Effect of cimetidine, ranitidine, famotidine and omeprazole on hepatocyte proliferation in vitro

Abstract

Recently reports have indicated that both cimetidine and ranitidine delay cell proliferation in rats following 70% partial hepatectomy and result in an increased mortality following this procedure. The present study was designed to determine whether three H2 blocking agents (cimetidine, ranitidine, famotidine) and a new, powerful antisecretory drug (omeprazole) specifically influence hepatocyte proliferation in primary culture. Hepatocytes were isolated from livers of normal male rats by the standard collagenase perfusion technique. Hepatic DNA synthesis and percent of labelled nuclei were determined after 48 h incubation. Hepatocytes in culture were incubated with the H2 blocking agents and omeprazole or with different concentrations of serum obtained from sham-operated or 70% hepatectomized rats treated or not with the same agents. Rats were injected intraperitoneally at 8:00 a.m. on two consecutive days. In hepatectomized rats, the first dose was injected at 8:00 a.m. immediately after surgery, the second, 24 h later. The serum of sham-operated or 70% hepatectomized rats that did not receive drugs served as control. No changes in DNA synthesis, percentage of labelled nuclei and transaminase were detected when the agents were added to the hepatocytes in culture at concentrations within the effective pharmacological dosage and 30 times higher. Similarly, no changes in these parameters were obtained when different concentrations of serum obtained from sham-operated rats treated with H2 blocking agents or omeprazole were added to the basal culture medium. However, a significant inhibition of DNA synthesis and of percentage of labelled nuclei was observed when hepatocytes were incubated in the presence of serum from 70% hepatectomized rats that had been treated with cimetidine or with ranitidine. The serum of 70% hepatectomized rats treated with famotidine and omeprazole had no effect on hepatocyte proliferation in vitro. No effect on transaminase was found in these conditions.

Fig. 1

Effect of cimetidine, ranitidine, famotidine and omeprazole on DNA synthesis of hepatocytes in primary culture. Hepatocyte isolation has been described under ‘Materials and Methods’. Cells were plated in MEM supplemented with 5% FCS and insulin (10⁻⁷ M). After 3 h, the medium and floating cells were discarded and serum-free medium plus insulin (10⁻⁷ M), EGF (10 ng/ml) and proline (0.26 mM) were added. H₂ blocking agents were added at different concentrations on the basis of their blood level after administration of a therapeutic dose; cimetidine, ranitidine, famotidine (0.62–30 μg/ml) and omeprazole (0.06–1.8 μg/ml). Cultures were exposed to 7.5 μCi [³H] thymidine for the last 24 h and processed after 48 h for determination of DNA synthesis. For each medium, triplicate culture dishes from 5 different animals were used. Results (mean ± S.D.) are expressed as cpm × 10⁻²/μg DNA.

Fig. 2

Effect of serum obtained from rats sham-operated and treated with H₂ blocking agents and omeprazole on DNA synthesis of hepatocytes in primary culture. The experimental conditions are described in ‘Materials and Methods’. Culture conditions were as in Fig. 1, with the exception that serum from rats treated with or without (as control) H₂ blocking agents were added instead of EGF. Four concentrations (10–50%) of serum were used. Results (mean ± S.D.) are expressed as cpm × 10⁻²/μg DNA. For each medium, triplicate culture dishes from 5 different experiments were used.

Fig. 3

Effect of different concentrations of serum obtained from 70% hepatectomized rats treated or not with cimetidine, ranitidine, famotidine and omeprazole on DNA synthesis, % labelling index and ALT of hepatocytes in primary culture. The experimental conditions are described in ‘Materials and Methods’. Culture conditions were as reported in Fig. 1, with the exception that serum from treated and not treated (as control) 70% hepatectomized rats was added instead of EGF. Four final concentrations (10, 20, 40 and 50%) of serum were used. Exposure to [³H] thymidine was for 24 h. Individual culture dishes were processed for autoradiography and DNA synthesis. A total of 1000 cells randomly selected were counted to obtain the percent of labelled cells. Results are presented as mean ± S.D. (DNA synthesis = cpm × 10⁻²/μg DNA; labelled nuclei = % of labelled nuclei; aminotransferase = U/ml). For each medium, triplicate culture dishes from 5 different experiments were used. Significantly different from the control value observed in each group: *P < 0.05; **P < 0.01.

Fig. 4

Cimetidine levels in sham-operated (●–––●) and PH (○– – –○) rats.