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and the overall synthesis of ranitidine ..
Based on these results, further experiments are in progress to assess hepatocyte proliferation in culture in the presence of cimetidine and ranitidine metabolites to completely clarify this phenomenon. In the interim, we emphasize that our results, confirming those found by Kanashima in vivo, focus attention on the need for caution and careful evaluation when this drug is administered to the patient immediately following hepatectomy.
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.
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depicts the effect of various concentrations of cimetidine, ranitidine, famotidine and omeprazole on the proliferation of hepatocytes cultured for 48 h in serum-free medium. Conditions were the same as in the previous experiment, with the exception of an addition of EGF to the medium which promoted hepatocyte proliferation. None of these drugs affected the hepatocyte proliferation stimulated by EGF. Further confirmation of these results is reported in , which lists the concentration of DNA and the percent labelled nuclei index of hepatocyte proliferation. This stimulation of hepatocyte proliferation over that observed in the culture incubated in the presence of insulin alone was not affected by the presence of drugs. The same results were obtained when, under the same conditions, serum of PH or non-PH rats was added. outlines the effect of various serum concentrations (10–50% final concentration) on the DNA synthesis of hepatocytes in culture. Serum was obtained from sham-operated animals which had received i.p. cimetidine, ranitidine, famotidine, omeprazole and saline. No statistical difference was found in DNA synthesis between any of these groups. The effect of serum, obtained from rats that underwent PH and received H2 blocking agents and omeprazole, on DNA synthesis and percentage of labelled nuclei is depicted in . The DNA synthetic activity of hepatocytes cultured in the presence of different concentrations of serum from hepatectomized rats treated with the drugs was compared with the values obtained using the serum from hepatectomized rats treated with saline. The serum of PH rats treated with cimetidine inhibited DNA synthesis and percent of labelled nuclei at all the concentrations of serum used. Although the inhibitory effect of cimetidine was partially covered by the different serum concentrations, the percent of the effect increased in relation to the amount of serum used (10% serum = 29% inhibition; 40% serum = 38% inhibition). The serum of PH rats injected with ranitidine inhibited hepatocyte proliferation only at the higher serum concentrations, i.e., 40–50%. No inhibitory effects were observed using the serum of PH animals treated with famotidine or omeprazole. The figure also shows the value of ALT in these conditions. No significant variation of ALT was observed in the presence of 40% serum concentrations.
However, a sig nificant 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.
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To further define their possible influence on hepatocyte proliferation, the effect of serum obtained from rats treated with H2 blocking agents or omeprazole was studied. Two sets of experiments were compared, one using the serum of sham-operated animals, the other using serum of 70% hepatectomized rats. The most significant results were observed when the serum of 70% hepatectomized rats treated with the drugs was added to the culture medium (). Serum from hepatectomized rats treated with cimetidine inhibited hepatic proliferation in a manner that was somewhat related to the concentration of serum used. Serum of PH animals treated with ranitidine resulted in inhibition of DNA synthesis only at very high concentrations, while the serum of PH animals treated with famotidine and omeprazole did not affect hepatic proliferation at any of the concentrations used. It is evident from these studies, therefore, that the significant inhibition of DNA synthesis and percent labelled nuclei that is modulated by cimetidine and ranitidine is not due to the actual drug but is manifested by their ability to induce some variation(s) in the composition of serum during the course of hepatic regeneration.
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