Synthesis and stability of 2'-deoxyguanosine 3'-monophosphate adducts of dimethyl sulfate, ethylene oxide and styrene oxide

Deoxyguanosine 3'-monophosphate (dGMP) was alkylated at the 7-position by dimethyl sulfate, ethylene oxide and styrene oxide in aqueous media and glacial acetic acid, respectively, to yield reasonable quantities of the products, which were purified by HPLC. dGMP adducts are needed as standards for the 32P-postlabelling assay. The stability of the adducts was studied at 37 degrees and neutral pH. The half-lives of disappearance of 7-methyl-dGMP and the beta-isomers of the styrene oxide adducts were about 250 min; 7-hydroxy-ethyl-dGMP and the alpha-isomers of the styrene oxide adducts had respective half-lives of 340 and 440 min. In all cases the main degradation product was the corresponding guanine adduct. The results demonstrate considerable lability of the 7-alkylation products of dGMP which has to be taken into consideration in devising the 32P-postlabelling assay.     

Preparation and properties of N6-monobutyryl adenosine 5'-monophosphate. A major hepatic metabolite of N6,O2'-dibutyryl cyclic adenosine 3':5'-monophosphate.

The synthesis and properties of N6-monobutyryl adenosine 5'-monophosphate are described. The properties of synthesized monobutyryl nucleotide have been compared to those of a metabolite recognized in previous studies (Castagna, M. C., Palmer, W.K., and Walsh, D.A. (1977) Arch. Biochem. Biophys. 181, 46-60) as the major radioactive product produced in the liver upon perfusion with N6,O2'-dibutyryl cyclic [3H]adenosine 3':5'-monophosphate. By the criteria of cochromatography on DEAE-cellulose and in three thin layer chromatographic systems and from equivalent rates of alkaline hydrolysis, N6-monobutyryl adenosine 5'-monophosphate has been identified as a major hepatic metabolite of N6,O2'-dibutyryl cyclic adenosine 3':5'-monophosphate.     

Mutagenicity studies with N6, O2'-dibutyryl cyclic adenosine 3',5'-monophosphate (DBcAMP)

N6,O2'-Dibutyryl cyclic adenosine 3,5-monophosphate (DBcAMP) was studied for mutagenicity using the rec assay, the Ames method, and in vitro cytogenetics. DBcAMP had no mutagenic effect on B. subtilis in the rec assay, or on S. typhimurium (TA1535, TA1537, TA1538, TA98 and TA100) or E. coli (WP2 uvrA). In the cytogenetic study, a significant increase in chromosomal aberrations was observed at a concentration of 50,000 micrograms/ml, but it was considered that this effect could be attributed to the secondary effect of the high osmotic pressure in the culture medium. These results suggest that DBcAMP has no mutagenic potential.     

Analysis of tamoxifen-DNA adducts in endometrial explants by MS and 32P-postlabeling

The nonsteroidal antiestrogen tamoxifen increases the risk of endometrial cancer; however, the mechanism for the induction of these tumors is not known. Recently, Sharma et al. [Biochem. Biophys. Res. Commun. 307 (2003) 157], using high performance liquid chromatography (HPLC) with online postcolumn photochemical activation and fluorescence detection, reported the presence of (E)-alpha-(deoxyguanosin- N2-yl)tamoxifen in DNA from human endometrial explants incubated with tamoxifen. Inasmuch as the methodology used by these investigators does not allow unambiguous characterization of tamoxifen-DNA adducts, we have used two additional techniques (HPLC coupled with electrospray ionization tandem mass spectrometry and 32P-postlabeling analyses) to assay for the presence of tamoxifen-DNA adducts in the human endometrial explant DNA. Tamoxifen-DNA adducts were not detected by either method.     

Quantitation of the 32P-postlabeling reaction using cyclic N1,N2 and C8 modified deoxyguanosine 3'-monophosphates as substrates.

The 32P-postlabeling technique was used to investigate the efficiency of phosphorylation reaction by T4 polynucleotide kinase using seven synthetic adducted deoxyguanosine 3'-monophosphates. The adducts included cyclic N1,N2 derivatives and the C8 adduct of 4-aminobiphenyl. The adducted substrates were detected at a subfemtomole sensitivity except for one of the diastereomeric propanoguanine derivatives. In general, the recommended conditions were found to be proper for an efficient phosphorylation of the adducts studied. Sensitivity of the adducts to the 3'-dephosphorylation reaction of nuclease P1 was also tested. All the complex cyclic adducts were resistant towards P1. However, the ethenoguanine and 4-aminobiphenyl adducts were relatively sensitive towards P1. No differences were noted between diastereomers.     

Enzymatic formation of inosine 3',5'-monophosphate and of 2'-deoxyguanosine 3',5'-monophosphate. Inosinate and deoxyguanylate cyclase activity.

Enzymes in particulate fractions from sea urchin sperm and in soluble fractions from rat lung were shown to catalyze the formation of inosine 3',5'-monophosphate (cyclic IMP) and of 2'-deoxyguanosine 3',5'-monophosphate (cyclic dGMP) from ITP and dGTP, respectively. With sea urchin sperm particulate fractions, Mn2+ was an essential metal cofactor for inosinate, deoxyguanylate, guanylate and adenylate cyclase activities. Heat-inactivation studies differentiated inosinate and deoxyguanylate cyclase activities from adenylate cyclase, but indicated an association of these activities with guanylate cyclase. Preincubation of sea urchin sperm particulate fractions with trypsin altered in a very similar manner guanylate, inosinate, and deoxyguanylate cyclase activities, and various metals and metal-nucleotide combinations protected the three cyclase activities to comparable degrees against trypsin. The relative guanylate, deoxyguanylate and inosinate cyclase activities at 0.1 mM nucleoside triphosphate were 1.0, 0.5 and 0.08, respectively. With these three cyclase activities, plots of reciprocal velocities against reciprocal Mn2+-nucleoside triphosphate concentrations were concave upward, suggesting positive homotropic effects. With rat lung soluble preparations, relative guanylate, deoxyguanylate, inosinate and adenylate cyclase activities at 0.09 mM nucleoside triphosphate were 1.0, 1.7, 0.1 and 0, respectively. MnGTP was a competitive inhibitor of deoxyguanylate cyclase activity (Ki equals 12.2 muM) and MndGTP was a competitive inhibitor of guanylate cyclase activity (Ki equals 16.2 muM). Inhibition studies using ITP were not conducted. When soluble fractions from rat lung were applied to Bio-Gel A 1.5 m columns, elution profiles of guanylate, deoxyguanylate and inosinate cyclase activities were similar. These results suggest that deoxyguanylate, guanylate and inosinate cyclase activities reside within the same protein molecule.     

Allyl protection of O6 position of 2'-deoxyguanosine

Allyl serves as the protecting group for O6 position of guanosine. The deblocking is easily performed by brief treatment with a palladium(O) catalyst and a nucleophile.     

Inhibition of gluconeogenesis and lactate formation from pyruvate by N6, O2'-dibutyryl adenosine 3':5'-monophosphate.

N6,O2-Dibutyryl adenosine 3':5'-monophosphate (Bt2cAMP) inhibits gluconeogenesis and lactate formation but increases ketogenesis by isolated liver cells incubated with high concentrations of pyruvate. The inhibitory effects can not be explained on the basis of an inhibition of the pyruvate dehydrogenase complex nor by a change in the NAD+ oxidation-reduction potential of the mitochondrial compartment. Both oleate and 3-hydroxybutyrate substantially increase the rates of gluconeogenesis and lactate formation from pyruvate but do not overcome the inhibition caused by Bt2cAMP. A decreased effectiveness of pyruvate kinase is proposed to account for the inhibition of both gluconeogenesis and lactate formation by Bt2cAMP. This enzyme catalyzes a step required in the transfer of reducing equivalents from the mitochondrial compartment to the cytoplasm and participates in the formation of glucose and lactate from pyruvate by the overall reaction: 2 pyruvate- + 2 NADHmito + 4 ATP4- + 4 H2O leads to 1/2 glucose + lactate- + 2 NAD+ mito + 4 ADP3- + 4 HPO4(2)- + H+. Inhibition of pyruvate kinase promotes gluconeogenesis with most substrates but inhibits gluconeogenesis from pyruvate for want of cytoplasmic reducing equivalents.     

Monitoring of dimethyl sulphate-induced N3-methyladenine, N7-methylguanine and O(6)-methylguanine DNA adducts using reversed-phase high performance liquid chromatography and mass spectrometry

This work describes the determination of N3-methyladenine, N7-methylguanine and O(6)-methylguanine adducts in dimethyl sulphate-treated salmon-testes DNA employing reversed-phase high performance liquid chromatography (RP-HPLC) with UV-vis detection, followed by mass-spectrometric verification using electrospray ionisation in positive mode ESI(+). Within validation parameters, accuracy, precision, calibration parameters, limit of detection (LOD) and quantitation (LOQ) as well as stability of standard stock solutions were tested and presented for UV/vis detection. The limit of detection (LOD) was found to be 0.1 ng/mL for N3-methyladenine and 0.2 ng/mL for both N7-methylguanine and O(6)-methylguanine (S/N=3). The limit of quantitation (LOQ) was found to be 0.5 ng/mL for all measured compounds, (S/N=10). Quantitative results were obtained for each substance based on eight-point calibration. Intra- and inter-day precisions were within 1.73-6.96 and 2.26-7.58%, respectively, and correlation coefficients of calibration curves (R(2)) ranged from 0.9992 to 0.9997. Relative proportion of N7-methylguanine was accounted for 61.53+/-2.97% (R.S.D.=4.8), N3-methyladenine for 38.19+/-2.99% (R.S.D.=9.6) and O(6)-methylguanine for 0.29+/-0.02% (R.S.D.=5.1), respectively. The application of the above-mentioned techniques provides a valuable contribution for simultaneous determination of methylated DNA adducts, and may represent a suitable approach for similar monitoring/screening studies.     

Acceleration of amphibian embryonic melanophore development by melanophore-stimulating hormone, N6,O2-dibutyryl adenosine 3',5'-monophosphate and theophylline.

Stage 14 (Gallien and Durocher, 1957)Pleurodeles waltlii embryos were treated with α- or β-melanophore-stimulating hormone (MSH), ACTH, dbc-AMP, c-AMP plus theophylline, theophylline, 5′-AMP, or 2′,3′-AMP. The development of melanophores was accelerated (appearing two stages earlier than in control embryos) by α- or β-MSH, ACTH, dbc-AMP, c-AMP plus theophylline, or theophylline alone. By the time embryos developed to stage 28, the control and treated embryos were indistinguishable in the number, distribution, and general morphology of their melanophores, suggesting these agents do not induce melanophore formation or mitosis. Cyclic AMP alone, 5′-AMP or 2′,3′-AMP were ineffective. Accelerated cytodifferentiation apparently requires preinduction by the invaginating chordo-mesoderm during stages 13 and 14 as presumptive neural plate explants cultured before chordo-mesoderm induction (stage 8) failed to produce melanophores with or without α-MSH or dbc-AMP. Explants from stage 14 embryos treated with α-MSH or dbc-AMP developed the same number of melanophores at the approximate time that treated whole embryos developed melanophores. Whole embryo experiments involving the faster developing embryos of Xenopus laevis were similar to those described for P. waltlii.     

A solid-phase extraction/high-performance liquid chromatography-based (32)P-postlabeling method for detection of cyclic 1,N(2)-propanodeoxyguanosine adducts derived from enals

The cyclic 1,N(2)-propanodeoxyguanosine (PdG) adducts are Michael addition products from reactions of deoxyguanosine (dG) with enals, including acrolein (Acr), crotonaldehyde (Cro), pentenal (Pen), heptenal (Hep), and 4-hydroxy-2-nonenal (HNE). Although this is a general reaction, only the PdG adducts derived from Acr, Cro, and HNE have been detected in vivo as endogenous DNA lesions. Our previous in vitro study demonstrated that PdG adducts of Acr, Cro, and Pen are predominantly derived from oxidation of omega-3 polyunsaturated fatty acids (PUFAs), whereas the long-chain Hep and HNE adducts are from omega-6 PUFAs. PdG adducts are important because they represent a new class of endogenous promutagenic DNA lesions with potential roles in carcinogenesis. Earlier, we developed a (32)P-postlabeling method for detecting PdG adducts from Acr and Cro and a modified method for the long-chain HNE adducts. Both methods require multiple high-performance liquid chromatography steps and, in some cases, time-consuming thin-layer chromatography for purification. There is a lack of a single, versatile, and efficient method for simultaneous detection of all five enal-derived PdG adducts. In this paper, we report an improved (32)P-postlabeling method which permits detection of Acr, Cro, Pen, Hep, and HNE adducts in a single DNA sample. This method relies on solid-phase extraction for adduct enrichment before and after (32)P-labeling; all five PdG adducts were converted to the ring-opened derivatives for confirmation of identities and quantification. The method was validated using the synthetic adducts and enal-modified DNA and was finally applied to rat liver DNA and rat liver DNA samples spiked with different amount of standards. The detection limit was determined to be as low as 0.5 fmol in 80 microg DNA, corresponding to 9 adducts/10(9) dG.     

Effects of human chorionic gonadotropin and N6,O2'-dibutyryladenosine 3' ,5'-monophosphate on phosphofructokinase activity in isolated rat testis.

Phosphofructokinase activity in rat testis is elevated by treatment in vitro with human chorionic godadotropin or N6,O2'-dibutyryladenosine 3' ,5'-monophosphate. Puromycin or actinomycin D suppresses the effect of the gonadotropin but does not affect the enzyme increase induced by the cyclic nucleotide. The possible cause for the divergent action of the two stimulatory agents are discussed.     

Simultaneous determination of O6-methyl-2'-deoxyguanosine, 8-oxo-7,8-dihydro-2'-deoxyguanosine, and 1,N6-etheno-2'-deoxyadenosine in DNA using on-line sample preparation by HPLC column switching coupled to ESI-MS/MS

O(6)-Methyl-2'-deoxyguanosine (O(6)-mdGuo), 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo), and 1,N(6)-etheno-2'-deoxyadenosine (epsilondAdo) are promutagenic DNA lesions originating from both endogenous and exogenous agents and actions (methylation, hydroxylation, lipid peroxidation products). A highly sensitive quantitative method was developed to measure these DNA adducts simultaneously, using liquid chromatography tandem mass spectrometry with column switching. Deuterated O(6)-[(2)H(3)]mdGuo was synthesized and used as internal standard. The limits of quantification for O(6)-mdGuo, 8-oxodGuo, and epsilondAdo were 24, 98, and 48 fmol on column, respectively. The method showed linearity in the range 0.24-125 pmol/ml, 0.98-125 pmol/ml, and 0.49-62.5 pmol/ml for the three adducts, respectively. The inter-day precision in the linear concentration range was between 1.7 and 9.3% for O(6)-mdGuo, 10.6 and 28.7% for 8-oxodGuo, and 6.2 and 10.4%, for epsilondAdo. In DNA isolated from liver of untreated 12-week-old female F344 rats, O(6)-mdGuo was above the limit of detection (37 adducts per 10(9) normal nucleosides) but could not be quantified. 8-oxodGuo and epsilondAdo showed background levels of 500 and 130 adducts per 10(9) normal nucleosides, respectively. DNA analyzed 1h after treatment of rats with dimethylnitrosamine by oral gavage of 50 microg/kg b.wt. did not affect the levels of 8-oxodGuo and epsilondAdo but resulted in 200 O(6)-mdGuo adducts per 10(9) normal nucleosides. The method developed will be of use to study the biological significance of exogenous DNA adducts as an increment to background DNA damage and the role of modulating factors, such as DNA repair.     

Styrene oxide-induced 2'-deoxycytidine adducts: implications for the mutagenicity of styrene oxide

The reaction between 2'-deoxycytidine and styrene 7,8-oxide (SO) resulted in alkylation at the 3-position and at the O(2)-position through the alpha- and beta-carbons of the epoxide but at the N(4)-position only through the alpha-carbon. The 3-alkylated adducts were found to deaminate to the corresponding 2'-deoxyuridine adducts (37 degrees C, pH 7.4) with half-lives of 6 min and 2.4 h for the alpha- and beta-isomers, respectively. The N(4)-alkylated products were stable at neutral pH. The O(2)-alkylated products were unstable being prone to depyrimidation and to isomerisation between alpha- and beta-isomers. In SO-treated double-stranded DNA, enzymatic hydrolysis allowed the identification of the beta3-deoxyuridine and alphaN(4)-deoxycytidine adducts (1.9 and 0.5% of total alkylation, respectively), in addition to the previously identified DNA-adducts. The 3-substituted uracil may have implications for the mutagenicity of SO.     

N-2 acetylation of 2'-deoxyguanosine by coffee mutagens, methylglyoxal and hydrogen peroxide

Coffee shows direct-acting mutagenicity in Salmonella typhimurium TA100 and most of this mutagenicity is due to the synergistic effects of methylglyoxal and hydrogen peroxide. The modifications of deoxyribonucleosides by methylglyoxal plus hydrogen peroxide were studied in vitro. When 2'-deoxyguanosine (6.25 mumole) was treated with methylglyoxal (125 mumole) and hydrogen peroxide (125 mumole) in 5 ml of 0.1 M phosphate buffer (pH 7.4) at 37 degrees C for 3 h, N2-acetyl-2'-deoxyguanosine was formed with a yield of 1.1%. Its formation increased time-dependently. By contrast, no appreciable modification of other deoxynucleosides was detected after their incubation with methylglyoxal and hydrogen peroxide under similar conditions. N2-Acetyl-2'-deoxyguanosine was also formed during incubation of 2'-deoxyguanosine with instant coffee.