In vivo biological behavior of a water-miscible fullerene: 14C labeling, absorption, distribution, excretion and acute toxicity.

BACKGROUND: Water-soluble fullerenes have recently been shown to exhibit considerable in vitro biological activity including cytotoxicity, site-selective DNA cleavage and inhibition of HIV protease. To assess the potential of these compounds as drugs, studies on the in vivo behavior of fullerenes are needed. We therefore set out to synthesize a radiolabeled, water-soluble fullerene, in order to obtain data on the oral absorption, distribution and excretion of this class of compounds. RESULTS: We synthesized a 14C-labeled water-soluble [60]fullerene using dipolar trimethylenemethane, which undergoes cycloaddition to [60]fullerene. When administered orally to rats, this compound was not efficiently absorbed and was excreted primarily in the feces. When injected intravenously, however, it was distributed rapidly to various tissues, and most of the material was retained in the body after one week. The compound was also able to penetrate the blood-brain barrier. Acute toxicity of the water-miscible fullerene was found to be quite low. CONCLUSIONS: Although the water-soluble fullerenes (and possibly their simple metabolites) are not acutely toxic, they are retained in the body for long periods, raising concerns about chronic toxic effects. The fact that fullerenes distribute rapidly to many tissues suggests that they may eventually be useful to deliver highly polar drugs through membranes to a target tissue, however, and they may even have applications in the delivery of drugs to the brain. Recent advances in fullerene synthetic chemistry may also make it possible to control fullerene absorption/excretion profiles in the future.

Light-induced oxidation of iron atoms in a photosensitive nitrile hydratase.

The photoactivation process of a photosensitive nitrile hydratase (NHase) from Rhodococcus sp. N-771 has been investigated by 57Fe Mössbauer spectroscopy and magnetic susceptibility measurements in order to clarify the behavior of iron atoms in the enzyme. Mössbauer spectra of inactive NHase gave two symmetric-doublet components indicating the presence of two iron species, while that of the active NHase gave a single symmetric doublet indicating the presence of a single iron species. Magnetic susceptibility measurements of the inactive and active HNase both showed small effective magnetic moments. These results led us to conclude that one of the two iron atoms incorporated in the NHase is oxidized during photoactivation, namely from a low spin ferrous to a low spin ferric state. This is the first observation of an intramolecular photooxidation phenomena involving iron in a single protein molecule.

Multitracer studies on the accumulation of radionuclides in mushrooms.

We used the multitracer technique to study the transfer of several radionuclides to two mushroom species. Radionuclides accumulated in the fruiting bodies of the mushrooms in the order of 83Rb > 65Zn > 54Mn > 22Na > 75Se and 85Sr > 60Co > 88Y, 102Rh, 139Ce, 143.144Pm, 146.153Gd and 173Lu > 175Hf. The concentration ratio values for 83Rb, 65Zn and 54Mn in the fruiting bodies were more than 10, whereas those for 60Co, 88Y, 102Rh, 121mTe, 175Hf and the rare earth elements were less than 1. There were major differences in the accumulations of the alkali elements.

Unique behavior of rubidium in mice under low oxygen atmosphere. Simultaneous tracing of Sc, Mn, Fe, Co, Zn, Se, Rb, Y, and Zr using off radioactive multitracer technique.

Relative tissue distributions of Sc, Mn, Fe, Co, Zn, Se, Rb, Y, and Zr tracers were studied in young mice housed under four different O2 atmospheres (10 percent, 20 percent, 80 percent, and 100 percent O2) using the radioactive multitracer technique developed at The Institute of Physical and Chemical Research (RIKEN). At 48 hr after injection of the multitracer, the tissue uptake rates were examined for 10 tissues (brain, cardiac muscle, lung, liver, spleen, pancreas, kidneys, skeletal muscle, bone, and blood) and expressed as the radioactivity percentage of the injected dose per gram of wet tissue (percent dose/g). Rubidium uptake rates for all soft tissues of mice housed under a 10 percent O2 atmosphere were found to be higher than those of the tissues of mice housed under the other three O2 atmospheres (20 percent, 80 percent, and 100 percent), but no difference was observed for the other elements (Sc, Mn, Fe, Co, Se, etc.) under any of the O2 atmospheres.

Biodistribution of trace elements in normal, aluminum-overloaded and cadmium-overloaded mice. An application of the radioactive multitracer technique.

The radioactive multitracer technique was applied to simultaneous evaluation of biobehavior of many trace elements in normal, Al-overloaded and Cd-overloaded mice. The tissue and subcellular distributions of Be, Sc, V, Mn, Fe, Co, Zn, As, Se, Rb, Sr, Y, Zr, Ru, and Rh were examined by using a multitracer solution, prepared from an Ag target irradiated with heavy ions, in 9 organs (brain, cardiac muscle, lung, liver, spleen, pancreas, kidney, skeletal muscle, and bone) and blood. Their tissue and subcellular distributions were evaluated by gamma-ray spectrometry in terms of tissue uptake rate (the radioactivity percentage of injected dose per gram of tissue, percent of dose/g) and fractionation percentage (the radioactivity percentage of nuclear fraction, mitochondrial fraction, microsomal fraction or supernatant fraction, percent), respectively. Comparative biobehavior of these elements in brain and liver is reported. Aluminium- and Cd-induced synergistic interactions were observed among these trace elements in their liver uptake behavior.

Bone uptake of vanadium in mice: simultaneous tracing of V, Se, Sr, Y, Zr, Ru and Rh using a radioactive multitracer.

The radioactive multitracer technique, which enables simultaneous tracing of a number of elements in a variety of bio-systems and an accurate comparison of their biobehavior, was employed in a study on uptake behavior of trace elements in various mice tissues, focusing on uptake of V by bone. We found that the bone uptake behavior of trace V closely resembles that of trace Sr and Y. It is suggested that V is a bone-seeking element which probably plays an important role in bone formation. The multitracer technique was thus shown to be useful for comparative studies of behavior of trace elements in living organisms.

Binding properties of various metals to blood components and serum proteins: a multitracer study.

The binding affinity of various trace elements to blood components and the pH-dependence of the binding affinity of the elements to serum proteins were examined using the radioactive multitracer technique. The binding affinity of 13 elements (Be, V, Mn, Zn, Se, Rb, Sr, Ce, Eu, Gd, Tm, Yb, and Lu) was simultaneously determined by gamma-ray spectrometry. The blood drawn from rats was separated into plasma, corpuscles, and erythrocyte ghosts. It was found that Be, Sr, Mn, and Zn bind highly to the plasma proteins. V and Se were highly bound to the corpuscles, and Se to the erythrocyte ghosts as well. Similar binding percentages of rare earth elements (Ce, Eu, Gd, Tm, Yb, and Lu) were found for each of the blood components, with the highest percentages being observed for plasma proteins. Albumin, beta-globulin, gamma-globulin, apotransferrin, and holotransferrin were examined in the study on the affinity of individual serum proteins. The pH dependence of the affinity of metal ions to the serum proteins in the pH range of 6.4-8.5 was examined using ultrafiltration and gamma-ray spectrometry. Each element showed a characteristic binding affinity to each serum protein, depending on pH. The results are discussed in terms of the chelating ability of metal ions and the nature of the serum proteins.

Study on the distribution of radioactive trace elements in vitamin D-overloaded rats using the multitracer technique.

The uptake and distribution of radioisotopes of beryllium, calcium, scandium, vanadium, chromium, manganese, iron, cobalt, nickel, zinc, gallium, arsenic, strontium and barium in vitamin D (VD)-overloaded rats were investigated and compared with those in control rats, using the multitracer technique. Each element revealed its characteristic distribution among various organs in control and VD-overloaded rats. For some elements, such as cobalt and chromium, the distribution patterns in them were significantly different. These results are discussed in terms of the metabolism of the elements in rats.

The use of a multitracer technique for the studies of the uptake and retention of trace elements in rats.

The uptake by, and distribution and retention of radioactive isotopes in various organs of Wistar rats were examined using the multitracer technique. A hydrocholoric acid solution (pH 3) of a carrier-free radioactive multitracer was prepared from gold foil irradiated with a 14N beam of 135 MeV nucleon. The solution was administered orally to 12 7-week-old male rats. Urine and faeces were collected and each group of three rats was killed at 1, 2, 3 or 6 days after administration. The percentage of administered dose of the 17 elements, Mn, Co, Zn, As, Rb, Sr, Y, Eu, Gd, Er, Tm, Yb, Lu, W, Re, Ir and Pt in the organs blood and excreta were determined using gamma-ray spectrometry. Each element revealed its characteristic distribution among the different organs, including the blood. These results are discussed and compared with those of single-tracer experiments, and the advantages of the multitracer technique are presented.

Multitracer study on uptake and excretion of trace elements in rats.

The multitracer technique was first applied to the investigation of the uptake and excretion behaviour of trace elements in rats. A multitracer solution, prepared by irradiation of a gold target with a 14N-beam from the RIKEN Ring Cyclotron, was orally administered to male Wistar rats. The uptake and excretion rates of 23 elements, Be, Mn, Co, Zn, As, Rb, Sr, Y, Zr, Ce, Pm, Eu, Gd, Tb, Er, Tm, Yb, Lu, Hf, W, Re, Ir and Pt, were simultaneously determined under strictly identical experimental conditions. For some of the elements, the results obtained were consistent with previous reports on uptake and excretion of the elements in animals. For the other elements, unique behaviour was revealed for the first time as described in the present work. These results show that the multitracer technique has excellent reliability and versatility for a comparative study of the uptake and excretion of many different elements in animals.