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D J Reed

Publications and source records attributed to D J Reed.

189 records · Page 11Linked to original sources
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In vitro dipeptide, nucleoside, and glutathione alkylation by S-(2-chloroethyl)glutathione and S-(2-chloroethyl)-L-cysteine.

S-(2-Chloroethyl)glutathione (CEG) and S-(2-chloroethyl)-L-cysteine (CEC) are putative glutathione-dependent metabolites of 1,2-dichloroethane bioactivation and have been shown to be direct-acting alkylating agents. A group of dipeptides, nucleosides, and glutathione were used as model compounds to investigate CEG and CEC alkylation events. The extent of glutathione and cysteinyltyrosine alkylation was much greater than histidyltyrosine greater than lysyltyrosine, glycyltyrosine, glycyltryptophan, and 2'-deoxyguanosine greater than 2'-deoxyadenosine, 2'-deoxycytidine, and thymidine for both CEG and CEC. The rate of S-alkylation of cysteinyltyrosine by CEG and CEC occurred at rates 54 and 72 times that for the N7 position of 2'-deoxyguanosine and 16 and 10 times that for histidyltyrosine imidazole nitrogen, respectively. The rate of S-alkylation of glutathione by CEG was found to be 27% faster than that for S-alkylation of cysteinyltyrosine whereas S-alkylation of glutathione by CEC was 22% slower than that for cysteinyltyrosine. Both CEG and CEC demonstrated a selectivity for cysteinyl thiol alkylation over a wide variety of other nucleophilic sites. These findings demonstrate a wide range of functional group reactivity that should be taken into consideration when assessing the alkylation of cellular macromolecules by such glutathione-derived metabolites of the 1,2-dihaloethanes in vivo.

Alkylation↗
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Utilization of glutathione during 1,2-dihaloethane metabolism in rat hepatocytes.

The metabolism of 1,2-dihaloethanes (DHEs) to glutathione-containing metabolites by freshly isolated rat hepatocytes was investigated. 1,2-Dichloroethane (DCE), 1,2-dibromoethane (DBE), and 1-bromo-2-chloroethane (BCE) were metabolized to S-(2-hydroxyethyl)glutathione (HEG), S-(carboxymethyl)glutathione (CMG), and S,S'-(1,2-ethanediyl)bis(glutathione) (GEG). The formation of these glutathione-containing metabolites was concomitant with the depletion of intracellular glutathione (GSH) and accounted for 58%, 84%, and 71% of the DCE-, BCE-, and DBE-induced loss of intracellular GSH, respectively. The covalent binding of [14C]DBE to hepatocyte protein reached 18.7 nmol/mL of cell suspension (7.8 nmol/mg of protein) within 2.0 h of incubation. Half of this covalent binding occurred within 0.5 h of incubation (4.0 nmol/mg of protein) in the presence of high levels of intracellular GSH (30% of initial GSH level at 0.5 h). Hepatocyte metabolism of 2-chloroacetic acid produced only CMG. 2-Chloroethanol metabolism gave rise to CMG and HEG in a 11.5:1.0 ratio; 2-chloroacetaldehyde produced almost equal amounts of CMG and HEG. GEG formation was increased significantly for DBE and BCE when GSH was added to the medium during treatment, suggesting that the GSH conjugates S-(2-haloethyl)glutathione are exported from the hepatocytes. These results indicate that the glutathione S-transferase-catalyzed conjugation of GSH with the DHEs is responsible for the majority of the DHE-induced GSH depletion. The S-(2-haloethyl)glutathione conjugates appear responsible for the extensive covalent binding to protein observed during [14C]DBE metabolism.

Animals↗
PubMedDate not supplied

Thioredoxin alkylation by a dihaloethane-glutathione conjugate.

Glutathione is a thiol-containing tripeptide which functions to protect cellular constituents from endogenous and xenobiotic electrophiles via conjugation and eventual excretion. In the case of compounds such as 1,2-dihaloethanes, however, conjugate formation results in bioactivation of the species rather than detoxification. The conjugate can then act as an alkylating agent toward cellular constituents including DNA, proteins, or lipids. Alkylation of protein thiols in cells exposed to dihaloethane may contribute substantially to the toxicity produced by these compounds. We examined the reactivity of the conjugate S-(2-chloroethyl)-glutathione (CEG) toward the model protein Escherichia coli thioredoxin. At physiological pH, treatment of thioredoxin by CEG resulted in the production of several bands visible on isoelectric focusing, which were determined by matrix-assisted laser desorption ionization (MALDI) mass spectrometry to be mono-, di-, tri-, and tetra-alkylated forms of thioredoxin. A concomitant loss of in vitro enzymatic activity was observed. These products were also observed when reaction was allowed to take place at pH 11.4. Treatment at pH 4.4 resulted in lesser alkylation of thioredoxin, with only the mono- and di-alkylated forms detected. Iodoacetic acid treatment of CEG-alkylated thioredoxin revealed that the iodoacetic acid-susceptible Cys32 was not carboxymethylated, suggesting that this is one of the sites alkylated by CEG.

Alkylating Agents↗
PubMedDate not supplied

Alkylation of oxytocin by S-(2-chloroethyl)glutathione and characterization of adducts by tandem mass spectrometry and Edman degradation.

S-(2-Chloroethyl)glutathione (CEG), an alkylating agent formed by glutathione conjugation with 1,2-dichloroethane (DCE), is able to alkylate DNA and proteins. As a prelude to identification of specific protein alkylation sites, the peptide oxytocin was alkylated by CEG, and tandem mass spectrometry was used to identify the alkylation sites. It was found that mono-, bis-, and tris-adducts can result from alkylation of reduced oxytocin and that tandem mass spectrometry differentiated (S-[2-(Cys1)ethyl]glutathione)oxytocin (mono-adduct Cys-1) from (S-[2-(Cys1,6)ethyl]glutathione)oxytocin (mono-adduct Cys-6). Manual Edman degradation was used to eliminate the possibility that alkylation has occurred at Tyr-2 rather than at Cys-1 in the case of (S-[2-(Cys1,6)ethyl]glutathione)oxytocin (bis-adduct) and mono-adduct Cys-1. A mono-adduct homodimer resulting from alkylation at Cys-6 and disulfide bridge formation through Cys-1 was also identified. Oxidized oxytocin formed two minor adducts, representing less than 5% of the oxytocin present in the reaction mixture. These findings demonstrate that alkylation of oxytocin by the episulfonium ion of CEG did occur, as evidenced by tandem mass spectrometry, and that characterization of these adducts will aid in the identification of alkylated amino acids in proteins exposed to CEG.

Alkylating Agents↗
PubMedDate not supplied

Alkylation of Escherichia coli thioredoxin by S-(2-chloroethyl)glutathione and identification of the adduct on the active site cysteine-32 by mass spectrometry.

Alkylation of reduced Escherichia coli thioredoxin by the episulfonium ion derived from S-(2-chloroethyl)glutathione (CEG) at physiologic pH resulted in at least three different alkylation products. These adducts were separated by reverse phase chromatography, digested with trypsin, and peptide-mapped. The peptide containing the active site cysteines was collected and sequenced by tandem mass spectrometry. Results indicate that the site of alkylation was at Cys-32 exclusively with no alkylation at Cys-35. Raising the pH above the pKa of Cys-35 to ionize the thiol before reacting with the episulfonium ion of CEG did not lead to alkylation at Cys-35, suggesting that a steric factor prevents the alkylating moiety of CEG from accessing this cysteine. A tryptic digest of a minor bis-adduct yielded an alkylated peptide which contained tyrosine, an amino acid known to be alkylated at its hydroxyl group by CEG. Sequencing by tandem mass spectrometry, however, was unsuccessful due to fragmentation of the alkylating moiety from the peptide. Results of this study confirm that the episulfonium ion of CEG can adduct thioredoxin at the active site and may have important toxicologic significance regarding the mechanism of 1,2-dichloroethane toxicity.

Alkylating Agents↗
PubMedDate not supplied

Isolation and measurement of 15N2 from respiratory gases of animals administered 15N-labeled substances.

A method is described for collection of metabolic 15N2 from in vitro preparations or intact rats administered 15N-containing compounds. The methods enables routine collection and mass spectrometric measurement of as little as 10 mumol 15N2 respired by a rat over a 24-h period. A device is described that includes either an animal chamber or a tissue reaction vessel in a closed recycling atmosphere, with automatic O2 replenishment and removal of CO2 and water. It is capable of sustaining moderate vacuum and is coupled to a high-vacuum manifold designed to process the contained atmosphere and respiratory gases. The starting atmosphere is an 80:20 mix of sulfur hexafluoride and O2. Recovery of 15N2 gas from the system without an animal present was 101.3 +/- 5.75%. When 15N2 gas was very slowly infused iv into an animal, recovery was 89.1 +/- 5.38%. Use of the method in studies of the fate of [15N]hydrazine in rats indicated that about 15% of the administered hydrazine is rapidly converted to 15N2, followed by slower conversion of an additional 7-10% over the next several hours.

Animals↗
PubMedDate not supplied

Metabolic fate of hydrazine.

Studies of the disposition of hydrazine administered to mammals have not succeeded in accounting for more than a modest fraction of the dose, nor have the excretory products been completely identified. We have utilized 15N-labeled hydrazine and conventional methods to account for about 75% of single doses of about 0.5 LD50 (1 mmol/kg). In 48 h, about 30% appeared in urine as hydrazine and about 20% emerged as a derivative that is acid-hydrolyzable to hydrazine. About 25% was converted to N2 gas, most of which appeared less than 30 min after administration. The percentage converted to N2 at 4 h increased only slightly with dose between 0.5 and 2.0 mmol/kg. Disappearance of hydrazine from blood was biphasic with half-times of 0.74 and 26.9 h.

Animals↗
PubMedDate not supplied

Urinary metabolites of 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea and 1-(2-chloroethyl)-3-(trans-4-methylcyclohexyl)-1-nitrosourea.

Urinary metabolites of ring 14C-labeled 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) and 1-(2-chloroethyl)-3-(trans-4-methylcyclohexyl)-1-nitrosourea (Methyl CCNU) from rats have been isolated and characterized by high-performance liquid chromatography and mass spectrometry. About 44% of the cyclohexyl moiety of CCNU was excreted in 24 hr and included approximately 10% of the excreted dose as free amines and 40% as conjugates that could be converted to amines by hydrolysis. Amine composition of free base plus hydrolyzable conjugates was 55% hydroxycyclohexylamines (3-trans, 3-cis, 4-cis, and 4-trans) and 30% cyclohexylamine. This strongly supports previous studies which indicated that CCNU is largely hydroxylated in vivo as well as in vitro. Rats pretreated with phenobarbital excreted high relative amounts of cis-4-hydroxy derivatives (41%), again showing a high degree of correlation between in vitro and in vivo results. Treatment of urine with beta-glucuronidase gave no apparent increase in free amines. However, sulfatase was about 25% as effective as alkaline hydrolysis for releasing free amines from whole urine. Major urinary metabolites were found to have m.w. of about 629, 413, 329, and 243 and represented 55%, 20%, 20%, and 5% of total excreted 14C, respectively. It was concluded that the higher m.w. metabolites may be conjugates of peptides possibly derived from active site-directed inactivation of specific enzymes. Previous work has shown that enzymes such as chymotrypsin and glutathione reductase are inhibited by isocyanates in this manner. Hydroxylated metabolites of Methyl CCNU had a pattern similar to that of CCNU. The major free (12%) and conjugated amine (54%) metabolites of Methyl CCNU in the urine in decreasing order of quantity present were cis-3-hydroxy-trans-4-methylcyclohexylamine, trans-4-methylcyclohexylamine, trans-4-hydroxymethylcyclohexylamine, and trans-3-hydroxy-trans-4-methyl-cyclohexylamine.

Amines↗