Mitochondrial pyruvate carriers are required for myocardial stress adaptation.

In addition to fatty acids, glucose and lactate are important myocardial substrates under physiologic and stress conditions. They are metabolized to pyruvate, which enters mitochondria via the mitochondrial pyruvate carrier (MPC) for citric acid cycle metabolism. In the present study, we show that MPC-mediated mitochondrial pyruvate utilization is essential for the partitioning of glucose-derived cytosolic metabolic intermediates, which modulate myocardial stress adaptation. Mice with cardiomyocyte-restricted deletion of subunit 1 of MPC (cMPC1-/-) developed age-dependent pathologic cardiac hypertrophy, transitioning to a dilated cardiomyopathy and premature death. Hypertrophied hearts accumulated lactate, pyruvate and glycogen, and displayed increased protein O-linked N-acetylglucosamine, which was prevented by increasing availability of non-glucose substrates in vivo by a ketogenic diet (KD) or a high-fat diet, which reversed the structural, metabolic and functional remodelling of non-stressed cMPC1-/- hearts. Although concurrent short-term KDs did not rescue cMPC1-/- hearts from rapid decompensation and early mortality after pressure overload, 3 weeks of a KD before transverse aortic constriction was sufficient to rescue this phenotype. Together, our results highlight the centrality of pyruvate metabolism to myocardial metabolism and function.

3,3'-Dichlorobiphenyl Is Metabolized to a Complex Mixture of Oxidative Metabolites, Including Novel Methoxylated Metabolites, by HepG2 Cells.

3,3'-Dichlorobiphenyl (PCB 11) is a byproduct of industrial processes and detected in environmental samples. PCB 11 and its metabolites are present in human serum, and emerging evidence demonstrates that PCB 11 is a developmental neurotoxicant. However, little is known about the metabolism of PCB 11 in humans. Here, we investigated the metabolism of PCB 11 and the associated metabolomics changes in HepG2 cells using untargeted high-resolution mass spectrometry. HepG2 cells were exposed for 24 h to PCB 11 in DMSO or DMSO alone. Cell culture media were analyzed with ultra-high-performance liquid chromatography coupled with high-resolution mass spectrometry. Thirty different metabolites were formed by HepG2 cells exposed to 10 µM PCB 11, including monohydroxylated, dihydroxylated, methoxylated-hydroxylated, and methoxylated-dihydroxylated metabolites and the corresponding sulfo and glucuronide conjugates. The methoxylated PCB metabolites were observed for the first time in a human-relevant model. 4-OH-PCB 11 (3,3'-dichlorobiphenyl-4-ol) and the corresponding catechol metabolite, 4,5-di-OH-PCB 11 (3',5-dichloro-3,4-dihydroxybiphenyl), were unambiguously identified based on liquid and gas chromatographic analyses. PCB 11 also altered several metabolic pathways, in particular vitamin B6 metabolism. These results demonstrate that complex PCB 11 metabolite profiles are formed in HepG2 cells that warrant further toxicological investigation, particularly since catechol metabolites are likely reactive and toxic.

Potential-Driven Electron Transfer Lowers the Dissociation Energy of the C-F Bond and Facilitates Reductive Defluorination of Perfluorooctane Sulfonate (PFOS).

The widespread environmental occurrence of per- and polyfluoroalkyl substances (PFAS) has attracted significant regulatory, research, and media attention because of their toxicity, recalcitrance, and ability to bioaccumulate. Perfluorooctane sulfonate (PFOS) is a particularly troublesome member of the PFAS family due to its immunity to biological remediation and radical-based oxidation. In the present study, we present a heterogeneous reductive degradation process that couples direct electron transfer (ET) from surface-modified carbon nanotube electrodes (under low potential conditions) to sorbed PFOS molecules using UV-generated hydrated electrons without any further chemical addition. We demonstrate that the ET process dramatically increases the PFOS defluorination rate while yielding shorter chain (C3-C7) perfluorinated acids and present both experimental and ab initio evidence of the synergistic relationship between electron addition to sorbed molecules and their ability to react with reductive hydrated electrons. Because of the low energy consumption associated with the ET process, the use of standard medium-pressure UV lamps and no further chemical addition, this reductive degradation process is a promising method for the destruction of persistent organic pollutants, including PFAS and other recalcitrant halogenated organic compounds.

ER Stress Inhibits Liver Fatty Acid Oxidation while Unmitigated Stress Leads to Anorexia-Induced Lipolysis and Both Liver and Kidney Steatosis.

The unfolded protein response (UPR), induced by endoplasmic reticulum (ER) stress, regulates the expression of factors that restore protein folding homeostasis. However, in the liver and kidney, ER stress also leads to lipid accumulation, accompanied at least in the liver by transcriptional suppression of metabolic genes. The mechanisms of this accumulation, including which pathways contribute to the phenotype in each organ, are unclear. We combined gene expression profiling, biochemical assays, and untargeted lipidomics to understand the basis of stress-dependent lipid accumulation, taking advantage of enhanced hepatic and renal steatosis in mice lacking the ER stress sensor ATF6α. We found that impaired fatty acid oxidation contributed to the early development of steatosis in the liver but not the kidney, while anorexia-induced lipolysis promoted late triglyceride and free fatty acid accumulation in both organs. These findings provide evidence for both direct and indirect regulation of peripheral metabolism by ER stress.

Identification of a sulfate metabolite of PCB 11 in human serum.

Despite increasing evidence for a major role for sulfation in the metabolism of lower-chlorinated polychlorinated biphenyls in vitro and in vivo, and initial evidence for potential bioactivities of the resulting sulfate ester metabolites, the formation of PCB sulfates in PCB exposed human populations had not been explored. The primary goal of this study was to determine if PCB sulfates, and potentially other conjugated PCB derivatives, are relevant classes of PCB metabolites in the serum of humans with known exposures to PCBs. In order to detect and quantify dichlorinated PCB sulfates in serum samples of 46 PCB-exposed individuals from either rural or urban communities, we developed a high-resolution mass spectrometry-based protocol using 4-PCB 11 sulfate as a model compound. The method also allowed the preliminary analysis of these 46 human serum extracts for the presence of other metabolites, such as glucuronic acid conjugates and hydroxylated PCBs. Sulfate ester metabolites derived from dichlorinated PCBs were detectable and quantifiable in more than 20% of analyzed serum samples. Moreover, we were able to utilize this method to detect PCB glucuronides and hydroxylated PCBs, albeit at lower frequencies than PCB sulfates. Altogether, our results provide initial evidence for the presence of PCB sulfates in human serum. Considering the inability of previously employed analytical protocols for PCBs to extract these sulfate ester metabolites and the concentrations of these metabolites observed in our current study, our data support the hypothesis that total serum levels of PCB metabolites in exposed individuals may have been underestimated in the past.

Cytochrome c adducts with PCB quinoid metabolites.

Polychlorinated biphenyls (PCBs) are a group of 209 individual congeners widely used as industrial chemicals. PCBs are found as by-products in dye and paint manufacture and are legacy, ubiquitous, and persistent as human and environmental contaminants. PCBs with fewer chlorine atoms may be metabolized to hydroxy- and dihydroxy-metabolites and further oxidized to quinoid metabolites both in vitro and in vivo. Specifically, quinoid metabolites may form adducts on nucleophilic sites within cells. We hypothesized that the PCB-quinones covalently bind to cytochrome c and, thereby, cause defects in the function of cytochrome c. In this study, synthetic PCB quinones, 2-(4'-chlorophenyl)-1,4-benzoquinone (PCB3-pQ), 4-4'-chlorophenyl)-1,2-benzoquinone (PCB3-oQ), 2-(3', 5'-dichlorophenyl)-1,4-benzoquinone, 2-(3',4', 5'-trichlorophenyl)-1,4-benzoquinone, and 2-(4'-chlorophenyl)-3,6-dichloro-1,4-benzoquinone, were incubated with cytochrome c, and adducts were detected by liquid chromatography-mass spectrometry (LC-MS) and matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI TOF). Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was employed to separate the adducted proteins, while trypsin digestion and liquid chromatography-tandem mass spectrometry (LC-MS/MS) were applied to identify the amino acid binding sites on cytochrome c. Conformation change of cytochrome c after binding with PCB3-pQ was investigated by SYBYL-X simulation and cytochrome c function was examined. We found that more than one molecule of PCB-quinone may bind to one molecule of cytochrome c. Lysine and glutamic acid were identified as the predominant binding sites. Software simulation showed conformation changes of adducted cytochrome c. Additionally, cross-linking of cytochrome c was observed on the SDS-PAGE gel. Cytochrome c was found to lose its function as electron acceptor after incubation with PCB quinones. These data provide evidence that the covalent binding of PCB quinone metabolites to cytochrome c may be included among the toxic effects of PCBs.

Tissue Distribution, Metabolism, and Excretion of 3,3'-Dichloro-4'-sulfooxy-biphenyl in the Rat.

Polychlorinated biphenyls (PCBs) with less chlorine atoms exhibit a greater susceptibility to metabolism than their more-chlorinated counterparts. Following initial hydroxylation of these less-chlorinated PCBs, metabolic sulfation to form PCB sulfates is increasingly recognized as an important component of their toxicology. Because procedures for the quantitative analysis of PCB sulfates in tissue samples have not been previously available, we have now developed an efficient, LC-ESI-MS/MS-based protocol for the quantitative analysis of 4-PCB 11 sulfate in biological samples. This procedure was used to determine the distribution of 4-PCB 11 sulfate in liver, kidney, lung, and brain as well as its excretion profile following its intravenous administration to male Sprague-Dawley rats. Following initial uptake of 4-PCB 11 sulfate, its concentration in these tissues and serum declined within the first hour following injection. Although biliary secretion was detected, analysis of 24 h collections of urine and feces revealed recovery of less than 4% of the administered 4-PCB 11 sulfate. High-resolution LC-MS analysis of bile, urine, and feces showed metabolic products derived from 4-PCB 11 sulfate. Thus, 4-PCB 11 sulfate at this dose was not directly excreted in the urine but was instead redistributed to tissues and/or subjected to further metabolism.

Chlorinated biphenyl quinones and phenyl-2,5-benzoquinone differentially modify the catalytic activity of human hydroxysteroid sulfotransferase hSULT2A1.

Human hydroxysteroid sulfotransferase (hSULT2A1) catalyzes the sulfation of a broad range of environmental chemicals, drugs, and other xenobiotics in addition to endogenous compounds that include hydroxysteroids and bile acids. Polychlorinated biphenyls (PCBs) are persistent environmental contaminants, and oxidized metabolites of PCBs may play significant roles in the etiology of their adverse health effects. Quinones derived from the oxidative metabolism of PCBs (PCB-quinones) react with nucleophilic sites in proteins and also undergo redox cycling to generate reactive oxygen species. This, along with the sensitivity of hSULT2A1 to oxidative modification at cysteine residues, led us to hypothesize that electrophilic PCB-quinones react with hSULT2A1 to alter its catalytic function. Thus, we examined the effects of four phenylbenzoquinones on the ability of hSULT2A1 to catalyze the sulfation of the endogenous substrate, dehydroepiandrosterone (DHEA). The quinones studied were 2'-chlorophenyl-2,5-benzoquinone (2'-Cl-BQ), 4'-chlorophenyl-2,5-benzoquinone (4'-Cl-BQ), 4'-chlorophenyl-3,6-dichloro-2,5-benzoquinone (3,6,4'-triCl-BQ), and phenyl-2,5-benzoquinone (PBQ). At all concentrations examined, pretreatment of hSULT2A1 with the PCB-quinones decreased the catalytic activity of hSULT2A1. Pretreatment with low concentrations of PBQ, however, increased the catalytic activity of the enzyme, while higher concentrations inhibited catalysis. A decrease in substrate inhibition with DHEA was seen following preincubation of hSULT2A1 with all of the quinones. Proteolytic digestion of the enzyme followed by LC/MS analysis indicated PCB-quinone- and PBQ-adducts at Cys55 and Cys199, as well as oxidation products at methionines in the protein. Equilibrium binding experiments and molecular modeling suggested that changes due to these modifications may affect the nucleotide binding site and the entrance to the sulfuryl acceptor binding site of hSULT2A1.

Attenuation of Leishmania infantum chagasi metacyclic promastigotes by sterol depletion.

The infectious metacyclic promastigotes of Leishmania protozoa establish infection in a mammalian host after they are deposited into the dermis by a sand fly vector. Several Leishmania virulence factors promote infection, including the glycosylphosphatidylinositol membrane-anchored major surface protease (MSP). Metacyclic Leishmania infantum chagasi promastigotes were treated with methyl-beta-cyclodextrin (MßCD), a sterol-chelating reagent, causing a 3-fold reduction in total cellular sterols as well as enhancing MSP release without affecting parasite viability in vitro. MßCD-treated promastigotes were more susceptible to complement-mediated lysis than untreated controls and reduced the parasite load 3-fold when inoculated into BALB/c mice. Paradoxically, MßCD-treated promastigotes caused a higher initial in vitro infection rate in human or murine macrophages than untreated controls, although their intracellular multiplication was hindered upon infection establishment. There was a corresponding larger amount of covalently bound C3b than iC3b on the parasite surfaces of MßCD-treated promastigotes exposed to healthy human serum in vitro, as well as loss of MSP, a protease that enhances C3b cleavage to iC3b. Mass spectrometry showed that MßCD promotes the release of proteins into the extracellular medium, including both MSP and MSP-like protein (MLP), from virulent metacyclic promastigotes. These data support the hypothesis that plasma membrane sterols are important for the virulence of Leishmania protozoa at least in part through retention of membrane virulence proteins.

Modification of the catalytic function of human hydroxysteroid sulfotransferase hSULT2A1 by formation of disulfide bonds.

The human cytosolic sulfotransferase hSULT2A1 catalyzes the sulfation of a broad range of xenobiotics, as well as endogenous hydroxysteroids and bile acids. Reversible modulation of the catalytic activity of this enzyme could play important roles in its physiologic functions. Whereas other mammalian sulfotransferases are known to be reversibly altered by changes in their redox environment, this has not been previously shown for hSULT2A1. We have examined the hypothesis that the formation of disulfide bonds in hSULT2A1 can reversibly regulate the catalytic function of the enzyme. Three thiol oxidants were used as model compounds to investigate their effects on homogeneous preparations of hSULT2A1: glutathione disulfide, 5,5'-dithiobis(2-nitrobenzoic acid), and 1,1'-azobis(N,N-dimethylformamide) (diamide). Examination of the effects of disulfide bond formation with these agents indicated that the activity of the enzyme is reversibly altered. Studies on the kinetics of the hSULT2A1-catalyzed sulfation of dehydroepiandrosterone (DHEA) showed the effects of disulfide bond formation on the substrate inhibition characteristics of the enzyme. The effects of these agents on the binding of substrates and products, liquid chromatography-mass spectrometry identification of the disulfides formed, and structural modeling of the modified enzyme were examined. Our results indicate that conformational changes at cysteines near the nucleotide binding site affect the binding of both the nucleotide and DHEA to the enzyme, with the specific effects dependent on the structure of the resulting disulfide. Thus, the formation of disulfide bonds in hSULT2A1 is a potentially important reversible mechanism for alterations in the rates of sulfation of both endogenous and xenobiotic substrates.

Urinary metabolomic markers of aminoglycoside nephrotoxicity in newborn rats.

BACKGROUND: Aminoglycoside exposure is a common cause of acute kidney injury (AKI). Delay in the diagnosis of AKI using conventional biomarkers has been one of the important obstacles in applying early effective interventions. We tested the hypothesis that urinary metabolomics could identify novel early biomarkers for toxic renal injury. METHODS: Three-day-old rats were divided into three groups; they received a single daily injection of vehicle (0.9% NaCl solution) or gentamicin at a dose of 10 or 20 mg/kg/d for 7 d. Urine and blood were collected after 3 and 7 d of injections. Urinary metabolites were evaluated using high-performance liquid chromatography and gas chromatography/mass spectrometry. RESULTS: A distinct urinary metabolic profile characterized by glucosuria, phosphaturia, and aminoaciduria was identified preceding changes in serum creatinine. At both the gentamicin doses, urinary tryptophan was significantly (P < 0.05) increased (fold change: 1.91 and 2.31 after 3 d; 1.81 and 1.93 after 7 d). Similarly, kynurenic acid, a tryptophan metabolite, showed a significant (P < 0.05) decrease (fold change: 0.26 and 0.24 after 3 d; 0.21 and 0.52 after 7 d), suggesting an interruption of the normal tryptophan metabolism pathway. CONCLUSION: We conclude that urinary metabolomic profiling provides a robust approach for identifying early and novel markers of gentamicin-induced AKI.

Identification of sulfated metabolites of 4-chlorobiphenyl (PCB3) in the serum and urine of male rats.

Polychlorinated biphenyls (PCBs) are legacy pollutants that exert toxicities through various mechanisms. In recent years exposure to PCBs via inhalation has been recognized as a hazard. Those PCBs with lower numbers of chlorine atoms (LC-PCBs) are semivolatile and have been reported in urban air, as well as in the indoor air of older buildings. LC-PCBs are bioactivated to phenols and further to quinone electrophiles with genotoxic/carcinogenic potential. We hypothesized that phenolic LC-PCBs are subject to conjugation and excretion in the urine. PCB3, often present in high concentrations in air, is a prototypical congener for the study of the metabolism and toxicity of LC-PCBs. Our objective was to identify metabolites of PCB3 in urine that could be potentially employed in the estimation of exposure to LC-PCBs. Male Sprague-Dawley rats (150-175 g) were housed in metabolism cages and received a single intraperitoneal injection of 600 µmol/kg body weight of PCB3. Urine was collected every 4 h; rats were euthanized at 36 h; and serum was collected. LC/MS analysis of urine before and after incubation with ß-glucuronidase and sulfatase showed that sulfate conjugates were in higher concentrations than glucuronide conjugates and free phenolic forms. At least two major metabolites and two minor metabolites were identified in urine that could be attributed to mercapturic acid metabolites of PCB3. Quantitation by authentic standards confirmed that approximately 3% of the dose was excreted in the urine as sulfates over 36 h, with peak excretion occurring at 10-20 h after exposure. The major metabolites were 4'PCB3sulfate, 3'PCB3 sulfate, 2'PCB3 sulfate, and presumably a catechol sulfate. The serum concentration of 4'PCB3 sulfate was 6.18 ± 2.16 µg/mL. This is the first report that sulfated metabolites of PCBs are formed in vivo. These findings suggest a prospective approach for exposure assessment of LC-PCBs by analysis of phase II metabolites in urine.

Mitochondrial superoxide and coenzyme Q in insulin-deficient rats: increased electron leak.

Mitochondrial superoxide is important in the pathogeneses of diabetes and its complications. However, there is uncertainty regarding the intrinsic propensity of mitochondria to generate this radical. Studies to date suggest that superoxide production by mitochondria of insulin-sensitive target tissues of insulin-deficient rodents is reduced or unchanged. Moreover, little is known of the role of the Coenzyme Q (CoQ), whose semiquinone form reacts with molecular oxygen to generate superoxide. We measured reactive oxygen species (ROS) production, respiratory parameters, and CoQ content in mitochondria from gastrocnemius muscle of control and streptozotocin (STZ)-diabetic rats. CoQ content did not differ between mitochondria isolated from vehicle- or STZ-treated animals. CoQ also was unaffected by weight loss in the absence of diabetes (induced by caloric restriction). Under state 4 or state 3 conditions, both respiration and ROS release were reduced in diabetic mitochondria fueled with succinate, glutamate plus malate, or with all three substrates (continuous TCA cycle). However, H(2)O(2) and directly measured superoxide production were substantially increased in gastrocnemius mitochondria of diabetic rats when expressed per unit oxygen consumed. On the basis of substrate and inhibitor effects, the mechanism involved multiple electron transport sites. More limited results using heart mitochondria were similar. ROS per unit respiration was greater in muscle mitochondria from diabetic compared with control rats during state 3, as well as state 4, while the reduction in ROS per unit respiration on transition to state 3 was less for diabetic mitochondria. In summary, ROS production is, in fact, increased in mitochondria from insulin-deficient muscle when considered relative to electron transport. This is evident on multiple energy substrates and in different respiratory states. CoQ is not reduced in diabetic mitochondria or with weight loss due to food restriction. The implications of these findings are discussed.

Oxidation of 4-chlorobiphenyl metabolites to electrophilic species by prostaglandin H synthase.

Hormonally sensitive tissues, like the prostate, ovary, and breast, increasingly studied as targets of environmental chemicals, are sources of an enzyme potentially capable of transforming and activating xenobiotics to highly reactive metabolites. Our study specifically addresses the question of whether prostaglandin H synthase (PGHS) can activate phenolic metabolites of polychlorinated biphenyls (PCBs). We found that human recombinant PGHS-2 catalyzed the oxidation of ortho (2',3'- and 3',4'-) and para (2',5'-) dihydroxy 4-chlorobiphenyl metabolites to their corresponding quinones. These were trapped in situ with N-acetyl cysteine, and the reaction products were isolated and characterized by liquid chromatography coupled mass spectrometry and (1)H and heteronuclear ((1)H-(13)C) nuclear magnetic resonance spectroscopy. Both mono- and di-N-acetyl cysteine Michael addition adducts were identified, with the 2',3'- and 2',5'-dihydroxy metabolites predominantly forming mono-N-acetyl cysteine adducts, while the 3',4'-dihydroxy predominantly formed disubstituted N-acetyl cysteine adducts. These studies clearly demonstrate that the phenolic metabolites of these environmental pollutants are activated by PGHS, as cosubstrates, to highly reactive electrophilic PCB quinones, with a potential for protein and DNA damage, especially in nonhepatic tissues where the enzyme is found.

Innovative application of fluoro tagging to trace airborne particulate and gas-phase polybrominated diphenyl ether exposures.

Polybrominated diphenyl ethers (PBDEs) are flame retardants applied as coatings to many consumer products, including household items. PBDEs are released and produce airborne vapors and dusts. Inhalation of particle-phase and/or gas-phase PBDEs is therefore a major route of exposure. In an attempt to mimic realistic airborne exposures, actual uptake, and deposition of particles and vapors, we prepared and characterized particles for future animal exposure studies. To trace the particles in environmental and biological systems, we employed fluoro tagging. We synthesized, characterized, and employed three PBDE congeners, 35, 47, and 99, and five fluoro-substituted PBDEs (F-PBDEs), 17-F5' 25-F5', 28-F3', 35-F5', 47-F3, and 99-F3', for this study. The PBDE congeners were selected because they are commonly found in house dust. For that reason, we coated spherical silica particles of 3 microm and C18 endcapped silica as representative and inert support materials, with 20, 30, and 40% PBDEs. We determined the particle size distributions by aerodynamic particle size spectrometry and the morphology by scanning electron microscopy. The suitability of the fluoro-tagged tracers to mimic their corresponding parent PBDEs was investigated by extraction studies from spiked blood serum. Our study is of fundamental importance to the development of xenobiotic tracers for monitoring routes of human exposure to PBDEs and understanding uptake of PBDEs from particles and vapors.

Inactivation of anthracyclines by serum heme proteins.

We have previously shown that the anticancer agent doxorubicin undergoes oxidation and inactivation when exposed to myeloperoxidase-containing human leukemia HL-60 cells, or to isolated myeloperoxidase, in the presence of hydrogen peroxide and nitrite. In the current study we report that commercial fetal bovine serum (FBS) alone oxidizes doxorubicin in the presence of hydrogen peroxide and that nitrite accelerates this oxidation. The efficacy of inactivation was dependent on the concentration of serum present; no reaction was observed when hydrogen peroxide or serum was omitted. Peroxidase activity assays, based on oxidation of 3,3',5,5'-tetramethylbenzidine, confirmed the presence of a peroxidase in the sera from several suppliers. The peroxidative activity was contained in the >10000 MW fraction. We also found that hemoglobin, a heme protein likely to be present in commercial FBS, is capable of oxidizing doxorubicin in the presence of hydrogen peroxide and that nitrite further stimulates the reaction. In contrast to intact doxorubicin, the serum + hydrogen peroxide + nitrite treated drug appeared to be nontoxic for PC3 human prostate cancer cells. Together, this study shows that (pseudo)peroxidases present in sera catalyze oxidation of doxorubicin by hydrogen peroxide and that this diminishes the tumoricidal activity of the anthracycline, at least in in vitro settings. Finally, this study also points out that addition of H2O2 to media containing FBS will stimulate peroxidase-type of reactions, which may affect cytotoxic properties of studied compounds.

Inactivation of anthracyclines by cellular peroxidase.

The anticancer anthracyclines, doxorubicin and daunorubicin, are highly cytotoxic to both cancer and normal cells. In this work, we have investigated the capacity of cellular myeloperoxidase to inactivate these agents. We show that incubation of human leukemia HL-60 cells with the anthracyclines in the presence of hydrogen peroxide and nitrite causes irreversible oxidation of the drugs, suggesting an extensive modification of their chromophores. Methimazole, 4-aminobenzoic acid hydrazide, or azide inhibits the reaction, suggesting that it is mediated by the cellular myeloperoxidase, an enzyme naturally present in large amounts in HL-60 cells. In contrast to the intact drugs, the oxidatively transformed anthracyclines were substantially less cytotoxic for HL-60 (assayed by apoptosis) and PC3 prostate cancer cells and H9c2 rat cardiac myoblasts in vitro (assayed by clonogenic survival), indicating that the oxidative metabolism of these agents leads to their inactivation. Using tandem mass spectrometry, we identified two specific metabolic products of the anthracycline degradation, 3-methoxyphthalic acid and 3-methoxysalicylic acid. These two metabolic products were obtained as authentic compounds and were nontoxic to HL-60 leukemic cells and cardiac myocytes. These findings may have important implications for the cellular pharmacology of anthracyclines and for clinical oncology.

Studies on the reactions between daptomycin and glyceraldehyde.

The objectives of this project were to determine the reaction pathways of daptomycin in the presence of glyceraldehyde in acidic solutions, and to quantitate the kinetics of the major pathways. In the presence of glyceraldehyde (pH range 1-7 at 25 to 60 degrees C), daptomycin formed two major products separable by RP-HPLC. The products were identified using UV spectroscopy, fluorimetry, mass spectrometry, and 2D-1H NMR. The reaction scheme involved the reversible formation of imine and anilide derivatives. Carbinolamine was believed to be a common intermediate in formation pathways of both products. The carbinolamine intermediate underwent either acid catalyzed dehydration resulting in imine formation or intramolecular hydrogen bonding and bond cleavage giving rise to anilide formation. In mild acid conditions, both products reversed to daptomycin. The reaction between daptomycin and glyceraldehyde was first-order with respect to both reactants. In a pH range of 1-7, the imine formation rate was pH dependent with a maximum rate at approximate pH values of 3-4. The observed pH dependence was consistent with the pH dependence of typical amine-aldehyde reactions.

20-hydroxyeicosatetraenoic acid (20-HETE) metabolism in coronary endothelial cells.

We have investigated the role of endothelial cells in the metabolism of 20-hydroxyeicosatetraenoic acid (20-HETE), a vasoactive mediator synthesized from arachidonic acid by cytochrome P450 omega-oxidases. Porcine coronary artery endothelial cells (PCEC) incorporated 20-[(3)H]HETE primarily into the sn-2 position of phospholipids through a coenzyme A-dependent process. The incorporation was reduced by equimolar amounts of arachidonic, eicosapentaenoic or 8,9-epoxyeicosatrienoic acids, but some uptake persisted even when a 10-fold excess of arachidonic acid was available. The retention of 20-[(3)H]HETE increased substantially when methyl arachidonoyl fluorophosphonate, but not bromoenol lactone, was added, suggesting that a Ca(2+)-dependent cytosolic phospholipase A(2) released the 20-HETE contained in PCEC phospholipids. Addition of calcium ionophore A23187 produced a rapid release of 20-[(3)H]HETE from the PCEC, a finding that also is consistent with a Ca(2+)-dependent mobilization process. PCEC also converted 20-[(3)H]HETE to 20-carboxy-arachidonic acid (20-COOH-AA) and 18-, 16-, and 14-carbon beta-oxidation products. 20-COOH-AA produced vasodilation in porcine coronary arterioles, but 20-HETE was inactive. These results suggest that the incorporation of 20-HETE and its subsequent conversion to 20-COOH-AA in the endothelium may be important in modulating coronary vascular function.