Chemosphere 139 (2015) 365–371
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Growth performance and oxidative damage in kidney induced by oral administration of Cr(III) in chicken Yanhan Liu, Cun Liu, Jia Cheng, Wentao Fan, Xiao Zhang, Jianzhu Liu ⇑ College of Veterinary Medicine, Research Center for Animal Disease Control Engineering Shandong Province, Shandong Agricultural University, Tai’an 271018, China
h i g h l i g h t s Excessive additive Cr
3+
for long time could cause related toxicity in chicken. given orally via water induced dose- and time-dependent toxicity in kidney. 3+ Cr toxicity could lead to nephritic oxidative damages and pathological lesions. 3+ Long-term Cr administration declined the growth performance in chicken. 3+
Cr
a r t i c l e
i n f o
Article history: Received 11 March 2015 Received in revised form 23 June 2015 Accepted 5 July 2015 Available online 24 July 2015 Keywords: Cr3+ Oral Oxidative damage Kidney Chicken
a b s t r a c t This study aimed to evaluate the effects of adding chromic chloride (CrCl3) in the drinking water of chickens. Hyland brown male chickens were randomly divided into four groups. Three groups orally received 1/2 LD50, 1/4 LD50, and 1/8 LD50 CrCl3 mg kg 1 body weight daily for 42 d. The fourth group was treated with water. The chickens were sacrificed at 14, 28, and 42 d post-treatment. The renal injury was examined through histological analysis, and kidney mass was determined. The effects on growth performance were assessed by measuring the weight of the body, chest muscles, and leg muscles. Oxidative damage was evaluated by determining the antioxidant defense levels in kidney homogenates. The body weight and the weight of tissues gained time-dependently, but significantly decreased compared with those in the control group (P < 0.05) at the same exposure time. Administering Cr3+ significantly increased the levels of malondialdehyde, glutathione, and hydrogen peroxide in the kidney compared with those in the control groups. Whereas, administering Cr3+ reduced the activities of superoxide dismutase, catalase, glutathione peroxidase, and total an-tioxidant capacity compared with those in the control group (P < 0.05) in a dose- and time-dependent manner. In conclusion, oral administration of CrCl3 decreases the growth performance of chickens, leads to the pathological lesions and affects nephritic antioxidant capacity in the kidney dose- and time-dependently. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction Chromium, a heavy transition metal, exists in different oxidation states; trivalent (Cr3+) and hexavalent (Cr6+) are the most common and stable forms of chromium (Zhang et al., 2008). A variety of organic chromium products have been used in feed, food and pharmaceutical industries. In a number of previous studies, chromium was reported to be associated with fargoing toxic effects including nephrotoxicity, hepatotoxicity, oxidative stress (Jin et al., 2015). Many researchers have suggested that Cr6+ is more toxic than Cr3+; Cr6+ can easily transfer across cellular membranes through non-specific anion carriers, whereas Cr3+ is poorly ⇑ Corresponding author. E-mail addresses: [email protected], [email protected] (J. Liu). http://dx.doi.org/10.1016/j.chemosphere.2015.07.032 0045-6535/Ó 2015 Elsevier Ltd. All rights reserved.
transported (Suwalsky et al., 2008; Lushchak et al., 2009b). After entering into the cell, Cr6+ is reduced to Cr3+ by cellular reductants to produce toxicity; hence, the toxicity of chromium on organisms rely on its form (Kumar et al., 2010; Das et al., 2015). Mammalian tissues have variable chromium contents, and its levels in human are affected by diet, age, and endocrine disorders (Real et al., 2008). Recently, the status of trivalent chromium as an essential element in humans and other animals has been challenged (Deng et al., 2015); however, Cr3+ is still an important trace element often added to livestock feed to improve animal production, growth, and meat quality (Debski et al., 2004). In low concentration, trivalent chromium can promote the growth and development of animals, thereby improving the quality of their meat (Piva et al., 2003). This is the primary reason why Cr3+ is added into animal feed. Nonetheless, in the actual process of adding Cr3+ into the feeds,
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people cannot control the concentration of trivalent chromium they put in, adversely causing such an element to be poisonous and dangerous for chicken. Renal damages can occur when animals are exposed or have ingested excessive trivalent chromium (Dayan and Paine, 2001). Therefore, the nutritional function of Cr3+ is still controversial. Poisoning could disrupt the physiological function of the body, thus generating free radicals and eventually results in lipid peroxidation (Liu et al., 2013). Sub-chronic chromium poisoning could damage the glomerulus in mouse kidney (Matos et al., 2009). Activities of antioxidant enzymes, such as superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX), gradually decrease with prolonged exposure to Cr(III), thus increasing the renal lipid peroxidation and damaging the kidney (Lushchak et al., 2009a). Animal experiments have confirmed that the absence of chromium could cause abnormal glucose tolerance, growth retardation, and hypercholesterolemia (Dallago et al., 2011; Tang et al., 2015). The relationship between chromium and the activities of antioxidant enzymes has been reported in research on mice. Chromium could significantly improve the activities of GSH-PX, catalase (CAT), and SOD in diabetic mice, thus enhancing their antioxidant capacities (Sundaram et al., 2013). Moreover, chromium could significantly increase the expression of SOD in the liver, confirming its ability to remove free radicals in mice (Wang et al., 2006). Food and water are the main conduits for chromium ingestion in general population. CrCl3, chromium tripicolinate, chromium nicotinate, chromium yeast, and chromium propionate have been used as supplements to livestock or poultry diets (Domínguez-Vara et al., 2009). However, the effects of adding excess CrCl3 on the growth performance and antioxidant capacity of livestock or poultry have not been reported. The kidney has the highest chromium concentration in the bodies of livestock and poultry (Mantovani et al., 2009). However, the toxicity of excess Cr3+ in the chicken kidney has not been reported. Therefore, the damages induced by Cr3+ via the oral route should be elucidated to evaluate its toxicity. This study was designed to investigate the toxicity of excess CrCl3, which was orally administered to chickens via their drinking water, focusing on growth performance, histological analysis, and oxidative damage. Enzymatic [SOD, total anti-oxidation (T-AOC), CAT, and GSH-PX] and non-enzymatic [malondialdehyde (MDA), GSH, and hydrogen peroxide (H2O2)] antioxidant defense capacity were evaluated in the renal homogenates. 2. Materials and methods 2.1. Experimental materials CrCl36H2O was purchased from Putian Tedia Company Inc. (Tai’an, China). CrCl3 (purity P 99.8%) was prepared using dechlorinated tap water. All other reagents used were of analytical grade. Commercial assay kits for T-AOC, SOD, GSH-PX, CAT, GSH, H2O2, and MDA were provided by Jiancheng Biotechnology Research Institute (Nanjing, China).
was 5013 mg kg 1 body weight for male chicken. A total of 72 Hyland brown male chickens (1 d old) were obtained from the Animal Center of Shandong Agriculture University (Shandong, China). The chickens were randomly assigned into four groups with 18 chickens in each group. The chickens were housed in four stainless steel cages based on their groups and acclimatized for 14 d prior to experiments. Cr3+ was orally administered via drinking water to the first three groups for 42 d with the following doses: 1/2 LD50 (high-dose group), 1/4 LD50 (middle-dose group), and 1/8 LD50 (low-dose group) mg kg 1 body weight daily. The fourth group was treated with water and designated as the control group. All chickens were provided with free access to standard diet. The chickens were housed in an air-conditioned room. The room temperature was regulated according to the chickens’ body temperature. After 14, 28 and 42 d, all chickens were fasted overnight and then euthanized. Six chickens from each group were weighed. The kidneys were immediately excised, rinsed with ice-cold 0.9% NaCl solution, dried using a filter paper, and then weighed. Chest and leg muscles were separated using the same method. The muscles were washed with deionized water and weighed to measure growth performance. 2.3. Histological analysis After necropsy, the kidneys were rapidly removed and rinsed with ice-cold saline. Small kidney pieces were freshly prepared and fixed with 10% neutral-buffered formaldehyde for 24 h. The fixed kidneys were dehydrated using a graded series of ethanol, cleared using xylene, and then embedded in paraffin. The specimens were sliced into 4 lm thick sections and stained with hematoxylin and eosin using routine methods (Li et al., 2013). The stained sections were examined using the optical microscope (XSP-BM16C, Optical Instrument Factory, Shanghai, China). 2.4. Preparation of homogenates The kidney fractions (300 mg) were homogenized using a glass Teflon homogenizer with 2700 mL of cold 0.9% NaCl solution (1:9). The homogenates were centrifuged at 3500–4000g for 10 min at 4 °C. The supernatants were carefully removed from the pellet and used directly for antioxidant assays. 2.5. Protein measurement Protein content was determined using Coomassie brilliant blue G-250 method. 2.6. Assay of total antioxidant capacity (T-AOC) The kidney contains many antioxidants that could reduce Fe3+ to Fe2+. Fe2+ combined with phenanthroline substances to form stable complexes. Absorbance was determined at 520 nm to express total antioxidant activity using the Uv–vis spectrophotometer (UV-2600, Optical Instrument Factory, Shanghai, China). 2.7. Antioxidant enzyme activities
2.2. Animals and treatment All experiments were carried out in accordance with the ethical guidelines of the National Institutes of Health Guide for the Care and in compliance with the rules on animal protection of the Shandong Agriculture University (SDAUA-2014-013). Before beginning the present study, we performed acute toxicity experiments to determine the oral 50% lethal dose (LD50) of CrCl3 for male chicken according to the Karber method (Bittenbender and Howell Jr., 1974) and confirmed that the LD50
2.7.1. Superoxide dismutase (SOD) Superoxide anion radicals oxidized hydroxylaminen to nitrite; these radicals were produced by xanthine and xanthine oxidase system. Nitrite produced a purple color under the action of chromogenic agent. Absorbance was determined with the Uv–vis spectrophotometer (UV-2600, Optical Instrument Factory, Shanghai, China) at 550 nm. One unit of SOD was defined as the amount of enzyme that exhibited 50% inhibition in 1 mg of protein tissues. SOD activity was expressed as U/mg protein.
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2.7.2. Catalase (CAT) CAT decomposed H2O2; the reaction was rapidly terminated by adding ammonium molybdate. The remaining H2O2 reacted with ammonium molybdate and produced a pale yellow clathrate. The production was determined at 405 nm to express CAT activity. CAT activity was expressed as U/mg protein. 2.7.3. Glutathione peroxidase (GSH-PX) GSH-PX catalyzed a portion of GSH to oxidize GSH, as well as H2O2 to H2O. The remaining GSH in the reaction reacted with dithiobis nitrobenzoic acid and produced a stable yellow substance. Absorbance was determined at 412 nm. One unit of GSH-PX activity was defined as the amount of the enzyme that lowered the concentration of GSH by 1 lmol/L per min at 37 °C per 1 mg of protein tissues. 2.8. Non-enzymatic antioxidant contents 2.8.1. Glutathione (GSH) GSH contains a mercapto group; the reaction between mercapto mix and dithiobis nitrobenzoic acid generated a yellow compound. The compound could be quantitatively determined using the Uv– vis spectrophotometer (UV-2600, Optical Instrument Factory, Shanghai, China) at 420 nm. 2.8.2. Hydrogen peroxide (H2O2) H2O2 reacted with molybdate to generate a clathrate. The amount of clathrate was determined at 405 nm using commercial kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). H2O2 activity was expressed as mmol/g protein. 2.8.3. Malondialdehyde (MDA) Lipid peroxidation in the kidney homogenates was determined using commercial kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer’s instructions. MDA was condensed with thiobarbituric acid and heated in a boiling water bath for 45 min. The reaction produced red substances, and their absorbance was determined at 535 nm. 2.9. Statistical analysis Data were initially encoded in Excel software and then analyzed using analysis of variance with SPSS 18.0 software (SPSS Inc, Chicago IL, USA) and LSD multiple comparison. P < 0.05 was considered statistically significant. Data were expressed as mean ± S.D. deviation. 3. Results 3.1. Effects of CrCl3 on the weight of the body, kidneys, chest muscles, and leg muscles The effect of CrCl3 on the body weight of chickens is shown in Fig. 1. The body weight time-dependently increased, however, the gain of body weight in the high-dose group was evidently slower (P < 0.05) than those of the middle- and low-dose groups. Moreover, the body weight in the three Cr-treated groups was significantly lower (P < 0.05) than that in the control group at the same exposure time. To investigate the effect of CrCl3 on growth performance, the absolute weight of the two kidneys, chest muscle and leg muscle from the four groups were determined at 14, 28, and 42 d (see Table. 1). The mean absolute weights of the kidneys and chest muscles were not significantly different (P > 0.05) among the groups after oral intake of CrCl3 at 14 d. At 28 d, the mean absolute
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weight in the high-dose group significantly decreased (P < 0.05). At 42 d, the mean absolute weights in the three Cr-treated groups significantly decreased (P < 0.05). The weight of leg muscles was significantly lower (P < 0.05) than that in the control group. The mean weights decreased with increased CrCl3 concentration and prolonged treatment, except in the middle group at 14 d. These results indicated that long-term administration of CrCl3 had negative effects on the growth performance of chicken. 3.2. Histopathological changes The histopathological lesions in kidney after exposure to CrCl3 were observed under optical microscope. The control and low-dose groups showed no toxic lesions at 14 d (pictures not shown). However, the other experimental groups presented similar damages, including slight swelling, nuclear condensation, and dissolution in the tubular epithelial cells. A part of the renal tubules showed severely swelling or enlarged lumen, and some membrane of epithelial cells dissolved or disappeared (Fig. 2B). In the highand middle-dose groups, the medulla units were hemorrhage, and renal tubule showed severe atrophy. Moreover, the structure of glomerulus was degenerative; and the renal capsule lumen significantly largened (Fig. 2A). Yellow substances were found in the renal distal tubules. These substances were speculated as renal granular casts, which were produced by the tubule damage caused by heavy metal poisoning. These histopathological changes indicated that CrCl3 induced toxic lesions in the kidney. 3.3. Changes in T-AOC activity The effects of CrCl3 on the T-AOC activity in the kidney are shown in Fig. 3. The T-AOC activities in kidney tissues significantly decreased (P < 0.05) in the high-dose groups compared with those in the control group at 28 and 42 d. Moreover, the T-AOC activities in kidney tissues significantly decreased (P < 0.05) as the experiment progressed. 3.4. Changes in antioxidant enzyme activities of SOD, CAT and GSH-PX SOD activities were not significantly different among the four groups at 14 d. However, SOD activities significantly decreased (P < 0.05) with increased CrCl3 concentration and prolonged time at 28 and 42 d. In the high-dose group, SOD activity significantly decreased (P < 0.05) compared with that in the control group (Fig. 4A). The high-dose group presented the lowest CAT levels in the kidney, followed by the middle-dose group and then the low-dose group. Each experimental group had significantly lower (P < 0.05) CAT levels than the control group at 14, 28, and 42 d (Fig. 4B). GSH-PX activities of the three Cr-treated groups at 14 d were significantly higher than those at 28 and 42 d. The GSH-PX activity showed no significant differences between the experimental groups and the control groups at 14 and 28 d, however, at 42 d GSH-PX activities of the three Cr-treated groups significantly decreased (P < 0.05) compared with that in the control group. GSH-PX activities decreased with increased CrCl3 concentration and prolonged treatment (Fig. 4C). 3.5. Changes in non-enzymatic antioxidant contents of GSH, H2O2 and MDA GSH levels were not significantly different among the experimental and control groups at 14 d; at 28 d, only in the high-dose group GSH levels significantly (P < 0.05) increased than that in the control group. Moreover, GSH levels in the three experimental groups were higher than that in the control group at 42 d. The GSH
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Fig. 1. The effects of chromic chloride on body weight. At 14 d, only the high-dose group was significantly lower than the control group and at 28 d both the high-dose group and the middle group were significantly lower than the control group, however, at 42 d all the three groups were significantly lower than the control group. Each value represented the mean ± SD. The different letters indicate that there are significant differences (P < 0.05) between any two groups.
levels in the kidney significantly increased in the experimental groups with increased CrCl3 concentration and prolonged time (Fig. 5A). H2O2 content in the high-dose group was significantly higher (P < 0.05) than that in the control group at 14 and 28 d; however, no significant differences were observed at 42 d. H2O2 content decreased daily in the experimental groups compared with that in the control group, and the H2O2 content in the high-dose group significantly decreased (P < 0.05). However, H2O2 content in the middle- and low-dose groups was lower than that in the control group at 28 d (Fig. 5B). The MDA levels in the kidney from each experimental group were significantly higher (P < 0.05) than that from the control group. The MDA levels increased daily in all experimental groups compared with that in the control group; the MDA levels in the high-dose group significantly increased (P < 0.05). However, the MDA levels in the middle-dose group were lower than those in the low-dose group at 42 d (Fig. 5C). 4. Discussion Chromium is often added into animal diet, particularly in the livestock industry. However, using chromium as a nutritional supplement remains debatable because excessive Cr3+ may cause pathologic lesions, particularly in the kidney. Several researchers have suggested that adding chromium in feeds can promote the growth and production performance of broilers (Shrivastava
et al., 2003; Debski et al., 2004). In the present study, long-term treatment with CrCl3 added in drinking water did not promote the growth and development of chickens. The body weight decreased after long-term exposure to CrCl3. The experimental groups had significantly lower increase (P < 0.05) in body weight than that in the control group. Higher CrCl3 concentration and prolonged treatment resulted in lower weight. The weight of the chest and leg muscles in the experimental groups was significantly (P < 0.05) lower than that in the control group. Apparently, promoting the growth of chicken by supplementing chromium in drinking water has not been reported. The role of chromium in animal nutrition remains ambiguous, and its advantages as a supplement remain debatable. The present study investigated the toxicity of Cr3+ exposure via the oral drinking route at relatively long duration using a chicken model. Previous study showed that intracellular reduction of Cr6+ to Cr3+ induces overproduction of reactive oxygen species (ROS), which is an important characteristic of Cr6+-induced toxicity in the kidney (Stohs et al., 2001). The kidney, one of the major sites for chromium accumulation in the organism, becomes the target of chromium during long-term exposure. Changes in the kidney as a response to trivalent chromium toxicity have been reported in rats, dogs, and goldfish (Carciofi et al., 2007; Lushchak et al., 2009a; Staniek et al., 2010). Exposure to chromium eventually results in its accumulation and thus causes severe histological changes in the chicken kidney. The present study demonstrated the effects of supplementing CrCl3 in drinking water on its accumulation in the kidney and on the histological structure. The characteristics of the pathological changes caused by chromium in kidney is in accordance with the findings in previous studies on other animals; consuming water supplemented with chromium increases the MDA content and suppresses the antioxidant defense mechanisms in kidney tissues (Gupta and Ballal, 2015). In the present study, the weight of the kidney evidently decreased after poisoning compared with that in the control group, indicating that the kidney function was severely injured. The renal function and the concentration of enzymes and proteins may be altered, and kidney necrosis can occur in cases administered with high doses. Chromium is widely distributed in the body but preferentially localized in the kidney; moreover, Cr has varying degrees of toxicity. Oxidative damage and biofilm injury may be the possible causes of nephritic damage induced by chromium which were observed in in vivo and in vitro experiment systems (Scibior et al., 2006; Ahmad et al., 2012). Oral chromium supplementation in the form of CrCl3 did not promote the growth performance of chickens in the present study. Chromium functioned as a toxic agent, impairing the cellular functions and integrity in the kidney.
Table 1 The mass of the two kidneys, chest and leg muscle of the chicken. Time of treatment
Group
Kidney (g)
Chest (g)
Leg muscle (g)
14 d
High Middle Low Control
3.48 ± 0.106a 3.49 ± 0.080a 3.49 ± 0.202a 3.50 ± 0.057a
11.93 ± 0.851a 12.27 ± 0.742a 11.90 ± 0.258a 11.30 ± 0.341a
15.70 ± 0.361a 18.20 ± 0.586b 17.67 ± 0.636b 22.03 ± 1.877c
28 d
High Middle Low Control
4.15 ± 0.290a 4.77 ± 0.225b 4.97 ± 0.167b 5.31 ± 0.272bc
16.30 ± 0.839a 17.63 ± 0.384b 18.10 ± 0.265bc 19.57 ± 0.203c
26.70 ± 3.963a 33.53 ± 2.591b 31.83 ± 0.984b 43.63 ± 1.488c
42 d
High Middle Low Control
4.41 ± 0.178a 5.33 ± 0.172b 5.64 ± 0.035c 5.85 ± 0.088cd
22.63 ± 0.504a 24.87 ± 0.176b 28.23 ± 0.285c 31.57 ± 0.825d
42.80 ± 0.693a 52.63 ± 0.684b 58.07 ± 1.097c 74.77 ± 1.802d
The data are the mean ± S.D. of the results obtained in the six chickens composing of each experiment group (n = 6). The different letters indicate that there are significant differences (P < 0.05) between any two groups in same column.
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Fig. 2. Photomicrograph of the kidney from experimental group, magnification 200, n = 6. (A) Glomerular lesions showed the glomerular cystic space largened and a little lymphocyte infiltrated. (B) Renal tubular lesions showed necrosis of the tubular epithelial cell, pycnosis and disintegration of the nucleus, the cell membrane became incomplete or even broken, vacuoles degeneration of the tubular epithelial cell extensively and tube type can be seen in a few renal tubular lumen. (C and D) were the renal tubular and glomerular from the control group.
Fig. 3. Effects of chromic chloride on T-AOC activities in the kidney. The different letters indicate that there are significant differences (P < 0.05) between any two groups. Each value represented the mean ± SD.
The effects of using chromium as a feed additive in the livestock industry on the micro-renal injury should be further investigated. Generating and eliminating free radicals are in a state of dynamic balance under normal physiological conditions (Sun et al., 2013). However, aging or poisoning can disrupt the body metabolism, resulting in free radical accumulation and lipid peroxidation (Kapun et al., 2012). If the body could not remove the excess free radicals, the integrity of the cell membranes would be damaged, thus decreasing immunity and resistance (Yao et al., 2013). Oxidative stress has an important role in the toxicity mechanism of numerous xenobiotics. T-AOC represents the antioxidant capacity of the body’s defense system, which comprehensively indicates the functional status of the antioxidant system. GSH-PX is an important antioxidant enzyme in the body; this enzyme specifically catalyzes GSH to remove H2O2 and reduce the occurrence of lipid peroxidation, thus protecting the structure and the complete function of cell membranes. SOD, a heavy metallic
enzyme in the body, is an important component in enzymatic systems. Free radical scavenging in the body is related to SOD content. Moreover, CAT is a combination enzyme with ferriporphyrin as the prosthetic group. CAT in erythrocytes or tissues can directly decompose H2O2 under specific conditions, causing the gradual decrease in its concentration in tissue fluids and thus protecting the body against damages (Zhang et al., 2013). This study showed that heavy metal poisoning in the body significantly decreased (P < 0.05) the total antioxidant capacity of the kidney. Therefore, with increased CrCl3 concentration and prolonged treatment, the activities of SOD, GSH-PX, and CAT significantly decreased (P < 0.05), particularly in the high-dose group at 28 and 42 d. The reduced SOD, GSH-PX, and CAT activities indicated that poisoning could release and accumulate reactive oxygen species. The accumulated reactive oxygen species could damage the antioxidant enzymatic systems, thus decreasing the damages and functions of the kidney. MDA is the end-product of lipid peroxidation caused by oxygen free radicals and enhanced oxygen free radicals in the kidney (Bento et al., 2013). The decreased antioxidant effect increased the MDA content. A study found that MDA can strongly react with various components in the cell membrane (Esmaeilnejad et al., 2014). MDA content can reflect the oxygen free radical metabolism in the kidney, whereas lipid peroxidation indirectly indicates the oxidative damage in the kidney. In this study, the MDA content in the chicken kidney significantly increased (P < 0.05) with increased CrCl3 concentration and prolonged treatment. The MDA content in the high-dose group in each treatment period was significantly higher (P < 0.05) than that in the control group. MDA content increased as the activities of antioxidant enzymes decreased. GSH is the most abundant non-protein thiol in the cells and is a key cellular antioxidant (Dimri et al., 2014). When cells produced a small amount of H2O2, GSH catalyzes H2O2 to form H2O under the action of GSH-PX, which could be oxidized also to GSSG. CrCl3 poisoning decreased the activity of GSH-PX; thus,
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Fig. 4. Effects of antioxidant enzyme activities of SOD, CAT and GSH-PX. A: Effects of chromic chloride on the SOD activities in the kidney. B: Effects of chromic chloride on the CAT activities in the kidney. C: Effects of chromic chloride on the GSH-PX activities in the kidney. The different letters indicate that there are significant differences (P < 0.05) between any two groups. Each value represented the mean ± SD.
Fig. 5. Changes in non-antioxidant enzyme contents of GSH, H2O2 and MDA. A: Effects of chromic chloride on the GSH levels in the kidney. B: Effects of chromic chloride on H2O2 content in the kidney. C: Effects of chromic chloride on MDA content in the kidney. The different letters indicate that there are significant differences (P < 0.05) between any two groups. Each value represented the mean ± SD.
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GSH could not be converted into GSSG. GSH content significantly increased (P < 0.05) in the high-dose group, in which GSH-PX activity was significantly reduced (P < 0.05). H2O2 is a harmful byproduct of many normal metabolic processes that can prevent damages to cells and tissues. H2O2 can be rapidly converted to other less dangerous substances. Thus, GSH-PX and CAT are frequently used by cells to rapidly catalyze the decomposition of H2O2 into less-reactive gaseous oxygen and water molecules (Heidarpour et al., 2012). The decrease in GSH-PX and CAT resulted in high H2O2 levels in the kidney. This experiment showed that oral uptake of CrCl3 through drinking water could abnormally change the stable oxidation and antioxidant mechanism in kidney cells; hence, the activities of antioxidant enzymes decreased, whereas that of lipid peroxide increased. Cell damages were exacerbated and eventually led to the injury in the structure and function of the chicken kidney. 5. Conclusion Summarizing, our results confirmed that long-term treatment with CrCl3 had negative effects on the growth performance of chickens. Moreover, the activities of the total antioxidant capacity (T-AOC) and antioxidant enzymes (SOD, CAT, and GSH-PX) significantly decreased, whereas MDA, GSH, and H2O2 contents significantly increased, and eventually destroyed the antioxidant defense system in kidney tissues. These findings may provide a new insight into the possible mechanism for the toxicity of using chromium as a long-term feed additive in the poultry industry. Conflicts of Interest The authors declare that they have no conflicts of interest concerning this article. Acknowledgment The study was supported by the earmarked fund Shandong Modern Agricultural Technology & Industry System (No. SDAIT-13-011-04). References Ahmad, M.K., Naqshbandi, A., Fareed, M., Mahmood, R., 2012. Oral administration of a nephrotoxic dose of potassium bromate, a food additive, alters renal redox and metabolic status and inhibits brush border membrane enzymes in rats. Food Chem. 134, 980–985. Bento, D.B., de Souza, B., Steckert, A.V., Dias, R.O., Leffa, D.D., Moreno, S.E., Petronilho, F., de Andrade, V.M., Dal-Pizzol, F., Romao, P.R., 2013. Oxidative stress in mice treated with antileishmanial meglumine antimoniate. Res. Vet. Sci. 95, 1134–1141. Bittenbender, H., Howell Jr., G.S., 1974. Adaptation of the Spearman-Karber method for estimating the T50 of cold stressed flower buds. J. Amer. Soc. Hortic. Sci. 99, 187–190. Carciofi, A.C., Vasconcellos, R.S., de Oliveira, L.D., Brunetto, M.A., Valério, A.G., Bazolli, R.S., Carrilho, E.N.V.M., Prada, F., 2007. Chromic oxide as a digestibility marker for dogs-A comparison of methods of analysis. Anim. Feed. Sci. Technol. 134, 273–282. Dallago, B.S., McManus, C.M., Caldeira, D.F., Lopes, A.C., Paim, T.P., Franco, E., Borges, B.O., Teles, P.H., Correa, P.S., Louvandini, H., 2011. Performance and ruminal protozoa in lambs with chromium supplementation. Res. Vet. Sci. 90, 253–256. Das, J., Sarkar, A., Sil, P.C., 2015. Hexavalent chromium induces apoptosis in human liver (HepG2) cells via redox imbalance. Toxicol. Rep. 2, 600–608. Dayan, A., Paine, A., 2001. Mechanisms of chromium toxicity, carcinogenicity and allergenicity: review of the literature from 1985 to 2000. Hum. Exp. Toxicol. 20, 439–451. Debski, B., Zalewski, W., Gralak, M.A., Kosla, T., 2004. Chromium-yeast supplementation of chicken broilers in an industrial farming system. J. Trace Elem. Med Biol. 18, 47–51. Deng, G., Wu, K., Cruce, A.A., Bowman, M.K., Vincent, J.B., 2015. Binding of trivalent chromium to serum transferrin is sufficiently rapid to be physiologically relevant. J. Inorg. Biochem. 143, 48–55. Dimri, U., Bandyopadhyay, S., Singh, S.K., Ranjan, R., Mukherjee, R., Yatoo, M.I., Patra, P.H., De, U.K., Dar, A.A., 2014. Assay of alterations in oxidative stress markers in
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Growth performance and oxidative damage in kidney induced by oral administration of Cr(III) in chicken Yanhan Liu, Cun Liu, Jia Cheng, Wentao Fan, Xiao Zhang, Jianzhu Liu ⇑ College of Veterinary Medicine, Research Center for Animal Disease Control Engineering Shandong Province, Shandong Agricultural University, Tai’an 271018, China
h i g h l i g h t s Excessive additive Cr
3+
for long time could cause related toxicity in chicken. given orally via water induced dose- and time-dependent toxicity in kidney. 3+ Cr toxicity could lead to nephritic oxidative damages and pathological lesions. 3+ Long-term Cr administration declined the growth performance in chicken. 3+
Cr
a r t i c l e
i n f o
Article history: Received 11 March 2015 Received in revised form 23 June 2015 Accepted 5 July 2015 Available online 24 July 2015 Keywords: Cr3+ Oral Oxidative damage Kidney Chicken
a b s t r a c t This study aimed to evaluate the effects of adding chromic chloride (CrCl3) in the drinking water of chickens. Hyland brown male chickens were randomly divided into four groups. Three groups orally received 1/2 LD50, 1/4 LD50, and 1/8 LD50 CrCl3 mg kg 1 body weight daily for 42 d. The fourth group was treated with water. The chickens were sacrificed at 14, 28, and 42 d post-treatment. The renal injury was examined through histological analysis, and kidney mass was determined. The effects on growth performance were assessed by measuring the weight of the body, chest muscles, and leg muscles. Oxidative damage was evaluated by determining the antioxidant defense levels in kidney homogenates. The body weight and the weight of tissues gained time-dependently, but significantly decreased compared with those in the control group (P < 0.05) at the same exposure time. Administering Cr3+ significantly increased the levels of malondialdehyde, glutathione, and hydrogen peroxide in the kidney compared with those in the control groups. Whereas, administering Cr3+ reduced the activities of superoxide dismutase, catalase, glutathione peroxidase, and total an-tioxidant capacity compared with those in the control group (P < 0.05) in a dose- and time-dependent manner. In conclusion, oral administration of CrCl3 decreases the growth performance of chickens, leads to the pathological lesions and affects nephritic antioxidant capacity in the kidney dose- and time-dependently. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction Chromium, a heavy transition metal, exists in different oxidation states; trivalent (Cr3+) and hexavalent (Cr6+) are the most common and stable forms of chromium (Zhang et al., 2008). A variety of organic chromium products have been used in feed, food and pharmaceutical industries. In a number of previous studies, chromium was reported to be associated with fargoing toxic effects including nephrotoxicity, hepatotoxicity, oxidative stress (Jin et al., 2015). Many researchers have suggested that Cr6+ is more toxic than Cr3+; Cr6+ can easily transfer across cellular membranes through non-specific anion carriers, whereas Cr3+ is poorly ⇑ Corresponding author. E-mail addresses: [email protected], [email protected] (J. Liu). http://dx.doi.org/10.1016/j.chemosphere.2015.07.032 0045-6535/Ó 2015 Elsevier Ltd. All rights reserved.
transported (Suwalsky et al., 2008; Lushchak et al., 2009b). After entering into the cell, Cr6+ is reduced to Cr3+ by cellular reductants to produce toxicity; hence, the toxicity of chromium on organisms rely on its form (Kumar et al., 2010; Das et al., 2015). Mammalian tissues have variable chromium contents, and its levels in human are affected by diet, age, and endocrine disorders (Real et al., 2008). Recently, the status of trivalent chromium as an essential element in humans and other animals has been challenged (Deng et al., 2015); however, Cr3+ is still an important trace element often added to livestock feed to improve animal production, growth, and meat quality (Debski et al., 2004). In low concentration, trivalent chromium can promote the growth and development of animals, thereby improving the quality of their meat (Piva et al., 2003). This is the primary reason why Cr3+ is added into animal feed. Nonetheless, in the actual process of adding Cr3+ into the feeds,
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people cannot control the concentration of trivalent chromium they put in, adversely causing such an element to be poisonous and dangerous for chicken. Renal damages can occur when animals are exposed or have ingested excessive trivalent chromium (Dayan and Paine, 2001). Therefore, the nutritional function of Cr3+ is still controversial. Poisoning could disrupt the physiological function of the body, thus generating free radicals and eventually results in lipid peroxidation (Liu et al., 2013). Sub-chronic chromium poisoning could damage the glomerulus in mouse kidney (Matos et al., 2009). Activities of antioxidant enzymes, such as superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX), gradually decrease with prolonged exposure to Cr(III), thus increasing the renal lipid peroxidation and damaging the kidney (Lushchak et al., 2009a). Animal experiments have confirmed that the absence of chromium could cause abnormal glucose tolerance, growth retardation, and hypercholesterolemia (Dallago et al., 2011; Tang et al., 2015). The relationship between chromium and the activities of antioxidant enzymes has been reported in research on mice. Chromium could significantly improve the activities of GSH-PX, catalase (CAT), and SOD in diabetic mice, thus enhancing their antioxidant capacities (Sundaram et al., 2013). Moreover, chromium could significantly increase the expression of SOD in the liver, confirming its ability to remove free radicals in mice (Wang et al., 2006). Food and water are the main conduits for chromium ingestion in general population. CrCl3, chromium tripicolinate, chromium nicotinate, chromium yeast, and chromium propionate have been used as supplements to livestock or poultry diets (Domínguez-Vara et al., 2009). However, the effects of adding excess CrCl3 on the growth performance and antioxidant capacity of livestock or poultry have not been reported. The kidney has the highest chromium concentration in the bodies of livestock and poultry (Mantovani et al., 2009). However, the toxicity of excess Cr3+ in the chicken kidney has not been reported. Therefore, the damages induced by Cr3+ via the oral route should be elucidated to evaluate its toxicity. This study was designed to investigate the toxicity of excess CrCl3, which was orally administered to chickens via their drinking water, focusing on growth performance, histological analysis, and oxidative damage. Enzymatic [SOD, total anti-oxidation (T-AOC), CAT, and GSH-PX] and non-enzymatic [malondialdehyde (MDA), GSH, and hydrogen peroxide (H2O2)] antioxidant defense capacity were evaluated in the renal homogenates. 2. Materials and methods 2.1. Experimental materials CrCl36H2O was purchased from Putian Tedia Company Inc. (Tai’an, China). CrCl3 (purity P 99.8%) was prepared using dechlorinated tap water. All other reagents used were of analytical grade. Commercial assay kits for T-AOC, SOD, GSH-PX, CAT, GSH, H2O2, and MDA were provided by Jiancheng Biotechnology Research Institute (Nanjing, China).
was 5013 mg kg 1 body weight for male chicken. A total of 72 Hyland brown male chickens (1 d old) were obtained from the Animal Center of Shandong Agriculture University (Shandong, China). The chickens were randomly assigned into four groups with 18 chickens in each group. The chickens were housed in four stainless steel cages based on their groups and acclimatized for 14 d prior to experiments. Cr3+ was orally administered via drinking water to the first three groups for 42 d with the following doses: 1/2 LD50 (high-dose group), 1/4 LD50 (middle-dose group), and 1/8 LD50 (low-dose group) mg kg 1 body weight daily. The fourth group was treated with water and designated as the control group. All chickens were provided with free access to standard diet. The chickens were housed in an air-conditioned room. The room temperature was regulated according to the chickens’ body temperature. After 14, 28 and 42 d, all chickens were fasted overnight and then euthanized. Six chickens from each group were weighed. The kidneys were immediately excised, rinsed with ice-cold 0.9% NaCl solution, dried using a filter paper, and then weighed. Chest and leg muscles were separated using the same method. The muscles were washed with deionized water and weighed to measure growth performance. 2.3. Histological analysis After necropsy, the kidneys were rapidly removed and rinsed with ice-cold saline. Small kidney pieces were freshly prepared and fixed with 10% neutral-buffered formaldehyde for 24 h. The fixed kidneys were dehydrated using a graded series of ethanol, cleared using xylene, and then embedded in paraffin. The specimens were sliced into 4 lm thick sections and stained with hematoxylin and eosin using routine methods (Li et al., 2013). The stained sections were examined using the optical microscope (XSP-BM16C, Optical Instrument Factory, Shanghai, China). 2.4. Preparation of homogenates The kidney fractions (300 mg) were homogenized using a glass Teflon homogenizer with 2700 mL of cold 0.9% NaCl solution (1:9). The homogenates were centrifuged at 3500–4000g for 10 min at 4 °C. The supernatants were carefully removed from the pellet and used directly for antioxidant assays. 2.5. Protein measurement Protein content was determined using Coomassie brilliant blue G-250 method. 2.6. Assay of total antioxidant capacity (T-AOC) The kidney contains many antioxidants that could reduce Fe3+ to Fe2+. Fe2+ combined with phenanthroline substances to form stable complexes. Absorbance was determined at 520 nm to express total antioxidant activity using the Uv–vis spectrophotometer (UV-2600, Optical Instrument Factory, Shanghai, China). 2.7. Antioxidant enzyme activities
2.2. Animals and treatment All experiments were carried out in accordance with the ethical guidelines of the National Institutes of Health Guide for the Care and in compliance with the rules on animal protection of the Shandong Agriculture University (SDAUA-2014-013). Before beginning the present study, we performed acute toxicity experiments to determine the oral 50% lethal dose (LD50) of CrCl3 for male chicken according to the Karber method (Bittenbender and Howell Jr., 1974) and confirmed that the LD50
2.7.1. Superoxide dismutase (SOD) Superoxide anion radicals oxidized hydroxylaminen to nitrite; these radicals were produced by xanthine and xanthine oxidase system. Nitrite produced a purple color under the action of chromogenic agent. Absorbance was determined with the Uv–vis spectrophotometer (UV-2600, Optical Instrument Factory, Shanghai, China) at 550 nm. One unit of SOD was defined as the amount of enzyme that exhibited 50% inhibition in 1 mg of protein tissues. SOD activity was expressed as U/mg protein.
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2.7.2. Catalase (CAT) CAT decomposed H2O2; the reaction was rapidly terminated by adding ammonium molybdate. The remaining H2O2 reacted with ammonium molybdate and produced a pale yellow clathrate. The production was determined at 405 nm to express CAT activity. CAT activity was expressed as U/mg protein. 2.7.3. Glutathione peroxidase (GSH-PX) GSH-PX catalyzed a portion of GSH to oxidize GSH, as well as H2O2 to H2O. The remaining GSH in the reaction reacted with dithiobis nitrobenzoic acid and produced a stable yellow substance. Absorbance was determined at 412 nm. One unit of GSH-PX activity was defined as the amount of the enzyme that lowered the concentration of GSH by 1 lmol/L per min at 37 °C per 1 mg of protein tissues. 2.8. Non-enzymatic antioxidant contents 2.8.1. Glutathione (GSH) GSH contains a mercapto group; the reaction between mercapto mix and dithiobis nitrobenzoic acid generated a yellow compound. The compound could be quantitatively determined using the Uv– vis spectrophotometer (UV-2600, Optical Instrument Factory, Shanghai, China) at 420 nm. 2.8.2. Hydrogen peroxide (H2O2) H2O2 reacted with molybdate to generate a clathrate. The amount of clathrate was determined at 405 nm using commercial kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). H2O2 activity was expressed as mmol/g protein. 2.8.3. Malondialdehyde (MDA) Lipid peroxidation in the kidney homogenates was determined using commercial kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer’s instructions. MDA was condensed with thiobarbituric acid and heated in a boiling water bath for 45 min. The reaction produced red substances, and their absorbance was determined at 535 nm. 2.9. Statistical analysis Data were initially encoded in Excel software and then analyzed using analysis of variance with SPSS 18.0 software (SPSS Inc, Chicago IL, USA) and LSD multiple comparison. P < 0.05 was considered statistically significant. Data were expressed as mean ± S.D. deviation. 3. Results 3.1. Effects of CrCl3 on the weight of the body, kidneys, chest muscles, and leg muscles The effect of CrCl3 on the body weight of chickens is shown in Fig. 1. The body weight time-dependently increased, however, the gain of body weight in the high-dose group was evidently slower (P < 0.05) than those of the middle- and low-dose groups. Moreover, the body weight in the three Cr-treated groups was significantly lower (P < 0.05) than that in the control group at the same exposure time. To investigate the effect of CrCl3 on growth performance, the absolute weight of the two kidneys, chest muscle and leg muscle from the four groups were determined at 14, 28, and 42 d (see Table. 1). The mean absolute weights of the kidneys and chest muscles were not significantly different (P > 0.05) among the groups after oral intake of CrCl3 at 14 d. At 28 d, the mean absolute
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weight in the high-dose group significantly decreased (P < 0.05). At 42 d, the mean absolute weights in the three Cr-treated groups significantly decreased (P < 0.05). The weight of leg muscles was significantly lower (P < 0.05) than that in the control group. The mean weights decreased with increased CrCl3 concentration and prolonged treatment, except in the middle group at 14 d. These results indicated that long-term administration of CrCl3 had negative effects on the growth performance of chicken. 3.2. Histopathological changes The histopathological lesions in kidney after exposure to CrCl3 were observed under optical microscope. The control and low-dose groups showed no toxic lesions at 14 d (pictures not shown). However, the other experimental groups presented similar damages, including slight swelling, nuclear condensation, and dissolution in the tubular epithelial cells. A part of the renal tubules showed severely swelling or enlarged lumen, and some membrane of epithelial cells dissolved or disappeared (Fig. 2B). In the highand middle-dose groups, the medulla units were hemorrhage, and renal tubule showed severe atrophy. Moreover, the structure of glomerulus was degenerative; and the renal capsule lumen significantly largened (Fig. 2A). Yellow substances were found in the renal distal tubules. These substances were speculated as renal granular casts, which were produced by the tubule damage caused by heavy metal poisoning. These histopathological changes indicated that CrCl3 induced toxic lesions in the kidney. 3.3. Changes in T-AOC activity The effects of CrCl3 on the T-AOC activity in the kidney are shown in Fig. 3. The T-AOC activities in kidney tissues significantly decreased (P < 0.05) in the high-dose groups compared with those in the control group at 28 and 42 d. Moreover, the T-AOC activities in kidney tissues significantly decreased (P < 0.05) as the experiment progressed. 3.4. Changes in antioxidant enzyme activities of SOD, CAT and GSH-PX SOD activities were not significantly different among the four groups at 14 d. However, SOD activities significantly decreased (P < 0.05) with increased CrCl3 concentration and prolonged time at 28 and 42 d. In the high-dose group, SOD activity significantly decreased (P < 0.05) compared with that in the control group (Fig. 4A). The high-dose group presented the lowest CAT levels in the kidney, followed by the middle-dose group and then the low-dose group. Each experimental group had significantly lower (P < 0.05) CAT levels than the control group at 14, 28, and 42 d (Fig. 4B). GSH-PX activities of the three Cr-treated groups at 14 d were significantly higher than those at 28 and 42 d. The GSH-PX activity showed no significant differences between the experimental groups and the control groups at 14 and 28 d, however, at 42 d GSH-PX activities of the three Cr-treated groups significantly decreased (P < 0.05) compared with that in the control group. GSH-PX activities decreased with increased CrCl3 concentration and prolonged treatment (Fig. 4C). 3.5. Changes in non-enzymatic antioxidant contents of GSH, H2O2 and MDA GSH levels were not significantly different among the experimental and control groups at 14 d; at 28 d, only in the high-dose group GSH levels significantly (P < 0.05) increased than that in the control group. Moreover, GSH levels in the three experimental groups were higher than that in the control group at 42 d. The GSH
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Fig. 1. The effects of chromic chloride on body weight. At 14 d, only the high-dose group was significantly lower than the control group and at 28 d both the high-dose group and the middle group were significantly lower than the control group, however, at 42 d all the three groups were significantly lower than the control group. Each value represented the mean ± SD. The different letters indicate that there are significant differences (P < 0.05) between any two groups.
levels in the kidney significantly increased in the experimental groups with increased CrCl3 concentration and prolonged time (Fig. 5A). H2O2 content in the high-dose group was significantly higher (P < 0.05) than that in the control group at 14 and 28 d; however, no significant differences were observed at 42 d. H2O2 content decreased daily in the experimental groups compared with that in the control group, and the H2O2 content in the high-dose group significantly decreased (P < 0.05). However, H2O2 content in the middle- and low-dose groups was lower than that in the control group at 28 d (Fig. 5B). The MDA levels in the kidney from each experimental group were significantly higher (P < 0.05) than that from the control group. The MDA levels increased daily in all experimental groups compared with that in the control group; the MDA levels in the high-dose group significantly increased (P < 0.05). However, the MDA levels in the middle-dose group were lower than those in the low-dose group at 42 d (Fig. 5C). 4. Discussion Chromium is often added into animal diet, particularly in the livestock industry. However, using chromium as a nutritional supplement remains debatable because excessive Cr3+ may cause pathologic lesions, particularly in the kidney. Several researchers have suggested that adding chromium in feeds can promote the growth and production performance of broilers (Shrivastava
et al., 2003; Debski et al., 2004). In the present study, long-term treatment with CrCl3 added in drinking water did not promote the growth and development of chickens. The body weight decreased after long-term exposure to CrCl3. The experimental groups had significantly lower increase (P < 0.05) in body weight than that in the control group. Higher CrCl3 concentration and prolonged treatment resulted in lower weight. The weight of the chest and leg muscles in the experimental groups was significantly (P < 0.05) lower than that in the control group. Apparently, promoting the growth of chicken by supplementing chromium in drinking water has not been reported. The role of chromium in animal nutrition remains ambiguous, and its advantages as a supplement remain debatable. The present study investigated the toxicity of Cr3+ exposure via the oral drinking route at relatively long duration using a chicken model. Previous study showed that intracellular reduction of Cr6+ to Cr3+ induces overproduction of reactive oxygen species (ROS), which is an important characteristic of Cr6+-induced toxicity in the kidney (Stohs et al., 2001). The kidney, one of the major sites for chromium accumulation in the organism, becomes the target of chromium during long-term exposure. Changes in the kidney as a response to trivalent chromium toxicity have been reported in rats, dogs, and goldfish (Carciofi et al., 2007; Lushchak et al., 2009a; Staniek et al., 2010). Exposure to chromium eventually results in its accumulation and thus causes severe histological changes in the chicken kidney. The present study demonstrated the effects of supplementing CrCl3 in drinking water on its accumulation in the kidney and on the histological structure. The characteristics of the pathological changes caused by chromium in kidney is in accordance with the findings in previous studies on other animals; consuming water supplemented with chromium increases the MDA content and suppresses the antioxidant defense mechanisms in kidney tissues (Gupta and Ballal, 2015). In the present study, the weight of the kidney evidently decreased after poisoning compared with that in the control group, indicating that the kidney function was severely injured. The renal function and the concentration of enzymes and proteins may be altered, and kidney necrosis can occur in cases administered with high doses. Chromium is widely distributed in the body but preferentially localized in the kidney; moreover, Cr has varying degrees of toxicity. Oxidative damage and biofilm injury may be the possible causes of nephritic damage induced by chromium which were observed in in vivo and in vitro experiment systems (Scibior et al., 2006; Ahmad et al., 2012). Oral chromium supplementation in the form of CrCl3 did not promote the growth performance of chickens in the present study. Chromium functioned as a toxic agent, impairing the cellular functions and integrity in the kidney.
Table 1 The mass of the two kidneys, chest and leg muscle of the chicken. Time of treatment
Group
Kidney (g)
Chest (g)
Leg muscle (g)
14 d
High Middle Low Control
3.48 ± 0.106a 3.49 ± 0.080a 3.49 ± 0.202a 3.50 ± 0.057a
11.93 ± 0.851a 12.27 ± 0.742a 11.90 ± 0.258a 11.30 ± 0.341a
15.70 ± 0.361a 18.20 ± 0.586b 17.67 ± 0.636b 22.03 ± 1.877c
28 d
High Middle Low Control
4.15 ± 0.290a 4.77 ± 0.225b 4.97 ± 0.167b 5.31 ± 0.272bc
16.30 ± 0.839a 17.63 ± 0.384b 18.10 ± 0.265bc 19.57 ± 0.203c
26.70 ± 3.963a 33.53 ± 2.591b 31.83 ± 0.984b 43.63 ± 1.488c
42 d
High Middle Low Control
4.41 ± 0.178a 5.33 ± 0.172b 5.64 ± 0.035c 5.85 ± 0.088cd
22.63 ± 0.504a 24.87 ± 0.176b 28.23 ± 0.285c 31.57 ± 0.825d
42.80 ± 0.693a 52.63 ± 0.684b 58.07 ± 1.097c 74.77 ± 1.802d
The data are the mean ± S.D. of the results obtained in the six chickens composing of each experiment group (n = 6). The different letters indicate that there are significant differences (P < 0.05) between any two groups in same column.
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Fig. 2. Photomicrograph of the kidney from experimental group, magnification 200, n = 6. (A) Glomerular lesions showed the glomerular cystic space largened and a little lymphocyte infiltrated. (B) Renal tubular lesions showed necrosis of the tubular epithelial cell, pycnosis and disintegration of the nucleus, the cell membrane became incomplete or even broken, vacuoles degeneration of the tubular epithelial cell extensively and tube type can be seen in a few renal tubular lumen. (C and D) were the renal tubular and glomerular from the control group.
Fig. 3. Effects of chromic chloride on T-AOC activities in the kidney. The different letters indicate that there are significant differences (P < 0.05) between any two groups. Each value represented the mean ± SD.
The effects of using chromium as a feed additive in the livestock industry on the micro-renal injury should be further investigated. Generating and eliminating free radicals are in a state of dynamic balance under normal physiological conditions (Sun et al., 2013). However, aging or poisoning can disrupt the body metabolism, resulting in free radical accumulation and lipid peroxidation (Kapun et al., 2012). If the body could not remove the excess free radicals, the integrity of the cell membranes would be damaged, thus decreasing immunity and resistance (Yao et al., 2013). Oxidative stress has an important role in the toxicity mechanism of numerous xenobiotics. T-AOC represents the antioxidant capacity of the body’s defense system, which comprehensively indicates the functional status of the antioxidant system. GSH-PX is an important antioxidant enzyme in the body; this enzyme specifically catalyzes GSH to remove H2O2 and reduce the occurrence of lipid peroxidation, thus protecting the structure and the complete function of cell membranes. SOD, a heavy metallic
enzyme in the body, is an important component in enzymatic systems. Free radical scavenging in the body is related to SOD content. Moreover, CAT is a combination enzyme with ferriporphyrin as the prosthetic group. CAT in erythrocytes or tissues can directly decompose H2O2 under specific conditions, causing the gradual decrease in its concentration in tissue fluids and thus protecting the body against damages (Zhang et al., 2013). This study showed that heavy metal poisoning in the body significantly decreased (P < 0.05) the total antioxidant capacity of the kidney. Therefore, with increased CrCl3 concentration and prolonged treatment, the activities of SOD, GSH-PX, and CAT significantly decreased (P < 0.05), particularly in the high-dose group at 28 and 42 d. The reduced SOD, GSH-PX, and CAT activities indicated that poisoning could release and accumulate reactive oxygen species. The accumulated reactive oxygen species could damage the antioxidant enzymatic systems, thus decreasing the damages and functions of the kidney. MDA is the end-product of lipid peroxidation caused by oxygen free radicals and enhanced oxygen free radicals in the kidney (Bento et al., 2013). The decreased antioxidant effect increased the MDA content. A study found that MDA can strongly react with various components in the cell membrane (Esmaeilnejad et al., 2014). MDA content can reflect the oxygen free radical metabolism in the kidney, whereas lipid peroxidation indirectly indicates the oxidative damage in the kidney. In this study, the MDA content in the chicken kidney significantly increased (P < 0.05) with increased CrCl3 concentration and prolonged treatment. The MDA content in the high-dose group in each treatment period was significantly higher (P < 0.05) than that in the control group. MDA content increased as the activities of antioxidant enzymes decreased. GSH is the most abundant non-protein thiol in the cells and is a key cellular antioxidant (Dimri et al., 2014). When cells produced a small amount of H2O2, GSH catalyzes H2O2 to form H2O under the action of GSH-PX, which could be oxidized also to GSSG. CrCl3 poisoning decreased the activity of GSH-PX; thus,
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Fig. 4. Effects of antioxidant enzyme activities of SOD, CAT and GSH-PX. A: Effects of chromic chloride on the SOD activities in the kidney. B: Effects of chromic chloride on the CAT activities in the kidney. C: Effects of chromic chloride on the GSH-PX activities in the kidney. The different letters indicate that there are significant differences (P < 0.05) between any two groups. Each value represented the mean ± SD.
Fig. 5. Changes in non-antioxidant enzyme contents of GSH, H2O2 and MDA. A: Effects of chromic chloride on the GSH levels in the kidney. B: Effects of chromic chloride on H2O2 content in the kidney. C: Effects of chromic chloride on MDA content in the kidney. The different letters indicate that there are significant differences (P < 0.05) between any two groups. Each value represented the mean ± SD.
Y. Liu et al. / Chemosphere 139 (2015) 365–371
GSH could not be converted into GSSG. GSH content significantly increased (P < 0.05) in the high-dose group, in which GSH-PX activity was significantly reduced (P < 0.05). H2O2 is a harmful byproduct of many normal metabolic processes that can prevent damages to cells and tissues. H2O2 can be rapidly converted to other less dangerous substances. Thus, GSH-PX and CAT are frequently used by cells to rapidly catalyze the decomposition of H2O2 into less-reactive gaseous oxygen and water molecules (Heidarpour et al., 2012). The decrease in GSH-PX and CAT resulted in high H2O2 levels in the kidney. This experiment showed that oral uptake of CrCl3 through drinking water could abnormally change the stable oxidation and antioxidant mechanism in kidney cells; hence, the activities of antioxidant enzymes decreased, whereas that of lipid peroxide increased. Cell damages were exacerbated and eventually led to the injury in the structure and function of the chicken kidney. 5. Conclusion Summarizing, our results confirmed that long-term treatment with CrCl3 had negative effects on the growth performance of chickens. Moreover, the activities of the total antioxidant capacity (T-AOC) and antioxidant enzymes (SOD, CAT, and GSH-PX) significantly decreased, whereas MDA, GSH, and H2O2 contents significantly increased, and eventually destroyed the antioxidant defense system in kidney tissues. These findings may provide a new insight into the possible mechanism for the toxicity of using chromium as a long-term feed additive in the poultry industry. Conflicts of Interest The authors declare that they have no conflicts of interest concerning this article. Acknowledgment The study was supported by the earmarked fund Shandong Modern Agricultural Technology & Industry System (No. SDAIT-13-011-04). References Ahmad, M.K., Naqshbandi, A., Fareed, M., Mahmood, R., 2012. Oral administration of a nephrotoxic dose of potassium bromate, a food additive, alters renal redox and metabolic status and inhibits brush border membrane enzymes in rats. Food Chem. 134, 980–985. Bento, D.B., de Souza, B., Steckert, A.V., Dias, R.O., Leffa, D.D., Moreno, S.E., Petronilho, F., de Andrade, V.M., Dal-Pizzol, F., Romao, P.R., 2013. Oxidative stress in mice treated with antileishmanial meglumine antimoniate. Res. Vet. Sci. 95, 1134–1141. Bittenbender, H., Howell Jr., G.S., 1974. Adaptation of the Spearman-Karber method for estimating the T50 of cold stressed flower buds. J. Amer. Soc. Hortic. Sci. 99, 187–190. Carciofi, A.C., Vasconcellos, R.S., de Oliveira, L.D., Brunetto, M.A., Valério, A.G., Bazolli, R.S., Carrilho, E.N.V.M., Prada, F., 2007. Chromic oxide as a digestibility marker for dogs-A comparison of methods of analysis. Anim. Feed. Sci. Technol. 134, 273–282. Dallago, B.S., McManus, C.M., Caldeira, D.F., Lopes, A.C., Paim, T.P., Franco, E., Borges, B.O., Teles, P.H., Correa, P.S., Louvandini, H., 2011. Performance and ruminal protozoa in lambs with chromium supplementation. Res. Vet. Sci. 90, 253–256. Das, J., Sarkar, A., Sil, P.C., 2015. Hexavalent chromium induces apoptosis in human liver (HepG2) cells via redox imbalance. Toxicol. Rep. 2, 600–608. Dayan, A., Paine, A., 2001. Mechanisms of chromium toxicity, carcinogenicity and allergenicity: review of the literature from 1985 to 2000. Hum. Exp. Toxicol. 20, 439–451. Debski, B., Zalewski, W., Gralak, M.A., Kosla, T., 2004. Chromium-yeast supplementation of chicken broilers in an industrial farming system. J. Trace Elem. Med Biol. 18, 47–51. Deng, G., Wu, K., Cruce, A.A., Bowman, M.K., Vincent, J.B., 2015. Binding of trivalent chromium to serum transferrin is sufficiently rapid to be physiologically relevant. J. Inorg. Biochem. 143, 48–55. Dimri, U., Bandyopadhyay, S., Singh, S.K., Ranjan, R., Mukherjee, R., Yatoo, M.I., Patra, P.H., De, U.K., Dar, A.A., 2014. Assay of alterations in oxidative stress markers in
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