ABSTRACT
A series of benzothienyloxy propylamines have been prepared and are demonstrated to be inhibitors of both serotonin and norepinephrine reuptake.
Subject(s)
Adrenergic Uptake Inhibitors/chemical synthesis , Antidepressive Agents/chemical synthesis , Norepinephrine/antagonists & inhibitors , Propylamines/chemical synthesis , Selective Serotonin Reuptake Inhibitors/chemical synthesis , Thiophenes/chemical synthesis , Adrenergic Uptake Inhibitors/pharmacology , Antidepressive Agents/pharmacology , Dopamine Plasma Membrane Transport Proteins , Humans , In Vitro Techniques , Membrane Glycoproteins/metabolism , Membrane Transport Proteins/metabolism , Microsomes, Liver/metabolism , Nerve Tissue Proteins/metabolism , Norepinephrine/metabolism , Norepinephrine Plasma Membrane Transport Proteins , Propylamines/pharmacology , Serotonin Plasma Membrane Transport Proteins , Selective Serotonin Reuptake Inhibitors/pharmacology , Stereoisomerism , Structure-Activity Relationship , Symporters/metabolism , Thiophenes/pharmacologyABSTRACT
Nano liquid chromatography (nanoLC) coupled to electrospray mass spectrometry (ES-MS) was evaluated for the analysis of DNA adducts in melphalan-treated Jurkat cells. The detection limit of the nanoLC-ES-MS-MS system was assessed using a dAMP-melphalan adduct. Compared to capillary liquid chromatography (capLC) ES-MS the absolute detection limit could be improved by a factor 10, leading to the detection of 395 fg dAMP-melphalan adduct under single-ion monitoring conditions at a S/N of 14. Minor adducts such as cross-linked adducts could be detected in in vitro solutions of 2'-deoxynucleotides (dNMP) treated with melphalan using column-switching nanoLC-ES-MS. These adducts were not found using capLC-ES-MS. More detailed structural information of the alkylation sites was obtained by examining the nanoLC-ES-MS-MS data. Jurkat cells were treated with melphalan, the modified DNA was isolated and enzymatically hydrolyzed. Several modified dinucleotides were identified, the most abundant adducts were pdG(Mel(Cl))pdC (m/z=453, t(r)=17.0 min) and pdG(Mel(OH)) pdC ring opened (m/z=453, t(r)=39.5 min).
Subject(s)
Chromatography, Liquid/methods , DNA Adducts/analysis , Melphalan/chemistry , Spectrometry, Mass, Electrospray Ionization/methods , Animals , Cattle , DNA/chemistry , DNA Adducts/chemistry , Humans , Jurkat CellsABSTRACT
Melphalan is a bifunctional alkylating agent that covalently binds with intracellular nucleophilic sites. A methodology using electrospray mass spectrometry was developed to detect and identify DNA adducts. Alkylation sites within a particular nucleotide were examined using electrospray tandem mass spectrometry hyphenated to capillary liquid chromatography in combination with a column switching system. In the reaction mixtures resulting from the interaction of 2'-deoxynucleotides and melphalan several base-aklylated adducts were found. In the case of 2'-deoxyadenosine monophosphate, thymidine monophosphate and 2'-deoxyguanosine phosphate alkylation was observed in the mononucleotide reaction mixtures but not in the DNA-hydrolysates. Calf thymus DNA was reacted in vitro with melphalan. The DNA pellet was isolated and enzymatically hydrolyzed with the aid of Nuclease P1. In this hydrolysate both mono-alkylated 2'-deoxynucleotides and dinucleotides were found. The most important adduct found was identified as the N-7 alklylated dGMP adduct. The alkylated dinucleotides were identified as a pdApdT/melphalan and pdGpdC/melphalan the latter being the most important.
Subject(s)
Chromatography, High Pressure Liquid/methods , DNA Adducts/analysis , Deoxyribonucleotides/analysis , Mass Spectrometry/methods , Melphalan/metabolism , Alkylation , Animals , Cattle , DNA/metabolism , Deoxyadenine Nucleotides/analysis , Deoxycytidine Monophosphate/analysis , Deoxyguanine Nucleotides/analysis , Melphalan/pharmacology , Sensitivity and Specificity , Thymidine Monophosphate/analysisABSTRACT
Calf thymus DNA was reacted in vitro with phenyl glycidyl ether (PGE) and was hydrolysed enzymatically, to the 5'-monophosphate nucleotides using deoxyribonuclease I (DNA-ase I) and nuclease P1. The adducts were concentrated using solid phase extraction (SPE), on a polystyrene divinylbenzene copolymer in order to remove the unmodified nucleotides. The adducts could be identified using capillary zone electrophoresis-electrospray tandem mass spectrometry (CZE ES-MS/MS), using sample stacking. In addition to the base alkylated 2'-deoxynucleotides present in the DNA-hydrolysate, also phosphate alkylated 2'-deoxynucleotide adducts were identified for TMP and dAMP. An additional adduct, dUMP alkylated on the uridine moiety was found originating from the hydrolytic deamination of dCMP alkylated on N3 of the cytosine moiety. Enzymatic hydrolysis using nuclease P1 was incomplete as shown by the presence of dinucleotides alkylated on the base moiety. They were successfully hydrolysed to the corresponding 2'-deoxynucleotides by snake venom phosphodiesterase (SVP). Data are shown indicating that alkylations on the pyrimidine bases were more resistant to enzymatic hydrolysis with nuclease P1 than the purine alkylated products.
Subject(s)
DNA Adducts/analysis , DNA/drug effects , Electrophoresis, Capillary/methods , Mass Spectrometry/methods , Phenyl Ethers/pharmacology , Alkylation , Animals , Cattle , Phosphates/metabolism , Spectrophotometry, Ultraviolet , Thymus Gland/drug effects , Thymus Gland/metabolismABSTRACT
In this work, the coupling of liquid nanochromatography to NanoFlow electrospray mass spectrometry was evaluated for the detection of DNA adducts. The NanoFlow ES LC/MS system was compared with the capillary and conventional ES LC/MS system by analyzing an in vitro reaction mixture resulting from the interaction of 2'-deoxyguanosine 5'-monophosphate with bisphenol A diglycidyl ether and by injecting 2'-deoxyadenosine. By using NanoFlow ES LC/MS, the mass sensitivity could be improved by a factor of 3300. Three different injection methods used in liquid nanochromatography, i.e., split, large-volume, and column-switching injections were compared in terms of sensitivity. Furthermore, NanoFlow ES LC/MS was used to detect 2'-deoxynucleotide adducts isolated from an in vitro mixture of calf thymus DNA and bisphenol A diglycidyl ether. Different 2'-deoxynucleotide adducts could be identified by monitoring typical product ions, diagnostic for the adducts.
