Mutation Research 780-781 (2015) 111–116
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Evaluation of the repeated-dose liver, bone marrow and peripheral blood micronucleus and comet assays using kojic acid Yosuke Ogiwara a,∗ , Mihoko Sugiura a , Kumiko Watanabe a , Junko Tawara a , Emi Endo a , Hiromi Maruyama a , Satoshi Tsuji a , Kenta Matsue a , Hisaharu Yamada a , Yumi Wako b , Kazufumi Kawasako b a b
Drug Safety, Taisho Pharmaceutical, Co., Ltd., 1-403, Yoshino-cho, Kita-ku, Saitama-shi, Saitama 331-9530, Japan LSI Medience Corporation, 14-1 Sunayama, Kamisu-shi, Ibaraki 314-0255, Japan
a r t i c l e
i n f o
Article history: Received 8 January 2015 Accepted 9 January 2015 Available online 12 January 2015 Keywords: Genotoxicity Micronucleus assay Comet assay Kojic acid Liver
a b s t r a c t The repeated-dose liver micronucleus assay has the potential to detect liver carcinogens and could be integrated into general toxicological studies. To assess the performance of this assay, kojic acid was tested in 14-day and 28-day liver micronucleus assays. We evaluated the incidence of micronucleated cells in liver, bone marrow and peripheral blood and performed comet assays in both the liver and peripheral blood (comet assay was performed only for 14-days). Kojic acid, a skin-whitening agent used in cosmetic products, was orally dosed in six-week-old male rats at 250, 500 and 1000 mg/kg/day for 14 days, and at 125, 250 and 500 mg/kg/day for 28 days. Organ weight and histopathology were examined at the end of the experiment. Neither a clear, positive response in micronucleus (MN) incidence nor changes in the percent of tail DNA in the comet assays was noted in liver and bone marrow. An increase of relative liver weight was observed in 1000 mg/kg/day for 14 days. In histopathology, minimal hypertrophy of hepatocytes was found at 1000 mg/kg/day for 14 days. The results of both the micronucleus assay and the comet assay indicate that 14-day and 28-day repeated dosing of kojic acid are non-genotoxic in the liver and bone marrow. Kojic acid has been known to act as a tumor-promoter in thyroid carcinogenesis but has not been shown to have initiation activities in liver carcinogenesis. Findings in this study are consistent with the evidence that kojic acid is not an apparent initiator of liver carcinogenesis. Therefore, the liver micronucleus assay is simple and sensitive to detect genotoxic liver carcinogens. © 2015 Elsevier B.V. All rights reserved.
1. Introduction This collaborative study by the Mammalian Mutagenicity Study (MMS) Group, a subgroup of the Japanese Environmental Mutagen Society (JEMS), was conducted to evaluate the suitability to detect liver carcinogens of the repeated-dose liver micronucleus assay using young adult rats. This study, using kojic acid (KA), was performed as a part of this collaborative study. KA is a natural substance produced by various strains of Aspergillus, Penicillium and Acetobacter. It has been widely used as a skin-whitening agent in a category of quasi-drugs or cosmetics. There are many reports of genotoxicity and carcinogenicity in studies with KA.
∗ Corresponding author. Tel.: +81 48 669 3073; fax: +81 48 652 7254. E-mail address: [email protected] (Y. Ogiwara). http://dx.doi.org/10.1016/j.mrgentox.2015.01.004 1383-5718/© 2015 Elsevier B.V. All rights reserved.
KA tested positive for genotoxicity in a bacterial reverse mutation test (with and without metabolic activation) and a chromosome aberration study in V79 cells (only cytotoxic dose without metabolic activation) [1]. However, negative results were obtained for KA genotoxicity in a gene mutation assay in V79 cells and in a mouse lymphoma assay [1]. In in vivo studies, KA treatment tested negative for genotoxicity in an unscheduled DNA synthesis study in rat hepatocytes, and negative results were reported in mouse bone marrow micronucleus assays [1]. In the LacZ transgenic mouse study, KA treatment was negative for genotoxicity in the liver [1]. In a previous collaborative study by MMS Group, KA was judged as negative in a liver micronucleus assay using a single administration to young rats [2]. Although positive results for KA carcinogenicity were obtained in the rodent thyroid [3,4], these could be attributed to KA’s interference with iodine uptake and organification, which causes a negative feedback of the pituitary–thyroid axis following the inhibition of thyroxin synthesis [5–7]. Other studies reported that
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KA treatment increased the number and area of glutathioneS-transferase placental-form positive foci in rat liver through repeated dosing toxicity studies. However, clear tumor induction was not observed in these livers [8–11]. It was suggested that KA may act as a tumor promoter rather than a tumor initiator in the liver [7–12]. In this study, we evaluated whether KA has the potential to induce micronuclei in the liver or bone marrow of rats after repeated dosing. Since it is very useful to evaluate multiple genotoxic endpoints in the same animals so as to reduce animal usage, we also tried to evaluate KA using the comet assay in the liver and peripheral blood. 2. Materials and methods
buffered formalin and centrifuged at 50 × g for 2 min. The hepatocyte pellet was resuspended in 10% neutral buffered formalin and stored in a refrigerator until analysis. The hepatocyte suspension was mixed with an equal volume of an acridine orange (AO: 500 g/mL) and 4 ,6-diamidino-2phenylindole dihydrochloride (DAPI: 10 g/mL) staining solution immediately before observation. Small aliquots (10-L) of this mixture were dropped onto glass slides and covered with a coverslip. The specimens were observed under a fluorescent microscope (BX51-34FL-1 and BX60-34FLB-1, Olympus Co., Ltd., Tokyo, Japan) with an ultraviolet excitation filter at 400× magnification and the number of MNHEPs per 2000 parenchymal hepatocytes (HEPs), including mono-, bi- and multi-nucleated cells, was counted for each animal. The number of mitotic phase cells among the 2000HEPs was also counted to determine the mitotic index (MI).
2.1. Chemicals Kojic acid (KA, CAS No. 501-30-4, >98% purity) was purchased from Sigma–Aldrich (St. Louis, USA). KA was suspended in 0.5% sodium carboxymethylcellulose (0.5% CMC-Na, Wako Pure Chemical Industries, Ltd., Osaka, Japan) before use. 2.2. Animals Male Crl:CD (SD) rats were purchased from Charles River Japan Inc. (Tsukuba, Japan), and they were six weeks old and weighed approximately 190–240 g at the beginning of the administration. The animals were housed one per cage in an air-conditioned room with a 12-h light/dark cycle. Food pellets and water were given ad libitum throughout the acclimatization and experimental periods. This study was conducted in accordance with the “Guideline for Animal Experimentation” specified by Research Center, Taisho Pharmaceutical Co., Ltd. 2.3. Dose levels and treatment In the three-month oral toxicity study in rats, only one of the 10 animals from the 1000 mg/kg/day group died in week three [13], allowing for 1000 mg/kg/day to be set as the highest dose and 500 and 250 mg/kg/day to be set as lower doses for the 14-day toxicity study, and 500 mg/kg/day to be set as the highest dose and 250 and 125 mg/kg/day to be set as lower doses for the 28-day toxicity study. Administration volume was 10 mL/kg. Animals receiving only the vehicle served as the negative control group, and no positive control group was established. KA was routinely administered orally by gavage once a day for 14 or 28 consecutive days. 2.4. Micronucleus (MN) assays 2.4.1. Liver MN assay Approximately 24 h after the 14th (14-day toxicity study) or 28th (28-day toxicity study) administration of KA or vehicle control, the rats were euthanized under anesthesia using isoflurane. The hepatocyte preparations were performed using a modified method of Narumi et al. [14]. The livers were excised, and approximately 1-g portions of the left lateral lobe were sliced into several 1-mm strips with a razor blade. These strips were rinsed with Hanks’ balanced salt solution (HBSS; GIBCO-Invitrogen, Carlsbad, CA, USA) and treated with HBSS containing 100 units/mL of collagenase (Collagenase Yakult-S, Yakult Pharmaceutical Industry, Co., Ltd., Tokyo, Japan) in a screw-capped flask with shaking at 37 ◦ C for 1 h. They were then shaken hard by hand approximately 50 times every half hour. The HBSS containing collagenase was removed, and fresh HBSS was added. The liver suspended in fresh HBSS was shaken hard to break apart cell clumps and forced through a cell strainer. The obtained cell suspension was then rinsed with 10% neutral
2.4.2. Bone marrow MN assay After removing the livers as described in the liver MN assay, the femurs were removed from the same animals, and the bone marrow cells were collected by washing these femurs with fetal bovine serum (FBS; GIBCO-Invitrogen). This mixture of bone marrow cells and FBS was centrifuged at 180 × g for 5 min, and the resulting supernatant was removed. The bone marrow cells were then suspended in a small amount of the remaining supernatant, and 4-L aliquots of this suspension were dropped and smeared onto glass slides. After the specimens had air-dried, they were fixed in methanol for 5 min. The specimens were stained with 40 g/mL AO solution, washed with Sörenzen’s phosphate buffer and covered with a cover slip. They were immediately observed under a fluorescent microscope (BX51-34FL-1, BX53-34FL-1 and BX60-34FLB-1, Olympus Co., Ltd., Tokyo, Japan) with a blue excitation filter at 1000× magnification. A total of 800 erythrocytes [immature erythrocyte (IME) + mature erythrocyte (ME)] from each animal were observed to determine the proportion of IMEs among erythrocytes. A total of 2000 IMEs from each animal was examined for micronucleated immature erythrocytes (MNIMEs), and the incidence of MNIMEs was determined. 2.4.3. Peripheral blood MN assay Peripheral blood was collected by piercing the ventral tail vein before the 4th day of KA administration (14-day toxicity study only) and again after the last administration of KA. A total of 5 L of peripheral blood was placed on an AO-coated glass slide and covered immediately with a cover slip. Erythrocytes were observed under a fluorescent microscope (BX51-34FL-1 and BX53-34FL-1, Olympus Co., Ltd., Tokyo, Japan) with a blue excitation filter at 1000× magnification. A total of 1000 erythrocytes [reticulocyte (RET) + mature erythrocyte (ME)] from each animal was observed to determine the proportion of RETs among erythrocytes. A total of 2000 RETs from each animal was examined for micronucleated reticulocytes (MNRETs), and the incidence of MNRETs was determined. 2.5. Comet assay Comet assays were conducted (14-day toxicity study only) according to the OECD guideline [15]. An extra administration was performed 21 h after the last administration (3 h before necropsy) prior to the comet assay. Following sampling for MN assays, a small piece of liver and a small volume of peripheral blood were collected from each animal. The livers were placed into an ice-cold mincing buffer (20 mM EDTA (disodium) and 10% dimethylsulfoxide in Hank’s balanced salt solution (HBSS) (Ca++ , Mg++ and phenol red free)), rinsed sufficiently with the cold mincing buffer to remove residual blood and minced with a pair of fine scissors to release the cells. The cell suspensions were strained through a
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Table 1 Results of the liver MN assay in rats treated with kojic acid. Dose level (mg/kg/day)
No. of animals
Administration period
0
5
14-day
250
5
14-day
500
5
14-day
1000
5
14-day
0
5
28-day
125
5
28-day
250
5
28-day
500
5
28-day
MNHEPs (%) individual data (mean ± SD)
Mitotic index (%) individual data (mean ± SD)
0.10, 0.15, 0.10, 0.00, 0.05 (0.08 ± 0.06) 0.05, 0.15, 0.10, 0.10, 0.15 (0.11 ± 0.04) 0.10, 0.00, 0.10, 0.00, 0.10 (0.06 ± 0.05) 0.05, 0.00, 0.05, 0.05, 0.05
0.10, 0.10, 0.05, 0.15, 0.15 (0.11 ± 0.04) 0.15, 0.00, 0.10, 0.10, 0.10 (0.09 ± 0.05) 0.20, 0.00, 0.05, 0.10, 0.05 (0.08 ± 0.08) 0.10, 0.00, 0.00, 0.00, 0.25
(0.04 ± 0.02) 0.05, 0.05, 0.15, 0.05, 0.10 (0.08 ± 0.04) 0.00, 0.10, 0.10, 0.05, 0.15 (0.08 ± 0.06) 0.05, 0.10, 0.00, 0.05, 0.00 (0.04 ± 0.04) 0.00, 0.10, 0.00, 0.05, 0.05 (0.04 ± 0.04)
(0.07 ± 0.11) 0.00, 0.00, 0.00, 0.00, 0.00 (0.00 ± 0.00) 0.00, 0.00, 0.00, 0.00, 0.05 (0.01 ± 0.02) 0.05, 0.00, 0.00, 0.00, 0.05 (0.02 ± 0.03) 0.00, 0.05, 0.00, 0.00, 0.00 (0.01 ± 0.02)
HEP: hepatocyte MNHEPs: micronucleated HEPs.
cell strainer to remove lumps and the remaining suspension was placed on ice. Peripheral blood was diluted to ten times the volume with mincing buffer and placed on ice. Comet slides were prepared using low melting point agarose and immersed in a chilled lysing solution (100 mM EDTA (disodium), 2.5 M sodium chloride, 10 mM tris hydroxymethyl aminomethane, with pH adjusted to 10.0 with sodium hydroxide, and 1% of Triton-X100 and 10% dimethylsulfoxide) overnight in a refrigerator under a light-proof condition. After the incubation period, comet slides were immersed in an alkaline solution (300 mM sodium hydroxide and 1 mM EDTA (disodium) in purified water, pH > 13) and left to unwind for 20 min. The slides were electrophoresed at 0.7 V/cm for 20 min with a constant voltage at approximately 300 mA. After completion of electrophoresis, the slides were immersed in the neutralization buffer (0.4 M tris hydroxymethyl aminomethane in purified water, pH 7.5) for 5 min and then dehydrated by immersion into absolute ethanol (99.5%) for 5 min. The slides were stained with SYBR Gold. Comets were measured via a digital camera linked to an image analyzer (Comet IV, Perceptive Instruments, Suffolk, UK) using a fluorescent microscope (BX53-34FL-1, Olympus Co., Ltd., Tokyo, Japan) with a blue excitation filter at 200× magnification. One hundred liver cells and 100 blood cells per animal were analyzed, respectively. The comet endpoint was % tail DNA.
2.6. Histopathological examination Upon euthanasia and after the isolation of HEPs, the residual liver tissue of the left lateral lobe was fixed with 10% phosphate buffered formalin, embedded in paraffin and then stained with hematoxylin and eosin (H.E.) according to the standard method. Histopathological examination was performed under a light microscope. 2.7. Statistical analysis Differences in the incidences of MNHEPs, MNIMEs and MNRETs between the test and the vehicle control groups were analyzed using the conditional binomial test reported by Kastenbaum and Bowman [16] at the upper-tailed significance levels of 5% and 1%. Organ weight, MI, proportion of IMEs, proportion of RETs and % tail DNA were analyzed for their statistical significance by the multiple comparison test, namely, homogeneity of variance was examined by the Bartlett’s test. When a homogeneous variance was demonstrated, one-way analysis of variance was applied, but when heterogeneous variance was demonstrated, the Kruskal–Wallis test was applied. When a statistical significance was demonstrated between groups, the difference was assessed by the Dunnett’s test
Table 2 Results of the bone marrow MN assay in rats with kojic acid. Dose level(mg/kg/day)
No. of animals
Administration period
MNIMEs (%) individual data (mean ± SD)
Proportion of IME (%) individual data (mean ± SD)
0
5
14-day
250
5
14-day
500
5
14-day
1000
5
14-day
0.15, 0.35, 0.10, 0.20, 0.30 (0.22 ± 0.10) 0.20, 0.25, 0.45, 0.35, 0.05 (0.26 ± 0.15) 0.25, 0.20, 0.05, 0.10, 0.35 (0.19 ± 0.12) 0.05, 0.25, 0.40, 0.05, 0.45 (0.24 ± 0.19)
69.5, 53.3, 64.9, 63.5, 55.4 (61.3 ± 6.8) 56.6, 55.8, 51.0, 55.8, 59.6 (55.8 ± 3.1) 48.6, 48.1, 44.0, 69.1, 63.6 (54.7 ± 11.0) 49.8, 54.1, 59.5, 54.6, 51.4 (53.9 ± 3.7)
0
5
28-day
125
5
28-day
250
5
28-day
500
5
28-day
0.35, 0.20, 0.25, 0.10, 0.30 (0.24 ± 0.10) 0.10, 0.15, 0.20, 0.15, 0.05 (0.13 ± 0.06) 0.10, 0.35, 0.20, 0.25, 0.05 (0.19 ± 0.12) 0.05, 0.30, 0.10, 0.20, 0.10 (0.15 ± 0.10)
46.8, 48.0, 59.5, 57.6, 60.0 (54.4 ± 6.4) 62.0, 63.8, 58.1, 59.5, 50.4 (58.8 ± 5.2) 67.1, 55.5, 44.5, 69.5, 52.5 (57.8 ± 10.4) 52.9, 51.5, 53.3, 54.0, 53.9 (53.1 ± 1.0)
IME: immature erythrocyte MNIMEs: micronucleated IMEs.
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Table 3 Results of the peripheral blood MN assay in rats treated with kojic acid. Dose level (mg/kg/day)
No. of animals
Administration period
Sampling day
MNRETs (%) individual data (mean ± SD)
Proportion of RET (%) individual data (mean ± SD)
0
5
14-day
Day 4
250
5
14-day
Day 4
500
5
14-day
Day 4
1000
5
14-day
Day 4
0
5
14-day
Day 15 (necropsy day)
250
5
14-day
Day 15 (necropsy day)
500
5
14-day
Day 15 (necropsy day)
1000
5
14-day
Day 15 (necropsy day)
0.25, 0.10, 0.15, 0.05, 0.35 (0.18 ± 0.12) 0.15, 0.25, 0.30, 0.15, 0.05 (0.18 ± 0.10) 0.00, 0.05, 0.05, 0.30, 0.10 (0.10 ± 0.12) 0.10, 0.30, 0.15, 0.20, 0.15 (0.18 ± 0.08) 0.15, 0.30, 0.10, 0.05, 0.05 (0.13 ± 0.10) 0.10, 0.20, 0.15, 0.10, 0.15 (0.14 ± 0.04) 0.25, 0.20, 0.15, 0.15, 0.05 (0.16 ± 0.07) 0.25, 0.25, 0.20, 0.15, 0.35 (0.24 ± 0.07)*
7.20, 4.00, 7.10, 4.90, 9.00 (6.44 ± 1.99) 6.10, 5.50, 4.30, 3.30, 5.50 (4.94 ± 1.13) 4.40, 7.70, 7.10, 8.10, 5.70 (6.60 ± 1.53) 6.20, 5.40, 6.00, 6.10, 6.60 (6.06 ± 0.43) 2.90, 2.20, 1.90, 1.70, 1.60 (2.06 ± 0.52) 2.20, 2.40, 2.00, 1.90, 2.40 (2.18 ± 0.23) 2.70, 2.90, 2.20, 2.30, 2.90 (2.60 ± 0.33) 2.60, 2.60, 2.50, 2.10, 2.20 (2.40 ± 0.23)
0
5
28-day
Day 29 (necropsy day)
125
5
28-day
Day 29 (necropsy day)
250
5
28-day
Day 29 (necropsy day)
500
5
28-day
Day 29 (necropsy day)
0.15, 0.10, 0.15, 0.05, 0.30 (0.15 ± 0.09) 0.15, 0.10, 0.25, 0.00, 0.30 (0.16 ± 0.12) 0.15, 0.20, 0.05, 0.00, 0.15 (0.11 ± 0.08) 0.05, 0.10, 0.10, 0.05, 0.20 (0.10 ± 0.06)
2.30, 2.20, 4.10, 2.80, 4.20 (3.12 ± 0.97) 2.80, 2.60, 2.70, 1.70, 3.00 (2.56 ± 0.50) 3.40, 2.20, 2.20, 2.00, 3.70 (2.70 ± 0.79) 3.10, 2.40, 2.80, 2.40, 2.00 (2.54 ± 0.42)
RETs: reticulocytes MNRETs: micronucleated RETs. Kastenbaum & Bowman’s statistical method. * p ≤ 0.05.
or Dunnett-type multiple comparison test. For statistical analysis, GLP computer systems (TOXstaff21, CTC Laboratory Systems Corp., Tokyo, Japan, and MiTOX Ver. 8, Mitsui Zosen Systems Research Inc., Tokyo, Japan) were used. 3. Results and discussion The results of liver and bone marrow MN assay are shown in Tables 1 and 2. KA did not increase the frequency of micronuclei in either the liver or the bone marrow. Furthermore, KA did not affect the liver mitotic index or the proportion of IMEs. In peripheral blood, KA slightly increased the frequency of micronuclei in the 1000 mg/kg group on day 15 (necropsy day) (p = 0.05), but not on days 4 and 29, and it did not affect the proportion of RETs on days 4, 15 and 29 (Table 3). However, this result was judged as equivocal because the increase in the frequency of micronu-
clei was very slight. These results indicate that KA might have a slight potential for induction of chromosomal damage in peripheral blood. The results of the comet assay show that KA did not induce DNA damage in the liver and peripheral blood (Table 4). KA treatment increased the relative liver weight in the 1000 mg/kg/day group (14-day toxicity study only) (p < 0.01, Table 5). Minimal hypertrophy of the hepatocytes was found in all animals in the 1000 mg/kg/day group (14-day toxicity study only) (Table 6). The techniques used in this liver MN assay were confirmed to be appropriate and robust from the positive results obtained elsewhere in this collaborative trial [17]. Skills and techniques of both the MN assay and the comet assay were confirmed from the previous results of these assays in our laboratory. Therefore, no positive control group was included in this study.
Table 4 Results of the comet assay in rats treated with kojic acid. Dose level (mg/kg/day)
No. of animals
Administration perioda
Organ
% tail DNA individual data (mean ± SD)
0
5
14-day
Liver
250
5
14-day
Liver
500
5
14-day
Liver
1000
5
14-day
Liver
0
5
14-day
Peripheral blood
250
5
14-day
Peripheral blood
500
5
14-day
Peripheral blood
1000
5
14-day
Peripheral blood
5.84, 6.94, 9.55, 9.75, 5.27 (7.47 ± 2.08) 6.94, 7.20, 9.54, 6.75, 10.09 (8.10 ± 1.58) 5.75, 4.68, 7.18, 13.28, 9.71 (8.12 ± 3.44) 2.73, 2.64, 7.25, 14.21, 9.41 (7.25 ± 4.87) 2.05, 1.98, 2.91, 2.38, 2.47 (2.36 ± 0.37) 2.41, 1.19, 1.78, 1.84, 3.39 (2.12 ± 0.83) 1.69, 1.31, 1.92, 0.50, 3.37 (1.76 ± 1.05) 2.68, 2.26, 1.76, 0.92, 2.45 (2.01 ± 0.70)
a An extra administration was performed 21 h after the last administration (3 h before necropsy) prior to the comet assay.
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Table 5 Results of the organ weight of the liver in rats treated with kojic acid. Dose level (mg/kg/day)
No. of animals
Administration period
Organ
Absolute organ weight (g) individual data (mean ± SD)
Relative organ weight (%) individual data (mean ± SD)
0
5
14-day
Liver
250
5
14-day
Liver
500
5
14-day
Liver
1000
5
14-day
Liver
8.88, 7.13, 8.78, 8.53, 9.59 (8.58 ± 0.90) 8.39, 9.77, 8.67, 8.15, 8.95 (8.79 ± 0.63) 8.32, 8.56, 8.61, 8.40, 8.69 (8.52 ± 0.15) 9.28, 9.23, 9.69, 8.44, 9.68 (9.26 ± 0.51)
3.10, 2.77, 3.16, 3.06, 3.17 (3.05 ± 0.16) 3.32, 3.33, 3.20, 3.08, 3.28 (3.24 ± 0.11) 3.30, 3.21, 3.10, 3.01, 3.21 (3.16 ± 0.11) 3.58, 3.85, 3.74, 3.39, 3.89 (3.69 ± 0.20)*
0
5
28-day
Liver
125
5
28-day
Liver
250
5
28-day
Liver
500
5
28-day
Liver
11.76, 10.95, 14.24, 12.71, 9.87 (11.91 ± 1.67) 11.67, 14.92, 10.98, 13.32, 8.75 (11.93 ± 2.34) 12.37, 10.74, 10.78, 12.54, 12.68 (11.82 ± 0.98) 11.37, 10.52, 11.01, 9.88, 11.81 (10.92 ± 0.75)
3.00, 2.88, 3.34, 2.87, 2.83 (2.98 ± 0.21) 2.80, 3.51, 2.93, 3.29, 2.60 (3.03 ± 0.37) 3.18, 2.85, 2.87, 2.98, 3.16 (3.01 ± 0.16) 3.12, 3.10, 2.86, 2.82, 2.95 (2.97 ± 0.14)
Dunnett’s comparison test. * p < 0.01. Table 6 Results of the histopathology in rats treated with kojic acid. Day 15 (necropsy day of 14-day study) Organ
Findings
Dose level (mg/kg/day)
0
Animal number
1 N –
Liver Hypertrophy, hepatocyte, diffuse
250 2 N –
3 N –
4 N –
5 N –
1 N –
2 N
3 N
4 N
5 N
1 N
500 2 N –
3 N –
4 N –
5 N –
1 N –
2 N
3 N
4 N
5 N
1 N
1000 2 N –
3 N –
4 N –
5 N –
1 P +
2 N
3 N
4 N
5 N
1 N
2 P +
3 P +
4 P +
5 P +
2 N
3 N
4 N
5 N
Day 29 (necropsy day of 28-day study) Organ
Findings
Liver
Dose level (mg/kg/day)
0
Animal number
1 N
125
250
500
N: no findings present. P: findings present. –: none, +: minimal, ++: mild, +++: moderate, ++++: severe.
Our results show that KA induced slight organ weight and histopathological changes in the rat liver. However, a clear positive response was not observed in the liver, bone marrow or peripheral blood in either the MN assay or the comet assay. Nohynek et al. reported negative results in the bone marrow MN assays which were performed in mice following 2-day and 5-day repeated intraperitoneal adminstrations at doses of 125, 250, 500 and 1000 mg/kg/day and 125, 250 and 500 mg/kg/day, respectively [1]. Our result in the bone marrow MN assay was consistent with their report. On the other hand, Suzuki et al. showed that KA was negative in the liver MN assays but positive in the peripheral blood MN assay in rats, 4 weeks of age, following single oral doses of 1000 and 2000 mg/kg [2]. The different results might be due to the different ages of the animals, suggesting that younger animals may be more sensitive when used in the peripheral blood MN assay [18]. KA treatment was associated with increased tumor incidence in the liver and thyroid, however, it might act as a tumor promoter rather than a tumor initiator [7–12]. Therefore, our results showing that KA does not have genotoxic potential in either the liver or bone marrow might be correspond to these reports. The liver MN assay is a simple and sensitive method to detect genotoxic compounds in the liver and could therefore detect hepatocarcinogens [14,17] which can not be detected in the other MN assays.
Conflict of interest The authors declare that there are no conflicts of interest.
Acknowledgment A part of this work was supported by the Health and Labour Sciences Research Grant H24-Chemistry-Designation-008 (Ministry of Health, Labour and Welfare). References [1] G.J. Nohynek, D. Kirkland, D. Marzin, H. Toutain, C. Leclerc-Ribaud, H. Jinnai, An assessment of the genotoxicity and human health risk of topical use of kojic acid [5-hydroxy-2-(hydroxymethyl)-4H-pyran-4-one], Food Chem. Toxicol. 42 (2004) 93–105. [2] H. Suzuki, N. Ikeda, K. Kobayashi, Y. Terashima, Y. Shimada, T. Suzuki, T. Hagiwara, S. Hatakeyama, K. Nagaoka, J. Yoshida, Y. Saito, J. Tanaka, M. Hayashi, Evaluation of liver and peripheral blood micronucleus assays with 9 chemicals using young rats. A study by the Collaborative Study Group for the Micronucleus Test (CSGMT)/Japanese Environmental Mutagen Society (JEMS)-Mammalian Mutagenicity Study Group (MMS), Mutat. Res. 583 (2005) 133–145. [3] N. Fujimoto, H. Watanabe, T. Nakatani, G. Roy, A. Ito, Induction of thyroid tumors in (C57BL/6N X C3H/N) F1 mice by oral administration of kojic acid, Food Chem. Toxicol. 36 (1998) 697–703. [4] T. Tamura, K. Mitsumori, Y. Totsuka, K. Wakabayashi, R. Kido, H. Kasai, M. Nasu, M. Hirose, Absence of in vivo genotoxic potential and tumor initiation activity of kojic acid in the rat thyroid, Toxicology 222 (2006) 213–224. [5] Y. Higa, A. Ohkubo, S. Kitajima, A. Hatori, K. Kariya, Studies on thyroid function in rats subjected to repeated oral administration with kojic acid, J. Toxicol. Sci. 25 (2000) 167–175. [6] Y. Higa, A. Ohkubo, S. Kitajima, M. Moriyasu, K. Kariya, Effect of kojic acid on thyroidal functions in rats by single-dose administration and in cultured rat thyroid cells (FRTL-5 cells), J. Toxicol. Sci. 27 (2002) 423–431. [7] T. Tamura, K. Mitsumori, H. Onodera, N. Fujimoto, K. Yasuhara, K. Takegawa, H. Takagi, M. Hirose, Dose-threshold for thyroid tumor-promoting effects of orally administered kojic acid in rats after initiation with N-bis(2-hydroxypropyl) nitrosamine, J. Toxicol. Sci. 26 (2001) 85–94.
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[8] Y. Ota, T. Imai, J. Onose, S. Takami, Y.M. Cho, M. Hirose, A. Nishikawa, A 55-week chronic toxicity study of dietary administered kojic acid (KA) in male F344 rats, J. Toxicol. Sci. 34 (2009) 305–313. [9] Y. Chusiri, R. Wongpoomchai, A. Kakehashi, M. Wei, H. Wanibuchi, U. Vinitketkumnuan, S. Fukushima, Non-genotoxic mode of action and possible threshold for hepatocarcinogenicity of kojic acid in F344 rats, Food Chem. Toxicol. 49 (2011) 471–476. [10] Y. Higa, M. Kawabe, K. Nabae, Y. Toda, S. Kitamoto, T. Hara, N. Tanaka, K. Kiriya, M. Takahashi, Kojic acid -absence of tumor-initiating activity in rat liver, and of carcinogenic and photo-genotoxic potential in mouse skin, J. Toxicol. Sci. 32 (2007) 143–159. [11] T. Takizawa, T. Imai, J. Onose, M. Ueda, T. Tamura, K. Mitsumori, K. Izumi, M. Hirose, Enhancement of hepatocarcinogenesis by kojic acid in rat two-stage models after initiation with N-bis(2-hydroxypropyl) nitrosamine or N-diethylnitrosamine, Toxicol. Sci. 81 (2004) 43–49. [12] T. Takizawa, K. Mitsumori, T. Tamura, M. Nasu, M. Ueda, T. Imai, M. Hirose, Hepatocellular tumor induction in heterozygous p53-deficient CBA mice by a 26-week dietary administration of kojic acid, Toxicol. Sci. 73 (2003) 287–293. [13] Scientific Committee on Consumer Products, opinion on kojic acid, SCCP/1182/08, (2008).
[14] K. Narumi, K. Ashizawa, R. Takashima, H. Takasawa, S. Katayama, Y. Tsuzuki, H. Tatemoto, T. Morita, M. Hayashi, S. Hamada, Development of a repeated-dose liver micronucleus assay using adult rats: an investigation of diethylnitrosamine and 2,4-diaminotoluene, Mutat. Res. 747 (2012) 234–239. [15] OECD guideline for the testing of chemicals in vivo mammalian alkaline comet assay, OECD, TG489, (2014). [16] M.A. Kastenbaum, K.O. Bowman, Tables for determining the statistical significance of mutation frequencies, Mutat. Res. 9 (1970) 527–549. [17] S. Hamada, W. Ohyama, R. Takashima, K. Shimada, K. Matsumoto, S. Kawakami, F. Uno, H. Sui, Y. Shimada, T. Imamura, S. Matsumura, H. Sanada, K. Inoue, S. Muto, I. Ogawa, A. Hayashi, T. Takayanagi, Y. Ogiwara, A. Maeda, E. Okada, Y. Terashima, H. Takasawa, K. Narumi, Y. Wako, K. Kawasako, M. Sano, N. Ohashi, T. Morita, H. Kojima, M. Honma, M. Hayashi, Evaluation of the repeated-dose liver and gastrointestinal tract micronucleus assays with 22 chemicals using young adult rats: Summary of the collaborative study by the Collaborative Study Group for the Micronucleus Test (CSGMT)/The Japanese Environmental Mutagen Society (JEMS)–Mammalian Mutagenicity Study Group (MMS), Mutat. Res. 780–781 (2015) 2–17. [18] S. Hamada, K. Nakajima, T. Serikawa, M. Hayashi, The effect of aging on the results of the rat micronucleus assay, Mutagenesis 18 (2003) 273–275.
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Mutation Research/Genetic Toxicology and Environmental Mutagenesis journal homepage: www.elsevier.com/locate/gentox Community address: www.elsevier.com/locate/mutres
Evaluation of the repeated-dose liver, bone marrow and peripheral blood micronucleus and comet assays using kojic acid Yosuke Ogiwara a,∗ , Mihoko Sugiura a , Kumiko Watanabe a , Junko Tawara a , Emi Endo a , Hiromi Maruyama a , Satoshi Tsuji a , Kenta Matsue a , Hisaharu Yamada a , Yumi Wako b , Kazufumi Kawasako b a b
Drug Safety, Taisho Pharmaceutical, Co., Ltd., 1-403, Yoshino-cho, Kita-ku, Saitama-shi, Saitama 331-9530, Japan LSI Medience Corporation, 14-1 Sunayama, Kamisu-shi, Ibaraki 314-0255, Japan
a r t i c l e
i n f o
Article history: Received 8 January 2015 Accepted 9 January 2015 Available online 12 January 2015 Keywords: Genotoxicity Micronucleus assay Comet assay Kojic acid Liver
a b s t r a c t The repeated-dose liver micronucleus assay has the potential to detect liver carcinogens and could be integrated into general toxicological studies. To assess the performance of this assay, kojic acid was tested in 14-day and 28-day liver micronucleus assays. We evaluated the incidence of micronucleated cells in liver, bone marrow and peripheral blood and performed comet assays in both the liver and peripheral blood (comet assay was performed only for 14-days). Kojic acid, a skin-whitening agent used in cosmetic products, was orally dosed in six-week-old male rats at 250, 500 and 1000 mg/kg/day for 14 days, and at 125, 250 and 500 mg/kg/day for 28 days. Organ weight and histopathology were examined at the end of the experiment. Neither a clear, positive response in micronucleus (MN) incidence nor changes in the percent of tail DNA in the comet assays was noted in liver and bone marrow. An increase of relative liver weight was observed in 1000 mg/kg/day for 14 days. In histopathology, minimal hypertrophy of hepatocytes was found at 1000 mg/kg/day for 14 days. The results of both the micronucleus assay and the comet assay indicate that 14-day and 28-day repeated dosing of kojic acid are non-genotoxic in the liver and bone marrow. Kojic acid has been known to act as a tumor-promoter in thyroid carcinogenesis but has not been shown to have initiation activities in liver carcinogenesis. Findings in this study are consistent with the evidence that kojic acid is not an apparent initiator of liver carcinogenesis. Therefore, the liver micronucleus assay is simple and sensitive to detect genotoxic liver carcinogens. © 2015 Elsevier B.V. All rights reserved.
1. Introduction This collaborative study by the Mammalian Mutagenicity Study (MMS) Group, a subgroup of the Japanese Environmental Mutagen Society (JEMS), was conducted to evaluate the suitability to detect liver carcinogens of the repeated-dose liver micronucleus assay using young adult rats. This study, using kojic acid (KA), was performed as a part of this collaborative study. KA is a natural substance produced by various strains of Aspergillus, Penicillium and Acetobacter. It has been widely used as a skin-whitening agent in a category of quasi-drugs or cosmetics. There are many reports of genotoxicity and carcinogenicity in studies with KA.
∗ Corresponding author. Tel.: +81 48 669 3073; fax: +81 48 652 7254. E-mail address: [email protected] (Y. Ogiwara). http://dx.doi.org/10.1016/j.mrgentox.2015.01.004 1383-5718/© 2015 Elsevier B.V. All rights reserved.
KA tested positive for genotoxicity in a bacterial reverse mutation test (with and without metabolic activation) and a chromosome aberration study in V79 cells (only cytotoxic dose without metabolic activation) [1]. However, negative results were obtained for KA genotoxicity in a gene mutation assay in V79 cells and in a mouse lymphoma assay [1]. In in vivo studies, KA treatment tested negative for genotoxicity in an unscheduled DNA synthesis study in rat hepatocytes, and negative results were reported in mouse bone marrow micronucleus assays [1]. In the LacZ transgenic mouse study, KA treatment was negative for genotoxicity in the liver [1]. In a previous collaborative study by MMS Group, KA was judged as negative in a liver micronucleus assay using a single administration to young rats [2]. Although positive results for KA carcinogenicity were obtained in the rodent thyroid [3,4], these could be attributed to KA’s interference with iodine uptake and organification, which causes a negative feedback of the pituitary–thyroid axis following the inhibition of thyroxin synthesis [5–7]. Other studies reported that
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KA treatment increased the number and area of glutathioneS-transferase placental-form positive foci in rat liver through repeated dosing toxicity studies. However, clear tumor induction was not observed in these livers [8–11]. It was suggested that KA may act as a tumor promoter rather than a tumor initiator in the liver [7–12]. In this study, we evaluated whether KA has the potential to induce micronuclei in the liver or bone marrow of rats after repeated dosing. Since it is very useful to evaluate multiple genotoxic endpoints in the same animals so as to reduce animal usage, we also tried to evaluate KA using the comet assay in the liver and peripheral blood. 2. Materials and methods
buffered formalin and centrifuged at 50 × g for 2 min. The hepatocyte pellet was resuspended in 10% neutral buffered formalin and stored in a refrigerator until analysis. The hepatocyte suspension was mixed with an equal volume of an acridine orange (AO: 500 g/mL) and 4 ,6-diamidino-2phenylindole dihydrochloride (DAPI: 10 g/mL) staining solution immediately before observation. Small aliquots (10-L) of this mixture were dropped onto glass slides and covered with a coverslip. The specimens were observed under a fluorescent microscope (BX51-34FL-1 and BX60-34FLB-1, Olympus Co., Ltd., Tokyo, Japan) with an ultraviolet excitation filter at 400× magnification and the number of MNHEPs per 2000 parenchymal hepatocytes (HEPs), including mono-, bi- and multi-nucleated cells, was counted for each animal. The number of mitotic phase cells among the 2000HEPs was also counted to determine the mitotic index (MI).
2.1. Chemicals Kojic acid (KA, CAS No. 501-30-4, >98% purity) was purchased from Sigma–Aldrich (St. Louis, USA). KA was suspended in 0.5% sodium carboxymethylcellulose (0.5% CMC-Na, Wako Pure Chemical Industries, Ltd., Osaka, Japan) before use. 2.2. Animals Male Crl:CD (SD) rats were purchased from Charles River Japan Inc. (Tsukuba, Japan), and they were six weeks old and weighed approximately 190–240 g at the beginning of the administration. The animals were housed one per cage in an air-conditioned room with a 12-h light/dark cycle. Food pellets and water were given ad libitum throughout the acclimatization and experimental periods. This study was conducted in accordance with the “Guideline for Animal Experimentation” specified by Research Center, Taisho Pharmaceutical Co., Ltd. 2.3. Dose levels and treatment In the three-month oral toxicity study in rats, only one of the 10 animals from the 1000 mg/kg/day group died in week three [13], allowing for 1000 mg/kg/day to be set as the highest dose and 500 and 250 mg/kg/day to be set as lower doses for the 14-day toxicity study, and 500 mg/kg/day to be set as the highest dose and 250 and 125 mg/kg/day to be set as lower doses for the 28-day toxicity study. Administration volume was 10 mL/kg. Animals receiving only the vehicle served as the negative control group, and no positive control group was established. KA was routinely administered orally by gavage once a day for 14 or 28 consecutive days. 2.4. Micronucleus (MN) assays 2.4.1. Liver MN assay Approximately 24 h after the 14th (14-day toxicity study) or 28th (28-day toxicity study) administration of KA or vehicle control, the rats were euthanized under anesthesia using isoflurane. The hepatocyte preparations were performed using a modified method of Narumi et al. [14]. The livers were excised, and approximately 1-g portions of the left lateral lobe were sliced into several 1-mm strips with a razor blade. These strips were rinsed with Hanks’ balanced salt solution (HBSS; GIBCO-Invitrogen, Carlsbad, CA, USA) and treated with HBSS containing 100 units/mL of collagenase (Collagenase Yakult-S, Yakult Pharmaceutical Industry, Co., Ltd., Tokyo, Japan) in a screw-capped flask with shaking at 37 ◦ C for 1 h. They were then shaken hard by hand approximately 50 times every half hour. The HBSS containing collagenase was removed, and fresh HBSS was added. The liver suspended in fresh HBSS was shaken hard to break apart cell clumps and forced through a cell strainer. The obtained cell suspension was then rinsed with 10% neutral
2.4.2. Bone marrow MN assay After removing the livers as described in the liver MN assay, the femurs were removed from the same animals, and the bone marrow cells were collected by washing these femurs with fetal bovine serum (FBS; GIBCO-Invitrogen). This mixture of bone marrow cells and FBS was centrifuged at 180 × g for 5 min, and the resulting supernatant was removed. The bone marrow cells were then suspended in a small amount of the remaining supernatant, and 4-L aliquots of this suspension were dropped and smeared onto glass slides. After the specimens had air-dried, they were fixed in methanol for 5 min. The specimens were stained with 40 g/mL AO solution, washed with Sörenzen’s phosphate buffer and covered with a cover slip. They were immediately observed under a fluorescent microscope (BX51-34FL-1, BX53-34FL-1 and BX60-34FLB-1, Olympus Co., Ltd., Tokyo, Japan) with a blue excitation filter at 1000× magnification. A total of 800 erythrocytes [immature erythrocyte (IME) + mature erythrocyte (ME)] from each animal were observed to determine the proportion of IMEs among erythrocytes. A total of 2000 IMEs from each animal was examined for micronucleated immature erythrocytes (MNIMEs), and the incidence of MNIMEs was determined. 2.4.3. Peripheral blood MN assay Peripheral blood was collected by piercing the ventral tail vein before the 4th day of KA administration (14-day toxicity study only) and again after the last administration of KA. A total of 5 L of peripheral blood was placed on an AO-coated glass slide and covered immediately with a cover slip. Erythrocytes were observed under a fluorescent microscope (BX51-34FL-1 and BX53-34FL-1, Olympus Co., Ltd., Tokyo, Japan) with a blue excitation filter at 1000× magnification. A total of 1000 erythrocytes [reticulocyte (RET) + mature erythrocyte (ME)] from each animal was observed to determine the proportion of RETs among erythrocytes. A total of 2000 RETs from each animal was examined for micronucleated reticulocytes (MNRETs), and the incidence of MNRETs was determined. 2.5. Comet assay Comet assays were conducted (14-day toxicity study only) according to the OECD guideline [15]. An extra administration was performed 21 h after the last administration (3 h before necropsy) prior to the comet assay. Following sampling for MN assays, a small piece of liver and a small volume of peripheral blood were collected from each animal. The livers were placed into an ice-cold mincing buffer (20 mM EDTA (disodium) and 10% dimethylsulfoxide in Hank’s balanced salt solution (HBSS) (Ca++ , Mg++ and phenol red free)), rinsed sufficiently with the cold mincing buffer to remove residual blood and minced with a pair of fine scissors to release the cells. The cell suspensions were strained through a
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Table 1 Results of the liver MN assay in rats treated with kojic acid. Dose level (mg/kg/day)
No. of animals
Administration period
0
5
14-day
250
5
14-day
500
5
14-day
1000
5
14-day
0
5
28-day
125
5
28-day
250
5
28-day
500
5
28-day
MNHEPs (%) individual data (mean ± SD)
Mitotic index (%) individual data (mean ± SD)
0.10, 0.15, 0.10, 0.00, 0.05 (0.08 ± 0.06) 0.05, 0.15, 0.10, 0.10, 0.15 (0.11 ± 0.04) 0.10, 0.00, 0.10, 0.00, 0.10 (0.06 ± 0.05) 0.05, 0.00, 0.05, 0.05, 0.05
0.10, 0.10, 0.05, 0.15, 0.15 (0.11 ± 0.04) 0.15, 0.00, 0.10, 0.10, 0.10 (0.09 ± 0.05) 0.20, 0.00, 0.05, 0.10, 0.05 (0.08 ± 0.08) 0.10, 0.00, 0.00, 0.00, 0.25
(0.04 ± 0.02) 0.05, 0.05, 0.15, 0.05, 0.10 (0.08 ± 0.04) 0.00, 0.10, 0.10, 0.05, 0.15 (0.08 ± 0.06) 0.05, 0.10, 0.00, 0.05, 0.00 (0.04 ± 0.04) 0.00, 0.10, 0.00, 0.05, 0.05 (0.04 ± 0.04)
(0.07 ± 0.11) 0.00, 0.00, 0.00, 0.00, 0.00 (0.00 ± 0.00) 0.00, 0.00, 0.00, 0.00, 0.05 (0.01 ± 0.02) 0.05, 0.00, 0.00, 0.00, 0.05 (0.02 ± 0.03) 0.00, 0.05, 0.00, 0.00, 0.00 (0.01 ± 0.02)
HEP: hepatocyte MNHEPs: micronucleated HEPs.
cell strainer to remove lumps and the remaining suspension was placed on ice. Peripheral blood was diluted to ten times the volume with mincing buffer and placed on ice. Comet slides were prepared using low melting point agarose and immersed in a chilled lysing solution (100 mM EDTA (disodium), 2.5 M sodium chloride, 10 mM tris hydroxymethyl aminomethane, with pH adjusted to 10.0 with sodium hydroxide, and 1% of Triton-X100 and 10% dimethylsulfoxide) overnight in a refrigerator under a light-proof condition. After the incubation period, comet slides were immersed in an alkaline solution (300 mM sodium hydroxide and 1 mM EDTA (disodium) in purified water, pH > 13) and left to unwind for 20 min. The slides were electrophoresed at 0.7 V/cm for 20 min with a constant voltage at approximately 300 mA. After completion of electrophoresis, the slides were immersed in the neutralization buffer (0.4 M tris hydroxymethyl aminomethane in purified water, pH 7.5) for 5 min and then dehydrated by immersion into absolute ethanol (99.5%) for 5 min. The slides were stained with SYBR Gold. Comets were measured via a digital camera linked to an image analyzer (Comet IV, Perceptive Instruments, Suffolk, UK) using a fluorescent microscope (BX53-34FL-1, Olympus Co., Ltd., Tokyo, Japan) with a blue excitation filter at 200× magnification. One hundred liver cells and 100 blood cells per animal were analyzed, respectively. The comet endpoint was % tail DNA.
2.6. Histopathological examination Upon euthanasia and after the isolation of HEPs, the residual liver tissue of the left lateral lobe was fixed with 10% phosphate buffered formalin, embedded in paraffin and then stained with hematoxylin and eosin (H.E.) according to the standard method. Histopathological examination was performed under a light microscope. 2.7. Statistical analysis Differences in the incidences of MNHEPs, MNIMEs and MNRETs between the test and the vehicle control groups were analyzed using the conditional binomial test reported by Kastenbaum and Bowman [16] at the upper-tailed significance levels of 5% and 1%. Organ weight, MI, proportion of IMEs, proportion of RETs and % tail DNA were analyzed for their statistical significance by the multiple comparison test, namely, homogeneity of variance was examined by the Bartlett’s test. When a homogeneous variance was demonstrated, one-way analysis of variance was applied, but when heterogeneous variance was demonstrated, the Kruskal–Wallis test was applied. When a statistical significance was demonstrated between groups, the difference was assessed by the Dunnett’s test
Table 2 Results of the bone marrow MN assay in rats with kojic acid. Dose level(mg/kg/day)
No. of animals
Administration period
MNIMEs (%) individual data (mean ± SD)
Proportion of IME (%) individual data (mean ± SD)
0
5
14-day
250
5
14-day
500
5
14-day
1000
5
14-day
0.15, 0.35, 0.10, 0.20, 0.30 (0.22 ± 0.10) 0.20, 0.25, 0.45, 0.35, 0.05 (0.26 ± 0.15) 0.25, 0.20, 0.05, 0.10, 0.35 (0.19 ± 0.12) 0.05, 0.25, 0.40, 0.05, 0.45 (0.24 ± 0.19)
69.5, 53.3, 64.9, 63.5, 55.4 (61.3 ± 6.8) 56.6, 55.8, 51.0, 55.8, 59.6 (55.8 ± 3.1) 48.6, 48.1, 44.0, 69.1, 63.6 (54.7 ± 11.0) 49.8, 54.1, 59.5, 54.6, 51.4 (53.9 ± 3.7)
0
5
28-day
125
5
28-day
250
5
28-day
500
5
28-day
0.35, 0.20, 0.25, 0.10, 0.30 (0.24 ± 0.10) 0.10, 0.15, 0.20, 0.15, 0.05 (0.13 ± 0.06) 0.10, 0.35, 0.20, 0.25, 0.05 (0.19 ± 0.12) 0.05, 0.30, 0.10, 0.20, 0.10 (0.15 ± 0.10)
46.8, 48.0, 59.5, 57.6, 60.0 (54.4 ± 6.4) 62.0, 63.8, 58.1, 59.5, 50.4 (58.8 ± 5.2) 67.1, 55.5, 44.5, 69.5, 52.5 (57.8 ± 10.4) 52.9, 51.5, 53.3, 54.0, 53.9 (53.1 ± 1.0)
IME: immature erythrocyte MNIMEs: micronucleated IMEs.
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Table 3 Results of the peripheral blood MN assay in rats treated with kojic acid. Dose level (mg/kg/day)
No. of animals
Administration period
Sampling day
MNRETs (%) individual data (mean ± SD)
Proportion of RET (%) individual data (mean ± SD)
0
5
14-day
Day 4
250
5
14-day
Day 4
500
5
14-day
Day 4
1000
5
14-day
Day 4
0
5
14-day
Day 15 (necropsy day)
250
5
14-day
Day 15 (necropsy day)
500
5
14-day
Day 15 (necropsy day)
1000
5
14-day
Day 15 (necropsy day)
0.25, 0.10, 0.15, 0.05, 0.35 (0.18 ± 0.12) 0.15, 0.25, 0.30, 0.15, 0.05 (0.18 ± 0.10) 0.00, 0.05, 0.05, 0.30, 0.10 (0.10 ± 0.12) 0.10, 0.30, 0.15, 0.20, 0.15 (0.18 ± 0.08) 0.15, 0.30, 0.10, 0.05, 0.05 (0.13 ± 0.10) 0.10, 0.20, 0.15, 0.10, 0.15 (0.14 ± 0.04) 0.25, 0.20, 0.15, 0.15, 0.05 (0.16 ± 0.07) 0.25, 0.25, 0.20, 0.15, 0.35 (0.24 ± 0.07)*
7.20, 4.00, 7.10, 4.90, 9.00 (6.44 ± 1.99) 6.10, 5.50, 4.30, 3.30, 5.50 (4.94 ± 1.13) 4.40, 7.70, 7.10, 8.10, 5.70 (6.60 ± 1.53) 6.20, 5.40, 6.00, 6.10, 6.60 (6.06 ± 0.43) 2.90, 2.20, 1.90, 1.70, 1.60 (2.06 ± 0.52) 2.20, 2.40, 2.00, 1.90, 2.40 (2.18 ± 0.23) 2.70, 2.90, 2.20, 2.30, 2.90 (2.60 ± 0.33) 2.60, 2.60, 2.50, 2.10, 2.20 (2.40 ± 0.23)
0
5
28-day
Day 29 (necropsy day)
125
5
28-day
Day 29 (necropsy day)
250
5
28-day
Day 29 (necropsy day)
500
5
28-day
Day 29 (necropsy day)
0.15, 0.10, 0.15, 0.05, 0.30 (0.15 ± 0.09) 0.15, 0.10, 0.25, 0.00, 0.30 (0.16 ± 0.12) 0.15, 0.20, 0.05, 0.00, 0.15 (0.11 ± 0.08) 0.05, 0.10, 0.10, 0.05, 0.20 (0.10 ± 0.06)
2.30, 2.20, 4.10, 2.80, 4.20 (3.12 ± 0.97) 2.80, 2.60, 2.70, 1.70, 3.00 (2.56 ± 0.50) 3.40, 2.20, 2.20, 2.00, 3.70 (2.70 ± 0.79) 3.10, 2.40, 2.80, 2.40, 2.00 (2.54 ± 0.42)
RETs: reticulocytes MNRETs: micronucleated RETs. Kastenbaum & Bowman’s statistical method. * p ≤ 0.05.
or Dunnett-type multiple comparison test. For statistical analysis, GLP computer systems (TOXstaff21, CTC Laboratory Systems Corp., Tokyo, Japan, and MiTOX Ver. 8, Mitsui Zosen Systems Research Inc., Tokyo, Japan) were used. 3. Results and discussion The results of liver and bone marrow MN assay are shown in Tables 1 and 2. KA did not increase the frequency of micronuclei in either the liver or the bone marrow. Furthermore, KA did not affect the liver mitotic index or the proportion of IMEs. In peripheral blood, KA slightly increased the frequency of micronuclei in the 1000 mg/kg group on day 15 (necropsy day) (p = 0.05), but not on days 4 and 29, and it did not affect the proportion of RETs on days 4, 15 and 29 (Table 3). However, this result was judged as equivocal because the increase in the frequency of micronu-
clei was very slight. These results indicate that KA might have a slight potential for induction of chromosomal damage in peripheral blood. The results of the comet assay show that KA did not induce DNA damage in the liver and peripheral blood (Table 4). KA treatment increased the relative liver weight in the 1000 mg/kg/day group (14-day toxicity study only) (p < 0.01, Table 5). Minimal hypertrophy of the hepatocytes was found in all animals in the 1000 mg/kg/day group (14-day toxicity study only) (Table 6). The techniques used in this liver MN assay were confirmed to be appropriate and robust from the positive results obtained elsewhere in this collaborative trial [17]. Skills and techniques of both the MN assay and the comet assay were confirmed from the previous results of these assays in our laboratory. Therefore, no positive control group was included in this study.
Table 4 Results of the comet assay in rats treated with kojic acid. Dose level (mg/kg/day)
No. of animals
Administration perioda
Organ
% tail DNA individual data (mean ± SD)
0
5
14-day
Liver
250
5
14-day
Liver
500
5
14-day
Liver
1000
5
14-day
Liver
0
5
14-day
Peripheral blood
250
5
14-day
Peripheral blood
500
5
14-day
Peripheral blood
1000
5
14-day
Peripheral blood
5.84, 6.94, 9.55, 9.75, 5.27 (7.47 ± 2.08) 6.94, 7.20, 9.54, 6.75, 10.09 (8.10 ± 1.58) 5.75, 4.68, 7.18, 13.28, 9.71 (8.12 ± 3.44) 2.73, 2.64, 7.25, 14.21, 9.41 (7.25 ± 4.87) 2.05, 1.98, 2.91, 2.38, 2.47 (2.36 ± 0.37) 2.41, 1.19, 1.78, 1.84, 3.39 (2.12 ± 0.83) 1.69, 1.31, 1.92, 0.50, 3.37 (1.76 ± 1.05) 2.68, 2.26, 1.76, 0.92, 2.45 (2.01 ± 0.70)
a An extra administration was performed 21 h after the last administration (3 h before necropsy) prior to the comet assay.
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Table 5 Results of the organ weight of the liver in rats treated with kojic acid. Dose level (mg/kg/day)
No. of animals
Administration period
Organ
Absolute organ weight (g) individual data (mean ± SD)
Relative organ weight (%) individual data (mean ± SD)
0
5
14-day
Liver
250
5
14-day
Liver
500
5
14-day
Liver
1000
5
14-day
Liver
8.88, 7.13, 8.78, 8.53, 9.59 (8.58 ± 0.90) 8.39, 9.77, 8.67, 8.15, 8.95 (8.79 ± 0.63) 8.32, 8.56, 8.61, 8.40, 8.69 (8.52 ± 0.15) 9.28, 9.23, 9.69, 8.44, 9.68 (9.26 ± 0.51)
3.10, 2.77, 3.16, 3.06, 3.17 (3.05 ± 0.16) 3.32, 3.33, 3.20, 3.08, 3.28 (3.24 ± 0.11) 3.30, 3.21, 3.10, 3.01, 3.21 (3.16 ± 0.11) 3.58, 3.85, 3.74, 3.39, 3.89 (3.69 ± 0.20)*
0
5
28-day
Liver
125
5
28-day
Liver
250
5
28-day
Liver
500
5
28-day
Liver
11.76, 10.95, 14.24, 12.71, 9.87 (11.91 ± 1.67) 11.67, 14.92, 10.98, 13.32, 8.75 (11.93 ± 2.34) 12.37, 10.74, 10.78, 12.54, 12.68 (11.82 ± 0.98) 11.37, 10.52, 11.01, 9.88, 11.81 (10.92 ± 0.75)
3.00, 2.88, 3.34, 2.87, 2.83 (2.98 ± 0.21) 2.80, 3.51, 2.93, 3.29, 2.60 (3.03 ± 0.37) 3.18, 2.85, 2.87, 2.98, 3.16 (3.01 ± 0.16) 3.12, 3.10, 2.86, 2.82, 2.95 (2.97 ± 0.14)
Dunnett’s comparison test. * p < 0.01. Table 6 Results of the histopathology in rats treated with kojic acid. Day 15 (necropsy day of 14-day study) Organ
Findings
Dose level (mg/kg/day)
0
Animal number
1 N –
Liver Hypertrophy, hepatocyte, diffuse
250 2 N –
3 N –
4 N –
5 N –
1 N –
2 N
3 N
4 N
5 N
1 N
500 2 N –
3 N –
4 N –
5 N –
1 N –
2 N
3 N
4 N
5 N
1 N
1000 2 N –
3 N –
4 N –
5 N –
1 P +
2 N
3 N
4 N
5 N
1 N
2 P +
3 P +
4 P +
5 P +
2 N
3 N
4 N
5 N
Day 29 (necropsy day of 28-day study) Organ
Findings
Liver
Dose level (mg/kg/day)
0
Animal number
1 N
125
250
500
N: no findings present. P: findings present. –: none, +: minimal, ++: mild, +++: moderate, ++++: severe.
Our results show that KA induced slight organ weight and histopathological changes in the rat liver. However, a clear positive response was not observed in the liver, bone marrow or peripheral blood in either the MN assay or the comet assay. Nohynek et al. reported negative results in the bone marrow MN assays which were performed in mice following 2-day and 5-day repeated intraperitoneal adminstrations at doses of 125, 250, 500 and 1000 mg/kg/day and 125, 250 and 500 mg/kg/day, respectively [1]. Our result in the bone marrow MN assay was consistent with their report. On the other hand, Suzuki et al. showed that KA was negative in the liver MN assays but positive in the peripheral blood MN assay in rats, 4 weeks of age, following single oral doses of 1000 and 2000 mg/kg [2]. The different results might be due to the different ages of the animals, suggesting that younger animals may be more sensitive when used in the peripheral blood MN assay [18]. KA treatment was associated with increased tumor incidence in the liver and thyroid, however, it might act as a tumor promoter rather than a tumor initiator [7–12]. Therefore, our results showing that KA does not have genotoxic potential in either the liver or bone marrow might be correspond to these reports. The liver MN assay is a simple and sensitive method to detect genotoxic compounds in the liver and could therefore detect hepatocarcinogens [14,17] which can not be detected in the other MN assays.
Conflict of interest The authors declare that there are no conflicts of interest.
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