Metabolic Incorporation Research Articles

Differential Incorporation of Ara-C, Gemcitabine, and Fludarabine Into Replicating and Repairing DNA in Proliferating Human Leukemia Cells

AbstractThe major actions of nucleoside analogs such as arabinosylcytosine (ara-C) and fludarabine occurs after their incorporation into DNA, during either replication or repair synthesis. The metabolic salvage and DNA incorporation of the normal nucleoside, deoxycytidine, is functionally compartmentalized toward repair synthesis in a process regulated by ribonucleotide reductase. The aim of this study was to investigate the metabolic pathways by which nucleoside analogs that do (fludarabine, gemcitabine) or do not (ara-C) affect ribonucleotide reductase are incorporated into DNA in proliferating human leukemia cells. Using alkaline density-gradient centrifugation to separate repaired DNA from replicating DNA and unreplicated parental DNA strands, approximately 60% of ara-C nucleotide in DNA was incorporated by repair synthesis in CCRF-CEM cells; the remainder was incorporated by replication. In contrast, fludarabine and gemcitabine, nucleosides that inhibit ribonucleotide reductase and decreased deoxynucleotide pools, were incorporated mainly within replicating DNA. Hydroxyurea also depleted deoxynucleotide pools and increased the incorporation of ara-C into DNA by replicative synthesis. Stimulation of DNA repair activity by UV irradiation selectively enhanced the incorporation of all nucleosides tested through repair synthesis. These findings suggest that the pathways by which therapeutically useful nucleoside analogs are incorporated into DNA are affected by cellular dNTP pools from de novo synthesis and by the relative activities of DNA repair and replication. The antitumor activity of these drugs may be enhanced by combination with either ribonucleotide reductase inhibitors to increase their incorporation into replicating DNA or with agents that induce DNA damage and evoke the DNA repair process.

Bcl-2 inhibits selective oxidation and externalization of phosphatidylserine during paraquat-induced apoptosis.

Oxidative stress is a potential component of the final common pathway leading to apoptosis following many diverse stimuli. Here, we document that the oxidant paraquat caused apoptosis in mouse 32D cells. We examined early paraquat-induced lipid peroxidation after metabolic incorporation of the oxidant-sensitive fluorescent fatty acid cis-parinaric acid (cis-PA) into phospholipids and high-performance liquid chromatography separation of specific phospholipid classes. Paraquat induced peroxidation of cis-PA primarily in phosphatidylserine (PS) and to a lesser extent in phosphatidylinositol (PI) within 2 h. The selective oxidation of PS occurred before signs of cytotoxicity and preceded the externalization of PS as assessed by annexin V binding. Overexpression of Bcl-2 afforded significant protection against paraquat-induced apoptosis, early PS and PI oxidation, and PS externalization but not the ultimate formation of high-molecular-weight DNA fragments. Therefore, both selective phospholipid peroxidation and DNA damage occurred after paraquat exposure, but only the former was specifically associated with apoptosis. We suggest Bcl-2 may inhibit oxidant-induced apoptosis by preventing the peroxidation of specific membrane phospholipids.

Kinetics of Fusion between Endoplasmic Reticulum Vesicles in Vitro

The endoplasmic reticulum (ER) is a highly dynamic organelle, continuously undergoing membrane fusion and fission. We have measured homotypic fusion between ER vesicles isolated from Chinese hamster ovary cells kinetically in vitro, using an assay based on the metabolic incorporation of pyrene-labeled fatty acids into the phospholipids of cellular membranes. An increase in pyrene-monomer fluorescence was observed after mixing labeled and unlabeled ER vesicles in the presence of ATP and GTP. The protein, temperature, and nucleotide dependence of the increase indicated that it was caused by membrane fusion rather than molecular transfer of labeled lipids to unlabeled membranes. This assay allowed the first kinetic measurements with virtually nonexchangeable probes of a homotypic membrane fusion event. At 37 degrees C, fusion started off immediately at a rate of 1.14 +/- 0.29%/min and reached a half-maximal level after 56 min. In the presence of guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS), or after treatment of the membranes with N-ethylmaleimide, fusion was reduced but not completely inhibited. Addition of GTP during a fusion reaction immediately accelerated, and GTPgammaS immediately slowed down the fusion reaction. Thus, these kinetic measurements indicate that G-proteins might act to rapidly enhance fusion beyond a basic level.

Indications for differential diagnosis of nontumor central nervous system diseases from tumors. A positron emission tomography study.

To accurately differentiate nontumor central nervous system (CNS) diseases from brain tumors, we retrospectively evaluated the cerebral circulation and metabolism in patients with nontumor CNS diseases using positron emission tomography (PET). Regional cerebral blood flow (rCBF), cerebral blood volume (rCBV), oxygen extraction fraction (rOEF), the metabolic rates of oxygen (rCMRO2), and of glucose (rCMRGI), and the uptake of 11C-methyl-L-methionine (11C-Met) were visually evaluated in lesions and compared with values for the contralateral white matter regions. PET findings were correlated with those of x-ray computed tomography (CT) and magnetic resonance imaging (MRI), and were analyzed for nontumor CNS diseases and cerebral gliomas. rCBF and rCBV were changeable from disease to disease or from stage to stage of disease progression. rOEF and rCMRO2 remained low in 5 and 6, respectively, of 9 nontumor CNS diseases examined, whereas these parameters were increased in CNS infections such as brain abscess. Overall, noteworthy was the locally increased rOEF and rCMRO2 in the patients with a brain abscess in contrast to the values for patients with gliomas. rCMRGI reflected biological characteristics of each disease, and correlated with cell density, whether reactive glial cells or inflammatory cells. 11C-Met was accumulated at a certain stage of nontumor CNS diseases, which implied uptake of the tracer as a result of disruption of the blood-brain barrier as well as metabolic incorporation.

A primary production deficit in the thrombocytopenia of equine infectious anemia

The purpose of this study was to identify the mechanisms responsible for the thrombocytopenia that develops following infection of horses by the lentivirus equine infectious anemia virus (EIAV). Immunocompetent Arabian foals and Arabian foals with severe combined immunodeficiency (SCID), which lack functional B and T lymphocytes, were experimentally infected with EIAV. Levels of viremia and a number of clinical and hematologic parameters were examined prior to and following infection. Thrombocytopenia was not dependent on the immune response: SCID foals were affected as severely as immunocompetent foals. Production of platelets, measured by metabolic incorporation of radioactive label, was significantly reduced. The decrease ranged from 35 to 89% in three SCID and two immunocompetent foals examined. Platelet survival, measured by 51Cr labeling, also declined following infection in both SCID and immunocompetent foals: 51 and 68%, respectively, relative to the preinfection life spans. The difference between immunocompetent and immunodeficient foals was not statistically significant. The number of megakaryocytes (MK) per square millimeter of bone marrow, determined by digitizing morphometry, was not significantly altered in either SCID or immunocompetent thrombocytopenic foals. Numbers of denuded MK nuclei per unit area increased, but the elevation was not statistically significant. No evidence for viral replication in MK was found. Three different parameters of intravascular coagulation (activated prothombin time, fibrin degradation products, and one-step prothombin time) remained normal until after platelet numbers had declined significantly, arguing against an important role for disseminated intravascular coagulation. The findings indicate that EIAV induces thrombocytopenia principally through an indirect, noncytocidal suppressive effect on platelet production, the mechanism of which is unknown. A shortening of platelet life span apparently contributes moderately to the platelet deficit as well. The shortening of platelet life span is multifactorial in origin, including both mechanisms that depend on an active immune response and those that do not.

Acid-labile adducts to protein can be used as indicators of the cysteine S-conjugate pathway of trichloroethene metabolism.

Covalent binding of radiolabel to tissue proteins following [14C]trichloroethene (TRI) exposure has been used as a measure of TRI activation. Gross binding of 14C label does not differentiate between alternate routes of metabolism and can be confounded when there is significant metabolic incorporation of radiolabel. We examined the covalent association of 14C label to hepatic and renal proteins in male F344 rats and B6C3F1 mice following oral treatment with [14C]TRI and three metabolites of TRI: [14C]trichloroacetate (TCA), [14C]dichloroacetate (DCA), and [14C]dichlorovinylcysteine (DCVC) in vivo. Association of radiolabel from [14C]TRI with hepatic proteins reached a maximum at 2 and 4 h in mouse and rat hepatic proteins, respectively. Association of radiolabel with renal proteins reached a maximum at 8 h in both species. An approach was developed based upon formation of protein adducts that release acetate and monochloroacetate (MCA) on acid hydrolysis. These adducts were found to be specifically associated with the activation of DCVC to reactive intermediates. Acetate and MCA were identified by using two different conditions of high-performance liquid chromatography (HPLC) separation with differing selectivity. Diethylmaleate and aminooxyacetic acid pretreatment inhibited the formation of these adducts from TRI, consistent with requirements for glutathione and beta-lyase. No evidence of these adducts was detected following [14C]TCA and [14C]DCA treatment. Renal acid-labile adduct formation from 25 mg/kg DCVC was approximately 12-fold greater in male B6C3F1 mice than in male F344 rats. They accounted for 7.8 and 4.6% of the total adducts to renal protein in rats and mice, respectively. Acid-labile adducts formed from 1000 mg/kg TRI were approximately two times greater in mice than rats. In this case, they accounted for 1.4 and 3.3% of the total adduct formed in renal proteins from TRI (corrected for metabolic incorporation), respectively. This greater dilution of adducts associated with DCVC in renal proteins of the rat suggests that covalent binding of TRI has less specificity for the DCVC pathway in rats than in mice.

Schistosoma mansoni: Changes in the Albumen Gland of Biomphalaria glabrata Snails Selected for Nonsusceptibility to the Parasite

The LAC-line strain of the snail Biomphalaria glabrata has very low susceptibility to the parasite Schistosoma mansoni and a very low reproductive potential. Upon examination of the reproductive tract of these snails, light and electron microscopy revealed obvious abnormalities in the albumen gland. Secretory cells that are normally cuboidal in susceptible NMRI (F0) snails were squamous in LAC-line snails. These LAC-line cells contained small secretory granules and negligible rough endoplasmic reticulum and Golgi, compared to large granules and an extensive array of both organelles in F0 albumen gland cells. Comparative analyses of soluble protein extracts of F0 and LAC-line albumen glands showed several qualitative differences. Among the most prominent was an 18-kDa protein in F0 snails that was remarkably reduced in the soluble protein extracts of LAC-line snails. Also, metabolic incorporation of [35S]-methionine was impaired in LAC-line albumen glands. Whether these albumen gland changes are caused by decreased susceptibility to parasitism is yet to determined.

Temperature dependence of cell-cell fusion induced by the envelope glycoprotein of human immunodeficiency virus type 1.

We investigated cell-cell fusion induced by the envelope glycoprotein of human immunodeficiency virus type 1 strain IIIB expressed on the surface of CHO cells. These cells formed syncytia when incubated together with CD4-positive human lymphoblastoid SupT1 cells or HeLa-CD4 cells but not when incubated with CD4-negative cell lines. A new assay for binding and fusion was developed by using fluorescent phospholipid analogs that were produced in SupT1 cells by metabolic incorporation of BODIPY-labeled fatty acids. Fusion occurred as early as 10 min after mixing of labeled SupT1 cells with unlabeled CHO-gp160 cells at 37 degrees C. When both the fluorescence assay and formation of syncytia were used, fusion of SupT1 and HeLa-CD4 cells with CHO-gp160 cells was observed only at temperatures above 25 degrees C, confirming recent observations (Y.-K. Fu, T.K. Hart, Z.L. Jonak, and P.J. Bugelski, J. Virol. 67:3818-3825, 1993). This temperature dependence was not observed with influenza virus-induced cell-cell fusion, which was quantitatively similar at both 20 and 37 degrees C, indicating that cell-cell fusion in general is not temperature dependent in this range. gp120-CD4-specific cell-cell binding was found over the entire 0 to 37 degrees C range but increased markedly above 25 degrees C. The enhanced binding and fusion were reduced by cytochalasins B and D. Binding of soluble gp120 to CD4-expressing cells was equivalent at 37 and 16 degrees C. Together, these data indicate that during gp120-gp41-induced syncytium formation, initial cell-cell binding is followed by a cytoskeleton-dependent increase in the number of gp120-CD4 complexes, leading to an increase in the avidity of cell-cell binding. The increased number of gp120-CD4 complexes is required for fusion, which suggests that the formation of a fusion complex consisting of multiple CD4 and gp120-gp41 molecules is a step in the fusion mechanism.

Partial characterization of a thyrotropin releasing hormone-sensitive glycosyl-phosphatidylinositol in pituitary lactotrophes

Metabolic labelling experiments performed with cultured pituitary lactotrophes revealed the presence of a glycosyl-phosphatidylinositol (GPtdlns) structurally related to GPtdlns lipids isolated from other cell types as demonstrated by: (i) metabolic incorporation of [ 3H]galactose, [ 3H]glucosamine and [ 3H]inositol into the polar inositolphosphoglycan moiety (InsPG) and [ 3H]myristate and [ 3H] palmitate into the diacylglycerol (DAG) backbone of GPtdlns; (ii) sensitivity of the [ 3H]labelled GPtdlns to nitrous acid deamination and; (iii) sensitivity of GPtdlns to phosphatidylinositol (Ptdlns)-specific phospholipase C (PLC) hydrolysis. In cultured pituitary cells labelled to isotopic steady state with 10 μCi/ml of [ 3H]glucosamine, treatment with hypothalamic TRH (10 −6 M) induced a rapid and transient hydrolysis (ca. 50%) of the labelled GPtdlns. Moreover, as demonstrated in [ 3H]inositol labelled cells, treatment with thyrotropin releasing hormone (TRH) elicited the cleavage of [ 3H]GPtdlns in a similar manner, and this effect was followed by the phosphoinositide (Ptdlns, PtdInsP and PtdInsP 2) hydrolysis 30 s later. These results suggest that the phosphodiesterase cleavage of GPtdlns could be an early event implicated in TRH action in pituitary lactotrophes.

A method for labeling cells for positron emission tomography (PET) studies

Positron emission tomography (PET) may provide an ideal means of monitoring the delivery of cells to normal and pathological tissue owing to its high resolution, specificity and three dimensional imaging capabilities. In order to implement such a technique, it is important to develop a labeling method for cells which provides a rapid and stable incorporation of the positron emitter without altering the viability or functional activity of the cells to be studied. Two approaches have been explored to achieve this end: metabolic incorporation of 2-[ 18F]fluorodeoxylglucose (2-[ 18F]FDG) or protein labeling with [ 11C]methyl iodide (MI). IL-2 activated mouse natural killer lymphocytes were expanded in culture for 7 days and used for these studies. Uptake of 2-[ 18F]FDG by the natural killer cells was found to occur rapidly and to level off after 30 min. The amount of cell-associated label was found to decay at a steady rate immediately following the procedure, with approximately 21% loss of label after 1 h. In contrast, labeling of cells with MI provided a stable association for 60 min which did not alter the viability or the cytotoxic activity of the cells. Injection of the labeled cells into a normal mouse followed by a full body scanning procedure demonstrated the accumulation of the cells in the lungs which corresponded to those seen by microscopy. These findings suggest that labeling lymphocyte populations with MI may provide a rapid and practical means to quantify systemic cell distribution by PET.

Identification of individual tyrosine sulfation sites within factor VIII required for optimal activity and efficient thrombin cleavage.

Factor VIII functions as an essential cofactor in the blood coagulation cascade for the factor IXa-mediated activation of factor X. Factor VIII contains 6 tyrosine residues at positions 346, 718, 719, 723, 1664, and 1680 that are modified by post-translational sulfation. This modification is required for full factor VIII procoagulant activity. We have employed site-directed mutagenesis to identify the individual sulfated tyrosines within factor VIII that influence activity. The molecules were expressed in COS-1 monkey cells by transient transfection, and the resultant proteins were characterized. Metabolic incorporation of [35S]sulfate demonstrated that all 6 tyrosine residues are sulfated in factor VIII. Sulfation at residues 346 and 1664 was required for full activity in a factor VIII clotting assay but did not affect factor VIII activity monitored by a factor Xa generation assay. The Tyr346-->Phe and Tyr1664-->Phe mutants displayed delayed thrombin activation that correlated with delayed cleavage at residues 372 and 1689, respectively. In contrast, these mutants were efficiently activated by factor Xa. A triple Tyr to Phe mutant at residues 718, 719, and 723 displayed both reduced factor VIII clotting activity and factor Xa generation activity. Finally, a Tyr1680-->Phe mutant factor VIII displayed a 5-fold reduced affinity for von Willebrand factor. The results demonstrate that 1) sulfation at tyrosine residues 346 and 1664 increases factor VIII activity by increasing the rate of thrombin activation and cleavage; 2) sulfation at tyrosine residues 718, 719, and 723 increases the intrinsic activity of factor VIIIa; and 3) sulfation at tyrosine residue 1680 increases the affinity for vWF. In addition, the results implicate that thrombin interacts with three distinct sites within factor VIII, two of which are required for proteolytic activation. The results demonstrate that the six sites of tyrosine sulfation modulate factor VIII activity through different mechanisms.

Combined effects of cadmium and linear alkyl benzene sulfonate on Lemna minor L.

: The effects of 0.1 ppm cadmium and 0.005% linear alkyl benzene sulfonates (LAS) on the uptake and metabolic incorporation of (14)C glycine by Lemna minor L., after 2, 24 and 48 h were studied for antagonistic/synergistic effects. Combined exposure was found to decrease the (14)C incorporation into proteins, DNA, RNA and phospholipids, to a greater extent than individual exposure. The presence of LAS increased the uptake of (109)Cd in the plants.

14] High-performance liquid chromatography of oligosaccharides

Publisher Summary This chapter proposes separation methods primarily for oligosaccharides that are radiolabeled by metabolic incorporation of 3H- or 14C-labeled sugar, metabolic incorporation of 35SO4 or 32PO4, or chemical incorporation of 3H at the reducing terminus by reduction with NaB3H4. The most common strategy is to first separate the radiolabeled oligosaccharides into different groups on the basis of their anionic character, using anion-exchange high-performance liquid chromatography (HPLC). Oligosaccharides differing in the number and/or character of their anionic groups are then further fractionated on the basis of size, using ion suppression-amine adsorption (ISAA) HPLC. The effects of chemical modifications and enzymatic digestions on individual species are subsequently monitored by anion-exchange HPLC and ISAA–HPLC along with other methods such as lectin affinity HPLC. Four methods for separation of anionic oligosaccharides are illustrated in the chapter. Fractionation of oligosaccharides bearing zero to four anionic moieties, using MicroPak AX-10 or AX-5 columns and a phosphate buffer (pH 4.0), is effective for oligosaccharides bearing terminal sialic acid. Oligosaccharides-bearing sulfate or phosphate can be fractionated under the same conditions; however, the concentration of the initial eluant is reduced to 2.5 mM in order to retain monosulfated species.

Changes in glycosylation of L1210 cells after exposure to various antimetabolites

This study establishes that antimetabolites do have the potency to change cellular glycosylation, as was suggested in our previous review ( Eur J Cancer 1990, 26, 516–523). Murine leukaemia L1210 cells were exposed to various antimetabolites under non-lethal conditions. The antimetabolites 5-fluorouracil (5FU), arabinofuranosylcytosine (AraC), methotrexate (MTX) and 6-mercaptopurine (6MP), but not 6-thioguanine, induced considerable changes in the metabolic incorporation of radioactively labelled monosaccharides. Each antimetabolite exhibited a different effect. Significant differences were found between the radioactivity incorporated from the monosaccharides glucosamine, fucose, mannose and galactose, relative to control values. Polyacrylamide gel electrophoresis indicated that changes were induced in the glycosylation of individual glycoproteins. 5FU, AraC, MTX and 6MP all influenced both pyrimidine- and purine-mediated sugar incorporation. This excludes, therefore, direct effects of the antimetabolites on their analogue nucleotide-sugars. The antimetabolite-induced changes in glycosylation did not directly correlate with the observed cell-cycle effects of the antimetabolites.

Adduction of hemoglobin and albumin in vivo by metabolites of trichloroethylene, trichloroacetate, and dichloroacetate in rats and mice

Adducts to macromolecules from trichloroethylene formed by in vivo and in vitro metabolism have been reported by many investigators. We examined the in vivo adduction of the blood proteins hemoglobin (Hb) and albumin in rats and mice dosed orally with [ 14C]trichloroethylene ([ 14C]TRI) to explore the development of a protein adduct biomarker of TRI exposure. We also examined the adduction of these two proteins from doses of [ 14C]trichloroacetate (TCA) and [ 14C]dichloroacetate (DCA), two metabolites of TRI. Association of label with albumin peaked at 4–8 hr in the rat (2480 nmol eq TRI/mg protein) and 2–4 hr in the mouse (1580 nmol eq TRI/mg protein). The decay was exponential with a half-life consistent with that of rat or mouse albumin (approx 24 hr). The time course of label with Hb was characterized by an early plateau at 8 hr in rat (28 nmol eq TRI/mg protein), 4 hr in mouse (7 nmol eq TRI/mg protein), and followed by a slow steady increase, peaking at 120 hr (54 nmol eq TRI/mg protein, rat; 38 nmol eq TRI/mg protein, mouse). This apparent binding was linear with dose in the rat, but was convex in the mouse albumin (mouse Hb label was below detection at low dose). We also found that a portion of the irreversibly associated label, referred to by previous investigators as “binding”, could be accounted for as metabolic incorporation of label into glycine and serine. The fraction accounted for by metabolic incorporation was constant in albumin (approximately 2 3 ), while in Hb, this portion was time dependent, approximately 30% at the early sampling time, 75% at the late time, implying the observed late increase could be accounted for by metabolic incorporation. TCA and DCA also formed Hb and albumin adducts. Portions of this binding was also due to metabolic incorporation. The pattern of the binding from TCA in albumin was different from that of TRI, implying a route to adduct from TRI which does not proceed through TCA.

A major oligomeric fibroblast proteoglycan identified as a novel large form of type-XII collagen.

Cultured chick embryo skin fibroblasts release a major component with a native molecular mass of about 1 MDa, which resolves into three polypeptide bands of about 300, 350 and 600 kDa upon reduction. We report here the purification of this oligomeric protein and show, by means of polyclonal and monoclonal antibodies, that its three polypeptide constituents are closely related. The 600-kDa polypeptide is likely to be a dimer of two smaller subunits which are cross-linked by non-reducible bonds. By electron microscopy, isolated oligomeric molecules exhibit a novel cruciform structure with a large central globular domain. One arm has the shape of a thin rod about 70 nm in length. The three other arms are thicker, longer (90 nm) and flexible, and carry a prominent double globule at their distal ends. Collagenase treatment of the oligomeric fibroblast protein yields two resistant fragments of about 270 kDa and 320 kDa. The intact 350-kDa and 600-kDa (but not the 300-kDa) polypeptides are chondroitinase sensitive and labeled by metabolic incorporation of [35S]sulfate; collagenase treatment does not remove any [35S] sulfate. Hence, the intact fibroblast protein has glycosaminoglycan chains attached to its non-collagenous domain. Three amino acid sequences obtained from chymotryptic fragments of the fibroblast protein correspond to sequences predicted for chick type-XII collagen from its full-length cDNA [Yamagata, M., Yamada, K. M., Yamada, S. S., Shinomura, T., Tanaka, H., Nishida, Y., Obara, M. & Kimata, K. (1991) J. Cell Biol. 115, 209-221]. However, the novel fibroblast protein described here differs significantly from previously isolated forms of type-XII collagen: its subunits are larger by one third, and it is a proteoglycan.

Dichloromethane (methylene chloride): Metabolism to formaldehyde and formation of DNA-protein cross-links in B6C3F1 mice and Syrian Golden hamsters

Dichloromethane (DCM) is metabolized via a glutathione transferase (GST)-dependent pathway to formaldehyde (HCHO), a mutagenic compound that could play an important role in the carcinogenic effects of DCM observed in the liver and lungs of B6C3F1 mice at 2000 and 4000 ppm. Syrian hamsters metabolize DCM more slowly than mice via this pathway, and hamsters exposed to 3500 ppm showed no apparent carcinogenic response. The possible formation of DNA-protein cross-links (DPX) from DCM in both species was examined. Male mice and hamsters were pre-exposed for 2 days (6 hr/day) to 4000 ppm of DCM and on the third day were exposed (6 hr) to a decaying concentration (4500 to 2500 ppm) of [ 14C]DCM. DPX were detected in mouse liver, but not in mouse lung, hamster liver, or hamster lung. The failure to detect DPX in mouse lung does not exclude their possible formation in a subpopulation of lung cells. Metabolic incorporation of 14C derived from [ 14C]DCM into DNA suggested a higher rate of turnover of some mouse lung cells than of hamster lung cells, but no large difference in the turnover rates of liver cells in the two species under these conditions. These results demonstrate that HCHO derived from DCM can form DNA-protein cross-links in the liver of the B6C3F1 mouse. The formation of DPX is dependent on the activity of the GST pathway, and species such as hamsters and humans having much lower rates of DCM metabolism via this pathway may not generate toxicologically significant concentrations of HCHO and DPX.

Adduction of Hemoglobin and Albumin in Vivo by Metabolites of Trichloroethylene, Trichloroacetate, and Dichloroacetate in Rats and Mice

Adducts to macromolecules from trichloroethylene formed by in vivo and in vitro metabolism have been reported by many investigators. We examined the in vivo adduction of the blood proteins hemoglobin (Hb) and albumin in rats and mice dosed orally with [14C]trichloroethylene ([14C]TRI) to explore the development of a protein adduct biomarker of TRI exposure. We also examined the adduction of these two proteins from doses of [14C]trichloroacetate (TCA) and [14C]dichloroacetate (DCA), two metabolites of TRI. Association of label with albumin peaked at 4–8 hr in the rat (2480 nmol eq TRI/mg protein) and 2–4 hr in the mouse (1580 nmol eq TRI/mg protein). The decay was exponential with a half-life consistent with that of rat or mouse albumin (approx 24 hr). The time course of label with Hb was characterized by an early plateau at 8 hr in rat (28 nmol eq TRI/ mg protein), 4 hr in mouse (7 nmol eq TRI/mg protein), and followed by a slow steady increase, peaking at 120 hr (54 nmol eq TRI/mg protein, rat; 38 nmol eq TRI/mg protein, mouse). This apparent binding was linear with dose in the rat, but was convex in the mouse albumin (mouse Hb label was below detection at low dose). We also found that a portion of the irreversibly associated label, referred to by previous investigators as “binding,” could be accounted for as metabolic incorporation of label into glycine and serine. The fraction accounted for by metabolic incorporation was constant in albumin (approximately ), while in Hb, this portion was time dependent, approximately 30% at the early sampling time, 75% at the late time, implying the observed late increase could be accounted for by metabolic incorporation. TCA and DCA also formed Hb and albumin adducts. Portions of this binding was also due to metabolic incorporation. The pattern of the binding from TCA in albumin was different from that of TRI, implying a route to adduct from TRI which does not proceed through TCA.

Metabolic flux and incorporation of [2- 13C]glycine into silk fibroin studied by 13C NMR in vivo and in vitro

13C NMR spectra in vivo and in vitro of Bombyx mori silkworm larva and the posterior silk gland were observed after administration of [2- 13C]glycine. In the in vivo 13C NMR spectra of larva, the administrated glycine flowed immediately from the midgut to the hemolymph and was then incorporated into the silk fibroin. This [2- 13C]glycine flux was analyzed with eight rate constants which were determined with a compartment model. The incorporation of [2- 13C]glycine into the silk fibroin was also observed by 13C NMR spectroscopy by circumfusion cultivation of the posterior silk gland. The rate constant for the incorporation of glycine in vitro was determined as 7.4 × 10 −4 min −1. This was 63% of the rate constant determined in vivo.

Mass spectrometric determination of isotopically labeled tyrosines and tryptophans in photosynthetic reaction centers of Rhodobacter sphaeroides R-26

A synthetic medium for growing Rhodobacter sphaeroides R-26 is developed. This medium opened the way to the preparation of photosynthetic reaction centers incorporated with l-[4′- 13C]tyrosine or l-[1′- 15N]tryptophan. Gas chromatography combined with mass spectroscopy was used to estimate the metabolic incorporation of the labeled amino acid into the protein. Conditions were found for near-quantitative incorporation of labeled aromatic amino acids into the reaction center.