Ferrous and ferric differentially deteriorate proliferation and differentiation of osteoblast-like UMR-106 cells

The association between iron overload and osteoporosis has been found in many diseases, such as hemochromatosis, β-thalassemia and sickle cell anemia with multiple blood transfusion. One of the contributing factors is iron toxicity to osteoblasts. Some studies showed the negative effects of iron on osteoblasts; however, the effects of two biological available iron species, i.e., ferric and ferrous, on osteoblasts are elusive. Since most intracellular ionized iron is ferric, osteoblasts was hypothesized to be more responsive to ferric iron. Herein, ferric ammonium citrate (FAC) and ferrous ammonium sulfate (FAS) were used as ferric and ferrous donors. Our results showed that both iron species suppressed cell survival and proliferation. Both also induced osteoblast cell death consistent with the higher levels of cleaved caspase 3 and caspase 7 in osteoblasts, indicating that iron induced osteoblast apoptosis. Iron treatments led to the elevated intracellular iron in osteoblasts as determined by atomic absorption spectrophotometry, thereby leading to a decreased expression of genes for cellular iron import and increased expression of genes for cellular iron export. Effects of FAC and FAS on osteoblast differentiation were determined by the activity of alkaline phosphatase (ALP). The lower ALP activity from osteoblast with iron exposure was found. In addition, ferric and ferrous differentially induced osteoblastic and osteoblast-derived osteoclastogenic gene expression alterations in osteoblast. Even though both iron species had similar effects on osteoblast cell survival and differentiation, the overall effects were markedly stronger in FAC-treated groups, suggesting that osteoblasts were more sensitive to ferric than ferrous.     

Chronic iron overload inhibits protein secretion by adult rat hepatocytes maintained in long-term primary culture

Short-term pure cultures and long-term cocultures of adult rat hepatocytes with rat liver epithelial cells, presumably derived from primitive biliary cells, were used to define in vitro models of iron overloaded hepatocytes in order to understand the molecular mechanism responsible for liver damage occurring in patients with hemochromatosis. In vitro iron overload was obtained by daily addition of ferric nitrilotriacetate to the culture medium. A concentration of 20 microM ferric salt induced hepatocyte iron overload with minimal cytotoxicity as evaluated by cell viability, morphological changes of treated cells and cytosolic enzyme leakage into the culture medium. The effects of iron overload on protein biosynthesis and secretion were studied in both short-term pure cultures and long-term cocultures of hepatocytes. The amounts of intracellular and newly synthesized proteins were never modified by the iron treatment. Furthermore, neither the relative amounts of transferrin and albumin mRNAs nor their translational products were altered by iron overload. Moreover, no change in the transferrin isomeric forms were observed in treated cells. In contrast, a prolonged exposure of cocultured hepatocytes to 20 microM ferric salt led to a significant decrease in the amount of proteins secreted in the medium. This decrease included the two major secreted proteins, namely albumin and transferrin, and probably all other secreted proteins. These results demonstrate that iron loading alters neither the total nor the liver specific protein synthesis activity of cultured hepatocytes. They suggest that chronic overload may impede the protein secretion process.     

高浓度葡萄糖通过p38MAPK-TXNIP/TRX-ROS通路抑制MC3T3-E1细胞成骨分化

目的：研究高浓度葡萄糖抑制MC3T3-E1细胞成骨分化的机理。方法：建立MC3T3-E1细胞成骨分化诱导体系,观察不同浓度葡萄糖（5.5mM和22mM）对MC3T3-E1细胞成骨分化的影响;用不同浓度的p38 MAPK抑制剂Fr167653（0.1μM、1.0μM和10μM）进行药物干预,观察MC3T3-E1细胞在22mM葡萄糖浓度下成骨分化的变化情况。通过钙含量检测、Real time PCR检测相关分化的变化;用Western Blot方法检测MC3T3-E1细胞分化过程中p38 MAPK磷酸化状态、TXNIP表达水平的变化;使用胰岛素二硫键还原法检测细胞内TRX活性水平;使用活性氧检测试剂盒检测细胞内自由氧生成水平。结果：体外诱导条件下,高浓度（22mM）葡萄糖通过升高p38 MAPK磷酸化水平,上调TXNIP表达水平,同时降低TRX活性,使细胞内自由氧生成增加,抑制MC3T3-E1细胞的成骨分化;Fr167653通过抑制p38 MAPK磷酸化,下调TXNIP表达同时升高TRX活性,抑制细胞内自由氧生成,解除高浓度葡萄糖对细胞成骨分化的抑制作用。结论：高浓度葡萄糖通过p38 MAPK-TXNIP/TRX-ROS信号通路抑制MC3T3-E1细胞成骨分化。     

Iron overload induces apoptosis of osteoblast cells via eliciting ER stress-mediated mitochondrial dysfunction and p-eIF2α/ATF4/CHOP pathway in vitro

Iron is an essential element for crucial biological function; whereas excess iron sedimentation impairs the main functions of tissues or organs. Cumulative researches have shown that the disturbances in iron metabolism, especially iron overload is closely concatenating with bone loss. Nevertheless, the specific process of iron overload-induced apoptosis in osteoblasts has not been thoroughly studied. In this study, our purpose is to elucidate the mechanism of osteoblast apoptosis induced by iron overload via the MC3T3-E1 cell line. Ferric ammonium citrate (FAC) was utilized to simulate iron overload conditions in vitro. These results showed that treatment with FAC dose-dependently induced the apoptosis of MC3T3-E1 cells at 48 h, dysfunction of iron metabolism, and increased intracellular reactive oxygen species (ROS) levels. Following, FAC does-dependently caused the calcium dyshomeostasis, decreased the calcium concentration in endoplasmic reticulum (ER), but increased the crosstalk between ER and mitochondria, and calcium concentration in the mitochondria. Moreover, FAC dose-dependently decreased mitochondrial membrane potential (MMP) and enhanced the expression of apoptosis related proteins (Bax, Cyto-C and C-caspase3). We furthermore revealed that FAC treatment activated the ER-mediated cell apoptosis via p-eIF2α/ATF4/CHOP pathway in MC3T3-E1 osteoblasts cells. In addition, pretreatment with the N-acetylcysteine (NAC) or Tauroursodeoxycholate Sodium (TUDC) attenuated cell apoptosis, ROS levels, mitochondria fragmentation and ER stress-related protein expression, and recovered the protein expression related to iron metabolism. In conclusion, our finding suggested that iron overload induced apoptosis via eliciting ER stress, which resulted in mitochondrial dysfunction and activated p-eIF2α/ATF4/CHOP pathway.     

Red blood cells upregulate cytoprotective proteins and the labile iron pool in dividing human T cells despite a reduction in oxidative stress

We have recently reported that red blood cells (RBC) promote T cell growth and survival by inhibiting activation-induced T cell death. In the present study, we have examined parameters of oxidative stress and intracellular iron in activated T cells and correlated these data with the expression of ferritin, heme oxygenase-1 (HO-1), and the transferrin receptor CD71. T cells growing in the presence of RBC had reduced levels of reactive oxygen species (ROS) and oxidatively modified proteins, suggesting that RBC efficiently counteracted ROS production on the activated T cells. Flow cytometry and immunodetection demonstrated that T cells dividing in the presence of RBC had increased levels of intracellular ferritin rich in L-subunits and HO-1 along with a downmodulation in CD71 expression. Finally, using the fluorescent iron indicator calcein and flow cytometry analysis, we were able to show that a relative amount of the labile iron pool (LIP) was upregulated in T cells growing in the presence of RBC. These findings are consistent with a typical response to iron overload. However, neither heme compounds nor ferric iron reproduced the levels of expansion and survival of T cells induced by intact RBC. Altogether, these data suggest that RBC inhibit apoptosis of activated T cells by a combination of ROS scavenging and upregulation of cytoprotective proteins such as ferritin and HO-1, which may counteract a possible toxic effect of the increased intracellular free iron.     

Iron overload threatens the growth of osteoblast cells via inhibiting the PI3K/AKT/FOXO3a/DUSP14 signaling pathway

Iron overload is a common stress in the development of cells. Growing evidence has indicated that iron overload is associated with osteoporosis. Therefore, enhancing the understanding of iron overload would benefit the development of novel approaches to the treatment of osteoporosis. The purpose of the present study was to analyze the effect of iron overload on osteoblast cells, via the MC3T3-E1 cell line, and to explore its possible underlying molecular mechanisms. Ferric ammonium citrate (FAC) was utilized to simulate iron overload conditions in vitro. FAC-induced iron overload strongly suppressed proliferation of osteoblast cells and induced apoptosis. Moreover, iron overload strongly suppressed the expression of dual-specificity phosphatase 14 (DUSP14). Additionally, overexpression of DUSP14 protected osteoblast cells from the deleterious effects of iron overload, and this protective effect was mediated by FOXO3a. Additionally, matrine rescued the function of DUSP14 in osteoblast cells. Most importantly, our analysis demonstrated the essential role of the PI3K/AKT/FOXO3a/DUSP14 signaling pathway in the defense against iron overload in osteoblast cells. Overall, our results not only elucidate deleterious effects of iron overload, but also unveil its possible signaling pathway in osteoblast cells.     

Iron accumulation, iron-mediated toxicity and altered levels of ferritin and transferrin receptor in cultured astrocytes during incubation with ferric ammonium citrate

The cellular uptake and storage of iron have to be tightly regulated in order to provide iron for essential cellular functions while preventing the iron-catalysed generation of reactive oxygen species (ROS). In contrast to cells in other organs, little is known about the regulation of iron metabolism in brain cells, particularly in astrocytes. To investigate the regulation of iron metabolism in astrocytes we have used primary astrocyte cultures from the brains of newborn rats. After application of ferric ammonium citrate (FAC), cultured astrocytes accumulated iron in a time- (0-48 h) and concentration-dependent (0.01-1 mm) manner. This accumulation was prevented if FAC was applied in combination with the iron-chelator deferoxamine (DFX). Application of FAC to astrocyte cultures caused a strong increase in the cellular content of the iron storage protein ferritin and a decrease in the amount of transferrin receptor (TfR), which is involved in the transferrin-mediated uptake of iron into cells. In contrast, application of DFX strongly increased the level of TfR. Both up-regulation of ferritin content by iron application and up-regulation of TfR content by DFX were prevented by the protein synthesis inhibitor cycloheximide (CHX). During incubation of astrocytes with FAC, a mild and transient increase in the extracellular activity of the cytosolic enzyme lactate dehydrogenase and in the concentration of intracellular ROS was observed. In contrast, prevention of protein synthesis by CHX during incubation with FAC resulted in significantly more cell loss and a persistent and intense increase in the production of intracellular ROS. These results demonstrate that both iron accumulation and deprivation modulate the synthesis of ferritin and TfR in astrocytes and that protein synthesis is required to prevent iron-mediated toxicity in astrocytes.     

Hepcidin increases intracellular Ca2+ of osteoblast hFOB1.19 through L-type Ca2+ channels

Hepcidin is a key player in the regulation of iron homeostasis. Several pathological conditions associated with iron overload are attributed to the depressed expression of hepcidin and are often associated with bone diseases including osteoporosis. Hepcidin was suggested to have anti-osteoporosis effects by preventing iron overload. We recently observed that hepcidin could increase intracellular calcium concentration in cultured osteoblast cells. The present study was designed to elucidate the source of the increased intracellular calcium following hepcidin activation. Cultured hFOB1.19 cells were used to test whether there was a dose dependent effect of hepcidin on increasing intracellular calcium. After finding the optimal concentration in increasing intracellular calcium, Cultured hFOB1.19 cells were then divided into three groups: (1) control group, (2) and (3) groups pretreated with either nimodipine (2 × 10(-5)mol/L) or EDTA (2 × 10(-3)mol/L) for 10 min before incubation with hepcidin (100 nmol/L). All cells were stimulated with hepcidin for 60 min and then stained with fluo-3/AM for 40 min before the intracellular calcium was observed using flow cytometry (FCM). As compared with controls, hepcidin treatment significantly increased intracellular calcium concentration. This effect was blocked by nimodipine and EDTA pretreatments which suggested that hepcidin-mediated calcium inflow was mainly through L-type Ca(2+) channels and that the release of intracellular calcium store was not significant. Hepcidin increases of intracellular calcium may be related to its anti-osteoporosis effect but this hypothesis needs further investigation.     

Gastrins, iron and colorectal cancer

This minireview explores the connections between circulating gastrins, iron status and colorectal cancer. The peptide hormone gastrin is a major regulator of acid secretion and a potent mitogen for normal and malignant gastrointestinal cells. Gastrins bind two ferric ions with μM affinity and, in the case of non-amidated forms of the hormone, iron binding is essential for biological activity. The ferric ion ligands have been identified as glutamates 7, 8 and 9 in the 18 amino acid peptide glycine-extended gastrin. An interaction between gastrin and transferrin was first demonstrated by covalent crosslinking techniques, and has been recently confirmed by surface plasmon resonance. We have therefore proposed that gastrins act as catalysts in the loading of transferrin with iron. Several recent lines of evidence, including the facts that the concentrations of circulating gastrins are increased in mice and humans with the iron overload disease haemochromatosis, and that transferrin saturation positively correlates with circulating gastrin concentrations, suggest that gastrins may be involved in iron homeostasis. In addition the recognition that ferric ions may play an unexpected role in the biological activity of non-amidated gastrins may assist in the development of new therapies for colorectal carcinoma.     

氧化还原对铁结合的大肠杆菌拓扑异构酶Ⅰ活性的调控

大肠杆菌拓扑异构酶 I（E. coli TopA）属于 I 型拓扑异构酶,在DNA复制、转录、重组和基因表达调控等过程中发挥关键作用。E. coli TopA 不仅能结合锌,还可以结合铁。细胞内过量铁可与锌竞争,通过与锌指结构域结合减弱其 DNA 结合能力和改变蛋白质空间构象,从而抑制TopA拓扑异构酶活性。然而,铁结合形式TopA的氧化还原特性以及氧化还原条件对其活性的影响仍不清楚。本研究通过紫外分光光谱和体外DNA拓扑异构酶活性分析,发现体外纯化得到的铁结合形式的 TopA 呈氧化状态,能够被二硫苏糖醇和连二亚硫酸钠还原,原本氧化状态下无活性的TopA在还原条件下,可恢复其拓扑异构酶活性。当还原剂被去除后,铁结合的TopA在空气中能够重新被氧化,且其活性重新受到抑制。这说明,氧化还原条件对铁结合的 TopA 功能具有可逆调节作用。通过金属 蛋白体外结合实验进一步发现,无金属结合的TopA蛋白(apo-TopA)在无氧条件下,与 Fe²⁺ 和 Fe³⁺ 均能结合,但与Fe²⁺ 结合能力较弱,并且TopA结合的Fe³⁺ 被还原成Fe²⁺ 后,结合力显著下降,能够被铁螯合指示剂菲咯嗪快速捕获。此外,蛋白质内源性荧光光谱分析实验表明,铁结合的TopA在氧化还原的不同状态时,其在330 nm左右的荧光值有显著差异。这提示,氧化还原条件可能通过影响铁离子与TopA的结合状态,引起蛋白质空间构象改变,从而对TopA的拓扑异构酶活性进行调节。此研究表明,铁结合TopA的拓扑异构酶活性会受到细胞内氧化还原信号的可逆调控,也提示I型拓扑异构酶可能是细胞铁超载通过氧化损伤引起细胞功能障碍（或铁死亡）的靶点之一。     

Gastrins, iron homeostasis and colorectal cancer

The peptide hormone gastrin has been identified as a major regulator of acid secretion and a potent mitogen for normal and malignant gastrointestinal cells. The importance of gastric acid in the absorption of dietary iron first became evident 50 years ago when iron deficiency anemia was recognized as a long-term consequence of partial gastrectomy. This review summarizes the connections between circulating gastrins, iron status and colorectal cancer. Gastrins bind two ferric ions with micromolar affinity and, in the case of non-amidated forms of the hormone, iron binding is essential for biological activity in vitro and in vivo. The demonstration of an interaction between gastrin and transferrin by biochemical techniques led to the proposal that gastrins catalyze the loading of transferrin with iron. Several lines of evidence, including the facts that the concentrations of circulating gastrins are increased in mice and humans with the iron overload disease hemochromatosis and that transferrin saturation positively correlates with circulating gastrin concentration, suggest the potential involvement of gastrins in iron homeostasis. Conversely, recognition that ferric ions play an unexpected role in the biological activity of gastrins may assist in the development of useful therapies for colorectal carcinoma and other disorders of mucosal proliferation in the gastrointestinal tract.     

Iron overload inhibits calcification and differentiation of ATDC5 cells

There is a little information about the effects of iron overload on cartilage metabolism. In the present study, we examined the effects of excess iron on the differentiation and mineralization of cultured chondrocytes, ATDC5 cells. We used ferric ammonium citrate (FAC) as a ferric ion donor and desferrioxamine (DFO) as a ferric ion chelator. Neither chemical affected the production of proteoglycan, a marker of an early stage of ATDC5 differentiation. In contrast, FAC inhibited the deposition of calcium, a late-stage event in chondrocyte differentiation, by ATDC5 cells in a dose-dependent manner, and DFO accelerated it. Energy dispersive X-ray spectroscopy/scanning electron microscope analysis revealed that the levels of iron and calcium in cells treated with FAC were increased and decreased, respectively. Furthermore, FAC inhibited the expression of matrix metalloproteinase 13 mRNA, another marker of late-stage chondrocyte differentiation. In addition, we found that the heavy and light chains of ferritin were expressed specifically at a late stage of ATDC5 differentiation, and the levels of both proteins were enhanced by the addition of iron. These results suggest that iron overload might give rise to osteopenia and arthritis by inhibiting chondrocyte differentiation and mineralization.     

Icariin protects against iron overload-induced bone loss via suppressing oxidative stress

Iron overload is common in patients with diseases such as hemoglobinopathies, hereditary hemochromatosis or elderly men and postmenopausal women. This disorder is frequently associated with bone loss and recently has been considered as an independent risk factor for osteoporosis. By excess reactive oxygen species (ROS) production through Fenton reaction, iron could induce osteoblast apoptosis, inhibit osteoblast osteogenic differentiation. Moreover, Iron could also promote osteoclasts differentiation and bone absorption. The goal of the study is to investigate whether icariin could reverse iron overload-induced bone loss in vitro and in vivo. Icariin is the major active ingredient of Herba Epimedii and has antioxidant, antiosteoporosis functions. In the current study, we demonstrated that oral administration of icariin significantly prevented bone loss in iron overloaded mice. Icariin could protect against iron overload-induced mitochondrial membrane potential dysfunction and ROS production, promote osteoblast survival and reverse the reduction of Runx2, alkaline phosphatase, and osteopontin expression induced by iron overload. Icariin also inhibited osteoclasts differentiation and function. Moreover, we also found that icariin remarkably reduced iron accumulation in bone marrow, suggesting that icariin has the ability to regulate systemic iron metabolism in vivo. These results indicated that icariin could be a potential natural resource for developing medicines to prevent or treat iron overload-induced osteoporosis.     

Growth-stimulating effect of transferrin on a hybridoma cell line: Relation to transferrin iron-transporting function

The relation of the growth-stimulating capacity of transferrin to its iron-transporting function was investigated in mouse hybridoma PLV-01 cells cultivated in a chemically defined medium. The cells were precultivated in protein-free medium supplemented either with ferric citrate (cells with a high intracellular iron level) or with iron-saturated transferrin (cells with a low intracellular iron level). Iron uptake was monitored after the application of 59Fe-labeled ferric citrate or pig transferrin. Cultivation of the cells at the optimum growth-stimulating concentration (500 microM) of ferric citrate resulted in an intracellular iron level about 100-fold higher than that of cells cultivated at the optimum transferrin concentration (5 micrograms/ml). Replacement of pig transferrin with bovine transferrin resulted in similar intracellular iron levels, but the growth-stimulating effect of bovine transferrin was more than one order of magnitude lower. Cells with a high intracellular iron level grew equally well when cultivated with iron-saturated transferrin or with apotransferrin + deferoxamine (2 micrograms/ml). On the other hand, cells with a low intracellular iron level required iron-saturated transferrin for further growth and apotransferrin + deferoxamine was ineffective. The results suggest that transferrin can act as a cell growth factor only in the iron-saturated form. However, several findings of this work indicate that supplying cells with iron cannot be accepted as the full explanation of the transferrin growth-stimulating effect.     

Colorimetric ferrozine-based assay for the quantitation of iron in cultured cells

The ferrozine-based colorimetric assay described here permits the quantitation of iron in cultured cells in amounts ranging between 0.2 and 30 nmol. Ferrous and ferric iron were detected equally well by the assay and the accuracy was unaffected by other divalent metal cations. This colorimetric assay was used to study iron accumulation in brain astrocytes that had been cultured in 24-well dishes. Iron complexed to cellular proteins was made accessible to ferrozine by treatment of cell lysates with acidic KMnO(4) solution. The basal amounts of iron in untreated astrocyte cultures were approximately 10 nmol iron per mg protein. Incubation of the cells with ferric ammonium citrate caused the total cellular iron content to increase in a concentration-dependent manner. The estimates of cellular iron content that were obtained with the ferrozine-based assay did not differ from those determined by atomic absorption spectroscopy. The colorimetric assay described here provides a sensitive, cheap, and reliable method for the quantitation of intracellular iron and for the investigation of iron accumulation in cultured cells.     

Age-dependent decline in bone nodule formation stimulating activity in rat serum is mainly due to the change in the corticosterone level

The replacement of fetal bovine serum with rat serum in a culture medium brought about a marked increase in the formation of mineralized bone nodules (BN) in primary cultures of rat calvarial cells. These effects of rat serum were most prominent when added during the early phase of the culture, indicating that the serum factor mainly acts on the cells during the growing phase. A significant increase in BN formation was observable at final rat serum concentration as low as 1%, and the effect was dependent on serum concentration, at least up to 10%. The addition of rat serum also increased alkaline phosphatase (ALP) activity, collagen synthesis, and DNA synthesis in calvarial cells. BN formation stimulating activity was extractable with ethyl acetate. The ethyl acetate extract was purified by TSK-GEL OH-120 column chromatography by monitoring the stimulation of ALP activity in ROS 17/2.8 cells. The chromatographic behavior of the ALP activity was found to be identical to that of corticosterone, the major glucocorticoid in rodents and the preincubation of the purified fraction with anticorticosterone antibody abolished the ALP stimulating activity. These results suggest that BN formation stimulating activity in rat serum is mainly attributable to corticosterone. The concentration of serum corticosterone decreased with age in parallel with BN formation stimulating activity, which suggests that the physiological level of corticosterone may have a regulatory role in the maintenance of osteoblast function.     

Chelation of intracellular iron enhances endothelial barrier function: A role for vitamin C?

Ascorbic acid improves endothelial barrier function by decreasing the permeability of endothelial cells cultured on semi-porous membrane filters. This decrease was not due to enhanced collagen synthesis and was mimicked by the collagen synthesis inhibitor ethyl-3,4-dihydroxybenzoic acid (EDHB). Since EDHB is known to chelate intracellular free iron, the effects of two membrane-permeant iron chelators were tested on endothelial permeability. Both 2,2′-dipyridyl and desferrioxamine decreased trans-endothelial permeability in a concentration-dependent manner. Increasing intracellular iron with a chelate of 8-hydroxyquinoline and ferric iron prevented effects of both EDHB and intracellular ascorbate. That EDHB and ascorbate did in fact chelate intracellular iron was supported by finding that they both decreased the cellular fluorescence quenching of the iron-sensitive dye Phen green SK. These results show that chelation of intracellular iron decreases endothelial barrier permeability and implicate this mechanism in the ability of EDHB and possibly intracellular ascorbate to tighten the endothelial barrier.     

Liver iron overload induced by tamoxifen in diabetic and non-diabetic female Wistar rats

Tamoxifen (TX), a drug used in the treatment of breast cancer, may cause hepatic changes in some patients. The consequences of its use on the liver tissues of rats with or without diabetes mellitus (DM) have not been fully explored. The purpose of this study was to evaluate the correlation between plasma hepatic enzyme levels and the presence of iron overload in the hepatic tissue of female Wistar rats with or without streptozotocin-induced DM and using TX. Female rats were studied in control groups: C-0 (non-drug users), C-V (sorbitol vehicle only) and C-TX (using TX). DM (diabetic non-drug users) and DM-TX (diabetics using TX) were the test groups. Sixty days after induced DM, blood samples were collected for glucose, alanine aminotransferase (ALT), aspartate aminotransferase (AST) alkaline phosphatase (ALP) and bilirubin measures. Hepatic fragments were processed and stained with hematoxylin and eosin, Masson’s trichrome, Perls. The hepatic iron content was quantified by atomic absorption spectrometry. AST, ALT and ALP levels were significantly elevated in the DM and DM-TX groups, with unchanged bilirubin levels. Liver iron overload using Perls stain and atomic absorption spectrometry were observed exclusively in groups C-TX and DM-TX. There was positive correlation between AST, ALT and ALP levels and microscopic hepatic siderosis intensity in group DM-TX. In conclusion, TX administration is associated with liver siderosis in diabetic and non-diabetic rats. In addition, TX induced liver iron overload with unaltered hepatic function in non-diabetic rats and may be a useful tool for investigating the biological control of iron metabolism.