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Timothy J Kinsella - One of the best experts on this subject based on the ideXlab platform.

  • 5 iodo 2 pyrimidinone 2 Deoxyribose mediated cytotoxicity and radiosensitization in u87 human glioblastoma xenografts
    International Journal of Radiation Oncology Biology Physics, 2007
    Co-Authors: Timothy J Kinsella, Michael T Kinsella, Yuji Seo, Gregory Berk
    Abstract:

    Purpose 5-Iodo-2-pyrimidinone-2′-Deoxyribose (IPdR) is a novel orally administered (p.o.) prodrug of 5-iododeoxyuridine. Because p.o. IPdR is being considered for clinical testing as a radiosensitizer in patients with high-grade gliomas, we performed this in vivo study of IPdR-mediated cytotoxicity and radiosensitization in a human glioblastoma xenograft model, U87. Methods and Materials Groups of 8 or 9 athymic male nude mice (6–8 weeks old) were implanted with s.c. U87 xenograft tumors (4 × 10 6 cells) and then randomized to 10 treatment groups receiving increasing doses of p.o. IPdR (0, 100, 250, 500, and 1000 mg/kg/d) administered once daily (q.d.) × 14 days with or without radiotherapy (RT) (0 or 2 Gy/d × 4 days) on days 11–14 of IPdR treatment. Systemic toxicity was determined by body weight measurements during and after IPdR treatment. Tumor response was assessed by changes in tumor volumes. Results IPdR alone at doses of ≥500 mg/kg/d resulted in moderate inhibition of tumor growth. The combination of IPdR plus RT resulted in a significant IPdR dose-dependent tumor growth delay, with the maximum radiosensitization using ≥500 mg/kg/d. IPdR doses of 500 and 1000 mg/kg/d resulted in transient 5–15% body weight loss during treatment. Conclusions In U87 human glioblastoma s.c. xenografts, p.o. IPdR given q.d. × 14 days and RT given 2 Gy/d × 4 days (days 11–14 of IPdR treatment) results in a significant tumor growth delay in an IPdR dose-dependent pattern. The use of p.o. IPdR plus RT holds promise for Phase I/II testing in patients with high-grade gliomas.

  • toxicology and pharmacokinetic study of orally administered 5 iodo 2 pyrimidinone 2 Deoxyribose ipdr x 28 days in fischer 344 rats impact on the initial clinical phase i trial design of ipdr mediated radiosensitization
    Cancer Chemotherapy and Pharmacology, 2007
    Co-Authors: Timothy J Kinsella, Michael T Kinsella, Seongwon Hong, Jerry Johnson, Brian L Burback, Patricia J Tosca
    Abstract:

    A toxicology and pharmacokinetic study of orally administered (po) IPdR (5-3iodo-2-pyrimidinone-2′Deoxyribose, NSC-726188) was performed in Fischer-344 rats using a once daily (qd) × 28 days dosing schedule as proposed for an initial phase I clinical trial of IPdR as a radiosensitizer. For the toxicology assessment, 80 male and female rats (10/sex/dosage group) were randomly assigned to groups receiving either 0, 0.2, 1.0 or 2.0 g kg−1day−1 of po IPdR × 28 days and one-half were observed to day 57 (recovery group). Animals were monitored for clinical signs during and following treatment with full necropsy of one-half of each dosage group at day 29 and 57. For the plasma pharmacokinetic assessment, 40 rats (10/sex/dosage group) were randomly assigned to groups receiving either 0.2 or 1.0 g kg−1day−1 of po IPdR × 28 days with multiple blood samplings on days 1 and 28 and single blood sampling on days 8 and 15. No drug-related deaths occurred. Higher IPdR doses resulted in transient weight loss and transient decreased hemoglobins but had no effect on white cells or platelets. Complete serum chemistry evaluation showed transient mild decreases in total protein, alkaline phosphatase, and serum globulin. Necropsy evaluation at day 29 showed minimal to mild histopathologic changes in bone marrow, lymph nodes and liver; all reversed by day 59. There were no sex-dependent differences in plasma pharmacokinetics of IPdR noted and the absorption and elimination kinetics of IPdR were found to be linear over the dose range studied. A once-daily dosing schedule of po IPdR for 28 days with doses up to 2.0 g kg−1day−1 appeared to be well tolerated in Fischer-344 rats. Drug-related weight loss and microscopic changes in bone marrow, lymph nodes and liver were observed. These changes were all reversed by day 57. IPdR disposition was linear over the dose range used. However, based on day 28 kinetics it appears that IPdR elimination is enhanced following repeated administration. These toxicology and pharmacokinetic data were used when considering the design of our initial phase I trial of po IPdR as a clinical radiosensitizer.

  • schedule dependent drug effects of oral 5 iodo 2 pyrimidinone 2 Deoxyribose as an in vivo radiosensitizer in u251 human glioblastoma xenografts
    Clinical Cancer Research, 2005
    Co-Authors: Yuji Seo, Tao Yan, Jane E Schupp, Tomas Radivoyevitch, Timothy J Kinsella
    Abstract:

    Purpose: 5-Iodo-2-pyrimidinone-2′-Deoxyribose (IPdR) is an oral prodrug of 5-iodo-2′-deoxyuridine (IUdR), an in vitro/in vivo radiosensitizer. IPdR can be rapidly converted to IUdR by a hepatic aldehyde oxidase. Previously, we found that the enzymatic conversion of IPdR to IUdR could be transiently reduced using a once daily (q.d.) treatment schedule and this may affect IPdR-mediated tumor radiosensitization. The purpose of this study is to measure the effect of different drug dosing schedules on tumor radiosensitization and therapeutic index in human glioblastoma xenografts. Experimental Design: Three different IPdR treatment schedules (thrice a day, t.i.d.; every other day, q.o.d.; every 3rd day, q.3.d.), compared with a q.d. schedule, were analyzed using athymic nude mice with human glioblastoma (U251) s.c. xenografts. Plasma pharmacokinetics, IUdR-DNA incorporation in tumor and normal proliferating tissues, tumor growth delay following irradiation, and body weight loss were used as end points. Results: The t.i.d. schedule with the same total daily doses as the q.d. schedule (250, 500, or 1,000 mg/kg/d) improved the efficiency of IPdR conversion to IUdR. As a result, the percentage of IUdR-DNA incorporation was higher using the t.i.d. schedule in the tumor xenografts as well as in normal small intestine and bone marrow. Using a fixed dose (500 mg/kg) per administration, the q.o.d. and q.3.d. schedules also showed greater IPdR conversion than the q.d. schedule, related to a greater recovery of hepatic aldehyde oxidase activity prior to the next drug dosing. In the tumor regrowth assay, all IPdR treatment schedules showed significant increases of regrowth delays compared with the control without IPdR (q.o.d., 29.4 days; q.d., 29.7 days; t.i.d., 34.7 days; radiotherapy alone, 15.7 days). The t.i.d. schedule also showed a significantly enhanced tumor growth delay compared with the q.d. schedule. Additionally, the q.o.d. schedule resulted in a significant reduction in systemic toxicity. Conclusions: The t.i.d. and q.o.d. dosing schedules improved the efficiency of enzymatic activation of IPdR to IUdR during treatment and changed the extent of tumor radiosensitization and/or systemic toxicity compared with a q.d. dosing schedule. These dosing schedules will be considered for future clinical trials of IPdR-mediated human tumor radiosensitization.

  • differential radiosensitization in dna mismatch repair proficient and deficient human colon cancer xenografts with 5 iodo 2 pyrimidinone 2 Deoxyribose
    Clinical Cancer Research, 2004
    Co-Authors: Yuji Seo, Tao Yan, Jane E Schupp, Valdir C Colussi, Kerri Taylor, Timothy J Kinsella
    Abstract:

    Purpose: 5-Iodo-2-pyrimidinone-2′-Deoxyribose (IPdR) is a pyrimidinone nucleoside prodrug of 5-iododeoxyuridine (IUdR) under investigation as an orally administered radiosensitizer. We previously reported that the mismatch repair (MMR) proteins (both hMSH2 and hMLH1) impact on the extent (percentage) of IUdR-DNA incorporation and subsequent in vitro IUdR-mediated radiosensitization in human tumor cell lines. In this study, we used oral IPdR to assess in vivo radiosensitization in MMR-proficient (MMR + ) and -deficient (MMR − ) human colon cancer xenografts. Experimental Design: We tested whether oral IPdR treatment (1 g/kg/d for 14 days) can result in differential IUdR incorporation in tumor cell DNA and subsequent radiosensitization after a short course (every day for 4 days) of fractionated radiation therapy, by using athymic nude mice with an isogenic pair of human colon cancer xenografts, HCT116 (MMR − , hMLH1 − ) and HCT116/3-6 (MMR + , hMLH1 + ). A tumor regrowth assay was used to assess radiosensitization. Systemic toxicity was assessed by daily body weights and by percentage of IUdR-DNA incorporation in normal bone marrow and intestine. Results: After a 14-day once-daily IPdR treatment by gastric gavage, significantly higher IUdR-DNA incorporation was found in HCT116 (MMR − ) tumor xenografts compared with HCT116/3-6 (MMR + ) tumor xenografts. Using a tumor regrowth assay after the 14-day drug treatment and a 4-day radiation therapy course (days 11–14 of IPdR), we found substantial radiosensitization in both HCT116 and HCT116/3-6 tumor xenografts. However, the sensitizer enhancement ratio (SER) was substantially higher in HCT116 (MMR − ) tumor xenografts (1.48 at 2 Gy per fraction, 1.41 at 4 Gy per fraction), compared with HCT116/3-6 (MMR + ) tumor xenografts (1.21 at 2 Gy per fraction, 1.20 at 4 Gy per fraction). No substantial systemic toxicity was found in the treatment groups. Conclusions: These results suggest that IPdR-mediated radiosensitization can be an effective in vivo approach to treat “drug-resistant” MMR-deficient tumors as well as MMR-proficient tumors.

  • preclinical study of the systemic toxicity and pharmacokinetics of 5 iodo 2 deoxypyrimidinone 2 Deoxyribose as a radiosensitizing prodrug in two non rodent animal species implications for phase i study design
    Clinical Cancer Research, 2000
    Co-Authors: Timothy J Kinsella, Jane E Schupp, Thomas W Davis, Suzanne E Berry, Hwashin Hwang, Kathy Warren, Frank M Balis, John Barnett, Howard Sands
    Abstract:

    We have demonstrated previously an improved therapeutic index for oral 5-iodo-2-deoxypyrimidinone-2′-Deoxyribose (IPdR) compared with oral and continuous infusion of 5-iodo-2′-deoxyuridine (IUdR) as a radiosensitizing agent using three different human tumor xenografts in athymic mice. IPdR is a prodrug that is efficiently converted to IUdR by a hepatic aldehyde oxidase, resulting in high IPdR and IUdR plasma levels in mice for ≥1 h after p.o. IPdR. Athymic mice tolerated oral IPdR at up to 1500 mg/kg/day given four times per day for 6–14 days without significant systemic toxicities. In anticipation of an investigational new drug application for the first clinical Phase I and pharmacology study of oral IPdR in humans, we studied the drug pharmacokinetics and host toxicities in two non-rodent, animal species. For the IPdR systemic toxicity and toxicology study, twenty-four male or female ferrets were randomly assigned to four IPdR dosage groups receiving 0, 15, 150, and 1500 mg/kg/day by oral gavage × 14 days prior to sacrifice on study day 15. All ferrets survived the 14-day treatment. Ferrets receiving 1500 mg/kg/day showed observable systemic toxicities with diarrhea, emesis, weight loss, and decreased motor activity beginning at days 5–8 of the 14-day schedule. Overall, both male and female ferrets receiving IPdR at 1500 mg/kg/day experienced significant weight loss (9 and 19%, respectively) compared with controls after the 14-day treatment. No weight loss or other systemic toxicities were observed in other IPdR dosage groups. Grossly, no anatomical lesions were noted at complete necropsy, although liver weights were increased in both male and female ferrets in the two higher IPdR dosage groups. Histologically, IPdR-treated animals showed dose-dependent microscopic changes in liver consisting of minimal to moderate cytoplasmic vacuolation of hepatocytes, which either occurred in the periportal area (high dosage group) or diffusely throughout the liver (lower dosage groups). Female ferrets in the highest IPdR dose group also showed decreased kidney and uterus weights at autopsy without any associated histological changes. No histological changes were found in central nervous system tissues. No significant abnormalities in blood cell counts, liver function tests, kidney function tests, or urinalysis were noted. Hepatic aldehyde oxidase activity was decreased to approximately 50 and 30% of control ferrets in the two higher IPdR dosage groups, respectively, after the 14-day treatment period. The % IUdR-DNA incorporation in ferret bone marrow at the completion of IPdR treatment was ≤0.05% in the two lower dosage groups and ≅2% in the 1500 mg/kg/day dosage group. The % IUdR-DNA in normal liver was ≤0.05% in all IPdR dosage groups. In a pharmacokinetic study in four Rhesus monkeys, we determined the plasma concentrations of IPdR after a single i.v. bolus of 50 mg/kg over 20 min. Using a two-compartment model to fit the plasma pharmacokinetic data, we found that IPdR was cleared in these non-human primates in a biexponential manner with an initial rapid distributive phase (mean T 1/2 α = 6.5 min), followed by an elimination phase with a mean T 1/2 β of 63 min. The mean maximum plasma concentration of IPdR was 124 ± 43 μm with a mean total body clearance of 1.75 ± 0.95 l/h/kg. IPdR was below detection ( We conclude that there are dose-limiting systemic toxicities to a 14-day schedule of p.o. IPdR at 1500 mg/kg/day in ferrets that were not found previously in athymic mice. However, no significant hematological, biochemical, or histopathological changes were found. Hepatic aldehyde oxidase activity was reduced in a dose-dependent in ferret liver, suggesting partial enzyme saturation by this IPdR schedule. The plasma pharmacokinetic profile in Rhesus monkeys showing biexponential clearance is similar to our published data in athymic mice. These data are being applied to the design of an initial clinical Phase I study of p.o. IPdR as a radiosensitizer.

Peter C Dedon - One of the best experts on this subject based on the ideXlab platform.

  • quantification of the 2 deoxyribonolactone and nucleoside 5 aldehyde products of 2 Deoxyribose oxidation in dna and cells by isotope dilution gas chromatography mass spectrometry differential effects of γ radiation and fe2 edta
    Journal of the American Chemical Society, 2010
    Co-Authors: Wan Chan, Koli Taghizadeh, Bingzi Chen, Lianrong Wang, Michael S Demott, Peter C Dedon
    Abstract:

    The oxidation of 2-Deoxyribose in DNA has emerged as a critical determinant of the cellular toxicity of oxidative damage to DNA, with oxidation of each carbon producing a unique spectrum of electrophilic products. We have developed and validated an isotope-dilution gas chromatography-coupled mass spectrometry (GC-MS) method for the rigorous quantification of two major 2-Deoxyribose oxidation products: the 2-deoxyribonolactone abasic site of 1’-oxidation and the nucleoside 5’-aldehyde of 5’-oxidation chemistry. The method entails elimination of these products as 5-methylene-2(5H)-furanone (5MF) and furfural, respectively, followed by derivatization with pentafluorophenylhydrazine (PFPH), addition of isotopically labeled PFPH derivatives as internal standards, extraction of the derivatives, and quantification by GC-MS analysis. The precision and accuracy of the method were validated with oligodeoxynucleotides containing the 2-deoxyribonolactone and nucleoside 5’-aldehyde lesions. Further, the well defined 2-Deoxyribose oxidation chemistry of the enediyne antibiotics, neocarzinostatin and calicheamicin γ1 I , was exploited in control studies, with neocarzinostatin producing 10 2-deoxyribonolactone and 300 nucleoside 5’-aldehyde per 10 6 nt per µM in accord with its established minor 1’- and major 5’-oxidation chemistry. Calicheamicin unexpectedly caused 1’-oxidation at a low level of 10 2-deoxyribonolactone per 10 6 nt per µM in addition to the expected predominance of 5’oxidation at 560 nucleoside 5’-aldehyde per 10 6 nt per µM. The two hydroxyl radical-mediated DNA oxidants, γ-radiation and Fe 2+ -EDTA, produced nucleoside 5’-aldehyde at a frequency of 57 per 10 6 nt per Gy (G-value 74 nmol/J) and 3.5 per 10 6 nt per µM, respectively, which amounted to 40% and 35%, respectively, of total 2-Deoxyribose oxidation as measured by a plasmid nicking assay. However, γ-radiation and Fe 2+ -EDTA produced different proportions of 2deoxyribonolactone at 7% and 24% of total 2-Deoxyribose oxidation, respectively, with

  • quantification of the 2 deoxyribonolactone and nucleoside 5 aldehyde products of 2 Deoxyribose oxidation in dna and cells by isotope dilution gas chromatography mass spectrometry differential effects of γ radiation and fe2 edta
    Journal of the American Chemical Society, 2010
    Co-Authors: Wan Chan, Koli Taghizadeh, Bingzi Chen, Lianrong Wang, Michael S Demott, Peter C Dedon
    Abstract:

    The oxidation of 2-Deoxyribose in DNA has emerged as a critical determinant of the cellular toxicity of oxidative damage to DNA, with oxidation of each carbon producing a unique spectrum of electrophilic products. We have developed and validated an isotope-dilution gas chromatography-coupled mass spectrometry (GC-MS) method for the rigorous quantification of two major 2-Deoxyribose oxidation products: the 2-deoxyribonolactone abasic site of 1'-oxidation and the nucleoside 5'-aldehyde of 5'-oxidation chemistry. The method entails elimination of these products as 5-methylene-2(5H)-furanone (5MF) and furfural, respectively, followed by derivatization with pentafluorophenylhydrazine (PFPH), addition of isotopically labeled PFPH derivatives as internal standards, extraction of the derivatives, and quantification by GC-MS analysis. The precision and accuracy of the method were validated with oligodeoxynucleotides containing the 2-deoxyribonolactone and nucleoside 5'-aldehyde lesions. Further, the well-defined 2-Deoxyribose oxidation chemistry of the enediyne antibiotics, neocarzinostatin and calicheamicin gamma(1)(I), was exploited in control studies, with neocarzinostatin producing 10 2-deoxyribonolactone and 300 nucleoside 5'-aldehyde per 10(6) nt per microM in accord with its established minor 1'- and major 5'-oxidation chemistry. Calicheamicin unexpectedly caused 1'-oxidation at a low level of 10 2-deoxyribonolactone per 10(6) nt per microM in addition to the expected predominance of 5'-oxidation at 560 nucleoside 5'-aldehyde per 10(6) nt per microM. The two hydroxyl radical-mediated DNA oxidants, gamma-radiation and Fe(2+)-EDTA, produced nucleoside 5'-aldehyde at a frequency of 57 per 10(6) nt per Gy (G-value 74 nmol/J) and 3.5 per 10(6) nt per microM, respectively, which amounted to 40% and 35%, respectively, of total 2-Deoxyribose oxidation as measured by a plasmid nicking assay. However, gamma-radiation and Fe(2+)-EDTA produced different proportions of 2-deoxyribonolactone at 7% and 24% of total 2-Deoxyribose oxidation, respectively, with frequencies of 10 lesions per 10(6) nt per Gy (G-value, 13 nmol/J) and 2.4 lesions per 10(6) nt per microM. Studies in TK6 human lymphoblastoid cells, in which the analytical data were corrected for losses sustained during DNA isolation, revealed background levels of 2-deoxyribonolactone and nucleoside 5'-aldehyde of 9.7 and 73 lesions per 10(6) nt, respectively. Gamma-irradiation of the cells caused increases of 0.045 and 0.22 lesions per 10(6) nt per Gy, respectively, which represents a approximately 250-fold quenching effect of the cellular environment similar to that observed in previous studies. The proportions of the various 2-Deoxyribose oxidation products generated by gamma-radiation are similar for purified DNA and cells. These results are consistent with solvent exposure as a major determinant of hydroxyl radical reactivity with 2-Deoxyribose in DNA, but the large differences between gamma-radiation and Fe(2+)-EDTA suggest that factors other than hydroxyl radical reactivity govern DNA oxidation chemistry.

  • The Chemical Toxicology of 2-Deoxyribose Oxidation in DNA
    Chemical research in toxicology, 2007
    Co-Authors: Peter C Dedon
    Abstract:

    Damage to DNA and RNA caused by oxidative mechanisms has been well-studied for its potential role in the development of human disease. Only recently, though, have we begun to appreciate that oxidation of the 2-Deoxyribose moiety in DNA is also a determinant of the genetic toxicology of oxidative stress and inflammation, with involvement in more than just “strand breaks”, such as complex DNA lesions, protein–DNA cross-links, and protein and DNA adducts. As an update to a 1992 review of 2′-Deoxyribose oxidation by bleomycin and the enediynes published in Chemical Research in Toxicology [Dedon, P. C., and Goldberg, I. H. (1992) Chem. Res. Toxicol. 5, 311–332], this review focuses on recent developments in the chemical biology, bioanalytical chemistry, and genetic toxicology of 2-Deoxyribose oxidation products in DNA under biologically relevant conditions.

  • gc ms methods to quantify the 2 deoxypentos 4 ulose and 3 phosphoglycolate pathways of 4 oxidation of 2 Deoxyribose in dna application to dna damage produced by γ radiation and bleomycin
    Chemical Research in Toxicology, 2007
    Co-Authors: Bingzi Chen, Koli Taghizadeh, Xinfeng Zhou, Jingyang Chen, Joanne Stubbe, Peter C Dedon
    Abstract:

    DNA oxidation plays a substantive role in the pathophysiology of human diseases, such as cancer. While the chemistry of nucleobase lesions has dominated studies of DNA damage, there is growing evidence that the oxidation of 2-Deoxyribose in DNA plays a critical role in the genetic toxicology of oxidative stress. As part of an effort to define the spectrum of 2-Deoxyribose oxidation products arising in vitro and in vivo, we now describe methods for quantifying products arising from 4′ oxidation of 2-Deoxyribose in DNA. The chemistry of 4′ oxidation partitions between either of two pathways to form either a 2-deoxypentos-4-ulose abasic site (oxAB) or a strand break comprised of a 3′-phosphoglycolate (3PG) residue and a 5′-phosphate, with the release of either malondialdehyde and free base or a base propenal. Highly sensitive gas chromatography/mass spectrometry (GC/MS) methods were developed to quantify both lesions. The abasic site was converted to a 3′-phosphoro-3-pyridazinylmethylate derivative by treatm...

Bingzi Chen - One of the best experts on this subject based on the ideXlab platform.

  • quantification of the 2 deoxyribonolactone and nucleoside 5 aldehyde products of 2 Deoxyribose oxidation in dna and cells by isotope dilution gas chromatography mass spectrometry differential effects of γ radiation and fe2 edta
    Journal of the American Chemical Society, 2010
    Co-Authors: Wan Chan, Koli Taghizadeh, Bingzi Chen, Lianrong Wang, Michael S Demott, Peter C Dedon
    Abstract:

    The oxidation of 2-Deoxyribose in DNA has emerged as a critical determinant of the cellular toxicity of oxidative damage to DNA, with oxidation of each carbon producing a unique spectrum of electrophilic products. We have developed and validated an isotope-dilution gas chromatography-coupled mass spectrometry (GC-MS) method for the rigorous quantification of two major 2-Deoxyribose oxidation products: the 2-deoxyribonolactone abasic site of 1’-oxidation and the nucleoside 5’-aldehyde of 5’-oxidation chemistry. The method entails elimination of these products as 5-methylene-2(5H)-furanone (5MF) and furfural, respectively, followed by derivatization with pentafluorophenylhydrazine (PFPH), addition of isotopically labeled PFPH derivatives as internal standards, extraction of the derivatives, and quantification by GC-MS analysis. The precision and accuracy of the method were validated with oligodeoxynucleotides containing the 2-deoxyribonolactone and nucleoside 5’-aldehyde lesions. Further, the well defined 2-Deoxyribose oxidation chemistry of the enediyne antibiotics, neocarzinostatin and calicheamicin γ1 I , was exploited in control studies, with neocarzinostatin producing 10 2-deoxyribonolactone and 300 nucleoside 5’-aldehyde per 10 6 nt per µM in accord with its established minor 1’- and major 5’-oxidation chemistry. Calicheamicin unexpectedly caused 1’-oxidation at a low level of 10 2-deoxyribonolactone per 10 6 nt per µM in addition to the expected predominance of 5’oxidation at 560 nucleoside 5’-aldehyde per 10 6 nt per µM. The two hydroxyl radical-mediated DNA oxidants, γ-radiation and Fe 2+ -EDTA, produced nucleoside 5’-aldehyde at a frequency of 57 per 10 6 nt per Gy (G-value 74 nmol/J) and 3.5 per 10 6 nt per µM, respectively, which amounted to 40% and 35%, respectively, of total 2-Deoxyribose oxidation as measured by a plasmid nicking assay. However, γ-radiation and Fe 2+ -EDTA produced different proportions of 2deoxyribonolactone at 7% and 24% of total 2-Deoxyribose oxidation, respectively, with

  • quantification of the 2 deoxyribonolactone and nucleoside 5 aldehyde products of 2 Deoxyribose oxidation in dna and cells by isotope dilution gas chromatography mass spectrometry differential effects of γ radiation and fe2 edta
    Journal of the American Chemical Society, 2010
    Co-Authors: Wan Chan, Koli Taghizadeh, Bingzi Chen, Lianrong Wang, Michael S Demott, Peter C Dedon
    Abstract:

    The oxidation of 2-Deoxyribose in DNA has emerged as a critical determinant of the cellular toxicity of oxidative damage to DNA, with oxidation of each carbon producing a unique spectrum of electrophilic products. We have developed and validated an isotope-dilution gas chromatography-coupled mass spectrometry (GC-MS) method for the rigorous quantification of two major 2-Deoxyribose oxidation products: the 2-deoxyribonolactone abasic site of 1'-oxidation and the nucleoside 5'-aldehyde of 5'-oxidation chemistry. The method entails elimination of these products as 5-methylene-2(5H)-furanone (5MF) and furfural, respectively, followed by derivatization with pentafluorophenylhydrazine (PFPH), addition of isotopically labeled PFPH derivatives as internal standards, extraction of the derivatives, and quantification by GC-MS analysis. The precision and accuracy of the method were validated with oligodeoxynucleotides containing the 2-deoxyribonolactone and nucleoside 5'-aldehyde lesions. Further, the well-defined 2-Deoxyribose oxidation chemistry of the enediyne antibiotics, neocarzinostatin and calicheamicin gamma(1)(I), was exploited in control studies, with neocarzinostatin producing 10 2-deoxyribonolactone and 300 nucleoside 5'-aldehyde per 10(6) nt per microM in accord with its established minor 1'- and major 5'-oxidation chemistry. Calicheamicin unexpectedly caused 1'-oxidation at a low level of 10 2-deoxyribonolactone per 10(6) nt per microM in addition to the expected predominance of 5'-oxidation at 560 nucleoside 5'-aldehyde per 10(6) nt per microM. The two hydroxyl radical-mediated DNA oxidants, gamma-radiation and Fe(2+)-EDTA, produced nucleoside 5'-aldehyde at a frequency of 57 per 10(6) nt per Gy (G-value 74 nmol/J) and 3.5 per 10(6) nt per microM, respectively, which amounted to 40% and 35%, respectively, of total 2-Deoxyribose oxidation as measured by a plasmid nicking assay. However, gamma-radiation and Fe(2+)-EDTA produced different proportions of 2-deoxyribonolactone at 7% and 24% of total 2-Deoxyribose oxidation, respectively, with frequencies of 10 lesions per 10(6) nt per Gy (G-value, 13 nmol/J) and 2.4 lesions per 10(6) nt per microM. Studies in TK6 human lymphoblastoid cells, in which the analytical data were corrected for losses sustained during DNA isolation, revealed background levels of 2-deoxyribonolactone and nucleoside 5'-aldehyde of 9.7 and 73 lesions per 10(6) nt, respectively. Gamma-irradiation of the cells caused increases of 0.045 and 0.22 lesions per 10(6) nt per Gy, respectively, which represents a approximately 250-fold quenching effect of the cellular environment similar to that observed in previous studies. The proportions of the various 2-Deoxyribose oxidation products generated by gamma-radiation are similar for purified DNA and cells. These results are consistent with solvent exposure as a major determinant of hydroxyl radical reactivity with 2-Deoxyribose in DNA, but the large differences between gamma-radiation and Fe(2+)-EDTA suggest that factors other than hydroxyl radical reactivity govern DNA oxidation chemistry.

  • gc ms methods to quantify the 2 deoxypentos 4 ulose and 3 phosphoglycolate pathways of 4 oxidation of 2 Deoxyribose in dna application to dna damage produced by γ radiation and bleomycin
    Chemical Research in Toxicology, 2007
    Co-Authors: Bingzi Chen, Koli Taghizadeh, Xinfeng Zhou, Jingyang Chen, Joanne Stubbe, Peter C Dedon
    Abstract:

    DNA oxidation plays a substantive role in the pathophysiology of human diseases, such as cancer. While the chemistry of nucleobase lesions has dominated studies of DNA damage, there is growing evidence that the oxidation of 2-Deoxyribose in DNA plays a critical role in the genetic toxicology of oxidative stress. As part of an effort to define the spectrum of 2-Deoxyribose oxidation products arising in vitro and in vivo, we now describe methods for quantifying products arising from 4′ oxidation of 2-Deoxyribose in DNA. The chemistry of 4′ oxidation partitions between either of two pathways to form either a 2-deoxypentos-4-ulose abasic site (oxAB) or a strand break comprised of a 3′-phosphoglycolate (3PG) residue and a 5′-phosphate, with the release of either malondialdehyde and free base or a base propenal. Highly sensitive gas chromatography/mass spectrometry (GC/MS) methods were developed to quantify both lesions. The abasic site was converted to a 3′-phosphoro-3-pyridazinylmethylate derivative by treatm...

Yuji Seo - One of the best experts on this subject based on the ideXlab platform.

  • 5 iodo 2 pyrimidinone 2 Deoxyribose mediated cytotoxicity and radiosensitization in u87 human glioblastoma xenografts
    International Journal of Radiation Oncology Biology Physics, 2007
    Co-Authors: Timothy J Kinsella, Michael T Kinsella, Yuji Seo, Gregory Berk
    Abstract:

    Purpose 5-Iodo-2-pyrimidinone-2′-Deoxyribose (IPdR) is a novel orally administered (p.o.) prodrug of 5-iododeoxyuridine. Because p.o. IPdR is being considered for clinical testing as a radiosensitizer in patients with high-grade gliomas, we performed this in vivo study of IPdR-mediated cytotoxicity and radiosensitization in a human glioblastoma xenograft model, U87. Methods and Materials Groups of 8 or 9 athymic male nude mice (6–8 weeks old) were implanted with s.c. U87 xenograft tumors (4 × 10 6 cells) and then randomized to 10 treatment groups receiving increasing doses of p.o. IPdR (0, 100, 250, 500, and 1000 mg/kg/d) administered once daily (q.d.) × 14 days with or without radiotherapy (RT) (0 or 2 Gy/d × 4 days) on days 11–14 of IPdR treatment. Systemic toxicity was determined by body weight measurements during and after IPdR treatment. Tumor response was assessed by changes in tumor volumes. Results IPdR alone at doses of ≥500 mg/kg/d resulted in moderate inhibition of tumor growth. The combination of IPdR plus RT resulted in a significant IPdR dose-dependent tumor growth delay, with the maximum radiosensitization using ≥500 mg/kg/d. IPdR doses of 500 and 1000 mg/kg/d resulted in transient 5–15% body weight loss during treatment. Conclusions In U87 human glioblastoma s.c. xenografts, p.o. IPdR given q.d. × 14 days and RT given 2 Gy/d × 4 days (days 11–14 of IPdR treatment) results in a significant tumor growth delay in an IPdR dose-dependent pattern. The use of p.o. IPdR plus RT holds promise for Phase I/II testing in patients with high-grade gliomas.

  • schedule dependent drug effects of oral 5 iodo 2 pyrimidinone 2 Deoxyribose as an in vivo radiosensitizer in u251 human glioblastoma xenografts
    Clinical Cancer Research, 2005
    Co-Authors: Yuji Seo, Tao Yan, Jane E Schupp, Tomas Radivoyevitch, Timothy J Kinsella
    Abstract:

    Purpose: 5-Iodo-2-pyrimidinone-2′-Deoxyribose (IPdR) is an oral prodrug of 5-iodo-2′-deoxyuridine (IUdR), an in vitro/in vivo radiosensitizer. IPdR can be rapidly converted to IUdR by a hepatic aldehyde oxidase. Previously, we found that the enzymatic conversion of IPdR to IUdR could be transiently reduced using a once daily (q.d.) treatment schedule and this may affect IPdR-mediated tumor radiosensitization. The purpose of this study is to measure the effect of different drug dosing schedules on tumor radiosensitization and therapeutic index in human glioblastoma xenografts. Experimental Design: Three different IPdR treatment schedules (thrice a day, t.i.d.; every other day, q.o.d.; every 3rd day, q.3.d.), compared with a q.d. schedule, were analyzed using athymic nude mice with human glioblastoma (U251) s.c. xenografts. Plasma pharmacokinetics, IUdR-DNA incorporation in tumor and normal proliferating tissues, tumor growth delay following irradiation, and body weight loss were used as end points. Results: The t.i.d. schedule with the same total daily doses as the q.d. schedule (250, 500, or 1,000 mg/kg/d) improved the efficiency of IPdR conversion to IUdR. As a result, the percentage of IUdR-DNA incorporation was higher using the t.i.d. schedule in the tumor xenografts as well as in normal small intestine and bone marrow. Using a fixed dose (500 mg/kg) per administration, the q.o.d. and q.3.d. schedules also showed greater IPdR conversion than the q.d. schedule, related to a greater recovery of hepatic aldehyde oxidase activity prior to the next drug dosing. In the tumor regrowth assay, all IPdR treatment schedules showed significant increases of regrowth delays compared with the control without IPdR (q.o.d., 29.4 days; q.d., 29.7 days; t.i.d., 34.7 days; radiotherapy alone, 15.7 days). The t.i.d. schedule also showed a significantly enhanced tumor growth delay compared with the q.d. schedule. Additionally, the q.o.d. schedule resulted in a significant reduction in systemic toxicity. Conclusions: The t.i.d. and q.o.d. dosing schedules improved the efficiency of enzymatic activation of IPdR to IUdR during treatment and changed the extent of tumor radiosensitization and/or systemic toxicity compared with a q.d. dosing schedule. These dosing schedules will be considered for future clinical trials of IPdR-mediated human tumor radiosensitization.

  • differential radiosensitization in dna mismatch repair proficient and deficient human colon cancer xenografts with 5 iodo 2 pyrimidinone 2 Deoxyribose
    Clinical Cancer Research, 2004
    Co-Authors: Yuji Seo, Tao Yan, Jane E Schupp, Valdir C Colussi, Kerri Taylor, Timothy J Kinsella
    Abstract:

    Purpose: 5-Iodo-2-pyrimidinone-2′-Deoxyribose (IPdR) is a pyrimidinone nucleoside prodrug of 5-iododeoxyuridine (IUdR) under investigation as an orally administered radiosensitizer. We previously reported that the mismatch repair (MMR) proteins (both hMSH2 and hMLH1) impact on the extent (percentage) of IUdR-DNA incorporation and subsequent in vitro IUdR-mediated radiosensitization in human tumor cell lines. In this study, we used oral IPdR to assess in vivo radiosensitization in MMR-proficient (MMR + ) and -deficient (MMR − ) human colon cancer xenografts. Experimental Design: We tested whether oral IPdR treatment (1 g/kg/d for 14 days) can result in differential IUdR incorporation in tumor cell DNA and subsequent radiosensitization after a short course (every day for 4 days) of fractionated radiation therapy, by using athymic nude mice with an isogenic pair of human colon cancer xenografts, HCT116 (MMR − , hMLH1 − ) and HCT116/3-6 (MMR + , hMLH1 + ). A tumor regrowth assay was used to assess radiosensitization. Systemic toxicity was assessed by daily body weights and by percentage of IUdR-DNA incorporation in normal bone marrow and intestine. Results: After a 14-day once-daily IPdR treatment by gastric gavage, significantly higher IUdR-DNA incorporation was found in HCT116 (MMR − ) tumor xenografts compared with HCT116/3-6 (MMR + ) tumor xenografts. Using a tumor regrowth assay after the 14-day drug treatment and a 4-day radiation therapy course (days 11–14 of IPdR), we found substantial radiosensitization in both HCT116 and HCT116/3-6 tumor xenografts. However, the sensitizer enhancement ratio (SER) was substantially higher in HCT116 (MMR − ) tumor xenografts (1.48 at 2 Gy per fraction, 1.41 at 4 Gy per fraction), compared with HCT116/3-6 (MMR + ) tumor xenografts (1.21 at 2 Gy per fraction, 1.20 at 4 Gy per fraction). No substantial systemic toxicity was found in the treatment groups. Conclusions: These results suggest that IPdR-mediated radiosensitization can be an effective in vivo approach to treat “drug-resistant” MMR-deficient tumors as well as MMR-proficient tumors.

Edward L Schwartz - One of the best experts on this subject based on the ideXlab platform.

  • thymidine phosphorylase and 2 Deoxyribose stimulate human endothelial cell migration by specific activation of the integrins α5β1 and αvβ3
    Journal of Biological Chemistry, 2003
    Co-Authors: Kylie A. Hotchkiss, Anthony W. Ashton, Edward L Schwartz
    Abstract:

    Abstract Thymidine phosphorylase is an angiogenic factor that is frequently overexpressed in solid tumors, in rheumatoid arthritis, and in response to inflammatory cytokines. Our previous studies showed that cells expressing thymidine phosphorylase stimulated endothelial cell migration in vitro. This was a consequence of the intracellular metabolism of thymidine by thymidine phosphorylase and subsequent extracellular release of 2-Deoxyribose. The mechanisms by which 2-Deoxyribose might mediate thymidine phosphorylase-induced cell migration in vitro, however, are obscure. Here we show that both thymidine phosphorylase and 2-Deoxyribose stimulated the formation of focal adhesions and the tyrosine 397 phosphorylation of focal adhesion kinase in human umbilical vein endothelial cells. Although similar actions occurred upon treatment with the angiogenic factor vascular endothelial growth factor (VEGF), thymidine phosphorylase differed from VEGF in that its effect on endothelial cell migration was blocked by antibodies to either integrin α5β1 or αvβ3, whereas VEGF-induced endothelial cell migration was only blocked by the αvβ3 antibody. Further, thymidine phosphorylase and 2-Deoxyribose, but not VEGF, increased the association of both focal adhesion kinase and the focal adhesion-associated protein vinculin with integrin α5β1 and, in intact cells, increased the co-localization of focal adhesion kinase with α5β1. Thymidine phosphorylase and 2-Deoxyribose-induced focal adhesion kinase phosphorylation was blocked by the antibodies to α5β1 and αvβ3, directly linking the migration and signaling components of thymidine phosphorylase and 2-Deoxyribose action. Cell surface expression of α5β1 was also increased by thymidine phosphorylase and 2-Deoxyribose. These experiments are the first to demonstrate a direct effect of thymidine phosphorylase and 2-Deoxyribose on signaling pathways associated with endothelial cell migration.

  • Thymidine phosphorylase and 2-Deoxyribose stimulate human endothelial cell migration by specific activation of the integrins α5β1 and αVβ3
    The Journal of biological chemistry, 2003
    Co-Authors: Kylie A. Hotchkiss, Anthony W. Ashton, Edward L Schwartz
    Abstract:

    Thymidine phosphorylase is an angiogenic factor that is frequently overexpressed in solid tumors, in rheumatoid arthritis, and in response to inflammatory cytokines. Our previous studies showed that cells expressing thymidine phosphorylase stimulated endothelial cell migration in vitro. This was a consequence of the intracellular metabolism of thymidine by thymidine phosphorylase and subsequent extracellular release of 2-Deoxyribose. The mechanisms by which 2-Deoxyribose might mediate thymidine phosphorylase-induced cell migration in vitro, however, are obscure. Here we show that both thymidine phosphorylase and 2-Deoxyribose stimulated the formation of focal adhesions and the tyrosine 397 phosphorylation of focal adhesion kinase in human umbilical vein endothelial cells. Although similar actions occurred upon treatment with the angiogenic factor vascular endothelial growth factor (VEGF), thymidine phosphorylase differed from VEGF in that its effect on endothelial cell migration was blocked by antibodies to either integrin alpha 5 beta 1 or alpha v beta 3, whereas VEGF-induced endothelial cell migration was only blocked by the alpha v beta 3 antibody. Further, thymidine phosphorylase and 2-Deoxyribose, but not VEGF, increased the association of both focal adhesion kinase and the focal adhesion-associated protein vinculin with integrin alpha 5 beta 1 and, in intact cells, increased the co-localization of focal adhesion kinase with alpha 5 beta 1. Thymidine phosphorylase and 2-Deoxyribose-induced focal adhesion kinase phosphorylation was blocked by the antibodies to alpha 5 beta 1 and alpha v beta 3, directly linking the migration and signaling components of thymidine phosphorylase and 2-Deoxyribose action. Cell surface expression of alpha 5 beta 1 was also increased by thymidine phosphorylase and 2-Deoxyribose. These experiments are the first to demonstrate a direct effect of thymidine phosphorylase and 2-Deoxyribose on signaling pathways associated with endothelial cell migration.