A Comparison of the Killing of Cultured Mammalian Cells Induced by Decay of Incorporated Tritiated Molecules at -196°C

The killing efficiency of tritium disintegrations in frozen mammalian cells labeled with tritiated uridine, histidine, and lysine was compared with the killing efficiency of incorporated tritiated thymidine. In each case, the distribution of tritium in the cells was determined by chemical fractionation as well as by radio-autography. Of all tritium disintegrations, by far the most effective were those occurring in DNA molecules within frozen cells; such incorporated tritium has a killing efficiency of 0.006. When cells were incubated with tritiated uridine for 10 min to label nuclear RNA, the killing efficiency was 0.0015. When the cells were pulse labeled with tritiated uridine and permitted to grow in nonradioactive media for 10 hr before freezing in order to incorporate tritium into cytoplasmic RNA, the killing efficiency was reduced to 0.0010. The results suggest that decay of tritium in nuclear RNA is more effective than that in cytoplasmic RNA. When the cells were labeled with tritiated histidine or lysine for 30 min, tritium atoms were found mainly in the acid soluble rather than in the protein fraction and the killing efficiency in each case was approximately 0.0007. The results of these suicide experiments indicate that the killing efficiency of tritium disintegrations depends on where tritium is located within the cells. Tritium disintegrations in the nucleus are more effective in killing the cell than that in cytoplasm; and tritium disintegrations on DNA in the nucleus is more effective in killing the cell than that of nuclear RNA.     

Tritioboration and synthesis of tritium-labeled polyunsaturated fatty acids

Methyl esters of polyunsaturated fatty acids labeled with tritium were prepared by partial stereoselective reduction of the corresponding acetylenic esters with tritiated disiamylborane, followed by protonolysis with tritiated acetic acid. The labeling was strictly specific, and the tritium atoms were located only at the carbon atoms forming the unsaturated bond(s). Synthesis of some tritiated fatty acid methyl esters with methylene-interrupted trans-cis double bonds is also reported.     

Tritiation of lysozyme by the free-radical interceptor method and determination of the tritium distribution among individual residues of the chromatographically homogeneous protein

Lysozyme has been tritiated by the free-radical interceptor method, which involves reaction of the carbon free radicals, resulting from gamma-irradiation of the lyophilized protein in a vacuum, with tritiated hydrogen sulfide. This technique resulted in a high yield of tritiated enzyme which was chromatographically indistinguishable from the native protein. To characterize this material with respect to its tritium distribution, it was first reduced, carboxymethylated, and digested with chymotrypsin. The peptides were separated and purified by ion exchange chromatography and electrophoresis and then analyzed to determine the specific activities of 78 of the 129 residues in the protein chain. The tritium was found to be broadly distributed, with only 12 residues apparently devoid of label. The relative specific activity, defined as the ratio of the specific activity of a given residue to the average specific activity for all residues of the same kind within the protein, did not exceed the value of 3.73 for isoleucine at position 88. A distinct spatial relationship is seen among the more heavily labeled residues when these results are related to the x-ray diffraction model of lysozyme. Thus, residues 16, 18, 19, 21, 23, 29, 31, and 32 may be grouped with 105, 110, 113, and 115; these highly labeled residues are close to a strongly hydrophobic region. Another heavily labeled area involves residues 50, 54, 57. 83, 84, and 88. In addition, residues 1 and 6 at the amino end of the chain are heavily labeled, as are 118, 121, and possibly 119 at the carboxyl end. This distribution of tritium appears to reflect the secondary free-radical distribution in the irradiated protein. These observations confirm previous indications that the secondary distribution is influenced by the conformation of the protein molecule.     

Glucose turnover in the young chicken (Gallus domesticus) using variously labeled (3H, U-14C) glucose tracers.

1. Various parameters of glucose metabolism--glucose replacement rate, percent recycling, mean transit time, and glucose mass were examined using various double labeled glucose tracers--2T-, U-14C; 3T-, U-14C; 4T-, U-14C; 5T-, U14C; and 6T-, U-14C. 2. Estimates of replacement rate were greatest for 2T-glucose (21.4 mg/min/kg), with 3T-, 4T-, 5T-, and 6T-glucose all having similar values (15.8, 15.6, 17.0, 16.0 mg/min/kg, respectively). 3. Calculated glucose mass based on all tritiated tracers (734-1086 mg/kg body weight) agreed closely with a direct determination of body glucose (969 mg/kg). 4. Reincorporation of tritium from 3H2O into glucose did not occur to any significant degree. 5. The young chick was found to have a very rapid rate of glucose turnover and high percent recycling compared to mammals.     

A rapid and simple radioassay for thymine 7-hydroxylase

This work describes a simple and convenient procedure for measuring the activity of thymine 7-hydroxylase. The principle of the procedure depends upon the conversion of tritiated thymine by the enzyme to 5-hydroxymethyluracil. This reaction simultaneously invokes the loss of a tritium atom and the formation of tritiated water. The quantity of tritiated water formed is stoichiometrically proportional to the amount of 5-hydroxymethyluracil produced.The sensitivity of this procedure was markedly improved when both catalase and BSA were included in the reaction mixture.     

Isotopic probes into pathways of ethanol metabolism

The relative extent of tritium labeling in glucose and water was determined when l-[2-³H]lactate or [(1R)1-³H]ethanol were the labeled substrates for rat liver parenchymal cells, incubated with 20 mm ethanol and 10 mml-lactate. From the relatively lower specific yield in glucose from the tritiated ethanol one can calculate a percentage contribution of a non-alcohol dehydrogenase-mediated pathway to total ethanol metabolism. This calculated value (about 35%) is somewhat higher than that determined by the use of pyrazole at 5 mm to inhibit alcohol dehydrogenase. The utilization of [(1R)1-³H]ethanol is slower than that of unlabeled ethanol, both in the absence and presence of 5 mm pyrazole, indicating isotope discrimination against tritium in both the alcohol dehydrogenase and non-alcohol dehydrogenase pathways.There was only a slight difference in the rate of utilization of normal and fully deuterated ethanol by rat liver cells in the absence of pyrazole. However, in the presence of 5 mm pyrazole, where essentially only the non-alcohol dehydrogenase pathway operates, deuterated ethanol was utilized at only about half the rate of nondeuterated ethanol. These findings are difficult to reconcile with a catalase-mediated pathway of ethanol metabolism in which the rate-limiting factor is the rate of H₂O₂ generation.     

Catabolism of Tritiated Thymidine by Aquatic Microbial Communities and Incorporation of Tritium into RNA and Protein

The incorporation of tritiated thymidine by five microbial ecosystems and the distribution of tritium into DNA, RNA, and protein were determined. All microbial assemblages tested exhibited significant labeling of RNA and protein (i.e., nonspecific labeling), as determined by differential acid-base hydrolysis. Nonspecific labeling was greatest in sediment samples, for which ≥95% of the tritium was recovered with the RNA and protein fractions. The percentage of tritium recovered in the DNA fraction ranged from 15 to 38% of the total labeled macromolecules recovered. Nonspecific labeling was independent of both incubation time and thymidine concentration over very wide ranges. Four different RNA hydrolysis reagents (KOH, NaOH, piperidine, and enzymes) solubilized tritium from cold trichloroacetic acid precipitates. High-pressure liquid chromatography separation of piperidine hydrolysates followed by measurement of isolated monophosphates confirmed the labeling of RNA and indicated that tritium was recovered primarily in CMP and AMP residues. We also evaluated the specificity of [2-³H]adenine incorporation into adenylate residues in both RNA and DNA in parallel with the [³H]thymidine experiments and compared the degree of nonspecific labeling by [³H]adenine with that derived from [³H]thymidine. Rapid catabolism of tritiated thymidine was evaluated by determining the disappearance of tritiated thymidine from the incubation medium and the appearance of degradation products by high-pressure liquid chromatography separation of the cell-free medium. Degradation product formation, including that of both volatile and nonvolatile compounds, was much greater than the rate of incorporation of tritium into stable macromolecules. The standard degradation pathway for thymidine coupled with utilization of Krebs cycle intermediates for the biosynthesis of amino acids, purines, and pyrimidines readily accounts for the observed nonspecific labeling in environmental samples.     

Germinal and somatic mutation induction in Drosophila after treatment of larvae with tritiated water.

The present study was carried out to evaluate the mutagenicity of tritium, administered as tritiated water, in Drosophila melanogaster. Larvae were fed on tritium-treated medium during their development. Germinal and somatic mutation induction was detected by means of the sex-linked recessive lethal and the wing spot tests, respectively. Our results show that beta-radiation from tritium is able to induce significant increases in the frequency of both germinal and somatic mutations.     

An isotopic assay of dUTPase activity based on coupling with thymidylate synthase

A new rapid, sensitive and convenient procedure is presented allowing determination of dUTPase activity. With [5-(3)H]dUTP used as the substrate, dUTPase, converts it to the corresponding monophosphate and is coupled with thymidylate synthase-catalyzed reaction, resulting in tritium release from [5-(3)H]dUMP. Following charcoal absorption of the labeled nuleotides, radioactivity of tritiated water is determined. The new assay was tested to show comparable results with a previously described assay, based on measuring dUTPase-catalyzed [5-(3)H]dUMP production.     

Determination de la transpiration au moyen de l'eau tritiee

Summary Tritiated water is used to measure the transpiration rate of trees in forest. A simple rapid method for extracting water from plant tissues was developed. To test the accuracy of the tritium method, the authors carried out a comparative study between this method and the weight-loss method. High correlations (r=0.99) between the two methods were obtained. But there was an increase of about 15% by the tritium method, assuming that transpiration values obtained by weight-loss were accurate.The data showed that the activity of tritium in twigs give a better transpiration rate than the activity determined only in needles.It is concluded that the tritiated water method is valid for measurement of tree transpiration.

     

Presence of Radiolabelled Metabolites in Release Studies Using [3H]γ-Aminobutyric Acid

Abstract: Most studies on γ-aminobutyric acid (GABA) release from nervous tissue have been conducted using radiolabelled GABA in the presence of aminooxyacetic acid (AOAA) to inhibit GABA: 2-oxoglutarate aminotransferase (GABA-T) to prevent conversion of labelled GABA to labeled catabolites. Here we present data showing that even in the presence of 10 μM-AOAA the spontaneous release of tritium from rat cortical synaptosomes prelabelled with 2,3-[³H]GABA is mainly in the form of tritiated water but that the increase in tritium release in the presence of unlabelled GABA or high potassium-ion concentrations is in the form of authentic [³H]GABA. Interpretation of results should take these facts into account.     

A metabolic derivation of tritium transfer coefficients in animal products

Tritium is a potentially important environmental contaminant originating from the nuclear industry, and its behaviour in the environment is controlled by that of hydrogen. Animal food products represent a potentially important source of tritium in the human diet and a number of transfer coefficient values for tritium transfer to a limited number of animal products are available. In this paper we present an approach for the derivation of tritium transfer coefficients which is based on the metabolism of hydrogen in animals. The derived transfer coefficients separately account for transfer to and from free (i.e. water) and organically bound tritium. A novel aspect of the approach is that tritium transfer can be predicted for any animal product for which the required metabolic input parameters are available. The predicted transfer coefficients are compared to available independent data. Agreement is good (R ²=0.97) with the exception of the transfer coefficient for transfer from tritiated water to organically bound tritium in ruminants. This may be attributable to the particular characteristics of ruminant digestion. We show that tritium transfer coefficients will vary in response to the metabolic status of an animal (e.g. stage of lactation, diet digestibility etc.) and that the use of a single transfer coefficient from diet to animal product is inappropriate. It is possible to derive concentration ratio values from the estimated transfer coefficients which relate the concentration of tritiated water and organically bound tritium in an animal product to their respective concentrations in the animals diet. These concentration ratios are shown to be less subject to metabolic variation and may be more useful radioecological parameters than transfer coefficients. For tritiated water the concentration ratio shows little variation between animal products ranging from 0.59 to 0.82. In the case of organically bound tritium the concentration ratios vary between animal products from 0.15 (goat milk) to 0.67 (eggs). Received: 28 May 2001 / Accepted: 20 August 2001     

Studying Liposomes by Tritium Bombardment

Bilayer liposomes from a mixture of dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylethanolamine (DPPC:DPPE=8:2, molar ratio) or DPPC labeled with ¹⁴C-DPPC (DPPC:¹⁴C-DPPC) were bombarded with thermally activated tritium atoms. The tritiated liposomes were hydrolyzed by phospholipase C, and the tritium incorporation into different parts of the bilayer along its thickness was determined. The tritium flux attenuation coefficients were calculated for the headgroup (k₁=0.176±0.032 Å^–1) and acylglycerol residue (k₂=0.046±0.004 Å^–1) layers indicating a preferential attenuation of the tritium flux in the headgroup region and relative transparence of the membrane hydrophobic part. The finding is potentially important to apply tritium bombardment for investigation of spatial organization of transmembrane proteins in their native lipid environment.     

The transfer of tritium-labeled organic material from grass into cow's milk

Two lactating cows were given tritiated hay containing organically bound tritium (OBT) only for about 4 weeks. Tritium activity was determined in milk fat, casein, lactose, milk water, and whole milk. In one cow, milk was sampled for approximately 450 days, covering two lactation periods. At steady state, specific tritium activities in casein, lactose, and milk water were 58, 10, and 11%, respectively, of those in milk fat. Some OBT was converted into THO during catabolism and entered the body water pool. This 3H source accounted for nearly 40% of tritium in lactose, but in casein and milk fat about 97% of tritium was derived from ingested OBT. Comparison of the specific activity of milk constituents with the specific activity of ingested hay showed the following values: 0.84 for milk fat, 0.49 for casein, 0.05 for lactose, 0.10 for milk water. About one-half of the tritium transferred to milk was found in organic milk constituents and the other half in milk water. Decrease of tritium activity with time could be represented by three components with different half-lives for the organic milk constituents. Those for milk fat and casein were quite similar, with a slow component of nearly 3 months.     

Tritium distribution in newborn mice after providing mother mice with drinking water containing tritiated thymidine

Throughout gestation pregnant mice received drinking water which contained [methyl-3H]thymidine (18.5 kBq/ml). The newborn mice were divided into two groups. One group was nursed by their own mothers, which were further supplied with tritiated thymidine until 4 weeks after delivery (Experiment I). The other group was nursed by "nonradioactive mothers" which were given no tritiated thymidine (Experiment II). Tritium incorporation into the small molecular components of the acid-soluble fraction, lipid, RNA, DNA, and protein was analyzed for the newborn mice at various ages. In Experiment II, total radioactivity per gram tissue decreased initially after birth with a half life of 2.5-2.9 days in spleen, liver, intestine, stomach, thymus, lung, kidney, heart, and brain. At about 2 weeks after birth, a slower component of tritium elimination due mainly to the DNA-bound tritium appeared. Specific activity of DNA at birth was organ specific, highest in heart and lowest in thymus. Cumulative absorbed dose in various organs was estimated for the first 4 weeks after birth based upon an assumption that total and DNA-bound tritium are uniformly distributed. The result showed that organ specificity of dose accumulation is obvious for DNA-bound tritium, highest in spleen (1.15 mGy) and lowest in brain (0.13 mGy). It was also shown that the tritium supply from mother's milk is of minor importance for dose accumulation of DNA-bound tritium in the cell nuclei of organs of suckling mice.     

Radiation quality of tritium beta-rays: microdosimetric distributions for nanometer-size targets in a medium containing tritiated water

To elucidate the characteristics of the action of tritium beta-rays, the following parameters are derived: electron slowing down spectra of primary electrons (beta-rays) and delta-rays in a medium containing tritiated water; and frequency distributions for the microdosimetric quantity j (number of effective primary events per track per target), fj, for nanometer-size targets exposed to tritiated water. Features of the radiation quality of tritium beta-rays are discussed by comparing the present results with those for 60Co gamma-rays and 7 MeV electrons. It is concluded that, although tritium beta-rays, 60Co gamma-rays, and 7 MeV electrons are classified as the same low l.e.t. radiation, the radiation quality of tritium beta-rays is considerably different from those of 60Co gamma-rays and 7 MeV electrons, and has specific features such as a high average l.e.t., a small total electron fluence per unit absorbed dose, and a different microdosimetric distribution, fj, for nanometer-size targets.     

Effect of lifetime intake of organically bound tritium and tritiated water on the oocytes of rats

Summary Rats were continuously exposed to constant activity of tritium in drinking water (HTO group) or to tritium organically bound in food (T-food group) in the period from conception ofF ₁ generation through maturity. Female offspring were killed at the age of 21 and 71 days and the oocytes in their ovaries were counted. Mean dose rates absorbed in the ovaries were for the HTO groups 7.25 ± 0.37 and 14.73 ± 0.79 mGy/day and for the T-food group 4.84 ± 0.25 mGy/day.Reduction in the oocyte number in the ovaries of females exposed to tritiated food was bigger than in the ovaries of females exposed to tritiated water. The dependence of the survival of small oocytes on the dose rate and the corresponding total accumulated dose had an exponential character. The damaging effect of tritium was for the period from conception to 21 days of age bigger than from 21 to 71 days of age. Of all stages of oocyte development, the highest sensitivity to tritium irradiation was observed in small oocytes and oocytes with one complete layer of follicle cells. As a result, relative number of the growing and large oocytes increased.     

Induction of mutations by tritiated water and 3H-thymidine in Drosophila melanogaster assayed by the somatic zeste-white eye mutation system

In order to study the mutagenic effect of exposure to tritium, Drosophila melanogaster larvae were treated with tritiated water (3H2O) or tritiated thymidine (3H-TdR) during development. Dose rates ranged from 0.0058 to 0.058 rad/h per nucleus for 3H-TdR and from 0.049 to 0.122 rad/h for 3H2O. Induction of mutations was measured by the appearance of somatic mutations in the eyes of an unstable strain of Drosophila melanogaster. Both substances caused a significant increase in mutation frequency. With the assumption that each mutation observed in this assay is caused by one DNA break, the effectiveness of tritium to create DNA breaks is estimated to be 0.20 breaks per decay for 3H-TdR and 0.27 breaks per decay for 3H2O.     

Metabolism of organically bound tritium in man

The classic methodology for estimating dose to man from environmental tritium assumes that all tritium, whether organically bound or free, enters directly into man's free body water compartment and is uniformly distributed as tritiated water. This methodology ignores the fact that organically bound tritium in foodstuffs may be directly assimilated in the bound compartment of tissues without previous oxidation. A four-compartment model consisting of a free body water compartment, two organic compartments, and a small, rapidly metabolizing compartment is proposed. The utility of this model lies in the ability to input organically bound tritium directly into organic compartments representing tissue solids. The model will be used to illustrate the potential importance of organically bound tritium to cumulative dose estimates. It is found that organically bound tritium in foodstuffs can increase cumulative total body dose by a factor of 1.7-4.5 times the free body water dose alone, depending on the bound-to-loose ratio of tritium in the diet.     

Movement of Water and Solutes in Sieve Tubes of Willow in Response to Puncture by Aphid Stylets. Evidence against a mass flow of solution

Experiments are described in which bark strips of willow were sealed to polythene tubes having two compartments. This allowed investigations to be made of the transport along the sieve tubes of tritiated water, ¹⁴C-labelled sugars, and ³²P-phosphates from one compartment, towards a stylet situated in the bark over the other compartment. Although activity from both ¹⁴C and ³²p was detected in the stylet exudate usually within 1 hour from isotope application, tritium activity was never detected even after a period of 8 hours in most experiments, though in certain cases, very low activities were detected after 4 hours. Subsequent experiments in which stylets were sited over both compartments showed that tritium activity moved laterally into the punctured sieve element more rapidly than either ¹⁴C or ³²P. Experiments using both live and dead bark in which stylets were not employed, showed that within 4 hours tritium activity had moved by diffusion along the whole length of a bark strip, therefore after this time tritium activity could have moved into the stylet exudate by a diffusional process. The lack of rapid longitudinal movement of tritiated water along the sieve tubes, indicates that the transport process is unlikely to be a mass flow of solution.