Diverse effects of chronic cobalt supplementation on iron metabolism during erythropoiesis

Diverse effects of chronic cobalt supplementation on iron metabolism during erythropoiesis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Diverse effects of chronic cobalt supplementation on iron metabolism during erythropoiesis Ekaterina Pavlova, Emilia Petrova, Alexey A. Tinkov, Olga P. Ajsuvakova, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4697764/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Cobalt (Co) is an essential trace element and its cellular uptake occurs in a similar to iron (Fe) profile. The aim was to assess the alterations in iron and Fe regulatory proteins concentrations - transferrin receptor 1 (TfR1), hepcidin and ferritin, and their effect on erythrocyte count (RBCs) in mice following chronic exposure to cobalt chloride (CoCl 2 ). Pregnant ICR mice were subjected to 125 mg/kg body weight CoCl 2 x6H 2 O daily 2–3 days prior delivery and treatment continued 90 days after birth. CoCl 2 was administrated with drinking water. Pups were sacrificed on postnatal days 18, 30, 45, 60 and 90. Exposure to CoCl 2 induced significant accumulation of Co ions in blood sera and RBCs. During long-term exposure the most Co was accumulated in the serum after 30 days of exposure and decreased by day 90 of dosing indicating that serum Co concentration is a reliable marker for recent exposure. Hemoglobin content increased in a time-dependent manner. Co administration significantly elevated serum Fe but decreased it in RBCs. Exposure to Co stimulated Fe storage, enhancing hepcidin production and ferritin concentrations, and reducing TfR1 expression. Chronic exposure to CoCl 2 resulted in a lower Fe content of mature mice compared to immature suggesting stimulated Fe release as a possible survival mechanism to counteract the toxic effects of Fe overload. cobalt chloride iron TfR1 hepcidin ferritin erythrocytes 1. Introduction Cobalt (Co) is an essential trace element that may cause severe health effects upon occupational or environmental overexposure. Cobalt ions stabilize hypoxia-inducible transcription factors (HIFs) that increase the expression of the erythropoietin (Epo) gene. In the past, cobalt chloride (CoCl 2 ) was administered to patients at daily doses of 25 to 300 mg as an anti-anaemic agent for stimulating erythropoiesis [ 1 ]. The existing data show that Co cellular uptake occurs through interaction with transferrin receptors and the uptake profile is similar to that of iron (Fe) [ 2 ]. Fe uptake, storage and intracellular trafficking are tightly controlled by the regulatory proteins – transferrin receptor 1 (TfR1), divalent metal transporter 1 (DMT1), hepatic hormone hepcidin (Hep), ferritin (Fer) and ferroportin (Fpn). Fe 3+ binds transferrin and is imported into cells through the membrane TfR1 and then is internalized in the endosome by clathrin-mediated endocytosis where it is reduced to ferrous iron by ferrireductase six-transmembrane epithelial antigen of the prostate 3 - STEAP3 [ 3 ]. Fe 2+ release from the endosome and transport into the cytosol is mediated by DMT1. Iron is then either utilized in metabolic processes, such as synthesis of hemoproteins and Fe-S cluster, sequestered and stored in cytosolic ferritin or exported by ferroportin. Hepcidin controls Fpn expression in the enterocytes, thus regulating whole body Fe absorption [ 4 ]. Although all cells may import, export or store iron, some have specific functions: e.g., erythroblasts are specialized in iron uptake, macrophages and enterocytes in iron export, and hepatocytes in iron storage. Within cells most iron is transferred to mitochondria for heme and Fe-S cluster production [ 5 ]. As Fe is primarily utilized by erythroid cells in the bone marrow of humans and vertebrates, a significant effect on erythrocyte renewal is expected. The differentiation from erythroblasts to erythrocytes is strongly Fe-dependent [ 6 ]. As erythropoietin, a hormone produced by the kidneys, stimulates red blood cell production, it also enhances the synthesis of erythroferrone (ERFE). ERFE is secreted by the erythroblasts and is the main erythroid regulator of hepatic hepcidin [ 7 ]. By decreasing the transcription of the gene encoding the iron-regulatory hormone hepcidin, ERFE stimulates iron absorption, as well as the release of iron from recycling macrophages and from stores in hepatocytes [ 8 ]. Dietary Fe is then used for heme and hemoglobin synthesis in the newly produced red blood cells. Besides Fe storage, ferritin also provides protection from oxidative damage [ 5 ]. Circulating differic transferrin (Tf) affects hepcidin production through binding to its second receptor - TfR2 [ 9 ]. The aim of the study is to assess the alterations in iron content and Fe regulatory protein (TfR1, hepcidin and ferritin) concentrations, and their effect on erythrocytes in mice following chronic exposure to cobalt chloride (CoCl 2 ). The study contributes to the elucidation for the role of CoCl 2 in regulating RBC production by increasing Fe availability for erythropoiesis and hemoglobin synthesis. It also demonstrates the kinetics of metal accumulation and distribution and that chronic treatment leads to significantly reduced Fe content in the erythrocytes compared to short-term administration, suggesting stimulated Fe release as a possible survival mechanism to counteract the toxic effects of Fe overload. 2. Materials and Methods 2.1. Animal model Mature ICR (Institute of Cancer Research) mice were purchased from the Experimental and breeding base for laboratory animals (EBBLA) - Slivnitza, Bulgaria. Animals were maintained in the Institute’s animal breeding facility at 23 ± 2°C and 12:12 h light/dark cycle. Mice were fed a standard diet TopMix (purchased from HL-TopMix Ltd., Kaloyanovo, Bulgaria) and had access to food ad libitum. Pregnant mice were placed into individual standard hard-bottom polypropylene cages and subjected to a daily dose of 125 mg cobalt chloride/kg body weight (CoCl 2 x6H 2 O) for 2–3 days prior delivery. The compound was dissolved and administered through drinking tap water. Our previous experiments and literature data show that while breastfeeding a mouse drinks approximately 15 ml water per day. For this reason, a 15 ml solution containing the assigned dose (125 mg/kg b.w.) was given to the mothers daily every morning. Mothers obtained the whole daily volume of the solution. Our previous experience showed no significant sex differences neither in body weight nor in haematological parameters and the experimental groups consisted of both male and female mice. At weaning age (day 25) the pups were separated and placed into individual cages and treatment continued until postnatal day 90. Pups were sacrificed by decapitation after etherization on postnatal days 18, 30, 45, 60 and 90. Blood serum was obtained and stored at -20°C for further analyses. Erythrocytic indices – red blood cell count (RBC), hemoglobin (Hb), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin content (MCHC), red blood cell distribution width (RDW) were analyzed in whole blood on automated hematological analyser BC2800Vt (Mindray, China). Age-matched mice obtaining regular tap water were used as control groups. The experimental design was carried out in accordance with to the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines and EU Directive 2010/63/EU for animal experiments. The study was approved by the Bulgarian Agency for Food Safety, Approval number 282 from 24.09.2020. 2.2. Enzyme-linked immunosorbent assay (ELISA) for TfR1, hepcidin and ferritin Blood sera from control and CoCl 2 -treated mice were analysed using mouse TfR1, hepcidin and ferritin ELISA kits (Elabscience Biotechnology Co., Ltd, China) according to the manufacturer’s instructions. The optical density was read at 450 nm on ELISA Reader GDV (GIO. DE VITA EC., Roma, Italy). The final concentrations were determined using Curve Expert 1.4 software and expressed in ng/g for TfR1 and ferritin, and in pg/g for hepcidin. 2.3. ICP-DRC-MS analyses of cobalt and iron content in RBCs and blood serum Prior to analysis 50–100 µl of the studied samples were subjected to digestion in concentrated HNO 3 (Sigma-Aldrich, Co., USA) in the Berghof SW-4 DAP-40 microwave system (Berghof Products + Instruments GmbH, Eningen, Germany). The digested samples were transferred into 15 mL polypropylene test tubes and adjusted to the final volume of 15 mL with deionized water (18 MΩ cm, Milli-Q, Millipore, Bedford, MA, USA) and thoroughly mixed up by shaking in the closed test tubes. Analysis of Co and Fe levels in the samples was performed using inductively-coupled plasma mass spectrometry with dynamic reaction cell technology (ICP-DRC-MS) at NexION 300D spectrometer (Perkin Elmer, USA) equipped with ESI SC-2 DX4 autosampler (Elemental Scientific Inc., Omaha, NE, USA). Co and Fe content in the studied samples was expressed as µg/ml. Calibration of the system was performed using standard solutions of iron and cobalt with different concentrations prepared from Universal Data Acquisition Standards Kits (PerkinElmer Inc., Shelton, CT 06484, USA. Internal online standardization was performed using 10 µg/L Yttrium (Y) and Rhodium (Rh) Pure Single-Element Standard (PerkinElmer Inc., Shelton, CT, USA) prepared on a matrix containing 8% 1-butanol (Merck KGaA, Gernsheim, Germany), 0.8% Triton X-100 (Sigma-Aldrich Co., St. Louis, MO, USA), 0.02% tetramethylammonium hydroxide (Alfa Aesar, Ward Hill, MA, USA) and 0.02% ethylenediaminetetraacetic acid (Sigma-Aldrich Co., St. Louis, MO, USA). Laboratory quality control was performed via permanent analysis of the certified reference material (GBW09101, Shanghai Institute of Nuclear Research, Shanghai, China). The recovery rates for Co and Fe were within the interval of 92–104% and 95–107%, respectively. 2.4. Statistical analysis The obtained data were processed using SPSS 16.0 for Windows. The results for TfR1, hepcidin and ferritin content are presented as mean value ± SD. Data on Co and Fe content in the studied samples are expressed as median and the respective 25 and 75 percentile boundaries (interquartile range). Statistical significance between the experimental groups was assessed using one-way ANOVA with Bonferonni post-hoc test at level of significance of p < 0.05. 3. Results 3.1. Co and Fe accumulation Exposure to CoCl 2 induced significant accumulation of Co ions in blood sera and RBCs of all experimental groups (Tables 1 , 2 ). The highest serum Co content was obtained for d30 mice - ~ 564.67-fold higher compared to the untreated control. Sera of d90 mice accumulated ~ 128.75-fold higher metal ions following chronic treatment. The results suggest that immature mice accumulate ~ 4.4-fold higher metal levels in comparison to mature animals. During long-term exposure, the most profound Co accumulation was observed during the first month of exposure. Similar to the serum, the highest Co content in erythrocytes was obtained for d30 mice, being 300-fold higher compared to the age-matched control group. The lowest Co accumulation was found in d90 mice, resulting in ~ 45.5-fold elevation in comparison to the untreated control. Cobalt content in RBCs was 6.59-fold higher in immature mice compared to mature showing the same tendency as for the serum Co content. Table 1 Co content (µg/ml) in serum of control and CoCl 2 -exposed mice Day Control (n = 5) 125 ml/kg CoCl 2 (n = 5) 18 0.003 ± 0.002 0.287 ± 0.215 * 30 0.003 ± 0.000 1.694 ± 0.241 * 45 0.005 ± 0.001 1.011 ± 0.318 * 60 0.004 ± 0.004 0.857 ± 0.241 * 90 0.004 ± 0.003 0.515 ± 0.178 * Each column represents the mean ± SD; * p < 0.05 Table 2 Co content (µg/g) in RBC of control and CoCl 2 -exposed mice Day Control (n = 5) 125 ml/kg CoCl 2 (n = 5) 18 0.001 ± 0.000 0.077 ± 0.035 * 30 0.002 ± 0.000 0.6 ± 0.357 * 45 0.002 ± 0.001 0.464 ± 0.322 * 60 0.002 ± 0.001 0.321 ± 0.067 * 90 0.002 ± 0.000 0.091 ± 0.022 * Each column represents the mean ± SD; * p < 0.05 In Co-exposed groups, Co level in serum of mature mice was ~ 5.66-fold higher than that in RBCs in day 90 mice, whereas in d45 mice it was higher by a factor of ~ 2.18. In immature mice serum Co content increased ~ 3.72-fold in day 18 Co-exposed mice and ~ 2.82-fold in day 30 cobalt-treated mice. In contrast, Fe content was significantly increased in sera and decreased in RBC following exposure to CoCl 2 (Tables 3 , 4 ). The increase in serum iron (sFe) content varied from 53% (1.53-fold) in d45 mice to 6.16-fold in d18 Co-exposed mice compared to the respective age-matched controls. In turn, RBC Fe content decreased in the range from 25% (1.25-fold) in d18 Co-exposed mice to 2.32-fold in d45 treated mice. Fe levels in blood serum and RBCs of mature Co-exposed mice (day 45, 60 and 90) were lower than those in immature animals (day 18 and 30). The erythrocytes of day 30 mice cobalt-treated mice contained ~ 2.74-fold more iron than day 45 mice. Surprisingly, iron content in RBCs of day 45 mice was similar to that of day 18 cobalt-exposed animals. Table 3 Serum Fe concentration (µg/ml) of control and CoCl 2 -exposed mice Day Control (n = 5) 125 ml/kg CoCl 2 (n = 5) 18 1.165 ± 0.71 7.175 ± 1.78 * 30 3.969 ± 1.729 7.445 ± 1.662 * 45 3.85 ± 2.062 5.905 ± 2.001 * 60 3.5 ± 5.442 6.82 ± 1.61 * 90 3.7 ± 2.625 6.709 ± 1.102 * Each column represents the mean ± SD; * p < 0.05 Table 4 Fe concentration (µg/g) in RBC of control and CoCl 2 -exposed mice Day Control (n = 5) 125 ml/kg CoCl 2 (n = 5) 18 342 ± 37.72 273.667 ± 57.757 30 647.797 ± 89.682 729.063 ± 26.748 45 657 ± 156.656 283 ± 49.78 * 60 689 ± 160.629 367.5 ± 41.869 * 90 671 ± 141.105 293.563 ± 101.167 * Each column represents the mean ± SD; * p < 0.05 3.2. Fe-regulatory proteins TfR1, hepcidin and ferritin expression Alterations in Fe content were associated with changes in expression of Fe-regulatory proteins, TfR1, hepcidin, and ferritin. Data obtained from ELISA assays demonstrated a decrease in TfR1 concentration (Table 5 ) in all experimental groups exposed to CoCl 2 except day 45 mice and increase in serum ferritin and hepcidin levels of CoCl 2 -exposed mice (Tables 6 , 7 ). The reduced serum TfR1 concentration corresponds to the significantly elevated serum Fe in the Co-exposed experimental groups. Table 5 TfR1 concentration (ng/g) of control and CoCl 2 -exposed mice. Each column represents the mean ± SD. Day Control (n = 5) 125 ml/kg CoCl 2 (n = 5) 18 6.13 ± 3.0 0.69 ± 0.20 30 0.84 ± 0.30 0.66 ± 1.10 45 0.35 ± 0.45 0.54 ± 0.55 60 2.51 ± 1.10 0.41 ± 0.26 90 18.82 ± 15.10 0.58 ± 0.43 Each column represents the mean ± SD Serum TfR1concentration reduction varied from ~ 32.45-fold in day 90 mice to 1.27-fold in day 30 cobalt-exposed mice. Table 6 Serum ferritin concentration (ng/g) of control and CoCl 2 -exposed mice Day Control (n = 5) 125 ml/kg CoCl 2 (n = 5) 18 3.7 ± 1.19 9.42 ± 11.12 30 4.44 ± 0.91 6.09 ± 4.34 45 4.83 ± 0.65 6.68 ± 3.79** 60 11.34 ± 3.52 5.94 ± 2.19** 90 6.29 ± 0 33.56 ± 18.49 Each column represents the mean ± SD; ** p < 0.01 Serum ferritin concentration was increased in all Co-treated groups except for day 60 mice where a significant ~ 1.9-fold decrease was observed. The highest concentration was found in day 90 mice which was a ~ 5.34-fold increase compared to the untreated control samples. Data correspond with the reduced RBCs iron content. Table 7 Hepcidin concentration (pg/g) of control and CoCl 2 -exposed mice. Day Control (n = 5) 125 ml/kg CoCl 2 (n = 5) 18 1323.9 ± 0 349.41 ± 66.09*** 30 299.09 ± 68.36 469.46 ± 320.32 45 406.51 ± 173.0 451.48 ± 224.66 60 1127.51 ± 239.91 230.71 ± 149.87*** 90 188.32 ± 0 359.74 ± 152.89 Each column represents the mean ± SD. *** p < 0.001 Chronic exposure to CoCl 2 increased serum hepcidin concentration in almost all experimental groups except day 18 and day 60 Co-treated mice where a significant decrease of ~ 3.79-fold and ~ 4.89-fold, respectively was found. The observed reduction is in agreement with the significantly elevated sFe concentrations in these groups. The highest increase - ~ 1.9-fold was found in day 90 cobalt-treated mice which corresponds to the lowest serum and RBC iron content and reduced TfR1 concentration found in that group. 3.3. Erythrocytic parameters Chronic exposure to CoCl 2 affected erythrocytic parameters, as well (Table 8 ). The effect was more pronounced for Hb as its content increased significantly in a time-dependent manner in CoCl 2 -treated mice with the highest content being measured in day 90 mice. RBCs increased in all exposed groups except in d45 and d90 which could explain the increased hepcidin concentration in these groups. The highest increase in RBC count was observed for d18 Co-exposed mice - ~ 1.86-fold compared to the untreated control. The Hb-related parameters - MCH and MCHC were also significantly increased in all cobalt-supplemented groups except for day 18 mice. Surprisingly, Co-exposure reduced RDW compared to the untreated age-matched controls making the RBCs more uniform in size. Significantly lower RDW was found in immature Co-treated mice compared to their age-matched untreated controls. The enhanced erythropoietic parameters in day 18 and day 60 cobalt-exposed mice could explain the decreased hepcidin activity in these groups. Table 8 Erythrocytic parameters in control and Co-treated mice. Experimental group RBC Hb MCV MCH MCHC RDW Day18 Control 3.68 ± 0.56 69 ± 12.7 55.41 ± 5.38 18.71 ± 2.23 344.13 ± 71.68 25.39 ± 5.84 CoCl 2 (n = 7) 6.88 ± 0.19 99 ± 3.5*** 54.78 ± 2.07 14.35 ± 0.51 *** 262.67 ± 4.23 ** 17.17 ± 0.68 ** Day30 Control 6.26 ± 0.43 78.8 ± 5.4 47.68 ± 2.48 12.57 ± 0.71 264.17 ± 5.04 29.48 ± 2.75 CoCl 2 (n = 7) 7.36 ± 0.84 101 ± 20.9* 48.36 ± 4.94 13.66 ± 2.18 282.21 ± 18.37 * 20.09 ± 2.11 *** Day45 Control 9.31 ± 078 116.14 ± 9.33 47.93 ± 2.0 12.44 ± 0.38 260.29 ± 5.15 22.93 ± 1.22 CoCl 2 (n = 7) 8.67 ± 10.06 125.14 ± 10.06 50.9 ± 3.51 14.44 ± 1.23 ** 284.57 ± 11.33 *** 21.46 ± 3.83 Day 60 Control 8.5 ± 0.94 111.57 ± 9.86 50.74 ± 3.32 13.11 ± 0.66 259.29 ± 6.47 17.36 ± 2.31 CoCl 2 (n = 9) 8.86 ± 0.76 127.89 ± 11.74* 49.78 ± 2.69 14.4 ± 0.98 * 290.22 ± 5.91*** 17.08 ± 2.27 Day 90 Control 9.64 ± 1.04 125.25 ± 11.95 49.48 ± 1.94 12.95 ± 0.24 262.75 ± 6.13 15.13 ± 0.64 CoCl 2 (n = 9) 8.99 ± 0.78 131.67 ± 11.91 50.44 ± 1.77 14.59 ± 0.39 *** 290.22 ± 7.31*** 15.39 ± 0.95 Each column represents the mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001 4. Discussion Exposure to cobalt may occur through diet, dietary supplements, occupational exposure, biomedical applications and medical devices [ 10 ]. Although not completely declared some dietary supplements are promoted as performance-boosting nutrients and contain soluble and bioavailable Co as their active ingredient [ 11 ]. As cobalt supplements may induce erythropoietin production thus affecting endurance performance, in 2017 the World Anti-Doping Agency included them in its prohibited list [ 12 ]. The erythropoietic effect of cobalt in our study was demonstrated by elevated Hb content and increased RBCs in almost all experimental groups. The expanded erythropoiesis may be due increased erythroferrone production as it is stimulated by hypoxia [ 13 ]. In addition, stimulated RBC production may be also due upregulation of GATA-1 known to enhance erythroid differentiation and to be highly expressed under hypoxia [ 14 , 15 ]. Although the dose of CoCl 2 sufficient to stimulate erythropoiesis is unknown, Hoffmeister et al. show that oral Co 2+ dosage of 10 mg/day for 5-day duration exerts erythropoietic effects [ 16 ]. In their earlier work the same authors find that a 3-week administration of 5 mg/day Co supplement significantly increased hemoglobin mass while the other hematological parameters were not significantly affected [ 17 ]. In serum Co 2+ binds to albumin, and the concentration of free, ionized Co 2+ is estimated at 5–12% of the total cobalt concentration. Our results for chronic treatment with CoCl 2 suggest that immature mice accumulate ~ 4.4-fold more metal compared to mature animals. Also, during long-term exposure the most Co is accumulated in serum after 30 days of exposure and decreases by day 90 of dosing. The decrease may be due to “auto-inhibition” by cobalt as suggested by Simonsen et al. [ 18 ], and increased excretion and/or suppressed absorption due to elevated body iron. Our results are in accordance with those of Reuber et al. [ 19 ] who demonstrate enhanced Co excretion during Co and/or iron supplementation. Our experimental data suggest that serum Co concentration is a reliable marker for a recent exposure - up to 30 days. Research data imply that during long-term cobalt exposure in vivo cobalt will be taken up practically irreversibly in the red cells during their 120 days life span [ 20 ]. Our results demonstrate significant cobalt accumulation in RBCs following chronic exposure to CoCl 2 . The highest concentration was found in day 30 mice and decreased by day 90 of exposure. Our results for the highest Co concentration in RBCs of day 30 mice are also similar to those of Bryan et al. [ 21 ] where cobalt concentration in erythrocytes increased with time to reach a plateau after 5–6 weeks of daily peritoneal exposure of rats. At systemic and cellular level Co is sequestered along the Fe-acquisition pathway. The behavior of the transferrin-Co complex is similar to that of iron with a fast interaction with the C-lobe and a very slow one with the N-lobe of TfR1 [ 22 ]. Iron is biologically essential but toxic in high concentrations. For this reason, its metabolism is tightly controlled at cellular and systemic level to prevent deficiency or overload [ 5 ]. In our study, chronic in vivo exposure to CoCl 2 significantly altered serum and erythrocyte Fe content. Similar results for significantly increased Co and Fe concentrations in the serum and RBCs of Co-exposed mice were obtained in our previous studies using lower daily dose of CoCl 2 [ 23 ]. Although Fe significantly increased in serum of Co-exposed mice, its content in mature mice remained lower compared to immature, with the largest difference of 9.9% between d30 and d90 mice. A possible explanation for the lower serum Fe content in mature mice is the elevated Hb content suggesting Fe utilization in heme synthesis. RBC iron decreased in Co-exposed mature mice but increased in d30 mice, being ~ 2.48-fold higher compared to d90 mice. Our results for reduced erythrocyte Fe content indicate that erythroid cells possibly export Fe to survive. This hypothesis is supported by Keel et al. [ 24 ] who demonstrate that the export of excess cytoplasmic heme from the erythroid precursors is mediated by the feline leukemia virus, subgroup C, receptor (FLVCR). Iron overload is known to damage RBC plasma membranes by increasing reactive oxygen species production and osmotic fragility [ 25 ]. A possible mechanism for erythrocyte Fe release in the blood plasma may be through increased ferroportin activity. Zhang et al. [ 25 ] demonstrate abundant ferroportin expression in mature RBCs and hypothesize that erythrocytes export Fe to avoid iron overload and hemolysis. In our studies we have observed stimulated ferroportin expression in the bone marrow of day 60 and day 90-cobalt treated mice (our unpublished data). In addition, cellular iron export is stimulated by ceruloplasmin. Low oxygen concentrations stimulate Fe release by ceruloplasmin who is also known to stabilize ferroportin on cell membrane [ 26 ]. Mature erythrocytes express ceruloplasmin receptors [ 27 ] and therefore its role in Fe reduction by RBCs should not be excluded. The insignificant change in cell volume also suggests that the erythrocytes likely export the excess Fe. According to McLaren et al. [ 28 ], cases of excess Fe are associated with RBC’s expanded size and higher cellular Hb concentration as a functional utilization of Fe within a non-toxic compartment. Within the cells Fe is stored in the form of ferritin. Ferritin is an iron-binding protein that indicates total body iron stores. The iron-storing capacity of ferritin is assisted by the ferroxidase activity of ferritin heavy chain, which converts reactive iron (Fe 2+ ) into inert, nucleated iron (Fe 3+ ), no longer available to catalyze the production of free radicals via Fenton chemistry [ 29 ]. It is an acute-phase protein that increases in response to inflammatory states, including malignancy, infection, and in liver, renal and autoimmune diseases. Serum ferritin is a clinical marker for Fe status as it is increased in cases of Fe overload and reduced in Fe deficiency [ 30 ]. Elevated serum ferritin also reflects macrophage ferritin content as ferritin is predominantly secreted in the circulation by the macrophages [ 5 ]. In our study serum ferritin was elevated almost in all experimental groups following chronic exposure to CoCl 2 . In day 90 exposed mice serum ferritin concentration was increased more than 5-fold also suggesting enhanced loss of Fe by the erythrocytes. Another key marker for Fe trafficking is TfR1. It can bind two different Fe-binding molecules: transferrin and ferritin [ 3 ]. Serum contains a soluble TfR (sTfR) molecule that circulates bound to transferrin that is cleaved from the whole TfR molecule and is a sensitive marker for erythropoiesis and iron deficiency [ 31 ]. The circulating sTfR level reflects total body TfR concentration, the major source of sTfR being bone marrow erythroid precursors [ 3 ]. The reduced serum TfR1 concentration in our study is in accordance with the increased serum Fe and ferritin concentrations. These results suggest downregulation of TfR due to increased intracellular Fe storage and total body Fe as TfR expression is shown to decrease in cases of excess Fe [ 3 ]. According to R’zik and Beguin [ 32 ] there is an inverse correlation between body iron stores and total TfR. In addition, the high serum Fe concentration may counteract erythropoietin production by the kidneys resulting in lower TfR expression. Another explanation for the reduced TfR1 may be that the increased serum ferritin delivers Fe to the tissues serving as an alternative pathway to the Tf-TfR one, as observed by Wang et al. [ 30 ]. Also, in vitro experiments have shown that the isoform of the second transferrin receptor - TfR2-α, incorporates Tf-bound iron into cells as effectively as TfR1, and it is expressed predominantly by erythroid progenitors but the expression of TfR2 mRNA does not show obvious changes upon iron loading or iron chelation [ 3 ]. In our previous study we also demonstrate stimulated TfR2 tissue expression in immature mice following Co exposure [ 33 ]. This suggests that the role of TfR2 should not be neglected in cases of stimulated erythropoiesis and/or elevated serum Fe. Increased serum iron levels upregulate hepcidin expression. Hepcidin acts as a negative regulator of Fe. In coordination with ferroportin hepcidin regulates Fe entry into plasma, its utilization and storage [ 6 ]. The elevated serum Fe in our study enhanced hepcidin production in almost all Co-exposed groups. The stimulated erythropoiesis in day 18 and day 60 mice may be a possible explanation for the significantly reduced serum hepcidin in these groups. In addition, erythroferrone which stimulated RBC production suppresses hepcidin expression in a time-dependent manner [ 13 ]. Stimulated erythropoiesis strongly suppresses the production of hepatic hepcidin in mice and humans, allowing more dietary iron and Fe from stores to enter blood plasma for heme and hemoglobin synthesis by developing erythrocytes in the marrow [5; 7]. The results indicate that Fe metabolism is tightly controlled/regulated by erythropoiesis through the suppression of hepcidin. In our study serum hepcidin concentration in mature mice was lower than in the immature. Possible explanation for this may be that during chronic exposure the high extracellular Fe concentration may suppress hepcidin production by a feedback mechanism. 5. Conclusions CoCl 2 supplementation exhibited diverse effects on Fe bioavailability in serum and erythrocytes of immature and mature mice. Chronic exposure to CoCl 2 resulted in a lower Fe content of mature mice compared to immature suggesting stimulated Fe release as a possible survival mechanism to counteract the toxic effects of Fe overload. Cobalt administration stimulated Fe storage, enhancing hepcidin production and ferritin concentrations, and reducing TfR1 expression. During long-term exposure the most Co was accumulated in the serum and erythrocytes after 30 days of treatment and decreased by day 90 of dosing indicating that whole blood Co concentration is a reliable marker for a recent exposure. Declarations Conflicts of Interest: The authors have no relevant financial or non-financial interests to disclose. Ethical Approval: The experimental protocol was performed at the Institute of Experimental Morphology, Pathology and Anthropology with Museum, Bulgarian Academy of Sciences, according to the ARRIVE guidelines and EU Directive 2010/63/EU for animal experiments. The study was approved by the Bulgarian Agency for Food Safety, Approval number 282 from 24.09.2020. Competing Interests Alexey A. Tinkov and Anatoly V. Skalny are members of the Editorial board Funding: This research was funded by Grants No. DNTS/Russia 02/1/14.06.2018 from the Bulgarian National Science Fund. AAT and AVS were supported by the Russian Ministry of Science and Higher Education (075-15-2024-550). Author Contribution E.P. (Ekaterina Pavlova) - methodology, investigation, visualization, writing - review and editing; E.P. (Emilia Petrova) - methodology, investigation, writing - review and editing; A.A.T. - methodology, investigation, formal analysis, writing - review and editing; O.P.A. - methodology, validation, formal analysis; P.R. - methodology, investigation; I.V. - methodology, investigation; A.V.S. - writing - review and editing, project administration; Y.G. - writing - review and editing All authors have read and agreed to the published version of the manuscript. Acknowledgements: This research was funded by Grants No. DNTS/Russia 02/1/14.06.2018 from the Bulgarian National Science Fund. AAT and AVS were supported by the Russian Ministry of Science and Higher Education (075-15-2024-550). Data Availability Statement: All data necessary to understand or reproduce this study are included in the manuscript. References Ebert B, Jelkmann W (2014) Intolerability of cobalt salt as erythropoietic agent. 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Int J Sports Med 37:82–84. https://doi:10.1055/s-0035-1569350 WADA (2018) List of prohibited substances and methods. Available online: https://www.wada-ama.org/sites/default/files/prohibited_list_2018_en.pdf Coffey R, Jung G, Olivera JD, Karin G, Pereira RC, Nemeth E, Ganz T (2022) Erythroid overproduction of erythroferrone causes iron overload and developmental abnormalities in mice. Blood 139:439–451. https://doi:10.1182/blood.2021014054 Liu F, Hu C, Ding J, Fu C, Wang S, Li T (2023) GATA-1 Promotes Erythroid Differentiation Through the Upregulation of miR-451a and miR-210-3p Expressions in CD34 + Cells in High-Altitude Polycythemia. High Alt Med Biol 24:59–67. https://doi:10.1089/ham.2022.0095 Bermudez D, Azad P, Figueroa-Mujíca R, Vizcardo-Galindo G, Corante N, Guerra-Giraldez C, Haddad GG, Villafuerte FC (2020) Increased hypoxic proliferative response and gene expression in erythroid progenitor cells of Andean highlanders with chronic mountain sickness. Am J Physiol Regul Integr Comp Physiol 318:R49–R56. https://doi:10.1152/ajpregu.00250.2019 Hoffmeister T, Schwenke D, Wachsmuth N, Krug O, Thevis M, Byrnes WC, Schmidt WFJ (2019) Erythropoietic effects of low-dose cobalt application. Drug Test Anal 11:200–207. https://doi:10.1002/dta.2478 Hoffmeister T, Schwenke D, Krug O, Wachsmuth N, Geyer H, Thevis M, Byrnes WC, Schmidt WFJ (2018) Effects of 3 weeks of oral low-dose cobalt on hemoglobin mass and aerobic performance. Front Physiol 9:1289. https://doi:10.3389/fphys.2018.01289 Simonsen LO, Brown AM, Harbak H, Kristensen BI, Bennekou P (2011) Cobalt uptake and binding in human red blood cells. Blood Cells Mol Dis 46:266–276. https://doi 10.1016/j.bcmd.2011.02.009 Reuber S, Kreuzer M, Kirchgessner M (1994) Interactions of cobalt and iron in absorption and retention. J Trace Elem Electrolytes Health Dis 8:151–158 Simonsen LO, Harbak H, Bennekou P (2012) Cobalt metabolism and toxicology-a brief update. Sci Total Environ 432. https://doi 10.1016/j.scitotenv.2012.06.009 . :210 – 215 Bryan SE, Morgan KS (1970) The effect of cobalt chloride on serum protein electrophoretic patterns in mice. FEBS Lett 9:277–280. https://doi:10.1016/0014-5793(70)80376-3 El Hage Chahine JM, Hémadi M, Ha-Duong NT (2012) Uptake and release of metal ions by transferrin and interaction with receptor 1. Biochim Biophys Acta 1820:334–347. https://doi:10.1016/j.bbagen.2011.07.008 Skalny AV, Gluhcheva Y, Ajsuvakova OP, Pavlova E, Petrova E, Rashev P, Vladov I, Shakieva RA, Aschner M, Tinkov AA (2021) Perinatal and early-life cobalt exposure impairs essential metal metabolism in immature ICR mice. Food Chem Toxicol 149:111973. https://doi:10.1016/j.fct.2021.111973 Keel SB, Doty RT, Yang Z, Quigley JG, Chen J, Knoblaugh S, Kingsley PD, De Domenico I, Vaughn MB, Kaplan J, Palis J, Abkowitz JL (2008) A heme export protein is required for red blood cell differentiation and iron homeostasis. Science 319:825–828. https://doi:10.1126/science.1151133 Zhang DL, Wu J, Shah BN, Greutélaers KC, Ghosh MC, Ollivierre H, Su XZ, Thuma PE, Bedu-Addo G, Mockenhaupt FP, Gordeuk VR, Rouault TA (2018) Erythrocytic ferroportin reduces intracellular iron accumulation, hemolysis, and malaria risk. Science 359:1520–1523. https://doi:10.1126/science.aal2022 De Domenico I, Ward DM, di Patti MC, Jeong SY, David S, Musci G, Kaplan J (2007) Ferroxidase activity is required for the stability of cell surface ferroportin in cells expressing GPI-ceruloplasmin. EMBO J 26:2823–2831. https://doi:10.1038/sj.emboj.7601735 Barnes G, Frieden E (1984) Ceruloplasmin receptors of erythrocytes. Biochem Biophys Res Commun 125:157–162. https://doi.org/10.1016/S0006-291X(84)80348-4 McLaren CE, Barton JC, Gordeuk VR, Wu L, Adams PC, Reboussin DM, Speechley M, Chang H, Acton RT, Harris EL, Ruggiero AM, Castro O (2007) Hemochromatosis and Iron Overload Screening Study Research Investigators. Determinants and characteristics of mean corpuscular volume and hemoglobin concentration in white HFE C282Y homozygotes in the hemochromatosis and iron overload screening study. Am J Hematol 82:898–905. https://doi:10.1002/ajh.20937 Soares MP, Hamza I (2016) Macrophages and iron metabolism. Immunity 44:492–504. https://doi 10.1016/j.immuni.2016.02.016 Wang W, Knovich MA, Coffman LG, Torti FM, Torti SV (2010) Serum ferritin: Past, present and future. Biochim Biophys Acta 1800:760–769. 10.1016/j.bbagen.2010.03.011 Shih YJ, Baynes RD, Hudson BG, Flowers CH, Skikne BS, Cook JD (1990) Serum transferrin receptor is a truncated form of tissue receptor. J Biol Chem 265:19077–19081 R’zik S, Beguin Y (2001) Serum soluble transferrin receptor concentration is an accurate estimate of the mass of tissue receptors. Exp Hematol 29. https://doi:10.1016/s0301-472x(01)00641-5 . :677 – 685 Gluhcheva Y, Pavlova E, Petrova E, Tinkov AA, Ajsuvakova OP, Skalnaya MG, Vladov I, Skalny AV (2020) The impact of perinatal cobalt chloride exposure on extramedullary erythropoiesis, tissue iron levels, and transferrin receptor expression in mice. Biol Trace Elem Res 194:423–431. 10.1007/s12011-019-01790-8 Additional Declarations Competing interest reported. Alexey A. Tinkov and Anatoly V. Skalny are members of the Editorial board Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4697764","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":326878001,"identity":"42ea4713-7eb8-4154-89a0-8fd8d7c48f4b","order_by":0,"name":"Ekaterina Pavlova","email":"","orcid":"","institution":"Institute of Experimental Morphology, Pathology and Anthropology with Museum, Bulgarian Academy of Sciences, Sofia, Bulgaria","correspondingAuthor":false,"prefix":"","firstName":"Ekaterina","middleName":"","lastName":"Pavlova","suffix":""},{"id":326878002,"identity":"ec21bc2f-1f3d-4b66-94e4-c28a610dff4e","order_by":1,"name":"Emilia Petrova","email":"","orcid":"","institution":"Institute of Experimental Morphology, Pathology and Anthropology with Museum, Bulgarian Academy of Sciences, Sofia, Bulgaria","correspondingAuthor":false,"prefix":"","firstName":"Emilia","middleName":"","lastName":"Petrova","suffix":""},{"id":326878003,"identity":"9282aa3f-9d6b-46cd-bd5a-3579716613f9","order_by":2,"name":"Alexey A. Tinkov","email":"","orcid":"","institution":"Orenburg State University","correspondingAuthor":false,"prefix":"","firstName":"Alexey","middleName":"A.","lastName":"Tinkov","suffix":""},{"id":326878004,"identity":"7c2a0db7-6dc7-4897-9cf8-7e872a4ea4c9","order_by":3,"name":"Olga P. Ajsuvakova","email":"","orcid":"","institution":"Orenburg State University","correspondingAuthor":false,"prefix":"","firstName":"Olga","middleName":"P.","lastName":"Ajsuvakova","suffix":""},{"id":326878005,"identity":"c25ce5db-d8e2-4445-b029-151dad189948","order_by":4,"name":"Pavel Rashev","email":"","orcid":"","institution":"Institute of Biology and Immunology of Reproduction “Acad. Kiril Bratanov”, Bulgarian Academy of Sciences, Sofia, Bulgaria","correspondingAuthor":false,"prefix":"","firstName":"Pavel","middleName":"","lastName":"Rashev","suffix":""},{"id":326878006,"identity":"3443511d-2984-4fa8-ad21-53debcc4cec6","order_by":5,"name":"Ivelin Vladov","email":"","orcid":"","institution":"Institute of Experimental Morphology, Pathology and Anthropology with Museum, Bulgarian Academy of Sciences, Sofia, Bulgaria","correspondingAuthor":false,"prefix":"","firstName":"Ivelin","middleName":"","lastName":"Vladov","suffix":""},{"id":326878007,"identity":"6950d7b2-4e0c-4577-a468-120b0caf4bec","order_by":6,"name":"Anatoly V. 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Alexey A. Tinkov and Anatoly V. Skalny are members of the Editorial board","formattedTitle":"Diverse effects of chronic cobalt supplementation on iron metabolism during erythropoiesis","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eCobalt (Co) is an essential trace element that may cause severe health effects upon occupational or environmental overexposure. Cobalt ions stabilize hypoxia-inducible transcription factors (HIFs) that increase the expression of the erythropoietin (Epo) gene. In the past, cobalt chloride (CoCl\u003csub\u003e2\u003c/sub\u003e) was administered to patients at daily doses of 25 to 300 mg as an anti-anaemic agent for stimulating erythropoiesis [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe existing data show that Co cellular uptake occurs through interaction with transferrin receptors and the uptake profile is similar to that of iron (Fe) [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Fe uptake, storage and intracellular trafficking are tightly controlled by the regulatory proteins \u0026ndash; transferrin receptor 1 (TfR1), divalent metal transporter 1 (DMT1), hepatic hormone hepcidin (Hep), ferritin (Fer) and ferroportin (Fpn). Fe\u003csup\u003e3+\u003c/sup\u003e binds transferrin and is imported into cells through the membrane TfR1 and then is internalized in the endosome by clathrin-mediated endocytosis where it is reduced to ferrous iron by ferrireductase six-transmembrane epithelial antigen of the prostate 3 - STEAP3 [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Fe\u003csup\u003e2+\u003c/sup\u003e release from the endosome and transport into the cytosol is mediated by DMT1. Iron is then either utilized in metabolic processes, such as synthesis of hemoproteins and Fe-S cluster, sequestered and stored in cytosolic ferritin or exported by ferroportin. Hepcidin controls Fpn expression in the enterocytes, thus regulating whole body Fe absorption [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAlthough all cells may import, export or store iron, some have specific functions: e.g., erythroblasts are specialized in iron uptake, macrophages and enterocytes in iron export, and hepatocytes in iron storage. Within cells most iron is transferred to mitochondria for heme and Fe-S cluster production [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. As Fe is primarily utilized by erythroid cells in the bone marrow of humans and vertebrates, a significant effect on erythrocyte renewal is expected. The differentiation from erythroblasts to erythrocytes is strongly Fe-dependent [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAs erythropoietin, a hormone produced by the kidneys, stimulates red blood cell production, it also enhances the synthesis of erythroferrone (ERFE). ERFE is secreted by the erythroblasts and is the main erythroid regulator of hepatic hepcidin [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. By decreasing the transcription of the gene encoding the iron-regulatory hormone hepcidin, ERFE stimulates iron absorption, as well as the release of iron from recycling macrophages and from stores in hepatocytes [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Dietary Fe is then used for heme and hemoglobin synthesis in the newly produced red blood cells. Besides Fe storage, ferritin also provides protection from oxidative damage [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Circulating differic transferrin (Tf) affects hepcidin production through binding to its second receptor - TfR2 [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe aim of the study is to assess the alterations in iron content and Fe regulatory protein (TfR1, hepcidin and ferritin) concentrations, and their effect on erythrocytes in mice following chronic exposure to cobalt chloride (CoCl\u003csub\u003e2\u003c/sub\u003e).\u003c/p\u003e \u003cp\u003eThe study contributes to the elucidation for the role of CoCl\u003csub\u003e2\u003c/sub\u003e in regulating RBC production by increasing Fe availability for erythropoiesis and hemoglobin synthesis. It also demonstrates the kinetics of metal accumulation and distribution and that chronic treatment leads to significantly reduced Fe content in the erythrocytes compared to short-term administration, suggesting stimulated Fe release as a possible survival mechanism to counteract the toxic effects of Fe overload.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Animal model\u003c/h2\u003e \u003cp\u003eMature ICR (Institute of Cancer Research) mice were purchased from the Experimental and breeding base for laboratory animals (EBBLA) - Slivnitza, Bulgaria. Animals were maintained in the Institute\u0026rsquo;s animal breeding facility at 23\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C and 12:12 h light/dark cycle. Mice were fed a standard diet TopMix (purchased from HL-TopMix Ltd., Kaloyanovo, Bulgaria) and had access to food ad libitum. Pregnant mice were placed into individual standard hard-bottom polypropylene cages and subjected to a daily dose of 125 mg cobalt chloride/kg body weight (CoCl\u003csub\u003e2\u003c/sub\u003ex6H\u003csub\u003e2\u003c/sub\u003eO) for 2\u0026ndash;3 days prior delivery. The compound was dissolved and administered through drinking tap water. Our previous experiments and literature data show that while breastfeeding a mouse drinks approximately 15 ml water per day. For this reason, a 15 ml solution containing the assigned dose (125 mg/kg b.w.) was given to the mothers daily every morning. Mothers obtained the whole daily volume of the solution. Our previous experience showed no significant sex differences neither in body weight nor in haematological parameters and the experimental groups consisted of both male and female mice. At weaning age (day 25) the pups were separated and placed into individual cages and treatment continued until postnatal day 90. Pups were sacrificed by decapitation after etherization on postnatal days 18, 30, 45, 60 and 90. Blood serum was obtained and stored at -20\u0026deg;C for further analyses. Erythrocytic indices \u0026ndash; red blood cell count (RBC), hemoglobin (Hb), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin content (MCHC), red blood cell distribution width (RDW) were analyzed in whole blood on automated hematological analyser BC2800Vt (Mindray, China). Age-matched mice obtaining regular tap water were used as control groups.\u003c/p\u003e \u003cp\u003e The experimental design was carried out in accordance with to the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines and EU Directive 2010/63/EU for animal experiments. The study was approved by the Bulgarian Agency for Food Safety, Approval number 282 from 24.09.2020.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Enzyme-linked immunosorbent assay (ELISA) for TfR1, hepcidin and ferritin\u003c/h2\u003e \u003cp\u003eBlood sera from control and CoCl\u003csub\u003e2\u003c/sub\u003e-treated mice were analysed using mouse TfR1, hepcidin and ferritin ELISA kits (Elabscience Biotechnology Co., Ltd, China) according to the manufacturer\u0026rsquo;s instructions. The optical density was read at 450 nm on ELISA Reader GDV (GIO. DE VITA EC., Roma, Italy). The final concentrations were determined using Curve Expert 1.4 software and expressed in ng/g for TfR1 and ferritin, and in pg/g for hepcidin.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. ICP-DRC-MS analyses of cobalt and iron content in RBCs and blood serum\u003c/h2\u003e \u003cp\u003ePrior to analysis 50\u0026ndash;100 \u0026micro;l of the studied samples were subjected to digestion in concentrated HNO\u003csub\u003e3\u003c/sub\u003e (Sigma-Aldrich, Co., USA) in the Berghof SW-4 DAP-40 microwave system (Berghof Products\u0026thinsp;+\u0026thinsp;Instruments GmbH, Eningen, Germany). The digested samples were transferred into 15 mL polypropylene test tubes and adjusted to the final volume of 15 mL with deionized water (18 MΩ cm, Milli-Q, Millipore, Bedford, MA, USA) and thoroughly mixed up by shaking in the closed test tubes.\u003c/p\u003e \u003cp\u003eAnalysis of Co and Fe levels in the samples was performed using inductively-coupled plasma mass spectrometry with dynamic reaction cell technology (ICP-DRC-MS) at NexION 300D spectrometer (Perkin Elmer, USA) equipped with ESI SC-2 DX4 autosampler (Elemental Scientific Inc., Omaha, NE, USA). Co and Fe content in the studied samples was expressed as \u0026micro;g/ml.\u003c/p\u003e \u003cp\u003eCalibration of the system was performed using standard solutions of iron and cobalt with different concentrations prepared from Universal Data Acquisition Standards Kits (PerkinElmer Inc., Shelton, CT 06484, USA. Internal online standardization was performed using 10 \u0026micro;g/L Yttrium (Y) and Rhodium (Rh) Pure Single-Element Standard (PerkinElmer Inc., Shelton, CT, USA) prepared on a matrix containing 8% 1-butanol (Merck KGaA, Gernsheim, Germany), 0.8% Triton X-100 (Sigma-Aldrich Co., St. Louis, MO, USA), 0.02% tetramethylammonium hydroxide (Alfa Aesar, Ward Hill, MA, USA) and 0.02% ethylenediaminetetraacetic acid (Sigma-Aldrich Co., St. Louis, MO, USA). Laboratory quality control was performed via permanent analysis of the certified reference material (GBW09101, Shanghai Institute of Nuclear Research, Shanghai, China). The recovery rates for Co and Fe were within the interval of 92\u0026ndash;104% and 95\u0026ndash;107%, respectively.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Statistical analysis\u003c/h2\u003e \u003cp\u003eThe obtained data were processed using SPSS 16.0 for Windows. The results for TfR1, hepcidin and ferritin content are presented as mean value\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. Data on Co and Fe content in the studied samples are expressed as median and the respective 25 and 75 percentile boundaries (interquartile range). Statistical significance between the experimental groups was assessed using one-way ANOVA with Bonferonni post-hoc test at level of significance of p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Co and Fe accumulation\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eExposure to CoCl\u003csub\u003e2\u003c/sub\u003e induced significant accumulation of Co ions in blood sera and RBCs of all experimental groups (Tables\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, \u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The highest serum Co content was obtained for d30 mice - ~ 564.67-fold higher compared to the untreated control. Sera of d90 mice accumulated\u0026thinsp;~\u0026thinsp;128.75-fold higher metal ions following chronic treatment. The results suggest that immature mice accumulate\u0026thinsp;~\u0026thinsp;4.4-fold higher metal levels in comparison to mature animals. During long-term exposure, the most profound Co accumulation was observed during the first month of exposure. Similar to the serum, the highest Co content in erythrocytes was obtained for d30 mice, being 300-fold higher compared to the age-matched control group. The lowest Co accumulation was found in d90 mice, resulting in ~\u0026thinsp;45.5-fold elevation in comparison to the untreated control. Cobalt content in RBCs was 6.59-fold higher in immature mice compared to mature showing the same tendency as for the serum Co content.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCo content (\u0026micro;g/ml) in serum of control and CoCl\u003csub\u003e2\u003c/sub\u003e-exposed mice\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDay\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eControl (n\u0026thinsp;=\u0026thinsp;5)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e125 ml/kg CoCl\u003csub\u003e2\u003c/sub\u003e (n\u0026thinsp;=\u0026thinsp;5)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.003\u0026thinsp;\u0026plusmn;\u0026thinsp;0.002\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.287\u0026thinsp;\u0026plusmn;\u0026thinsp;0.215\u003cb\u003e*\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.003\u0026thinsp;\u0026plusmn;\u0026thinsp;0.000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.694\u0026thinsp;\u0026plusmn;\u0026thinsp;0.241\u003cb\u003e*\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.005\u0026thinsp;\u0026plusmn;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.011\u0026thinsp;\u0026plusmn;\u0026thinsp;0.318\u003cb\u003e*\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.004\u0026thinsp;\u0026plusmn;\u0026thinsp;0.004\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.857\u0026thinsp;\u0026plusmn;\u0026thinsp;0.241\u003cb\u003e*\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.004\u0026thinsp;\u0026plusmn;\u0026thinsp;0.003\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.515\u0026thinsp;\u0026plusmn;\u0026thinsp;0.178\u003cb\u003e*\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c3\" namest=\"c1\"\u003e \u003cp\u003eEach column represents the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD; * p\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCo content (\u0026micro;g/g) in RBC of control and CoCl\u003csub\u003e2\u003c/sub\u003e-exposed mice\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDay\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eControl (n\u0026thinsp;=\u0026thinsp;5)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e125 ml/kg CoCl\u003csub\u003e2\u003c/sub\u003e (n\u0026thinsp;=\u0026thinsp;5)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.001\u0026thinsp;\u0026plusmn;\u0026thinsp;0.000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.077\u0026thinsp;\u0026plusmn;\u0026thinsp;0.035\u003cb\u003e*\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.002\u0026thinsp;\u0026plusmn;\u0026thinsp;0.000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.357\u003cb\u003e*\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.002\u0026thinsp;\u0026plusmn;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.464\u0026thinsp;\u0026plusmn;\u0026thinsp;0.322\u003cb\u003e*\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.002\u0026thinsp;\u0026plusmn;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.321\u0026thinsp;\u0026plusmn;\u0026thinsp;0.067\u003cb\u003e*\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.002\u0026thinsp;\u0026plusmn;\u0026thinsp;0.000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.091\u0026thinsp;\u0026plusmn;\u0026thinsp;0.022\u003cb\u003e*\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c3\" namest=\"c1\"\u003e \u003cp\u003eEach column represents the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD; * p\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eIn Co-exposed groups, Co level in serum of mature mice was ~\u0026thinsp;5.66-fold higher than that in RBCs in day 90 mice, whereas in d45 mice it was higher by a factor of ~\u0026thinsp;2.18. In immature mice serum Co content increased\u0026thinsp;~\u0026thinsp;3.72-fold in day 18 Co-exposed mice and ~\u0026thinsp;2.82-fold in day 30 cobalt-treated mice.\u003c/p\u003e \u003cp\u003eIn contrast, Fe content was significantly increased in sera and decreased in RBC following exposure to CoCl\u003csub\u003e2\u003c/sub\u003e (Tables\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, \u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The increase in serum iron (sFe) content varied from 53% (1.53-fold) in d45 mice to 6.16-fold in d18 Co-exposed mice compared to the respective age-matched controls. In turn, RBC Fe content decreased in the range from 25% (1.25-fold) in d18 Co-exposed mice to 2.32-fold in d45 treated mice. Fe levels in blood serum and RBCs of mature Co-exposed mice (day 45, 60 and 90) were lower than those in immature animals (day 18 and 30). The erythrocytes of day 30 mice cobalt-treated mice contained\u0026thinsp;~\u0026thinsp;2.74-fold more iron than day 45 mice. Surprisingly, iron content in RBCs of day 45 mice was similar to that of day 18 cobalt-exposed animals.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSerum Fe concentration (\u0026micro;g/ml) of control and CoCl\u003csub\u003e2\u003c/sub\u003e-exposed mice\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDay\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eControl (n\u0026thinsp;=\u0026thinsp;5)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e125 ml/kg CoCl\u003csub\u003e2\u003c/sub\u003e (n\u0026thinsp;=\u0026thinsp;5)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.165\u0026thinsp;\u0026plusmn;\u0026thinsp;0.71\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7.175\u0026thinsp;\u0026plusmn;\u0026thinsp;1.78\u003cb\u003e*\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3.969\u0026thinsp;\u0026plusmn;\u0026thinsp;1.729\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7.445\u0026thinsp;\u0026plusmn;\u0026thinsp;1.662\u003cb\u003e*\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3.85\u0026thinsp;\u0026plusmn;\u0026thinsp;2.062\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5.905\u0026thinsp;\u0026plusmn;\u0026thinsp;2.001\u003cb\u003e*\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3.5\u0026thinsp;\u0026plusmn;\u0026thinsp;5.442\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6.82\u0026thinsp;\u0026plusmn;\u0026thinsp;1.61\u003cb\u003e*\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3.7\u0026thinsp;\u0026plusmn;\u0026thinsp;2.625\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6.709\u0026thinsp;\u0026plusmn;\u0026thinsp;1.102\u003cb\u003e*\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c3\" namest=\"c1\"\u003e \u003cp\u003eEach column represents the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD; * p\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eFe concentration (\u0026micro;g/g) in RBC of control and CoCl\u003csub\u003e2\u003c/sub\u003e-exposed mice\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDay\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eControl (n\u0026thinsp;=\u0026thinsp;5)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e125 ml/kg CoCl\u003csub\u003e2\u003c/sub\u003e (n\u0026thinsp;=\u0026thinsp;5)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e342\u0026thinsp;\u0026plusmn;\u0026thinsp;37.72\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e273.667\u0026thinsp;\u0026plusmn;\u0026thinsp;57.757\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e647.797\u0026thinsp;\u0026plusmn;\u0026thinsp;89.682\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e729.063\u0026thinsp;\u0026plusmn;\u0026thinsp;26.748\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e657\u0026thinsp;\u0026plusmn;\u0026thinsp;156.656\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e283\u0026thinsp;\u0026plusmn;\u0026thinsp;49.78\u003cb\u003e*\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e689\u0026thinsp;\u0026plusmn;\u0026thinsp;160.629\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e367.5\u0026thinsp;\u0026plusmn;\u0026thinsp;41.869\u003cb\u003e*\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e671\u0026thinsp;\u0026plusmn;\u0026thinsp;141.105\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e293.563\u0026thinsp;\u0026plusmn;\u0026thinsp;101.167\u003cb\u003e*\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c3\" namest=\"c1\"\u003e \u003cp\u003eEach column represents the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD; * p\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Fe-regulatory proteins TfR1, hepcidin and ferritin expression\u003c/h2\u003e \u003cp\u003eAlterations in Fe content were associated with changes in expression of Fe-regulatory proteins, TfR1, hepcidin, and ferritin. Data obtained from ELISA assays demonstrated a decrease in TfR1 concentration (Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e) in all experimental groups exposed to CoCl\u003csub\u003e2\u003c/sub\u003e except day 45 mice and increase in serum ferritin and hepcidin levels of CoCl\u003csub\u003e2\u003c/sub\u003e-exposed mice (Tables\u0026nbsp;\u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, \u003cspan refid=\"Tab7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). The reduced serum TfR1 concentration corresponds to the significantly elevated serum Fe in the Co-exposed experimental groups.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eTfR1 concentration (ng/g) of control and CoCl\u003csub\u003e2\u003c/sub\u003e-exposed mice. Each column represents the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDay\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eControl (n\u0026thinsp;=\u0026thinsp;5)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e125 ml/kg CoCl\u003csub\u003e2\u003c/sub\u003e (n\u0026thinsp;=\u0026thinsp;5)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6.13\u0026thinsp;\u0026plusmn;\u0026thinsp;3.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.69\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.84\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.66\u0026thinsp;\u0026plusmn;\u0026thinsp;1.10\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.35\u0026thinsp;\u0026plusmn;\u0026thinsp;0.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.54\u0026thinsp;\u0026plusmn;\u0026thinsp;0.55\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.51\u0026thinsp;\u0026plusmn;\u0026thinsp;1.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.41\u0026thinsp;\u0026plusmn;\u0026thinsp;0.26\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e18.82\u0026thinsp;\u0026plusmn;\u0026thinsp;15.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.58\u0026thinsp;\u0026plusmn;\u0026thinsp;0.43\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c3\" namest=\"c1\"\u003e \u003cp\u003eEach column represents the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eSerum TfR1concentration reduction varied from ~\u0026thinsp;32.45-fold in day 90 mice to 1.27-fold in day 30 cobalt-exposed mice.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab6\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 6\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSerum ferritin concentration (ng/g) of control and CoCl\u003csub\u003e2\u003c/sub\u003e-exposed mice\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDay\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eControl (n\u0026thinsp;=\u0026thinsp;5)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e125 ml/kg CoCl\u003csub\u003e2\u003c/sub\u003e (n\u0026thinsp;=\u0026thinsp;5)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3.7\u0026thinsp;\u0026plusmn;\u0026thinsp;1.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e9.42\u0026thinsp;\u0026plusmn;\u0026thinsp;11.12\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4.44\u0026thinsp;\u0026plusmn;\u0026thinsp;0.91\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6.09\u0026thinsp;\u0026plusmn;\u0026thinsp;4.34\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4.83\u0026thinsp;\u0026plusmn;\u0026thinsp;0.65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6.68\u0026thinsp;\u0026plusmn;\u0026thinsp;3.79**\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e11.34\u0026thinsp;\u0026plusmn;\u0026thinsp;3.52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5.94\u0026thinsp;\u0026plusmn;\u0026thinsp;2.19**\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6.29\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e33.56\u0026thinsp;\u0026plusmn;\u0026thinsp;18.49\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c3\" namest=\"c1\"\u003e \u003cp\u003eEach column represents the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD; ** p\u0026thinsp;\u0026lt;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eSerum ferritin concentration was increased in all Co-treated groups except for day 60 mice where a significant\u0026thinsp;~\u0026thinsp;1.9-fold decrease was observed. The highest concentration was found in day 90 mice which was a\u0026thinsp;~\u0026thinsp;5.34-fold increase compared to the untreated control samples. Data correspond with the reduced RBCs iron content.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab7\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 7\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eHepcidin concentration (pg/g) of control and CoCl\u003csub\u003e2\u003c/sub\u003e-exposed mice.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDay\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eControl (n\u0026thinsp;=\u0026thinsp;5)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e125 ml/kg CoCl\u003csub\u003e2\u003c/sub\u003e (n\u0026thinsp;=\u0026thinsp;5)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"1\" nameend=\"c4\" namest=\"c4\"\u003e\u0026nbsp;\u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1323.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e349.41\u0026thinsp;\u0026plusmn;\u0026thinsp;66.09***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c4\" namest=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e299.09\u0026thinsp;\u0026plusmn;\u0026thinsp;68.36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e469.46\u0026thinsp;\u0026plusmn;\u0026thinsp;320.32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c4\" namest=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e406.51\u0026thinsp;\u0026plusmn;\u0026thinsp;173.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e451.48\u0026thinsp;\u0026plusmn;\u0026thinsp;224.66\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c4\" namest=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1127.51\u0026thinsp;\u0026plusmn;\u0026thinsp;239.91\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e230.71\u0026thinsp;\u0026plusmn;\u0026thinsp;149.87***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c4\" namest=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e188.32\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e359.74\u0026thinsp;\u0026plusmn;\u0026thinsp;152.89\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c4\" namest=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"4\" nameend=\"c4\" namest=\"c1\"\u003e \u003cp\u003eEach column represents the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. *** p\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eChronic exposure to CoCl\u003csub\u003e2\u003c/sub\u003e increased serum hepcidin concentration in almost all experimental groups except day 18 and day 60 Co-treated mice where a significant decrease of ~\u0026thinsp;3.79-fold and ~\u0026thinsp;4.89-fold, respectively was found. The observed reduction is in agreement with the significantly elevated sFe concentrations in these groups. The highest increase - ~ 1.9-fold was found in day 90 cobalt-treated mice which corresponds to the lowest serum and RBC iron content and reduced TfR1 concentration found in that group.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Erythrocytic parameters\u003c/h2\u003e \u003cp\u003eChronic exposure to CoCl\u003csub\u003e2\u003c/sub\u003e affected erythrocytic parameters, as well (Table\u0026nbsp;\u003cspan refid=\"Tab8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). The effect was more pronounced for Hb as its content increased significantly in a time-dependent manner in CoCl\u003csub\u003e2\u003c/sub\u003e-treated mice with the highest content being measured in day 90 mice. RBCs increased in all exposed groups except in d45 and d90 which could explain the increased hepcidin concentration in these groups. The highest increase in RBC count was observed for d18 Co-exposed mice - ~ 1.86-fold compared to the untreated control. The Hb-related parameters - MCH and MCHC were also significantly increased in all cobalt-supplemented groups except for day 18 mice. Surprisingly, Co-exposure reduced RDW compared to the untreated age-matched controls making the RBCs more uniform in size. Significantly lower RDW was found in immature Co-treated mice compared to their age-matched untreated controls. The enhanced erythropoietic parameters in day 18 and day 60 cobalt-exposed mice could explain the decreased hepcidin activity in these groups.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab8\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 8\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eErythrocytic parameters in control and Co-treated mice.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eExperimental\u003c/p\u003e \u003cp\u003egroup\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRBC\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHb\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMCV\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMCH\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eMCHC\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eRDW\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"7\" nameend=\"c7\" namest=\"c1\"\u003e \u003cp\u003eDay18\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eControl\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3.68\u0026thinsp;\u0026plusmn;\u0026thinsp;0.56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e69\u0026thinsp;\u0026plusmn;\u0026thinsp;12.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e55.41\u0026thinsp;\u0026plusmn;\u0026thinsp;5.38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e18.71\u0026thinsp;\u0026plusmn;\u0026thinsp;2.23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e344.13\u0026thinsp;\u0026plusmn;\u0026thinsp;71.68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e25.39\u0026thinsp;\u0026plusmn;\u0026thinsp;5.84\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eCoCl\u003c/b\u003e\u003csub\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sub\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e(n\u0026thinsp;=\u0026thinsp;7)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6.88\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e99\u0026thinsp;\u0026plusmn;\u0026thinsp;3.5***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e54.78\u0026thinsp;\u0026plusmn;\u0026thinsp;2.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e14.35\u0026thinsp;\u0026plusmn;\u0026thinsp;0.51 ***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e262.67\u0026thinsp;\u0026plusmn;\u0026thinsp;4.23 **\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e17.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.68 **\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"7\" nameend=\"c7\" namest=\"c1\"\u003e \u003cp\u003eDay30\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eControl\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6.26\u0026thinsp;\u0026plusmn;\u0026thinsp;0.43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e78.8\u0026thinsp;\u0026plusmn;\u0026thinsp;5.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e47.68\u0026thinsp;\u0026plusmn;\u0026thinsp;2.48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e12.57\u0026thinsp;\u0026plusmn;\u0026thinsp;0.71\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e264.17\u0026thinsp;\u0026plusmn;\u0026thinsp;5.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e29.48\u0026thinsp;\u0026plusmn;\u0026thinsp;2.75\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eCoCl\u003c/b\u003e\u003csub\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sub\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e(n\u0026thinsp;=\u0026thinsp;7)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7.36\u0026thinsp;\u0026plusmn;\u0026thinsp;0.84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e101\u0026thinsp;\u0026plusmn;\u0026thinsp;20.9*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e48.36\u0026thinsp;\u0026plusmn;\u0026thinsp;4.94\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e13.66\u0026thinsp;\u0026plusmn;\u0026thinsp;2.18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e282.21\u0026thinsp;\u0026plusmn;\u0026thinsp;18.37 *\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e20.09\u0026thinsp;\u0026plusmn;\u0026thinsp;2.11 ***\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"7\" nameend=\"c7\" namest=\"c1\"\u003e \u003cp\u003eDay45\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eControl\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e9.31\u0026thinsp;\u0026plusmn;\u0026thinsp;078\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e116.14\u0026thinsp;\u0026plusmn;\u0026thinsp;9.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e47.93\u0026thinsp;\u0026plusmn;\u0026thinsp;2.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e12.44\u0026thinsp;\u0026plusmn;\u0026thinsp;0.38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e260.29\u0026thinsp;\u0026plusmn;\u0026thinsp;5.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e22.93\u0026thinsp;\u0026plusmn;\u0026thinsp;1.22\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eCoCl\u003c/b\u003e\u003csub\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sub\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e(n\u0026thinsp;=\u0026thinsp;7)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8.67\u0026thinsp;\u0026plusmn;\u0026thinsp;10.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e125.14\u0026thinsp;\u0026plusmn;\u0026thinsp;10.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e50.9\u0026thinsp;\u0026plusmn;\u0026thinsp;3.51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e14.44\u0026thinsp;\u0026plusmn;\u0026thinsp;1.23 **\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e284.57\u0026thinsp;\u0026plusmn;\u0026thinsp;11.33 ***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e21.46\u0026thinsp;\u0026plusmn;\u0026thinsp;3.83\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"7\" nameend=\"c7\" namest=\"c1\"\u003e \u003cp\u003eDay 60\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eControl\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.94\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e111.57\u0026thinsp;\u0026plusmn;\u0026thinsp;9.86\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e50.74\u0026thinsp;\u0026plusmn;\u0026thinsp;3.32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e13.11\u0026thinsp;\u0026plusmn;\u0026thinsp;0.66\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e259.29\u0026thinsp;\u0026plusmn;\u0026thinsp;6.47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e17.36\u0026thinsp;\u0026plusmn;\u0026thinsp;2.31\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eCoCl\u003c/b\u003e\u003csub\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sub\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e(n\u0026thinsp;=\u0026thinsp;9)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8.86\u0026thinsp;\u0026plusmn;\u0026thinsp;0.76\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e127.89\u0026thinsp;\u0026plusmn;\u0026thinsp;11.74*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e49.78\u0026thinsp;\u0026plusmn;\u0026thinsp;2.69\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e14.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.98 *\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e290.22\u0026thinsp;\u0026plusmn;\u0026thinsp;5.91***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e17.08\u0026thinsp;\u0026plusmn;\u0026thinsp;2.27\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"7\" nameend=\"c7\" namest=\"c1\"\u003e \u003cp\u003eDay 90\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eControl\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e9.64\u0026thinsp;\u0026plusmn;\u0026thinsp;1.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e125.25\u0026thinsp;\u0026plusmn;\u0026thinsp;11.95\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e49.48\u0026thinsp;\u0026plusmn;\u0026thinsp;1.94\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e12.95\u0026thinsp;\u0026plusmn;\u0026thinsp;0.24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e262.75\u0026thinsp;\u0026plusmn;\u0026thinsp;6.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e15.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.64\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eCoCl\u003c/b\u003e\u003csub\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sub\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e(n\u0026thinsp;=\u0026thinsp;9)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8.99\u0026thinsp;\u0026plusmn;\u0026thinsp;0.78\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e131.67\u0026thinsp;\u0026plusmn;\u0026thinsp;11.91\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e50.44\u0026thinsp;\u0026plusmn;\u0026thinsp;1.77\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e14.59\u0026thinsp;\u0026plusmn;\u0026thinsp;0.39 ***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e290.22\u0026thinsp;\u0026plusmn;\u0026thinsp;7.31***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e15.39\u0026thinsp;\u0026plusmn;\u0026thinsp;0.95\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"7\" nameend=\"c7\" namest=\"c1\"\u003e \u003cp\u003eEach column represents the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. * p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, ** p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, *** p\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eExposure to cobalt may occur through diet, dietary supplements, occupational exposure, biomedical applications and medical devices [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Although not completely declared some dietary supplements are promoted as performance-boosting nutrients and contain soluble and bioavailable Co as their active ingredient [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. As cobalt supplements may induce erythropoietin production thus affecting endurance performance, in 2017 the World Anti-Doping Agency included them in its prohibited list [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. The erythropoietic effect of cobalt in our study was demonstrated by elevated Hb content and increased RBCs in almost all experimental groups. The expanded erythropoiesis may be due increased erythroferrone production as it is stimulated by hypoxia [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. In addition, stimulated RBC production may be also due upregulation of GATA-1 known to enhance erythroid differentiation and to be highly expressed under hypoxia [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Although the dose of CoCl\u003csub\u003e2\u003c/sub\u003e sufficient to stimulate erythropoiesis is unknown, Hoffmeister et al. show that oral Co\u003csup\u003e2+\u003c/sup\u003e dosage of 10 mg/day for 5-day duration exerts erythropoietic effects [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. In their earlier work the same authors find that a 3-week administration of 5 mg/day Co supplement significantly increased hemoglobin mass while the other hematological parameters were not significantly affected [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn serum Co\u003csup\u003e2+\u003c/sup\u003e binds to albumin, and the concentration of free, ionized Co\u003csup\u003e2+\u003c/sup\u003e is estimated at 5\u0026ndash;12% of the total cobalt concentration. Our results for chronic treatment with CoCl\u003csub\u003e2\u003c/sub\u003e suggest that immature mice accumulate\u0026thinsp;~\u0026thinsp;4.4-fold more metal compared to mature animals. Also, during long-term exposure the most Co is accumulated in serum after 30 days of exposure and decreases by day 90 of dosing. The decrease may be due to \u0026ldquo;auto-inhibition\u0026rdquo; by cobalt as suggested by Simonsen et al. [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], and increased excretion and/or suppressed absorption due to elevated body iron. Our results are in accordance with those of Reuber et al. [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] who demonstrate enhanced Co excretion during Co and/or iron supplementation. Our experimental data suggest that serum Co concentration is a reliable marker for a recent exposure - up to 30 days.\u003c/p\u003e \u003cp\u003eResearch data imply that during long-term cobalt exposure \u003cem\u003ein vivo\u003c/em\u003e cobalt will be taken up practically irreversibly in the red cells during their 120 days life span [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Our results demonstrate significant cobalt accumulation in RBCs following chronic exposure to CoCl\u003csub\u003e2\u003c/sub\u003e. The highest concentration was found in day 30 mice and decreased by day 90 of exposure. Our results for the highest Co concentration in RBCs of day 30 mice are also similar to those of Bryan et al. [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] where cobalt concentration in erythrocytes increased with time to reach a plateau after 5\u0026ndash;6 weeks of daily peritoneal exposure of rats.\u003c/p\u003e \u003cp\u003eAt systemic and cellular level Co is sequestered along the Fe-acquisition pathway. The behavior of the transferrin-Co complex is similar to that of iron with a fast interaction with the C-lobe and a very slow one with the N-lobe of TfR1 [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIron is biologically essential but toxic in high concentrations. For this reason, its metabolism is tightly controlled at cellular and systemic level to prevent deficiency or overload [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn our study, chronic \u003cem\u003ein vivo\u003c/em\u003e exposure to CoCl\u003csub\u003e2\u003c/sub\u003e significantly altered serum and erythrocyte Fe content. Similar results for significantly increased Co and Fe concentrations in the serum and RBCs of Co-exposed mice were obtained in our previous studies using lower daily dose of CoCl\u003csub\u003e2\u003c/sub\u003e [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAlthough Fe significantly increased in serum of Co-exposed mice, its content in mature mice remained lower compared to immature, with the largest difference of 9.9% between d30 and d90 mice. A possible explanation for the lower serum Fe content in mature mice is the elevated Hb content suggesting Fe utilization in heme synthesis. RBC iron decreased in Co-exposed mature mice but increased in d30 mice, being ~\u0026thinsp;2.48-fold higher compared to d90 mice.\u003c/p\u003e \u003cp\u003eOur results for reduced erythrocyte Fe content indicate that erythroid cells possibly export Fe to survive. This hypothesis is supported by Keel et al. [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] who demonstrate that the export of excess cytoplasmic heme from the erythroid precursors is mediated by the feline leukemia virus, subgroup C, receptor (FLVCR). Iron overload is known to damage RBC plasma membranes by increasing reactive oxygen species production and osmotic fragility [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. A possible mechanism for erythrocyte Fe release in the blood plasma may be through increased ferroportin activity. Zhang et al. [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] demonstrate abundant ferroportin expression in mature RBCs and hypothesize that erythrocytes export Fe to avoid iron overload and hemolysis.\u003c/p\u003e \u003cp\u003eIn our studies we have observed stimulated ferroportin expression in the bone marrow of day 60 and day 90-cobalt treated mice (our unpublished data). In addition, cellular iron export is stimulated by ceruloplasmin. Low oxygen concentrations stimulate Fe release by ceruloplasmin who is also known to stabilize ferroportin on cell membrane [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Mature erythrocytes express ceruloplasmin receptors [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] and therefore its role in Fe reduction by RBCs should not be excluded. The insignificant change in cell volume also suggests that the erythrocytes likely export the excess Fe. According to McLaren et al. [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], cases of excess Fe are associated with RBC\u0026rsquo;s expanded size and higher cellular Hb concentration as a functional utilization of Fe within a non-toxic compartment.\u003c/p\u003e \u003cp\u003eWithin the cells Fe is stored in the form of ferritin. Ferritin is an iron-binding protein that indicates total body iron stores. The iron-storing capacity of ferritin is assisted by the ferroxidase activity of ferritin heavy chain, which converts reactive iron (Fe\u003csup\u003e2+\u003c/sup\u003e) into inert, nucleated iron (Fe\u003csup\u003e3+\u003c/sup\u003e), no longer available to catalyze the production of free radicals via Fenton chemistry [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. It is an acute-phase protein that increases in response to inflammatory states, including malignancy, infection, and in liver, renal and autoimmune diseases. Serum ferritin is a clinical marker for Fe status as it is increased in cases of Fe overload and reduced in Fe deficiency [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Elevated serum ferritin also reflects macrophage ferritin content as ferritin is predominantly secreted in the circulation by the macrophages [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. In our study serum ferritin was elevated almost in all experimental groups following chronic exposure to CoCl\u003csub\u003e2\u003c/sub\u003e. In day 90 exposed mice serum ferritin concentration was increased more than 5-fold also suggesting enhanced loss of Fe by the erythrocytes.\u003c/p\u003e \u003cp\u003eAnother key marker for Fe trafficking is TfR1. It can bind two different Fe-binding molecules: transferrin and ferritin [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Serum contains a soluble TfR (sTfR) molecule that circulates bound to transferrin that is cleaved from the whole TfR molecule and is a sensitive marker for erythropoiesis and iron deficiency [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. The circulating sTfR level reflects total body TfR concentration, the major source of sTfR being bone marrow erythroid precursors [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe reduced serum TfR1 concentration in our study is in accordance with the increased serum Fe and ferritin concentrations. These results suggest downregulation of TfR due to increased intracellular Fe storage and total body Fe as TfR expression is shown to decrease in cases of excess Fe [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. According to R\u0026rsquo;zik and Beguin [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e] there is an inverse correlation between body iron stores and total TfR. In addition, the high serum Fe concentration may counteract erythropoietin production by the kidneys resulting in lower TfR expression.\u003c/p\u003e \u003cp\u003eAnother explanation for the reduced TfR1 may be that the increased serum ferritin delivers Fe to the tissues serving as an alternative pathway to the Tf-TfR one, as observed by Wang et al. [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Also, \u003cem\u003ein vitro\u003c/em\u003e experiments have shown that the isoform of the second transferrin receptor - TfR2-α, incorporates Tf-bound iron into cells as effectively as TfR1, and it is expressed predominantly by erythroid progenitors but the expression of TfR2 mRNA does not show obvious changes upon iron loading or iron chelation [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. In our previous study we also demonstrate stimulated TfR2 tissue expression in immature mice following Co exposure [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. This suggests that the role of TfR2 should not be neglected in cases of stimulated erythropoiesis and/or elevated serum Fe.\u003c/p\u003e \u003cp\u003eIncreased serum iron levels upregulate hepcidin expression. Hepcidin acts as a negative regulator of Fe. In coordination with ferroportin hepcidin regulates Fe entry into plasma, its utilization and storage [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. The elevated serum Fe in our study enhanced hepcidin production in almost all Co-exposed groups. The stimulated erythropoiesis in day 18 and day 60 mice may be a possible explanation for the significantly reduced serum hepcidin in these groups. In addition, erythroferrone which stimulated RBC production suppresses hepcidin expression in a time-dependent manner [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Stimulated erythropoiesis strongly suppresses the production of hepatic hepcidin in mice and humans, allowing more dietary iron and Fe from stores to enter blood plasma for heme and hemoglobin synthesis by developing erythrocytes in the marrow [5; 7].\u003c/p\u003e \u003cp\u003eThe results indicate that Fe metabolism is tightly controlled/regulated by erythropoiesis through the suppression of hepcidin. In our study serum hepcidin concentration in mature mice was lower than in the immature. Possible explanation for this may be that during chronic exposure the high extracellular Fe concentration may suppress hepcidin production by a feedback mechanism.\u003c/p\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eCoCl\u003csub\u003e2\u003c/sub\u003e supplementation exhibited diverse effects on Fe bioavailability in serum and erythrocytes of immature and mature mice. Chronic exposure to CoCl\u003csub\u003e2\u003c/sub\u003e resulted in a lower Fe content of mature mice compared to immature suggesting stimulated Fe release as a possible survival mechanism to counteract the toxic effects of Fe overload. Cobalt administration stimulated Fe storage, enhancing hepcidin production and ferritin concentrations, and reducing TfR1 expression. During long-term exposure the most Co was accumulated in the serum and erythrocytes after 30 days of treatment and decreased by day 90 of dosing indicating that whole blood Co concentration is a reliable marker for a recent exposure.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eConflicts of Interest:\u003c/h2\u003e \u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eEthical Approval:\u003c/h2\u003e \u003cp\u003e The experimental protocol was performed at the Institute of Experimental Morphology, Pathology and Anthropology with Museum, Bulgarian Academy of Sciences, according to the ARRIVE guidelines and EU Directive 2010/63/EU for animal experiments. The study was approved by the Bulgarian Agency for Food Safety, Approval number 282 from 24.09.2020.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003cp\u003eAlexey A. Tinkov and Anatoly V. Skalny are members of the Editorial board\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding:\u003c/h2\u003e \u003cp\u003eThis research was funded by Grants No. DNTS/Russia 02/1/14.06.2018 from the Bulgarian National Science Fund. AAT and AVS were supported by the Russian Ministry of Science and Higher Education (075-15-2024-550).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eE.P. (Ekaterina Pavlova) - methodology, investigation, visualization, writing - review and editing; E.P. (Emilia Petrova) - methodology, investigation, writing - review and editing; A.A.T. - methodology, investigation, formal analysis, writing - review and editing; O.P.A. - methodology, validation, formal analysis; P.R. - methodology, investigation; I.V. - methodology, investigation; A.V.S. - writing - review and editing, project administration; Y.G. - writing - review and editing All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgements:\u003c/h2\u003e \u003cp\u003eThis research was funded by Grants No. DNTS/Russia 02/1/14.06.2018 from the Bulgarian National Science Fund. AAT and AVS were supported by the Russian Ministry of Science and Higher Education (075-15-2024-550).\u003c/p\u003e\u003ch2\u003eData Availability Statement:\u003c/h2\u003e \u003cp\u003eAll data necessary to understand or reproduce this study are included in the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eEbert B, Jelkmann W (2014) Intolerability of cobalt salt as erythropoietic agent. Drug Test Anal 6:185\u0026ndash;189. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi:10.1002/dta.1528\u003c/span\u003e\u003cspan address=\"https://doi:10.1002/dta.1528\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSmith TA (2005) Human serum transferrin cobalt complex: stability and cellular uptake of cobalt. 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Biol Trace Elem Res 194:423\u0026ndash;431. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s12011-019-01790-8\u003c/span\u003e\u003cspan address=\"10.1007/s12011-019-01790-8\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"cobalt chloride, iron, TfR1, hepcidin, ferritin, erythrocytes","lastPublishedDoi":"10.21203/rs.3.rs-4697764/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4697764/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCobalt (Co) is an essential trace element and its cellular uptake occurs in a similar to iron (Fe) profile. The aim was to assess the alterations in iron and Fe regulatory proteins concentrations - transferrin receptor 1 (TfR1), hepcidin and ferritin, and their effect on erythrocyte count (RBCs) in mice following chronic exposure to cobalt chloride (CoCl\u003csub\u003e2\u003c/sub\u003e). Pregnant ICR mice were subjected to 125 mg/kg body weight CoCl\u003csub\u003e2\u003c/sub\u003ex6H\u003csub\u003e2\u003c/sub\u003eO daily 2\u0026ndash;3 days prior delivery and treatment continued 90 days after birth. CoCl\u003csub\u003e2\u003c/sub\u003e was administrated with drinking water. Pups were sacrificed on postnatal days 18, 30, 45, 60 and 90. Exposure to CoCl\u003csub\u003e2\u003c/sub\u003e induced significant accumulation of Co ions in blood sera and RBCs. During long-term exposure the most Co was accumulated in the serum after 30 days of exposure and decreased by day 90 of dosing indicating that serum Co concentration is a reliable marker for recent exposure. Hemoglobin content increased in a time-dependent manner. Co administration significantly elevated serum Fe but decreased it in RBCs. Exposure to Co stimulated Fe storage, enhancing hepcidin production and ferritin concentrations, and reducing TfR1 expression. Chronic exposure to CoCl\u003csub\u003e2\u003c/sub\u003e resulted in a lower Fe content of mature mice compared to immature suggesting stimulated Fe release as a possible survival mechanism to counteract the toxic effects of Fe overload.\u003c/p\u003e","manuscriptTitle":"Diverse effects of chronic cobalt supplementation on iron metabolism during erythropoiesis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-31 15:31:31","doi":"10.21203/rs.3.rs-4697764/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"14c8d576-6f47-445b-9ebc-a0ae148e9662","owner":[],"postedDate":"July 31st, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-09-17T00:18:54+00:00","versionOfRecord":[],"versionCreatedAt":"2024-07-31 15:31:31","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4697764","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4697764","identity":"rs-4697764","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}