Role of rate-limiting enzymes of nucleotide metabolism in taurocholate-induced DNA synthesis inhibition.
OBJECTIVE
In previous studies we have shown the ability of bile acids to reduce the rate of thymidine incorporation into DNA by the regenerating rodent liver. The aim of the present work was to investigate the sensitivity of the key pathways involved in thymidine metabolism to taurocholate.


RESULTS
Incubation of [14C]-thymidine with mouse liver extracts revealed that addition of taurocholate to the reaction medium induced significant dose-dependent inhibition in the activity of the salvage nucleotide pathway rate-limiting enzyme, thymidine kinase, while other steps of nucleotide metabolism machinery, such as the rate-limiting enzyme of de novo deoxyribonucleotide synthesis, ribonucleotide reductase and the rate-limiting enzyme of thymidine catabolism, dihydropyrimidine dehydrogenase were found to be insensitive to inhibition by taurocholate. Additional experiments were carried out on isolated perfused rat livers whose regeneration was induced by two-thirds hepatectomy and synchronized by intravenous administration of reversible ribonucleic reductase inhibitor hydroxyurea (bolus: 170 mumol/100 g body weight, plus 10 h infusion: 2.0 mumol/min per 100 g body weight, from 14 to 24 h after hepatectomy). Hydroxyurea treatment was interrupted and liver perfusions were carried out 0, 2, 4 or 8 h later. Thymidine incorporation into DNA over 30 min perfusion with media containing [14C]-thymidine was measured after separating DNA from acid-soluble fraction. A marked increase in DNA synthesis was observed up to 4 h after stopping ribonucleotide reductase inhibition. At this time, reduced relevance of the salvage pathway can be expected as compared with the de novo released pathway. In contrast with the inhibitory effect observed when taurocholate was added to the perfusate of untreated regenerating livers, taurocholate was found to have no effect on DNA synthesis, at the peak of synchronized DNA synthesis, although taurocholate-induced alteration in thymidine metabolism was suggested from h.p.l.c. analysis of acid-soluble fraction.


CONCLUSIONS
These results suggest that effects on the nucleotide metabolism machinery, and hence changes in deoxyribonucleotide phosphate pools may underlie the ability of taurocholate to affect DNA synthesis by the regenerating rodent liver.
Monte
J M
JM
Department of Physiology and Pharmacology, Faculty of Pharmacy, University of Salamanca, Spain.
Barbero
E R
ER
Villanueva
G R
GR
Serrano
M A
MA
Marin
J J
JJ
D000818:Animals; D004247:DNA / Q000037:antagonists & inhibitors* / Q000096:biosynthesis; D004798:Enzymes / Q000378:metabolism*; D008099:Liver / Q000187:drug effects* / Q000378:metabolism*; D008115:Liver Regeneration / Q000502:physiology*; D008297:Male; D051379:Mice; D008815:Mice, Inbred Strains; D009711:Nucleotides / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D013656:Taurocholic Acid / Q000494:pharmacology*; D013936:Thymidine / Q000378:metabolism
D004798:Enzymes; D009711:Nucleotides; D013656:Taurocholic Acid; D004247:DNA; D013936:Thymidine