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.     

Rapid microspectrofluorometric studies in EL2 cells following intracellular accumulation of dibenzocarbazoles.

Microspectrofluorometric observations were carried out in EL2 ascites cancer cells and dibenzo(a,e)fluoranthene (diB(a,e)F)-grown EL2 cells, following treatment (5 min) with three dibenzocarbazoles (1,2,7,8; 1,2,5,6 and 3,4,5,6). After microinjection of glucose-6-P leading to reduction of NAD(P), a sequence of difference spectra (after substrate minus before) is recorded. In dibenzocarbazole-untreated cells, maximum (NAD(P) reduction (emission maximum at 465-475 nm) is attained within 5 s, followed by a gradual return to initial fluorescence within 20 to 200 s (faster in the diB(a,e)F-grown). In dibenzocarbazole-treated cells there is a rather regular increase in the intensity of the difference spectrum up to approximately 300-500 s. Initially the increase is more predominant in the region around 460-470 nm, but it gains later prominence in the shorter wavelength region (420-430 nm) characteristic of the hydrocarbon (higher and steadier increase in the 3,4,5,6, dibenzocarbazole-treated diB(a,e)F-grown). Subsequently there is a gradual decrease of fluorescence which may or may or not return to initial level. The observed increase spectra require evaluation in terms of possible components (e.g. a mixture of NAD(P)H and hydrocarbon, binding changes, succession of fluorescent metabolites).     

Rapid microspectrofluorometric studies in EL2 cells following intracellular accumulation of dibenzocarbazoles

Summary Microspectrofluorometric observations were carried out in EL2 ascites cancer cells and dibenzo(a,e)fluoranthene (diB(a,e)F)-grown EL2 cells, following treatment (5 min) with three dibenzocarbazoles (1,2,7,8; 1,2,5,6 and 3,4,5,6). After microinjection of glucose-6-P leading to reduction of NAD(P), a sequence of difference spectra (after substrate minus before) is recorded. In dibenzocarbazole-untreated cells, maximum NAD(P) reduction (emission maximum at 465–475 nm) is attained within 5 s, followed by a gradual return to initial fluorescence within 20 to 200 s (faster in the diB(a,e)F-grown). In dibenzocarbazole-treated cells there is a rather regular increase in the intensity of the difference spectrum up to 300–500 s. Initially the increase is more predominant in the region around 460–470 nm, but it gains later prominence in the shorter wavelength region (420–430 nm) characteristic of the hydrocarbon (higher and steadier increase in the 3,4,5,6, dibenzocarbazole-treated diB(a,e)F-grown). Subsequently there is a gradual decrease of fluorescence which may or may not return to initial level. The observed increase spectra require evaluation in terms of possible components (e.g. a mixture of NAD(P)H and hydrocarbon, binding changes, succession of fluorescent metabolites).     

The effect of atebrine and an acridine analog (BCMA) on the coenzyme fluorescence spectra of cultured melanoma and Ehrlich ascites (EL2) cells.

Coenzyme fluorescence spectra of single living cells are due to free pyridine nucleotides (folded configuration), bound pyridine nucleotides (unfolded configuration) and a third component, possibly a mixture or flavins. Such spectra can be used to recognize possible differences in coenzyme composition between cell lines or changes of metabolic pathways due to chemicals acting at levels below or above cytotoxicity, by high resolution spectrofluorometry. A study of spectra recorded from cultured Ehrlich ascites (EL2), and Harding Passey melanoma cells (HPM-67 and HPM-73 line) grown under comparable conditions, shows that free NAD(P)H predominates in HPM-67 and EL2, while this coenzyme is bound in HPM-73. The free/bound ratio may be profoundly modifed by chemicals, e.g. in the HPM-73 increase of free and decrease of bound NAD(P)H occurred upon treatment with 10(-6) oligomycin. When atebrine at levels (10(-6) M) below cytotoxicity was added, there was a decrease of the free NAD(P)H spectrum possibly through energy transfer from NAD(P)H to atebrine. Consideration of long range energy transfer i.e., excitation of atebrine by fluorescence of NAD(P)H vs. short range transfer of excitation energy from free NAD(P)H to atebrine, favors the latter mechanism. A transient (reversible) increase in atebrine fluorescence is seen following intracellular microinjection of substrate (e.g. glucose-6-P) leading to an increase in free NAD(P)H. At cytotoxic levels of atebrine (e.g 2 x 10(-5) M) an irreversible increase of atebrine fluorescence is seen. The microspectrofluorometric technique appears therefore well suited to study physiological processes at the level of intracellular coenzymes, as well as possible processes of intermolecular energy transfer in the microenvironment.     

The effect of atebrine and an acridine analog (BCMA) on the coenzyme fluorescence spectra of cultured melanoma and Ehrlich ascites (EL2) cells

Summary Coenzyme fluorescence spectra of single living cells are due to free pyridine nucleotides (folded configuration), bound pyridine nucleotides (unfolded configuration) and a third component, possibly a mixture of flavins. Such spectra can be used to recognize possible differences in coenzyme composition between cell lines or changes of metabolic pathways due to chemicals acting at levels below or above cytotoxicity, by high resolution spectrofluorometry.A study of spectra recorded from cultured Ehrlich ascites (EL2), and Harding Passey melanom a cells (HPM-67 and HPM-73 line) grown under comparable conditions, shows that free NAD(P)H predominates in HPM-67 and EL2, while this coenzyme is bound in HPM-73. The free/bound ratio may be profoundly modified by chemicals, e.g. in the HPM-73 increase of free and decrease of bound NAD(P)H occurred upon treatment with 10^–6 oligomycin.When atebrine at levels (10^–6 M) below cytotoxicity was added, there was a decrease of the free NAD(P)H spectrum possibly through energy transfer from NAD(P)H to atebrine. Consideration of long range energy transfer i.e., excitation of atebrine by fluorescence of NAD(P)H vs. short range transfer of excitation energy from free NAD(P)H to atebrine, favors the latter mechanism. A transient (reversible) increase in atebrine fluorescence is seen following intracellular microinjection of substrate (e.g. glucose-6-P) leading to an increase in free NAD(P)H. At cytotoxic levels of atebrine (e.g. 2×10^–5 M) an irreversible increase of atebrine fluorescence is seen.The microspectrofluorometric technique appears therefore well suited to study physiological processes at the level of intracellular coenzymes, as well as possible processes of intermolecular energy transfer in the microenvironment.     

Isolation of nuclei from epidermis cells of larvae of the blowfly Calliphora vicina R.-D

Rapid microspectrofluorometry has been used to evaluate 1-pyrene-butyric acid as an oxygen probe in single living EL2 ascites tissue culture cells. Despite instrumental conditions preventing detection of the pyrene butyric acid maxima at 380 and 400 nm, the probe having penetrated the cell can be easily identified (maximum around 440 nm in unconnected spectra) from the fluorescence emission spectrum, as compared with NAD(P)H emission in controls (maximum around 460 nm). Fluorescence changes during gradually increasing anaerobiosis under nitrogen flow, are compatible with a linear relationship between the reciprocal of the fluorescence intensity and the intracellular oxygen concentration (increase in 430, 434, 442/461 nm ratios at anaerobiosis). The cells having absorbed the probe continue to catabolize glycolytic substrate, but some inhibition is noticeable (e.g. from the amplitude of the NAD(P)H fluorescence increase spectrum due to intracellular addition of glucose-6-P). In principle rapid microspectrofluorometry allows a multiprobe (e.g. 1-pyrene-butyric acid for oxygen, vs NAD(P)H for metabolism) exploration of the living cell.     

The effect of intracellular oxygen on the metabolization of Benzo(a)pyrene and Benzo(k)Fluoranthene. A microspectrofluorometric analysis in single living cells

Summary The fluorescence increase which accompanies the injection of glycolytic intermediates to Benzo(a)pyrene (BP) and Benzo(k) Fluoranthene-B(k)F treated EL2 ascites cancer cells, under aerobic and anaerobic conditions, has been studied in a microspectrofluorometer. In the carcinogen-treated cells the altered fluorescence increase pattern (in reference to control cells) which is observed at aerobiosis and attributed to BP or B(k)F metabolization, is not any more observable at anaerobiosis, in which case the fluorescence increase of the carcinogen-treated cells resembles that of the controls. This difference in behavior is discussed and a comparison is initiated between the response to injection in cells treated with BP (compound with K region) or B(k)F (compound without K region).     

NAD(P)H oscillates in pollen tubes and is correlated with tip growth

The location and changes in NAD(P)H have been monitored during oscillatory growth in pollen tubes of lily (Lilium formosanum) using the endogenous fluorescence of the reduced coenzyme (excitation, 360 nm; emission, >400 nm). The strongest signal resides 20 to 40 microm behind the apex where mitochondria (stained with Mitotracker Green) accumulate. Measurements at 3-s intervals reveal that NAD(P)H-dependent fluorescence oscillates during oscillatory growth. Cross-correlation analysis indicates that the peaks follow growth maxima by 7 to 11 s or 77 degrees to 116 degrees, whereas the troughs anticipate growth maxima by 5 to 10 s or 54 degrees to 107 degrees. We have focused on the troughs because they anticipate growth and are as strongly correlated with growth as the peaks. Analysis of the signal in 10-microm increments along the length of the tube indicates that the troughs are most advanced in the extreme apex. However, this signal moves basipetally as a wave, being in phase with growth rate oscillations at 50 to 60 microm from the apex. We suggest that the changes in fluorescence are due to an oscillation between the reduced (peaks) and oxidized (troughs) states of the coenzyme and that an increase in the oxidized state [NAD(P)(+)] may be coupled to the synthesis of ATP. We also show that diphenyleneiodonium, an inhibitor of NAD(P)H dehydrogenases, causes an increase in fluorescence and a decrease in tube growth. Finally, staining with 5-(and-6)-chloromethyl-2',7'-dichlorohydrofluorescein acetate indicates that reactive oxygen species are most abundant in the region where mitochondria accumulate and where NAD(P)H fluorescence is maximal.     

Ultraviolet-induced autofluorescence characterization of normal and tumoral esophageal epithelium cells with quantitation of NAD(P)H.

Cellular autofluorescence was characterized in normal human esophageal cells and in malignant esophageal epithelial cells. The study was performed under excitation at 351 nm where the cell fluorescence is mainly due to the reduced pyridine nucleotides (NAD(P)H) with a very small contribution from the oxidized flavins (FMN, FAD) or lipopigments. The autofluorescence emission of squamous cell carcinoma, adenocarcinoma on Barrett's mucosa and normal cells was characterized by microspectrofluorimetry on monolayers and by spectrofluorimetry on cell suspensions. The relative contribution of each fluorophore to the fluorescence emission of the different cell types was evaluated by a curve-fitting analysis. A statistically highly significant difference was observed between the average intensity of the raw spectra of the different cell types. Tumoral cells had a fluorescence intensity approximately twice as high as that of normal cells. The results of the NAD(P)H quantitation analyzed by microspectrofluorimetry on single living cells and spectrofluorimetry on cell suspensions were consistent with those obtained by biochemical cycling assays, showing that the amount of intracellular NAD(P)H is higher in tumoral cells than in normal cells. Bound NAD(P)H concentration was found to be quite stable whatever the cell type while the amount of free NAD(P)H showed a very important increase in tumoral cells.     

Neither total culture fluorescence nor intracellular fluorescence are indicative of NAD(P)H levels in Escherichia coli MG 1655

Summary The blue fluorescence emitted by microbial cells irradiated with UV light at 360 nm is usually supposed to provide a good estimate of the cell NAD(P)H content. Here we present an example of a microbial fermentation in which culture fluorescence, both in the cells and in the medium, was almost exclusively due to the presence of a fluorophore that displayed an emission spectrum very similar to that of NAD(P)H but that we show by biochemical studies to be a different compound. Our results demonstrate that studies on the redox state of cells should be based on on-line fluorescence data only after appropriate control experiments to establish a definitive correlation between fluorescence and NAD(P)H levels. Offprint requests to: J. E. Bailey     

Use of 1,4 diacetoxy-2,3 dicyanobenzol in microspectrofluorometry for determining the intracellular pH of isolated living cells

Microspectrofluorometry allows to obtain the fluorescence spectrum of an isolated living cell. When cells are preincubated with 1,4 diacetoxy-2,3 dicyanobenzol the cellular fluorescence spectrum can be resolved in its components i.e. the characteristic fluorescence spectrum of each ionized forms of the probe and the intrinsic cell fluorescence spectrum due to NAD(P)H. This allows the determination of the intracellular pH with good accuracy. Furthermore, comparison between the intensity of the intrinsic cell fluorescence and the probe fluorescence intensity offers us an opportunity to monitor the intracellular amount of the drug.     

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.     

The UV fading of hydrocarbon fluorescence and its prevention for observations in single living cells.

Excitation intensities used for standard microspectrofluorometric observations of natural cell fluorescence, i.e. NAD(P)H, lead to fading of hydrocarbon (polycyclic aromatic, heterocyclic) fluorescence in EL2 cells incubated with such compounds. The disappearance of hydrocarbon fluorescence under excitation at 366 nm seems to be an exponential function of time. The fading prevents studies on hydrocarbon metabolization in correlation with intracellular microelectrophoretic injection of substrate, e.g. glucose-6-P. A return to 8-10 times less intense excitation conditions used in an earlier prototype microspectrofluorometer, has allowed the observation of sequential changes in the difference spectra (after glucose-6-P minus before) of hydrocarbon-treated cells (e.g. benzo(a)pyrene, dibenzocarbazols). The possible relative contributions of NAD(P)H and hydrocarbon metabolites (or alterations) to such sequential spectra are still under consideration, but the main obstacle to their observation, fading, is removed by less intense excitation.     

Non-photochemical quenching of chlorophyll a fluorescence by oxidised plastoquinone: new evidences based on modulation of the redox state of the endogenous plastoquinone pool in broken spinach chloroplasts

Twenty-five years ago, non-photochemical quenching of chlorophyll fluorescence by oxidised plastoquinone (PQ) was proposed to be responsible for the lowering of the maximum fluorescence yield reported to occur when leaves or chloroplasts were treated in the dark with 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), an inhibitor of electron flow beyond the primary quinone electron acceptor (Q(A)) of photosystem (PS) II. Since then, the notion of PQ-quenching has received support but has also been put in doubt, due to inconsistent experimental findings. In the present study, the possible role of the native PQ-pool as a non-photochemical quencher was reinvestigated, employing measurements of the fast chlorophyll a fluorescence kinetics (from 50 micros to 5 s). The about 20% lowering of the maximum fluorescence yield F(M), observed in osmotically broken spinach chloroplasts treated with DCMU, was eliminated when the oxidised PQ-pool was non-photochemically reduced to PQH(2) by dark incubation of the samples in the presence of NAD(P)H, both under anaerobic and aerobic conditions. Incubation under anaerobic conditions in the absence of NAD(P)H had comparatively minor effects. In DCMU-treated samples incubated in the presence of NAD(P)H fluorescence quenching started to develop again after 20-30 ms of illumination, i.e., the time when PQH(2) starts getting reoxidized by PS I activity. NAD(P)H-dependent restoration of F(M) was largely, if not completely, eliminated when the samples were briefly (5 s) pre-illuminated with red or far-red light. Addition to the incubation medium of HgCl(2) that inhibits dark reduction of PQ by NAD(P)H also abolished NAD(P)H-dependent restoration of F(M). Collectively, our results provide strong new evidence for the occurrence of PQ-quenching. The finding that DCMU alone did not affect the minimum fluorescence yield F(0) allowed us to calculate, for different redox states of the native PQ-pool, the fractional quenching at the F(0) level (Q(0)) and to compare it with the fractional quenching at the F(M) level (Q(M)). The experimentally determined Q(0)/Q(M) ratios were found to be equal to the corresponding F(0)/F(M) ratios, demonstrating that PQ-quenching is solely exerted on the excited state of antenna chlorophylls.     

Multichannel microspectrofluorometry for topographic and spectral analysis of NAD(P)H fluorescence in single living cells.

Starting from a previously described prototype microspectrofluorometer a more versatile apparatus has been developed with rapid optional operation on a topographic mode for the simultaneous multisite evaluation of NAD(P) reduction-reoxidation transients or on a spectral mode for the analysis of natural and exogenous fluorochromes, in single living cells. On the topographic mode, adetailed kinetic analysis of NAD or NAD P-linked dehydrogenases can be made from 50-100 cell points imultaneously via automatic recording of topographic scans upt to 16 times a second, in correlation with microelectrophoretic intracellular inuection of metabolites (e.g. nearly immediate response to glucose 6-phosphate, 20-25 s delay for 6-phosphogluconate). Rapid shifts from topographic to spectral operation make possible the detection of a change in fluorescence intensity at a specific intracellular site and the immediate verification of its nature (NAD(P)H or exogenous fluorochrome) by spectral observations.     

NADH fluorescence and oxygen uptake responses of hybridoma cultures to substrate pulse and step changes

A fiber-optic probe was interfaced to an analytical spectrofluorophotometeru and used to measure NAD(P)H fluorescence of hybridoma cells in a bioreactor. NAD(P)H fluorescence was found to qualitatively represent metabolic state during various induced metabolic transitions. NAD(P)H fluorescence increased immediately following aerobic-anaerobic transitions, and decreased immediately upon transition back to aerobic metabolism. Pulsing of glucose to glucose-depleted cultures caused NAD(P)H fluorescence to first increase immediately after the pulse, and then decrease gradually before reaching a constant level. Pulsing of glutamine to glutamine-depleted cultures resulted in a gradual increase in NAD(P)H fluorescence which lagged a simultaneous increase in oxygen uptake. ATP production and oxygen uptake also varied with metabolic state. The decrease in oxidative phosphorylation following transition from aerobic to anaerobic metabolism was found to be only partially compensated by the concomitant increase in substrate-level phosphorylation, as shown by decreases of 35-52% in calculated total specific ATP production rates. The specific oxygen uptake rate decreased by 6-38% following glucose pulses of between 0.2 and 0.5 g/L, respectively, and by 50% following glutamine depletion. Subsequent pulsing of glutamine after depletion caused oxygen uptake to increase by 50%.     

Metabolic cofactors NAD(P)H and FAD as potential indicators of cancer cell response to chemotherapy with paclitaxel

Paclitaxel, a widely used antimicrotubular agent, predominantly eliminates rapidly proliferating cancer cells, while slowly proliferating and quiescent cells can survive the treatment, which is one of the main reasons for tumor recurrence and non-responsiveness to the drug. To improve the efficacy of chemotherapy, biomarkers need to be developed to enable monitoring of tumor responses. In this study we considered the auto-fluorescent metabolic cofactors NAD(P)H and FAD as possible indicators of cancer cell response to therapy with paclitaxel. It was found that, among the tested parameters (the fluorescence intensity-based redox ratio FAD/NAD(P)H, and the fluorescence lifetimes of NAD(P)H and FAD), the fluorescence lifetime of NAD(P)H is the most sensitive in tracking the drug response, and is capable of indicating heterogeneous cellular responses both in cell monolayers and in multicellular tumor spheroids. We observed that metabolic reorganization to a more oxidative state preceded the morphological manifestation of cell death and developed faster in cells that were more responsive to the drug. Our results suggest that noninvasive, label-free monitoring of the drug-induced metabolic changes by noting the NAD(P)H fluorescence lifetime is a valuable approach to characterize the responses of cancer cells to anti-cancer treatments and, therefore, to predict the effectiveness of chemotherapy.     

Spectral characterization of NAD(P)H fluorescence in intact isolated chloroplasts and leaves: Effect of chlorophyll concentration on reabsorption of blue-green fluorescence

In the present communication we report a spectral analysis of the blue-green fluorescence related to changes in NAD(P) redox state in chloroplasts and leaves. To assess the contribution of reabsorption and the inner filter effect, we compared transmission and fluorescence at different chloroplast concentrations, and showed that reabsorption by the photosynthetic pigments (chlorophylls and carotenoids) was at the origin of the two peaks in the emission spectrum in vivo. The absence of potential green-emitting fluorophores in chloroplasts was determined by measuring variable and time-resolved fluorescence at different wavelengths. We defined the conditions which optimize the UV-excited blue-green fluorescence signal dependent on NAD(P)H, and we present an example of monitoring of NAD(P)H fluorescence in intact leaves.     

Rapid microspectrofluorimetry for biochemical and metabolic studies in single living cells

A rapid multichannel microspectrofluorometer (e.g., to NAD(P)H, fluorescent probes) can be operated on a topographic mode for the evaluation of intracellular metabolic topography or on a spectral mode for the individual or simultaneous intracellular spectral analysis of various fluorochromes. The fluorescence emission spectra of the living cells, as well as difference spectra (spectra after intracellular microelectrophoretic addition of substrate minus before)_are analyzed under various conditions, and provide a direct proof that the fluorescence observed is that of NAD(P)H. The spectral changes which accompany treatment with substrate (e.g., glucose-6-P) can be further followed in cells incubated with other probes (e.g., acridine orange). Repeated and quite reversible transients of NAD(P) reduction—reoxidation may be observed in cells having absorbed acridine orange following repetitive additions of substrate. The spectral response to substrate is also comparatively studied in cells grown in presence of agents affecting the cell cycle (e.g., dibutyryl cyclic AMP, bleomycin).     

NAD(P)H,a primary target of 1O2 in mitochondria of intact cells

Direct reaction of NAD(P)H with oxidants like singlet oxygen ((1)O(2)) has not yet been demonstrated in biological systems. We therefore chose different rhodamine derivatives (tetramethylrhodamine methyl ester, TMRM; 2',4',5',7'-tetrabromorhodamine 123 bromide; and rhodamine 123; Rho 123) to selectively generate singlet oxygen within the NAD(P)H-rich mitochondrial matrix of cultured hepatocytes. In a cell-free system, photoactivation of all of these dyes led to the formation of (1)O(2), which readily oxidized NAD(P)H to NAD(P)(+). In hepatocytes loaded with the various dyes only TMRM and Rho 123 proved suited to generating (1)O(2) within the mitochondrial matrix space. Photoactivation of the intracellular dyes (TMRM for 5-10 s, Rho 123 for 60 s) led to a significant (29.6 +/- 8.2 and 30.2 +/- 5.2%) and rapid decrease in mitochondrial NAD(P)H fluorescence followed by a slow increase. Prolonged photoactivation (> or =15 s) of TMRM-loaded cells resulted in even stronger NAD(P)H oxidation, the rapid onset of mitochondrial permeability transition, and apoptotic cell death. These results demonstrate that NAD(P)H is the primary target for (1)O(2) in hepatocyte mitochondria. Thus NAD(P)H may operate directly as an intracellular antioxidant, as long as it is regenerated. At cell-injurious concentrations of the oxidant, however, NAD(P)H depletion may be the event that triggers cell death.