Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 1991 Apr 1;275(Pt 1):247–252. doi: 10.1042/bj2750247

Localization and differential induction of cytochrome P450IVA and acyl-CoA oxidase in rat liver.

D R Bell 1, R G Bars 1, G G Gibson 1, C R Elcombe 1
PMCID: PMC1150039  PMID: 2018479

Abstract

The peroxisome proliferators are structurally diverse chemicals which induce hyperplasia, hypertrophy and the proliferation of peroxisomes in the rodent liver. Cytochrome P450IVA1 and peroxisomal enzymes, such as acyl-CoA oxidase, are induced and are early markers of treatment with peroxisome proliferators. In this study, rats were dosed intraperitoneally with the potent peroxisome proliferator methylclofenapate and the hepatic induction response was studied. There was no significant change in the enzyme activities of laurate hydroxylase (cytochrome P450IVA1) or acyl-CoA oxidase in the first 8 h after treatment, but the activities had doubled at 24 h, suggesting that these enzymes are not involved in the mediation of early events in peroxisome proliferation. Hepatic cytochrome P450IVA1 mRNA was significantly increased at 6 and 8 h after treatment, rising to 15-fold above control values at 30 h. In contrast, acyl-CoA oxidase mRNA showed no significant change in the first 8 h, but increased to 13-fold above control values at 24 and 30 h, thereby demonstrating different kinetics of induction of the two mRNAs. In order to determine whether cytochrome P450IVA1 and peroxisomal enzymes were included in the same cells, rats were treated daily with sub-maximal (2 or 5 mg/kg) and maximal (25 mg/kg) inducing doses of methylclofenapate for 4 days. The lobular distribution of induced proteins was determined immunocytochemically with antibodies raised against P450IVA1 and acyl-CoA oxidase. Livers from control animals showed minimal staining for both proteins. However, in the livers of animals treated with 2 or 5 mg of methylclofenapate/kg, both acyl-CoA and P450IVA immunostaining was increased, mainly in the centrilobular area. Immunostaining of serial sections revealed that these proteins were induced in the same region of the lobule. A maximal inducing dose of methylclofenapate (25 mg/kg) caused panlobular induction of both proteins. The results demonstrate that these proteins are induced in a dose-dependent manner in the same, spatially distinct, sensitive region of the liver lobule.

Full text

PDF
247

Images in this article

249Fig. 1.
on p.249
249Fig. 2.
on p.249
251Fig. 5.
on p.251

Selected References

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

  1. Bains S. K., Gardiner S. M., Mannweiler K., Gillett D., Gibson G. G. Immunochemical study on the contribution of hypolipidaemic-induced cytochrome P-452 to the metabolism of lauric acid and arachidonic acid. Biochem Pharmacol. 1985 Sep 15;34(18):3221–3229. doi: 10.1016/0006-2952(85)90338-7. [DOI] [PubMed] [Google Scholar]
  2. Bars R. G., Mitchell A. M., Wolf C. R., Elcombe C. R. Induction of cytochrome P-450 in cultured rat hepatocytes. The heterogeneous localization of specific isoenzymes using immunocytochemistry. Biochem J. 1989 Aug 15;262(1):151–158. doi: 10.1042/bj2620151. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Cathala G., Savouret J. F., Mendez B., West B. L., Karin M., Martial J. A., Baxter J. D. A method for isolation of intact, translationally active ribonucleic acid. DNA. 1983;2(4):329–335. doi: 10.1089/dna.1983.2.329. [DOI] [PubMed] [Google Scholar]
  4. Chatterjee B., Murty C. V., Olson M. J., Roy A. K. Cloning and expression of the rat liver cDNA for peroxisomal enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase in lambda GT11. Transcriptional regulation of enzyme activity by Wy-14643 in primary cultures of rat hepatocytes. Eur J Biochem. 1987 Jul 15;166(2):273–278. doi: 10.1111/j.1432-1033.1987.tb13511.x. [DOI] [PubMed] [Google Scholar]
  5. Earnshaw D., Dale J. W., Goldfarb P. S., Gibson G. G. Differential splicing in the 3' non-coding region of rat cytochrome P-452 (P450 IVA1) mRNA. FEBS Lett. 1988 Aug 29;236(2):357–361. doi: 10.1016/0014-5793(88)80055-3. [DOI] [PubMed] [Google Scholar]
  6. Gibson G. G., Orton T. C., Tamburini P. P. Cytochrome P-450 induction by clofibrate. Purification and properties of a hepatic cytochrome P-450 relatively specific for the 12- and 11-hydroxylation of dodecanoic acid (lauric acid). Biochem J. 1982 Apr 1;203(1):161–168. doi: 10.1042/bj2030161. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Hardwick J. P., Song B. J., Huberman E., Gonzalez F. J. Isolation, complementary DNA sequence, and regulation of rat hepatic lauric acid omega-hydroxylase (cytochrome P-450LA omega). Identification of a new cytochrome P-450 gene family. J Biol Chem. 1987 Jan 15;262(2):801–810. [PubMed] [Google Scholar]
  8. Harley C. B. Hybridization of oligo(dT) to RNA on nitrocellulose. Gene Anal Tech. 1987 Mar-Apr;4(2):17–22. doi: 10.1016/0735-0651(87)90013-6. [DOI] [PubMed] [Google Scholar]
  9. Horie S., Ishii H., Suga T. Changes in peroxisomal fatty acid oxidation in the diabetic rat liver. J Biochem. 1981 Dec;90(6):1691–1696. doi: 10.1093/oxfordjournals.jbchem.a133645. [DOI] [PubMed] [Google Scholar]
  10. Kimura S., Hanioka N., Matsunaga E., Gonzalez F. J. The rat clofibrate-inducible CYP4A gene subfamily. I. Complete intron and exon sequence of the CYP4A1 and CYP4A2 genes, unique exon organization, and identification of a conserved 19-bp upstream element. DNA. 1989 Sep;8(7):503–516. doi: 10.1089/dna.1.1989.8.503. [DOI] [PubMed] [Google Scholar]
  11. Kimura S., Hardwick J. P., Kozak C. A., Gonzalez F. J. The rat clofibrate-inducible CYP4A subfamily. II. cDNA sequence of IVA3, mapping of the Cyp4a locus to mouse chromosome 4, and coordinate and tissue-specific regulation of the CYP4A genes. DNA. 1989 Sep;8(7):517–525. doi: 10.1089/dna.1.1989.8.517. [DOI] [PubMed] [Google Scholar]
  12. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  13. Lalwani N. D., Fahl W. E., Reddy J. K. Detection of a nafenopin-binding protein in rat liver cytosol associated with the induction of peroxisome proliferation by hypolipidemic compounds. Biochem Biophys Res Commun. 1983 Oct 31;116(2):388–393. doi: 10.1016/0006-291x(83)90534-x. [DOI] [PubMed] [Google Scholar]
  14. Lock E. A., Mitchell A. M., Elcombe C. R. Biochemical mechanisms of induction of hepatic peroxisome proliferation. Annu Rev Pharmacol Toxicol. 1989;29:145–163. doi: 10.1146/annurev.pa.29.040189.001045. [DOI] [PubMed] [Google Scholar]
  15. Melton D. A., Krieg P. A., Rebagliati M. R., Maniatis T., Zinn K., Green M. R. Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter. Nucleic Acids Res. 1984 Sep 25;12(18):7035–7056. doi: 10.1093/nar/12.18.7035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Mitchell A. M., Lhuguenot J. C., Bridges J. W., Elcombe C. R. Identification of the proximate peroxisome proliferator(s) derived from di(2-ethylhexyl) phthalate. Toxicol Appl Pharmacol. 1985 Aug;80(1):23–32. doi: 10.1016/0041-008x(85)90097-3. [DOI] [PubMed] [Google Scholar]
  17. Miyazawa S., Hayashi H., Hijikata M., Ishii N., Furuta S., Kagamiyama H., Osumi T., Hashimoto T. Complete nucleotide sequence of cDNA and predicted amino acid sequence of rat acyl-CoA oxidase. J Biol Chem. 1987 Jun 15;262(17):8131–8137. [PubMed] [Google Scholar]
  18. Myers R. M., Larin Z., Maniatis T. Detection of single base substitutions by ribonuclease cleavage at mismatches in RNA:DNA duplexes. Science. 1985 Dec 13;230(4731):1242–1246. doi: 10.1126/science.4071043. [DOI] [PubMed] [Google Scholar]
  19. Orton T. C., Parker G. L. The effect of hypolipidemic agents on the hepatic microsomal drug-metabolizing enzyme system of the rat. Induction of cytochrome(s) P-450 with specificity toward terminal hydroxylation of lauric acid. Drug Metab Dispos. 1982 Mar-Apr;10(2):110–115. [PubMed] [Google Scholar]
  20. Osmundsen H. Peroxisomal beta-oxidation of long fatty acids: effects of high fat diets. Ann N Y Acad Sci. 1982;386:13–29. doi: 10.1111/j.1749-6632.1982.tb21404.x. [DOI] [PubMed] [Google Scholar]
  21. Osumi T., Ishii N., Miyazawa S., Hashimoto T. Isolation and structural characterization of the rat acyl-CoA oxidase gene. J Biol Chem. 1987 Jun 15;262(17):8138–8143. [PubMed] [Google Scholar]
  22. Reddy J. K., Azarnoff D. L., Hignite C. E. Hypolipidaemic hepatic peroxisome proliferators form a novel class of chemical carcinogens. Nature. 1980 Jan 24;283(5745):397–398. doi: 10.1038/283397a0. [DOI] [PubMed] [Google Scholar]
  23. Reddy J. K., Goel S. K., Nemali M. R., Carrino J. J., Laffler T. G., Reddy M. K., Sperbeck S. J., Osumi T., Hashimoto T., Lalwani N. D. Transcription regulation of peroxisomal fatty acyl-CoA oxidase and enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase in rat liver by peroxisome proliferators. Proc Natl Acad Sci U S A. 1986 Mar;83(6):1747–1751. doi: 10.1073/pnas.83.6.1747. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Studencki A. B., Wallace R. B. Allele-specific hybridization using oligonucleotide probes of very high specific activity: discrimination of the human beta A- and beta S-globin genes. DNA. 1984;3(1):7–15. doi: 10.1089/dna.1.1984.3.7. [DOI] [PubMed] [Google Scholar]
  25. Styles J. A., Kelly M., Pritchard N. R., Elcombe C. R. A species comparison of acute hyperplasia induced by the peroxisome proliferator methylclofenapate: involvement of the binucleated hepatocyte. Carcinogenesis. 1988 Sep;9(9):1647–1655. doi: 10.1093/carcin/9.9.1647. [DOI] [PubMed] [Google Scholar]
  26. Voigt J. M., Guengerich F. P., Baron J. Localization of a cytochrome P-450 isozyme (cytochrome P-450 PB-B) and NADPH-cytochrome P-450 reductase in rat nasal mucosa. Cancer Lett. 1985 Jul;27(3):241–247. doi: 10.1016/0304-3835(85)90180-6. [DOI] [PubMed] [Google Scholar]
  27. Wolf C. R., Moll E., Friedberg T., Oesch F., Buchmann A., Kuhlmann W. D., Kunz H. W. Characterization, localization and regulation of a novel phenobarbital-inducible form of cytochrome P450, compared with three further P450-isoenzymes, NADPH P450-reductase, glutathione transferases and microsomal epoxide hydrolase. Carcinogenesis. 1984 Aug;5(8):993–1001. doi: 10.1093/carcin/5.8.993. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES