ToxSci Advance Access originally published online on December 22, 2003
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Toxicological Sciences 77, 307-314 (2004)
Copyright © 2004 by the Society of Toxicology
IN VITRO TOXICOLOGY AND ALTERNATIVE TESTING |
DNA Adduct Formation in Precision-Cut Rat Liver and Lung Slices Exposed to Benzo[a]pyrene
,3
* Department of Pharmacology and Toxicology, State University of New York at Buffalo, Buffalo, New York 14214,
Department of Molecular Pharmacology and Cancer Therapeutics, Roswell Park Cancer Institute, Buffalo, New York 14263; and
Department of Molecular and Cellular Biophysics, Roswell Park Cancer Institute, Buffalo, New York 14263
ABSTRACT
Chemical-DNA adducts provide an integrated measure of exposure, absorption, bioactivation, detoxification, and DNA repair following exposure to a genotoxic agent. Benzo[a]pyrene (BaP), a prototypical polycyclic aromatic hydrocarbon (PAH), can be bioactivated by cytochrome P-450s (CYPs) and epoxide hydrolase to genotoxic metabolites which form covalent adducts with DNA. In this study, we utilized precision-cut rat liver and lung slices exposed to BaP to investigate tissue-specific differences in chemical absorption and formation of DNA adducts. To investigate the contribution of bioactivating CYPs (such as CYP1A1 and CYP1B1) on the formation of BaP–DNA adducts, animals were also pretreated in vivo with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin) prior to in vitro incubation of tissue slices with BaP. Furthermore, the tissue distribution of BaP and BaP–DNA adduct levels from in vivo studies were compared with those from the in vitro tissue slice experiments. The results indicate a time- and concentration-dependent increase in tissue-associated BaP following exposure of rat liver and lung tissue slices to BaP in vitro, with generally higher levels of BaP retained in lung tissue. Furthermore, rat liver and lung slices metabolized BaP to reactive intermediates that formed covalent adducts with DNA. Total BaP–DNA adducts increased with concentration and incubation time. Adduct levels (fmol adduct/µg DNA) in lung slices were greater than liver at all doses. Liver slices contained one major and two minor adducts, while lung slices contained two major and 3 minor adducts. The tissue-specific qualitative profile of these adducts in tissue slices was similar to that observed from in vivo studies, further validating the use of this model. Pretreatment of animals with TCDD prior to in vitro incubation with BaP potentiated the levels of DNA adduct formation. TCDD pretreatment altered the adduct distribution in lung but not in liver slices. Together, the results suggest that tissue-specific qualitative and quantitative differences in BaP–DNA adducts could contribute to the lung being a target tissue for BaP carcinogenesis. Furthermore, the results validate the use of precision-cut tissue slices incubated in dynamic organ culture as a useful model for the study of chemical–DNA adduct formation.
Key Words: benzo(a)pyrene; DNA adduct; cytochrome P-450; TCDD; tissue slices.
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