Human hepatocellular carcinoma HepG2 cell lines were purchased from the Korean Cell Line Bank (Seoul, Korea). Cells were routinely maintained in minimum essential medium [Life technologies (Molecular Probes), Carlsbad, CA, USA], supplemented with 10% fetal bovine serum (FBS) and antibiotics (50 U/ml penicillin and 50 μg/ml streptomycin; Sigma-Aldrich Co. LLC., St. Louis, MO, USA) at 37°C in a humidified atmosphere containing 5% CO₂.

Equol, daidzein and daidzin were purchased from LC Laboratories^® (Woburn, MA, USA) and synthesized O-DMA was a gift from Dr Lee (Professor of Chemistry Department, Duksung Women’s University, Republic of Korea), these were dissolved in dimethylsulfoxide (final concentration 0.1% in medium).

Cytotoxicity was evaluated by lactate dehydrogenase (LDH) release and an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cells were plated at a density of 1×10⁵ cells/well in a 96-well tissue culture plate (Corning Incorporated Life Sciences, Tewksbury, MA, USA) and incubated at 37°C for 24 h. Plated cells were treated with indicated concentrations of the molecules, O-DMA, equol, daidzein and daidzin. Following 72 h treatment, to determine the LDH release, 100 μl/well supernatant medium was transferred into corresponding wells of an optically clear 96-well flat bottom microtiter plate and an LDH cytotoxicity detection kit (Takara Bio Inc., Shiga, Japan) was used. Following treatment and incubation, plated cells were incubated with MTT (Sigma, St. Louis, MO, USA; 0.5 mg/ml final concentration) for 4 h at 37°C. When all medium from the plates had been discarded, 100 μl DMSO was added to each well. The plates were placed at room temperature for 5 min with agitation, so that complete dissolution of formazan was achieved. The absorbance of MTT formazan was determined at 540 nm by a ultraviolet/visible spectrophotometric plate reader (Emax; Molecular Devices, Sunnyvale, CA, USA).

Catalase activity was assayed according to Aebi (11). Catalase activity was calculated as nmol of H₂O₂ decomposed/min/mg/protein. Superoxide dismutase (SOD) activity was assayed according to the pyrogallol autoxidation method of Marklund and Marklund (12). Each unit of SOD activity was defined as the quantity of enzyme that inhibited the auto-oxidation of pyrogallol by 50% under experimental conditions. Protein concentration was determined by a Bradford protein assay kit II (Bio-Rad Laboratories, Hercules, CA, USA).

Cells were lysed in RIPA buffer [1% NP-40, 150 mM NaCl, 0.05% deoxycholic acid, 1% SDS and 50 mM Tris (pH 7.5)] containing protease inhibitor at 4°C for 1 h. The supernatant was separated by centrifugation and protein concentration was determined by a Bradford protein assay kit II. Proteins (25 μg/well) denatured with sample buffer were separated by 10% SDS-polyacrylamide gel. Proteins were transferred onto nitrocellulose membranes (0.45 μm). The membranes were blocked with a 1% bovine serum albumin solution for 3 h and washed twice with phosphate-buffered saline containing 0.2% Tween-20 and incubated with the primary antibody at 4°C overnight. Antibodies against catalase, CuZn-, Mn-SOD and β-actin were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA) and used to probe the separate membranes. The following day, the immunoreaction was continued with the secondary goat anti-rabbit horseradish peroxidase-conjugated antibody Santa Cruz Biotechnology, Inc. following washing for 2 h at room temperature. The specific protein bands were detected with an Opti-4CN Substrate kit (Bio-Rad Laboratories).

Samples were homogenized with TRIzol (Gibco-BRL, Carlsbad, CA, USA) and mRNA was extracted according to the manufacturer’s instructions. First-strand cDNA was synthesized using SuperScript First-Strand Synthesis system (Invitrogen Life Technologies, Carlsbad, CA, USA). Each target mRNA expression was quantified by quantitative PCR with the use of CFB-3120 MiniOpticon™ system (Bio-Rad Laboratories, Inc.). The CFB-3120 MiniOpticon system uses an array of 48-light-emmitting diodes, which efficiently excite fluorescent dyes with absorption spectra between 470 and 505 nm. PCR reactions were performed with 2X SYBR^®-Green mix (Finnzymes, Vantaa, Finland). Each mRNA level was calculated by means of the comparative cycle threshold (Ct) method using 2^−ΔΔCt, according to the manufacturer’s instructions. GAPDH was used as an endogenous control (internal control). The fold change in target gene relative to the endogenous control was determined as fold change = 2^−ΔΔCt ; where ΔΔCt = (Ct_target − Ct_endogenous)_{treated group} − (Ct_target − Ct_endogenous)_{control group}. The untreated sample (control group) was defined as the calibrator in this experiment. Therefore, the quantities of catalase, CuZn-SOD and Mn-SOD transcripts in the other samples were assigned arbitrary units relative to the levels in the calibrator sample.

All values are expressed as means ± standard deviation. Data were analyzed by unpaired Student’s t-test or one-way analysis of variance followed by Dunnett’s multiple comparison test (SigmaStat, Jandel Corporation, San Rafael, CA, USA). For all comparisons, P<0.05 was considered to indicate a statistically significant difference.

The cytotoxicity of O-DMA, equol, daidzein and daidzin was assessed by LDH release and MTT assays in HepG2 human hepatocellular carcinoma cells exposed to each compound at concentrations of 5–200 μM for 24, 48 or 72 h. As shown in Fig. 1, O-DMA and equol did not affect LDH release. By contrast, daidzein and daidzin resulted in an increase in LDH release by 10–28% following exposure for 72 h; however, the difference was only statistically significant for daidzin at a concentration of 200 μM. In terms of daidzein and daidzin, this result was in agreement with the increased growth of HepG2 induced by daidzein and daidzin (7 and 18% for daidzein and daidzin, respectively, at 200 μM for 72 h). O-DMA and equol at >75 μM significantly inhibited the viability of HepG2 cells. At concentrations <100 μM, cell growth was not altered by the addition of O-DMA or equol.

When antioxidant function was investigated in HepG2 cells exposed to 100 μM of each compound, all showed significant activation of antioxidant activity compared with control levels (Fig. 2A, P<0.05). Catalase activity was significantly increased by O-DMA and equol (each 4.7-fold compared with control). Daidzein increased catalase activity by 6.2-fold. Daidzin also increased catalase activity, although it had no effect on total SOD. This result was supported by the mRNA (Fig. 2B) and protein expression data (Fig. 2C).

Antioxidant activities were assayed in cells exposed to O-DMA or equol at 5, 10, 50 and 100 μM for 72 h (Figs. 3 and 4). O-DMA significantly increased the activity and expression of catalase at concentrations >10 μM. Total SOD activity was increased by >2.0-fold at 200 μM alone and CuZn-SOD expression was more pronounced compared with Mn-SOD. By contrast, 200 μM equol significantly increased the catalase activity by 5.6-fold compared with control levels. In addition, total SOD activity was significantly increased in a dose-dependent manner at concentrations between 10 and 100 μM. The increased CuZn-SOD expression induced by equol showed a similar pattern to total SOD activity and Mn-SOD expression was markedly increased with the addition of 200 μM equol.