Production Of NADP Research Articles

Staphylococcus aureus NAD kinase is required for envelop and antibiotic stress responses

Global burden of infectious diseases and antimicrobial resistance are major public health issues calling for innovative control measures. Bacterial NAD kinase (NADK) is a crucial enzyme for production of NADP(H) and growth. In Staphylococcus aureus, NADK promotes pathogenesis by supporting production of key virulence determinants. Here, we find that knockdown of NADK by CRISPR interference sensitizes S. aureus to osmotic stress and to stresses induced by antibiotics targeting the envelop as well as replication, transcription and translation. Thus, NADK represents a promising target for the development of inhibitors which could be used in combination with current antibiotics.

NADK-mediated de novo NADP(H) synthesis is a metabolic adaptation essential for breast cancer metastasis

Metabolic reprogramming and metabolic plasticity allow cancer cells to fine-tune their metabolism and adapt to the ever-changing environments of the metastatic cascade, for which lipid metabolism and oxidative stress are of particular importance. NADPH is a central co-factor for both lipid and redox homeostasis, suggesting that cancer cells may require larger pools of NADPH to efficiently metastasize. NADPH is recycled through reduction of NADP+ by several enzymatic systems in cells; however, de novo NADP+ is synthesized only through one known enzymatic reaction, catalyzed by NAD+ kinase (NADK). Here, we show that NADK is upregulated in metastatic breast cancer cells enabling de novo production of NADP(H) and the expansion of the NADP(H) pools thereby increasing the ability of these cells to adapt to the challenges of the metastatic cascade and efficiently metastasize. Mechanistically, we found that metastatic signals lead to a histone H3.3 variant-mediated epigenetic regulation of the NADK promoter, resulting in increased NADK levels in cells with metastatic ability. Together, our work presents a previously uncharacterized role for NADK and de novo NADP(H) production as a contributor to breast cancer progression and suggests that NADK constitutes an important and much needed therapeutic target for metastatic breast cancers.

Synthesis and structure-activity relationship studies of original cyclic diadenosine derivatives as nanomolar inhibitors of NAD kinase from pathogenic bacteria

Nicotinamide adenine dinucleotide kinases (NAD kinases) are essential and ubiquitous enzymes involved in the production of NADP(H) which is an essential cofactor in many metabolic pathways. Targeting NAD kinase (NADK), a rate limiting enzyme of NADP biosynthesis pathway, represents a new promising approach to treat bacterial infections. Previously, we have produced the first NADK inhibitor active against staphylococcal infection. From this linear di-adenosine derivative, namely NKI1, we designed macrocyclic analogues. Here, we describe the synthesis and evaluation of an original series of cyclic diadenosine derivatives as NADK inhibitors of two pathogenic bacteria, Listeria monocytogenes and Staphylococcus aureus. The nature and length of the link between the two adenosine units were examined leading to sub-micromolar inhibitors of NADK1 from L. monocytogenes, including its most potent in vitro inhibitor reported so far (with a 300-fold improvement compared to NKI1).

Loss of chloroplast-localized NAD kinase causes ROS stress in Arabidopsis thaliana.

Chloroplast-localized NAD kinase (NADK2) is responsible for the production of NADP+, which is an electron acceptor in the linear electron flow of photosynthesis. The Arabidopsis T-DNA-inserted mutant of NADK2 (nadk2) showed delayed growth and pale-green leaves under continuous light conditions. Under short-day conditions (8h light / 16h dark), the nadk2 mutant showed more severe growth inhibition.The genomic fragment containing the promoter and coding region of NADK2 complemented the phenotypes of nadk2 obtained under continuous light and short-day conditions. The nadk2 mutant produced higher amounts of H2O2 and O2-, which were reduced in the complementary line. Under short-day conditions, the nadk2 mutant accumulated more H2O2 than under continuous light conditions. The accumulation of ascorbate and up-regulation of the PDF1.2 and PR1 genes indicated that the nadk2 mutant is under ROS stress and responding to keep its living activities.

Postharvest Dehydration Temperature Modulates the Transcriptomic Programme and Flavonoid Profile of Grape Berries.

Raisins are a popular and nutritious snack that is produced through the dehydration of postharvest grape berries under high temperature (HT). However, the response of the endogenous metabolism of white grape varieties to postharvest dehydration under different temperature have not been fully elucidated to date. In this study, the white grape cultivar ‘Xiangfei’ was chosen to investigate the effect of dehydration at 50 °C, 40 °C, and 30 °C on the transcriptomic programme and metabolite profiles of grape berries. Postharvest dehydration promoted the accumulation of soluble sugar components and organic acids in berries. The content of gallic acid and its derivatives increased during the dehydration process and the temperature of 40 °C was the optimal for flavonoids and proanthocyanidins accumulation. High-temperature dehydration stress might promote the accumulation of gallic acid by increasing the expression levels of their biosynthesis related genes and regulating the production of NADP+ and NADPH. Compared with that at 30 °C, dehydration at 40 °C accelerated the transcription programme of 7654 genes and induced the continuous upregulation of genes related to the heat stress response and redox homeostasis in each stage. The results of this study indicate that an appropriate dehydration temperature should be selected and applied when producing polyphenols-rich raisins.

Preparation and enzymatic activity of Fe3O4-IDA-Ni/NAD kinase magnetic catalyst

The use of oxidoreductases as biocatalysts for industrial production of valuable compounds has a strong demand for NADP. Herein, we prepared superparamagnetic NAD kinase catalyst to synthesize NADP in vitro. First, Fe3O4 particles were synthesized through a solvothermal method, followed by the chemical modification with epichloro-hydrin, iminodiacetic acid, and Ni2+ to yield functional Fe3O4 sub-microspheres. Subsequently, NAD kinase of Escherichia coli was overexpressed and immobilized on to the surface of magnetic sub-microspheres. The immobilized NAD kinase was used to catalyze the conversion of NAD to NADP in a cell-free system. Under optimal condition, the conversion ratio of NAD reached 91.7% and remained at 86.3% after repeated use for five times. Our study revealed that the novel magnetic NAD kinase catalyst possessed favorable properties for magnetic manipulation and NADP production.

Enzymatic Characteristics of a Polyphosphate/ATP-NAD Kinase, PanK, from Myxococcus xanthus.

NAD kinase is a crucial enzyme for production of NADP+. Myxococcus xanthus is a gram-negative soil bacterium that forms fruiting bodies and spores under starvation, and it accumulates polyphosphate (poly(P)) during early development. We found that M. xanthus NAD kinase (PanK) utilized both ATP and poly(P) as phosphoryl donors; therefore, PanK was designated as a poly(P)/ATP-NAD kinase. Unlike other poly(P)/ATP-NAD kinases, PanK hardly exhibited NADH kinase activity. The NAD kinase activity of PanK was inhibited by NADPH, but not NADH. Replacement of Thr-90 in the GGDGT motif of PanK with Asn decreased both ATP- and poly(P)-dependent NAD kinase activities; however, poly(P)-dependent NAD kinase activity was further decreased by approximately 6- to 10-fold compared with ATP-dependent NAD kinase activity, suggesting that Thr-90 in the GGDGT motif of PanK may be important for poly(P) utilization. PanK preferred ATP and short-chain poly(P) as phosphoryl donors. The Km of PanK for ATP, poly(P)4, and poly(P)10-15 was 0.66mM, 0.08mM, and 0.71mM, respectively, and the catalytic efficiency (kcat/Km) for poly(P)4 was 2.4-fold higher than that for ATP, suggesting that M. xanthus under starvation conditions may be able to efficiently generate NADP+ using PanK, ATP, and poly(P).

Prolyl hydroxylase inhibition protects the kidneys from ischemia via upregulation of glycogen storage

Hypoxia-inducible factor (HIF) mediates protection via hypoxic preconditioning in both, invitro and invivo ischemia models. However, the underlying mechanism remains largely unknown. Prolyl hydroxylase domain proteins serve as the main HIF regulator via hydroxylation of HIFα leading to its degradation. At present, prolyl hydroxylase inhibitors including enarodustat are under clinical trials for the treatment of renal anemia. In an invitro model of ischemia produced by oxygen-glucose deprivation of renal proximal tubule cells in culture, enarodustat treatment and siRNA knockdown of prolyl hydroxylase 2, but not of prolyl hydroxylase 1 or prolyl hydroxylase 3, significantly increased the cell viability and reduced the levels of reactive oxygen species. These effects were offset by the simultaneous knockdown of HIF1α. In another invitro ischemia model induced by the blockade of oxidative phosphorylation with rotenone/antimycin A, enarodustat-enhanced glycogen storage prolonged glycolysis and delayed ATP depletion. Although autophagy is another possible mechanism of prolyl hydroxylase inhibition-induced cytoprotection, gene knockout of a key autophagy associated protein, Atg5, did not affect the protection. Enarodustat increased the expression of several enzymes involved in glycogen synthesis, including phosphoglucomutase 1, glycogen synthase 1, and 1,4-α glucan branching enzyme. Increased glycogen served as substrate for ATP and NADP production and augmented reduction of glutathione. Inhibition of glycogen synthase 1 and glutathione reductase nullified enarodustat's protective effect. Enarodustat also protected the kidneys in a rat ischemia reperfusion injury model and the protection was partially abrogated by inhibiting glycogenolysis. Thus, prolyl hydroxylase inhibition protects the kidney from ischemia via upregulation of glycogen synthesis.

Epigenetic loss of AOX1 expression via EZH2 leads to metabolic deregulations and promotes bladder cancer progression.

Advanced Bladder Cancer (BLCA) remains a clinical challenge that lacks effective therapeutic measures. Here, we show that distinct, stage-wise metabolic alterations in BLCA are associated with the loss of function of aldehyde oxidase (AOX1). AOX1 associated metabolites have a high predictive value for advanced BLCA and our findings demonstrate that AOX1 is epigenetically silenced during BLCA progression by the methyltransferase activity of EZH2. Knockdown (KD) of AOX1 in normal bladder epithelial cells re-wires the tryptophan-kynurenine pathway resulting in elevated NADP levels which may increase metabolic flux through the pentose phosphate (PPP) pathway, enabling increased nucleotide synthesis, and promoting cell invasion. Inhibition of NADP synthesis rescues the metabolic effects of AOX1 KD. Ectopic AOX1 expression decreases NADP production, PPP flux and nucleotide synthesis, while decreasing invasion in cell line models and suppressing growth in tumor xenografts. Further gain and loss of AOX1 confirm the EZH2-dependent activation, metabolic deregulation, and tumor growth in BLCA. Our findings highlight the therapeutic potential of AOX1 and provide a basis for the development of prognostic markers for advanced BLCA.

The effector AvrRxo1 phosphorylates NAD in planta.

Gram-negative bacterial pathogens of plants and animals employ type III secreted effectors to suppress innate immunity. Most characterized effectors work through modification of host proteins or transcriptional regulators, although a few are known to modify small molecule targets. The Xanthomonas type III secreted avirulence factor AvrRxo1 is a structural homolog of the zeta toxin family of sugar-nucleotide kinases that suppresses bacterial growth. AvrRxo1 was recently reported to phosphorylate the central metabolite and signaling molecule NAD in vitro, suggesting that the effector might enhance bacterial virulence on plants through manipulation of primary metabolic pathways. In this study, we determine that AvrRxo1 phosphorylates NAD in planta, and that its kinase catalytic sites are necessary for its toxic and resistance-triggering phenotypes. A global metabolomics approach was used to independently identify 3’-NADP as the sole detectable product of AvrRxo1 expression in yeast and bacteria, and NAD kinase activity was confirmed in vitro. 3’-NADP accumulated upon transient expression of AvrRxo1 in Nicotiana benthamiana and in rice leaves infected with avrRxo1-expressing strains of X. oryzae. Mutation of the catalytic aspartic acid residue D193 abolished AvrRxo1 kinase activity and several phenotypes of AvrRxo1, including toxicity in yeast, bacteria, and plants, suppression of the flg22-triggered ROS burst, and ability to trigger an R gene-mediated hypersensitive response. A mutation in the Walker A ATP-binding motif abolished the toxicity of AvrRxo1, but did not abolish the 3’-NADP production, virulence enhancement, ROS suppression, or HR-triggering phenotypes of AvrRxo1. These results demonstrate that a type III effector targets the central metabolite and redox carrier NAD in planta, and that this catalytic activity is required for toxicity and suppression of the ROS burst.

Biochemical Characterization of Heterodimeric Isocitrate Dehydrogenase I Using a Novel High‐throughput Mass Spectrometry‐based Assay

In several types of human tumors, a gain‐of‐function mutation within a single allele of isocitrate dehydrogenase 1 (IDH1) results in the production of a known oncometabolite, 2‐hydroxyglutarate (2‐HG). IDH1 is enzymatically active as a dimer, and both homodimeric and wt:mutant heterodimeric proteins are produced in cells. Since the heterodimer is comprised of both the mutant and wild‐type subunits, it therefore catalyzes both the canonical (a‐ketoglutarate generating) and the neomorphic (2‐HG generating) reactions. Traditionally, IDH1 assays monitor the reduction or oxidation of NADP/H, a common co‐factor substrate for both the mutant and wild‐type subunits. This, in conjunction with an extremely low predicted NADPH Km, greatly complicates the kinetic characterization of heterodimeric IDH1. Here we present a novel mass‐spectrometry‐based IDH1 assay. Not only are we able to selectively monitor 2‐HG production by the mutant subunit, but incorporation of a unique NADPH regeneration system allows us to study the reaction at nanomolar NADPH concentrations. Using this platform we determined that the IDH1 heterodimer exhibits a significantly lower Km for both a‐KG (314 μM) and NADPH (26 nM) than the mutant homodimeric protein (a‐KG Km = 2800 μM and NADPH Km = 173 nM). To further characterize the heterodimer and determine if the wild‐type subunit catalyzes production of NADP+ and isocitrate, we assessed the activity of a catalytically impaired wild‐type subunit (Y139D) and also monitored the heterodimeric reaction by NMR. Our findings indicate that under the conditions of our assay, the wild‐type subunit makes minimal contributions to total NADP+ production.

Conferring the ability to utilize inorganic polyphosphate on ATP-specific NAD kinase

NAD kinase (NADK) is a crucial enzyme for production of NADP+. ATP-specific NADK prefers ATP to inorganic polyphosphate [poly(P)] as a phosphoryl donor, whereas poly(P)/ATP-NADK utilizes both ATP and poly(P), and is employed in industrial mass production of NADP+. Poly(P)/ATP-NADKs are distributed throughout Gram-positive bacteria and Archaea, whereas ATP-specific NADKs are found in Gram-negative α- and γ-proteobacteria and eukaryotes. In this study, we succeeded in conferring the ability to utilize poly(P) on γ-proteobacterial ATP-specific NADKs through a single amino-acid substitution; the substituted amino-acid residue is therefore important in determining the phosphoryl-donor specificity of γ-proteobacterial NADKs. We also demonstrate that a poly(P)/ATP-NADK created through this method is suitable for the poly(P)-dependent mass production of NADP+. Moreover, based on our results, we provide insight into the evolution of bacterial NADKs, in particular, how NADKs evolved from poly(P)/ATP-NADKs into ATP-specific NADKs.

Synthetic Lethal and Biochemical Analyses of NAD and NADH Kinases in Saccharomyces cerevisiae Establish Separation of Cellular Functions

Production of NADP and NADPH depends on activity of NAD and NADH kinases. Here we characterized all combinations of mutants in yeast NAD and NADH kinases to determine their physiological roles. We constructed a diploid strain heterozygous for disruption of POS5, encoding mitochondrial NADH kinase, UTR1, cytosolic NAD kinase, and YEF1, a UTR1-homologous gene we characterized as encoding a low specific activity cytosolic NAD kinase. pos5 utr1 is a synthetic lethal combination rescued by plasmid-borne copies of the POS5 or UTR1 genes or by YEF1 driven by the ADH1 promoter. Respiratory-deficient and oxidative damage-sensitive defects in pos5 mutants were not made more deleterious by yef1 deletion, and a quantitative growth phenotype of pos5 and its arginine auxotrophy were repaired by plasmid-borne POS5 but not UTR1 or ADH1-driven YEF1. utr1 haploids have a slow growth phenotype on glucose not exacerbated by yef1 deletion but reversed by either plasmid-borne UTR1 or ADH1-driven YEF1. The defect in fermentative growth of utr1 mutants renders POS5 but not POS5-dependent mitochondrial genome maintenance essential because rho-utr1 derivatives are viable. Purified Yef1 has similar nucleoside triphosphate specificity but substantially lower specific activity and less discrimination in favor of NAD versus NADH phosphorylation than Utr1. Low expression and low intrinsic NAD kinase activity of Yef1 and the lack of phenotype associated with yef1 suggest that Utr1 and Pos5 are responsible for essentially all NAD/NADH kinase activity in vivo. The data are compatible with a model in which there is no exchange of NADP, NADPH, or cytoplasmic NAD/NADH kinase between nucleocytoplasmic and mitochondrial compartments, but the cytoplasm is exposed to mitochondrial NAD/NADH kinase during the transit of the molecule.

Evidence that feedback inhibition of NAD kinase controls responses to oxidative stress.

Formation of NADP+ from NAD+ is catalyzed by NAD kinase (NadK; EC 2.7.1.23). Evidence is presented that NadK is the only NAD kinase of Salmonella enterica and is essential for growth. NadK is inhibited allosterically by NADPH and NADH. Without effectors, NadK exists as an equilibrium mixture of dimers and tetramers (KD = 1.0 +/- 0.8 mM) but is converted entirely to tetramers in the presence of the inhibitor NADPH. Comparison of NadK kinetic parameters with pool sizes of NADH and NADPH suggests that NadK is substantially inhibited during normal growth and, thus, can increase its activity greatly in response to temporary drops in the pools of inhibitory NADH and NADPH. The primary inhibitor is NADPH during aerobic growth and NADH during anaerobic growth. A model is proposed in which variation of NadK activity is central to the adjustment of pyridine nucleotide pools in response to changes in aeration, oxidative stress, and UV irradiation. It is suggested that each of these environmental factors causes a decrease in the level of reduced pyridine nucleotides, activates NadK, and increases production of NADP(H) at the expense of NAD(H). Activation of NadK may constitute a defensive response that resists loss of protective NADPH.

Establishment of a Mass-Production System for NADP Using Bacterial Inorganic Polyphosphate/ATP-NAD Kinase.

The inorganic polyphosphate/ATP-dependent NAD kinase (Ppnk) of Mycobacterium tuberculosis was applied to the mass-production of NADP from NAD and inorganic polyphosphate (metaphosphate). When Ppnk purified from recombinant Escherichia coli cells overexpressing the M. tuberculosis Ppnk was used, 30 mM (27 g/l) NADP was produced from 50 mM NAD and 100 mg/ml metaphosphate at 37 degrees C and pH 7.0. The recombinant E. coli cells were immobilized in polyacrylamide gel, and treated with acetone to render the cells permeable to substrates and products. When acetone-treated immobilized cells were used, 16 mM (14 g/l) NADP was produced from 50 mM NAD and 100 mg/ml metaphosphate at 37 degrees C and pH 7.0. The isolation of NADP formed in the reaction mixture was easy because of the absence of by-products (ATP degradation compounds), and this NADP production system using purified Ppnk or immobilized recombinant E. coli cells expressing Ppnk is thought to be feasible in the production of NADP on an industrial scale.

Establishment of a mass-production system for NADP using bacterial inorganic polyphosphate/ATP-NAD kinase

The inorganic polyphosphate/ATP-dependent NAD kinase (Ppnk) of Mycobacterium tuberculosis was applied to the mass-production of NADP from NAD and inorganic polyphosphate (metaphosphate). When Ppnk purified from recombinant Escherichia coli cells overexpressing the M. tuberculosis Ppnk was used, 30 mM (27 g/ l) NADP was produced from 50 mM NAD and 100 mg/ml metaphosphate at 37°C and pH 7.0. The recombinant E. coli cells were immobilized in polyacrylamide gel, and treated with acetone to render the cells permeable to substrates and products. When acetone-treated immobilized cells were used, 16 mM (14 g/ l) NADP was produced from 50 mM NAD and 100 mg/ml metaphosphate at 37°C and pH 7.0. The isolation of NADP formed in the reaction mixture was easy because of the absence of by-products (ATP degradation compounds), and this NADP production system using purified Ppnk or immobilized recombinant E. coli cells expressing Ppnk is thought to be feasible in the production of NADP on an industrial scale.

Isotachophoretic analysis of the dihydrofolate reductase reaction in the presence of methotrexate and ascorbic acid.

The antifolate methotrexate (MTX) is widely used in cancer chemotherapy. In this study, we show that MTX (MTX-Glu1) and MTX-polyglutamates (MTX-Glu2-5) strongly inhibited the growth of the leukemic cell line MOLT-4. This effect, however, was mitigated by ascorbic acid. We investigated whether ascorbic acid is able to reduce dihydrofolic acid (DHF) to tetrahydrofolic acid (THF) directly or by circumventing the MTX inhibition of dihydrofolate reductase (DHFR). The inhibition of this NADPH-dependent reduction of DHF by MTX-Glun in the absence or presence of ascorbate, was determined by analytical isotachophoresis. Using 0.01 M HCl/histidine, pH 6.0, as a leading electrolyte (L) and 0.005 M 2-(N-morpholino)ethanesulfonic acid (MES)/histidine, pH 6.0, as a terminating electrolyte (T), MTX-Glun derivatives including MTX-Glu1 could be easily separated, whereas the quantitative estimation of THF was not possible. A quantitative characterization of the DHFR reaction by measuring NADPH, NADP+ and ascorbate was achieved with another system (L: 0.01 M HCI/beta-alanine, pH 3.73; T: 0.01 M caproic acid, pH 3.27). Nanomolar concentrations of MTX-Glu1-5 inhibited consumption of NADPH and production of NADP+. Ascorbic acid was not able to reduce DHF, neither directly nor after inhibition of DHFR by MTX. However, ascorbic acid seemed to diminish the oxidation of THF and this may account for its capacity to reduce the inhibitory effect of MTX on MOLT-4 cells.

Coenzyme production using immobilized enzymes. II. Factors affecting the continuous production of NADP

The effects of certain factors on the continuous production of NADP + with immobilized NAD + kinase were studied. In a column reactor, the maximum NADP + productivity was experienced at pH 8.0. The presence of 10–12 mM Mg 2+ ion appeared to be optimum for maximum productivity. K + ion at 1–5 mM behaved as an activator. Mn 2+ and Zn 2+ ions proved effective activators of the immobilized NAD + kinase. Systems of up to three or four columns were most effective in NADP + production.

Activities of the Pentose Phosphate Pathway and Enzymes of Proline Metabolism in Legume Root Nodules

Based on localization and high activities of pyrroline-5-carboxylate reductase and proline dehydrogenase activities in soybean nodules, we previously suggested two major roles for pyrroline-5-carboxylate reductase in addition to the production of the considerable quantity of proline needed for biosynthesis; namely, transfer of energy to the location of biological N(2) fixation, and production of NADP(+) to drive the pentose phosphate pathway. The latter produces ribose-5-phosphate which can be used in de novo purine synthesis required for synthesis of ureides, the major form in which biologically fixed N(2) is transported from soybean root nodules to the plant shoot. In this paper, we report rapid induction (in soybean nodules) and exceptionally high activities (in nodules of eight species of N(2)-fixing plants) of pentose phosphate pathway and pyrroline-5-carboxylate reductase. There was a marked increase in proline dehydrogenase activity during soybean (Glycine max) ontogeny. The magnitude of proline dehydrogenase activity in bacteroids of soybean nodules was sufficiently high during most of the time course to supply a significant fraction of the energy requirement for N(2) fixation. Proline dehydrogenase activity in bacteroids from nodules of other species was also high. These observations support the above hypothesis. However, comparison of pentose phosphate pathway and pyrroline-5-carboxylate reductase activities of ureide versus amide-exporting nodules offers no support. The hypothesis predicts that pyrroline-5-carboxylate and pentose phosphate pathway activities should be higher in ureide-exporting nodules than in amide-exporting nodules. This predicted distinction was not observed in the results of in vitro assays of these activities.

Oxidant-induced changes in the cellular energy homeostasis: A study with 3,5-dimethyl N-acetyl- p-benzoquinone imine and isolated hepatocytes

Exposure of isolated hepatocytes to 400 μM 3,5-dimethyl N-acetyl- p-benzoquinone imine (3,5-diMe NAPQI), rapidly induced the formation of plasma membrane blebs. More than 50% of the viable cells were afected after 1 min incubation with 3,5-diMe NAPQI. Rapid loss of mitochondrial ATP, and sequential increases in ADP and AMP accompanied hepatocyte blebbing. 3,5-diMe NAPQI also induced a propounced elevation of mitochondrial NADP level, whereas the NAD concentration was unaffected. Similar alterations in the adenine and pyridine nucleotide pools were found to occur in the cytosol, although at slower rates. During the initial phase of ATP loss and NADP production, there was also a concomitant decrease in the oxygen uptake of the hepatocytes. The decreases in energy substrates occurred in parallel to an increased uptake of trypan blue into the cells. Treatment of the hepatocytes with dithiothreitol, following 4 min exposure of the cells to 3,5-diMe NAPQI, reversed the quinone imine-induced changes in nucelotide levels and reduced the cytotoxicity. It is concluded that alteration of mitochondrial function, which results in changes in the cellular energy homeostasis, is an important event in the development of cytotoxicity caused by 3,5-diMe NAPQI.