Analysis of 5-iodo-2'-deoxyuridine incorporation in murine melanoma for photon activation therapy.

Quantitative evaluation of the dose enhancement obtained with analog nucleoside agents such as iododeoxyuridine (IdUrd) requires knowledge of the degree to which the thymidine (Thd) in DNA is replaced by IdUrd. In the present investigation, mice were infused with IdUrd using an intravenous infusion apparatus capable of delivering continuous multi-day infusions without restraining the mice. The absolute incorporation of IdUrd in DNA was measured by 125IdUrd label, both in whole tissue and extracted DNA, showing a good correlation between levels observed in DNA and whole tissue. Replacement in a Harding-Passey murine melanoma tumor carried in BALB/c mice approached 10%. In addition, a Neutron Activation Analysis (NAA) technique was developed which showed in vitro, a sensitivity sufficient to evaluate the % replacement of Thd by IdUrd in small biological samples with a sensitivity greater than 0.1 ppm, at 1% replacement in mg samples. This method can provide information on iodine substitution in DNA in humans where the use of a radioactive DNA-seeking substance would be undesirable. Analyses of IdUrd incorporation in cultured cells by NAA and 125I counting showed good agreement.

Residual toxicity in hematopoietic cells following a single dose of methylnitrosourea.

The residual injury to the proliferation capability of hemopoietic stem cells (CFU-S) which results from their exposure to leukemogenic agents was evaluated in mice given a single leukemogenic dose of methyl nitrosourea (MNU 50 mg/kg body weight, i.v.). Bone marrow cellularity, splenic weight, number of CFU-S and the proportion of cycling to noncycling CFU-S were measured in an effort to detect and acute and residual injury to the CFU-S from mice given MNU 21 and 3 days earlier. Marrow cells were also transferred into lethally irradiated mice to observe the self-renewal capability of the CFU-S in the recipient spleen and bone marrow. The results of these measurements show that the CFU-S in marrow from mice given 50 mg/kg of MNU 21 days earlier still have a defective ability for self-renewal, although the total cellularity, number of CFU-S and proportion of cycling and noncycling CFU-S in the donor have returned to the normal range. The relationship of this self-renewal defect to the development of leukemia after this leukemogenic dose of MNU is not known.

The distribution of tritium among the amino acids of proteins obtained from mice exposed to tritiated water.

The distribution of tritium among the amino acids of serum proteins in mice chronically exposed to tritiated water was determined by ion exchange chromatography of the protein hydrolysate. The specific activity of nonexchangeable tritium in these amino acids relative to the specific activity of tritium in the tissue water of mice ranged from 0.04 for phenylalanine and threonine to 1.0 for glycine and alanine. Since tritium from tissue water can enter the nonexchangeable positions of amino acids only as the result of metabolic processing, the relative specific activity of tritium in each amino acid is an indicator of the extent of such processing. The tritium content of tyrosine and all the amino acids required in the diet for survival is quite low, except for histidine, and can be entirely accounted for by transamination or, in the case of methionine, by transmethylation. The tritium content of the other amino acids is too high to result from such minor processing and must reflect primarily the fraction synthesized de novo. The implications of these findings with respect to the radiobiological consequences of a diet containing tritiated proteins are discussed.

The oxygen enhancement ratio for single- and double-strand breaks induced by tritium incorporated in DNA of cultured human T1 cells. Impact of the transmutation effect.

The effect of oxygen, expressed as the oxygen enhancement ratio (OER), on the number of single-strand breaks (SSB) and double-strand breaks (DSB) induced in DNA by the radioactive decay of tritium was measured in human T1 cells whose DNA had been labeled with tritium at carbon atom number 6 of thymidine. Decays were accumulated in vivo under aerobic conditions at 0-1 degrees C and at -196 degrees C and in a nitrogen atmosphere at 0-1 degrees C. The number of SSB and DSB produced was analyzed by sucrose gradient centrifugation. For each tritium decay there were 0.25 DSB in cells exposed to air at 0-1 degrees C and 0.07 in cells kept under nitrogen, indicating an OER of 3.6, a value expected for such low-LET radiation. However, for each tritium decay there were 1.25 SSB in cells exposed to air at 0-1 degrees C and 0.76 in cells kept under nitrogen indicating an OER of only 1.7. The corresponding values for 60Co gamma radiation, expressed as SSB per 100 eV absorbed energy, were 4.5 and 1.0, giving an OER of 4.5. The low OER value found for SSB induced by tritium decay can be explained if 31% of the total SSB produced in air result from transmutation by a mechanism which does not produce DSB and is unaffected by oxygen.

Histone turnover within nonproliferating cells.

The turnover of DNA and histones in the livers and brains of mice has been determined. These mice had been exposed to constant levels of tritiated water from conception until they were 8 months old. At this point, exposure to tritium was discontinued, and the tritium remaining in DNA and histones was measured at various intervals afterward. The half-lives calculated for these components (with 95% confidence limits given in parentheses) were 117 (85-188) days for liver histone, 318 (241-466) days for liver DNA, 159 (129-208) days for brain histone and 593 (376-1406) days for brain DNA. The difference between histone and DNA turnover is statistically significant for both tissues and indicates that histone turnover within tissues cannot be solely accounted for by cell turnover within the tissue but also must include histone turnover within living cells. The half-life of histone within cells is estimated to be 117 (88-178) days in liver and 223 (187-277) days in brain.

Radiotoxicity of intranuclear 125I atoms not bound to DNA.

The radiotoxicity of 125I covalently bound to DNA is unusually high. This has been attributed both to the Auger electrons which result from the electron capture process accompanying 125I decay and to local transmutation effects which cause extensive damage to nearby structures. We introduced 125I into cell nuclei in the form of iodoantipyrine, a molecule which diffuses freely through cells, and we have compared the survival of these cells to those exposed to radiation from extracellular 125I-labelled albumin or 55Fe-labelled transferrin. We found a value for D0 of 34 rad for 125I decays occurring within the cell nucleus compared to 362 rad for extracellular 125I and 277 rad for extracellular 55Fe. Since transmutation effects are very short range and 125I was distributed uniformly throughout the nucleus rather than bound to DNA, most of the radiotoxicity of intranuclear 125I-labelled iodoantipyrine must be due to Auger electrons.

The genetic and late somatic effects of chronic tritium ingestion in mice.

The long term somatic and genetic effects of chronic tritium (3H) ingestion have been measured in mice. Second generation animals, who with their parents were maintained on drinking water containing 3 muCi/ml of HTO, have been bred and the number of dominant lethal mutations measured. The increase in dominant lethal mutations in treated animals as compared to tap water controls is significant at the 1% level. The stem cell content of the bone marrow has been measured throughout the lifetime of the animals. A slight reduction in colony forming units in the bone marrow of treated animals is evident beginning after approximately 12 weeks, increasing in severity throughout the lifetime of the animals. The distribution of tritium in tissue water protein fractions was determined, and the relative contribution to tissue dose calculated. The differential incorporation into protein fractions was not found to be significant.

Antiviral activity and side effects of polyriboinosinic-cytidylic acid complexes as affected by molecular size.

The decrease of the molecular size of poly(I.C) to less than 10(6) decreases its ability to induce interferon, protect mice against virus, or enhance the immune response. Immune adjuvant activity appeared more sensitive to molecular weight than the other protective activities. The composition of the complex-the molecular size of the individual homopolymers when one was large and the other small-did not affect antiviral activity; the activity of a complex made from large poly(I) and small poly(C) was similar to one made from small poly(I) and large poly(C). Molecular size of the complex did not profoundly alter the side effects of poly(I.C). At 2 mg/kg, none of the complexes markedly altered phagocytic function. Only the largest complex sensitized the mouse to endotoxin. However, all of the complexes studied profoundly inhibited drug metabolism by the liver microsomal enzymes between 24 and 72 hr after their inoculation. Decreasing the molecular weight did not alter this inhibition.