Skip to main content

Advertisement

An autoradiographic study of cellular proliferaton, DNA synthesis and cell cycle variability in the rat liver caused by phenobarbital-induced oxidative stress: The protective role of melatonin

Abstract

The protective effect of melatonin against phenobarbital-induced oxidative stress in the rat liver was measured based on lipid peroxidation levels (malondialedyde and 4-hydroxyalkenals). Cellular proliferation, DNA synthesis and cell cycle duration were quantitated by the incorporation of 3H-thymidine, detected by autoradiography, into newly synthesized DNA. Two experiments were carried out in this study, each on four equal-sized groups of male rats (control, melatonin [10 mg/kg], phenobabital [20 mg/kg] and phenobarbital plus melatonin). Experiment I was designed to study the proliferative activity and rate of DNA synthesis, and measure the levels of lipid peroxidation, while experiment II was for cell cycle time determination. Relative to the controls, the phenobarbital-treated rats showed a significant increase (P < 0.01) in the lipid peroxidation levels (30.7%), labelling index (69.4%) and rate of DNA synthesis (37.8%), and a decrease in the cell cycle time. Administering melatonin to the phenobarbital-treated rats significantly reduced (P < 0.01) the lipid peroxidation levels (23.5%), labelling index (38.2%) and rate of DNA synthesis (29.0%), and increased the cell cycle time. These results seem to indicate that the stimulatory effect of phenobarbital on the oxidized lipids, proliferative activity, kinetics of DNA synthesis and cell cycle time alteration in the liver may be one of the mechanisms by which the non-genotoxic mitogen induces its carcinogenic action. Furthermore, melatonin displayed powerful protection against the toxic effect of phenobarbital.

Abbreviations

GC/N:

grain count per labelled nucleus

4-HAD:

4-hydroxyalkenals

LI:

labelling index

LPO:

lipid peroxidation

MDA:

malondialdehyde

PB:

phenobarbital

ROS:

reactive oxygen species

References

  1. 1.

    Peraino, C., Fry, R.J.M. and Staffeldt, E. Enhancement of spontaneous hepatic tumorigenesis in C3H mice by dietary Phenobarbital. J. Natl. Cancer Inst. 51 (1973) 1349–1350.

  2. 2.

    Clemmesen, J. and Hjalgrim-Jensen, S. Is Phenobarbital carcinogenic? A follow-up of 8078 epileptics. Ecotoxicol. Environ. Safety 1 (1978) 457–470.

  3. 3.

    Jirtle, R.L., Meyer, S.A. and Brockenbrough, J.S. Liver tumor promoter Phenobarbital: a biphasic modulator or hepatocyte proliferation. Prog. Clin. Biol. Res. 369 (1991) 209–216.

  4. 4.

    Tsai, W.H. and DeAngelo, A.B. Responsiveness hepatocytes from dichloroacetic acid or phenobarbital treated mice to growth factors in primary cultures. Cancer Lett. 99 (1996) 177–183.

  5. 5.

    Jones, H.B., Clarke, N.A. and Barras, N.C. Phenobarbital-induced hepatocellular proliferation: anti-bromodeoxyuridine and anti-proliferating cell nuclear antigen immunocytochemistry. J. Histochem. Cytochem. 41 (1993) 21–27.

  6. 6.

    Peraino, C., Fry, R.J.M., Staffeldt, E. and Christopher, J.P. Comparative enhancing effects of Phenobarbital, amobarbital, diphenylhydantoin and dichlorodiphenyl-trichloroethane on 2-acetylaminofluorene induced hepatic tumourigenesis in rat. Cancer Res. 35 (1975) 2884–2890.

  7. 7.

    Shelby, M.D. and Zieger, E. Activity of human carcinogens in the salmonella and rodent bone-marrow cytogenetics tests. Mutat. Res. 234 (1990) 257–261.

  8. 8.

    Butterworth, B.E., Popp, J.A., Conolly, R.B. and Goldsworthy, T.L. Chemically induced cell proliferation in carcinogenesis. in: Mechanisms of Carcinogenesis in Risk Identification. Scientific Publications (Vainio, H., Magee, P.N., McGregor, D.B.and McMichael, A.J. Eds.), Lyon, France, 1992, 279–305.

  9. 9.

    Schulte-Hermann, R., Bursch, W., Grasl-Kraupp, B., Torok, L., Ellinger, A. and Mullauer, L. Role of active cell death (apoptosis) in multi-stage carcinogenesis. Toxicol. Lett. 82/83 (1995) 143–148.

  10. 10.

    Butterworth, B.E., Conolly, R.B. and Morgan, K.T. A strategy for establishing mode of action of chemical carcinogens as a guide for approaches to risk assessments. Cancer Lett. 93 (1995) 129–146.

  11. 11.

    Melnick, R. and Huff, J. Liver carcinogenesis is not a predicted outcome of chemically induced hepatocyte proliferation. Toxicol. Indust. Health 9 (1993) 415–438.

  12. 12.

    Columbano, A., Ledda-Columbano, G., Coni, P., Pichiri-Coni, G., Curto, M. and Pani, P. Chemically induced cell proliferation and carcinogenesis: Differential effect of compensatory cell proliferation and mitogen-induced direct hyperplasia on hepatocarcinogenesis in the rat. in: Chemically Induced Cell Proliferation: Implications for Risk Assessment (Butterworth, B., Slaga, T., Farland, W. and McClain, M. Eds.), Wiley-Liss, New York. 1991, 217–225.

  13. 13.

    Bursch, W., Fesus, L. and Schulte-Hermann, R. Apoptosis (’Programmed’ cell death) and its relevance in liver injury and carcinogenesis. in: Tissue-Specific Toxicity Biochemical Mechanisms (Dekant, W. and Neumann, H. Eds.), Academic Press, London. 1992, 95–115.

  14. 14.

    Goldsworthy, T., Fransson-Steen, R. and Maronpot, R. Importance of and approaches to quantification of hepatocyte apoptosis. Toxicol. Pathol. 24 (1996) 24–35.

  15. 15.

    Stark, A.A., Russell, J.J., Langenbach, R., Pagano, D.A., Zeiger, E. and Huberman, E. Localization of oxidative damage by a glutathione-γ-glutamyl transpeptidase system in neoplastic lesions in sections of livers from carcinogen-treated rats. Carcinogenesis 15 (1994) 343–348.

  16. 16.

    Imaoka, S., Osada, M., Minamiyama, Y., Yukimura, T., Toyokuni, S., Takemura, S., Hiroi, T. and Funae, Y. Role of Phenobarbital-induced cytochrome P450s as a source of active oxygen species in DNA-oxidation. Cancer Lett. 203 (2004) 117–125.

  17. 17.

    Diez-Fernandez, C., Sanz, N., Alvarez, A.M., Wolf, A. and Cascales, M. The effect of non-genotoxic carcinogens, phenobarbital and clofibrate, on the relationship between reactive oxygen species, antioxidant enzyme expression and apoptosis. Carcinogenesis 19 (1998) 1715–1722.

  18. 18.

    Venditti, R., Daniele, C.M., Deleo, T. and DiMeo, S. Effect of phenobarbital treatment on characteristics determining susceptibility to oxidants of homogenates, mitochondria and microsomes from rat liver. Cell Physiol. Biochem. 8 (1998) 328–338.

  19. 19.

    Aniya, Y., Shimoji, M. and Naito, A. Increase in liver microsomal glutathione-S-transferase activity by phenobarbital treatment of rats: possible involvement of oxidative activation via cytochrome P450. Biochem. Pharmacol. 46 (1993) 1741–1747.

  20. 20.

    Saintot, M., Astre, C., Pujol, H. and Gerber, M. Tumor progerssion and oxidant antioxidant status. Carcinogenesis 17 (1996) 1267–1271.

  21. 21.

    Wolfle, D. and, Marquardt, H. Antioxidants inhibit the enhancement of malignant cell transformation induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin, Carcinogenesis 17 (1996) 1273–1278.

  22. 22.

    Whisler, R.L., Goyette, M.A., Grants, J.S. and Newhouse, Y.G. Sublethal levels of oxidant stress stimulate multiple serine/threonine kinases and suppress protein phosphatases in jurkat T cells. Arch. Biochem. Biophys. 319 (1995) 23–25.

  23. 23.

    Kass, G.E.N.,. Free radical induced changes in cell signal transduction. in: Free radical Toxicology (Wallace, K.B. Ed.), Taylor and Francis, Washington, DC, 1997, 349–374.

  24. 24.

    Reiter, R.J. Oxidative damage in the central nervous system: Protection by melatonin. Prog. Neurobiol. 56 (1998) 359–384.

  25. 25.

    Reiter, R.J. Melatonin: Lowering the high price of free radicals. News Physiol. Sci. 15 (2000) 246–250.

  26. 26.

    El-Sokkary, G.H. Melatonin protects against oxidative stress induced by the kidney arcinogen KBrO3. Neuroendocrinol. Lett. 21 (2000) 461–468.

  27. 27.

    El-Sokkary, G.H. Inhibition of 2-nitropropane-induced cellular proliferation, DNA synthesis and histopathological changes by melatonin. Neuroendocrinol. Lett. 23 (2002) 335–340.

  28. 28.

    El-Sokkary, G.H.; Reiter, R.J. and Abdel Ghaffar, S.Kh. Melatonin supplementation restores cellular proliferation and DNA synthesis in the splenic and thymic lymphocytes of old rats. Neuroendocrinol. Lett. 24 (2003) 215–223.

  29. 29.

    El-Sokkary, G.H., Abdel-Rahman, G.H. and Kamel, E.S. Melatonin protects against lead-induced hepatic and renal toxicity in male rats. Toxicology 213 (2005) 25–33.

  30. 30.

    Abd-Elghaffar, S.Kh, El-Sokkary, G.H. and Sharkawy, A.A. Aluminium-induced neurotoxicity and oxidative damage in rabbits: Protective effect of melatonin. Neuroendocinol. Lett. 26 (2005) 609–616.

  31. 31.

    Martinez-Cruz, F., Osuna, C. and Guerrero, J.M. Mitochondrial damage induced by fetal hyperphenylalaniemia in the rat brain and liver: Its prevention by melatonin, vitamin E and vitamin C. Neurosci. Lett. 392 (2006) 1–4.

  32. 32.

    Hamatani, K., Kawahara, A. and Amano, M. Quantitative study of deoxycytidine incorporation in large and small lymphocytes of the mouse. Cell Tissue Kinet. 16 (1983) 557–570.

  33. 33.

    Sadava, D.E. Cell cycle. in: Cell Biology, Organelle Structure and Function. Jones and Bartlett Publishers, 1993, 442-496.

  34. 34.

    Jirtle, R.L. and Meyer, S.A. Liver tumor promotion: effect of phenobarbital on EGF and protein kinase C signal transduction and TGFß1 expression. Dig. Dis. Sci. 36 (1991) 659–668.

  35. 35.

    Guppy, M.J., Wilton, J.C., Sharma, R. and Chipman, J.K. Modulation of phenobarbitone induced loss of gap junctional intercellular communication in hepatocyte couplets. Carcinogenesis 15 (1994) 1917–1921.

  36. 36.

    Bock, K.W., Lipp, H.P. and Bock-Hennig, B.S. Induction of drug-metabolizing enzymes by xenobiotics. Xenobiotica 20 (1990) 1101–1111.

  37. 37.

    Murkofsky, R.L., Glover, S.E., Miller, R.T., Popp, J.A. and Cattley, R.C. Effect of regeneration and hyperplasia on levels of DNA base oxidation in rat liver. Cancer Lett. 70 (1993) 51–56.

  38. 38.

    Kinoshita, A., Wanibuchi, H., Imaoka, S., Ogawa, M., Masuda, C., Morimura, K., Funae, Y. and Fukushima, S. Formation of 8-hydroxydeoxyguanosine and cell-cycle arrest in the rat liver via generation of oxidative stress by phenobarbital: association with expression profiles of p21 WAF1/Cip1, cyclin D1 and Ogg1. Carcinogenesis 23 (2002) 341–349.

  39. 39.

    Kaufmann, W.K., Ririe, D.G. and Kaufman, D.G. Phenobarbital-dependent proliferation of putative initiated rat hepatocytes. Carcinigenesis 9 (1988) 779–782.

  40. 40.

    Arora, V.K., Bhatia, A. and Sood, S.K. Synergistic promoter effect of diethylstilbesterol & phenobarbitone in diethylnitrosamine induced hepatic neoplasia in rats. Indian J. Med. Res. 90 (1989) 9–16.

  41. 41.

    Weghorst, C.M. and Klaunig, J.E. Phenobarbital promotion in diethylnitrosamine-initiated infant B6C3F1 mice. Influence of gender. Carcinogenesis 10 (1989) 609–612.

  42. 42.

    Debiec-Rychter, M. and Wang, C.Y. Induction of urinary bladder of the F344 rat. Toxicol. Appl. Pharmacol. 105 (1990) 345–349.

  43. 43.

    Morsi, G.A. Autoradiographic studies on the effect of sodium phenobarbital on the kinetics of DNA synthesis in the hepatocytes of male albino rat. Egypt. J. Histol. 14 (1991) 511–523.

  44. 44.

    Gonzales, J.A., Christensen, J.G., Preston, R.J., Goldsworthy, T.L., Tlsty, T.D. and Fox, T.R. Attenuation of G1 checkpoint function by the non-genotoxic carcinogen Phenobarbital. Carcinogenesis 19 (1998) 1173–1183.

  45. 45.

    Hartwell, L.H. and Weinhert, T.A. Checkpoints: Controls that ensure the order of cell cycle events. Science 246 (1989) 629–633.

  46. 46.

    Kaufmann, W.K. and Wilson, S.R. G1 arrest during cell cycle dependent clastogenesis in UV-irradiated human fibroblasts. Mutat. Res. 314 (1994) 67–76.

  47. 47.

    Livingstone, L.R., White, A., Sprouse, J., Livanos, E., Jacks, T. and Tlsty, T.D. Altered cell cycle arrest and gene amplification potential accompany loss of wild-type p53. Cell 70 (1992) 923–935.

  48. 48.

    White, A.E., Livanos, E.M. and Tlsty, T.D. Differential disruption of genomic integrity and cell cycle regulation in normal human fibroblasts by the HPV oncoproteins. Genes Dev. 8 (1994) 666–677.

  49. 49.

    Hartwell, L. Defects in a cell cycle checkpoint may be responsible for the genomic instability of cancer cells. Cell 71 (1992) 543–546.

  50. 50.

    Tan, D.X., Chen, L.D., Poeggeler, B., Manchester, L.C. and Reiter, R.J. Melatonin: a potent, endogenous hydroxyl radical scavenger. Endocr. J. 1 (1993) 57–60.

  51. 51.

    Reiter, R.J., Tan, D.X., Qi, W., Manchester, L.C., Karbownik, M. and Calvo, J.R. Pharmacology and physiology of melatonin in reduction of oxidative stress in vivo. Biol. Signals Recept. 9 (2000) 160–171.

  52. 52.

    Cardinali, D.P., Borfman, G. P., Liotta, G., Perez Floret, S., Albornoz, L.E., Cutrera, R.A., Batista, J. and Ortiga, G.B. A multifactorial approach employing melatonin to accelerate resynchronization of sleep-wake cycle after a 12 timezone westerly transmeridian flight in elite soccer athletes. J. Pineal Res. 32 (2002) 41–46.

  53. 53.

    Cardinali, D.P. Clinical perspectives for the use of melatonin as a neuroprotective chronobiotic in Alzheimer’s disease. Aktual. Neurol. 3 (2003) 188–204.

  54. 54.

    Blask, D.E., Sauer, L.A. and Dauchy, R.T. Melatonin as a chronobiotic/anticancer agent. Curr. Top. Med. Chem. 2 (2002) 113–132.

  55. 55.

    Cos, S. and Sanchez-Barcelo, E.J. Melatonin inhibition of MCF-7 human breast cancer cells growth: influence of cell proliferation rate. Cancer Lett. 93 (1995) 207–212.

  56. 56.

    Persengiev, S.P. and Kyurkchiev, S. Selective effect of melatonin on the proliferation of lymphoid cells. Int. J. Biochem. 25 (1993) 441–444.

  57. 57.

    Malamud, D. The cell cycle and cancer. (Baserga, R. Ed.), Cold Spring Harbor, New York. 1971, 132–141.

  58. 58.

    Crespo, D., Fernandez-Viadero, C., Verduga, R., Ovejero, V. and Cos, S. Interaction between melatonin and estradiol on morphological and morphometric features of MCF-7 human breast cancer cells. J. Pineal Res. 16 (1994) 215–222.

  59. 59.

    Jayat, C. and Ratinaud, M.H. Cell cycle analysis by flow cytometry: Principles and Applications. Biol. Cell 78 (1993) 15–25.

  60. 60.

    Cos, S., Blask, D.E., Lemus-Wilson, A. and Hill, A.B. Effects of melatonin on the cell cycle kinetics and estrogen rescue of MCF-7 human breast cancer cells in culture. J. Pineal Res. 10 (1991) 36–43.

Download references

Author information

Correspondence to Gamal H. El-Sokkary.

Rights and permissions

Reprints and Permissions

About this article

Key words

  • Phenobarbital
  • Melatonin
  • Lipid peroxidation
  • Cell proliferation
  • DNA synthesis
  • Cell cycle