The in vivo metabolism of Benzo(a)pyrene studied by chromatography in combination with mass spectrometry

Sammanfattning: Polycyclic aromatic hydrocarbons (PAHs) constitute a large class of chemicalswith widespread occurrence in the environment. It is essentially impossible to avoidexposure to these substances on a daily basis. Benzo[a]pyrene (BP), a model PAH,is a powerful mutagenic and carcinogenic agent in various experimental systems andis also suspected to be of a significant health risk to humans. To date, the majorityof studies on the characterization of BP metabolites has been performed in vitro.The analysis of the integral metabolism of BP occurring in vivo has been hampereddue to methodological difficulties in isolating and identifying the myriad of traceBP metabolites formed in vivo owing to their low relative abundance and instability. The thorough characterization of in vivo metabolites of BP in urine from germfreerats, given a single intraperitoneal dose of BP, is described in this thesis. Ionexchange chromatography gives information on the nature of the conjugation. Six fractionswere collected: the NEUTRAL FRACTION, containing unconjugated BP, and FRACTIONS I-Vcontaining BP conjugates. The metabolites in NEUTRAL FRACTION were analyzed by HPLCand gas chromatography/mass spectrometry (GC/MS). A trans-11,12-diol was found asa major metabolite of BP in this fraction. Trace levels of metabolites probably relatedto the well-known activation pathway leading to BP-7,8-dihydrodiol-9,10-epoxide (BPDE)were detected, e.g. the trans-7,8-diol and tetrols. The presence of a high abundanceof trihydroxy-BPs may indicate a hitherto unsuspected action of dihydrodiol dehydrogenase(DDH) in the in vivo metabolism of BP. A novel strategy was developed using liquidchromatography/mass spectrometry (LC/MS) to analyze BP conjugates. Bile acid andsteroid conjugates in urine, separated by the ion exchange procedure, were used asmodel compounds. The LC/electrospray MS method was then applied to the analysis ofconjugated metabolites of BP (FRACTIONS II-V). The major metabolite in FRACTION IIwas tentatively characterized as a pentahydroxy-BP or a structural isomer. Conjugatesof tetrahydrotrihydroxy-, dihydrodihydroxy- and dihydrotrihydroxy-BPs with N-acetylcysteineand a BP-O-glucuronide were formed in smaller amounts. The predominant compoundswere oxygenated at C-7,8,9,10. Metabolites detected in FRACTION m included monohydroxy-BP-O-sulfates,dihydrodihydroxy-BP-O-sulfates and a BP-O,O' -diglucuronide. BP-O,O'-disulfates werefound in FRACTION IV. Only a trace level of a tetrahydro-trihydroxy-BP-glutathioneconjugate was detected in FRACTION V. The GC/MS and LC/MS strategy was designed tobe generally applicable to the analysis of lipophilic endogenous substances, drugs,xenobiotics and their metabolites in biological matrices. ISBN 91-628-2640-9