Carcinogenesis, Vol 18, 1055-1062, Copyright © 1997 by Oxford University Press
HH Schmeiser, E Frei, M Wiessler and M Stiborova
Aristolochic acid I (AAI) and aristolochic acid II (AAII), the two major
components of the carcinogenic plant extract aristolochic acid (AA), are
known to be mutagenic and to form DNA adducts in vivo. According to the
structures of the major DNA adducts identified in animals and humans,
nitroreduction is the crucial pathway in the metabolic activation of these
naturally occurring nitroarenes to their ultimate carcinogenic species.
Using the nuclease P1-enhanced version of the 32P-post-labelling assay we
investigated the formation of DNA adducts by AAI and AAII in different in
vitro activation systems in order to determine the most suitable in vitro
system mimicking target tissue activation. Although DNA adducts resulting
from oxidative activation of AAs have not yet been identified both
reductive and oxidative in vitro systems were employed. In vitro
incubations were conducted under standardized conditions (0.3 mM AAs; 4 mM
dNp as calf thymus DNA) using rat liver microsomes, xanthine oxidase (a
mammalian nitroreductase), horseradish peroxidase, lactoperoxidase and
chemical reduction by zinc. Enzymatic incubations were performed under
aerobic and anaerobic conditions. A combination of two independent
chromatographic systems (ion-exchange chromatography and reversed-phase
HPLC) with reference compounds was used for the identification of DNA
adducts detected by the 32P-post-labelling assay. The two known major
adducts of AAI or AAII found in vivo were generated by all in vitro systems
except for incubations with AAII and horseradish peroxidase where two
unknown adducts predominated. Irrespective of the in vitro activation
system used, the majority of adduct spots obtained were identified as the
previously characterized four AA-DNA adducts: dA-AAI, dA-AAII, dG-AAI and
dG-AAII. This indicates that both reductive and peroxidative activation of
AAI or AAII resulted in chromatographically indistinguishable DNA adducts.
Thus, peroxidase mediated activation of AAs led to the formation of the
same adducts that had been observed in vivo and upon reductive activation
in several in vitro systems. Quantitative analyses of individual adducts
formed in the various in vitro systems revealed relative adduct labelling
(RAL) values over a 100,000-fold range from 4 in 10(3) for activation of
AAII to deoxyadenosine adducts by zinc to only 3 in 10(8) for activation of
AAII by lactoperoxidase. The extent of DNA modification by AAI was higher
than by AAII in all enzymatic in vitro systems. Only activation by zinc
resulted in higher total binding to exogenous DNA by AAII than by AAI.
Aerobic incubations with rat liver microsomes generated AAI- and AAII-DNA
adduct profiles reproducing profiles in target tissue (forestomach) of
rats, thus providing the most appropriate activation among the in vitro
systems tested.
ARTICLES
Comparison of DNA adduct formation by aristolochic acids in various in vitro activation systems by 32P-post-labelling: evidence for reductive activation by peroxidases
Department of Molecular Toxicology, German Cancer Research Center, Heidelberg, Germany.
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