Biol Trace Elem Res DOI 10.1007/s12011-015-0357-9

The Role of Oxidative Stress in Gastrointestinal Tract Tissues Induced by Arsenic Toxicity in Cocks Ying Guo 1 & Panpan Zhao 1 & Guangyang Guo 1 & Zhibo Hu 1 & Li Tian 1 & Kexin Zhang 1 & Wen Zhang 1 & Mingwei Xing 1

Received: 5 February 2015 / Accepted: 26 April 2015 # Springer Science+Business Media New York 2015

Abstract Arsenic (As) is a widely distributed trace element which is known to be associated with numerous adverse effects on human beings and animals. Arsenic trioxide (As2O3) is an inorganic arsenical-containing toxic compound. The effect of excessive amounts of As2O3 exposure on gastrointestinal tract tissue damage in cocks is still unknown. This study was conducted to investigate the effect of As2O3 exposure on gastrointestinal tract tissue damage in cocks. In total, 72 1day-old male Hyline cocks were randomly divided into four groups and fed either a commercial diet or an As2O3 supplement diet containing 7.5, 15, and 30 mg/kg As2O3. The experiment lasted for 90 days and gastrointestinal tract tissue samples (gizzard, glandular stomach, duodenum, jejunum, ileum, cecum, and rectum) were collected at 30, 60, and 90 days. Catalase (CAT), glutathione (GSH), and glutathione peroxidase (GSH-Px) activities; malondialdehyde (MDA) contents; and hydroxyl radical (OH·)-mediated inhibition were examined. Furthermore, the results demonstrated that MDA content in the gastrointestinal tract was increased, while the activities of CAT, GSH, and GSH-Px and the ability to resist OH· was decreased in the As2O3 treatment groups. Ying Guo and Panpan Zhao contributed equally to this work. * Wen Zhang zhangwenwake[email protected] * Mingwei Xing [email protected] Ying Guo [email protected] Panpan Zhao [email protected] 1

College of Wildlife Resources, Northeast Forestry University, Harbin 150040, Heilongjiang Province, China

Extensive damage was observed in the gastrointestinal tract. These findings indicated that As2O3 exposure caused oxidative damage in the gastrointestinal tract of cocks due to alterations in antioxidant enzyme activities and elevation of free radicals. Keywords Arsenic toxicity . Gastrointestinal tract tissues . Cocks . Oxidative damage

Introduction Exposure to arsenic trioxide (As2O3) is an important public health concern for As2O3 is a poisonous chemical substance for people and animals. As2O3 is a trivalent inorganic arsenical compound that affects a variety of organs including the liver, kidney, skin, gastrointestinal tract, and circulatory system [1, 2]. Organisms are inevitably exposed to As2O3 which widely exists in contaminated water, food, soil, and some occurring minerals [3, 4], and As2O3 toxicity could affect a wide variety of organisms [5]. Drinking water that chronically contains arsenic leads to dermal lesions (e.g., hyperpigmentation, hyperkeratosis, desquamation, and hair loss) [6], peripheral neuropathy, skin cancer, and peripheral vascular disease. As2O3 hardly has any positive effects on human health, and its interaction with intracellular reactive oxygen species (ROS) causes cell damage [7]. Arsenate is similar to phosphate; it can replace phosphorus in the body, which can lead to phosphorus replacement in the bone [2]. Arsenic can also perturb the DNA repair process [8] and can therefore increase susceptibility to cancer (e.g., skin cancer). Arsenite (As+3) can reportedly suppress the human and murine immune system [9, 10]; however, As2O3 in the bird gastrointestinal tract has been reported less often.

Guo et al.

Arsenic (As) uptake from water and food can have a significant negative influence on human and animal health. As+3 is reportedly a contributing factor in acute peroxide toxicity in laboratory animals [11]. Meanwhile, As +3 disrupts oxidative phosphorylation and increases ROS generation at the cellular level [12]. Oxidative stress can cause many negative effects including loss of ions, DNA strand breaks, and membrane peroxidation. However, it remains unknown how ROS influences the gastrointestinal tract of birds. One of the primary antioxidant enzymes is catalase (CAT), which is commonly found in most organisms. CAT is composed of four polypeptide chains with more than 500 amino acids each [13] and can catalyze the decomposition of H2O2 to H2O without a reducing substance [14]. Glutathione peroxidase (GSHPx) is also an antioxidant enzyme and exists widely in many organs especially in intestinal tissues. It can protect membrane lipids from oxidative damage [15]. Another related factor to antioxidant enzymes is malondialdehyde (MDA), which is produced when membrane polyunsaturated fatty acids undergo peroxidation [16], and its content will reflect the degree of lipid peroxidation damage. In recent years, numerous studies on As compounds have been reported; however, almost all of them are focused on humans. Moreover, studies on the effects of As compounds on the gastrointestinal tract of birds are few. Therefore, we designed the experiment to investigate the changes in histopathology, ROS, and antioxidant defense enzyme systems induced by As2O3 exposure in the gastrointestinal tracts of cocks. In this study, an arsenicinduced toxicity model was constructed by serving the male Hyline cocks an arsenic-supplemented diet and measuring the effects of arsenic-induced toxicity on the gastrointestinal tract by detecting CAT, GSH, and GSH-Px activities; MDA content; and OH· inhibition using assay kits. This study provided a theoretical foundation for clarifying the molecular mechanism of arsenic-induced toxicity of the gastrointestinal tract.

Materials and Method Animal Care and Experimental Design All of the procedures used in this research were authorized by the Institutional Animal Care and Use Committee of Northeast Forest University. In total, 72 1-day-old male Hyline cocks were separated randomly and divided into four groups (18 cocks per group) including a low-As group (L group), a middle-As group (M group), a high-As group (H group), and the control group (C group). The C group was fed basic diet, the L group was fed the basic diet plus 7.5 mg/kg As2O3, the M group was fed the basic diet plus

15 mg/kg As2O3, and the M group was fed the basic diet plus 30 mg /kg As2O3, which represents 1/20, 1/40, and 1/18 of the median lethal dose (LD50) for cocks, respectively. The cocks were maintained in the Laboratory Animal Center, College of Wildlife Resource, Northeast Forest University, China. Over the entire experimental period, food and water were supplied sufficiently. On the 30th, 60th, and 90th day of the experiment, 15 cocks in each group were selected. The cocks were euthanized with sodium pentobarbital, and the gastrointestinal tract (including muscular stomach, glandular stomach, duodenum, jejunum, ileum, cecum, and rectum) was immediately excised, blotted, and rinsed with ice-cold 0.9 % NaCl solution. They were dried with filter paper and weighed. Gastrointestinal tract (500 mg) tissues were homogenized using a glass Teflon homogenizer in cold 0.9 % NaCl solution. The homogenates were centrifuged at 3000×g for 5 min, and the supernatant was collected and used for the experiments. The remaining tissues were frozen immediately in liquid nitrogen and stored at −80 °C until required. Histological Observations Gastrointestinal tract tissue samples were collected, cut to approximately 0.5 cm3, and fixed in 4 % paraformaldehyde for 24 h at room temperature. Then, tissues were routinely embedded in paraffin wax. Tissues were sectioned at 5 μm, and the sections were stained with hematoxylin and eosin (HE) for light microscopy. Determination of Oxidative Damage Parameters in the Gastrointestinal Tract Tissues CAT activity was measured in gastrointestinal tract tissue homogenates after isolation from cocks according to the manufacturer’s protocol of the CAT detection kit (Nanjing Jiancheng Bioengineering Institute, China). Total CAT activity was measured at 405 nm. GSH content was measured in gastrointestinal tract tissue homogenates after isolation from cocks according to the manufacturer’s GSH content assay kit instructions (Nanjing Jiancheng Bioengineering Institute, China). Total GSH content was measured at 420 nm. GSH-Px activity was measured in gastrointestinal tract tissue homogenate supernatants after isolation from cocks using a GSH-Px detection kit (Nanjing Jiancheng Bioengineering Institute, China) according to the manufacturer’s protocol (Nanjing Jiancheng Bioengineering Institute, China). Total GSH-Px content was measured at 412 nm. The MDA contents in gastrointestinal tract tissue homogenates were assayed using kits (Nanjing Jiancheng

The Role of Oxidative Stress in Gastrointestinal Tract Tissues

Fig. 1 Histopathological damage of the gastrointestinal tract. a Gizzard of the C group (×60 magnification). HE stain. b Glandular stomach of the C group. c Duodenum of the C group. d Jejunum of the C group. e Ileum of the C group (×60 magnification). HE stain. f Cecum of the C group. g

Rectum of the C group. a Gizzard of the H group. b Glandular stomach of the H group. c Duodenum of the H group. d Jejunum of the H group. e Ileum of the H group. f Cecum of the H group. g Rectum of the H group

Bioengineering Institute, PR China) according to the manufacturer’s protocol. Supernatant absorbance was measured at 532 nm. The OH· inhibition ability in the homogenates of gastrointestinal tract tissues was measured using a hydroxy radical detection kit according to the manufacturer’s protocol (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Absorbance of the supernatant was measured at 600 nm.

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The Role of Oxidative Stress in Gastrointestinal Tract Tissues Induced by Arsenic Toxicity in Cocks.

Arsenic (As) is a widely distributed trace element which is known to be associated with numerous adverse effects on human beings and animals. Arsenic ...
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