Study on Acute and Subchronic Toxicity, Cytotoxicity and In Vitro Developmental Toxicity of Safflower Extracts of “IL 111” and “LRV 51 51” Cultivars

1. Introduction

   IL 111 and LRV 51 51, two of 30 different cultivars of safflower, Carthamus tinctorius L, Asteraceae, are commonly cultivated in Iran [1]. IL 111 has red-colored florets, without spiny leaves, while LRV 51 51 has yellow florets and spiny leaves. Safflower cultivar crops are universally used for coloring (red dyes, yellow dyes), flavoring in foods and medicines. Safflower oil is used as cooking oil, in salad dressing and for the production of margarine, as well as a nutritional supplement. The safflower flowers are used as a cheaper substitute for saffron [2, 3]. A transgenic safflower plant is currently being used in the pharmaceutical industry (SemBioSysl) to produce human insulin. In traditional medicine, safflower has been indicated for its sedative, laxative and anti-inflammatory properties [4, 5]. Potential benefits of safflower have also been demonstrated in hypercoagulopathy, hypertension, hepatoprotection, neuroprotec-tion, inhibition of melanin production as well as antioxidant and anti-tumor activities [6-8]. On the other hand, due to its immunosup-pressant effects, Safflower is recommended to be cautiously utilized in situations where the immune system is disturbed [9, 10]. Although Safflower is reported to be useful for menstrual problems, it is forbidden in pregnancy because of uterine stimulant property [11]. Flavonoides, phenylethanoid glycosides, coumarins, fatty acids, steroids and polysaccharides are among the safflower floret components [6], and might be responsible for the effects observed.

      Botanical properties of safflower and its medical application have encouraged the improvement of its cultivars all over the world; therefore, evaluation of acute and subchronic toxicity, potential mutagenicity, cytotoxicity and teratogenicity of these medicinal plants are important. In our previous study, we observed that IL 111 did not elicit a significant level of mutagenic activity in TA98 and TA100 strains of Salmonella typhimurium using Ames assay [12]. In this study, we aimed to evaluate the acute and subchronic toxicity of IL 111 and LRV 51 51 in mice and rats, their potential cytotoxicity on murine fibroblasts, and their direct teratogenic effects on embryonic cells.

 Table 1. Haematological and biochemical study rats treated with safflower extracts for 9-weeks.

*Significantly difference from control group (p < 0.05 ); (n=6); WBC, white blood cell count; RBC, red blood cell count; Hb, Hemoglobin; Hct, Hematocrit; MCV, mean corpuscular volume; MCH, mean corpuscular haemoglobin; MCHC, mean corpuscular haemoglobin concentration; PT, prothrombin time; AST, aspartate aminotransferase; ALT, alanine aminotransferase; ALP, alkaline phophatase; LDH, lactate dehydrogenase; CPK, creatine phosphokinase.

2. Materials and methods

2.1. Chemicals

        Dulbecco’s modified Eagle’s medium (DMEM), penicillin-streptomycin, fetal bovine serum (FBS), trypsin-EDTA, DMEM- F12 and L-glutamine were supplied by Gibco, UK. MTT (3-(4, 5 dimethylthiazol-2yl) 2, 5 diphenyl tetrazolium) was provided by Sigma, USA. Nylon mesh was obtained from Portex, UK.

2.2. Preparation of extracts

      IL 111 (red florets) and LRV 51 51 (yellow florets), obtained from the Seed and Plant Improvement Institute (Karaj, Iran), and authenticated at the gene bank of ministry of Agriculture of Iran where their voucher specimens are deposited as tn-79813 and tn-79812, respectively. Legitimate [authenticated and negative for aflatoxines] dried florets (20 g) were minced and macerated in ethanolic solution (70%) for 3 days at room temperature. The corresponding extracts were filtered, desiccated by freeze dryer (Labconco, USA), and stored at 4-8 ºC. Ethanol was totally removed during freeze-drying and the solutions of extracts for each experiment were prepared every week.

 Figure 1. Effects of C. tinctorius (IL 111 & LRV 51 51) on food consumption and body weight (n=6). The rats were treated with IL 111 and LRV 51 51 extracts for 9 weeks. A: Animals were monitored daily and the average amount of food/rat/week was calculated. B: Animals were weighted individually and the results are depicted against time (week). There is not a statistically significant difference between the extracts and control groups.

Figure 2. Cytotoxicity of C. tinctorius (IL 111 and LRV 51 51) in Balb/C fibroblast cells. Cells were exposed to different concentration of extracts for 72 h and MTT assay was applied to study the cytotoxicity. Both of the extracts caused a dose dependent reduction in cell viability ( p < 0.05 ); however there is not a statistically significant difference between the extracts (two-way ANOVA; n=6).

Table 2. Final body weight and organ index in rats treated with safflower extracts for 9-weeks.

No significant difference was observed between treatment and control groups (n=6).

2.3. Animals

     Wistar albino rats and Balb/c mice (Pasteur, Iran), were maintained in animal house of Tehran University of Medical Sciences in 12/12 h light/dark cycle at 21±2 °C. Food and water were available ad libitum. After 10 days of acclimatization and ensuring about their healthiness, animal studies were initiated. The experimental protocol was approved by the Research and Ethics Committee of Tehran University of Medical Sciences.

2.4. Acute toxicity

    Forty five male Wistar rats (weight: 200±20 g; age: 3 months) and 45 male Balb/c mice (weight: 25±5 g; age: 2 months) were divided into 9 groups consisted of 5 rats and 5 mice. Overnight fasted animals received 0.5, 1, 15 and 17 g/kg of extracts dissolved in normal saline, whereas control groups were treated only with normal saline. The extracts were administered orally in a total volume of 0.2 ml for mice and 0.5 ml for rats. Mortality rate, clinical and behavioral presentations such as stereotype activities were monitored for 48 h as described previously [13].

2.5. Subchronic toxicity

     Male Wistar rats (weight: 130±20 g; age: 2 months) were randomly divided into control and treatment groups; according to acute toxicity study, treatment groups received 1 g/kg/day of extracts by gavage, 5 days a week for 9 weeks [14, 15]. During the experiment, mortality, behavioral changes, body weight, water and food consumption were monitored daily except for weekends. Three rats in each group were euthanized by cervical dislocation after 4 weeks. The remaining 6 animals were euthanized at the end of treatment for laboratory investigations; abdominal aorta blood samples were collected for hematological examination using an autohematology analyzer (PE-6000; Procan Electronics Inc.) and biochemical evaluation (Pars Azmoon enzymatic kits, Iran). Liver, kidneys and spleen were weighed, slices were prepared using Rotary microtome, Slee,

 Germany, and submitted to haematoxylin-eosin staining; pathological examinations were accomplished with Olympus BX-100 [13].

     Figure 3. Effects of C. tinctorius (IL 111 and LRV 51 51) on differentiation and viability of limb bud cells (n=8). Limb bud cells were prepared and exposed to extracts for 72 h. At the end of treatment, A: differentiation was studied by Alcian blue staining method; B: cytotoxicity was evaluated by Trypan blue dye exclusion; C: the ratio of differentiation to cytotoxicity was determined to study the teratogenicity. Although the extracts caused a dose dependent effect in differentiation and viability of limb bud cells, normalization of differentiation to viability revealed that they cannot be considered as biologically teratogen.

2.6. Cytotoxicity assay in vitro

     Assessment of cytotoxicity was based on the reduction of tetrazolium salt by mitochondrial dehydrogenase. Balb/C 3T3 fibroblast cells (Pasteur Institute, Iran) were cultivated in DMEM, supplemented with 4.5 g/l glucose, 2 mM glutamine, penicillin (100 IU/ml), streptomycin (100 µg /ml) and 10% FBS. Cells were plated at 1×10⁴ in 96-well (Nunc, Denmark) and permitted to proliferate at 37 °C in a humidified atmosphere with 5% CO₂ to get their logarithmic phase of growth and were then subjected to different treatments. At the end of each experiment, MTT was added and cells were incubated at 37 °C for another 4 h; formazan crystals were finally dissolved in DMSO and optical density was measured at 550 nm. Trypan blue dye exclusion test were also performed to evaluate the validity of indirect assay. IC₅₀ values were then calculated and the data are expressed as percent of control.

2.7. Micromass culture and Alcian blue staining for chondrogenic foci

   Micromass culture was accomplished as previously described [16]. Briefly, at the day 13th of gestation Wistar rats embryo limb bud tissues were removed, equilibrated in dissociation medium and disaggregated by triturating through a Pasteur pipette and passaging through a 50 µm nylon filter mesh. The resulting cell suspension was subsequently utilized for micromass culture in DMEM-F12 containing FBS (10%) and L-glutamine (455 µg/l).

      Cells were fixed with 10% formalin containing 1% cetylpyridinum HCl (w/v) for 20 min. at room temperature. The number of stained foci was subsequently designated according to Flint and Orton in which chemicals are considered as potent teratogen when the ratio of differentiation/ cytotoxicity values are equal to 0.5 or less where the control group is attributed 1 [17]. Retinoic acid (90 µg/ml), which can hinder more than 90% of limb bud differentiation, was used as positive control [18].

2.8. Statistical analysis

     The data are represented as the means±SD; one way ANOVA followed by tukey test was applied to study the differences in hematological and biochemical factors in subchronic toxicity study. The effects of the increasing concentrations of safflower extracts and the differences between the two extracts in the in vitro studies were assessed by two-way ANOVA; p values less than 0.05 were considered statistically significant. All statistical computations were performed with SigmaPlot Version 11 package.

3. Results

3.1. Acute toxicity study

     Acute toxicity studies were conducted for both IL 111 and LRV 51 51 extracts in mice and rats. Neither death, nor alteration in stereotype activities was observed during the 48 h observation period after administration of different doses of both extracts up to 17 g/kg. Therefore, a single dose of both extracts (1 g/kg/day) was selected for sub chronic investigation [15].

3.2 Subchronic toxicity study

    During a 9-week subchronic administration of IL 111 and LRV 51 51 extracts, no change was observed in animal’s death rate, behavioural signs, food consumption or body weight (Figure 1). Haematological and biochemical factors showed no remarkable difference between control and LRV 51 51 group, but we observed a significant decrease in haemoglobin concentration as well as prolongation of prothrombin time and a notable increase in serum concentration of lactate dehydrogenase (LDH), creatine phosphokinase (CPK), triglycerides (TG) and cholesterol in rats which received IL 111 (p

     Absolute organ weight, organ:body weight ratios or macroscopic sight of the organs were not noticeably affected by any of the C. tinctorius extracts (Table 2). On the other hand, microscopic evaluation of the tissues showed some abnormalities in liver, spleen and kidney in both IL 111 and LRV 51 51 groups which are described in Table 3.

3.3. Cytotoxicity in vitro

    Treatment of Balb/c 3T3 cells with IL 111 and LRV 51 51 extracts led to a dose dependent decline in the viability of cells compared to control (pC. tinctorius extracts for 72 h and MTT assay revealed that the IC₅₀ values in Balb/c cells were 394 µg/ml and 429 µg/ml for IL 111 and LRV 51 51 variants, respectively (Figure 2). However, Two-way ANOVA revealed no statistically significant difference between IL 111 and LRV 51 51 cytotoxicity in Balb/c cells (p=0.898).

3.4. Effects of C. tinctorius on the viability and differentiation of limb bud cells

       On the other hand, when the number of stained cells was normalized to the cytotoxicity of extracts, it was revealed that none of the extracts caused a remarkable level of teratogenicity which is characterized by values equal to 0.5 or less in differentiation/cytotoxicity ratio where the control group is designated 1 (Figure 3 C).

4. Discussion

      With either extracts, there were no effects on survival, clinical signs, or behavioral changes, in mice and rats, after a single oral administration up to 17 g/kg. Therefore, Iranian safflowers can be categorized as “relatively harmless” in acute toxicity category [14]. Subchronic administration of IL 111 resulted in a decrease in hemoglobin concentration as well as prolongation of prothrombin time and an increase in LDH, CPK, TG and cholesterol (Table 1). Liu et al. (2004), in a 90-day subchronic toxicity study of hydroxysafflower yellow A (180 mg/kg/day) in male Sprague-Dawley rats, the dominant fragment of the safflower yellow pigments, reported no significant alteration of the levels glucose, cholesterol, alkaline phosphatase (ALP), total bilirubin (TBIL), blood urea nitrogen (BUN), total protein (TP), albumin, alanine aminotransferase (ALT), aspirate aminotransferase (AST), creatinine, magnesium, calcium, natrium and potassium. However, it was observed to prolong blood coagulation time, increase kidney and liver indices (organ/body weight) and cause slight nephrotoxicity [19]. This is somewhat consistent with our histopathologic findings of kidney and liver abnormalitites by C. tinctorius extracts.

  Zhu et al. have shown that safflower could have a neuroprotecive effect at doses as low as 6.0 mg/kg [7] in rats, thus one must consider that the moderate toxicity that we observed has occurred in much higher doses. In addition, Grimm et al. demonstrated an immunosuppression property for safflower oil (9 g fat/Kg b.w./day) [10].

      We observed the IC50 values of IL 111 and LRV 51 51 extracts to be 394 and 429 µg/ml in balb/c 3T3 cells, correspondingly, and the rat embryo limb bud cells appeared to be more vulnerable to the toxic effects of both extracts than are the balb/c 3T3 cells. A mixture of ginseng and Carthamus tinctorius has also been demonstrated to hamper proliferation and elicit apoptosis in breast cancer cell lines [20]. On the other hand, Kinobeon A which can be isolated from safflower diminishes the production of thiobarbituric acid reactive species in NADPH-induced microsomal lipid peroxidation scheme and improves survival of Madin-Darby bovine kidney cells when exposed to oxidative reagents [21].

       The mammalian embryo can itself be cultured for a limited period of time and the limb bud cells of rat embryos can differentiate into a number of cell types including cartilage, which makes it possible to examine the effects of teratogens [22, 23]. In this in vitro embryotoxicity assay, 50% reduction of dif-ferentiation is considered as an indication of biological difference for teratogenicity; with this approach 93% of teratogens and 89% of non-teratogens have been correctly identified [24-27].

         The decrement in limb bud differentiation by C. tinctorius might indicate a teratogenic potential, however, these results confounded by the reduction of cell number, reflect a cytotoxic effect of IL 111 and LRV 51 51 on embryonic cells rather than a differentiation challenge and the reported teratogenic effects for safflower in animal studies may emanate from the effects of extracts on placenta or an indirect impact on embryo.

    We observed that IL 111 extract produced higher biochemical and histological toxicity in subchronic study as well as higher potency in declining the number of differentiated limb bud cells. Fan et al. have found remarkable variations in the content of different compounds in safflower samples gathered from different areas in China [6]. Although the chemical composition of safflower extracts from India [28] and China [6] are investigated and it is described that flavonoides, phenylethanoid glycosides, coumarins, fatty acids, steroids and polysaccharides are main safflower floret components, such a study is necessary for Iranian safflower in order to explain the differences in toxicity of IL 11 and LRV 51 51.

       In our previous study we have demonstrated that IL 111 extracts did not induce mutagenicity in TA98 and TA100 strains of Salmonella typhimurium using Ames assay [12]. On the other hand, Nobakht et al. administered the extract by gavage on days 0 to 8 of gestation and described that water extracts of safflower at doses of 1.6 and 2 mg/kg/day elicited embryos abortion, whereas at lower doses it altered cellular dimension, orientation and degeneration in the neural tube of GD 13 embryos [29]. They have also described that safflower extracts have elicited a dose-dependent cytotoxicity in B65 rat nervous cell line [29]. Bahmanpour et al. reported that C. tinctorius might affect the early development of the embryo due to its induction of congenital malformations like exencephally, spina bifida and tail and limb necrosis [30]. Both of the above-mentioned studies have treated pregnant mice with safflower extract and evaluated its in vivo effects on embryo, and may reflect an influence of safflower on placenta rather than the fetus itself. In addition, they have mainly observed the effects of C. tinctorius on nervous system, whereas our results more likely reflect the direct impacts of IL 111 and LRV 51 51 extracts on differentiation of limb bud cells, in the absence of maternal (i.e. metabolism, toxicity) and placental influences.

5. Conclusion

      According to our results, IL 111 and LRV 5151 as dominant cultivars of Iranian safflower was relatively harmless in the acute toxicity paradigm described; they induce slight hematological, biochemical and pathological changes in subchronic exposure; they provoke a level of cytotoxixity in Balb/C 3T3 and limb bud cells but cannot be categorized as teratogenic.

Acknowledgments

      This project has been financially supported by the deputy of research of Tehran University of Medical Sciences. The authors would like to appreciate Mr. A. H. Omidi’s efforts in identification and collection of safflower cultivars, and express their gratitude to the staff of animal lab and cell culture lab for their help and support.