Biol Trace Elem Res DOI 10.1007/s12011-015-0425-1
The Effects of Lithium Administration on Oxidant/Antioxidant Status in Rats: Biochemical and Histomorphological Evaluations Selmin Toplan 1 & Semra Ozdemir 1 & Gamze Tanriverdi 2 & M. Can Akyolcu 1 & Dervis Ozcelik 1 & Nuran Darıyerli 3
Received: 18 March 2015 / Accepted: 26 June 2015 # Springer Science+Business Media New York 2015
Abstract Present study was planned to determine possible dose-dependent effects of lithium (Li) on oxidantantioxidant status and histomorphological changes in liver and kidney tissues. For this purpose, twenty-four Wistar male rats were equally divided into three groups: the rats in group I served as controls, drinking tap water without lithium. Groups II and III received 0.1 and 0.2 % lithium carbonate (Li2CO3) through their drinking water, respectively, for 30 days. At the end of the experimental period, lithium concentrations, levels of malondialdehyde (MDA) and glutathione (GSH) and superoxide dismutase (SOD) activities were measured in considered tissues. Histomorphological study was also performed on liver and kidney tissues. Compared to controls, MDA was significantly higher but GSH level lower in groups II and III. SOD activity was higher in group III, but no difference was determined in group II in liver tissue. In kidney tissue, there was no difference determined in MDA and GSH levels between control and experimental groups but SOD activity in groups II and III was significantly higher. In histologic sections of both experimental liver and kidney tissues, specific degenerations were observed. The results of the present study
* Semra Ozdemir [email protected]; [email protected] 1
Department of Biophysics, Cerrahpasa Medical Faculty, Istanbul University, Istanbul, Turkey
2
Department of Histology and Embryology, Cerrahpasa Medical Faculty, Istanbul University, Istanbul, Turkey
3
Department of Physiology, Cerrahpasa Medical Faculty, Istanbul University, Istanbul, Turkey
show that treatment with lithium carbonate may result in liver and kidney tissue abnormalities and oxidative damage.
Keywords Lithium . Oxidative stress . Antioxidants . Kidney . Liver fibrosis
Introduction Lithium salts are commonly used in the treatment of bipolar diseases. Readily absorbed from the gastrointestinal tract, lithium is initially distributed in the extracellular fluid and then accumulates in certain tissues. Although highly effective in reducing symptoms of manic depression, lithium induces structural and functional disturbances after prolonged treatment at therapeutic doses in organism [1]. There are various reports reflecting the toxic effects of lithium on liver structure and function [2, 3]. Lithium is also considered of being nephrotoxic. Renal insufficiency in long-term lithium treatment has also been reported recently [4]. Different results have been reported related to applied lithium dose and duration. There are also differences between studies that investigated lithiuminduced oxidative damage. Under normal physiological conditions, the oxidants and antioxidants generated during normal aerobic metabolism [1, 5]. Depending on specific situations considered, oxidant-antioxidant status may be broken down and tissue damage may arise. While it has been showed by few researchers that lithium has protective effect in some specific tissues [6, 7], some other researchers reported that higher dose application of lithium leads to toxicity in tissues [8, 9]. Present study was conducted to investigate effects of different doses of lithium on oxidant antioxidant status and histological changes in liver and kidney tissues.
Materials and Methods
inductively coupled plasma-optical emission spectroscopy (ICP-OES).
Experimental Protocol The Animal Care and Use Committee of Laboratory Animal Service, Istanbul University approved the study protocol and methodology. Twenty four Wistar male rats weighing between 160 and 200 g were selected for the study. All rats were kept in metal free cages at an ambient temperature of 22 ± 1 °C, controlled lighting with alternate dark (18.00 – 06.00 h) and light (06.00–18.00 h) cycles, and were fed standard rat diet. Lithium carbonate (Li2CO3) that applied in our study was purchased from Sigma. The dose and time of lithium carbonate administration selection was based on our previously data where significant changes were observed [10]. The rats were equally divided into three groups: Group I acted as controls, receiving normal tap water without lithium; group II received 0.1 % Li2CO3 (1 g/L drinking water) while group III received 0.2 % Li2CO3 (2 g/L drinking water) during 30 days [10, 11]. Specimen Collection At the end of the 30-day experimental period, rats were sacrificed under ketamine anesthesia. The liver and kidney tissue samples were collected and tissue homogenates (10 % (w/v)) were prepared in 0.1 mol/L Tris-HCl buffer, pH = 7.4. Supernatants were obtained by centrifugation at 3000×g for 15 min using a Hettich Universal centrifuge. In supernatants, lithium concentrations, malondialdehyde (MDA) levels as lipid peroxidation indicator, and superoxide dismutase (SOD) activities and glutathione (GSH) levels as antioxidant defense system indicator were measured. Biochemical Analysis Measurement of GSH was performed by using the Bioxytech GSH-400 kit. Supernatants were processed with metaphosphoric acid (MPA) for protein denaturation. After centrifugation, the chromogenic reagent was added to the supernatant and the absorbance of a formed thione was measured at 400 nm. GSH levels were calculated as molar concentrations (in micromoles per liter) [12]. The measurement of SOD activity was done by using the Northwest kit (NWKSOD 02). The Cu-ZnSOD activity was determined by the method of inhibition of hematoxylin autoxidation to hematein monitored at 560 nm using a standard curve obtained with purified bovine blood enzyme [13]. Malondialdehyde levels were measured by using the NWK-MDA01 kit. This method is based on the reaction of MDA with TBA forming a MDATBA2 adducts that has a large extinction coefficient at 532 nm [14]. Tissue Li concentrations were measured by using
Histological Evaluation From the sacrificed animals, liver and kidney tissues were taken and fixed in 10 % neutral buffered formalin for 24 h and then embedded in paraffin and cut into 5-μm sections for histological examinations. The kidney slides were stained with hematoxylin + eosin (H+E) and periodic acid Schiff (PAS) using conventional methods for light microscopic examination. A semiquantitative score was developed to evaluate the degree of the damage. A minimum of 20 glomeruli (range 20 to 60) in each specimen was examined, and the severity of the lesion was graded from 0 to 4+ according to the percentage of glomerular involvement. Thus, a 1+ lesion represented an involvement of 25 % of the glomerulus, while a 4+ lesion indicated that 100 % of the glomerulus was involved. An injury score was then obtained by multiplying the degree of damage (0 to 4+) by the percentage of the glomeruli with the same degree of injury, that is, increase in mezengial matrix material or glomerulosclerosis. The extent of the injury for each individual tissue specimen was then obtained by the addition of these scores. For example, if 5 of 20 glomeruli had a lesion of 1+ and 5 of 20 had a lesion of 3+, the final injury score for that specimen would be (1 × 5/20) + (3 × 5/ 20) × 100 = 100. The injury score for individual tissue specimens derived by each investigator varied from 11 % in the specimens with minimal changes (0 to 1+) up to 18 % in specimens with more severe and widespread (2 to 4+) injury. The scores obtained by the two investigators were averaged [15]. The liver slides were stained with H+E and Sirius red (SR). H+E is a staining method used for detecting the tissue morphology. Sirius red staining was used to demonstrate collagen fiber architecture. Fibrosis and fatty degenerations were scored by two independent researchers. The section was scored as described by Kaya Dagistanli [16] as follows: 0— intact liver; 1—centrilobular necrosis and fatty degeneration; 2—centrilobular and midlobular fatty degeneration, perivenular fibrosis; 3—septal fibrosis, pseudolobule formation; and 4—regenerative nodule formation, cirrhosis. Photographs of sections were taken at different magnifications in an Olympus BX61 research microscope, fitted with Olympus DP72 model digital camera. Histological evaluations of tissues were performed by light microscope. Statistical Analysis The values were reported as means ± SD. Comparison between groups was performed with the Mann-Whitney U test using SPSS v.10.0 statistical software. Significance was set at p < 0.05.
The Effects of Lithium Administration on Oxidant/Antioxidant
Results Biochemical Analysis SOD activity, GSH and MDA levels, and tissue lithium concentrations of the experimental and control groups obtained from the measurements of related tissues are given in Table 1. The liver tissue MDA levels of both experimental groups were found to be significantly higher than MDA levels of the control group (p < 0.01). GSH levels of the groups II and III were significantly lower than those of the control group (p < 0.01) and III (p < 0.05). While there was no difference detected in the activity of SOD between controls and group II, it was found to be significantly higher in group III (p < 0.05) (Table 1). In the kidney tissue, there was no difference in the MDA and GSH levels between control and experimental groups. Compared to controls, the SOD activities of both experimental groups were significantly higher (p < 0.01) (Table 1). Liver and kidney tissue lithium concentrations were found to be significantly elevated related to dose applied (Table 1). The body weights were found to be significantly reduced only in group III when compared to controls (p < 0.05). Histological Results Kidney and liver tissue sections of control group had a normal architecture under microscopic observations (Fig. 1a, b and Fig. 2a, b). But in histological sections of both experimental groups, tissue-specific degenerations were observed. Compared to controls, the kidney tissues of group II showed degenerative glomeruli and tubule structures. Increase in mesangial matrix (Fig. 1c) and proximal and distal tubule Table 1 Effects of lithium exposure on MDA and GSH levels and SOD activities in liver and kidney tissues of control and experimental groups of rats and their total body weights
damages were obvious (Fig. 1d). Similar degenerations were also observed in group III but the observed damage was greater than the damage in group II (Fig. 1e, f). Scoring for mesangial matrix increase has been shown in Table 2. The histopathological results showed that lithium treatment caused significant and dose-dependent tissue damages in the liver of both group rats. Disorganization in hepatic cords and vacuolization in hepatocytes were also observed. The amount of collagen fibers was increased among the central vein, portal areas, and hepatocyte cords in both groups (Fig. 2c–f). But all histopathological changes were more prominent at group III which received higher dose lithium compared to group II samples (Fig. 2e, f). Scoring for fibrosis and fatty degenerations has been given in Table 3. Discussion The present study evidently demonstrated effects of lithium on histological structures and oxidant antioxidant status in liver and kidney tissues. Lipid peroxidation changes have been reported by different researchers related to effects of lithium on liver and kidney tissues. Kielczykowska et al. [17] showed that MDA concentration was depressed in rat liver tissue giving the evidence of the Li protective effect. Li et al. [18] reported that lithium exerted the divergent effect on lipid peroxidation in rat liver. According to researchers, lower doses resulted in inhibition, whereas the higher concentrations showed a stimulating influence. Joshi et al. [9] showed that lithium treatment led to a significant increase in concentration of lipid peroxidation product, i.e., malondialdehyde. Our results indicate that liver tissue MDA levels were significantly increased in both experimental group animals when exposed to lithium. This significant increase can be explained by
Group I
Group II
Group III
(n = 8)
(n = 8)
(n = 8)
(control)
(0.1 % Li2CO3)
(0.2 % Li2CO3)
Liver Li (mEq/L) Kidney Li (mEq/L) Liver MDA (nmol/g) Kidney MDA (nmol/g) Liver SOD activity (U/mg protein) Kidney SOD activity (U/mg protein)
0.00 0.00 8.84 ± 0.56 11.78 ± 1.44 56.42 ± 9.71 67.0 ± 5.74
0.31 1.36 10.53 11.98 59.01 81.04
1.42 2.96 10.93 12.48 72.74 92.21
Liver GSH (μmol/L) Kidney GSH (μmol/L) Body weight (g)
22.09 ± 2.47 17.44 ± 0.42 190 ± 15
18.08 ± 1.00** 18.27 ± 0.99 179 ± 19
Parameters
± ± ± ± ± ±
0.15 0.70 1.33** 1.05 11.48 8.89**
± ± ± ± ± ±
0.53## 1.22# 1.57** 1.68 10.40* 14.17**
19.75 ± 0.86* 18.92 ± 2.05 169 ± 15*
Data are the means ± SD GSH glutathione, MDA malondialdehyde, SOD superoxide dismutase *p < 0.05; **p < 0.01 versus control and experimental groups; # p < 0.01; group III
##
p < 0.001 versus group II and
Fig. 1 PAS staining was applied for identifying the effects of lithium exposure on the expansion of mezengial matrix scoring in kidney tissues of the rats. Matrix expansion was detectable in both groups (c,
d), especially in the second group (e, f). In control group, histological structure was normal (a, b). Magnification, a–f: ×400
damage due to oxidative stress. However, Li administration for a period of 30 days caused no change in kidney tissue MDA. Glutathione is considered to be an important determinant to evaluate the tissue injury caused by various chemicals and toxins, as it is an important constituent of the intracellular protective system. Lipid peroxidation causes glutathione to be consumed by the glutathione-related enzymes as detoxifying peroxides. Chadha et al. [3] declared that lithium treatment to animals resulted in a significant increase in the levels of hepatic lipid peroxide and SOD activity for the different durations of 1, 2, and 4 months. They detected a significant
decrease in the levels of GSH. Younes and Siegers [19] have also observed that depletion of GSH enhances lipid peroxidation, supporting the fact that GSH has an association with lipid peroxidation. Our results showed that lithium application led to statistically significant decrease in GSH level of liver in groups II and III. These significant changes in MDA and GSH levels in the studied tissues can be possibly due to oxidative stress. Decrease in GSH level in our study may be accepted as an indicator of increased lipid peroxidation, and these injurious symptoms were found to be directly related to the dose effect. In such a situation, there is only one
Fig. 2 Sirius red staining was applied for identifying the effects of lithium exposure on the collagen accumulation in liver tissues of the rats. a–f Collagen accumulation was increased around central vein and
hepatocyte cords in both of the experimental groups (c–f). In control group, histological structure was normal (a, b). Magnification, a–f: ×400
The Effects of Lithium Administration on Oxidant/Antioxidant Table 2 Mesangial matrix scoring in kidney tissues of control and experimental groups Parameters
Group I (n = 6) (control)
Group III Group II (n = 6) (n = 6) (0.1 % Li2CO3) (0.2 % Li2CO3)
Mesangial matrix 26.25 ± 8.76 73.75 ± 9.84* score
124.58 ± 20.15*,#
*p < 0.001 versus control and experimental groups; # p < 0.001 versus group II and group III
possibility: GSH has been utilized by detoxifying peroxides in liver tissue. Several authors have documented that lithium intoxication is dose related and is responsible for side effects in the renal system such as acute renal failure, nephrotic syndrome, and polyuria for humans [20] and animals [21, 22]. Kielczykowska et al. [17] observed that lower doses of lithium administration caused no changes of MDA and GSH levels in kidney tissue in their study which is to be analog results of our study. Chimielnicka and Nasiadek [23] showed that oral administration of lithium carbonate induces renal toxicity in the rat as well as the injurious symptoms which were found to be directly related to the dose effect. SOD is an important antioxidant enzyme which primarily catalyzes the dismutation of superoxide anion and thus acts as a first-line antioxidant defense. Our results show that there was no difference in SOD enzyme activity between liver tissue of controls and group II, but significant higher activity was detected in group III compared to controls (p < 0.05). The increased SOD enzyme activity in group III may be an indicator of high-dose lithium application-induced oxidative stress in liver tissue. Tandon et al. [2] observed a significant decrease in the activity of SOD after lithium treatment in the liver. Nciri et al. [24] found that lipid peroxidation level and activities of SOD and GPX were increased in liver, lithium treatment, especially at the highest dose for 28 days. According to our results, SOD activity in both experimental groups was found to be higher compared to control group in kidney tissue. The increased activity of antioxidative enzyme SOD in the kidney can be explained as sign of success of defense mechanism against lipid peroxidation because kidney tissue contains antioxidants that prevent damage from excessive oxygen metabolites; they Table 3 Fibrosis and fatty degeneration scoring in liver tissues of control and experimental groups Parameters
Group I (n = 6) (control)
Group III Group II (n = 6) (n = 6) (0.1 % Li2CO3) (0.2 % Li2CO3)
Fibrosis and fatty 0.20 ± 0.07 1.008 ± 1.17* degeneration score
1.41 ± 0.28*,#
*p < 0.001 versus control and experimental groups; # p < 0.001 versus group II and group III
act either by decomposing peroxide or trapping the free radicals. Nciri et al. [25] found that the lipid peroxidation and SOD activity were increased in the kidney when low doses of lithium carbonate are injected into mice. According to Nciri, SOD levels typically decrease when stress conditions are reduced. By contrast, an increase in oxidative stress levels induces elevated SOD, mostly associated with elevation in cell hydrogen peroxide concentration. Results of our study show that lithium administration caused many deformities and histological alterations in liver and kidney tissues of rats. In histologic sections in both experimental groups, tissue-specific degenerations were observed. Mezengial matrix increase and proximal tubule damage was obvious. According to general literature knowledge, in general lithium treatment experimental studies, so far, there was no increase of mesangial matrix in the kidney and fibrosis findings in liver tissues have been reported. We esteem that such a determined histomorphological change due to lithium application in our study is an important point. In our study, disorganization in hepatic cords and vacuolization in hepatocytes were observed. The amount of collagen fiber was increased in the central vein, portal areas, and hepatocyte cords in both groups. The histopathological results show that lithium treatment caused dose-dependent tissue damages in the liver of both group rats. But all histopathological changes were more prominent at higher dose. Ahmad et al. [26] reported that LiCl treatment caused significant and dose-dependent tissue damages in the liver and kidney. The results of our study are consistent with their findings as far as histopathological changes are concerned. Li mostly causes tubulointerstitial nephropathy; however, severe damage of the mitochondria and endoplasmic reticulum in electron microscope examinations has been documented implicating renal ischemia as a pathogenetic mechanism. Aurell M. [27] and Sharma and Iqbal [8] suggested that the small doses of lithium induce toxicity in rats and the histopathological appearance of the kidney tissues revealed many deformative alterations. Kumarguru et al. [28] showed a significant chronic tubulointerstitial nephropathy (CTIN) and a considerable glomerular pathology in the kidneys. His results appear to be the first time that mast cells were demonstrated in a case of lithium-induced nephropathy in humans. It may be hypothesized that mast cells may possibly play a role in lithium-induced nephropathy as a concurrent mechanism. At doses in the toxic range, the proximal tubules may also be affected. Therefore, low dose (and usually chronic) toxicity is associated with distal tubular abnormalities and high dose toxicity characteristically manifests as proximal tubular damage, when a prerenal effect is often also seen [29, 30]. The results of the present study show that the degree of oxidative stress is dose dependent and that the higher the dose of lithium has a greater toxic effect. In sum, we conclude that high doses of lithium may cause liver and kidney tissue
function abnormalities and increased oxidative damage possibly resulting in damage to liver. According to our study, even though no oxidative stress was detected in kidney tissue due to lithium exposure, we detected significant histopathological changes though. To explain cell deformation in kidneys after lithium administration without lipid peroxidation may need further studies using the other markers in order to draw a conclusion for oxidative damage and to understand if such effects are originated from lithium exposure itself or from other possible mechanism due to lithium toxicity.
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17. Conflict of Interest The authors declare that they have no competing interests. Funding This work was supported by the Research Fund of the Istanbul University (project number, BYP 10226).
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The Effects of Lithium Administration on Oxidant/Antioxidant Status in Rats: Biochemical and Histomorphological Evaluations Selmin Toplan 1 & Semra Ozdemir 1 & Gamze Tanriverdi 2 & M. Can Akyolcu 1 & Dervis Ozcelik 1 & Nuran Darıyerli 3
Received: 18 March 2015 / Accepted: 26 June 2015 # Springer Science+Business Media New York 2015
Abstract Present study was planned to determine possible dose-dependent effects of lithium (Li) on oxidantantioxidant status and histomorphological changes in liver and kidney tissues. For this purpose, twenty-four Wistar male rats were equally divided into three groups: the rats in group I served as controls, drinking tap water without lithium. Groups II and III received 0.1 and 0.2 % lithium carbonate (Li2CO3) through their drinking water, respectively, for 30 days. At the end of the experimental period, lithium concentrations, levels of malondialdehyde (MDA) and glutathione (GSH) and superoxide dismutase (SOD) activities were measured in considered tissues. Histomorphological study was also performed on liver and kidney tissues. Compared to controls, MDA was significantly higher but GSH level lower in groups II and III. SOD activity was higher in group III, but no difference was determined in group II in liver tissue. In kidney tissue, there was no difference determined in MDA and GSH levels between control and experimental groups but SOD activity in groups II and III was significantly higher. In histologic sections of both experimental liver and kidney tissues, specific degenerations were observed. The results of the present study
* Semra Ozdemir [email protected]; [email protected] 1
Department of Biophysics, Cerrahpasa Medical Faculty, Istanbul University, Istanbul, Turkey
2
Department of Histology and Embryology, Cerrahpasa Medical Faculty, Istanbul University, Istanbul, Turkey
3
Department of Physiology, Cerrahpasa Medical Faculty, Istanbul University, Istanbul, Turkey
show that treatment with lithium carbonate may result in liver and kidney tissue abnormalities and oxidative damage.
Keywords Lithium . Oxidative stress . Antioxidants . Kidney . Liver fibrosis
Introduction Lithium salts are commonly used in the treatment of bipolar diseases. Readily absorbed from the gastrointestinal tract, lithium is initially distributed in the extracellular fluid and then accumulates in certain tissues. Although highly effective in reducing symptoms of manic depression, lithium induces structural and functional disturbances after prolonged treatment at therapeutic doses in organism [1]. There are various reports reflecting the toxic effects of lithium on liver structure and function [2, 3]. Lithium is also considered of being nephrotoxic. Renal insufficiency in long-term lithium treatment has also been reported recently [4]. Different results have been reported related to applied lithium dose and duration. There are also differences between studies that investigated lithiuminduced oxidative damage. Under normal physiological conditions, the oxidants and antioxidants generated during normal aerobic metabolism [1, 5]. Depending on specific situations considered, oxidant-antioxidant status may be broken down and tissue damage may arise. While it has been showed by few researchers that lithium has protective effect in some specific tissues [6, 7], some other researchers reported that higher dose application of lithium leads to toxicity in tissues [8, 9]. Present study was conducted to investigate effects of different doses of lithium on oxidant antioxidant status and histological changes in liver and kidney tissues.
Materials and Methods
inductively coupled plasma-optical emission spectroscopy (ICP-OES).
Experimental Protocol The Animal Care and Use Committee of Laboratory Animal Service, Istanbul University approved the study protocol and methodology. Twenty four Wistar male rats weighing between 160 and 200 g were selected for the study. All rats were kept in metal free cages at an ambient temperature of 22 ± 1 °C, controlled lighting with alternate dark (18.00 – 06.00 h) and light (06.00–18.00 h) cycles, and were fed standard rat diet. Lithium carbonate (Li2CO3) that applied in our study was purchased from Sigma. The dose and time of lithium carbonate administration selection was based on our previously data where significant changes were observed [10]. The rats were equally divided into three groups: Group I acted as controls, receiving normal tap water without lithium; group II received 0.1 % Li2CO3 (1 g/L drinking water) while group III received 0.2 % Li2CO3 (2 g/L drinking water) during 30 days [10, 11]. Specimen Collection At the end of the 30-day experimental period, rats were sacrificed under ketamine anesthesia. The liver and kidney tissue samples were collected and tissue homogenates (10 % (w/v)) were prepared in 0.1 mol/L Tris-HCl buffer, pH = 7.4. Supernatants were obtained by centrifugation at 3000×g for 15 min using a Hettich Universal centrifuge. In supernatants, lithium concentrations, malondialdehyde (MDA) levels as lipid peroxidation indicator, and superoxide dismutase (SOD) activities and glutathione (GSH) levels as antioxidant defense system indicator were measured. Biochemical Analysis Measurement of GSH was performed by using the Bioxytech GSH-400 kit. Supernatants were processed with metaphosphoric acid (MPA) for protein denaturation. After centrifugation, the chromogenic reagent was added to the supernatant and the absorbance of a formed thione was measured at 400 nm. GSH levels were calculated as molar concentrations (in micromoles per liter) [12]. The measurement of SOD activity was done by using the Northwest kit (NWKSOD 02). The Cu-ZnSOD activity was determined by the method of inhibition of hematoxylin autoxidation to hematein monitored at 560 nm using a standard curve obtained with purified bovine blood enzyme [13]. Malondialdehyde levels were measured by using the NWK-MDA01 kit. This method is based on the reaction of MDA with TBA forming a MDATBA2 adducts that has a large extinction coefficient at 532 nm [14]. Tissue Li concentrations were measured by using
Histological Evaluation From the sacrificed animals, liver and kidney tissues were taken and fixed in 10 % neutral buffered formalin for 24 h and then embedded in paraffin and cut into 5-μm sections for histological examinations. The kidney slides were stained with hematoxylin + eosin (H+E) and periodic acid Schiff (PAS) using conventional methods for light microscopic examination. A semiquantitative score was developed to evaluate the degree of the damage. A minimum of 20 glomeruli (range 20 to 60) in each specimen was examined, and the severity of the lesion was graded from 0 to 4+ according to the percentage of glomerular involvement. Thus, a 1+ lesion represented an involvement of 25 % of the glomerulus, while a 4+ lesion indicated that 100 % of the glomerulus was involved. An injury score was then obtained by multiplying the degree of damage (0 to 4+) by the percentage of the glomeruli with the same degree of injury, that is, increase in mezengial matrix material or glomerulosclerosis. The extent of the injury for each individual tissue specimen was then obtained by the addition of these scores. For example, if 5 of 20 glomeruli had a lesion of 1+ and 5 of 20 had a lesion of 3+, the final injury score for that specimen would be (1 × 5/20) + (3 × 5/ 20) × 100 = 100. The injury score for individual tissue specimens derived by each investigator varied from 11 % in the specimens with minimal changes (0 to 1+) up to 18 % in specimens with more severe and widespread (2 to 4+) injury. The scores obtained by the two investigators were averaged [15]. The liver slides were stained with H+E and Sirius red (SR). H+E is a staining method used for detecting the tissue morphology. Sirius red staining was used to demonstrate collagen fiber architecture. Fibrosis and fatty degenerations were scored by two independent researchers. The section was scored as described by Kaya Dagistanli [16] as follows: 0— intact liver; 1—centrilobular necrosis and fatty degeneration; 2—centrilobular and midlobular fatty degeneration, perivenular fibrosis; 3—septal fibrosis, pseudolobule formation; and 4—regenerative nodule formation, cirrhosis. Photographs of sections were taken at different magnifications in an Olympus BX61 research microscope, fitted with Olympus DP72 model digital camera. Histological evaluations of tissues were performed by light microscope. Statistical Analysis The values were reported as means ± SD. Comparison between groups was performed with the Mann-Whitney U test using SPSS v.10.0 statistical software. Significance was set at p < 0.05.
The Effects of Lithium Administration on Oxidant/Antioxidant
Results Biochemical Analysis SOD activity, GSH and MDA levels, and tissue lithium concentrations of the experimental and control groups obtained from the measurements of related tissues are given in Table 1. The liver tissue MDA levels of both experimental groups were found to be significantly higher than MDA levels of the control group (p < 0.01). GSH levels of the groups II and III were significantly lower than those of the control group (p < 0.01) and III (p < 0.05). While there was no difference detected in the activity of SOD between controls and group II, it was found to be significantly higher in group III (p < 0.05) (Table 1). In the kidney tissue, there was no difference in the MDA and GSH levels between control and experimental groups. Compared to controls, the SOD activities of both experimental groups were significantly higher (p < 0.01) (Table 1). Liver and kidney tissue lithium concentrations were found to be significantly elevated related to dose applied (Table 1). The body weights were found to be significantly reduced only in group III when compared to controls (p < 0.05). Histological Results Kidney and liver tissue sections of control group had a normal architecture under microscopic observations (Fig. 1a, b and Fig. 2a, b). But in histological sections of both experimental groups, tissue-specific degenerations were observed. Compared to controls, the kidney tissues of group II showed degenerative glomeruli and tubule structures. Increase in mesangial matrix (Fig. 1c) and proximal and distal tubule Table 1 Effects of lithium exposure on MDA and GSH levels and SOD activities in liver and kidney tissues of control and experimental groups of rats and their total body weights
damages were obvious (Fig. 1d). Similar degenerations were also observed in group III but the observed damage was greater than the damage in group II (Fig. 1e, f). Scoring for mesangial matrix increase has been shown in Table 2. The histopathological results showed that lithium treatment caused significant and dose-dependent tissue damages in the liver of both group rats. Disorganization in hepatic cords and vacuolization in hepatocytes were also observed. The amount of collagen fibers was increased among the central vein, portal areas, and hepatocyte cords in both groups (Fig. 2c–f). But all histopathological changes were more prominent at group III which received higher dose lithium compared to group II samples (Fig. 2e, f). Scoring for fibrosis and fatty degenerations has been given in Table 3. Discussion The present study evidently demonstrated effects of lithium on histological structures and oxidant antioxidant status in liver and kidney tissues. Lipid peroxidation changes have been reported by different researchers related to effects of lithium on liver and kidney tissues. Kielczykowska et al. [17] showed that MDA concentration was depressed in rat liver tissue giving the evidence of the Li protective effect. Li et al. [18] reported that lithium exerted the divergent effect on lipid peroxidation in rat liver. According to researchers, lower doses resulted in inhibition, whereas the higher concentrations showed a stimulating influence. Joshi et al. [9] showed that lithium treatment led to a significant increase in concentration of lipid peroxidation product, i.e., malondialdehyde. Our results indicate that liver tissue MDA levels were significantly increased in both experimental group animals when exposed to lithium. This significant increase can be explained by
Group I
Group II
Group III
(n = 8)
(n = 8)
(n = 8)
(control)
(0.1 % Li2CO3)
(0.2 % Li2CO3)
Liver Li (mEq/L) Kidney Li (mEq/L) Liver MDA (nmol/g) Kidney MDA (nmol/g) Liver SOD activity (U/mg protein) Kidney SOD activity (U/mg protein)
0.00 0.00 8.84 ± 0.56 11.78 ± 1.44 56.42 ± 9.71 67.0 ± 5.74
0.31 1.36 10.53 11.98 59.01 81.04
1.42 2.96 10.93 12.48 72.74 92.21
Liver GSH (μmol/L) Kidney GSH (μmol/L) Body weight (g)
22.09 ± 2.47 17.44 ± 0.42 190 ± 15
18.08 ± 1.00** 18.27 ± 0.99 179 ± 19
Parameters
± ± ± ± ± ±
0.15 0.70 1.33** 1.05 11.48 8.89**
± ± ± ± ± ±
0.53## 1.22# 1.57** 1.68 10.40* 14.17**
19.75 ± 0.86* 18.92 ± 2.05 169 ± 15*
Data are the means ± SD GSH glutathione, MDA malondialdehyde, SOD superoxide dismutase *p < 0.05; **p < 0.01 versus control and experimental groups; # p < 0.01; group III
##
p < 0.001 versus group II and
Fig. 1 PAS staining was applied for identifying the effects of lithium exposure on the expansion of mezengial matrix scoring in kidney tissues of the rats. Matrix expansion was detectable in both groups (c,
d), especially in the second group (e, f). In control group, histological structure was normal (a, b). Magnification, a–f: ×400
damage due to oxidative stress. However, Li administration for a period of 30 days caused no change in kidney tissue MDA. Glutathione is considered to be an important determinant to evaluate the tissue injury caused by various chemicals and toxins, as it is an important constituent of the intracellular protective system. Lipid peroxidation causes glutathione to be consumed by the glutathione-related enzymes as detoxifying peroxides. Chadha et al. [3] declared that lithium treatment to animals resulted in a significant increase in the levels of hepatic lipid peroxide and SOD activity for the different durations of 1, 2, and 4 months. They detected a significant
decrease in the levels of GSH. Younes and Siegers [19] have also observed that depletion of GSH enhances lipid peroxidation, supporting the fact that GSH has an association with lipid peroxidation. Our results showed that lithium application led to statistically significant decrease in GSH level of liver in groups II and III. These significant changes in MDA and GSH levels in the studied tissues can be possibly due to oxidative stress. Decrease in GSH level in our study may be accepted as an indicator of increased lipid peroxidation, and these injurious symptoms were found to be directly related to the dose effect. In such a situation, there is only one
Fig. 2 Sirius red staining was applied for identifying the effects of lithium exposure on the collagen accumulation in liver tissues of the rats. a–f Collagen accumulation was increased around central vein and
hepatocyte cords in both of the experimental groups (c–f). In control group, histological structure was normal (a, b). Magnification, a–f: ×400
The Effects of Lithium Administration on Oxidant/Antioxidant Table 2 Mesangial matrix scoring in kidney tissues of control and experimental groups Parameters
Group I (n = 6) (control)
Group III Group II (n = 6) (n = 6) (0.1 % Li2CO3) (0.2 % Li2CO3)
Mesangial matrix 26.25 ± 8.76 73.75 ± 9.84* score
124.58 ± 20.15*,#
*p < 0.001 versus control and experimental groups; # p < 0.001 versus group II and group III
possibility: GSH has been utilized by detoxifying peroxides in liver tissue. Several authors have documented that lithium intoxication is dose related and is responsible for side effects in the renal system such as acute renal failure, nephrotic syndrome, and polyuria for humans [20] and animals [21, 22]. Kielczykowska et al. [17] observed that lower doses of lithium administration caused no changes of MDA and GSH levels in kidney tissue in their study which is to be analog results of our study. Chimielnicka and Nasiadek [23] showed that oral administration of lithium carbonate induces renal toxicity in the rat as well as the injurious symptoms which were found to be directly related to the dose effect. SOD is an important antioxidant enzyme which primarily catalyzes the dismutation of superoxide anion and thus acts as a first-line antioxidant defense. Our results show that there was no difference in SOD enzyme activity between liver tissue of controls and group II, but significant higher activity was detected in group III compared to controls (p < 0.05). The increased SOD enzyme activity in group III may be an indicator of high-dose lithium application-induced oxidative stress in liver tissue. Tandon et al. [2] observed a significant decrease in the activity of SOD after lithium treatment in the liver. Nciri et al. [24] found that lipid peroxidation level and activities of SOD and GPX were increased in liver, lithium treatment, especially at the highest dose for 28 days. According to our results, SOD activity in both experimental groups was found to be higher compared to control group in kidney tissue. The increased activity of antioxidative enzyme SOD in the kidney can be explained as sign of success of defense mechanism against lipid peroxidation because kidney tissue contains antioxidants that prevent damage from excessive oxygen metabolites; they Table 3 Fibrosis and fatty degeneration scoring in liver tissues of control and experimental groups Parameters
Group I (n = 6) (control)
Group III Group II (n = 6) (n = 6) (0.1 % Li2CO3) (0.2 % Li2CO3)
Fibrosis and fatty 0.20 ± 0.07 1.008 ± 1.17* degeneration score
1.41 ± 0.28*,#
*p < 0.001 versus control and experimental groups; # p < 0.001 versus group II and group III
act either by decomposing peroxide or trapping the free radicals. Nciri et al. [25] found that the lipid peroxidation and SOD activity were increased in the kidney when low doses of lithium carbonate are injected into mice. According to Nciri, SOD levels typically decrease when stress conditions are reduced. By contrast, an increase in oxidative stress levels induces elevated SOD, mostly associated with elevation in cell hydrogen peroxide concentration. Results of our study show that lithium administration caused many deformities and histological alterations in liver and kidney tissues of rats. In histologic sections in both experimental groups, tissue-specific degenerations were observed. Mezengial matrix increase and proximal tubule damage was obvious. According to general literature knowledge, in general lithium treatment experimental studies, so far, there was no increase of mesangial matrix in the kidney and fibrosis findings in liver tissues have been reported. We esteem that such a determined histomorphological change due to lithium application in our study is an important point. In our study, disorganization in hepatic cords and vacuolization in hepatocytes were observed. The amount of collagen fiber was increased in the central vein, portal areas, and hepatocyte cords in both groups. The histopathological results show that lithium treatment caused dose-dependent tissue damages in the liver of both group rats. But all histopathological changes were more prominent at higher dose. Ahmad et al. [26] reported that LiCl treatment caused significant and dose-dependent tissue damages in the liver and kidney. The results of our study are consistent with their findings as far as histopathological changes are concerned. Li mostly causes tubulointerstitial nephropathy; however, severe damage of the mitochondria and endoplasmic reticulum in electron microscope examinations has been documented implicating renal ischemia as a pathogenetic mechanism. Aurell M. [27] and Sharma and Iqbal [8] suggested that the small doses of lithium induce toxicity in rats and the histopathological appearance of the kidney tissues revealed many deformative alterations. Kumarguru et al. [28] showed a significant chronic tubulointerstitial nephropathy (CTIN) and a considerable glomerular pathology in the kidneys. His results appear to be the first time that mast cells were demonstrated in a case of lithium-induced nephropathy in humans. It may be hypothesized that mast cells may possibly play a role in lithium-induced nephropathy as a concurrent mechanism. At doses in the toxic range, the proximal tubules may also be affected. Therefore, low dose (and usually chronic) toxicity is associated with distal tubular abnormalities and high dose toxicity characteristically manifests as proximal tubular damage, when a prerenal effect is often also seen [29, 30]. The results of the present study show that the degree of oxidative stress is dose dependent and that the higher the dose of lithium has a greater toxic effect. In sum, we conclude that high doses of lithium may cause liver and kidney tissue
function abnormalities and increased oxidative damage possibly resulting in damage to liver. According to our study, even though no oxidative stress was detected in kidney tissue due to lithium exposure, we detected significant histopathological changes though. To explain cell deformation in kidneys after lithium administration without lipid peroxidation may need further studies using the other markers in order to draw a conclusion for oxidative damage and to understand if such effects are originated from lithium exposure itself or from other possible mechanism due to lithium toxicity.
13.
14.
15.
16.
17. Conflict of Interest The authors declare that they have no competing interests. Funding This work was supported by the Research Fund of the Istanbul University (project number, BYP 10226).
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