Reutilization of 125I-UdR during growth of a solid mammary carcinoma: implications for the 125I-UdR loss technique.

Reutilization of thymidine (TdR) and 5-iodo-2'-deoxyuridine (I-UdR) released by dying tumour cells was assayed in the syngeneic adenocarcinoma EO 771 by injecting heat killed, labelled tumour cells into tumours. 3H and 125I liberation from labelled breakdown products was measured in tumours of various sizes without or with separation of tumours into viable and necrotic portions. Internal reutilization of 3H-TdR was considerably greater than that of 125I-UdR. 125I-UdR released by dying tumour cells was reutilized at about 10%. There was no significant increase in 125I-UdR reutilization during tumour growth. It is concluded that measurements of radioactivity loss by the 125I-UdR technique can result in underestimating the real cell loss depending on the amount of internal reutilization by the tumours investigated. Compared with 3H-TdR 125I-UdR is the tracer of choice for long term studies of cell loss.

Cell loss from viable and necrotic tumor regions after local gamma irradiation measured by 125I-UdR.

Using a tracer technique, loss of cells from perivascular and average tumor cells of the syngeneic mammary adenocarcinoma EO 771 in male C57Bl/6J mice may be measured in the living animal, by the use of 125-labelled 5-iodo-2'-deoxyuridine (125I-UdR). It was the purpose of this paper to compare measurements in vivo with those made in vitro following local 60Co-gamma irradiation in the absorbed dose range from 10 to 27.5 Gy, incorporation of radioactivity into DNA of tumor cells and activity loss from labelled tumor cells were measured externally by a special scintillation counter device. In addition, by injecting the vital dye "light green" into the mice the I-125-activity of the stained viable and unstained necrotic regions were separately measured for loss of activity following gamma irradiation. A comparison was made between radiation induced growth delay and the depression of 125I-UdR incorporation into DNA of the proliferating tumor cells. After local tumor irradiation with a dose of 27.5 Gy 60Co gamma rays an enhancement of the activity loss by 0.5% per hour was externally observed for the perivascular tumor cell population. A lower enhancement of 0.4% per hour was externally registered in the average tumor cell population. Both values were evaluated relative to sham-irradiated control tumors. The measurements on isolated tumors were in comparatively good agreement with the external values. The activity loss rate from the viable, euoxic tissue increased by 0.4% per hour after 27.5 Gy 60Co gamma rays and by 0.3% per hour in the average cell population, the latter representing a mixture of euoxic and hypoxic cells. The results demonstrate, that the external measurements are a good indicator for radiation effects under in vivo-conditions.

Analytical techniques for boron and boron 10 analysis in a solid experimental tumor EO. 771.

If a tumor can be preferentially loaded with a suitable boron-10 compound and irradiated with thermal neutrons, malignant cells can be selectively destroyed via the alpha-particle + Li 7-nucleus from the reaction 10B(n, alpha)7Li. Neutron capture therapy with two boron-10 amino acid analogs of low toxicity has been tested in recent years: (a) trimethylamine-carboxyborane, (A3) and (b) amine-carboxyborane, (A7). Now the boron-10 glycineamide analog (A8), amineboryl-carboxamide has been synthesized; it contains 13.81% boron (90% Boron 10 + 10% Boron 11) and shows a very low toxicity in mice. The effects of this compound were tested on the syngeneic solid adenocarcinoma EO 771 on the right hind leg of male C57 BL/6J mice under standard conditions, by measuring tumor volume growth delay and cell cycle changes using flow cytometry. Boron distribution between tumor and muscle was analyzed by emission spectroscopy with inductively coupled plasma (ICP) following injection of a suspension of peanut oil emulsion. In addition, boron-10 concentration in the tumor were analyzed with prompt gamma-activation analysis and neutron capture radiography (Kodak-Pathé LR 115) at the MRR reactor in Brookhaven after i.p. injection of 0.4 mg/g A8. Application of A8 alone (0.4 mg/g i.p.) or thermal neutron irradiation of the tumor EO. 771 produced a tumor growth delay of 1-2 days for tumor volume doubling. Application of the boron 10 glycine-amide analog A8 i.p. plus 5 X 10(12)n/cm2 resulted in a growth delay of 3-6 days. In contrast intratumoral application of A8 plus 4 X 10(12)n/cm2 neutrons gave a growth delay of 7-14 days; the fraction of (G2 + M) cells rose from 35% (neutrons alone) to 52%, as evaluated from flow cytometry.

Non-invasive investigations of the growth kinetics of a solid experimental tumor (sarcoma-180).

The investigations reported were performed to test applicability of non-invasive methods for the measurement of the parameters determining the growth of a solid experimental tumor and to measure these data for the tumor system sarcoma-180/NMRI-mice. It could be shown that non-invasive methods can be used for the measurement of tumor growth, especially the fraction of proliferating cells (growth fraction) which is of special importance for tumor therapy. For the tumor system under investigation, the growth is completely determined by an exponential decrease of the growth fraction.

Evaluation of radio- and chemotoxic effects of 125I-UdR on tumour growth and host survival.

The toxicity of 5-iodo-2'-deoxyuridine (I-UdR) was assayed in male C57 BL/6J mice bearing the syngeneic mammary adenocarcinoma EO 771 by injecting different doses of 'cold' I-UdR or 125-iodine labelled I-UdR. Host survival, tumour growth, DNA-precursor incorporation, whole-body retention and tumour activity loss rates were chosen as biological end points. There was no measurable effect on host survival up to doses of 5 micrograms I-UdR or 50 microCi 125I-UdR per mouse during a mean life-span of 25 days. Adjusted to a constant amount of 0.55 micrograms I-UdR/mouse, radiotoxicity of 125I-UdR on tumour growth (up to 17 days after implantation), tracer incorporation, whole-body and tumour retention (up to 12 days after 125I-UdR injection) could be excluded up to a dosage of 50 microCi 125I-UdR/mouse. It is concluded that in situ evaluation of tumour activity loss rates in carcinoma EO 771 is not disturbed by toxic effects of I-UdR or 125I-UdR within the dose limits mentioned.

Acute and temporary inhibition of thymidine kinase in mouse bone marrow cells after low-dose exposure.

Low-dose whole-body exposure of mice to less than 0.01 Gy gamma-rays causes inhibition of incorporation of thymidine or 5-iodo-2-deoxyuridine into DNA of bone-marrow cells in vitro; the effect is maximal in cells at 4 hours after exposure and then subsides within about 10 hours. This is due to the inhibition of cellular thymidine kinase, which gradually develops to a maximum at 4 hours after exposure and again subsides within the next 10 hours. This inhibition involves only 35 per cent of the entire cellular enzyme activity and, analogous to the depression of thymidine incorporation into the cells, is only seen when the cells are collected into medium that is buffered to 7.2-7.4 and contains about 1350 mg NaHCO3 per litre. Addition of NaHCO3 to the cell homogenate or to the high speed supernatant containing the enzyme, but not to intact cells, failed to produce enzyme inhibition. There is also no depression of 5-iodo-2-deoxyuridine uptake into the intact cells in vitro when the mice are irradiated either shortly before or after i.v. injection of 0.02 mg of procain chloride. The reversibility of the effect in vivo and in vitro suggests a particular enzyme control mechanism that may be non-specifically triggered by intracellular charges, such as peroxides, and may enhance repair.

Cell loss from viable and necrotic tumour regions measured by 125I-UdR.

Loss of cells from vital and necrotic areas of the syngeneic mammary adenocarcinoma EO 771 in male C57 BL/6J mice may be measured by use of 125I-labelled 5-iodo-2'-deoxyuridine (125I-UdR). Later than 50 hr after an intraperitoneal injection of 20 muCi 125I-UdR the incorporated activity of the entire tumour was externally measured and found to decrease with time after injection. The injected amount was neither chemo- nor radiotoxic. By injecting the vital dye 'light green', unstained necrotic and stained viable regions were separately excised and measured for loss of activity throughout the natural development of the labelled tumour. With the appearance of necrotic regions, labelled viable cells became necrotic, and activity was slowly eliminated. With increasing proportions of necrosis during tumour growth, the rate of loss of activity of the whole tumour decreased. Loss of activity from viable tumour regions reflected cell death and exceeded the loss rates of the whole tumour by a factor of 2 to 3. The data show that loss of activity from the whole tumour results from a superposition of different elimination rates of viable and necrotic tumour regions and is not an immediate consequence of cell death in the course of undisturbed tumour development.

Effect of blood serum from irradiated mice on the incorporation of DNA, RNA and protein precursor in L929 cells. A sensitive assay for measuring low dose response.

Serum from whole-body irradiated mice inhibits incorporation of DNA precursors into DNA of L929 cells in culture in a dose-dependent way. The humoral factor interfering with the incorporation of 3H-thymidine and 125I-iododeoxyuridine is identical to thymidine. The degree of depression of 125I-iododeoxyuridine-uptake is more sensitive than that of 3H-thymidine. Irradiation of donor mice does not confer a toxic effect of blood serum on cell growth in culture. Incorporation of 3H-leuchine into protein and 3H-cytidine into DNA and RNA is not affected by the serum of irradiated mice; there is no effect on the incorporation of 3H-cytidine from the intracellular precursor pool into DNA or RNA either. The present findings demonstrate the specificity and high sensitivity of the assay system for measuring thymidine concentration in mouse blood serum and point to possible applications of analysing abnormalities in DNA metabolism resulting in, or from, disturbances of the thymidine reutilization pathway.

Acute non-stochastic effect of very low dose whole-body exposure, a thymidine equivalent serum factor.

Whole-body irradiation of mice causes the dose-dependent appearance of a humoral factor in blood serum which inhibits incorporation of 125-IUdR into tissue culture cells. This factor appears even at doses below 0.01 Gy gamma irradiation and thus is probably not related to cell death. Data are presented relating this humoral factor to thymidine. Since at low doses the target size for this effect was calculated to be the entire cell, a cellular effect is postulated linking the site of few primary absorption events, anywhere in the cell, with the cellular membrane, thus causing changes in membrane charge, structure and/or fluidity. This may lead to blocking thymidine acceptance by the cell, and thus would cause a pile-up of thymidine in the reutilization pathway in peripheral blood and would give rise to the observed effect. The effect appears as a temporary disturbance of the physiological equilibrium and should not be related at present to any cellular damage. The acute low-dose effect described has implications for the measurement of low-dose exposure by biological dosimeters and on basic research on membrane function.

Selective delivery of liposome-associated cis-dichlorodiammineplatinum(II) by heat and its influence on tumor drug uptake and growth.

In an attempt to optimize the chemotherapeutic treatment of mouse tumor Sarcoma 180, liposomes containing cis-dichlorodiammineplatinum(II) (PDD), having transition temperatures of few degrees higher than the rectal temperature of mice, were used in combination with local hyperthermia. The uptake of radioactive PDD by tumors heated for 1 hr at 42 degrees was almost four-fold greater when the drug was associated in liposomes than if administered as free drug. Uptake of liposome-administered radioactive platinum by liver was twice that obtained with free PDD, whereas its incorporation by the kidney was the same by either method of drug administration. The effect of various combinations of hyperthermia, drug-containing liposomes, and free PDD on tumor growth was also studied. Treatment with liposome-associated PDD plus local heating resulted in a dose-modifying factor of 7 when compared with free drug and no hyperthermia. The dose-modifying factor was 2.5 when PDD liposomes and heat were compared within free drug and heat. Thus, PDD could be specifically released from liposomes by heat and resulted in both a greater drug uptake and a delayed tumor growth following treatment. Potential normal tissue toxicity problems, however, still need to be resolved before clinical application of this combined modality will be possible.

Low dose effect of ionizing radiation on incorporation of iododeoxyuridine into bone marrow cells.

The reduction of the incorporation of IUdR in bone marrow cells depends on the time after irradiation and on te microenvironment of the cells. The strongest effect is observed 4 hours after irradiation. For absorbed doses above 40 rad, whole-body irradiated mice were more sensitive with respect to depression of IUdR incorporation in bone marrow cells, when the bone marrow cells were labelled in vivo, and less sensitive for in vitro labelling. The converse was observed for very small doses of 1 rad and below. Such small doses resulted in a significant depression of IUdR incorporation after in vivo irradiation and in vitro labelling and showed no effect at all after in vivo irradiation and in vivo labelling. The least effect of radiation was observed after both irradiation and labelling in vitro. Although the mechanisms are not fully understood, the biological results and microdosimetric considerations indicate that at the smallest doses the effect is due to functional changes of cellular organelles which control intracellular mechanisms. A working hypothesis is proposed for the reduction of IUdR incorporation at low doses as being due to functional changes of the cellular membranes.

Measurements of in vivo effects of an electron-affinic radiosensitizer using a double tracer technique.

In sarcoma-180 of NMRI mice, euoxic tumour cells were labeled with 131I-iododeoxyuridine, and the average tumour cells were labeled with 125I-iododeoxyuridine. An external counting technique permitted in vivo measurements of radiation effects on rates of loss of incorporated activity from the different labeled tumour areas. The technique was employed to study the effect of tumour exposure to gamma irradiation with and without chemical radiosensitization by misonidazole (La Roche 07-0582). Injecting 500 mg/kg of Ro-07-0582 15 minutes before irradiation reduced the measurable oxygen effect by a factor of 1.54. The dose modifying factor evaluated from the 50% tumour regression at 100 days was 1.57.

[In-vivo-examinations of the relative radiosensitivity of hypoxic tumor cells (author's transl)]. / In-vivo-Untersuchungen über die relative Strahlenempfindlichkeit hypoxischer Tumorzellen

The rate of cellular losses of hypoxic respectively euoxic cells of the solid experimental tumor "sarcoma 180" was examined in vivo after irradiations with gamma rays, 15 MeV neutrons, and alpha particles. The tumor cells were labeled in vivo, at first with 125I-UdR and after 50 hours with 131I-UdR. After a further interval of about 20 hours the tumor was irradiated. This method of double labeling makes it possible to determine externally the rates of cellular losses in the labeled zones of tumor cells presenting different partial oxygen pressures. The increase of the rates of cellular losses among the euoxic and hypoxic tumor cells induced by the gamma radiation differs by 2.6; it decreases, however, to 1.5 after injection of nitrofurazone prior to the irradiation. After irradiation with 15 MeV neutrons, a difference of only 1.4 was observed. If the tumors were irradiated internally with alpha particles from the reaction 10B(n,alpha) 7Li, there was no difference between the two rates of cellular losses. As far as the above mentioned kinds of radiation are concerned, the ratios from the increase of the rates of cellular losses induced by radiation are well corresponding to the oxygen enhancement found by other authors during their examinations in vitro.

[DNA Turnover in the Whole Body of Tumour Bearing Mice (author's transl)]. / DNA-Umsatz im Ganzkörper tumortragender Mäuse

UNLABELLED: During the last 3 decades several authors have found in tumour-bearing animals an increase of synthesis and content of DNA in various organs which were free from neoplastic cells (Griffin, 1957;Kelly and Jones, 1950; Morgan and Cameron, 1973; Cerecedo et al., 1951; Lombardo et al., 1952). 5-iodo-2'-deoxyuridine (IUdR) is a thymidine analogue and specifically incorporated into DNA. When it is labelled with 125I or 131I it permits to reinvestigate these findings by measuring the rate of precursor incorporation into DNA and the rate of loss of labelled DNA in the living animal by means of counting the gamma emission from the incorporated iodine isotopes. In this paper, therefore, an attempt is made to analyse the DNA turnover in the whole body of liver tumour-bearing mice. MATERIAL AND METHODS: 5-iodo-2'-deoxyuridine (IUdR) labelled with 125iodine was used as DNA precursor. It is a thymidine analogue 5% of which is specifically incorporated into the DNA of those proliferating cells which are in the phase of DNA synthesis at the moment of tracer application. Non-incorporated IUdR (about 95% of the injected amount) is rapidly degraded and excreted within 24 hours. The tracer remains bound to the cellular DNA druing the life span of the labelled cells. After cell death only about 5% of IUdR from DNA breakdown is reutilized. 125I has a half live of 60 days and therefore allows, over periods of weeks, external measurements of the DNA turnover in the living animal without disturbing the physiological environment. The measured loss of DNA-bound 125I reflects almost exclusively the turnover of the labelled cells. Female albino NMRI mice, 2 months old, bearing sarcoma-180 implanted into the right hind leg were intraveneously injected with 2 muCi 125I-UdR. At the time of injection, the tumour had reached in one group of mice an average volume of about 25 mm3 and in another group an average volume of nearly 850 mm3. When implanted into subcutaneous tissue sarcoma-180 rarely produces metastases in parenchymal organs, never in the spleen and--within the first 30 days after implantation--only in ca. 10% of the animals a small metastasis in a single lymphnode (Deodhar and Crile, 1969; Franchi et al., 1968). Whole body measurements were carried out immediately after tracer injection and then daily during the first week and every second or third day in the following 2 weeks in a NaI well counter with a single channel pulse height analyser. The tumour activity was also determined in vivo by a special counting device. RESULTS: In the normal mouse 4 to 6% of injected 125I-UdR is retained in the whole body 24 hours after tracer injection. During the following five days the 125I activity rapidly declines to 0.8% of that of day O immediately after injection. Thereafter the rate of loss of activity greatly diminishes (Fig. I). The first component of the turnover curve reflects an average daily cell loss of approximately 30% and involves about 90% of the incorporated activity...