14C-orotic Acid Research Articles

Glutathione-induced growth of embryogenic tissue of white spruce correlates with changes in pyrimidine nucleotide metabolism

Exogenous applications of reduced glutathione (GSH) and oxidized glutathione (GSSG) promote growth of embryogenic tissue of white spruce during a 7-day subculture period. Compared to control tissue, a statistically significant increase in fresh weight, as well as RNA and DNA content, was observed in the presence of GSH and GSSG during the last days in culture. The effects of these two metabolites on pyrimidine nucleotide metabolism was investigated by following the metabolic fate of 14C-orotic acid, a precursor of the de novo synthesis, and 14C-uridine and 14C-uracil, intermediates of the respective salvage and degradation pathways. Compared to control embryos, GSH-treated tissue was able to utilize a larger fraction of supplied orotic acid for UMP production, possibly due to the increased activity of orotate phosphoribosyltransferase (OPRT). The activity of this enzyme increased markedly in tissue cultured with GSH during the last days in culture. Salvage of uridine for nucleotide and nucleic acid synthesis was observed in all treatments, especially in GSSG-treated tissue at day 7. The increased salvage activity in this tissue correlated with the increase in activities of the two uridine salvage enzymes, uridine kinase (URK) and nucleoside phosphotransferase measured with uridine (NPT(uridine)). Compared to control tissue, tissue treated with either GSH or GSSG was able to utilize a large fraction of uracil for nucleotide synthesis, denoting a better ability to divert this precursor from degradation. Nucleotide and nucleic acid analyses revealed that in both GSH and GSSG-treated tissue, the endogenous levels of UTP, CTP, as well as those of RNA and DNA, were increased compared to those of control tissue. Overall, the results from this study suggest that GSH and GSSG can induce growth of embryogenic tissue of white spruce through distinct metabolic changes of pyrimidine nucleotides.

The effect of Lentinan on proliferative processes in parenchymal organs of rats — I. The effect on pyrimidine and nucleic acid syntheses

The present study investigated the effect of Lentinan on the biochemical events associated with the pyrimidine and nucleic acid syntheses in the liver, kidney, thymus and spleen of rats. Lentinan was used at a dose of 4 mg/kg/day (twice) and in a single dose of 20 mg/kg. The following changes were observed. 1. (1) The utilization of ( 14C)orotic acid for the synthesis of uridine components of liver acid-soluble extract and RNA uracil was activated after the administration of both doses of the drug. The specific activity of cytidine components of the acid-soluble extract and RNA were, on the other hand, not affected. The same holds true for the kidney. The ratio of the specific activity of cytidine: uridine components of the acid soluble extract as well as RNA decreased after the administration of both doses of the drug. The specific activity of DNA cytosine and thymine are slightly suppressed in the liver after the administration of a high dose of Lentinan; no effect was observed in the kidney. 2. (2) The uptake of ( 14C)cytidine by the liver was not affected; the specific activity of DNA cytosine and thymine were increased after the administration of a high dose of Lentinan. 3. (3) The uptake of ( 14C)thymidine by the liver was not affected; the specific activity of liver DNA thymine was increased after the administration of both doses of the drug. In the thymus an increase of specific activity of DNA thymine has also been observed. 4. (4) Repeated doses of the drug (4 mg/kg for 6 consecutive days) increased te weight of the spleen. The specific activity of DNA thymine of the liver and spleen were significantly increased.

Biosynthesis of pyrimidine nucleotides and level of cytochrome P-450 in rat liver and kidney after clofibrate administration (an in vivo study).

Like other lipid-soluble xenobiotics, clofibrate (ethyl-2-(4-chlorophenoxy)-2-methylpropanoate) increased the level of microsomal cytochrome P-450 in liver and decreased the utilization of 14C-orotic acid for the synthesis of hepatic cytidine nucleotides. This phenomenon was associated with the increased (a) uptake of 14C-cytidine, (b) total content of cytidine components of the acid-soluble extract and (c) utilization of this nucleoside for the synthesis of RNA. No changes were observed in uridine components. Clofibrate also increased the level of cytochrome P-450 in kidney microsomes; the degree of induction was almost the same as in the liver. The variations of renal pyrimidine metabolism after administration of the drug were analogous to those observed in the liver.

Effect of maternal dietary deficiency on the metabolism of high-molecular weight, soluble and transfer RNA in the liver of the 21-day-old offspring of rats

The 21-day-old male progeny of dietary-restricted rats were administered 14C-orotic acid intraperitoneally to study the metabolism of high-molecular weight soluble (sRNA) and transfer RNA (tRNA) in their livers, which were removed at various time intervals ranging from 6 to 120 minutes after the injection. DNA and total RNA were extracted from each liver sample, and nucleic acids were fractionated with 2M NaCl into insoluble (high-molecular weight) and soluble (DNA and tRNA) fractions. All the preparations were analyzed by sucrose density gradient sedimentation, and the distribution of radioactivity was determined. Dietary-restricted progeny experienced significant increases of RNA (p<0.001), including high-molecular weight sRNA (p<0.001) and tRNA components (p<0.001). The greatest increases were apparent in the tRNA and 4–18s RNA fractions. Dietary restriction during the period of growth, gestation and lactation caused a significant reduction in total body weight and liver weight in the 21-day-old progeny. It also enhanced the rate of RNA synthesis in the livers of these animals. The ratios of DNA/total RNA were not affected by dietary deprivation of the progeny, inferring and confirming the observation noted above that the progeny must have experienced augmented RNA degradation and elevated biosynthesis of various forms of RNA.

The effect of epinephrine and serotonin on hepatic poly (A) + RNA synthesis

In vivo administration of epinephrine or serotonin has been shown to stimulate the incorporation of 14C-orotic acid into Poly(A) + RNA. However, only epinephrine and not serotonin could stimulate DNA dependent RNA polymerase activity of isolated hepatic nuclei in in vitro experiments.

In vitro effects of aflatoxin B1 on the uptake of 14C-orotic acid into kidney, liver and muscle tissue of the Mongolian gerbil, Meriones unguiculatus.

Aflatoxins are a mixture of highly toxic metabolites produced by certain strains of the mold Aspergillu8 ~avu~ a contaminant of groundnuts and other grains. The chemical structures for these potent hepatotoxic and hepatocarcinogenic agents have been studied extensively and aflatoxin B I (AFB I) has been found to be the most abundant and toxic metabol~te (B~TLER 1966). A major area of study involving the aflatoxins has dealt with their mechanism of action. Often severe inhibition of protein synthesis by the cell can be measured by uptake and incorporation of various labeled precursors of protein and nucleic acids (SMITH 1963, CLIFFORD & REES 1966, 1967). One investigator reported inhibition Of nuclear and nucl~olar RNA synthesis (30% and 85%, respectively) as measured by 14C-orotic acid incorporation in Other work with rats showed rat livers (LEFARGE et al. 1966). i~ no inhibition of incorporation of C-orotic acid in in vivo studies of the nucleotide pool, but immediate inhibition in in vitro studies. However, the authors did find inhibition of nucleotide precursors into nuclear RNA and postulated inhibition of RNA polymerase as a possible explanation (CLIFFORD & REES 1967). Later work subsequently explored this hypothesis corroborating it for RNA polymerase and implicating metabolites of AFB I as the active intermediate forms responsible for the inhibitmon (AKINRIMIST et al. 1974). A metabolite of AFB I was also suggested as the actual inhibitor of RNA synthesis in another work where the incorporation of RNA precursors, 14C-orotic acid and 3H-orotic acid, was markedly inhibited by low levels of AFH I and AFGI, but not AFB 2 and AFG 2 (MCINTOSH et al. 1976). Most of the literature involving measurement of uptake and incorporation of precursors has dealt m~inly with the effects on rats and few recent studies have used 14C-orotic acid. It was the purpose of this study to determine the effects of AFB_ on the uptake of 14C-orotic acid into kidney, liver and muscle tissue of the Mongolian gerbil, Meriones unguicula~s. This animal appears to be less sensitive to the toxin than the rat (GODY & NEAL 1976, LLEWELLYN & THOMEN 1978).

Liver RNA metabolism in adrenalectomized and intact whole-body irradiated and 5-fluoroorotic acid treated rats

Newly synthesized RNA as measured by 30 min uptake of (6- 14C)orotic acid was determined in various liver cell fractions of control, irradiated, 5-FOA treated and 5-FOA plus irradiated adrenalectomizedand intact rats. The level of RNA synthesis was elevated1 hr post-irradiation in adrenal intact rats and peaked at approximately 6 hr except in the nucleus. When labelled RNA precursor was injected for longer times prior to killing similar specific radioactivity results were obtained. Administration of 5-FOA prevented much of the increased incorporation of label into liver RNA of irradiated adrenal intact rats. Synthesis of RNA in adrenalectomized rats (30 min pulse uptake) was reduced by irradiation. However, when the isotope was administered for periods of 3 hr or longer prior to killing the RNA specific radioactivities were greater than those values obtained for controls. These data can be interpreted as follows: (1) the increase in liver RNA synthesis following whole-body irradiation is probably mediated via the adrenal gland; (2) catabolic activity is apparently decreased by irradiation in adrenalectomized rats; (3) when combined with irradiation 5-FOA is neither additive nor synergistic.

Electrophoretic analysis of liver rRNA maturation in irradiated and 5-fluoroorotic acid treated rats

Gel electrophoretic patterns of nuclear and cytoplasmic ribosomal RNA (rRNA) from livers of intact and adrenalectomized rats were studied after exposure to gamma radiation (1800 rad) and/or 5-fluoroorotic acid (5-FOA) treatment. By 6 hr after either 5-FOA administration alone, or in combination with irradiation, incorporation of (6- 14C )orotic acid into 45S rRNA precursors was elevated because maturation was blocked and appearance of 28S and 18S subunits delayed. By 12 hr post-irradiation the 45S block was firmly established. In adrenalectomized rats 5-FOA administration alone produced a block within 0.5 hr and after 6 hr following exposure to radiation. The results obtained in the current study are consistent with the conclusion that radiation selectively inhibits rRNA maturation in liver cells; and the response is influenced by the presence of adrenal glands.

Topographical differences of RNA labelling in rat brain after intraventricular administration of labelled RNA precursors.

14C-uridine or 14C-orotic acid was injected into the third or fourth brain ventricle of adult rats. The rate of incorporation of these precursors into the RNA of various brain regions was studied by autoradiography. 0.5 hour, 1 hour, 2 hours and 4 hours after the injection of labelled uridine and/or orotic acid into the third ventricle, a very uneven labelling of different brain regions was observed. The highest grain density was found over the ventricular walls and in the closely adjacent brain tissue; the intensity of labelling decreased sharply with distance from the ventricular lumen. 24 hours after intraventricular injection, a medio-lateral gradient of grain density was no longer observed. An intense labelling of leptomeninx (especially at the base of the brain) and of ependymal cells was observed at all time intervals investigated. At time intervals 0.5-2 hours the grain density of these structures surpassed by a considerable amount the grain density over neurona, glial cells or neuropile. Two hours after the injection of 14C-orotic acid into the fourth ventricle, the grains were mainly localised over leptomeningeal cells and vessels at the base of the brain and in the adjacent narrow strip of brain tissue. The rest of the brain was only very faintly labelled.

Pyrimidine biosynthesis and its regulation in embryos of the sea urchin, Arbacia punctulata

1. 1. Evidence for the occurrence of the orotate pathway for the de novo biosynthesis of pyrimidines in the developing sea urchin embryo was provided by the incorporation of ( 14C) NaHCO 3, ( 14C) carbamoylaspartate, and (6- 14C) orotic acid into the pyrimidines of RNA, in vivo. 2. 2. This interpretation was supported by the observation that the radio-label from ( 14C) NaHCO 3 appeared in the C-2 position of the uracil ring and by the demonstration of the activity of all of the enzymes of the orotate pathway in cell-free extracts of sea urchin eggs. 3. 3. Evidence was obtained for the regulation of the de novo pathway by end-product inhibition.

The effect of the number of daily training sessions on skeletal muscle protein synthesis

The purpose of this investigation was to examine the effects of training either once or three times/day on the principal stages of skeletal muscle. The experiments were carried out on male albino rats fed either 3 or 5 times/day a diet containing 20% protein. Experimental animals swam either once or 3 times/day, 6 days/week for 10 weeks with weights attached. The total duration of daily activity was equal for both groups and at 10 weeks each animal was swimming for 60 min/day with 3% of his body weight attached. All the animals were examined at rest after the 10-week training programs. The adequacy of the weight-loading and training schedules was estimated by body weight dynamics and such energy metabolites as creatine phosphate and glycogen. Skeletal muscle RNA and protein synthesis were studied by means of 14C-orotic acid and 14C-leucine incorporation, respectively. Quadriceps and gastrocnemius muscles were used for analysis in all experiments. It was found that the increase in the number of daily training sessions resulted in an increased content and intensity of synthesis of skeletal muscle proteins as evidenced by an increase in the content of amino acids in the muscle, an increased synthesis of both microsomal and ribosomal RNA, an increased stability of (poly-A)-containing messenger RNA, and an increased synthesis of all skeletal muscle protein fractions: myofibrillar, sarcoplasmic, and myostromal.

Synergistic toxic effects of citrinin, ochratoxin A and penicillic acid in mice

G. A. Sansing, E. B. Lillehoj, R. W. Detroy and M. A. Miller. Synergistic toxic effects of citrinin, ochratoxin A and penicillic acid in mice. Toxicon 14, 213–220, 1976.—The ld 50's in mice of citrinin, ochratoxin A, and penicillic acid injected intraperitoneally were 89, 22, and 100 mg per kg of body weight, respectively. Paired combinations of the mycotoxins, citrinin: ochratoxin A (CI:OA), ochratoxin A:penicillic acid (OA:PA), and penicillic acid:citrinin (PA:CI) elicited synergistic lethal responses. After administration of citrinin, 14C-orotic acid incorporation into both liver and kidney increased significantly by 27 hr with a return to control levels at 51 hr. Treatment with penicillic acid also increased orotic acid incorporation into liver ribonucleic acid (RNA) at 27 hr and at 4 hr in the kidney. Ochratoxin A inhibited orotic acid incorporation into both liver and kidney RNA 6 hr after toxin injection with a subsequent return to control levels at 27 hr. Orotic acid incorporation was inhibited in both kidney and liver 6 hr after treatment with the toxin combination CI:OA, PA:CI stimulated precursor incorporation into kidney RNA at 27 hr and inhibited the function in liver. The OA:PA combination inhibited orotic acid incorporation in both organs 15–27 hr after toxin treatment.

Neonatal hypothyroidism: Enhanced incorporation of precursors into cerebral RNA [formula omitted] and normalizing effect of a semi-acute injection of thyroxine

The incorporation of radioactivity from intracisternally injected (6− 14C) orotic acid into cerebral RNA and precursor pool was markedly elevated in 25 day-old hypothyroid rats compared to controls. Similar results were achieved with (2− 14C) uridine as the isotopic precursor. The increase in specific radioactivity of cerebral ribosomal RNA and the purified nucleotide precursor pool was essentially restored to normal by a single injection of L-thyroxine at 22 days of age (72 hours prior to sacrifice). The results are compatible with an increased intracellular uptake (transport) of RNA precursors and reflect the participation of thyroid hormones in the regulation of events at the membrane during brain development.

MECHANISM OF ACTION OF ALDOSTERONE IN ADRENALECTOMIZED MOUSE KIDNEY

Time-course of effect of aldosterone on the incorporation of 14C-orotic acid into kidney RNA from adrenalectomized and normal mice was examined with the following results.1. Stimulatory effect of aldosterone (0.55 μg/kg, s.c.) on 14C-orotic acid incorporation into RNA from kidney homogenate and its nuclear fraction was observed in early stages (1.53 hr) after aldosterone administration in adrenalectomized mice, however, the incorporation was not changed at 5 hr after aldosterone. RNA content in homogenate and subcellular fractions did not change in any cases.2. After the administration of larger dose of aldosterone (50μg/kg) to adrenalectomized mice, 14C-orotic acid incorporation into kidney RNA was not changed until 3 hr.3. Cortisol had no effect on RNA specific radioactivity and RNA amount in adrenalectomized mouse kidney. 4. 14C-orotic acid incorporation into kidney RNA was not changed until 5 hr between normal and adrenalectomized mice.5. 14C-orotic acid incorporation into kidney nuclear RNA from normal mice was increased until 3 hr after aldosterone (2 and 20μg/kg) administration and the maximum effect was observed at 1.5 hr later.6. The early effects of aldosterone on RNA synthesis in adrenalectomized mouse kidney are agreed with the general concept in “induction hypothesis” which has been offered by the study using adrenalectomized rat kidney and/or urinary bladder of toad.

Nucleic acid precursor synthesis by Plasmodium lophurae parasitizing chicken erythrocytes

Chicken erythrocytes parasitized by Plasmodium lophurae were cultured in vitro in the presence of glycine-2- 14C, glycine-U- 14C, and 14C-Na-formate for 10–16 hr. Purines isolated from the acid-soluble fraction (ASF), RNA and DNA of parasitized blood exposed to 14C-glycine contained specific activities equivalent to those of uninfected erythrocyte purines. However, parasitized blood samples incorporated 14C-Na-formate into ASF, RNA and DNA purines to a much greater extent than uninfected blood; the ratio of incorporated formate vs glycine by infected blood samples indicated the absence of a complete de novo purine pathway, but failed to rule out the existence of a partial de novo purine pathway in the host-parasite complex. Adenine-8- 14C and 14C-orotic acid served as purine and pyrimidine nucleotide precursors, respectively, in the P. lophurae-chicken erythrocyte complex; 14C-uracil did not serve as an effective pyrimidine nucleotide precursor under in vitro conditions. Autoradiographic studies failed to demonstrate either the in vivo or in vitro incorporation of 3H-thymidine or 3H-uridine into the nucleic acids of intraerythrocytic stages of P. lophurae.

RNA metabolism in the cortex of newly weaned rats following first exposure to light

Newly weaned rats which had been kept in the darl from birth were injected intraventricularly with (6- 14C) orotic acid. Experimental rats were exposed to light for 2 h and dark controls were returned to the dark environment for 2 h. It was found that after this period the relative specific activity of the RNA in the visual cortex of the former was significantly (p<0.001) higher than that of the RNA in the visual cortex of the latter. There was no significant difference in the labelling of the frontal cortex. In a second group of experiments light deprived newly weaned rats were exposed to light for periods ranging from 0–15 h prior to being given a 1 h pulse of (6- 14C) orotic acid. After 1–2 h after first exposure to light the labelling of the RNA in the visual cortex was significantly increased (p<0.001) but after 3 h the labelling was not significantly different from the dark control value. This transient increase in RNA labelling after first exposure to light was not found in the frontal cortex.

A comparison of RNA metabolism in the visual cortex of sighted rats and rats with retinal degeneration

The RNA metabolism in the visual cortex of visually stimulated adult blind and sighted rats has been compared. The blind rats belonged to a strain suffering from an inherited retinal dystrophy. This condition renders them blind after the age of 3–4 weeks. There is no gross difference in the metabolic rate of the blind and sighted strains. No significant differences were seen in the RNA, DNA, and protein content per wet weight of the two types of visual cortex but the RNA DNA ratio was significantly lower in the blind animals. Intraventricularly administered ( 14C) orotic acid was incorporated into RNA to a lesser extent in the visual cortex of the blind animals. This could not be wholly attributed to a difference in the size of the acid soluble pool. Chromatographic studies on RNA hydrolysates and the acid soluble precursor pool indicated that the reduced labeling of RNA in the blind visual cortex was not due to a reduction in the rate of conversion of orotic acid to UTP and thus it would appear that the lowered rate of incorporation of label into RNA is due to a reduced rate of RNA synthesis. This was restricted to the visual cortex, no differences were observed in the frontal cortex. In vitro studies indicated that there were no differences in the total ribonuclease activity in the blind and sighted visual cortex but the RNA polymerase activity (but not the Poly C synthetase activity) was slightly lower in the visual cortex of the blind animals.

Effect of adrenalectomy and of adrenocortical hormones on RNA turnover in a variety of subcellular fractions of rat liver.

(1) The turnover of RNA was examined by following the loss of radioactivity after a single injection of 14C-orotic acid in nuclei, mitochondria, smooth endoplasmic reticulum, rough endoplasmic reticulum, free polysomes, total ribosomes, and sRNA from rat liver. The half-lives of the RNA of various rat liver fractions were found to be 8.7, 6.5, 6.4, 5.4, 4.4, 4.9, and 4.8 days, respectively.(2) In adrenalectomized rats there was a significant decrease in turnover rate. This suggests a slower synthesis of RNA under steady-state conditions. The corresponding values were 12.0, 8.5, 6.5, 6.9, 5.8, 6.1, and 5.9 days.(3) Daily administration of corticosterone or hydrocortisone to adrenalectomized rats restored the turnover rate to normal. Aldosterone was without effect.(4) Aggregate RNA polymerase of rat liver nuclei was decreased by adrenalectomy and restored by corticosterone or hydrocortisone. Aldosterone was without effect.

Incorporation of orally administered radioactive precursors into nucleic acids and ribosomes of housefly ovaries

After emergence female houseflies were fed for 4 days on a diet containing 14C-orotic acid and 3H-thymidine-5-triphosphate, or 3H-leucine. Nucleic acids and ribosomes were then isolated from the ovaries and studied by MAK column chromatography and sedimentation analysis respectively. The ultraviolet absorption and radioactivity of the fractions were also measured. After MAK column chromatography, the u.v. elution pattern showed that only tow distinct peaks, corresponding to tRNA and rRNA were present. A similar elution pattern was obtained by measuring the 14C from 14C-orotic acid incorporated into the RNA. Because of the small quantity present, DNA was not measurable by u.v. absorption, but by determining the incorporation of 3H from 3H-TTP, its presence was clearly evident. Sedimentation analysis of ovarian ribosomes revealed four polymeric forms besides subunits and monomers. The incorporation of 14C-orotic acid and 3H-leucine into the ribosomes was used to follow the synthesis of rRNA and rProtein respectively. Sucrose density gradient centrifugation of the rRNA indicated that the ovarian rRNA consisted primarily of 28 and 18 S particles.

Pentagastrin-stimulated incorporation of 14 C-orotic acid into RNA of gastric and duodenal mucosa.

SummaryFasted rats were injected 3 times over a 24 hr period with either pentagastrin, saline or histamine. 14C-Orotic acid was injected 1 hr prior to sacrifice. The incorporation of orotic acid into phenol extracted RNA was determined for gastric and duodenal mucosa and the liver. Pentagastrin injected animals incorporated 3 times as much orotic acid into RNA of the gastric and twice as much into RNA of the duodenal mucosa when compared to saline or histamine injected controls. Incorporation of orotic acid into liver RNA was not affected by pentagastrin.We conclude that pentagastrin stimulates the incorporation of orotic acid into mucosal RNA of the upper gastrointestinal tract and that is probably indicative of induction of RNA synthesis. These findings support the current evidence and hypothesis (3) that gastrin is a trophic hormone for gastric and duodenal mucosa.