Chem. Res. Toxicol. 1990,3, 340-343
340
Identification of an in Vivo Chrysene Diol Epoxide Adduct in Human Hemoglobin Billy W. Day,? Paul L. Skipper,+John S. Wishnok,t Jacalyn Coghlin,* S. Katherine Ha"ond,* Peter Gann,* and Steven R. Tannenbaum**t Department of Chemistry and Division of Toxicology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, and D e p a r t m e n t of Family and Community Medicine, University of Massachusetts Medical Center, Worcester, Massachusetts 01655 Received February 16, 1990
Human blood samples were analyzed by fluorescence and mass spectrometry for the presence of polycyclic aromatic hydrocarbon metabolites covalently bound to hemoglobin. Globin was prepared by HC1-acetone precipitation and enzymatically digested. The polycyclic aromatic alcohols generated from ester adduds during digestion were concentrated by monoclonal antibody immunoaffinity chromatography and separated by C-18 HPLC. Analysis of collected fractions by room temperature fluorescence spectroscopy revealed the presence of the anti-tetrahydrotetrols of benzo[a]pyrene and another component possessing a phenanthrene chromophore. Gas chromatographic-mass spectral analysis of the HF'LC fractions revealed that the latter compound was r-l,t-2,t-3,~-4-tetrahydroxy-1,2,3,4-tetrahydrochrysene, arising from a hemoglobin-chrysene diol epoxide adduct.
Introduction
were purchased from the NCI Chemical Carcinogen Respository
maintained by the Midwest Research Institute (KansasCity, MO). The tetrahydro anti-diol epoxide metabolite of benzoThe cis- and tram-tetrols were obtained by hydrolysis (12 h, 25 [alpyrene (Be)'is apparently formed in humans exposed "C) of the corresponding epoxide in 0.9% aqueous formic acidto ambient BaP since the products of its reaction with tetrahydrofuran (99:1), extraction into ethyl acetate, and puriDNA and hemoglobin can be found in blood and placental fication by HLPC on a (2-18column with the gradient described tissue (1,2). The product of the reaction with hemoglobin below (see ref 6 for basis of stereochemical assignments). Ben~~[clphenanthrene 1,2,3,4-tetrahydrotetrolsderived from the is a carboxylic ester which hydrolyzes to form two epimeric anti-diol epoxide (7) were a gift from Dr. A. Dipple of the tetrahydrotetrols when the protein is enzymatically diBRI-Basic Research Program at the NCI-Frederick Cancer gested (2,3). The ease of hydrolysis has been attributed Research Facility. Trisyl "Z"[ 1.5 mequiv of N-(trimethylsily1)to structural features of the ester: the acylated oxygen i m i h l e / m L of pyridine], purchased from Pierce (Rockford, IL), atom is attached to a benzylic carbon atom on its alkyl side was used as the silylation reagent at room temperature (10 pL, (2). Many carcinogenic metabolites of polycyclic aromatic reaction time
