title: S - and N-alkylating agents diminish the fluorescence of fluorescent dye-stained DNA.
abstract: Sulfur mustard (SM), a chemical warfare agent, causes DNA alkylation, which is believed to be the main cause of its toxicity. SM DNA adducts are commonly used to verify exposure to this vesicant. However, the required analytical state-of-the-art mass-spectrometry methods are complex, use delicate instruments, are not mobile, and require laboratory infrastructure that is most likely not available in conflict zones. Attempts have thus been made to develop rapid detection methods that can be used in the field. The analysis of SM DNA adducts (HETE-G) by immunodetection is a convenient and suitable method. For a diagnostic assessment, HETE-G levels must be determined in relation to the total DNA in the sample. Total DNA can be easily visualized by the use of fluorescent DNA dyes. This study examines whether SM and related compounds affect total DNA staining, an issue that has not been investigated before. After pure DNA was extracted from human keratinocytes (HaCaT cells), DNA was exposed to different S- and N-alkylating agents. Our experiments revealed a significant, dose-dependent decrease in the fluorescence signal of fluorescent dye-stained DNA after exposure to alkylating agents. After mass spectrometry and additional fluorescence measurements ruled out covalent modifications of ethidium bromide (EthBr) by SM, we assumed that DNA crosslinks caused DNA condensation and thereby impaired access of the fluorescent dyes to the DNA. DNA digestion by restriction enzymes restored fluorescence, a fact that strengthened our hypothesis. However, monofunctional agents, which are unable to crosslink DNA, also decreased the fluorescence signal. In subsequent experiments, we demonstrated that protons produced during DNA alkylation caused a pH decrease that was found responsible for the reduction in fluorescence. The use of an appropriate buffer system eliminated the adverse effect of alkylating agents on DNA staining with fluorescent dyes. An appropriate buffer system is thus crucial for DNA quantification with fluorescent dyes in the presence of alkylating compounds.
authors.affiliation: Bundeswehr Institute of Pharmacology and Toxicology, 80937 Munich, Germany.
authors.forename: Robert
authors.initials: R
authors.lastname: Giesche
authors.affiliation: Bundeswehr Institute of Pharmacology and Toxicology, 80937 Munich, Germany.
authors.forename: Harald
authors.initials: H
authors.lastname: John
authors.affiliation: Walther Straub Institute of Pharmacology and Toxicology, Ludwig-Maximilians-Universität München, 80336 Munich, Germany; Bundeswehr Medical Academy, 80937 Munich, Germany.
authors.forename: Kai
authors.initials: K
authors.lastname: Kehe
authors.affiliation: Bundeswehr Institute of Pharmacology and Toxicology, 80937 Munich, Germany.
authors.forename: Annette
authors.initials: A
authors.lastname: Schmidt
authors.affiliation: Bundeswehr Institute of Pharmacology and Toxicology, 80937 Munich, Germany; Walther Straub Institute of Pharmacology and Toxicology, Ludwig-Maximilians-Universität München, 80336 Munich, Germany.
authors.forename: Tanja
authors.initials: T
authors.lastname: Popp
authors.affiliation: Comprehensive Pneumology Center Munich (CPCM), German Center for Lung Research, 81377 Munich, Germany; Bundeswehr Medical Command, 56070 Koblenz, Germany.
authors.forename: Frank
authors.initials: F
authors.lastname: Balzuweit
authors.affiliation: Bundeswehr Institute of Pharmacology and Toxicology, 80937 Munich, Germany.
authors.forename: Horst
authors.initials: H
authors.lastname: Thiermann
authors.affiliation: Walther Straub Institute of Pharmacology and Toxicology, Ludwig-Maximilians-Universität München, 80336 Munich, Germany; Comprehensive Pneumology Center Munich (CPCM), German Center for Lung Research, 81377 Munich, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Gudermann
authors.affiliation: Bundeswehr Institute of Pharmacology and Toxicology, 80937 Munich, Germany; Walther Straub Institute of Pharmacology and Toxicology, Ludwig-Maximilians-Universität München, 80336 Munich, Germany. Electronic address: dirksteinritz@bundeswehr.org.
authors.forename: Dirk
authors.initials: D
authors.lastname: Steinritz
mesh_terms: D000477:Alkylating Agents / Q000737:chemistry*; D002460:Cell Line; D002619:Chemical Warfare Agents / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid; D004247:DNA / Q000737:chemistry*; D018736:DNA Adducts / Q000032:analysis; D004996:Ethidium / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry*; D006801:Humans; D009151:Mustard Gas / Q000737:chemistry*; D053719:Tandem Mass Spectrometry
chemical_list: D000477:Alkylating Agents; D002619:Chemical Warfare Agents; D018736:DNA Adducts; D005456:Fluorescent Dyes; D004247:DNA; D004996:Ethidium; D009151:Mustard Gas
keywords: Chemical warfare agents; DNA staining; Ethidium bromide; Fluorescent dyes; Mass spectrometry; Sulfur mustard