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

Polycyclic aromatic hydrocarbons (PAHs) constitute a large class of chemicals with widespread occurrence in the environment. It is essentially impossible to avoid exposure 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 and is also suspected to be of a significant health risk to humans. To date, the majority of 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 hampered due to methodological difficulties in isolating and identifying the myriad of trace BP metabolites formed in vivo owing to their low relative abundance and instability.

The thorough characterization of in vivo metabolites of BP in urine from germfree rats, given a single intraperitoneal dose of BP, is described in this thesis. Ion exchange chromatography gives information on the nature of the conjugation. Six fractions were collected: the NEUTRAL FRACTION, containing unconjugated BP, and FRACTIONS I-V containing BP conjugates. The metabolites in NEUTRAL FRACTION were analyzed by HPLC and gas chromatography/mass spectrometry (GC/MS). A trans-11,12-diol was found as a major metabolite of BP in this fraction. Trace levels of metabolites probably related to 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 abundance of 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 liquid chromatography/mass spectrometry (LC/MS) to analyze BP conjugates. Bile acid and steroid conjugates in urine, separated by the ion exchange procedure, were used as model compounds. The LC/electrospray MS method was then applied to the analysis of conjugated metabolites of BP (FRACTIONS II-V).

The major metabolite in FRACTION II was tentatively characterized as a pentahydroxy-BP or a structural isomer. Conjugates of tetrahydrotrihydroxy-, dihydrodihydroxy- and dihydrotrihydroxy-BPs with N-acetylcysteine and a BP-O-glucuronide were formed in smaller amounts. The predominant compounds were 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 were found in FRACTION IV. Only a trace level of a tetrahydro-trihydroxy-BP-glutathione conjugate was detected in FRACTION V. The GC/MS and LC/MS strategy was designed to be generally applicable to the analysis of lipophilic endogenous substances, drugs, xenobiotics and their metabolites in biological matrices.