Enhanced cytotoxic activity of macrophages and suppressed tumor metastases in mice irradiated with low doses of X- rays.

We showed in our previous report that a single exposure of mice to 0.1 or 0.2 Gy X-rays led to the significant inhibition of the development of artificial tumor metastases in the lungs and that the effect was related to the enhanced activity of natural killer cells. In the present study, a possible involvement of cytotoxic macrophages in the anti-metastatic effect of the low-level X-ray exposures was investigated. We now demonstrate that irradiation of mice with either of the two low doses of X-rays significantly stimulates the macrophage-mediated cytolysis of the susceptible tumor targets and that the effect coincides with the enhanced production of nitric oxide in the collected effector cells. We also show that suppression of the in vivo function of macrophages by carrageenan eliminates the inhibitory effect of the two low doses of X-rays on the development of pulmonary tumor colonies as well as significantly suppresses the macrophage-mediated cytotoxicity and nitric oxide production. Finally, aminoguanidine added to the culture medium of the assayed macrophages not only shuts down the nitric oxide synthesis in these cells but also significantly suppresses their cytolytic activity. Overall, the obtained results indicate that inhibition of the tumor metastases by a single exposure of mice to 0.1 or 0.2 Gy X-rays results, to a large extent, from the radiation-induced stimulation of the cytocidal activity of macrophages which secrete enhanced amounts of nitric oxide.

Single low doses of X rays inhibit the development of experimental tumor metastases and trigger the activities of NK cells in mice.

There is evidence indicating that low-level exposures to low- LET radiation may inhibit the development of tumors, but the mechanism of this effect is virtually unknown. In the present study, BALB/c mice were irradiated with single doses of 0.1 or 0.2 Gy X rays and injected intravenously 2 h later with syngeneic L1 sarcoma cells. Compared to the values obtained for sham-irradiated control mice, the numbers of pulmonary tumor colonies were significantly reduced in the animals exposed to either 0.1 or 0.2 Gy X rays. Concurrently, a significant stimulation of NK cell-mediated cytotoxic activity was detected in splenocyte suspensions obtained from irradiated mice compared to sham-exposed mice. Intraperitoneal injection of the NK-suppressive anti-asialo GM1 antibody totally abrogated the tumor inhibitory effect of the exposures to 0.1 and 0.2 Gy X rays. These results indicate that single irradiations of mice with either 0.1 or 0.2 Gy X rays suppress the development of experimental tumor metastases primarily due to the stimulation of the cytolytic function of NK cells by radiation.

(99m)Tc human IgG radiolabelled by HYNIC. Biodistribution and scintigraphy of experimentally induced inflammatory lesions in animal model.

BACKGROUND: Our goal was the efficient labelling of highly purified human gammaglobulin. This radioactive protein fraction can be used as a basic compound of radiopharmaceutical formulation for inflammation lesion diagnosis. This application was experimentally illustrated in animal models with artificially induced inflammatory lesions after turpentine oil injection into mouse leg muscle. MATERIALS AND METHODS: Hydrazine nicotinamine derivative of human gammaglobulin (IgG-HYNIC) was synthesized according to Abrams method. The radionuclide: technetium (99m)Tc has been introduced into protein molecules by indirect method incorporation in phosphate buffer, pH 7.4, in the presence of stannous chloride as a reducing agent for sodium pertechnetate, and EDTA as a coligand. Radiochemical purity was estimated by thin layer chromatography. The stability of labelled IgG-HYNIC derivative in human serum in presence of copper, cobalt, iron and manganum salts was analyzed by HPLC method (BioSEP SEC 4000, eluent: 0.1 mol/L phosphate). Inflammation lesions were induced in Balb/3 mice muscles by injection of 0.2 ml turpentine oil into the leg muscle. Five days later, inflammation lesions were visualized by hIgG-HYNIC- (99m)Tc injections. The tracer accumulation in tissue was evaluated by gamma camera at 1 to 24 hour intervals. RESULTS: Efficiency of technetium99m Tc human gammaglobulin labelling (pH 7.4, temp. 37 degrees C) was strictly dependant on ligand and coligand presence in the reaction mixture. Labelling of IgG molecules without any supplements resulted in very low efficiency, never exceeding the range of 5%. Presence of EDTA or hydrazine nicotinamide (HYNIC) conjugated with IgG increased radiolabelling efficiency to 50%. IgG-HYNIC derivative in EDTA presence enables us to reach value above 95% radiochemical purity. Stability of IgG-HYNIC derivative labelled with technetium (99m) Tc decreased rapidly in serum in time--up to 70% of initial value in 30 minutes and only 20% during further 4 hr incubation. This means that as much as 80% of radiotracer present in IgG molecules has been dissociated during incubation with serum. This forced us to find proper conditions for improving the stability of radioactive IgG-HYNIC conjugate in circulating serum for at least six hours. It was achieved by using a reaction medium supplement with divalent metal cations in the following compounds: MgCl2, CoSO4, Cu (NO3)2 and FeCl2 in equimolar ratio to EDTA. Scintigraphy of (99m)Tc gammaglobulin in artificially induced inflammatory lesions of mouse thigh muscle showed a 4 times higher accumulation of the tracer after 6 hours post injection, and 6 times higher after 24 hours. CONCLUSIONS: A human gammaglobulin derivative (hIgG-HYNIC) labelled with technetium (99m)Tc by indirect method with high radiochemical purity can be a basic compound of formulation for infection/inflammation scintigraphy.