Genotoxicity evaluation of So-ochim-tang-gamibang (SOCG), a herbal medicine

The SOCG was prepared from six crude herbs, Cyperi Rhizoma (Cyperus rotundus L.), Lindera Radix (Lindera strychnifolia Fern.-Vill.), Aucklandiae Radix (Auckladia lappa Decne.), Glycyrrhizae Radix (Glycyrrhiza uralensis Fisch.), Aurantii Fructus (Citrus aurantium L.) and Platycodi Radix (Platycodon grandiflorus Jacq. A.DC), which were purchased from Dong Kyung Pharmaceutical (Seoul, Korea). The SOCG was formulated by boiling the six herbs (ratio 8: 4: 1: 1: 4: 4, in the order given above. This ratio is the standard formula in clinical application.) in distilled water at 100 °C for 2 h. The volume of distilled water was 10 times the total herb weight (110 kg/1100 L, w/v). This decoction was evaporated under reduced pressure at 60 °C for 5 h and freeze-dried. The extracted SOCG powder was stored at − 20 °C. Voucher specimens (No. 194A079–85) of the collected herb samples were deposited in the herbarium of Han Kook Shin Yak (Nonsan, Korea). The SOCG batch used in this study was identical with the batch used in a previous study [4]. The SOCG powder was reconstituted in distilled water (Choongwae Pharma, Korea) just before use.

Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 and Escherichia coli strain WP2uvrA(pKM101) were used as the tester strains. The TA98 and TA1537 strains were used to detect frame-shift mutagens and TA100, TA1537 and WP2uvrA(pKM101) strains were used for detecting base-pair substitution mutagens. These strains were purchased from Molecular Toxicology, Inc. (MOLTOX™, U.S.A.) on October 28, 2011.

The Chinese Hamster Lung cell line was purchased from the American Type Culture Collection (ATCC, U.S.A.) on Nov. 24, 2011. The modal chromosome number of this cell line is 25. The doubling time is approximately 15 h. Cells were cultured in Eagle’s Minimum Essential Medium (EMEM, Lonza Walkersville, U.S.A.) supplemented with 10% fetal bovine serum (FBS, Gibco, U.S.A.) and incubated in a 5% CO₂ incubator (MCO-20AIC, SANYO, Japan) at 37 °C. The cell line was evaluated for contamination of mycoplasma with the Hoechst Stain Kit (MPBIOMEDICALS, Japan). Sub-culturing was done approximately twice a week.

S9 consisting of phenobarbital and 5,6-benzoflavone-induced S9 fraction and cofactor were purchased from Oriental Yeast in Japan. The S9 mix was used as a metabolic activation system. The cofactor for the Ames test contained 0.4 mol/L MgCl₂, 1.65 mol/L KCl, 1.0 mol/L glucose-6-phosphate, 0.1 mol/L NADPH, 0.1 mo/L NADH, 0.2 mol/L sodium phosphate buffer (pH 7.4) and purified water. The cofactor for the chromosomal aberration test contained 50 mmol/L MgCl₂, 330 mmol/L KCl, 50 mmol/L glucose-6-phosphate, 40 mmol/L NADP, 20 mmol/L HEPES buffer (pH 7.2) and purified water. The protein content of S9 was 20.3–23.2 mg/mL S9, and the cytochrome P450 content was 2.40–2.86 nmol/mg microsomal protein. In the Ames test, the final concentration of S9 in the S9 mix was 10% v/v. In the chromosomal aberration test, the final concentration of S9 in the culture medium was approximately 5% v/v. Fresh S9 mix was prepared before use.

The Ames test was conducted in accordance with the MFDS guideline (Notification No.2013–121). Histidine auxotroph mutants of Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 and a tryptophan auxotroph mutant of Escherichia coli strain WP2uvrA(pKM101) were cultured in nutrient broth No.2 medium for 8.5 h in a shaking water bath (37 °C, 130 rpm). The bacteria strains are widely used for Ames test due to the high sensitivity to mutagens. MFDS, OECD, and ICH guidelines suggest the strains for bacterial reverse mutation test [2, 8, 9]. The turbidity of the cultures was measured with a UV/VIS spectrophotometer (660 nm, V-550, Jasco, Japan). Cultures with a density greater than 1 × 10⁹ cells/mL were used in this test. Positive controls used in this test were 2-Nitrofluorene (2-NF, Sigma-Aldrich, U.S.A.), Sodium azide (SA, Sigma-Aldrich, U.S.A.), 9-Aminoacridine (9-AA, Sigma-Aldrich, U.S.A.), 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide (AF2, Wako, Japan) and 2-Aminoanthracene (2-AA, Sigma-Aldrich, U.S.A.); the negative control was water for injection (Choongwae Pharma, Korea).

In the presence of metabolic activation, 100 μL of each the SOCG, strain-specific positive control, or negative control were mixed with 500 μL of S9 mix and 100 μL of pre-incubated bacterial suspension. These mixtures were incubated in a shaking water bath at 37 °C for 20 min. Then, 2 mL of warmed top agar for Salmonella typhimurium were added to the TA98, TA100, TA1535 and TA1537 strains, and 2 mL of top agar for Escherichia coli were added to the WP2uvrA(pKM101) strain. Finally, these mixtures were poured onto minimal glucose agar plates. In the absence of metabolic activation, 500 μL of 0.1 mol/L sodium phosphate buffer (pH 7.4) instead of S9 mix were added, and the rest of procedure was carried out with the same method as above. After the top agar was solidified, the plates were cultured in an incubator at 37 °C for 48 h. A preliminary dose range-finding study showed that SOCG did not have growth inhibition to 5000 μg/plate, which is the highest dose level for the Ames test. Triplicate plates were used per dose in the test. The result of the Ames test was considered to be positive when the number of revertant colonies for any strain at one or more doses was increased at least two times when compared to the negative control value and there was a dose dependency or reproducibility as the dose increased. In addition, the growth inhibition was terminated when there was a clearing or diminution of the background lawn, the appearance of micro-colonies or a decrease of more than 50% in the number of revertant colonies compared to the negative control.

The chromosomal aberration test was conducted in accordance with the MFDS guideline (Notification No.2013–121). We used Chinese hamster lung cells for our study. This cell line is suggested by MFDS, OECD, and ICH guidelines, and widely used for in vitro mammalian chromosomal aberration test due to the high sensitivity [2, 9‐11]. The experimental protocol was performed with three treatment systems: short duration treatments with and without metabolic activation and continuous treatment without metabolic activation. A preliminary cell growth inhibition study was done to determine the highest dose levels for the chromosomal aberration test [12] and 5000 μg/mL was set the highest dose for all the systems. The calculated 50% growth inhibitory concentrations (IC₅₀) were approximately 3438.1 μg/mL and 2287.5 μg/mL for the short duration treatments with and without metabolic activation, respectively, and 709.0 μg/mL for the continuous treatment without metabolic activation. Based on the results, the highest dose levels were as 3500 μg/mL, 2300 μg/mL, and 710 μg/mL for each system. Positive controls used in this test were mitomycin C (Sigma-Aldrich, U.S.A.) and benzo[α]pyrene (Sigma-Aldrich, U.S.A.); the negative control was distilled water for injection (Choongwae Pharma., Korea). Each treatment was performed in duplicate. For the short duration treatments, the cells were treated with the SOCG for 6 h, and cell culture medium was exchanged to fresh. The cells were cultured for an additionally 18 h. For the continuous treatment, the cells were treated with the test substance for 24 h. Chromosome slides were prepared for a 1.5 cell cycle length after the start of treatment. Two hours prior to the culture completion, colcemid (0.2 μg/mL, Gibco, U.S.A.) was added. Following culture completion, the collected cells were treated with 0.075 mol/L KCl solution (prewarmed at 37 °C) at 37 °C for 20 min and fixed with an ice-cold fixative mixture (methanol:acetic acid = 3:1). The cells were dropped onto clean dry slides and stained with a 3% Giemsa solution for 20 min. Two hundred metaphases per dose were observed with a light microscope (× 400–600 magnification, BX51, Olympus, Japan). The chromosomal aberration was classified into structural aberration, numerical aberration and other. Structural aberrations that were recorded included chromatid break (ctb), chromatid exchange (cte), chromosome break (csb), chromosome exchange (cse), gap and fragmentation (frg). Numerical aberrations that were recorded included polyploidy (pol) and endoreduplication (end). For the above aberrations, any cell with one or more aberrations was counted as one aberrant cell. For gap, the number of cells excluding gap was recorded and calculated. The identification of a chromosome aberration was carried out according to JEMS-MMS [13]. The result of the in vitro mammalian chromosomal aberration test was evaluated in accordance with the criteria of Toshio Sofuni [14]. For the positive controls, the frequency of cells with aberrations was above 10%; for equivocal, it was above 5% and below 10%, and for negative, it was below 5%.

The micronucleus test was conducted in accordance with the MFDS guideline (Notification No.2013–121). SOCG was administered once by gavage to male ICR mice at doses of 1250, 2500, and 5000 mg/kg with 10 mL/kg volume. Mice in the negative control group received only the vehicle (water for injection; Choongwae Pharma, Korea) by oral gavage. Mitomycin C (Sigma-Aldrich, U.S.A.) was administered by intraperitoneal injection at a dose of 2 mg/kg as a positive control. The animals were randomly assigned to 5 groups (N = 5/each group). All animals were observed daily for clinical signs, and individual body weights were recorded just prior to administration and prior to harvesting the bone marrows cells. The bone marrow cells were harvested at 24 h after administration. Immediately following sacrifice by cervical dislocation, femurs were dissected from each animal and trimmed. Bone marrow cells were collected by rinsing the canal with fetal bovine serum 200 μL and centrifuged at 1000 rpm for 5 min (4 °C). The precipitate was mixed well and placed onto a clean dry slide and spread with 1 drop of cell suspension. The slides were air-dried, fixed with methanol for 5 min, and stained with a 3% Giemsa staining solution (0.01 mol/L Sörenson phosphate buffer solution, pH 6.8) for 30 min. The stained slides were washed with 0.01 mol/L Sörenson phosphate buffer solution (pH 6.8) and 0.004% citric acid solution. Slides were evaluated under a microscope (BX51, Olympus, Japan) at 600–1000-fold magnification. A total of 2000 polychromatic erythrocytes (PCE) per animal were scored for determining the frequency of micronucleus polychromatic erythrocytes (MNPCEs). To evaluate the myelotoxic effects, 500 erythrocytes were scored per animal to determine the ratio of PCE to the total number of erythrocytes. The result of the in vivo micronucleus test was considered to be positive when the frequency of the MNPCEs was statistically significantly increased according to the criteria of Kastenbaum & Bowman [15].

The statistical analysis was done with the SAS program (version 9.3, SAS Institute, U.S.A.). For the aberration cell data from the in vitro chromosomal aberration test, Fisher’s exact test was used for the comparison of the negative control group with the test substance groups or the positive control group (significance levels: 0.05 and 0.01, two-tailed). The ratio of PCE among total erythrocytes and body weight data were analyzed with Bartlett’s test for homogeneity of variance (significance level: 0.05). Data showing homogeneous variance were analyzed with one-way analysis of variance (ANOVA) for homogeneous data (significance level: 0.05).

The result of the bacterial reverse mutation test is shown in Table 1. The mean number of revertant colonies from the test substance was less than twice that of the negative control value in the presence and absence of metabolic activation at any dose level in all the strains. In the positive control group, the mean number of revertant colonies was markedly increased when compared to the negative control value. Growth inhibition by the test substance and precipitation were not observed at any dose level in all the strains. The number of revertant colonies in the negative and positive control groups was within the range of the historical control data.

In the preliminary cell growth inhibition study, greater than 50% cytotoxicity was observed in the short duration treatments with and without metabolic activation and the continuous treatment without metabolic activation (Fig. 1). The dose levels of the in vitro mammalian chromosomal aberration test were chosen by considering the cytotoxicity of the SOCG. The result is shown in Table 2. In the slide preparation, 200 metaphase cells were not evident at high dose levels in the short duration treatments with and without metabolic activation. Therefore, three dose levels excluding the high dose were selected for the observation of chromosome aberrations. In the continuous treatment without metabolic activation, 200 metaphase cells were evident at high dose levels. Therefore, three dose levels including the high dose were selected for the observation of chromosome aberrations. The frequency of cells with chromosome aberrations was less than 5% for the short duration treatments with and without metabolic activation and for the continuous treatment without metabolic activation. There was no statistically significant difference in the frequency of cells with chromosome aberrations at any dose level of the test substance compared to the negative control group. In the positive control group, the frequency of cells with structural chromosomal aberrations was statistically significantly increased compared to the negative control group (p < 0.01, Fisher’s exact test). The frequency of cells with chromosome aberrations for the positive and negative controls was within the range of the historical control data.

In the preliminary dose range finding study, no signs of systemic toxicity were recorded in SOCG-treated mice. The sampling time of the bone marrow cells was chosen based on a preliminary study (data not shown). The result of the in vivo micronucleus test is shown in Table 3. There was no significant increase in the incidence of MNPCEs in the SOCG-treated groups when compared to the negative control group. There was no significant difference in the ratio of PCE among the total erythrocytes in any of the SOCG-treated groups when compared to the negative control group. The induction of MNPCEs in the negative and positive control groups was within the range of the historical control data.