Chemical carcinogenesis. I. Rat liver tRNA ethylation by ethionine

The mechanism of biological action of the powerful carcinogen ethionine is still unknown at the present. Here the "in vivo" tRNA ethylation after administration of radioactive ethionine to rats has been reinvestigated. In particular, the radioactive "pyrimidine nucleotides" fraction was examined: chromatographic and ultraviolet-spectral analyses indicated the presence of imidazole-ring-opened derivatives of guanosine in this fraction, the identification of which is reported in the accompanying paper. These data appear particularly interesting especially when considering the recently advanced hypothesis (6,7) of a transversion purine----pyrimidine as the initial precancerous biochemical lesion in chemical carcinogenesis.     

Origin and formation of 1,7-dimethylguanosine in tRNA chemical and enzymatic methylation

tRNA chemical methylation: 1. 1,7-Dimethylguanosine was found in in vivo methylated tRNA from liver and kidney of rat after exposure to a low dose of dimethylnitrosamine (4 mg/kg body weight). 2. At 4 h after dimethylnitrosamine administration, the 1,7-dimethylguanosine:7-methylguanine ratio (product ratio) for liver and kidney tRNA was 0.017 and 0.091, respectively. At 24 h after dimethylnitrosamine administration, the product ratio was lower in both hepatic and renal tRNA. 3. When dimethylnitrosamine was given in four separate daily injections, the product ratio in hepatic tRNA 4 h after the last dose was the same as for the same total dose given by a single injection, but in renal tRNA it was lower. No dialkyl compound was found in liver and kidney tRNA 24 h after the last multiple injection. tRNA enzymatic methylation: 1. Base analyses of Escherichia coli B tRNA methylated in vitro, by using S-adenosylmethionine as physiological methyl donor and enzyme preparations from liver and kidney of normal rat, indicated that 1,7-dimethylguanosine was also a product of enzymatic methylation. 2. The amount of 1,7-dimethylguanosine formed by kidney enzyme preparation was 3-times that produced by the liver extract. 3. A second type of enzymatic methylation assay where chemically methylated tRNA was used as substrate indicated that the 7-methylguanosine residues in the nucleic acid are not the substrate of the methylase activity forming the 1,7-dimethylguanosine moieties. Analogous data were obtained for the origin of 1,7-dimethylguanosine residues in tRNA chemical methylation by dimethyl sulphate.     

tRNA chemical methylation. In vitro and in vivo formation of 1,7-dimethylguanosine at high concentrations of methylating agents

The methylation patterns produced in Escherichia coli B tRNA by a range of concentrations of the weak carcinogen dimethyl sulphate were examined with the following results: 1. 1,7-Dimethylguanosine was found to be formed in high amounts in the tRNA methylation reaction at high concentrations of methylating agent. 2. The dialkylated compound was recovered mainly in the form of derivatives, the spectral and chromatographic behaviour of which varied according to the procedures used for their isolation. Similar results were obtained for the in vivo methylation of rat-liver tRNA: after administration of a very high dose of the powerful carcinogen dimethylnitrosamine, 1,7-dimethylguanosine was found in rat-liver tRNA. Moreover, the analysis of the time-course of nucleic acid methylation indicated that this dialkylated product was still present in rat-liver tRNA when the major product of alkylation, 7-methylguanine, had almost completely disappeared.     

Effects of ethionine on tRNA methylation in male and female rats

Administration of hepatocarcinogens to rats results in an increase in tRNA methyltransferase activity in the target tissues. Ethionine is active as a carcinogen only in female rats and only in females is this increase in enzyme activity seen. However, ethionine also causes the formation of methyl-deficient tRNA in the liver. Other hepatocarcinogens do not do this. Ethionine is equally effective in this action in males and females. Thus, the two actions of ethionine are completely separable, and the methyl-deficiency of tRNA is caused by an activity not identical with the carcinogenic one.     

Selective inhibition of uracil tRNA methylases of E. coli by ethionine.

L-ethionine has been found to inhibit uracil tRNA methylating enzymes in vitro under conditions where methylation of other tRNA bases is unaffected. No selective inhibitor for uracil tRNA methylases has been identified previously. 15 mM L-ethionine or 30 mM D,L-ethionine caused about 40% inhibition of tRNA methylation catalyzed by enzyme extracts from E. coli B or E. coli M3S (mixtures of methylases for uracil, guanine, cytosine, and adenine) but did not inhibit the activity of preparations from an E. coli mutant that lacks uracil tRNA methylase. Analysis of the 14CH3 bases in methyl-deficient E. coli tRNA after its in vitro methylation with E. coli B3 enzymes in the presence or absence of ethionine showed that ethionine inhibited 14CH3 transfer to uracil in tRNA, but did not diminish significantly the 14CH3 transfer to other tRNA bases. Under similar conditions 0.6 mM S-adenosylethionine and 0.2 mM ethylthioadenosine inhibited the overall tRNA base methylating activity of E. coli B preparations about 50% but neither of these ethionine metabolites preferentially inhibited uracil methylation. Ethionine was not competitive with S-adenosyl methionine. Uracil methylation was not inhibited by alanine, valine, or ethionine sulfoxide. It is suggested that the thymine deficiency that we found earlier in tRNA from ethionine-treated E. coli B cells, resulted from base specific inhibition by the amino acid, ethionine, of uracil tRNA methylation in vivo.     

Chemical probes for tRNA tertiary structure: Comparative alkylation of tRNA with methylnitrosourea, ethylnitrosourea and dimethylsulfate

The tertiary structure of tRNA in solution can be proved by chemical modification experiments. Three reagents, N-ethyl-N-nitrosourea, N-methyl-N-nitrosourea and dimethylsulfate which are known to alkylate nucleic acids at nucleophilic centers were compared. It is found that N-ethyl-N-nitrosourea and N-methyl-N-nitrosourea mainly react with phosphate residues and dimethylsulfate only with the bases. With dimethylsulfate the extent of alkylation of guanosines is about one order of magnitude higher than that of the phosphates by the nitrosocompounds.     

Structural alterations in deoxyribonucleic acid on chemical ethylation.

Ethylation of DNA by diethyl sulfate gave 7-ethylguanine as the major product. Dimethyl sulfate was much more reactive than diethyl sulfate in forming 7-alkylguanine. The hydrodynamic properties of DNA did not change as a direct consequence of ethylation. On incubation at 37 °C, the viscosity of ethylated DNA decreased at a rate similar to that of methylated DNA. The rate of depurination of 7-ethylguanine from ethylated DNA was the same as that of 7-methylguanine from methylated DNA. These results demonstrate that ethyl groups have identical effects as methyl groups on the secondary structure and stability of DNA.     

Aggregation and particle formation of tRNA by dendrimers

Major attention has been focused on dendrimer-DNA complexes because of their applications in gene delivery systems. Dendrimers are also used to transport miRNA and siRNA in vitro. We examine the interaction of tRNA with several dendrimers of different compositions, mPEG-PAMAM (G3), mPEG-PAMAM (G4), and PAMAM (G4) under physiological conditions using constant tRNA concentration and various dendrimer contents. FTIR, UV-visible, and CD spectroscopic methods as well as atomic force microscopy (AFM) were used to analyze the macromolecule binding mode, the binding constant, and the effects of dendrimer complexation on RNA stability, aggregation, particle formation, and conformation. Structural analysis showed that dendrimer-tRNA complexation occurred via RNA bases and the backbone phosphate group with both hydrophilic and hydrophobic contacts. The overall binding constants of K(mPEG-G3) = 7.6 (± 0.9) × 10(3) M(-1), K(mPEG-G4) = 1.5 (± 0.40) × 10(4) M(-1), and K(PAMAM-G4) = 5.3 (± 0.60) × 10(4) M(-1) show stronger polymer-RNA complexation by PAMAM-G4 than pegylated dendrimers. RNA remains in the A-family structure, whereas biopolymer aggregation and particle formation occurred at high polymer concentrations.     

Alterations of tRNA modification in mammalian systems: the effect of ethionine.

The relationship between the modification of tRNA and its ability to act as a substrate for homologous tRNA modification enzymes in vitro was studied. The tRNA extracted from the livers of rats was active as a substrate for in vitro methylation with extracts from normal rat liver 19 h after treatment with L-ethionine (35 mg/100 g/24 h). After 4 weeks of feeding a diet containing o.25% DL-ethionine, the tRNA was a poor substrate for methylation in vitro, even though it was deficient in methylated nucleosides. Only 18% and 7% of the available sites could be methylated after 67 h and 4 weeks, respectively, of ethionine treatment. 3-(3-amino-3-carboxypropyl)uridine, a nucleoside that is also synthesized from S-adenosylmethionine, was assayed in individual tRNAs by their reactivity with the N-hydroxysuccinimide ester of phenoxyacetic acid. The reactivity of tRNAIle, tRNAAsn, and tRNAThr was decreased by treatment with ethionine at 67 h as well as at 2 and 4 weeks, although no difference could be detected at 19 h.     

Functionally impaired tRNA from ethionine treated rats as detected in injected Xenopus oocytes.

Treatment of rats with ethionine was found to cause severe impairment in the aminoacylation capacity of tRNA. This effect was only observed when assayed in injected oocytes, while invitro assays of aminoacylation failed to detect differences between normal tRNA and tRNA from ethionine treated animals. The effect of ethionine on the tRNA population was not uniform and differed for various amino acid specific tRNAs. Thus liver tRNA from ethionine treated rats showed a decreased capacity for phenylalanine aminoacylation, while no change was found in the case of leucine. On the other hand, the level of histidine aminoacylation was higher for tRNA from ethionine treated animals. An even more complex response was observed with methionine aminoacylation where tRNA from ethionine treated animals showed an initially faster rate than control tRNA. With more prolonged incubation periods, the methionyl-tRNA from ethionine treated animals was deacylated at an accelerated rate while the level of normal methionyl-tRNA remained almost constant.In addition to the aminoacylation reaction, the participation of aminoacyl-tRNA in protein synthesis was severely impaired. In this case, both the injected oocyte system and the cell-free wheat germ assay revealed these differences which were manifested with various mRNA and viral RNA preparations.     

Catabolism of glucose in pea stem section during root formation and its inhibition by kinetin and ethionine

The catabolism of specifically¹⁴C-labelled glucose during the root formation and its inhibition by kinetin and ethionine in the etiolated pea stem sections were studied. The formation of root meristematic foci in the pericycle region of sections was accompanied by the decrease of the C₆/C₁ ratio. Such a result and activation of pentose phosphate cycle, which was also checked by another method, suggested increased participation of pentose phosphate cycle in glucose oxidation. The above mentioned changes were also found after the prevention of root formation by ethionine and, therefore, do not seem to be specific for the meristematic foci formation. The growth of newly formed roots was closely connected with the rise of C₆/C₁ values. The increase of CO₂ release from the first carbon atom of glucose molecule was recorded after the inhibition of root formation by ethionine. The rise of C₆/C₁ values and decrease of pentose phosphate cycle activity was observed after the treatment of pea stem sections by kinetin in the first 64 hours after sectioning. In this case root formation was prevented and the growth of lateral buds was stimulated. The secondary xylem formation which took place later was accompanied by the activation of the pentose phosphate cycle. These phenomena are discussed in relation to cell division and biosynthesis of lignin-precursors.     

Dynamics of amino acids in pea stem sections during root formation and its inhibition by kinetin and ethionine

An investigation was conducted to study the interrelation of free amino acid metabolism and root formation in etiolated pea stem sections as dependent on time and on inhibition of root formation by kinetin and ethionine. The rise in the level of aspartic acid and increase in the rate of conversion of¹⁴C-labeled glucose to free amino acids were found to be characteristic features of the formation of foci of meristematic cells in pericyclo region. The formation of roots was reflected, in general, much more in the rate of conversion of labeled glucose to free amino acids than in the levels of corresponding amino acids. The total amount of free amino acids was not significantly changed during incubation of stem sections in a solution of kinetin (5×10^?5 m). A rapid fall in their level was recorded in the next 24 hours. The incorporation of¹⁴C from glucose into a precursor of lignin, phenylalanine, was completely inhibited by kinetin which stimulated simultanously the growth of adjacent buds. Stimulation of secondary xylem formation, which appeared later, was accompanied by the resumption of¹⁴C-incorporation into phenylalanine. Inhibition of root formation by ethionine resulted in the rapid fall of the level of most amino acids and in a significant decrease in the rate of incorporation of¹⁴C from glucose into amino acids. A decreasing level of ethionine in tissues during cultivation of ethionine-treated stem sections was accompanied by a gradual rise in the individual amino acids and in the rate of conversion of glucose into free amino acids.     

Fractionation of proteins of pea stem sections during root formation and its inhibition by kinetin and ethionine

Changes in the protein constituents in pea stem sections during root formation and its inhibition by kinetin and ethionine were studied. Only quantitative differences in the protein fractions separated on DEAE-cellulose column were noted. The formation of foci of meristematic cells in pericycle was accompanied by an increase in the amount of fraction “l”. This fraction disappeared rapidly in sections where root formation either did not occur (internodial sections without nodes) or was inhibited by ethionine (stem sections with basal and apical nodes). Incubation of stem sections in a kinetin solution for 16 hours after cutting of stems partially preserved fraction “l”. The increase in the amount of fraction “l” was one of the first metabolic changes in root-forming pea stem sections after cutting of stems.     

Ribosomal binding and dipeptide formation by misacylated tRNA(Phe),S

Eight structurally modified peptidyl-tRNA(Phe),s were employed to study P-site binding and peptide bond formation in a cell-free system involving Escherichia coli ribosomes programmed with poly(uridylic acid). It was found that the two analogues (N-acetyl-D-phenylalanyl-tRNA(Phe) and N-acetyl-D-tyrosyl-tRNA(Phe] containing D-amino acids functioned poorly as donors in the peptidyltransferase reaction and that two N-acetyl-L-phenylalanyl-tRNA(Phe)'s differing from the prototype substrate in that they contained 2'- or 3'-deoxyadenosine at the 3'-terminus failed to form dipeptide at all when L-phenylalanyl-tRNA(Phe) was the acceptor tRNA. Interestingly, all four of these peptidyl-tRNA's bound to ribosomes to about the same extent as tRNA's that functioned normally as donors in the peptidyltransferase reaction, at least in the absence of competing peptidyl-tRNA species. Two peptidyl-tRNA's lacking an amino group were also tested. In comparison with N-acetyl-L-phenylalanyl-tRNA(Phe) it was found that trans-cinnamyl-tRNA(Phe) and 3-phenylpropionyl-tRNA(Phe)'s formed dipeptides to the extent of 53 and 80%, respectively, when L-phenylalanyl-tRNA(Phe)was used as the acceptor tRNA. N-Acetyl-beta-phenylalanyl-tRNA(Phe) was found to be the most efficient donor substrate studied. Both isomers transferred N-acetyl-beta-phenylalanine to L-phenylalanyl-tRNA(Phe); the nature of the dipeptides formed in each case was verified by HPLC in comparison with authentic synthetic samples. Further, the rate and extent of peptide bond formation in each case exceeded that observed with the control tRNA, N-acetyl-L-phenylalanyl-tRNA(Phe).