Please use this identifier to cite or link to this item: http://adhlui.com.ui.edu.ng/jspui/handle/123456789/855
Title: THE ROLE OF KUPFFER CELLS IN ETHANOL AND AFLATOXIN B₁-INDUCED p53 GENE MUTATION IN HEPATOCARCINOGENESIS
Authors: OWUMI, S. E.
Keywords: Kupffer cells
Ethanol
Aflatoxin B₁
DNA synthesis
Hepatocarcinogenesis
Issue Date: Jun-2009
Abstract: Kupffer cells (KC) play an important role in hepatic homeostasis, and in acute and chronic response of the liver to endotoxins. The KC also mediate the release of cykokines and reactive oxygen species (ROS) involved in signaling pathways, implicated in gene expression, cell growth and apoptosis, KC activation via ethanol of (EtOH)-induced endotoxemia induces ROS and cytokine release, modulating cell fate and potential hepatocyte injury. Its impact on hepatocytes deoxyribonucleic acid (DNA) synthesis and potential effect on the replication on the replication of B₁ mutated p53 gene has not been well investigated. This study was carried out to examine the role of KC in EtOH-induced endotoxemia and associated hepatic DNA synthesis. The rate of hepatocyte DNA synthesis (in vitro and in vivo) was used as an index for cell growth. In vitro primary and percoll purified hepatocyte isoiates (3.25 x 10⁶cells) were exposed to EtOH for 24-48 hrs. In vivo, KC clodronate liposomes-depleted C57BL6 mice (KC-) and KC-competent mice (KC+) were fed EtOH-diet (3%w/v EtOH). Synthesis of DNA was assayed by using 5-bromo-2'-deoxyuridine (BrdU) incorporation into DNA of hepatocytes by BrdU-immunohistochemistry. Hepatic injury was assessed by histopathology of Iiver and level of transaminases in serum. Cytokine transcript (Tumour necrosis factor-alpha; TNF-α) was assayed by reverse transcriptase-polymerase in reaction (RT-PCR) and agarose gel-electrophoresis. Serum alcohol dehydrogenase was assayed for by standard laboratory method. Apoptosis was assessed by terminal deoxynucleotidyl transferase (dUTP) nick end labeling (TUNEL) assay. Protien phosphorylation of extracellular signal-regulated kinase (ERK) ERK1 (p42) and ERK2 (p-44) was assayed by western blotting. Aflatoxin B₁ induced p53 mutation was assessed by restriction fragment analysis using Hac III restriction enzyme on DNA isolate. The Ievels of scrum transaminases were within normal range in EtOH and Aflatoxin B₁ treatment groups (ATS ≤ 150 IU/L; GGT ≤25 lU/L). Terminal group mean body weight was reduced by EtOH (10.0%) and mean liver weight (5.0%) compared to control. Kupffer cells were effectively depleted by percoll purification in vitro ( 85.0-90.0%) and with clodronate liposomes in vivo 80.0-90.0%) as indicated by F4/80 immunocytochemical assay. KC+ mice fed Et0H-diet, DNA synthesis increased by 183.0% relative to control (KC+). In KC- fed EtOH-diet, there was a 50.0% decrease relative to control (KC-). The number of cells per field of view increased by 20 0% compared to control in vivo. There was however 148.0% decrease in comparison with KC+ fed EtOH. TNF-α increascd 148.0% in KC+ fed EtOH, 69.0% in KC- and 72.0% in KC fed EtOH compared with the control KC+. Phosphorylation of ERK1/ERK2 (p42/p44) increased in the KC+ fed EtOH in comparison with KC- fed EtOH. Alcohol dehydrogenase level was also elevated in KC+ by 120.0% over control. Restriction analysis showed Aflatoxin B₁ induced mutation at p53 gene locus compared to control. Hepatocytes/mm² also increased by 20.0%. Depletion of KC modulates the effect of EtOH-induced endotoxemia probably by reduction of TNF-α and ROS release with concomitant effect on mitogen activated protein kinase signaling pathway. This accounts for hepatocyte DNA synthesis and apoptotic cell death.
Description: A Thesis in the Department of Biochemistry submitted to the Faculty of Basic Medical Sciences, College of Medicine, University of Ibadan, in partial fulfillment of the requirements for the degree of Doctor of Philosophy of the University of Ibadan, Nigeria.
URI: http://adhlui.com.ui.edu.ng/jspui/handle/123456789/855
Appears in Collections:Theses in Biochemistry

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