title: Acrolein mercapturates: synthesis, characterization, and assessment of their role in the bladder toxicity of cyclophosphamide.
abstract: Acrolein is the metabolite of cyclophosphamide (CP) believed to be involved in the bladder toxicity associated with this anticancer drug. The mechanism by which this extremely reactive intermediate is delivered to the bladder is not known. Glutathione (GSH) readily conjugates with acrolein, and the acrolein mercapturate S-(3-hydroxypropyl)-N-acetylcysteine (3-hydroxy-PrMCA) has been found in the urine of animals and man given CP. The objectives of this study were to prepare and characterize synthetic standards of the GSH acrolein adduct (3-oxopropyl)glutathione (3-oxoPrGSH), the acrolein mercapturates S-(3-oxopropyl)-N-acetylcysteine (3-oxoPrMCA) and 3-hydroxyPrMCA, and the S-oxidation product of 3-oxoPrMCA (3-oxoPrMCA S-oxide). In addition, the release of acrolein from, and the bladder toxicity of, these conjugates was determined. 3-OxoPrGSH and 3-oxoPrMCA were prepared with a 99% yield by condensing acrolein with GSH and N-acetylcysteine, respectively. 3-HydroxyPrMCA was prepared with a 63% yield by refluxing 3-chloropropanol and N-acetylcysteine in a basic medium. Oxidation of 3-oxoPrMCA with H2O2 was used to prepare 3-oxoPrMCA S-oxide. By decreasing the reaction time to 1 h, and adjusting the ratio of 3-oxoPrMCA to H2O2, the yield of 3-oxoPrMCA S-oxide was increased to 96%. The anhydrous aldehyde, 3-oxoPrMCA, afforded characteristic aldehydic proton resonances (1H NMR) in deuterated dimethyl sulfoxide. New resonances were observed in deuterated water, indicating a 75% hydration of the aldehyde to the corresponding geminal diol. This phenomenon was enhanced with 3-oxoPrMCA S-oxide where approximately 100% hydration of the aldehyde to the corresponding geminal diol was observed. When incubated at 25 degrees C in 100 mM potassium phosphate buffer containing 1 M KCl, pH 8.0, 3-oxoPrMCA released approximately 6% and 3-oxoPrMCA S-oxide released approximately 16-18% of the theoretical maximum yield of acrolein after 30 min, as indicated by an increase in absorbance at 210 nm and confirmed by trapping this aldehyde as a semicarbazone. There was less than a 2% yield of acrolein from 3-hydroxyPrMCA or 3-oxoPrGSH under similar conditions. At pH 7.4 the release of acrolein from 3-oxoPrMCA and 3-oxoPrMCA S-oxide was decreased by 50%. An assay where aldehydes are reacted with m-aminophenol in acid media produced fluorescence consistent with 72%, 46%, 23%, and 1% yields of acrolein from 3-oxoPrMCA S-oxide, 3-oxoPrMCA, 3-oxoPrGSH, and 3-hydroxyPrMCA, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)
authors.affiliation: Division of Pharmacology and Toxicology, College of Pharmacy, University of Texas at Austin 78712-1074, USA.
authors.forename: K
authors.initials: K
authors.lastname: Ramu
authors.forename: L H
authors.initials: LH
authors.lastname: Fraiser
authors.forename: B
authors.initials: B
authors.lastname: Mamiya
authors.forename: T
authors.initials: T
authors.lastname: Ahmed
authors.forename: J P
authors.initials: JP
authors.lastname: Kehrer
mesh_terms: D000111:Acetylcysteine / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000737:chemistry / Q000633:toxicity; D000171:Acrolein / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000737:chemistry / Q000633:toxicity; D000818:Animals; D002855:Chromatography, Thin Layer; D003520:Cyclophosphamide / Q000737:chemistry / Q000633:toxicity*; D006454:Hemoglobins / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008297:Male; D013058:Mass Spectrometry; D051379:Mice; D008813:Mice, Inbred ICR; D010659:Phenylhydrazines / Q000737:chemistry; D012664:Semicarbazones / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D001745:Urinary Bladder Diseases / Q000139:chemically induced* / Q000473:pathology
chemical_list: D006454:Hemoglobins; D010659:Phenylhydrazines; D012664:Semicarbazones; C004787:2,4-dinitrophenylhydrazine; D000171:Acrolein; D003520:Cyclophosphamide; D000111:Acetylcysteine