Abstract
Background
Excess reactive oxygen species related to neoplasia of liver has been established. Essentially, the human body has developed different antioxidant systems for defence against these attacks. To evaluate the redox status in hepatocellular carcinoma (HCC) induced by hepatitis B virus (HBV), the most important aetiological factor in Taiwan, changes in O2 . generation, lipid peroxidation as well as antioxidant status in the blood of HCC patients with HBV carriers for more than 20 years were measured.
Methods
Superoxide anion radical (O2 .−) generation and the levels of malondialdehyde (MDA) served as an index of lipid peroxidation along with the analyses of activities of superoxide dismutase (SOD), glutathione peroxidase (GPx) and glutathione reductase (GRx); also, glutathione status, including reduced glutathione (GSH) and oxidized glutathione (GSSG), and the levels of vitamins A, C and E were determined.
Results
In 54 patients, the levels of O2 .−, MDA and GSSG, and the activities of SOD and GRx of blood were significantly higher than those of 57 controls. Conversely, the levels of GSH and total GSH, and GSH/GSSG ratio, and vitamins A and C were significantly decreased. Additionally, there were no significant changes in the activity of GPx and the levels of vitamin E.
Conclusions
Our data suggest that the redox statuses in patients with HBV-associated HCC were elevated or decreased in certain parameters. However, the increased activities of antioxidant enzymes may be a compensatory up-regulation and the decrease antioxidant statuses were responses to the enhanced oxidative stress in those patients.
Introduction
Hepatocellular carcinoma (HCC) is one of the most common cancers in the Chinese population. 1 It is also one of the most deadly diseases, with a five-year survival rate of less than 5% without treatment. Commonly, any chronic inflammatory liver disease has the potential to develop HCC. Meanwhile, based on the present knowledge, the most common pathophysiological process associated with the carcinogenesis is liver cirrhosis, which could be caused by hepatitis B or C virus infection or alcohol consumption. 2 Among these risk factors, chronic hepatitis B virus (HBV) infection was considered as the most important aetiological factor in Taiwan. 3 In addition, it was estimated that around 350 million people worldwide suffered from chronic HBV infection, and many of them may eventually develop HCC. 4
Reactive oxygen species (ROS), including superoxide anion radical (O2 .−), plays an important role in carcinogenesis such as leukaemia, breast cancer, etc. 5,6 ROS has the potential to react with polyunsaturated fatty acids, and then releases toxic and reactive aldehyde metabolites such as malondialdehyde (MDA), one of the bio-markers of lipid peroxidation. MDA has also been reported to be involved in tumour promotion as it can interact with the functional groups of a variety of cellular compounds. 7 Different antioxidant systems, including antioxidant enzymes such as superoxide dismutase (SOD), glutathione peroxidase (GPx) and glutathione reductase (GRx), and non-enzymatic antioxidants such as glutathione (GSH) and vitamins A, C and E within the human body are required to lessen the damage of ROS. 8
Our previous study had revealed that GSH statuses in HCC patients were significantly decreased, and this evidence implied that free radical cytotoxicity played an important role in the pathophysiology of HCC. 9 The balance of the redox status is complicated in the human body, which is affected by many aforementioned parameters. 8 These parameters are often inextricably linked and, as a result, it is hard to judge the true meaning of the balance of the redox system by one of these parameters alone. Even though Teufelhofer et al. 10 had documented that the status of O2 .− could be associated with HCC in mice; evidence of the detailed assessment of the redox status in HCC patients is still limited. Therefore, we launched this study to assess changes in oxidant status, including O2 .− and MDA, in the blood of HBV-associated HCC patients who had been carriers of HBV for more than 20 years. Furthermore, the activities of SOD, GPx and GRx, GSH status, and the levels of vitamins A, C and E were evaluated.
Patients and methods
Patients
The present study was based on 54 newly diagnosed HBV-associated HCC patients (mean age 57 ± 29 years, ranging from 31 to 81 years old, male/female = 38/16), with a history of being HBV carriers for over 20 years, recruited from the Department of General Surgery, Kaohsiung Medical University Chung-Ho Memorial Hospital. In order to reduce the complexity, hepatitis C virus (HCV) infection cases and co-infection cases of HBV and HCV were excluded from this study. A group of 57 controls (male/female = 37/20) without HBV infection who were recruited from the Department of Physical Examination in the same hospital served as control subjects with a mean age of 58 ± 10, ranging from 46 to 74 years old. The general characteristics of the study subjects are listed in Table 1. Both cases and controls did not have the following habits of taking any dietary supplements of vitamins A, C or E, cigarette smoking, or alcohol consumption before blood sampling. Regarding smoking habits, all cases of HBV-associated HCC patients claimed that they quit smoking after having been diagnosed if they had been smokers before. These patients were clinically classified as stage I (38 patients), stage II (11 patients), stage III (5 patients) and stage IV (0 patient) according to the TNM system. In most of the patients, tumour cells were moderately differentiated and invaded nearby vessels in 10 cases of all. None was diagnosed as a recurrent case. In addition, any volunteer controls who suffered from diseases such as diabetes mellitus, rheumatoid arthritis or any other malignancies, which had the potential to affect the results of oxidant or antioxidant status, were excluded from this study. The study was approved by the ethical committee of Kaohsiung Medical University and informed consent was obtained from all the participants.
General characteristics of patients with HBV-associated HCC and controls
HBV, hepatitis B virus; HCC, hepatocellular carcinoma; SD, standard deviation
*Assessed by Chi-square test
Blood sample collection and preparation
Fasting blood samples were collected from patients after diagnosis and right before any substantial treatment, especially chemo- or radiotherapy, and also from controls. Heparinized blood samples obtained from the study subjects were processed immediately to measure O2 .− generation. In addition, we collected another set of blood samples in ethylenediamine tetraacetic acid (EDTA) tubes. The activities of GPx, SOD and GRX were measured by using EDTA-whole blood. The plasma was separated by centrifugation at 3000 rpm for 10 min at 4°C and used for the analysis of MDA and vitamins A, C and E. After the separation of plasma in EDTA blood, the buffy coat layer was removed and erythrocytes (RBC) were washed three times with normal saline. Aliquots of the washed RBC were prepared for the GSH status assay. After the initial treatment, the whole blood, plasma and haemolysate of EDTA blood were stored at −20°C until assay, except for GSH status, which was stored at −80°C. 11 Haemolysed samples were excluded from the study.
Reagents and chemicals
All the chemicals and reagents used in the study were of analytical grade. Lucigenin, thiobarbituric acid (TBA), boric acid, metaphosphoric acid (MPA), GSH, oxidized glutathione (GSSG), trichloroacetic acid (TCA) and Tricine were obtained from Sigma (St Louis, MO, USA). SOD, GPx and GRx commercial kits were obtained from Randox Laboratories (Antrim, UK).
Measurement of O2 .− generation
O2 .− generation was measured by lucigenin-based chemiluminescence, which had been described in our previous study. 12 Briefly, 0.5 mL of whole blood was mixed with 1 mL of 2 mmol/L lucigenin solution. After being gently mixed, the reaction mixture was placed in a flat-bottomed reaction cuvette at an appropriate position in the black-box unit of an ultraweak chemiluminescence analyser with a high-sensitivity detector (3.3 × 10−15 W/(cm2 × count)) from Jye Horn Co. (Taipei, Taiwan). Photon emission was counted at 37°C for 10 min under atmospheric conditions.
Measurement of MDA
Products of lipid peroxidation in plasma were estimated by the TBA method. 13 MDA, which is a stable end product of fatty acid peroxidation, reacts with TBA at acidic conditions to form a complex that has maximum absorbance at 532 nm. Three hundred microlitres of the sample were mixed with 1.5 mL of 0.05 mmol/L HCl and 0.5 mL of 0.67% TBA, and then mixed and boiled well in heated water at 95°C for 30 min. After cooling, the products were extracted in 2 mL of 15% butanol and centrifuged at 2500 rpm at 4°C for 30 min. The absorbance of the supernatant was determined at 532 nm.
Measurement of SOD activity
The method of SOD assay is based on the generation of O2 .− produced by xanthine and xanthine oxidase (XOD), which react with 2-(4-iodophenyl)-3-(4-nitrophenol)-5-phenyltetrazolium chloride (INT) to form a red formazan dye measured by a spectrophotometer (Beckman Coulter, CA, USA). SOD activity was measured by the degree of inhibition of this reaction. The results were expressed as U/g Hb.
Measurement of GPx activity
GPx activity was measured by commercial Ransel kits (Randox Laboratories, Antrim, UK) and determined according to the method of Paglia and Valentine. 14 Briefly, because GPx catalyses the oxidation of GSH by cumene hydroperoxide, in the presence of GRx and NADPH, GSSG is immediately converted to the reduced form with concomitant oxidation of NADPH to NADP+, which could be measured by a spectrophotometer (Beckman Coulter, CA, USA). The change in absorption was observed at 340 nm. The results were expressed as U/g Hb.
Measurement of GRx activity
GRx activity was measured by commercial GR kits (Randox Laboratories, Antrim, UK). The method is based on the fact that GRx catalyses GSSG in the presence of NADPH, which is oxidized to NADP+. The decrease in absorbance at 340 nm was measured by a spectrophotometer (Beckman Coulter, CA, USA). The results were expressed as U/g Hb.
GSH status analysis
GSH status analysis was assayed according to the method of Lin et al. 11 Briefly, 100 μL aliquots of washed RBC were added to 300 μL ice-cold 5% MPA. To precipitate proteins completely, the samples were vortexed and incubated on ice for 10 min. After centrifugation at 12,000 rpm for 10 min at 4°C, the supernatants were filtered through a 0.2 μm filter and diluted five times before being injected into the P/ACE-MDQ capillary electrophoresis system (Beckman Coulter, CA, USA), equipped with a fixed wavelength UV detector. Beckman P/ACE MDQ software was used for instrument control. Data were quantified on the basis of corrected peak areas with migration time.
Measurement of vitamin A
Plasma vitamin A was determined by a modified fluorometric method. 15 A total of 100 μL of plasma was drawn into a new Pyrex tube; 1 mL of double-distilled water was added and mixed; and 3 mL of absolute ethanol was added and vortexed. We used 5 mL of hexane to extract vitamin A by vortexing for 1 min. After centrifugation at 2500 rpm for 10 min at 4°C, the supernatant was drawn to a crystal cuvette to determine its concentration by a fluorescence spectrophotometer (Hitachi, Tokyo, Japan). The excitation wavelength was 340 nm and the emission wavelength was 480 nm. Serial dilutions of 4000 μg/mL vitamin A acetate solution and a solution of double-distilled water were applied as standards and a blank.
Measurement of vitamin E
Plasma vitamin E was also determined by a modified fluorometric method. 15 In all, 100 μL of plasma was mixed with 1 mL of double-distilled water; 3 mL of absolute ethanol was added and vortexed. We used 5 mL of hexane to extract vitamin E. After centrifugation, the supernatant was adopted to determine the concentration of vitamin E with a fluorescence spectrophotometer (Hitachi, Tokyo, Japan). The excitation wavelength was 295 nm and the emission wavelength was 340 nm. Serial dilutios of 200 μg/dL of vitamin E solution and a solution of double-distilled water were applied as standards and a blank.
Measurement of vitamin C
Plasma vitamin C was determined by the method of Lee et al. 16 Briefly, 200 μL of plasma was drawn into a microcentrifuge tube that contained 50 μL of 25 μmmol/L dithiothreitol, and 100 μL of 12% TCA was placed in the tube. To precipitate proteins completely, the samples were vortexed and incubated for 10–30 min. After centrifugation at 12,000 rpm for 10 min at 4°C, a 200 μL supernatant was drawn into a new microcentrifuge tube. Finally, we used 200 μL of ether to extract vitamin C. After centrifugation, the liquid in the lower layer was filtered through a 0.2 μm filter before being injected into the P/ACE-MDQ capillary electrophoresis system (Beckman Coulter, CA, USA), equipped with a fixed wavelength UV detector to determine the concentration of vitamin C. In all, 100 μg/mL isoascorbic acid was applied as the internal standard. Beckman P/ACE MDQ software was used for instrument control. Data were quantified on the basis of corrected peak areas with migration time.
Statistical analysis
All statistical analyses were conducted by using SPSS Version 10.0. Data were expressed as mean ± standard deviation. Statistical significance was assessed with Student's t-test. Two-side P < 0.05 were considered to be significant.
Results
Levels of O2 .−
As shown in Figure 1, the levels of O2 .− in the blood of HBV-associated HCC patients were significantly higher than those of controls (P < 0.05). Meanwhile, the levels of O2 .− in the blood of HBV-associated HCC patients were nearly 1.36 times higher than those of controls (404 ± 145 versus 304 ± 138 counts/10 s × 103 WBC).
Statistical analyses of the superoxide anion radical levels in the blood of the 54 patients and 57 controls with Student's t-test (P < 0.05)
Levels of MDA
The levels of MDA in the blood of HBV-associated HCC patients were also significantly higher than those of controls, as shown in Figure 2 (P < 0.01). In addition, the levels of MDA in the blood of HBV-associated HCC patients were almost twofolds those of controls (2.74 ± 0.63 versus 1.34 ± 0.88 μmmol/L).
Statistical analyses of the malondialdehyde levels in the blood of the 54 patients and 57 controls with Student's t-test (P < 0.01)
Activity of antioxidant enzymes
The activities of SOD and GRx in the blood of HBV-associated HCC patients were significantly higher than those of controls, as demonstrated in Figure 3a–c (P < 0.01 for both); nevertheless, there were no statistically significant changes in the activities of GPx between the two groups.
Analyses of the (a) superoxide dismutase (SOD), (b) glutathione peroxidase (GPx) and (c) glutathione reductase (GRx) activities in the blood of the 54 patients and 57 controls with Student's t-test. Both the SOD and GRx activities were significantly different (P < 0.01 and P < 0.05, respectively), while there were no significant changes in the GPx activity
GSH status
As shown in Figure 4b, the levels of GSSG in the blood of HBV-associated HCC patients were significantly higher (P < 0.01); however, the levels of GSH (Figure 4a) and the ratio of GSH/GSSG (Figure 4c) were significantly lower (P < 0.05 and 0.01, respectively) than those of controls.
Analyses of the levels of (a) glutathione (GSH) and (b) oxidized glutathione (GSSG), (c) GSH/GSSG ratio and (d) total GSH status in the blood of the 54 patients and 57 controls with Student's t-test (P < 0.01 for GSH, GSSG, GSH/GSSG ratio, and P < 0.05 for total GSH status). aTotal GSH are expressed as equivalent GSH + 2GSSG
Levels of vitamins A, C and E
As shown in Figure 5a–c, the levels of vitamins A and C in the blood of HBV-associated HCC patients were significantly lower than those of controls (P < 0.01 for both). However, there were no statistically significant changes in the levels of vitamin E between the two groups.
Analyses of the (a) vitamin A, (b) C and (c) E levels in the blood. The vitamins A, C and E levels of the 54 patients and 57 controls were compared with Student's t-test. Both the vitamins A and C levels were significantly different (P < 0.01), while the vitamin E level did not show significant changes
Discussion
It is fully established that excessive generation of ROS with exhausted antioxidant defence systems can cause oxidative damage. 7 Meanwhile, a growing body of evidence has indicated that this disproportionate oxidative damage is involved in carcinogenesis. 5–10 With the production of oxygen species and over-production, higher rates of lipid peroxidation have been found in some neoplastic lesions, including liver cancer. 17 We followed the aforementioned lead and launched this present study to evaluate the radical-related status and antioxidant defence system in the blood of HBV-associated HCC patients. Interestingly, most of the laboratory data in this study consistently showed that patients could suffer from oxidative stress in their disease process, and therefore they seemed to consume more antioxidants.
MDA, a product of lipid peroxidation (which is the oxidation of polyunsaturated fatty acids in membranes induced by free radicals), is an important indicator of oxidative damage. The present study showed that both the rates of O2 .− production and the levels of MDA were significantly higher in the blood of our patients than in those of controls. Thus, those patients had obviously experienced oxidative stress. Increased MDA levels in cancerous lesions or plasma have been reported in many previous studies including breast cancer 6,18 and lung cancer, 18 which were also consistent with our results.
In addition, in this study, the different changes in the levels of GSH and GSSG in HBV-associated HCC patients indicated that the patients had undergone oxidative stress. GSH, abundant in most cells, is an important substrate for GPx and glutathione transferase and acts by quenching free radicals; 19 therefore, evaluation of the GSH system in different tumours can reveal its potential usefulness in a clinical setting. It is implicated in the cellular defence against xenobiotics and deleterious compounds, such as free radicals and hydroperoxides. 20 A decrease in blood GSH in circulation has been reported in several diseases, including malignancies. 21 The significant decrease in the GSH level and increase in the GSSG level, an oxidized form of GSH, of HCC patients were also consistently revealed in our data.
In the antioxidative enzyme defence system, SOD as well as GPx and GRx play an important role in defending against oxidative stress. From previous studies, SOD or GPx activity was lower in HCC than in normal liver cells. 22 The present study revealed that the blood SOD and GRx activities of our patients were significantly increased, which implied that patients endured oxidative stress as well. In addition, blood GPx activity was increased; however, not significantly.
It has been documented that lower antioxidant capacity and oxidant–antioxidant imbalance could play important roles in multi-stage carcinogenesis. 23 Vitamin E is a free radical quencher present in cell membranes and plasma lipoproteins. It blocks the chain reaction of lipid peroxidation by scavenging peroxyl radicals and is simultaneously converted to a tocopherol radical. 24 The tocopherol radical can be reconverted back to α-tocopherol by the consumption of vitamin C, which should be supplied from food. Thus, the levels of vitamin C serve as an important indicator of oxidative stress in the human body. In addition, it is well known that vitamin A can also directly scavenge free radicals. 24 In the present study, the levels of vitamins A and C were significantly decreased in the blood of HBV-associated HCC patients. The reason more antioxidants being consumed could be explained by the higher oxidative stress in HCC patients. The levels of vitamin E were also decreased, although not significantly.
There remain some limitations on this study. First of all, the blood samples of the HCC patients were drawn after their diagnosis and right before treatment. The higher oxidative status was measured after HBV-associated HCC had developed. Therefore, from the results of the present study, we cannot make any inference to assert that oxidative damage caused liver neoplasm. However, we believe that HBV-associated HCC patients experienced more oxidative stress in their disease process from this study. Secondly, dietary differences have been reported to influence oxidative statuses. 25 Even though we did not document any dietary information from all the study subjects, HCC patients and all the contributor controls lived in southern Taiwan, which is a relatively homogeneous environment. Therefore, differences in dietary intake may be negligible or not likely to be significant.
In summary, changes in the levels of O2 .−, MDA, GSH status and antioxidants consistently demonstrated that HBV-associated HCC patients had undergone oxidative damage during their clinical disease progression. Meanwhile, the increased activities of erythrocyte antioxidant enzymes may be the compensation in response to this enhanced oxidative stress. It deserves further investigation to determine whether a poorer oxidative status could affect the prognosis of HCC.
DECLARATIONS