Increased metabolizing activities of the tricarboxylic acid cycle and decreased drug metabolism in hepatocellular carcinoma

Hiroyuki Fukuda1,4, Masaaki Ebara1, Mitsunobu Okuyama2, Nobuyuki Sugiura1, Masaharu Yoshikawa1, Hiromitsu Saisho1, Ryo Shimizu2, Naomi Motoji2, Akiyo Shigematsu2 and Takaho Watayo3

1 Department of Medicine and Clinical Oncology, Graduate School of Medicine, Chiba University, Inohana 1-8-1, Chuo-ku, Chiba-shi, Chiba 260-0856, Japan,
2 Institute of Whole Body Metabolism, Nauchi 340-2, Shiroi, Chiba 270-1407, Japan and
3 Department of Surgery, Tokyo Metropolitan Ebara General Hospital, Higashiyukigaya 4-5-10, Ohta-ku, Tokyo 145-0065, Japan


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The aim of this study was to determine the metabolizing activities in the liver of patients with hepatocellular carcinoma (HCC). Thin-layer chromatography autoradioluminography was used to measure metabolizing activities. Succinate-producing activity (SPA) was used as an indicator of metabolizing activity of the tricarboxylic acid (TCA) cycle in mitochondria, and diazepam-N-demethylating activity (DZ-DA), diazepam-3-hydroxylating activity (DZ-HA) and tolbutamide-methyl-hydroxylating activity (TB-HA) as indicators of drug metabolizing activities by P-450. SPA and drug-metabolizing activity of HepG2 cells were examined to compare with those of human liver specimens. Liver biopsy specimens of 30 patients and surgical specimens of eight patients with HCC were studied. SPA of HepG2 cells was as high as that of human tumor tissue, and DZ-DA, DZ-HA and TB-HA were undetectable in HepG2 cells. SPA and DZ-HA of non-tumor tissue in biopsy samples were significantly higher than those in resected liver specimens (P < 0.05). In biopsy specimens, SPA was significantly higher in tumor tissue than in non-tumor tissue (P < 0.05), and DZ-DA, DZ-HA and TB-HA were significantly lower in tumor tissue (P < 0.01). SPA was significantly higher in large tumors (>=30 mm) than in small tumors <30 mm (P < 0.05), and TB-HA was significantly lower in large tumors than in small tumors (P < 0.01). DZ-DA and TB-HA significantly decreased with the progression of the tumor differentiation (P < 0.05). In conclusion, HCC has increased metabolizing activities of the TCA cycle and decreased drug-metabolizing activities.

Abbreviations: DMA, drug-metabolizing activity; DZ-DA, diazepam-N-demethylating activity; DZ-HA, diazepam-3-hydroxylating activity; G-6-P, glucose-6-phosphate; HCC, hepatocellular carcinoma; SPA, succinateproducing activity; TB-HA, tolbutamide-methyl-hydroxylating activity; TCA, tricarboxylic acid; TLC-ARLG, thin-layer chromatography autoradioluminography.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Hepatocellular carcinoma (HCC) is one of the most common cancers in the world (13). HCC is generally considered to be a hypervascular tumor, and vascular endothelial growth factor contributes to the angiogenesis of HCC (4,5). It is thought that tumors require oxygen and nutrients, which are supplied through neovascularization. However, there are only a few studies on the metabolism of rat hepatocytes and HCC (6). In regenerating rabbit liver after hepatectomy, the mitochondrial respiration is elevated for increased production of ATP for growth (7). Mitochondria, isolated from rat HCC, oxidize many NAD-linked respiratory substrates at rates 1.5–4 times faster than those from normal rat liver (8). The activities of cytochrome P-450s of cirrhotic liver or hyperplastic nodules in the rat liver are lower than those in normal controls (9,10). It is therefore speculated that human HCC may have higher mitochondrial tricarboxylic acid (TCA) cycle activities to produce large amounts of ATP for growth compared with non-tumor liver parenchyma and lose the original drug metabolizing activities of hepatocytes. However, there is no report evaluating both mitochondrial oxidative phosphorylation and cytochrome P-450 activities in fresh human HCC cells and non-tumor tissue simultaneously, because of the technical difficulties with a limited amount of fresh tissue.

In pharmacokinetic studies, HPLC (1113), GC (14) and GC-MS (15,16) have been used for the identification and quantification of metabolites. However, these methods are complicated and difficult to perform. Even though large samples can be obtained from resected specimens, the tissue metabolism in these samples is greatly reduced (17,18). Even though fresh biopsy samples can be obtained, the amount of tissue is too small for measurement by the former methods. However, because of the high sensitivity of thin-layer chromatography autoradioluminography (TLC-ARLG), tissue metabolism can now be measured with only a very small quantity of radioisotope and a liver sample obtained by biopsy in rats (1922). TLC-ARLG also has the advantage of repeated use of imaging plates (23).

In this study, we investigated both the metabolism of [14C]acetate using human liver samples from normal control, tumor and non-tumor tissue from livers with HCC, to evaluate TCA cycle activities, and the activities of P-450 simultaneously.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Cell culture of human hepatocellular carcinoma
HepG2 cells were obtained from American Type Culture Collection (Washington DC) and were maintained in monolayer culture in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Rockville, MD) containing 10% fetal bovine serum, 100 U/ml penicillin, 100 µg/ml streptomycin, 1% non-essential amino acids and 1 mM pyruvic acid at 37°C in culture dishes in a 5% CO2 incubator.

Patients
Between March 1996 and December 1999, biopsies of tumor and non-tumor tissue were performed in 30 liver cirrhosis patients with HCC for differential diagnosis. We performed liver biopsies two times, to obtain tumor and non-tumor specimens, in all HCC patients. Non-tumor tissue means the surrounding liver parenchyma, collected from patient with HCC. One part of each specimen was used for this study. Liver resection was performed in eight liver cirrhosis patients with HCC. Surgically resected specimens of normal liver from eight patients with metastatic liver tumors were examined as normal control. Normal tissue means liver without liver disease. Patients gave informed consent to all clinical investigations, which were performed in accordance with the principles embodied in the Declaration of Helsinki.

The patients consisted of 20 men and 10 women, with a mean age of 62.7 ± 7.6 (mean ± SD) years. The average tumor diameter was 37.4 ± 29.9 mm (mean ± SD), ranging from 14 to 150 mm. The diagnosis of HCC was confirmed by histologic examination of sonographically guided biopsy specimens from every lesion. All HCC patients were considered to have associated liver cirrhosis according to the findings of biopsy, ultrasonography, magnetic resonance imaging, computed tomography and other clinical examinations. The grade of liver dysfunction in the 30 HCC patients was classified as Child A in 18 (60%), Child B in nine (30%) and Child C in three (10%) patients. The etiology of liver cirrhosis was considered to be hepatitis B virus infection (HBs-Ag positive) in two (6.7%), hepatitis C virus (HCV) infection (HCV-Ab positive) in 23 (76.6%), both HBs-Ag and HCV-Ab positive in two (6.7%), and both HBs-Ag and HCV-Ab negative in three (10%) patients. The last group included two (6.7%) patients with alcoholic cirrhosis.

Histological findings
Specimens from HCC and liver parenchyma were obtained using a 21-gauge biopsy needle (Sonopsy, Hakko, Tokyo, Japan) under sonographic guidance. The diagnosis of HCC was made histologically on hematoxylin-eosin-stained sections, according to WHO criteria (24). The diagnosis of chronic hepatitis was made histologically on hematoxylin–eosin stained sections, according to the new European classification (25). Samples mainly consisting of necroses or other connective tissue were not included.

Radiolabeled compounds and other chemicals
[1,2-14C]Acetate (2.07 GBq/mmol) was supplied by American Radiolabeled Chemicals (St Louis, MO). [Ring-U-14C]Tolbutamide (TB; 2.22 GBq/mmol) and [2-14C]diazepam (DZ; 2.04 GBq/mmol) were supplied by Amersham (Amersham, UK). NADP+, glucose-6-phosphate (G-6-P) and G-6-P dehydrogenase were obtained from Oriental Yeast (Osaka, Japan), and the other chemicals used were of analytical grade.

Measurement of succinate-producing activity and drug-metabolizing activity
HepG2 cells were centrifuged for 10 min at 300 g at 37°C, and succinate-producing activity (SPA) and drug-metabolizing activity (DMA) in 5 mg wet weight HepG2 cells were measured.

In the resection cases, the liver was cut into small pieces and put into ice-cold Hanks’ solution containing 0.5% bovine serum albumin. Fine liver specimens aspirated using a 21-gauge biopsy needle (Sonopsy) were tested without homogenization.

In biopsy cases, SPA and DMA in liver samples were measured directly after rinsing in Hanks’ solution without homogenization. Acetate is immediately converted in mitochondria to acetyl-CoA, which enters the TCA cycle, and succinate is produced via this cycle. Then, 2–10 µl Hanks’ solution, containing the 14C-labeled substrate (20 µM acetate, 2 µM each of TB and DZ) and an NADPH-regenerating system (15 mM MgCl2, 1 mM NADP+, 10 mM G-6-P, 1 U/ml G-6-P dehydrogenase) for TB and DZ was added to 1–5 mg of the liver sample, and total volume of the incubation was twice the volume of the wet liver sample. After incubation at 37°C for 30 (acetate) or 60 min (TB and DZ), the reaction was stopped by twice the volume of methanol, and the reaction mixture was centrifuged for 5 min. A 5 µl portion of the supernatant was developed by TLC in the upper layer of a vigorously shaken mixture of ethyl acetate/toluene/water/formic acid (6/2/2/1.5) for acetate, and chloroform/ethyl acetate/acetic acid (70/30/1) for TB and DZ, using a silica gel TLC plate (Merck, Frankfurt, Germany). The resultant TLC plate was put directly on an imaging plate (IP, Fuji Photo Film, Tokyo, Japan) and the radioactivity in the succinate or metabolite fractions was analyzed using a Bio-imaging analyzer (BAS 2000, Fuji Photo Film) followed by calculation of SPA, DZ-DA, DZ-HA and TB-HA as described previously (2022). A minimum sample of liver of 1 mg was necessary for the determination of SPA and DMA.

Statistical analysis
The measured values were expressed as mean ± standard error. Comparison between two groups was performed using Mann–Whitney rank sum test or Wilcoxon rank sum test. Comparison between multiple groups was performed using one-way ANOVA with Scheffe’s method. A P value <0.05 was considered statistically significant.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
SPA and DMA of HepG2 cells were examined to compare with those of human specimens. SPA of HepG2 cells (n = 7) was 9.56 ± 1.07 pmol/min/g tissue and the activity of DZ-DA, DZ-HA and TB-HA (n = 7) was below detection.

To investigate the influence of resection on liver samples, we compared SPA and DMA in biopsy samples with those in resected liver specimens. Figure 1Go shows the results of TLC-ARLG of radioactive metabolites in biopsy specimens and resected liver specimens. Radioactive succinate, 3-hydroxy-DZ, N-demethyl-DZ and hydroxymethyl-TB were detected more strongly in biopsy specimens than in resected liver specimens.



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Fig. 1. TLC-ARLG of metabolites of 14C-labeled substrates in biopsy and resected specimens. The amount of tissue was 3 mg wet weight in biopsy specimen, and 5 mg wet weight in resected liver specimen. The developing system was the upper layer of ethyl acetate/toluene/water/formic acid (6/2/2/1.5). SUC, succinate; 3-OH-DZ, 3-hydroxy-diazepam; N-DM-DZ, N-demethyl-diazepam; M-OH-TB, hydroxymethyl-tolbutamide.

 
In non-tumor tissue, SPA and DZ-HA in biopsy samples were significantly higher than those in resected liver specimens (P < 0.01, P < 0.05) (Figure 2a and bGo). DZ-DA in biopsy samples was higher than that in resected liver specimens, but the difference was not significant. There was no significant difference in TB-HA between biopsy and resected liver specimens. In tumor tissue, SPA in biopsy samples was higher than that in resected liver specimens, but the difference was not statistically significant. DZ-DA, DZ-HA and TB-HA in biopsy specimens were significantly higher than those in resected specimens (P < 0.01, P < 0.01, P < 0.05) (Figure 2a and bGo).



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Fig. 2. Comparison of SPA and DMA in non-tumor and tumor tissue. In biopsy samples, SPA and DMA were compared in non-tumor tissue and tumor tissue (a). In resected samples, SPA and DMA were compared in normal control, non-tumor tissue and tumor tissue (b). Values are expressed as mean ± standard error. *P < 0.05, **P < 0.01.

 
Although the metabolizing activity in biopsy cases was evaluated only in non-tumor and tumor tissue, in resected cases it was evaluated in normal liver, non-tumor and tumor tissue. In biopsy cases, SPA in tumor tissue was significantly higher than that in non-tumor tissue (P < 0.05) (Figure 2aGo). In contrast, DZ-DA, DZ-HA and TB-HA in tumor tissue were significantly lower than those in non-tumor tissue (P < 0.01) (Figure 2aGo). In resection cases, SPA in tumor tissue was significantly higher than that in normal liver and non-tumor tissue (P < 0.05) (Figure 2bGo). There was no significant difference between normal liver and non-tumor tissue (Figure 2bGo). DZ-DA and DZ-HA in tumor tissue were significantly lower than those in normal liver and non-tumor tissue in resection cases (P < 0.05, P < 0.01, P < 0.01, P < 0.05), but there was no significant difference between normal liver and non-tumor tissue (Figure 2bGo). TB-HA in tumor tissue was significantly lower than that in normal liver and non-tumor tissue in resection cases (P < 0.01), but there was no significant difference between normal liver and non-tumor tissue (Figure 2bGo).

Since SPA was found to be higher while DZ-DA, DZ-HA as well as TB-HA were lower in tumor as compared with non-tumor tissue, these metabolic activities were evaluated in relation to the tumor size. SPA in tumors >=30 mm in diameter was significantly higher than that in tumors <30 mm (P < 0.05) (Figure 3Go). In contrast, DZ-DA and DZ-HA in tumors >=30 mm in diameter were lower than those in tumors <30 mm, but the difference was not significant (Figure 3Go). TB-HA in tumors >=30 mm in diameter was significantly lower than that in tumors <30 mm (P < 0.01) (Figure 3Go).



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Fig. 3. SPA and DMA according to tumor size. SPA and DMA in tumors >=30 mm in diameter and those in tumors <30 mm were compared. Values are expressed as mean ± standard error. *P < 0.05, **P < 0.01.

 
Figure 4Go shows SPA, DZ-DA, DZ-HA and TB-HA in relation to histological findings. SPA in well-differentiated HCC was lower than that in moderately and poorly differentiated HCC, but the difference was not significant (Figure 4Go). However, DZ-DA and TB-HA in well-differentiated HCC were significantly higher than those in moderately and poorly differentiated HCC (P < 0.05) (Figure 4Go). DZ-HA in well-differentiated HCC was higher than that in moderately and poorly differentiated HCC, but the difference was not significant (Figure 4Go).



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Fig 4. SPA and DMA according to histological findings of HCC. SPA and DMA in well differentiated HCC and those in moderately and poorly differentiated HCC were compared. Values are expressed as mean ± standard error. *P < 0.05.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
It has been very difficult to carry out reliable quantitative measurements of liver metabolism in a living person. Until now, it was only possible to speculate from indirect data, and the process was very complicated. However, with the recent introduction of the TLC-ARLG method, which has a sufficiently high sensitivity, tissue metabolism can be measured with only a minimal quantity of sample. We previously applied this system to the rat liver and proved its usefulness (1922). Because there is no report about the metabolism of HCC, we applied this method in order to study both the activity of TCA cycle and P-450 simultaneously, and to study the change of the metabolism with the progression of the tumor differentiation.

Up to the present, the trypan blue exclusion test (17) and the erythrocin exclusion test (18) have been used to measure liver tissue viability. However, these tests are also complicated because the tissue must be minced. Other methods for measuring liver viability use lactate dehydrogenase (LDH) and ATP levels (26). Leakage of LDH from hepatocytes is a reliable indicator of cell damage and can be easily measured. However, the LDH level does not purely reflect the metabolic activity of hepatocytes. A large sample is required for the measurement of ATP level. Vons et al. (18) reported that the viability of biopsy material, evaluated by trypan blue exclusion test, was better than that of resected liver. In our study, we evaluated the activity of the TCA cycle and drug metabolism in liver biopsy specimens and resected liver, and compared the two. Similarly, by this method, the activity in liver biopsy specimens was higher than that in resected liver. This may be explained by the fact that liver biopsy specimens are obtained very quickly, ruling out possible ischemic damage to the sample, and further, the sample does not require slicing or incubation, so the liver cells are fresh. Olinga et al. (27) evaluated the influence of cold storage on drug metabolism in hepatocytes and slices of monkey liver, and concluded that the total phase I metabolism, as well as the formation of 7-hydroxycoumarin glucuronide, was decreased after cold storage. Similarly, in non-tumor tissue, DZ-DA in resected samples was lower than that in biopsy samples.

The ability to maintain an increased rate of mitochondrial oxidative phosphorylation and glycolysis under aerobic condition is thought to be important for rapidly growing tumors and regenerating liver. In comparison with normal cells, the activity of hexokinase in the glycolytic pathway was elevated in hepatoma cells (28). In regenerating liver, it is speculated that ATP is consumed for active biosynthesis of components such as DNA and protein, causing an increased demand for energy. As a result, in regenerating rabbit liver, the rate of mitchondrial respiration is increased after major hepatectomy (7,29). Several reports indicate that pyruvate is oxidized efficiently by many experimental tumors (3032). Cederbaum and Rubin (6) reported increased metabolizing activitiy of TCA cycle in Becker HCC H-252 in male AC1 rats. Dietzen and Davis (8) also isolated mitochondria from normal liver and hepatoma, and evaluated them with regard to their bioenergetic and metabolic properties. Hepatoma mitochondria oxidized many NAD-linked respiratory substrates at rates 1.5–4 times faster than those from liver. HCC are generally hypervascular (4,5), and it is speculated that they require a large amount of oxygen and nutrients. In our study, SPA in tumor tissue was significantly higher than that in non-tumor tissue and normal liver, both in biopsy specimens and resected liver. Similarly, SPA in HepG2 cells was also as high as that in human tumor tissue and higher than that in normal liver. Because the vascularity of HCC increases with tumor enlargement (33), it is also speculated that large HCC need more energy than small ones. And, it is reported that advanced-stage HCC have rapid growth (34). In this study, SPA in tumors >=30 mm in diameter was higher than that in tumors <30 mm.

CYP1A2, 2C9, 2C19, 2D6, 2E1 and 3A4 are important in phase I metabolism. Because only CYP1A2 is not able to be detected because of lack of a 14C-labeled compound, we measured CYP 2C9, 2C19, 2D6, 2E1 and 3A4. The activities of CYP 2C9, 2C19 and 3A4 were always detectable, so we estimated these three activities in this study. Because the allelic frequency of CYP2C19 poor metabilizers in Asian population is 13–23% (35) and there are some genotypes of CYP2C19, we compared tumor tissue with non-tumor tissue in the same patients.

Guengerich and Turvy (9) compared the levels of human microsomal cytochrome P-450 enzymes in normal and cirrhotic liver samples. Futhermore, Degawa et al. (10) studied changes in cytochrome P-450 isozymes in the early stage of hepatocarcinogenesis in male F344 rats. These studies demonstrated a decreased level of microsomes in cirrhotic liver compared with that in normal liver. Similarly, in our study, DMA by P-450 in HCC was lower than that in surrounding liver parenchyma, and DMA decreased with the tumor size and the degree of histological differentiation. DMA was not detected in HepG2 cells.

In conclusion, HCC has increased metabolizing activities of the TCA cycle and decreased DMA.


    Notes
 
4 To whom correspondence should be addressed Email: fukuhiro{at}ho.chiba-u.ac.jp Back


    Acknowledgments
 
We are indebted to Dr N.Higashi (Institute of Whole Body Metabolism) and Dr Y.Momose (Institute of Whole Body Metabolism) for technical assistance. We are also grateful to Dr Y.Shino (Department of Molecular Virology, Graduate School of Medicine, Chiba University) and Dr H.Tokita (Chiba Cancer Center) for guidance on cell cultivation. We thank Professor Emeritus K.Okuda (Department of Medicine and Clinical Oncology, Graduate School of Medicine, Chiba University) for his suggestions and critical review of the manuscript.


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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
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Received December 18, 2001; revised August 28, 2002; accepted August 28, 2002.