Geoffrey H. Moyer

Binding of N-hydroxy acetylaminofluorene to eu- and heterochromatin fractions of rat liver in vivo

The in vivo binding of N-hydroxy acetylaminofluorene (N-OH AAF) to rat liver DNA was studied in hetero- and euchromatin fractions prepared by sedimentation through sucrose gradients. The greater transcriptional capacity of the slowly sedimenting (euchromatin) fractions was confirmed by their enhanced incorporation of radioactive precursors into RNA in vivo and their increased template activity for in vitro RNA synthesis by purified RNA polymerase. N-OH AFF was bound in 4- to 5-fold greater amounts to euchromatin DNA than to heterochromatin 2 h after a single injection of the compound. However, the bound carcinogen appeared to be eliminated more rapidly from euchromatin than from heterochromatin by DNA repair processes.

Quantitative and qualitative changes in RNA metabolism in rats fed N-2-acetylaminofluorene

Abstract The effects of N-2 acetylaminofluorene (AAF) on hepatic RNA metabolism were studied in rats fed the carcinogen for 3 to 31 days, a period when early events in neoplastic transformation are thought to occur. Although the liver cells remained normal microscopically, the rate of RNA synthesis was inhibited by 55% during the first week of AAF feeding. This resulted in a substantial decrease in both nuclear and cytoplasmic RNA. Shifts also occurred in the distribution of cytoplasmic RNA among various subcellular fractions. Sedimentation profiles showed a small but consistent reduction in polysome size and in the amount of RNA in the larger polysomal aggregates. All of these parameters returned to normal or near normal after 1 month of AAF feeding, indicating the development of a form of resistance to AAF. RNA-DNA hybridization experiments revealed that hepatic transcription of moderately reiterated genes throughout the first month of AAF feeding was qualitatively similar to that in normal liver. Likewise, transport of these classes of RNA to the cytoplasm was unaltered. The initial quantitative derangements in RNA metabolism and their subsequent recovery in the face of continued administration of the chemical may be important in the induction of hepatic cancer by AAF.

The acute effects of N-hydroxy-acetylaminofluorene on rat liver protein synthesis

Abstract A single intraperitoneal injection of N-hydroxy-acetylaminofluorene (N-hydroxy-AAF) at a dosage of 30 mg/kg significantly inhibited rat liver protein synthesis within 15 min. Marked alterations in the subcellular distribution of hepatic RNA accompanied the decline in protein synthesis in treated rats. These changes included decreases in nuclear and bound polysomal RNA and increases in free polysomal and non-sedimentable RNA. Heavy polysomal aggregates, both free and bound, were almost completely degraded to monomers and dimers during this period. Sedimentation profiles of total cytoplasmic RNA revealed no evidence of gross RNA breakdown in N-hydroxy-AAF-treated animals. To determine the mechanisms responsible for the inhibition of protein synthesis by N-hydroxy-AAF, cellular components involved in protein synthesis were purified from control and treated animals and examined in two cell-free systems. In a system which measures polypeptide chain elongation and release, the incorporation of amino acids into protein was reduced by 35% using polysomes from N-hydroxy-AAF treated animals compared with controls. By contrast, the function of the pH 5 fraction (containing aminoacylating enzymes and tRNA) from the carcinogen-treated animals was unimpaired. A wheat germ lysate system was used to determine the ability of mRNA to program polypeptide chain initiation and elongation. Cytoplasmic poly(A) + RNA from N-hydroxy-AAF treated rats showed reduced capacity to stimulate protein synthesis in wheat germ lysates compared with similar preparations from DMSO-injected control rats. The rapid inhibition of protein synthesis by N-hydroxy-AAF may be an important contributing factor to other toxic effects of the carcinogen, including the inhibition of rRNA synthesis.

A novel method for the small scale synthesis of N-acetoxy-2-acetylaminofluorene and its adducts

The carcinogen N-acetoxy-2-acetylaminofluorene (N-acetoxy-AAF) can be synthesized effectively in milligram amounts by reacting N-hydroxy-2-acetylaminofluorene (N-hydroxy-AAF) with acetic anhydride in a continuously mixed, closed 2-phase system consisting of ethyl ether/petroleum ether (2 : 1 v/v) and dilute sodium bicarbonate. The organic phase containing N-acetoxy-AAF can be added directly to 10 mm sodium citrate/20% ethanol to synthesize nucleoside or nucleotide adducts without the need to crystallize or resuspend N-acetoxy-AAF in a polar solvent. The advantage of the method is that it makes possible the synthesis of nucleic acid derivatives of N-acetoxy-AAF with high specific activity in small quantities.

Inhibition of RNA synthesis by derivatives of the carcinogen 2-acetylaminofluorene.

Summary The way in which the carcinogen N-hydroxy-N-2-acetylaminofluorene inhibits hepatic RNA synthesis was studied using the reactive ester N-acetoxy-N-2-acetylaminofluorene (N-acetoxy AAF). Preincubation of isolated nuclei with N-acetoxy AAF inhibited the ability of those nuclei to carry out RNA synthesis using endogeneous template or an exogenous template poly d(AT). Likewise, the template ability of DNA for RNA synthesis and the transcriptional capacity of RNA polymerases I and II were inhibited by preincubation of these macromolecules in vitro with N-acetoxy AAF. Inactivation of enzyme or template by N-acetoxy AAF was associated with and presumably caused by binding of AAF derivatives to these macromolecules. AAF binding required to inactivate polymerase or DNA in vitro was greater than that observed in vivo under conditions where RNA synthesis was inhibited to a comparable extent suggesting that additional mechanisms may operate in vivo to sensitize enzyme and template to inactivation by AAF binding.

Inhibition of RNA Polymerase III by N-Hydroxy-2-Acetylaminofluorene

Abstract Parenteral administration of the potent hepatocarcinogen N-hydroxy-2-acetylaminofluorene to rats results in a substantial inhibition of hepatic RNA polymerase III(C), the enzyme responsible for the synthesis of tRNA and 5 S RNA. The minor component of RNA polymerase III extractable from nuclei shows a 45-65% reduction in activity while the major component of the enzyme extractable from cytoplasm shows a 40-65% inhibition. This reduction of RNA polymerase III activity is sufficient to account for the inhibition of rRNA synthesis produced by administration of the carcinogen in vivo.

STUDIES ON THE INHIBITION OF TRANSCRIPTION BY THE HEPATOCARCINOGEN N-HYDROXY-2-ACETYIAMINOFLUORENE

The mechanisms of inhibition of hepatic RNA synthesis by the carcinogen N-OH AAF have been examined. The activity of purified RNA polymerases I and II is reduced while template effects do not appear to be important at dosages Commonly employed. Inhibition of protein synthesis may contribute to the reduction in rRNA synthesis.