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Investigation of tritium incorporation of tritium incorporation by means of excreted metabolites.

The commonly accepted urine analysis by liquid scintillation method was applied for whole body dose estimating. After the separation of metabolite fractions the organically bound tritium in urine could be measured. Urine samples from workers repeatedly exposed to tritium incorporation during the chemical processing of various labeled compounds have been collected and analyzed. The time dependence of tritium activity in certain metabolites was found to be characteristic, significantly differing from the 3H concentration curve of the native or treated urine sample.

Creatinine

The incorporation of tritium from tritium-enriched water into UDP-N-acetylglucosamine and UDP-N-acetylmannosamine catalyzed by UDP-N-adetylglucosamine 2-epimerase from Escherichia coli.

Uridine diphosphate N-acetylglucosamine 2-epimerase from Escherichia coli 014 K7 H- catalyzes the reversible epimerization of uridine diphosphate N-acetylglucosamine to uridine diphosphate N-acetylmannosamine. During epimerization, tritium from tritium-enriched water is incorporated into both uridine diphosphate N-acetylglucosamine and uridine diphosphate N-acetylmannosamine. The position of incorporation is C-2 of the N-acetylhexosamine moieties.

Carbohydrate Epimerases

Metabolism in normal and virus-transformed chick embryo fibroblasts as observed with glucose labeled with 14C and tritium and with tritium-labeled water.

Glucose metabolism in normal and virus-transformed chick embryo fibroblast cells in culture was observed by allowing the cells to metabolize [U-14C]glucose plus glucose labeled with tritium in the C-1, C-3, and C-6 positions. Similarities and differences between normal and transformed cells were observed and measured. Both normal and transformed cells are found to metabolize about 20% of the glucose via the oxidative pentose phosphate cycle, with the rates being about twice as much for transformed cells as for normal cells under the chosen conditions. Nevertheless, the ratio of glucose metabolized via oxidative pentose cycle to the net flow of that metabolized directly to fructose 6-phosphate is about the same in normal and transformed cells. Although the rate of flow of [14C]glucose into the tricarboxylic acid cycle intermediates and amino acids derived from them appears to be the same in normal and transformed cells, the rate of tritium incorporation from H3HO into these intermediates seems to be much higher in normal cells.

Animals

Biokinetic aspects of tissue-bound tritium in algae.

For the estimate of the radiation exposure of man and for the calculation of the risk of artificial tritium from nuclear power plants, organic tissue-bound tritium is of decisive importance. In model experiments, a tritium incorporation of 61 to 71% was found from tritiated water (HTO) into organic matter of planctonic algae under under reproducible conditions and this was related to the theoretical value. In further experiments the tritium release from these high tritiated algae was of interest. Kept in darkness in tritium-free, non-sterile river water, so that autolytic processes and bacterial decomposition could occur, the concentration of HTO was measured over a period of three weeks. A relatively long half-life of tissue-bound tritium was found under various temperature conditions. Therefore it must be considered that a significant retention of tritium in biological matter has to be taken into account in a natural ecosystem. In streams into which the cooling water of a nuclear reactor is released all conditions are found already for a long turnover and cycling of artificial tritium in living organisms as well as the conditions for a favourable transport of tritium by food chains to man.

Ecology

Comparative incorporation of tritium from tritiated water versus tritiated thymidine, uridine or leucine.

Whereas the radiotoxicity of tritium has been extensively studied, comparatively little information exists on its long-term effects as a potential environmental pollutant, particularly at small dosage. This investigation was primarily aimed at assessing comparatively a possible carcinogenic potency of tritiated water versus radioactive precursors of DNA, RNA and proteins, namely tritiated thymidine, uridine and leucine in C57 Black mice. Tritium is largely released in the environment in the form of tritiated water. There are many uncertainties, however, as to how tritium is incorporated from tritiated water into cell constituents quantitively and qualitatively. In 1965, we reported on the carcinogenic effect of tritium in the form of tritiated thymidine on newborn C57 BL mice in the dose range of 0.3--1.5 muCi/g [Mewissen. 1965]. Hence the selection of tritiated water, and of tritiated precursors, in an attempt to evaluate their respective role in the tritium transfer process and to correlate their possible late effects with their specific patterns or sites of incorporation. This study deals with tritium incorporation from tritiated water and various precursors at the 1 or 10 muCi level. RSA values, i.e., the ratio of organically bound tritium per hydrogen content of dry tissue over aqueous tritium per hydrogen content of water, were estimated for newborn, juvenile and adult mice, at various time intervals (1, 8, 15, 22 and 29 days) following single administration of tritiated water, tritiated thymidine, uridine or leucine. The data available at this time show that administration of tritiated water (or precursors) result in a complex time dependent and age dependent residual activity dynamics both in the organic component and in the aqueous fraction of tissue. A few preliminary conclusions can be made. Following a single acute or brief exposure to tritiated water, values of activity become exceedingly small after a relatively short time period. In a steady state equilibrium, resulting from chronic exposure to tritiated drinking water, RSA values tend to stabilize. However, wide variations between various organs are to be expected, as suggested by their respective RSA values following a single exposure. In view of these observations, it would seem that a realistic estimate of the internal dose to the radiosensitive nucleus must take into consideration the age dependent incorporation of tritium from tritiated water, as well as the variation between organs. The carcinogenic risk has often been estimated from a uniform dose dependency model. The influence of time and space microdistribution of dose within tissues and more particularly at specific sites (such as DNA, RNA or protein) has received, as yet, little attention, as well as the relative contributions of the time sequence of dose absorption during the usually long latency period. Such factors, among others, may be critical in carcinogenesis from internal irradiation...

Age Factors

Distribution of tritiated compounds (tritiated thymidine and tritiated water) in the mother-fetus system and its consequences for the radiotoxic effect of tritium.

The incorporation and distribution of tritiated thymidine (3H-TdR) and tritiated water (HTO) have been measured in newborn rats exposed to various levels of tritium by continuous infusion into pregnant rats from day 9 until term. In the animals exposed to HTO, the tritium activity was homogeneously distributed, while 3H-TdR led to accumulation of DNA-bound and homogeneously distributed tritium. The incorporated activity and the specific activity of DNA from ovaries which showed a reduction of total oocyte number by approximately 50% were used to estimate the dose absorbed by the ovarian cell nuclei in both systems. From the absorbed dose, a factor of 3.7 was calculated for the "internal relative biological effectiveness" of DNA-bound tritium as compared to homogeneously distributed 3H under the restrictive assumption that the static description of the system at birth reflects the situation during the time of dynamic development of the ovaries when the toxic effect occurs. The influence of these dynamic factors of changing nuclear size and tritium incorporation during the sensitive period is weighed against the possibility that the continuous 3H-TdR infusion during pregnancy might represent a model in which DNA-bound tritium shows a higher effectiveness than homogeneously distributed tritium.

Animals

Rate of tritium labeling of specific purines in relation to nucleic acid and particularly transfer RNA conformation.

The kinetics of the incorporation of tritium into the C-8 positions of purine units in nucleic acids has been studied. The polymers investigated include poly(A), poly(A): poly (U) duplex, a double-stranded viral RNA, tRNA, and DNA. In the random coil state, the kinetics of incorporation of tritium into the purine sites of the polymers are identical with those for the corresponding purine mononucleotides. When the nucleic acids are in their native conformations, however, the purine labeling rates are reduced below that expected for the free mononucleotides. The magnitude of the effect is remarkably dependent upon the particular nucleic acid. For example, at 37 degrees C the purines in double-stranded DNA label at a rate two- to threefold slower than the corresponding mononucleotides, but in a double-stranded viral RNA, a 30- to 40-fold effect is found. The data suggest a strong influence of microscopic helix structure on the rate of tritium incorporation. First-order rate constants for the exchange of tritium into specific purine sites in yeast tRNAPhe were also determined. This was done by partially labeling the nucleic acid in tritiated water, and subsequently removing free and loosely bound tritium. Under conditions where exchange-out does not occur, the nucleic acid was digested with specific nucleases; chromatographic separation then enabled specific activities of purines from specific sites to be obtained. The rate constants for these sites show a large variation. They are markedly reduced for those residues occurring in cloverleaf helical sections and, in certain cases, for those known from crystallographic data to be involved in tertiary interactions. As examples of bases that can participate in tertiary interactions, the crystal structures show A14 and G15 in special base-pairing arrangements. Both purines (A14 and G15) occur in single-stranded sections of the cloverleaf; both show markedly reduced C-8 hydrogen-exchange rates. On the other hand, rate constants for bases and regions known to be on the outside of the moleculesuch as the anticodon loop and the 3' terminusāre perturbed the least. In one instance, a base in the dihydrouridine loop believed to be involved in tertiary interactions, according to crystallographic studies, incorporates tritium as if it were relatively unperburbed by the tRNA structure. The structural interactions of this base may be partially or completely broken at 37 degrees C in solution.

Base Sequence

Tritium release from Ti-T layers in air, in aqueous media and in animal experiments.

In connection with Ti-T incorporation hazard to which operators of neutron generators are exposed the release of tritium from Ti-T preparations of different ages was studied in experiments carried out in air, in aqueous media and in living animals. Samples were prepared with activities from 10 to 30 mCi and the effect of storage on the tritium release rate was also observed. In 250 days a fraction of 10(-3) of the tritium activity was absorbed by aqueous liquids. In air the release varied from 10(-6) to 10(-7) per hour. The Ti-T samples of different ages, introduced surgically into the abdominal cavity of rats, showed the tritium release rate to decrease with time. The tritium activity observable in the circulation was 5 to 6 orders of magnitude smaller compared with the introduced value. The observations permit the inference that in the case of Ti-T incorporation only a minor fraction of the tritium burden can be assessed from the activity measured in the urine.

Air Pollutants, Radioactive

Hydrogen-tritium exchange titration of the histidine residues in bovine heart cytochrome c and analysis of their microenvironment.

Microenvironments of the three histidine residues located at the positions 18, 26, and 33 from the amino terminus in bovine heart cytochrome c were analysed in solution by the hydrogen-tritium exchange titration method, which has been developed in this laboratory. Histidine-18, which is liganded to the heme iron, and histidine-26 did not incorporate tritium in native state, indicating that the two are located in solvent inaccessible hydrophobic regions. Histidine-33 was labeled with tritium to an appreciable extent and seemed to be partially buried in the molecule. The pKa value estimated for histidine-33 was 6.1 at 37 degrees by the tritium exchange titration, suggesting that the residue interacts very weakly with a neighboring cationic group. These results seem to be compatible with the tertiary structure of the protein deduced from the X-ray crystallographic analysis.

Amino Acid Sequence

Fundamental study on decontamination of tritium from gaseous phases by copper oxide.

Some fundamental studies on removal of tritium from gaseous streams by a small column of copper oxide were carried out. An about 10 cm length of column packed with 100 g of copper oxide was examined on the conversion ratios of hydrogen and tritium into water. The experiment with hydrogen of 0.1% in argon showed that the logarithmic plot of remaining ratio of hydrogen by passing through the column is proportional to the reciprocal of gas flow rate and to the length of oxide column. The oxide column was also used for treating tritium-bearing waste gas from an evacuating system of alpha D-T neutron generator. It was observed that at column temperatures lower than 450 degrees C the conversion ratio of tritium in the waste gas into water is considerably smaller than that of hydrogen of 0.1% in argon.

Copper

Metabolism of 1-3H-ethanol by isolated liver cells. Time-course of the transfer of tritium from R,S-1-3H-ethanol to lactate and beta-hydroxybutyrate.

Parenchymal cells isolated from the liver of 24 h fasted rats were incubated with 65 mM R,S-1-3H -ethanol plus 3 mM pyruvate as substrates in the absence and presence of 1.7 mM 4-methylpyrazole. Metabolite levels and the time-course of the transfer of tritum from ethanol to lactate and beta-hydroxybutyrate was measured during the first 15 min of ethanol metabolism. The time-course of the loss of tritium from 2-3H-L-lactate and 3-3H-beta-D-hydroxybutyrate in experiments identical to the above-mentioned was estimated. A GLC method for the isolation of lactate and beta-hydroxybutyrate and the preparation of 2-3H-L-lactate and O-3H-beta-D-hydroxybutyrate is described. The incorporation rate of tritium from ethanol into lactate and beta-hydroxybutyrate decreased with time. Addition of 4-methyl-pyrazole decreased the incorporation rate roughly proportional to the decrease in ethanol and acetaldehyde metabolism. The observed incorporation rates of tritium to lactate were corrected for the detritiation rates measured in experiments with I-3H-L-lactate and 3-3H-beta-D-hydroxybutyrate as substrates. The rate of extramitochondrial acetaldehyde oxidation was calculated from the corrected initial rates of incorporation of tritium into lactate to 0-0.4 mumol min-1 (ml of cells)-1.

Acetaldehyde

Tritium retention in rat after administration of various doses of tritiated water.

Hooded inbred rats were given subcutaneously HTO doses ranging from 1.8 muCi/g b.w. to 115 muCi/g b.w. Samples of liver, lung, kidney, skin muscle, small and large intestine were taken at 1, 3, and 5 days intervals within 21 days of the experiment. The concentrations of tritium in free water and bound in particular tissues have been determined, and the biological half-lifes (Tb) of tritium were calculated by means of least squares. The biological half-life of free-water tritium varied from 3.6 days in kidney to 4.8 days in muscle for the control group being slightly lower in the groups with higher HTO doses. The retention of tissue-bound tritium varied in each group of animals. Tb values varied from 10.3 days in the large intestine to 85.0 days in the muscle of the control group and were significantly lower at higher HTO doses. This result is opposite to the effect observed after external X-ray irradiation for the same radiation dose level.

Animals

Distribution of tritium in the different organs of calves and pigs after ingestion of various tritiated feeds.

A research programme on the transfer of tritium in the food chain has been in progress for several years on the experimental farm of the Nuclear Energy Research Center at Mol. The studies reported here are related to the distribution of tritium in the organs of farm animals contaminated in various ways. Two young male calves ingested tritiated milk; the daily intake of 3H-organic form was about 15 muCi for each calf and the total activity ingested until the sacrifice was 482 muCi. Three male pigs from the same litter and about 7 weeks old were used for each experiment on the administration of tritium under different forms: (a) single intraperitoneal injection of 39.3 mCi HTO (P2, P3, P4). (b) daily ingestion of 28.4 muCi HTO. The total activity ingested was respectively 569 muCi (P5) and 766.8 muCi (P6). (c) ingestion of tritiated potatoes. The total activity ingested was respectively 21 muCi (P8), 40.3 muCi (P9) and 48.1 muCi (P10). (d) ingestion of tritiated milk powder. The total activity ingested was respectively 60.6 muCi (P13), 110.4 muCi (P11) and 154.5 muCi (P12). After slaughtering of each animal various organs were removed and analyzed for the 3H content in the tissue water and in the organic matter. We could verify that the chemical form of 3H present in the food is of great importance for the incorporation of 3H in the organic matter of the animal organs. The total incorporation increases by a factor 5.6 when 3H is ingested as tritiated milkpowder by pigs as compared to HTO and with a factor 15 for calves. When tritiated potatoes were ingested by pigs a factor 15.6 was found. The transfer of 3H from HTO and milk feed ingested in the organic fraction of organs is lower for pig than for calf. When we consider the 3H in the tissue water of organs the specific activity (SA) is a little lower than the SA of ingested HTO and after ingestion of tritiated milk feed the activity is very low and no difference due to the species is found. After fractionation of liver and spleen tissue following the technique of Schmidt--Thannhauser radioactivity was found in all liver and spleen constituent lipids--RNA--DNA and proteins, but after isolation and purification of DNA following the original methods, we have not been able to demonstrate that tritium is really incorporated into DNA molecules of a non-dividing organ such as the liver nor of an actively dividing organ such as the spleen.

Animal Feed