Hyperosmolarity caused mitochondrial DNA oxidative damage in
HCECs
In nuclear and mitochondrial DNA, 8-hydroxy-2'-deoxyguanosine (8-OHdG) is one of the
predominant forms of free radical-induced oxidative lesions, and has therefore been widely
used as a biomarker for oxidative stress [30]. Aconitase-2, one of the enzymes participating in
the tricarboxylic acid cycle, acts as a biosensor for oxidative stress and preserves mitochondrial
DNA oxidative damage [31–33]. As evaluated by Western blot analysis, aconitase-2 protein
was found to increase markedly at an osmolarity-dependent manner in HCECs exposed to
hyperosmotic media with 400 and 450 mOsM for 24 hours when compared to that in isosmolar
medium, as well as house-keeping protein β-actin (Fig 3A). The immunohistochemical
staining (Fig 3B) further showed that the punctate staining of aconitase-2 in cytoplasm, representing
the mitochondria; and the density and intensity of the punctate staining of aconitase-2
were markedly increased in the cells exposed to hypertonic media at 400–450 mOsM when
compared with cells in isosmolar medium. Interestingly, the markedly increased red brown
staining of an oxidized DNA product 8-OHdG was also observed to be localized in cytoplasm
or nuclear compartment of HCECs exposed to hyperosmotic media (400–450 mOsM) for 24hours while the most cells were stained negative in isosmolar media (Fig 3B). Furthermore, the
enlarged single cell images of 8-OHdG staining showed its homogenous staining in nuclear,
while a punctate pattern in cytoplasm. The density and intensity of 8-OHdG punctate staining
in cytoplasm were markedly increased in the cells exposed to hypertonic media at 400 and 450
mOsM, indicating mitochondrial DNA damage.