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Antioxidant therapy of cobalt and vitamin E in hemosiderosis
Antioxidant therapy of cobalt and vitamin E in hemosiderosis (~IGDEM iNAN, KAMER KILIN(~, ESIN KOTILOGLU,HASAN ORHAN AKMAN, ILKNUR KILI~, and JOSEF MICHL ANKARA, TURKEY,and BROOKLYN,NEW YORK
The protective effects of cobalt and vitamin E in iron overloaded rats were investigated. Rats were divided into four groups: group I as control, group 2 received only iron; group 3 iron and cobalt, group 4 iron and vitamin E. All injections were given 3 times per week for 3 weeks. Biochemical and histopathologic studies were done on samples of blood and liver, spleen, and intestine. The results showed that the administration of iron with cobalt or vitamin E decreased lipid peroxidation and the levels of hypoxanthine in all tissues (P < .001). Tissue associated myeloperoxidase (MPO) activity was increased in all iron-overloaded animals. However, vitamin E and cobalt decreased MPO activity (P < .001) in all tissues with the e x c e p tion of the intestines, where cobalt was ineffective. Cobalt therapy increased hemoglobin, hematocrit, and MCV (P < .05). In contrast to SGPT activity, SGOT activity was significantly increased in all groups but more so in group 3 animals. The increased activity of serum SGOT levels might be related to the mechanical injury by cardiac puncture. The most striking histopathologic finding was the presence of granulomas in the livers of 71% of the animals of group 2 and in 66.6% of group 3. Interestingly, granulomas developed in only 33.3% of group 4 animals, whereas no granulomas were found in the livers of control animals (group I). In this article we report that cobalt is as effective as vitamin E in significantly reducing ironinduced b i o c h e m i c a l changes in an iron-overload in vivo model. We further describe for the first time the presence of extensive granuloma formation in ironoverloaded liver tissue and the greater efficiency of vitamin E over cobalt in protecting against granuloma formation in iron overload. (J Lab Clin Med 1998;132:157-65)
Abbreviations: ATP = adenosine triphosphate; GSH = glutathione; Hb = hemoglobin; Hct = hematocrit; MCV = mean cell volume; MNC : mononuclear celL; MPO : myeloperoxidase; PMN = polymorphonuclear leukocyte; RBC : red blood cell; SGOT = serum glutamate oxaloacetate transaminase; SGPT= serum glutamate pyruvate transaminase; TBARS= thiobarbituric acid-reactive substance; WBC : white blood cell
From the Department of Pediatrics, Hacettepe Children's Hospital; the Department of Biochemistry and the Department of Pediatrics, Division of Pediatric Pathology, Hacettepe Children's Hospital, Hacettepe University, Ankara; and the Departments of Pathology, Anatomy, and Cell Biology, SUNY Health Science Center at Brooklyn. Submitted for publication February 18, 1998; accepted March 24, 1998. Reprint requests: ~i~dem Juan, MD, SUNY Health Science Center at Brooklyn, Division of Pediatric Neurology, Box 118, 450 Clmkson Avenue, Brooklyn, NY 11203. Copyright © 1998 by Mosby, Inc. 0022-2143/98 $5.00 + 0 5/1/91322
p
r i m a r y and s e c o n d a r y i r o n - o v e r l o a d disorders are characterized by the d e v e l o p m e n t o f the accumulation o f iron in m o n o n u c l e a r phagocytes and parenchymal cells of the liver, spleen, heart, pancreas, and other organs. Consequently iron overload has been associated with injury, fibrosis, and ultimately cirrhosis in the target tissue. 1-5 The m e c h a n i s m s underlying the cytotoxicity of iron in the liver as well as several other tissues are not completely understood. Previous studies suggest that o x y g e n free radicals are involved in iron toxicity, and iron-mediated lysosomal
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disruption may p l a y a role in initiating cell injury.2, 6-8 Iron occupies a central role in oxy-radical chemistry, because it can initiate oxygen radical formation. Iron is a catalyst in the Haber-Weiss reaction and is involved in the initiation and propagation of lipid peroxidation. 7-tl Iron o v e r l o a d is still an o u t s t a n d i n g p r o b l e m , part i c u l a r l y in p a t i e n t s u n d e r g o i n g l o n g - t e r m t r a n s f u sion, and the use o f d e f e r o x a m i n e as a chelator is still the only effective and available treatment to decrease the iron o v e r l o a d in the tissues to prevent the initiation o f i r r e v e r s i b l e changes. 12 C o n s i d e r i n g the r o l e o f iron in l i p i d p e r o x i d a t i o n r e a c t i o n s , the q u e s t i o n is w h e t h e r a n t i o x i d a n t c o m p o u n d s m a y have a d d i t i o n a l t h e r a p e u t i c value b e c a u s e o f their s u p p o r t o f antioxidant defenses a t t h e cellular level. In fact, the protective effect o f vitamin E in iron overload is well studied and d o c u m e n t e d by m a n y researchers. 13-t8 In this study we a i m e d to find the effect o f cobalt as an a n t i o x i d a n t cation on iron overload. C o b a l t c h l o r i d e had b e e n i n i t i a l l y r e p o r t e d to i n c r e a s e the rate o f l i p i d p e r o x i d a t i o n in vivo 19 and to i n d u c e h e p a t i c l i p i d p e r o x i d a t i o n as a c o n s e q u e n c e o f acute c o b a l t c h l o r i d e toxicity. 2° P r e v i o u s l y it was shown that c o b a l t i n h i b i t e d n o n e n z y m a t i c l i p i d p e r o x i d a t i o n in d i f f e r e n t s y s t e m s c o n t a i n i n g iron but d i d not affect reduced nicotinamide adenine dinucleotide phosp h a t e o x i d a t i o n or i r o n r e d u c t i o n in m i c r o s o m e s . 21 The inhibitory action o f c o b a l t requires the presence o f i r o n c o m p l e x in p e r o x i d i z i n g s y s t e m s . C o b a l t c o m p e t e s with iron on the a n i o n i c o x y g e n o f p h o s phate groups in p h o s p h o l i p i d s to inhibit lipid peroxidation. 21-23 C o n s e q u e n t l y w e i n v e s t i g a t e d w h e t h e r c o b a l t has any p r o t e c t i v e effects in iron o v e r l o a d in vivo a l o n g with the w e l l - k n o w n n a t u r a l a n t i o x i d a n t vitamin E. METHODS Animal model. Adult male Wistar rats weighing 145 to 150 g were allowed free access to diet and water. The animals were divided into 4 groups. In group 1, rats (n = 6) were injected subcutaneously with only normal saline solution, whereas those in group 2 (n = 8) were injected with iron dextran (Sigma Chemical Co). Animals in group 3 (n = 12) received iron and cobalt, whereas those in group 4 (n = 12) were injected with iron and vitamin E. Each rat was injected subcutaneously only in the right inguinal area with 125 mg iron dextran per kilogram of body weight as described previously by Furuya and Williams. 24 Immediately thereafter group 3 was injected intraperitoneally with 15 mg/kg body weight of cobalt chloride (COC12.6H20) purchased from Sigma, and group 4 was injected intramuscularly with 100 mg/kg body weight of vitamin E (Ephynal) that was provided by Roche, Istanbul, Turkey. Injections of cobalt and vitamin E were given 3 times (every other day) a week for 3
weeks. One day after the last injection, blood samples were obtained by cardiac puncture under ether anesthesia, and then the rats were killed immediately by cervical dislocation. Although the liver is the predominantly affected organ in experimental hemosiderosis and in human disease, other organs become involved during disease progression. Therefore, to establish any potentially protective effect of cobalt or vitamin E on iron-induced cell injury in our model, in addition to the liver, the spleen and intestines of all animals were taken out and blood was withdrawn from the vascular structure by flushing with 4°C normal saline solution. Then tissues were sampled for biochemical and histopathologic studies. Tissues for biochemical analysis were immediately deep frozen. For histopathology the tissues were immediately fixed in 10% buffered formalin and embedded in paraffin, from which sections were prepared and stained with hematoxylin-eosin and Prussian blue for examination by light microscopy.
Biochemical analysis Blood. Samples were analyzed for WBC and RBC counts,
Hct, Hb, and MCV, and SGOT and SGPT activities by using routine clinical methods. Tissues. Frozen tissues were weighed, and 10% tissue homogenates were prepared in 50 mmol/L ice-cold K-phosphate buffer with a glass-glass tissue homogenizer. In the homogenates, iron levels (rag/100 ml tissue homogenate) were measured by the colorimetric method of Bothwell and Mallett. 25 The Ellman reaction was used to determine tissue GSH levels.26 Lipid peroxidation was measm'ed indirectly by quantitation of TBARS levels (gmol/g wet tissue) present in the tissues as described by Uchiyama and Mihara. 27 Consumption of ATP was evaluated by determining hypoxanthine levels (gmol/g wet tissue) through the xanthine oxidase-catalyzed conversion of hypoxanthine to uric acid. 28 Tissue associated MPO activity was measured as described by Grisham et al. ~9 The reaction was initiated by the addition of H202, and the rate of reaction was recorded spectrophotometrically. Considering the initial and linear phase of the reaction, we measured the absorbance change per minute, and the enzyme activity (IU/g wet tissue) was expressed as the amount of enzyme producing a change of 1 absorbance unit per minute. Statistics. Statistical analyses were done by using SPSS for Windows (version 5.0), and the results were presented as oneway variance analysis _+SD. RESULTS Hemosiderosis model. W h e n tissue iron levels were measured in liver and spleen homogenates, the results confirmed that the injection o f iron dextran at 125 mg/kg body weight in accordance with the protocol as d e s c r i b e d b y F u r u y a and W i l l i a m s 24 leads within 3 weeks to the generation of hemosiderosis. As shown in Table I, the injection o f iron dextran resulted in a 16fold and 5-fold increase in the amount o f iron found in the liver and spleen, respectively, o f the animals in
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Fig. 1. Light microscopy of Wistar rat control liver tissues. A, Hematoxylin and eosin staining. B, Prussian blue staining. (Original magnification x40.)
Table I. Tissue iron levels after iron o v e r l o a d
group 2 as compared with those in group 1. A similar accumulation of iron was observed in the livers (16.5fold) and spleens (2.3-fold) of the animals in group 3. Because of insufficient amounts of tissue specimens available after the biochemical analysis of tissue samples from group 4, quantitation of iron could be done in only 2 animals. Nevertheless, the results show an accumulation of iron in the tissues of group 4 that is very similar to that found in group 2, although the small sample number did not permit a statistical comparison. Histopathologic findings. The presence of hemosiderosis in groups 2 and 3 as well as in group 4 and its absence in the animals of group 1 was proven by direct histologic examination of liver tissue sections. As can be seen in the Prussian blue-stained liver sections, the iron deposits are present throughout the tissue in Kupffer cells and hepatocytes, while no deposits can be found in the sections of group 1 (Fig. 1, A and B). Liver. Mononuclear inflammation was minimal in the portal tracts. In 2 of the 6 animals in group 1, a few foci of minute spotty necrosis were indicated by the presence of MNCs and PMNs. No evidence of iron accumulation in the liver was found. In contrast, in 5 of the 7 animals in group 2, portal inflammation was increased, as indicated by the presence of extravascular MNCs. As expected, the effect of overloading with iron is expressed in the Prussian blue staining of numerous Kupffer cells and histiocytes rather than hepatocytes. In this group, granulomas (1 to 17 per section) could be found that consisted of hemosiderin-laden macrophages, lymphocytes, plasma cells, and sparse PMNs. In group 3, deposition of hemosiderin occurred predominantly in Kupffer ceils and histiocytes. Furthermore, in 2 of the 12 animals in this group, hemosiderin deposition was also observed in hepatocytes but to a lesser degree than in Kupffer cells. Although the extent of portal inflammation in group 3 appeared to be similar to that seen in group 2, granulomas were found in 8 animals of group 3 (1 to 27 granulomas per section).
Liver (mg/100 ml)
Group Group Group Group
1 2 3 4
161.33 2577.5 2668.5 2572.5
__.39.5* +_ 852.91_ 693.8t + 743.81
Spleen (mg/100 ml) 216.5 1077.7 490.5 1058.1
+__134.5 _+ 581.6:l: +- 106.7 -+ 370.1
*Values are expressed as mean _+SD. 1Different from group 1 mean, P< .05. SDifferent from groups 1 and 3 means, P < ,05.
In the animals of group 4, hemosiderin was predominantly deposited in Kupffer cells and histiocytes. Portal inflammation was similar to that seen in groups 2 and 3. Granulomas (1 to 12 granulomas per section), however, were found in only 4 of the 12 animals in group 4 (Fig. 2, A and B). It is interesting that a characteristic feature of the granulomas in group 3 and 4 was their frequently centrilobular location and the presence of the nuclear dust of PMNs within the granulomas in group 3 (Fig. 2, C and D). Spleen. In contrast to the restricted presence of ironcontaining MNCs within the red pulp of the spleens of the control animals, there was evidence of increased deposition of iron within the red and white pulp of the spleens of all the animals in the 3 iron-injected groups. It is interesting to note the presence of large numbers of Prussian blue-positive macrophages within the white pulp of the spleens of group 2 and 3 and the apparently organized migration of iron-laden macrophages into the white pulp in groups 3 and 4. In addition, areas defined as white pulp appeared to be considerably enlarged in the spleens of the iron-overloaded animals of groups 2, 3, and 4. Treatment with neither vitamin E nor cobalt appeared to have any effect on the distribution of iron in the spleens of the respective animals, although cobalt treatment seems to have resulted in a reduction in the total amount of iron in the spleens of group 3 animals (Table I).
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Fig. 2. Granulomaformation.A, C, Hematoxylinand eosin staining. B, D, Prussian blue staining. Granuloma is composedof hemosiderin-filledhistiocytes, lymphocytes,plasma cells, and PMNs. Note the granulomas in the liver, with the presence of central nuclear dust material in group 3. (Original magnificationx80.) Intestine. Minimal sparse deposition of iron was seen in control group 1 and in experimental groups 2 and 3. Some moderate infiltration of lymphocytes, plasma ceils, and PMNs also occurred that consisted predominantly of eosinophilic leukocytes in the lamina propria of the intestines of all groups. Interestingly, virtually no iron-containing cells, including Peyer's patches, were found in the intestines of the iron-overloaded and vitamin E-treated animals in group 4. Biochemical analysis. Recently cobalt was reported to increase the GSH level in the liver of rats intoxicated with high doses of acetaminophen. 30 Because the increase in GSH levels is suggested to provide protection against oxidant stress, we decided to measure the amount of GSH in the livers of our experimental animals (groups 2 thorugh 4) and controls (group 1). The results showed virtually no difference between the 4 groups, with 8.22 _+ 0.40 mmol/g tissue for group 1, 8.10 _+ 0.73 mmol/g tissue for group 2, 8.14 _+ 1.12 mmol/g tissue for group 3, and 8.22 + 0.67 mmol/g tissue for group 4. Subsequent measurements of GSH in spleen homogenates also showed no difference between groups 2 through 4 (4.34 _+0.28 mmol/g tissue, 4.31 + 0.37 mmol/g tissue, and 4.36 _+ 0.30 mmol/g tissue, respectively) and group 1 (4.32 -+ 0.25 mmol/g tissue). Suprisingly, however, the GSH value in the intestines of the animals in group 4 treated with iron and vitamin E was 5.75 _+0.42 mmol/g tissue, statistically different (P < .05) from those measured for groups 1 (5.20 _ 0.11
mmol/g tissue), 2 (5.22 + 0.25 mmol/g tissue), and 3 (5.12 _+0.40 mmol/g tissue). In the next evaluation we were concerned with the extent of lipid peroxidation as a measure of the cytotoxicity of iron overload and the effect of cobalt and vitamin E on this process. First, tissue hypoxanthine in all groups was determined; the results are presented in Fig. 3. In contrast to results in group 1, iron overload in group 2 increased the hypoxanthine level in the liver 3.8 times (0.498 _+0.076 gmol/g vs 0.129 _+0.013 gmol/g), that in the spleen 2 times (0.331 _+ .0021 gmol/g vs 0.159 _+ 0.012 gmol/g), and that in the intestinal tissues 1.7 times (0.204 _+0.018 mmol/g vs 0.115 + 0.008 gmol/g). The administration of cobalt (group 3) decreased the levels of hypoxanthine in the liver, spleen, and intestine (0.350 _+0.044 gmol/g, 0.273 _+0.036 gmol/g, and .164 _+0.021 gmol/g, respectively) of iron-overloaded animals when compared with results in group 2 (see above) (P < .001). Similarly, treatment with vitamin E of iron-overloaded animals significantly reduced hypoxanthine levels in all tissues measured (liver, 0.352 + 0.076 gmol/g; spleen, 0.244 _+0.029 gmol/g; intestine, 0.164 _+0.016 gmol/g) as compared with group 2 (P < .001). Although cobalt was as effective as vitamin E in reducing hypoxanthine levels in liver and intestine, vitamin E was more effective in the spleen (P < .001). Lipid peroxidation in the rat fiver, spleen, and intestine was determined by the amount of TBARS present in the tissue homogenates as described by Uchiyama et al.27
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versus group 1. *P < .001 for hypoxanthinelevels as comparedwith group 2; there is no difference between values of group 3 and 4 in the liver and intestines. §P < .001 for hypoxanthinelevels as comparedwith group2 and group4. q[P< .001 for the effectof vitaminE as comparedwith group 2 and 3.
The results of this analysis are summarized in Fig. 4. It is clear that a hemosiderotic liver as generated in this model in group 2 contains 3.5 times the amount of TBARS (0.312 +_ 0.069 gmol/g) than a normal liver (group 1, 0.087 + 0.011 gmol/g). Although not quite as extensive, the increases in TBARS in the spleen (1.8 times) and in the intestine (1.8 times) of group 2 were still significant when compared with those in group 1 (P < .001). Cobalt as well as vitamin E effectively prevented the accumulation of TBARS in the livers of group 3 (0.156 _+ 0.033 gmol/g) and group 4 (0.142 + 0.019 gmol/g). Although cobalt and vitamin E also prevented the accumulation of TBARS in the spleen and intestines of experimental groups 3 and 4, respectively, vitamin E was more effective than cobalt (P < .001)(Fig. 4). It has been well documented that inflammatory cells, in particular macrophages and polymorphonuclear leukocytes, play an important role in the initiation and maintenance of lipid peroxidation during an inflammatory process. Consequently we measured MPO activity in the tissue homogenates of all 4 groups. The results, summarized in Fig. 5, clearly show that as expected, iron overload leads to a 4.3fold increase in MPO activity in the liver (16.8 ± 2.9 IU/g vs 3.83 ± 1.5 IU/g), a 1.9-fold increase in the spleen (34.6 ± 5.0 IU/g vs 17.9 _+2.1 IU/g), and a 2.1fold increase in the intestine (46.9 _+ 6.1 IU/g vs 22.1
+ 1.1 IU/g). Fig. 4 further shows that both cobalt and vitamin E decreased tissue MPO activity in rat liver by 43% and 55%, respectively, and in the spleen by 20% and 33%, respectively (P < .001). As already seen above, vitamin E again appears to be more effective than cobalt. With respect to the intestine, vitamin E decreased MPO activity by 24%, to 36.1 +_4.3 IU/g, while cobalt had no effect (40.5 _+ 7.3 IU/g vs 46.9 -!_ 6.1 IU/g [group 2]). Biochemical and hematologic analysis. The results of the biochemical determinations on blood samples are summarized in Table II. Despite a large increase in hepatic lipid peroxide levels, iron overload did not increase SGPT levels (P < .05). Although SGOT levels were high in all animals including those in group 1, SGOT values in group 3 and 4 were significantly higher but statistically different in group 3 when compared with those measured for groups 1 and 2 (P < .05). In contrast to vitamin E, cobalt administration increased Hb, Hct, and MCV in the animals of group 3 as compared with those in groups 1 and 2 (P < .05) (Table II). On the other hand, in all animals given iron, neither cobalt nor vitamin E affected WBC or RBC counts.
DISCUSSION It has been reported that iron overload stimulates
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Fig. 4. Tissue lipid peroxide levels. Values are expressed as mean + 1 SD. #P < .001 for lipid peroxide levels as compared with group 1. *p < .001 for lipid peroxide levels as compared with group 2. §P < .001 for lipid peroxide levels as compared with group 2. q[P < .001 for effect of vitamin E as compared with groups 2 and 3.
lipid peroxidation by facilitating the decomposition of lipid peroxides and the formation of OH radical from H202 and O -2 in tissues and through the generation of 0 .2 and H202 by accelerating the non-enzymatic oxidation of several molecules. 7-11 As a potent antioxidant, vitamin E prevents iron-induced peroxidati0n.9,13-18 In contrast, little is known about the anti-peroxidative actions of cobalt in vivo. The results of this study show that in vivo cobalt in addition to vitamin E can function as a potent antioxidant in the amelioration of ironinduced lipid peroxidation. In our study, lipid peroxidation was measured as TBARS in rat tissue, which was stimulated by iron overload. Because the highest TBARS value was observed in the liver, it might be considered to be the most sensitive of the organs to biochemical changes in iron overload, and accordingly, a major site of iron deposition. In the liver, cobalt and vitamin E reduced lipid peroxide levels to 50% of the values measured in group 2 (Fig. 4, columns 3 and 4). Both cobalt and vitamin E were also effective in reducing lipid peroxidation in the spleen and intestine, but in the intestine, vitamin E was more effective than cobalt. Thus both antioxidant agents protected all tissues examined against the peroxidative effect of iron. The increased production of free oxygen radicals by iron causes peroxidative decomposition of the polyunsaturated fatty acids of mitochondrial and microsomal membranes as well as of the plasma membrane. 1,2,7 Pre-
sumably these membrane damages will cause specific structural and functional disturbances in cell integrity, especially in mitochondria. 31-34 Iron overload causes a 70% decrease in cytochrome c oxidase activity, leading to reduced ATP synthesis. 35-3s Accordingly, increased ATP demand causes the ATP degradation to hypoxanthine and the formation of several new reactive oxygen species. The concentration of hypoxanthine in the liver was found to be increased 3-fold to 4-fold over the control values, depending on the duration of the hypoxiaischemia that was correlated with the decreased ATP and ADP levels. 39 In our study, iron accumulation caused a several-fold elevation of tissue hypoxanthine in the animals of group 2 versus those in group 1 (Fig. 3, columns 1 and 2). It is important to note that establishing normal hypoxanthine levels in either tissue or blood results in considerably varied values when existing methods are used. 39-44 On the other hand, our control values were similar to the results reported by others. 39 Treatment with either vitamin E or cobalt decreased tissue hypoxanthine levels in iron overload significantly (Fig. 3, columns 3 and 4). Because vitamin E is a powerful inhibitor of lipid peroxidation in biologic membranes, 14-18 its action as an antioxidant on hypoxanthine levels is expected. Interestingly, cobalt presented the same protective effect in the tissues that correlated with its effect on iron-induced lipid peroxidation. According to our knowledge, this study is the first to describe a cor-
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Fig. 5. Effect of cobalt and vitamin E on tissue M P O activities. #P < .001 for MPO activities as compared with group 1. *P < .001 for tissue M P O activities as compared with group 2. ~[P < .001 for the effect of vitamin E as compared with group 2.
Table II. C o m p l e t e blood cell and serum transaminase values WBC (xl031mm 3) Group Group Group Group
1 2 3 4
13.5 19.7 14.4 13.6
_+ 3.9 _+ 8.9 _+ 6.4 _+ 9.2
RBC (xl0elmm 3) 6.9 7.5 7.3 7.7
_+ 0.9 _+ 0.5 +_ 0.9 + 1.3
Hb (gldl) 12.9 14.2 16.2 14.5
_+ 1.6" __ 1.1 + 1.07* + 1.7
Hot (%) 36.8 39.3 45.6 41.3
MCV (fl)
_+ 5.3 +_ 2.2 ___4.4* _+ 6.7
53.1 54.5 57.9 53.9
-+ 1.5 _+2.2 _+ 5.7* -+ 1.8
SGOT (IUIL) 152.0 140.0 283.6 209.6
_+ 26.5 _+ 22.8 + 156.4" _+ 39.6
SGPT (IUIL) 53.6 42.5 59.5 51.0
_+ 9.6 _+ 15.9 -+ 28 __ 16.5
*P < .05 for the values c o m p a r e d with the other groups,
relation of tissue hypoxanthine levels with lipid peroxidation in iron overload. When increased production of free oxygen radicals stimulates lipid peroxidation in biologic membranes, peroxidation of membrane lipids increases lysosomal fragility and the levels of cytoplasmic Ca +2. Ca +2 accumulation, via activation of phospholipase A a, increases the synthesis of eicosanoids that are chemoattractants. As a consequence, PMN infiltration into the damaged site is increased, resulting in the additional production of reactive oxygen species by activated PMNs, the release of hydrolytic enzymes, and the exacerbation of tissue damage. 45-48 In a previous study of the effect of hypoxia in newborn rats, we showed decreased lipid peroxidation and MPO activity during the oxidative stress. 49 In the present study, in addition to the effect of vitamin E, cobalt decreased iron-induced MPO activity in the liver and spleen with little effect in the intes-
tine (Fig. 5). The decrease in MPO activity caused by antioxidants suggests that they may lead to a reduction of inflammatory reactions caused by iron. Therefore, histopathologic analyses were performed with the aim of identifying the consequences of these biochemical changes. To our surprise, the liver sections of 50% of animals with iron overload showed the previously unreported formation of granulomas containing large numbers of iron-stuffed MNCs, Kupffer cells, and PMNs. Although this may suggest an immunologic process as the pathogenesis of the granulomas, previous investigations have shown that the dextran used as vector in the iron-dextran preparation for the present study is immunologically inert and therefore cannot explain this granuloma formation.50, 51 Consequently the large amount of iron present in the tissues may play a central role in the formation of these granulomas. The frequently unique centrilobular location of the granulomas fur-
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ther supports a chemical rather than immunologic pathogenesis. 52 On the other hand, the presence of numerous iron-laden macrophages in the seemingly enlarged splenic follicles of the animals in groups 2, 3, and 4 prevents us from totally excluding an immunologic process as the basis of the formation o f these granulomas. As stated in the introduction, the aim of the present study was to investigate the antioxidant effect of cobalt in iron overload and to compare this effect with that of the wellknown antioxidant vitamin E. The presence of the granuloma was unexpected; therefore, pinpointing the pathophysiologic process of this serendipitious finding will require a separate investigation. In this context it is important to note that treatment with vitamin E and cobalt reduced MPO production not only in the liver but also in the spleen and intestines of iron-overloaded animals. Although this finding suggests that a decrease in the production o f cytotoxic oxygen radicals as a consequence of reduced M P O production may be responsible for the slightly reduced numbers of granulomas in the presence of cobalt and especially of vitamin E, additional experiments are necessary to ascertain this observation. The absence of significant changes in serum SGPT activity suggests that most liver tissue cells remained intact. It is possible, on the other hand, that the increased activity of serum SGOT levels has been caused b y mechanical injury such as cardiac puncture, as reported in other in vivo studies. Despite increased lipid peroxidation, tissue-associated M P O activity, and hypoxanthine levels, no significant change was detected in the amount of tissue G S H that functions as another important natural antioxidant. Recently it was reported that v i t a m i n E and cobalt affect G S H level during tissue injury.143 ° In the present study we used the Ellman reaction to determine tissue G S H levels. Because this reaction is not completely specific for glutathione, p o s s i b l e interference with sulphydryl groups of amino acids or proteins (eg, metallothionein) during the assay could explain the absence of change in "glutathione. ''26 Besides the biochemical and histopathologic changes in the tissues, the effect of the cobalt on Hb and Hct can provide an advantage in the assessment of treatment with cobalt. 53,54 Unlike our understanding regarding vitamin E, our k n o w l e d g e of how cobalt is treated by and processed in different tissues is less extensive. Although cobalt is known to be a cardiotoxic ion, a few animal studies have analyzed the potential protective effect of cobalt on cephaloridine-induced lipid peroxidation, 55 poisoning by potassium cyanide, 56 and anoxic damage in rat hippocampal slices. 57 Unfortunately, iron overload affects morbidity by causing many acute and long-term side effects and organ failure. The results o f the present study suggest that antioxidant agents extend significant protective
effects against iron-induced tissue injury. A l t h o u g h cobalt was considered an oxidative cation until recently, in this study cobalt was shown to have a strong antioxidant effect on i r o n - i n d u c e d lipid p e r o x i d a t i o n and subsequent b i o c h e m i c a l changes in tissues when c o m p a r e d with vitamin E. Further investigations will establish the effective therapeutic dose versus tissue toxicity regarding the use o f cobalt in the clinical setting. We thank Professor ~i~dem Altay, from the Department of Pediatrics, Hacettepe Children's Hospital, for her continuous support and guidance in this study. We also thank Professor Safiye G6~tis, from the Department of Pediatrics, Division of Pediatric Pathology, Hacettepe Children's Hospital, for her evaluation and comments on the histopathologic results. REFERENCES
1. Gordeuk VR, Bacon BR, Brittenham GM. Iron overload: cause and consequences. Ann Rev Nutr 1987;7:485-508. 2. Figueiredo MS, Baffa O, Neto JB, Zago MA. Liver injury and generation of hydroxyl free radicals in experimental secondary hemochromatosis. Res Exp Med 1993;193:27-37. 3. Wheby MS. Liver damage in disorders of iron overload. Arch Intern Med 1984;144:621-2. 4. Carthew P, Dorman BM, Edwards RE, Francis JE, Smith AG. A unique rodent model for both the cardiotoxic and hepatotoxic effects of prolonged iron overload. Lab Invest 1993;69:217-22. 5. MacDonald RA, Pechet GS. Experimental hemochromatosis in rats. Am J Pathol 1964;46:85-102. 6. Rachmilewitz EA, Lubin BH, Shohet SB. Lipid membrane peroxidation in [3-thalassemia major. Blood 1976;47:495505. 7. Bacon BR, Tavill A, Brittenham GM, Park CH. Hepatic lipid peroxidation in vivo in rats with chronic iron overload. J Clin Invest 1983;71:429-39. 8. Golberg L, Martin LE, Batchelor A. Biochemical changes in tissues of animals injected with iron. Biochem J 1962;83:291-8. 9. Andersen HJ, Chen H, Pellett LJ, Tappel AL. Ferrous-ironinduced oxidation in chicken liver slices as measured by hemichrome formation and thiobarbimric acid-reactive substances: effects of dietary vitamin E and B-carotene. Free Radical Biol Med 1993;15:37-48. 10. Halliwell B, Gutteridge MC. Iron and free radical reactions: two aspects of antioxidant protection. Trends Biochem Sci 1986;11:372-5. 11. Wu W, Meydani M, Meydani SN, Burklund PM, Blumberg JF, Munro HN. Effect of dietary iron overload on lipid peroxidation, prostaglandin synthesis and lymphocyte proliferation in young and old rats. J Nutr 1990;120:280-9. 12. Ehlers KH, Giardina PJ, Lesser ML, Engle MA, Hilgartner MW. Prolonged survival in patients with beta-thalassemia major treated with deferoxamine. J Pediatr 1991;118:540-5. 13. Dillard CJ, Gavino VC, Tappel AL. Relative antioxidant effectiveness of c~-tocopheroland T-tocopherol in iron-loaded rats. J Nutr 1983;113:2266-73. 14. Pascoe GA, Fariss MW, OlafsdoUir K, Reed DJ. A role of vitamin E in protection against cell injury: maintenance of intracellular glutathione precursors and biosynthesis. Eur J Biochem 1987;166:241-7.
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15. Hershko C, Link G, Pinson A. Modification of iron uptake and lipid peroxidation by hypoxia, ascorbic acid, and ~-tocopherol in iron-loaded rat myocardial cell cultures. J Lab Clir~ Med 1987;110:355-61. 16. Bacon BR, Britton RS, O'Neill R. Effects of vitamin E deficiency on hepatic mitochondrial lipid peroxidation and oxidative metabolism in rats with chronic dietary iron overload. Hepatology 1989;9:398-404. 17. Sharma BK, Bacon BR, Britton RS, Park CH, Mageirea CJ, O'Neill R, et al. Prevention of hepatocyte injury and lipid peroxidation by iron chelators and c~-tocopherol in isolated iron-loaded rat hepatocytes. Hepatology 1990;12:31-9. 18. Fukuzawa K, Chida H, Tokumura A, Tsukatani H. Antioxidative effect of c~-tocopherol incorporation into lecithin liposomes on ascorbic acid-Fe2+-induced lipid peroxidation. Arch Biochem Biophys 1980;206:173-80. 19. Hasan M, Ali SF. Effects of thallium, nickel, and cobalt administration of the lipid peroxidationin different regions of the rat brain. Toxicol Appl Pharmacol 1981 ;57:8-13. 20. Sundermen FW Jr, Zaharia O. Hepatic lipid peroxidation in CoC12 treated rats, evidenced by elevated concentrations of thiobarbituric acid chromogens. Res Commun Chem Pathol Pharmacol 1988;59:69-78. 21. Tampo Y, Yonaha M. Mechanism of cobalt (II) ion inhibition of iron supported phospholipid peroxidation. Arch Biochem Biophys 1991;289:26-32. 22. Kilin~ K, Rouhani R. Cobaltous ion inhibition of lipid peroxidation in biological membranes. Biophys Acta 1992;1125:189-95. 23. Tampo Y, Yonaha M. Mechanism of cobalt (II) ion inhibition of iron-supported phospholipid. Arch Biochem Biophys 1991;289:26-32. 24. Furuya K, Williams GM. Neoplastic conversion in rat liver by the antihistamine methapyrilene demonstrated by a sequential syncarsinogenic effect with N-2-fluorenlacetamide. Pharmacology 1984;74:63-9. 25. Bothwell TH, Mallett B. The determination of iron in plasma or serum. Biochem J 1955;59:599-602. 26. Ellman GL. Tissue sulphydryl groups. Arch Biochem Biophys 1959;82:70-7. 27. Uchiyama M, Mihara M. Determination of malonaldehyde precursor in tissues by thiobarbituric acid test. Anal Biochem 1977;86:271-8. 28. Karaguzel G, Tanyel FC, Kilinc K, Buyukpamukcu N, Hicsonmez A. The preventive role of chemical sympathectomy on contralateral testicular hypoxic parameters encountered during unilateral testicular torsion. Br J Urol 1994;74:507-10. 29. Grisham MB, Hernandez LA, Granger DN. Xanthine oxidase and neutrophyl infiltration in intestine ischemia. Am J Physiol 1986;251:G567-74. 30. Roberts SA, Price VF, Jollow DJ. The mechanism of cobalt chloride-induced protection against acetaminophen hepatotoxicity. Drug Metab Dispos 1986;14:25-33. 31. Bacon BR, Park CH, Brittenham GM, O'Neill R, Tavill AS. Hepatic mithochondrial oxidative in rats with chronic dietary iron overload. Hepatology 1985;5:789-97. 32. Bacon BR, O'Neill R, Park CH. Iron induced peroxidative injury to isolated rat hepatic mitochondria. J Free Radic Biol Med 1986;2:339-47. 33. Bacon BR, Brittenham GM, O'Neill R. Effects of vitamin E deficiency on hepatic mithochondrial lipid peroxidation and oxidative metabolism in rats with chronic dietary iron. Hepatology 1990;11:93-7. 34. Manev H, Cagnoli CM, Atabay C, Kharlamov E, Ikanomovfc MD, Grayson DR. Neuronal apoptosis in an in-vitro model
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of photochemically induced oxidative stress. Exp Neural 1995:198-206. Britteubam GM, O'Neill R, Bacon BR. Hepatic mitochondrial malondialdehyde metabolism in rats with chronic iron overload. Hepatology 1990;11:93-7. Bacon BR, O'Neill R, Brittenham GM. Hepatic mitochondrial energy production in rats with chronic iron overload. Gastroenterology 1993;105:1134-40. Wolff SP, Garner A, Dean RT. Free radicals, lipids and protein degradation. Trends Biochemical Sci 1986;11:27-31. Davies KJA. Protein damage and degradation by oxygen radicals. I. General aspects. J Biol Chem 1987;262:9895-901. Koboyashi H, Nonami T, Kurokawa T, Kitahara S, Harada A, Nakao A, et al. Changes in the glutathione redox system during ischemia and reperfusion in rat liver. Scand J Gastroenterol 1992;27:711-6. Kleihue P, Kobayashi K, Hossmann KA. Purine nucleotide metabolism in the cat brain after one hour of complete ischemia. J Neurochem 1974;23:417-25. Sangstad OD, Schrader H. The determination of inosine and hypoxantine in rat brain during normothermic and hypothermic anoxia. Acta Neurol Scand 1978;57:281-8. Sangstad OD, Aasen AO. Plasma hypoxantine concentrations in pigs as prognostic aids in hypoxia. Eur Surg Res 1980;12:123-9. Cumingham SK, Keaveny TV. Splanchic organ adenine nucleotides and their metabolites in haemorrhagic shock. Ir J Med Sci 1977;146:136-43. Saugstad OD. Hypoxantine as an indicator of hypoxia: its role in health and disease through free radical prodiaction. Pediatr Res 1988;23:143-9. McCord JM. Oxygen derived radicals in postischemic tissue injury. N Engl J Med 1985;312:159-63. Vannucci RC. Experimental biology of cerebral hypoxiaischemia relation to perinatal brain damage. Pediatr Res 1990;27:317-26. Panganamala RV, Conwell DG. The effects of lypooxygenation of arachidonic acid in leukocytes. Nature 1982;288:183-5. Ode N. The role of reperfusion induced injury in the pathogenesis of the crush syndrome. N Engl J Med 1991;324:1417-21. Inan ~, Kiliq I, Kilinq K, Kotiloglu E, Kalayci 0. The effect of high dose antenatal vitamin E on hypoxia induced changes in newborn rats. Pediatr Res 1995;38:685-9. Denk H, Scheuer PJ, Bapista A, Bianchi L, Callea F, Groote J, et al. Guidelines for the diagnosis and interpretation of hepatic granulomas. Histopatol 1994;25:209-18. Haddow A, Horning ES. On the carcinogenicity of an irondextran complex. J Natl Cancer Inst 1960;24:109-47. Robins SR, Cotran RS~ Pathologic basis of disease. 2nd ed. Philadelphia: WB Saunders, 1979:530-5. Edwards MS, Curtis JR. Use of cobaltous chloride in anemia of malntenance-hemodialysis patients. Lancet 1971; 2:582-3. Anonymous. Cobalt in severe renal failure [editorial]. Lancet 1976;2:26-7. Cojocel C, Laeschke KH, Inselmann G, Baumann K. Inhibition of cephaloridine-induced lipid peroxidation. Toxicology 1985;35:295-305. Bovykin BA. The use of complex cobalt compound as a cyanide antidote. Eksperimentalnaia i Klinicheskaia. Farmakologiia 1994;57:57-60. Kass IS, Cotrell JE, Chambers G. Magnesium and cobalt, not nimodipine, protect neurons against anoxic damage in rat hippocampal slice. Anesthesiology 1988;69:710-5.
The protective effects of cobalt and vitamin E in iron overloaded rats were investigated. Rats were divided into four groups: group I as control, group 2 received only iron; group 3 iron and cobalt, group 4 iron and vitamin E. All injections were given 3 times per week for 3 weeks. Biochemical and histopathologic studies were done on samples of blood and liver, spleen, and intestine. The results showed that the administration of iron with cobalt or vitamin E decreased lipid peroxidation and the levels of hypoxanthine in all tissues (P < .001). Tissue associated myeloperoxidase (MPO) activity was increased in all iron-overloaded animals. However, vitamin E and cobalt decreased MPO activity (P < .001) in all tissues with the e x c e p tion of the intestines, where cobalt was ineffective. Cobalt therapy increased hemoglobin, hematocrit, and MCV (P < .05). In contrast to SGPT activity, SGOT activity was significantly increased in all groups but more so in group 3 animals. The increased activity of serum SGOT levels might be related to the mechanical injury by cardiac puncture. The most striking histopathologic finding was the presence of granulomas in the livers of 71% of the animals of group 2 and in 66.6% of group 3. Interestingly, granulomas developed in only 33.3% of group 4 animals, whereas no granulomas were found in the livers of control animals (group I). In this article we report that cobalt is as effective as vitamin E in significantly reducing ironinduced b i o c h e m i c a l changes in an iron-overload in vivo model. We further describe for the first time the presence of extensive granuloma formation in ironoverloaded liver tissue and the greater efficiency of vitamin E over cobalt in protecting against granuloma formation in iron overload. (J Lab Clin Med 1998;132:157-65)
Abbreviations: ATP = adenosine triphosphate; GSH = glutathione; Hb = hemoglobin; Hct = hematocrit; MCV = mean cell volume; MNC : mononuclear celL; MPO : myeloperoxidase; PMN = polymorphonuclear leukocyte; RBC : red blood cell; SGOT = serum glutamate oxaloacetate transaminase; SGPT= serum glutamate pyruvate transaminase; TBARS= thiobarbituric acid-reactive substance; WBC : white blood cell
From the Department of Pediatrics, Hacettepe Children's Hospital; the Department of Biochemistry and the Department of Pediatrics, Division of Pediatric Pathology, Hacettepe Children's Hospital, Hacettepe University, Ankara; and the Departments of Pathology, Anatomy, and Cell Biology, SUNY Health Science Center at Brooklyn. Submitted for publication February 18, 1998; accepted March 24, 1998. Reprint requests: ~i~dem Juan, MD, SUNY Health Science Center at Brooklyn, Division of Pediatric Neurology, Box 118, 450 Clmkson Avenue, Brooklyn, NY 11203. Copyright © 1998 by Mosby, Inc. 0022-2143/98 $5.00 + 0 5/1/91322
p
r i m a r y and s e c o n d a r y i r o n - o v e r l o a d disorders are characterized by the d e v e l o p m e n t o f the accumulation o f iron in m o n o n u c l e a r phagocytes and parenchymal cells of the liver, spleen, heart, pancreas, and other organs. Consequently iron overload has been associated with injury, fibrosis, and ultimately cirrhosis in the target tissue. 1-5 The m e c h a n i s m s underlying the cytotoxicity of iron in the liver as well as several other tissues are not completely understood. Previous studies suggest that o x y g e n free radicals are involved in iron toxicity, and iron-mediated lysosomal
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disruption may p l a y a role in initiating cell injury.2, 6-8 Iron occupies a central role in oxy-radical chemistry, because it can initiate oxygen radical formation. Iron is a catalyst in the Haber-Weiss reaction and is involved in the initiation and propagation of lipid peroxidation. 7-tl Iron o v e r l o a d is still an o u t s t a n d i n g p r o b l e m , part i c u l a r l y in p a t i e n t s u n d e r g o i n g l o n g - t e r m t r a n s f u sion, and the use o f d e f e r o x a m i n e as a chelator is still the only effective and available treatment to decrease the iron o v e r l o a d in the tissues to prevent the initiation o f i r r e v e r s i b l e changes. 12 C o n s i d e r i n g the r o l e o f iron in l i p i d p e r o x i d a t i o n r e a c t i o n s , the q u e s t i o n is w h e t h e r a n t i o x i d a n t c o m p o u n d s m a y have a d d i t i o n a l t h e r a p e u t i c value b e c a u s e o f their s u p p o r t o f antioxidant defenses a t t h e cellular level. In fact, the protective effect o f vitamin E in iron overload is well studied and d o c u m e n t e d by m a n y researchers. 13-t8 In this study we a i m e d to find the effect o f cobalt as an a n t i o x i d a n t cation on iron overload. C o b a l t c h l o r i d e had b e e n i n i t i a l l y r e p o r t e d to i n c r e a s e the rate o f l i p i d p e r o x i d a t i o n in vivo 19 and to i n d u c e h e p a t i c l i p i d p e r o x i d a t i o n as a c o n s e q u e n c e o f acute c o b a l t c h l o r i d e toxicity. 2° P r e v i o u s l y it was shown that c o b a l t i n h i b i t e d n o n e n z y m a t i c l i p i d p e r o x i d a t i o n in d i f f e r e n t s y s t e m s c o n t a i n i n g iron but d i d not affect reduced nicotinamide adenine dinucleotide phosp h a t e o x i d a t i o n or i r o n r e d u c t i o n in m i c r o s o m e s . 21 The inhibitory action o f c o b a l t requires the presence o f i r o n c o m p l e x in p e r o x i d i z i n g s y s t e m s . C o b a l t c o m p e t e s with iron on the a n i o n i c o x y g e n o f p h o s phate groups in p h o s p h o l i p i d s to inhibit lipid peroxidation. 21-23 C o n s e q u e n t l y w e i n v e s t i g a t e d w h e t h e r c o b a l t has any p r o t e c t i v e effects in iron o v e r l o a d in vivo a l o n g with the w e l l - k n o w n n a t u r a l a n t i o x i d a n t vitamin E. METHODS Animal model. Adult male Wistar rats weighing 145 to 150 g were allowed free access to diet and water. The animals were divided into 4 groups. In group 1, rats (n = 6) were injected subcutaneously with only normal saline solution, whereas those in group 2 (n = 8) were injected with iron dextran (Sigma Chemical Co). Animals in group 3 (n = 12) received iron and cobalt, whereas those in group 4 (n = 12) were injected with iron and vitamin E. Each rat was injected subcutaneously only in the right inguinal area with 125 mg iron dextran per kilogram of body weight as described previously by Furuya and Williams. 24 Immediately thereafter group 3 was injected intraperitoneally with 15 mg/kg body weight of cobalt chloride (COC12.6H20) purchased from Sigma, and group 4 was injected intramuscularly with 100 mg/kg body weight of vitamin E (Ephynal) that was provided by Roche, Istanbul, Turkey. Injections of cobalt and vitamin E were given 3 times (every other day) a week for 3
weeks. One day after the last injection, blood samples were obtained by cardiac puncture under ether anesthesia, and then the rats were killed immediately by cervical dislocation. Although the liver is the predominantly affected organ in experimental hemosiderosis and in human disease, other organs become involved during disease progression. Therefore, to establish any potentially protective effect of cobalt or vitamin E on iron-induced cell injury in our model, in addition to the liver, the spleen and intestines of all animals were taken out and blood was withdrawn from the vascular structure by flushing with 4°C normal saline solution. Then tissues were sampled for biochemical and histopathologic studies. Tissues for biochemical analysis were immediately deep frozen. For histopathology the tissues were immediately fixed in 10% buffered formalin and embedded in paraffin, from which sections were prepared and stained with hematoxylin-eosin and Prussian blue for examination by light microscopy.
Biochemical analysis Blood. Samples were analyzed for WBC and RBC counts,
Hct, Hb, and MCV, and SGOT and SGPT activities by using routine clinical methods. Tissues. Frozen tissues were weighed, and 10% tissue homogenates were prepared in 50 mmol/L ice-cold K-phosphate buffer with a glass-glass tissue homogenizer. In the homogenates, iron levels (rag/100 ml tissue homogenate) were measured by the colorimetric method of Bothwell and Mallett. 25 The Ellman reaction was used to determine tissue GSH levels.26 Lipid peroxidation was measm'ed indirectly by quantitation of TBARS levels (gmol/g wet tissue) present in the tissues as described by Uchiyama and Mihara. 27 Consumption of ATP was evaluated by determining hypoxanthine levels (gmol/g wet tissue) through the xanthine oxidase-catalyzed conversion of hypoxanthine to uric acid. 28 Tissue associated MPO activity was measured as described by Grisham et al. ~9 The reaction was initiated by the addition of H202, and the rate of reaction was recorded spectrophotometrically. Considering the initial and linear phase of the reaction, we measured the absorbance change per minute, and the enzyme activity (IU/g wet tissue) was expressed as the amount of enzyme producing a change of 1 absorbance unit per minute. Statistics. Statistical analyses were done by using SPSS for Windows (version 5.0), and the results were presented as oneway variance analysis _+SD. RESULTS Hemosiderosis model. W h e n tissue iron levels were measured in liver and spleen homogenates, the results confirmed that the injection o f iron dextran at 125 mg/kg body weight in accordance with the protocol as d e s c r i b e d b y F u r u y a and W i l l i a m s 24 leads within 3 weeks to the generation of hemosiderosis. As shown in Table I, the injection o f iron dextran resulted in a 16fold and 5-fold increase in the amount o f iron found in the liver and spleen, respectively, o f the animals in
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Fig. 1. Light microscopy of Wistar rat control liver tissues. A, Hematoxylin and eosin staining. B, Prussian blue staining. (Original magnification x40.)
Table I. Tissue iron levels after iron o v e r l o a d
group 2 as compared with those in group 1. A similar accumulation of iron was observed in the livers (16.5fold) and spleens (2.3-fold) of the animals in group 3. Because of insufficient amounts of tissue specimens available after the biochemical analysis of tissue samples from group 4, quantitation of iron could be done in only 2 animals. Nevertheless, the results show an accumulation of iron in the tissues of group 4 that is very similar to that found in group 2, although the small sample number did not permit a statistical comparison. Histopathologic findings. The presence of hemosiderosis in groups 2 and 3 as well as in group 4 and its absence in the animals of group 1 was proven by direct histologic examination of liver tissue sections. As can be seen in the Prussian blue-stained liver sections, the iron deposits are present throughout the tissue in Kupffer cells and hepatocytes, while no deposits can be found in the sections of group 1 (Fig. 1, A and B). Liver. Mononuclear inflammation was minimal in the portal tracts. In 2 of the 6 animals in group 1, a few foci of minute spotty necrosis were indicated by the presence of MNCs and PMNs. No evidence of iron accumulation in the liver was found. In contrast, in 5 of the 7 animals in group 2, portal inflammation was increased, as indicated by the presence of extravascular MNCs. As expected, the effect of overloading with iron is expressed in the Prussian blue staining of numerous Kupffer cells and histiocytes rather than hepatocytes. In this group, granulomas (1 to 17 per section) could be found that consisted of hemosiderin-laden macrophages, lymphocytes, plasma cells, and sparse PMNs. In group 3, deposition of hemosiderin occurred predominantly in Kupffer ceils and histiocytes. Furthermore, in 2 of the 12 animals in this group, hemosiderin deposition was also observed in hepatocytes but to a lesser degree than in Kupffer cells. Although the extent of portal inflammation in group 3 appeared to be similar to that seen in group 2, granulomas were found in 8 animals of group 3 (1 to 27 granulomas per section).
Liver (mg/100 ml)
Group Group Group Group
1 2 3 4
161.33 2577.5 2668.5 2572.5
__.39.5* +_ 852.91_ 693.8t + 743.81
Spleen (mg/100 ml) 216.5 1077.7 490.5 1058.1
+__134.5 _+ 581.6:l: +- 106.7 -+ 370.1
*Values are expressed as mean _+SD. 1Different from group 1 mean, P< .05. SDifferent from groups 1 and 3 means, P < ,05.
In the animals of group 4, hemosiderin was predominantly deposited in Kupffer cells and histiocytes. Portal inflammation was similar to that seen in groups 2 and 3. Granulomas (1 to 12 granulomas per section), however, were found in only 4 of the 12 animals in group 4 (Fig. 2, A and B). It is interesting that a characteristic feature of the granulomas in group 3 and 4 was their frequently centrilobular location and the presence of the nuclear dust of PMNs within the granulomas in group 3 (Fig. 2, C and D). Spleen. In contrast to the restricted presence of ironcontaining MNCs within the red pulp of the spleens of the control animals, there was evidence of increased deposition of iron within the red and white pulp of the spleens of all the animals in the 3 iron-injected groups. It is interesting to note the presence of large numbers of Prussian blue-positive macrophages within the white pulp of the spleens of group 2 and 3 and the apparently organized migration of iron-laden macrophages into the white pulp in groups 3 and 4. In addition, areas defined as white pulp appeared to be considerably enlarged in the spleens of the iron-overloaded animals of groups 2, 3, and 4. Treatment with neither vitamin E nor cobalt appeared to have any effect on the distribution of iron in the spleens of the respective animals, although cobalt treatment seems to have resulted in a reduction in the total amount of iron in the spleens of group 3 animals (Table I).
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Fig. 2. Granulomaformation.A, C, Hematoxylinand eosin staining. B, D, Prussian blue staining. Granuloma is composedof hemosiderin-filledhistiocytes, lymphocytes,plasma cells, and PMNs. Note the granulomas in the liver, with the presence of central nuclear dust material in group 3. (Original magnificationx80.) Intestine. Minimal sparse deposition of iron was seen in control group 1 and in experimental groups 2 and 3. Some moderate infiltration of lymphocytes, plasma ceils, and PMNs also occurred that consisted predominantly of eosinophilic leukocytes in the lamina propria of the intestines of all groups. Interestingly, virtually no iron-containing cells, including Peyer's patches, were found in the intestines of the iron-overloaded and vitamin E-treated animals in group 4. Biochemical analysis. Recently cobalt was reported to increase the GSH level in the liver of rats intoxicated with high doses of acetaminophen. 30 Because the increase in GSH levels is suggested to provide protection against oxidant stress, we decided to measure the amount of GSH in the livers of our experimental animals (groups 2 thorugh 4) and controls (group 1). The results showed virtually no difference between the 4 groups, with 8.22 _+ 0.40 mmol/g tissue for group 1, 8.10 _+ 0.73 mmol/g tissue for group 2, 8.14 _+ 1.12 mmol/g tissue for group 3, and 8.22 + 0.67 mmol/g tissue for group 4. Subsequent measurements of GSH in spleen homogenates also showed no difference between groups 2 through 4 (4.34 _+0.28 mmol/g tissue, 4.31 + 0.37 mmol/g tissue, and 4.36 _+ 0.30 mmol/g tissue, respectively) and group 1 (4.32 -+ 0.25 mmol/g tissue). Suprisingly, however, the GSH value in the intestines of the animals in group 4 treated with iron and vitamin E was 5.75 _+0.42 mmol/g tissue, statistically different (P < .05) from those measured for groups 1 (5.20 _ 0.11
mmol/g tissue), 2 (5.22 + 0.25 mmol/g tissue), and 3 (5.12 _+0.40 mmol/g tissue). In the next evaluation we were concerned with the extent of lipid peroxidation as a measure of the cytotoxicity of iron overload and the effect of cobalt and vitamin E on this process. First, tissue hypoxanthine in all groups was determined; the results are presented in Fig. 3. In contrast to results in group 1, iron overload in group 2 increased the hypoxanthine level in the liver 3.8 times (0.498 _+0.076 gmol/g vs 0.129 _+0.013 gmol/g), that in the spleen 2 times (0.331 _+ .0021 gmol/g vs 0.159 _+ 0.012 gmol/g), and that in the intestinal tissues 1.7 times (0.204 _+0.018 mmol/g vs 0.115 + 0.008 gmol/g). The administration of cobalt (group 3) decreased the levels of hypoxanthine in the liver, spleen, and intestine (0.350 _+0.044 gmol/g, 0.273 _+0.036 gmol/g, and .164 _+0.021 gmol/g, respectively) of iron-overloaded animals when compared with results in group 2 (see above) (P < .001). Similarly, treatment with vitamin E of iron-overloaded animals significantly reduced hypoxanthine levels in all tissues measured (liver, 0.352 + 0.076 gmol/g; spleen, 0.244 _+0.029 gmol/g; intestine, 0.164 _+0.016 gmol/g) as compared with group 2 (P < .001). Although cobalt was as effective as vitamin E in reducing hypoxanthine levels in liver and intestine, vitamin E was more effective in the spleen (P < .001). Lipid peroxidation in the rat fiver, spleen, and intestine was determined by the amount of TBARS present in the tissue homogenates as described by Uchiyama et al.27
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F i g . 3. T i s s u e h y p o x a n t h i n e levels. Values are e x p r e s s e d as m e a n _+ 1 SD. # P < .001 for h y p o x a n t h i n e levels
versus group 1. *P < .001 for hypoxanthinelevels as comparedwith group 2; there is no difference between values of group 3 and 4 in the liver and intestines. §P < .001 for hypoxanthinelevels as comparedwith group2 and group4. q[P< .001 for the effectof vitaminE as comparedwith group 2 and 3.
The results of this analysis are summarized in Fig. 4. It is clear that a hemosiderotic liver as generated in this model in group 2 contains 3.5 times the amount of TBARS (0.312 +_ 0.069 gmol/g) than a normal liver (group 1, 0.087 + 0.011 gmol/g). Although not quite as extensive, the increases in TBARS in the spleen (1.8 times) and in the intestine (1.8 times) of group 2 were still significant when compared with those in group 1 (P < .001). Cobalt as well as vitamin E effectively prevented the accumulation of TBARS in the livers of group 3 (0.156 _+ 0.033 gmol/g) and group 4 (0.142 + 0.019 gmol/g). Although cobalt and vitamin E also prevented the accumulation of TBARS in the spleen and intestines of experimental groups 3 and 4, respectively, vitamin E was more effective than cobalt (P < .001)(Fig. 4). It has been well documented that inflammatory cells, in particular macrophages and polymorphonuclear leukocytes, play an important role in the initiation and maintenance of lipid peroxidation during an inflammatory process. Consequently we measured MPO activity in the tissue homogenates of all 4 groups. The results, summarized in Fig. 5, clearly show that as expected, iron overload leads to a 4.3fold increase in MPO activity in the liver (16.8 ± 2.9 IU/g vs 3.83 ± 1.5 IU/g), a 1.9-fold increase in the spleen (34.6 ± 5.0 IU/g vs 17.9 _+2.1 IU/g), and a 2.1fold increase in the intestine (46.9 _+ 6.1 IU/g vs 22.1
+ 1.1 IU/g). Fig. 4 further shows that both cobalt and vitamin E decreased tissue MPO activity in rat liver by 43% and 55%, respectively, and in the spleen by 20% and 33%, respectively (P < .001). As already seen above, vitamin E again appears to be more effective than cobalt. With respect to the intestine, vitamin E decreased MPO activity by 24%, to 36.1 +_4.3 IU/g, while cobalt had no effect (40.5 _+ 7.3 IU/g vs 46.9 -!_ 6.1 IU/g [group 2]). Biochemical and hematologic analysis. The results of the biochemical determinations on blood samples are summarized in Table II. Despite a large increase in hepatic lipid peroxide levels, iron overload did not increase SGPT levels (P < .05). Although SGOT levels were high in all animals including those in group 1, SGOT values in group 3 and 4 were significantly higher but statistically different in group 3 when compared with those measured for groups 1 and 2 (P < .05). In contrast to vitamin E, cobalt administration increased Hb, Hct, and MCV in the animals of group 3 as compared with those in groups 1 and 2 (P < .05) (Table II). On the other hand, in all animals given iron, neither cobalt nor vitamin E affected WBC or RBC counts.
DISCUSSION It has been reported that iron overload stimulates
162
J Lab Clin Med August 1998
Jnan et al.
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0.3
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0.2
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0.15
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I
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I
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Fig. 4. Tissue lipid peroxide levels. Values are expressed as mean + 1 SD. #P < .001 for lipid peroxide levels as compared with group 1. *p < .001 for lipid peroxide levels as compared with group 2. §P < .001 for lipid peroxide levels as compared with group 2. q[P < .001 for effect of vitamin E as compared with groups 2 and 3.
lipid peroxidation by facilitating the decomposition of lipid peroxides and the formation of OH radical from H202 and O -2 in tissues and through the generation of 0 .2 and H202 by accelerating the non-enzymatic oxidation of several molecules. 7-11 As a potent antioxidant, vitamin E prevents iron-induced peroxidati0n.9,13-18 In contrast, little is known about the anti-peroxidative actions of cobalt in vivo. The results of this study show that in vivo cobalt in addition to vitamin E can function as a potent antioxidant in the amelioration of ironinduced lipid peroxidation. In our study, lipid peroxidation was measured as TBARS in rat tissue, which was stimulated by iron overload. Because the highest TBARS value was observed in the liver, it might be considered to be the most sensitive of the organs to biochemical changes in iron overload, and accordingly, a major site of iron deposition. In the liver, cobalt and vitamin E reduced lipid peroxide levels to 50% of the values measured in group 2 (Fig. 4, columns 3 and 4). Both cobalt and vitamin E were also effective in reducing lipid peroxidation in the spleen and intestine, but in the intestine, vitamin E was more effective than cobalt. Thus both antioxidant agents protected all tissues examined against the peroxidative effect of iron. The increased production of free oxygen radicals by iron causes peroxidative decomposition of the polyunsaturated fatty acids of mitochondrial and microsomal membranes as well as of the plasma membrane. 1,2,7 Pre-
sumably these membrane damages will cause specific structural and functional disturbances in cell integrity, especially in mitochondria. 31-34 Iron overload causes a 70% decrease in cytochrome c oxidase activity, leading to reduced ATP synthesis. 35-3s Accordingly, increased ATP demand causes the ATP degradation to hypoxanthine and the formation of several new reactive oxygen species. The concentration of hypoxanthine in the liver was found to be increased 3-fold to 4-fold over the control values, depending on the duration of the hypoxiaischemia that was correlated with the decreased ATP and ADP levels. 39 In our study, iron accumulation caused a several-fold elevation of tissue hypoxanthine in the animals of group 2 versus those in group 1 (Fig. 3, columns 1 and 2). It is important to note that establishing normal hypoxanthine levels in either tissue or blood results in considerably varied values when existing methods are used. 39-44 On the other hand, our control values were similar to the results reported by others. 39 Treatment with either vitamin E or cobalt decreased tissue hypoxanthine levels in iron overload significantly (Fig. 3, columns 3 and 4). Because vitamin E is a powerful inhibitor of lipid peroxidation in biologic membranes, 14-18 its action as an antioxidant on hypoxanthine levels is expected. Interestingly, cobalt presented the same protective effect in the tissues that correlated with its effect on iron-induced lipid peroxidation. According to our knowledge, this study is the first to describe a cor-
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inan et al.
163
60
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Fig. 5. Effect of cobalt and vitamin E on tissue M P O activities. #P < .001 for MPO activities as compared with group 1. *P < .001 for tissue M P O activities as compared with group 2. ~[P < .001 for the effect of vitamin E as compared with group 2.
Table II. C o m p l e t e blood cell and serum transaminase values WBC (xl031mm 3) Group Group Group Group
1 2 3 4
13.5 19.7 14.4 13.6
_+ 3.9 _+ 8.9 _+ 6.4 _+ 9.2
RBC (xl0elmm 3) 6.9 7.5 7.3 7.7
_+ 0.9 _+ 0.5 +_ 0.9 + 1.3
Hb (gldl) 12.9 14.2 16.2 14.5
_+ 1.6" __ 1.1 + 1.07* + 1.7
Hot (%) 36.8 39.3 45.6 41.3
MCV (fl)
_+ 5.3 +_ 2.2 ___4.4* _+ 6.7
53.1 54.5 57.9 53.9
-+ 1.5 _+2.2 _+ 5.7* -+ 1.8
SGOT (IUIL) 152.0 140.0 283.6 209.6
_+ 26.5 _+ 22.8 + 156.4" _+ 39.6
SGPT (IUIL) 53.6 42.5 59.5 51.0
_+ 9.6 _+ 15.9 -+ 28 __ 16.5
*P < .05 for the values c o m p a r e d with the other groups,
relation of tissue hypoxanthine levels with lipid peroxidation in iron overload. When increased production of free oxygen radicals stimulates lipid peroxidation in biologic membranes, peroxidation of membrane lipids increases lysosomal fragility and the levels of cytoplasmic Ca +2. Ca +2 accumulation, via activation of phospholipase A a, increases the synthesis of eicosanoids that are chemoattractants. As a consequence, PMN infiltration into the damaged site is increased, resulting in the additional production of reactive oxygen species by activated PMNs, the release of hydrolytic enzymes, and the exacerbation of tissue damage. 45-48 In a previous study of the effect of hypoxia in newborn rats, we showed decreased lipid peroxidation and MPO activity during the oxidative stress. 49 In the present study, in addition to the effect of vitamin E, cobalt decreased iron-induced MPO activity in the liver and spleen with little effect in the intes-
tine (Fig. 5). The decrease in MPO activity caused by antioxidants suggests that they may lead to a reduction of inflammatory reactions caused by iron. Therefore, histopathologic analyses were performed with the aim of identifying the consequences of these biochemical changes. To our surprise, the liver sections of 50% of animals with iron overload showed the previously unreported formation of granulomas containing large numbers of iron-stuffed MNCs, Kupffer cells, and PMNs. Although this may suggest an immunologic process as the pathogenesis of the granulomas, previous investigations have shown that the dextran used as vector in the iron-dextran preparation for the present study is immunologically inert and therefore cannot explain this granuloma formation.50, 51 Consequently the large amount of iron present in the tissues may play a central role in the formation of these granulomas. The frequently unique centrilobular location of the granulomas fur-
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ther supports a chemical rather than immunologic pathogenesis. 52 On the other hand, the presence of numerous iron-laden macrophages in the seemingly enlarged splenic follicles of the animals in groups 2, 3, and 4 prevents us from totally excluding an immunologic process as the basis of the formation o f these granulomas. As stated in the introduction, the aim of the present study was to investigate the antioxidant effect of cobalt in iron overload and to compare this effect with that of the wellknown antioxidant vitamin E. The presence of the granuloma was unexpected; therefore, pinpointing the pathophysiologic process of this serendipitious finding will require a separate investigation. In this context it is important to note that treatment with vitamin E and cobalt reduced MPO production not only in the liver but also in the spleen and intestines of iron-overloaded animals. Although this finding suggests that a decrease in the production o f cytotoxic oxygen radicals as a consequence of reduced M P O production may be responsible for the slightly reduced numbers of granulomas in the presence of cobalt and especially of vitamin E, additional experiments are necessary to ascertain this observation. The absence of significant changes in serum SGPT activity suggests that most liver tissue cells remained intact. It is possible, on the other hand, that the increased activity of serum SGOT levels has been caused b y mechanical injury such as cardiac puncture, as reported in other in vivo studies. Despite increased lipid peroxidation, tissue-associated M P O activity, and hypoxanthine levels, no significant change was detected in the amount of tissue G S H that functions as another important natural antioxidant. Recently it was reported that v i t a m i n E and cobalt affect G S H level during tissue injury.143 ° In the present study we used the Ellman reaction to determine tissue G S H levels. Because this reaction is not completely specific for glutathione, p o s s i b l e interference with sulphydryl groups of amino acids or proteins (eg, metallothionein) during the assay could explain the absence of change in "glutathione. ''26 Besides the biochemical and histopathologic changes in the tissues, the effect of the cobalt on Hb and Hct can provide an advantage in the assessment of treatment with cobalt. 53,54 Unlike our understanding regarding vitamin E, our k n o w l e d g e of how cobalt is treated by and processed in different tissues is less extensive. Although cobalt is known to be a cardiotoxic ion, a few animal studies have analyzed the potential protective effect of cobalt on cephaloridine-induced lipid peroxidation, 55 poisoning by potassium cyanide, 56 and anoxic damage in rat hippocampal slices. 57 Unfortunately, iron overload affects morbidity by causing many acute and long-term side effects and organ failure. The results o f the present study suggest that antioxidant agents extend significant protective
effects against iron-induced tissue injury. A l t h o u g h cobalt was considered an oxidative cation until recently, in this study cobalt was shown to have a strong antioxidant effect on i r o n - i n d u c e d lipid p e r o x i d a t i o n and subsequent b i o c h e m i c a l changes in tissues when c o m p a r e d with vitamin E. Further investigations will establish the effective therapeutic dose versus tissue toxicity regarding the use o f cobalt in the clinical setting. We thank Professor ~i~dem Altay, from the Department of Pediatrics, Hacettepe Children's Hospital, for her continuous support and guidance in this study. We also thank Professor Safiye G6~tis, from the Department of Pediatrics, Division of Pediatric Pathology, Hacettepe Children's Hospital, for her evaluation and comments on the histopathologic results. REFERENCES
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