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Biomedical subjects

A J Varghese

Publications and source records attributed to A J Varghese.

At least 19 recordsLinked to original sources
PubMed1995

Defective DNA-dependent protein kinase activity is linked to V(D)J recombination and DNA repair defects associated with the murine scid mutation.

Murine cells homozygous for the severe combined immune deficiency mutation (scid) and V3 mutant hamster cells fall into the same complementation group and show similar defects in V(D)J recombination and DNA double-stranded break repair. Here we show that both cell types lack DNA-dependent protein kinase (DNA-PK) activity owing to defects in DNA-PKcs, the catalytic subunit of this enzyme. Furthermore, we demonstrate that yeast artificial chromosomes containing the DNA-PKcs gene complement both the DNA repair and recombination deficiencies of V3 cells, and we conclude that DNA-PKcs is encoded by the XRCC7 gene. As DNA-PK binds to DNA ends and is activated by these structures, our findings provide novel insights into V(D)J recombination and DNA repair processes.

Amino Acid Sequence↗
PubMed1994

A DNA repair defect in Chinese hamster ovary cells affects V(D)J recombination similarly to the murine scid mutation.

Lymphocyte antigen receptor variable regions are encoded by gene segments that are assembled by the site-specific variable (diversity) joining (V(D)J) recombination process. We have assayed the V-3 Chinese hamster ovary cell line, which has a double-strand DNA break repair (dsbr) defect, for the ability to carry out V(D)J recombination following transfection of constructs that encode the RAG-1 and RAG-2 proteins necessary to confer V(D)J recombination activity to non-lymphoid cells. The V-3 cells had substantially impaired ability to undergo V(D)J recombination of transiently introduced test substrates. Although these cells can initiate V(D)J recombination by introducing endonucleolytic scissions at the junctions of V(D)J recombination signal sequences and the flanking coding sequences, they have a greatly impaired ability to rejoin the coding sequences. Detailed characterization of attempted coding joins recovered from these cells indicated that the V(D)J recombination defect in V-3 is similar in phenotype to the murine severe combined immunodeficient (scid) defect and quite distinct from that found in other Chinese hamster ovary dsbr mutant cell lines. Somatic cell complementation analyses between homozygous scid mutant fibroblasts and V-3 cells confirmed that these mutations fall into the same genetic complementation group.

Animals↗
PubMed1989

Cell cycle responses of two X-ray sensitive mutants defective in DNA repair.

Both the xrs and V-3 lines of Chinese hamster ovary cells exhibit marked sensitivity to ionizing radiation. They are also sensitive to agents such as bleomycin and H2O2 but exhibit normal responses to ultraviolet light and mitomycin C. Both cell lines are defective in split-dose repair and repair of double-strand breaks in DNA. Analysis of response to radiation as a function of age in the cell cycle indicates that both cell lines exhibit a marked sensitivity in late G1 and early S phase with more limited sensitization throughout the remainder of the cell cycle.

Animals↗
PubMed1986

Identification of a reactive glutathione conjugate as a metabolite of SR-2508 in CHO cells.

Reaction between GSH and the hydroxylamine derivative of SR-2508 results in the formation of two stable conjugates identified as 2-amino-4-S-glutathionyl and 2-amino-5-S-glutathionyl imidazoles. These stable conjugates are apparently formed from a reactive derivative of the hydroxylamine that is sufficiently stable to be isolated after HPLC separation. The physical and chemical properties of this derivative are consistent with it being a GSH conjugate in which the glutathionyl residue is attached to the 2-amino nitrogen of the imidazole moiety through sulphur. With excess GSH, under physiological conditions, it forms a mixture of the two stable GSH conjugates. In CHO cells exposed to SR-2508 under hypoxic conditions, this unstable GSH conjugate has been detected and suggests the possibility of GSH functioning as a carrier of a toxic metabolite of 2-nitroimidazoles under certain conditions.

Animals↗
PubMed1986

Reaction of 2-nitroimidazole metabolites with guanine and possible biological consequences.

Nitroimidazoles, under hypoxic conditions, undergo reduction reactions producing a variety of reactive species; at the same time, they exert a variety of biological effects. The purpose of the present study was to investigate the reduction chemistry of 2-nitroimidazoles, the reaction of the reduced metabolites with nucleic acid constituents and the possible biological significance of these reactions. Earlier studies had demonstrated that, following reduction, 2-nitroimidazoles react with guanine derivatives to produce identical to that seen on reaction of glyoxal with the same guanine derivatives. We report here the identification of this product in the nucleic acid of cells exposed to 2-nitroimidazoles under hypoxic conditions. Using glyoxal as a model compound, we also demonstrate that cellular formation of such a product could account for a number of the biological properties of 2-nitroimidazoles under hypoxic conditions.

Animals↗
PubMed1985

Properties of 2-hydroxylaminoimidazoles and their implications for the biological effects of 2-nitroimidazoles.

In aqueous solution, in the presence of ammonium chloride, N1-substituted 2-nitroimidazoles are readily reduced to the corresponding hydroxylamines. In air, under neutral conditions, analogous to the reactions of aromatic hydroxylamines, 2-hydroxylaminoimidazoles are converted to the azoxy derivatives via a base-catalyzed condensation reaction between the hydroxylamine and its oxidation product, the nitroso derivative. In nitrogen, rearrangement to form the 2-amino-4(5)hydroxyimidazole derivative followed by addition of water across the C4-C5 double bond to yield isomers of a 4,5-dihydro-4,5-dihydroxy derivative appears to be a major reaction. 2-hydroxylaminoimidazoles undergo a complex series of reactions with glutathione. The initial reaction is the formation of a labile conjugate involving an N-S-linkage. Subsequently in the presence of excess GSH, under neutral conditions, two stable conjugates identified as 2-amino-4-S-glutathionyl- and 2-amino-5-S-glutathionyl imidazoles are formed. Nucleophilic attack by GSH on the imidazole ring of a nitrenium ion is postulated as the initial step in the formation of the stable GSH conjugates as well as the 2-amino-4,5-dihydro dihydroxy derivative. The results provide a molecular mechanism for many of the biological effects of N1-substituted 2-nitroimidazoles in hypoxic mammalian cells.

Amines↗
PubMed1984

Misonidazole-glutathione conjugates in CHO cells.

Misonidazole, after reduction to the hydroxylamine derivative, reacts with glutathione (GSH) under physiological conditions. The reaction product has been identified as a mixture of two isomeric conjugates. When water soluble extracts of CHO cells exposed to misonidazole under hypoxic conditions are subjected to HPLC analysis, misonidazole derivatives, having the same chromatographic properties as the GSH-MISO conjugates, were detected. The identity of the synthetic and cellular products was further confirmed by identification of the amine derivative of misonidazole after desulfurization with Raney Nickel. When CHO cells were incubated with misonidazole in the presence of added GSH, a substantial increase in the amount of the conjugate was detected. When extracts of CHO cells exposed to misonidazole under hypoxia were subsequently exposed to GSH, an increased formation of the conjugate was observed. A rearrangement product of the hydroxylamine derivative of misonidazole is postulated as the reactive intermediate responsible for the formation of the conjugate.

Animals↗
PubMed1984

Detection of a reactive metabolite of misonidazole in human urine.

Chemical studies have indicated that, following reduction of misonidazole to the hydroxylamine derivative, reaction with guanosine leads to the formation of a 2-carbon addition product of guanosine. In this study, the formation of the guanosine product is used to detect the presence of a reactive metabolite of misonidazole in the urine of patients treated with misonidazole. Urine samples were incubated with [14C]guanosine and the guanosine product was separated by HPLC analysis. The quantities of product vary as much as 10-fold from patient to patient and it is suggested that the assay might be useful as a predictor of patients susceptible to the development of peripheral neuropathy or other effects of misonidazole.

Combined Modality Therapy↗
PubMed1984

Detection of a reactive metabolite of misonidazole in hypoxic mammalian cells.

A misonidazole metabolite capable of reacting with guanosine has been detected in extracts of Chinese hamster ovary cells exposed to misonidazole under hypoxic conditions. A misonidazole metabolite with identical chromatographic properties and reactivity with guanosine has been detected in earlier studies with misonidazole reduced to the hydroxylamine state by chemical, radiolytic, or electrolytic means. The proposed structure of the guanosine product involves the addition of a two-carbon fragment between the N1 and N2 positions of guanosine. Rearrangement of the N-hydroxy derivative of misonidazole to a C-hydroxy derivative is postulated as the initial step in the reaction scheme.

Animals↗
PubMed1983

Glutathione conjugates of misonidazole.

The hydroxylamine derivative of misonidazole reacts with glutathione under physiological conditions to form two isomeric conjugates. Based on physical and chemical properties, the two conjugates have been identified as 1-[2-amino-(4-glutathion-S-yl)-1-imidazolyl]-3-methoxypropanol and 1-[2-amino-(5-glutathion-S-yl)-1-imidazolyl]-3-methoxypropanol. The formation of the glutathione conjugates of reduced misonidazole offers a molecular mechanism for the depletion of GSH in mammalian cells after exposure to misonidazole under hypoxic conditions.

Chromatography, High Pressure Liquid↗
PubMed1983

Modification of guanine derivatives by reduced 2-nitroimidazoles.

Misonidazole, after reduction to the hydroxylamine derivative, was found to react with guanosine in aqueous solution at pH 7. The guanosine product was isolated and was assigned a structure having a new 5-membered ring with a -CHOH-CHOH-linkage between the N-1 and N-2 positions of guanine. Removal of the sugar residue from the guanosine product by acid hydrolysis resulted in the corresponding guanine derivative, which was also made by reacting guanine with reduced misonidazole. In aqueous solution at pH 11, the guanine product was quantitatively converted to guanine within 20 min. A number of N-1-substituted 2-nitroimidazoles and 2-nitroimidazole reacted with guanosine in an analogous manner, giving rise to the same product as misonidazole, indicating that the C-4-C-5 fragment from the imidazoles is involved in the modification. Neither misonidazole nor its amine or hydrazo derivatives reacted with guanosine. Reduced misonidazole reacted with N-2-methyl guanosine, whereas with N-1-methyl guanosine a reaction was not detected. The identity of Structure I was confirmed by comparison with an authentic sample of Structure I that was prepared by reacting guanosine with glyoxal. Reactions such as the modification of guanine provide a possible molecular mechanism for the cytotoxic and neurotoxic properties of misonidazole.

Chemical Phenomena↗
PubMed1981

Cellular and chemical reduction products of misonidazole.

Misonidazole is readily reduced by zinc dust in aqueous solution in the presence of ammonium chloride. High pressure liquid chromatographic (HPLC) separation of the reduction mixture revealed the presence of three products. These were identified as the hydroxylamine, amine and the hydrazo derivative of misonidazole. There is evidence that the azoxy derivative was an intermediate in the reduction process. When the reduction was carried out in dilute solution (0.1 mg/ml), the hydroxylamine was the only product. In concentrated solution (20 mg/ml), the hydrazo derivative was the major product. When misonidazole was reduced with hydrogen using palladium as catalyst, the amine was the only detectable product. Of the three products, only the hydroxylamine was found to bind covalently to bovine albumin. In Chinese hamster ovary (CHO) cells under hypoxic conditions the amine was confirmed as one of the metabolites. There was no evidence for the presence of detectable amounts of the hydroxylamine in the cell extracts. These studies suggest that the hydroxylamine is probably the reactive reduction metabolite responsible for the in vivo and in vitro binding of misonidazole to cellular macromolecules.

Animals↗
PubMed1980

Binding to cellular macromolecules as a possible mechanism for the cytotoxicity of misonidazole.

Reduction of the nitro group occurred when [14C]misonidazole was treated with zinc dust in aqueous solution in the presence of ammonium chloride. When the reduction mixture was allowed to react with calf thymus DNA or bovine albumin, radioactivity was bound to both DNA and protein. Under the same conditions, misonidazole did not bind to these macromolecules. Analysis of the reduction mixture indicated that the hydroxylamine, amine, and hydrazo derivatives of mizonidazole were the major products. In a number of tissues of C3H mice after administration of [14C]misonidazole, radioactivity was detected in the DNA, RNA, and protein fractions. Similar results were also obtained with Chinese hamster ovary cells incubated with the drug in the absence of oxygen. It is postulated that nitroreduction and binding of the nitroreduction products to macromolecules is a probable mechanism for the mutagenic and cytotoxic properties of misonidazole.

Ammonium Chloride↗
PubMed1980

Binding of nitroreduction products of misonidazole to nucleic acids and protein.

[14C]Misonidazole was reduced with zinc dust in aqueous solution in the presence of ammonium chloride. When the reduction mixture was allowed to react with calf thymus DNA and the reaction mixture was dialyzed, radioactivity was detected in the DNA fraction. Bovine albumin, after reaction with the reduction mixture and subsequent Bio-Gel P-2 column chromatography, was also found to be radioactive. The hydroxylamine derivative of misonidazole was detected in the reduction mixture. It is postulated that binding of nitroreduction products of misonidazole to cellular macromolecules is a probable mechanism for the cytotoxicity of misonidazole.

Ammonium Chloride↗