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. 1992 Aug 15;286(Pt 1):289–294. doi: 10.1042/bj2860289

Effect of pyrazole, cobalt and phenobarbital on mouse liver cytochrome P-450 2a-4/5 (Cyp2a-4/5) expression.

B Hahnemann 1, P Salonpää 1, M Pasanen 1, J Mäenpää 1, P Honkakoski 1, R Juvonen 1, M A Lang 1, O Pelkonen 1, H Raunio 1
PMCID: PMC1133053  PMID: 1520280

Abstract

Pyrazole, cobalt and phenobarbital increase the activity of coumarin 7-hydroxylase (COH) in mouse liver. To study the mechanism of this increase, we measured the expression of the cytochrome P-450 2a-4/5 (Cyp2a-4/5) complex, which mediates testosterone 15 alpha-hydroxylase and COH activities, as a function of dose and time after the treatment of C57BL/6 (B6) and DBA/2 (D2) male mice with the inducers. COH activity and Cyp2a-4/5 steady-state mRNA levels were increased in both strains in response to the inducers. No marked effect occurred with testosterone 15 alpha-hydroxylase or activities associated with Cyp1a-1 or Cyp2e-1. A 2-7-fold increase in response to the inducers was seen in the amount of P-450Coh (cytochrome P-450 isoenzyme catalysing coumarin 7-hydroxylation) protein in Western immunoblots. PCR amplification of a 1 kb region in Cyp2a-4/5-mRNA-derived cDNA, followed by cutting at the diagnostic PstI site, showed that most of the steady-state mRNA consisted of Cyp2a-5, which is also the form most affected by pyrazole. Nuclear run-off analysis revealed no increase in the transcription rate of Cyp2a-4/5 after pyrazole or cobalt treatment, whereas a 2-3-fold increase occurred after phenobarbital pretreatment in B6 mice. Together with previous reports [Aida & Negishi (1991) Biochemistry 30, 8041-8045], the current data suggest that both pyrazole and cobalt increase COH catalytic activity by affecting Cyp2a-5 by post-transcriptional mechanisms in mice.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Adesnik M., Atchison M. Genes for cytochrome P-450 and their regulation. CRC Crit Rev Biochem. 1986;19(3):247–305. doi: 10.3109/10409238609084657. [DOI] [PubMed] [Google Scholar]
  2. Aida K., Negishi M. Posttranscriptional regulation of coumarin 7-hydroxylase induction by xenobiotics in mouse liver: mRNA stabilization by pyrazole. Biochemistry. 1991 Aug 13;30(32):8041–8045. doi: 10.1021/bi00246a023. [DOI] [PubMed] [Google Scholar]
  3. Aitio A. A simple and sensitive assay of 7-ethoxycoumarin deethylation. Anal Biochem. 1978 Apr;85(2):488–491. doi: 10.1016/0003-2697(78)90245-2. [DOI] [PubMed] [Google Scholar]
  4. Albretsen C., Haukanes B. I., Aasland R., Kleppe K. Optimal conditions for hybridization with oligonucleotides: a study with myc-oncogene DNA probes. Anal Biochem. 1988 Apr;170(1):193–202. doi: 10.1016/0003-2697(88)90108-x. [DOI] [PubMed] [Google Scholar]
  5. Anderson L. M., Angel M. Induction of dimethylnitrosamine demethylase activity in mouse liver by polychlorinated biphenyls and 3-methylcholanthrene. Biochem Pharmacol. 1980 May 15;29(10):1375–1383. doi: 10.1016/0006-2952(80)90433-5. [DOI] [PubMed] [Google Scholar]
  6. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  7. Burke M. D., Prough R. A., Mayer R. T. Characteristics of a microsomal cytochrome P-448-mediated reaction. Ethoxyresorufin O-de-ethylation. Drug Metab Dispos. 1977 Jan-Feb;5(1):1–8. [PubMed] [Google Scholar]
  8. Burke M. D., Thompson S., Elcombe C. R., Halpert J., Haaparanta T., Mayer R. T. Ethoxy-, pentoxy- and benzyloxyphenoxazones and homologues: a series of substrates to distinguish between different induced cytochromes P-450. Biochem Pharmacol. 1985 Sep 15;34(18):3337–3345. doi: 10.1016/0006-2952(85)90355-7. [DOI] [PubMed] [Google Scholar]
  9. Burkhart B. A., Harada N., Negishi M. Sexual dimorphism of testosterone 15 alpha-hydroxylase mRNA levels in mouse liver. cDNA cloning and regulation. J Biol Chem. 1985 Dec 5;260(28):15357–15361. [PubMed] [Google Scholar]
  10. Chirgwin J. M., Przybyla A. E., MacDonald R. J., Rutter W. J. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry. 1979 Nov 27;18(24):5294–5299. doi: 10.1021/bi00591a005. [DOI] [PubMed] [Google Scholar]
  11. Conney A. H. Induction of microsomal enzymes by foreign chemicals and carcinogenesis by polycyclic aromatic hydrocarbons: G. H. A. Clowes Memorial Lecture. Cancer Res. 1982 Dec;42(12):4875–4917. [PubMed] [Google Scholar]
  12. Hahnemann B., Kühn B., Heubel F., Legrum W. Selective induction of the coumarin hydroxylase by N-containing heteroaromatic compounds. Arch Toxicol Suppl. 1989;13:297–301. doi: 10.1007/978-3-642-74117-3_54. [DOI] [PubMed] [Google Scholar]
  13. Hawkes R., Niday E., Gordon J. A dot-immunobinding assay for monoclonal and other antibodies. Anal Biochem. 1982 Jan 1;119(1):142–147. doi: 10.1016/0003-2697(82)90677-7. [DOI] [PubMed] [Google Scholar]
  14. Juvonen R. O., Autio S., Lang M. A. Pyrazole as a modifier of liver microsomal monooxygenase in DBA/2N and AKR/J mice. Biochem Pharmacol. 1987 Nov 15;36(22):3993–3997. doi: 10.1016/0006-2952(87)90469-2. [DOI] [PubMed] [Google Scholar]
  15. Juvonen R. O., Kaipainen P. K., Lang M. A. Selective induction of coumarin 7-hydroxylase by pyrazole in D2 mice. Eur J Biochem. 1985 Oct 1;152(1):3–8. doi: 10.1111/j.1432-1033.1985.tb09156.x. [DOI] [PubMed] [Google Scholar]
  16. Juvonen R. O., Shkumatov V. M., Lang M. A. Purification and characterization of a liver microsomal cytochrome P-450 isoenzyme with a high affinity and metabolic capacity for coumarin from pyrazole-treated D2 mice. Eur J Biochem. 1988 Jan 15;171(1-2):205–211. doi: 10.1111/j.1432-1033.1988.tb13777.x. [DOI] [PubMed] [Google Scholar]
  17. Kocer Z., Raunio H., Pasanen M., Arvela P., Raiskila T., Honkakoski P., Lang M. A., Negishi M., Pelkonen O. Comparison between cobalt and pyrazole in the increased expression of coumarin 7-hydroxylase in mouse liver. Biochem Pharmacol. 1991 Feb 1;41(3):462–465. doi: 10.1016/0006-2952(91)90547-i. [DOI] [PubMed] [Google Scholar]
  18. Kyhse-Andersen J. Electroblotting of multiple gels: a simple apparatus without buffer tank for rapid transfer of proteins from polyacrylamide to nitrocellulose. J Biochem Biophys Methods. 1984 Dec;10(3-4):203–209. doi: 10.1016/0165-022x(84)90040-x. [DOI] [PubMed] [Google Scholar]
  19. Lang M. A., Juvonen R., Järvinen P., Honkakoski P., Raunio H. Mouse liver P450Coh: genetic regulation of the pyrazole-inducible enzyme and comparison with other P450 isoenzymes. Arch Biochem Biophys. 1989 May 15;271(1):139–148. doi: 10.1016/0003-9861(89)90264-6. [DOI] [PubMed] [Google Scholar]
  20. Legrum W., Netter K. J. Characteristics of coumarin metabolism by liver microsomes from cobalt-pretreated mice. Xenobiotica. 1980 Apr;10(4):271–279. doi: 10.3109/00498258009033755. [DOI] [PubMed] [Google Scholar]
  21. Legrum W., Stuehmeier G., Netter K. J. Cobalt as a modifier of microsomal monooxgenases in mice. Toxicol Appl Pharmacol. 1979 Apr;48(2):195–204. doi: 10.1016/0041-008x(79)90024-3. [DOI] [PubMed] [Google Scholar]
  22. Lindberg R., Burkhart B., Ichikawa T., Negishi M. The structure and characterization of type I P-450(15) alpha gene as major steroid 15 alpha-hydroxylase and its comparison with type II P-450(15) alpha gene. J Biol Chem. 1989 Apr 15;264(11):6465–6471. [PubMed] [Google Scholar]
  23. Maines M. D. New developments in the regulation of heme metabolism and their implications. Crit Rev Toxicol. 1984;12(3):241–314. doi: 10.3109/10408448409021604. [DOI] [PubMed] [Google Scholar]
  24. Mangoura S. A., Strack A., Legrum W., Netter K. J. Indium selectively increases the cytochrome P-450 dependent O-dealkylation of coumarin derivatives in male mice. Naunyn Schmiedebergs Arch Pharmacol. 1989 May;339(5):596–602. doi: 10.1007/BF00167267. [DOI] [PubMed] [Google Scholar]
  25. Nebert D. W., Gonzalez F. J. P450 genes: structure, evolution, and regulation. Annu Rev Biochem. 1987;56:945–993. doi: 10.1146/annurev.bi.56.070187.004501. [DOI] [PubMed] [Google Scholar]
  26. Nebert D. W., Nelson D. R., Coon M. J., Estabrook R. W., Feyereisen R., Fujii-Kuriyama Y., Gonzalez F. J., Guengerich F. P., Gunsalus I. C., Johnson E. F. The P450 superfamily: update on new sequences, gene mapping, and recommended nomenclature. DNA Cell Biol. 1991 Jan-Feb;10(1):1–14. doi: 10.1089/dna.1991.10.1. [DOI] [PubMed] [Google Scholar]
  27. Negishi M., Lindberg R., Burkhart B., Ichikawa T., Honkakoski P., Lang M. Mouse steroid 15 alpha-hydroxylase gene family: identification of type II P-450(15)alpha as coumarin 7-hydroxylase. Biochemistry. 1989 May 16;28(10):4169–4172. doi: 10.1021/bi00436a007. [DOI] [PubMed] [Google Scholar]
  28. OMURA T., SATO R. THE CARBON MONOXIDE-BINDING PIGMENT OF LIVER MICROSOMES. I. EVIDENCE FOR ITS HEMOPROTEIN NATURE. J Biol Chem. 1964 Jul;239:2370–2378. [PubMed] [Google Scholar]
  29. Okey A. B. Enzyme induction in the cytochrome P-450 system. Pharmacol Ther. 1990;45(2):241–298. doi: 10.1016/0163-7258(90)90030-6. [DOI] [PubMed] [Google Scholar]
  30. Raunio H., Kojo A., Juvonen R., Honkakoski P., Järvinen P., Lang M. A., Vähäkangas K., Gelboin H. V., Park S. S., Pelkonen O. Mouse hepatic cytochrome P-450 isozyme induction by 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene, pyrazole, and phenobarbital. Biochem Pharmacol. 1988 Nov 1;37(21):4141–4147. doi: 10.1016/0006-2952(88)90108-6. [DOI] [PubMed] [Google Scholar]
  31. Raunio H., Syngelmä T., Pasanen M., Juvonen R., Honkakoski P., Kairaluoma M. A., Sotaniemi E., Lang M. A., Pelkonen O. Immunochemical and catalytical studies on hepatic coumarin 7-hydroxylase in man, rat, and mouse. Biochem Pharmacol. 1988 Oct 15;37(20):3889–3895. doi: 10.1016/0006-2952(88)90070-6. [DOI] [PubMed] [Google Scholar]
  32. Song B. J., Gelboin H. V., Park S. S., Yang C. S., Gonzalez F. J. Complementary DNA and protein sequences of ethanol-inducible rat and human cytochrome P-450s. Transcriptional and post-transcriptional regulation of the rat enzyme. J Biol Chem. 1986 Dec 15;261(35):16689–16697. [PubMed] [Google Scholar]
  33. Squires E. J., Negishi M. Reciprocal regulation of sex-dependent expression of testosterone 15 alpha-hydroxylase (P-450(15 alpha)) in liver and kidney of male mice by androgen. Evidence for a single gene. J Biol Chem. 1988 Mar 25;263(9):4166–4171. [PubMed] [Google Scholar]
  34. Theodorsson-Norheim E. Kruskal-Wallis test: BASIC computer program to perform nonparametric one-way analysis of variance and multiple comparisons on ranks of several independent samples. Comput Methods Programs Biomed. 1986 Aug;23(1):57–62. doi: 10.1016/0169-2607(86)90081-7. [DOI] [PubMed] [Google Scholar]
  35. Waxman D. J., Ko A., Walsh C. Regioselectivity and stereoselectivity of androgen hydroxylations catalyzed by cytochrome P-450 isozymes purified from phenobarbital-induced rat liver. J Biol Chem. 1983 Oct 10;258(19):11937–11947. [PubMed] [Google Scholar]
  36. Wood A. W., Conney A. H. Genetic variation in coumarin hydroxylase activity in the mouse (Mus musculus). Science. 1974 Aug 16;185(4151):612–614. doi: 10.1126/science.185.4151.612. [DOI] [PubMed] [Google Scholar]
  37. Wood A. W. Genetic regulation of coumarin hydroxylase activity in mice. Biochemical characterization of the enzyme from two inbred strains and their F1 hybrid. J Biol Chem. 1979 Jul 10;254(13):5641–5646. [PubMed] [Google Scholar]

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