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conjectured that the decreased DNA synthesis observed in MLA 144 cells treated
with Oph and Proto could be due to a toxic effect of these chemicals. The plasma
membrane integrity of MLA 144 cells subjected to trypan blue exclusion
(cell viability) and chromium release (membrane permeability) was therefore
assessed. Although treatment with Proto and Oph for 3 h in darkness caused growth
arrest, cell viability was not significantly reduced as evidenced by trypan blue
exclusion. Indeed, the total cell number did not decrease in Oph or Proto treated
cells. Proto-treated (0.05-0.1 mM) cell viability after 3 and 24 h of incubation was
similar to controls.
A chromium release assay was also used as a test of membrane permeability.
A combination of ALA and Oph did not induce significant chromium release from
cells, during 3 h of incubation with ALA (1-4 mM) and Oph (0.75-3 mM) (Rebeiz
et al. 2001 ). Thus, at 0.75-3 mM, Oph did not appear to be toxic to MLA 144 cells
and did not induce cell lysis during 3 h of dark of incubation.
However, after 24 h of treatment with Oph (1.5-3 mM), cell membranes no
longer excluded trypan blue (15-16 % viability) (Rebeiz et al. 2001 ). Also,
non-chelating isomers of Oph significantly decreased cell viability after 3 and
24 h of incubation.
19.4.4
Induction of Apoptosis by Oph
Internucleosomal cleavage of DNA was first measured to confirm the occurrence of
apoptosis. MLA 144 cells that had been serum-deprived for 3 h exhibited slight
DNA fragmentation in comparison to cells at time zero (Rebeiz et al. 2001 ).
However, treatment with Oph at either 0.75 mM or 1.5 mM induced a dramatic
increase in DNA cleavage after 3 h of dark incubation. A similar DNA laddering
pattern was observed when cells were treated with a combination of ALA and Oph.
Internucleosomal cleavage of DNA was also evident after 6 h incubation with Oph.
Treatment with ALA or Proto alone did not induce DNA fragmentation at either
3 or 6 h. Thus, although Proto did induce growth arrest, unlike Oph, it did not
induce apoptosis.
Cells undergoing apoptosis usually exhibit a characteristic hypodiploid peak of
DNA when stained with the DNA intercalating dye PI and analyzed by flow
cytometry. Thus MLA 144 cells were treated for 3 h with ALA, Oph, Proto, or
medium, in a serum-free system prior to ethanol permeabilization and staining with
PI. Cell cycle analysis was determined by analysis with MPLUS software. Basal
level of apoptosis was 8
3.7 % in medium-treated cells (Rebeiz et al. 2001 ).
Treatment with 1.5 mM Oph induced a significant level of apoptosis (45
1.8 %,
p
0.05), and a distinct subdiploid peak (apoptotic) became apparent. In addition,
the proportion of cells accumulating in early S phase increased in comparison
to medium-treated cells (60
<
0.05).
At 0.75 mM Oph, which induced the highest level of internucleosomal DNA
cleavage, the majority of the cells became apoptotic (81
2.5 % vs. 48
2.5 % respectively, p
<
5.6 %), and most of
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