fine structural study. J. Cell Biol. 43: 506–520; 1969.

    Article  PubMed  CAS  Google Scholar 

  28. Le Cam, A.; Guillouzo, A.; Freychet, P. Ultrastructural and biochemical studies of isolated adult rat hepatocytes prepared under hypoxic conditions. Exp. Cell Res. 98: 382–395; 1976.

    Article  PubMed  Google Scholar 

  29. Williams, G. M.; Bermudez, E.; Scaramuzzino, D. Rat hepatocyte primary cell cultures. III. Improved dissociation and attachment techniques and the enhancement of survival by culture medium. In Vitro 13: 809–817; 1977.

    Article  PubMed  CAS  Google Scholar 

  30. Beers, R. F. Jr.; Sizer, I. W. A spectrophotometric method for measuring the breakdown of hydrogen peroxide by catalase. J. Biol. Chem. 195: 133–140; 1952.

    PubMed  CAS  Google Scholar 

  31. Lowry, O. H.; Rosenbrough, N. J.; Farr, A. L.; Randall, R. J. Protein measurement with Folin phenol reagent. J. Biol. Chem. 193: 265–275; 1951.

    PubMed  CAS  Google Scholar 

  32. Novikoff, A. B.; Novikoff, P. M.; Davis, C.; Quintana, N. Studies on microperoxisomes. II. A cytochemical method for light and electron microscopy. J. Histochem. Cytochem. 20: 1006–1023; 1972.

    PubMed  CAS  Google Scholar 

  33. Brinkley, B. R.; Murphy, P.; Richardson, L. C. Procedure for embedding in situ selected cells cultured in vitro. J. Cell Biol. 35: 279–283; 1967.

    Article  PubMed  CAS  Google Scholar 

  34. Novikoff, A. B.; Novikoff, P. M.; Davis, C.; Quintana, N. Studies on microperoxisomes. V. Are microperoxisomes ubiquitous in mammalian cells? J. Histochem. Cytochem. 21: 737–755; 1973.

    PubMed  CAS  Google Scholar 

  35. Stäubli, W.; Schweizer, W.; Suter, J.; Weibel, E. R. The proliferative response of hepatic peroxisomes of neonatal rats to treatment with SU-13,437 (nafenopin). J. Cell Biol. 74: 665–689; 1977.

    Article  PubMed  Google Scholar 

  36. Svoboda, D.; Grady, H.; Azarnoff, D. L. Microbodies in experimentally altered cells. J. Cell Biol. 35: 127–152; 1967.

    Article  PubMed  CAS  Google Scholar 

  37. Legg, P. G.; Wood, R. L. New observations on microbodies. A cytochemical study on CPIB-treated rat liver. J. Cell Biol. 45: 118–129; 1970.

    Article  PubMed  CAS  Google Scholar 

  38. Kolde, G.; Roessner, A.; Themann, H. Effects of clofibrate (alpha-p-clorophenoxyisobutyryl-ethylester) on male rat liver. Correlated ultrastructural-morphometric and biochemical investigations. Virchows Arch. [Cell Pathol.] B22: 73–87; 1976.

    Google Scholar 

  39. Svoboda, D.; Azarnoff, D. L. Response of hepatic microbodies to a hypolipidemic agent, ethyl chlorophenoxyisobutylate (CPIB). J. Cell Biol. 30: 442–450; 1966.

    Article  PubMed  CAS  Google Scholar 

  40. Markwell, M. A. K.; Bieber, L. L.; Tolbert, E. Differential increase of hepatic peroxisomal, mitochondrial and microsomal carnitine acyltransferase in clofibrate-fed rats. Biochem. Pharmacol. 26: 1697–1703; 1977.

    Article  PubMed  CAS  Google Scholar 

  41. Lazarow, P. B.; De Duve, C. A fatty acyl-CoA oxidizing system in rat liver peroxisomes: enhancement by clofibrate, a hypolipidemic drug. Proc. Natl. Acad. Sci. USA 73: 2043–2046; 1979.

    Article  Google Scholar 

  42. Reddy, M. K.; Qureshi, S. A.; Hollenberg, P. F.; Reddy, J. K. Immunochemical identity of peroxisomal enoyl-CoA hydratase with the peroxisome proliferation associated 80,000 mol. wt. polypeptide in rat liver. J. Cell Biol. 89: 406–417; 1981.

    Article  PubMed  CAS  Google Scholar 

  43. Sirica, A. E.; Richards, W.; Tsukada, Y.; Sattler, C. A.; Pitot, H. C. Fetal phenotypic expression in primary cultures of adult rat hepatocytes on collagen gel/nylon meshes. Proc. Natl. Acad. Sci. USA 76: 283–287; 1979.

    Article  PubMed  CAS  Google Scholar 

  44. Edwards, A. M. Regulation of γ-glutamyltransferase in rat hepatocyte monolayer cultures. Cancer Res. 42: 1107–1115; 1982.

    PubMed  CAS  Google Scholar 

  45. Rosenberg, M. R.; Strom, S. C.; Michalopoulos, G. Effect of hydrocortisone and nicotinamide on gamma glutamyltransferase in primary cultures of rat hepatocytes. In Vitro 18: 775–782; 1982.

    PubMed  CAS  Google Scholar 

  46. Ivarie, R. D.; Morris, J. A.; Eberhardt, N. L. Hormonal domains of response: actions of glucocorticoid and thyroid hormone in regulating pleiotropic responses in cultured cells. Recent Prog. Horm. Res. 36: 195–235; 1980.

    PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Pathology, Cancer Research Institute, Sapporo Medical College, Nishi-17-chome, Minami-1-jo, Chuo-ku, 060, Sapporo, Japan

    Kazunori Furukawa, Yohichi Mochizuki & Norimasa Sawada

Authors
  1. Kazunori Furukawa
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Yohichi Mochizuki
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Norimasa Sawada
    View author publications

    You can also search for this author in PubMed Google Scholar

Additional information

This work was supported in part by Grants-in-Aid for Scientific Research from Ministry of Education, Science and Culture, Japan, 448143, 50168, 501069, and 577196, and by a Grant-in-Aid from Hokkaido Geriatrics Research Institute.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Furukawa, K., Mochizuki, Y. & Sawada, N. Properties of peroxisomes and their induction by clofibrate in normal adult rat hepatocytes in primary culture. In Vitro 20, 573–584 (1984). https://doi.org/10.1007/BF02639773

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF02639773

Key words

Search

Navigation