mean of the MED from SSR exposure in the AM was 51.43 with
standard error of the mean of 8.09, while the mean of the MED
from SSR exposure in the PM was 86.14 with standard error of
the mean of 19.74.
XPA
Western blot was performed to determine if there was a difference
in XPA expression between the AM and PM irradiated
skin and compare them to unirradiated skin biopsy of subject.
The unirradiated skin was taken in the PM. Western blot and
quantification of data are shown in Figure 4. Actin was used as
a loading control. XPA was barely detectable in the unirradiated
(-UV) sample. XPA was clearly expressed in the PM UV-irradiated
tissue, but showed significantly more expression in the AM
UV-irradiated skin as demonstrated in Figure 4A. Compared to
the unirradiated (-UV) sample, SSR exposure in the AM resulted
in a 20-fold increase in XPA expression, while SSR exposure
in the PM showed a less than 5 fold increase in XPA expression
(Figure 4B).
Immunohistochemistal staining for XPA was performed on the
corresponding biopsy sample to determine whether XPA was
localized in the nucleus where it can participate in NER. As depicted
in Figure 5, there was more nuclear fluorescence for XPA
in the sample from the AM exposure versus the sample from
the PM exposure. Both had a higher fluorescence signal compared
to the unirradiated (-UV) sample.
Quantification using MetaMorph software confirms that the
amount of XPA in the nuclei was significantly higher in the AM
versus PM (Figure 5D). Nuclear localization was 46% higher in
the AM irradiated skin compared to the unirradiated (-UV) skin,
and 26% higher in the PM irradiated skin compared to the unirradiated
(-UV) skin. Nuclear localization of XPA was 27% higher
in the AM compared to the PM.
CPD
Immunohistochemical staining for CPD was performed on the
corresponding biopsy sample to evaluate the level of UV-induced
DNA damage in the nucleus. As seen in Figure 6A and
6B, there was significantly more CPD in the sample from the
AM exposure versus the sample from the PM exposure. The unirradiated
(-UV) sample showed slight autofluorescence with no
visible CPD fluorescence in the nuclei (Figure 6C).
Quantification using MetaMorph software confirmed that the
amount of CPD in the nuclei was significantly higher in the AM
versus the PM exposure (Figure 6D). Nuclear localization was
31% higher in the AM irradiated skin compared to the unirradiated
(-UV) skin, and only slightly higher (10%) in the PM
compared to the unirradiated (-UV) skin. Nuclear localization of
CPD was 25% higher in the AM compared to the PM.
