Genotoxicity evaluation of Oryeong-san water extract using in vitro and in vivo tests

Background Oryeong-san, a mixture of five herbal plants, is a well-known therapy for renal-associated diseases such as those manifesting edema, dysuria, and oliguria. Methods In the present study, we investigatee the potential genotoxic effects of a water extract of Oryeong-san (ORSE) in three mutagenicity assays (an in vitro bacterial reverse mutation assay (Ames test) with Salmonella typhimurium and Escherichia coli strains, an in vitro mammalian chromosomal aberration test using Chinese hamster lung cells, and an in vivo micronucleus test using ICR mice bone marrow). Results ORSE showed no genotoxicity in the Ames test up to 5000 μg/plate; the in vitro chromosome aberration test showed no significant structural aberrations with and without the S9 mix up to 5000 μg/mL, or the in vivo micronucleus test up to 2000 mg/kg body weight. Conclusions In conclusion, under the current test conditions, ORSE seems safe for use; however, other genotoxicity tests (e.g. sister-chromatid exchange or Comet) or chronic toxicity tests are warranted.


Background
The global demand for herbal medicinal products has increased significantly in recent years. "By 2003 in the United States alone, over 1500 herbal products sold were nutraceuticals, which are exempt from extensive preclinical efficacy and toxicity testing by the US Food and Drug Administration" [1]. Obidike and Salawu reported that despite the growing market demand for herbal medicines, there are still concerns associated with not only their use, but also their safety. The primary purpose of toxicological assessment of any herbal medicine is to identify side effects and to determine limits of exposure at which such effects occur [2]. Less than 10% of herbal products in the world market are truely standardized to known major active components and quality control measures are not always diligently adhered to [3]. The traditional herbal medicine Oryeong-san (also known as Wulingsan in traditional Chinese medicine and as Gorei-san in Japanese Kampo medicine) is a mixture of five herbal preparations (Alisma orientale (Sam) Juzepzuk, Poria cocos, Atractylodes japonica Koidzumi et Kitagawa, Polyporus umbellatus Fries, and Cinnamomum cassia J.Presl). Oryeong-san is well known for the treatment of renal diseases, dysuria, manifesting edema, and oliguria [4]. Oryeong-san also has antihypertensive [5], antidiabetic [6], antioxidative [7], and antigastric [8] properties, and confers hepatic protection.
Genotoxicity is a special type of toxicity, because it is often the most difficult to detect. The aim of a genotoxic test is to detect mutagenic carcinogens as well as to detect germ cell mutagens with the goal of limiting human exposure to these potentially dangerous chemicals [9]. The purpose of the present study was to evaluate the safety of an aqueous extract of Oryeong-san (ORSE) and its potential genotoxicity. We assessed these properties using a standard battery of tests recommended by the Korea Food and Drug Administration: a bacterial reverse mutation test (Ames test), a chromosome aberration test, and an in vivo micronucleus (MN) test.

Reagents and materials
The reference standards, cinnamaldehyde and coumarin, were purchased from Wako (Osaka, Japan) and Sigma-Aldrich (St. Louis, CA, USA). The purity of the two reference standards was ≥98.0% by HPLC analysis. The HPLC-grade solvents, acetonitrile and water, methanol, were obtained from J.T. Baker (Phillipsburg, NJ, USA). The Oryeong-san samples used in this study consisted of five herbal medicines (Table 1)

Preparation of standard and sample solutions
Standard stock solutions of coumarin and cinnamaldehyde were dissolved in methanol at 1.0 mg/mL and stored below 4°C. A decoction of Oryeong-san, which was composed of 5 herbal medicines (Table 1), was prepared in SungIl Bioex Co. Ltd. (Hwaseong, Korea). Briefly, the mixture (120.0 kg. i.e. about 4270.46 times of composition of single dose) of 5 herbal medicine was extracted in distilled water (1200 L) at 80°C for 2 h by reflux. The solution was freeze-dried to about 21.8 kg of water extract powder (yield: 18.17%). Lyophilized Oryeong-san extract (200 mg) was dissolved in distilled water (20 mL). The solution was filtered through a 0.2 μm membrane filter (Woongki Science, Seoul, Korea).

Bacterial reverse mutation assay (Ames test)
Bacterial reverse mutation assays were conducted as described previously [10]. Salmonella typhimurium strains TA98 and TA1537 (to detect frame-shift mutagens), TA100, and TA1535, and Escherichia coli strain WP2uvrA (to detect base pair-substitution mutagens) were obtained from Molecular Toxicology Inc. (Boone, NC, USA) and were used as the tester strains. To evaluate the toxicity and solubility (precipitation) of ORSE, a pilot experiment was performed with all bacterial strains (data not shown). The positive control factors were 2-nitrofluorene (2-NF), 2-aminoanthracene (2-AA), 9-aminoacridine (9-AA), 4-nitroquinoline X-oxide (4NQO), and benzo (a) pyrene (BP). A dose range-finding test was performed to determine the highest concentration for the present study, which was performed with the five tester strains at concentrations of 0, 1250, 2500, and 5,000 μg/plate with and without the S9 mixture. The number of revertant colonies did not increase to more than twice the value observed in the controls for any of the tester stains. However, there were increased numbers of revertant colonies of TA1535 with the S9 mixture. Based on these results, a dose of 5,000 μg/ plate was selected as the maximum dose. Briefly, various concentrations of ORSE were incubated with the tester strains at 37°C for 48 h in the presence or absence of metabolic activation by the S9 mixture along with vehicle and positive controls containing the following combinations of substances and doses: 2-AA at 2 μg/plate vs. TA1535 with or without the S9 mixture and at 4 μg/plate vs. WP2uvrA with the S9 mixture; 9-AA at 50 μg/plate vs. TA1537 without the S9 mixture; BP at 2 μg/plate vs. TA98 with or without the S9 mixture and vs. TA100 and TA1537 with the S9 mixture); 2-NF at 2 μg/plate vs. TA98 without the S9 mixture; 4NQO at 0.5 μg/plate vs. WP2uvrA without the S9 mixture; and sodium azide at 0.5 μg/plate vs. TA100 and TA1535 without the S9 mixture. Each concentration of ORSE was tested in triplicate. A result was deemed positive if there was a concentration-related increase over the range tested and/or a reproducible increase at one or more concentrations in the number of revertant colonies per plate in at least one strain with or without the S9 mixture. An antibacterial effect (cytotoxicity) was defined as a clearing or diminution of the background lawn, the appearance of microcolonies, and/or a decrease of > 50% in the number of colonies compared with the relevant vehicle control.

Chromosome aberration test
Chromosome aberration tests were conducted as described previously [10] with minor modification as described by Ishidate et al. [11] and Dean and Danford [12]. Chinese hamster lung (CHL) cells were obtained from American Type Culture Collection (Manassas, VA, USA) in 2004. The cells were thawed in culture medium and then grown for more than 7 days as a monolayer. Cells were cultured in reconstituted MEM (Gibco-Invitrogen, USA) supplemented with 2.2 g of sodium bicarbonate, 292 mg of l-glutamine, streptomycin sulfate (100 μg/ mL), penicillin G · Na (10 5 units), and 10% (v/v) fetal bovine serum (FBS; Gibco-Invitrogen, USA) per liter. The cultures were incubated at 37°C in a humidified atmosphere with 1.5% CO 2 . A preliminary dose rangefinding study was performed to determine the highest concentration for this study. Using the results from the dose range-finding study, the dose range for the present study was designed to quantify the solubility and cytotoxicity of GBT. Ethyl methanesulfonate (EMS) was used as a positive control substance without metabolic activation and cyclophosphamide (CPA) with metabolic activation. Cells were trypsinized and counted, and the relative cell count (RCC) was calculated. The cells were centrifuged at~1000 rpm for 5 min and resuspended in 5 mL of 75 mM KCl solution. After 10 min at room temperature, 5 mL of fixative (methanol:glacial acetic acid = 3:1 v/v) was added to the cell suspension, and the suspension was refrigerated for~20 min. The fixative was changed twice by centrifugation at~1500 rpm for 5 min. Two slides were prepared from each fixedcell suspension. The slides were air-dried, stained with 3% Giemsa solution, washed in tap water and distilled water, dried, and mounted in DPX (Fluka) for chromosome aberration scoring. Chromosome aberrations were identified morphologically according to the principles described in the ' Atlas of chromosome aberration by chemicals' (JEMS-MMS, 1988). Cells with more than four of the same type of aberration were scored as multiple aberrations. Any metaphase with one or more aberrations, regardless of the type, was classified as an aberration metaphase. Slides were scanned systemically, and each set of metaphases was examined at 1000× magnification. Structural chromosome aberrations were evaluated in 100 well-spread metaphases, each containing 23-27 chromosomes. The microscopic stage coordinates and each type and number of aberration were recorded for each aberrant metaphase. The results are expressed as the number of findings per 100 metaphases. Regardless of the presence of aberrations, an additional 100 metaphases were examined to determine the frequency of diploidy (DP), polyploidy (PP, > 37 chromosomes), and endoreduplication (ER).
In vivo MN test MN tests using mice was conducted as described previously [10]. Specific pathogen-free male CrljOri:CD1

Statistical analyses
The statistical analyses used for the study were selected based on the methods used in published reports [13] using SAS software (version 9.1.3, SAS Institute Inc., Cary, NC, USA). Each metaphase was classified as a normal metaphase or aberrant metaphase with one or more aberrations, and the frequency of aberrant metaphase was analyzed statistically. The numerical aberrations were classified into DP, PP, and ER, and the frequencies of PP + ER were analyzed. The χ 2 test and Fisher's exact test were performed to compare the vehicle control and ORSE-treated groups [14]. Fisher's exact test was used to compare the vehicle and positive control groups. Differences were regarded as significant at P < 0.05The in vivo MN results were evaluated as described previously [10] using the method of Lovell et al. [15] with minor modification. Data with heterogeneous variances were analyzed using Kruskal-Wallis analysis of variance followed by multiple comparisons using Dunnett's test [16] The significance was accepted when all of the PCE/(PCE + NCE) ratios were > 0.1. The result was judged as positive when there was a significant and dose-related increase or a reproducible increase in the frequency of MNPCEs or aberrant metaphases at one or more dose levels. Differences were regarded as significant at P < 0.05.

HPLC analysis of ORSE
Using established conditions, three components were eluted within 35 min in a sample analysis using mobile phases consisting of solvent A (water) and solvent B (acetonitrile). A typical HPLC chromatogram for ORSE is shown in Fig. 1. The retention times of the two components were 11.60 min (coumarin) and 14.88 min (cinnamaldehyde). The regression equations for coumarin and cinnamaldehyde were y = 42536.98x -4343.49 and y = 107074.20x -46100.12. The correlation coefficients (r 2 ) of the calibration curves for the two constituents were 1.0000 and 0.9999. These results showed that the calibration curves showed good linearity. The contents of two components identified in ORSE were 0.37 mg/g and 0.05 mg/g ( Table 2).

Bacterial reverse mutation assay (Ames test)
No positive mutagenic response was observed in any of the S. typhimurium or E. coli strains tested compared with concurrent vehicle control groups regardless of the presence (Fig. 2a) or absence (Fig. 2b) of the S9 mix up to 5000 μg/plate.

Chromosome aberration tests
According to our preliminary study (data not shown), ORSE neither inhibited cell growth nor killed Chinese hamster lung (CHL) cells. We examined the concentration range of 1250, 2500, and 5000 μg/mL, which was most compatible with a good cell-proliferating ability and which produced a sufficient number of metaphases for the confirmatory assay. Therefore, we used 5000 μg/ mL as the highest exposure level and serial dilutions for further dose-response tests. There were no statistically significant increase in the number of metaphase cells with structural aberrations at   (Table 3) compared with the vehicle control group (P < 0.01). In each positive control group, the number of metaphases with structural aberrations in the vehicle and positive control groups was within the range established in historical data of the Korea Institute of Toxicology (KIT, 2009). These findings confirm that the methodologies used in this study were valid. Therefore, under the conditions of this test, ORSE showed a positive response in the chromosomal aberration test.

MN test
As shown

Discussion
The present study demonstrated that ORSE, a traditional herbal medicine for the treatment of various renal diseases, was not genotoxic using an in vitro chromosomal abbreviation test, an in vitro bacterial reverse mutation assay, or an in vivo MN test. ORSE did not exhibit any genotoxic potential in any of the test systems employed. An in vitro chromosome aberration test using CHL cells was performed to determine whether ORSE affects the genotoxicity. The in vitro chromosome aberration test is used to identify agents that induce structural   S. typhimurium strains TA100, TA1535, TA98, and TA1537 and the tryptophan auxotroph strain E. coli WP2 uvrA were used in the bacterial reverse mutation test [18,19]. These strains have been shown to be sensitive to the mutagenic activity of a wide range of chemical classes [20].
In the bacterial reverse mutation test (Ames test), we used the histidine auxotroph S. typhimurium strains TA100, TA1535, TA98, and TA1537 and the tryptophan auxotroph strain E. coli WP2 uvrA [18,19]. In the present study, no mutagenic effects of ORSE in S. typhimurium strains TA100, TA1535, TA98, or TA1537, or the E. coli WP2 uvrA was observed at 555.6, 1666.7, and 5000 μg/plate. The present tested strains have been shown to be sensitive to the mutagenic activity of a wide range of chemical classes. Mutation of genes results in a deficient DNA repair system and greatly enhances the sensitivity of these strains to certain mutagens [21]. Therefore, ORSE did not appear to mutate any genes in vitro. MN tests have been employed for genotoxicity and mutagenicity detection of materials that induce the formation of DNA fragments [22][23][24]. The increased MN frequency is related to cancer, because MN can be a target of carcinogenesis [25,26]. In the present study, there was no significant or dose-related increase in the number of MNPCEs per 2000 PCEs at any ORSEtreatment dose level. An elevated frequency of micronucleated PCEs(MNPCEs) indicates chromosomal damage (Fench, Krishna). Also, no abnormal signs and body weights were observed in any of the gropus. Therefore, ORSE is associated with a low risk for carcinogenesis.

Conclusion
On the basis of our results, the ORSE did not exert any genotoxicity in the battery of three assessments, in vitro bacterial reverse mutation assay, or an in vivo MN test.
To our knowledge, this is the first published study to demonstrated traditional herbal medicine genotoxicity of the ORSE. Significantly different from the control at p < 0.05