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A E Maccubbin

Publications and source records attributed to A E Maccubbin.

At least 19 recordsLinked to original sources
PubMed2000

Double-base lesions are produced in DNA by free radicals.

Evidence has been accumulating at the oligomer level that free radical-initiated DNA damage includes lesions in which two adjacent bases are both modified. Prominent examples are lesions in which a pyrimidine base is degraded to a formamido remnant and an adjacent guanine base is oxidized. An assay has been devised to detect double-base lesions based on the fact that the phosphoester bond 3' to a nuclesoside bearing the formamido lesion is resistant to hydrolysis by nuclease P1. The residual modified dinucleoside monophosphates obtained from a nuclease P1 (plus acid phosphatase) digest of DNA can be (32)P-postlabeled using T4 polynucleotide kinase. Using this assay the formamido single lesion and the formamido-8-oxoguanine double lesion were detected in calf thymus DNA after X-irradiation in oxygenated aqueous solution. The lesions were measured in the forms d(P(F)pG) and d(P(F)pG(H)), where P(F) stands for a pyrimidine nucleoside having the base degraded to a formamido remnant and G(H) stands for 8-oxo-deoxyguanosine. The yields in calf thymus DNA irradiated 60 Gy were 8.6 and 3.2 pmol/microgram DNA, respectively.

Adenosine Triphosphate↗
PubMed1999

Assay for reactive oxygen species-induced DNA damage: measurement of the formamido and thymine glycol lesions.

A 32P-postlabeling assay has been developed for the simultaneous detection of the thymine glycol lesion and the formamido remnant of pyrimidine bases in DNA exposed to reactive oxygen species (ROS). The formamido lesion is a principal lesion produced in X-irradiated DNA oligomers when oxygen is available to mediate the damage process. Production of the well-known thymine glycol lesion is less dependent on the concentration of oxygen. These two lesions have the common property that they make the phosphoester bond 3' to the modified nucleoside resistant to hydrolysis by nuclease P1. Our assay uses 32P-postlabeling to measure these lesions in the form of modified dimers obtained from DNA by nuclease P1 digestion. Appropriate carriers and internal standards have been chemically synthesized to improve the reliability and accuracy of the assay. The measurements were accomplished on 1-microgram samples of DNA.

Acid Phosphatase↗
PubMed1998

Detection of mitomycin C-DNA adducts in vivo by 32P-postlabeling: time course for formation and removal of adducts and biochemical modulation.

Mitomycin C (MMC) is a DNA cross-linking agent that has been used in cancer chemotherapy for over 20 years, yet little is known either qualitatively or quantitatively about MMC-induced DNA adduct formation and repair in vivo. As an initial means of investigating this, we used a recently developed 32P-postlabeling assay to examine the formation and loss of MMC-DNA adducts in the tissues of a simple in vivo model test system, the chick embryo, following treatment with a chemotherapeutic dose of MMC. As early as 15 min after MMC treatment, four adducts could be detected in the liver which were tentatively identified as the (CpG) N2G-MMC-N2G interstrand cross-link, the bifunctionally activated MMC-N2G monoadduct, and two isomers (alpha and beta) of the monofunctionally activated MMC-N2G monoadduct. The (GpG) N2G-MMC-N2G intrastrand cross-link appears to be a poor substrate for nuclease P1 and/or T4 kinase and was not evaluable by this assay. Levels of all four detectable adducts increased substantially within the first 2 h after MMC treatment, reached maximal levels by 6 h, and decreased progressively thereafter through 24 h, although low levels of certain adducts persisted beyond 24 h. Lung and kidney had comparable levels of total MMC adducts, which were approximately 60% those of the liver, and there were no significant differences in the proportion of specific adducts among the three tissues. The interstrand cross-link represented approximately 13-14% of the total MMC adducts, which is approximately 5-fold greater than the proportion of CpG sites in the genome. In addition, the interstrand cross-link was selectively decreased after 16 h relative to the three monoadducts, suggesting preferential repair. The effect of modulating different components of the Phase I and Phase II drug metabolism on MMC adduct formation, using either glutethimide, 3,4,3',4'-tetrachlorobiphenyl, dexamethasone, buthionine sulfoximine, ethacrynic acid, or N-acetylcysteine pretreatments, was examined to characterize the possible pathways of MMC metabolism and adduct formation in vivo. Surprisingly, none of these pretreatments had a significant effect on individual or total adducts with the exception of dexamethasone, which caused an almost 2-fold proportional increase in all four adducts in the liver.

Acetylcysteine↗
PubMed1997

Mutations induced by monofunctional and bifunctional phosphoramide mustards in supF tRNA gene.

The relative mutagenicity, nature of the mutations and the sequence specificity of mutations induced by the bifunctional alkylating agent, phosphoramide mustard (PM) and a monofunctional derivative, dechloroethyl phosphoramide mustard (dePM), were analyzed by the Ames test and by an in vitro shuttle vector mutagenesis assay. Both PM and dePM increased the mutation frequency above background in either assay. However, on an equimolar basis, dePM was less mutagenic than PM. In the in vitro shuttle vector mutagenesis assay, sequencing demonstrated that about 40% of the mutant plasmids contained more than one mutation in the supF tRNA gene segment of the plasmid. About 70% of the mutations observed in dePM-treated plasmids were single base substitutions with A:T and G:C base pairs being mutated at equivalent rates. In contrast, only about 50% of the mutations observed in PM-treated plasmids were single base substitutions, 80% of which involved G:C base pairs. Single base deletions and insertions were found in approximately equal proportions with both compounds; however, these lesions were in greater abundance in PM-treated plasmids. Putative hot-spots for mutation in the supF tRNA gene included base pairs at position 102 and 110 for PM and positions 170 and 171 for dePM.

Alkylating Agents↗
PubMed1997

Mutations induced in a shuttle vector plasmid exposed to monofunctionally activated mitomycin C.

Reductive activation of mitomycin C leads to its covalent binding to DNA, forming monoadducts and cross-links. The cytotoxicity of mitomycin C has been attributed to cross-link formation, whereas monoadducts are assumed to cause mutagenicity. We have developed a 32P-postlabeling technique to measure mitomycin C DNA adducts. Using this technique, we have measured monoadduct formation in the shuttle vector plasmid pSP189 and have determined mutations induced by monoadduct formation. The shuttle vector plasmid was incubated with mitomycin C under conditions favoring monofunctional activation of mitomycin C. The plasmid was then replicated in human Ad293 cells, rescued in bacteria, and analyzed for mutations in the supF tRNA gene sequence of pSP189. One major mitomycin C/DNA adduct was observed by 32P-postlabeling and was characterized as a monoadduct of guanine. When pSP189 was exposed to monofunctionally activated mitomycin C, increases in adduct levels and mutation frequency were found to be related to mitomycin C concentration. The majority of the mutations involved single bases, with base substitutions making up 59.1% of the total mutations observed. Of the base substitutions, 67.2% were transversions and 32.8% were transitions, with nearly 80% of all base substitutions involving G:C base pairs. Deletions, either as single bases or large deletions, also involved G:C base pairs the majority of the time. The observed bias of mutations at G:C and the formation of a mitomycin C/DNA monoadduct involving guanine suggests that monoadduct formation may be responsible for the mutations.

Base Sequence↗
PubMed1995

Mutations induced by dacarbazine activated with cytochrome P-450.

The mutagenicity of the antitumor drug dacarbazine (DTIC) is due to alkylation of cellular DNA by metabolites resulting from the metabolism of this drug by the mixed function oxidase system. In the present study, we used an in vitro shuttle vector assay to study the base and sequence specificity of mutagenesis by DTIC. The shuttle vector plasmid pSP189 was treated with DTIC (1-2.5 mM) in vitro in a reconstituted cytochrome P-450 system at 37 degrees C for either 30 or 60 min. SupF tRNA gene insert contained in the plasmid was sequenced after replication of the drug-treated plasmid in human Ad 293 cells followed by amplification in indicator bacteria. Mutagenesis of DTIC in this system was dependent upon the presence of the cytochrome P-450 reconstituted system and NADPH. Mutations induced by DTIC included single base substitutions (35%), single base deletions (30.5%), single base insertions (19.4%) and large deletions (13.8%). Among the substitutions, transversions and transitions were in the ratio of 1:0.7. Base pairs 108 and 127 in the SupF tRNA of the pSP189 were identified as mutational hot spots.

Biotransformation↗
PubMed1995

DNA damage in UVB-irradiated keratinocytes.

A DNA lesion which results from the breakdown of a pyrimidine base leaving a formamido remnant has been associated with oxidative stress. This lesion is shown to be produced in keratinocytes irradiated in culture with UVB light. The amount of formamido lesion produced is comparable to the amount of the 8-hydroxyguanine lesion. The two lesions were measured by 32P-postlabeling and electrochemical detection methods respectively.

Animals↗
PubMed1995

Free radical-induced double base lesions.

Evidence is presented for the formation of products in irradiated dinucleoside monophosphates in which both bases are damaged. The dinucleoside monophosphates d(GpT), d(GpC), d(TpG) and d(CpG) were X-irradiated in oxygenated aqueous solution. Product identification was by NMR spectroscopy. In products containing double base lesions, guanine is converted to 8-hydroxyguanine and the pyrimidine base is degraded to a formamido remnant.

Chromatography, High Pressure Liquid↗
PubMed1994

Mutations in a shuttle vector exposed to activated mitomycin C.

The cytotoxicity of the potent antibiotic and antitumor agent mitomycin C (MMC) is due to its irreversible binding to DNA. Alkylating species generated by bioreductive activation of MMC are known to cause monoadducts and cross-links in DNA by specifically binding to guanine residues. To gain insight into how these lesions lead to base- and sequence-specific mutations, shuttle vector pSP189 was treated with MMC chemically reduced by treatment with sodium borohydride, replicated in human Ad293 cells, rescued in bacteria, and analyzed for mutations in the supF tRNA gene sequence. The MMC-induced mutations were predominantly base substitutions. Eighty-four percent of the base substitutions were transversions, with G:C-->T:A the major transversion. Single base deletions were the other major mutational event, and 77% of these were G:C deletions. Base positions 115, 123, and 163 were mutational hot spots based on the frequency of independent mutations. Identification of a single MMC adduct (presumed to be a modified G on the basis of its Rf value) and clustering of MMC-induced mutations at three GC-rich areas (nt 100-123, 152-163, and 168-176) suggested that the mutational spectrum we found was due to binding of MMC to guanine on either strand of the plasmid DNA.

Alkylation↗
PubMed1994

Formation of 8-hydroxyguanine in DNA during mitomycin C activation.

DNA damage caused indirectly via reactive oxygen species generated during reductive activation of mitomycin C was evaluated. This oxidative DNA damage was measured by determining the formation of 8-hydroxyguanine in DNA exposed to chemically or enzymatically activated mitomycin C. The level of 8-hydroxyguanine was measured indirectly by determining formamidopyrimidine-DNA glycosylase-sensitive sites induced in plasmid DNA exposed to mitomycin C and directly by a 32P-postlabeling assay for the modified base. Activation of mitomycin C by sodium borohydride in air, by H2/Pt, or xanthine oxidase in N2 caused increases in the level of 8-hydroxyguanine. The extent of the increase varied according to the incubation conditions with the greatest increase being observed in DNA exposed to mitomycin C activated under hypoxic conditions. These results support a possible indirect mechanism for DNA damage caused by mitomycin C that is mediated by reactive oxygen species.

Anaerobiosis↗
PubMed1993

The differential lysis of phosphoester bonds by nuclease P1.

The hydrolysis by nuclease P1 of the 16 common deoxydinucleoside monophosphates was examined. The rates of hydrolysis of phosphodiester bond differ by more than two orders of magnitude; dinucleotide monophosphates of the type d(TpN) being most resistant and d(GpN) being next most resistant. The profiles of a mixture of the 16 common dinucleoside monophosphates and of DNA after partial hydrolysis by nuclease P1 and simultaneous treatment with acid phosphatase were compared. The resultant profiles are very similar, except for the appearance of 5-methyldeoxycytidine in the latter. Similar profiles are also obtained from a mixture of dinucleoside monophosphates and from DNA exposed to ionizing radiation beforehand. The 8-hydroxyguanine lesion and a formamido remnant of thymine appear in both profiles as a modified nucleoside and as modified dinucleoside monophosphate respectively. These results suggest that certain radiation induced DNA lesions can be selectively postlabelled based on their resistance to hydrolysis by nuclease P1. The nature of the nuclease P1-substrate interaction is discussed.

Animals↗
PubMed1993

32P-postlabeling assay for free radical-induced DNA damage: the formamido remnant of thymine.

A prominent lesion in DNA exposed to oxidative free radicals results from the degradation of thymine leaving a formamido remnant. A 32P-postlabeling assay has been developed for the detection of the formamido lesion. The assay is based on the circumstance that the lesion prevents hydrolysis by nuclease P1 of the phosphoester bond 3' to the damaged nucleoside. Thus, a nuclease P1 plus acid phosphatase digest of DNA generates mostly nucleosides whereas the formamido lesion is rendered as a modified dinucleoside monophosphate. Dinucleoside monophosphates, but not nucleosides, are apt substrates for 32P-postlabeling by polynucleotide kinase. The assay was applied to calf thymus DNA X-irradiated in oxygenated solution. The formamido lesion could be detected down to a dose of a few Gy.

Animals↗
PubMed1993

Characterization of the products of dinucleoside monophosphates d(GpN) irradiated in aqueous solutions.

The dinucleoside monophosphates d(GpT), d(GpC), and d(GpA) were X-irradiated in oxygenated aqueous solution. The principal products were identified in the intact modified dinucleoside monophosphates using NMR spectroscopy and FAB mass spectrometry. The 8-hydroxyguanine modification is a major product in each of the d(GpN). The d(GpN), where N is a pyrimidine nucleoside, also yield products in which the pyrimidine base is degraded to a formamido remnant. The most interesting product is one bearing two base damages, 8-hydroxylation of the guanine base and degradation of the pyrimidine base to a formamido remnant. This double lesion was observed in both d(GpN) where N is a pyrimidine base.

Chromatography, High Pressure Liquid↗
PubMed1992

32P-postlabeling of acrolein-deoxyguanosine adducts in DNA after nuclease P1 digestion.

In order to study the relationship between the level of acrolein-DNA adducts and their biological effects, sensitive methods are needed to quantitate DNA adducts. 32P-postlabeling is one such method that has been widely used and we have adapted the technique to detect acrolein-deoxyguanosine adducts. Adducts formed by the reaction of acrolein and deoxyguanosine-3'-monophosphate were isolated by HPLC. Based on their UV spectra and cochromatography with standards after dephosphorylation with acid phosphatase, these adducts were identified as the nucleotide equivalents of cyclic 1,N2-propanodeoxyguanosine adducts formed by acrolein that have been described by Chung et al. [15]. As nucleotides, the adducts were good substrates for polynucleotide kinase-mediated transfer of phosphate from ATP and were able to be detected by 32P-postlabeling. These adducts were resistant to the activity of nuclease P1 and dinucleoside monophosphates in the form d(G*pN) where G* is the acrolein-guanine adduct also resisted digestion by nuclease P1. Digestion of DNA by nuclease P1 and acid phosphatase resulted in the conversion of normal nucleotides to nucleosides and selective enrichment of the adducts as dinucleoside monophosphates. Using nuclease P1/acid phosphatase digestion, followed by 32P-postlabeling and TLC separation, levels of the two adducts in acrolein-treated DNA were found to be about 6185 and 19,222 nmol/mol.

Acid Phosphatase↗
PubMed1992

Characterization of two radiation-induced lesions from DNA: studies using nuclease P1.

Exposure of DNA oligomers to ionizing radiation in oxygenated solution reveals that the two lesions formed in highest yield are the 8-hydroxy modification of guanine and the formamido remnant of thymine. The effect of these lesions on the hydrolysis of the phosphodiester bond by nuclease P1 was studied. Whereas the 8-hydroxyguanine lesion does not affect hydrolysis of the adjacent 3' phosphoester bond by nuclease P1, the formamido lesion markedly retards hydrolysis. Consistent with the oligomer results, digestion of irradiated DNA polymer by nuclease P1 plus acid phosphatase yielded the 8-hydroxyguanine lesion, obtained as the modified nucleoside, and the formamido lesion, obtained as a modified dinucleotide.

DNA↗
PubMed1992

Analysis of DNA damage at the dinucleoside monophosphate level: application to the formamido lesion.

The dinucleoside monophosphates d(TpG), d(TpC), and d(TpT) were X-irradiated in oxygenated solution. In each case the modification of the dinucleoside in which the thymine base is degraded to a formamido remnant was observed as a principal product. The hydrolysis of the phosphoester bond of formamido-modified dinucleosides is much slower than that of the corresponding unmodified dinucleosides. This effect is also observable in the hydrolysis of irradiated DNA, where hydrolysis by nuclease P1 (plus acid phosphatase) generates the modified dinucleosides d(TFpN), TF being the modified thymidine. The total yield of the formamido lesion in all its forms, d(TFpN), exceeds the yield of any other base modification.

Chromatography, High Pressure Liquid↗
PubMed1991

A cyclophosphamide/DNA phosphoester adduct formed in vitro and in vivo.

The antitumor activity of cyclophosphamide is thought to be due to the alkylating activity of phosphoramide mustard, a metabolite of cyclophosphamide. Reaction of 2'-deoxyguanosine 3'-monophosphate and phosphoramide mustard resulted in the formation of several adducts that could be detected by high performance liquid chromatography (HPLC). One of these adducts, isolated and purified by HPLC, could be detected by 32P postlabeling. This product was identified by UV, nuclear magnetic resonance, and mass spectrometry and by acid, base, and enzymatic hydrolysis to be 2'-deoxyguanosine 3'-monophosphate 2-(2-hydroxyethyl)aminoethyl ester. A combination of HPLC fractionation of digested DNA and 32P postlabeling was used to detect this adduct in calf thymus DNA incubated in vitro with metabolically activated cyclophosphamide and in DNA from the liver of mice treated with cyclophosphamide. In these DNA samples the adduct occurred at a level of 1/10(5) and 1/3 x 10(7) nucleotides, respectively.

Alkylation↗