Responses of Human Lens Epithelial Cells to Quercetin and DMSO

Abstract

purpose. Oxidative stress is an initiating factor in the development of maturity-onset cataract. Diet has a significant impact on cataract development, and individual dietary components responsible for the protective effect include flavonoids, of which quercetin is the most important. The purpose of this study was to investigate the protective effect of quercetin and its toxicity for human lens epithelial cells (HLECs).

methods. HLECs in culture were incubated for 48 hours with either 1% (vol/vol) dimethyl sulfoxide (DMSO) alone or with this concentration of DMSO and between 0.1 and 100 μM of quercetin. Nonstimulated cells served as control cultures. The viability of HLECs was measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) colorimetric assay. Gene expression was assessed with reverse transcription-polymerase chain reaction (RT-PCR). Cellular apoptosis was examined by in situ immunocytochemistry using terminal deoxynucleotidyl transferase-mediated biotin-dUTP nicked labeling (TUNEL) and by flow cytometry, using annexin V-FITC apoptosis detection.

results. DMSO (1% vol/vol) decreased cell viability, increased cellular apoptosis, and upregulated Bax in these cells; 0.1 μM quercetin inhibited these effects and protected HLECs from the toxicity of DMSO. Higher concentrations of quercetin the viability of HLECs decreased. In a dose-dependent response to quercetin, cellular apoptosis increased and the change correlated with upregulation of Bax and decreased cell viability.

conclusions. Quercetin, at a low concentration (0.1 μM), protects HLECs and reverses the toxic effects of DMSO (1% vol/vol). However, at higher concentrations, quercetin is toxic to HLECs with an LD₅₀ of 90.85 μM. Quercetin induced apoptosis and upregulates apoptotic genes in HLECs in a dose-dependent manner.

Cataract is defined as any opacity of the lens that affects vision. It is the major cause of blindness worldwide. Oxidative stress is an initiating factor in the development of maturity-onset cataract. ¹ Evidence suggests that H₂O₂ is a major oxidant involved in cataract formation in humans. ²  

The flavonoids are a group of polyphenolic compounds with diverse chemical structures and characteristics that exert a wide range of antioxidant properties. ³ They are found ubiquitously in plants and are major components of human dietary antioxidant intake. ⁴ There is epidemiologic evidence that a sufficient intake of fruit and vegetables can lower the risk of cataract in humans. ⁵ Furthermore, researchers have shown that quercetin, a major dietary flavonoid, is an effective inhibitor of H₂O₂-induced lens opacification. ⁶  

In investigating the role of quercetin in relation to cataract development, it is important to understand both the antioxidant property and the toxicity of quercetin in human lens epithelial cells (HLECs). The purpose of this research was to investigate the toxicity and the damage mechanism of quercetin on HLECs. 

Materials and Methods

Media and reagents included Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen-Gibco, Grand Island, NY), fetal bovine serum (FBS; Invitrogen-Gibco), phosphate-buffered saline (PBS), Hanks’ balanced salt solution (HBSS), trypsin-EDTA solution, penicillin, streptomycin, dimethyl sulfoxide (DMSO; -Invitrogen-Gibco BRL), and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT). A carbon dioxide (CO₂) incubator (Heraeus; Pacific Laboratory Products, Blackburn, VIC, Australia), a sterile bench, a phase-contrast microscope (Olympus, Tokyo, Japan), a centrifuge, a spectrophotometer, pipettes, culture flasks, and tubing (Corning, Corning, NY) were used. All the other reagents were obtained from Sigma unless specified otherwise. Quercetin was a kind gift from Yi Chen, MD (People’s Hospital of Peking University, Beijing, China). 

Cell Culture

HLECs (SRA 01/04 ⁷ ) were cultured at 37°C in DMEM supplemented with 20% FBS, 100 U/mL penicillin, and 100 U/mL streptomycin in a humidified atmosphere containing 5% CO₂. Cells were cultured and passed in 1:4 split ratio (approximately 5 × 10⁴ cells/cm²) after confluence using 0.025% trypsin, 0.02% EDTA in Ca²⁺, and Mg²⁺-free HBSS, and were harvested by trypsin-EDTA digestion at the times indicated. The length of time between passages was 4 days. 

Preparation and Incubation of Quercetin on HLECs and Description of Groups

Quercetin in DMSO or DMSO alone was added at the concentration indicated in Table 1 , and the final concentration of DMSO was 1% (vol/vol). Control HLECs remained in medium alone for the duration of the experiment. HLECs were maintained in medium for 24 hours before exposure to the indicated final concentrations of quercetin and DMSO at 37°C in 5% CO₂ environment for 48 hours. Nonstimulated cells served as the control. At the indicated time points, the cells were collected and evaluated for viability, incidence of apoptosis, and occurrence of specific gene transcripts. 

Description of Groups

The study groups were as follows: Control group (N): nonstimulated HLECs; group 0: HLECs in 1% (vol/vol) DMSO; group 0.1: HLECs in 0.1 μM quercetin and 1% (vol/vol) DMSO; group 1: HLECs in 1 μM quercetin and 1% (vol/vol) DMSO; group 10: HLECs in 10 μM quercetin and 1% (vol/vol) DMSO; group 50: HLECs in 50 μM quercetin and 1% (vol/vol) DMSO; and group 100: HLECs in 100 μM quercetin and 1% (vol/vol) DMSO. 

Measurement of Cell Viability

The MTT assays, which are frequently used to measure cell proliferation and cytotoxicity, were used to verify the viability of HLECs incubated with quercetin. Immediately after cells in a 96-well plate were incubated with quercetin for 48 hours (concentrations shown in Table 1 ), 20 μL of stock MTT solution (5 mg/mL) was added to each well, and cells were incubated for 4 hours at 37°C in a 5% CO₂ environment. The medium was aspirated from the well without disturbing the formazan crystals. DMSO (100 μL) was added to each well, the plates were shaken for 10 minutes on a plate shaker, and the absorbance at 570 nm was measured by ELISA reader. Cell viability was calculated from the absorbance ratios in the control group and the sample group. 

In Situ Apoptosis Detection

Apoptotic cells were detected using a cell-viability kit (In Situ Cell Death Detection Kit, POD; Roche, Indianapolis, IN), according to the manufacturer’s instructions. This type of assay is often referred to as the TUNEL method. 

Detection of Apoptosis by Flow Cytometry

For evaluation of apoptosis, floating and carefully trypsinized cells were collected and washed in PBS. Staining with an annexin V-FITC apoptosis detection kit (Peking University Center for Human Disease Genomics, Beijing, China), according to the manufacturer’s instructions, and with a combination of fluoresceinated annexin V (annexin V-FITC) and propidium iodide (PI). Ten thousand events per sample were acquired and analyzed by flow cytometry (FACSCalibur; BD Biosciences, Franklin Lanes, NJ). 

RNA Isolation and RT-PCR

The total cellular RNA was isolated (TRIzol reagent; Invitrogen-Gibco). RNA was reverse transcribed by using a cDNA synthesis kit (Promega Corp. Madison, WI). Then, the cDNA was amplified in a 20-μL reaction mixture by PCR the following conditions: denaturation for 60 seconds at 94°C, annealing for 45 seconds at 60°C, and extension for 60 seconds at 72°C, for 35 cycles in a thermal controller (model PTC-200, MJ Research, Watertown, MA). The primer sequences specific for the genes examined and predicted sizes are in Table 2 . Each PCR product was run on an agarose gel to confirm its product size. DNA size markers (Promega Corp.) were run in parallel, to validate the predicted sizes of the amplified bands. Analysis of quantification and melting curve was performed (Gel-pro Analyzer 4.5; Media Cybernetics, Silver Spring, MD). 

Statistical Analysis

Results are expressed as means ± SD. Statistical significance was determined by Student’s t-test to determine specific differences between groups, and regression analysis was calculated by linear regression. The median lethal dose (LD₅₀) was calculated by probit analysis (SPSS ver. 11.5; SPSS, Chicago, IL). P < 0.05 was considered statistically significant. 

Results

Effects of DMSO on the Viability of HLECs Incubated with Quercetin

Compared to control cells, the viability of cells incubated with 1% (vol/vol) DMSO decreased ≥10%, which showed that DMSO was toxic for HLECs (t = −3.055; P = 0.038). Incubation with 0.1 μM quercetin reversed the effect of 1% (vol/vol) DMSO on the HLECs (t= −3.323; P = 0.029). However, as the concentration of quercetin in the medium was increased, the viability of the HLECs decreased (Fig. 1). 

In Situ Apoptosis Detection

Figure 2shows that the number of TUNEL-positive nuclei (green fluorescence) increased with increasing concentration of quercetin. Of course, there were more apoptotic cells in samples incubated with DMSO than in control cells. 

Detection of Apoptosis by FCM

The apoptotic ratio in control cells was 2.90% (Fig. 3 , group N). Although there was an obvious increase of apoptotic cells induced by DMSO (Fig. 3 , group 0), incubation with 0.1 μM quercetin prevented cellular apoptosis (Fig. 3 , group 0.1). With an increasing final concentration of quercetin in the medium, the apoptotic cells ratio of HLECs increased. The computed data (SPSS ver. 11.5; SPSS) showed that there was a linear relationship between the apoptotic ratio of HLECs and the final concentration of quercetin in the medium (correlation coefficient R ² = 0.873, t-test t= 5.238, P = 0.006). 

Expression of mRNA by Apoptotic Genes

We determined whether incubation with quercetin and DMSO could influence the expression of apoptotic genes in HLECs. As shown in Figure 4 , expression of Bax mRNA increased with increasing final concentration of quercetin in the medium. Bcl-2 and Bcl-xl mRNA was not expressed in any group. 

Discussion

Many studies have indicated that dietary quercetin and its metabolites are active in inhibiting oxidative damage to the lens by inhibiting aldose reductase and thus may have a role in prevention of cataract formation, such as sugar cataract and hydrogen peroxide-induced cataract. ^{8 9 10 11 12 13} Quercetin (10 μM) and 3′-O-methyl quercetin (10 μM) inhibit hydrogen peroxide (500 μM)-induced sodium and calcium influx, and lens opacification. ¹² A previous study showed that individuals with high plasma levels of vitamin C, vitamin E, and carotenoids appear to have a reduced risk of cataract, ¹⁴ and several studies have demonstrated that diets rich in fruit and vegetables, and therefore flavonoids, protect against cataract formation. ^{15 16} In one study, tea, a major source of dietary quercetin and other flavonoids, was also shown to be protective. ¹⁷  

Quercetin is fat soluble, and DMSO is the most commonly used hydrotrope to help quercetin dissolve in water. DMSO can be used to conserve cells in liquid nitrogen and can protect cells at low temperature. Moreover, DMSO can trap the free radical hydroxide and is often used as a free radical scavenger in experiments. At the same time, DMSO is poisonous to cells at normal temperature. Earlier studies have reported that cataracts occur in guinea pigs and rabbits administered DMSO cutaneously or subcutaneously, and a peculiar transient cataract was also observed in dogs. ^{18 19} In our study, the final concentration of DMSO in the medium was 1% (vol/vol), which is higher than that used in the previous studies in which the concentrations of DMSO were 0.1%–0.25% (vol/vol). ^{6 20} Our study showed that HLECs in 1% (vol/vol) DMSO had lower viability and a higher level of apoptosis, with upregulated expression of the apoptotic promoter Bax. Previous studies on quercetin did not mention any toxic effect of DMSO on HLECs incubated with quercetin. 

Our study showed that 0.1 μM quercetin could reverse the detrimental effects of 1% (vol/vol) DMSO on HLECs. HLECs that were incubated with 0.1 μM quercetin and 1% (vol/vol) DMSO had almost the same viability and only a slight difference in cellular apoptosis from that in the control cells, but they had a higher expression of Bax than did the control cells. This finding was evidence that 0.1 μM quercetin could protect HLECs from the effects of DMSO at 37°C. We used a very low concentration of quercetin compared with that used in previous studies. It also may be related to the fact that our study used HLECs, and previous studies used rodent whole lens, which has a capsule that could prevent or retard the entry of quercetin into the LECs. 

As DMSO is a free radical scavenger, the toxicity of DMSO for HLECs was not due to oxyradicals. Previous studies reported that DMSO could induce apoptosis in some cells, such as EL-4 and PSV1, which might be due to the effects of DMSO in modulating the cell cycle. ^{21 22} Spin-trapping studies have indicated that fresh butadiene soot in a buffered aqueous solution containing DMSO oxidizes the DMSO, leading to formation of the CH₃ radical. ²³ The protection of quercetin against the detrimental effects of DMSO on HLECs needs further investigation. 

In our study, the viability of HLECs incubated with quercetin decreased in a dose-dependent manner, and cellular apoptosis increased. On the basis of the debility of HLECs incubated with quercetin, the LD₅₀ of quercetin on HLECs with 1% (vol/vol) DMSO was 90.85 μM (95% confidence limits: 65.0, 158.6 μM), which is very low compared with the results of previous studies. ^{13 14 15} There may be several reasons for this result. First, we used HLECs, and so the concentration of quercetin was in the cells equal to that in the intercellular substance of whole lens, not equal to that in the medium. Second, HLECs were incubated with quercetin for 48 hours in our study, much longer than in the previous studies. As quercetin in our bodies is persistent, the long-term toxicity of quercetin is worthy of study. Third, the use of different species was a very important factor. 

Previous studies have shown that quercetin is an inhibitor of heat-shock protein (HSP). ²⁴ It could decrease the expression of HSP-27, -70, and -90, and heat shock factors (HSF)-2 and -4. Quercetin pretreatment significantly reversed the decrease in caspase-1, -6, and -8 activity and the antiapoptotic effect of a proteasome inhibitor on IFN-γ-treated LECs. Some in vitro studies have revealed that quercetin interacted directly with DNA, to stabilize its secondary structure, but prolonged exposure to quercetin could result in disruption of the double-helical configuration. ²⁵ Under mutagenic conditions (aerobic, Cu²⁺, or Fe³⁺), Sahu and Washington, ^{26 27 28} demonstrated that quercetin damaged the DNA of isolated rat liver nuclei. However, in the presence of certain antioxidants, such as mannitol and catalase, DNA was protected from quercetin-induced damage. By contrast, other antioxidants such as glutathione and diallyl sulfide stimulated quercetin-related damage to DNA, whereas superoxide dismutase had no effect. These observations suggest that hydroxyl radicals were responsible for quercetin-related damage to DNA. Damage was not restricted to DNA. Sahu and Washington ²⁷ found that, under the same experimental conditions, the pro-oxidant activity of quercetin enhanced lipid peroxidation of rat liver nuclei, particularly in the presence of Fe³⁺. Furthermore, lipid peroxidation by quercetin was inhibited by mannitol or glutathione. Moreover, catalase had no effect on quercetin-induced lipid peroxidation, but superoxide dismutase, ascorbic acid and diallyl sulfide stimulated quercetin-induced peroxidation. This behavior suggested strongly that the pro-oxidant or antioxidant activity of quercetin depends on the redox state of its biological environment. 

Our study showed that cellular apoptosis was increased very close to the debility of HLECs incubated with quercetin in a dose-dependent manner, and so we hypothesize that the debility of HLECs incubated with quercetin is the result of the HLEC apoptosis induce by quercetin. The gradually upregulated expression of Bax mRNA in HLECs incubated with quercetin served as evidence. 

In summary, we showed that quercetin at a low concentration (0.1 μM), could protect HLECs and reverse the toxic effects of 1% (vol/vol) DMSO. Our study suggests that quercetin has toxicity for HLECs with an LD₅₀ of 90.85 μM. Quercetin induced apoptosis and upregulated apoptotic genes in HLECs in a dose-dependent manner. 

 Supported by Grant 2001BA611B-07 from the National Key Technologies R&D Program China.

 Submitted for publication October 30, 2006; revised January 9, February 26, and March 14, 2007; accepted May 15, 2007.

 Disclosure: X.-G. Cao, None; X.-X. Li, None; Y.-Z. Bao, None; N.-Z. Xing, None; Y. Chen, None

 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

 

Table 1.

 

The Final Concentrations of Quercetin and DMSO

Groups	Control	0	0.1	1	10	50	100
Quercetin (μM)	0	0	0.1	1	10	50	100
DMSO % (vol/vol)	0	1	1	1	1	1	1

Table 2.

 

Primer Sequences Specific for the Genes Examined and Predicted Sizes

Gene	Primers			Expected Product Sizes (bp)
GADPH	5′-GCAGGGGGGAGCCAAAAGGG-3′	5′-CTGTCCCTGCTGCACTTTTGT-3′	566
Bax	5′-TGCCAGCCCCAGCGTCAAAG-3′	5′-TCTTCCGCCGGTTGGTCTGTT-3′	365
Bcl-2	5′-ACCAAGAAGCTGAGCGAGTGTC-3′	5′-TCGTCTGCATCTCACTCAT-3′	247
Bcl-xl	5′-ACAAAGATGGTCACGGTCTGCC-3′	5′-GATAAATCTCTGCCTCACG-3′	777

Figure 1.

 

MTT showing quercetin and DMSO-induced viability and debility changes in HLECs. HLECs were maintained in (group N) DMEM alone; (group 0) incubated with 1% (vol/vol) DMSO; (group 0.1) incubated with 0.1 μM quercetin and 1% (vol/vol) DMSO; (group 1) incubated with 1 μM quercetin and 1% (vol/vol) DMSO; (group 10) incubated with 10 μM quercetin and 1% (vol/vol) DMSO; (group 50) incubated with 50 μM quercetin and 1% (vol/vol) DMSO; and (group 100) incubated with 100 μM quercetin and 1% (vol/vol) DMSO for 48 hours at 37°C. Data are expressed as the mean ± SD; n ≥ 3.

Figure 2.

 

TUNEL labeling showing quercetin and DMSO-induced apoptosis of HLECs, which were cultured in the solutions described in Figure 1for 48 hours at 37°C (DM 4000 B/M fluorescence microscope, Leica, Heidelberg, Germany). Magnification ×400.

Figure 3.

 

Flow cytometry graphs showing quercetin and DMSO-induced apoptosis of HLECs which were maintained in the solutions described in Figure 1 . for 48 hours at 37°C. Right: E, the apoptotic ratio of control and treated HLECs.

Figure 4.

 

Expression of mRNA by apoptotic genes for control and treated HLECs. Lane M, DNA marker. The remaining lanes represent the groups and cell culture solutions described in Figure 1 . GAPDH was used as a standard. Similar results were obtained from three independent experiments.