Renato Baserga

Thorotrast-induced cancer in man

The case history is presented of a malignant tumor in man attributed to the injection of Thorotrast. 55 references. (C.H.)

TWO-EMULSION RADIOAUTOGRAPHY

Two-emulsion radioautography can distinguish between beta particles emitted from C14 atoms and those originating in H3 atoms. It uses two layers of sensitized emulsion separated by an inert layer of celloidin: the C14 beta particles, because of their longer range, are recorded in the second emulsion, but the beta particles from tritium are arrested by the first emulsion. Experiments have shown that the combination of two NTB emulsions separated by a celloidin layer is the most satisfactory. The total thickness of the radioautograph is 19 µ. The first emulsion plus the celloidin layer measure together 12 µ in thickness and decrease by 38% the flux of C14 beta particles reaching the second emulsion. Staining of the specimen with Mayers hematoxylin and eosin and celloidin coating follow the exposure and processing of the first emulsion. The second emulsion is applied after the celloidin coating and processed after a suitable second exposure. By using a tritiated precursor of DNA and a C14-labeled precursor of RNA or proteins, or viceversa, two-emulsion radioautography can be applied to investigate two distinct metabolic processes occurring at the same time in the same cell.

Factors which Affect Efficiency of Autoradiography with Tritiated Thymidine

The overall efficiency of autoradiography with tritium-labeled thymidine has been found to be influenced by the following conditions: (1) exposure in an atmosphere of CO2 and the use of the stripping-film technique, both of which increase the autoradiographic efficiency when compared to exposure in air or to dip-coating technique; (2) latent image fading, which increases with increasing exposure. Up to 2 wk of exposure, however, this disadvantage is counterbalanced by the fading of the mechanical background produced during stripping or dip-coating; (3) the thickness of the inert coating interposed between the labeled locus and the sensitized emulsion. A layer of inert coating can be obtained that will arrest all beta particles from tritium, while having no effect on more energetic emitters like C14; (4) the amount of tritiated thymidine given, with relatively large amounts producing an increase in the mean grain count per labeled cell but not in the percentage of cells identifiable as labeled; and (5) the...

Uptake of radioactive thymidine and cytidine by Ehrlich ascites tumor cells in different stages of growth

SummaryIn Strong A female mice, the Ehrlich ascites tumor inoculated into the peritoneal cavity grows exponentially for the first 7 days with a doubling time of about 36 hours. The tumor enters then into a late stage during which the number of tumor cells in the peritoneal cavity does not increase. The uptake of intraperitoneally injected thymidine decreases from the exponential to the late stage, mostly because of a decrease in the fraction of cells in DNA synthesis. During the exponential phase, the uptake of thymidine is a function of the amount of radioactive thymidine injected per tumor cell, the utilization decreasing with increasing cell dose. The uptake of intraperitoneally injected cytidine decreases slightly with time after inoculation although the fraction of tumor cells in RNA synthesis remains constant.

Further Observations on Induction of Tumors in Mice with Radioactive Thymidine

Summary Thymidine labeled with tritium or with carbon-14, when incorporated into DNA of proliferating cells, produces a selective mode of irradiation of cells and cell nuclei. Single and multiple injections of radioactive thymidine have been found to shorten the life span and to produce an increased incidence of tumors in CaFl mice. The incidence of tumors increased with dose and it was greater in animals injected as young adults than in animals injected later in life. Labeling of embryos in utero with tritiated thymidine likewise increased the incidence of malignant tumors.

A radioatographic study of the uptake of [14C]leucine by tumor cells in deoxyribonucleic acid synthesis

Abstract Ehrlich ascites tumor cells from mice injected simultaneously with [3H]thymidine and l -[ i -14C]leucine were autoradiographed with a two-emulsion technique, which allows the study of two distinct metabolic processes occurring at the same time in the same cell. The results show that the uptake of [14C]leucine is highest in Ehrlich ascites tumor cells in DNA synthesis. As DNA synthesis ceases, the uptake of leucine decreases considerably, until it reaches a minimum during actual division.

RNA synthesis in liver and heart of growing and adult mice

Abstract Cells of liver and heart of 12-day-old mice actively synthesize DNA, whereas in 5-month-old mice, the rate of DNA synthesis in these two organs is reduced to a negligible level. Livers and hearts of 12-day- and 5-month-old mice were therefore compared in order to determine differences in some aspects of RNA synthesis. RNA synthesis was studied by determining nucleic acid concentration, incorporation in vivo of radioactive cytidine into whole cells and into subcellular fractions, the size of the endogenous cytidine pool, RNA turnover over a period of 30 days, the effect of actinomycin D and sucrose gradient analysis of phenol-extracted RNA. The rate of synthesis of rapidly-labeled RNA was higher in 12-day-old than in 5-month-old tissues (50 % higher in liver and 2.4 times higher in heart). The specific activity of the cytidine precursor pool was doubled in the adult tissues. In addition, the turnover of RNA was decreased in the adult heart, and the concentration of RNA per cell in the adult liver was almost twice the amount found in growing liver. Other parameters of RNA synthesis were substantially similar in growing and adult livers an hearts.

The use of standard slides in semiquantitative radioautography with tritiated compounds.

Essentially identical smears can be prepared from a pooled suspension of Ehrlich ascites tumor cells labeled with tritiated thymidine. In these smears, the percentage of labeled cells and the mean grain count per labeled cell vary within narrow limits: in the same slide, within 2 to 5% of the mean of the slide. In smears processed together in the same manner, the percentage of labeled cells (calculated on 1,000 cells) varies within 2%, and the mean grain count per labeled cell (calculated on 50 cells) between 15 and 20% of the mean of all smears. Once the limits of variability are known, smears from the same cell population can be used to ensure the accuracy of procedures performed on separate batches of radio-autographs that have been processed separately but in a presumably identical manner. A comparison of the mean grain counts of standard smears from separate batches will indicate if variations, not otherwise detectable by the experimenter, have actually occurred during the radioautographic procedure.

THE EFFICIENCY OF AUTORADIOGRAPHIC STRIPPING FILM APPLIED TO TISSUE SECTIONS CONTAINING TRITIATED THYMIDINE

Tritium-labeled thymidine has been extensively used in recent years to investigate the kinetics of cellular proliferation in a variety of normal and pathological tissues. It is known that thymidine is incorporated into the nuclei of cells synthesizing DNA prior to mitosis [1] but little quantitative information is available on its uptake by single cells. Also dependent upon quantitative data is the problem of radiation dosimetry, from intranuclear tritium, a problem with more than theoretical significance since it has been shown that tritiated thymidine can produce radiation damage and death in some of the labeled cells [2, 3]. For these purposes, measurements of total tritium activity in tissue samples [4] are inadequate, because of the nonuniform distribution of thymidine in living tissue and the short range of the tritium beta particle. Under these conditions, 90% of the energy of the disintegrations is dissipated within the nucleus [5] and the customary calculation of radiation dose in terms of energy absorbed per unit mass of tissue is inadequate. In order to correlate dose with biological effect the distribution of dose on a microscopic scale must be considered.