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Developmental toxicity and genotoxicity studies of wogonin
Regulatory Toxicology and Pharmacology 60 (2011) 212–217
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Developmental toxicity and genotoxicity studies of wogonin Li Zhao a,1, Zhen Chen b,1, Qing Zhao a, Daidi Wang c, Rong Hu a, Qidong You d,⇑, Qinglong Guo a,⇑ a
Jiangsu Key Laboratory of Carcinogenesis and Intervention, China Pharmaceutical University, China Department of Pharmacology, China Pharmaceutical University, China c Jiangsu Institute for Food and Drug Control, China d Department of Medicinal Chemistry, China Pharmaceutical University, China b
a r t i c l e
i n f o
Article history: Received 26 May 2010 Available online 1 April 2011 Keywords: Wogonin Developmental toxicity Ames test Chromosome aberration Micronucleus
a b s t r a c t We studied the developmental toxicities and genotoxic potency of a widely bioactive plant medicinewogonin in vivo and in vitro. In the in vivo developmental experiments, high dose of wogonin (40 mg/kg, intravenous injection) significantly induced the maternal weight gains and affected fetus including bodyweight, resorptions, live birth index and fetal skeletal alterations. In Ames test, no concentration-dependently increased TA98, TA100, and TA102 revertants were detected in wogonin groups whether in presence of metabolic activating enzymes or not. In the chromosome aberration test, wogonin dose-dependently increased structural chromosomal aberrations in CHL cells both with and without S9, even the effect was all judged ( ). In micronucleus assay, no significant changes of MNPCE/PCE and PCE/NCE were found on mouse bone marrow micronucleus in wogonin groups. We concluded that wogonin induced developmental toxicities on pregnant mice and fetus, and the genotoxicities were positive. However no significant malformation was observed and only in vitro potency of chromosome aberration was weak, which suggested us wogonin could be a relatively safe drug in clinic. Ó 2011 Elsevier Inc. All rights reserved.
1. Introduction Scutellaria baicalensis Georgi is a traditional Chinese medicinal herb mainly harvested from the northern part of China. S. baicalensis Georgi radix has been used as a traditional drug in oriental countries for various purposes (Lim et al., 2003). One of the active components in the plant is 5,7-dihydroxy-8-methoxyflavone, also known as wogonin (Fig. 1). Wogonin is a flavonoid and has been shown to exert antioxidant (Gao et al., 1999), antiviral (Ma et al., 2002; Guo et al., 2007), antithrombotic (Kimura et al., 1997) and anti-inflammatory (Park et al., 2001) activities. Recently, more and more studies focused on its antiproliferative and apoptosis inducing activity in human tumor cells (Chow et al., 2008; Lee et al., 2008; Li-Weber, 2009). In addition, it’s differentiation inducing effect (Zhang et al., 2008) and potential effect of overcoming multidrug resistance (Lee et al., 2009) were reported. Based on the wide use and pharmacological research, the acute and subchronic toxicities and plasma pharmacokinetic of wogonin were determined (Peng et al., 2009; Qi et al., 2009) in our lab. The LD50 of 286.15 mg/kg on mice and some heart injury in rats by long period of wogonin treatment were shown. Besides, a wide ⇑ Corresponding authors. Address: 24 Tongjia Xiang, Nanjing 210009, China. Fax: +86 25 3271055. E-mail addresses: [email protected] (Q. You), [email protected] (Q. Guo). 1 These authors contributed equally to this article. 0273-2300/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.yrtph.2011.03.008
margin of safety and no significant organ toxicity in dogs for a long time intravenous administration was detected. For advanced studies on toxicities of wogonin, the developmental toxicities and genotoxicities were investigated in this paper. The studies were followed and conducted in accordance with ‘Chemical Toxicity Test Technique Guideline’ issued by State Food and Drug Administration (SFDA of China). We hope the results presented in this paper should be helpful to establish the safe dosage, frequency and therapeutic duration in clinical applications of this natural product. 2. Materials and methods 2.1. Test material Wogonin was prepared at China Pharmaceutical University, Nanjing, China (purity:>99%). It was dissolved at various concentrations in DMSO or physiological saline before use. For in vivo experiments, wogonin was administrated intravenously (i.v.) once a day. 2.2. Chemicals Fetal bovine serum (FBS), penicillin/streptomycin, RPMI-1640, and trypsin were obtained from Gibco BRL (Grand Island, NY). 3methylcholanthrene, 4-nitroquinoline-N-oxide (4-NQO), 2-aminoanthracene (2AA), sodium azide, colcemid and 2-aminofluorene
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dose was determined as the half of the LD50 value of wogonin on mice (Qi et al., 2009). In 12–72 h after injection, at every time point (12, 18, 24, 48 and 72 h), 2 mice (1 male and 1 female) were sacrificed. No frequency of micronuclei in polychromatic was detected. So the time point of 24 h and the doses of 143.0, 71.5 and 35.8 mg/kg were selected. 2.5. Experimental procedures Fig. 1. Chemical structure of wogonin. Molecular formula: C16H12O5; molecular weight: 284.26.
(2-AF) were obtained from Sigma Chemical Co. Ltd. Cyclophosphamide (CP), mitomycin c (MMC), daunorubicin, acrichine was supplied by Jiangsu Heng Rui medicine Co., Ltd. (China). 2.3. Animal husbandry For developmental toxicity study, female Sprague–Dawley (SD) rats weighing 230 ± 20 g and male SD rats weighing 300–400 g were obtained from Shanghai Sipper-bk Animal Co., Ltd. (China). They were maintained in pathogen-free stainless steel cages at 24 ± 2 °C with a standard 12 h light/12 h dark cycle and allowed free access to tap water and food. For Ames test, Wistar rats (200 ± 20 g) were used for preparation of the liver microsomal (S9) fraction. For the erythrocyte micronucleus studies, healthy Kunming albino mice of both sexes with body weight of 25 ± 1 g were used. All the Wistar rats and Kunming albino mice were provided by Animal Facility at China Pharmaceutical University. They were maintained under the same conditions as described above and were allowed free access to tap water and food. All the housing conditions and test operations were carried out according to GLPs of the People’s Republic of China. 2.4. Dose level selection A pilot developmental toxicity study was conducted to assist in setting dose levels for the main developmental study. Three groups of pregnant female rats were exposed to concentrations of 80, 60 and 40 mg/kg of wogonin over days 6–15 of gestation, two rats in every group; the day of confirmed mating was designated as gestation day 0 (GD0). Except for slightly reduced weight gains, no adverse effects were observed at 40 mg/kg. At 80 mg/kg, one animal died at the 11th day of pregnancy (GD11). Significant maternal toxicities such as increased number of resorptions and reduced live fetuses were observed at 60 mg/kg. So the concentrations of 40, 13.3 and 4.4 mg/kg were selected for the developmental toxicity study. For genotoxicity studies in vitro (Ames test), dose levels of wogonin in preliminary test were 5000, 2500, 1250, 625, 313, 156, and 0 lg/plate. In the groups of 5000 and 2500 lg/plate, the significant bacteriostatic actions were observed (data were not shown). Based on the results, dose levels of 1250, 125, 12.5, 1.25 and 0.125 lg/plate were used. For chromosomal aberration test, Chinese hamster lung fibroblast (CHL) cells were cultured in RPMI-1640 medium supplemented with 10% heat-inactivated FBS. In the direct method, cells were seeded at a density of 2.2 104 cells per dish on the third day, the cells were treated with wogonin for 48 h. An IC50 of 15.697 lg/ml was calculated by probit analysis. Based on the results, the high dose was determined as 16 lg/ml, the middle and low doses were 8 and 4 lg/ml. For micronucleus studies, two groups of mice, with 10 each (5 male and 5 female) were exposed to concentrations of 143.0 and 71.5 mg/kg of wogonin once by intravenous injection. The high
2.5.1. Developmental toxicities in rats Female rats were mated overnight at the proportion of three females to each male. Vaginal smears were collected daily and examined in optics microscopy for the presence of sperm. The day of sperm detected in vaginal smears was designated as gestation day 0 (GD0). The mated females were randomly assigned to different experimental groups (15 rats per-group) and treated by 40, 13.3 and 4.4 mg/kg of wogonin and vehicle alone once everyday by i.v. in a volume of 2 ml/kg from day 6 to day 15. The maternal body weights were measured on 0, 3, 7, 10, 13, 16 and 20 days of pregnancy gestation. Food consumption was measured on days 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, and 20 of gestation. Individual clinical observations were recorded each morning and each afternoon of the exposure period. At gestation day 20, the rats were killed and the fetuses were removed immediately. The following data were recorded: total litter size, birth weights, live birth index [(number of live offspring/ number of offspring delivered) 100%], sex ratio and external alterations (Muller et al., 2009). Approximately one-half of the live fetuses were decapitated prior to dissection and fixed in Bouin’s solution and subsequently examined for visceral alterations using a free-hand sectioning technique (Wilson, 1965). The remaining fetuses were fixed in ethanol, eviscerated, macerated in 1% aqueous potassium hydroxide solution, stained with alizarin red S, and examined for skeletal alterations (Staples and Schnell, 1964). 2.5.2. S9 fraction preparation Rat liver S9 used for metabolic activation was prepared as described previously (Maron and Ames, 1983). To obtain the liver microsomal fraction, each Wistar rats was administered by intraperitoneal injection with 3-methylcholanthrene (30 mg/kg) every day, and 4 days later the rats were killed by cervical dislocation. The livers were homogenated, diluted 1:4 with 0.15 M KCl, and centrifuged for 10 min at 9000g. The supernatant was pulled and diluted (giving a protein concentration of 30 mg/ml), frozen in small aliquots, and stored at 196 °C until use. The final preparation of the metabolizing system (S9 mixture) was made in accordance with the protocol of (Ames et al., 1975). The composition and final concentrations of the S9 mix were as follows: glucose-6-phosphate, 4.4 mM; nicotinamide-adenine dinucleotide phosphate (NADP), 0.84 mM; KCl, 30 mM; NaHCO3, 0.032%; and S9 fraction, 10% (Cheng et al., 2004). 2.5.3. In vitro genotoxicity studies (Ames test) The method followed the recommendations of Maron and Ames (Maron and Ames, 1983). The Salmonella typhimurium bacteria and histidine auxotrophic strains TA97, TA98, TA100, and TA102 were obtained from Shanghai Institute for Drug Control and grown for 14 h at 35 ± 2 °C with continuous shaking. Bacteria were grown to a density of 2 109 cells/ml with OD600 absorbance of 0.2– 0.3. Top agar, containing 2 ml of heated agar, 0.1 ml of test chemical, 0.1 ml of bacteria, and 0.5 ml of S9 solution, was mixed up and added to three different minimal glucose agar plates. All plates were incubated at 37 °C for 48 h, and the number of bacteria colonies was determined. The entire experiment was replicated again on a different day with a total of six plates for each concentration of wogonin with and without S9. S9 liver cell extracts contain
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enzymes that may activate the potential mutagen. Each tester strain was routinely checked to confirm its features for optimal response to known mutagenic chemicals as follows: 4-NQO (0.5 mg/ plate), MMC (0.5 mg/plate), and 2AA (5 mg/plate). Daunorubicin (400 lg/plate), acrichine (750 lg/plate) and sodium azide (6 lg/ plate) were used as positive controls without S9, and 2-AF (80 lg/plate) was as positive control with S9. A test compound was judged to be mutagenic in the plate test if it produced, in at least one concentration and one strain, a response equal to twice (or more) of the control incidence with a dose–response relationship considered to be positive (de Serres and Shelby, 1979). The only exception was strain TA102, which has a relatively high spontaneous revertant number, where an increase by a factor of 1.5 above the control level was taken as an indication of a mutagenic effect. 2.5.4. Chromosomal aberration test The chromosomal aberration test was carried out according to the protocol previously reported (Matsuoka et al., 1993). CHL cells were treated with DMSO (solvent), MMC (0.8 lg/ml), CP (12 lg/ ml), and various concentrations of wogonin for 6 h with or without S9. After treatment, the reaction mixture was replaced with fresh medium and cells were harvested after an additional culture for 18 h. 2 h before harvest, 0.2 lg/ml colcemid was administered, and metaphase chromosomes were prepared as described (Tsutsui et al., 1983). Chromosome preparation cultures were made and stained with Giemsa solution. The test was repeated three times and the data were summarized as the mean number of chromosome aberrations. The slides were coded, but not scored, blind. One observer scored for aberrations. The number of cells with chromosomal aberrations among 100 well-spread metaphases was recorded. The types of aberrations were divided into eight groups (Cheng et al., 2004): chromosome-type gap (G), chromosome-type break (B), chromosome-type ring (R), chromosometype dicentric (D), chromatid-type gap (g), chromatid-type break (b), chromatid-type exchange (e) and ployploid (p). Achromatic lesions greater than the width of the chromatid were scored as gaps unless there was displacement of the broken piece of chromatid. If there was displacement, it was recorded as a break. If the frequency of CHL cells with structural or numerical aberrations in both untreated and solvent-treated negative controls did not exceed 4%, we judged ( ). The positive (+) result was decided by that the frequency of aberrant cells or polyploidy was P10%. The uncertain range between negative and positive, i.e. 5–9%, we termed inconclusive (±). We determined a result was (++) if the frequency P20%. The overall evaluation for each chemical was made by judging individual results in different dose groups. When the outcome was evaluated as inconclusive, the experiment was repeated. Statistical analysis was conducted using the Cochran–Armitage test.
2.5.6. Statistical analyses Data are expressed as the mean ± SEM for the number of experiments indicated. Statistical analysis of the data was performed by Student’s t-test, P-values less than 0.05were considered significant. The values of IC50 were calculated and obtained from five regression lines; each regression line was constructed of at least five points. The values of inhibition of these points ranged from 20 to 80%. 3. Results 3.1. Developmental toxicities in rats As shown in Fig. 2, in the first 7 days of pregnancy, there were no obvious changes in the maternal body weights among different groups, while from the 10th day, a dose-related inhibition of maternal body weights was observed. At 40 mg/kg, the reduction persisted until the end of the study. At the lower exposure levels, 13.3 and 4.4 mg/kg, the decrease was not significant. At the 20th day, the average maternal body weight of control group reached 450 g, while those of 40.0, 13.3 and 4.4 mg/kg wogonin groups were 401, 432 and 445 g, respectively. The mean final body weight in the high dose group was about 11% lower than that of the control group, and the middle dose group was 4% lower than that of control group. There was no significant inhibition on maternal body weight gain in the low dose group. No significant changes on food consumption and clinical observations were detected in all groups. Effects of wogonin on fetuses were showed in Table 1 there was no influence on total litter size and sex ratio in treatment groups. While a concomitant increased resorptions in wogonin treatment groups was observed. At 40 mg/kg, there was about 123% higher than that in control group, and in the middle and low group, there were 38.5% and 84.6% increase respectively. Meanwhile, a 5.81% reduction in live birth rate was detected at high dose group. And slight reductions were shown at 13.3 and 4.4 mg/kg group (2.32% and 3.56%). In addition, high dose of wogonin reduced the mean birth weights to 98%, which was statistically different from control. No such effect was observed at 13.3 and 4.4 mg/kg groups. 550
Solvent control Wognin 40.0mg/kg Wognin 13.3mg/kg
500
Wognin 4.4mg/kg
450
Body weight (g)
214
400
350
2.5.5. Erythrocyte micronucleus studies Four groups of mice (10 pre-group, half male and half female) received a single intravenous injection of 0 (solvent control), 35.8, 71.5, or 143.0 mg/kg wognin. Another group received a single 30 mg/kg dose of CP by i.v. as the positive control for micronuclei formation. At 24 h after dosing, animals were sacrificed, a bone marrow sample was collected from femurs immediately. The slides were coded, scored blindly to control for bias, and decoded upon completion. Two thousand polychromatic erythrocytes (PCE) were examined from each animal and the number of micronucleated polychromatic erythrocytes (MN-PCE) was recorded. The ratio PCE/NCE was calculated by counting a total of 500 erythrocytes (Gonzalez Borroto et al., 2003).
300
250 0
3
7
10
13
16
20
Pregnancy days Fig. 2. Effects of wogonin on maternal body weights. Pregnant rats were treated with wogonin (40, 13.3 and 4.4 mg/kg, i.v.) and vehicle alone once everyday from day 6 to day 15 of pregnancy. The maternal body weights were measured on 0, 3, 7, 10, 13, 16 and 20 days of pregnancy. The results are presented as mean ± SEM (n = 15).
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L. Zhao et al. / Regulatory Toxicology and Pharmacology 60 (2011) 212–217 Table 1 Effects of wogonin on fetuses. Group
Dose (mg/kg)
Total litter size
Resorptions
Live birth index
Birth weights (g) (X ± SEM)
Sexual ratio (female/male)
Solvent control
– 40.0 13.3 4.4
230 252 214 259
13 29 18 24
93.91% 88.10% 91.59% 90.35%
3.96±0.44 3.90±0.43* 3.95±0.46 4.08±0.43
101/115 129/93 93/103 109/125
Wogonin
Each data was tested with control group data. P < 0.05.
*
Table 2 Effects of wogonin on fetal malformations. Group
Solvent control Wogonin
Dose
External alterations
(mg/kg)
Total live fetuses
No. featus with malformations
Visceral alterations Total fetuses examined
No. featus with malformations
– 40.0 13.3 4.4
216 222 196 234
0 0 0 0
108 111 98 117
0 0 0 0
Each data was tested with control group data.
Table 3 Effects of wogonin on skeletal alterations. Group
Dose (mg/kg)
Total fetuses examined
Skull, retarded ossification
Rib, fused
Sternebra, retarded ossification
Vertebra, retarded ossification
Control
– 40.0 13.3 4.4
111 116 101 121
0 20** 9** 2
0 0 0 0
1 4** 1 0
1 3** 0 0
Wogonin
Each data was tested with control group data. P < 0.01.
**
No influence in fetal malformations on external alterations and visceral alterations was observed at all groups (Table 2). While statistically significant increases in fetal skeletal alterations consisting of delayed skull, sternebral ossification and retarded ossifications of vertebra at 40 mg/kg group were detected (Table 3). At 13.3 mg/kg, only the increase in skull was statistically significant. There were no compound-related effects on fetal skeletal alterations at 4.4 mg/kg. At all doses, no rudimentary cervical ribs were observed. The effects of wogonin on fetus skeletal alterations were shown by a dose-dependent manner.
creased in cells treated with CP (12 lg/ml; 25.0%, p < 0.01) and MMC (0.8 lg/ml; 31.5%, p < 0.01) with and without S9, respectively. Both in absence and presence of S9, wogonin (4, 8, and 16 lg/ml) concentration dependently increased structural chromosomal aberrations at 6 h of treatment. We also found that wogonin (16 lg/ml) induced chromosomal aberration was potentiated up to 2-fold with and without S9. Even so, the aberrant effect of wogonin was slight. By our judgment, the aberrant rates of every dose of wogonin were all lower than 4%, which were judged ( ).
3.2. Mutagenicity of wogonin
3.4. In vivo induction of micronuclei by wogonin
The mutagenicity of wogonin was examined by Ames method (Maron and Ames, 1983). The assay was carried out in vitro using four histidine-requiring strains of Salmonella typhimurium (TA97, TA98, TA100, and TA102) with and without a metabolicactivating enzyme (S9). Each strain of Salmonella was treated with wogonin at 1250, 125, 12.5, 1.25 and 0.125 mg/plate, respectively. Our results showed that wogonin did not increase colony formation in strains TA97, TA98, TA100, and TA102 with or without S9 mix (Table 4) at concentrations of up to 1250 mg/plate. An isolated positive Ames result is normally considered to be of no significant mutagenicity of wogonin.
Results of micronucleus assay of Kunming albino mice treated with different doses of wogonin and positive control are shown in Table 6. Mice injected (i.v.) with CP (30 mg/kg body weight) showed a significant increase in the frequency of micronucleated PCE (MN-PCE/1000) and decrease in the PCE/NCE ratio (0.51 ± 0.11) at 24 h (32.50 ± 9.42), indicating significant effect of the treatment on erythropoiesis. While there were no significant differences from controls in the frequency of MN-PCE or in the mean percent PCE for mice treated with doses of wogonin.
3.3. Induction of chromosome aberrations by wogonin in CHL cells
Flavonoids are regular edible constituents of our ordinary diet and have become active medicine clinically. Examination of their genotoxic effects has received increasing attention in recent years. Wogonin is one of the active flavonoids in S. baicalensis Georgi radix and presents extensive pharmacological actions. In the present
The effect of wogonin on chromosomes was studied with CHL cells, and the results were given in Table 5. The incidence of CHL cells with structural chromosomal aberrations significantly in-
4. Discussion
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Table 4 Revertants in three strains of Salmonella typhimurium treated with different concentrations of wogonin in the absence and presence of a metabolic-activating enzyme (S9). Without S9 Dose (lg/plate)
Group
Revertants (his+/plate) TA97
Control
– 1250 125 12.5 1.25 0.125 750 400 6
Wogonin
Acrichine Daunorubicin Sodium azide With S9 Control
2-AF
29.0 ± 3.0 26.2 ± 3.1 29.7 ± 3.3 32.0 ± 2.8 30.5 ± 2.7 28.3 ± 3.7
TA100
TA102
157.3 ± 11.7 116.0 ± 17.6 153.7 ± 15.2 161.7 ± 13.8 157.5 ± 12.6 164.3 ± 15.2
263.0 ± 15.4 228.3 ± 11.7 259.2 ± 16.2 265.3 ± 14.3 267.3 ± 16.7 273.2 ± 13.8 1031.3 ± 129.9**
293.7 ± 39.1** 939.3 ± 86.2**
– 1250 125 12.5 1.25 0.125 80
Wogonin
TA98
128.2 ± 6.6 98.3 ± 15.9 122.2 ± 14.7 120.5 ± 17.4 127.5 ± 14.9 129.0 ± 17.0 1152.0 ± 171.7**
127.0 ± 11.3 101.8 ± 12.5 125.2 ± 9.2 123.5 ± 14.5 126.0 ± 14.2 132.2 ± 14.6 1057.3 ± 76.1**
32.5 ± 3.6 27.3 ± 3.2 34.8 ± 4.3 33.5 ± 5.0 36.7 ± 3.9 31.7 ± 4.5 1209.3 ± 143.8**
162.6 ± 131.1 123.5 ± 11.4 158.0 ± 14.7 155.8 ± 14.8 166.5 ± 13.7 160.8 ± 16.8 774.3 ± 112.4**
269.3 ± 16.3 246.8 ± 11.7 278.3 ± 14.6 274.5 ± 12.8 268.2 ± 13.7 265.7 ± 15.0 447.0 ± 52.3**
Values are presented as the mean ± SEM (n = 6). The negative control consisted of 100 ll DMSO/plate. Each data was tested with control group data. ** P < 0.01.
Table 5 Chromosome aberrations of CHL cells treated with wogonin. With S9 Group DMSO Wogonin CP Without S9 DMSO Wogonin MMC
Dose (lg/ml) 5uL/ml 16 8 4 12
Cells observed 200 200 200 200 200
Aberrant cells(%) 1.5 ± 0.1 3.5 ± 0.8** 3.0 ± 0.5** 2.5 ± 0.3* 25.0 ± 2.6**
Aberrant type GBp GBE GBE GBE GBRE
++
5 lL/ml 16 8 4 0.8
200 200 200 200 200
1.5 3.0 2.5 1.5 31.5
GRp GBp GBp GBp GBE
++
± 0.1 ± 0.7** ± 0.4* ± 0.1 ± 3.1**
Judgement
Values are presented as the mean ± SEM (n = 3). The negative control consisted of 5 ll DMSO/ml. CP was the positive control in the medium with S9 and MMC was the positive control in the medium without S9. Each data was tested with DMSO group data. * P < 0.05. ** P < 0.01.
Table 6 Effects of wogonin on mouse bone marrow micronucleus. Group
Dose(mg/kg)
MNPCE/PCE (%)
PCE/NCE
Control
– 143.0 71.5 35.8 30
1.20 ± 1.14 1.50 ± 0.97 1.30 ± 0.95 1.40 ± 1.07 32.50± 9.42**
0.81 ± 0.20 0.74 ± 0.19 0.80 ± 0.16 0.77±0.16 0.51±0.11**
wogonin CP
Values are presented as the mean ± SEM (n = 10). Each data was tested with control group data. ** P < 0.01.
studies, we examined the developmental toxicities and genotoxicities of this compound. In developmental toxicity study, SD rats were dosed by intravenous injection from gestation day 6 though 15. Maternal toxicity was observed at 40 mg/kg of wogonin as a significant decline in body weight gain. From GD10, a statistical significance compared with the control group appeared in the high dose group and continued to GD20. At the meantime, there was no significant evi-
dence of a compound-related toxicity at middle and low dose group. At the last day, the mean body weight in the high dose group was about 11% lower than that of the control group, and the decrease was 4% and 1% in the middle and low dose group, respectively. While in our previous reports (Peng et al., 2009; Qi et al., 2009), no significant changes in the body weights of the beagle dogs and male SD rats were observed, even if such higher dose affected to heart were administered. It was suggested that the body weight of pregnant animals would be more sensitive to intravenous injection with wogonin than that of non-pregnant animals. The fetuses body weights were also decreased in the high group, which might be related with the maternal toxicity. Besides, exposure to high dose of wogonin caused increased resorptions, decreased live birth index and fetal skeletal alterations such as delayed skull, sternebral ossification and retarded ossifications of vertebra. However, 40 mg/kg of wogonin did not cause any fetal malformations including external and visceral alterations. In summary, there were no significant developmental toxicities at middle and low doses as evidenced by the lack of significant effects on maternal body weight gain, fetal malformations, total fetal size, live birth index, birth weights and fetal skeletal alterations.
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Over the years, Ames test has been used worldwide as an initial screening tool to determine the mutagenic potential of new chemicals and drugs, because there is a high predictive value for rodent carcinogenicity when a mutagenic response is obtained (Zeiger et al., 1990). Our data showed that wogonin was not mutagenic to Salmonella strains TA97, TA98, TA100, and TA102 at concentrations up to 1250 mg/plate. The results agreed with previous report where more than 70 flavonoids have been tested for mutagenicity in different strains of S. typhimurium by the Ames test, only aglycone flavonoids exhibited appreciable mutagenic activity (Brown and Dietrich, 1979; Middleton et al., 2000). DNA breaks and the formation of clastogens could be detected by in vitro CHL cells in the chromosome aberration test and in vivo micronucleus assay. In the chromosome aberration test, wogonin (4, 8, and 16 lg/ml) concentration-dependently increased structural chromosomal aberrations at 6 h of treatment both in the absence and presence of S9. The slight mutagenicity of wogonin may attribute to flavonoid. Wogonin is one of flavonoids, which shows the characteristics of a ‘‘Janus’’ compound (Tavares et al., 2006). In fact, flavonoids may present antimutagenic activity mainly due to their ability to scavenge free radicals, with this being one of the most important mechanisms of antimutagenesis and anticarcinogenesis. On the other hand, pro-oxidant effects of flavonoids have been reported, which would cause damage to the genetic material (Halliwell et al., 2005). So, there is mechanistic evidence indicating that some compounds can both induce and prevent damage. For that reason, before establishing a chemopreventive strategy, it is necessary to know under what conditions a compound promotes health and prevents genome damage. In the micronucleus assay, we can not rule out the possibility that wogonin was an aneugen in this study with or without metabolic activation, which was consistent with the previous report that flavonoids do not appear to be mutagenic in mammals in vivo (Middleton et al., 2000). In conclusion, wogonin induced developmental toxicities on pregnant mice and fetus, and the genotoxicities were positive. However no significant malformation was observed and only in vitro potency of chromosome aberration was weak, which suggested us wogonin could be a relatively safe drug in clinic. Conflict of interest statement The authors have no conflicts to declare. Acknowledgments This work was supported by the International Cooperation Program of China (No. 2008DFA32120), the National Natural Science Foundation of China (No. 30701032), the Science and Technology Development Program supported by the devision of Science and Technology, Jiangsu (No. BE2009674) References Ames, B.N., McCann, J., Yamasaki, E., 1975. Methods for detecting carcinogens and mutagens with the Salmonella/mammalian-microsome mutagenicity test. Mutat. Res. 31, 347–364. Brown, J.P., Dietrich, P.S., 1979. Mutagenicity of plant flavonols in the Salmonella/ mammalian microsome test: activation of flavonol glycosides by mixed glycosidases from rat cecal bacteria and other sources. Mutat. Res. 66, 223–240.
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Guo, Q., Zhao, L., You, Q., Yang, Y., Gu, H., Song, G., Lu, N., Xin, J., 2007. Anti-hepatitis B virus activity of wogonin in vitro and in vivo. Antiviral Res. 74, 16–24. Halliwell, B., Rafter, J., Jenner, A., 2005. Health promotion by flavonoids, tocopherols, tocotrienols, and other phenols: direct or indirect effects? Antioxidant or not? Am. J. Clin. Nutr. 81, 268S–276S. Kimura, Y., Okuda, H., Ogita, Z., 1997. Effects of flavonoids isolated from scutellariae radix on fibrinolytic system induced by trypsin in human umbilical vein endothelial cells. J. Nat. Prod. 60, 598–601. Lee, D.H., Kim, C., Zhang, L., Lee, Y.J., 2008. Role of p53, PUMA, and Bax in wogonin-induced apoptosis in human cancer cells. Biochem. Pharmacol. 75, 2020–2033. Lee, E., Enomoto, R., Koshiba, C., Hirano, H., 2009. Inhibition of P-glycoprotein by wogonin is involved with the potentiation of etoposide-induced apoptosis in cancer cells. Ann. N. Y. Acad. Sci. 1171, 132–136. Li-Weber, M., 2009. New therapeutic aspects of flavones: the anticancer properties of Scutellaria and its main active constituents Wogonin, Baicalein and Baicalin. Cancer Treat. Rev. 35, 57–68. Lim, B.O., Choue, R.W., Lee, H.Y., Seong, N.S., Kim, J.D., 2003. Effect of the flavonoid components obtained from Scutellaria radix on the histamine, immunoglobulin E and lipid peroxidation of spleen lymphocytes of Sprague-Dawley rats. Biosci. Biotechnol. Biochem. 67, 1126–1129. Ma, S.C., Du, J., But, P.P., Deng, X.L., Zhang, Y.W., Ooi, V.E., Xu, H.X., Lee, S.H., Lee, S.F., 2002. Antiviral Chinese medicinal herbs against respiratory syncytial virus. J. Ethnopharmacol. 79, 205–211. Maron, D.M., Ames, B.N., 1983. Revised methods for the Salmonella mutagenicity test. Mutat. Res. 113, 173–215. Matsuoka, A., Horikawa, K., Yamazaki, N., Sera, N., Sofuni, T., Tokiwa, H., 1993. Chromosomal aberrations induced in vitro by 3, 7- and 3, 9-dinitrofluoranthene. Mutat. Res. 298, 255–259. 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Toxicological studies of wogonin in experimental animals. Phytother. Res. 23, 417–422. Staples, R.E., Schnell, V.L., 1964. Refinements in rapid clearing technic in the KohAlizarin red S method for fetal bone. Stain technology 39, 61–63. Tavares, D.C., Mazzaron Barcelos, G.R., Silva, L.F., Chacon Tonin, C.C., Bastos, J.K., 2006. Propolis-induced genotoxicity and antigenotoxicity in Chinese hamster ovary cells. Toxicol. In Vitro 20, 1154–1158. Tsutsui, T., Maizumi, H., McLachlan, J.A., Barrett, J.C., 1983. Aneuploidy induction and cell transformation by diethylstilbestrol: a possible chromosomal mechanism in carcinogenesis. Cancer Res. 43, 3814–3821. Wilson, J.G., 1965. Embryological Considerations in Teratology. Ann. N. Y. Acad. Sci. 123, 219–227. Zeiger, E., Haseman, J. K., Shelby, M. D., Margolin, B. H., Tennant, R. W. (1990). Evaluation of four in vitro genetic toxicity tests for predicting rodent carcinogenicity: confirmation of earlier results with 41 additional chemicals. 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Regulatory Toxicology and Pharmacology journal homepage: www.elsevier.com/locate/yrtph
Developmental toxicity and genotoxicity studies of wogonin Li Zhao a,1, Zhen Chen b,1, Qing Zhao a, Daidi Wang c, Rong Hu a, Qidong You d,⇑, Qinglong Guo a,⇑ a
Jiangsu Key Laboratory of Carcinogenesis and Intervention, China Pharmaceutical University, China Department of Pharmacology, China Pharmaceutical University, China c Jiangsu Institute for Food and Drug Control, China d Department of Medicinal Chemistry, China Pharmaceutical University, China b
a r t i c l e
i n f o
Article history: Received 26 May 2010 Available online 1 April 2011 Keywords: Wogonin Developmental toxicity Ames test Chromosome aberration Micronucleus
a b s t r a c t We studied the developmental toxicities and genotoxic potency of a widely bioactive plant medicinewogonin in vivo and in vitro. In the in vivo developmental experiments, high dose of wogonin (40 mg/kg, intravenous injection) significantly induced the maternal weight gains and affected fetus including bodyweight, resorptions, live birth index and fetal skeletal alterations. In Ames test, no concentration-dependently increased TA98, TA100, and TA102 revertants were detected in wogonin groups whether in presence of metabolic activating enzymes or not. In the chromosome aberration test, wogonin dose-dependently increased structural chromosomal aberrations in CHL cells both with and without S9, even the effect was all judged ( ). In micronucleus assay, no significant changes of MNPCE/PCE and PCE/NCE were found on mouse bone marrow micronucleus in wogonin groups. We concluded that wogonin induced developmental toxicities on pregnant mice and fetus, and the genotoxicities were positive. However no significant malformation was observed and only in vitro potency of chromosome aberration was weak, which suggested us wogonin could be a relatively safe drug in clinic. Ó 2011 Elsevier Inc. All rights reserved.
1. Introduction Scutellaria baicalensis Georgi is a traditional Chinese medicinal herb mainly harvested from the northern part of China. S. baicalensis Georgi radix has been used as a traditional drug in oriental countries for various purposes (Lim et al., 2003). One of the active components in the plant is 5,7-dihydroxy-8-methoxyflavone, also known as wogonin (Fig. 1). Wogonin is a flavonoid and has been shown to exert antioxidant (Gao et al., 1999), antiviral (Ma et al., 2002; Guo et al., 2007), antithrombotic (Kimura et al., 1997) and anti-inflammatory (Park et al., 2001) activities. Recently, more and more studies focused on its antiproliferative and apoptosis inducing activity in human tumor cells (Chow et al., 2008; Lee et al., 2008; Li-Weber, 2009). In addition, it’s differentiation inducing effect (Zhang et al., 2008) and potential effect of overcoming multidrug resistance (Lee et al., 2009) were reported. Based on the wide use and pharmacological research, the acute and subchronic toxicities and plasma pharmacokinetic of wogonin were determined (Peng et al., 2009; Qi et al., 2009) in our lab. The LD50 of 286.15 mg/kg on mice and some heart injury in rats by long period of wogonin treatment were shown. Besides, a wide ⇑ Corresponding authors. Address: 24 Tongjia Xiang, Nanjing 210009, China. Fax: +86 25 3271055. E-mail addresses: [email protected] (Q. You), [email protected] (Q. Guo). 1 These authors contributed equally to this article. 0273-2300/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.yrtph.2011.03.008
margin of safety and no significant organ toxicity in dogs for a long time intravenous administration was detected. For advanced studies on toxicities of wogonin, the developmental toxicities and genotoxicities were investigated in this paper. The studies were followed and conducted in accordance with ‘Chemical Toxicity Test Technique Guideline’ issued by State Food and Drug Administration (SFDA of China). We hope the results presented in this paper should be helpful to establish the safe dosage, frequency and therapeutic duration in clinical applications of this natural product. 2. Materials and methods 2.1. Test material Wogonin was prepared at China Pharmaceutical University, Nanjing, China (purity:>99%). It was dissolved at various concentrations in DMSO or physiological saline before use. For in vivo experiments, wogonin was administrated intravenously (i.v.) once a day. 2.2. Chemicals Fetal bovine serum (FBS), penicillin/streptomycin, RPMI-1640, and trypsin were obtained from Gibco BRL (Grand Island, NY). 3methylcholanthrene, 4-nitroquinoline-N-oxide (4-NQO), 2-aminoanthracene (2AA), sodium azide, colcemid and 2-aminofluorene
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213
dose was determined as the half of the LD50 value of wogonin on mice (Qi et al., 2009). In 12–72 h after injection, at every time point (12, 18, 24, 48 and 72 h), 2 mice (1 male and 1 female) were sacrificed. No frequency of micronuclei in polychromatic was detected. So the time point of 24 h and the doses of 143.0, 71.5 and 35.8 mg/kg were selected. 2.5. Experimental procedures Fig. 1. Chemical structure of wogonin. Molecular formula: C16H12O5; molecular weight: 284.26.
(2-AF) were obtained from Sigma Chemical Co. Ltd. Cyclophosphamide (CP), mitomycin c (MMC), daunorubicin, acrichine was supplied by Jiangsu Heng Rui medicine Co., Ltd. (China). 2.3. Animal husbandry For developmental toxicity study, female Sprague–Dawley (SD) rats weighing 230 ± 20 g and male SD rats weighing 300–400 g were obtained from Shanghai Sipper-bk Animal Co., Ltd. (China). They were maintained in pathogen-free stainless steel cages at 24 ± 2 °C with a standard 12 h light/12 h dark cycle and allowed free access to tap water and food. For Ames test, Wistar rats (200 ± 20 g) were used for preparation of the liver microsomal (S9) fraction. For the erythrocyte micronucleus studies, healthy Kunming albino mice of both sexes with body weight of 25 ± 1 g were used. All the Wistar rats and Kunming albino mice were provided by Animal Facility at China Pharmaceutical University. They were maintained under the same conditions as described above and were allowed free access to tap water and food. All the housing conditions and test operations were carried out according to GLPs of the People’s Republic of China. 2.4. Dose level selection A pilot developmental toxicity study was conducted to assist in setting dose levels for the main developmental study. Three groups of pregnant female rats were exposed to concentrations of 80, 60 and 40 mg/kg of wogonin over days 6–15 of gestation, two rats in every group; the day of confirmed mating was designated as gestation day 0 (GD0). Except for slightly reduced weight gains, no adverse effects were observed at 40 mg/kg. At 80 mg/kg, one animal died at the 11th day of pregnancy (GD11). Significant maternal toxicities such as increased number of resorptions and reduced live fetuses were observed at 60 mg/kg. So the concentrations of 40, 13.3 and 4.4 mg/kg were selected for the developmental toxicity study. For genotoxicity studies in vitro (Ames test), dose levels of wogonin in preliminary test were 5000, 2500, 1250, 625, 313, 156, and 0 lg/plate. In the groups of 5000 and 2500 lg/plate, the significant bacteriostatic actions were observed (data were not shown). Based on the results, dose levels of 1250, 125, 12.5, 1.25 and 0.125 lg/plate were used. For chromosomal aberration test, Chinese hamster lung fibroblast (CHL) cells were cultured in RPMI-1640 medium supplemented with 10% heat-inactivated FBS. In the direct method, cells were seeded at a density of 2.2 104 cells per dish on the third day, the cells were treated with wogonin for 48 h. An IC50 of 15.697 lg/ml was calculated by probit analysis. Based on the results, the high dose was determined as 16 lg/ml, the middle and low doses were 8 and 4 lg/ml. For micronucleus studies, two groups of mice, with 10 each (5 male and 5 female) were exposed to concentrations of 143.0 and 71.5 mg/kg of wogonin once by intravenous injection. The high
2.5.1. Developmental toxicities in rats Female rats were mated overnight at the proportion of three females to each male. Vaginal smears were collected daily and examined in optics microscopy for the presence of sperm. The day of sperm detected in vaginal smears was designated as gestation day 0 (GD0). The mated females were randomly assigned to different experimental groups (15 rats per-group) and treated by 40, 13.3 and 4.4 mg/kg of wogonin and vehicle alone once everyday by i.v. in a volume of 2 ml/kg from day 6 to day 15. The maternal body weights were measured on 0, 3, 7, 10, 13, 16 and 20 days of pregnancy gestation. Food consumption was measured on days 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, and 20 of gestation. Individual clinical observations were recorded each morning and each afternoon of the exposure period. At gestation day 20, the rats were killed and the fetuses were removed immediately. The following data were recorded: total litter size, birth weights, live birth index [(number of live offspring/ number of offspring delivered) 100%], sex ratio and external alterations (Muller et al., 2009). Approximately one-half of the live fetuses were decapitated prior to dissection and fixed in Bouin’s solution and subsequently examined for visceral alterations using a free-hand sectioning technique (Wilson, 1965). The remaining fetuses were fixed in ethanol, eviscerated, macerated in 1% aqueous potassium hydroxide solution, stained with alizarin red S, and examined for skeletal alterations (Staples and Schnell, 1964). 2.5.2. S9 fraction preparation Rat liver S9 used for metabolic activation was prepared as described previously (Maron and Ames, 1983). To obtain the liver microsomal fraction, each Wistar rats was administered by intraperitoneal injection with 3-methylcholanthrene (30 mg/kg) every day, and 4 days later the rats were killed by cervical dislocation. The livers were homogenated, diluted 1:4 with 0.15 M KCl, and centrifuged for 10 min at 9000g. The supernatant was pulled and diluted (giving a protein concentration of 30 mg/ml), frozen in small aliquots, and stored at 196 °C until use. The final preparation of the metabolizing system (S9 mixture) was made in accordance with the protocol of (Ames et al., 1975). The composition and final concentrations of the S9 mix were as follows: glucose-6-phosphate, 4.4 mM; nicotinamide-adenine dinucleotide phosphate (NADP), 0.84 mM; KCl, 30 mM; NaHCO3, 0.032%; and S9 fraction, 10% (Cheng et al., 2004). 2.5.3. In vitro genotoxicity studies (Ames test) The method followed the recommendations of Maron and Ames (Maron and Ames, 1983). The Salmonella typhimurium bacteria and histidine auxotrophic strains TA97, TA98, TA100, and TA102 were obtained from Shanghai Institute for Drug Control and grown for 14 h at 35 ± 2 °C with continuous shaking. Bacteria were grown to a density of 2 109 cells/ml with OD600 absorbance of 0.2– 0.3. Top agar, containing 2 ml of heated agar, 0.1 ml of test chemical, 0.1 ml of bacteria, and 0.5 ml of S9 solution, was mixed up and added to three different minimal glucose agar plates. All plates were incubated at 37 °C for 48 h, and the number of bacteria colonies was determined. The entire experiment was replicated again on a different day with a total of six plates for each concentration of wogonin with and without S9. S9 liver cell extracts contain
L. Zhao et al. / Regulatory Toxicology and Pharmacology 60 (2011) 212–217
enzymes that may activate the potential mutagen. Each tester strain was routinely checked to confirm its features for optimal response to known mutagenic chemicals as follows: 4-NQO (0.5 mg/ plate), MMC (0.5 mg/plate), and 2AA (5 mg/plate). Daunorubicin (400 lg/plate), acrichine (750 lg/plate) and sodium azide (6 lg/ plate) were used as positive controls without S9, and 2-AF (80 lg/plate) was as positive control with S9. A test compound was judged to be mutagenic in the plate test if it produced, in at least one concentration and one strain, a response equal to twice (or more) of the control incidence with a dose–response relationship considered to be positive (de Serres and Shelby, 1979). The only exception was strain TA102, which has a relatively high spontaneous revertant number, where an increase by a factor of 1.5 above the control level was taken as an indication of a mutagenic effect. 2.5.4. Chromosomal aberration test The chromosomal aberration test was carried out according to the protocol previously reported (Matsuoka et al., 1993). CHL cells were treated with DMSO (solvent), MMC (0.8 lg/ml), CP (12 lg/ ml), and various concentrations of wogonin for 6 h with or without S9. After treatment, the reaction mixture was replaced with fresh medium and cells were harvested after an additional culture for 18 h. 2 h before harvest, 0.2 lg/ml colcemid was administered, and metaphase chromosomes were prepared as described (Tsutsui et al., 1983). Chromosome preparation cultures were made and stained with Giemsa solution. The test was repeated three times and the data were summarized as the mean number of chromosome aberrations. The slides were coded, but not scored, blind. One observer scored for aberrations. The number of cells with chromosomal aberrations among 100 well-spread metaphases was recorded. The types of aberrations were divided into eight groups (Cheng et al., 2004): chromosome-type gap (G), chromosome-type break (B), chromosome-type ring (R), chromosometype dicentric (D), chromatid-type gap (g), chromatid-type break (b), chromatid-type exchange (e) and ployploid (p). Achromatic lesions greater than the width of the chromatid were scored as gaps unless there was displacement of the broken piece of chromatid. If there was displacement, it was recorded as a break. If the frequency of CHL cells with structural or numerical aberrations in both untreated and solvent-treated negative controls did not exceed 4%, we judged ( ). The positive (+) result was decided by that the frequency of aberrant cells or polyploidy was P10%. The uncertain range between negative and positive, i.e. 5–9%, we termed inconclusive (±). We determined a result was (++) if the frequency P20%. The overall evaluation for each chemical was made by judging individual results in different dose groups. When the outcome was evaluated as inconclusive, the experiment was repeated. Statistical analysis was conducted using the Cochran–Armitage test.
2.5.6. Statistical analyses Data are expressed as the mean ± SEM for the number of experiments indicated. Statistical analysis of the data was performed by Student’s t-test, P-values less than 0.05were considered significant. The values of IC50 were calculated and obtained from five regression lines; each regression line was constructed of at least five points. The values of inhibition of these points ranged from 20 to 80%. 3. Results 3.1. Developmental toxicities in rats As shown in Fig. 2, in the first 7 days of pregnancy, there were no obvious changes in the maternal body weights among different groups, while from the 10th day, a dose-related inhibition of maternal body weights was observed. At 40 mg/kg, the reduction persisted until the end of the study. At the lower exposure levels, 13.3 and 4.4 mg/kg, the decrease was not significant. At the 20th day, the average maternal body weight of control group reached 450 g, while those of 40.0, 13.3 and 4.4 mg/kg wogonin groups were 401, 432 and 445 g, respectively. The mean final body weight in the high dose group was about 11% lower than that of the control group, and the middle dose group was 4% lower than that of control group. There was no significant inhibition on maternal body weight gain in the low dose group. No significant changes on food consumption and clinical observations were detected in all groups. Effects of wogonin on fetuses were showed in Table 1 there was no influence on total litter size and sex ratio in treatment groups. While a concomitant increased resorptions in wogonin treatment groups was observed. At 40 mg/kg, there was about 123% higher than that in control group, and in the middle and low group, there were 38.5% and 84.6% increase respectively. Meanwhile, a 5.81% reduction in live birth rate was detected at high dose group. And slight reductions were shown at 13.3 and 4.4 mg/kg group (2.32% and 3.56%). In addition, high dose of wogonin reduced the mean birth weights to 98%, which was statistically different from control. No such effect was observed at 13.3 and 4.4 mg/kg groups. 550
Solvent control Wognin 40.0mg/kg Wognin 13.3mg/kg
500
Wognin 4.4mg/kg
450
Body weight (g)
214
400
350
2.5.5. Erythrocyte micronucleus studies Four groups of mice (10 pre-group, half male and half female) received a single intravenous injection of 0 (solvent control), 35.8, 71.5, or 143.0 mg/kg wognin. Another group received a single 30 mg/kg dose of CP by i.v. as the positive control for micronuclei formation. At 24 h after dosing, animals were sacrificed, a bone marrow sample was collected from femurs immediately. The slides were coded, scored blindly to control for bias, and decoded upon completion. Two thousand polychromatic erythrocytes (PCE) were examined from each animal and the number of micronucleated polychromatic erythrocytes (MN-PCE) was recorded. The ratio PCE/NCE was calculated by counting a total of 500 erythrocytes (Gonzalez Borroto et al., 2003).
300
250 0
3
7
10
13
16
20
Pregnancy days Fig. 2. Effects of wogonin on maternal body weights. Pregnant rats were treated with wogonin (40, 13.3 and 4.4 mg/kg, i.v.) and vehicle alone once everyday from day 6 to day 15 of pregnancy. The maternal body weights were measured on 0, 3, 7, 10, 13, 16 and 20 days of pregnancy. The results are presented as mean ± SEM (n = 15).
215
L. Zhao et al. / Regulatory Toxicology and Pharmacology 60 (2011) 212–217 Table 1 Effects of wogonin on fetuses. Group
Dose (mg/kg)
Total litter size
Resorptions
Live birth index
Birth weights (g) (X ± SEM)
Sexual ratio (female/male)
Solvent control
– 40.0 13.3 4.4
230 252 214 259
13 29 18 24
93.91% 88.10% 91.59% 90.35%
3.96±0.44 3.90±0.43* 3.95±0.46 4.08±0.43
101/115 129/93 93/103 109/125
Wogonin
Each data was tested with control group data. P < 0.05.
*
Table 2 Effects of wogonin on fetal malformations. Group
Solvent control Wogonin
Dose
External alterations
(mg/kg)
Total live fetuses
No. featus with malformations
Visceral alterations Total fetuses examined
No. featus with malformations
– 40.0 13.3 4.4
216 222 196 234
0 0 0 0
108 111 98 117
0 0 0 0
Each data was tested with control group data.
Table 3 Effects of wogonin on skeletal alterations. Group
Dose (mg/kg)
Total fetuses examined
Skull, retarded ossification
Rib, fused
Sternebra, retarded ossification
Vertebra, retarded ossification
Control
– 40.0 13.3 4.4
111 116 101 121
0 20** 9** 2
0 0 0 0
1 4** 1 0
1 3** 0 0
Wogonin
Each data was tested with control group data. P < 0.01.
**
No influence in fetal malformations on external alterations and visceral alterations was observed at all groups (Table 2). While statistically significant increases in fetal skeletal alterations consisting of delayed skull, sternebral ossification and retarded ossifications of vertebra at 40 mg/kg group were detected (Table 3). At 13.3 mg/kg, only the increase in skull was statistically significant. There were no compound-related effects on fetal skeletal alterations at 4.4 mg/kg. At all doses, no rudimentary cervical ribs were observed. The effects of wogonin on fetus skeletal alterations were shown by a dose-dependent manner.
creased in cells treated with CP (12 lg/ml; 25.0%, p < 0.01) and MMC (0.8 lg/ml; 31.5%, p < 0.01) with and without S9, respectively. Both in absence and presence of S9, wogonin (4, 8, and 16 lg/ml) concentration dependently increased structural chromosomal aberrations at 6 h of treatment. We also found that wogonin (16 lg/ml) induced chromosomal aberration was potentiated up to 2-fold with and without S9. Even so, the aberrant effect of wogonin was slight. By our judgment, the aberrant rates of every dose of wogonin were all lower than 4%, which were judged ( ).
3.2. Mutagenicity of wogonin
3.4. In vivo induction of micronuclei by wogonin
The mutagenicity of wogonin was examined by Ames method (Maron and Ames, 1983). The assay was carried out in vitro using four histidine-requiring strains of Salmonella typhimurium (TA97, TA98, TA100, and TA102) with and without a metabolicactivating enzyme (S9). Each strain of Salmonella was treated with wogonin at 1250, 125, 12.5, 1.25 and 0.125 mg/plate, respectively. Our results showed that wogonin did not increase colony formation in strains TA97, TA98, TA100, and TA102 with or without S9 mix (Table 4) at concentrations of up to 1250 mg/plate. An isolated positive Ames result is normally considered to be of no significant mutagenicity of wogonin.
Results of micronucleus assay of Kunming albino mice treated with different doses of wogonin and positive control are shown in Table 6. Mice injected (i.v.) with CP (30 mg/kg body weight) showed a significant increase in the frequency of micronucleated PCE (MN-PCE/1000) and decrease in the PCE/NCE ratio (0.51 ± 0.11) at 24 h (32.50 ± 9.42), indicating significant effect of the treatment on erythropoiesis. While there were no significant differences from controls in the frequency of MN-PCE or in the mean percent PCE for mice treated with doses of wogonin.
3.3. Induction of chromosome aberrations by wogonin in CHL cells
Flavonoids are regular edible constituents of our ordinary diet and have become active medicine clinically. Examination of their genotoxic effects has received increasing attention in recent years. Wogonin is one of the active flavonoids in S. baicalensis Georgi radix and presents extensive pharmacological actions. In the present
The effect of wogonin on chromosomes was studied with CHL cells, and the results were given in Table 5. The incidence of CHL cells with structural chromosomal aberrations significantly in-
4. Discussion
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Table 4 Revertants in three strains of Salmonella typhimurium treated with different concentrations of wogonin in the absence and presence of a metabolic-activating enzyme (S9). Without S9 Dose (lg/plate)
Group
Revertants (his+/plate) TA97
Control
– 1250 125 12.5 1.25 0.125 750 400 6
Wogonin
Acrichine Daunorubicin Sodium azide With S9 Control
2-AF
29.0 ± 3.0 26.2 ± 3.1 29.7 ± 3.3 32.0 ± 2.8 30.5 ± 2.7 28.3 ± 3.7
TA100
TA102
157.3 ± 11.7 116.0 ± 17.6 153.7 ± 15.2 161.7 ± 13.8 157.5 ± 12.6 164.3 ± 15.2
263.0 ± 15.4 228.3 ± 11.7 259.2 ± 16.2 265.3 ± 14.3 267.3 ± 16.7 273.2 ± 13.8 1031.3 ± 129.9**
293.7 ± 39.1** 939.3 ± 86.2**
– 1250 125 12.5 1.25 0.125 80
Wogonin
TA98
128.2 ± 6.6 98.3 ± 15.9 122.2 ± 14.7 120.5 ± 17.4 127.5 ± 14.9 129.0 ± 17.0 1152.0 ± 171.7**
127.0 ± 11.3 101.8 ± 12.5 125.2 ± 9.2 123.5 ± 14.5 126.0 ± 14.2 132.2 ± 14.6 1057.3 ± 76.1**
32.5 ± 3.6 27.3 ± 3.2 34.8 ± 4.3 33.5 ± 5.0 36.7 ± 3.9 31.7 ± 4.5 1209.3 ± 143.8**
162.6 ± 131.1 123.5 ± 11.4 158.0 ± 14.7 155.8 ± 14.8 166.5 ± 13.7 160.8 ± 16.8 774.3 ± 112.4**
269.3 ± 16.3 246.8 ± 11.7 278.3 ± 14.6 274.5 ± 12.8 268.2 ± 13.7 265.7 ± 15.0 447.0 ± 52.3**
Values are presented as the mean ± SEM (n = 6). The negative control consisted of 100 ll DMSO/plate. Each data was tested with control group data. ** P < 0.01.
Table 5 Chromosome aberrations of CHL cells treated with wogonin. With S9 Group DMSO Wogonin CP Without S9 DMSO Wogonin MMC
Dose (lg/ml) 5uL/ml 16 8 4 12
Cells observed 200 200 200 200 200
Aberrant cells(%) 1.5 ± 0.1 3.5 ± 0.8** 3.0 ± 0.5** 2.5 ± 0.3* 25.0 ± 2.6**
Aberrant type GBp GBE GBE GBE GBRE
++
5 lL/ml 16 8 4 0.8
200 200 200 200 200
1.5 3.0 2.5 1.5 31.5
GRp GBp GBp GBp GBE
++
± 0.1 ± 0.7** ± 0.4* ± 0.1 ± 3.1**
Judgement
Values are presented as the mean ± SEM (n = 3). The negative control consisted of 5 ll DMSO/ml. CP was the positive control in the medium with S9 and MMC was the positive control in the medium without S9. Each data was tested with DMSO group data. * P < 0.05. ** P < 0.01.
Table 6 Effects of wogonin on mouse bone marrow micronucleus. Group
Dose(mg/kg)
MNPCE/PCE (%)
PCE/NCE
Control
– 143.0 71.5 35.8 30
1.20 ± 1.14 1.50 ± 0.97 1.30 ± 0.95 1.40 ± 1.07 32.50± 9.42**
0.81 ± 0.20 0.74 ± 0.19 0.80 ± 0.16 0.77±0.16 0.51±0.11**
wogonin CP
Values are presented as the mean ± SEM (n = 10). Each data was tested with control group data. ** P < 0.01.
studies, we examined the developmental toxicities and genotoxicities of this compound. In developmental toxicity study, SD rats were dosed by intravenous injection from gestation day 6 though 15. Maternal toxicity was observed at 40 mg/kg of wogonin as a significant decline in body weight gain. From GD10, a statistical significance compared with the control group appeared in the high dose group and continued to GD20. At the meantime, there was no significant evi-
dence of a compound-related toxicity at middle and low dose group. At the last day, the mean body weight in the high dose group was about 11% lower than that of the control group, and the decrease was 4% and 1% in the middle and low dose group, respectively. While in our previous reports (Peng et al., 2009; Qi et al., 2009), no significant changes in the body weights of the beagle dogs and male SD rats were observed, even if such higher dose affected to heart were administered. It was suggested that the body weight of pregnant animals would be more sensitive to intravenous injection with wogonin than that of non-pregnant animals. The fetuses body weights were also decreased in the high group, which might be related with the maternal toxicity. Besides, exposure to high dose of wogonin caused increased resorptions, decreased live birth index and fetal skeletal alterations such as delayed skull, sternebral ossification and retarded ossifications of vertebra. However, 40 mg/kg of wogonin did not cause any fetal malformations including external and visceral alterations. In summary, there were no significant developmental toxicities at middle and low doses as evidenced by the lack of significant effects on maternal body weight gain, fetal malformations, total fetal size, live birth index, birth weights and fetal skeletal alterations.
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Over the years, Ames test has been used worldwide as an initial screening tool to determine the mutagenic potential of new chemicals and drugs, because there is a high predictive value for rodent carcinogenicity when a mutagenic response is obtained (Zeiger et al., 1990). Our data showed that wogonin was not mutagenic to Salmonella strains TA97, TA98, TA100, and TA102 at concentrations up to 1250 mg/plate. The results agreed with previous report where more than 70 flavonoids have been tested for mutagenicity in different strains of S. typhimurium by the Ames test, only aglycone flavonoids exhibited appreciable mutagenic activity (Brown and Dietrich, 1979; Middleton et al., 2000). DNA breaks and the formation of clastogens could be detected by in vitro CHL cells in the chromosome aberration test and in vivo micronucleus assay. In the chromosome aberration test, wogonin (4, 8, and 16 lg/ml) concentration-dependently increased structural chromosomal aberrations at 6 h of treatment both in the absence and presence of S9. The slight mutagenicity of wogonin may attribute to flavonoid. Wogonin is one of flavonoids, which shows the characteristics of a ‘‘Janus’’ compound (Tavares et al., 2006). In fact, flavonoids may present antimutagenic activity mainly due to their ability to scavenge free radicals, with this being one of the most important mechanisms of antimutagenesis and anticarcinogenesis. On the other hand, pro-oxidant effects of flavonoids have been reported, which would cause damage to the genetic material (Halliwell et al., 2005). So, there is mechanistic evidence indicating that some compounds can both induce and prevent damage. For that reason, before establishing a chemopreventive strategy, it is necessary to know under what conditions a compound promotes health and prevents genome damage. In the micronucleus assay, we can not rule out the possibility that wogonin was an aneugen in this study with or without metabolic activation, which was consistent with the previous report that flavonoids do not appear to be mutagenic in mammals in vivo (Middleton et al., 2000). In conclusion, wogonin induced developmental toxicities on pregnant mice and fetus, and the genotoxicities were positive. However no significant malformation was observed and only in vitro potency of chromosome aberration was weak, which suggested us wogonin could be a relatively safe drug in clinic. Conflict of interest statement The authors have no conflicts to declare. Acknowledgments This work was supported by the International Cooperation Program of China (No. 2008DFA32120), the National Natural Science Foundation of China (No. 30701032), the Science and Technology Development Program supported by the devision of Science and Technology, Jiangsu (No. BE2009674) References Ames, B.N., McCann, J., Yamasaki, E., 1975. Methods for detecting carcinogens and mutagens with the Salmonella/mammalian-microsome mutagenicity test. Mutat. Res. 31, 347–364. Brown, J.P., Dietrich, P.S., 1979. Mutagenicity of plant flavonols in the Salmonella/ mammalian microsome test: activation of flavonol glycosides by mixed glycosidases from rat cecal bacteria and other sources. Mutat. Res. 66, 223–240.
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