Effect of phenobarbital on hepatic cell proliferation and apoptosis in mice deficient in the p50 subunit of NF-kappaB

Abstract

Phenobarbital (PB) is a nongenotoxic tumor promoter in the liver. One mechanism by which PB may exert its tumor promoting activity is by inducing oxidative stress. We previously found that PB administration increased hepatic NF-kappaB DNA binding activity. In this study we examined the hypothesis that the effects of PB on cell proliferation and apoptosis are dependent on NF-kappaB. We used a mouse model that is deficient in the p50 subunit of NF-kappaB; previous studies had found that p50-/- mice were less sensitive to the induction of hepatic cell proliferation by PCBs or peroxisome proliferators. Mice (p50-/- and wild-type B6129) were fed a control diet or one containing 0.05% PB for 3, 10 or 34 days. At the end of the experiment, the mice were euthanized and livers removed and processed. PB increased cell proliferation at 3 and 10 days (but not at 34 days), but the deletion of the NF-kappaB p50 subunit did not inhibit these increases. p50-/- Mice had higher cell proliferation at the 3 day (only in mice fed PB) and 34-day timepoints. PB decreased hepatocyte apoptosis after 3 days, slightly decreased it after 10 days, and did not affect it after 34 days. The deletion of the NF-kappaB p50 subunit did not influence PB's effect on apoptosis. In p50-/- mice, apoptosis was increased after 3 or 10 days compared to wild-type mice, but no effect was seen after 34 days. The hepatic expression of the NF-kappaB-regulated gene TNF-alpha correlated more with the hepatic cell proliferation data than with hepatic apoptosis, and was not decreased by the deletion of the p50 subunit. These findings show that the p50 subunit of NF-kappaB is not required for the alteration of hepatocyte proliferation or apoptosis by PB up to 34 days after its administration.

Figure 1

Effect of PB administration on hepatocyte proliferation in wild-type and p50 -/- mice. A. 3-day administration. B. 10-day administration. C. 34 day administration. Mice were administered a single dose of BrdU two hours before euthanasia. Histological sections for the liver were immunohistochemically stained for BrdU, and labeling indexes were determined in hepatocytes to determine the rate of DNA synthesis. Data are expressed as means ± standard errors. *Significant effect of phenobarbital; ^†Significant effect of p50 deletion.

Figure 1

Effect of PB administration on hepatocyte proliferation in wild-type and p50 -/- mice. A. 3-day administration. B. 10-day administration. C. 34 day administration. Mice were administered a single dose of BrdU two hours before euthanasia. Histological sections for the liver were immunohistochemically stained for BrdU, and labeling indexes were determined in hepatocytes to determine the rate of DNA synthesis. Data are expressed as means ± standard errors. *Significant effect of phenobarbital; ^†Significant effect of p50 deletion.

Figure 1

Effect of PB administration on hepatocyte proliferation in wild-type and p50 -/- mice. A. 3-day administration. B. 10-day administration. C. 34 day administration. Mice were administered a single dose of BrdU two hours before euthanasia. Histological sections for the liver were immunohistochemically stained for BrdU, and labeling indexes were determined in hepatocytes to determine the rate of DNA synthesis. Data are expressed as means ± standard errors. *Significant effect of phenobarbital; ^†Significant effect of p50 deletion.

Figure 2

Effect of PB administration on hepatocyte apoptosis in wild-type and p50 -/- mice. A. 3-day administration. B. 10-day administration. C. 34 day administration. Histological sections for the liver were used for TUNEL staining, and apoptotic indexes were determined in hepatocytes to determine the rate of apoptosis. Data are expressed as means ± standard errors. *Significant effect of phenobarbital; ^†Significant effect of p50 deletion.

Figure 2

Effect of PB administration on hepatocyte apoptosis in wild-type and p50 -/- mice. A. 3-day administration. B. 10-day administration. C. 34 day administration. Histological sections for the liver were used for TUNEL staining, and apoptotic indexes were determined in hepatocytes to determine the rate of apoptosis. Data are expressed as means ± standard errors. *Significant effect of phenobarbital; ^†Significant effect of p50 deletion.

Figure 2

Effect of PB administration on hepatocyte apoptosis in wild-type and p50 -/- mice. A. 3-day administration. B. 10-day administration. C. 34 day administration. Histological sections for the liver were used for TUNEL staining, and apoptotic indexes were determined in hepatocytes to determine the rate of apoptosis. Data are expressed as means ± standard errors. *Significant effect of phenobarbital; ^†Significant effect of p50 deletion.

Figure 3

Effect of PB administration on TNF-α expression in wild-type and p50 -/- mice. A. 3-day administration. B. 10-day administration. C. 34 day administration. Real-time PCR was performed on RNA obtained from liver homogenates for both TNF-α and ß-actin. Results are expressed as a fraction of ß-actin expression. Data are expressed as means ± standard errors. *Significant effect of phenobarbital; ^†Significant effect of p50 deletion.

Figure 3

Effect of PB administration on TNF-α expression in wild-type and p50 -/- mice. A. 3-day administration. B. 10-day administration. C. 34 day administration. Real-time PCR was performed on RNA obtained from liver homogenates for both TNF-α and ß-actin. Results are expressed as a fraction of ß-actin expression. Data are expressed as means ± standard errors. *Significant effect of phenobarbital; ^†Significant effect of p50 deletion.

Figure 3

Effect of PB administration on TNF-α expression in wild-type and p50 -/- mice. A. 3-day administration. B. 10-day administration. C. 34 day administration. Real-time PCR was performed on RNA obtained from liver homogenates for both TNF-α and ß-actin. Results are expressed as a fraction of ß-actin expression. Data are expressed as means ± standard errors. *Significant effect of phenobarbital; ^†Significant effect of p50 deletion.

Figure 4

Effect of 3-day PB administration on the DNA binding activity of NF-κB in wild-type and p50 -/- mice. Nuclear extracts were prepared from mouse livers and used in EMSAs with a radiolabeled oligonucleotide containing an NF-κB binding site. After electrophoresis, gels were dried and subjected to autoradiography. Three mice were used for each of the four experimental treatments, and each lane represents a single mouse. Lane abbreviations: WT, wild-type mice; PB, phenobarbital; p50, p50 -/- mice.

Figure 5

Effect of 10-day PB administration on the DNA binding activity of NF-κB in wild-type and p50 -/- mice. Nuclear extracts were prepared from mouse livers and used in EMSAs with a radiolabeled oligonucleotide containing an NF-κB binding site. After electrophoresis, gels were dried and subjected to autoradiography. Three mice were used for each of the four experimental treatments, and each lane represents a single mouse. Lane abbreviations: WT, wild-type mice; PB, phenobarbital; p50, p50 -/- mice.

Figure 6

Effect of 34-day PB administration on the DNA binding activity of NF-κB in wild-type and p50 -/- mice. Nuclear extracts were prepared from mouse livers and used in EMSAs with a radiolabeled oligonucleotide containing an NF-κB binding site. After electrophoresis, gels were dried and subjected to autoradiography. Three mice were used for each of the four experimental treatments, and each lane represents a single mouse. Lane abbreviations: WT, wild-type mice; PB, phenobarbital; p50, p50 -/- mice.