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Original Paper
Pharmacology 2008;82:127–137 DOI: 10.1159/000142728
Comparative Study of GastrointestinalTract and Liver Toxicity of Ferrous Sulfate,Iron Amino Chelate and Iron PolymaltoseComplex in Normal Rats
Jorge E. Toblli Gabriel Cao Leda Olivieri Margarita Angerosa
Laboratory of Experimental Medicine, Hospital Alemán, Buenos Aires , Argentina
villus were increased (p ! 0.01) in the FS and iron AC groups versus the IPC and control groups. Ferritin was elevated(p ! 0.01) in the IPC group versus FS and iron AC in the small intestine and liver. The oxidative stress markers were all sig-nificantly (p ! 0.01) altered in the FS and iron AC groups ver-sus the IPC and control groups in the intestinal mucosa and liver. Conclusion: FS exhibited important acute toxicity as well as early and late GI tract and liver toxicity. Despite show-ing similar LD 50 as IPC, iron AC presented differences regard-ing early and late GI tract and liver toxicity versus IPC.
Copyright © 2008 S. Karger AG, Basel
Introduction
Iron is an essential nutrient for a large number of cellular activities including oxygen transport, electron transfer and gene regulation. It is involved in the struc-ture and proper functioning of hemoglobin, myoglobin, cytochromes, and several enzymes active in porphyrin synthesis, oxygen regulation and immunity [1] .
According to the World Health Organization, iron de-ficiency anemia is the leading form of nutritional anemia throughout the world affecting more than 1 billion peo-ple. Young children from birth to 12 years of age, adoles-cents and females at child-bearing age are the most sus-ceptible groups affected by iron deficiency anemia. Fur-thermore, anemia in older individuals is associated with
Key Words
Oral iron compounds � Oxidative stress � Gastrointestinal tract � Liver � Iron polymaltose complex
Abstract
Iron deficiency is a common worldwide problem leading to several morbidities including anemia. Although oral iron is the first choice in iron deficiency therapy, it may produce gastrointestinal (GI) and liver disorders. The aim of our study was to evaluate: (1) acute toxicity (LD 50 ) in different oral iron compounds such as ferrous sulfate (FS), iron amino chelate (AC) and iron polymaltose complex (IPC) and (2) possible differences in early and late toxicity in the GI tract and liver between them. Methods: Hematological variables, liver en-zymes, oxidative stress markers (thiobarbituric-acid-reactive substances, reduced glutathione, catalase, glutathione per-oxidase, CuZn superoxide dysmutase) in intestinal mucosa and liver homogenates, and morphological parameters (gross anatomy, histology) were evaluated in non-anemic rats. Results: LD 50 was lower (p ! 0.01) in FS versus iron AC and IPC. The liver enzymes were increased in the FS group(p ! 0.05). The FS group presented gastric mucosal erosions and the iron AC group showed submucosal hemorrhages in the lower GI tract (colon and rectum) versus the IPC and con-trol groups. In the small intestine, the villi/crypt ratio and goblet cells per villus were significantly (p ! 0.01) reduced in the FS and iron AC groups versus IPC. The eosinophils per
Received: November 15, 2007 Accepted after revision: February 11, 2008 Published online: July 8, 2008
Jorge E. Toblli, MD, PhD Laboratory of Experimental Medicine, Hospital Alemán Av. Pueyrredon 1640 Buenos Aires 1118 (Argentina) Tel. +54 11 4827 7000, Fax +54 11 4805 6087, E-Mail [email protected]
© 2008 S. Karger AG, Basel0031–7012/08/0822–0127$24.50/0
Accessible online at:www.karger.com/pha
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a very wide range of complications including increased risk of mortality, cardiovascular disease, cognitive dys-function, longer hospitalization for elective procedures and comorbid conditions. Additionally, it causes reduced bone density, falls and fractures; and it has a significant effect on the quality of life in the elderly. Since iron defi-ciency in general and iron deficiency anemia in particu-lar are morbidities present in a huge population world-wide, the importance of correcting this situation through iron therapy is essential in public health terms [2] .
On the other hand, excess cellular iron can be toxic due to production of reactive oxygen species such as O2
– and HO, which damage proteins, lipids and DNA [3–5] .
It is well known that oral iron therapy may produce a variable degree of gastrointestinal (GI) toxicity. More-over, the liver is the primary site for iron storage; there-fore, it is a particular risk for iron-induced oxidative stress and tissue damage. Because oral iron therapy is the first line option in order to replace the iron deficiency state, it is mandatory to evaluate the potential GI and liv-er toxicity of different oral iron preparations for a better selection criterion. Consequently, the aim of the present study was, first, to evaluate acute toxicity by LD 50 in dif-ferent oral iron preparations such as ferrous sulfate (FS), iron amino chelate (AC) and iron polymaltose complex (IPC) and, secondly, to establish possible differences in early (4 weeks) and late (4 months) toxicity in the GI tract and liver between these oral iron compounds.
Methods
Animals and Treatments All the experiments were approved by the Hospital Alemán
Ethics Committee and the Teaching and Research Committee and according to the NIH Guide for the Care and Use of Labora-tory Animals. Rats were housed in a temperature-controlled room (22 8 2 ° C) with free access to tap water and fed standard rat chow (Cooperación, Argentina) ad libitum throughout the study.
Experiment 1: LD 50 Groups of 6 male and 6 female Sprague-Dawley rats weighing
200–220 g were used for this experiment. In order to evaluate LD 50 , each iron compound in a single oral dose was administered by a stomach tube after the animal had been fasted for about 18 h prior to dosing. Diet was withheld for 3 h after dosing. Increasing doses of (1) FS, (2) AC, and (3) IPC were used. A group of rats which received an equivalent volume of vehicle by stomach tube was included as control. Clinical signs of the animals were ob-served 4 times on the dose day and thereafter once a day. The number of deaths was counted at 24 h after treatment.
Experiment 2: Early Toxicity in GI Tract and Liver Twenty-four male and 24 female Sprague-Dawley rats weigh-
ing 200–220 g were randomized into 4 groups of 12 animals each with equal male-female proportions. The rats were housed in a temperature-controlled room (22 8 2 ° C) with free access to tap water and fed standard rat chow (Cooperación, Argentina) ad li-bitum throughout the study. During 4 weeks each group received a daily dose of oral iron preparation, which was equal to the 10% of LD 50 of the corresponding compound as indicated in the fol-lowing schedule:
FS group (n = 12, 6 males and 6 females) with FS = 25 mg/kg/day; iron AC group (n = 12, 6 males and 6 females) with iron AC = 280 mg/kg; IPC group (n = 12, 6 males and 6 females) with IPC = 280 mg/kg, and control group (n = 12, 6 males and 6 fe-males) with regular tap water. Each iron compound was given in drinking water. With the purpose of administering the right dose of iron preparations, a daily evaluation of water consumption and body weight determination were performed in all animals. In or-der to collect 24-hour urine, the animals were placed in meta-bolic cages. The rats from each group were sacrificed at 4 weeks. Previously, blood samples were obtained for biochemistry deter-mination. The liver, esophagus, stomach, bowel and kidneys were perfused with ice-cold saline and then removed for microscopy and immunohistochemical techniques.
Experiment 3: Late Toxicity in GI Tract and Liver Twenty-four male and 24 female Sprague-Dawley rats weigh-
ing 200–220 g were randomized into 4 groups of 12 animals each with equal male-female proportions. The rats were housed and fed in similar conditions to experiment 2. During 4 months, each group received a daily dose of oral iron preparation which was equal to the 10% of LD 50 of the corresponding compound, as in-dicated in the following schedule: FS group (n = 12, 6 males and 6 females) with FS = 25 mg/kg/day; iron AC group (n = 12, 6 males and 6 females) with iron AC = 280 mg/kg; IPC group (n = 12, 6 males and 6 females) with IPC = 280 mg/kg, and control group (n = 12, 6 males and 6 females) with regular tap water. Each iron compound was given in drinking water.
With the purpose of administering the right dose of iron prep-arations, a daily evaluation of water consumption and body weight determination were performed in all animals. The rats from each group were sacrificed at the fourth month of treatment. The liver and intestine were removed for oxidative stress parameter evalu-ation in fresh tissue.
Biochemical Procedures After 14-hour fasting, rat blood samples were collected from
the tail vein in capillary tubes. Hemoglobin was determined by SYSMEX XT 1800i, Roche Diagnostics. Serum iron and liver en-zymes, like aspartate aminotransferase, alanine aminotransfer-ase and alkaline phosphatase, were assessed by colorimetric and UV methods respectively, using an autoanalyzer Modular P800, Roche Diagnostics, with the correspondent reagents (Roche Di-agnostics GmbH, Mannheim, Germany). Aliquots of sera and urine were assayed for creatinine using the enzymatic UV meth-od (Randox Laboratories Ltd., Crumlin, UK). Creatinine clear-ance was determined by the standard formula. Serum transferrin was evaluated by radial immunodiffusion (Diffu-Plate, Biocien-tifica, S.A.). Transferrin saturation (TSAT; percent) was obtained using chemical methods.
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Oxidative Stress Parameter Evaluation in Small Intestine Mucosa and Liver Homogenate Preparation A fraction of the whole liver and small intestine mucosa were
homogenized (1: 3, w/v) in ice-cold 0.25 mol/l sucrose. Reduced glutathione (GSH) levels were determined in the 10,000 g super-natant following methods as previously described [6, 7] . Another fraction of each corresponding perfused tissue was homogenized (1: 10, w/v) in 0.05 mol/l sodium phosphate buffer, pH 7.4, and was directly used for the determination of malondialdehyde in order to evaluate lipoperoxidation by thiobarbituric-acid-reactive sub-stances (TBARS) [8] or it was centrifuged at 4 ° C during 15 min at 9,500 g . The resulting supernatant was used for measuring catalase activity [9] . Finally, another portion of each corresponding per-fused tissue fraction was homogenized (1: 3, w/v) in ice-cold su-crose (0.25 mol/l). The supernatant obtained after centrifugation at 105,000 g for 90 min was used for measuring CuZn superoxide dismutase (CuZn-SOD) and glutathione peroxidase (GPx) activity [10, 11] . Enzyme units were defined as the amount of enzyme pro-ducing 1 nmol of product or consuming 1 nmol of substrate (cata-lase) under the standard incubation conditions. Specific activity was expressed as units/milligram protein [12] . One unit of CuZn-SOD was defined as the amount of CuZn-SOD capable of inhibit-ing the rate of NADH oxidation measured in the control by 50%.
Gross Anatomy at GI Tract The lesion index was determined as the sum of erosion length
per rat. Iron-compound-induced lesion was assessed and scored for severity according to: (0) absence of lesion, (1) superficial, 1–5 hemorrhagic points, (2) superficial, 6–10 hemorrhagic points, (3) submucosal hemorrhagic lesions with small erosions and (4) se-vere hemorrhagic lesion and some invasive lesions.
Light Microscopy Portions of the esophagus, stomach, upper and lower GI tract,
liver, spleen and kidney were cut and fixed in phosphate-buffered 10% formaldehyde (pH 7.2) and embedded in paraffin. Three-mi-crometer sections were cut and stained with hematoxylin-eosin. Prussian blue (iron detection technique) was performed accord-ing to the classic Perl description. All observations in light mi-croscopy were performed using a Nikon E400 light microscope (Nikon Instrument Group, Melville, N.Y., USA), and the observer was blind to the animal group.
Immunohistochemical Study Immunolabeling of specimens was carried out by a modified
avidin-biotin-peroxidase complex technique, Vectastain ABC kit (Universal Elite, Vector Laboratories, Calif., USA). Following deparaffinization and rehydration, the sections were washed in phosphate-buffered saline (PBS) for 5 min. Quenching of endog-enous peroxidase activity was achieved by incubating the sections in 1% hydrogen peroxide in methanol for 30 min. After washing them in PBS, pH 7.2, for 20 min, they were incubated with block-ing serum during 20 min. Thereafter, the sections were incubated with the primary antibody, rinsed in PBS and incubated with bio-tinylated universal antibody during 30 min. After washing them in PBS, they were incubated with Vectastain Elite ABC reagent (Vector Laboratories) for 40 min, and exposed to 0.1% diamino-benzidine (Polyscience, Warrington, Pa., USA) and 0.2% hydro-gen peroxide in 50 m M Tris buffer, pH 8, for 5 min. Tissue ferritin
was quantified using antiferritin (Biogen, San Román, Calif., USA) monoclonal antibody.
Morphometric Analysis Histological sections were studied in each animal by an image
analyzer, Image-Pro Plus version 4 for Windows (Media Cybernet-ics, LP, Silver Spring, Md., USA). Morphological analyses were per-formed at a magnification of ! 100 or ! 400 depending on the tis-sue evaluated with the observer blind to the animal group, and the data were averaged. In the upper GI tract (small intestine), we eval-uated: (a) villi/crypt ratio; (b) number of eosinophils per villus, and (c) number of goblet cells per villus. Ferritin immuno staining in the small intestine was evaluated by a semiquantita tive score ac-cording to the following schedule: 0 = no staining; 1 = mild positive staining per villus; 2 = moderate positive staining per villus; 3 = intense positive staining per villus, and 4 = very intense positive staining per villus. In the liver we evaluated (a) iron deposits by the percentage of positive Prussian blue staining/square millimeter, and (b) the amount of tissue ferritin, expressed by positive ferritin immunostaining/square millimeter expressed as a percentage.
Statistical Method Values were expressed as means 8 SD. All statistical analyses
were performed using absolute values and processed through GraphPad Prism, version 2.0 (GraphPad Software, Inc., San Di-ego, Calif., USA). For parameters with Gaussian distribution, comparisons among groups were carried out using ANOVA; for parameters like histological data with non-Gaussian distribution comparisons were performed by Kruskal-Wallis test (nonpara-metric ANOVA) and Dunn’s multiple comparison test. A value of p ! 0.05 was considered significant.
Results
Experiment 1: LD 50 FS showed the lowest LD 50 in comparison with the
other oral iron preparations. On the other hand, iron AC and IPC presented similar values with a remarkably high LD 50 (FS = 255 mg Fe/kg/body weight; iron AC 1 2,800 mg Fe/kg/body weight; IPC 1 2,800 mg Fe/kg/body weight). It is worth mentioning that in the AC and IPC groups, because the increasing amount of iron compound solution exceeded the rat stomach capacity throughout the process for achieving LD 50 , we decided to take the highest tested dose as a cutoff one in these cases. As a re-sult, these data indicate a low acute toxicity of iron AC and IPC. There were not significant differences between the sexes in the groups with respect to LD 50 .
Experiment 2: Early Toxicity in GI Tract and Liver Body Weight, Food Intake, Water Consumption, Urine Output, Bowel Movement There were not significant differences between the
sexes in the groups with respect to the variables explored.
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At baseline all animals presented no differences with re-spect to body weight. However, while the control group exhibited normal growth throughout the study, the ani-mals which received FS showed a significantly lower weight. At the same time, the rats from the iron AC group presented significant differences with respect to the IPC and control groups, as represented in figure 1 a. Food in-take was equivalent between the groups at baseline and during the first 2 weeks. On the other hand, the FS group presented a significantly lower food consumption in comparison with the other groups in the third and fourth weeks. Interestingly, during the last week of treatment, while the animals from the IPC and control groups showed similar food intake, those from the iron AC group presented a significantly lower value, as indicated in fig-ure 1 b.
Bowel movement was significantly diminished in the animals from the FS group in the fourth week, indicating some degree of constipation in these rats ( fig. 2 ). No dif-ferences were observed concerning water consumption and urine output between the groups throughout the study ( fig. 2 ).
28 days0
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Fig. 1. Body weight evolution ( a ) and food consumption ( b ) throughout the study. No differences were found in the first weeks, however, significant variation was observed in the last weeks between the groups. a p ! 0.01: vs. all groups; b p ! 0.01: vs. IPC and control.
28 daysa Baseline 7 days 14 days 21 days0
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Fig. 2. Water consumption ( a ), urine output ( b ) and bowel move-ment ( c ). a p ! 0.01: vs. IPC and control; b p ! 0.01: vs. all groups.
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Hemoglobin, Serum Iron and TSAT There were not significant differences between the
sexes in the groups with respect to the variables explored. No significant variation was observed in hemoglobin concentration and hematocrit between the groups in the fourth week ( table 1 a). At the end of the experiment, the animals from the FS and iron AC groups presented a sig-nificantly (p ! 0.01) higher level of serum iron concentra-tion as well as the percentage of TSAT compared with the IPC and control groups, which showed similar values be-tween them, as indicated in table 1 a.
Renal Function Parameters There were not significant differences between the
sexes in the groups with respect to the variables explored. Creatinine clearance presented no variation between the groups at baseline and at the end of the experiment. Sig-nificant proteinuria was not observed in any group throughout the experiment.
Liver Function Parameters The liver enzymes (aspartate aminotransferase, ala-
nine aminotransferase and alkaline phosphatase) were increased (p ! 0.05) in the FS group in the fourth week when compared with the other groups, which did not present different values between them throughout the study ( table 1 b). However, although the iron AC group did not reach a higher value with statistical significance in liver enzymes with respect to the IPC and control groups, it is worth mentioning that this group showed higher enzyme values, which may suggest some inflam-matory reaction ( table 1 b).
Gross Anatomy of GI Tract No lesions were observed in the esophagus in any
group at the end of the study. On the other hand, the an-imals from the FS group presented gastric mucosal ero-sions, as illustrated in figure 3 . The other groups did not display any lesions in the stomach.
Whereas no macroscopic damage was observed in the upper GI tract (small intestine) in any group, the rats from the FS and iron AC groups presented a different le-sion degree from a simple mucosal edema and congestion to submucosal hemorrhages in the lower GI tract (colon and rectum), which indicates intestinal mucosa irritation of the corresponding iron compound throughout the lower GI tract, as illustrated in figure 4 . On the contrary, the IPC and control groups showed no lesions at the gross anatomy.
Microscopy Findings and Immunohistochemical Study No lesions were observed in the esophagus in any
group at the end of the study. The animals from the FS group presented inflammatory cell infiltration at the submucosa in the stomach. The other groups showed no microscopy lesions in the stomach. In the small intestine, the villi/crypt ratio was significantly lower in the FS and iron AC groups when compared with the IPC and control groups ( fig. 5 ). Moreover, the number of goblet cells per villus, which are involved in mucus secretion, was mark-edly reduced in these groups relative to the IPC and con-trol group ( fig. 6 a). Finally, the number of eosinophilsper villus was increased in the FS and iron AC groupsin comparison with the IPC and control groups ( fig. 6 b).
Table 1. Hematological and liver enzyme data at 4-week treatment (means 8 SD)
FS (n = 12) Iron AC (n = 12) IPC (n = 12) Control (n = 12)
a Hematological parametersHemoglobin, g/dl 15.880.3 15.780.3 16.080.2 15.680.4Hematocrit, % 47.281.1 46.381.2 47.080.9 46.181.3Serum iron, �g/dl 287.1814.9b 274.8819.3b 229.5811.4 203.3816.1TSAT, % 59.787.7b 58.886.9b 41.285.7 36.185.6
b Liver enzymesAspartate aminotransferase, IU/l 189.6822.3a 134.1815.7 114.6813.2 110.18 12.1Alanine aminotransferase, IU/l 78.986.4a 55.788.4 46.687.8 42.384.8Alkaline phosphatase, IU/l 631.1823.9a 578.9842.3 541.7830.2 530.5842.9
a p < 0.05 versus all groups; b p < 0.01 versus IPC and control.
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Control
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Control0
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Fig. 3. a Lesion score in the stomach at 4 weeks. a p ! 0.01 vs. all groups. b Gastric erosion in an animal of the FS group (circle). Fig. 4. a Lesion score in the low GI tract at 4 weeks. a p ! 0.05: vs. all groups; b p ! 0.05: vs. IPC and control. b Mucosal erosion in animals of the FS and AC groups (arrows). No lesions in the IPC and control groups.
Fig. 5. a Villi/crypt ratio in the small intestine at 4 weeks in all groups. a p ! 0.01: vs. all groups; b p ! 0.01: vs. IPC and control. b Micrographs of the small intestine in all groups. Note edemain villi and a reduced villi/crypt ratio especially in the FS group. No lesion in the IPC and control groups.
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Control ControlFS
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Fig. 6. a Number of goblet cells/villus in the small intestine at 4 weeks in all groups. b Number of eosinophils/villus in the small intestine at 4 weeks in all groups. a p ! 0.01: vs. all groups; b p ! 0.01: vs. IPC and control. c Micrographs of the small intestine in
all groups. Arrows indicate eosinophils and circles goblet cells in villi. Note the large number of eosinophils in the FS and AC groups along with a reduced amount of goblet cells in comparison with the IPC and control groups.
ControlFS
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Iron AC
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Fig. 7. a Semiquantitative score of ferritin immunostaining in the small intestine at 4 weeks in the different groups. a p ! 0.01: vs. all groups; b p ! 0.01: vs. FS and iron AC. b Microscopic images of the small intestine from all groups; arrows indicate ferritin de-posits. Original magnification ! 400.
Fig. 8. a Staining by Prussian blue in the liver at 4 weeks in the different groups. a p ! 0.01: vs. all groups; b p ! 0.01: vs. FS and iron AC. b Microscopic images of the liver in all groups; arrows indicate iron deposits. Note important iron deposits in the IPC in comparison with the other groups. Prussian blue. Original mag-nification ! 100.
ControlFS Iron AC IPC0
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All these findings suggest a variable degree of injury to the small intestine in these groups. A different degree of congestion, in the colon and rectum, was observed in ves-sels near to the mucosa in the rats belonging to the FS and iron AC groups. No lesions were found in the IPC and control groups.
Concerning ferritin immunostaining in the small in-testine, the rats from the FS and iron AC groups showed a lower score of immunostaining. In contrast, the ani-mals from the IPC group presented the highest value(p ! 0.01), as represented in figure 7 . In the liver, the IPC group exhibited a large area of positive staining for iron in interstitial cells (Kupffer’s cell) in comparison with the other groups, as illustrated in figure 8 .
Finally, ferritin immunostaining in the liver revealed that an important and significant (p ! 0.01) amount of tissue ferritin was observed in the IPC group compared with the other groups ( fig. 9 ).
Experiment 3: Late Toxicity in GI Tract and Liver Oxidative Stress Evaluation in Small Intestine Mucosa and Liver The animals from the FS and iron AC groups showed
a significant (p ! 0.01) increase in TBARS (lipoperoxida-tion), in small intestine mucosa as well as in the liver when compared with the IPC and control groups, which did not present any differences between them ( tables 2 and 3 , respectively). At the same time, the antioxidant enzymes, catalase and CuZn-SOD, were reduced in the rats from the FS and iron AC groups, together with a re-markable decrease in GSH (p ! 0.01), not only in intestine mucosa but also in the liver, in comparison with the IPC and control groups ( tables 2 and 3 ), indicating a high lev-el of oxidative stress presented in the former groups.
Finally, GPx activity, which is involved in removing H 2 O 2 using GSH, was significantly elevated (p ! 0.01) in both FS and iron AC, compared with the IPC and control groups, as shown in tables 2 and 3 .
ControlFS Iron AC IPC0
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Fig. 9. a Percentage of ferritin immunostaining in the liver at 4 weeks in the different groups. a p ! 0.01: vs. all groups; b p ! 0.01: vs. FS and iron AC. b Microscopic images of the liver in all groups;
arrows indicate ferritin deposits (in brown). A wide area of ferritin immunostaining in the IPC group in comparison with the other groups is observed. Ferritin. Original magnification ! 100.
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Discussion
In the present study using biochemical, gross anato-my, light microscopy and immunohistochemistry tech-niques, important differences were found between FS, iron AC and IPC concerning GI and liver toxicity. The most toxic compound regarding acute toxicity was FS ac-cording to the LD 50. On the other hand, iron AC and IPC presented a similarly high LD 50 .
Although no substantial variations with respect to he-moglobin concentration were observed among the groups throughout the study, the FS and iron AC groups pre-sented a significant increase in serum iron concentration together with a higher TSAT% when compared with the IPC and control groups. These findings suggest an over-saturation of the mechanism involved in iron transport. As a potential consequence, this situation may be leading to iron overload in various tissues, producing deleterious effects, probably through an oxidative stress pathway.
With respect to early toxicity in the GI tract, the ani-mals from the FS and iron AC groups presented lower food consumption along with a significantly lower body weight in relation to the IPC and control groups at the end of the experiment. Moreover, the bowel movement in rats from the FS group was markedly reduced in comparison
with the other groups. All these results suggest disar-rangements in the GI tract. Both gross anatomy and mi-croscopy evaluation confirm the inflammatory status in the GI tract in the FS and iron AC groups, especially at the small intestine and lower GI tract. Notably, the in-creased number of eosinophils in villi of animals from the FS and iron AC groups suggests some allergic compo-nent in the small intestine injury mechanism in these an-imals. Benoni et al. [13] reported a variable impact of oral FS (10–30 mg/kg) on the GI environment after single and repeated administration. Although these authors did not observe any substantial damage to GI structures, in the chronic experiment at lower doses, they found a strong alteration in fecal flora. In addition, other investigators such as Abraham et al. [14] reported erosive or ulcerative mucosal injury in most patients receiving FS therapy. De-spite the fact that FS presented the highest level of GI damage, iron AC, which is today considered as a safe compound [15] , also showed a considerable degree of GI toxicity, as observed in our results.
Both the FS and iron AC groups presented a signifi-cant increase in oxidative stress markers in the intestinal mucosa when compared with IPC. As already mentioned above, more rapid iron absorption from the intestine in the FS and iron AC groups, as suggested by a higher
Table 3. Oxidative stress parameters in the liver at 4-month treatment
FS (n = 12) Iron AC (n = 12) IPC (n = 12) Control (n = 12)
TBARS, nmol/MDA/mg tissue protein 4.080.2a 2.880.3a 1.080.1 0.880.2GSH, nmol/mg tissue protein 6.281.1a 7.981.2b 13.881.7 15.180.9Catalase, U/mg tissue protein 7.880.8a 6.280.5b 3.580.3 3.180.5GPx, U/mg tissue protein 10.981.4a 8.781.0b 1.780.5 1.480.4CuZn-SOD, U/mg tissue protein 99.387.5a 73.588.6b 35.185.4 28.183.9
Values are means 8 SD. MDA = Malondialdehyde. a p < 0.01: versus all groups; b p < 0.01: versus IPC and control.
Table 2. Oxidative stress parameters in small intestine mucosa at 4-month treatment
FS (n = 12) Iron AC (n = 12) IPC (n = 12) Control (n = 12)
TBARS, nmol/MDA/mg tissue protein 4.980.9a 3.680.3a 1.380.4 1.180.5GSH, nmol/mg tissue protein 3.881.0a 6.081.2b 11.480.6 12.881.7Catalase, U/mg tissue protein 6.780.5a 5.480.3b 3.280.2 2.980.3GPx, U/mg tissue protein 12.381.6a 10.281.0b 6.381.4 5.981.2CuZn-SOD, U/mg tissue protein 83.187.5a 70.685.1b 25.183.8 17.085.0
Values are means 8 SD. MDA = Malondialdehyde. a p < 0.01: versus all groups; b p < 0.01: versus IPC and control.
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GI and Liver Toxicity in Different Oral Iron Compounds
Pharmacology 2008;82:127–137 137
TSAT% compared with IPC, might explain the reason why these compounds cause free iron and then trigger off the cascade of oxidative stress.
The ferritin deposits in the small intestine were sig-nificantly increased in the IPC group in opposition to the other groups. We think that this is a relevant finding, which may help understand why IPC presents a con-trolled serum iron concentration without oversaturation in the iron transport pathway and, therefore, less possi-bility of developing iron toxicity. Our data confirm the clinical findings observed by Jacobs et al. [16] , who pos-tulated, in clinical studies, that IPC formulation more closely approximates the way in which intestinal cells handle dietary iron and thus physiologic regulatory mechanisms would be expected to reciprocally slow down absorption as stores expand.
Liver function was affected in the FS group, as indi-cated by the significant increase in liver enzymes in these animals. It is worth mentioning that, while the animals from the FS and iron AC groups presented a reduced
amount of iron in hepatic reticular cells (Prussian blue) and ferritin deposits (immunostaining), the rats from the IPC group showed a significantly higher value of both in the liver. Since ferritin within the cells is the physiological pathway of iron storage, a reduced expression of this pro-tein, as observed in the explored tissue by immunohisto-chemistry, may indicate that most free iron from the cir-culation was not stored as ferritin. This situation may lead to a different degree of inflammatory response, as found in our experiments. In order to illustrate this con-cept, all markers of oxidative stress evaluated after 4 months of treatment were significantly altered in the FS and iron AC groups with respect to the IPC and control groups.
Finally, information provided in the current study suggests that FS presents an important acute toxicity based on its LD 50 , and a substantial early GI tract and liver toxicity. Despite showing a similar LD 50 with respect to IPC, iron AC presents considerable differences regard-ing early GI tract toxicity.
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