Biol Trace Elem Res DOI 10.1007/s12011-016-0640-4

Oxidative Stress and Histological Alterations of Chicken Brain Induced by Oral Administration of Chromium(III) Jia Cheng 1 & Wentao Fan 1 & Xiaona Zhao 1 & Yanhan Liu 1 & Ziqiang Cheng 1 & Yongxia Liu 2 & Jianzhu Liu 1

Received: 24 November 2015 / Accepted: 3 February 2016 # Springer Science+Business Media New York 2016

Abstract This experiment was conducted to investigate the oxidative stress in chickens exposed to different concentrations of chromium trichloride (CrCl3) in drinking water. Seventy-two Hylan Brown male chickens were randomly divided into four groups: three experimental groups and one control group. The experimental groups were exposed to three different doses (50 % LD50, 25 % LD50, and 12.5 % LD50) of CrCl3 mg/kg body weight for 42 days, while the control group was given the equivalent water. The activities of antioxidant enzymes (superoxide dismutase, catalase, and glutathione peroxidase) and non-enzymatic index (glutathione, total antioxidant capacity, malondialdehyde, and hydrogen peroxide) were measured after obtaining the brain samples. Results suggested that 50 % LD 50 chromium(III) significantly increased (P < 0.05) the contents of malondialdehyde and hydrogen peroxide. The antioxidant enzyme activities, total glutathione concentration, and total antioxidant capacity decreased significantly (P < 0.05) compared with those of the controls and were consistent with the increase in dosage and time. Additionally, extensive histological alterations were observed in the chicken brain, such as the vacuolization and nuclear condensation of the neurons. These results indicated that

exposure to high-dose CrCl3 for a certain time could induce the occurrence of oxidative stress and histological alterations. Keywords Chicken . Brain . CrCl3 . Oxidative damage· . Histological alteration

Abbreviations CrCl3 Chromium trichloride CAT Catalase GSH Glutathione GPX Glutathione peroxidase GSSG Oxidized glutathione Hydrogen peroxide H2O2 LD50 50 % lethal dose MDA Malondialdehyde ROS Reactive oxygen species T-SOD Total superoxide dismutase T-AOC Total antioxidant capacity TBA Thiobarbituric acid

Introduction * Yongxia Liu [email protected] * Jianzhu Liu [email protected]

1

College of Veterinary Medicine, Research Center for Animal Disease Control Engineering Shandong Province, Shandong Agricultural University, Tai’an 271018, China

2

Research Center for Animal Disease Control Engineering, Shandong Agricultural University, Tai’an, Shandong 271018, China

Chromium (Cr) is abundant in the earth’s crust, and trivalent chromium [Cr(III)] and hexavalent chromium [Cr(VI)] are the most stable forms [1]. The trivalent form of chromium has a strong tendency to form coordination compounds, whereas the hexavalent form is usually linked with oxygen (CrO42−, chromate; Cr2O72−, dichromate). Cr(VI) enters many types of cells and could be reduced intracellularly under physiological conditions by several methods to produce reactive intermediates and, ultimately, Cr(III) [2, 3]. Cr(VI) is a major environmental toxin and a pollutant emitted from cigarette smoke, automobile emissions, and hazardous wastes [4]. Cr(III) has

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been identified as the active ingredient of Bglucose tolerance factor^ (GTF) [5], and some studies suggested that Cr(III) has a positive effect on the growth rate and feed efficiency of growing poultry under stress condition [6, 7]. However, chromium has not been considered an essential element for mammals, but rather has its effects as a pharmacological agent [8]. Therefore, Cr(III) may also have toxicity because it has been postulated to cause oxidative stress. For example, a study concluded that Cr(III) exposure could induce oxidative stress in goldfish and fails to enhance the efficiency of the antioxidant system in the organ. Moreover, a comparison of the Cr(III) effects on goldfish metabolism indicated that the trivalent ion induces strong oxidative stress [9, 10]. Chromium picolinate of high concentrations could cause cell death through an apoptotic and necrotic mechanism, whereas apoptotic death is the main mechanism of oxidative stress that induces mitochondrial dysfunction [11]. Furthermore, although Cr(III) is not yet classified as a human carcinogenic, it can induce DNA damage [12]. Certain researchers have suggested that Cr(III) has genotoxic effect on the sperm of mice and could cause sperm deformity [13]. In mouse, chromium trichloride adversely affects the mouse oocyte by inhibiting the extrusion of the first polar body, affecting the quality and viability of the oocyte, as well as reducing the number of oocytes of superovulation [14]. Therefore, Cr(III) is harmful to many systems at high concentrations, although it has been used in many dietary supplements at low concentrations [15, 16]. Chromium is often applied as an additive to animal feeding, and certain studies have shown that Cr(III) could significantly improve the function of the humoral immune and carcass quality of broilers [17]. However, excessive intake in chickens directly leads to oxidative damage and histopathological changes [18, 19]. Although extensive studies have been carried out on Cr(III) in many organs, few investigations have been conducted in the brain tissue [10]. In this study, the oxidative stress and histopathological alterations of chicken brain induced by Cr(III) were measured.

Materials and Methods Reagents Chromium chloride (CrCl3·6H2O) was purchased from Putian Tedia Company Inc. in Tai’an, China. CrCl3·6H2O (purity ≥99.8 %) was prepared in dechlorinated tap water. All other reagents were of analytical grade. The catalase (CAT), glutathione peroxidase (GPX), superoxide dismutase (SOD), hydrogen peroxide (H 2 O 2 ), glutathione (GSH), and malondialdehyde (MDA), as well as the total antioxidant capacity (T-AOC) and total protein quantification (Coomassie

Brilliant Blue) detection kits, were purchased from Nanjing Jiancheng Bioengineering Institute (Jiangsu Province, China). Animals and Chromium Exposure Seventy-two Hylan Brown male chicks (1-day old), obtained from the Hylan Breeder Company (Tai’an, China), were used in this study. They were maintained under standard laboratory conditions of 33 ± 2 °C temperature and 24 h light. The animals had free access to water and commercial standard pellet diet from Liuhe Jingwei Farming and Animal Husbandry Co., Ltd. Feedstuff Factory (Tai’an, China). After being supplied food and water ad libitum for a week, the chicks were randomly assigned to four groups with 18 chicks in each group. These chicks were maintained in an air-conditioned room at 25 ± 1 °C. The oral 50 % lethal dose (LD50) of CrCl3 for chicken was determined as 5013 mg/kg body weight according the Karber method [20]. The exposed groups received CrCl3 orally in water with different concentrations of 12.5 % LD50 (low-dose), 25 % LD50 (middle-dose), and 50 % LD50 (high-dose) mg/kg body weight daily. The control group was given equivalent water. All animals were given standard diet. Animal experiments were performed in accordance with institutional guidelines and the guiding principles in the use of animals in toxicology on animal protection of the Shandong Agriculture University. Harvesting of Samples Every six chickens were slaughtered respectively on the 14th, 28th, and 42th day. Brain tissues were removed and frozen in liquid nitrogen. Small sections of the cerebrum were freshly prepared and fixed in 10 % neutral-buffered formaldehyde for at least 24 h. Detection of Chromium Content Brain tissue samples (approximately 0.20 g of wet mass) were digested with concentrated HNO3 (65 % Merck) and H2O2 (30 % Merck) for 30 min in a Microwave Digestion System (MARS-5, CEM), and this process was repeated two to three times. All digested samples were diluted with de-ionized water so that the analyte was within the calibration range. Content of Cr was evaluated by an inductively coupled plasma mass spectrometer (ICP-MS; Hewlett-Packard, HP-4500, Avondale, PA, USA) according to the manufacturer’s recommendation. The accuracy of the determination of Cr was assured using the certified reference material mussel tissue ERM®-CE278 (ERM), and the detection limits (LOD, ng/ ml) determined by the measurement of blank solution was 0.04.

Oxidative Stress and Histological Alterations of Chicken Brain

Preparation of Tissue Homogenates

Non-enzymatic Antioxidants

The frozen brain fractions (0.5 g) were removed and weighed accurately and then diluted with 0.9 % sodium chloride solution (4.5 ml) according to the ratio (1:9) between quality and volume. The mixtures were centrifuged at 12,000 rpm for 15 min at 4 °C using the high speed freezing centrifuge (HITACHI-CR21G, RiLi, Japan), and the supernatant was measured.

T-AOC

Protein Quantification The protein content in the chicken brain was firstly determined using the method of Coomassie Brilliant Blue. The developed blue color of the resulting reaction was measured at 420 nm with a spectrophotometer (UV-7504, Xinmao, China).

Enzymatic Antioxidants

The T-AOC activity was detected by a colorimetry kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The resulting reaction was measured at 520 nm with a spectrophotometer. Other procedures were carried out following the manufacturer’s protocols, and the detection limits were 0.2–55.2 U/ml. MDA The MDA contents in the brain homogenates were determined using commercial kits by the method described by Jena et al. [22]. In brief, thiobarbituric acid reactive substance was produced after mixing trichloroacetic acid with the homogenate and centrifuging, and it was a red substance. The developed red color of the resulting reaction was measured at 532 nm with a spectrophotometer. The detection limits of MDA were 0–113.0 nmol/ml.

SOD H2O2 SOD activity was determined using nitro blue tetrazolium (NBT) method [21]. This assay relies on the capability of the enzyme to inhibit the phenazine methosulfate-mediated reduction of NBT dye. The NBT method is adequate to determine the forms of SOD, MnSOD, and Cu/ZnSOD. The developed blue color was measured at 550 nm with a spectrophotometer. The enzyme activity was expressed as U/g brain, and the detection limits were 0.5–122.1 U/ml.

Catalase CAT activity was spectrophotometrically measured. One unit of CAT activity was defined as 1 mg of tissue protein consumed by 1 μmol H2O2 at 405 nm. The CAT activity was expressed as U/mg brain, and the detection limits were 0.2– 24.8 U/ml.

Glutathione Peroxidase GPX activity was determined using a GPX kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). A series of enzymatic reactions was activated by GPX in the homogenate, subsequently leading to the conversion of reduced glutathione (GSH) to oxidized glutathione (GSSG). The change in absorbance during the conversion of GSH to GSSG was recorded spectrophotometrically at 420 nm. The activity of GPX was expressed as mU/g brain, and the detection limits were 20–330 U.

The H2O2 content in chicken brain was spectrophotometrically assessed. H2O2 bound with molybdenic acid to form a clathrate, which was measured at 405 nm. H2O2 content was expressed as mmol/g protein. The H2O2 content was then calculated. GSH The GSH content in the brain was determined using a GSH detection kit by the method described by Jones [23]. The method was based on the development of a yellow color; the absorbance was measured at 420 nm. The total GSH content was expressed as μg/g tissue, and the detection limits were 0.3–147.1 mg GSH/L. Histological Studies The samples of chicken cerebrum were immediately fixed in formalin solution (10 %) and processed in a series of graded ethanol solutions. Cerebrum samples were stained with hematoxylin–eosin. These sections intended for the histological examination by light microscopy at a magnification of ×400. Statistical Analysis Statistical analysis was performed using one-way analysis of variance with the SPSS software (Version 11.0, SPSS Inc., USA). All values were expressed as mean ± SEM. All

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measurements were replicated three times. The differences were considered significant if P < 0.05.

Results Chromium Content in the Chicken Brain After the chickens were exposed to CrCl3 for 14, 28, or 42 days, the content of Cr was higher (P < 0.05) compared with those of the control group in the brain. The contents of Cr in the high-dose group showed significant (P < 0.05) changes on 14 days; at 28 days, the Cr contents in the three Cr-exposed groups were all higher (P < 0.05) compared with those of the control group, while a similar trend was also found at 42 days (Fig. 1). Antioxidant and Associated Enzymes In this experiment, several parameters like non-enzymatic antioxidants (GSH, MDA, T-AOC, and H2O2) and enzymatic antioxidants (CAT, SOD, and GPX) were determined in the brain homogenates. The effects on a variety of parameters were monitored in the brain.

irregular variation at the same dose with prolonged time. No significant changes in T-AOC value were observed among the samples in the low-dose group at 14, 28, and 42 days; however, significant differences (P < 0.05) in T-AOC activities were observed in the high-dose group at 42 days (Fig. 2). In our experiment, the GSH levels significantly decreased (P < 0.05) with middle-dose and high-dose CrCl3 treatments at 14, 28, and 42 days. A significant decrease (P < 0.05) in the GSH levels was observed in the brain of experimental groups compared with those of the control group at 42 days (Fig. 3c). A significant increase (P < 0.05) in MDA contents of all experimental groups was found among the experimental groups at 42 days. However, after 14 days of treatment, the MDA contents decreased, but the difference was not significant (P < 0.05). In a practical term, MDA contents in the same group showed increase with the growing time (Fig. 3a). The value of H2O2 in the chicken brain of experimental groups showed significant increase (P < 0.05) with increasing dose of chromium at 14 days. However, the H2O2 values in the same group decreased constantly with prolonged time and all H2O2 values in experimental groups were higher than that of the control (Fig. 3b).

Non-enzymatic Index

Enzymatic Antioxidants

Cr(III) exposure did not alter the protein content. Therefore, the content of protein had no evident increase or decrease when animals were exposed to CrCl3 (figures not shown). The effect of CrCl3 on the T-AOC activities in the brain was shown in Fig. 2. The T-AOC activities of all the experimental groups were lower than that of the control group. Meanwhile, the T-AOC activities in the same group showed

Our results revealed that in CrCl3-treated chickens, the activities of SOD and GPX exhibited a consistent trend in the brain homogenates (Fig. 4). In fact, the GPX activities decreased when the chickens were exposed to Cr(III), as compared to the control group. Moreover, the high-dose group had significant lower (P < 0.05) GPX activity than the control group at 14, 28,

Fig. 1 The chromium concentrations in the brain at different chromium exposition for an increasing time (μg/g). The data are shown as means ± SEM, n = 6. * indicates being significantly different from the corresponding control (P < 0.05)

Fig. 2 Effects of Cr(III) on T-AOC activity in chicken brain. Data are expressed as means ± SEM, n = 6. * indicates being significantly different from the corresponding control (P < 0.05)

Oxidative Stress and Histological Alterations of Chicken Brain Fig. 3 Effects of Cr(III) on nonenzymatic antioxidants in chicken brain. a MDA content, b H2O2 content, c total GSH content. Data are expressed as means ± SEM, n = 6. * indicates being significantly different from the corresponding control (P < 0.05)

and 42 days (Fig. 4b). The GPX activities of chicken brain samples in the same group had an evident decrease with the growing time. After CrCl3 treatment, the SOD activities also decreased. In addition, these decreases showed an outstanding difference

(P < 0.05) at 42 days. The SOD activities also had an obvious decrease (P < 0.05) in the high-dose and middle-dose group (Fig. 4c). The CAT activities showed a different profile (Fig. 4a). The CAT values of the experimental groups were decreased

Cheng et al. Fig. 4 Effects of Cr(III) on enzymatic antioxidants in chicken brain. a CAT activity, b GPX activity, c SOD activity. Data are expressed as means ± SEM, n = 6. * indicates being significantly different from the corresponding control (P < 0.05)

immediately at 14 days (P < 0.05). However, in the same group, the CAT value had a slight increase with prolonged time, especially in the low-dose and middle-dose group. The

CAT activities showed a significant difference (P < 0.05) compared to the control. Nevertheless, all CAT activities were lower compared with that of the control.

Oxidative Stress and Histological Alterations of Chicken Brain Fig. 5 Cerebrum histological alterations of chickens induced by different dose of Cr(III) at 42 days. a Controls, b low-dose group, c middle-dose group, d high-dose group. Optic microscopy: HE (×400)

Histological Results Figure 5 showed that cerebrum histological alterations of chickens were induced by three different doses Cr(III) at 42 days. As shown in the figure, cytoplasmic vacuolation of neurons and nuclear condensation were discovered, which resulted in the neuronal necrosis. Additionally, the interstitial edema was appeared in Cr(III) exposure groups (Fig. 5). These pathological alterations indicated that CrCl3 could result in toxic lesion in the chicken brain.

Discussion Chromium is one kind of transition metal and trace element. If excessive, it will cause great harm to the body. An increasing body of evidence indicated that transition metals might cause oxidative damages in exposed organisms [24]. The present study investigated the oxidative stress in chicken brain exposed to different CrCl3 concentrations via the oral drinking. The results showed that long-term exposure to CrCl3 resulted in oxidative stress and histological alterations to chicken brain. Our results were consistent with previous studies of Travacio et al. [25], who explained the decrease of antioxidant enzymes using their activation or synthesis in the long-term exposure of chromium. The brain tissue is a soft organ that is particularly susceptible to free radical attack, and it contains large amounts of polyunsaturated fatty acids and consumes 20 % of the body’s oxygen. In addition, the brain is characterized by highly intensive oxidative metabolism because of high concentrations of specific compounds, which are catalytically implicated in

the production of free radical [26]. Thus, the brain tissue has a relatively poor antioxidant defense system. Studies have suggested that the oxidative stress induced by heavy metals results in an increase in reactive oxygen species (ROS), stimulating an increase in antioxidant enzyme activity [27]. GSH is the most abundant non-protein factor in the cells and is a key cellular antioxidant [28]. In our study, the brain GSH status was significantly impaired following chromium intoxication. The significant decrease (P < 0.05) in the total GSH status concentration in chicken brain (Fig. 3C) indicated an enhanced consumption/degradation or reduced synthesis of the tripeptide under Cr(III) treatment. These changes in GSH pools were usually associated with oxidative stress [29]. When cells produced a small amount of free radical by CrCl3, GSH can be combined with the free radicals under the action of GPX, which could be oxidized to GSSG. Actually, in order to remove the free radicals, the GSH and GPX were consumed. Therefore, GSH content significantly decreased (P < 0.05) in the high-dose group, in which GPX activities were also significantly reduced (P < 0.05). 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. MDA is the product of oxidative stress. Therefore, the increase in MDA content is an important indication of oxidation. In our study, MDA content in the chicken brain significantly increased (P < 0.05) with increased CrCl3 concentration at 42 days. In our study, CAT activity, especially in the high-dose group, significantly decreased (P < 0.05) in comparison with that in the control group. H2O2 is a harmful by-product of many normal metabolic processes in cells and tissues. CATs are frequently used by cells to rapidly catalyze the

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decomposition of H2O2 into less-reactive gaseous oxygen and water molecules [30]. Thus, the decrease in CAT activity led to the increase in H2O2 content. However, to our surprise, the CAT activity in experimental groups at 28 days was higher than that at 14 days (Fig. 4a), which resulted in the unusual changes of H2O2 content (Fig. 3B). Previous study suggested that Cd2+ resulted in low CAT activity by possible mechanisms, which might direct the metal-mediated structural alteration of the enzyme and depression of CAT synthesis [31]. The generation of ROS by heavy metal stresses directly affects the activity of antioxidant enzymes, especially CAT [32]. The maximum generation of O2− and •OH causes the maximum depression of CAT activity during the metal toxicity [33]. Thus, we guessed that Cr(III) may bind with the CAT by possible mechanisms in 14 days, thereby reducing CAT activity. Certain experimenters believe that heavy metals are directly related to the decrease in CAT. However, whether the decrease in CAT was directly related to the metal needs further experiment. Histological studies also provided an important evidence for the biochemical analysis. Under microscopic examination, severe distortions in the cellular architecture were observed at 42 days (Fig. 5). The brain tissue of the experimental group at 14 and 28 days had no evident changes compared with that in the control group (pictures not shown). The histological changes were seen in the cerebrum of the CrCl3-treated chickens after 42 days which were characterized by cytoplasmic vacuolization. As a study showed, the increased amount of ROS product might attack the membranes and enhance their permeability, leading to vacuolization [34]. According to these studies, the enhanced permeability of the cell membranes could lead to an increase in intracellular water, which sufficiently produced cytoplasmic vacuolization (Fig. 5). This experiment investigated that being exposed to different CrCl3 concentrations via oral drinking could result in oxidative stress and histological alterations in chicken brain. Therefore, long-term exposure to CrCl3 results in the decrease of the antioxidant enzymes activities and the increase of lipid peroxide.

Conclusion Exposure to Cr(III) can cause changes in the antioxidant enzymes and non-enzymatic index in chicken brain. Excessive Cr(III) induced oxidative stress in the chicken brain with doseand time-dependent manners. Furthermore, exposure to excessive Cr(III) for a certain time can result in histological damage in the chicken brain. Therefore, this study can be served as a scientific evidence for the long-term application of Cr(III) on feed production in poultry industry, and it provided an important reference for further studies on Cr(III)mediated brain injury mechanisms in chicken.

Acknowledgments The study was supported by the earmarked fund Shandong Modern Agricultural Technology and Industry System (No. SDAIT-13-011-04). Compliance with ethical standards Conflicts of interest The authors declare that they have no conflicts of interest concerning this article

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