Cyclooxygenase 2 and Prostaglandin E2 are not Involved in N-Nitrosodiethylamine-Initiated Early Rat Hepatocarcinogenesis

Abstract

The present study was undertaken to investigate the effect of dietary supplementation with nimesulide or eugenol on N-nitrosodiethylamine (DEN)-initiated early hepatocarcinogenesis in F344 male rats. Both compounds did not alter the expression of cytochrome P450 (CYP) 2E1, the enzyme that plays a major role in the activation of DEN to genotoxic products; however, nimesulide induced the expression of CYP1A1. Western blot analysis revealed that COX-1 and COX-2 protein expressions were not modulated by DEN compared with normal controls. Furthermore, post-initiation feeding with nimesulide or eugenol did not modulate COX-2 protein expression in normal or DEN-treated rats, whereas eugenol significantly increased the liver prostaglandin E(2) (PGE(2)) levels of DEN-injected animals compared with the DEN controls. Ultimately, nimesulide or eugenol did not modify DEN-induced hepatocarcinogenesis as evidenced by insignificant changes in the number and size of preneoplastic placental glutathione S-transferase (GST-P) positive liver foci compared with the DEN controls. These results suggest that COX-2, as well as prostaglandin E(2), may play no role in the post-initiation development of DEN-induced rat hepatocarcinogenesis at an early stage.

Keywords: cyclooxygenases; eugenol; liver; nimesulide; rat.

Fig. 1

Experimental protocol. A total of 42 six-week-old F344 male rats were used throughout this study. A group of thirty-three rats (each animal was injected with DEN) and a group of nine rats (each rat was injected with saline) were further subdivided each into three equally-sized groups as shown. The rats were sequentially sacrificed two or four weeks following DEN injection or 4 weeks following saline injection. : Diethylnitrosamine (DEN), 200 mg/Kg b.w., i.p. : Saline, i.p. : Nimesulide 400 ppm in diet, : Eugenol 6000 ppm in diet, : Basal diet. S: Sacrifice.

Fig. 2

A representative western blot showing the expression of cytochrome P450 (CYP) 1A1/1A2 and 2E1 proteins in the livers of F344 male rats two weeks after DEN injection and treatment with nimesulide (NIM) or eugenol (EUG). The histogram shows the relative densities of bands normalized to β-actin (means ± SE). * Significantly different from the DEN controls at P<0.05 by LSD test.

Fig. 3

A representative western blot showing the expression of cyclooxygenase (COX)-1 and COX-2 proteins in the livers of F344 male rats four weeks following DEN injection and treatment with nimesulide (NIM) or eugenol (EUG). The histogram shows the relative densities of bands normalized to β-actin (means ± SE).

Fig. 4

A representative western blot showing the expression of COX-1 and COX-2 proteins in the livers of normal F344 males, livers of those treated with nimesulide or eugenol and livers of DEN-injected rats. The histogram shows the relative densities of bands normalized to β-actin (means ± SE).

Fig. 5

Representative hematoxylin and eosin staining (A, C, E, G, I and K) and immunohistochemical findings of GST-P positive liver preneoplastic foci (B, D, F, H, J and L) of the DEN controls two weeks (A and B) and four weeks (C and D) after DEN injection (200 mg/Kg b.w.) and following treatment with nimesulide (E and F, G and H) or eugenol ( I and J, K and L) for one or three weeks , respectively (×20). Arrows indicate foci.

Fig. 6

Statistical comparison of the number of GST-P positive liver cell foci (No./cm²) in F344 male rats two and four weeks after DEN injection and treatment with nimesulide (NIM) or eugenol (EUG), as compared with their counterpart DEN controls (values are means ± SE).

Fig. 7

Statistical comparison of the areas of GST-P positive liver cell foci (mm²/cm²) in F344 male rats two and four weeks following DEN injection and treatment with nimesulide (NIM) or eugenol (EUG), as compared with their counterpart DEN controls (values are means ± SE).