Large basophilic cells in the bone marrow in iron deficiency anemia

Summary Many large cells with a strongly basophilic cytoplasm and large nuclei with a delicate chromatin pattern and large nucleoli were studied in 1 sections of core biopsies in 8 patients with iron deficiency anemia and in 5 patients with pernicious anemia. In 2 normal controls these cells were rare. Possible features of granulocytic differentiation were assessed with the Giemsa stain and with the naphtol-ASD-chloroacetate esterase reaction which is indicative of early granule formation: the large cells were constantly negative.In thin sections the morphological appearances of the large basophilic cells are rather similar in such different haematological disorders as pernicious anemia or iron deficiency anemia. A clear difference can be demonstrated by karyometry, which shows that the large basophilic cells in pernicious anemia are significantly larger than those in iron deficiency anemia or in the normal control.     

Histogenesis of dieldrin and DDT-induced hepatocellular carcinoma in Balb/c mice

Male, Balb/c mice were fed diets containing dieldrin (10 ppm) and DDT (100-175 ppm) for 75 weeks. Control and treated mice were serially killed and their livers analyzed by histological and histochemical procedures after 2, 4, 8, 16, 36, 52 and 75 weeks of exposure. Mice administered both chlorinated hydrocarbons initially responded with centrolobular hepatocytomegaly. The cells were characterized by decreased glucose-6-phosphatase and succinate dehydrogenase activity. At later periods 52 through 75 weeks, foci of phenotypically-altered hepatocytes were noted. The cells of these lesions were basophilic or clear-staining in hematoxylin and eosin-stained sections and displayed increased gamma glutamyl transpeptidase activity. In mice preloaded with iron dextran, cells of foci were negative for iron when the surrounding parenchyma was siderotic. Hepatocellular adenomas (HA) and carcinomas (HPC) were composed of cells with increased gamma glutamyl transpeptidase and glucose-6-phosphate dehydrogenase and decreased glucose-6-phosphatase and succinate dehydrogenase activity. In iron loaded mice, the cells of HA and HPC did not stain for iron in otherwise siderotic surroundings. Both hepatocellular foci and adenomas may be potential precursors of mouse hepatocellular carcinomas.     

Iron distribution and histopathological characterization of the liver and heart of β-thalassemic mice with parenteral iron overload: Effects of deferoxamine and deferiprone

The liver and heart are the major target organs for iron accumulation and iron toxicity in β-thalassemia. To mimic the phenomenon of heavy iron overload resulting from repeated blood transfusions, a total of 180 mg of iron dextran was intraperitoneally injected into C57BL/6J mice (WT) and heterozygous β-globin knockout mice (^muβ^th-3/+, BKO). The effects of deferiprone and deferoxamine in this model were investigated. The iron was distributed homogenously throughout the 4 liver lobes (left, caudate, right and median) and was present in hepatocytes, Kupffer cells and the sinusoidal space. Iron accumulation in phagocytic macrophages, recruitment of hepatic lymphocytes and nucleus membrane degeneration were observed as a result of iron overload in the WT and BKO mice. However, the expansion of hepatic extramedullary hematopoiesis was observed only in the BKO mice with iron overload. In the heart, the iron accumulated in the cardiac interstitium and myocytes, and moderate hypertrophy of the myocardial fibers and cardiac myocyte degeneration were observed. Although the total liver iron was not significantly altered by iron chelation therapy, image analysis demonstrated a difference in the efficacies of two iron chelators. The major site of chelation was the extracellular compartment, but treatment with deferiprone also resulted in intracellular iron chelation. Interestingly, iron chelators reversed the pathological changes resulting from iron overload in WT and BKO mice despite being used for only a short treatment period. We suggest that some of these effects may be secondary to the anti-inflammatory activity of the chelators.     

Morphologic and cytochemical properties of mouse liver neoplasms induced by diethylnitrosamine and promoted by 4,4'-dichlorodiphenyltrichloroethane, chlordane, or heptachlor

The relationships between the gross appearance, histologic types, and cytochemical characteristics of hepatocellular neoplasms were studied in B6C3F1 mice given the liver carcinogen diethylnitrosamine either alone or followed by the organochlorine pesticides, 4,4'-dichlorodiphenyltrichloroethane, chlordane, or heptachlor as promoting agents. Hepatocellular neoplasms were categorized according to their cytoplasmic staining properties with hematoxylin and eosin. Acidophilic neoplasms more often displayed increased activity of alkaline phosphatase than did basophilic neoplasms. The activities of glucose-6-phosphatase and adenosine triphosphatase were decreased in both acidophilic and basophilic neoplasms. There was no difference in the activities of these enzymes or gamma-glutamyltranspeptidase between adenomas and carcinomas, although most neoplasms did not display gamma-glutamyltranspeptidase. Chlordane or heptachlor exposure increased the alkaline phosphatase activity in neoplastic cells, but not that of other enzymes. The majority of neoplasms displayed a deficiency of iron accumulation. The macroscopic appearance of neoplasms was closely related to their cytoplasmic staining properties and cytochemical characteristics.     

Distribution of ferritin in the rat hippocampus after kainate-induced neuronal injury

A gradual increase in iron occurs in the lesioned hippocampus after neuronal injury induced by the excitotoxin kainate, and the present study was carried out to investigate whether this increase in iron might be associated with changes in expression of the iron binding protein, ferritin. An increase in ferritin immunoreactivity was observed in glial cells of the hippocampus, as early as three days after intracerebroventricular injections of kainate. The number of ferritin positive cells peaked four weeks after the kainate injection, and decreased eight and twelve weeks after injection. They were found to be mostly microglia and oligodendrocytes by double immunofluorescence labeling with glial markers. A number of ferritin-labeled endothelial cells were also observed via electron microscopy. The decline in ferritin immunoreactivity four weeks after the injection of kainate is accompanied by an increase in the number of ferric and ferrous iron positive cells in the lesioned tissue. A substantial non-overlap between ferritin and iron-containing cells was observed. In particular, spherical ferric or ferrous iron-laden cells in the degenerating hippocampus were unlabeled for ferritin for long time periods after the kainate injection. An increase in iron, together with a reduced expression of iron binding proteins such as ferritin at long time intervals after kainate lesions, could result in a relative decrease in ferritin-induced ferroxidase activity and the presence of some of the iron in the ferrous form. It is postulated that this may contribute to chronic neuronal injury, following acute kainate-induced neurodegeneration.     

The way we teach general pathology in Oslo, Norway

Hepatocellular carcinoma was induced in rats by administering aflatoxin B₁ (AFB₁) for 6 weeks. Malignant tumours were preceded by foci and nodules of altered hepatocytes of three histological types, composed of basophilic, eosinophilic, and vacuolated cells. In addition, there were areas of altered hepatocytes that were considered as hyperplastic. Lectins were used as histochemical markers to compare the expression of membrane glycoproteins in hepatocellular carcinomas and hepatic nodules with non-nodular or control hepatocytes. There were marked changes in the lectin-binding patterns of the hepatocellular carcinoma cells and the eosinophilic nodules. The lectin-binding patterns of basophilic nodules, vacuolated nodules, and hyperplastic areas were similar to non-nodular or untreated hepatocytes. The similarity in the lectin-binding changes of the eosinophilic nodules and hepatocellular carcinomas suggests that the eosinophilic nodules may be an early stage in the development of carcinoma.     

PROCESS OF CARBONYL IRON ABSORPTION FROM THE RAT DUODENAL MUCOSA

The process of carbonyl iron absorption from rat duodenal mucosa was investigated histologically with Perls' iron staining and immunostaining for ferritin and transferrin. The change in iron and ferritin distribution in the liver after oral administration of carbonyl iron was also studied. After oral administration of carbonyl iron (100 mg iron/kg body weight), stainable iron appeared not only in the duodenal lumina but also in the epithelial cell cytoplasm, intercellular spaces of epithelial cells and subepithelial stroma. Some of the stainable iron in the epithelial and their intercellular spaces was in the form of fine granules smaller than carbonyl iron particles. Mucosal ferritin increased after administration of carbonyl iron but its distribution was different from that of stainable iron. The amount and the distribution of transferrin in the duodenal mucosa were not changed after carbonyl iron administration. These observations suggested that some carbonyl iron was absorbed without the participation of ferritin and transferrin. In the liver, fine granular stainable iron appeared in the peripheral zones of the hepatic lobules around the portal vein, and liver ferritin increased after oral administration of carbonyl iron. Increased liver ferritin was detected in some of the perisinusoidal cytoplasm of hepatocytes. ACTA PATHOL JPN 38: 1377–1390. 1988.     

Histochemical studies on the early proliferative lesion induced in the rat liver by aflatoxin

Male inbred Fischer rats were fed a diet containing 5 p.p.m. aflatoxin for 1, 3, 4½ and 6 weeks at which times groups were killed for histological and histochemical study. Aflatoxin produced a scattered individual cell necrosis of parenchymal cells by 1 week. At 3 weeks small basophilic proliferative foci were seen which increased in size and abundance to 6 weeks. These foci showed starvation-resistant glycogen, variable depletion of glucose-6-phosphatase, succinic dehydrogenase, aniline hydrogenase, membrane ATPase and acid phosphatase. At 6 weeks the foci showed the presence of gamma glutamyl transpeptidase and glucose-6-phosphate dehydrogenase. The basophilic foci were not preceded by other focal histological and histochemical change. The basophilic proliferative lesions are observed when an irreversible change has been induced in the liver. The role of such lesions in the histogenesis of hepatocellular carcinoma is discussed.     

Increased iron staining in the cerebral cortex of cholesterol fed rabbits

The link between hypercholesterolemia and neuronal damage is not clear. In the present study, we studied some of the possible effects of hypercholesterolemia on the brain, using the cholesterol fed New Zealand White rabbit as a model. An increase in the number of iron positive cells (i.e. oligodendrocytes) was observed in the brain parenchyma, in rabbits treated with a high cholesterol diet for 8 weeks. At this time, no neuronal death was observed, indicating that the increased iron did not occur as a consequence of neuronal injury. No heme oxygenase-1 (HO-1) or bilirubin immunoreactivity was observed in the brains in these rabbits, indicating that the iron accumulation did not occur as a consequence of increased breakdown of heme. It is postulated that cholesterol could have subtly damaged brain endothelial cells, resulting in increased iron transport across brain endothelial cells. Hypercholesterolaemia is known to be associated with increased plasma lipid peroxidation which might contribute to such damage.     

Transferrin Receptor Expression in Normal, Iron-deficient and Iron-overloaded Rats

The distribution of transferrin receptor (TfR) positive cells and their staining intensity were examined in the liver, duodenum, pancreas, spleen, kidney and brain of iron deficient, iron-overloaded and normal Wistar rats to elucidate the regulatory effects of iron on TfR expression in vivo. Iron deficiency was produced by an iron deficient food and water regimen, and iron overload by repeated intraperitoneal injections of ferric nitrilotriacetate (Fe³-NTA) for 12 weeks. In iron deficient rats, levels of hemoglobin (Hb= 5.9 ± 0.7) and serum iron (Sl = 29.9 ± 4.4) were lower, and total iron binding capacity (TIBC = 624.4 ± 72.7) was higher than in normal rats (Hb = 15.6 ± 0.9, Sl = 206.5 # 20.5, TIBC = 416.0 ± 56.0), and Wee versa for SI (217.7 ± 15.5) and TIBC (307.1 ± M45.4) in iron-overloaded rats. In normal rats, TfR positive granules were observed in hepatocytes and Kupffer cells of the liver, absorptive epithelium of the duodenum, acinar and Langerhans islet cells of the pancreas, macrophages and red pulp erythro-blasts of the spleen, and distal convoluted tubular epithelium of the kidney. Although the tissue distribution pattern of TfR positive cells was similar in normal, iron deficient and iron overloaded rats, the staining intensity and number of TfR positive cells were obviously higher in iron deficient, and lower in iron overloaded rats. We conclude that TfR expression is negatively regulated by the tissue concentration of iron. Acta Pathol Jpn 39: 759-764, 1989.     

第3～5周人胚肝的细胞特征和生长因子及受体表达的研究

用第 3～ 5周人胚 ,石蜡切片 ,免疫组化染色 ,光镜下观察人胚肝的细胞特征和HGF、IGF -I、TGFβ1等生长因子及其受体、PCNA、AFP、CK19等的表达。结果发现第 3周末肝芽形成 ,第 4周肝索开始形成 ,第 3～ 4周人胚肝由同一类具有幼稚细胞形态学特征的细胞构成。这些细胞为AFP、c Met阳性反应。第 5周时肝索细胞的数量增加 ,开始出现PCNA的表达 ,仍仅为同一类细胞。第 5周肝索细胞呈IGF -I、TGFβ1及其受体免疫反应阳性 ,HGF阴性 ,其周围的心肌细胞及间充质细胞为HGF阳性反应。结果提示第 3～ 5周 ,组成肝芽和肝索的细胞属于肝干细胞 ,其形态和因子表达的差异说明肝干细胞可能处于不同的发育阶段 ,AFP、c Met可以作为此阶段肝干细胞的标记物 ,HGF、IGF -I、TGFβ1及其受体可能参与对早期人胚肝发育的调节。     

Behavioral effects of iron deficiency in the adult rat

Adult hooded rats were placed on an iron deficient diet for 2 (Stage 1), 8 (Stage 2) or 12 (Stage 3) weeks. Iron levels were monitored by a number of indices, including hemoglobin concentration, serum iron level, transferrin saturation, total and latent iron binding capacity and brain and liver nonheme iron level. All three dietary conditions yielded depressed hemoglobin levels. The most significant observation was a marked reduction in liver and brain iron levels for Stage 1 animals. All animals were trained on a single trial taste-aversion task. The taste of saccharin solution was paired with either an immediate or delayed intragastric injection of lithium chloride. Memory for the association was measured 24 hr or 4 days after training and was found to be significantly below normal for Stage 1 and not significantly different from normal for either Stage 2 or Stage 3 animals. Learning remained at normal levels and was even enhanced at some stages of deficiency (Stages 2 and 3). Activity levels decreased with increasing iron deficiency. There appeared to be a direct association between memory for the taste aversion task and liver and brain iron levels.     

Allogeneic Transplantation,Fas Signaling,and Dysregulation of Hepcidin

Hepatic iron overload is common in patients undergoing hematopoietic cell transplantation. We showed previously in a murine model that transplantation of allogeneic T cells induced iron deposition and down-regulation of hepcidin (Hamp) in hepatocytes. We hypothesized that hepatic injury was related to disrupted iron homeostasis triggered by the interaction of Fas-ligand, expressed on activated T cells, with Fas on hepatocytes. In the current study, we determined the effects of modified expression of the Flice inhibitory protein (FLIP long [FLIPL]), which interferes with Fas signaling, on the impact of Fas-initiated signals on the expression of IL-6 and Stat3 and their downstream target, Hamp. To exclude a possible contribution by other pathways, we used agonistic anti-Fas antibodies (rather than allogeneic T cells) to trigger Fas signaling. Inhibition of FLIPL by RNA interference resulted, as expected, not only in enhanced hepatocyte apoptosis in response to Fas signals, but also in decreased levels of IL-6, Stat3, and Hamp. In contrast, overexpression of FLIPL protected hepatocytes against agonistic anti-Fas antibody-mediated apoptosis and increased the levels of IL-6 and Stat3, thereby maintaining the expression of Hamp in an NF-κB–dependent fashion. In vivo overexpression of FLIPL in the liver via hydrodynamic transfection, similarly, interfered with Fas-initiated apoptosis and prevented down-regulation of IL-6, Stat3, and Hamp. These data indicate that Fas-dependent signals alter the regulation of iron homeostasis and suggest that signals initiated by Fas may contribute to peritransplantation iron accumulation.     

Anti-hepatoma effect of safrole from Cinnamomum longepaniculatum leaf essential oil in vitro

The aim of this study was to study the anti-hepatoma effect of safrole and elucidate its molecular mechanism, the human hepatoma BEL-7402 cells were incubated with various concentrations (40, 80, 160, 320 and 640 μg/ml) of safrole and the cell proliferation and apoptosis were evaluated. The results showed that both the cell proliferation determined by 3-(4,5-dimethyl-thiazolyl-2)-2,5-diphenyl tetrazolium brominde (MTT) assay and cell colony determined by soft agar assay were significantly suppressed by safrole in a dose-time-dependent manner. Characteristic morphological and biochemical changes associated with apoptosis, including cells shrinkage, deformation and vacuolization of mitochondria, nuclear chromatin condensation and fragmentation, formation of apoptotic bodies were observed when treated with safrole for 24 h and 48 h. Cell cycle changes evaluated by flow cytometry analysis showed that the safrole could induce accumulation of cells arrested at G₁ and S phases of the cell cycle. These results demonstrated that safrole is potent anti-hepatoma agent and the underlying mechanism may be attributed to suppress tumor cell growth by inducing cell apoptosis.     

Increased hepatic iron deposition resulting from treatment of chronic hepatitis C with ribavirin

Increased levels of hepatic iron may impair the response of patients with chronic hepatitis C to treatment with interferon-alfa, but combination therapy with ribavirin has demonstrated efficacy in the treatment of hepatitis C. When used alone or with interferon-alfa, ribavirin may cause a dose-dependent reversible hemolytic anemia. We compared the extent and cellular localization of iron deposition in liver tissue from biopsy specimens obtained before and after 36 weeks of therapy with ribavirin or placebo for 59 patients with chronic hepatitis C. Paired slides were available for review from 26 ribavirin and 27 placebo recipients. Iron deposition was assessed using coded slides stained with Perls Prussian blue and was semi-quantitated in hepatocytes, Kupffer cells, and areas of fibrosis. The overall iron score fell by 0.96 in the placebo group and increased 1.69 in the ribavirin recipients. Iron was deposited mainly in hepatocytes; the hepatocyte iron score increased from 2.19 to 3.81 in the ribavirin group. The amount of iron staining in Kupffer cells declined in the placebo group and increased slightly in the ribavirin group. Iron changes in areas of fibrosis were minor and did not differ between groups. Increased total hepatic iron deposition occurred during a 9-month course of ribavirin. Ribavirin-associated hemolysis deposits iron preferentially in hepatocytes. This increased deposition of hepatic iron does not seem to affect the biochemical or histologic response to ribavirin therapy but may have implications for hepatocyte susceptibility to future injury.     

去铁胺诱导的自噬增加线粒体内铁的含量并促进三阴性乳腺癌细胞 MDA-MB-231 的迁移

癌细胞转移是乳腺癌患者死亡的主要原因,铁代谢异常造成的铁超载能促进乳腺癌细胞的增殖和迁移。去铁胺（DFO）是被广泛使用的铁螯合剂,能抑制多种肿瘤细胞的增殖,降低雌激素受体（ER）阳性人类乳腺癌细胞中的铁含量,但增加了三阴性乳腺癌（TNBC）细胞中的铁含量。此外,DFO 可以增加线粒体内铁的含量,然而线粒体内铁的来源尚不清楚。该研究通过免疫荧光、蛋白质印迹及电感耦合等离子质谱,分析侵袭性乳腺癌 MDA-MB-231 和非侵袭性乳腺癌 MCF-7中的铁代谢和自噬相关蛋白,利用自噬抑制剂探究线粒体铁增多的机制。该研究发现,DFO 能通过诱导铁蛋白自噬促进线粒体内铁积累增加,从而促进 TNBC 细胞的上皮-间质转化与迁移。经过 DFO 处理,线粒体钙离子单向转运蛋白（MCU）和线粒体铁转运蛋白 1（Mfrn1）可能参与了铁从细胞质向线粒体的转运。该研究为临床中探寻新的靶向铁代谢治疗三阴性乳腺癌的方式提供了研究基础。     

Liver cell carcinomas in the medaka (Oryzias latipes) induced by methylazoxymethanol-acetate

Medaka (Oryzias latipes) were treated with 0.3 ppm methylazoxymethanol-acetate (MAM-A) for 3 days (group I) and with 0.1 ppm MAM-A for 2 weeks (group II) and the effects on the medaka liver cells up to 6 weeks after commencement of the treatment and the induction of liver tumours at the 18th or 24th weeks were observed by light and electron microscopy. As an early effect, PAS-positive granules and basophilic liver cells appeared at the fourth or fifth week in group II. In group I, PAS-positive granules, basophilic liver cells and eosinophilic liver cells were observed from the second week. They tended to increase in number and formed aggregate bodies and basophilic liver cell foci. The aggregate bodies of PAS-positive granules resembled melano-macrophage centres (MMC). However, there were ultrastructural differences between these aggregate bodies and MMC. Basophilic liver cells contained much rough-surfaced endoplasmic reticulum and eosinophilic liver cells contained much smooth-surfaced endoplasmic retriculum and mitochondria by electron microscopical observation. Liver cell nodules appeared in 3 out of 21 fish but liver tumours were not found within 24 weeks in group II. In group I, basophilic liver cell foci progressed to liver cell nodules in 5 out of 9 fish. Furthermore, they progressed to liver cell adenomas in 3 and carcinomas in 3 of 9 fish at 18 weeks. The sequential morphological alterations observed in the present experiment were similar to those obtained in rats. Basophilic liver cell foci appeared to be an early aspect of hepatocarcinogenesis in medaka. Considering the susceptibility of medaka to the carcinogenic effect of MAM-A in a relatively short time and the low concentration of this material required for induction of tumours, medaka seems to be a useful animal for research on neoplasms and pre-screening carcinogenicity of chemicals.     

Seasonal siderosis in female eider nesting in Svalbard

Specimens of female and male eider (Somateria mollissima) were collected in Svalbard. Atomic absorption analyses revealed mean hepatic iron concentrations of 280 micrograms per g wet weight 2 to 3 weeks before egg laying, 2620 micrograms per g after 2 to 3 weeks brooding and 800 micrograms per g 2 weeks after hatching. At the highest concentration, there was massive siderosis with the iron located in both parenchymal and non-parenchymal cells. No signs of liver injury were seen. The female eider fasts completely from the start of egg laying to the end of hatching. The non-parenchymal iron deposits are probably the result of catabolism of blood and lean tissue, i.e., translocation of body iron. The parenchymal siderosis can hardly be explained by liver weight loss alone. An increased iron absorption preceding the egg laying is suggested.     

Hereditary hemochromatosis of a young girl: detection of early iron deposition in liver cell lysosomes using transmission electron microscopy and electron energy loss spectroscopy

A 14-year-old girl demonstrated increased iron concentration and transferrin saturation, suggesting iron overload of unknown origin. Liver biopsy showed no fibrosis or hepatocytic atrophia. Nevertheless, Prussian blue reaction for histochemical detection of iron demonstrated very weak positive granules in a few hepatocytes on the periphery of hepatic lobules in close connection to bile capillaries. This very early stage of hemochromatosis was confirmed by TEM and EELS for iron accumulation inside hepatocytic lysosomes and residual bodies. Such siderosomes were scarce in number and iron content, compared to a case of manifested hemochromatosis and liver cirrhosis (Jonas L, Fulda G, Salemeh T, et al. Ultrastruct Pathol . 2001; 25: 111-118.). Liver iron concentration as measured by inductively coupled plasma-mass spectrometry (ICP-MS) and atomic absorption spectrometry (AAS) yielded 2.005 mg/g tissue dry weight, which was considered not significantly increased. In the absence of known causes for secondary iron overload, the early diagnosis was evidenced by genotyping, revealed homozygosity for the HFE gene C282Y mutation, demonstrating the presence of hereditary hemochromatosis.     

DNA adducts from alkoxyallylbenzene herb and spice constituents in cultured human (HepG2) cells

Alkoxy derivatives of allylbenzene, including safrole, estragole, methyleugenol, myristicin, dill apiol, and parsley apiol, are important herb and spice constituents. Human exposure occurs mainly through consumption of food and drinks. Safrole, estragole, and methyleugenol are weak animal carcinogens. Experimental data reveal the genotoxicity and/or carcinogenicity of some allylbenzenes; however, except for safrole, the potential capacity of allylbenzenes for forming adducts in human cellular DNA has not been investigated. In the present study, we have exposed metabolically competent human hepatoma (HepG2) cells to three concentrations (50, 150, and 450 muM) of each of the six aforementioned allylbenzenes and shown by the monophosphate (32)P-postlabeling assay that each compound formed DNA adducts. With the exception of methyleugenol, DNA adduction was dose dependent, decreasing in the order, estragole > methyleugenol > safrole approximately myristicin > dill apiol > parsley apiol. These results demonstrate that safrole, estragole, methyleugenol, myristicin, dill apiol, and parsley apiol are capable of altering the DNA in these cells and thus may contribute to human carcinogenesis.