Bile acid-induced modifications in DNA synthesis by the regenerating perfused rat liver,

Abstract

Liver cell proliferation is a complex process that can be affected by a large number of factors such as bile acids, which have been reported to be associated to the pathogenesis of liver cancer. In this work, bile acid-induced modifications in DNA synthesis by regenerating perfused rat liver were investigated. Two-thirds hepatectomy was carried out 24 hr before perfusion of liver with recirculating, erythrocyte-free Krebs-Henseleit solution. The viability of the preparations was maintained under all experimental conditions, as indicated by bile flow, oxygen uptake, perfusion pressure, perfusion flow and release of lactate dehydrogenase and potassium into the perfusate. Livers received (min 10 to min 60) bile acid infusion at a rate of 25 nmol/min/gm liver (i.e., maximal secretion rate/2) in regenerating livers as calculated for taurocholate in separate experiments). Trace amounts of [methyl-14C]thymidine were added to the perfusate at min 30. At the end of the experiments (min 60) the livers were washed, removed, weighed and homogenized to determine radioactivity in whole tissue, in DNA and in non-DNA-related fractions. Taurocholate and, to a lesser extent, taurodeoxycholate and dehydrocholate (but not ursodeoxycholate) were found to reduce 14C incorporation into DNA. This was not due to changes in the content of 14C in whole, regenerating liver tissue. Taurocholate, taurodeoxycholate, dehydrocholate and ursodeoxycholate had no effect on thymidine uptake; moreover, the proportion of 14C found in bile was negligible. However, bile acid-induced modification in the fate of intracellular thymidine was observed. In regenerating livers receiving no bile acid, the 14C carried by thymidine metabolites accounted for about 60% of 14C in whole liver tissue. Taurocholate markedly increased this proportion to about 80%. Reverse-phase high-pressure liquid chromatography revealed that most of this 14C (about 80%) was recovered at the elution time, corresponding to thymidine catabolites rather than to DNA precursors. These results suggest that bile acids induce enhancement of thymidine catabolism that reduces its incorporation into DNA; inhibition in the process of DNA synthesis itself, leading to a subsequent increase in the metabolism of DNA precursors; or both. Moreover, from the diversity in this property for bile acid species it might be inferred that changes in the composition and size of the bile acid pool during liver carcinogenesis or regeneration play a role in the modulation of the proliferative process.

Bile secretion by the rat liver during synchronized regeneration

“…Plasma and bile samples were collected every 10 minutes and bile flow, oxygen uptake, perfusion pressure, perfusion flow, potassium and lactate dehydrogenase activity released into the perfusate were determined during the experiments to assess the viability of the preparations. The values obtained were similar to those previously found by us in experiments on isolated nonregenerating (Marin et al 1991a) and regenerating (Marin et al 1993) liver preparations (data not shown).…”

“…The hepatic artery was ligated and the common bile duct, portal vein and inferior vena cava were cannulated. Livers were perfused in situ in a prewarmed (388C) cabinet by an adaptation (Marin et al 1993) of the method of Miller et al (1951) for regenerating livers. The recirculating perfusion medium was 100 ml of a modified Krebs-Henseleit solution containing the following in mM: 120 NaCl, 5 KCl, 1.29 CaCl 2 , 0.65 MgSO 4 , 25NaHCO 3 , 1.17 KH 2 PO 4 , 5.0 D-glucose, 3% BSA and gentamicin sulphate (100 mg/l).…”

“…Since Higgins and Anderson (1931) started to use twothirds PH in rats as an experimental model to study LR, this protocol has been widely used by others and ourselves in the investigation of liver cell proliferation (Marin et al 1993;Barbero et al 1995) as well as of the changes occurring in enzymatic activities and liver functions during this process (Marin et al, 1991b;Perez-Barriocanal et al 1986b).In this work we used a combinationof this with another old but still useful model; i.e. the isolated in situ perfused rat liver.…”

“…the isolated in situ perfused rat liver. Appropriately adapted (Marin et al 1993), this preparation has the advantages of both maintaining organ integrity and allowing perfusate manipulations, which are very useful in the performance of experiments designed to study bile secretion.…”

Salamanca, Spain Symposium

“…Using different experimental approaches the effect of bile acids on DNA synthesis by the regenerating rodent liver has been investigated. Radiolabelled thymidine incorporation into DNA was found to be inhibited by taurocholate, both in the mouse in vivo (Barbero et al 1995) and in regenerating perfused rat liver (Marin et al 1993). Therefore, whereas a role for paracrine and autocrine factors in this effect cannot be ruled out, it is suggested that changes in the blood supply of nutrients and the presence of extrahepatic factors are not mandatory for bile acid-induced inhibition of DNA synthesis during liver regeneration.…”