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For the purpose to define the mechanism of heavy metal intoxication by inhalation, morphologic observations were made on rat lungs after nasal instillation of iron colloid particles of positive and negative electric charges. Histochemical observation was also made on the liver and spleen of these animals. The instilled iron colloid particles reach the alveolar cavity easily, as can be seen in the tissue sections stained by Prussian blue reaction. Alveolar macrophages do take up them avidly both of positive and negative charges, though much less the positive particles than negative ones. In contrast, the alveolar epithelial cells take up solely positive particles by phagocytosis but not negative ones. Electron microscope observation revealed that the positive particles are ingested by Type I epithelial cells by pinocytosis and by Type II cells by phagocytosis as well. Then the iron colloid particles are transferred into the basement membrane by exocytosis. Travelling through the basement membrane they are again taken up by capillary endothelial cells by phagocytosis. Some particles were found in the intercellular clefts of capillary endothelial cells but not any iron colloid particles in the intercellular spaces of epithelial cells and in the capillary lumen. However, the liver and spleen tissues of the animals given iron colloid showed a strong positive iron reaction. On the basis of these observations, the mechanism of acute intoxication by inhaling heavy metal dusts like lead fume is discussed from the view point of selective uptake of alveolar epithelial and capillary endothelial cells for the particles of the positive electric cha'rge.

Constitutional lipid peroxidation in randomly selected 32 cases of clinically advanced carcinoma from human gastrointestinal tract (20 cases), breast (8 cases) and kidney (4 cases) was examined histochemically in frozen sections using cold Schiff's reagent. Only two cases of gastrointestinal carcinoma were positive by the reagent. Non-cancerous parenchymal cells were negative. These findings suggest that detectable constitutional lipid peroxidation seldom occurs in either cancerous or normal tissues. The capacity for normal and neoplastic tissues to undergo lipid peroxidation was also studied by incubation with an iron-NADPH pro-oxidant system. Normal parenchymal cells showed, to various degrees, a positive reactivity. In gastrointestinal carcinoma, 6 out of 7 cases of well differentiated adenocarcinoma reacted positively, whereas 2 out of 8 cases of moderately to poorly differentiated adenocarcinoma disclosed weakly positive reactions. Mucinous adenocarcinomas (4 cases) were all negative. Signet-ring cell carcinoma (1 case) was positive. One out of 8 cases of breast cancer also showed positive reaction. Four renal cell carcinomas were all negative. Cancer cells have lower capacity to undergo lipid peroxidation than normal cells, when the iron-NADPH pro-oxidant system was employed. In gastrointestinal carcinoma, the ability to undergo lipid peroxidation by the iron-NADPH pro-oxidant seems to be correlated with their histological differentiation. This fact may suggest that differences in lipid composition or the NADPH enzyme system exist between well differentiated and poorly differentiated gastrointestinal malignancies.

1. In an attempt to see how fatty acid iron will be absorbed from the intestine, a single administration of fatty acid iron was given and when histological observations were done with lapse of time, it was found that the iron compound was first split into iron and fatty acid and each of them was then absorbed by the intestines by a different mechanism as described in the first report. The present experiment further confirmed these findings. 2. Following the first experiment, another attempt was made to determine how iron was absorbed in the animals given successive oral administration under various conditions or a single intravenous injection of colloidal fatty acid iron, and it was demonstrated that under a certain condition the presence of fat in the feed accelerates the iron absorption from the intestine but its mechanism remains unclarified.

It has been indicated that, when fatty acid iron is administered orally, the iron compound is split into iron and fatty acid and absorbed by different mechanisms.

Based on our original concept, a fibroblast-inhibiting agent, chloroquine, was used against various animal tumors. Among transplanted animal tumors, the drug was most effective on relatively connective tissue-rich Bashford and Brown-Pearce tumors, as reflected by prolongation of life span, inhibition of tumor growth, inhibition of lowering of liver catalase activity, improvement of iron metabolism, increase of tumor necrosis, inhibition of connective tissue formation, and decrease of acid mucopolysaccharide. On the other hand, it was of little advantage in Ehrlich, Yoshida and MH134 tumors which contain little connective tissue, except for a decrease of the amount of ascites and ascites tumor cells in the former two tumors. These results indicate that chloroquine suppress the growth of the tumors relatively rich in connective tissue. This effect of chloroquine appears to be due to the primary attack of the stromal connective tissue of tumors being followed by the degeneration of tumor cells, though its probable anti-tumor activity by the indirect effects through its anti-inflammatory and systemic humoral activities should be taken into consideration.

A granuloma pouch was formed on the back of rats by the original method of SELYE. Seven days when granuloma tissue reached its maximum, 35S labeled ChS, 59Fe labeled ChS-Fe, labeled ferric ammoninum citrate and colloidal 198Au were injected into the pouch and their absorption and organ distribution examined and compared with the results in the case where 59Fe labeled ferric ammoninum citrate and colloidal 198Au were injected into the gluteal muscle. 1. When 35S labeled ChS was injected into the granuloma pouch, radioactivity of the organs per gram tissue was high in the kidney, liver, bone marrow and spleen, in descending order. The maximum activity was seen 12 to 24 hours after injection, which is slow compared to the results obtained by KISHIDA in intraperitoneal and oral administration. 2. The absorption of Ch S-Fe by pouch where the iron is enveloped by the large ChS molecule, is slower than that of ferric ammonium citrate, an inorganic compound. 3. The uptake of Fe from the blood by bone marrow is larger when the increase of blood Fe ion concentration is slow, rater than when the increase is rapid. 4. When conoidal 198Au is injected into the pouch and injected into the" gluteal muscle, the 198Au is phargocytozed by the reticuloendothelial system organs, the liver showing the largest uptake among all organs. 5. In the intramuscular injection of colloidal 198Au and 59Fe labeled ferric ammonium citrate, radioactivity of pouch fluid is lower than that of blood. However, the difference between the two is less in the case of colloidal 198Au. 6. In the granuloma ponch, radioactivity of the abdominal wall proves to be greater than that of the dorsal wall.

Following Fibrin Plate Method of SZOLLOSY and RENGEI² , and ASTRUP and MULLERTZ³, the author conducted a series of experiments in an attempt to identify human blood by detecting the proactivator believed to be one of the enzyme proteins contained abundantly in human blood. As the results it has been found that with 0.1 mg. % SK-solution human blood alone responds to the reaction, showing almost absolute species-specificity within 4 hours but not with blood of monkey. In addition, the sensitivity is so high that it responds positively up to the dilution of 1: 8,000 to 1: 10,000 (human blood: physiological saline solution). By means of this method using 0.1 mg% SK-solution it has been clearly demonstated that the identification of human blood is possible in a variety of conditions and states as may be encountered in practical legal medicine such as with blood stains in cloth, wood, stone, leaves of tree even with a trace of blood stain, old human blood stain left standing for 20 to 30 years, old blood mixed with iron rust, blood stains soaked in various oils, and even the blood stained cloth washed thoroughly and left standing in room temperature for 6 months. Therefore, this Fibrin Plate Method seems to be the excellent one for the identification of human blood.

For the purpose of revealing the role of the reticuloendothelial system (RES) for the antibody formation, the rats which received the repeated intraperitoneal and subcutaneous injections of a vast amount of PVP were challenged by bovine serum albumin (BSA) introducing through 2 routes of intramuscular and intravenous, and then antibody formation was observed. Blood cell count and clearance rate of radiogold were observed for the purpose of obtaining the information of blockade grade of the RES by PVP. Phagocytic activity of macrophages ingesting PVP against iron colloid were also observed in vitro. 1. A severe anemia was induced by the administration of a vast amount of PVP, 15 ml of 3% solution daily or every other day for 63 days. Histological picture indicated the suppressed erythropoiesis probably by iron deficiency or the lowered iron transporting activity of the RES, as the anemia recovered after intraperitoneal iron injections. 2. With the generalized and marked swelling of the RES, the cells in germinal center of spleen and lymph nodes were extremely swollen and lymphocytes disappeared completely, suggesting that the macrophages in germinal center play an important role in reproduction and differentiation of lymphocytes. 3. The phagocytic activity of the RES as understood from the clearance rate of radiogold was suppressed only slightly even by a heavy deposition of PVP after the repeated injections. The state of blockade or the suppressed phagocytic activity persisted for 48 hours or more after the several PVP injections. However, complete blockade of the RES or inactivation of the phagocytic activity by PVP injection was not attained. 4. A prolonged treatment of animals with PVP caused delay in the appearance of circulating antibody but the final titration reached the same level as that of control. The data suggest that the blockade of the RES by PVP induces the delay in the transmittance of the information for the antibody formation from the macrophages to the immunologically competent cells but no delay in the ingesting antigen by the macrophages.

The rats which received the repeated intra peritoneal or intravenous　injections of methyl palmitate showed a marked depressed phagocytic activity　of the RES as shown by the clearance test with radioactive iron as　well as by histological observations and a significantly suppressed antibody　formation against the challenge by BSA.　Differing from the cases of the blockade of the RES made by PVP or　radiogold, the injection of methyl palmitate did not result in any injurious　effect on the lymph follicles of lymph nodes and spleen and the plasma　cells proliferation as revealed by the histological observation. Histochemical observations of iron phagocytosis of the RES done by　Perls stain revealed that methyl palmitate　suppressed the phagocytic activity　of the Kupffer cells of the liver dramatically and also suppressed the　phagocytic activity of the sinus-lining cells in spleen to a lesser degree.　The result indicates that the injection of methyl palmitate attacks the phagocytic　function of the RES selectively and induces the reduced immune　response of the organism without giving any damages to the proliferation of immunologically com petent cells.　The fact suggests that the RES lowered in their phagocytic activity　fails to produce the informational substance for immune　response, showing　a lower level in the antibody formation even in the presence of antigen　and proliferating immunologically competent cells.

For the purpose to clarify the mechanism of phagocytosis or pinocytosis, the observations on the tumor ascites, including the macrophages as well as the tumor cells, were carried out by incubating with the iron colloid with or without pretreatment by several inhibibitors of glycolysis, oxidative phosphorylation and respiration, or under hypotonic or cold environments. The results have demonstrated that there are three steps in the phagocytosis. The first step is the adhesion of the substance to the cell surface, which is not an energy-requiring process. The second step is the engulfing which proceeds by using the energy supplied by glycolysis. The third is the accumulation of the substance into the vesicles through the canaliculi connecting the cell surface with the vesicles. The discussion was made on the existence of the active site on the cell surface to which the substance can be adhered, and the accumulation mechanism of the material into the phagocytic vesicles by the membrane flow, the flowing movement of the outer lipid layer of a unit membrane through the canaliculi which connect the cell surface to the phagocytic vesicles.

Non-hemin iron content in gastric juice was examined in 46 patients with various blood diseases, especially idiopathic hypochromic anemia and in 26 healthy controls. 1. The iron content in gastric juice was found to be 290 μg/ dl in healthy controls, a lower value of 110 μg/ dl in idiopathic hypochromic anemia and a higher value of 550 μg / dl in aplastic anemia. These values were in a close correlation with serum iron or sideroblasts. 2. In idiopathic hypochromic anemia there was also a close correlation between the iron content in gastric juice and hemoglobin. In the course of treating idiopathic hypochromic anemia (stage of recovery of anemia) the iron content in gastric juice showed a marked increase over the value in healthy controls as well as a transient increase after an intravenous iron tolerance test. This condition may be interpreted as an "iron-losing anemia". Iron excretion of gastric mucosa in various blood diseases and its changes in the course of treating idiopathic hypochromic anemia in relation to the cause of this anemia were discussed.

For the investigation of iron metabolism in the intestinal mucosa in various　blood diseases, intestinal biopsy (duodenum) was performed on 10 healthy controls　and 35 cases with various blood diseases. The following are　the results　of the studies on distribution of stainable　iron, amounts of non-hemin iron in the　biopsied materials, and iron uptake of the intestinal epithelial cells.　1) An evaluation of distribution of stainable iron by Berlin　blue reaction　showed none or very mild degree, if any,　inhealthy controls, an increase in　aplastic anemia,　pernicious anemia, some of leukemias and in iron deficiency anemia following iron therapy, and a decrease in　idiopathic hypochromic anemia, anchylostomiasis anemia,　anemia with cancer, myxedema, hemolytic anemia,　and in　some of leukemias. Some of anemia with cancer, however,　showed an　increase of a certain degree. In iron absorption tests, no changes were found other than a very mild increase in aplastic anemia. 2) Non-hemin iron was 70-112γ/g in healthy controls, increased in aplastic anemia approximately to 100-200γ/g, ranging 40-130γ/g in leukemia, and decreased in idiopathic hypochromic anemia and in anemia with cancer ranging 30-60γ/g and 30-50γ/g respectively. Amounts of non-hemin iron and serum iron or sideroblasts show a fair correlation. The fractionation of nonhemin iron in aplastic anemia didn't show any difference in relationship of each fraction from healthy controls despite the increased amount in the former. 3) A radioautographic evaluation of iron uptake by intestinal epithelium was performed by our device for evaluation of intestinal absorption capacity. The iron uptake was mild in healthy controls, almost none in aplastic anemia, and marked in iron deficiency anemia where it was decreased approximately to the level of healthy controls following iron therapy. 4) The intestinal tissue iron showed a series of changes similar to those of iron present in the serum or erythroblasts, and the non-hemin iron in the intestinal mucosa is inversely correlated with iron uptake of epithelium and is considered to regulate the absorption according to its amount.

A chronic epileptic focus was induced by a microinjection of ferric chloride solution into the sensorimotor cortex of rats. Two types of somatosensory evoked potentials (SEPs) were recorded from the cortex near the injection site. In animals showing an initial positive-negative biphasic SEP, spikes appeared in electrocorticograms (ECoGs) more frequently on the side ipsilateral to the injection site than on the contralateral side, whereas in animals showing an initial negative monophasic SEP, spikes appeared more frequently on the contralateral side.

A previous study has shown that a single injection of ferric nitrilotriacetate (Fe-NTA) produces hepatic parenchymal iron loading in rats. The present paper reports on iron uptake by rat liver and iron toxicity in the liver after a single injection of Fe-NTA (7.5 mg Fe/kg B.W.). Iron uptake was examined with 59Fe-NTA and Fe-[14C]-NTA. Thirty percent of the injected 59Fe was incorporated in the liver non-heme iron fraction at 3 h and retained for 240 h. Only 1% of the 14C injected as Fe-[14C]-NTA was taken up by the liver at 3 h. Gel filtration with a Sephadex G-25 column of the supernatant fraction of the liver obtained 3 h after the injection showed two peaks of 14C activity. One was eluted in the void volume, and the other corresponded to [14C]-NTA. The former had a molecular weight of 5,000-10,000 as determined with a Sephacryl S-300 column and also had 59Fe activity. The electron spin resonance spectra showed that the generation of a free radical in the liver was initiated within 1 h of the iron administration. The free radical generated in the serum by Fe-NTA was revealed to be superoxide by the spin trapping method. These results suggest that Fe-NTA transfers iron to transferrin in the serum and induces hepatic iron loading. Small amounts of the injected iron were taken up by the liver as Fe-NTA and generated superoxide which may have induced lipid peroxidation of the cellular membranes.

A microinjection of ferric chloride solution into the left frontal cortex of rats induced epileptic discharges which were recorded by electrocorticography. In animals having such electrographic seizure activity 30 to 60 days after the injection, the accumulation of cyclic AMP elicited by norepinephrine was examined in slices from four cortical regions. The accumulation was significantly greater in the left anterior area, into which region the ferric chloride solution was injected, than in the right anterior area. There was also a tendency for greater norepinephrine-elicited accumulation of cyclic AMP to occur in the left posterior area than in the right posterior area.

An epileptic focus not resulting in generalized convulsions was induced by a microinjection of ferric chloride solution into the left anterior cortex of rats. The formation of the epileptic focus was confirmed by the appearance of bilateral spike and slow wave complexes as well as focal isolated spikes in electrocorticograms (ECoGs). The effect of glutamate on cyclic AMP accumulation was examined in incubated slices prepared from four quadrants of the epileptic cortex. In animals showing isolated spikes 8 to 10 days after the microinjection, the effect of glutamate on cyclic AMP accumulation was stimulatory. It was greatest in the left anterior quadrant which included the injection site, but only slight in the left and right posterior quadrants. In animals showing spike and slow wave complexes 30 to 60 days after the microinjection, the stimulatory effect of glutamate was also most pronounced in the left anterior quadrant. In the right anterior and the left posterior quadrants glutamate had almost no effect, while in the right posterior quadrant, glutamate was inhibitory.

The present study showed that many neurons in the adult rat brain possessed a perineuronal sulfated proteoglycan surface coat which reacted to cationic iron colloid and aldehyde fuchsin. This surface coat was stained supravitally with Ehrlich's methylene blue and doubly stained with Ehrlich's methylene blue and aldehyde fuchsin. The surface coat was also stained with Gomori's ammoniacal silver and doubly stained with Gomori's ammoniacal silver and cationic iron colloid. The surface coat was usually expressed together with a nerve cell surface glycoprotein net detectable with lectin Wisteria floribunda agglutinin. These findings indicate that the perineuronal proteoglycan surface coat is identical to Cajal's superficial reticulum and contains some collagenous elements. It was further demonstrated that collagenase digestion erased Gomori's ammoniacal silver impregnation within the perineuronal proteoglycan surface coat.

<p>Iron plays a critical role in the production of activated oxygen species and the activity of chelated iron in the biological system depends on the chemical forms of the chelators. In the present study, we used ferric nitrolotriacetate (Fe-NTA, molar ratio of iron to chelators = 1:3), ferric ethylenediaminetetraacetate (Fe-EDTA, 1:3 complex) and ferric Desferal (Fe-Des, 1:1.1 complex) to see their "free" iron content in aqueous solutions in vitro and in the serum obtained after a single intraperitoneal injection of the chelates to rats (7.5 mg of iron/kg). "Free" iron was measured by the bleomycin-assay system. When Fe-NTA was dissolved in water, "free" iron increased linearly with total iron concentration up to 10 microM, whereas Fe-EDTA and Fe-Des showed no "free" iron with corresponding iron concentrations. When these three ferric chelates were dissolved in normal rat serum, "free" iron in Fe-NTA increased abruptly between 40 microM and 60 microM iron concentrations, then increased slowly up to 100 microM. Fe-Des did not show any "free" iron at comparable iron concentrations. Fe-EDTA had an intermediate "free" iron level in the serum. Among the ferric chelate complexes, Fe-NTA showed a much faster increase of and a higher content of "free" iron in the serum than the other two complexes after a single injection of the chelates into rats.(ABSTRACT TRUNCATED AT 250 WORDS)</p>

Head injury or hemorrhagic cortical infarction results in extravasation of blood and breakdown of red blood cells and hemoglobin. Iron liberated from hemoglobin, and hemoglobin itself, are associated with the generation of reactive oxygen species (ROS) and reactive nitrogen species (RNS). ROS and RNS have been demonstrated to be involved in the mechanism of seizures induced by iron ions in the rat brain, an experimental animal model for posttraumatic epilepsy (PTE). ROS are responsible for the induction for peroxidation of neural lipids, i.e., an injury of neuronal membranes, and also could induce disorders in the excitatory and inhibitory neurotransmitters. Antioxidants, such as a phosphate diester of vitamin E and C (EPC-K1) and antiepileptic zonisamide, have been known to prevent the epileptogenic focus formation, or to attenuate seizure activities in the iron-injected rat brain. Natural antioxidants, such as alpha-tocopherol, and condensed tannins, including (-)-epigallocatechin and (-)-epigallocatechin-3-O-gallate, adenosine and its derivative, melatonin, uyaku (Lindera Strychnifolia), fermented papaya preparations, Gastrodia elata BI., and Guilingji, have been demonstrated to scavenge ROS and/or RNS and to be prophylactic for the occurrence of epileptic discharge in the iron-injected rat brain.

Peripheral blood lymphocytes and the various lymphocyte fractions from patients with cancer of the colon were cultivated with target cells (P-4788) derived from the colon cancer. Changes in the surface ultrastructure during tumor cell destruction were studied by scanning electron microscopy (SEM). P-4788 cells adhering to the coverslip showed various surface activity. The surfaces of some cells were relatively flat; others were smooth or had fine granules. Still other cells were villous, round or had marked blebs. When host lymphocytes were added to the target cells, adhesion of the two cell groups began by many fine projections. After incubation for 6 h, some lymphocytes had adhered to the target cells. Many lymphocytes had adhered to the target tumor cells by 24--48 h incubation. Ultimately the tumor cells became swollen and disrupted. Most lymphocytes adherent to the target cells had few microvilli. Lymphocytes after elimination of phagocytes by carbonyl iron treatment also adhered readily. Some target cells showed adhesion with lymphocytes passed through nylon-wool columns, although the number of lymphocytes adhering was fewer than in the case of lymphocytes not passed through nylon-wool columns. T cells were collected from lymphocytes that form rosettes with SRBC by isolation with NH4Cl. They had markedly elongated microvilli which in places were sparsely scattered and tended to be localized on the side, a finding which suggests loss of cell activity by the time of SEM. Only a few T cells adhered to target cells and they seemed to be T cells without activity. It was thought that there are cytotoxic cells among T cells and that the co-existence of T cells, non-T cells and monocytes caused target cell destruction.