|
The carcinogenic Cr(VI) is transported into cells through the
anion transport system and is reduced intracellularly,
generating Cr(V), Cr(IV), Cr(III), and reactive oxygen species.
All the Cr(VI) metabolites may be potent agents that damage DNA.
Ascorbic acid (AsA) is a major cellular reductant for Cr(VI)
metabolism. In this study, we have adopted a shuttle vector
system to determine the molecular mutagenicity of untreated and
Cr(VI)/AsA-treated shuttle vector pSP189 during replication in
Fanconi's anemia (FA) cells that have defects in repairing DNA
cross-links and oxidative damage.The pSP189 vector, containing a
bacterial suppressor tRNA gene, supF, was transfected into FA
cells for DNA replication to take place. The progeny plasmids
were then rescued, purified, and introduced into an indicator E.
coli that had an amber mutation in the β-galactosidase gene.
The mutations generated during replication in FA cells were
compared with that obtained in human 293 cells (Tsou et al.,
1998).The supF mutation frequency obtained in untreated plasmids
replication in FA cells was 2.6-fold higher than that obtained
in 293 cells. DNA sequencing analysis of 87 spontaneous supF
mutants derived from FA cells showed that 47% were base
substitutions, and 46% were large deletions and rearrangements.
In contrast, the majority of spontaneous supF mutations obtained
in 293 cells were base substitutions (71%). FA cells may have
high level of reactive oxygen species that attack DNA and
thereby generating high frequency of large deletions and
rearrangements. The supF mutation frequency obtained in Cr(VI)/
AsA-treated plasmids replication in FA cells was significantly
higher than that obtained in 293 cells (p < 0.02). DNA
sequencing analysis of 113 Cr(VI)/AsA-induced mutants showed
that 75% were base substitutions, 12% were single-base
deletions, and 12% were deletions. Almost all (94%) the base
substitutions occurred at G‧C base pairs, and the major base
substitutions were G‧C → T‧A transversions (62%). Most of
the base substitutions were located at 5'GG and 5'GG sites.The
types of mutations induced by Cr(VI)/AsA-treated plasmids
replication in FA cells were similar to that obtained in 293
cells, however, different hot spots were induced in these two
cell lines. 8-Hydroxydeoxyguanosine (8-OHdG) adducts and DNA-
interstrand cross-links were induced by Cr(VI)/AsA in a Hepes
buffer. During DNA replication, 8-OHdG adducts could result in
G‧C → T‧A transversions, whereas DNA-interstrand cross-links
could block DNA synthesis. Administrating mannitol to FA cells
significantly reduced the mutation frequency occurred
spontaneously (p < 0.02) or induced by Cr(VI)/AsA (p <0.02
).Taken together, these results suggest that 8-OHdG adducts,
resulting from hydroxyl radicals, may be the mutagenic lesions
in the Cr(VI)/AsA system, and that the high mutation frequency
observed in FA cells may due to high amounts of intracellular
reactive oxygen species, and not derived from DNA-interstrand
cross-links.
|