A possible mechanism of inhibition of protein synthesis by dimethylnitrosamine

1. The incorporation of [¹⁴C]leucine into liver proteins of rats was measured in vivo at various times after treatment of the animals with dimethylnitrosamine and was correlated with the state of the liver ribosomal aggregates. Inhibition of incorporation ran parallel with breakdown of the aggregates. 2. Inhibition of leucine incorporation into protein and breakdown of ribosomal aggregates were not preceded by inhibition of incorporation of [¹⁴C]orotate into nuclear RNA of the liver. 3. Evidence was obtained of methylation of nuclear RNA in the livers of rats treated with [¹⁴C]dimethylnitrosamine. 4. Zonal centrifugation analysis of radioactive, nuclear, ribosomal and transfer RNA from livers of rats treated with [¹⁴C]dimethylnitrosamine revealed labelling of all centrifugal fractions to about the same extent. 5. It is suggested that methylation of messenger RNA might occur in the livers of dimethylnitrosamine-treated rats and the possible relation of this to inhibition of hepatic protein synthesis is discussed.     

Role of endogenous regucalcin in nuclear regulation of regenerating rat liver: suppression of the enhanced ribonucleic acid synthesis activity

The role of endogenous regucalcin in the regulation of ribonucleic acid (RNA) synthesis activity in the nucleus of normal and regenerating rat livers was investigated. Nuclear RNA synthesis was measured by the incorporation of [(3)H]-uridine 5'-triphosphate into the nuclear RNA in vitro. The presence of regucalcin (0.25 or 0.5 microM) in the reaction mixture caused a significant decrease in nuclear RNA synthesis of normal rat liver. alpha-Amanitin (10(-8)-10(-6) M), an inhibitor of RNA polymerase II and III, decreased significantly nuclear RNA synthesis activity. The effect of regucalcin (0.25 microM) in decreasing nuclear RNA synthesis activity was not seen in the presence of alpha-amanitin (10(-6) M). The calcium chloride (10 microM)-increased nuclear RNA synthesis activity was significantly suppressed by the addition of regucalcin (0.25 microM). RNA synthesis activity was significantly enhanced in the nuclei of regenating rat liver obtained at 24, 48, or 72 h after partial hepatectomy. This enhancement was significantly inhibited in the presence of PD98059 (10(-5) M), staurosporine (10(-6) M), or vanadate (10(-3) M). Western analysis of the nuclei of regenerating liver obtained at 24, 48, or 72 h after partial hepatectomy showed a significant increase in regucalcin protein as compared with that of sham-operated rats. The presence of anti-regucalcin monoclonal antibody (25 or 50 ng/ml) in the reaction mixture caused a significant increase in nuclear RNA synthesis activity of normal rat liver. This increase was completely blocked by the addition of regucalcin (1.0 microM). The effect of anti-regucalcin monoclonal antibody (50 ng/ml) in increasing nuclear RNA synthesis activity was significantly enhanced in the nuclei of regenerating liver obtained at 24, 48, or 72 h after partial hepatectomy. This enhancement was significantly suppressed by the addition of alpha-amanitin (10(-6) M), PD98059 (10(-5) M), staurosporine (10(-6) M), or vanadate (10(-3) M) in the reaction mixture. The present study demonstrates that endogenous regucalcin has a suppressive effect on the enhancement of RNA synthesis activity in the nucleus of regenerating rat liver with proliferative cells.     

Quantitative isolation of oligo- and polyadenosine-diphosphoribosylated proteins by affinity chromatography from livers of normal and dimethylnitrosamine-treated Syrian hamsters. In vivo and in vitro metabolism of the homopolymer

Polyadenosine- and adenosine-diphosphoribosylated proteins of hamster liver were quantitatively isolated with the aid of m-aminophenyl boronic acid glutaryl hydrazide polyacrylamide affinity resin by selective adsorption at pH 8.2 and elution at pH 4.0. Polymer-free proteins, DNA, and RNA are readily separated from adenosine-diphosphoribosylated proteins. The total quantity of proteins that is covalently modified by the homopolymer is 14.3 micrograms/mg of DNA or 37.4 micrograms/g of liver in controls and 38.7 micrograms/mg of DNA or 116 micrograms/g of liver in dimethylnitrosamine-treated hamsters. Polymer content increases from 9 to 15 nmol/mg of DNA to 42 to 118 nmol/mg of DNA following treatment with dimethylnitrosamine. Pulse labeling with [14C]ribose results in a parallel doubling in dimethylnitrosamine-treated animals of the specific activities of adenosine- diphosphoribose and NAD+ and of the [14C]ribose content of polyadenosine-diphosphoribose of chain length between 20 and 40, indicating chain elongation of pre-existing larger polymers. Two groups of proteins that are isolated as polyadenosine-diphosphoribose adducts are increased significantly after treatment with dimethylnitrosamine, one minor component of a mass between 100-112 X 10(3) daltons, and a major group exhibiting a mass of 158-162 X 10(3) daltons. Polyadenosine-diphosphoribose synthetase activity of isolated hepatic nuclei is increased by 32-37% after dimethylnitrosamine treatment, and since the change in glycohydrolase activity is negligible relative to the increase in synthetase, the augmentation of polyadenosine-diphosphoribosylated proteins can be explained by the increased synthetase of nuclei. The molecular size distribution of DNA in liver nuclei of control and dimethylnitrosamine-treated hamsters is indistinguishable.     

Vaccinia virus RNA polymerase associated with nuclei of infected HeLa cells.

Though vaccinia virus DNA and RNA replication take place predominantly in the cytoplasm of an infected cell, virus formation requires the presence of a functional nucleus in a yet undefined manner. When the nuclei from cells infected for 3 h are isolated and purified, they are found to synthesize five times more RNA in vitro than do corresponding nuclei from noninfected cells. Fifty percent of the RNA synthesized in vitro by nuclei from infected cells is vaccinia specific, and this vaccinia RNA synthesis is resistant to alpha-amanitin concentrations up to 100 micrograms/ml. Furthermore, when the RNA polymerase activities of these nuclei are separated on DEAE-Sephadex columns, 56% of the total nuclear enzyme activity is found to be the vaccinia-specific RNA polymerase known to be alpha-amanitin resistant. The nucleus associated vaccinia RNA polymerase represents 18% of the total cellular vaccinia RNA polymerase. This synthesis of vaccinia RNA in the nucleus may explain the nuclear requirement for vaccinia virus maturation.     

The absorption and metabolism in rats of small oral doses of dimethylnitrosamine. Implication for the possible hazard of dimethylnitrosamine in human food.

1. Groups of rats were given one dose of the carcinogen dimethylnitrosamine by gastric intubation. The dose was varied between 10mg/kg body wt. and 1 microgram/kg body wt. 2. The dose was rapidly absorbed. 3. The methylation of liver DNA resulting from the administration of this carcinogen was proportional to dose. This suggests that small doses are absorbed from the gut with no more loss than large doses. 4. As the dose was decreased there was a disproportionately greater decrease in the alkylation of kidney DNA, and when the dose was less than 40 microgram/kg body wt. the methylation of kidney DNA was no longer detectable. This possibly explains why small amounts of dimethylnitrosamine in the diet do not induce kidney tumours. 5. Comparison of the relative alkylation of liver DNA and kidney DNA resulting from an oral and from an intravenous dose of dimethylnitrosamine suggest that small amounts of dimethylnitrosamine absorbed into the portal blood from the gut are completely metabolized by the liver and do not enter the general circulation. 6. The implications of these results for the possible hazard of dimethylnitrosamine in human food is discussed.     

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.     

Effects of alpha-amanitin on RNA synthesis by mouse embryos in culture

Investigations were conducted to test the effects of alpha-amanitin on RNA synthesis in preimplantation mouse embryos. Exposure of embryos in culture to 1-100 microgram/ml alpha-amanitin produced a dose- and time-dependence suppression of total RNA synthesis as measured by incorporation of [3H]uridine. Synthesis of polyadenylated RNA in blastocyst-stage embryos was abolished by alpha-amanitin-treatment at concentrations and exposure times that suppressed total RNA synthesis by less than 15%. DNA-dependent RNA polymerase activity was measured in lysates of embryos at several stages of preimplantation development. alpha-Amanitin suppressed total polymerase activity assayed under ionic conditions favorable to the detection of RNA polymerase II. Electrophoretic analyses revealed that preincubation of blastocysts in 100 microgram/ml alpha-amanitin reduced labelling of cytoplasmic 28S and 18S RNA by inhibition of both synthesis and maturation of nucleolar 45SrRNA-precursor. This action of alpha-amanitin on nucleolar RNA synthesis cannot be correlated with the minimal suppression of nucleolar RNA polymerase activity and suggests that the synthesis and processing of rRNA may be under control of nucleoplasmic gene products.     

Transcription of specific genes in isolated nuclei from HeLa cells in vitro.

Isolated HeLa cell nuclei were employed to catalyze the synthesis of RNA in vitro. In the presence of low concentrations of alpha-amanitin (1 mug/ml), used to suppress the formation heterogeneous nRNA, these nuclei synthesize RNA very efficiently for extended periods of time (at least 60 min) at an elongation rate of about seven nucleotides per second. The product, analyzed on sucrose density gradients and polyacrylamide gels was found to exist of two predominant size classes. Synthesis of the 45-S ribosomal precursor was completely resistant even to high concentrations of alpha-amanitin (150 mug/ml) and hence was catalyzed by enzyme A (or I). A limited degree of processing of the 45-S precursor occurred in vitro. In addition, a second RNA class of low molecular weight (4-8 S) was synthesized by HeLa cell nuclei in the presence of 1 mug/ml alpha-amanitin in vitro. Analysis on 8% polyacrylamide gels resolved the RNA into four distinct components. Their synthesis was resistant to low (1 mug/ml) but clearly sensitive to high (150 mug/ml) concentrations of alpha-amanitin. Consequently the synthesis of all these small-molecular-weight RNA species is catalyzed by RNA polymerase C (or III). For the assessment of the initiation frequency of the individual classes of RNA, a new technique was developed independent of labelling the 5' end of the RNA molecule with the gamma-phosphate of the initiating nucleotide. It employs the double labelling of an RNA molecule with two different isotopes added sequentially at different stages of completion of the chain. From the incorporation ratio of the two isotopes into a particular class of RNA, conclusions can be drawn concerning their initiation frequency. The results obtained have shown a high reinitiation frequency for the small-molecular-weight RNA species at all stages of the incubation reaction. In contrast, reinitiation of the 45-S precursor RNA occurs only to a limited extent in isolated HeLa cell nuclei in vitro.     

Some studies of the effects of aflatoxin B1 in vivo and in vitro on nucleic acid synthesis in rat and mouse.

The mouse compared with the rat, is more resistent to the acute toxic action of aflatoxin B1 and is refractory to its hepatocarcinogenic properties. Aflatoxin B1 inhibits DNA synthesis more strongly than RNA synthesis in the rat, and both nucleic acid syntheses more strongly in rat than in the mouse. Mouse hepatic microsomes, like those of the rat, are capable of metabolizing aflatoxin B1 in vitro in the presence of NADPH, to an active form which binds to DNA both in solution and in intact nuclei and also inhibits nuclear RNA synthesis. Non NADPH-dependent binding of aflatoxin B1 to nuclei is not effective in inhibiting RNA polymerase and is largely removed by washing with lipid solvents. Mouse nuclear RNA polymerases particularly Mn 2+ (NH4)2SO4 primed acitivity are more resistant to inhibition in vitro by activated aflatoxin B1 than are the corresponding enzyme activities in rat liver nuclei. This would appear to be due to the bound aflatoxin B1 being less efficient in the case of the mouse nucleus, in inhibiting RNA synthesis. Mouse liver slices exhibit a much lesser degree of inhibition of RNA synthesis by aflatoxin B1 than do rat liver slices. Accompanying this is a lower level of binding of aflatoxin B1 to subcellular particulate fractions in the mouse liver slice compared to the rat, this disparity being most marked in the case of the nuclear fraction. The suggestion is made that the resistance of RNA synthesis in the mouse liver, to aflatoxin B1, and perhaps also resistance to its toxicity, is dependent, not on a lower capacity to activate the toxin, but (a) on a less efficient inhibition of RNA synthesis by nuclear bound toxin, and (b) a detoxifying mechanism at least partially situated in the cytosol fraction.     

Synthesis of low molecular weight RNA components A, C and D by polymerase II in alpha-amanitin-resistant hamster cells.

In an attempt to establish which RNA polymerase catalyzes the synthesis of the low molecular weight RNA components A, C and D, Ama 1 cells (mutant Chinese hamster cells) were used in experiments with addition of alpha-amanitin. Ama 1 cells contain an altered RNA polymerase II which is 800 times more resistant towards inhibition by alpha-amanitin than the wild type enzyme. Alpha-amanitin (up to 200 microgram/ml) added to these cells does not affect the synthesis of the low molecular weight RNAs A, C and D. These data together with our previous data showing that alpha-amanitin (0.5 - 5.0 microgram/ml) preferentially inhibits the synthesis of A, C and D in normal cells indicate that RNA polymerase II catalyzes the synthesis of the low molecular weight RNA components A, C and D.