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Oxidative stress plays an important role in carcinogenesis because of induction of DNA damage and its effects on intracellular signal transduction pathways. Here, we investigated the relationship between the defence against oxidative stress and human renal cell carcinoma that originates from proximal tubular epithelium. Oxygen insensitivity of the histochemical assay of glucose-6-phosphate dehydrogenase (G6PD) activity is a diagnostic tool for the detection of carcinomas. Its mechanism is based on high G6PD activity, reduced superoxide dismutase activity and reduced numbers of peroxisomes in the cancer cells. Five out of the 8 renal carcinomas studied here demonstrated oxygen insensitivity. These carcinomas showed high G6PD activity, whereas the other 3 carcinomas contained lower G6PD activity and were oxygen sensitive like non-cancer cells. Oxygen insensitivity did not correlate with tumour grade, staging or presence of metastases. Electron microscopy and immunofluorescence of catalase showed large numbers of peroxisomes in epithelial cells of proximal tubules of normal human kidney, whereas these organelles were completely absent in cancer cells of all carcinomas. As a consequence of the absence of peroxisomes in cancer cells, fatty acid metabolism is disturbed in addition to the altered glucose metabolism that is generally observed in cancer cells. Therefore, therapeutic approaches should focus on metabolism in addition to other strategies targeting signal transduction and angiogenesis. (C) 2009 Published by Elsevier GmbH

Cell cycle regulation is dependent on multiple cellular and molecular events. Cell proliferation requires metabolic sources for the duplication of DNA and cell size. However, nucleotide reservoirs are not sufficient to support cell duplication and, therefore, biosynthetic pathways should be upregulated during cell cycle. Here, we reveal that glucose-6-phosphate dehydrogenase (G6PDH) and transketolase (TKT), the 2 key enzymes of oxidative and nonoxidative branches of the pentose phosphate pathway (PPP), respectively, which is necessary for nucleotide synthesis, are enhanced during cell cycle progression of the human colon cancer cell line HT29. These enhanced enzyme activities coincide with an increased ratio of pentose monophosphate to hexose monophosphate pool during late G1 and S phase, suggesting a potential role for pentose phosphates in proliferating signaling. Isotopomeric analysis distribution of nucleotide ribose synthesized from 1,2-(13)C(2)-glucose confirms the activation of the PPP during late G1 and S phase and reveals specific upregulation of the oxidative branch. Our data sustain the idea of a critical oxidative and nonoxidative balance in cancer cells, which is consistent with a late G1 metabolic check point. The distinctive modulation of these enzymes during cell cycle progression may represent a new strategy to inhibit proliferation in anticancer treatments.

Understanding of the possible toxicity associated with hypervitaminosis A becomes increasingly important in view of the popularity of vitamin A supplementation. Hypervitaminosis A for many years may eventually lead to hepatocellular damage. In the present study, rats were treated for 7 days with high doses of retinol to study the early effects on the metabolism of different types of liver cells using (enzyme) histochemistry, immunohistochemistry and electron microscopy. Excessive intake of vitamin A activates Kupffer cells and induces accumulation of lipid droplets in fat-storing cells as well as proliferation of these cells. Moreover, it affects the metabolic heterogeneity in the liver lobules, but does not lead to apparent cell damage. Based on the changes in marker enzymes for different metabolic processes, it is concluded that the capacity for breakdown of purines, the antioxidant capacity, the potential for phagocytosis and the regulation of ammonia levels were largely decreased. Increased alkaline phosphatase activity in hepatocytes pointed to an activated process of transport of retinol esters over the bile canalicular membrane. The possible causes of these metabolic changes have been described in the discussion.

Liver regeneration after partial hepatectomy was studied in four groups of rats: control rats (n = 12), rats with 1 week of common bile duct obstruction (n = 11), rats with restoration of bile flow after 1 week of obstruction (n = 9) and a sham-operated group (n = 7). Parameters of DNA synthesis--thymidine kinase activity and in vivo bromodeoxyuridine incorporation--were measured at partial hepatectomy (T = 0), and 24 and 48 h after partial hepatectomy. During common bile duct obstruction, DNA synthesis was already stimulated at T = 0, but partial hepatectomy in common bile duct obstruction rats induced a delayed DNA synthesis. After 1 week of restoration of bile flow, normal DNA synthesis had returned at T = 0, but DNA synthesis after partial hepatectomy was still delayed. The sham-operated rats showed a normal regeneration response after partial hepatectomy assessed by bromodeoxyuridine incorporation but delayed as assessed by thymidine kinase activity, partly due to the impaired physical condition of the animals. The present data support the hypothesis that during cholestasis, regeneration promoting, and inhibitory factors accumulate in the liver, their balance determining whether regeneration after partial hepatectomy will occur in a normal, enhanced or delayed way.

(Pre)neoplastic lesions in livers of rats induced by diethylnitrosamine are characterized by elevated activity of the first irreversible enzyme of the oxidative branch of the pentose phosphate pathway (PPP), glucose-6-phosphate dehydrogenase (G6PD), for production of NADPH. In the present study, the activity of G6PD, and the other NADPH-producing enzymes, phosphogluconate dehydrogenase (PGD), isocitrate dehydrogenase (ICD) and malate dehydrogenase (MD) was investigated in (pre)neoplastic lesions by metabolic mapping. Transketolase (TKT), the reversible rate-limiting enzyme of the non-oxidative branch of the PPP, mainly responsible for ribose production, was studied as well. Activity of G6PD in (pre)neoplastic lesions was highest, whereas activity of PGD and ICD was only 10% and of MD 5% of G6PD activity, respectively. Glucose-6-phosphate dehydrogenase activity in (pre)neoplastic lesions was increased 25 times compared with extralesional parenchyma, which was also the highest activity increase of the four NADPH-producing dehydrogenases. Transketolase activity was 0.1% of G6PD activity in lesions and was increased 2.5-fold as compared with normal parenchyma. Transketolase activity was localized by electron microscopy exclusively at membranes of granular endoplasmic reticulum in rat hepatoma cells where G6PD activity is localized as well. It is concluded that NADPH in (pre)neoplastic lesions is mainly produced by G6PD, whereas elevated TKT activity in (pre)neoplastic lesions is responsible for ribose formation with concomitant energy supply by glycolysis. The similar localization of G6PD and TKT activity suggests the channelling of substrates at this site to optimize the efficiency of NADPH and ribose synthesis.

Targeted therapies have demonstrated clinical benefit with limited impact on long-term disease specific survival in the treatment of renal cell cancer (RCC). New opportunities for the treatment of tumors that are resistant or have relapsed, are needed. Increased anaerobic glucose fermentation to lactate (aerobic glycolysis), leading to oxygen- and mitochondria-independent ATP generation is a hallmark of aggressive cancer growth. This metabolic shift results in increased lactate production via cycling through the pentose phosphate pathway (PPP), and plays an important role in tumor immune escape, progression and resistance to immune-, radiation- and chemo-therapy. This study explored the activity and impact of the oxidative and nonoxidative branches or the PPP on RCC to evaluate new therapeutic options. Activity was determined in the oxidative branch by glacose-6-phosphate-dehydrogenase (G6PD) activity, and in the nonoxidative branch by the total transketolase activity and the specific expression of the transketolase-like-1 (TKTL1) protein. Transketolase and G6PD activity were intensely elevated in tumor tissues. Transketolase, but not G6PD activity, was more elevated in metastasizing tumors and TKTL1 protein was significantly overexpressed in progressing tumors (p = 0.03). Lethal tumors, where surrogate parameters such as grading and staging had failed to predict progression, showed intensive TKTL1 protein expression. RCC was found to have activated oxidative and nonoxidative glucose metabolism through the PPP, displaying a bioenergetic shift toward nonoxiclative glucose fermentation in progressing tumors. The coexistence of cancer cells with differentially regulated energy supplies provides new insights in carcinogenesis and novel anticancer targets. (C) 2008 Wiley-Liss, Inc

Biosynthesis of steroid hormones in the cortex of the adrenal gland takes place in smooth endoplasmic reticulum and mitochondria and requires NADPH. Four enzymes produce NADPH: glucose-6-phosphate dehydrogenase (G6PD), the key regulatory enzyme of the pentose phosphate pathway, phosphogluconate dehydrogenase (PGD), the third enzyme of that pathway, malate dehydrogenase (MDH), and isocitrate dehydrogenase (ICDH). However, the contribution of each enzyme to NADPH production in the cortex of adrenal gland has not been established. Therefore, activity of G6PD, PGD, MDH, and ICDH was localized and quantified in rat adrenocortical tissue using metabolic mapping, image analysis, and electron microscopy. The four enzymes have similar localization patterns in adrenal gland with highest activities in the zona fasciculata of the cortex. G6PD activity was strongest, PGD, MDH, and ICDH activity was ∼60%, 15%, and 7% of G6PD activity, respectively. The Kmvalue of G6PD for glucose-6-phosphate was two times higher than the Kmvalue of PGD for phosphogluconate. As a consequence, virtual flux rates through G6PD and PGD are largely similar. It is concluded that G6PD and PGD provide the major part of NADPH in adrenocortical cells. Their activity is localized in the cytoplasm associated with free ribosomes and membranes of the smooth endoplasmic reticulum, indicating that NADPH-demanding processes related to biosynthesis of steroid hormones take place at these sites. Complete inhibition of G6PD by androsterones suggests that there is feedback regulation of steroid hormone biosynthesis via G6PD.

Oxygen insensitivity of carcinoma cells and oxygen sensitivity of non-cancer cells in the histochemical assay of glucose-6-phosphate dehydrogenase (G6PD) enables detection of carcinoma cells in unfixed cell smears or cryostat sections of biopsies. The metabolic background of oxygen insensitivity is still not understood completely. In the present study, rat hepatocytes, rat hepatoma cells (FTO-2B), and human colon carcinoma cells (HT29) were used to elucidate these backgrounds. The residual activity in oxygen was 0%, 55%, and 80% in hepatocytes, hepatoma cells, and colon carcinoma cells, respectively. N-ethylmaleimide (NEM), a blocker of SH-groups, did not affect G6PD activity in both carcinoma cell types but reduced G6PD activity in hepatocytes by 40%. Ultrastructural localization of G6PD activity was exclusively in the cytoplasm of carcinoma cells, but in hepatocytes both in cytoplasm and peroxisomes. NEM abolished peroxisomal G6PD activity only. Histochemical assay of catalase activity demonstrated absence of peroxisomes in both carcinoma cell lines. It is concluded that absence of SH-sensitive G6PD activity in peroxisomes in cancer cells is responsible for the oxygen-insensitivity phenomenon.

Metabolic mapping of enzyme activities (enzyme histochemistry) is an important tool to understand (patho)physiological functions of enzymes. A new enzyme histochemical method has been developed to detect transketolase activity in situ in various rat tissues and its ultrastructural localization in individual cells. In situ detection of transketolase is important because this multifunctional enzyme has been related with diseases such as cancer, diabetes, Alzheimer's disease, and Wernicke-Korsakoff's syndrome. The proposed method is based on the tetrazolium salt method applied to unfixed cryostat sections in the presence of polyvinyl alcohol. The method appeared to be specific for transketolase activity when the proper control reaction is performed and showed a linear increase of the amount of final reaction product with incubation time. Transketolase activity was studied in liver, small intestine, trachea, tongue, kidney, adrenal gland, and eye. Activity was found in liver parenchyma, epithelium of small intestine, trachea, tongue, proximal tubules of kidney and cornea, and ganglion cells in medulla of adrenal gland. To demonstrate transketolase activity ultrastructurally in liver parenchymal cells, the cupper iron method was used. It was shown that transketolase activity was present in peroxisomes and at membranes of granular endoplasmic reticulum. This ultrastructural localization is similar to that of glucose-6-phosphate dehydrogenase activity, suggesting activity of the pentose phosphate pathway at these sites. It is concluded that the method developed for in situ localization of transketolase activity for light and electron microscopy is specific and allows further investigation of the role of transketolase in (proliferation of) cancer cells and other pathophysiological processes.

>> SUMMARY Since the introduction of cyano-ditolyl-tetrazolium chloride (CTC), a tetrazolium salt that gives rise to a fluorescent formazan after reduction, it has been applied to quantify activity of dehydrogenases in individual cells using flow cytometry. Confocal laser scanning microscopy (CLSM) showed that the fluorescent formazan was exclusively localized at the surface of individual cells and not at intracellular sites of enzyme activity. In the present study, the technique has been optimized to localize activity of glucose-6-phosphate dehydrogenase (G6PD) intracellularly in individual cells. Activity was demonstrated in cultured fibrosarcoma cells in different stages of the cell cycle. Cells were incubated for the detection of G6PD activity using a medium containing 6% (w/v) polyvinyl alcohol, 5 mM CTC, magnesium chloride, sodium azide, the electron carrier methoxyphenazine methosulphate, NADP, and glucose-6-phosphate. Before incubation, cells were permeabilized with 0.025% glutaraldehyde. Fluorescent formazan was localized exclusively in the cytoplasm of fibrosarcoma cells. The amount of fluorescent formazan in cells increased linearly with incubation time when measured with flow cytometry and CLSM. When combining the Hoechst staining for DNA with the CTC method for the demonstration of G6PD activity, flow cytometry showed that G6PD activity of cells in S phase and G2/M phase is 27 6 4% and 43 6 4% higher, respectively, than that of cells in G1 phase. CLSM revealed that cells in all phases of mitosis as well as during apoptosis contained considerably lower G6PD activity than cells in interphase. It is concluded that posttranslational regulation of G6PD is responsible for this cell cycle–dependent activity. (J Histochem Cytochem 54:47–52, 2006)

Lentiviral vectors can stably transduce dividing and nondividing cells in vivo and are best suited to long-term correction of inherited liver diseases. Intraportal administration of lentiviral vectors expressing green fluorescent protein (Lenti-GFP) in mice resulted in a higher transduction of nonparenchymal cells than hepatocytes (7.32 +/- 3.66% vs 0.22 +/- 0.08%, respectively). Therefore, various treatments were explored to increase lentiviral transduction of hepatocytes. Lenti-GFP was injected into the common bile duct, which led to transduction of biliary epithelium and hepatocytes at low efficiency. Transient removal of the sinusoidal endothelial cell layer by cyclophosphamide to increase accessibility to hepatocytes did not improve hepatocyte transduction (0.42 +/- 0.36%). Inhibition of Kupffer cell function by gadolinium chloride led to a significant decrease in GFP-positive nonparenchymal cells (2.15 +/- 3.14%) and a sevenfold increase in GFP-positive hepatocytes compared to nonpretreated mice (1.48 +/- 2.01%). These findings suggest that sinusoidal endothelial cells do not significantly limit lentiviral transduction of hepatocytes, while Kupffer cells sequester lentiviral particles thereby preventing hepatocyte transduction. Therefore, the use of agents that inhibit Kupffer cell function may be important for lentiviral vector treatment of liver disease.

Proteases are essential for protein catabolism, regulation of a wide range of biological processes, and in the pathogenesis of many diseases. Several techniques are available to localize activity of proteases in tissue sections or cell preparations. For localization of the activity of matrix metalloproteinases, in situ zymography was introduced some decades ago. The procedure is based on zymography using SDS polyacrylamide gels containing gelatin, casein, or fibrin as substrate. For in situ zymography, either a photographic emulsion containing gelatin or a fluorescence-labeled proteinaceous macromolecular substrate is brought into contact with a tissue section or cell preparation. After incubation, enzymatic activity is revealed as white spots in a dark background or as black spots in a fluorescent background. However, this approach does not allow precise localization of proteinase activity because of limited sensitivity. A major improvement in sensitivity was achieved with the introduction of dye-quenched (DQ-)gelatin, which is gelatin that is heavily labeled with FITC molecules so that its fluorescence is quenched. After cleavage of DQ-gelatin by gelatinolytic activity, fluorescent peptides are produced that are visible against a weakly fluorescent background. The incubation with DQ-gelatin can be combined with simultaneous immunohistochemical detection of a protein on the same section. To draw valid conclusions from the findings with in situ zymography, specific inhibitors need to be used and the technique has to be combined with immunohistochemistry and zymography. In that case, in situ zymography provides data that extend our understanding of the role of specific proteinases in various physiological and pathological conditions. (J Histochem Cytochem 52:711–722, 2004)

Various proteases are involved in cancer progression and metastasis. In particular, gelatinases, matrix metalloproteinase-2 (MMP-2) and NIMP-9, have been implicated to play a role in colon cancer progression and metastasis in animal models and patients. In the present review, the clinical relevance and the prognostic value of messenger ribonucleic acid (mRNA) and protein expression and proenzyme activation of MMP-2 and MMP-9 are evaluated in relation to colorectal cancer. Expression of tissue inhibitors of MMPs (TIMPs) in relation with MMP expression in cancer tissues and the relevance of detection of plasma or serum levels of MMP-2 and/or MMP-9 and TIMPs for prognosis are also discussed. Furthermore, involvement of MMP-2 and MMP-9 in experimental models of colorectal cancer is reviewed. In vitro studies have suggested that gelatinase is expressed in cancer cells but animal models indicated that gelatinase expression in non-cancer cells in tumors contributes to cancer progression. In fact, interactions between cancer cells and host tissues have been shown to modulate gelatinase expression in host cells. Inhibition of gelatinases by synthetic MMP inhibitors has been considered to be an attractive approach to block cancer progression. However, despite promising results in animal models, clinical trials with MMP inhibitors have been disappointing so far. To obtain more insight in the (patho)physiological functions of gelatinases, regulation of MMP-2 and MMP-9 expression is discussed. Mitogen activated protein kinase (MAPK) signalling has been shown to be involved in regulation of gelatinase expression in both cancer cells and non-cancer cells. Expression can be triggered by a variety of stimuli including growth factors, cytokines and extracellular matrix (ECM) components. On the other hand, MMP-2 and MMP-9 activity regulates bioavailability and activity of growth factors and cytokines, affects the immune response and is involved in angiogenesis. Because of the multifunctionality of gelatinases, it is unpredictable at what stage of cancer development and in which processes gelatinase activity is involved. Therefore, it is concluded that the use of MMP inhibitors to treat cancer should be considered carefully. (C) 2004 Elsevier B.V, All rights reserved

Colon cancer preferentially metastasizes to the liver. To determine cellular backgrounds of this preference, we generated an enhanced green fluorescent protein (eGFP)-expressing rat adenocarcinoma cell line (CC531s) that forms metastases in rat liver after administration to the portal vein. Intravital videomicroscopy (IVVM) was used to visualize early events in the development of tumors in livers of live animals from the time of injection of the cancer cells up to 4 days afterward. Based on information obtained with IVVM, tissue areas were selected for further analysis using confocal laser scanning microscopy (CLSM), electron microscopy (EM), and electron tomography. It was shown that initial arrest of colon cancer cells in sinusoids of the liver was due to size restriction. Adhesion of cancer cells to endothelial cells was never found. Instead, endothelial cells retracted rapidly and interactions were observed only between cancer cells and hepatocytes. Tumors developed exclusively intravascularly during the first 4 days. In conclusion, initial steps in the classic metastatic cascade such as adhesion to endothelium and extravasation are not essential for colon cancer metastasis in liver.

Glucose-6-phosphate dehydrogenase (G6PD; EC 1.1.1.49) is the key regulatory enzyme of the pentose phosphate pathway and produces NADPH and riboses. In this study, the kinetic properties of G6PD activity were determined in situ in chemically induced hepatocellular carcinomas, and extralesional and control parenchyma in rat livers and were directly compared with those of the second NADPH-producing enzyme of the pentose phosphate pathway, phosphogluconate dehydrogenase (PGD). Distribution patterns of G6PD activity, protein, and mRNA levels were also compared to establish the regulation mechanisms of G6PD activity. In (pre)neoplastic lesions, the Vmaxof G6PD was 150-fold higher and the Kmfor G6P was 10-fold higher than in control liver parenchyma, whereas in extralesional parenchyma, the Vmaxwas similar to that in normal parenchyma but the Kmwas fivefold lower. This means that virtual fluxes at physiological substrate concentrations are 20-fold higher in lesions and twofold higher in extralesional parenchyma than in normal parenchyma. The Vmaxof PGD was fivefold higher in lesions than in normal and extralesional liver parenchyma, whereas the Kmwas not affected. Amounts of G6PD protein and mRNA were similar in lesions and in extralesional liver parenchyma. These results demonstrate that G6PD is strongly activated post-translationally in (pre)neoplastic lesions to produce NADPH.

Summary In principle, enzyme histochemistry should be performed on unfixed tissues and cells to avoid inhibition of enzyme activity by chemical fixation. For EM enzyme histochemistry, unfixed tissue specimens include fresh tissue blocks, non-frozen tissue chopper sections, cryostat sections and cell preparations. Studies on localization of enzyme activity at the ultrastructural level in unfixed specimens, be it fresh or frozen, are reviewed here. Preservation of ultrastructural morphology is discussed with special attention to the effects of freezing. It is concluded that unfixed cryostat sections are the best alternative for EM histochemistry of tissues, when interposing a semipermeable membrane in between cryostat section and gelled incubation medium. It is an adequate method to preserve structural integrity of unfixed tissue on the one hand and to avoid inactivation of the enzyme by chemical fixation on the other. For EM cytochemistry on individual cells, a better preservation of ultrastructure may be obtained because freezing can be avoided, but mild pretreatment with a fixative or detergent may be necessary to permeabilize cellular membranes for demonstration of intracellular enzyme activity.

Metabolic control analysis predicts that effects on tumor growth are likely to be obtained with lower concentrations of drug, if an enzyme with a high control coefficient on tumor growth is being inhibited. Here we measure glucose-6-phosphate dehydrogenase (G6PDH) control coefficient on in vivo tumor growth using mice bearing Ehrlich ascites tumor cells. We used dehydroepiandrosterone-sulphate (DHEA-S), an uncompetitive inhibitor of this enzyme and the in situ cytochemical method to measure the enzyme activity changes that accompany changes on tumor cell growth. This method ensures that the enzyme activity determined is the one existing in the in situ conditions and enables computing a control coefficient in in situ conditions. From the data obtained on tumor cell number and the in situ enzyme activities in absence and presence of DHEA-S, a control coefficient of 0.41 for G6PDH on tumor cell growth was computed. This value is approximately the half of the transketolase control coefficient value of 0.9 previously reported. Moreover, the use of in situ methods to assess enzyme activities, applied for first time for the calculation of control coefficients in this study, opens new avenues to the use of uncompetitive inhibitors for the measurement of in situ control coefficients.

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 49:565–571, 2001)

Glucose-6-phosphate dehydrogenase activity has been localized ultrastructurally in fixed tissues. Activity was found in particular in association with ribosomes of granular endoplasmatic reticulum. Biochemical studies indicated that glucose-6-phosphate dehydrogenase activity is also present in the cytoplasm and in peroxisomes. Fixation may be held responsible for selective inactivation of part of glucose-6-phosphate dehydrogenase activity. In the present study, we applied the ferricyanide method for the demonstration of glucose-6-phosphate dehydrogenase activity in unfixed cryostat sections of rat liver in combination with the semipermeable membrane technique and in isolated rat liver parenchymal cells. Isolated liver parenchymal cells were permeabilized with 0.025% glutaraldehyde after NADP+ protection of the active site of glucose-6-phosphate dehydrogenase. This treatment resulted in only slight inactivation of glucose-6-phosphate dehydrogenase activity. The composition of the incubation medium was optimized on the basis of rapid light microscopical analysis of the formation of reddish-brown final reaction product in sections. With the optimized method, electron dense reaction product was observed in cryostat sections on granular endoplasmic reticulum, in mitochondria and at the cell border. However, the ultrastructural morphology was rather poor. In contrast, the morphology of incubated isolated cells was preserved much better. Electron dense precipitate was found on ribosomes of the granular endoplasmic reticulum, in peroxisomes and the cytoplasm, particularly at the periphery of cells. In conclusion, our ultrastructural study clearly demonstrates that it is essential to use mildly-fixed cells to allow detection of glucose-6-phosphate dehydrogenase activity in all cellular compartments where activity is present.

NADH coenzyme Q reductase (EC 1.6.5.3) has been suggested in the literature to be inactivated by ischaemia. In the present study, NADH coenzyme Q reductase activity was localized in unfixed cryostat sections of ischaemic rat livers and quantified using image analysis. In vitro ischaemia was induced by storage of rat liver fragments for 30, 60, and 120 min at 37°C. In vivo ischaemia was provoked by clamping the afferent vessels of median and left lateral liver lobes for 60 min followed by 30, 60 and 180 min of reperfusion. NADH coenzyme Q reductase activity was demonstrated with the tetrazolium salt method in the presence of polyvinyl alcohol. Final reaction product was found in liver parenchymal cells and its distribution was homogeneous within liver lobules. Only low amounts of final reaction product were formed when the incubation was performed in the absence of the substrate NADH. A non-linear relation was found between the absorbance and incubation time when the reaction was performed in the presence of NADH. Therefore, the initial velocity was taken as the true rate of enzyme activity. A linear relationship was found for the initial velocity and section thickness up to 6 µm followed by a levelling off. Electron microscopically, NADH coenzyme Q reductase activity was localized at the outer and inner membranes of mitochondria. In vitro ischaemia up to 120 min did not affect NADH coenzyme Q reductase activity. At 30 min reperfusion after in vivo ischaemia for 60 min enzyme activity was slightly decreased in certain foci which also showed diminished lactate dehydrogenase activity. A further decrease of enzyme activities in foci was observed at 180 min reperfusion after ischaemia. It is concluded that NADH coenzyme Q reductase activity is not sensitive to ischaemia. Furthermore, it is likely that the enzyme leaks from liver parenchymal cells into the circulation during reperfusion after ischaemia.

Pretransplant rinse solutions have been shown to reduce reperfusion injury in cold-stored liver grafts, especially at the nonparenchymal level in sinusoidal endothelial cells (SEC). In this study, different rinse temperatures were tested in a rat liver preservation model. Livers were washed out in situ via the portal vein with cold (4 degrees C) University of Wisconsin (UW) solution, and after hepatectomy (t0), were stored for 8, 16, or 24 h of cold ischemia time (CIT). After storage, livers were flushed with UW solution at either 4 degrees C, 20 degrees C, or 37 degrees C and reperfused for 90 min (37 degrees C). Control livers were reperfused at t0 without preflush. Levels of hyaluronic acid (HA), purine nucleoside phosphorylase (PNP), AST, and LDH were measured in the reperfusion medium. Bile production was monitored during reperfusion. At the end of reperfusion, liver biopsies were taken for enzyme hystochemistry (5'-nucleotidase and LDH). After 8-h CIT and a flush at 4 degrees C, a release of endogenous HA (-7%) was observed, whereas uptake of exogenous HA occurred after the 20 degrees C flush (2%, P = NS) and after the 37 degrees C flush (24%, p < 0.001). HA release occurred at all three preflush temperatures after the 16-h CIT but was significantly lower when flushed at 37 degrees C (-10%) that at 4 degrees C and 20 degrees C (-64% and -17%, respectively, p = 0.05). After the 24-h CIT, the release of endogenous HA increased in the 4 degrees C and 20 degrees C preflush groups, but not in the 37 degrees C group. Levels of PNP and AST increased until the 24-h CIT in all groups but were significantly lower after preflush at 37 degrees C. Release of LDH did not increase with increasing periods of cold storage in any of the flush series. Compared to control livers, mean bile production during reperfusion was significantly decreased following preflush at 4 degrees C or 37 degrees C after all periods of CIT. No differences in mean bile production could be demonstrated in the three preflush groups after any period of CIT. LDH activity in liver tissue was best preserved after the 8 and 16-h CIT in combination with the 37 degrees C preflush, indicating less hepatocellular damage. In conclusion, in cold stored rat livers flushed at 37 degrees C before reperfusion, SEC and hepatocellular damage is attenuated

The involvement of hepatocytes in proliferation of bile ductule-like structures during cholestasis remains controversial. The present study was an attempt to address the issue of whether hepatocytes transform into ductular epithelial cells in response to cholestasis and, if so, which mechanisms are involved. Cholestasis was induced by common bile duct-ligation (CBDL) in rat liver for 2, 7, and 14 days. Immunofluorescence microscopy of cytokeratin 19 (CK19) was performed to assess the proliferation of bile ductules. Hepatocellular filamentous actin (F-actin) was studied using fluorescence microscopy of 7-nitrobenzene-2-oxa-1,3-diazole phallacidin and electron microscopy. Double labeling of F-actin and laminin was performed to reveal the relationship between rearrangement of F-actin and deposition of the extracellular matrix protein. The results showed that the localization of F-actin in hepatocytes underwent considerable changes after CBDL, from an even distribution at the entire plasma membrane in control liver to a more concentrated localization at one domain of the plasma membrane. This was followed by formation of rosette-like structures in pericentral and midzonal areas of the parenchyma. CK19 was expressed, as in the control liver, in the epithelial cells of proliferated bile ductules in enlarged portal tracts but not in rosette-like structures of CBDL livers. Furthermore, CBDL induced increasing amounts of laminin in the basal lamina of bile ducts and in connective tissue of portal tracts. In parenchyma, the newly deposited laminin was in close association with the rosette-like structures. It is concluded that the rearrangement of hepatocellular F-actin after CBDL precedes the formation of rosette-like structures. It is speculated that the altered F-actin contracts at one side of hepatocytes leading to tubular structures. Laminin may play an important role in this transformation process.

Vectorial sorting of plasma membrane protein-containing vesicles is essential for the establishment and maintenance of cell polarity. In the present study, the involvement of altered vesicle transport in the redistribution of membrane-bound Ca2+, Mg2+-ATPase resulting from cholestasis was investigated in hepatocytes. Cholestasis was induced in rat liver by common bile duct ligation. Ca2+, Mg2+-ATPase activity was demonstrated histochemically at the light and electron microscopical levels. Microtubules, an important factor for transcellular transport of vesicles, were studied in situ by immunofluorescence microscopy and electron microscopy in detergent-extracted preparations. The results showed that microtubules underwent significant changes after common bile duct ligation. The most pronounced alteration was focal accumulation of β-tubulin in the cytoplasm of hepato cytes after 7 days of common bile duct ligation. At the electron microscopical level, the number of microtubules was increased considerably. In control livers, the activity of Ca2+, Mg2+-ATPase was localized only at the apical plasma membrane of hepatocytes, but it was also present at the basolateral plasma membrane after common bile duct ligation. The number of intracellular vesicles containing Ca2+, Mg2+-ATPase activity was increased strikingly, and some of them were associated with lateral membrane domains in which Ca2+, Mg2+-ATPase activity was found. It is concluded that common bile duct ligation induces the rearrangement of microtubules, which may disturb vectorial transport of Ca2+, Mg2+-ATPase-containing vesicles in hepatocytes, leading to the redistribution of Ca2+, Mg2+-ATPase. © 1998 Chapman & Hall

Treatment with peroxisome proliferators induces increased numbers and alterations in the shape of peroxisomes in liver. It ultimately leads to hepatocellular carcinomas induced by the persistent production of high amounts of H2O2 as a result of a dramatical increase in acyl-CoA oxidase activity. The effects of peroxisome proliferators on other peroxisomal oxidase activities are less well documented. In the present study, the distribution patterns of the activity of SdD-amino acid oxidase, SlD-alpha-hydroxy acid oxidase, polyamine oxidase, urate oxidase and catalase activities were investigated in unfixed cryostat sections of liver, kidney and duodenum of rats treated with either clofibrate or bis(2-ethylhexyl)phthalate. The activities of xanthine oxidoreductase, which produces urate, a potent anti-oxidant, and xanthine oxidase, which produces oxygen radicals, were studied as well. The liver was the only organ that was affected by treatment. The number of peroxisomes increased considerably. SdD-Amino acid oxidase and polyamine oxidase activities were completely abolished by the treatment, whereas SlD-alpha-hydroxy acid oxidase activity decreased and urate oxidase activity increased periportally and decreased pericentrally. Total catalase activity increased because of the larger numbers of peroxisomes, but it decreased per individual peroxisome. Xanthine oxidoreductase activity decreased, whereas the percentage of xanthine oxidase remained constant. We conclude that oxidases in rat liver are affected differentially, indicating that the expression of activity of each oxidase is regulated individually. © 1998 Chapman & Hall

Animal models of liver ischaemia and reperfusion are frequently used to study the consequences on liver cells of transient oxygen deprivation. In 3 different rat models we studied ischaemia/reperfusion effects on liver cell membrane integrity, cytoplasmic enzyme proteins and enzyme activities by in situ histochemical techniques. In vivo ischaemia, as well as no-flow hypoxia, or N2-induced hypoxia in isolated perfused livers, reduced the activity of 5'-nucleotidase, a sensitive marker for plasma membrane damage in hepatocytes. As little as 2 minutes of reoxygenation in each model resulted in leakage of soluble enzymes from parenchymal and non-parenchymal liver cells, as shown by decreased protein level and activity of cytoplasmic enzymes. Whereas a multifocal decrease was observed after in vivo reperfusion, a decrease was found in all periportal and midzonal cells after blood-free reoxygenation. As judged by alkaline phosphatase activity and immunohistochemistry, an influx of inflammatory cells was not found in the in vivo model. Our findings indicate that reoxygenation itself, rather than restoration of flow, accounts for the loss of soluble enzymes from liver cells after a period of hypoxia. In situ detection of enzyme protein and activity proved useful for the examination of very early ischaemia/reperfusion effects on rat liver cells.

Summary 3,39-Diaminobenzidine, in the presence of manganese and cobalt ions, was applied for the detection of superoxide anions in unfixed cryostat sections of rat oesophagus, trachea, skin and intact mesenterium. In all connnective tissues, a blue final reaction product was found in a granular form in mast cells. The amount of final reaction product formed after incubation with diaminobenzidine and cobalt ions was increased by the addition of manganese ions. Electron microscopical analysis revealed that the electron-dense final reaction product was exclusively present in the granules of mast cells and on elastin fibres. It was found that the constitutive spontaneous formation of final reaction product in mast cells was enzymatic and dependent on the presence of oxygen in the medium. Of all the enzyme inhibitors and free radical scavengers tested, only azide strongly reduced the amount of final reaction product. It was concluded that the reaction was partly caused by peroxidase activity, but that superoxide anions are also constitutively and spontaneously produced in mast cell granules. The exact enzymatic source could not be established. Whether this property of mast cell granules plays an antimicrobial role in connective tissues can only be speculated.

Superoxide anion radicals have been implicated in a variety of pathological processes. Under physiological conditions, superoxide dismutase (SOD) is effectively able to disproportionate superoxide anions into hydrogen peroxide and dioxygen. Until now, no techniques have been available to localize SOD activity within tissues. In the present study, SOD activity was detected in different rat tissues using a thin film of xanthine oxidase between the glass slide and the unfixed cryostat section and a medium containing hypoxanthine as a source of electrons for the production of superoxide anions. The incubation medium also contained cerium ions to precipitate the hydrogen peroxide product and polyvinyl alcohol to prevent leakage of soluble and/or loosely bound enzymes from the sections into the incubation medium. The cerium perhydroxides that are formed were visualized for the light microscope in a second step using an incubation medium consisting of 3,3′-diaminobenzidine, cobalt ions, and hydrogen peroxide, which results in oxidation of the diaminobenzidine to the final insoluble blue reaction product. By this methodology, high enzyme activity was found not only in endothelial cells of liver and kidney but also in hepatocytes of liver, myocytes of heart, smooth and striated cells of muscle, acinar cells of pancreas, epithelial cells of kidney ducts, and epithelial cells of the small intestine and colon. These findings were largely in agreement with immunohistochemical data obtained using antibodies against the Cu/Zn- and Mn-SODs. However, high activity was also detected extracellularly at the surface of epithelia of trachea, esophagus, small intestine, and colon and at the extracellular matrices, cartilage, and connective tissues. We conclude from these latter data that the activity of the extracellular form of the dismutase is localized. The present method allows the analysis of all three types of known SOD activity (Cu/Zn, Mn, and extracellular) in different tissues and cell compartments. Copyright © 1996 Elsevier Science Inc.

In the present study, the endogenous formation of reactive oxygen species was localized in rat liver and small intestine. The 3,3′-diaminobenzidine (DAB)-Mn2+ technique in which cobalt ions were included in the incubation medium was applied to unfixed cryostat sections of intact tissues. Addition of manganese ions to the DAB-Co2+- containing medium greatly increased the amounts of final reaction product formed compared with incubations with only DAB and cobalt ions. In liver, a blue final reaction product was deposited, particularly in hepatocytes surrounding portal tracts. In the small intestine, the DAB--cobalt complex was mainly found at the basal side of enterocytes. Goblet cells remained unstained. Electron microscopical images revealed that an electron-dense reaction product was exclusively present at both inner and outer membranes and at the intermembrane space in mitochondria of liver parenchymal cells and duodenal enterocytes. It was shown that the spontaneous formation of final reaction product was enzymatic and dependent on the presence of oxygen in the medium. Sulphide decreased the reaction, which may indicate that cytochrome c oxidase was partially involved. Benzoquinone and histidine, which are scavengers of superoxide anions and singlet oxygen respectively, reduced the amount of final reaction product considerably. Furthermore, the formation of final reaction product was sensitive to specific inhibitors of NADH:coenzyme Q reductase and aldehydeoxidase, indicating that these enzymes were at least partly responsible for the generation of superoxide anions and singlet oxygen and for the formation of the DAB--cobalt complex.

Activity of xanthine oxidoreductase (total xanthine dehydrogenase plus xanthine oxidase) and xanthine oxidase was determined cytophotometrically in periportal and pericentral areas of livers of rats under various (patho)physiological conditions that are known to affect the content of reduced glutathione. For this purpose, rats were either normally fed or fasted for 24 hours, fasted for 24 hours, and treated with diethylmaleate that depleted glutathione or treated by in vivo ischemia for 2 hours in the livers. Xanthine oxidoreductase activity was shown histochemically with the use of a tetrazolium salt procedure, and xanthine oxidase activity was localized with a cerium- diaminobenzidine-cobalt-hydrogen peroxide technique in unfixed cryostat sections of the livers. Cytophotometric measurements showed that total xanthine oxidoreductase activity was decreased after fasting and ischemia, whereas only ischemia caused reduced xanthine oxidase activity. Moreover, the percentage of xanthine oxidase of total xanthine oxidoreductase activity was constant in both periportal and pericentral areas at the level of approximately 4% in normally fed and 24-hour fasted and diethylmaleate-treated rats. Ischemia reduced this percentage in both areas of the liver to 2%. It was concluded that the amount of endogenous reduced glutathione did not affect the percentage of xanthine oxidase. The low percentage of xanthine oxidase as determined in the present in situ histochemical study indicates that in vivo the percentage oxidase in rat liver is lower than is assumed on the basis of biochemical assays in liver homogenates even after strictly controlled homogenization procedures. Apparently, conversion of xanthine dehydrogenase into xanthine oxidase may occur in vitro to yield percentages of xanthine oxidase of 10%-20% as are reported in the literature. The latter increase in the percentage of xanthine oxidase may be caused by changes in the local environment of the enzymes, which is left completely intact in histochemical assays. The finding of this low percentage of xanthine oxidase further stresses that the main function of xanthine oxidoreductase in the liver is not the production of superoxide anion radicals and/or hydrogen peroxide but rather the metabolism of xanthine to uric acid, which can act as a potent antioxidant. (Hepatology 1996 Nov;24(5):1179-84)

The uptake of hyaluronic acid (HA) was used to assess preservation damage to sinusoidal endothelial cells (SEC) during cold storage and subsequent normothermic reperfusion of rat livers. After 8, 16, 24, and 48 h storage in University of Wisconsin (UW) solution, livers were gravity-flushed via the portal vein with a standard volume of cold UW solution containing 50 micrograms/l HA. The effluent was collected for analysis of HA, aspartate aminotransferase (AST), and lactate dehydrogenase (LDH). The mean uptake of HA at 0 h was 59.1% +/- 4.6% (mean +/- SEM). After 8 h of storage, HA uptake was similar (55.5% +/- 7.3%), whereas after 16 h of storage it was reduced to 34.7% +/- 5.8%. At 24 and 48 h of storage, no uptake of HA was found. In a second series of experiments, livers were stored in UW solution and subsequently reperfused for 90 min with a Krebs-Henseleit solution (37 degrees C) in a recirculating system containing 150 micrograms/l HA. Following 8 h of storage, 34.6% +/- 8.0% of the initial HA concentration was taken up from the perfusate. After 16 and 24 h of storage, no uptake of HA was found. The results of this study indicate that damage to SEC occurs progressively during storage, leading to zero uptake of HA by the rat livers at 24 h of cold ischemia time. Additional reperfusion injury to the SEC was demonstrated by the reduced ability of the SEC to take up HA following normothermic reperfusion. The uptake of exogenous HA in preserved livers, used as a tool to assess SEC injury, enables the detection of early preservation damage.

This study was performed to clarify the mechanisms underlying post-resection changes in liver cell proliferation and metabolism. To assess the role of gut-derived endotoxaemia and endogenous cytokines in these changes, the effects of peri-operative treatment with either the lipopolysaccharide-neutralizing bactericidal/permeability-increasing protein or interleukin-1 receptor antagonist were investigated at 24 h after two-thirds hepatectomy in rats. Peri-operative treatment with either agent caused enhanced expression of proliferating cell nuclear antigen (PCNA) and reduced lipid accumulation. Activity of the hexose monophosphate shunt was significantly decreased after partial hepatectomy and restored by interleukin-1 receptor antagonist only. After partial hepatectomy, bile canalicular alkaline phosphatase activity was significantly increased in pericentral zones and redistributed to both bile canalicular and sinusoidal membranes of hepatocytes. These effects were not significantly influenced by either treatment. It is concluded that endotoxin restricts liver cell proliferation and leads to lipid accumulation following partial hepatectomy, and that interleukin-1 is a principal mediator in these processes. Furthermore, interleukin-1 mediates a repression of the pentose phosphate pathway. These changes may be of significance with respect to liver function, at least in the early phase after partial hepatectomy.

In the present study a technique was developed to demonstrate 5′-nucleotidase activity in unfixed cryostat sections of rat liver at the light- and electron-microscope level using a semipermeable membrane. In order to retain the ultrastructure of the unfixed material as much as possible, incubations were also performed at 4°C rather than at 37°C. The optimized incubation medium contained 300 mm Tris-maleate buffer, pH 7.2, 5 mm adenosine monophosphate as substrate, 30 mm cerium chloride as capturing agent for liberated phosphate, 10 mm magnesium chloride as activator and 1.5% agar. At the light-microscope level, similar localizations of 5′-nucleotidase activity were obtained when incubations were performed at 37°C and 4°C. Enzyme activity was present mainly at bile canalicular membranes and at sinusoidal membranes of hepatocytes; total activity was higher in pericentral than in periportal areas. Cytophotometric analyses revealed that specific formation of final reaction product (FRP) (test minus control reaction) at 37°C followed a hyperbolic curve with time. A linear relationship was found between specific amounts of FRP and section thickness up to 8μm. 5′-Nucleotidase activity was about three-fold higher after incubation for 30 min at 37°C than at 4°C. At the electron-microscope level, it was demonstrated that the ultrastructure of rat liver was rather well-preserved after incubating unfixed cryostat sections attached to a semipermeable membrane and electron-dense FRP was found at bile canalicular and sinusoidal plasma membrane of hepatocytes. The most distinct changes in ultrastructure after incubation at 37°C, in comparison with that at 4°C, were the appearance of multi-lamellar structures at bile canaliculi at 37°C. We conclude that the present method is valid for the demonstration of 5′-nucleotidase activity in unfixed cryostat sections of rat liver at both the light- and electron-microscope levels and that hypothermic incubations improve ultrastructural morphology substantially.

Precise localization of xanthine oxidase activity might elucidate physiological functions of the enzyme, which have not been established so far. Because xanthine oxidase is sensitive to chemical (aldehyde) fixation, we have localized its activity in unfixed cryostat sections of rat duodenum, oesophagus and tongue mounted on a semipermeable membrane. Previous studies had shown that this procedure enables the exact localization of activities of peroxisomal oxidases with maintenance of acceptable ultrastructure. Moreover, leakage and/or diffusion of enzyme molecules was prevented with this method. The incubation medium to detect xanthine oxidase activity contained hypoxanthine as substrate and cerium ions as capturing agent for hydrogen peroxide. After incubation, reaction product in the sections was either visualized for light microscopy or sections were fixed immediately and processed for electron microscopy. At the ultrastructural level, crystalline reaction product specifically formed by xanthine oxidase activity was found to be present in the cytoplasmic matrix of enterocytes and goblet cells and in mucus of duodenum. Moderate activity was found in the cytoplasm of apical cell layers of epithelia of oesophagus and tongue, with highest activity in the cornified layer. Moreover, large amounts of reaction product were found to surround bacteria present between cell remnants of the cornified layer of the oesophagus. Many bacteria surrounded by the enzyme showed signs of destruction and/or cell death. The intracellular localization of xanthine oxidase activity in the cytoplasm of epithelial cells as well as the extracellular localization suggest that the enzyme plays a role in the lumen of the digestive tract, for instance in the defence against microorganisms.

Glycogen phosphorylase activity has been demonstrated at the ultrastructural level in liver and heart tissue of fasted rats. Unfixed cryostat sections were incubated by mounting them on a semipermeable membrane stretched over a gelled incubation medium. The medium contained a high concentration of glucose 1-phosphate which enables indirect detection of glycogen phosphorylase activity on the basis of the synthesis of glycogen. Tissue fixation, dehydration and embedding for electron microscopical study were performed after the incubation had been completed. The ultrastructure of both liver and heart tissue was rather well preserved. Glycogen granules resulting from glycogen phosphorylase activity were found in the cytoplasmic matrix of both hepatocytes and cardiomyocytes; no relationship with membranous structures could be detected. It is concluded that the semipermeable membrane method is well suited for localizing cytosolic enzyme activities at the ultrastructural level without prior tissue fixation; this opens further perspectives for correlations between histochemical and biochemical data.

The tetrazolium salt method previously developed for the detection of xanthine oxidoreductase activity in unfixed cryostat sections has been validated for quantitative purposes. The specificity of the enzyme reaction was studied by incubating unfixed cryostat sections of rat liver in test medium containing the substrate hypoxanthine, in control medium that lacked the substrate, and in medium containing substrate and allopurinol, a specific inhibitor of xanthine oxidoreductase activity. The specific reaction rate was determined cytophotometrically by subtracting the amount of final reaction product generated in the control reaction from that formed in the test reaction. Highest specific enzyme activity in rat liver was found when the incubation medium contained 18% (w/v) polyvinyl alcohol, 100 mM phosphate buffer, pH 7.8, 0.45 mM 1-methoxyphenazine methosulfate, 5 mM tetranitro BT, and 0.5 mM hypoxanthine. Enzyme activity was present in liver parenchymal cells and in sinusoidal cells (endothelial and Kupffer cells) and was completely inhibited by allopurinol. A linear relationship was observed between the specific amount of final reaction product generated at 37 degrees C and incubation time at least up to 21 min, as well as section thickness up to 12 microns. Xanthine oxidoreductase activity, expressed as mumoles substrate converted per cm3 tissue/min, was 1.61 +/- 0.34 in pericentral areas and 1.24 +/- 0.16 in periportal areas. These values are similar to biochemical data reported in the literature. In conclusion, the tetrazolium method to detect xanthine oxidoreductase activity in unfixed cryostat sections of rat liver gives a reliable reflection of in situ activity.

Data concerning the substrate specificity and the exact intracellular localization of the polyamine-catabolizing enzyme polyamine oxidase are conflicting. Biochemical studies have shown that N1-acetylation of spermine and spermidine dramatically increases the specificity of these compounds for peroxisomal polyamine oxidase to produce spermidine and putrescine, respectively. On the other hand, polyamine oxidase activity was demonstrated histochemically both in peroxisomes and in cytoplasm of several tissues, using spermidine and/or spermine as substrate. To elucidate the in situ substrate specificity of polyamine oxidase and the localization of its activity, enzyme activity was detected in rat liver, kidney, and duodenum at the light and electron microscopic levels. For this purpose, unfixed cryostat sections were applied to avoid changes in enzyme activity owing to chemical fixation. Spermine, spermidine, their N1-acetylated forms, and putrescine were used as substrates, and cerium ions as capturing agent for H2O2. Control reactions were performed in the absence of substrate or in the presence of substrate and specific oxidase inhibitors. At the light microscopic level, final reaction product specifically generated by polyamine oxidase activity was found exclusively in a granular form in hepatocytes, epithelial cells of proximal tubules of the kidney, and epithelial cells of duodenal villi with N1-acetylspermidine or N1-acetylspermine as substrates. Final reaction product was not observed in any of the tissues after incubation in the presence of putrescine, spermidine, or spermine. Formation of specific final reaction product was prevented by incubation in the presence of a specific polyamine oxidase inhibitor, but it was not affected by a diamine oxidase inhibitor. Ultrastructural studies revealed that polyamine oxidase activity is localized exclusively to the matrix of peroxisomes of kidney and liver and to microperoxisomes of the duodenum. The localization patterns obtained with unfixed tissues are in agreement with biochemical data. Strong intraperoxisomal, interperoxisomal, and intercellular heterogeneity in polyamine oxidase activity was found in all tissues investigated.

A recently developed histochemical technique to demonstrate xanthine oxidase activity in milk globules of bovine mammary gland and in epithelial cells of rat small intestine using cerium ions and a semipermeable membrane was slightly modified. The semipermeable membrane method was replaced by the addition of 10% (w/v) polyvinyl alcohol to the incubation medium. This technically more simple procedure enabled detection of xanthine oxidase activity in unfixed cryostat sections of rat liver. Both methods gave qualitatively and quantitatively similar results. Activity was found in sinusoidal cells and in liver parenchymal cells, with 50% higher activity in pericentral than in periportal areas. The specificity of the reaction was proven by the generation of only small amounts of final reaction product on incubation either in the absence of the substrates hypoxanthine or oxygen or in the presence of hypoxanthine and allopurinol. Allopurinol is a specific inhibitor of xanthine oxidase activity. The amount of final reaction product, as measured cytophotometrically in rat liver, increased linearly with incubation time (15-90 min) and with section thickness (up to 12 microns). By varying the hypoxanthine concentrations, a Km value of 0.05 mM was found. Addition of dithiothreitol to the incubation medium reduced the amount of final reaction product by 85%, which was caused by conversion of reversible xanthine oxidase into xanthine dehydrogenase. This histochemical method can be used for quantitative analysis of in situ xanthine oxidase activity.

The aim of this study was to test whether conversion of xanthine dehydrogenase into xanthine oxidase as induced by fasting, ischemia of the liver or both is an in vivo process or only occurs in vitro in homogenates. For this purpose, the conversion rate of xanthine dehydrogenase into xanthine oxidase was studied in liver homogenates obtained from rats after normal feeding or 24 hr of fasting followed or not by 2 hr of ischemia of the liver. In fed rats, the conversion rate of xanthine dehydrogenase into xanthine oxidase was studied as well in liver homogenates after different periods of reperfusion after 2 hr of ischemia. Homogenization was carried out under strictly controlled conditions, after which the supernatants were incubated at 37 degrees C in buffer for 0 to 5 hr. Enzyme activities were assayed spectrophotometrically by measuring urate production at 295 nm. Conversion started only after 2 to 3 hr of incubation of supernatants of control fed livers, whereas conversion started immediately after 24 hr of fasting. The percentage oxidase activity of total xanthine oxidoreductase activity in ischemic livers from fed animals was slightly higher (26.7% +/- 1.7%; p < 0.05) than in control livers (19.3% +/- 1.4%), whereas the percent oxidase activity in ischemic livers from fasted animals (16.7% +/- 1.0%) was not different from that in control animals (16.8% +/- 1.1%). Ischemia for 2 hr caused in vitro a substantial increase in the conversion rate in supernatants of livers of fed and fasted rats as compared with their controls.(ABSTRACT TRUNCATED AT 250 WORDS)

Localization of the activity of both the dehydrogenase and oxidase forms of xanthine oxidoreductase were studied in biopsy and postmortem specimens of various human tissues with a recently developed histochemical method using unfixed cryostat sections, poly-(vinyl alcohol) as tissue stabilizator, 1-methoxyphenazine methosulphate as intermediate electron acceptor and Tetranitro BT as final electron acceptor. High enzyme activity was found only in the liver and jejunum, whereas all the other organs studied showed no activity. In the liver, enzyme activity was found in sinusoidal cells and both in periportal and pericentral hepatocytes. In the jejunum, enterocytes and goblet cells, as well as the lamina propria beneath the basement membrane showed activity. The oxidase activity and total dehydrogenase and oxidase activity of xanthine oxidoreductase, as determined biochemically, were found in the liver and jejunum, but not in the kidney and spleen. This confirmed the histochemical results for these organs. Autolytic rat livers several hours after death were studied to exclude artefacts due to postmortem changes in the human material. These showed loss of activity both histochemically and biochemically. However, the percentage activity of xanthine oxidase did not change significantly in these livers compared with controls. The findings are discussed with respect to the possible function of the enzyme. Furthermore, the low conversion rate of xanthine dehydrogenase into xanthine oxidase during autolysis is discussed in relation to ischemia-reperfusion injury.

Many enzymes are distributed heterogeneously within the liver lobule. The factors that play a determining role in the establishment and maintenance of these heterogeneous expression patterns have not yet been identified. To investigate whether the composition of the afferent hepatic blood plays a crucial role in the maintenance of the heterogeneity of gene expression of the parenchymal cells within the liver lobule, we changed the source of the afferent hepatic blood by microsurgical techniques. Three different groups of experimental animals were studied: rats with livers that are perfused with portal blood only (ligation of the hepatic artery), with caval blood only (portocaval transposition and ligation of the hepatic artery) and arterial blood only (portocaval shunt, arterialization of the distal end of the portal vein and ligation of the hepatic artery). To study differences in gene expression patterns, we chose enzymes that have a heterogeneous expression pattern within the liver lobule: the periportally located enzymes carbamoylphosphate synthase, succinate dehydrogenase, phosphoenolpyruvate carboxylkinase and the pericentrally located enzymes glutamine synthase, glutamate dehydrogenase and NADPH–cytochrome P-450 reductase. To eliminate the potential interference of the long half-lives of some of these proteins on the interpretation of the results, we also studied the distribution of the mRNAs of carbamoylphosphate synthase, glutamine synthase, glutamate dehydrogenase and phosphoenolpyruvate carboxykinase. The animals were studied 2 wk after the operations. On the basis of their changes in body weight the animals were in steady state for at least a week. The patterns of gene expression of the enzymes studied did not change, regardless of the source of the altered afferent hepatic blood. The changes in gene expression that were observed in animals that did not regain their preoperative weight were shown to be caused by a limited intake of food. This study demonstrates that the physiological position of the liver within the circulation (i.e., between the gastrointestinal tract and the systemic circulation) is not as critical as is often stated and is certainly not essential for the maintenance of liver cell heterogeneity. The data suggest that the direction of the bloodstream (i.e., the existence of an upstream and a downstream compartment) is a major determinant of zonation of gene expression. (HEPATOLOGY 1993;18:1144-1153).

d-Amino acid oxidase activity was demonstrated in peroxisomes of rat liver using unfixed cryostat sections and a histochemical technique using cerium ions as capture reagent for hydrogen peroxide and diaminobenzidine, cobalt ions and exogenous hydrogen peroxide to visualize the final reaction product for light microscopical analysis. Cytophotometric analysis of liver sections revealed similar zero-order reaction velocities of d-amino acid oxidase with activity twice as high in periportal areas as in pericentral areas of liver lobuli when using either d-proline or d,l-thiazolidine-2-carboxylic acid as substrates. On the other hand, a 4–5 times higher KM value was found for d-proline than for d,l-thiazolidine-2-carboxylic acid. The KM values in periportal and pericentral areas were similar for each substrate. These findings support the suggestion that the physiological substrate for d-amino acid oxidase may be d,l-thiazolidine-2-carboxylic acid, the adduct of cysteamine and glyoxylic acid. d-Amino acid oxidase may play a role in vivo in the production of oxalate which may participate in metabolic control processes as an intracellular messenger molecule.

Summary Methods based on the use of cerium to detect the activity of oxidases and phosphatases are rapidly expanding. Both classes of enzymes can be demonstrated with cerium at the electron and light microscopical level. The in situ detection of H2O2 production with cerium is another application that has great potential, particularly in experimental pathological research. The fine precipitate of the cerium-containing final reaction product, cerium perhydroxide or cerium phosphate, enables a very precise localization. With such techniques, important advances have been made in cell biology, such as the discovery of new organelles, functional subcompartmentization of peroxisomes, tubular lysosomes and the elucidation of the function of extracellular ATPases. At the light microscopical level, the activity of enzymes can be quantified in situ because the production of final reaction product in the cerium methods is proportional to enzyme activity in tissue sections or cells. Cerium precipitates have strong reflectance properties and this enables their use in confocal scanning laser microscopy. In the present review, the principles of cerium methods are outlined and applications in cell biology and pathology are discussed.

Xanthine oxidoreductase exists in two functionally distinct forms. Under normal conditions, the larger part of the enzyme occurs as an NAD(+)-dependent dehydrogenase form which produces NADH and urate. The dehydrogenase can be transformed under various (patho)physiological conditions to an oxygen-dependent oxidase form which produces oxygen radicals and/or hydrogen peroxide and urate. Tetrazolium salts are used to demonstrate the total activity of both the dehydrogenase and the oxidase form of the enzyme. We have developed a procedure to detect the oxidase form only in unfixed cryostat sections with the use of cerium on the basis of the semipermeable membrane technique. The incubation medium contained hypoxanthine as substrate, cerium ions, and sodium azide to inhibit catalase and peroxidase activity. In a second-step reaction, diaminobenzidine was polymerized in the presence of cobalt ions by decomposition of cerium perhydroxide. Large amounts of final reaction product were found in milk droplets in the acini of lactating bovine mammary gland, whereas milk-secreting epithelial cells contained hardly any final reaction product. In rat duodenum, enzyme activity was found in the cytoplasm of enterocytes and goblet cells but not in the mucus. Control reactions performed in the absence of substrate or in the presence of substrate and allopurinol, a specific inhibitor of xanthine oxidase, were completely negative in both tissues, with the exception of polymorphonuclear leukocytes in the lamina propria of duodenum. The positive nonspecific reaction in these cells was caused by myeloperoxidase activity. We conclude that the present method is specific for the detection of xanthine oxidase activity. Moreover, conversion of the dehydrogenase form into the oxidase form can be prevented by omission of chemical fixation of the tissue in the present procedure.

The activity and localization of the plasma membrane-bound enzyme 5′-nucleotidase (5′-NT) in liver tissue are sensitive parameters of ischemic damage. The value of 5′-NT as a marker of liver graft viability was studied in relation to liver preservation. In six mongrel dogs, the main right and left branches of the portal vein were cannulated and flushed separately in situ with cold University of Wisconsin (UW) solution and Euro-Collins (EC) solution, respectively. After hepatectomy, the right and left liver lobes were split and stored at 5°C in either of the two solutions. 5′-NT activity was demonstrated in cryostat sections of liver tissue using the lead salt method. After 48 h of storage in EC solution, the 5′-NT score had decreased to 31%±16% (n=6), whereas in UW solution the 5′-NT score was 76%±10% (n=6). Significantly (P5 days). pretransplant 5′-NT scores ranged from 61% to 100%. The 72-h-preserved livers (n=5) did not show life-supporting function. Pretransplant 5′-NT scores (33%±12%, n=5) were significantly (P

In 36 male Wistar rats extrahepatic cholestasis was induced by ligation and transsection of the common bile duct. After 1, 2 and 3 weeks of cholestasis the bile flow was restored by means of a Roux-en-Y choledochojejunostomy. Plasma levels of bilirubin, alkaline phosphatase, GOT and clotting factor X were measured weekly. Liver biopsies were taken at the time of restored bile flow as well as 3 and 8 weeks thereafter. Histochemical reaction for lactate dehydrogenase activity and Sirius Red F3BA staining were used as measure for functional liver parenchyma and collagen, respectively. Acid phosphatase, alkaline phosphatase and 5'-nucleotidase activities as well as the glycogen content were demonstrated in cryostat sections of the same biopsies. After 1, 2 and 3 weeks of common bile duct obstruction, levels of bilirubin, alkaline phosphatase and GOT significantly increased, whereas levels of clotting factor X decreased. RBF resulted in normalization of all these levels to control range. The volume density of functional parenchyma was found to be reduced to 90%, 73% and 64% of the control values following 1, 2 and 3 weeks of common bile duct obstruction respectively, returning to 96%, 94% and 88% at 8 weeks, respectively, after restored bile flow. The collagen content increased significantly during cholestasis up to 5-fold after 3 weeks of common bile duct obstruction. After restored bile flow, a slight decrease of collagen was measured in some animals but in none of the three groups a return to normal values appeared. Cholestasis induced an alteration in localization and/or activity of the three enzymes analyzed as well as a depletion of glycogen stores. All changes in activity and distribution pattern of the three enzymes, as well as the glycogen depletion during common bile duct obstruction normalised after restored bile flow was performed. However, the longer common bile duct obstruction had existed, the longer period was needed for full recovery. In conclusion, even after 3 weeks of common bile duct obstruction the parenchyma/stroma relationship grossly normalized after restored bile flow with an almost complete restoration of the parenchyma and a concomitant recovery of liver function. However, collagen once formed, did not disappear but remained as more condensed septa, which apparently did not interfere with normal function.

The localization of xanthine oxidoreductase activity was investigated in unfixed cryostat sections of various rat tissues by an enzyme histochemical method which specifically demonstrates both the dehydrogenase and oxidase forms of xanthine oxidoreductase. High activity was found in epithelial cells from skin, vagina, uterus, penis, liver, oral and nasal cavities, tongue, esophagus, fore-stomach and small intestine. In addition activity was demonstrated in sinusoidal cells of liver and adrenal cortex, endothelial cells in various organs and connective tissue fibroblasts. Xanthine oxidoreductase produces urate which is a scavenger of oxygen-derived radicals. Because the enzyme is found in epithelial and endothelial cells which are subject to relatively high oxidant stress, it is postulated that in these cells xanthine oxidoreductase is involved in the antioxidant enzyme defense system. In addition, a possible role for the enzyme in proliferation and differentiation processes is discussed.