Int J Clin Exp Med 2015;8(7):10568-10576 www.ijcem.com /ISSN:1940-5901/IJCEM0008402 Original Article Effects of repeated high dosage of chloral hydrate and pentobarbital sodium anesthesia on hepatocellular system in rats Jianhong Yu1*, Xuehui Sun2*, Guifeng Sang3 1Department of Anesthesiology, Yuhuangding Hospital, Yantai 264000, China; 2Department of Rheumatology, Yuhuangding Hospital, Yantai 264000, China; 3Operating Room, Yuhuangding Hospital, Yantai 264000, China. *Equal contributors. Received March 24, 2015; Accepted July 2, 2015; Epub July 15, 2015; Published July 30, 2015 Abstract: This study aims to investigate the possible effects of repeated high dosage of chloral hydrate and pen- tobarbital sodium anesthesia on hepatocellular system in rats. Thirty Sprague Dawley rats were randomly divided into 3 groups: control group (group A), chloral hydrate group (group B) and pentobarbital sodium group (group C). Antioxidant enzymes superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), glutathione s transferase (GST) and catalase (CAT) activities and thiobarbituric acid-reactive substances (TBARS) level as well as serum biochemi- cal parameters alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP) and total bilirubin (T-BIL) were determined. Liver histopathological examinations were performed at termination. Furthermore, Bax and Bcl-2 expression, and caspase-3 activity were also evaluated. The SOD, GSH-Px, GST and CAT activities significantly decreased but TBARS levels increased in group B and C compared with group A. Hepatic injury was evidenced by a significant increase in serum ALT, AST and ALP activities in group B and C, which also con- firmed by the histopathological alterations. Moreover, administration of chloral hydrate and pentobarbital sodium could induce certain hepatic apoptosis accompanied by the upregulated Bax expression, the downregulated Bcl-2 expression and Bcl-2/Bax ratio, and the increase of caspase-3 activity. Repeated high dosage of chloral hydrate and pentobarbital sodium anesthesia could produce hepatotoxicity. Keywords: Chloral hydrate, pentobarbital sodium, anesthesia, hepatotoxicity, oxidative damage, antioxidant en- zymes, serum biochemistry, histopathological alterations, apoptosis, Bcl-2/Bax ratio Introduction great damage to the body of laboratory animals [1]. Pentobarbital sodium, a barbiturate, also A large number of evidences in the research acts as an important anesthetic agent in animal area of laboratory animals suggest that differ- experiment. Previous reports have demonstrat- ent experimental methods can cause animal ed that it provide a “protective” effect for the stress response [1]. If not control or intervene brain in regional [7-13], as well as global isch- in time, it will cause negative effects on the emia [14, 15]. Moreover, it also acts as an body and even affect the scientific results. important anesthetic agent in animal experi- Chloral hydrate is frequently used as a hypnotic ment. Owing to its high pH value, pentobarbital agent at low dosage, it also acts as an anes- sodium given with high dosage has a low safety thetic agent at higher dosages [2]. Side effects margin due to the potent respiratory and cardio- of chloral hydrate with high dosage mainly vascular depressant effects [1], and recovery occurred in abdominal organs. Many investiga- might be prolonged, with convulsive move- tors have confirmed that chloral hydrate given ments and “paddling”, severe tissue reactions at a high dosage can cause inflammation of the [16]. splenic capsule, gastric ulcers [3], severe ady- namic ileus [4], peritonitis, and even death [5, Although chloral hydrate and pentobarbital 6]. Besides, it also has a certain inhibitory sodium are not the first-choice anesthetic for effect on respiratory system and can cause surgical procedures and negative effects were Chloral hydrate and pentobarbital sodium anesthesia on hepatocellular system also observed, laboratories have been using sia according to the rat weight at 4.5 mL/kg, these drugs to establish their research model. group C were anesthetized by intraperitoneal Recent observations demonstrated a complex injection of 1% pentobarbital sodium according influence of chloral hydrate and pentobarbital to the rat weight at 6 mL/kg. The animals in sodium on the immune status, on the integrity group A were administered with 6 mL/kg nor- of various organ systems and on the post- mal saline. All the above administrations were anaesthetic outcome of different diseases, repeated for 5 consecutive days. After the last especially at high dosages or concentrations administration the rats were sacrificed by cervi- [17]. However, up to now there were no con- cal dislocation. They were disinfected abdomi- trolled studies focused on repeated high dos- nally, and then the abdomen was cut open to ages of chloral hydrate and pentobarbital sodi- expose abdominal veins, 2.0 mL blood was col- um anesthesia on the hepatocellular systems. lected into ethylenediamine tetraacetic acid Therefore, present study aims to explore the (EDTA) treated tubes. The livers of the rats were possible effects of repeated high dosage of quickly removed which was immediately divid- chloral hydrate and pentobarbital sodium anes- ed into two portions. Part of each liver was thesia on hepatocellular system by evaluating immediately placed in 10% (v/v) formaldehyde antioxidant enzymes, serum biochemical para- solution for electron microscopic examination meters and histopathological changes and and the rest of the livers were kept at -80°C for some apoptosis factors. subsequent assays. Materials and methods SOD, CAT, GSH-Px and GST activities and TBARS level Animals Antioxidant components in the hepatic tissue Sprague Dawley rats (330 ± 20 g, male) were samples were analyzed in the experimental purchased from the Experimental Animal rats. Liver tissues were washed with cold deion- Center of Suzhou Aiermaite technology Co. Ltd. ized water to discard blood and then homoge- (SPF grade, Certificate No. SCXK20140007). nized in 50 mM Tris-HCl, pH = 7.4 (1:10, w/v). All rats were housed in groups of five with free After centrifugation at 2400 × g for 15 min at access to food and water and kept in a regu- 4°C, the upper clear layer was taken and used lated environment with a 12 h light/dark cycle for the analyses. In this fraction thiobarbituric at 23 ± 3°C and 40-70% humidity. Each rat was acid-reactive substances (TBARS) levels were examined for clinical signs of ill health on determined by using the thiobarbituric acid receipt and observed within 7 days of arrival. method [18]. A part of the homogenate was All procedures were in accordance with the extracted in ethanol/chloroform mixture (5:3, Guidelines of the Animal Care and Use of v/v) to discard the lipid fraction, which caused Laboratory Animals from the Association of interferences in the activity measurements of Laboratory Animal Science and the Center for superoxide dismutase (SOD), glutathione per- Laboratory Animal Science of Yantai University. oxidase (GSH-Px), glutathione s transferase (GST) and catalase (CAT). The SOD, GSH-Px, Experimental design GST and CAT activities which reflected as com- mon indexes of antioxidant status of tissues Thirty Sprague Dawley rats were randomly were determined with the corresponding ELISA divided into three groups according to different kits (Nanjing Jiancheng Bioengineering Insti- methods of anesthesia: control group (group A, tute, Nanjing, China) according to the manufac- n = 10), chloral hydrate intraperitoneal injec- turer’s instructions. The results were expressed tion group (group B, n = 10) and pentobarbital as U/mg protein, IU/mg protein, IU/mg protein sodium intraperitoneal injection group (group and IU/mg protein, respectively. C, n = 10). Chloral hydrate and pentobarbital sodium were all purchased from Sinopharm Serum biochemistry Chemical Reagent Co., Ltd. (Shanghai, China). Nothing was done for group A. All rats were fed Blood samples were centrifuged at 3000 × g with the same laboratory diet during the study. for 10 min to separate plasma. Serum bio- Rats in group B were treated with intraperito- chemical parameters including alanine amino- neal injection of 10% chloral hydrate anesthe- transferase (ALT), aspartate aminotransferase 10569 Int J Clin Exp Med 2015;8(7):10568-10576 Chloral hydrate and pentobarbital sodium anesthesia on hepatocellular system Figure 1. Effects of different treatment on SOD, GSH-Px, GST and CAT activities and TBARS level in liver homogenate. A: SOD activity; B: GSH-Px activity; C: GST activ- ity; D: CAT activity; E: TBARS level. Data are presented as mean ± SD for 10 rats in each group. *P < 0.05 and **P < 0.01 vs the con- trol group. (AST), alkaline phosphatase (ALP) and total bili- malin solution, and were routinely processed rubin (T-BIL) were analyzed using a biochemical (embedded in paraffin and sectioned at 3-5 analyzer (TBA-FR40; TOSHIBA Co., Ltd., Osaka, μm). The sections were stained with hematoxy- Country). All the kits mentioned above were lin and eosin (H&E) stain for microscopic purchased from Zhongsheng Beikong Bio- examination. technology and Science Inc. (Beijing, China). The results were expressed in U/L. Western blot analysis Histopathological examination The levels of Bcl-2 and Bax were detected by western blot analysis. The hepatic tissue sam- Histopathological analysis was performed from ples were homogenized in lysis buffer contain- three animals per group and was randomly ing complete