Effect of dichloromethylene diphosphonate on liver regeneration following thioacetamide-induced necrosis in rats

Abstract

Aim: To study the effect of dichloromethylene diphosphonate (DMDP), a selective Kupffer cell toxicant in reference to liver damage and postnecrotic liver regeneration in rats induced by sublethal dose thioacetamide (TA).

Methods: Rats, intravenously (iv) pre-treated with a single dose of DMDP (10 mg/kg), were intraperitoneally (ip) injected with TA 6.6 mmol/kg (per 500 mg/kg body weight). Hepatocytes were isolated from rats at 0, 24, 48 and 72 h following TA intoxication and blood and liver samples were obtained. To evaluate the mechanisms involved in the postnecrotic regenerative state, DNA distribution and ploidy time course were assayed in isolated hepatocytes. Circulating cytokine tumor necrosis factor-alpha (TNF-α) was assayed in serum and determined by reverse transcriptase-polymerase chain reaction in liver extract.

Results: The effect of DMDP induced noticeable changes in postnecrotic regeneration, causing an increased percentage of hepatocytes in the cell cycle S phase. The increase at 24 h in S1 population in rats pretreated with DMDP + TA was significantly (P < 0.05) different compared with that of the TA group (18.07% vs 8.57%). Hepatocytes increased their proliferation as a result of these changes. Also, TNF-α expression and serum level were increased in rats pre-treated with DMDP. Thus, DMDP pre-treatment reduced TA-induced liver injury and accelerated postnecrotic liver regeneration.

Conclusion: These results demonstrate that Kupffer cells are involved in TA-induced liver, as well as in postnecrotic proliferative liver states.

Keywords: Cell cycle; Dichloromethylene diphosphonate; Hepatotoxicity; Kupffer cells; Thioacetamide.

Figure 1

Enzymatic activity after dichloromethylene diphosphonate pre-treatment in rats intoxicated with one sublethal dose of thioacetamide. A: Effect of dichloromethylene diphosphonate (DMDP) pre-treatment on aspartate aminotransferase (AST) activity in the serum of rats intoxicated with one sublethal dose of thioacetamide (TA); B: Illustrates the effect of DMDP pre-treatment on isocitrate dehydrogenase activity in the serum of rats intoxicated with one sublethal dose of TA. Samples were obtained at 0, 12, 24, 48 and 72 h following TA 6.6 mmol (per 500 mg/kg body weight). The results, expressed as nmol per min per mL of serum, are the mean ± SD of four determinations in duplicate from four rats. ^aP < 0.05 vs the respective control, ^cP < 0.05 vs differences due to DMDP.

Figure 2

Effects of dichloromethylene diphosphonate in protein levels, gene expression and messenger levels tumor necrosis factor-alpha. A: Effects of dichloromethylene diphosphonate (DMDP) pre-treatment on serum tumor necrosis factor-alpha (TNF-α) levels determined by enzyme-linked immunosorbent assay tests on serum samples. Columns and vertical bars represent mean ± SD evaluated in four determinations from four rats. ^aP < 0.05 vs control group; ^cP < 0.05 vs the DMDP-treated group; B: Effects of DMDP in gene expression profile of TNF-α assayed by reverse transcriptase-polymerase chain reaction analysis; C: Illustrates the effect of DMDP pre-treatment on the levels of TNF-α messenger RNA (mRNA) in liver homogenates of rats intoxicated with a sublethal dose of thioacetamide (TA). Samples were obtained at 0, 24, 48 and 72 h. The results, expressed in arbitrary units, are the mean ± SD of four determinations from four rats. ^aP < 0.05 vs the respective control, ^cP < 0.05 vs differences due to DMDP.