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PubMed2006

Exploring the cellular activity of camptothecin-triple-helix-forming oligonucleotide conjugates.

Topoisomerase I is a ubiquitous DNA-cleaving enzyme and an important therapeutic target in cancer chemotherapy for camptothecins (CPTs). These drugs stimulate DNA cleavage by topoisomerase I but exhibit little sequence preference, inducing toxicity and side effects. A convenient strategy to confer sequence specificity consists of the linkage of topoisomerase poisons to DNA sequence recognition elements. In this context, triple-helix-forming oligonucleotides (TFOs) covalently linked to CPTs were investigated for the capacity to direct topoisomerase I-mediated DNA cleavage in cells. In the first part of our study, we showed that these optimized conjugates were able to regulate gene expression in cells upon the use of a Photinus pyralis luciferase reporter gene system. Furthermore, the formation of covalent topoisomerase I/DNA complexes by the TFO-CPT conjugates was detected in cell nuclei. In the second part, we elucidated the molecular specificity of topoisomerase I cleavage by the conjugates by using modified DNA targets and in vitro cleavage assays. Mutations either in the triplex site or in the DNA duplex receptor are not tolerated; such DNA modifications completely abolished conjugate-induced cleavage all along the DNA. These results indicate that these conjugates may be further developed to improve chemotherapeutic cancer treatments by targeting topoisomerase I-induced DNA cleavage to appropriately chosen genes.

Antineoplastic Agents, Phytogenic↗
PubMed2006

[Retarded excision of pyrimidine dimers in human unstimulated lymphocytes].

Using immuno-labelling of cyclobutane pyrimidine dimers (CPDs) in nuclei of peripheral lymphocytes after their UVC-irradiation and cultivation, we have found that within the first four hours of cultivation the CPD-specific fluorescent signal from cell nuclei increased. Earlier, a similar increase in binding of antibody specific for pyrimidine (6-4) pyrimidone photoproducts to undenatured DNA isolated from UV-irradiated Chinese hamster ovary cells was reported (Mitchell et al., 1986). Our experiments showed that nucleotide excision repair enzyme might induce such of DNA modification in lymphocyte nuclei that increased specific antibody binding to DNA fragments with lesions. We suggest that enzymatic formation of open structures in DNA predominated qualitatively over dual-incision and excision of these fragments, and resulted in the enhanced exposure of the pyrimidine dimers in nuclei to specific antibodies. The results evidence that nucleotid excision repair in unstimualted human lymphocytes being deficient in dual incision and removal of UV-induced DNA lesions appear to be capable of performing chromatin relaxation and pre-incision uncoiling of DNA fragments with lesions.

Cell Nucleus↗
PubMed1986

DNA damage in L5178YS cells following exposure to benzene metabolites.

Because DNA modification may be a prerequisite for chemical carcinogenesis, the DNA-damaging potential of benzene and its metabolites was examined in order to identify the proximate DNA-damaging agent associated with benzene exposure. A DNA synthesis inhibition assay previously identified p-benzoquinone as the most potent overall cellular toxin and inhibitor of DNA synthesis, but failed to discriminate among the hydroxylated metabolites. Therefore, the ability of benzene and its metabolites to induce DNA strand breaks in the mouse lymphoma cell line, L5178YS, was examined in order to provide a more accurate indication of the DNA damage associated with benzene and its metabolites. Cells were exposed to benzene, hydroquinone, catechol, phenol, 1,2,4-benzenetriol, or p-benzoquinone over a 1000-fold concentration range (1.0 microM-1.0 mM). Concentrations of benzene, phenol, or catechol as high as 1.0 mM did not increase the percentage of single-stranded DNA observed. Concentrations of hydroquinone as high as 0.1 mM were also ineffective. In contrast, both p-benzoquinone and 1,2,4-benzenetriol produced DNA breaks in a dose-related fashion. Of the two, benzoquinone proved to be more potent with an ED50 of approximately equal to 2.5 microM compared with 55.0 microM for benzenetriol. The DNA damage induced by 6.0 microM benzoquinone was maximal within 3 min of exposure and yielded approximately 70% single-stranded DNA after alkaline denaturation. By contrast, the single-stranded DNA observed after benzenetriol exposure required 60 min of exposure to achieve the same extent of damage as that found with benzoquinone. These results suggest that the benzene metabolites, benzenetriol and benzoquinone, may cause DNA damage and that the mechanisms responsible for the damage associated with these two compounds may be different.

Animals↗
PubMed1990

Sequence-specific chemical modification of chromatin DNA with reactive derivatives of oligonucleotides.

Chemical modification of the chromatin DNA with alkylating derivatives of oligothymidylate (pT)16 and oligoadenylate (pA)16 bearing 4-(N-2-chloroethyl-N-methylamino)benzylphosphamide group at the 5'-phosphate has been investigated. It was found that the derivatives do react with DNA in chromatin. The reactions occur presumably at the complementary sequences of the DNA since the reaction of the oligothymidylate derivative is inhibited by oligonucleotide (pT)16 taken in excess and is not influenced by hexadecanucleotide of a random structure. Isolated DNA does not react with the oligothymidylate derivative. It is concluded that in chromatin, DNA is partially unwound or possesses some sites which can be opened easily in the presence of complementary oligonucleotides.

Alkylating Agents↗
PubMed2004

Is modification sufficient to protect a bacterial chromosome from a resident restriction endonuclease?

It has been generally accepted that DNA modification protects the chromosome of a bacterium encoding a restriction and modification system. But, when target sequences within the chromosome of one such bacterium (Escherichia coli K-12) are unmodified, the cell does not destroy its own DNA; instead, ClpXP inactivates the nuclease, and restriction is said to be alleviated. Thus, the resident chromosome is recognized as 'self' rather than 'foreign' even in the absence of modification. We now provide evidence that restriction alleviation may be a characteristic of Type I restriction-modification systems, and that it can be achieved by different mechanisms. Our experiments support disassembly of active endonuclease complexes as a potential mechanism. We identify amino acid substitutions in a restriction endonuclease, which impair restriction alleviation in response to treatment with a mutagen, and demonstrate that restriction alleviation serves to protect the chromosome even in the absence of mutagenic treatment. In the absence of efficient restriction alleviation, a Type I restriction enzyme cleaves host DNA and, under these conditions, homologous recombination maintains the integrity of the bacterial chromosome.

Adenosine Triphosphatases↗
PubMed2003

Preferential carcinogen-DNA adduct formation at codons 12 and 14 in the human K-ras gene and their possible mechanisms.

In the ras gene superfamily, codon 12 (-TGGTG-) of the K-ras gene is the most frequently mutated codon in human cancers. Recently, we have found that bulky chemical carcinogens preferentially form DNA adducts at codons 12 and 14 (-CGTAG-) in the K-ras gene in normal human bronchial epithelial (NHBE) cells. Furthermore, DNA adducts formed at codon 12 of the K-ras gene are poorly repaired compared with those at other codons including codon 14. These results suggest that targeted carcinogen-DNA adduct formation is a major reason for the observed high mutation frequency at codon 12 of the K-ras gene in human cancers. This preferential carcinogen-DNA adduct formation at codons 12 and 14 could result from effects of (1) primary sequences of these codons and their surrounding codons in the K-ras gene, (2) the chromatin structure, and/or (3) epigenetic factors such as C5 cytosine methylation or other DNA modifications at these codons and their surrounding codons. To distinguish these possibilities, we have introduced modifications with benzo[a]pyrene diol epoxide, N-hydroxy-2-aminofluorene, and aflatoxin B1 8,9-epoxide in (1) naked intact genomic DNA isolated from NHBE cells, (2) fragmented genomic DNA digested by restriction enzymes, and (3) in vitro synthesized DNA fragments containing the K-ras gene exon 1 sequence with or without methylation of the cytosines at CpG sites and the cytosines pairing with the guanines of codons 12 and 14. The distribution of carcinogen-DNA adducts in the K-ras gene was mapped at the nucleotide sequence level using the UvrABC nuclease incision method with or without the ligation-mediated polymerase chain reaction technique. We have found that carcinogens preferentially form adducts at codons 12 and 14 in the K-ras gene exon 1 in intact as well as in fragmented genomic DNA. In contrast, this preferential DNA adduct formation at codons 12 and 14 was not observed in PCR-amplified DNA fragments containing the K-ras gene exon 1 sequence. Methylation of the cytosine at the CpG site of codon 14, or the cytosine pairing with guanine of codon 14, greatly enhanced carcinogen-DNA adduct formation at codon 14 but did not affect carcinogen-DNA adduct formation at codon 12. Methylation of the cytosine pairing with the guanine of codon 12 also did not enhance carcinogen-DNA adduct formation at codon 12. Furthermore, we found that the cytosine at the CpG site of codon 14 is highly methylated in NHBE cells. These results suggest that cytosine methylation at the CpG site is the major reason for the preferential DNA damage at codon 14 and that epigenetic modification(s) other than cytosine methylation may contribute to the preferential DNA damage at codon 12 of the K-ras gene.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1978

DNA methylation at a CCGG sequence in the large intron of the rabbit beta-globin gene: tissue-specific variations.

We have analysed DNA modification in a HapII site (CCGG) present in the major intron of the discontinuous rabbit beta-globin gene. In most somatic tissues, including erythroid and non-erythroid tissues, about 50% of the DNA is resistant to cleavage at this site by HapII, though 100% cleavage is found with the isoschizomer MspI. Since the former enzyme is unable to cleave CCGG sites if the internal C residue is 5-methyl C (and since methylation is the only form of CpG modification documented in animal DNA), while the latter enzyme cleaves DNA irrespective of methylation at this residue, we infer that 50% of the CCGG sites in the beta-globin gene intron are methylated in these tissues. The same site appears to be 100% methylated (judged by the same criterium) in sperm DNA and about 80% methylated in brain DNA. DNA from the rabbit SIRC cell line is entirely unmethylated at this site.

Animals↗
PubMed2005

siRNA induced transcriptional gene silencing in mammalian cells.

Epigenetics is the study of meiotically and mitotically heritable changes in gene expression which are not coded for in the DNA. Three distinct mechanisms appear to be intricately related and implicated in initiating and/or sustaining epigenetic modifications; DNA methylation, RNA-associated silencing, and histone modifications. It has recently become clear in human cells that RNA plays a far more profound and complex role in regulating the expression of the gene. This regulatory effect is through RNA-associated silencing, can be transcriptional in nature, and is operable through an RNA interference based mechanism (RNAi) that is specifically mediated by small-interfering RNAs (siRNAs). Specifically, the recent observations by both our groups that siRNAs can silence target genes at the level of the chromatin in mammalian cells. We discuss here siRNA mediated transcriptional gene silencing and directed DNA methylation as well as the putative mechanism involved in human cells. Undoubtedly, the ramifications from this paradigm shift of RNA regulating the expression of the gene are immeasurable both therapeutically (i.e., directed control of a genes expression) and biologically in understanding the evolution of the cell.

Animals↗
PubMed1991

Biological aspects of cytosine methylation in eukaryotic cells.

The existence in eukaryotes of a fifth base, 5-methylcytosine, and of tissue-specific methylation patterns have been known for many years, but except for a general association with inactive genes and chromatin the exact function of this DNA modification has remained elusive. The different hypotheses regarding the role of DNA methylation in regulation of gene expression, chromatin structure, development, and diseases, including cancer are summarized, and the experimental evidence for them is discussed. Structural and functional properties of the eukaryotic DNA cytosine methyltransferase are also reviewed.

Animals↗
PubMed1993

Performance of 181 chemicals in a Drosophila assay predominantly monitoring interchromosomal mitotic recombination.

An evaluation is presented of the effects of 181 chemicals in the (white/white+) (w/w+) eye mosaic assay, an in vivo short-term test measuring genetic damage in somatic cells of Drosophila after treatment of larvae. The genetic principle of this system is loss of heterozygosity for the wild-type reporter gene w+, an event predominantly resulting from homologous interchromosomal mitotic recombination between the two X chromosomes of female genotypes. The w/w+ eye mosaic test detects a broad spectrum of DNA modifications, since all distinct classes of genotoxins are monitored. Non-DNA-reactive chemicals are in principle not detected by this system. Occasional positive responses obtained for chemicals such as amitrole, ethionine and hexachloeroethane are probably not related to the mechanism responsible for their tumorigenicity. The principle outcome of this analysis is the necessity for classification of responses into three categories. (i) Positive, '++'. The 92 chemicals (Tables II and III) falling into this category were clearly recombinagenic in the assay, meaning that dose-response relations were obtained (or could have been established as was evident from the strong responses obtained at one or two exposure doses). Among the 92 chemicals were 49 promutagens including volatile chemicals such as vinyl bromide and vinyl chloride. (ii) Marginally positive, '+w'. The definition of a weakly positive response is the absence of a dose-response relationship due to the fact that a weak but reproducible effect, in most cases no more than a doubling of the spontaneous clone frequency, is inherently related to toxicity. The 40 chemicals (Tables IV and V) belonging to this category mainly represented four distinct types. (a) Procarcinogens, such as 2-acetylaminofluorene, dibenz[a,h]anthracene, p-dimethylaminoazobenzene, 2-naphthylamine and safrole, for which metabolic conversion was the apparent problem in the assay. (b) Electrophilic chemicals of high nucleophilic selectivity: acrolein, acrylamide, acrylonitrile, epichlorohydrin, chloroethylisocyanate, 1,2-epoxybutane, N-methyl-n-vinylacetamide, methyl vinylketone, 2-methyl-2-vinyloxirane and methyl vinylsulfone. These chemicals have a relatively low DNA reactivity. Chloroethylisocyanate was active only in the absence of excision repair, suggesting that efficient DNA repair is the cause for the weak genotoxic effectiveness of these genotoxins. (c) Spindle poisons (Table V) were active at rather low but toxic exposure levels. Irregularities in the structure of ommatidia were seen at dose levels producing no more than 2- to 3-fold increases in clone frequencies. (d) The fourth group consisted of chemicals generally regarded as non-genotoxic carcinogens: amitrole, ethionine, ethylurea, tetrachloroethylene and thiourea. Their weak responses were always accompanied by signs of toxicity.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1984

Synthesis, secretion and processing of alpha-factor-interferon fusion proteins in yeast.

A gene fusion consisting of 960 base pairs of 5'-flanking region of the yeast MF alpha 1 gene, 257 base pairs coding for alpha-factor prepro sequence, and a modified human IFN-alpha 1 gene was constructed. MAT alpha cells containing the chimeric gene synthesized and secreted active IFN-alpha 1 into the growth medium. The secreted interferon molecules contained the last 4 amino acids of alpha-factor prepro sequence and the amino acids encoded by the DNA modifications introduced at the beginning of IFN-alpha 1 gene. DNA sequences coding for these amino acids were removed by oligonucleotide-directed in vitro mutagenesis. Yeast cells transformed with expression plasmids containing the altered junction synthesized and secreted human IFN-alpha 1 with the natural NH2-terminus.

Base Sequence↗
PubMed2006

Bacillus subtilis SbcC protein plays an important role in DNA inter-strand cross-link repair.

BACKGROUND: Several distinct pathways for the repair of damaged DNA exist in all cells. DNA modifications are repaired by base excision or nucleotide excision repair, while DNA double strand breaks (DSBs) can be repaired through direct joining of broken ends (non homologous end joining, NHEJ) or through recombination with the non broken sister chromosome (homologous recombination, HR). Rad50 protein plays an important role in repair of DNA damage in eukaryotic cells, and forms a complex with the Mre11 nuclease. The prokaryotic ortholog of Rad50, SbcC, also forms a complex with a nuclease, SbcD, in Escherichia coli, and has been implicated in the removal of hairpin structures that can arise during DNA replication. Ku protein is a component of the NHEJ pathway in pro- and eukaryotic cells. RESULTS: A deletion of the sbcC gene rendered Bacillus subtilis cells sensitive to DNA damage caused by Mitomycin C (MMC) or by gamma irradiation. The deletion of the sbcC gene in a recN mutant background increased the sensitivity of the single recN mutant strain. SbcC was also non-epistatic with AddAB (analog of Escherichia coli RecBCD), but epistatic with RecA. A deletion of the ykoV gene encoding the B. subtilis Ku protein in a sbcC mutant strain did not resulted in an increase in sensitivity towards MMC and gamma irradiation, but exacerbated the phenotype of a recN or a recA mutant strain. In exponentially growing cells, SbcC-GFP was present throughout the cells, or as a central focus in rare cases. Upon induction of DNA damage, SbcC formed 1, rarely 2, foci on the nucleoids. Different to RecN protein, which forms repair centers at any location on the nucleoids, SbcC foci mostly co-localized with the DNA polymerase complex. In contrast to this, AddA-GFP or AddB-GFP did not form detectable foci upon addition of MMC. CONCLUSION: Our experiments show that SbcC plays an important role in the repair of DNA inter-strand cross-links (induced by MMC), most likely through HR, and suggest that NHEJ via Ku serves as a backup DNA repair system. The cell biological experiments show that SbcC functions in close proximity to the replication machinery, suggesting that SbcC may act on stalled or collapsed replication forks. Our results show that different patterns of localization exist for DNA repair proteins, and that the B. subtilis SMC proteins RecN and SbcC play distinct roles in the repair of DNA damage.

Bacillus subtilis↗
PubMedDate not supplied

[Solid phase immunoenzyme analysis of the immunospecificity of DNA from calf spleen alkylated by thiophosphamide].

DNA binding activity of rabbit antiserum against calf spleen DNA's modified by thiophosphamide (DNA-T) was studied by means of solid enzyme immunoassays (ELISA). The studies demonstrated the preferential binding of the immobilized DNA-T compared to immobilized single-stranded DNA (ss-DNA) and only small preference compared to native DNA. Two antisera against DNA-T were purified by affinity chromatography on a ss-DNA-CNBr agarose from antibodies to calf spleen ss-DNA. They interacted only with the immobilized DNA-T, but not with ss-DNA or native DNA. These results demonstrated that DNA modification by thiophosphamide, decreases the immunogenicity of usual nitrogen-containing DNA bases, but detected new immunogenic specificity for adducts. Detection of new immunogenic specificity in DNA's alkylated by thiophosphamide, resulted in the development of a sensitive enzyme immunoassay for the detection of these adducts in nucleic acids, in monitoring their formation, persistence and repair damages in DNA.

Alkylation↗
PubMed1999

Interaction of chemically modified antisense oligonucleotides with sense DNA: a label-free interaction study with reflectometric interference spectroscopy.

Antisense oligonucleotides (ON) are regarded as potential therapeutic agents for controlling gene expression at the mRNA level. The strength of the interaction with the target sequence is one critical factor for the therapeutic efficiency of an ON. Herein, the results of studies on antisense 15mer and 20mer ONs against mdr1b-mRNA are described. The mdr1b is a member of the group that encodes the P-glycoprotein (Pgp), responsible for the phenomenon of multidrug resistance. The effects of backbone modification (DNA, phosphorothioate (PTO)), terminal modifications (hexadecyl, cholesteryl, tocopherol, polyethylenglycol, 2'-O-methyl-modified RNA) and base sequence misalignments (1 to 3 bases) on interaction kinetics and binding strength were investigated. The interaction of an immobilized sense strand with the dissolved antisense ON was monitored with a label-free optical transducer based on thin film interference (RIfS). Association kinetics were detected at a low density of immobilized ON. Thermodynamics were investigated by homogeneous phase titration of sense and antisense ON and subsequent quantification of equilibrium concentrations of unbound ON at a transducer highly loaded with sense ON. Association rate constants varied from 3.1 (+/- 0.2) x 10(4) M-1 s-1 (poly(ethylene glycol)-modified DNA strand) to 4.3 (+/- 0.1) x 10(4) M-1 s-1 (hexadecyl-modified strand). Binding constants varied from 1.9 (+/- 0.1) x 10(8) M-1 (cholesteryl modification) to 5 (+/- 0.4) x 10(7) M-1 (tocopherol modification). Phosphorothioate ON showed a reduction in binding strength of more than 1 order of magnitude. The data presented give valuable information for the efficiency of modified antisense oligonucleotides.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2003

DNA methylation-related chromatin remodeling in activity-dependent BDNF gene regulation.

In conjunction with histone modifications, DNA methylation plays critical roles in gene silencing through chromatin remodeling. Changes in DNA methylation perturb neuronal function, and mutations in a methyl-CpG-binding protein, MeCP2, are associated with Rett syndrome. We report that increased synthesis of brain-derived neurotrophic factor (BDNF) in neurons after depolarization correlates with a decrease in CpG methylation within the regulatory region of the Bdnf gene. Moreover, increased Bdnf transcription involves dissociation of the MeCP2-histone deacetylase-mSin3A repression complex from its promoter. Our findings suggest that DNA methylation-related chromatin remodeling is important for activity-dependent gene regulation that may be critical for neural plasticity.

Animals↗
PubMed1991

Sulfur-containing proreactive intermediates: hydrolysis and mutagenicity of halovinyl 2-nitrophenyl disulfides.

Chemical cleavage of the sulfur-sulfur bond in halovinyl and fluoroalkyl 2-nitrophenyl disulfides is expected to yield halovinyl and fluoroalkyl thiols identical to those formed by cysteine conjugate beta-lyase catalyzed cleavage of the corresponding cysteine S-conjugates. To study the potential use of disulfides as precursors for these thiols, whose transformation to acylating agents is most likely responsible for cysteine S-conjugate mutagenicity, we determined the mutagenicity of several halovinyl and fluoroalkyl 2-nitrophenyl disulfides and identified products formed by hydrolysis of these disulfides, 1,2,3,4,4-Pentachlorobutadienyl 2-nitrophenyl disulfide, 1,2,2-trichlorovinyl 2-nitrophenyl disulfide, 1-fluro-2,2-dichlorovinyl 2-nitrophenyl disulfide and 1,2-dichloro-3,3,3-trifluropropenyl 2-nitrophenyl disulfide were mutagenic in nitroreductase deficient strains of Salmonella typhimurium TA100; as haloalkyl cysteine S-conjugates, 1,1-difluoro-2,2-dichloroethyl 2-nitrophenyl disulfide and 1-chloro-1,2,2-trifluroethyl 2-nitrophenyl disulfide were not mutagenic. Hydrolysis of 1,2,3,4,4-pentachlorobutadienyl 2-nitrophenyl disulfide and 1,2,2-trifluorethyl 2-nitrophenyl disulfide in presence of diethylamine resulted in tetrachlorothiobutenoic acid diethylamide and chlorofluorothionoacetic acid diethylamide. The differences in mutagenicity between halovinyl and fluoroalkyl disulfides are most likely responsible to their different abilities to react with DNA-constituents. Products formed from the mutagenic 1,2,3,4,4-pentachlorobutadienyl 2-nitrophenyl disulfide modified 2'-deoxyguanosine-3'-monophosphate and DNA as detected by 32Phosphorus-postlabeling, whereas products formed from the nonmutagenic 1-chloro-1,2,2-trifluoroethyl 2-nitrophenyl disulfide did not result in detectable 2'-deoxyguanosine-3'-monophosphate and DNA modification.

DNA↗
PubMed1989

A rapid protocol for the purification of mitochondrial DNA suitable for studying restriction fragment length polymorphisms.

When analyzing mitochondrial DNA (mtDNA) from various normal and malignant human tissues, it became necessary to enhance mtDNA isolation for improved yields and quality. The method described here consists of rapid and simple-to-perform steps, avoiding complicated instrumentation. It was designed for preparation of undegraded mtDNA and is highly useful when limited amounts of tissues, cells and unique biopsies of tumors (fresh or frozen) are available. The resulting mtDNA is sufficiently pure for restriction analysis, subcloning, labeling and various types of hybridization. Using Sau3A and MspI, restriction analysis revealed new restriction-fragment length polymorphisms for Caucasians, independent of the DNA source, and hence excluding tissue-specific DNA modifications.

Centrifugation↗
PubMed2004

Quantification of 1,N6-etheno-2'-deoxyadenosine in human urine by column-switching LC/APCI-MS/MS.

1,N6-etheno-2'-deoxyadenosine (epsilondA) is one of several promutagenic DNA modifications arising from cellular oxidative metabolism. It is believed that these background DNA lesions may contribute to various diseases, such as cancer. Therefore, human biomonitoring of epsilondA in urine could be a potential marker for oxidative stress-related DNA damage. Existing methods for quantifying urinary epsilondA use 32P postlabeling. We have developed a nonradioactive, fast, and easier method based on column-switching liquid chromatography with atmospheric pressure chemical ionization tandem mass spectrometry (LC/APCI-MS/MS) in the positive mode. Differences in column temperatures were used to influence analyte retention and sample focusing. With multiple reaction monitoring (MRM) mode the afforded limit of detection was about 0.7 pM when starting with 3 ml of urine. The urinary excretion rates of epsilondA from 28 nonsmoking and 5 smoking men were 10.0-99.6 pmol/24 h, and did not correlate with body weight, age, or plasma vitamin C concentration. The 5 smokers excreted 30.5 +/-8.5 and the 28 nonsmokers excreted 38.6 +/- 2.4 pmol epsilondA per 24 h, p=.37 (mean +/- SEM). The demonstrated level of performance suggests the future applicability of this method to studies of cancer and other diseases related to oxidative stress in humans.

Chromatography, Liquid↗