|Molecular Vision 2006;
Received 31 October 2005 | Accepted 31 March 2006 | Published 4 April 2006
Estradiol attenuates mitochondrial depolarization in polyol-stressed lens epithelial cells
James M. Flynn, Patrick R.
Department of Cell Biology and Genetics, University of North Texas Health Science Center at Fort Worth, Fort Worth, TX
Correspondence to: Dr. Patrick R. Cammarata, Department of Cell Biology and Genetics, University of North Texas Health Science Center at Fort Worth, 3500 Camp Bowie Boulevard, Fort Worth, TX, 76107; Phone: (817) 735-2045; FAX: (817) 735-2610; email: email@example.com
Purpose: This study examined the state of mitochondrial physiology subsequent to exposing lens epithelium to high ambient galactose (Gal), which upon conversion to galactitol (GalOH) and resultant intracellular accumulation thereof, leads to profound destabilization of mitochondrial membrane potential (Δψm). Further, we determined whether the aldose reductase (AR) inhibitor, Sorbinil, or estrogen (17β-E2, and its isomer, 17α-E2, which exhibits marginal binding affinity for estrogen receptor), administered prior to and concomitant with Gal exposure might prevent or delay mitochondrial membrane depolarization.
Methods: Secondary cultures of bovine lens epithelial cells (BLECs), as well as a virally-transformed human lens epithelial cell line (HLE-B3), were maintained in 40 mM galactose (Gal) for up to seven days in the presence and absence of Sorbinil, 17β-E2 or 17α-E2. Endogenous accumulation of reactive oxygen species (ROS) was assessed by loading cells with H2DCF-DA, which upon oxidation in the presence of ROS transitions to the fluorescent compound, DCF. To assess Δψm, confocal microscopy was employed in conjunction with the potentiometric dye, JC-1. Intracellular polyol content was determined by gas chromatography. Cells were monitored for apoptosis and necrosis as determined by annexin V-propidium iodide staining and visualized by confocal fluorescence microscopy.
Results: BLECs, more so than HLE-B3 cells, accumulate high intracellular levels of GalOH upon exposure to high ambient Gal. BLECs were significantly depolarized while HLE-B3 cells showed little depolarization over the same course of Gal exposure. The addition of either 17α-E2 or 17β-E2 to BLECs, over a dose range of 0.01 μM to 1.0 μM, prevented mitochondrial membrane depolarization as did the addition of 0.1 mM Sorbinil. The polyol content in BLECs after 3 days of exposure to Gal was 282 nmol/mg protein. Co-addition of Sorbinil during the 3-day exposure period prevented any significant accumulation of GalOH. Co-administration of either isoform of estrogen did not block GalOH synthesis and the level of attained intracellular accumulation was similar to that of Gal alone. The observed accumulation of ROS from HLE-B3 cells subsequent to 3 days of Gal exposure was negligible and consistent with that of control cells maintained in physiological medium. Intracellular accumulation of ROS with 3-day, Gal-maintained BLECs, exhibited a marginal but statistically significant increase over control cells maintained in physiological medium (5.5 mM glucose) and similar levels of ROS were generated irrespective of the presence of estrogen with Gal. Bolus addition of 100 μM hydrogen peroxide to 3-day, Gal plus Sorbinil-maintained BLECs failed to induce a change in mitochondrial membrane potential. Evidence of apoptosis or necrosis was negligible through 7 days of sustained exposure to high ambient Gal.
Conclusions: Polyol accumulation promotes mitochondrial membrane depolarization and the decrease in Δψm is prevented by prior addition and co-administration of Sorbinil or estrogen with Gal. Unlike Sorbinil, estrogens' mode of action is not via the inhibition of aldose reductase activity. The data supports the theory that with Gal plus estradiol-treated cells, at a given intracellular polyol load, a larger portion of the mitochondrial population retains Δψm, and hence continues to function relative to Gal-treated cells. Results with 17α-E2 indicate that maintaining Δψm, in the face of chronic polyol accumulation, is likely to be mediated via a nuclear estrogen receptor-independent mechanism. The failure of supraphysiological levels of hydrogen peroxide added to Gal plus Sorbinil-maintained BLECs to depolarize mitochondria indicates that polyol accumulation, not ROS generation, is the causative factor responsible for the loss of mitochondrial membrane potential.
One complication of the diabetic condition is the formation of cataract in the lens. While surgical procedures can correct vision loss with the implantation of an intraocular lens; this presents a large financial burden on national health care systems. Thus, there is a need for pharmaceutical agents which prevent or delay the onset of cataract . The study described herein examined the protective effects of estrogen in lens epithelial cells, and, more to the point, the prevention of mitochondrial membrane depolarization in spite of a rapid and sustained rise in intracellular polyol content. These data establish a new mechanistic role for sex steroid hormones on lenticular pathophysiology and identifies the novel approach of targeting mitochondrial function to reduce the detrimental effects of polyol stress.
The ocular lens absorbs aldose sugars in a non-insulin dependant manner. Sugar levels within the lens cell are therefore determined by the ambient extracellular aldose concentration, thus making the tissue vulnerable to elevated blood glucose levels. The damage to tissues under hyperglycemic diabetic conditions is thought to be mediated in part through aldose influx via the polyol pathway. Via aldose reductase, this NADPH-dependant enzymatic reaction converts elevated levels of glucose or galactose to the polyols, sorbitol and galactitol, respectively. Polyols may promote damage to the cell due to osmotic stress or by the generation of reactive oxygen species, but may also exert further secondary stress through the loss (or significant lowering) of NADPH. For instance, use of the NADPH cofactor slows the regeneration of cellular glutathione pools causing oxidative stress .
A growing body of literature has established that estrogen functions as a cytoprotectant by activating genomic and non-genomic mechanisms in disparate tissues . Further support for the cytoprotective nature of estrogen may be found in a number of studies examining type two diabetes and hormone replacement therapy [4-6]. Sex hormone receptors and the mechanism of action of hormone receptors in the eye have been reported (for a review of the topic refer to ). Using cultured lens epithelium, we recently reported that estradiol prevents the collapse of mitochondrial membrane potential in the face of acute oxidative stress [8,9]. That data led us to examine the effect of polyol accumulation on mitochondrial function and to probe estradiol's cytoprotective ability to prevent the collapse of mitochondrial membrane potential in the face of chronic polyol stress.
The current study demonstrates that the potentiometric dye, JC-1, can be utilized to detect mitochondrial depolarization caused by intracellular polyol accumulation. We compared the effects of hyperglycemia on cultured human lens epithelial cells (HLE-B3) and bovine lens epithelial cells (BLECs). The comparison is not trivial as we have clearly shown in the past that intracellular polyol is dramatically increased in BLECs maintained in 40 mM galactose, the polyol level being greater than 325 nmol/mg protein . By contrast, intracellular galactitol content in HLECs was more than five times lower. The difference in accumulated polyol content probably reflects the relative levels of AR activity between the two species of cultured cells. Herein we show that HLECs exposed to Gal for up to five days are largely unaffected insofar as mitochondrial depolarization is concerned, whereas BLECs show extensive depolarization, preventable by aldose reductase inhibition. The role of polyols in destabilizing mitochondrial membrane potential is thus unquestionably established. Moreover, we further demonstrate that both 17β-E2 and 17α-E2 if co-administered with Gal delays, if not prevents, polyol-generated mitochondrial depolarization in BLECs despite the presence of elevated intracellular GalOH, consistent with the hormone's inherent capability to directly (or indirectly) stabilize mitochondria and increase mitochondrial tolerance to the polyol insult.
HLE-B3 cells, a human lens epithelial cell line immortalized by SV-40 viral transformation , were obtained from Usha Andley (Washington University School of Medicine, Department of Ophthalmology, St. Louis, MO). Cells were maintained in Eagle's minimal essential medium (MEM) supplemented with 20% fetal bovine serum (FBS; Hyclone Laboratories, Logan, UT), 2 mM L-glutamine, nonessential amino acids and 0.02 g/l gentamycin solution (Sigma Chemical Co., St. Louis, MO) at 37 °C and 5% CO2/95% O2. All experiments were performed with monolayers of HLE-B3 cells that did not exceed passage 20. Primary cultures of bovine lens epithelial cells (BLECs) were established from the aseptic dissection of bovine (Bos taurus) lenses and cultures were maintained in MEM supplemented with 10% bovine calf serum (BCS; Hyclone Laboratories, Logan, UT), 2 mM L-glutamine, nonessential amino acids and 0.02 g/l gentamycin solution as previously described . All experiments were performed with monolayers of BLECs that did not exceed passage 3. Cells were maintained in either hyperglycemic MEM containing 40 mM galactose (Gal) or normoglycemic minimal essential medium (MEM) containing 5.5 mM glucose (supplemented with 34.5 mM fructose to equalize the osmolarity).
1,3,5(10) estratrien-3, 17β-diol (17β-E2) and its stereoisomer, 17α-E2, were purchased from Steraloids, Inc. (Newport, RI). 17β-E2 is the native ligand for estrogen receptors while 17α-E2 exhibits little to no binding capacity to estrogen receptors . For all experiments, the estrogens were dissolved in 100% ethanol to a final stock concentration of 10 mM which was further diluted to achieve the desired concentration of steroid. The diluted estrogen solution was added to the cell cultures at 1 μl drug solution per ml of MEM resulting in a final working concentration from 0.01 μM to 1 μM. Cells receiving 17α-E2 or 17β-E2 were pre-incubated for at least 18 h prior to the addition of 40 mM galatctose. Control cells received an equivalent aliquot of ethanol.
In an alternative set of experiments, the aldose reductase inhibitor, Sorbinil (Pfizer Inc., Groton, CT) was also administered to the cells. The drug was directly dissolved into MEM and then placed on the cells at a final concentration of 0.1 mM. The cells were replaced with fresh Gal with or without estrogens or Sorbinil, or fresh MEM on a daily basis.
JC-1 staining and confocal microscopy
Following experimental treatments, cells seeded on 60 mm dishes were stained with the cationic dye, 5,5',6,6'-tetrachloro1,1',3,3'-tetraethyl-benzimidazolylcarbocyanine iodide (JC-1; Molecular Probes, Eugene, OR) as previously described  in order to determine the state of mitochondrial membrane potential. JC-1 is a potentiometric dye which exhibits a membrane potential dependent loss as J-aggregates (polarized mitochondria) transition to JC-1 monomers (depolarized mitochondria) as indicated by fluorescence emission shift from red to green . Therefore, mitochondrial depolarization is indicated by an increase in the green/red fluorescence intensity ratio. Briefly, culture medium was removed from adherent HLE-B3 and BLECs and the monolayers were rinsed one time with Dulbecco's Modified Eagle's Medium (DMEM) without phenol red (Sigma-Aldrich, St. Louis, MO). Cell monolayers were incubated with DMEM containing 10% serum and 5 μg/ml JC-1 at 37 °C for 30 min. Following the incubation, cells were rinsed two times with DMEM and images were obtained using a 10x objective on a confocal microscope (Zeiss LSM410) excited at 488 nm (for JC-1) set to simultaneously detect green emissions (510-525 nm) and red emissions (590 nm) channels using a dual band-pass filter.
The fluorescent dye, H2-DCFDA (Sigma-Aldrich, St. Louis, MO), was used to detect the presence of intracellular reactive oxygen species (ROS). H2-DCFDA is a nonfluorescent and nonpolar compound which upon diffusion into cells is hydrolyzed by cellular esterases to a polar compound and upon doing so is trapped within the cells. Intracellular DCFH is rapidly converted to the fluorescent compound, DCF in the presence of ROS. Cells were plated into 60 mm dishes and treated for three days under normoglycemic conditions as described above or with hyperglycemic conditions in the presence or absence of estrogens or aldose reductase inhibitor. After three days of experimental treatment the media was removed and the cells rinsed and then replaced with Dulbecco's Modified Eagle's Medium (DMEM) without phenol red. H2-DCFDA was dissolved in DMSO to a final 50 mM stock solution which was further diluted in DMEM to a final concentration of 50 μM. The cells were then incubated for 5 min at 37 °C and subsequently rinsed two additional times with DMEM and imaged immediately. Upon oxidation via interaction with ROS, the dye is cleaved forming the fluorescent compound DCF, excited at 488 nm and detected at 530 nm. This compound is also cleaved by exposure to UV light, thus permitting the use of UV excitation as a positive control to check for efficient dye loading into the cells, as previously reported by Chen et al., 2003 .
Polyol determination by gas chromatography
Cells were cultured on 100 mm dishes and treated as described above in hyperglycemic media with or without Sorbinil or estrogens or control MEM; after 3 days of incubation, the media was removed and the cells rinsed with PBS. The cells were then dispersed with trypsin and collected in 5 ml of PBS. The cells were subsequently centrifuged at 2,500 rpm for 4.5 min. The supernatant was removed and 500 μl of zinc sulfate was added to the cell pellet, the mixture snap frozen with liquid nitrogen and thawed at 30 °C four times and subsequently homogenized. The resulting suspension was transferred to a 1.6 ml tube and spun at 14,000 rpm for twenty min to pellet the cellular proteins. The supernatant was poured off and the protein pellet saved for protein determination to normalize the polyol content of the cells. To the supernatant was added 500 μl of barium hydroxide followed by centrifugation at 2,500 rpm for 8 min. After centrifugation, the resulting supernatant was lyophilized, reconstituted in Deriva-sil, and polyol content determined as described by Ramana et al., 2003 . It should be noted that this method does not distinguish between sorbitol and galactitol, samples are therefore reported as total polyol so that the data is recorded in nanomoles of polyol/milligram of protein.
Annexin V-FITC/propidium iodide cell death detection
To assess lens cell viability, a cell death detection assay was used which detects both apoptosis and necrosis. The Biovision annexin V-FITC apoptosis detection kit (Mountain View, CA) was used according to the manufacturer's directions in conjunction with confocal microscope imaging. Briefly, cells were grown in 35 mm tissue culture dishes and maintained under normoglycemic or high galactose conditions as described in the text. Cells were stained using both a fluorescent labeled annexin V conjugate and propidium iodide. Live cells were imaged using a band-pass filter for detection of the green FITC (excitation 488 nm, emission 530 nm) and the red propidium iodide (excitation 488 nm, emission 633 nm) wavelengths. Fluorescent green staining of the plasma membrane indicates apoptosis by the release of annexin V to the outer leaflet of the plasma membrane. Red staining of DNA with propidium iodide, in conjunction with green annexin V staining, indicates a loss of plasma membrane integrity typical of necrotic cells.
Statistics and image analysis
Statistical analysis for significant differences between treatment groups was performed with the statistics program SPSS (version 12; SPSS, Chicago, IL). The two-tailed student's t-test for independent samples was used for the comparison of data sets. All error bars are shown as the mean±SEM. A p value less than 0.05 was considered significant. The confocal images were analyzed with MetaMorph image analysis software (version 6.1; Molecular Devices Corporation, Downingtown, PA). The entire field of an image was utilized for quantitation of average fluorescence after the individual fluorescence channels for red and green were adjusted for background fluorescence and the data was evaluated as green to red ratio. Therefore, in a typical bar graph, the greater the ratio bar the larger the degree of mitochondrial membrane depolarization.
Polyol accumulation prompts depolarization of mitochondrial membrane potential
BLECs and HLE-B3 cells were cultured in either normoglycemic control media (minimal essential medium, MEM, consisting of 5.5 mM glucose adjusted with 34.5 mM fructose to equalize osmolarity), 40 mM Gal or Gal supplemented with aldose reductase inhibitor for five days. After five days of continuous exposure to Gal, cells were stained with JC-1 to determine the state of mitochondrial membrane potential. The HLE-B3 cells showed little change in their membrane potential. The bovine cells were severely depolarized as typified by the shift from red to green fluorescence (Figure 1A). Co-administration of Sorbinil in the culture medium effectively prevented the depolarization caused by exposure to 40 mM galactose. In contrast to HLE-B3 cells, which showed little mitochondrial membrane depolarization in the presence of Gal, BLECs, which were markedly depolarized in the presence of Gal, showed statistically significant stabilization against depolarization compared to Gal-exposed cells not treated with aldose reductase inhibitor (Figure 1B).
Effect of 17β-E2 and 17α-E2 on Δψm in BLECs
To elucidate whether estrogen lessens the degree of depolarization of mitochondrial membrane potential in cultured BLECs consequential to polyol insult, 17β-E2 and 17α-E2 were administered over a range of concentration which spanned 0.01 μM to 1.0 μM over a time course of three days with fresh administration of media and hormone daily. At the end of three days the cells were examined with the mitochondrial membrane potentiometric dye, JC-1. By three days, the mitochondria were clearly depolarized in Gal media relative to control MEM; both 17β-E2 and 17α-E2 over the entire dose range tested prevented depolarization (Figure 2A). The ratio of green to red fluorescence based on multiple random field confocal images is shown in Figure 2B. The data clearly demonstrates statistically significant prevention of mitochondrial depolarization using both the 17α- and 17β-isoforms of estradiol over the dose range employed.
Estradiol does not act as an aldose reductase inhibitor
Three day Gal-maintained BLECs had a polyol level of 282 nmol/mg protein compared to MEM control which contained only 36 nmol/mg protein (recall that the method employed to detect and quantify polyols does not distinguish between sorbitol and galactitol, hence, the amount of observed polyol reflects sorbitol accumulation). The concomitant administration of Sorbinil to the Gal medium completely blocked the accumulation of GalOH. BLECs exposed to Gal medium supplemented with 1 μM 17α-E2 or 17β-E2 contained a polyol level similar to that of Gal (Table 1).
Generation of reactive oxygen species
The production of ROS after three days of cell exposure to Gal was observed as the fluorescence generated upon rapid oxidation of DCFH to DCF within cells using confocal microscopy. Figure 3A depicts HLE-B3 cells maintained in Gal alone or supplemented with Sorbinil, 17α-E2, or 17β-E2. In all cases little difference in fluorescence intensity was observed from that of control cells maintained in physiological medium. In contrast, as a positive control, cells exposed to constant ultra-violet Laser (350 nm) and fluorescence were markedly stimulated indicating positive dye-loading quality. Statistical analysis of the images confirmed that there was no significant accumulation of ROS in the HLE-B3 cells exposed to hyperglycemic conditions over that of control cells (Figure 3B).
The DCF staining of the bovine lens cell cultures showed a small variation upon visual examination of the confocal images (Figure 4A). Cells exposed to Gal or Gal supplemented with 17α-E2 or 17β-E2 showed a slight increase in fluorescence intensity over that of control cells or cells maintained in Gal supplemented with aldose reductase inhibitor, which appeared to have a fluorescence intensity similar to that of control cells. Statistical analysis of the images verified that there was a small but statistically significant increase in the accumulation of ROS within cells exposed to 40 mM galactose media for three days (Figure 4B). As indicated in Figure 4A, and verified by statistical analysis, addition of either 17α-E2 or 17β-E2 to the hyperglycemic cultures did not attenuate this small generation of ROS as detected by DCF staining. The administration of Sorbinil, however, did reduce the average fluorescence generated from the DCF, to a level that was similar to that observed with cells maintained in control medium. Taken together, these observations suggested that ROS accumulation is linked to polyol accumulation such that when polyol generation is prevented by aldose reductase inhibition, ROS generation is also inhibited. Moreover, estradiol does not appear to function as a scavenger of free radicals.
In order to determine whether the low level of ROS generation observed with Gal-exposed BLECs was sufficiently high enough to affect mitochondrial membrane potential, the following experiment was performed. Cells maintained in control, physiological medium were treated with Sorbinil, 24 h prior to switching to Gal medium supplemented with fresh Sorbinil. Two days later, the medium was replaced with fresh Gal/Sorbinil and the cells maintained an additional 24 h (i.e., 3 days of Gal/Sorbinil supplementation) and a parallel set of plates were maintained in Gal medium alone. Under these conditions, the Gal/Sorbinil treated cells synthesize no polyols and generate little to no reactive oxygen species, whereas the Gal-treated cells generate a low but detectable level of ROS as shown above (Figure 4A,B). To each of two plates maintained in Gal/Sorbinil was added a bolus supplementation of either 25 μM, 50 μM, or 100 μM hydrogen peroxide and 90 min later, all cells were monitored for intracellular ROS by DCF staining and for the state of mitochondrial membrane potential by JC-1 staining as described in the methods. The relative amount of intracellular reactive oxygen species, as detected by DCF staining, clearly increased with Gal/Sor-treated cells supplemented with 25 μM hydrogen peroxide relative to Gal/Sor-treated cells alone (Figure 5A). The assay, having reached the saturation limits of detection, showed no further increase with additional supplementation to 50 μM or 100 μM hydrogen peroxide. However, the relative amount of ROS generation observed with Gal-treated cells was well below the level attained by the addition of 25 μM hydrogen peroxide to Gal/Sorbinil-treated cells. The state of mitochondrial membrane potential observed up through 100 μM bolus hydrogen peroxide was similar to Gal/Sorbinil-treated cells (Figure 5B). That is, the addition of 100 μM hydrogen peroxide, which produced an intracellular level of ROS which far exceeded the level of peroxide generated in Gal-exposed cells, failed to initiate mitochondrial membrane depolarization in cultured BLECs.
Apoptosis/necrosis marginally occurs in Gal-maintained BLECs
To determine whether cell death was the cause of the Gal-induced collapse in mitochondrial membrane potential, we examined annexin V-propidium iodide staining levels in Gal-sustained BLECs through seven days of exposure. Addition of high ambient Gal to BLEC cultures resulted in negligible annexin V and propidium iodide staining relative to control cells maintained in physiological medium (Figure 6), indicative of little apoptotic and necrotic cell death attributable to Gal or GalOH intracellular accumulation. BLECs supplemented with Gal plus 17α-E2 or 17β-E2, likewise, appeared similar to control cells, indicating that neither Gal or estrogen prompted cell death via apoptosis or necrosis.
Hyperglycemia is held to be the cause of many diabetic complications including retinopathy, neuropathy, nephropathy, macrovascular and microvascular injury, and cataract. Oxidative stress resulting from hyperglycemia-induced elevation in the production of mitochondrial ROS is alleged to play a major role in the etiology of diabetic complications. Nishikawa et al.  provided compelling data as to the role of ROS generation in arbitrating hyperglycemic-induced cell damage. Hyperglycemia activated the transcription factor, NF-κB and subsequently, intracellular reactive oxygen species, protein kinase C (PKC) activity and polyol levels increased. Interruption of mitochondrial ROS generation prevented each of the hyperglycemic-induced effects mentioned above. To achieve the disruption of mitochondrial ROS generation these researchers resorted to "poisoning electron transport" in order to demonstrate that hyperglycemic exposure elicits said effects so that, therefore, "collapsing mitochondrial function" prevents the onset of ROS generation, PKC activation, and polyol accumulation. Their data suggested that hyperglycemic-induced oxidative stress is an early onset result of elevated glucose and that activation of NF-κB represents an initial signaling incident which then goes on to galvanize other pathways which ultimately leads to cell damage and eventual cell death. Our experimental approach advances their original observation, in that, we were able to demonstrate that estrogen stabilizes mitochondrial membrane potential albeit, "without collapsing mitochondrial function", in the face of hyperglycemic exposure, likely by increasing mitochondrial tolerance to reactive oxygen species and/or elevated intracellular polyols. Indeed, our data clearly established similar GalOH levels in 40 mM galactose-exposed cells regardless of whether estrogen was present or absent (Table 1).
Reactive oxygen species cause profound injury to a diverse number of intracellular macromolecules in eukaryotes. This damage includes lipid peroxidation, protein alteration, breakage of covalent bonds of carbohydrates and cleavage of DNA strands. Mitochondria have been found to be particularly susceptible to oxidative damage. As stated by Crawford et al. , "Release of calcium, depletion of ATP, lipid peroxidation, protein oxidation, DNA damage, loss of electron transport capacity, and other types of oxidant-induced mitochondrial damage have been reported." Down-regulation of a number of mitochondrial gene transcripts has been described by several laboratories [19,20] as a result of oxidative-induced injury.
The last stage of cellular oxidative metabolism develops on the electron transport chain of mitochondria. Electron transport is the process wherein electrons shuttle along respiratory enzymes of the inner mitochondrial membrane and in doing so manifest the generation of ATP. This modus operandi is generally well-organized and efficient but if compromised by hyperglycemic insult, results in the production of elevated levels of intracellular reactive oxygen species (ROS). The over-accumulation of ROS probably leads to irreversible injury to components of crucial cell functions. Indeed, there is evidence linking oxidative stress with cataractogenesis. ROS-induced damage is associated with alteration to cation levels and severe inhibition of lens Na,K-ATPase . Inability to detoxify H2O2 due to the inhibition of the glutathione redox cycle results in the disturbance of membrane cytoskeleton, the formation of distinct vacuoles in the anterior region of the lens at the germinative zone between the epithelium and lens fibers and formation of epithelial cell blebs .
Our experimental findings permitted us to make the distinction as to whether mitochondrial membrane depolarization in Gal-exposed BLECs was initiated solely by polyol accumulation, the small but observable increase in ROS accumulation or a synergistic interplay between both polyol and ROS insult. Administration of supraphysiological doses of hydrogen peroxide to Gal/Sorbinil-treated cells failed to bring about mitochondrial depolarization (Figure 5B.) This result was anticipated as we had previously reported that BLECs contain substantial intracellular levels of GSH and that only by the addition of L-buthionine sulfoximine (an inhibitor of glutathione biosynthesis) could marked depolarization of mitochondria be induced by bolus peroxide supplement . However, the inability of bolus peroxide to prompt mitochondrial depolarization in Gal/Sorbinil-treated cells, coupled to the observation that relative ROS generation in Gal-exposed cells (which were depolarized) was well below the detection level of intracellular ROS seen with the addition of 25 μM hydrogen peroxide to Gal/Sorbinil-treated cells (Figure 5A) sanctions the conclusion that ROS accumulation in Gal-exposed BLECs played little to no part in mitochondrial depolarization. We hasten to add that this conclusion may or may not hold true for all species of cultured lens epithelia as intracellular GSH levels likely differ from species to species. Our results, however, clearly establish that estrogen stabilized mitochondrial membrane potential in spite of the continued presence of elevated polyols (Figure 2A,B, Table 1).
Studies using tissue culture and animal models have suggested beneficial effects of estrogen in lens to prevent or delay the onset of cataractogenesis. In a lens culture system, estrogen protected lenses against cataracts induced by transforming growth factor-β (TGFβ) . A transgenic mouse model expressing a dominant-negative form of estrogen receptor α, which inhibits estrogen receptor α function, was recently used to show spontaneously formed cortical cataracts in female mice after puberty which progressed with age, suggesting that repression of (nuclear) estrogen action induces cortical cataract . Estrogen has also been reported to exert protective effects in a rat model of age-related cataracts induced by methylnitrosourea (MNU) . The reported effects of estrogen do not appear to require estrogen receptors strongly suggesting that estrogen exerts antioxidant activities through estrogen receptor-independent mechanisms . Data presented in the current study with 17α-E2, which exhibits negligible receptor binding , further supports the notion that cytoprotection by estrogen (against polyol-induced mitochondrial depolarization) potentially does not require nuclear estrogen receptors (Figure 2A,B). The reader should note that we choose to specify "nuclear" estrogen receptors (see below). The action of estrogen to stabilize mitochondria against depolarization, apparently independent of nuclear estrogen receptor, is particularly interesting considering that we have recently shown that the wild type estrogen receptor β resides in mitochondria, as well as in the nucleus [27,28]. Warner and Gustafsson  recently stated, "There has been much written on whether the nongenomic effects of E2 are mediated by membrane-bound ERs and whether or not these receptors are identical to the nuclear ERs. Since the plasma membrane is not a barrier for E2 entry into cells, it is not necessary for estrogen receptors (whatever their nature) to be membrane-bound in order for them to be activated by E2 and trigger changes in ion channels or kinases at the cell surface. However, the nature and location of the receptor might have a profound effect on its affinity for E2 and this might explain why many rapid effects of E2 are observed at concentrations higher than 1 nM, which is the concentration of E2 at which maximal activity of the nuclear receptor is achieved." Therefore, the reader is cautioned, that interpretation of results using 17α-E2 presented in the current study, with regards to nuclear estrogen receptor dependency, is based upon the use of pharmacological doses (>1 nM) of estradiol.
However, we previously reported that 17α-estradiol and 17β-estradiol equipotently increased the amount of Ca2+ or H2O2 required to collapse mitochondrial membrane potential in human lens epithelial cells, effectively stabilizing mitochondrial integrity and preserving function under pathogenic conditions . This effect did not require prolonged exposure to the estrogens, as we have shown that 5 and 30 min incubations elicit a response, providing suggestive evidence for rapid, non-genomic action of the estrogens.
The cytoprotective stabilization of mitochondrial membrane potential by estrogens may be attributed to consolidation of several mechanisms of action. The restraint of Δψm collapse might be explained by a repression of Ca2+ uptake into the mitochondria, increased tolerance to mitochondrial calcium sequestration, increased Ca2+ efflux from the mitochondria, increased resorption of Ca2+ into endoplasmic reticulum, increased efflux of Ca2+ via the plasma membrane and/or direct mitochondrial membrane stabilization via binding of estradiol. The exact mechanism(s) regarding how estrogen exerts its beneficial effects against hyperglycemia is unknown to date. In addition to maintaining mitochondrial integrity as discussed above, it is also plausible that estrogen may oppose or protect against the toxic action polyols and/or ROS (i.e., oxidative stress) accumulation by indirect (mitochondrial) membrane stabilization via activated signal transduction pathways. H2O2 has been shown to activate signaling pathways such as the stress activated protein kinase (SAPK)/c-Jun NH2-terminal kinase (JNK) pathway in human lens epithelial cells  and the p38 pathway (also activated during cell stress) in human leukemia cells . H2O2 addition to culture medium induces apoptosis or programmed cell death . Activation of these types of "stress pathways" is associated with apoptosis and suppressing these pathways either via direct inhibition or by stimulation of "survival" pathways like the phosphatidylinositol 3-OH kinase (PI3-K)-Akt pathway has been observed to regulate apoptotic progression . Estrogen activation of "survival" pathways has, to date, not been explored in great detail but nevertheless represents a potential mechanism for protection against polyol-induced and ROS-induced apoptosis. We recently reported that 17β-E2 prevented H2O2 induced injury to several oxidant susceptible components of the cellular ATP generating machinery, including abundances of mitochondrial gene transcripts encoding respiratory chain subunits and cytochrome c, the glycolytic pathway enzyme, glceraldehyde-3-phosphate dehydrogenase, and the energy-shuttling creatine kinase system, as well as mitochondrial membrane potential, thereby preserving the driving force for ATP synthesis . Moreover, we have shown that 17β-E2 acts as a positive regulator of the survival signal transduction pathway, MAPK which, in turn, acts to stabilize Δψm. In effect, the relative degree of ERK phosphorylation positively correlated with attenuation of the extent of depolarization of mitochondrial membrane potential regardless of acute oxidative stress . Whether stimulation of ERK 1/2 likewise influences mitochondrial stability in the face of chronic polyol stress is currently under active investigation in this laboratory.
Moreover, that mitochondrial membrane potential decreased during an early stage of Gal exposure in BLECs, before significant cell death was evident, indicates that mitochondrial membrane depolarization is an early-onset, and likely crucial, event in the cell death pathway.
In summary, our data demonstrates that estrogen protects against mitochondrial membrane depolarization despite the continuous generation of polyols resulting from exposure of bovine lens epithelial cells to hyperglycemic conditions in cell culture. It is to be expected that the protective mechanism by which 17β-E2 operates will prove to be multifactorial such that activation of both genomic and non-genomic pathways integrate so as to put forth a combinatorial mitochondrial defensive condition in order to resist oxidative insult. Our goal is to demonstrate that estrogens or phytoestrogens, which are estrogenic-like compounds which lack estrogenic activity, could provide useful therapies for the delay or prevention of hyperglycemic-induced cataracts in post-menopausal women and that non-feminizing estrogen could provide similar protection in men because of the lack of undesirable side effects. Moreover, we are interested in further characterizing the heretofore unappreciated association between estradiol-stimulated activation of specific signal transduction pathways as it relates to the mechanism of stabilization of mitochondrial membrane potential recently uncovered in our laboratory . Targeting mitochondrial function so as to increase tolerance to polyol and/or ROS insult characterizes a novel concept which will contribute to innovative regimens to prevent or delay onset of the adverse consequences of hyperglycemia.
This work was supported by a Public Health National Service Award, EY05570 (PRC). The authors thank Dr. Satish Srivastava and co-workers (University of Texas Medical Branch, Galveston, TX) for polyol determinations. This work was first reported at the 2005 ARVO meeting in Fort Lauderdale, FL.
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