Pharmacokinetic Properties of Orally Administered 4'-Cyano-2'-deoxyguanosine, a Novel Nucleoside Analog Inhibitor of the Hepatitis B Virus, in Viral Liver Injury Model Rats.

A nucleoside analog, 4'-cyano-2'-deoxyguanosine (CdG), which was developed as an inhibitor of the chronic hepatitis B virus (HBV), exhibited a superior antiviral activity against both wild-type and drugs-resistant HBV to marketed nucleoside analogs. In addition to previous pharmacokinetic studies of CdG in healthy rats, this study reports on an evaluation of the pharmacokinetic characteristics of CdG in a rat model of viral liver injury (VLI) induced by treatment with concanavalin A. Following an intravenous administration of CdG at a dose of 1 mg/kg, the plasma concentration profile of CdG in VLI model rats was found to be similar to that of healthy rats with no significant difference in kinetic parameters. However, when CdG was orally administered at a dose of 1 mg/kg, the maximum blood concentration was much lower in VLI model rats than in healthy rats. Interestingly, the amount of residual food in the stomachs in VLI model rats was significantly larger than that in healthy rats, indicating that the adsorption of CdG in the gastrointestinal tract was inhibited in the presence of food as well as other marketed nucleoside analogs. As observed in healthy rats, CdG was largely distributed to the liver compared to the kidney in the VLI model. These results suggest that liver pathology has only a minor effect on the pharmacokinetic properties of CdG, but the influence of food on CdG absorption needs to be considered.

Recent technical and biological development in the analysis of biomarker N-deoxyguanosine-C8-4-aminobiphenyl.

4-Aminobiphenyl (4-ABP) which is primarily formed during tobacco combustion and overheated meat is a major carcinogen responsible for various cancers. Its adducted form, N-deoxyguanosine-C8-4-aminobiphenyl (dG-C8-4-ABP), has long been employed as a biomarker for assessment of the risk for cancer. In this review, the metabolism and carcinogenisity of 4-ABP will be discussed, followed by a discussion of the current common approaches of analyzing dG-C8-4-ABP. The major part of this review will be on the history and recent development of key methods for detection and quantitation of dG-C8-4-ABP in complex biological samples and their biological applications, from the traditional 2P-postlabelling and immunoassay methods to modern liquid chromatography-mass spectrometry (LC-MS) with the latter as the focus. Many vital biological discoveries based on dG-C8-4-ABP have been published by using the nanoLC-MS with column switching platform in our laboratory, which has also been adopted and further improved by many other researchers. We hope this review can provide a perspective of the challenges that had to be addressed in reaching our present goals and possibly bring new ideas for those who are still working on the frontline of DNA adducts area.

Pharmacokinetics studies of 4'-cyano-2'-deoxyguanosine, a potent inhibitor of the hepatitis B virus, in rats.

OBJECTIVES: 4'-cyano-2'-deoxyguanosine (CdG), a novel nucleoside analogue, has a high degree of antiviral activity against the chronic hepatitis B virus (HBV). The objective of this study was to develop an analytical method for quantitatively determining CdG levels in biological samples by liquid chromatography-mass spectrometry (LC/MS) and to investigate the pharmacokinetic properties of CdG in rats after intravenous and oral administration. METHODS: An analytical method using a UPLC system interfaced with a TOF-MS system was developed and validated. The pharmacokinetic properties after the intravenous and oral administration of CdG to rats were evaluated. In vivo pharmacokinetic interactions between CdG and entecavir were also investigated. KEY FINDINGS: A rapid, simple and selective method for the quantification of CdG in biological samples was established using LC/MS with solid-phase extraction. In vivo pharmacokinetic studies of CdG in rats demonstrated that CdG is highly bioavailable, is rapidly absorbed from the intestinal tract, is then distributed to the liver rather than kidney and is ultimately excreted via the urine in an unchanged form. The co-administration of CdG and entecavir led to pharmacokinetic interactions with each other. CONCLUSIONS: The data generated in this study provide support for the clinical development of CdG for use in the treatment of HBV.

The stability of the oxidative stress marker, urinary 8-hydroxy-2'- deoxyguanosine (8-OHdG), when stored at room temperature.

We examined the stabilities of urinary 8-hydroxy-2'-deoxyguanosine (8-OHdG) stored at room temperature (25 degrees C) for 24 h or at -80 degrees C for 800 days. Subjects were 19 males and 17 females aged 23-58 yr for the 24-h study, and 9 males and 4 females aged 24-54 yr for the 800-day study. We obtained information on the subjects by questionnaires and interviews. The level of urinary 8-OHdG was measured by HPLC using two-step separations. There were no significant changes of amount of urinary 8-OHdG under either storage conditions. We conclude that urine samples can be stored at 25 degrees C and below for 24 h, when the research purpose includes the determination of urinary 8-OHdG. Urinary 8-OHdG was also stable for over two years when stored at -80 degrees C.

Syntheses of (2')3'-15N-amino-(2')3'-deoxyguanosine and determination of their pKa values by 15N NMR spectroscopy.

2'-Amino-2'-deoxyguanosine and 3'-amino-3'-deoxyguanosine are valuable probes for investigating the metal ion interactions at the active site of the group I ribozyme. However, these experiments require a thorough understanding of the protonation state of the amino group at a specific pH. Here, we describe the first syntheses of 2'-15N-amino-2'-deoxyadenosine, 2'-15N-amino-2'-deoxyguanosine, and 3'-15N-amino-3'-deoxyguanosine. The 15N-enriched nucleus allows convenient and accurate determination of the amine pKa by 15N NMR.

Single-turnover kinetic analysis of the mutagenic potential of 8-oxo-7,8-dihydro-2'-deoxyguanosine during gap-filling synthesis catalyzed by human DNA polymerases lambda and beta.

In the presence of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) damage, many DNA polymerases exhibit a dual coding potential which facilitates efficient incorporation of matched dCTP or mismatched dATP. This also holds true for the insertion of 8-oxodGTP opposite template bases dC and dA. Employing single-turnover kinetic methods, we examined human DNA polymerase beta and its novel X-family homolog, human DNA polymerase lambda, to determine which nucleotide and template base was preferred when encountering 8-oxodG and 8-oxodGTP, respectively. While DNA polymerase beta preferentially incorporated dCTP over dATP, DNA polymerase lambda did not modulate a preference for either dCTP or dATP when opposite 8-oxodG in single-nucleotide gapped DNA, as incorporation proceeded with essentially equal efficiency and probability. Moreover, DNA polymerase lambda is more efficient than DNA polymerase beta to fill this oxidized single-nucleotide gap. Insertion of 8-oxodGTP by both DNA polymerases lambda and beta occurred predominantly against template dA, thereby reiterating how the asymmetrical design of the polymerase active site differentially accommodated the anti and syn conformations of 8-oxodG and 8-oxodGTP. Although the electronegative oxygen at the C8 position of 8-oxodG may induce DNA structural perturbations, human DNA ligase I was found to effectively ligate the incorporated 8-oxodGMP to a downstream strand, which sealed the nicked DNA. Consequently, the erroneous nucleotide incorporations catalyzed by DNA polymerases lambda and beta as well as the subsequent ligation catalyzed by a DNA ligase during base excision repair are a threat to genomic integrity.

Exogenous 8-oxo-dG is not utilized for nucleotide synthesis but enhances the accumulation of 8-oxo-Gua in DNA through error-prone DNA synthesis.

7,8-Dihydro-8-oxoguanine (8-oxo-Gua) and its nucleoside in cytosol are derived from the repair of oxidative DNA and the cleanup of oxidatively damaged DNA precursors, respectively. While the harmful effects of 8-oxo-Gua present in DNA have been studied extensively, few have reported its effects on cytosolic function. Our previous study showed that the addition of 8-oxo-dG to culture media caused an accumulation of 8-oxo-Gua in nuclear DNA in several leukemic cells including KG-1, which lack 8-oxoguanine glycosylase 1 (OGG1) activity due to mutational loss. However, the mechanism underlying 8-oxo-Gua level increases in DNA has not been addressed. In this study, we elucidated the metabolic fate of 8-oxo-Gua-containing nucleotide and the effect of exogenous 8-oxo-dG on DNA synthesis in KG-1 cells. We found that 8-oxo-dGMP was rapidly dephosphorylated to 8-oxo-dG rather than phosphorylated to 8-oxo-dGDP, thus indicating that 8-oxo-Gua-containing molecule is not used as a substrate for DNA synthesis in KG-1 cells. In fact, radiolabeled 8-oxo-dG was incubated but radioactivity was not detected in nuclear DNA of KG-1 cells, showing that 8-oxo-dG is not directly incorporated into DNA. Interestingly, the activity of DNA polymerase beta, which synthesize DNA with low fidelity increased in KG-1 cells treated with 8-oxo-dG, whereas the expression of DNA polymerase alpha decreased. In addition, the accumulation of 8-oxo-Gua in KG-1 DNA was completely inhibited by a specific inhibitor of DNA polymerase beta. Thus, our findings address that the insertion of 8-oxo-dG into KG-1 DNA is not due to the direct incorporation of exogenous 8-oxo-dG, but rather to the inaccurate incorporation of endogenous 8-oxo-dGTP by DNA polymerase beta. It further suggests that 8-oxo-dG in the cytosol may function as an active molecule itself and perturb the well-defined DNA synthesis.

O6-3-[125I]iodobenzyl-2'-deoxyguanosine ([125I]IBdG): synthesis and evaluation of its usefulness as an agent for quantification of alkylguanine-DNA alkyltransferase (AGT).

The development of O(6)-(3-[(125)I]iodobenzyl)-2'-deoxyguanosine ([(125)I]IBdG), the glycosylated analogue of the O(6)-3-iodobenzylguanine (IBG), as an agent for the in vivo mapping of the DNA repair protein alkylguanine-DNA alkyltransferase (AGT) is described. Synthesis of its tin precursor, O(6)-3-trimethylstannylbenzyl-2'-deoxyguanosine (TBdG) was achieved in four steps from deoxyguanosine. Radioiodination of TBdG in a single step gave [(125)I]IBdG in 70-85% isolated radiochemical yield. [(125)I]IBdG bound specifically to pure AGT with an IC(50) of 7.1 microM. From paired-label assays, [(125)I]IBdG showed a 2- to 3-fold higher cellular uptake than [(131)I]IBG in DAOY medulloblastoma, TE-671 rhabdomyosarcoma, SK-Mel-28 melanoma, and HT-29 colon carcinoma human cell lines. Uptake of both labeled compounds in these cell lines decreased with increasing concentrations of unlabeled O(6)-benzylguanine (BG) when BG was present in the medium during incubation with the labeled compounds. Compared to BG, unlabeled IBdG diminished the uptake of [(125)I]IBdG and [(131)I]IBG in DAOY cells more efficiently (IC(50)<1 microM vs >10 microM for BG). There was no significant change in cell-bound activity of [(125)I]IBdG and [(131)I]IBG when BG was removed from the incubation medium before incubating cells with the tracers, suggesting that only a very small portion of radioactivity taken up by the cells is AGT bound. This was corroborated by gel-electrophoresis performed on extracts from cells treated with varying amounts of BG and then incubated with [(125)I]IBdG in the presence of BG. No radiolabeled AGT band was discernable by phosphor-imaging, signifying low cellular AGT binding of the radiotracer. In contrast, when cell extracts were prepared from BG pre-treated cells and aliquots were incubated with [(125)I]IBdG subsequently, the intensity of radiolabeled AGT band decreased linearly as a function of BG concentration. This suggests that the low level of [(125)I]IBdG that binds to AGT does so in a concentration dependent manner. These data suggest that IBdG is transported across the cell membrane to a higher degree than IBG. However, to be a practical tracer for quantifying cellular AGT, considerable localization of such derivatives need to occur within the cell nucleus where AGT is present predominantly.

The novel nucleoside transport system exhibited by NB4 cells, csg, transports deoxyguanosine analogues, including ara-G.

We studied acceptance of various deoxyguanosine analogues by the unique guanosine preferring nucleoside transport system exhibited by NB4 cells, csg. Indirect assessment of acceptance using transport inhibition assays revealed that both 1-beta-D-arabinofuranosylguanine (ara-G) and 4'-thio-beta-D-xylofuranosylguanine (thio-xyl-G) compete with guanosine for the csg system, inhibiting guanosine flux by approximately 50%. Direct examination of [3H]-ara-G transport revealed total transport was equally allocated to csg, and es systems and a total transport rate similar to that determined for guanosine [Flanagan and Meckling-Gill, J Biol Chem 1997;272:18026-32]. Cytotoxicity assays revealed that although both ara-G and thio-xyl-G were capable of competing with guanosine for the csg system, neither analogue elicited cytotoxic effects at physiologically relevant concentrations. The analog, 4'-thio-beta-D-arabinofuranosylguanine does not gain entry to NB4 cells via the csg transport system. Competition assays revealed that this analogue potentiated the inward flux of guanosine and was capable of killing NB4 cells with potency similar to the conventional leukemia drug, ara-C.

Kinetics of hydrolysis of 8-(arylamino)-2'-deoxyguanosines.

The 8-(arylamino)-2'-deoxyguanosines, or C-8 adducts, are the major adducts formed by reaction of N-arylnitrenium ions derived from carcinogenic and mutagenic amines with 2'-deoxyguanosine (d-G) and guanosine residues of DNA. The hydrolysis kinetics of three C-8 adducts 1a-c were determined by UV and HPLC methods at 20 degrees C under acidic, neutral, and mildly alkaline conditions. At pH < 2 the dominant hydrolysis process is spontaneous cleavage of the C-N bond of the doubly protonated substrate, 1H(2)(+2) (Scheme 2). The C-8 adducts are 2- to 5-fold more reactive than d-G under these conditions. At 3 < pH < 6 the hydrolysis kinetics are dominated by cleavage of the C-N bond of the monoprotonated nucleoside 1H(+). Under these conditions the hydrolysis kinetics are accelerated by 40- to 1300-fold over that of d-G. The rate increase appears to be caused by a combination of steric acceleration of C-N bond cleavage and a decrease in the ionization constant of 1H(+), K(a1), due to the electron-donating properties of the arylamino C-8 substituent. Under neutral pH conditions a slow (k(obs) approximately 10(-8) s(-1) to 5 x 10(-7) s(-1)) spontaneous cleavage of the C-N bond of the neutral nucleoside, 1, occurs that has not been previously reported for simple purine nucleosides. Finally, under mildly alkaline conditions a process consistent with spontaneous decomposition of the anion 1(-) or OH(-)-induced decomposition of 1 is observed. The latter process has been observed for other purine nucleosides, including the closely related 1d, and involves nucleophilic attack of OH(-) on C-8 to cleave the imidazole ring of the purine.

Role of TP53 in repair of N-(deoxyguanosin-8-yl)-4-aminobiphenyl adducts in human transitional cell carcinoma of the urinary bladder.

The global genomic repair of DNA adducts was examined in human papillary transitional cell carcinoma (TCC) cell lines after exposure to N:-hydroxy-4-acetylaminobiphenyl (N-OH-AABP), the proximate carcinogenic metabolite of the human bladder carcinogen 4-aminobiphenyl (ABP). (32)P-post-labeling analysis of TCC cultures exposed to N-OH-AABP revealed a major adduct, identified as the 3',5'-bisphosphate derivative of N-(deoxyguanosin-8-yl)-4-aminobiphenyl (dG-C8-ABP). The amount of adduct formation in TCC10 was dependent upon the dose and the duration of exposure and ranged between 1 and 5 adducts/10(7) nucleotides. To test if p53 regulates repair of the dG-C8-ABP adduct in genomic DNA, an isogeneic set of cell lines was obtained by infection of the TCC10 cultures with a retroviral construct expressing a trans-dominant mutant of p53, namely a Val-->Ala mutation at codon 143. The TDM143-TCC10 line expressing the mutant form of p53 was selected. The rate of repair of dG-C8-ABP was compared between TCC10 and TDM143-TCC10 cultures after treatment with 15 microM N-OH-AABP. The rate of disappearance of the adduct was monitored over a period of time after chemical treatment. (32)P-post-labeling analysis of dG-C8-ABP in parental TCC10 showed its rapid removal, the majority of adducts disappearing within 48 h. In contrast to TCC10, TDM143-TCC10 was relatively slower in removal of dG-C8-ABP. After 24 h DNA repair TDM143-TCC10 showed an approximately 3-fold greater amount of dG-C8-ABP compared with TCC10. These results imply that p53 plays a role in the repair of ABP adducts and that in p53 null cells the unrepaired DNA damage could cause accumulation of mutations, which might contribute to increased genomic instability and neoplastic progression.

The biochemistry and pharmacology of PD 116124 (8-amino-2'-nordeoxyguanosine), an inhibitor of purine nucleoside phosphorylase (PNP).

PD 116124 (8-amino-2'-nordeoxyguanosine; 2,8-diamino-1,9-dihydro-9- ([2-hydroxy-1-(hydroxymethyl)ethoxy]methyl)-6H-purin-6-one) is a competitive, reversible inhibitor of human purine nucleoside phosphorylase with an apparent inhibition constant of 0.41 microM. In a cell line system using human MOLT-4 and CEM T lymphoblasts and human MGL-8 and NC-37 B lymphoblasts, PD 116124 failed to inhibit [3H]thymidine uptake at concentrations up to 500 microM. However, in the presence of 10 microM 2'-deoxyguanosine (dGuo), a noninhibitory dGuo concentration by itself, PD 116124 produced potent inhibition of growth of both T cell lines but not of either B cell line. Significant elevation of intracellular 2'-deoxyguanosine triphosphate was observed in both inhibited T cell lines but not in either B cell line. Greater and more sustained accumulation of 2'-deoxyguanosine triphosphate was observed in T lymphoblasts cultured with PD 116124 plus dGuo than with dGuo only. PD 116124 was only weakly inhibitory in human mixed lymphocyte cultures (IC50 approximately equal to 1420 microM), but in the presence of 10 microM dGuo, the IC50 for PD 116124 was reduced to 108.7 microM. Administration of PD 116124 p.o. to normal male Wistar rats caused dose-dependent elevation of plasma inosine up through 500 mg/kg. Maximal inosine elevation occurred at 30 min after dosing, and elevation was significant even 24 hr after dosing. Guanosine was also elevated, although not in a dose-dependent manner. Administration of PD 116124 i.v. produced marked and statistically significant elevation of both inosine and guanosine.(ABSTRACT TRUNCATED AT 250 WORDS)

Hydroxylation and deglycosylation of 2'-deoxyguanosine in the presence of magnesium and nickel.

Nickel (Ni), a carcinogenic and genotoxic metal, has been shown to enhance deglycosylation and hydroxylation of 2'-deoxyguanosine (dG) that has been caused by ascorbic acid and H2O2. There is evidence that Mg is a competitive antagonist of the toxicological effects of Ni. A factorial design was used to examine the interactive influence of Mg and Ni on the deglycosylation and hydroxylation of dG under a range of pH conditions in which ascorbate (Ascb) and H2O2 were added. Formation of guanine (Gu) (deglycosylation) and 8-hydroxy-2'-deoxyguanosine (8-OH-dG) (hydroxylation) appeared in large amounts in samples in which both H2O2 and Ascb were present. The largest amounts of Gu appeared where both Ni or magnesium (Mg) were present. When Mg alone was present, the amounts of Gu was intermediate between these two. Slightly less 8-OH-dG was formed where only Mg was present. The reaction mixtures were more sensitive to the pH than to the respective presence or absence of metals. At slightly acid or neutral pH (6.2-7.0) large amounts of both Gu and 8-OH-dG were formed. Gu formation decreased dramatically between pH 7.0 and 7.2. There was no 8-OH-dG formed at pH 7.8 and only small amounts at pH 7.6. The formation of 8-OH-dG was generally less where Mg was present. When Ni was absent, 8-OH-dG formation was greater in the pH 6.8 mixtures. The formation of Gu and 8-OH-dG from 2'-deoxyguanosine are directly a function of pH. Slight changes in pH greatly effected the formation of these biomarkers of oxidatively damaged DNA. Additional research is needed to determine if this is a cause or effect, i.e. does pH enhance toxicity conditions, thus permitting formation of 8-OH-dG, or does pH permit the reaction to proceed.