| Summary: | 碩士 === 國立臺灣大學 === 獸醫學系 === 86 === The objective of the study was to investigate the possible role of
apoptosis in T-2 toxin (T-2), aflatoxin B1 (AFB1), and fumonisin B1 (FB1)-
induced cytotoxicity in swine peripheral blood mononuclear cells (SPBMC)
(mainly lymphocytes) and bronchoalveolar lavage cells (SBALC) (mainly
alveolar macrophages). Following incubation with various concentrations of
T-2, AFB1, and FB1 for different time periods, the cytocidal effect of
each mycotoxin on SPBMC and SBALC was measured by an MTT colorimetric
assay. Extraction of DNA for gel electrophoresis, analysis of cellular
light scattering properties and quantitation of TUNEL positive cells by
flow cytometry, and examination of morphological alterations by light
microscopy were performed to detect apoptosis. When SPBMC or SBALC were
exposed to 0.01, 0.1, and 1 ug/ml T-2 for 4 hours, a strong and rapid
cytocidal effect was observed in a dose and time-dependent manner. DNA
regular fragmentation, a decrease in FSC and an increase in SSC, and a
dose and time-dependent increase in TUNEL positive ratio were found when
SPBMC exposed to 0.01, 0.1, and 1 ug/ml T-2 for 2, 10, and 4-10 hours,
respectively. After incubated with 0.01 and 0.1 ug/ml T-2 for 6 hours, DNA
regular fragmentation was observed in SBALC; reduction in FSC and SSC and
a dose and time-dependent increase in TUNEL positive ratio were also
observed following 6-12 hours of incubation. There were about 30% of SPBMC
showing characteristic morphological changes of apoptosis when exposed to
0.1 ug/ml T-2 for 10 hours. When incubated with T-2 at the same
concentration for 11 hours, more than 80% of SBALC underwent necrosis but
morphological evidence of apoptosis could also be found in some of the
remaining alveolar macrophages. The results indicate that T-2 is capable
of causing apoptosis in SPBMC and inducing necrosis concomitant with mild
apoptosis in SBALC. For AFB1, the cell viability of SPBMC was increased
when incubated at concentrations of 1 and 10 ug/ml for 12 hours; however,
a significant cytocidal effect was observed when SPBMC were exposed to 100
ug/ml AFB1 for 6 hours. For SBALC, an evident dose and time-dependent
cytocidal effect was observed when incubated with AFB1 at a concentration
more than 10 ug/ml for 6 hours. Although the spontaneous DNA fragmentation
of SPBMC was inhibited when exposed to 10 and 100 ug/ml AFB1 for 6-8 and
2-8 hours, respectively, the same concentrations of AFB1 induced DNA
laddering fragmentation in SBALC after 6 hours of incubation. When
incubated with 10 and 100 ug/ml AFB1 for 24-48 hours, the SPBMC showed
decreased FSC and increased SSC. The TUNEL positive ratio of SPBMC was
increased in a time-dependent manner when exposed to 10 and 100 ug/ml AFB1
for 10-48 hours with 10 ug/ml AFB1 more effective in apoptosis induction.
At the same concentrations, AFB1 caused a decrease in both FSC and SSC and
an increase in TUNEL positive ratio in SBALC following 12-24 hours of
incubation. As SPBMC, 10 ug/ml AFB1 was also more capable of inducing
apoptosis in SBALC than was 100 ug/ml AFB1. When exposed to 50 ug/ml AFB1
for 24 hours, there were 60-70% of SPBMC showed the characteristic
morphological changes of apoptosis; however, the same concentration of
AFB1 caused necrosis in 50% of the SBALC and apoptosis in more than 20% of
the alveolar macrophages by 48 hours. The result indicates that AFB1 is
able to cause apoptosis in SPBMC but induces necrosis and apoptosis in
SBALC simultaneously. Instead of cytocidal effect, the cell viability was
increased when SPBMC were exposed to 1, 10, and 100 ug/ml FB1 for 6 hours.
When incubated with 1, 10, and 100 ug/ml FB1, the cell viability of SBALC
was markedly enhanced in the first day, but a significant time and dose-
dependent reduction in cell viability appeared on the second day and
thereafter. The spontaneous DNA fragmentation in SPBMC was completely
inhibited by 10 and 100 ug/ml FB1 during the incubation period of 1-4
hours. The same concentrations of FB1 caused DNA regular fragmentation in
SBALC after 2 days of exposure and the intensity of DNA fragmentation was
increased with time. Following incubation with 100 ug/ml FB1 for 24-48 and
48 hours, there were a decrease in FSC and an increase in SSC and an
increase in the TUNEL positive ratio in SPBMC, respectively. Conversely,
the ratio of apoptosis in SPBMC was reduced by 10 ug/ml FB1 during the
incubation period of 24-48 hours. The changes in cellular light scattering
properties in SBALC were reduced when exposed to 10 and 100 ug/ml FB1 for
48 hours. The same concentrations of FB1 were capable of reducing the
TUNEL positive ratio of SBALC in the first day of exposure, but caused a
dose and time-dependent decrease from day 2 to 5. There was no obvious
difference in the morphological changes of SPBMC between the control and
cells treated with 50 ug/ml FB1 for 24 and 48 hours; by day 4, there were
15% of SBALC with morphological changes of apoptosis. The results indicate
that FB1 can cause apoptosis in both SPBMC and SBALC. Thus, the present
study has demonstrated that apoptosis is involved in T-2, AFB1, and FB1-
induced cell injuries in both SPBMC and SBALC.
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