ARTICLE |
Correspondence to: Wilma M. Frederiks, Dept. of Cell Biology and Histology, Academic Medical Center, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands. E-mail: w.m.frederiks@amc.uva.nl
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Summary |
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Oxygen insensitivity of the histochemical assay to detect glucose-6-phosphate dehydrogenase (G6PD) activity with NT as tetrazolium salt has been proved to be a powerful tool to discriminate various types of adenocarcinoma from normal tissues. Here we investigated whether this phenomenon can also be applied to differentiate between chemically induced hepatocellular (pre)neoplasms and normal liver tissue in rats. Residual activity (percentage of the amount of final reaction product that is generated in oxygen and that is generated in nitrogen) was 60% in (pre)neoplastic cells and 6% in normal liver parenchymal cells. This means that the oxygen insensitivity test is a useful tool to distinguish (pre)neoplasms from normal rat liver tissue. N-Ethylmaleimide, a blocker of SH groups, did not affect G6PD activity in (pre)neoplastic cells, whereas activity in normal cells was reduced by half. Therefore, the absence of essential SH groups in G6PD in (pre)neoplastic cells is held responsible for the oxygen insensitivity phenomenon. We conclude that oxygen insensitivity of the histochemical assay for G6PD activity is a fast, easy, and cheap tool to diagnose (pre)neoplasms in rat liver. Discrimination is likely to be based on altered properties of the enzyme in (pre)neoplastic cells. (J Histochem Cytochem 49:565571, 2001)
Key Words: hepatocellular carcinoma, glucose-6-phosphate, dehydrogenase, enzyme histochemistry, neotetrazolium, diagnosis, oxygen insensitivity test, image analysis
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Introduction |
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HEPATOCELLULAR CARCINOMA (HCC) is one of the most common human malignancies, occurring mainly in Asia and Africa. The majority of patients with HCC have a longstanding history of liver cirrhosis due to, among others, aflatoxin exposure, chronic hepatitis B and/or C viral infection, or alcohol abuse (
To discriminate between malignant cells and non-malignant cells, the histochemical assay of G6PD is performed with the tetrazolium salt neotetrazolium chloride (NT) (
Certain metabolic changes in cancer cells are held responsible for the oxygen insensitivity phenomenon, such as a combination of elevated G6PD activity, decreased superoxide dismutase activity, and decreased lipid peroxidation capacity (
In this study we investigated in rats whether the oxygen insensitivity of the histochemical assay of G6PD activity could be used for diagnosis of HCC. Furthermore, we investigated further chemical backgrounds of the oxygen insensitivity test in these cells and focused on a possible role of changes in SH groups in the active site of G6PD.
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Materials and Methods |
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Induction of HCC
HCC was induced in livers of 15 male Wistar rats by administration of 0.01% diethylnitrosamine (Sigma: www.sigma-aldrich.com, St Louis, MO) via drinking water over 6, 9, or 12 weeks (
Enzyme Histochemical Localization of G6PD Activity
Incubation media for the demonstration of G6PD activity were prepared as previously described (
Involvement of SH Groups and GSH in G6PD Activity
To establish the involvement of SH groups in G6PD activity, 10 mM N-ethylmaleimide (NEM; BDH: www.bdh.com, Poole, UK), a blocker of SH groups, was added to incubation media (1 cm2. The slides were air-dried at room temperature (RT). Then tissue sections were mounted on top of the GSH film and stored in the cryostat cabinet (-25C) until use.
Histochemical Localization of GSH
GSH was localized using a solution of 5.0 mM Mercury Orange (Sigma) in toluene. Mercury Orange forms a complex with SH groups that are present in GSH and proteins. When short incubation times are used, GSH is preferentially stained (
Image Analysis to Quantify Histochemical Reactions
Formazan production in sections was measured by image analysis according to
Biochemical Assays
Spectrophotometric analysis of G6PD activity was performed using 3 ml of a buffered 5% PVA solution containing NADP, G6P, mPMS, and NT in the concentrations as described for the histochemical assay. Solutions were saturated with either nitrogen or oxygen. After saturation, 30 µl of a 1:100 diluted G6PD solution (1000 U/ml, from Leuconostoc mesenteroides; Boehringer) in distilled water was added to start the reaction. Cuvettes were covered with glass slides to ensure that the solutions were kept in a saturated oxygen or nitrogen environment. The involvement of SH groups in the reaction was studied by adding 10 mM NEM or 0.1 or 1.0 mM GSH to the media. Increases in absorbance were measured at 585 nm using an Ultrospec III spectrophotometer (Pharmacia: www.apbiotech.com, Uppsala, Sweden).
Statistical Analysis
Statistical processing of data was performed using Excel 97 (Microsoft: www.microsoft.com, San Jose, CA) and SPSS 8.0 for Windows (SPSS: www.spss.com, Chicago, IL). Differences were considered statistically significant when p<0.05.
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Results |
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Oxygen Insensitivity of HCC
In all livers investigated, foci of undifferentiated cells were found in hematoxylineosin-stained sections. Occasionally these foci were surrounded by connective tissue. Most foci were positive after PAS staining, indicating that these cells contained glycogen (Fig 1A). This means that these cells were preneoplastic and not neoplastic foci (
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Formazan formation due to G6PD activity, as demonstrated with the oxygen sensitive tetrazolium salt NT in the presence of nitrogen, was high in foci and low in normal liver tissue (Fig 2A). When the assay was performed in the presence of oxygen, formazan was produced in foci of preneoplastic and neoplastic cells but not in normal cells (Fig 2B). In nitrogen, G6PD activity in foci of (pre)neoplastic cells was fourfold higher than in normal cells. The RA was only 6% (95% CI 1.79.9) in normal liver tissue and 60% (95% CI 45.574.5) in (pre)neoplasms. These differences were significantly different and demonstrate that G6PD activity was oxygen sensitive in normal liver tissue and partly oxygen insensitive in (pre)neoplastic foci (Table 1).
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Involvement of SH Groups in G6PD Activity
Incubations in NEM-containing media showed that the activity in normal liver tissue, but not in (pre)neoplastic foci, was sensitive to the blocker of SH groups. Activity in the presence of NEM was half of that in the absence of NEM in normal tissue, whereas in foci, G6PD activity was not affected by NEM (Table 2), indicating that the chemical properties of G6PD are different in normal tissue and (pre)neoplastic foci.
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Fig 2C shows intense fluorescence in (pre)neoplastic tissue but not in normal hepatic tissue due to complex formation of GSH and Mercury Orange. Films made of a solution of 0.1 mM GSH underneath the cryostat sections did not affect the amounts of formazan produced by G6PD activity in control liver tissue and foci in the presence of nitrogen or oxygen, whereas those made of a solution of 1.0 mM GSH abolished most formazan production in all cases (Table 3).
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Involvement of SH Groups in G6PD Activity as Determined Biochemically
The involvement of SH groups in the histochemical assay of G6PD activity in the presence and absence of oxygen was investigated further in in vitro experiments. Formation of formazan was followed in cuvettes over time as measured with a spectrophotometer. Activity of purified G6PD was sensitive to oxygen to the same extent as G6PD in normal liver tissue (RA 4%). Moreover, NEM reduced G6PD activity in nitrogen with 86%. This was even stronger than in the histochemical assay applied to normal liver tissue.
GSH (1 mM) inhibited formazan production by G6PD activity by 90%. Formazan formation in the presence of oxygen was too low to draw valid conclusions with respect to effects of NEM or GSH.
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Discussion |
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The present study shows that oxygen insensitivity of the histochemical assay for G6PD activity occurs only in (pre)neoplastic cells and not in normal liver tissue in rats (Fig 2A and Fig 2B; Table 1). Therefore, it can be used to detect (pre)neoplasm in rat liver. Oxygen insensitivity was demonstrated previously for cancer cells of human breast, bronchus, stomach, colon, and pancreas (
The extremely low RA of 6% in control liver is clearly below the limit of 20% that was proposed as borderline between non-malignant tissue (<20%) and malignant tissue (>20%) by
The present study also shows that foci of (pre)neoplastic cells contain very high G6PD activity (Fig 1B), which was shown previously by others (
Previous studies indicated that decreased SOD activity and lipid peroxidation capacity, in combination with high G6PD activity in cancer cells, were responsible for the oxygen insensitivity of G6PD in cancer cells (
First, we observed high amounts of GSH in (pre)neoplastic foci in comparison with normal liver tissue (Fig 2C). We hypothesized that GSH plays a role in the oxygen insensitivity phenomenon by prevention of lipid peroxidation due to capture of hydrogen peroxide. It is known that lipid peroxidation products inhibit G6PD activity (
We next hypothesized that changes in the G6PD protein render (pre)neoplastic foci oxygen insensitive, and therefore we investigated involvement of SH groups in the activity of G6PD. We found that NEM, as blocker of SH groups, reduced G6PD activity in control liver tissue in situ as well as in vitro. In contrast, G6PD activity in (pre)neoplastic cells was not affected by NEM. This shows that G6PD in normal cells, but not in (pre)neoplastic cells, is dependent on SH groups for its activity. Gel electrophoresis of microdissected tissue should establish whether the properties of the enzyme in normal tissues and (pre)neoplastic foci differ also in other aspects.
The consequences of our results for the chemical backgrounds of the oxygen insensitivity phenomenon are summarized in Fig 3. It is a simplification of the scheme proposed by
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We conclude that the oxygen insensitivity of the histochemical assay for G6PD activity is a fast, easy, and cheap, tool to diagnose (pre)neoplastic cells in rat liver. The absence of essential SH groups in G6PD in (pre)neoplastic cells may be held responsible for the oxygen insensitivity of these cells. Our data warrant further investigations to determine whether the assay can be used for detection of (pre)neoplasm in human liver.
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Acknowledgments |
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We thank C.G. Wu for donating the animal tissues, Ms K.S. Bosch for technical assistance, Mr J. Peeterse for preparation of the figures, and Ms T.M.S. Pierik for careful checking of the manuscript.
Received for publication July 19, 2000; accepted January 8, 2001.
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