Comparison of the structure and catabolism of rat thymus DNA exposed to tritium oxide and gamma-radiation in equal doses

A decrease in DNA concentration and in the molecular mass of single-stranded DNA, an increase in the PDN content, and activation of acid DNAses in rat thymus were observed after a single administration of tritium oxide in a dose of 22 mBq/g (a cumulative dose of 7.8 Gy) and gamma-irradiation at a corresponding dose-rate and value of the cumulative dose. These changes were most pronounced during the period of dose accretion, i.e. during 14-30 days after the beginning of irradiation. The degree to which the indices under study varied from the controls was 2-3 times in rats given tritium oxide than in those exposed to gamma-irradiation.     

Relative biological effectiveness of tritium oxide based on nucleic acid metabolic indices

Relative biological effectiveness (RBE) of tritium oxide, as compared to gamma-rays (137Cs), with regard to LD50/30 is 2,32 +/- 0,69 for rats. The RBE coefficients for tritium oxide are obtained with regard to some indices of nucleic acid metabolism in the thymus and spleen during the dose formation (0-14 days). The RBE of tritium oxide increases with a decrease in radiation dose as determined according to the concentration and content of DNA per organ and activity of thymus DNAases.     

Nucleic acid metabolism in rat hematopoietic tissues during and after prolonged administration of different doses of tritium oxide

During the long-term administration to rats of tritium oxide in doses of 0.37, 0.925 and 1.85 MBq/g body mass the content of karyocytes and nucleic acids in the bone marrow and spleen was decreased, the rate of their biosynthesis changed, the DNA structure impaired, the content of salt-soluble polydeoxynucleotides increased, and DNAases activated. The observed changes were function of dose. After the end of the administration of the isotope the animals which had received a lesser tritium dose exhibited a more rapid and complete recovery.     

Study of the structure and metabolism of the rat thymus gland during long-term administration of tritium oxide in different doses

During-3-month administration of tritium oxide (0.37, 0.925 and 1.85 MBq/g body weight) to rats one could observe the loss of the thymus weight, decrease in the concentration, content and production of DNA and RNA per organ, reduction of the molecular weight of single-stranded DNA, increase in the content of the salt-soluble polydeoxyribonucleotides, and activation of acidic DNAases. These changes were function of tritium dose daily administered to rats.     

Nucleic acid metabolism in rat liver after single and prolonged exposure to tritium oxide

It was shown that after a single administration of tritium oxide in a dose of 22.2 MBq/g body mass the liver mass increased, the concentration of nucleic acids decreased and the biosynthesis rate increased during a one-month observation. By the end of the observation period (the first year) the parameters under study were normalized. The long-term administration of tritium oxide in daily doses of 0.37, 0.925 and 1.85 MBq/g body mass caused changes in the nucleic acid metabolism which were less manifest (at early times), than in the case of a single injection. At the same time, the long-term administration of tritium oxide in the dose of 0.925 MBq/g caused a substantial disturbance of the nucleic acid metabolism at later times (after 2-9 months).     

The significance of immunity disorders in the development of tumorous effects during the long-term action of tritium in mice

In experiments with CBA mice it was shown that the long-term influence of tritium oxide administered with drinking water daily during 180 days (370 kBq.g-1 of body mass, cumulative dose 8.7 Gy, dose rate 4.5 cGy.day-1) causes the development of immunity deficiency 90 days after the onset of the administration that persists for up to 270 days. There is a 34% decrease in the average life of irradiated animals and an increase in the number of malignant neoplasms (postmortem examination was performed after natural death or killing on days 250, 350 and 450). There is a direct relationship between the occurrence of malignant neoplasms and the immunity deficiency.     

Effect of tritium oxide in a wide range of doses on the nucleic acid metabolism of the rat spleen

A single injection of tritium oxide in a dose of 1.1 MBq/g (0.5 Gy for 30 days) was shown to impair the nucleic acid metabolism in the rat spleen. The changes in the indices under study (e.g. mass, nucleic acid content and biosynthesis) increased with the dose, and the recovery started later and was incomplete. Qualitative differences were found in the effects of tritium oxide and gamma radiation with regard to the rate of DNA biosynthesis: 24 h following the injection of the radionuclide specific activity of DNA increased with dose, whereas this function was inverse in the case of gamma irradiation as it was reported in the literature.     

Dependence of the damage and recovery of nucleic acid metabolism on the dose rate in tritium oxide exposure

Disturbance and normalization of nucleic acid metabolism in rat thymus was studied after the effect of tritium oxide delivered in similar cumulative doses but at different dose rates. Both the disturbance and normalization were shown to be a function of dose rate, the slightest damage and the complete recovery being registered at the lowest dose rate (the amount of tritium oxide administered being 0.37 MBq/g/day). The rate of restoration was also a function of dose rate; with tritium oxide dose of 1.85 MBq/g/day (the dose rate at the stage of the equilibrium tritium content in the aqueous phase being 0.38 Gy/day) it was 9 times as high as that after a dose of 0.37 MBq/g/day (0.11 Gy/day dose rate).     

Normal killer function in CBA mice as affected by long-term intake of tritium oxide

A study was made of the cytotoxic function of normal killers of CBA mice during long-term administration of tritium oxide in potable water (a cumulative absorbed dose of 8.7 Gy, dose-rate, 4.5 Gy/day) and at different times after termination of the radionuclide injection. A mean decrease of 20-30% in the lytic effect of the effectors from spleen of the experimental mice on cell-targets of K-562 human erythroleukosis was demonstrated by a 17-19-hour test with 51Cr. At later times after termination of action of tritium oxide, the cytotoxic effect of normal killers increased by 1.8 times as compared to intact controls of the same age.     

Theoretical foundation and experimental proof of the accumulating transfer of tritium from water into DNA and other biomolecules in vitro and in vivo

Heisenberg's uncertainty principle, which describes a fundamental property of matter, predicts the light hydrogen isotope to prefer the strong hydrogen bridge positions (with large local uncertainty). In contrast, the heavy hydrogen isotope tritium originally localized in water, should finally be found in the exchangeable hydrogen bridge positions of proteins, carbohydrates and nucleotides which are definitely less strong than those of water. The fractionation factor of tritium and protium measured between water and DNA as well as the mixture of biomolecules of liver confirm this conclusion and show in addition, a tritum accumulation also in the hydration sheets of the biomolecules. Furthermore, the larger intrinsic rate of natural increase of tritium than of hydrogen in tissue solids of growing maize and barley confirm the accumulating tritium transfer from water to biomolecules taking place in vivo.     

Relative biological effectiveness of tritium oxide based on DNA destruction and thymus depletion in rats

As estimated by DNA destruction and thymus depletion for 21 days of observation, relative biological effectiveness (RBE) of tritium oxide (RBE HTO), as compared to gamma-quanta (137Cs), was 3.1 +/- 0.2 and 3.1 +/- 0.3, respectively. On days 1, 3, 7, 10, 14, and 21 RBE HTO, for the same indices at minimum radiation doses, was about 6-2. As determined by T1/2 period of double-stranded DNA diminution and thymus cell depletion RBE HTO was 5.5 +/- 0.4.     

Comparative evaluation of the carcinogenic effects of chronic exposure to tritium oxide and external gamma-radiation

In experiments of on rats a study was made of late effects of chronic action of tritium oxide (3HOH) during 6 months (37 X 10(4) Bq/g/day) and external gamma-radiation (137Cs) which were delivered in comparable daily and cumulative doses. It was shown that 3HOH produced a more pronounced blastomogenic effect. The RBE coefficient of tritium oxide approximated 1, with a reference to the average life shortening, and 4.2 and 2.5, with a reference to the incidence of malignant tumors and leukoses, respectively.     

The frequency of chromosome aberrations in rat bone marrow cells in the late period after a single uptake of tritium

A prolonged self-maintenance of haemopoietic tissue cells with stable chromosome rearrangements following a single intake of tritium oxide in the amount of 24 MBq/g of body weight (absorbed dose of 11 Gy) is shown. Mutant cells revealed long after the radionuclide exposure are descendants of stem-cell precursors, bearing stable chromosome aberrations during the period of formation of radiation injury after the radionuclide administration.     

Prostaglandin H synthase catalyzes regiospecific release of tritium from labeled estradiol

Prostaglandin H synthase (PHS) from ram seminal vesicle microsomes was found to catalyze the release of tritium (3H) from estradiol (E2) regiospecifically labeled in position C-2 or C-4 of ring A but not from positions C-17 alpha, C-16 alpha, or C-6,7. Formation of 3H2O from ring A of E2 is dependent upon native enzyme supplemented with either arachidonic acid, eicosapentaenoic acid, or hydrogen peroxide and proceeds very rapidly as do other cooxidation reactions catalyzed by PHS-peroxidase. The 3H-loss from ring A of E2 reflecting oxidative displacement of this isotope by PHS increases linearly up to 100 microM under our conditions (8-45 nmol/mg x 5 min). Loss of tritium in various blanks is negligible by comparison. Indomethacin (0.07 and 0.2 mM) inhibited the PHS-dependent release of 3H2O from estradiol but less efficiently than it inhibited DES-cooxidation measured in parallel incubations under similar conditions. Addition of EDTA (0.5 mM) had no effect on the regiospecific transfer of 3H from E2 or on DES-oxidation; ascorbic acid (0.5 mM) or NADH (0.33 mM) clearly inhibited both reactions and to a similar extent. These data suggest that estradiol-2/4-hydroxylation can be catalyzed by PHS in vitro probably via its peroxidase activity and point to PHS as an enzyme that could contribute to catechol estrogen formation in vitro by tissue preparations in the presence of unsaturated fatty acids or peroxides.     

The immunity indices of rats after the long-term action of tritium oxide or gamma irradiation

In experiments with Wistar rats, a study was made of the content of antibody-forming cells and cytotoxic activity of normal killers after long-term administration of tritium oxide (3HOH) (370 kBq.g-1 of body mass daily, cumulative dose, 8.1 Gy, and dose rate, 8.5 cGy/day), and after gamma irradiation with corresponding doses. The long-term radiation effect caused a decrease in the immunity indices: the impairment of the immune reactions was more pronounced after the effect of 3HOH than after gamma irradiation. Damages to the immune system of mice and rats after irradiation with similar doses were compared.     

Occurrence of mammary tumors in rats after exposure to tritium beta rays and 200-kVp X rays

The RBE for tritium was estimated in reference to 200-kVp X rays, using acceleration of breast tumor appearance in the female Sprague-Dawley rat as the end-point. Chronic X-ray doses of 0.3-2.0 Gy were delivered over 10 days. Intraperitoneal injections of tritiated water ranging in concentrations from 45 to 370 MBq/100 g body wt were administered, followed by four additional injections at 2-day intervals and half of the initial concentrations. Seventy-five percent of the total tritium dose was delivered to the mammary gland within the first 10 days and 95% within the first 20 days after the start of the tritium exposure. RBE estimations were based on various criteria including the tumor incidence per Gy at 450 days postirradiation and the time required to induce tumors in 50% of the animals at risk. The results suggest that tritium beta rays are about 1.1-1.3 times more effective than chronic 200-kVp X rays for acceleration of the appearance of rat mammary tumors. However, the uncertainties involved in these calculations are such that the effects of tritium beta rays could not be reliably distinguished from those of chronic 200-kVp X rays. Measured differences in RBE values were slightly larger for the comparison between acute and chronic X rays than for the comparison between chronic tritium beta rays and chronic X rays.     

The consequences of the isotope effect on proline dehydrogenation rates estimated by the tritium loss method.

Loss of tritium from a substrate is often used to estimate the rate of dehydrogenation. However, loss of 3H may be much slower than loss of H because of the tritium isotope effect. In order to assess the impact of the tritium isotope effect, loss of 3H from the C-5 position of proline during dehydrogenation by rat liver mitochondria and bacteroids from soybean (Glycine max [L.] Merrill) nodules was compared with appearance of 14C in products of [14C]proline dehydrogenation. Incubations were carried out in the presence of o-aminobenzaldehyde (added to trap the initial product, delta 1-pyroline-5-carboxylate). The fraction of total 14C products trapped by o-aminobenzaldehyde varied from 0.07 to 0.75 depending upon experimental conditions. With rat liver mitochondria, dehydrogenation of [14C]proline was between 3.27 and 9.25 times faster than dehydrogenation of 3H proline, depending upon assay conditions. Soybean nodule bacteriods dehydrogenated [14C]proline about 5 times faster than [3H]proline. We conclude the following: (i) the rate of proline dehydrogenation may be greatly underestimated by the tritium assay because of the tritium isotope effect, and (ii) the 14C assay may underestimate the rate of proline dehydrogenation if it is assumed that o-aminobenzaldehyde quantitatively traps delta 1-pyrroline-5-carboxylate under all conditions. The simplicity of the tritium assay makes it attractive for routine use. However, its use requires determination of the tritium isotope effect, under the specific conditions of the assay, in order to correct the results. The considerations discussed here have broad applicability to any dehydrogenase assay employing tritium loss.     

The solid-state catalytic synthesis of tritium labeled amino acids,peptides and proteins

Summary New catalytic reaction between a solid bioorganic compound and activated spillover tritium (ST), based on High-temperature Solid-state Catalytic Isotopic Exchange (HSCIE) was examined. The HSCIE mechanism and determination of the reactivity of hydrogen atoms in amino acids, peptides and proteins was investigated. Quantum mechanical calculations of the reactivity of hydrogen atoms in amino acids in the HSCIE reaction were done. The carbon atom with a greater proton affinity undergoes a greater exchange of hydrogen for tritium in HSCIE. The electrofilic nature of spillover hydrogen in the reaction of HSCIE was revealed. The isotope exchange between ST and the hydrogen of the solid organic compound proceeds with a high degree of configuration retention at the carbon atoms. The HSCIE reaction enables to synthesize tritium labeled proteins with a specific activity of 20–30 mCi/mg and kept biological activity.Presented at the 3rd International Congress on Amino Acids, Peptides and Analogues. Vienna, August, 23–27, 1993     

In vivo covalent binding of retronecine-labelled [3H]seneciphylline and [3H]senecionine to DNA of rat liver, lung and kidney

Retronecine-labelled [3H]seneciphylline ([3H]SPH) and [3H]senecionine ([3H]SON) of high specific radioactivity (22 and 49 mCi/mmol, respectively) were prepared biosynthetically with seedlings of Senecio vulgaris L. using [2,3-3H]putrescine as precursor. [2,3-3H]Putrescine was synthesized by Gabriel synthesis of 1,4-diamino-2-butene from 1,4-dibromo-2-butene and catalytic hydrogenation of the product with tritium gas. Rats of both sexes were treated with the labelled pyrrolizidine alkaloids (PAs) (75-215 microCi SPH or 40-485 microCi SON/kg body wt.) and killed after 6 h or 4-5 days. SON-treated females excreted 83.4 +/- 0.2% of applied radioactivity in faeces and urine within 4 days whereas equally treated males excreted 90.9 +/- 3.2% in the same time. Excretion of 3H-activity from SPH-treated females was completed within 5 days (104.7 +/- 6.4%). Corresponding with these results, tissue levels were highest in SON-treated females. DNA and proteins were isolated from liver, lungs and kidneys and covalent binding of the alkaloids to DNA was determined. A Covalent Binding Index (CBI, mumol alkaloid bound per mol nucleotides/mmol alkaloid administered per kg body wt.) of 210 +/- 12 was found for the liver from SON-treated females whereas binding to liver DNA of males was lower by a factor of 4. The DNA damage determined six hours after treatment persisted during the following 4 days. Administration of [3H]SPH to female and male rats resulted in a CBI of 69 +/- 7 and 73/92, respectively, for the liver DNA. Furthermore we found binding of both alkaloids to DNA of lungs and kidneys in male and female rats. The in vivo formation of [3H]SON derived DNA adducts could be proved by HPLC analysis of hydrolyzed DNA.     

Alpha-secondary tritium kinetic isotope effects indicate hydrogen tunneling and coupled motion occur in the oxidation of L-malate by NAD-malic enzyme

The NAD-malic enzyme from Ascaris suum catalyzes the divalent metal ion-dependent oxidative decarboxylation of L-malate to give pyruvate and CO2, with NAD+ as the oxidant. Alpha-secondary tritium kinetic isotope effects were measured with NAD+ or APAD+ and L-malate-2-H(D) and several different divalent metal ions. The alpha-secondary tritium kinetic isotope effects are slightly higher than 1 with NAD+ and L-malate as substrates, much larger than the expected inverse isotope effect for a hybridization change from sp2 to sp3. The alpha-secondary tritium kinetic isotope effects are reduced to values near 1 with L-malate-2-D as the substrate, regardless of the metal ion that is used. Data suggest the presence of quantum mechanical tunneling and coupled motion in the malic enzyme reaction when NAD+ and malate are used as substrates. Isotope effects were also measured using the D/T method with NAD+ and Mn2+ as the substrate pair. A Swain-Schaad exponent of 2.2 (less than the value of 3.26 expected for strictly semiclassical behavior) is estimated, suggesting the presence of other slow steps along the reaction pathway. With APAD+ and Mn2+ as the substrate pair, inverse alpha-secondary tritium kinetic isotope effects are observed, and a Swain-Schaad exponent of 3.3 is estimated, consistent with rate-limiting hydride transfer and no quantum mechanical tunneling or coupled motion. Data are discussed in terms of the malic enzyme mechanism and the theory developed by Huskey for D/T isotope effects as an indicator of tunneling [Huskey, W. P. (1991) J. Phys. Org. Chem. 4, 361-366].