Environmental Toxicology and Pharmacology 41 (2016) 219–224
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Environmental Toxicology and Pharmacology journal homepage: www.elsevier.com/locate/etap
Subacute effects of low dose lead nitrate and mercury chloride exposure on kidney of rats Fatma Gökc¸e Apaydın a,∗ , Hatice Bas¸ b , Suna Kalender c , Yusuf Kalender a a b c
Gazi University, Faculty of Science, Department of Biology, Ankara 06500, Turkey Bozok University, Faculty of Arts and Science, Department of Biology, Yozgat 66100, Turkey Gazi University, Faculty of Gazi Education, Department of Science, Ankara 06500, Turkey
a r t i c l e
i n f o
Article history: Received 14 September 2015 Received in revised form 30 November 2015 Accepted 13 December 2015 Available online 17 December 2015 Keywords: Lead nitrate Mercury chloride Oxidative stress Biochemical parameters Histopathology Kidney
a b s t r a c t Lead nitrate and mercury chloride are the most common heavy metal pollutants. In the present study, the effects of lead and mercury induced nephrotoxicity were studied in Wistar rats. Lead nitrate (LN, 45 mg/kg b.w/day) and mercury chloride (MC, 0.02 mg/kg b.w/day) and their combination were administered orally for 28 days. Four groups of rats were used in the study: control, LN, MC and LN plus MC groups. Serum biochemical parameters, lipid peroxidation, antioxidant enzymes and histopathological changes in kidney tissues were investigated in all treatment groups. LN and MC caused severe histopathological changes. It was shown that LN, MC and also co-treatment with LN and MC exposure induced significant increase in serum urea, uric acid and creatinine levels. There were also statistically significant changes in antioxidant enzyme activities (SOD, CAT, GPx and GST) and lipid peroxidation (MDA) in all groups except control group. In this study, we showed that MC caused more harmful effects than LN in rats. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Metals and their compounds found in living organisms have many important roles. Nevertheless, when their exposure is excessive, they may cause toxic effects (Janicka et al., 2015). Heavy metals are known to be toxic to organisms (Durak et al., 2010). They are a major class of suspected carcinogens. At low concentrations, heavy metals such as lead and mercury, can cause harmfull effects and tend to accumulate in the food chain (Garcia-Nino and Pedraza-Chaverri, 2014). These metals enter to the body by ingestion, inhalation or through the skin and their presence may induce serious toxicity in animals and humans (Alissa and Ferns, 2011). For many years lead has been recognized as a highly toxic metal to both humans and animals. It can accumulate in soils and sediments through aerial deposition from metal industries. Other main sources of lead have been leaded gasoline in cars and shotgun pellets (Rainio et al., 2015). After absorption, most Pb binds to proteins in erythrocytes and is distributed to soft tissues and bone (GarciaNino and Pedraza-Chaverri, 2014). In the case of mercury, the highest exposition occurs through vapors from amalgam dental fillings and diet, based on seafood and fish which deliver high amounts
∗ Corresponding author. Tel.: +90 312 202 1205; fax: +90 312 2122279. E-mail address: [email protected] (F.G. Apaydın). http://dx.doi.org/10.1016/j.etap.2015.12.003 1382-6689/© 2015 Elsevier B.V. All rights reserved.
of methyl mercury (Janicka et al., 2015). Lead has three main biochemical properties that support to its toxic effects. First, it has high affinity to SH groups and inhibits SH dependent enzymes which are essential for biological processes. Second, lead acts in a manner similar to calcium and competitively inhibits actions of calcium in substantial areas such as mitochondrial oxidative phosphorylation and intracellular messenger system. Third, lead can affect the transcription of DNA by interaction with nucleic acid binding proteins with potential consequences for gene regulation (Gordon et al., 2002). Mercury is a highly toxic metal that can exert multiple adverse effects (Aslanturk et al., 2014). The mercuric ion produces systemic toxicity at much lower concentrations through inhibition of many enzymes, especially those containing the SH group. It can induce suppress humoral and cellular immunity and increase tumor frequency (Yole et al., 2007). Mercury may also induce autoimmune disease in genetically susceptible animals (Warfvinge et al., 1995). Heavy metals are persistent and widespread pollutants that affect the structure and function of several organs by generating oxidative stress (Garcia-Nino and Pedraza-Chaverri, 2014). They generate reactive oxygen species (ROS) (Sarkar et al., 2014). These species cause oxidative injury via different mechanisms. ROS may induce cell injury or death. Extreme production of ROS causes alterations in subcellular structures. These alterations are modifications of proteins and DNA, lipid peroxidation (LPO) of polyunsaturated
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Fig. 1. Schematic overview for design of the experiment.
fatty acids and changes of cellular antioxidant system. Malondialdehyde (MDA) is the end product of LPO, and thus increased MDA content is an important indicator of LPO (Uzun and Kalender, 2013; Kalender et al., 2013). Cells may stimulate antioxidant and detoxification responses to counter heavy metal damages. The involvement of antioxidant enzymes such as glutathione peroxidase (GPx), glutathione-S-transferase (GST), superoxide dismutase (SOD) and catalase (CAT) play a considerable mission in protecting cells from oxidative stress (Sarkar et al., 2014). Previous studies, many experimental works reported that heavy metals can cause histopathological alterations in many tissues (Dai et al., 2013; Yuan et al., 2014). Heavy metals have toxic effects to renal cells of mammalian. The relationship between metals and toxicity of the kidney has been demonstrated in previous experiments (Renugadevi and Prabu, 2009). Kidneys are major organs which maintain the internal environment of the body. They are sensitive to injury by drugs and environmental chemicals. Metals can cause renal injury. Nephrotoxicity of metals was described by Matos et al. (2009) and Liu et al. (2010). The purpose of the present study was to investigate the biochemical and pathological effectiveness of low dose LN and MC in rats and also to evaluate value of MDA, serum urea, uric acid and creatinine levels, the activities of SOD, CAT, GPx and GST, in the kidneys. Becasue of kidneys are primary target organ, in which MC and LN are intensively accumulated following chronic exposure (WHO, 1991).
Gazi University Laboratory Animals Growing and Experimental Research Center. They were allowed to get into the habit of new situation for 10 days. The animals were housed at 18–22 ◦ C and they were given water and food ad libitum, while a 12-h on/12-h off light cycle was maintained. The protocol was approved by the Gazi University Animal Experiments Local Ethics Committee (Protocol no: G.Ü.ET–13.011) and experiments were performed accordancing to the international guidelines for care and use of laboratory animals. Animals were randomly divided into 4 groups as follows (n = 6 for each group). 1. Control group (1 ml/kg b.w distilled water) 2. Animals exposed to lead nitrate (45 mg/kg b.w, 1/50 LD50, LN) 3. Animals exposed to mercury chloride (0.02 mg/kg bw., 1/50 LD50, MC) 4. Animals exposed to lead nitrate + mercury chloride (45 mg/kg b.w LN + 0.02 mg/kg b.w MC) Distilled water, LN and MC were exposed to rats orally via gavage. At the end of the 28 days, the rats were dissected using ketamin + xylazin than the kidneys were removed for investigations about MDA levels, antioxidant enzyme activities and light microscopic examinations. Blood samples of the animals were taken from the heart and collected into sterile tubes for analysing serum urea, uric acid and creatinine levels. Fig. 1 shows a schematic diagram for he design of the experiment. 2.2. Test chemicals
2. Materials and methods 2.1. Animals and studied groups Twenty four adult albino Wistar rats (300–320 g, 90 days old) were used throughout the study. They were obtained from the
LN, MC and all the other chemicals were obtained from Sigma Aldrich. LN and MC were dissolved in distilled water (Yole et al., 2007; Sharma et al., 2010). The chemicals were administered in the morning (between 09:00 and 10:00) to non-fasted rats. During 28 days, 1 ml/kg b.w (body weight) distilled water for control group,
F.G. Apaydın et al. / Environmental Toxicology and Pharmacology 41 (2016) 219–224
221
4 Week
500 400
a, b, c
a, b
a
SOD (nmol/mgprotein)
600
300 200 100 0 control
lead nitrate
mercury chloride
lead nitrate+mercury chloride
Fig. 2. Effects of subacute treatment of LN and MC on SOD activities in the kidney tissues of rats. Each bar represents mean ± SEM of six animals in each group. Significance at P < 0.05. (a) Comparison of control and other groups. (b) Comparison of lead nitrate group and other groups. (c) Comparison of mercury chloride group and other groups.
0.02 mg/kg b.w (1/50 LD50 ) MC (Yole et al., 2007) for MC treatment groups and 45 mg/kg b.w (1/50 LD50 ) LN (Sharma et al., 2010) for lead treatment groups were given to rats daily via gavage.
2.3. Biochemical analysis 2.3.1. MDA levels and antioxidant enzyme activities Kidneys were washed with sodium phosphate buffer (pH 7.2) then they were homogenized with Heidolph Silent Crusher M. The homogenates were centrifuged for 15 min at 4 ◦ C. Activities of antioxidant enzymes and MDA levels of kidneys were detected using a spectrophotometer (Shimadzu UV 1700, Kyoto, Japan). The protein concentration was determined by the method of Lowry et al. (1951). The activity of SOD was obtained by Marklund and Marklund’s study (1974) at 440 nm and the enzyme activity was expressed as nmol/mg protein. Renal CAT activity was estimated following the method of Aebi (1984) at 240 nm and the activity was defined as mol/mg protein. According to the study of Habig et al. (1974), activity of GST was determined at 340 nm and the data was given as nmol/mg protein. Activity of GPx was measured by Paglia and Valentine’s method (1987). At 340 nm, the reaction was monitored and value was stated as nmol/mg protein. MDA level of kidney was analysed at 532 nm by thiobarbituric acid method described by Ohkawa et al. (1979) and it was defined as nmol/mg protein.
2.3.2. Evaluation of serum urea, uric acid and creatinine levels Blood samples which were taken by heart were centrifuged at 3500 rpm then serum was separated. Urea, uric acid and creatinine levels of serum were determined using a spectrophotometricenzymatic kit (Thermo Trace-BECGMAN, Germany) and analyzed by an autoanalyzer (Bayer ope-RA).
2.4. Pathological evaluation Rats were dissected and the kidneys were removed and placed into the fixative bouin. After, kidney samples were dehydrated in ascending grades of ethanol and embedded in paraffin and paraffin block of the tissues were prepared. The specimens were cut in 4–6 m slices and were stained with Hematoxylin–Eosin (H&E). Then the tissues were evaluated under a light microscope (Olympus BX51, Tokyo, Japan) and photographed with a camera (Olympus E-330, Olympus Optical Co., Ltd., Japan). Ten slides were prepared from each kidney tissue.
2.5. Statistical analysis The data were expressed as mean ± SEM. The data of all groups were compared with each other. Groups were compared using one-way analysis of variance (ANOVA) test followed by Tukey. Significance was accepted at P < 0.05. 3. Results 3.1. Biochemical results 3.1.1. MDA levels and antioxidant enzyme activities Treatment with LN and MC, decreased the activities of SOD, CAT, GPx and GST in kidney tissues but MC showed more toxicity than LN except GST activity. In combination with LN and MC induced more damages than use of them alone (Figs. 2–5) (P < 0.05). Levels of MDA were determined in kidney tissues of rats. At the end of the experimental period, all of the treatment groups showed increasing of MDA level compared to control. We observed more increased in MC group than LN group. Treated with LN + MC caused more harmfull effects than use of them alone (Fig. 6) (P < 0.05). 3.1.2. Serum urea, uric acid and creatinine levels Treatment with LN and MC increased the levels of urea, uric acid and creatinine levels compared to control group. There is no important differences between MC, LN and MC + LN groups except urea levels (Table 1) (P < 0.05). 3.2. Histological results of kidney In our study histopathological examination of rat kidneys were investigated using light microscope. The histological examination of the kidney tissues of the control treated rats showed normal structure. Rats treated with LN alone exhibited tubular degeneration. MC treated group showed tubular dilatation and Table 1 Urea, uric acid and creatinine levels of control and experimental rats. Groups
Urea (mg/dL)
Control Lead nitrate Mercury chloride LN + MC
21.80 25.53 27.91 25.53
± ± ± ±
0.98 0.99a 1.69a , b 1.91a , c
Uric acid (mg/dL) 0.48 0.69 0.74 0.62
± ± ± ±
0.03 0.11a 0.07a 0.08a
Creatinine (mg/dL) 0.29 0.32 0.34 0.33
± ± ± ±
0.01 0.02a 0.02a 0.11a
Values are mean ± SEM of six rats in each group. Significance at P < 0.05. a Comparison of control and other groups. b Comparison of LN-treated group and other groups. c Comparison of MC-treated group and other group.
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4 Week
200 150 100
a, b, c
a, b
a
CAT (µmol/mgprotein)
250
50 0 control
lead nitrate
mercury chloride
lead nitrate+mercury chloride
Fig. 3. Effects of subacute treatment of LN and MC on CAT activities in the kidney tissues of rats. Each bar represents mean ± SEM of six animals in each group. Significance at P < 0.05. (a) Comparison of control and other groups. (b) Comparison of lead nitrate group and other groups. (c) Comparison of mercury chloride group and other groups.
4 Week
lead nitrate
mercury chloride
40 30
a, b, c
a, b
50
a
GPx (nmol/mgprotein)
60
20 10 0 control
lead nitrate+mercury chloride
Fig. 4. Effects of subacute treatment of LN and MC on GPx activities in the kidney tissues of rats. Each bar represents mean ± SEM of six animals in each group. Significance at P < 0.05. (a) Comparison of control and other groups. (b) Comparison of lead nitrate group and other groups. (c) Comparison of mercury chloride group and other groups.
glomerular lobulation. In LN + MC treated rats tubular degeneration was shown, too. There are no significant differences among LN, MC and LN plus MC groups (Fig. 7A–D). 4. Discussion Many toxicological studies demonstrated that environmental heavy metals are very harmful to humans and wildlife (Nasiadek
et al., 2014). Previous studies have already reported that the heavy metals cause different organ damages like liver in rats under different experimental conditions (Apaydin et al., 2015; Liu et al., 2015). Mercury chloride and lead nitrate can easily cross the brain-blood barrier and placenta (WHO, 1991). Many authors reported that mercury and lead cause oxidative stress and stimulate ROS (Kalender et al., 2013, 2015). The reaction of these ROS with cellular biomolecules result in LPO and also
4 Week
35 a
30
a
25
a, b, c
GST (nmol/mgprotein)
40
20 15 10 5 0 control
lead nitrate
mercury chloride
lead nitrate+mercury chloride
Fig. 5. Effects of subacute treatment of LN and MC on GST activities in the kidney tissues of rats. Each bar represents mean ± SEM of six animals in each group. Significance at P < 0.05. (a) Comparison of control and other groups. (b) Comparison of lead nitrate group and other groups. (c) Comparison of mercury chloride group and other groups.
4 Week
a, b
25 20
a
MDA (nmol/mgprotein)
30
223
a, b, c
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15 10 5 0 control
lead nitrate
mercury chloride
lead nitrate+mercury chloride
Fig. 6. Effects of subacute treatment of LN and MC on MDA levels in the kidney tissues of rats. Each bar represents mean ± SEM of six animals in each group. Significance at P < 0.05. (a) Comparison of control and other groups. (b) Comparison of lead nitrate group and other groups. (c) Comparison of mercury chloride group and other groups.
Fig. 7. (A) Kidney sections of control rats, (B) kidney sections of MC rats: tubular dilatation ( ( ). (D) Kidney sections of LN plus MC rats: Tubular degeneration ( ) X200, H&E.
membrane damage (Amamou et al., 2015). Antioxidants have been shown to scavenge reactive oxygen species (ROS) (Apaydın et al., 2014; Kalender et al., 2014). CAT activity is often correlated to SOD activity. SOD and CAT act like the first line of defence system. GPx converts H2 O2 to H2 O and GST catalyzes the conjugation of several electrophilic substrates to the thiol group of GSH. MDA is an important indicator of LPO which is primarily oxidation product of peroxidized polyunsaturated fatty acids (Demir et al., 2011). In this study we show inhibition of SOD, CAT, GPx and GST activities and increasing in MDA levels in MC and LN treated rats. It has been proposed that the enhancement of LPO by MC and LN may be due to consequence of inhibition of antioxidant enzymes. Antioxidant enzyme activity changes reported in the present study may be due to the production of ROS. Also, previous studies have shown that lead and mercury can change antioxidant activities of enzymes by inhibiting functional sulfhydryl (SH) groups, because it has high
), glomerular lobulation (
). (C) Kidney sections of LN rats: tubular degeneration
affinity for SH groups in these enzymes (Janicka et al., 2015; Rainio et al., 2015). We show that these changes were greater MC than LN. It may be due to LD50 value of MC which is more toxic than LN. It is known that exposure to toxic metals like mercury may induce apoptosis and necrosis in different cell types (Vergilio et al., 2015). Furthermore, mercury causes severe alterations in the tissues of experimental animals (Kalender et al., 2013). Histopathological studies have been widely used as biomarkers for the toxicological investigations and also heavy metal toxicities (Apaydin et al., 2015). In this study, we observed various histopathological changes in kidney tissues such as tubular degeneration. These pathological changes in LN, MC and LN plus MC treated groups may be due to increased LPO and ROS production which in turn oxidative stress. In addition, light microscopic findings support the result of the biochemical assays.
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It is reported that MC and LN are very harmful nephrotoxic pollutant and has been increased plasma urea concentration (Merzoug et al., 2009). Urea, uric acid and creatinine are waste products of metabolism that have to be excreated by the kidney (Tootian et al., 2012). So they are the most sensitive biochemical markers employed in the diagnosis of renal damage. El-Demerdash et al. (2004) reported that elevated blood urea is known to be correlated with an increased protein catabolism in mammals and/or the conversion of ammonia to urea as a result of increased synthesis of arginase enzyme involved in urea production. Also, increasing in plasma urea, uric acid and creatinine concentrations in the present experiment may be due to kidney dysfunction as suggested by pathological changes and oxidative stress. Similarly, investigators indicated that the significant elevation in urea and creatinine levels is closely related to the impairment of renal function. Also plasma creatinine increases in renal diseases gave prognostic significance than those of other nitrogenous substances (Amin et al., 2010). In conclusion, the present study demonstrated that low doses of MC and LN exposure induced the nephrotic oxidative stress response characterized by the histopathological damage and alterations of serum biochemical parameters. Mercury chloride has more effective toxicity than lead nitrate. Despite the treatment of LD50 dose of lead and mercury, mercury caused more harmful effects on kidneys. References Aebi, H., 1984. Catalase in vitro. Methods Enzymol. 105, 121–126. Alissa, E., Ferns, G., 2011. Heavy metal poisoning and cardiovascular disease. J. Toxicol. 2011, 1–21. Amamou, F., Nemmiche, S., Meziane, R., Didi, A., 2015. Protective effect of olive oil and colocynth oil against cadmium-induced oxidative stress in the liver of wistar rats. Food Chem. Toxicol. 78, 177–184. Amin, K.A., Hameid, H.A., Elsstar, A.H., 2010. Effect of food azo dyes tartrazine and carmoisine on biochemical parameters related to renal, hepatic function and oxidative stress biomarkers in young male rats. Food Chem. Toxicol. 48, 2994–2999. Apaydın, F.G., Kalender, S., Demir, F., Bas, H., 2014. Effects of sodium selenite supplementation on lead nitrate-induced oxidative stress in lung tissue of diabetic and non-diabetic rats. GU. J. Sci. 27 (2), 847–853. Apaydin, F.G., Kalender, S., Bas, H., Demir, F., Kalender, Y., 2015. Lead nitrate induced testicular toxicity in diabetic and non-diabetic rats:protective role of sodium selenite. Braz. Arch. Biol. Technol. 58, 68–74. Aslanturk, A., Uzunhisarcikli, M., Kalender, S., Demir, F., 2014. Sodium selenite and vitamin E in preventing mercuric chloride induced renal toxicity in rats. Food Chem. Toxicol. 70, 185–190. Dai, S., Yin, Z., Yuan, G., Lu, H., Jia, R., Xu, S., Li, L., Shu, Y., Liang, X., He, C., Lu, C., Zhan, W., 2013. Quantification of metallothionein on the liver and kidney of rats by subchronic lead and cadmium in combination. Environ. Toxicol. Pharmacol. 36, 1207–1216. Demir, F., Uzun, F.G., Durak, D., Kalender, Y., 2011. Subacute chlorpyrifos-induced oxidative stress in rat erythrocytes and the protective effects of catechin and quercetin. Pestic. Biochem. Physiol. 99, 77–81. Durak, D., Kalender, S., Uzun, F.G., Demir, F., Kalender, Y., 2010. Mercury chlorideinduced oxidative stress and the protective effect of vitamins C and E in human erythrocytes in vitro. Afr. J. Biotechnol. 9 (4), 488–495. El-Demerdash, F., Yousef, M.I., Kedwany, F.S., Bghdadi, H.H., 2004. Cadmium-induced changes in lipid peroxidation, blood hematology, biochemical parameters and semen quality of male rats: protective role of vitamin E and b-carotene. Food Chem. Toxicol. 42, 1563–1571. Garcia-Nino, W.R., Pedraza-Chaverri, J., 2014. Protective effect of curcumin against heavy metals-induced liver damage. Food Chem. Toxicol. 69, 182–201.
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Contents lists available at ScienceDirect
Environmental Toxicology and Pharmacology journal homepage: www.elsevier.com/locate/etap
Subacute effects of low dose lead nitrate and mercury chloride exposure on kidney of rats Fatma Gökc¸e Apaydın a,∗ , Hatice Bas¸ b , Suna Kalender c , Yusuf Kalender a a b c
Gazi University, Faculty of Science, Department of Biology, Ankara 06500, Turkey Bozok University, Faculty of Arts and Science, Department of Biology, Yozgat 66100, Turkey Gazi University, Faculty of Gazi Education, Department of Science, Ankara 06500, Turkey
a r t i c l e
i n f o
Article history: Received 14 September 2015 Received in revised form 30 November 2015 Accepted 13 December 2015 Available online 17 December 2015 Keywords: Lead nitrate Mercury chloride Oxidative stress Biochemical parameters Histopathology Kidney
a b s t r a c t Lead nitrate and mercury chloride are the most common heavy metal pollutants. In the present study, the effects of lead and mercury induced nephrotoxicity were studied in Wistar rats. Lead nitrate (LN, 45 mg/kg b.w/day) and mercury chloride (MC, 0.02 mg/kg b.w/day) and their combination were administered orally for 28 days. Four groups of rats were used in the study: control, LN, MC and LN plus MC groups. Serum biochemical parameters, lipid peroxidation, antioxidant enzymes and histopathological changes in kidney tissues were investigated in all treatment groups. LN and MC caused severe histopathological changes. It was shown that LN, MC and also co-treatment with LN and MC exposure induced significant increase in serum urea, uric acid and creatinine levels. There were also statistically significant changes in antioxidant enzyme activities (SOD, CAT, GPx and GST) and lipid peroxidation (MDA) in all groups except control group. In this study, we showed that MC caused more harmful effects than LN in rats. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Metals and their compounds found in living organisms have many important roles. Nevertheless, when their exposure is excessive, they may cause toxic effects (Janicka et al., 2015). Heavy metals are known to be toxic to organisms (Durak et al., 2010). They are a major class of suspected carcinogens. At low concentrations, heavy metals such as lead and mercury, can cause harmfull effects and tend to accumulate in the food chain (Garcia-Nino and Pedraza-Chaverri, 2014). These metals enter to the body by ingestion, inhalation or through the skin and their presence may induce serious toxicity in animals and humans (Alissa and Ferns, 2011). For many years lead has been recognized as a highly toxic metal to both humans and animals. It can accumulate in soils and sediments through aerial deposition from metal industries. Other main sources of lead have been leaded gasoline in cars and shotgun pellets (Rainio et al., 2015). After absorption, most Pb binds to proteins in erythrocytes and is distributed to soft tissues and bone (GarciaNino and Pedraza-Chaverri, 2014). In the case of mercury, the highest exposition occurs through vapors from amalgam dental fillings and diet, based on seafood and fish which deliver high amounts
∗ Corresponding author. Tel.: +90 312 202 1205; fax: +90 312 2122279. E-mail address: [email protected] (F.G. Apaydın). http://dx.doi.org/10.1016/j.etap.2015.12.003 1382-6689/© 2015 Elsevier B.V. All rights reserved.
of methyl mercury (Janicka et al., 2015). Lead has three main biochemical properties that support to its toxic effects. First, it has high affinity to SH groups and inhibits SH dependent enzymes which are essential for biological processes. Second, lead acts in a manner similar to calcium and competitively inhibits actions of calcium in substantial areas such as mitochondrial oxidative phosphorylation and intracellular messenger system. Third, lead can affect the transcription of DNA by interaction with nucleic acid binding proteins with potential consequences for gene regulation (Gordon et al., 2002). Mercury is a highly toxic metal that can exert multiple adverse effects (Aslanturk et al., 2014). The mercuric ion produces systemic toxicity at much lower concentrations through inhibition of many enzymes, especially those containing the SH group. It can induce suppress humoral and cellular immunity and increase tumor frequency (Yole et al., 2007). Mercury may also induce autoimmune disease in genetically susceptible animals (Warfvinge et al., 1995). Heavy metals are persistent and widespread pollutants that affect the structure and function of several organs by generating oxidative stress (Garcia-Nino and Pedraza-Chaverri, 2014). They generate reactive oxygen species (ROS) (Sarkar et al., 2014). These species cause oxidative injury via different mechanisms. ROS may induce cell injury or death. Extreme production of ROS causes alterations in subcellular structures. These alterations are modifications of proteins and DNA, lipid peroxidation (LPO) of polyunsaturated
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Fig. 1. Schematic overview for design of the experiment.
fatty acids and changes of cellular antioxidant system. Malondialdehyde (MDA) is the end product of LPO, and thus increased MDA content is an important indicator of LPO (Uzun and Kalender, 2013; Kalender et al., 2013). Cells may stimulate antioxidant and detoxification responses to counter heavy metal damages. The involvement of antioxidant enzymes such as glutathione peroxidase (GPx), glutathione-S-transferase (GST), superoxide dismutase (SOD) and catalase (CAT) play a considerable mission in protecting cells from oxidative stress (Sarkar et al., 2014). Previous studies, many experimental works reported that heavy metals can cause histopathological alterations in many tissues (Dai et al., 2013; Yuan et al., 2014). Heavy metals have toxic effects to renal cells of mammalian. The relationship between metals and toxicity of the kidney has been demonstrated in previous experiments (Renugadevi and Prabu, 2009). Kidneys are major organs which maintain the internal environment of the body. They are sensitive to injury by drugs and environmental chemicals. Metals can cause renal injury. Nephrotoxicity of metals was described by Matos et al. (2009) and Liu et al. (2010). The purpose of the present study was to investigate the biochemical and pathological effectiveness of low dose LN and MC in rats and also to evaluate value of MDA, serum urea, uric acid and creatinine levels, the activities of SOD, CAT, GPx and GST, in the kidneys. Becasue of kidneys are primary target organ, in which MC and LN are intensively accumulated following chronic exposure (WHO, 1991).
Gazi University Laboratory Animals Growing and Experimental Research Center. They were allowed to get into the habit of new situation for 10 days. The animals were housed at 18–22 ◦ C and they were given water and food ad libitum, while a 12-h on/12-h off light cycle was maintained. The protocol was approved by the Gazi University Animal Experiments Local Ethics Committee (Protocol no: G.Ü.ET–13.011) and experiments were performed accordancing to the international guidelines for care and use of laboratory animals. Animals were randomly divided into 4 groups as follows (n = 6 for each group). 1. Control group (1 ml/kg b.w distilled water) 2. Animals exposed to lead nitrate (45 mg/kg b.w, 1/50 LD50, LN) 3. Animals exposed to mercury chloride (0.02 mg/kg bw., 1/50 LD50, MC) 4. Animals exposed to lead nitrate + mercury chloride (45 mg/kg b.w LN + 0.02 mg/kg b.w MC) Distilled water, LN and MC were exposed to rats orally via gavage. At the end of the 28 days, the rats were dissected using ketamin + xylazin than the kidneys were removed for investigations about MDA levels, antioxidant enzyme activities and light microscopic examinations. Blood samples of the animals were taken from the heart and collected into sterile tubes for analysing serum urea, uric acid and creatinine levels. Fig. 1 shows a schematic diagram for he design of the experiment. 2.2. Test chemicals
2. Materials and methods 2.1. Animals and studied groups Twenty four adult albino Wistar rats (300–320 g, 90 days old) were used throughout the study. They were obtained from the
LN, MC and all the other chemicals were obtained from Sigma Aldrich. LN and MC were dissolved in distilled water (Yole et al., 2007; Sharma et al., 2010). The chemicals were administered in the morning (between 09:00 and 10:00) to non-fasted rats. During 28 days, 1 ml/kg b.w (body weight) distilled water for control group,
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4 Week
500 400
a, b, c
a, b
a
SOD (nmol/mgprotein)
600
300 200 100 0 control
lead nitrate
mercury chloride
lead nitrate+mercury chloride
Fig. 2. Effects of subacute treatment of LN and MC on SOD activities in the kidney tissues of rats. Each bar represents mean ± SEM of six animals in each group. Significance at P < 0.05. (a) Comparison of control and other groups. (b) Comparison of lead nitrate group and other groups. (c) Comparison of mercury chloride group and other groups.
0.02 mg/kg b.w (1/50 LD50 ) MC (Yole et al., 2007) for MC treatment groups and 45 mg/kg b.w (1/50 LD50 ) LN (Sharma et al., 2010) for lead treatment groups were given to rats daily via gavage.
2.3. Biochemical analysis 2.3.1. MDA levels and antioxidant enzyme activities Kidneys were washed with sodium phosphate buffer (pH 7.2) then they were homogenized with Heidolph Silent Crusher M. The homogenates were centrifuged for 15 min at 4 ◦ C. Activities of antioxidant enzymes and MDA levels of kidneys were detected using a spectrophotometer (Shimadzu UV 1700, Kyoto, Japan). The protein concentration was determined by the method of Lowry et al. (1951). The activity of SOD was obtained by Marklund and Marklund’s study (1974) at 440 nm and the enzyme activity was expressed as nmol/mg protein. Renal CAT activity was estimated following the method of Aebi (1984) at 240 nm and the activity was defined as mol/mg protein. According to the study of Habig et al. (1974), activity of GST was determined at 340 nm and the data was given as nmol/mg protein. Activity of GPx was measured by Paglia and Valentine’s method (1987). At 340 nm, the reaction was monitored and value was stated as nmol/mg protein. MDA level of kidney was analysed at 532 nm by thiobarbituric acid method described by Ohkawa et al. (1979) and it was defined as nmol/mg protein.
2.3.2. Evaluation of serum urea, uric acid and creatinine levels Blood samples which were taken by heart were centrifuged at 3500 rpm then serum was separated. Urea, uric acid and creatinine levels of serum were determined using a spectrophotometricenzymatic kit (Thermo Trace-BECGMAN, Germany) and analyzed by an autoanalyzer (Bayer ope-RA).
2.4. Pathological evaluation Rats were dissected and the kidneys were removed and placed into the fixative bouin. After, kidney samples were dehydrated in ascending grades of ethanol and embedded in paraffin and paraffin block of the tissues were prepared. The specimens were cut in 4–6 m slices and were stained with Hematoxylin–Eosin (H&E). Then the tissues were evaluated under a light microscope (Olympus BX51, Tokyo, Japan) and photographed with a camera (Olympus E-330, Olympus Optical Co., Ltd., Japan). Ten slides were prepared from each kidney tissue.
2.5. Statistical analysis The data were expressed as mean ± SEM. The data of all groups were compared with each other. Groups were compared using one-way analysis of variance (ANOVA) test followed by Tukey. Significance was accepted at P < 0.05. 3. Results 3.1. Biochemical results 3.1.1. MDA levels and antioxidant enzyme activities Treatment with LN and MC, decreased the activities of SOD, CAT, GPx and GST in kidney tissues but MC showed more toxicity than LN except GST activity. In combination with LN and MC induced more damages than use of them alone (Figs. 2–5) (P < 0.05). Levels of MDA were determined in kidney tissues of rats. At the end of the experimental period, all of the treatment groups showed increasing of MDA level compared to control. We observed more increased in MC group than LN group. Treated with LN + MC caused more harmfull effects than use of them alone (Fig. 6) (P < 0.05). 3.1.2. Serum urea, uric acid and creatinine levels Treatment with LN and MC increased the levels of urea, uric acid and creatinine levels compared to control group. There is no important differences between MC, LN and MC + LN groups except urea levels (Table 1) (P < 0.05). 3.2. Histological results of kidney In our study histopathological examination of rat kidneys were investigated using light microscope. The histological examination of the kidney tissues of the control treated rats showed normal structure. Rats treated with LN alone exhibited tubular degeneration. MC treated group showed tubular dilatation and Table 1 Urea, uric acid and creatinine levels of control and experimental rats. Groups
Urea (mg/dL)
Control Lead nitrate Mercury chloride LN + MC
21.80 25.53 27.91 25.53
± ± ± ±
0.98 0.99a 1.69a , b 1.91a , c
Uric acid (mg/dL) 0.48 0.69 0.74 0.62
± ± ± ±
0.03 0.11a 0.07a 0.08a
Creatinine (mg/dL) 0.29 0.32 0.34 0.33
± ± ± ±
0.01 0.02a 0.02a 0.11a
Values are mean ± SEM of six rats in each group. Significance at P < 0.05. a Comparison of control and other groups. b Comparison of LN-treated group and other groups. c Comparison of MC-treated group and other group.
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4 Week
200 150 100
a, b, c
a, b
a
CAT (µmol/mgprotein)
250
50 0 control
lead nitrate
mercury chloride
lead nitrate+mercury chloride
Fig. 3. Effects of subacute treatment of LN and MC on CAT activities in the kidney tissues of rats. Each bar represents mean ± SEM of six animals in each group. Significance at P < 0.05. (a) Comparison of control and other groups. (b) Comparison of lead nitrate group and other groups. (c) Comparison of mercury chloride group and other groups.
4 Week
lead nitrate
mercury chloride
40 30
a, b, c
a, b
50
a
GPx (nmol/mgprotein)
60
20 10 0 control
lead nitrate+mercury chloride
Fig. 4. Effects of subacute treatment of LN and MC on GPx activities in the kidney tissues of rats. Each bar represents mean ± SEM of six animals in each group. Significance at P < 0.05. (a) Comparison of control and other groups. (b) Comparison of lead nitrate group and other groups. (c) Comparison of mercury chloride group and other groups.
glomerular lobulation. In LN + MC treated rats tubular degeneration was shown, too. There are no significant differences among LN, MC and LN plus MC groups (Fig. 7A–D). 4. Discussion Many toxicological studies demonstrated that environmental heavy metals are very harmful to humans and wildlife (Nasiadek
et al., 2014). Previous studies have already reported that the heavy metals cause different organ damages like liver in rats under different experimental conditions (Apaydin et al., 2015; Liu et al., 2015). Mercury chloride and lead nitrate can easily cross the brain-blood barrier and placenta (WHO, 1991). Many authors reported that mercury and lead cause oxidative stress and stimulate ROS (Kalender et al., 2013, 2015). The reaction of these ROS with cellular biomolecules result in LPO and also
4 Week
35 a
30
a
25
a, b, c
GST (nmol/mgprotein)
40
20 15 10 5 0 control
lead nitrate
mercury chloride
lead nitrate+mercury chloride
Fig. 5. Effects of subacute treatment of LN and MC on GST activities in the kidney tissues of rats. Each bar represents mean ± SEM of six animals in each group. Significance at P < 0.05. (a) Comparison of control and other groups. (b) Comparison of lead nitrate group and other groups. (c) Comparison of mercury chloride group and other groups.
4 Week
a, b
25 20
a
MDA (nmol/mgprotein)
30
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a, b, c
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15 10 5 0 control
lead nitrate
mercury chloride
lead nitrate+mercury chloride
Fig. 6. Effects of subacute treatment of LN and MC on MDA levels in the kidney tissues of rats. Each bar represents mean ± SEM of six animals in each group. Significance at P < 0.05. (a) Comparison of control and other groups. (b) Comparison of lead nitrate group and other groups. (c) Comparison of mercury chloride group and other groups.
Fig. 7. (A) Kidney sections of control rats, (B) kidney sections of MC rats: tubular dilatation ( ( ). (D) Kidney sections of LN plus MC rats: Tubular degeneration ( ) X200, H&E.
membrane damage (Amamou et al., 2015). Antioxidants have been shown to scavenge reactive oxygen species (ROS) (Apaydın et al., 2014; Kalender et al., 2014). CAT activity is often correlated to SOD activity. SOD and CAT act like the first line of defence system. GPx converts H2 O2 to H2 O and GST catalyzes the conjugation of several electrophilic substrates to the thiol group of GSH. MDA is an important indicator of LPO which is primarily oxidation product of peroxidized polyunsaturated fatty acids (Demir et al., 2011). In this study we show inhibition of SOD, CAT, GPx and GST activities and increasing in MDA levels in MC and LN treated rats. It has been proposed that the enhancement of LPO by MC and LN may be due to consequence of inhibition of antioxidant enzymes. Antioxidant enzyme activity changes reported in the present study may be due to the production of ROS. Also, previous studies have shown that lead and mercury can change antioxidant activities of enzymes by inhibiting functional sulfhydryl (SH) groups, because it has high
), glomerular lobulation (
). (C) Kidney sections of LN rats: tubular degeneration
affinity for SH groups in these enzymes (Janicka et al., 2015; Rainio et al., 2015). We show that these changes were greater MC than LN. It may be due to LD50 value of MC which is more toxic than LN. It is known that exposure to toxic metals like mercury may induce apoptosis and necrosis in different cell types (Vergilio et al., 2015). Furthermore, mercury causes severe alterations in the tissues of experimental animals (Kalender et al., 2013). Histopathological studies have been widely used as biomarkers for the toxicological investigations and also heavy metal toxicities (Apaydin et al., 2015). In this study, we observed various histopathological changes in kidney tissues such as tubular degeneration. These pathological changes in LN, MC and LN plus MC treated groups may be due to increased LPO and ROS production which in turn oxidative stress. In addition, light microscopic findings support the result of the biochemical assays.
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It is reported that MC and LN are very harmful nephrotoxic pollutant and has been increased plasma urea concentration (Merzoug et al., 2009). Urea, uric acid and creatinine are waste products of metabolism that have to be excreated by the kidney (Tootian et al., 2012). So they are the most sensitive biochemical markers employed in the diagnosis of renal damage. El-Demerdash et al. (2004) reported that elevated blood urea is known to be correlated with an increased protein catabolism in mammals and/or the conversion of ammonia to urea as a result of increased synthesis of arginase enzyme involved in urea production. Also, increasing in plasma urea, uric acid and creatinine concentrations in the present experiment may be due to kidney dysfunction as suggested by pathological changes and oxidative stress. Similarly, investigators indicated that the significant elevation in urea and creatinine levels is closely related to the impairment of renal function. Also plasma creatinine increases in renal diseases gave prognostic significance than those of other nitrogenous substances (Amin et al., 2010). In conclusion, the present study demonstrated that low doses of MC and LN exposure induced the nephrotic oxidative stress response characterized by the histopathological damage and alterations of serum biochemical parameters. Mercury chloride has more effective toxicity than lead nitrate. Despite the treatment of LD50 dose of lead and mercury, mercury caused more harmful effects on kidneys. References Aebi, H., 1984. Catalase in vitro. Methods Enzymol. 105, 121–126. Alissa, E., Ferns, G., 2011. Heavy metal poisoning and cardiovascular disease. J. Toxicol. 2011, 1–21. Amamou, F., Nemmiche, S., Meziane, R., Didi, A., 2015. Protective effect of olive oil and colocynth oil against cadmium-induced oxidative stress in the liver of wistar rats. Food Chem. Toxicol. 78, 177–184. Amin, K.A., Hameid, H.A., Elsstar, A.H., 2010. Effect of food azo dyes tartrazine and carmoisine on biochemical parameters related to renal, hepatic function and oxidative stress biomarkers in young male rats. Food Chem. Toxicol. 48, 2994–2999. Apaydın, F.G., Kalender, S., Demir, F., Bas, H., 2014. Effects of sodium selenite supplementation on lead nitrate-induced oxidative stress in lung tissue of diabetic and non-diabetic rats. GU. J. Sci. 27 (2), 847–853. Apaydin, F.G., Kalender, S., Bas, H., Demir, F., Kalender, Y., 2015. Lead nitrate induced testicular toxicity in diabetic and non-diabetic rats:protective role of sodium selenite. Braz. Arch. Biol. Technol. 58, 68–74. Aslanturk, A., Uzunhisarcikli, M., Kalender, S., Demir, F., 2014. Sodium selenite and vitamin E in preventing mercuric chloride induced renal toxicity in rats. Food Chem. Toxicol. 70, 185–190. Dai, S., Yin, Z., Yuan, G., Lu, H., Jia, R., Xu, S., Li, L., Shu, Y., Liang, X., He, C., Lu, C., Zhan, W., 2013. Quantification of metallothionein on the liver and kidney of rats by subchronic lead and cadmium in combination. Environ. Toxicol. Pharmacol. 36, 1207–1216. Demir, F., Uzun, F.G., Durak, D., Kalender, Y., 2011. Subacute chlorpyrifos-induced oxidative stress in rat erythrocytes and the protective effects of catechin and quercetin. Pestic. Biochem. Physiol. 99, 77–81. Durak, D., Kalender, S., Uzun, F.G., Demir, F., Kalender, Y., 2010. Mercury chlorideinduced oxidative stress and the protective effect of vitamins C and E in human erythrocytes in vitro. Afr. J. Biotechnol. 9 (4), 488–495. El-Demerdash, F., Yousef, M.I., Kedwany, F.S., Bghdadi, H.H., 2004. Cadmium-induced changes in lipid peroxidation, blood hematology, biochemical parameters and semen quality of male rats: protective role of vitamin E and b-carotene. Food Chem. Toxicol. 42, 1563–1571. Garcia-Nino, W.R., Pedraza-Chaverri, J., 2014. Protective effect of curcumin against heavy metals-induced liver damage. Food Chem. Toxicol. 69, 182–201.
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