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Journal of Labelled Compounds 07/2011; 54(9):607 - 612.
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Michael P Gamcsik,
M Daniel Clark,
Susan M Ludeman, James B Springer,
Michael A D'Alessandro,
Nicholas E Simpson,
Roxana Pourdeyhimi,
C Bryce Johnson,
Stephanie D Teeter,
Stephen J Blackband,
Peter E Thelwall
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ABSTRACT: The cysteine precursor L-2-oxothiazolidine-4-carboxylate (OTZ, procysteine) can raise cysteine concentration, and thus glutathione levels, in some tissues. OTZ has therefore been proposed as a prodrug for combating oxidative stress. We have synthesized stable isotope labeled OTZ (i.e. L-2-oxo-[5-(13)C]-thiazolidine-4-carboxylate, (13)C-OTZ) and tracked its uptake and metabolism in vivo in rat brain by (13)C magnetic resonance spectroscopy. Although uptake and clearance of (13)C-OTZ was detectable in rat brain following a bolus dose by in vivo spectroscopy, no incorporation of isotope label into brain glutathione was detectable. Continuous infusion of (13)C-OTZ over 20 h, however, resulted in (13)C-label incorporation into glutathione, taurine, hypotaurine and lactate at levels sufficient for detection by in vivo magnetic resonance spectroscopy. Examination of brain tissue extracts by mass spectrometry confirmed only low levels of isotope incorporation into glutathione in rats treated with a bolus dose and much higher levels after 20 h of continuous infusion. In contrast to some previous studies, bolus administration of OTZ did not alter brain glutathione levels. Even a continuous infusion of OTZ over 20 h failed to raise brain glutathione levels. These studies demonstrate the utility of in vivo magnetic resonance for non-invasive monitoring of antioxidant uptake and metabolism in intact brain. These types of experiments can be used to evaluate the efficacy of various interventions for maintenance of brain glutathione.
Neurochemical Research 03/2011; 36(3):443-51. · 2.24 Impact Factor
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Journal of Labelled Compounds 02/2007; 50(2):115 - 122.
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Journal of Labelled Compounds 01/2007; 50(2):79 - 84.
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ABSTRACT: It is well-established that at pH 7.4, intramolecular 1,3-N-alkylation reactions in isophosphoramide mustard (IPM) and phosphoramide mustard (PM) produce electrophilic alkylating agents with aziridinyl moieties. To investigate the role of 1,5-intramolecular cyclizations in the chemistry of IPM and PM, the five-membered ring phospholidine products of these reactions were independently synthesized and characterized by (31)P NMR. In 0.33 M BisTris, pH 7.4, 37 degrees C, the intramolecular O-alkylation product of IPM [2-(2-chloroethylamino)-2-tetrahydro-2H-1,3,2-oxazaphospholidine-2-oxide (11)] had a chemical shift of delta 33.0 and a half-life of 3.3 h. The O-alkylation product of PM [2-amino-3-(2-chloroethyl)tetrahydro-2H-1,3,2-oxazaphospholidine-2-oxide (12)] displayed a chemical shift of delta 30.6 and a half-life of 26.9 h. For both IPM and PM, 1,5-N-alkylation provides the same product [1-(2-chloroethyl)-2-hydroxy-tetrahydro-2H-1,3,2-diazaphospholidine-2-oxide (13)]. Because of its instability, 13 was generated in situ and was not isolated; however, the chemical shift (delta 33.0) and reactivity (half-life 0.3 h at 25 degrees C) of the species attributed to 13 were consistent with the assigned structure. Resonances with (31)P NMR chemical shifts indicative of 11 or 12 did not appear in reaction solutions of IPM or PM. The compound assigned as 13 gave hydrolysis products that were not found in reaction solutions of IPM or PM. The collective data supported the conclusion that intramolecular 1,5-alkylations do not contribute to the chemistry of IPM or PM in aqueous solutions at pH 7.4, 37 degrees C. Conversely, 11 and 12 were found to be the major if not exclusive products formed in DMSO solutions of the respective cyclohexylammonium salts of IPM and PM. Both 11 and 12 were relatively noncytotoxic against a series of cell lines, but there were differences in mutagenicities. Chinese hamster ovary cells were exposed to 11 or 12 for one half-life of each compound; 11 was nonmutagenic up to 500 microM, while 12 (500 microM) was mutagenic with 246 mutant colonies/10(6) surviving cells.
Chemical Research in Toxicology 10/2004; 17(9):1217-26. · 3.78 Impact Factor
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ABSTRACT: O6-benzylguanine (BG) is a unique purine analog that has been shown to influence nitrogen mustard activity and increase cytotoxicity. Ifosfamide is a nitrogen mustard with growing clinical applications; effective modulation may lead to improved efficacy. We thus undertook a preliminary investigation of BG's effects on ifosfamide and ifosfamide derivatives in vitro.
BG's effect on ifosfamide toxicity was studied in CHO cells transfected with O6-alkylguanine-DNA alkyltransferase (AGT) (CHOwtAGT) or control plasmid pcDNA3 (CHOpcDNA) using five ifosfamide derivatives and two control compounds: 4-hydroperoxyifosfamide (4HI), isophosphoramide mustard (IPM), phenylketoifosfamide (PKIF), 4-hydroperoxydidechloroifosfamide (4HDI), chloroacetaldehyde (CAA), didechloroisophosphoramide mustard (d-IPM), didechlorophenylketoifosfamide (d-PKIF). To further explore the mechanism of interaction, BG's effect on apoptosis (annexin V-FITC) and cell cycle distribution in cells exposed to ifosfamide was also analyzed.
BG substantially enhanced cytotoxicity induced only by agents that produce IPM (4HI, IPM, PKIF) in both CHOwtAGT and CHOpcDNA cell lines. BG did not modulate 4HDI or CAA cytotoxicity. The addition of BG to IPM in CHO cells increased the percentage of apoptotic cells from 5.5% to 28.9% at 72 h after treatment. Cell cycle analysis showed that BG exposure was associated with G1 arrest. At 16 h following treatment with IPM, PKIF, or phosphoramide mustard (PM), BG increased the percentage of cells in G1 from 16-20% to 29-64%.
BG's ability to increase 4HI-, IPM-, and PKIF-mediated cytotoxicity in cells devoid of AGT activity suggests a novel AGT-independent mode of action that is associated with increased apoptosis and may involve G1 arrest. BG selectively enhanced IPM toxicity without enhancement of acrolein and CAA toxicity. The data strongly support further investigation into combinations of BG and nitrogen mustards.
Cancer Chemotherapy and Pharmacology 11/2003; 52(4):291-302. · 2.83 Impact Factor
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ABSTRACT: Differentiation agents that induce neoplastic cells to regain a normal phenotype and/or cause growth arrest without significantly affecting normal cells represent an attractive option for cancer treatment. Analogues of short chain fatty acids, such as phenylbutyrate (PB), have been studied as clinically relevant agents. In an attempt to improve its pharmacokinetic profile, structural modifications of PB and other fatty acids have been studied. We hypothesize that strategic isotopic modification of PB would result in a longer half-life and thus translate into a more potent differentiation agent for clinical use. Using a colon cancer model, we demonstrated that 2,2,3,3-tetradeuterated PB (D4PB) significantly increased induction of apoptosis and inhibition of cell proliferation as compared with PB and butyrate. Difference in potency could not be explained by the effect of D4PB on the expression of specific regulatory proteins of the apoptotic cascade or from the inhibitory effect of D4PB on histone deacetylase activity. Interestingly, exposure of HT-29 colon cancer cells to D4PB resulted in a slowing of S transit, in contrast to butyrate and PB, which induced a G2/M cell cycle block. This difference in cell cycle effect may explain the differences seen in the potency of the phenotypic changes seen with treatment with D4PB. Further studies are needed to elucidate the mechanisms underlying effects of D4PB on the cell cycle.
Journal of Pharmaceutical Sciences 05/2002; 91(4):1054-64. · 3.06 Impact Factor
