J Korean Med Sci.  2011 Feb;26(2):222-230. 10.3346/jkms.2011.26.2.222.

Effects of Benzo(a)pyrene on the Expression of Heat Shock Proteins, Pro-inflammatory Cytokines and Antioxidant Enzymes in Hepatic Tumors Induced by Rat Hepatoma N1-S1 Cells

• Zheng Z
• Park SY
• Lee M
• Phark S
• Won NH
• Kang HS
• Sul D

• KMID: 1782110
• DOI: http://doi.org/10.3346/jkms.2011.26.2.222

Abstract

Benzo(a)pyrene (BaP) is a polycyclic aromatic hydrocarbon (PAH) that is easily introduced to humans via consumption of grilled or smoked meat. BaP causes harmful oxidative effects on cell development, growth and survival through an increase in membrane lipid peroxidation, oxidative DNA damage and mutagenesis. Therefore, the present study was conducted to evaluate the synergistic effects of BaP on oxidative stress in hepatic tumors. In this study, we established a hepatic tumor model by injecting rat hepatoma N1-S1 cells into healthy rats. Changes in the abundance of heat shock proteins (HSPs), antioxidant enzymes and pro-inflammatory cytokines were then investigated by western blot analysis. In addition, we examined changes in oxidative stress levels. Injection of N1-S1 cells or concomitant injection of BaP and N1-S1 cells resulted in the formation of hepatic tumors at the injection site. Evaluation of rat plasma reveals that hepatic tumors induced by BaP and N1-S1 cells expresses higher levels of Hsp27, superoxide dismutase (SOD), and tumor necrosis factor-alpha (TNF-alpha) when compared to those induced by N1-S1 cells only. The collective results of this study suggest that BaP exerts synergistic effects on the expression of HSP, cytokines and antioxidant enzymes in hepatic tumors induced by rat hepatoma N1-S1 cells.

Keyword

Liver Neoplasms; N1-S1 Rat Hepatoma Cells; Benzo(a)pyrene; Heat-Shock Proteins; Oxidative Stress; Cytokines

MeSH Terms

Animals
Antioxidants/*metabolism
Benzo(a)pyrene/*pharmacology
Carcinoma, Hepatocellular/metabolism/pathology
Cell Line, Tumor/*drug effects
Cytokines/*metabolism
Heat-Shock Proteins/*metabolism
Humans
Liver Neoplasms/*enzymology/*metabolism/pathology
Male
Neoplasms, Experimental/metabolism/pathology
Oxidative Stress/drug effects
Rats
Rats, Sprague-Dawley

Figure

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  Fig. 1 Development of tumors in rats injected with N1-S1 cells. Rats injected with N1-S1 cells were sacrificed one, two, four weeks after injection. (A) No tumors are observed in rats injected with vehicle controls. (B) Small tumors are found one week after injection of N1-S1 cells. (C) Tumors two weeks after injection of N1-S1 cells. A well-defined tumor mass with a multinodular appearance is found. (D) Tumors four weeks after injection of N1-S1 cells. The main tumor exhibits a multinodular appearance with depressed septations. Two satellite lesions are noted. Microscopically, all tumors revealed similar histology.

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  Fig. 2 Histology of tumors induced by concomitant injection of BaP plus N1-S1 cells. Resected livers were fixed in formalin, and sections were stained with hematoxylin and eosin. (A) Most of tumors shows tumoral necrosis (H&E, × 100). (B) Tumors are composed of anaplastic round cells with pushing borders (H&E, × 200), including (C) a small 0.4 cm diameter tumors exhibiting tumoral necrosis associated with extensive fibrosis (H&E, × 100). (D) In necrotic areas, dystrophic calcifications, fibrosis and foreign body type giant cell reaction are observed (H&E, × 200).

• 

  Fig. 3 Expression levels of HSPs in rats with N1-S1 cells or concomitant injection of BaP and N1-S1 cells. Plasma collected from rats with hepatic tumors was evaluated for HSPs and was compared to control-injected and BaP-injected rats. (A) Western blot analysis of HSPs was performed using anti-Hsp27, Hsp90 and GRP78 antibodies. Equal amounts of total protein were loaded into each lane. (B-D) Graphs illustrating the changes in the levels of Hsp27, Hsp90, and GRP78 in the plasma from control-injected rats, BaP-injected rats, and rats with hepatic tumors induced by injection of N1-S1cells or concomitant injection of BaP and N1-S1 cells. Densitometric values are expressed as a ratio to control-injected rats. All data represent the mean ± SD of five different rats. P < 0.05 compared to the control rats, P < 0.05 compared to BaP-injected rats, P < 0.05 compared to rats with N1-S1 cell-induced hepatic tumors, P < 0.05 compared to rats with hepatic tumors induced by concomitant injection of BaP and N1-S1 cells. †, ‡, §, significant difference between control and BaP, N1-S1, and N1-S1 + BaP groups. *, significantly different from control; †, significantly different from BaP; ‡, significantly different from N1-S1; §, significantly different from N1-S1 + BaP.

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  Fig. 4 Levels of antioxidant enzymes and protein oxidation in rats with hepatic tumors induced by injection of N1-S1 cells or concomitant injection of BaP and N1-S1 cells. Plasma collected from rats with hepatic tumors was evaluated for heat shock proteins and was compared to controls and BaP-injected rats. (A) Western blot analysis of antioxidant enzymes was performed using anti-SOD-1 and GST antibodies. Equal amounts of total protein were loaded into each lane. (B, C) Graphs showing changes in the levels of SOD-1 and GST in plasma from controls, BaP-injected rats, or rats with hepatic tumors induced by injection of N1-S1 cells or concomitant injection of BaP and N1-S1 cells. (D) Western blot analysis of carbonyl content was performed using an anti-dinitrophenylhydrazine antibody. Equal amounts of total protein were loaded into each lane. Arrow: a significant increase in protein oxidation compared to vehicle-treated control rats. (E) Graph showing changes in the levels of protein oxidation in plasma from controls, BaP-injected rats, or rats with hepatic tumors induced by injection of N1-S1 cells or concomitant injection of BaP and N1-S1 cells. Densitometric values are expressed as a ratio to control rats. All data represent the mean ± SD of five different rats. P < 0.05 compared to the control rats, P < 0.05 compared to BaP-injected rats, P < 0.05 compared to rats with N1-S1 cell-induced hepatic tumors, P < 0.05 compared to rats with hepatic tumors induced by concomitant injection of BaP and N1-S1 cells. *, significantly different from control; †, significantly different from BaP; ‡, significantly different from N1-S1; §, significantly different from N1-S1 + BaP.

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  Fig. 5 Expression levels of cytokines in rats with hepatic tumors induced by injection of N1-S1 cells or concomitant injection of BaP and N1-S1 cells. Plasma collected from rats with hepatic tumors was evaluated for cytokines and was compared to controls and BaP-injected rats. (A) Western blot analysis of cytokines was performed using anti-TNF-α, IL-1β and IL-6 antibodies. Equal amounts of total protein were loaded into each lane. (B-D) Graphs showing changes in the levels of TNF-α, IL-1β and IL-6 in plasma from controls, BaP-injected rats or rats with hepatic tumors induced by injection of N1-S1 cells or concomitant injection of BaP and N1-S1 cells. Densitometric values are expressed as a ratio to control rats. All data represent the mean ± SD of five different rats. P < 0.05 compared to the control rats, P < 0.05 compared to BaP-injected rats, P < 0.05 compared to rats with N1-S1 cell-induced hepatic tumors, P < 0.05 compared to rats with hepatic tumors induced by concomitant injection of BaP and N1-S1 cells. *, significantly different from control; †, significantly different from BaP; ‡, significantly different from N1-S1; §, significantly different from N1-S1 + BaP.

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