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Japanese Journal of Cancer Research : Gann logoLink to Japanese Journal of Cancer Research : Gann
. 1989 Feb;80(2):126–131. doi: 10.1111/j.1349-7006.1989.tb02279.x

Changes in the Quantity and Activity of Cytochrome P‐450 Isozymes in Primary Cultured Rat Hepatocytes

Masayuki Namiki 1, Masakuni Degawa 1, Takashi Masuko 1, Yoshiyuki Hashimoto 1,
PMCID: PMC5917698  PMID: 2498247

Abstract

Hepatocytes from male Spragne‐Dawley rats pretreated with a cytochrome P‐450 inducer, 3‐methoxy‐4‐aminoazobenzene (3‐MeO‐AAB), 3‐methylcholanthrene (MC) or phenobarbital (PB), were cultured in vitro, and changes in the quantity and activity of microsomal cytochrome P‐450 isozymes in the cells were determined by means of immunochemical methods and a bacterial mutation test, respectively. The results of enzyme‐linked immunosorbent assay using monoclonal antibodies against rat P‐450 isozymes ‐ test using Salmonella typhimurium TA 98 and carcinogenic aromatic amines. These results indicate that microsomal cytochrome P‐450c in primary cultured rat hepatocytes is more stable in culture, in terms of both quantity and activity, than cytochrome P‐450d and P‐450b/e.

Keywords: Rat hepatocytes, Cytochrome P‐450, Anti‐P‐450 monoclonal antibodies, Cytochrome P‐450 inducers

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REFERENCES

  • 1. ) Degawa , M. , Kojima , M. and Hashimoto , Y.Species difference between rats and mice in activities of enzymes activating aromatic amines: effect of dietary 3‐methoxy‐4‐aminoazobenzene . Gann , 75 , 966 – 975 ( 1984. ). [PubMed] [Google Scholar]
  • 2. ) Degawa , M. , Hishinuma , T. , Yoshida , H. and Hashimoto , Y.Species, sex and organ differences in induction of a P‐450 isozyme responsible for carcinogen activation: effect of dietary hepatocarcinogenic tryptophan pyrolysate components in mice and rats . Carcinogenesis , 8 , 1913 – 1918 ( 1987. ). [DOI] [PubMed] [Google Scholar]
  • 3. ) Hashimoto , Y. , Degawa , M. , Kojima , M. and Hishinuma , T.Induction of carcinogen activation enzyme(s) by feeding of a carcinogenic tryptophan pyrolysate correlates to sex difference in the carcinogenesis of the mouse . Gann , 73 , 508 – 510 ( 1982. ). [PubMed] [Google Scholar]
  • 4. ) Degawa , M. , Kojima , M. , Hishinuma , T. and Hashimoto , Y.Sex‐dependent induction of hepatic enzymes for mutagenic activation of a tryptophan pyrolysate component, 3‐amino‐l,4‐dimethyl‐5H‐pyrido[4,3‐6]indole, by feeding in mice . Cancer Res. , 45 , 96 – 102 ( 1985. ). [PubMed] [Google Scholar]
  • 5. ) Miller , J. A. , Miller , E. C. and Finger , G. C.Further studies on the carcinogenicity of dyes related to 4‐methylaminoazobenzene. The requirement for an unsub‐stituted 2‐position . Cancer Res. , 17 , 387 – 398 ( 1957. ). [PubMed] [Google Scholar]
  • 6. ) Williams , G. M.Detection of chemical carcinogens by unscheduled DNA synthesis in rat liver primary cell culture . Cancer Res. , 37 , 1845 – 1851 ( 1977. ). [PubMed] [Google Scholar]
  • 7. ) Hashimoto , Y. , Masuko , T. , Itoh , K. , Yagita , H. , Hishinuma , T. , Degawa , M. , Kamataki , T. and Kato , R.Monoclonal antibodies against a high spin form of rat cytochrome P‐448 . Biochem. Biophys. Res. Commun. , 131 , 600 – 606 ( 1985. ). [DOI] [PubMed] [Google Scholar]
  • 8. ) Degawa , M. , Ohta , A. , Namiki , M. , Masuko , T. and Hashimoto , Y.In vivo selection of a low spin form of cytochrome P‐448 from 3‐methylchoIanthrene‐induced rat cytochrome P‐450 isozymes by carbon tetrachloride . Biochem. Pharmacol. , 36 , 3315 – 3317 ( 1987. ). [DOI] [PubMed] [Google Scholar]
  • 9. ) Masuko , T. , Sugahara , K. , Nakajima , S. and Hashimoto , Y.Altered expression of immunohistochemically detected P‐450 component(s) in nitrosamine‐induced rat urinary bladder lesion . Jpn. J. Cancer Res. , 78 , 840 – 845 ( 1987. ). [PubMed] [Google Scholar]
  • 10. ) Hishinuma , T. , Degawa , M. , Masuko , T. and Hashimoto , Y.Induction of cytochrome P‐448 isozyme(s) in primary cultured rat hepatocytes by drugs which induced different isozymes in vivo . Biochem. Biophys. Res. Commun. , 148 , 947 – 953 ( 1987. ). [DOI] [PubMed] [Google Scholar]
  • 11. ) Lowry , O. H. , Rosebrough , N. J. , Farr , A. L. and Randall , R. J.Protein measurement with the Folin phenol reagent . J. Biol. Chem. , 193 , 265 – 275 ( 1951. ). [PubMed] [Google Scholar]
  • 12. ) Omura , T. and Sato , R.The carbon monooxide binding pigment of liver microsomes. I. Evidence for its nemoprotein nature . J. Biol. Chem. , 239 , 2370 – 2378 ( 1964. ). [PubMed] [Google Scholar]
  • 13. ) Degawa , M. , Kojima , M. , Masuko , T. , Hishinuma , T. and Hashimoto , Y.3‐Methoxy‐4‐aminoazobenzene, a selective inducer for a high spin form of cytochrome P‐448 in rat liver microsomes . Biochem. Biophys. Res. Commun. , 133 , 1072 – 1077 ( 1985. ). [DOI] [PubMed] [Google Scholar]
  • 14. ) Degawa , M. , Kojima , M. , Sato , Y. and Hashimoto , Y.Induction of a high spin form of microsomal cytochrome P‐448 in rat liver by 4‐aminoazobenzene derivatives . Biochem. Pharmacol , 35 , 3565 – 3570 ( 1986. ). [DOI] [PubMed] [Google Scholar]
  • 15. ) Thomas , P. E. , Reik , L. M. , Ryan , D. E , and Levin , W.Induction of two immunochemically related rat liver P‐450 isozymes, cytochromes P‐450c and P‐450d, by structurally diverse xenobiotics . J. Biol Chem. , 258 , 4590 – 4598 ( 1983. ). [PubMed] [Google Scholar]
  • 16. ) Guengerich , F. P. , Dannan , G. A. , Wright , S. T. , Martin , M. V. and Kaminsky , L. S.Purification and characterization of liver microsomal cytochromes P‐450: electrophoretic, spectral, catalytic, and immunochemical properties and inducibility of eight isozymes isolated from rats treated with phenobarbital or β‐naphthoflavone . Biochemistry , 21 , 6019 – 6030 ( 1982. ). [DOI] [PubMed] [Google Scholar]
  • 17. ) Degawa , M. , Kojima , M. and Hashimoto , Y.3‐Methoxy‐4‐aminoazobenzene, a unique carcinogenic aromatic amine as a substrate for P‐450‐mediated mutagenesis . Mutat. Res. , 152 , 125 – 129 ( 1985. ). [DOI] [PubMed] [Google Scholar]
  • 18. ) Kamataki , T. , Maeda , K. , Yamazoe , Y. , Matsuda , N. , Ishii , K. and Kato , R.A high spin form of P‐450 highly purified from PCB‐treated rats: catalytic characterization and irnmunochemical quantitation in liver microsomes . Mol. Pharmacol. , 24 , 146 – 155 ( 1983. ). [PubMed] [Google Scholar]
  • 19. ) Degawa , M. , Ueno , H. , Miura , S. , Ohta , A. and Namiki , M.A simple method for assessment of rat cytochrome P‐448 isozymes responsible for the mutagenic activation of carcinogenic chemicals . Mutat. Res. , 203 , 333 – 338 ( 1988. ). [DOI] [PubMed] [Google Scholar]
  • 20. ) Fahl , W. E. , Michalopoulos , G. , Sattler , G. L. , Jefcoate , C. R. and Pitot , H. C.Characteristics of microsomal enzyme controls in primary cultures of rat hepatocytes . Arch, Biochem. Biophys. , 192 , 61 – 72 ( 1979. ). [DOI] [PubMed] [Google Scholar]
  • 21. ) Steward , A. R. , Dannan , G. A. , Guzellian , P. S. and Guengerich , F. P.Changes in the concentration of seven forms of cytochrome P‐450 in primary cultures of adult rat hepatocytes . Mol. Pharmacol. , 27 , 125 – 132 ( 1985. ). [PubMed] [Google Scholar]
  • 22. ) Steward , A. R. , Wrighton , S. A. , Pasco , D. S. , Pagan , J. B. , Li , D , and Guzelian , P. S.Synthesis and degradation of 3‐methylcholanthrene‐inducible cytochromes P‐450 and their mRNAs in primary monolayer cultures of adult rat hepatocytes . Arch. Biochem. Biophys. , 241 , 494 – 508 ( 1985. ). [DOI] [PubMed] [Google Scholar]
  • 23. ) Grant , M. H. , Smith , S. J. and Burke , M. D.Strain differences in the maintenance of cytochrome P‐450 and mixed‐function‐oxidase activities in cultured rat hepatocytes . Biochem. J. , 239 , 785 – 788 ( 1986. ). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24. ) Dickens , M. and Peterson , R. E.Effects of a hormone‐supplemented medium on cytochrome P‐450 content and monooxygenase activities of rat hepatocytes in primary culture . Biochem. Pharmacol. , 29 , 1231 – 1238 ( 1982. ). [DOI] [PubMed] [Google Scholar]
  • 25. ) Lake , B. G. and Paine , A. J.The effect of hepatocyte culture condition on cytochrome linked drug metabolizing enzymes . Biochem. Pharmacol. , 31 , 2141 – 2144 ( 1982. ). [DOI] [PubMed] [Google Scholar]
  • 26. ) Paine , A. J. and 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. ). [DOI] [PubMed] [Google Scholar]

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