Biological and Pharmaceutical Bulletin
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Genotoxicity of Rice Bran Oil Extracted by Supercritical CO2 Extraction
Jae-Suk ChoiEun Jin CheonTae-Uk KimWoi-Sook MoonJoo-Wan KimMi-Ryung Kim
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2014 Volume 37 Issue 12 Pages 1963-1970

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Abstract

Rice bran oil extracted by supercritical CO2 extraction (RB-SCE) reportedly exhibits pharmacological activities such as antioxidant and in vivo hair growth-inducing effects. Such activities raise the possibility of the development of novel hair growth-inducing agents using RB-SCE. The aim of this study was to investigate the potential genotoxic effects of RB-SCE in three short-term mutagenicity assays (bacterial reverse mutation assay, in vitro mammalian chromosomal aberration test, and in vivo micronucleus assay). RB-SCE showed no genotoxicity in the bacterial reverse mutation assay up to 5000 mg/plate and in the in vivo micronucleus test up to 600 mg/kg body weight. However, at 120 µg/mL with S9 mix and 200 µg/mL without S9 mix RB-SCE showed significantly different genotoxicity than the negative control in the in vitro chromosome aberration test. The induction of chromosomal aberrations under the present conditions may have no biological significance. We have herein demonstrated that RB-SCE can be regarded as a non-genotoxic material based on the available in vivo and in vitro results.

Rice bran is the largest by-product of the rice milling process, comprising 8% of milled rice. Approximately 20% to 30% of rice bran is used for oil production, and the remainder is discarded or used in livestock feed or fertilizer.1) Although rice bran has not been highly utilized in the food industry, it has various health benefits, including antioxidant,1) anticancer,2) and antihyperlipidemic effects.3) Additionally, rice bran is a potent inducer of hair growth4) due to its inhibition of 5-alpha-reductase.5)

Supercritical CO2 extraction (SCE), which is conducted at low temperatures using supercritical CO2 as the solvent, has been introduced as an alternative one-step method of oil extraction. Oil extraction at low temperatures minimizes the thermal degradation of antioxidants, proteins, and other health-benefical components. Additionally, supercritical CO2 has the advantages of being environmentally friendly, nontoxic, nonflammable, inexpensive, and easily removed from the final product.6)

Rice bran oil extracted by SCE (RB-SCE) is generally thought to be safe. A few researchers have investigated the toxicological safety of RB-SCE by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay in RAW264.7 cells as well as the single-oral-dose toxicity in rat.7) Despite its therapeutic potential, no reports have described the genotoxicity of RB-SCE. Genotoxicity assays may be either in vivo or in vitro and are designed to detect compounds that directly or indirectly induce genetic damage by various mechanisms. However, no single assay is capable of detecting all relevant genotoxic agents.

The usual approach to the evaluation of genotoxicity should include three-battery testing of in vitro and in vivo genotoxicity assays.813) The following standard battery for pharmaceutical genotoxicity testing is recommended from ICH Harmonized Tripartite Guideline14): 1) assay for bacterial gene mutation, 2) in vitro cytogenic assay of chromosomal damage with mammalian cells, and 3) in vivo assay of chromosomal damage using rodent hematopoietic cells.

Therefore, to evaluate the genotoxic potential of RB-SCE, we performed a bacterial reverse mutation assay, in vitro chromosomal aberration test using Chinese hamster lung (CHL) cells, and in vivo micronucleus assay using male ICR mice.

MATERIALS AND METHODS

Test Material

RB-SCE (rice bran, Oryza sativa L. var. japonica) was prepared in a semi-continuous flow-type apparatus with a 3-L extractor (Choi et al.4)). Briefly, CO2 was pumped into the extractor, and a flow rate of 135 g of CO2 per minute was used for extraction. The extraction vessel was loosely packed with glass wool, and a 1-kg rice bran sample was added and distributed throughout the packing. Extractions were performed at 32°C and 270 bar for 240 min. The RB-SCE yield from 1 kg of rice bran was 13%. After storage at –80°C, the RB-SCE was allowed to thaw in a water bath circulator (MCB-3011D, Mono-Tech Eng Co., Ltd., Daegu, Korea) at 40°C. The appropriate dose was then weighed and dissolved in the vehicle, dimethylsulfoxide (#472301; Sigma-Aldrich Co., St. Louis, MO, U.S.A.) by vortexing. The treatment solutions with RB-SCE were prepared just before use.

Chemicals

Most chemicals, including the positive controls [2-aminoanthracene (2-AA), benzo[a]pyrene (B[a]P), sodium azide (SA), 2-nitrofluorene (2-NF), 4-nitroquinoline 1-oxide (4-NQO), acridine mutagen ICR 191 (ICR-191), ethylmethanesulfonate (EMS), and cyclophosphamide monohydrate (CPA)], were purchased from Sigma-Aldrich Co. Fetal bovine serum, Minimum Essential Medium, and penicillin-streptomycin were obtained from GIBCO-Invitrogen (Carlsbad, CA, U.S.A.). S9, which was prepared from male Sprague-Dawley (SD) rats induced with Aroclor 1254, was obtained from Molecular Toxicology Inc. (Boone, NC, U.S.A.). Cofactor-I for the S9 mix was obtained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan).

Bacterial Strains and Culture for Reverse Mutation Test

Test strains (Salmonella typhimurium TA100, 1535, 98, and 1537; Escherichia coli WP2 uvrA) were obtained from Molecular Toxicology, Inc. and subcultured in the Preclinical Research Center of ChemOn, Inc. (Suwon, Korea). For the reverse mutation test, the frozen stocks were thawed and cultured for 10 h to prepare master plates of test strains. A portion of each bacterial culture was used to confirm genotypes. After confirming the genetic characteristics of the strains, the master plates were used as the bacterial source for the mutagenicity assays.

Cell Line and Culture for Chromosomal Aberration Assay

The in vitro chromosomal aberration assay was conducted using CHL fibroblasts, which were obtained from the American Type Culture Collection (Manassas, VA, U.S.A.). This cell line has been demonstrated to be sensitive to the clastogenic activity of various chemical agents. The cells were cultured in Minimum Essential Medium supplemented with 100 U penicillin, 100 mg/mL streptomycin, 2 mM L-glutamine, and 10% fetal bovine serum. Cultures were incubated in a humidified incubator (Forma 311 and 3111) at 37°C±1°C in 5% CO2 in air. Subculture was conducted every 2 to 3 d using 0.1% trypsin solution to prevent overgrowth.

Bacterial Reverse Mutation Assay

To detect frame-shift mutagens, Salmonella typhimurium strains TA98 and TA1537 were used as test strains. And to detect base-pair substitution mutagens, strains TA100 and TA1535, and E. coli strain WP2 uvrA were used as test strains. The mutation assay was conducted following to the method described by Maron and Ames15) with slight modifications.8) A 0.1-mL aliquot of RB-SCE containing 5 to 5000 µg/plate per plate, a 0.1-mL inoculum of the tester strain and 0.5 mL of S9 mix (or sodium phosphate buffer, pH 7.4 for the S9 negative group) were added to each tube containing 2 mL of top agar. The contents of the test tubes were mixed well, and the mixtures were poured onto the Vogel–Bonner minimal agar plates. The plates were then incubated at 37°C for 48 h. Triplicate plates were run for each assay. 2-AA and B[a]P were used as positive controls without S9 mix, and SA, 2-NF, ICR-191, and 4NQO were used as positive controls with S9 mix. The increase in revertants was calculated as the number of colonies of treated plates divided by the number of colonies of negative control plates. Materials with a factor of <0.5 were determined to be cytotoxic, and materials with a factor of >1.5 were determined to be genotoxic.

In Vitro Chromosomal Aberration Assay

The in vitro chromosomal aberration assay was conducted using CHL cells as described by Dean and Danford16) and Ishidate et al.17) with slight modifications.8) The assay comprised short-term (6-h treatment/18-h recovery in the presence and absence of S9 mix) and continuous treatments (24-h treatment/0-h recovery in the absence of S9 mix). In the range-finding test, cells were treated with 8 levels of RB-SCE ranging 5 to 5000 µg/mL, and the treatments were repeated in the narrower ranges. The treatment conditions were grossly observed at the start and end of the treatment. Precipitation was defined as settlement of fine particles of RB-SCE at the end of the treatment. Cells were removed from flasks and counted approximately 24 h after treatment. The relative cell count (RCC) was calculated as follows and used as an indicator of concurrent cytotoxicity:   

In the range-finding test, turbidity was observed at 120 µg/mL and higher doses until the end of the treatment. Cytotoxicity was observed in all of the treatment series. The highest doses expected to produce an RCC of <50% were selected with the aid of a linear regression equation, and the dose levels for this study were determined as shown in Tables 2–4, respectively. Approximately 22 h after treatment, colchicine was added to each culture at a final concentration of 1 µM and incubated for 2 h for mitotic arrest. Slides of CHL cells were prepared by the hypotonic methanol–glacial acetic acid–flame drying–Giemsa schedule for metaphase plate analysis. Two slides were prepared for each culture.

Morphological classification and counting of chromosomal aberrations were performed according to the Japanese Environmental Mutagen Society-Mammalian Mutagenicity Study Group (JEMS-MMS).18) In total, 200 metaphases (100 metaphases from each duplicate culture) were selected and analyzed for each treatment group under 1000× magnification using a light microscope (Nikon Ni-U, Tokyo, Japan). Metaphases with 23 to 27 centromeres were evaluated for chromosomal aberrations; if any were present, the type, number, and location of the aberrations in the slide were recorded. Chromosomal aberrations were classified into chromosome-type deletions (csd)/exchange (cse) and chromatid-type deletions (ctd)/exchange (cte). The frequency of metaphases with aberrations of each culture, both inclusive and exclusive of gaps, was determined. A metaphase with >10 aberrations (multiple aberrations including gaps) or with chromosomal fragmentation was classified as “other” and counted as one aberration. Regardless of the presence of aberrations, 100 additional metaphases were examined to determine the frequencies of diploidy (23–36 centromeres), polyploidy (≥37 centromeres), and endoreduplication. The results were expressed as the mean numbers of aberrant metaphases, excluding gaps, per 100 metaphases. The result was considered to be positive if a dose-related increase in the number of aberrant metaphases or a reproducible increase in at least one dose level was present. However, the statistical significance was not the only factor determining a positive response; the biological relevance, frequency of aberrant metaphases, and presence of cytotoxicity were also considered.

Animals

Seven-week-old SPF male ICR mice weighing 28.72 to 32.33 g were purchased from Koatech Co., Ltd. (Pyeongtaeki, Korea). The animals were housed in stainless steel cages. An ambient temperature of 23°C±3°C, relative humidity of 55%±15%, and photoperiod of 12 h (08 : 00–20 : 00) with 150 to 300 lx luminous intensity and 10 to 20 air exchanges per hour were maintained throughout the study. A pellet diet (Harlan Laboratories Inc., Indianapolis, IN, U.S.A.) and water disinfected by UV ultrafiltration were provided ad libitum. The animals’ clinical signs were checked and recorded once a day during a 5-d period of quarantine and acclimatization. All animals used in this study were cared for in accordance with the principles outlined in the Guide for the Care and Use of Laboratory Animals.19)

In Vivo Micronucleus Assay

RB-SCE was administered by gastric gavage to groups of six mice at doses of 150, 300, and 600 mg/kg. Mice in the negative control group received only corn oil as a vehicle. Cyclophosphamide in normal saline (10 mL/kg) was administered to six mice by intraperitoneal injection at 70 mg/kg and served as the positive control. After 24 h after the last administration which showed the peak induction of micronuclei, mice were euthanized. This study was approved by Institutional Animal Care and Use Committee (IACUC) of Nonclinical Research Institute, Chemon Inc. (Serial No.: 13-M169; May 6, 2013).

Observing the induction of micronuclei, bone marrow was prepared as described previously.8,9,20) One femur of each mouse was excised intact after euthanasia. The bone marrow was expelled from the bone cavity by repeated gentle aspiration and flushing with fetal bovine serum. The cell suspension was centrifuged at 1000 rpm for 5 min. The supernatant was discarded and the pellet was re-suspended in an appropriate volume of fetal bovine serum. Two or more slides of the cell suspension per mouse were made. The air-dried slides were stained with 0.05% acridine orange solution. After 2 min, the fluorescence was checked and counting was started after confirming vivid fluorescence. The slides were then examined using a fluorescence microscope at 400× magnification (model Ni-U with a B-2A fluorescence filter set; Nikon, Tokyo, Japan).

Micronuclei were identified according to Hayashi et al.21) Polychromatic erythrocytes (PCEs) appeared red, and normochromatic erythrocytes (NCEs) appeared dark gray. A micronucleus was observed as a green spot on the red background. The frequency of micronucleated PCE (MNPCEs) was determined by counting the number of MNPCEs per 2000 PCEs per animal. Micronuclei were defined as small, round- to oval-shaped structures that stained similarly to cell nuclei and were generally 1/5 to 1/20 the size of PCEs. In total, 2000 PCEs were scored per animal by the same observer to determine the frequencies of MNPCEs. The PCE/red blood cell (RBC) ratio was calculated by counting 500 RBCs (PCEs+NCEs) per animal.

Statistical Analysis

The statistical analyses of the in vitro chromosomal aberration results were conducted using the Statistical Analysis System software as described by Richardson et al.22) Fisher’s exact test was performed to evaluate the number of aberrant metaphases (excluding gaps) and the number of cells exhibiting polyploidy+endoreduplication in the negative control and RB-SCE groups. If a statistically significant increase in the number of aberrant cells was noted, the dose-responsiveness was tested by the linear-by-linear association chi-squared test. Statistical evaluation of the in vivo micronucleus results was performed according to Lovell et al.23) with a minor modification. Data showing heterogeneous variances were analyzed by the Kruskal–Wallis analysis of variance followed by multiple comparisons using Dunnett’s test. The study results were accepted when all of the PCE/(PCE+NCE) ratios were >0.1.24) Results were judged as positive when a statistically significant and dose-related increase or a reproducible increase in the frequency of MNPCEs (in vivo MN assay) or aberrant metaphases (in vitro CA assay) was observed in at least one dose level. The result of the statistical evaluation was regarded as significant at a p value of <0.05. No statistical analysis was performed on the Ames results.

RESULTS

Bacterial Reverse Mutation Assay of RB-SCE

Histidine-requiring mutants of S. typhimurium (TA98, TA100, TA1535, and TA1537) and tryptophan-requiring mutants of E. coli WP2 uvrA with and without metabolic activation (S9) were used for point-mutation tests. In TA1535 and TA98, growth inhibition was observed at 5000 g/plate, but there were no increases in revertant colonies at any dose levels of test strains treated with RB-SCE. Neither an increase in revertants nor cytotoxicity in S. typhimurium TA100 or TA1537 or E. coli WP2 uvrA was observed at any dose level of RB-SCE with or without metabolic activation. In WP2 uvrA, there was neither an increase in colonies nor cytotoxicity at any dose level of RB-SCE with or without metabolic activation. The mean revertant of the positive control for each test strain was clearly higher than that of the vehicle control for that strain. The viable cell counts of test strains was 0.53 to 2.47×109 (TA strains) and 1.75×109 (E. coli) colony forming unit (CFU)/mL, and at least 0.5×108 CFU of bacteria per plate were plated (Table 1).

Table 1. Reverse Mutagenicity Assay Results
Tester strainChemical treatedDose (µg/plate)Colonies/plate [factor]a)
With S9 mixWithout S9 mix
TA98RB-SCE027±224±1
1523±2[0.9]21±3[0.9]
5023±3[0.9]20±3[0.8]
15021±2[0.8]21±3[0.9]
50021±2[0.8]19±1[0.8]
150015±1[0.6]17±3[0.7]
500013±1[0.5]12±2[0.5]
TA100RB-SCE0107±3104±2
15105±3[1.0]110±11[1.1]
50103±5[1.0]110±9[1.1]
150107±7[1.0]102±6[1.0]
500104±15[1.0]104±11[1.0]
150089±13[0.8]104±9[1.0]
500096±2[0.9]91±15[0.9]
TA1535RB-SCE014±312±2
1513±6[1.0]11±3[0.9]
5011±1[0.8]13±3[1.1]
15011±5[0.8]12±3[1.0]
5007±1[0.5]9±1[0.8]
15008±1[0.6]7±1[0.6]
50007±1[0.5]6±2[0.5]
TA1537RB-SCE015±310±3
1513±3[0.8]10±3[1.0]
5011±2[0.7]9±3[0.9]
15013±4[0.8]8±1[0.7]
50013±3[0.8]8±2[0.8]
150013±3[0.9]7±2[0.7]
500011±3[0.7]6±2[0.6]
E. coli WP2 uvrARB-SCE025±318±2
1523±3[0.9]17±1[0.9]
5020±3[0.8]17±3[0.9]
15020±4[0.8]15±2[0.8]
50021±3[0.8]17±3[0.9]
150019±4[0.8]14±1[0.8]
500018±4[0.7]14±2[0.8]
PositiveControls
TA98B[a]P1.0188±23[7.1]
TA1002-AA1.0543±20[5.1]
TA15352-AA2.0148±13[10.9]
TA15372-AA1.0115±12[7.5]
WP2 uvrA2-AA6.0114±7[4.6]
TA982-NF2.0339±22[14.1]
TA100SA0.5322±12[3.1]
TA1535SA0.5130±15[10.9]
TA1537ICR-1910.5182±10[17.6]
WP2 uvrA4NQO0.5117±11[6.5]

a) Three plates/dose were used. No. of colonies on treated plate/No. of colonies on negative control plate. b) 2-AA, 2-aminoanthracene; SA, sodium azide; B[a]P, benzo[a]pyrene; ICR-191, acridine mutagen ICR 191; 4NQO, 4-nitroquinoline N-oxide; 2-NF, 2-nitrofluorene.

In Vitro Mammalian Chromosomal Aberration Assay of RB-SCE

An in vitro chromosome aberration test was performed using CHL cells in the presence and absence of an exogenous metabolic activation system (S9 mix) to evaluate the potential of RB-SCE to induce structural chromosomal aberrations. In total, 100 metaphases per culture (200 metaphases per dose) were evaluated for chromosome aberrations. The results were expressed as the mean frequency of metaphases with structural or numerical aberrations per 100 metaphases.

At 6 h of treatment and 18 h of recovery in the presence of S9 mix, turbidity in the RB-SCE treated mixture was observed at over 120 µg/mL, and the RCC decreased with an increasing dose. In the presence of S9 mix, the frequency of metaphases with structural aberrations was 0.0, 1.0, 0.5, 3.0, and 9.0 in the negative control at 30, 60, 120, and 140 µg/mL RB-SCE, respectively, and the values at 120 and 140 µg/mL were significantly increased in a dose-dependent manner (p<0.05 and p<0.01, respectively). In the positive control, there was a statistically significant (p<0.01) increase in the mean frequency of aberrant metaphases (25.0) (p<0.01) (Table 2).

Table 2. In Vitro Chromosome Aberration Test with S9 Mix (6-h Treatment)
Dose (µg/mL)No. of examined cellsAberrationsPP+ERNo. of aberrant metaphasea)RCC (%)
Chromosome typeChromatid typeOthersGapsNo.Decision+Gaps−Gaps
csdcsectdcteNo.No.Decision
01000.00.00.00.00.00.00.0Negative(0.0)(0.0)Negative100
30100(0.5)(0.0)(0.0)(0.5)(0.0)(0.0)(0.0)Negative(1.0)(1.0)Negative92
60100(0.0)(0.0)(0.0)(1.5)(0.0)(0.0)(1.5)Negative(0.5)(0.5)Negative88
120T100(0.0)(1.0)(0.5)(2.5)(0.0)(0.5)(3.5)*Positive(3.0)(3.0)*Positive55
140T100(0.0)(0.0)(2.0)(12.0)(0.0)(1.0)(13.0)**Positive(9.5)(9.0)**Positive40
20B[a]P100(0.0)(0.0)(4.0)(41.0)(0.0)(0.5)(1.0)Negative(25.0)(25.0)**Positive72

Test article: RB-SCE (rice bran oil extracted by supercritical CO2 extraction). a) Inclusive/exclusive gaps, means of duplicate cultures. One-hundred metaphases were examined per culture. T: turbid at the end of the treatment, PP: polyploid, ER: endoreduplication, B[a]P: benzo[a]pyrene. RCC: relative cell count=(cell count of treated flask/cell count of control flask)×100%. Gaps: chromosome type+chromatid type gaps, csd: chromosome type deletions, cse: chromosome type exchanges, ctd: chromatid type deletions, cte: chromatid type exchanges, Others: metaphases with more than 10 aberrations (including gaps) or with chromosome fragmentation. * Significantly different from the negative control at p<0.05 (Fisher’s exact test). ** Significantly different from the negative control at p<0.01 (Fisher’s exact test).

At 6 h of treatment and 18 h of recovery in the absence of S9 mix, turbidity of the treatment mixture was observed over 200 µg/mL. The frequency of metaphases with numerical aberrations was 0.0, 0.0, 0.0, 8.0, and 6.0 in the negative control at 50, 100, 200, and 220 µg/mL RB-SCE, respectively, and the values at 200 and 220 µg/mL of the test article were significantly increased (p<0.01). The mean frequency of metaphases with structural aberrations in the negative control and all dose levels of the RB-SCE were ≤2.0, and there was no statistically significant increase at any dose level of RB-SCE. In the positive control, there was a statistically significant (p<0.01) increase in the mean frequency of aberrant metaphases (18.5) (Table 3).

Table 3. In Vitro Chromosome Aberration Test without S9 Mix (6-h Treatment)
Dose (µg/mL)No. cells examined (mean)AberrationsPP+ERNo. aberrant metaphasea)RCC
Chromosome typeChromatid typeOthers (mean)Gaps (mean)No. (mean)Decision+Gaps−Gaps
csd (mean)cse (mean)ctd (mean)cte (mean)No. (mean)No. (mean)Decision
01000000000+0Negative00Negative100
1000000000+000
(100)(0.0)(0.0)(0.0)(0.0)(0.0)(0.0)(0.0)(0.0)(0.0)
501000000000+0Negative00Negative96
1000000000+000
(100)(0.0)(0.0)(0.0)(0.0)(0.0)(0.0)(0.0)(0.0)(0.0)
1001000000010+0Negative10Negative89
1000100000+011
(100)(0.0)(0.5)(0.0)(0.0)(0.0)(0.5)(0.0)(1.0)(0.5)
200T1000001000+7Positive11Negative54
1000021020+922
(100)(0.0)(0.0)(1.0)(1.0)(0.0)(1.0)(8.0)**(1.5)(1.5)
220T1000004000+6Positive22Negative38
1000004000+622
(100)(0.0)(0.0)(0.0)(4.0)(0.0)(0.0)(6.0)**(2.0)(2.0)
800EMS10020416010+0Negative2020Positive76
10000222000+01717
(100)(1.0)(0.0)(3.0)(19.0)(0.0)(0.5)(0.0)(18.5)(18.5)**

Test article: RB-SCE (rice bran oil extracted by supercritical CO2 extraction). a) Inclusive/exclusive gaps, means of duplicate cultures. One-hundred metaphases were examined per culture. T: turbid at the end of the treatment, PP: polyploid, ER: endoreduplication, EMS: ethylmethanesulfonate (positive control article), RCC: relative cell count=(cell count of treated flask/cell count of control flask)×100%. Gaps: chromosome type+chromatid type gaps, csd: chromosome type deletions, cse: chromosome type exchanges, ctd: chromatid type deletions, cte: chromatid type exchanges, Others: metaphases with more than 10 aberrations (including gaps) or with chromosome fragmentation. * Significantly different from the negative control at p<0.05 (Fisher’s exact test). ** Significantly different from the negative control at p<0.01 (Fisher’s exact test).

At 24 h of treatment and 0 h of recovery in the absence of S9 mix, turbidity of the treatment mixture was observed over 120 µg/mL. The mean frequency of metaphases with numerical aberrations in the negative control and at all dose levels of the test article was 0.0, and there was no statistically significant increase at any dose level of RB-SCE. In the positive control, there was a statistically significant (p<0.01) increase in the frequency of aberrant metaphases (21.5) (Table 4).

Table 4. In Vitro Chromosome Aberration Test without S9 Mix (24-h Treatment)
Dose (µg/mL)No. cells examined (mean)AberrationsPP+ERNo. aberrant metaphasea)RCC
Chromosome typeChromatid typeOthers (mean)Gaps (mean)No. (mean)Decision+Gaps−Gaps
csd (mean)cse (mean)ctd (mean)cte (mean)No. (mean)No. (mean)Decision
01000000000+0Negative00Negative100
1000000000+000
(100)(0.0)(0.0)(0.0)(0.0)(0.0)(0.0)(0.0)(0.0)(0.0)
301000000000+0Negative00Negative102
1000000000+000
(100)(0.0)(0.0)(0.0)(0.0)(0.0)(0.0)(0.0)(0.0)(0.0)
601000000010+0Negative10Negative85
1000000000+000
(100)(0.0)(0.0)(0.0)(0.0)(0.0)(0.5)(0.0)(0.5)(0.0)
120T1000000000+0Negative00Negative54
1000000000+000
(100)(0.0)(0.0)(0.0)(0.0)(0.0)(0.0)(0.0)(0.0)(0.0)
150T1000000000+0Negative00Negative43
1000000010+010
(100)(0.0)(0.0)(0.0)(0.0)(0.0)(0.5)(0.0)(0.0)(0.0)
600EMS10001424000+0Negative2323Positive71
10000522000+02020
(100)(0.0)(0.5)(4.5)(23.0)(0.0)(0.0)(0.0)(21.5)(21.5)**

Test article: RB-SCE (rice bran oil extracted by supercritical CO2 extraction). a) Inclusive/exclusive gaps, means of duplicate cultures. One-hundred metaphases were examined per culture. T: turbid at the end of the treatment, PP: polyploid, ER: endoreduplication, EMS: ethylmethanesulfonate (positive control article), RCC: relative cell count=(cell count of treated flask/cell count of control flask)×100%. Gaps: chromosome type+chromatid type gaps, csd: chromosome type deletions, cse: chromosome type exchanges, ctd: chromatid type deletions, cte: chromatid type exchanges, Others: metaphases with more than 10 aberrations (including gaps) or with chromosome fragmentation. ** Significantly different from the negative control at p<0.01 (Fisher’s exact test).

In Vivo Micronucleus (MN) Assay of RB-SCE

The number of MNPCEs per 2000 PCEs was 0.33, 0.83, 0.17, and 0.83 for the negative control at 0, 150, 300, and 600 mg/kg/d, respectively. There was no statistically significant increase in the frequencies of MNPCE at any dose level of the RB-SCE compared with the negative control. The positive control material, cyclophosphamide monohydrate (CPA10), induced a statistically significant (p<0.01) increase in MNPCE (55.17) when compared with the negative control. The PCE : RBC ratio, an indicator of cytotoxicity, was 0.54, 0.51, 0.51, and 0.44 for the negative control at 0 150, 300, and 600 mg/kg/d, respectively. The ratio at 600 mg/kg/d was significantly lower than that in the negative control group (p<0.01). The PCE : RBC ratio in the positive control group (0.45) was significantly (p<0.01) lower than that in the negative control (Table 5). Additionally, there was no statistically significant difference in the body weights of animals among all groups. No mortality was observed at any of the dose levels. No abnormal clinical signs were observed in any groups (data not shown).

Table 5. Observation of Micronucleus and the PCE : RBC Ratio
Dose (mg/kg/d)Number of dosingAnimals per doseMNPCE/2000 PCE (mean±S.D.)PCE : RBC ratio% Control
ControlNormal260.33±0.520.54±0.02100
CPA 70 (mg/kg)1655.17±2.64**0.45±0.03**83
RB-SCE0
150260.83±0.750.51±0.0494
300260.17±0.410.51±0.0294
600260.83±1.170.44±0.05**81

Vehicle (corn oil) and Test article (rice bran extract by supercritical carbon dioxide) were orally administered to mice for two consecutive days. CPA was intraperitoneally administered to mice once on the day of the 2nd admin. Bone marrow smears were prepared about 24 h after the final administration. PCE: Polychromatic erythrocyte, RBC: Red blood cells (polychromatic erythrocyte+normochromatic erythrocyte), MNPCE: Micronucleated polychromatic erythrocyte, CPA: Cyclophosphamide monohydrate. ** Significantly different from the negative control group at p<0.01.

DISCUSSION

RB-SCE reportedly has various beneficial health activities, such as antioxidant, anticancer, antihyperlipidemic, and in vivo hair growth-inducing effects.14) Despite its increasing interest and use, very little data on the potential genotoxicity of RB-SCE are available.25) Thus, the genotoxic effects of RB-SCE were evaluated using the standard three-test battery recommended by the International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use.14)

The histidine auxotroph 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.15,26) These test strains are among those recommended by the test guidelines of KFDA10) and OECD TG 471.11) These strains have been shown to be sensitive to the mutagenic activity of a wide range of chemical classes. The rfa mutation in TA strains results in the partial loss of the lipopolysaccharide barrier of the cell wall, making it more permeable to certain classes of large molecules. The uvrA or uvrB gene is essential for excision repair of the test strain. Mutations of these genes result in a deficient DNA repair system and greatly enhance the sensitivity of these strains to certain mutagens. The presence of plasmid pKM101 further increases the sensitivity of these strains to some mutagens.

In the bacterial reverse mutation test, there were no increases in the number of revertants per plate in all test strains in either the presence or absence of S9 mix, and the experimental results failed to meet the criteria for positivity. Therefore, we concluded that the RB-SCE did not induce reverse mutations in the test strains used in this study.

An in vitro chromosome aberration test using CHL cells was performed to determine whether RB-SCE affects the genotoxicity tendency. CHL cells exhibit a stable karyotype and short generation time and are easy to maintain. In the 6-h treatment with the metabolic activation system (+S), there were statistically significant increases in the frequency of metaphase with structural aberrations at 120 µg/mL (3.0%) and 140 µg/mL (9.0%). The RCC values at these doses were 55% and 40%, respectively. At the same dose levels, there were statistically significant increases in the frequencies of metaphase with numerical aberrations (endoreduplications, 3.5% and 13.0%). In the 6-h treatment without the metabolic activation system (−S), there were statistically significant increases in the frequency of metaphase with numerical aberrations (endoreduplications) at 200 µg/mL (8.0%) and 220 µg/mL (6.0%). The RCC values at these doses were 54% and 38%, respectively. In these in vitro chromosome aberration test with 6-h treatments (+S and −S), the differences of RCC values at doses with turbidity are most likely a result of the cytotoxicity induced by various soluble components of RB-SCE even in the generation of the precipitate. Further researches are needed to elucidate the reason.

The OECD test guidelines are being revised. In the draft guidelines for the in vitro chromosomal aberration test (TG 473; 29 Oct. 2012)12) and in vivo micronucleus test (TG 474; 29 Oct. 2012),13) the relative increase in the cell count (RICC) is the recommended indicator of cytotoxicity; the RCC is no longer recommended. In both of the draft guidelines, 55%±5% is the recommended cytotoxicity range.

In our study, the RCC at 120 and 140 µg/mL (+S) was 55% and 40%, which are equivalent to 45% and 60% cytotoxicity, respectively. When the RCC-based values are converted to RICC-based values, they are 12% and −17% and are equivalent to 88% and 117% cytotoxicity. This shows that these dose levels are extremely cytotoxic. Therefore, the unusual structural chromosomal aberrations observed at these dose levels were most likely a result of severe cytotoxicity. The increases in numerical aberrations (endoreduplication) observed in both of the 6-h treatment series may also be explained by the severe cytotoxicity and cell growth inhibition. The RCC values at 200 and 220 µg/mL in the second treatment series were equal to 22% RICC at 200 µg/mL and −5% RICC at 220 µg/mL, equivalent to 78% and 105% cytotoxicity, again showing severe cytotoxicity. These results indicate that RB-SCE induces chromosomal aberrations only at doses extremely cytotoxic in CHL/IU cells, but the induction of chromosomal aberrations under the present experimental conditions may have no biological significance. Because the cytotoxicity of RB-SCE in CHL cells were most likely results of damage of CHL cell membrane and cell growth inhibition rather than the harmful effect of RB-SCE on chromosome itself. For that reason, it is judged that the possibility on these cytotoxicities of RB-SCE in vivo may be low.

An in vivo micronucleus assay was performed using male ICR mice to confirm the in vitro RB-SCE genotoxicity results. There was no statistically significant or dose-related increase in the number of MNPCEs per 2000 PCEs at any dose level. This result did not meet the criteria for positivity. We conclude that RB-SCE did not induce micronuclei in the bone marrow cells of ICR mice used in this study under the present experimental conditions.

CONCLUSION

Our results indicate that RB-SCE acts as a nongenotoxic material based on the available in vivo and in vitro results. Therefore, RB-SCE can be used as a promising source of functional foods, cosmetics, and pharmaceuticals.

Acknowledgment

This work was supported by a Grant (No. 311014-03) from the Ministry for Food, Agriculture, Forestry and Fisheries, Republic of Korea.

REFERENCES
 
© 2014 The Pharmaceutical Society of Japan
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