DISTINCT TISSUE DISTRIBUTION OF METABOLITES OF THE NOVEL GLUTATHIONE-ACTIVATED THIOPURINE PRODRUGS<i>CIS</i>-6-(2-ACETYLVINYLTHIO)PURINE AND<i>TRANS</i>-6-(2-ACETYLVINYLTHIO)GUANINE AND 6-THIOGUANINE IN THE MOUSE

Gunnarsdottir, Sjöfn; Elfarra, Adnan A.

doi:10.1124/dmd.31.6.718

Cited by 18 publications

(12 citation statements)

References 16 publications

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“…The cytotoxicity of the thiopurine drugs involved the formation of S G nucleotide upon metabolic activation and its subsequent incorporation into DNA (29). In DNA, S G can be methylated by S-adenosyl-L-methionine to form S 6 mG (9) and converted to SO3H G upon UVA irradiation (18).…”

Section: Discussionmentioning

confidence: 99%

Mutagenic and Cytotoxic Properties of 6-Thioguanine, S-Methylthioguanine, and Guanine-S6-sulfonic Acid

Yuan

Wang

2008

Journal of Biological Chemistry

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Thiopurine drugs, including 6-thioguanine ( S G), 6-mercaptopurine, and azathioprine, are widely employed anticancer agents and immunosuppressants. The formation of S G nucleotides from the thiopurine prodrugs and their subsequent incorporation into nucleic acids are important for the drugs to exert their cytotoxic effects.S G in DNA can be methylated by S-adenosyl-L-methionine to give S 6 -methylthioguanine (S 6 mG) and oxidized by UVA light to render guanine-S 6 -sulfonic acid ( SO3H G). Here, we constructed single-stranded M13 shuttle vectors carrying a S G, S 6 mG, or SO3H G at a unique site and allowed the vectors to propagate in wild-type and bypass polymerasedeficient Escherichia coli cells. Analysis of the replication products by using the competitive replication and adduct bypass and a slightly modified restriction enzyme digestion and post-labeling assays revealed that, although none of the three thionucleosides considerably blocked DNA replication in all transfected E. coli cells, both S 6 mG and SO3H G were highly mutagenic, which resulted in G3 A mutation at frequencies of 94 and 77%, respectively, in wild-type E. coli cells. Deficiency in bypass polymerases does not result in alteration of mutation frequencies of these two lesions. In contrast to what was found from previous steady-state kinetic analysis, our data demonstrated that 6-thioguanine is mutagenic, with G3 A transition occurring at a frequency of ϳ10%. The mutagenic properties of 6-thioguanine and its derivatives revealed in the present study offered important knowledge about the biological implications of these thionucleosides.

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Section: Discussionmentioning

confidence: 99%

Mutagenic and Cytotoxic Properties of 6-Thioguanine, S-Methylthioguanine, and Guanine-S6-sulfonic Acid

Yuan

Wang

2008

Journal of Biological Chemistry

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“…All samples were first centrifuged at 10,000g for 5 min. Total GSH (GSH ϩ GSSG) levels and GSSG levels alone were then determined as described by Gunnarsdottir and Elfarra (2003). Standard curves were run in all experiments to allow for quantitation of the results.…”

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confidence: 99%

l-Methionine Toxicity in Freshly Isolated Mouse Hepatocytes Is Gender-Dependent and Mediated in Part by Transamination

Dever¹,

Elfarra²

2008

J Pharmacol Exp Ther

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L-Methionine (Met) has been implicated in parenteral nutritionassociated cholestasis in infants and, at high levels, it causes liver toxicity by mechanisms that are not clear. In this study, Met toxicity was characterized in freshly isolated male and female mouse hepatocytes incubated with 5 to 30 mM Met for 0 to 5 h. In male hepatocytes, 20 mM Met was cytotoxic at 4 h as indicated by trypan blue exclusion and lactate dehydrogenase leakage assays. Cytotoxicity was preceded by reduced glutathione (GSH) depletion at 3 h without glutathione disulfide formation. Exposure to 30 mM Met resulted in increased cytotoxicity and GSH depletion. It is interesting to note that female hepatocytes were resistant to Met-induced cytotoxicity at these concentrations and showed increased cellular GSH levels compared with hepatocytes exposed to medium alone. The effects of amino-oxyacetic acid (AOAA), an inhibitor of Met transamination, and 3-deazaadenosine (3-DA), an inhibitor of the Met transmethylation pathway enzyme S-adenosylhomocysteine hydrolase, on Met toxicity in male hepatocytes were then examined. Addition of 0.2 mM AOAA partially blocked Met-induced GSH depletion and cytotoxicity, whereas 0.1 mM 3-DA potentiated Met-induced toxicity. Exposure of male hepatocytes to 0.3 mM 3-methylthiopropionic acid (3-MTP), a known Met transamination metabolite, resulted in cytotoxicity and cellular GSH depletion similar to that observed with 30 mM Met, whereas incubations with D-methionine resulted in no toxicity. Female hepatocytes were less sensitive to 3-MTP toxicity than males, which may partially explain their resistance to Met toxicity. Taken together, these results suggest that Met transamination and not transmethylation plays a major role in Met toxicity in male mouse hepatocytes.

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“…The primary activation pathway of the thiopurine drugs is through the formation of thioguanine nucleotide (TGN) and its subsequent incorporation into DNA (1,5). Although extensive studies on 6-TG metabolism have been carried out (2,6), the biochemical mechanisms for its cytotoxicity remain largely unclear, partially because of the lack of a strong mutagenic effect of 6-TG (7,8). On the other hand, further transformation of 6-TG in DNA might be involved in the cytotoxicity of the thiopurine drugs.…”

Section: Introductionmentioning

confidence: 99%

In vitro replication and thermodynamic studies of methylation and oxidation modifications of 6-thioguanine

Wang

2007

Nucleic Acids Research

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The cytotoxic effects of thiopurine drugs are mostly exerted through the formation of thioguanine nucleotide and its subsequent incorporation into DNA. The 6-thioguanine (6-TG) in DNA can be converted to S6-methylthio-2-aminopurine (2-AP-6-SCH3) and 2-aminopurine-6-sulfonic acid (2-AP-6-SO3H) upon reaction with S-adenosyl-L-methionine and irradiation with UVA light, respectively. Here we prepared oligodeoxynucleotides (ODNs) harboring a 6-TG, 2-AP-6-SCH3 or 2-AP-6-SO3H at a defined site and examined, by using LC-MS/MS, the in vitro replication of these substrates with yeast polymerase η and Klenow fragment (KF−). Our results revealed that 2-AP-6-SCH3 could be bypassed by KF−, with significant misincorporation of thymine opposite the lesion. The 2-AP-6-SO3H, however, blocked markedly the nucleotide insertion by KF−. Yeast pol η could bypass all three modified nucleosides; although dCMP was inserted preferentially, we found substantial misincorporation of dTMP and dAMP opposite 2-AP-6-SCH3 and 2-AP-6-SO3H, respectively. Moreover, both KF− and yeast pol η induced a considerable amount of -2 frameshift products from the replication of 2-AP-6-SCH3- and 2-AP-6-SO3H-bearing substrates. Our results also underscored the importance of measuring the relative ionization efficiencies of replication products in the accurate quantification of these products by LC-MS/MS. Moreover, thermodynamic studies revealed that 2-AP-6-SCH3 and 2-AP-6-SO3H could cause more destabilization to duplex DNA than 6-TG. Taken together, the results from this study shed important new light on the biological implications of the two metabolites of 6-TG.

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DISTINCT TISSUE DISTRIBUTION OF METABOLITES OF THE NOVEL GLUTATHIONE-ACTIVATED THIOPURINE PRODRUGSCIS-6-(2-ACETYLVINYLTHIO)PURINE ANDTRANS-6-(2-ACETYLVINYLTHIO)GUANINE AND 6-THIOGUANINE IN THE MOUSE

Cited by 18 publications

References 16 publications

Mutagenic and Cytotoxic Properties of 6-Thioguanine, S-Methylthioguanine, and Guanine-S6-sulfonic Acid

Mutagenic and Cytotoxic Properties of 6-Thioguanine, S-Methylthioguanine, and Guanine-S6-sulfonic Acid

l-Methionine Toxicity in Freshly Isolated Mouse Hepatocytes Is Gender-Dependent and Mediated in Part by Transamination

In vitro replication and thermodynamic studies of methylation and oxidation modifications of 6-thioguanine

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