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Feb 20, 2009 - Abstract Altered copper homeostasis and oxidative stress have been observed in patients with hepatocellular carcinoma. Non-ceruloplasmin ...
Biol Trace Elem Res (2009) 130:229–240 DOI 10.1007/s12011-009-8338-5

Relevance of Non-ceruloplasmin Copper to Oxidative Stress in Patients with Hepatocellular Carcinoma Arumugam Geetha & Panneerselvam Saranya & Sam Annie Jeyachristy & Rajagopal Surendran & Arunachalam Sundaram

Received: 18 January 2009 / Accepted: 3 February 2009 / Published online: 20 February 2009 # Humana Press Inc. 2009

Abstract Altered copper homeostasis and oxidative stress have been observed in patients with hepatocellular carcinoma. Non-ceruloplasmin copper, the free form, is a potent prooxidant than the protein bound copper. The aim of the present study was to evaluate which form of copper can be correlated with the oxidative stress in the circulation and in the malignant liver tissues of hepatocellular carcinoma patients. Hepatocellular carcinoma patients (grades II and III, n=18) were enrolled in this study. Serum levels of total, free and bound copper, ceruloplasmin, iron, iron-binding capacity, lipid peroxidation products, and enzymatic and non-enzymatic antioxidants were quantified in serum and in malignant liver tissues and compared with those of normal samples (n=20). A significant positive correlation between the serum non-ceruloplasmin copper and lipid peroxidation products and negative correlation with antioxidants were observed in hepatocellular carcinoma patients. In liver tissue, glutathione peroxidase, superoxide dismutase, and catalase activity were significantly decreased with concomitant elevation in oxidative stress markers. Our experiment revealed that the elevation in non-ceruloplasmin copper has high relevance with the oxidative stress than the bound copper. Keywords Hepatocellular carcinoma . Ceruloplasmin . Non-ceruloplasmin copper . Oxidative stress . Antioxidants

A. Geetha (*) : P. Saranya : S. Annie Jeyachristy Department of Biochemistry, Bharathi Women’s College, Chennai 600 108, Tamil Nadu, India e-mail: [email protected] R. Surendran Department of Surgical Gastroenterology and Proctology, Stanley Medical College and Hospital, Chennai 600 108, Tamil Nadu, India A. Sundaram Department of Pathology, Stanley Medical College and Hospital, Chennai 600 108, Tamil Nadu, India

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Introduction Hepatocellular carcinoma (HCC) ranks as fifth most common cancer in the world with increasing incidence of new cases every year [1]. This tumor is increasingly associated worldwide with estimates of hepatitis B and hepatitis C prevalence [2]. Cirrhosis may be premalignant irrespective of etiology [3]. The hepatocyte necrosis and mitosis favor nodular regeneration which, under appropriate circumstances, is followed by hepatocyte dysplasia and carcinoma [4]. Excessive formation of reactive oxygen species and reactive nitrogen species are reported in various stages of neoplastic transformation [5]. Pro-oxidant/antioxidant imbalance and thereby oxidative stress has been found in patients with chronic liver diseases including carcinoma. Few reports also showed that hepatocellular carcinoma is associated with free radical production and their defective clearance by antioxidants [6, 7]. Iron and copper are essential components of cellular metabolism. Many enzymes and protein require iron and copper for their normal functions. These metals in their “free” and “unbound form” initiate free radical reaction and act as pro-oxidants to propagate the formation of toxic oxygen-free radicals which alter the functions of important biomolecules such as lipids and proteins [8]. The levels of the “free copper” also known as nonceruloplasmin copper (NCC) and iron are finely controlled by ceruloplasmin and ironbinding proteins, respectively, in the circulation. It has been reported that copper content of plasma and hepatocytes elevated in liver malignancy [9]. There are ample number of evidences stating that copper is angiogenic and pro-oxidative in nature [10, 11]. But the study on the relative levels of free and bound copper (BC) and their relative influence on free radical formation in HCC is limited. Hence, the present study was undertaken to emphasize the influence of free copper on free-radical-mediated oxidative stress in hepatocellular carcinoma patients. For this study, we have carried out simultaneous measurement of different forms of copper, lipid peroxides, enzymatic, and non-enzymatic antioxidants in the blood and in the malignant liver tissues obtained from hepatocellular carcinoma patients.

Subjects and Methods Materials Thiobarbituric acid (TBA), malondialdehyde, xylenol orange, butylated hydroxytoluene, Griess reagent, dithionitrobenzoic acid, phenazine methosulphate (PMS), nitroblue tetrazolium (NBT), reduced nucleotide adenine dinucleotide (NADH), Tris–HCl, and reduced glutathione (GSH) were purchased from Sisco Research Laboratories, Mumbai, India. Rabbit anti-mouse nitrotyrosine antibody was purchased from Sigma-Aldrich Chemicals, Bangalore, India, and goat anti-rabbit IgG secondary antibody was purchased from Genei Laboratories, Bangalore, India. Subjects Blood samples were collected from 18 patients with hepatocellular carcinoma and age- and sex-matched normal volunteers after obtaining informed consent. Hepatocellular carcinoma patients who underwent hepatic resection at Stanley Medical College and Hospital, Chennai and affiliated hospitals of Dr. M.G.R. Medical University, Chennai were the subjects of this study. The clinical history of the patients is given in Table 1. Liver biopsy samples were

Relevance of Non-ceruloplasmin Copper to Oxidative Stress

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Table 1 Clinical Characteristics of Patients Characteristics

Number of patients HCC patients

Normal

Total number

18

20

Male/Female ratio

15/3

15/5

Age

30–50

30–50

Types of tumor Adenocarcinoma

10

Cholangiocarcinoma (intrahepatic) Hepatic angiosarcoma Grade

3

– –

I

5 –

II

3

III

15

collected from the patients. We could collect only eight autopsied normal liver samples during postmortem. Blood Sampling A portion of blood was collected in sterile vials containing EDTA as anticoagulant and centrifuged at 1,000×g for 15 min. Plasma was carefully separated. Buffy coat was removed and the packed cells were washed thrice with 0.89% saline. Another portion of the blood was collected without any anticoagulant and serum was separated. Biochemical estimations were carried out immediately. A part of the resected tissue was fixed in 10% formalin–saline, embedded in paraffin, processed for histopathological observation, and stained with hematoxylin–eosin. Another part of the tissue was quickly washed in isotonic buffer and homogenized in 0.1 ml Tris– HCl buffer, pH 7.5, and used for the assay of lipid peroxides and antioxidant enzymes. Remaining part of the tissue was processed for copper estimation. Methods Total copper (TC) was determined by flame atomic absorption spectroscopy (FAAS). Sample preparation for serum copper determination was done by the method of Evenson and Warren [12] by diluting the serum tenfold with 10 mM nitric acid to give a final pH of about 3.0, and tissue copper was determined by the method of Carthew and Dey [13]. This is a simple method using tissue extraction of copper in 8 M nitric acid for 45 min at 100°C followed by sample dilution and quantification by FAAS. Iron and ceruloplasmin were quantified by standard methods of Ramsay [14] and Sunderman and Nomoto [15], respectively. NCC and BC levels were calculated based on the formula given by Walshe [16] and Gaffney et al [17]. Serum iron-binding capacity was determined by the method of Ramsay [18]. Lipid peroxides in terms of thiobarbituric acid reacting substances (TBARS) and lipid hydroperoxides (LHP) were measured by the methods of Draper and Hadley [19] and Jiang et al. [20], respectively. Serum concentrations of nitric oxide (NO) [21], nitrosothiol (NOSO) [22], and nitroprotein [23] were also determined.

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Superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) were assayed in red blood cells (RBC) by the methods of Kakkar et al. [24], Sinha [25], and Flohe and Gunzler [26], respectively. SOD assay was done by a modified method from that of Mishra and Fridovich [27]. In the modified method, NBT/PMS/NADH system was used as superoxide generator instead of xanthine/xanthine oxidase in the original method. Red blood cells (1–5×106) were washed with 10 ml ice-cold phosphate buffered saline in a 100nm dish. After keeping the dish on ice, 1 ml lysis buffer (10 mM Tris, pH 7.5, 150 mM NaCl, 0.1 mM EDTA, 0.5% Triton 100) was added and incubated on ice for 10 min. Cells were collected with a rubber policeman, transferred to a microfuge tube, and centrifuged at 12,000×g for 10 min. The supernatant lysate was used as the enzyme source. The enzymes were also assayed in liver homogenate. For homogenization, 5–10 ml of the same buffer was used per gram tissue and centrifuged at 12,000×g for 10 min. The supernatant was used as the enzyme source. The total sulphydryl content was determined by the method of Moron et al. [28]. Vitamins E and C, the major non-enzymatic antioxidants, were also determined by the method of Baker et al. [29] and Jacob [30], respectively. The method of Bradford [31] was used for the estimation of proteins. Data were analyzed using a commercially available statistic software package (SPSS for Windows, version 7.5). Dunnett’s test was used to calculate the level of significance. Spearman’s rank test was carried out for correlation analyses. Results were presented as mean±SD, and the p value