Aqueous-soluble Metabolites Research Articles

Viscum album mother tinctures: Harvest conditions and host trees influence the plant metabolome and the glycolytic pathway of breast cancer cells.

Viscum album is a semi-parasitic plant used for over one hundred years in complementary cancer therapy. The main commercial drugs used in cancer patients' treatment are derived from the aqueous V. album extracts, whose cytotoxic potential is mostly attributed to the aqueous soluble antitumoral metabolites. On the counterpart, ethanol solvents must be used to obtain V. album mother tinctures. This methodology permits better solubilization of phenolic compounds, among others, which present antitumoral bioactivity. Recently, the metabolomics approach revealed the influence of the host tree on the V. album subspecies differentiation. To increase the scientific information about the chemical differences related to the host trees and to clarify the seasonal influences, in this study, the metabolome of 50V. album mother tinctures from three subspecies (abietis, album, austriacum) and five host trees (Malus domestica, Quercus sp., Ulmus carpinifolia, Pinus sylvestris, Abies alba) was evaluated using summer and winter plant harvests. The in vitro cytotoxic activities were investigated in breast cancer cells (MDA-MB-231) and immortalized normal human keratinocytes (HaCaT). The summer V. album mother tinctures presented higher cytotoxic activity than winter ones. Among the summer samples, those prepared with V. album subsp. album were more cytotoxic than V. album subsp. abietis and subsp. V. album subsp. austriacum. The V. album harvested from Quercus petraea and Abies alba inhibited the key-glycolytic enzymes: hexokinase (HK), phosphofructokinase (PFK), pyruvate kinase (PK). This activity was related to a reduction in glucose uptake and lactate production, which were host-tree-time-dose-dependent. The untargeted metabolomic approach was able to discriminate the mother tinctures according to respective botanical classes and harvest season. A total of 188 metabolites were annotated under positive and negative modes. Fourteen compounds were responsible for the samples differentiation, and, to the best of our knowledge, eight were described in the Viscum album species for the first time. Our study shows the interruption of the Warburg effect as a novel antitumoral mechanism triggered by V. album mother tinctures, which is related to their metabolite profile. These results bring scientific evidence that encourages the use of V. album mother tinctures as a natural product for cancer therapy.

Investigation of Obesity-Alleviation Effect of Eurycoma longifolia on Mice Fed with a High-Fat Diet through Metabolomics Revealed Enhanced Decomposition and Inhibition of Accumulation of Lipids.

The metabolic and bioactivity effects of Eurycoma longifolia (Eucalyptus longifolia) in obesity treatment were studied in mice fed with a high-fat diet using a metabolomics approach. Aqueous extracts of E. longifolia were obtained via grinding, dissolving, and freeze-drying. The hepatic steatosis effect of E. longifolia was characterized by hematoxylin and eosin histological staining. External performance of the obesity-alleviation effect was monitored by measuring body and food weight. In addition, the metabolomics analysis of the E. longifolia-mice interaction system was performed using the established platform combining liquid chromatography-tandem mass spectrometry with statistical analysis. The presence and spatial distribution patterns of differential molecules were further evaluated through desorption electrospray ionization-mass spectrometry imaging. The results showed that E. longifolia played a vital role in downregulating lipid accumulation (especially triacylglycerols) and fatty acids biosynthesis together with enhanced lipid decomposition and healing in Bagg albino mice. During such a process, E. longifolia mainly induced metabolomic alterations of amino acids, organic acids, phospholipids, and glycerolipids. Moreover, under the experimental concentrations, E. longifolia induced more fluctuations of aqueous-soluble metabolites in the plasma and lipids in the liver than in the kidneys. This study provides an advanced alternative to traditional E. longifolia-based studies for evaluating the metabolic effects and bioactivity of E. longifolia through metabolomics technology, revealing potential technological improvement and clinical application.

Phytochemical analysis and in vitro anti-proliferative activity of Viscum album ethanolic extracts

BackgroundViscum album L. (Santalaceae), commonly known as mistletoe, is a hemiparasitic plant traditionally used in complementary cancer treatment. Its antitumor potential is mostly attributed to the presence of aqueous soluble metabolites; however, the use of ethanol as solvent also permits the extraction of pharmacological compounds with antitumor potential. The clinical efficacy of mistletoe therapy inspired the present work, which focuses on ethanolic extracts (V. album “mother tinctures”, MT) prepared from different host trees.MethodsSamples from three European subspecies (album, austriacum, and abietis) were harvested, and five different V. album-MT strains were prepared. The following phytochemical analyses were performed: thin layer chromatography (TLC), high-performance liquid chromatography (HPLC) and liquid chromatography-high resolution mass spectrometry (LC-HRMS). The proliferation assay was performed with WST-1 after incubation of tumor (Yoshida and Molt-4) and fibroblast cell lines (NIH/3 T3) with different MT concentrations (0.5 to 0.05% v/v). The cell death mechanism was investigated by flow cytometry (FACS) using Annexin V-7AAD.ResultsChemical analyses of MT showed the presence of phenolic acids, flavonoids and lignans. The MT flavonoid and viscotoxin contents (mg/g fresh weight) were highest in Quercus robur (9.67 ± 0.85 mg/g) and Malus domestica (3.95 ± 0.58 mg/mg), respectively. The viscotoxin isoform proportions (% total) were also different among the VA subspecies with a higher content of A3 in V. album growing on Abies alba (60.57 ± 2.13). The phytochemical compounds as well as the viscotoxin contents are probably related to the antitumor effects of MT. The cell death mechanisms evaluated by colorimetric and FACS methodologies involved necrotic damage, which was host tree-, time- and dose- dependent, with different selectivity to tumor cells. Mother tincture from V. album ssp. abietis was the most effective at inducing in vitro cellular effects, even when incubated at the smallest concentration tested, probably because of the higher content of VT A3.ConclusionOur results indicate the promising antitumor potential of Viscum album ethanolic extracts and the importance of botanical and phytochemical characterization for in vitro anti-proliferative effects.

Concentration of Metabolites from Low-density Planktonic Communities for Environmental Metabolomics using Nuclear Magnetic Resonance Spectroscopy

Environmental metabolomics is an emerging field that is promoting new understanding in how organisms respond to and interact with the environment and each other at the biochemical level. Nuclear magnetic resonance (NMR) spectroscopy is one of several technologies, including gas chromatography-mass spectrometry (GC-MS), with considerable promise for such studies. Advantages of NMR are that it is suitable for untargeted analyses, provides structural information and spectra can be queried in quantitative and statistical manners against recently available databases of individual metabolite spectra. In addition, NMR spectral data can be combined with data from other omics levels (e.g. transcriptomics, genomics) to provide a more comprehensive understanding of the physiological responses of taxa to each other and the environment. However, NMR is less sensitive than other metabolomic techniques, making it difficult to apply to natural microbial systems where sample populations can be low-density and metabolite concentrations low compared to metabolites from well-defined and readily extractable sources such as whole tissues, biofluids or cell-cultures. Consequently, the few direct environmental metabolomic studies of microbes performed to date have been limited to culture-based or easily defined high-density ecosystems such as host-symbiont systems, constructed co-cultures or manipulations of the gut environment where stable isotope labeling can be additionally used to enhance NMR signals. Methods that facilitate the concentration and collection of environmental metabolites at concentrations suitable for NMR are lacking. Since recent attention has been given to the environmental metabolomics of organisms within the aquatic environment, where much of the energy and material flow is mediated by the planktonic community, we have developed a method for the concentration and extraction of whole-community metabolites from planktonic microbial systems by filtration. Commercially available hydrophilic poly-1,1-difluoroethene (PVDF) filters are specially treated to completely remove extractables, which can otherwise appear as contaminants in subsequent analyses. These treated filters are then used to filter environmental or experimental samples of interest. Filters containing the wet sample material are lyophilized and aqueous-soluble metabolites are extracted directly for conventional NMR spectroscopy using a standardized potassium phosphate extraction buffer. Data derived from these methods can be analyzed statistically to identify meaningful patterns, or integrated with other omics levels for comprehensive understanding of community and ecosystem function.

Functional properties and substrate characterization of human CYP26A1, CYP26B1, and CYP26C1 expressed by recombinant baculovirus in insect cells

Introduction The cytochrome P450 CYP26 family of retinoic acid (RA) metabolizing enzymes, comprising CYP26A1, CYP26B1, and CYP26C1 is critical for establishing patterns of RA distribution during embryonic development and retinoid homeostasis in the adult. All three members of this family can metabolize all trans-RA. CYP26C1 has also been shown to efficiently metabolize the 9 -cis isomer of RA. Methods We have co-expressed each of the CYP26 enzymes along with the NADPH-cytochrome P450 oxidoreductase using a baculovirus/Sf9 insect cell expression system to determine the enzymatic activities of these enzymes in cell free preparations and have established an in vitro binding assay to permit comparison of binding affinities of the three CYP26 enzymes. Results We demonstrated that the expressed enzymes can efficiently coordinate heme, as verified by spectral-difference analysis. All CYP26s efficiently metabolized all -trans-RA to polar aqueous-soluble metabolites, and in competition experiments exhibited IC 50 values of 16, 27, and 15 nM for CYP26A1, B1, and C1 respectively for all- trans-RA. Furthermore, this metabolism was blocked with the CYP inhibitor ketoconazole. CYP26C1 metabolism of all trans-RA could also be effectively competed with 9- cis RA, with IC 50 of 62 nM, and was sensitive to ketoconazole inhibition. Discussion CYP26 enzymes are functionally expressed in microsomal fractions of insect cells and stably bind radiolabeled RA isomers with affinities respecting their substrate specificities. We demonstrated that compared to CYP26A and CYP26B, only CYP26C1 was able to bind with high affinity to 9- cis-RA. These assays will be useful for the screening of synthetic substrates and inhibitors of CYP26 enzymes and may be applicable to other cytochrome P450s and their respective substrates.

Effect of the Histone Deacetylase Inhibitor Trichostatin A on the Metabolome of Cultured Primary Hepatocytes

Trichostatin A (TSA) is a histone deacetylase inhibitor that has antiproliferative and differentiation-inducing effects on cancer cells, and in cultures of primary hepatocytes has been shown to maintain xenobiotic metabolic capacity. Using an NMR-based metabolic profiling approach, we evaluated if the endogenous metabolome was stabilized and the normal metabolic phenotype retained in this model. Aqueous soluble metabolites were extracted from isolated rat hepatocytes after 44 and 92 h exposure to TSA (25 muM) together with time-matched controls and measured by (1)H NMR spectroscopy. Multivariate analysis showed a clear difference in the global metabolic profile over time in control samples, while the TSA treated group was more closely clustered at both time points, suggesting that treatment reduced the time related effect on metabolism that was observed in the control. TSA treatment was associated with decreases in glycerophosphocholine, 3-hydroxybutyric acid, glycine and adenosine, an increase in glycogen, and a reduction in the decrease of inosine, hypoxanthine, and glutathione over time. Collectively, our data suggest that TSA treatment reduces the loss of a normal metabolic phenotype in cultured primary hepatocytes, improving the model as a tool to study endogenous liver metabolism, xenobiotic metabolism, and potentially affecting the accuracy of all biological assays in this system.

Calcitroic Acid Is a Major Catabolic Metabolite in the Metabolism of 1α-Dihydroxyvitamin D2

Calcitroic acid (1α-hydroxy-23 carboxy-24,25,26,27-tetranorvitamin D3) is known to be the major water-soluble metabolite produced during the deactivation of 1α,25-dihydroxyvitamin D3. This deactivation process involves a series of oxidation reactions at C24 and C23 leading to side-chain cleavage and, ultimately, formation of the calcitroic acid. Like 1α,25-dihydroxyvitamin D3, 1α,25-dihydroxyvitamin D2 is also known to undergo side-chain oxidation; however, to date there has been no evidence suggesting that 1α,25-dihydroxyvitamin D2 undergoes side-chain cleavage. To investigate this possibility, we studied 1α,25-dihydroxyvitamin D2 metabolism in HPK1A-ras cells as well as the well characterized perfused rat kidney system. Lipid and aqueous-soluble metabolites were prepared for characterization. Aqueous-soluble metabolites were subjected to reverse-phase HPLC analysis. The major aqueous-soluble metabolite from both the kidney and cell incubations comigrated with authentic calcitroic acid on two reverse-phase HPLC columns of different chemistry. The putative calcitroic acid from the cell and kidney incubations was methylated and found to comigrate with methylated authentic standard on straight-phase and reverse-phase HPLC columns. The identity of the methylated metabolite from cell incubations was also confirmed by mass spectral analysis. These data show, for the first time, that calcitroic acid is a major terminal product for the deactivation of 1α,25-dihydroxyvitamin D2. Intermediates leading to the formation of the calcitroic acid in the 1α,25-dihydroxyvitamin D2 metabolism pathway are currently being studied.

Rapid hepatic metabolism of 7-ketocholesterol in vivo: implications for dietary oxysterols

7-Ketocholesterol is a major dietary oxysterol and the predominant non-enzymically formed oxysterol in human atherosclerotic plaque. We tested the hypothesis that 7-ketocholesterol is preferentially retained by tissues relative to cholesterol in vivo. To ensure rapid tissue uptake, acetylated low density lipoprotein, labeled with esters of [(14)C]-7-ketocholesterol and [(3)H]cholesterol, was injected into rats via a jugular catheter. At timed intervals (2 min to 24 h) rats (n = 48 total) were exsanguinated and tissues were dissected and assayed for radioactivity. In two experiments the majority of both radiolabels appeared in the liver after 2 min. In all tissues, (14)C appeared transiently and did not accumulate. Rather, it was metabolized in the liver and excreted into the intestine mainly as aqueous-soluble metabolites (presumably bile acids). By 9 h, (14)C in the liver had decreased to 10% of the injected dose while 36% was present in the intestine. In contrast, at 9 h 38% of (3)H was evident in the liver while only 5% was found in the intestine. Unlike [(3)H]cholesterol, little (14)C was found to re-enter the circulation, indicating that enterohepatic recycling of 7-ketocholesterol was negligible. This is the first report of the distribution of an oxysterol relative to cholesterol, administered simultaneously, in a whole animal model. The finding that [(14)C]-7-ketocholesterol is rapidly metabolized and excreted by the liver suggests that diet may not be a major source of oxysterols in atherosclerotic plaque, and that perhaps dietary oxysterols make little or no contribution to atherogenesis.

Elimination of sediment-associated benzo[a]pyrene and its metabolites by polychaete worms exposed to 3-methylcholanthrene

Polychaete worms can be important vectors for the accumulation and transfer of sediment-associated organic contaminants, including polycyclic aromatic hydrocarbons (PAHs). Differences between species in ability to metabolize and eliminate organic compounds may contribute to differences in steady state body burdens of parent compound and metabolites. In this study, three species of polychaete worms, Leitoscoloplos fragilis, Marenzellaria viridis, and Nereis diversicolor, were exposed to trace quantities of the radiolabelled PAH, benzo[a]-pyrene (BaP) in sediment, with or without 3-methylcholanthrene (3MC), a compound known to induce metabolism of PAHs in other species. After exposure, worms were transferred to clean sediment and the rate of elimination of parent compound and disposition of metabolites was followed for up to 17 days. Elimination of parent compound was slowest in L. fragilis (half-life = 10.3 d), the species with the most limited ability to metabolize BaP. Elimination of parent BaP was more rapid in M. viridis (half-life = 5.1 d) and N. diversicolor (3.7 d), species which metabolize BaP extensively. Aqueous-soluble metabolites appeared to be eliminated very slowly in M. viridis. Treatment with 3MC did not to enhance the rate elimination of parent compound, suggesting that metabolism of BaP was not induced by exposure to 3MC.

The toxicokinetics of benzo[a]pyrene in juvenile coho salmon, Oncorhynchus kisutch, during smoltification

The activities of Phase I and Phase II biotransformation enzymes in the livers of yearling coho salmon (Oncorhynchus kisutch), were measured biweekly from February until the release date from the hatchery in mid-June, in order to observe any alterations in baseline levels during smoltification. Peak enzyme activities occurred in February and March and then declined through to June. Total cytochrome P450 levels ranged from 0.024±0.009 to 0.095±0.010 nmol mg-1 microsomal protein, ethoxyresorufin O-deethylase activity ranged from 2.74±0.75 to 9.94±0.85 pmol min-1 mg-1 microsomal protein, and glutathione S-transferase activity ranged from 0.07±0.01 to 0.33±0.01 µmol min-1 mg-1 cytosolic protein during this period. Following an intraperitoneal injection of [3H]benzo[a]pyrene (B[a]P), elimination occurred rapidly (>71% excreted into the bile within 24h) from February to June. Although the distribution of B[a]P in tissues changed through the sampling period, the highest leels of B[a]P-derived radioactivity were found in the liver, bile and fat. Analysis of the bile revealed that 55 to 63% of the radioactivity was Phase I metabolites, 16 to 24% glucuronide conjugates, 8% sulfate conjugates, 7% other conjugates and 6% aqueous-soluble metabolites. These findings suggest that the transformation from freshwater adapted coho ‘parr’ to ‘smolts’, can significantly alter biotransformation enzyme activities and the distribution and elimination of xenobiotics such as benzo[a]pyrene in these fish.

Transformation and tolerance of TNT (2,4,6-trinitrotoluene) by fungi

Eight fungi were tested for the ability to transform and tolerate TNT (2,4,6-trinitrotoluene) in liquid culture. In contrast to most previous studies, we used high concentrations of TNT (200 ppm), short incubation times, nonlignolytic conditions and taxonomically diverse fungi. Virtually no TNT was detectible in cultures of Cladosporium resinae and Cunninghamella echinulata var. elegans after three days growth. All fungi produced azoxy-tetranitrotoluene, amino-dinitrotoluene and hydroxylamino-dinitrotoluene metabolites. The fate of [ 14C]TNT fed to cultures was followed. Cunninghamella echinulata var. elegans, Trichoderma viride, Schizophyllum commune and Cladosporium resinae converted significant amounts (27%, 19%, 18% and 8%, respectively) to aqueous-soluble metabolites. No fungi produced 14CO 2 or 14C-volatile metabolites. Trichoderma viride and C. resinae were most tolerant of TNT, as determined by the inhibition of radial growth on agar. Imperfect fungi, native to soil and able to tolerate TNT and transform it rapidly, could be used to increase the efficiency of mineralization of TNT by Phanerochaete chrysosporium.

Liver slices in dynamic organ culture. II. Anin vitrocellular technique for the study of integrated drug metabolism using human tissue

1. Precision cut human liver slices in dynamic organ culture have been used to study the integrated metabolism of 7-ethoxycoumarin and the conjugation of 7-hydroxycoumarin. 2. The metabolism of 7-ethoxycoumarin and 7-hydroxycoumarin was monitored for 6 h. For both substrates there was a time-dependent increase in metabolites present in the incubation medium. The low levels of free 7-hydroxycoumarin found in the medium when 7-ethoxycoumarin was the substrate suggests good coupling of phase I and phase II metabolism. 3. With suitable incubation conditions, i.e. change of medium containing new substrate every 2 h, the metabolism of both 7-ethoxycoumarin and 7-hydroxycoumarin by human liver slices was found to proceed at similar rates for up to 24 h. This was demonstrated using five separate human liver preparations. 4. Human liver slices also metabolized mono-chlorobenzene and o-, m- and p-dichlorobenzene to aqueous soluble metabolites. There was a time-dependent increase in the appearance of aqueous soluble metabolites present in the incubation medium. Metabolites were not retained by the liver slices. 5. A cold-storage transit buffer has been described and used to maintain the levels of drug metabolism in both rat and human tissue for periods of up to 6 h. 6. The use of human liver slices in dynamic organ culture as a suitable method for the direct assessment of integrated hepatic drug metabolism is proposed.

Liver slices in dynamic organ culture. I. An alternativein vitrotechnique for the study of rat hepatic drug metabolism

1. Precision-cut liver slices in dynamic organ culture, a novel in vitro technique, is described and applied to the study of hepatic drug metabolism in the rat. 2. These slices catalysed the oxidative O-deethylation of the substrate, 7-ethoxycoumarin, over 6 h incubation. In addition, the direct conjugation of 7-hydroxycoumarin with either sulphate or glucuronic acid was maintained over 6 h. 3. The formation of 7-hydroxycoumarin and the presence of the sulphate and glucuronide conjugates in slices exposed to 7-ethoxycoumarin demonstrated integrated phase I and phase II drug metabolizing activities in this system. 4. Minor modifications of the incubation system allowed for the metabolism of four volatile chlorinated benzenes: monochlorobenzene 1,2-, 1,3-, and 1,4-dichlorobenzenes to aqueous soluble metabolites. 5. The use of liver slices in dynamic organ culture as an alternative preparation for the study of xenobiotic metabolism is discussed.

Quantitative brain autoradiography of [9, 10‐3H]palmitic acid incorporation into brain lipids

The distribution of radioactivity within brain metabolic compartments was examined following the intravenous injection of [9,10-3H]palmitate into awake rats. Brain radioactivity reached a maximum value by 15 min after [9,10-3H]palmitate injection and remained unchanged for at least 4 hr. Regional differences in radioactivity could be determined with high resolution by quantitative autoradiography, at the level of cell layers within the hippocampus and cerebral cortex, and between striosomes of the caudate nucleus. Regional brain radioactivities were converted to normalized regional radioactivities (k) by dividing them by the integrated plasma fatty acid radioactivity (integrated over the time course of the experiment). These values reflected incorporation mainly into brain phospholipids; radioactivity due to nonlipid components was minimal. Indeed, about 85% of brain radioactivity was within lipids between 5 min and 4 hr postinjection, the remainder being equally divided between protein-associated pellet and aqueous-soluble metabolites. The major lipids labeled were phospholipids, particularly phosphatidylcholine, which contained about 75% of phospholipid radioactivity. The results show that [9,10-3H]palmitate can be used to examine incorporation of plasma palmitate into individual brain regions via quantitative autoradiography. Furthermore, the tracer is a rather selective marker for phosphatidylcholine and can be used to examine turnover and synthesis of this phospholipid. [9,10-3H]palmitate has advantages over [U-14C]palmitate for autoradiographic studies of incorporation; following the 14C-tracer, significant label even at 4 hr after injection is in nonlipid compartments (glutamate and aspartate), and the long path length of 14C limits resolution at the cell layer level.

Thermal modulation of benzo[ a]pyrene metabolism by the gulf toadfish, Opsanus beta

Gulf toadfish, Opsanus beta, were exposed to initial [ 14 C] benzo[a] pyrene (BaP) concentrations of 5 μg 1 −1 at acclimation temperatures (18 or 28°C) or following an acute temperature change (18 to 28°C or 28 to 18°C) in a simple static exposure system. At 24 h, fish were sacrificed and the bile was analyzed by HPLC for Phase I and Phase II metabolites of BaP. Toadfish can metabolize BaP rapidly, as very low levels of parent compound were detected, and the overall rate of uptake and metabolic processing of [ 14C]BaP was higher in fish exposed at high temperatures. There were no significant differences between temperature treatments in the proportions of organic soluble and aqueous soluble metabolites, which contributed approximately 7.8 and 92%, respectively. A variety of Phase I metabolites were produced by toadfish, and the major Phase I metabolite identified was benzo[ a]pyrene 7,8-dihydrodiol. There were significant quantitative differences between temperature treatments in the classes of Phase I metabolites produced. Fish acclimated to high temperature produced more triols and tetrols (breakdown products of highly carcinogenic BaP diol epoxides) than did fish acclimated at low temperature regardless of exposure temperature. Thus, in addition to increasing rate of uptake, higher temperatures appear to lead to a shift in metabolism towards potentially more carcinogenic intermediates.

Intravenous injection of [1‐14C]arachidonate to examine regional brain lipid metabolism in unanesthetized rats

We examined the metabolic disposition and brain distribution of an unsaturated fatty acid, [1-14C]arachidonate, between 5 and 240 min following its intravenous bolus injection in unanesthetized adult rats. Injected [1-14C]arachidonate was cleared rapidly from plasma, with less than 10% remaining by 2 min. Total brain radioactivity, 0.2% of the injected dose, was near maximal by 5 min, reached a peak by 15 min, then slowly declined. Radioactivity in brain lipids constituted greater than 82% of the total brain radioactivity at all times. Radioactivity in aqueous-soluble metabolites was greatest at 5 min (13% of total) and declined to 5% by 240 min. Protein pellet-associated radioactivity gradually rose to a peak of 7% by 120 min. Within the lipid fraction, more than 92% of radioactivity was in glycerolipids, with greater than 81% in phospholipids. Radioactivity in inositol phosphoglyceride was maximal at 5 min (47% of phospholipid radioactivity); and declined to 34% by 20 min, whereas radioactivity in choline phosphoglyceride peaked at 15 min (41% of phospholipid radioactivity) and was constant thereafter. In contrast, radioactivity in ethanolamine phosphoglycerides increased from 7 to 17% during the course of the experiment. Quantitative autoradiography of brain sections indicated incorporation of [1-14C]arachidonate into gray-matter regions was 1.5- to threefold that into white-matter regions. The data were analyzed in terms of a model for brain fatty acid uptake from plasma. Estimates of unidirectional transfer constants, k, for [1-14]arachidonate from plasma to brain regions with an intact blood-brain barrier ranged from 0.0005 to 0.0015 ml.sec-1.g-1 and were correlated with those for [9,10-3H]palmitate. The results indicate that brain phospholipid metabolism in awake animals can be examined regionally and quantitatively using intravenous injection of [1-14C]-arachidonate combined with quantitative autoradiography and biochemical analysis.

Secretagogues for lung surfactant increase lung uptake of alveolar phospholipids.

This study evaluated the effect of known secretagogues for lung surfactant on the uptake of phospholipid from the aveolar space. Synthetic liposomes containing tracer [choline-methyl-3H]dipalmitoyl phosphatidylcholine (DPPC) were instilled endotracheally in anesthetized rats. Lungs were then isolated and perfused under control conditions or with addition of terbutaline (0.1 mM), ATP (1 mM), or a phorbol ester, tradecanoyl phorbol acetate (TPA, 30 ng/ml). Uptake of liposomes was defined as lavage-resistant accumulation of radioactivity in the lung. Uptake at 2 h was 18.2 +/- 0.93% of instilled radioactivity per minute (mean +/- SE, n = 4) under control conditions and was increased by 56-82% in the presence of each of the agonists (P less than 0.05). At 5 min after addition of terbutaline, uptake was unchanged but secretion of phosphatidylcholine (PC) into the alveolar space was significantly stimulated. Internalized diphosphatidylcholine was degraded to aqueous soluble metabolites and also converted to PC containing an unsaturated fatty acid, and this metabolism was significantly stimulated in the presence of each of the secretagogues. These results indicate that known secretagogues for surfactant increase uptake and metabolism of phospholipid, suggesting linkage of these processes in a physiologically regulated surfactant cycle.

Degradation and reutilization of alveolar phosphatidylcholine by rat lungs.

We have shown previously that phospholipids instilled through the trachea are removed from the air spaces in isolated rat lungs by a process that is stimulated by beta-adrenergic agonists. In this study, we evaluated the fate of radiolabeled lipid vesicles [50% [3H]dipalmitoyl phosphatidylcholine (DPPC), 25% phosphatidylcholine (PC), 15% cholesterol, and 10% phosphatidylglycerol (PG)]. Vesicles were instilled through the trachea of anesthetized rats, and the lungs removed for perfusion. The percent of instilled 3H that could not be removed from lungs by extensive lung lavage increased progressively; at 3 h this fraction was 25.8 +/- 0.63% (mean +/- SE; n = 8). The percent of dpm in the lung homogenate accounted for by PC decreased progressively while dpm in lyso-PC, unsaturated PC, and aqueous soluble metabolites [choline, choline phosphate, glycerophosphorycholine, and cytidine 5'-diphosphate (CDP) choline (CDP-choline) increased. The dpm in microsomal and lamellar body fractions isolated from lung homogenates also increased progressively with time of perfusion. The presence of 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP) significantly stimulated both uptake of DPPC and the appearance of radioactivity in metabolites and subcellular organelles. This effect of 8-BrcAMP was not due to stimulation of phospholipase A activity. These results indicate that exogenous phospholipids instilled into the air spaces of rat lungs are internalized and degraded by a process that is stimulated by cAMP.(ABSTRACT TRUNCATED AT 250 WORDS)

The in vitro metabolism and bioactivation of 1,2-dibromoethane (ethylene dibromide) by human liver.

The nematocide, grain fumigant, and gasoline additive 1,2-dibromoethane (DBE) is both a cellular and a genetic toxin that is metabolically activated in rats and mice by mixed function oxidases (MFO) as well as glutathione 5-transferases (GST). The purpose of this study was to determine whether DBE is similarly metabolized and bioactivated by human liver in vitro. Human liver microsomal and cytosolic metabolism of DBE was monitored by the production of aqueous-soluble metabolites from [14-C]-DBE. Reactive intermediates were detected as irreversibly bound adducts to protein or DNA. 1,2-Dibromoethane was metabolized by human liver cytosolic GST, microsomal GST, and microsomal MFO. Cytosolic GST activity (9 +/- 2 nmol/20 min/mg protein) was about four times greater than the other two activities. Only MFO activity resulted in adducts irreversibly bound to protein (1.5 +/- .4 nmol/20 min/mg protein) and was inhibited by the presence of glutathione. Both MFO and GST activity resulted in irreversibly bound adducts to DNA. Microsomal and cytosolic GST activity each produced about twice as many DNA adducts as microsomal MFO activity. These results suggest that human liver, like rat and mouse liver, metabolizes DBE to aqueous-soluble metabolites by both MFO and GST activity. Furthermore, each of these activities produces reactive metabolites that can irreversibly bind to cellular macromolecules.

Mechanism of chloroform nephrotoxicity: III. Renal and hepatic microsomal metabolism of chloroform in mice

In vitro studies with male ICR mouse renal cortical slices have indicated that chloroform (CHCl 3) is metabolized by the kidney to a nephrotoxic intermediate, possibly by a cytochrome P-450-dependent mechanism similar to that occurring in the liver. In this investigation, metabolism of 14CHCl 3 by microsomes prepared from renal cortex and liver provided definitive evidence for a role of cytochrome P-450 in the renal metabolism and toxicity of CHCl 3. 14CHCl 3 was metabolized to 14CO 2 and covalently bound radioactivity by male renal cortical microsomes; metabolism required oxygen, a NADPH regenerating system, was dependent on incubation time, microsomal protein concentration, and substrate concentration, and was inhibited by carbon monoxide. Consistent with the absence of CHCl 3 nephrotoxicity in female mice, little or no metabolism of 14CHCl 3 by female renal cortical microsomes was detected. CHCl 3 produced a type I binding spectrum with oxidized male renal cortical and hepatic microsomes. Incubation of glutathione with microsomes and 14CHCl 3 increased the amount of aqueous soluble metabolites detected with a concomitant decrease of metabolism to 14CO 2 and covalently bound radioactivity, suggesting the formation of a phosgene conjugate as has been described for hepatic CHCl 3 metabolism. These data support the hypothesis that renal cytochrome P-450 metabolizes CHCl 3 to a nephrotoxic intermediate.