Human NAD(P)H:quinone oxidoreductase inhibition by flavonoids in living cells

Procedures for assessing enzyme inhibition in living cells are an important tool in the study of the relevance of enzyme-catalyzed reactions and interactions in the human body. This paper presents the effects of flavonoids on NAD(P)H:quinone oxidoreductase 1 (NQO1) activity, by a newly developed method to measure NQO1 inhibition in intact cells. The principle of this method is based on the resorufin reductase activity of NQO1. The change in fluorescence in time was used to determine NQO1 activity in intact Chinese hamster ovary (CHO) cells genetically engineered to overexpress human NQO1. Applying this method to determine the inhibitory effects of reported in vitro NQO1 inhibitors (dicoumarol, 7,8-dihydroxyflavone, chrysin) showed that for all inhibitors tested, the IC50 in intact cells was at least 3 orders of magnitude higher than the IC50 in cell lysates. This result demonstrates that in vitro studies with purified NQO1 or with extracts from disrupted tissues are of limited value for obtaining insight into the situation in living cells. Possible factors underlying this discrepancy are being discussed. For the first time, we determined NQO1 inhibition by flavonoids in cells without disruption of the cells or addition of cofactors, enabling the assessment of enzymatic activity and the interaction of modulators of enzymatic activity in an intracellular situation.     

A preliminary microspectrofluorometric study of NAD(P) reduction in dibenzo(a, e) fluoranthene-treated single living cells.

The fluorescence increase, due to NAD(P) reduction, following microelectrophoretic injection of glucose 6-P (G6P) into EL2 and NCTC 8739 single living cells treated with diBenzo(ae) Fluoranthene (diB(ae)F) and non-treated, has been studied with a rapid microspectrofluorometer. This study shows the enhanced capacity of treated cells to utilize larger doses (6-10 times more) of G6P than control cells. The time course of the return to the initial fluorescence level is essentially related to the magnitude of the injection dose. There are alterations (e.g. red & blue shifts) in the fluorescence spectrum of diB(ae)F-treated cells before injection and in the increase spectrum after injection of G6P, as compared to the same spectra in the diB(ae)F-untreated cells. This is discussed in reference to the metabolization of diB(ae)F as an alternative pathway for the reoxidation of NAD(P)H.     

Two-photon fluorescence spectroscopy and microscopy of NAD(P)H and flavoprotein

Two-photon (2P) ratiometric redox fluorometry and microscopy of pyridine nucleotide (NAD(P)H) and flavoprotein (FP) fluorescence, at 800-nm excitation, has been demonstrated as a function of mitochondrial metabolic states in isolated adult dog cardiomyocytes. We have measured the 2P-excitation spectra of NAD(P)H, flavin adenine dinucleotide (FAD), and lipoamide dehydrogenase (LipDH) over the wavelength range of 720-1000 nm. The 2P-excitation action cross sections (sigma2P) increase rapidly at wavelengths below 800 nm, and the maximum sigma2P of LipDH is approximately 5 and 12 times larger than those of FAD and NAD(P)H, respectively. Only FAD and LipDH can be efficiently excited at wavelengths above 800 nm with a broad 2P-excitation band around 900 nm. Two autofluorescence spectral regions (i.e., approximately 410-490 nm and approximately 510-650 nm) of isolated cardiomyocytes were imaged using 2P-laser scanning microscopy. At 750-nm excitation, fluorescence of both regions is dominated by NAD(P)H emission, as indicated by fluorescence intensity changes induced by mitochondrial inhibitor NaCN and mitochondria uncoupler carbonyl cyanide p-(trifluoromethoxy) phenyl hydrazone (FCCP). In contrast, 2P-FP fluorescence dominates at 900-nm excitation, which is in agreement with the sigma2P measurements. Finally, 2P-autofluorescence emission spectra of single cardiac cells have been obtained, with results suggesting potential for substantial improvement of the proposed 2P-ratiometric technique.     

Penetration and localization of furocoumarins in single living cells studied by microspectrofluorometry

The microspectrofluorometric technique has been used to study the penetration and the localization of psoralen, 4,5′,8-trimethylpsoralen and 4′-aminomethyltrioxsalen in single living L-cells. The concentration of the different compounds inside the cell reached a plateau in 2 min with psoralen and aminomethyltrioxsalen and in 20 min with trioxsalen. Washing of the cells with culture medium produced only a partial removal of the three furocoumarins, distributed apparently in equivalent amount in the nucleus and cytoplasm.     

A topographic analysis of metabolic pathways in single living cells by multisite microfluorometry.

Multichannel (multisite) microfluorometry in conjunction with microinjection of metabolic intermediates (e.g. glycolytic phosphate esters) was used for in situ topographic analysis of metabolic transients (e.g. NAD(P)NAD(P)H) in correlation to structure and compartmentalization of single living EL2 and L cells. In cells submitted to repeated microinjections with different doses of substrate (glucose-6-P or -1-P, 6-phosphogluconate, allosteric factors) rate laws were derived by a power or exponential approximation. On this basis intracellular metabolic rates were evaluated topographically and the multisite-multicomponent control of enzyme pathways in integrated biochemical systems was assessed. From different simulation trials it appeared that the transients observed are best simulated by a difference of exponentials, accounting for NAD(P) reducing and reoxidizing pathways. The determination of intracellular metabolic rate laws, their multisite-multicomponent control and the extent to which topographic discrimination of compartmentalization is possible provide the basis for application to specific problems in cell physiology, specialized cell function or the understanding of multicellular steady states.     

A preliminary microspectrofluorometric study of NAD(P) reduction in diBenzo(a,e) Fluoranthene-treated single living cells

Summary The fluorescence increase, due to NAD(P) reduction, following microelectrophoretic injection of glucose 6-P (G6P) into EL2 and NCTC 8739 single living cells treated with diBenzo(ae)Fluoranthene (diB(ae)F) and non-treated, has been studied with a rapid microspectrofluorometer. This study shows the enhanced capacity of treated cells to utilize larger doses (6–10 times more) of G6P than control cells. The time course of the return to the initial fluorescence level is essentially related to the magnitude of the injection dose. There are alterations (e.g. red & blue shifts) in the fluorescence spectrum of diB(ae)F-treated cells before injection and in the increase spectrum after injection of G6P, as compared to the same spectra in the diB(ae)F-untreated cells. This is discussed in reference to the metabolization of diB(ae)F as an alternative pathway for the reoxidation of NAD(P)H.     

Monitoring microaerobic denitrification of Pseudomonas aeruginosa by online NAD(P)H fluorescence

Defined as the transition conditions in which the organism(s) performs simultaneous aerobic and anaerobic respiration or fermentation, microaerobic conditions are commonly present in the nature. Microaerobic metabolism of microorganisms is however poorly characterized. Being extremely sensitive to the change in cellular electron-accepting mechanisms, NAD(P)H fluorescence provides a useful ways for online monitoring of microaerobic metabolism. Its application to studies of microbial nitrate respiration and particularly, denitrification of Pseudomonas aeruginosa is reviewed here, centering on four topics: (1) online monitoring of anaerobic nitrate respiration by NAD(P)H fluorescence, (2) effects of denitrification on P. aeruginosa phenotypes, (3) microaerobic denitrification of P. aeruginosa in continuous culture, and (4) correlation between NAD(P)H fluorescence and denitrification-to-respiration ratio. Online NAD(P)H fluorescence is shown to sensitively detect the changes of cellular metabolism. For example, it revealed the intermediate nitrite accumulation in C-limited Escherichia coli performing anaerobic nitrate respiration via dissimilative ammonification, by exhibiting two-stage profiles with intriguing fluorescence oscillation. When applied to continuous culture studies of P. aeruginosa (ATCC 9027), the online fluorescence helped to identify that the bacterium conducted denitrification even at DO > 1 mg/l. In addition, the fluorescence profile showed a unique correlation with the fraction of electrons accepted by denitrification (out of all the electrons accepted by aerobic and anaerobic respiration). The applicability of online NAD(P)H fluorescence in monitoring and quantitatively describing the sensitive microaerobic state of microorganisms is clearly demonstrated.     

Monitoring microaerobic denitrification of Pseudomonas aeruginosa by online NAD(P)H fluorescence

Defined as the transition conditions in which the organism(s) performs simultaneous aerobic and anaerobic respiration or fermentation, microaerobic conditions are commonly present in the nature. Microaerobic metabolism of microorganisms is however poorly characterized. Being extremely sensitive to the change in cellular electron-accepting mechanisms, NAD(P)H fluorescence provides a useful ways for online monitoring of microaerobic metabolism. Its application to studies of microbial nitrate respiration and particularly, denitrification of Pseudomonas aeruginosa is reviewed here, centering on four topics: (1) online monitoring of anaerobic nitrate respiration by NAD(P)H fluorescence, (2) effects of denitrification on P. aeruginosa phenotypes, (3) microaerobic denitrification of P. aeruginosa in continuous culture, and (4) correlation between NAD(P)H fluorescence and denitrification-to-respiration ratio. Online NAD(P)H fluorescence is shown to sensitively detect the changes of cellular metabolism. For example, it revealed the intermediate nitrite accumulation in C-limited Escherichia coli performing anaerobic nitrate respiration via dissimilative ammonification, by exhibiting two-stage profiles with intriguing fluorescence oscillation. When applied to continuous culture studies of P. aeruginosa (ATCC 9027), the online fluorescence helped to identify that the bacterium conducted denitrification even at DO > 1 mg/l. In addition, the fluorescence profile showed a unique correlation with the fraction of electrons accepted by denitrification (out of all the electrons accepted by aerobic and anaerobic respiration). The applicability of online NAD(P)H fluorescence in monitoring and quantitatively describing the sensitive microaerobic state of microorganisms is clearly demonstrated.

     

Measurements of mitochondrial deficiencies in living cells by microspectrofluorometry.

Microspectrofluorometric methods were developed for detection of mitochondrial metabolites and marker molecules in living cells. After excitation in the near UV and blue spectral ranges, respiratory-deficient strains of Saccharomyces cerevisiae showed higher levels of intrinsic fluorescence than corresponding wild types. This may be attributed to an increased emission by NADH and flavin molecules of the mutants. After incubation with the mitochondrial marker rhodamine 123, there was a strong indication that an energy transfer from flavin to rhodamine molecules occurred, which was more pronounced for the respiratory-deficient yeast strains. Skin fibroblasts obtained from patients with mitochondrial diseases showed approximately the same levels of autofluorescence and energy transfer but higher variances than a control cell line. These higher variances may result from a coexistence of intact and defective mitochondria.     

Correlation between cellular fluorescence and NAD(P)H levels in Alcaligenes eutrophus JMP 134

The culture fluorescence of Alcaligenes eutrophus JMP 134 was determined on-line by an Ingold Fluorosensor and correlated to the intracellular concentrations of reduced nicotinamide adenine dinucleotide (phosphate). The data were obtained from aerobic cultures of the strain growing chemostatically on phenol, phenol+sodium formate and fructose, as well as from aerobic/anaerobic transitions and substrate pulse experiments. The total culture fluorescence was corrected to take into account the inner filter effect of cells. Upon analysing the intracellular concentration of the dinucleotides using HPLC, it became evident that both NADH and NADPH contribute significantly to the fluorescence signal. A linear relationship between the sum of NAD(P)H and the net culture fluorescence was obtained from these data with a correlation factor of r=0.82. These investigations indicate that the measurement of culture fluorescence is a practicable tool for monitoring the redox state of a cellular culture, provided the total fluorescence signal is adjusted and the investigations are supported by direct measurements of intracellular levels of reduced dinucleotides.The authors are very grateful to the Deutsche Forschungsgemeinschaft for supporting this work (B1 345/I-2) and to Prof. T. Scheper (Institute of Biochemistry, University of Münster) for his generous assistance.     

Vanadate-stimulated oxidation of NAD(P)H

Vanadate stimulates the oxidation of NAD(P)H by biological membranes because such membranes contain NAD(P)H oxidases which are capable of reducing dioxygen to O₂⁻ and because vanadate catalyzes the oxidation of NAD(P)H by O₂⁻, by a free radical chain mechanism. Dihydropyridines, such as reduced nicotinamide mononucleotide (NMNH), which are not substrates for membrane-associated NAD(P)H oxidases, are not oxidized by membranes plus vanadate unless NAD(P)H is present to serve as a source of O₂⁻. When [NMNH] greatly exceeds [NAD(P)H], in such reaction mixtures, one can observe the oxidation of many molecules of NMNH per NAD(P)H consumed. This reflects the chain length of the free radical chain mechanism. We have discussed the mechanism and significance of this process and have tried to clarify the pertinent but confusing literature.     

A nomogram method for calculating the NAD(P)+/NAD(P)H ratio in cell compartments

A new method to calculate the ratios of free NAD+/NADH and NADP+/NADPH [NAD(P)+/NAD(P)H] in the cytoplasm and mitochondria of cells by means of nomographs is suggested. The method permits estimating the redox state of the tissue with allowance for the content of metabolites in the dehydrogenase systems. The method may be used widely in the biochemical and medical practice.     

Periodic fluctuations in oxygen consumption comparing HeLa (cancer) and CHO (non-cancer) cells and response to external NAD(P)+/NAD(P)H

Oxygen consumption in the presence of cyanide was utilized as a measure of plasma membrane electron transport in Chinese hamster ovary (CHO) and human cervical carcinoma (HeLa) cell lines. Both intact cells and isolated plasma membranes carry cyanide-insensitive NADH(P)H oxidases at their external membrane surfaces (designated ECTO-NOX proteins). Regular oscillatory patterns of oxygen consumption with period lengths characteristic of those observed for rates of NADH oxidation by ECTO-NOX proteins were observed to provide evidence for transfer of protons and electrons to reduce oxygen to water. The oscillations plus the resistance to inhibition by cyanide identify the bulk of the oxygen consumption as due to ECTO-NOX proteins. With intact CHO cells, oxygen consumption was enhanced by but not dependent upon external NAD(P)H addition. With intact HeLa cells, oxygen consumption was inhibited by both NADH and NAD⁺ as was growth. The results suggest that plasma membrane electron transport from internal donors to oxygen as an external acceptor is mediated through ECTO-NOX proteins and that electron transport to molecular oxygen may be differentially affected by external pyridine nucleotides depending on cell type.     

Bioluminescence analysis of NAD(P)H and NAD(P)+ preventing mutual interference by selective nucleotide destruction and enhanced specific light emission.

Improved bioluminescence analysis of pyridine nucleotides has been designed based on the fact that the luminescence intensity expresses the velocity of the light formation. The bacterial luciferase system is, in principle, composed of two reactions with two different velocities, one for energy supply by the oxidation of NAD(P)H and the other for the subsequent light generation. The rate setting can be arranged such that an emission maximum is produced 30 to 40 s after mixing the sample with the light-yielding solution, hence providing for a convenient analytical performance. The maximal intensity which is easily recorded, e.g., by a tracking volt-meter, is proportional to the concentration of the reduced nucleotide. Discriminative analysis of the various pyridine nucleotides is facilitated by selective destruction of the oxidized forms with alkali and the reduced forms with acid. Erroneous conversion of NAD(P)H to NAD(P)+ may be induced by haemoglobin in a tissue sample but this is prevented by the presence of 2 mM ascorbic acid at the instant of the acidification. Simultaneous coupling of the ongoing reduction of a pyridine nucleotide to the oxidation in the bacterial luciferase system generates a light-yielding cycle which offers important advantages. With NAD(P)+ as the analytic target compound, direct measurement replaces a preceding separate conversion to NAD(P)H. The four nucleotide forms become determinable in a sample by combining selective destruction of either the reduced or oxidized species with a nucleotide-specific reduction in the cycle. Discriminative analyses are furthermore facilitated by the enhanced emission which is due to the energy derived from the continuous specific reduction, whereas initial light signals from side reactions fade out. It is often possible to suppress disturbing analytical errors by the design of the light-yielding cycle. If the rate of the dehydrogenase reactions is kept low compared with the overall rate of the luciferase system, moderately impaired function of some of its components may only give rise to a slight and tolerable decrease in emission intensity. Kinetic evaluations and model experiments are presented and supplemented with applications to tissue samples.