Archives of Toxicology

, Volume 67, Issue 9, pp 622–628 | Cite as

Assessment of the influence of subacute phenobarbitone administration on multi-tissue cell proliferation in the rat using bromodeoxyuridine immunocytochemistry

  • Huw B. Jones
  • Noel A. B. Clarke
Original Investigations


The effects of daily administration of phenobarbitone on the mitotic rates of several tissues were investigated by bromodeoxyuridine (BrdU) immunocytochemistry. Phenobarbitone (80 mg/kg per day) was dosed to AP Wistar male rats for up to 7 days and BrdU (10 mg/ml) was given by infusion at a rate of 10 μl/h via subcutaneously implanted osmotic minipumps for 2 days prior to necropsy on days 1, 2, 3, 5 and 7. BrdU-labelled nuclei were visualised by peroxidase-antiperoxidase immunocytochemistry and counts of the numbers of labelled cells (labelling index, LI%) made from at least 1000 cells per tissue section(s). The LIs of several tissues (testis, adrenal cortex and medulla, kidney distal convoluted tubule and exocrine pancreas) showed no statistical difference by comparison with controls. Several tissues exhibited characteristic responses to phenobarbitone administration. Pituitary and endocrine pancreas LIs were decreased while those of thyroid, liver and kidney proximal convoluted tubule were increased. The pattern of LI increase was unique to each tissue with liver (median and lateral lobes) increased two-fold on day 3 and returning to control levels thereafter while kidney proximal tubule LI rose gradually with time and remained elevated on day 7. Thyroid LI on day 1 was almost double that of day 0 control and increased steadily thereafter. These data illustrate the varied responses of different tissues to phenobarbitone exposure, namely, depression and stimulation of mitosis. The causation of these functional changes is discussed in relation to direct and indirect effects on functional parameters, especially enzyme induction, alterations in hormonal and growth factor status and receptor regulation.

Key words

Cell proliferation Phenobarbitone Immunocytochemistry Bromodeoxyuridine (BrdU) Liver Kidney Pituitary Adrenal Testis Thyroid Pancreas 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Ames BN, Gold LS (1990 a) Chemical carcinogenesis: too many rodent carcinogens. Proc Natl Acad Sci USA 87: 7772–7776PubMedGoogle Scholar
  2. Ames BN, Gold LS (1990 b) Too many rodent carcinogens: mitogenesis increases mutagenesis. Science 249: 970–971PubMedGoogle Scholar
  3. Bars RG, Elcombe CR (1991) Dose-dependent acinar induction of P450IA1 in rat liver. Biochem J 277: 577–580PubMedGoogle Scholar
  4. Bars R, Mitchell AM, Wolf CR, Elcombe CR (1989) Heterogeneous induction of cytochromes P-450 in hepatocytes in vitro and in vivo. In: Schuster I (ed.) Cytochrome P-450: biochemistry and biophysics. Taylor and Francis, London, pp 841 -844Google Scholar
  5. Bolt HM, Bolt M, Kappus H (1977) Interaction of rifampicin treatment with pharmacokinetics and metabolism of ethinyloestradiol in man. Acta Endocrinol 85: 189–197PubMedGoogle Scholar
  6. Boobis AR, Caldwell J, De Matteis F, Elcombe CR (1985) Microsomes and drug oxidations. Taylor and Francis, London PhiladelphiaGoogle Scholar
  7. Bucher NLR, Swaffield MN (1975 a) Regulation of hepatic regeneration in rats by synergistic action of insulin and glucagon. Proc Natl Acad Sci USA 72: 1157–1160PubMedGoogle Scholar
  8. Bucher NLR, Swaffield MN (1975 b) Synergistic action of glucagon and insulin in regulation of hepatic regeneration. Adv Enzyme Regul 13: 281–293PubMedGoogle Scholar
  9. Butterworth BE (1991) Chemically induced cell proliferation as a predictive assay for potential carcinogenicity. Prog Clin Biol Res 369: 457–467PubMedGoogle Scholar
  10. Chin WW, Shupnik MA, Ross DS, Habener JF, Ridgway EC (1985) Regulation of the α- and thyrotropin β-subunit messenger ribonucleic acids by thyroid hormones. Endocrinology 116: 873–878PubMedGoogle Scholar
  11. Clayson DB, Nera EA, Lok E (1989) The potential for the use of cell proliferation studies in carcinogen risk assessment. Regul Toxicol Pharmacol 9: 284–295PubMedGoogle Scholar
  12. Cohen SM, Ellwein LB (1990) Cell proliferation in carcinogenesis. Science 249: 1007–1011PubMedGoogle Scholar
  13. Coltrera MD, Gown AM (1991) PCNA/Cyclin expression and BrdU uptake define different subpopulations in different cell lines. J Histochem Cytochem 39: 23–30PubMedGoogle Scholar
  14. Cunningham ML, Matthews HB (1991) Relationship of hepatocarcinogenicity and hepatocellular proliferation induced by mutagenic noncarcinogens vs carcinogens. Toxicol Appl Pharmacol 110: 505–513PubMedGoogle Scholar
  15. Cunningham ML, Foley J, Maronpot RR, Matthews HB (1991) Correlation of hepatocarcinogenicity and hepatocellular proliferation induced by two mutagenic diaminotoluenes. Toxicol Appl Pharmacol 107: 562–567PubMedGoogle Scholar
  16. De Sandro V, Chevrier M, Boddaert A, Melcion C, Cordier A, Richert L (1991) Comparison of the effects of propylthiouracil, amiodarone, diphenylhydantoin, phenobarbital and 3-methylcholanthrene on hepatic and renal T4 metabolism and thyroid gland function in rats. Toxicol Appl Pharmacol 111: 263 -278PubMedGoogle Scholar
  17. Hoermann R, Saller B, Mann K (1989) Insulin and insulin-like growth factor (IGF I) modulate the effects of bTSH on3H-thymidine incorporation in human thyroid cells in primary culture. Klin Wochenschr 67: 976–979PubMedGoogle Scholar
  18. Jirtle RL, Meyer SA (1991) Liver tumor promotion: effect of phenobarbital on EGF and protein kinase C signal transduction and transforming growth factor-β1 expression. Digest Dis Sci 36: 659–668PubMedGoogle Scholar
  19. Jones HB, Clarke NAB, Barrass NC (1993) Phenobarbitone-induced hepatocellular proliferation: anti-bromodeoxyuridine and anti-proliferating cell nuclear antigen immunocytochemistry. J Histochem Cytochem 41: 21–27PubMedGoogle Scholar
  20. Kan M, Zhang G, Zarnegar R, Michalopoulos G, Myoken Y, McKeehan WL, Stevens JI (1991) Hepatocyte growth factor/hepatopoietin A stimulates the growth of rat kidney proximal tubule cells (RPTE), rat non-parenchymal liver cells, human melanoma cells, mouse keratinocytes and stimulates anchorage-independent growth of SV-40 transformed RPTE. Biochem Biophys Res Commun 174: 331–337PubMedGoogle Scholar
  21. Kute TE, Quadri Y (1991) Measurement of proliferation nuclear and membrane markers in tumor cells by flow cytometry. J Histochem Cytochem 39: 1125–1128PubMedGoogle Scholar
  22. Levin W, Welch RM, Conney AH (1974) Increased liver microsomal androgen metabolism by phenobarbital: correlation with decreased androgen action on the seminal vesicles of the rat. J Pharmacol Exp Ther 188: 287–292PubMedGoogle Scholar
  23. Lu AYH, West SB (1978) Reconstituted mammalian mixed-function oxidases: requirements, specificities and other properties. Pharmacol Ther A 2: 337–358Google Scholar
  24. Marsmann DS, Cattley RC, Conway JG, Popp JA (1988) Relationship of hepatic peroxisome proliferation and replicative DNA synthesis to the hepatocarcinogenicity of the peroxisome proliferators di (2-ethylhexyl) phthalate and [4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio] acetic acid (Wy-14, 643) in rats. Cancer Res 48: 6739–6744PubMedGoogle Scholar
  25. McClain RM (1989) The significance of hepatic microsomal enzyme induction and altered thyroid function in rats: implications for thyroid gland neoplasia. Toxicol Pathol 17: 294–306PubMedGoogle Scholar
  26. McClain RM (1990) Mouse liver tumors and microsomal enzyme-inducing drugs: experimental and clinical perspectives with phenobarbital. Prog Clin Biol Res 331: 345–365PubMedGoogle Scholar
  27. McClain RM, Posch RC, Bosakowski T, Armstrong JM (1988) Studies on the mode of action for thyroid gland tumor promotion in rats by phenobarbital. Toxicol Appl Pharmacol 94: 254–265PubMedGoogle Scholar
  28. McClain RM, Levin AA, Posch R, Downing JC (1989) The effect of phenobarbital on the metabolism and excretion of thyroxine in rats. Toxicol Appl Pharmacol 99: 216–228PubMedGoogle Scholar
  29. Mirsalis JC, Steinmetz KL (1990) The role of hyperplasia in liver carcinogenesis. Prog Clin Biol Res 369: 149–161Google Scholar
  30. Raghavan R, Steart PV, Weller RO (1990) Cell proliferation patterns in the diagnosis of astrocytomas, anaplastic astrocytomas and glioblastoma multiforme: a Ki-67 study. Neuropathol Appl Neurobiol 16: 123–129PubMedGoogle Scholar
  31. Schwartz M, Peres G, Buchmann H, Friedberg T, Waxman DJ, Kunz W (1987) Phénobarbital induction of cytochrome P-450 in normal and preneoplastic liver: comparison of enzyme and mRNA expression as detected by immunocytochemistry and in situ hybridization. Carcinogenesis 5: 143–153Google Scholar
  32. Turner NA, Wilson NM, Jefcoate CR, Pitot HC (1988) The expression and metabolic activity of cytochrome P-450 isozymes in control and phenobarbital-induced primary cultures of rat hepatocytes. Arch Biochem Biophys 263: 204–215PubMedGoogle Scholar
  33. Werk EE, Thrasher BS, Sholiton LJ, Olinger C, Choi Y (1971) Cortisol production in epileptic patients treated with diphenylhydantoin. Clin Pharmacol Ther 12: 698–703PubMedGoogle Scholar

Copyright information

© Springer-Verlag 1993

Authors and Affiliations

  • Huw B. Jones
    • 1
  • Noel A. B. Clarke
    • 1
  1. 1.Safety of Medicines DepartmentZeneca PharmaceuticalsMacclesfieldUK

Personalised recommendations