title: Assay and time course of 5-fluorouracil incorporation into RNA of L1210/0 ascites cells in vivo.
abstract: A method for determination of levels of incorporation of nonradiolabeled 5-fluorouracil (FUra) into RNA (F-RNA) in tissue samples is shown to be applicable to tissues in vivo. BDF1 mice bearing L1210 ascites cells were injected intraperitoneally with [14C]FUra, 100 mg/kg. The time course of F-RNA levels in L1210 cells was determined by following the radiolabeled drug, and by NaB3H4 labeling of isolated and derivatized nucleoside. RNA ribonucleotides were obtained by KOH hydrolysis of perchloric acid precipitates of cell sonicates. FUMP nucleotides were separated from remaining nucleotides by DEAE-cellulose chromatography. FUMP fractions were treated with alkaline phosphatase, and FUrd was separated from non-FUrd nucleoside contaminants by additional DEAE-cellulose chromatography. FUrd was quantitated by periodate oxidation of ribose and NaB3H4 reduction of the resulting nucleoside dialdehydes. Isolation of tritiated FUrd-trialcohol from remaining tissue contaminants and background radioactivity was done by silica gel thin layer chromatography. Comparison of results obtained by isolation of [14C]FUrd with results of NaB3H4 labeling of the same samples showed parallel results with comparable biological standard deviations, although the tritium method consistently gave slightly lower values. The peak level of F-RNA at 3 hr was 1 base substitution per 174 normal nucleotides. The level of F-RNA after 3 hr declined slowly, so that at 96 hr there still remained 1 FUra base per 597 normal nucleotides. Serial determinations of RNA content showed marked decreases, on the basis of either DNA or protein level, that continued up to 96 hr after FUra administration. These biochemical effects are among the most prolonged reported for FUra, suggesting the possibility that F-RNA represents a storage compartment for release of toxic metabolites and emphasize the need for additional study of RNA effects at long time points. Our method for assay of F-RNA appears to be suitable for study of biopsy specimens of tumors and normal tissues following nonradiolabeled-FUra administration.
authors.forename: C P
authors.initials: CP
authors.lastname: Spears
authors.forename: J
authors.initials: J
authors.lastname: Shani
authors.forename: A H
authors.initials: AH
authors.lastname: Shahinian
authors.forename: W
authors.initials: W
authors.lastname: Wolf
authors.forename: C
authors.initials: C
authors.lastname: Heidelberger
authors.forename: P V
authors.initials: PV
authors.lastname: Danenberg
mesh_terms: D000818:Animals; D002250:Carbon Radioisotopes; D004247:DNA / Q000032:analysis; D005260:Female; D005472:Fluorouracil / Q000378:metabolism*; D007700:Kinetics; D007939:Leukemia L1210 / Q000378:metabolism*; D051379:Mice; D008811:Mice, Inbred DBA; D012313:RNA / Q000032:analysis / Q000096:biosynthesis*
chemical_list: D002250:Carbon Radioisotopes; D012313:RNA; D004247:DNA; D005472:Fluorouracil