Candidate: Po-Chang Chiang

Degree of: Doctor of Philosophy

Department: Chemistry

Title: Quantification of Benzo[a]pyrene-Guanine Adducts in In Vitro and In Vivo Tissue Samples by LC Tandem Mass Spectrometry

Committee:
Dr. Jay Means, Chair
Dr. Subra Muralidharan
Dr. James Howell
Dr. Elke Schoffers
Dr. Charles Ide

Date: Tuesday, May 1, 2001, 4:00 p.m. - 6:00 p.m.
1728 Wood Hall

Abstract:
Benzo[a]pyrene, after metabolic activation, is known as one of the most potent PAH (polycyclic aromatic hydrocarbon) that causes carcinogenesis effect (fourth annual report on carcinogens,1985). It is believed that when benzo[a]pyrene is activated by one-electron oxidation to its radical cation, it binds the C-6 of benzo[a]pyrene to either the C-8 or N-7 of guanine on DNA and forms DNA adducts (Rogan et al, 1990). They are 8-(benzo[a]pyrene-6-yl)guanine and 7-(benzo[a]pyrene-6-yl)guanine (Rogan et al., 1988).  Adduct formation is considered the critical step in the process of a tumor's formation (Miller, 1970).

A sensitive and rugged liquid chromatography/tandem mass spectrometry/ multiple reaction monitoring coupled with a stable isotope internal standard method was developed for detection and quantitative analysis of 8-(benzo[a]pyrene-6-yl)guanine (BaP-C8Gua) and 7-(benzo[a]pyrene-6-yl)guanine (BaP-N7Gua) as DNA adducts. Both in vitro (horseradish peroxidase catalyzed reaction of benzo[a]pyrene with DNA) and in vivo (dietary exposure of benzo[a]pyrene to animal) samples were analyzed by our method.

 Separation and analysis were performed using HPLC coupled in-line to an electrospray ionization (ESI) triple quadrupole mass spectrometer.  BaP-C8Gua, and BaP-N7Gua were synthesized and used as external standards and BaP-C8Gua-d₁₁ and BaP-N7Gua-d₁₁ were synthesized and used as internal standards. The method provides structural confirmation of the adduct as well as quantitative analysis with the accuracy and precision to measure biologically relevant levels in small tissue sample sizes (<1g). The application of this method was demonstrated by measuring the DNA adduct levels in rat and fish samples after exposure to benzo[a]pyrene.

Our method has proven to be well suited for these analyses, providing low limit of detection, quick turnaround time, additional specificity, and structure confirmation.  We believe that more effort should be made in this area and the importance of liquid chromatography/tandem mass spectrometry in future research cannot be overemphasized.

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