TECHNICAL NOTE |
Correspondence to: Erhard Severin, Institut für Strahlenbiologie, Universität Münster, Robert-Koch-Str. 43, D-48149 Münster, Germany.
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Summary |
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The tetrazolium salt 5-cyano-2,3-di-p-toluyl-tetrazolium chloride (CTC), yielding a fluorescent formazan on reduction, was used to measure NAD(P)H oxidoreductase activity. In this study, optimal conditions for the flow cytometric technique were determined empirically with tissue culture cell lines and mouse Ehrlich ascites cells. Applying a coupled reaction procedure, NADH and NADPH as substrates of the oxidoreductases to be measured are generated endogenously by lactate or glucose-6-phosphate dehydrogenase, respectively. The results were evaluated by combining spectrophotometry and flow cytometry. We obtained integral activities for each group of NADH and NADPH oxidoreductases. Furthermore, by counterstaining the DNA with DAPI, followed by bivariate analysis of flow cytometric data, our assay gives a detailed distribution of enzyme activities of all cells, even in subgroups present in heterogeneous cell populations. Therefore, this protocol permits the study of NAD(P)H oxidoreductase activities in ex vivo tumor samples in which mixed cellular populations may be present. (J Histochem Cytochem 46:761765, 1998)
Key Words: NAD(P)H oxidoreductases, flow cytometry, fluorescent formazan, tetrazolium salt CTC, enzyme activity, cultured endothelial cells
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Introduction |
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The NAD(P)H oxidoreductases catalyze reactions between two substrates, thereby oxidizing the nucleotides NADH and NADPH and simultaneously reducing an acceptor, e.g., a cytochrome or a quinone moiety. Considerable interest has focused on this powerful enzyme system, which is linked to the mitochondrial and the microsomal (cytosolic) electron transport chains (reviewed in
The natural acceptors of electrons in normal dehydrogenase reactions in vivo are flavine enzymes and/or quinones such as ubiquinone. The cytochemical demonstration of these reactions was made possible by the discovery that the artificial electron carrier phenazine methosulfate could bypass flavine enzymes and transfer electrons directly to the tetrazolium salt (
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Materials and Methods |
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Reagents
L-lactate (Lac) and glucose-6-phosphate (G6P) were from Sigma (Deisenhofen, Germany) NAD and NADP as sodium salts were from Boehringer (Mannheim, Germany). Phenazinemethosulfate (PMS) was from Sigma and methoxyPMS (MPMS) from Serva (Heidelberg, Germany). The tetrazolium salt 5-cyano-2,3-di-p-toluyl-tetrazolium chloride (CTC) was synthesized by ourselves (
Cells and Culture
The permanent endothelial cell line eEnd2 (
Cell Incubations for Enzyme Demonstration
Oxygen interferes with CTC formazan production (
Assay of Lactate (LDH) and Glucose-6-phosphate (G6PDH) Dehydrogenases. The cell membranes were permeabilized with 0.05% glutaraldehyde for 1 min. The cells were washed then with PBS and centrifuged at less than 100 x g. Two million pretreated cells were resuspended in 0.2 ml PBS and KCN (0.5 mmol/liter, pH adjusted to 7.4) was added to inhibit cytochrome oxidases. The electron carrier PMS or MPMS (0.1 mmol/liter), the co-enzyme NAD or NADP (0.5 mmol/liter), and the substrate lactate (20.0 mmol/liter) or G6P (2.0 mmol/liter) were added, resulting in a working volume of 1 ml. The reaction was started by adding CTC (2.0 mmol/liter). The substrates were omitted in control reactions.
All concentrations are given in final values.
Assay of NAD(P)H-d.
Because these enzyme systems may be inhibited by aldehydes (
Measurements
The cell samples were placed on ice and centrifuged at 100 x g for 5 min. The pellet was resuspended in 1 ml cold PBS and fixed by adding 0.075 ml of 4% paraformaldehyde, pH 7.4, for 1 hr at 4C. Thereafter, the cells were washed again, and 0.5 ml PBS and 0.03 ml of 5% Tween 20 was added, followed by DAPI (10 mmol/liter final concentration) 5 min later. After adding standard fluorescent beads (Polysciences) bivariate flow cytometry applying sequential two-wavelength illumination (
The cell numbers in gated subgroups, discernible in the histogram by their DNA content, were counted and their median formazan fluorescence intensities were estimated in relation to those of the fluorescence beads.
In a second step, the amount of formazan in an aliquot of each cell sample was measured in a Zeiss PMQII spectrophotometer (slit width 20 µm, wavelength 450 nm) after elution with ethanol. From the known number of cells that were present in the aliquot before, the median enzyme activity per single cell was calculated according to the LambertBeer equation (molar excitation coefficient of CTC formazan: = 20200) as described earlier (
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Results |
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Enzyme-positive cells carry bright orange fluorescent formazan crystals (Figure 2), the amount of which depends on the activity of the cellular redox process under investigation (
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The amount of formazan produced in a single cell can be measured flow cytometrically even in heterogeneous samples when cellular DNA is stained with DAPI. The range of enzyme activities in each subgroup is also revealed by this method. An example is given in Figure 3. After prolonged culture, the endothelial cell line eEnd2 produced a transformed subline with short doubling times and high DNA content, which overgrew the original, almost DNA diploid cells (compare top left histogram of Figure 3). The lactate dehydrogenase activity (Row 2), as an example of NAD-reducing potential, and the activity of NADH-d (Row 3) are higher in the faster growing subline (b vs a) by a factor of 1.4, i.e., 44.8 vs 32.2 fmol per average active G0/G1-phase cell for 10 min. This difference, however, does not appear to be related to cell volume (ratio of 2.3:1). On the other hand, the NADPH-d activity of the newly transformed endothelial cells (Row 5, column b, 7.7 fmol) is lower than that of the original subline (Row 5, column a, 13.9 fmol), whereas the G6PDH reaction (Row 4) which supplied the reduced nucleotides for demonstrating the diaphorases in the coupled reaction procedure is stronger in the high DNA subline (30.1 vs. 23.9 fmol per cell for 10 min). These data were calculated from three independent experiments using the LambertBeer equation after simultaneously performed spectrophotometry of the cell material as described previously (
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The group of NADH-d generates more than three times more CTC formazan than that of NADPH-d in cells during log phase growth. No further experiments were undertaken to identify the enzyme actually responsible for NAD(P)H oxidizing activity in our cell lines. Instead, the oxidoreductases were measured only according to their general specifity for NADH or NADPH.
The enzyme activities measured depend on the per-meabilization procedure of cell membranes. The activities after digitonin are higher than those after glutaraldehyde because of the release of intracellular compounds into the incubation medium and the consequent production of floating formazan crystals, especially in the LDH reaction. These free crystals were included in spectrophotometric readings but not in flow cytometry. The aldehyde fixation, however, affects the diaphorase activity (data not shown). Therefore, the LDH and G6PDH activity was measured in glutaraldehyde-pretreated cells, whereas all diaphorase measurements were performed with cells treated with digitonin.
Lecithin, bovine serum albumin, Tween 20, and Triton X-100 were not stimulating in our assays, in contrast to results reported in the literature (
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Discussion |
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Adapting the coupled reaction procedure of histochemistry to flow cytometry, we determined NAD(P)H-d activities in single cells within subgroups of cell samples. In addition, the range of enzyme activities in the subgroups is shown. This is not possible in spectrophotometric assays. In the present study, the tetrazolium salt CTC was used, which produces brightly fluorescent formazan crystals on reduction. This property enables the succesful combination of tetrazolium salt technique and flow cytometry. Previously we measured the activity of several dehydrogenases in ascites cells (
For histochemical demonstration of DT-diaphorase activity, the tetrazolium salts NBT and TNBT were usually used as indicators and the reduced co-enzymes were directly added to generate a one-step reaction (e.g.,
The final concentrations of reagents in our experiments are in most cases lower than recommended in histochemistry. In modern textbooks, the inclusion of macromolecular additives is demanded, e.g., polyvinyl alcohol or agarose, to prevent leakage of the intracellular compounds out of the tissue sections into the medium, or the interposition of semipermeable membranes. Without these additives few (structurally bound) enzymes are believed to be reliably localizable. However, tissue stabilizers have their limitations, e.g., they show some fluorescence and are difficult to remove because of their high viscosity. Semipermeable membranes are also not advantageous with cell suspensions. Furthermore, they are not necessary in our incubations of whole but slightly permeabilized cells. Therefore, our protocol with low concentrations of co-enzyme and, in dehydrogenase assays, intermediate electron acceptors considerably reduces the problem of unspecific formazan production.
All intact cells produce CTC formazan crystals on their surface by the activities of intracellular or plasma membrane-linked enzymes (
The formazan amount increases in aerobically incubated cells when cyanide is present (
The only way to visualize the activity of a specific NAD(P)H-d is the immunocytochemical approach, but no quantitative determination by this method has been published until now. In our opinion, it is not a drawback that we were unable to measure each member of the NAD(P)H oxidoreductases separately but instead obtained the activities of both the groups of NADH or NADPH-oxidizing enzymes, because each enzyme can compensate, at least in part, for the function of the other enzymes in the same group. Therefore, only the integral activity obtained from these enzymes is important for the assessment of cellular efficiency in the detoxification of damaging compounds and in the activation of certain drugs.
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Acknowledgments |
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We are indebted to Ms M. Volkermann for skillful technical assistance and to Dr W. Risau (Bad Homburg, Germany) for providing the endothelial cell line eEnd2. We thank Dr P. Cullen, Münster, for improving the English of the manuscript.
Received for publication May 7, 1997; accepted January 7, 1998.
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