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Evaluation of the toxicity of mastic gum with 13 weeks dietary administration to F344 rats
Food and Chemical Toxicology 45 (2007) 494–501 www.elsevier.com/locate/foodchemtox
Evaluation of the toxicity of mastic gum with 13 weeks dietary administration to F344 rats Jin Seok Kang, Hideki Wanibuchi, Elsayed I. Salim, Anna Kinoshita, Shoji Fukushima
*
Department of Pathology, Osaka City University Medical School, 1-4-3 Asahi-machi, Abeno-ku, Osaka 545-8585, Japan Received 28 November 2005; accepted 24 September 2006
Abstract Dietary toxicity of mastic gum, a natural food additive, was studied in male and female F344 rats fed 0%, 0.22%, 0.67% and 2% levels mixed into powdered basal diet for 13 weeks. No mortality or obvious clinical signs were observed in any of the animals throughout the experimental period. Body weights were significantly reduced in the high dose-treated group from week 2 to the end of the experiment in males, and at weeks 8 and 13 in females. There were increased absolute and relative liver weights in a dose-related manner or limited to the high dose group males or females, along with changes in hematological parameters, including increased WBC and platelet in high dose males. Altered serum biochemistry parameters included increases of total proteins, albumin, and total cholesterol in both sexes, and c-GTP in females only. However, macroscopic examination at necropsy revealed no gross lesions, and microscopic examination also revealed no treatment-related findings in any organs examined. As dietary treatment of mastic gum for 13 weeks in the present study caused decreased body weights at the high dose, especially in males, and increased liver weights in a dose-related manner in both genders without any morphological findings, it is concluded that the administration of it has a no observed adverse effect level (NOAEL) of 0.67% in the diet. Ó 2006 Elsevier Ltd. All rights reserved. Keywords: Mastic gum; Toxicity; F344 rats
1. Introduction Food additives are chemicals which are added to food during processing, either as coloring agents, preservatives, antioxidants, sweeteners or flavoring materials. In the modern society, with the advent of processed foods, there has been an inevitable use of food additives. Most commonly employed food additives are safe, but some have demonstrated unexpected toxicity or carcinogenicity Abbreviations: (A/G), albumin/globulin ratio; (ALT), alanine aminotransferase; (ALP), alkaline phosphatase; (AST), aspartate aminotransferase; (BUN), blood urea nitrogen; (c-GTP), c-glutamyl transpeptidase; (Hb), hemoglobin concentration; (Hct), hematocrit; (MCV), mean corpuscular volume; (MCH), mean corpuscular hemoglobin; (MCHC), mean corpuscular hemoglobin concentration; (NOAEL), no observed adverse effect level; (RBC), red blood cell count; (WBC), white blood cell count. * Corresponding author. Tel.: +81 6 6645 3735; fax: +81 6 6646 3093. E-mail address: [email protected] (S. Fukushima). 0278-6915/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2006.09.013
(Parke and Lewis, 1992; Johnson, 2002), and the usage of some is associated with allergies and asthma (Lucas et al., 2001; Simon, 2003). Therefore, considerable controversy has arisen regarding the benefits and potential hazards of food additives, and achievement of some degree of consensus is necessary for safety evaluation (Poulsen, 1991; Adams and Smith, 2004). Due to a general lack of toxicological data for some natural food additives, risk assessment and safety evaluation often cannot be undertaken. Therefore, a need exists to increase the toxicological database for natural food additives. Mastic gum is a hard, transparent resin collected from the mastic tree (Pistacia lentiscus) found chiefly in Mediterranean countries. The resin may be chewed as a gum or it may be added to bakery goods or alcoholic beverages (Ford et al., 1992). Mastic gum is known to have some pharmacological activities. It has been used for the
J.S. Kang et al. / Food and Chemical Toxicology 45 (2007) 494–501
treatment of stomach disorders, and recent research suggests it may have potential benefit for the treatment of oral, gastric and duodenal ailments (Al-Habbal et al., 1984; AlSaid et al., 1986; Huwez et al., 1998; Takahashi et al., 2003), although there have been contrary reports (Bebb et al., 2003; Loughlin et al., 2003). Toxicological data of mastic gum have been reported concerning acute toxicity, skin irritation and phototoxicity in animals and humans (Spott and Shelley, 1970; Keynan et al., 1987, 1997; Ford et al., 1992). In the present experiment, the general toxicity of mastic gum was evaluated with 13 weeks dietary administration to male and female F344 rats. 2. Material and methods 2.1. Animals and the test chemical Five-week-old F344 rats (40 males and 40 females) were obtained from Charles River Japan, Inc. (Atsugi, Japan), and housed in a room maintained on a 12 h light/dark cycle, at constant temperature (23 ± 1 °C) and humidity (50 ± 5%). They were allowed free access to powdered CRF-1 basal diet (Oriental Yeast, Tokyo, Japan) and tap water throughout the experiment. Mastic gum was obtained from Nakamura Chiro Association (Tokyo, Japan). For setting of the high dose, we carried out a preliminary test by adding mastic gum at concentrations of 0%, 0.56%, 1.67% and 5% into powdered CRF-1 basal diet (Oriental Yeast, Tokyo, Japan), which was then fed to F344 rats for seven days. Since decreased body weight and increased liver weight were evident at the highest-dose in males, along with increased liver weight in females, 2% was selected as the highest dose for the 13 weeks toxicity test. For dietary administration, mastic gum was mixed at concentrations of 0%, 0.22%, 0.67% and 2% into a CRF-1 powdered basal diet. The stability in the powder diet was evaluated and no decomposition was confirmed after storage for 56 days at room temperature. Diets were therefore prepared twice during this experiment.
2.2. Experimental design Male and female, six-week-old animals were randomly allocated to eight groups. Groups 1–4 (males) and groups 5–8 (females), ten rats per group, received diets containing mastic gum at doses of 0%, 0.22%, 0.67% and 2%, respectively, for 13 weeks. All procedures were approved by the Institutional Animal Care and Use Committee of Osaka City University Medical School. The animals were observed for clinical signs and mortality daily. Body weights, food consumption and water intake were measured every week, and the mean food consumption or water intake/animal/day was calculated per group in each sex. At the end of the experiment, all animals were fasted overnight and euthanized by exsanguination under ether anesthesia. Blood was taken from the abdominal aorta for hematology and blood chemistry analyses. Hematological examinations were performed for the following parameters: white blood cell count (WBC), red blood cell count (RBC), hemoglobin concentration (Hb), hematocrit (Hct), platelet count, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC) and differential WBC counts. Serum biochemistry was performed for the following parameters: total proteins, albumin, albumin/globulin ratio (A/G), aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), c-glutamyl transpeptidase (c-GTP), creatinine, triglycerides, total cholesterol, total bilirubin, blood urea nitrogen (BUN), sodium, potassium, chloride, calcium and inorganic phosphorus.
495
Analyses for hematological examination and serum chemistry were conducted at Mitsubishi Kagaku Bio-Clinical Laboratories, Inc., using automatic analyzing machines, SE-9000/Sysmex and AU5200/Olympus, for blood and serum analyses, respectively. All animals were subjected to a complete necropsy. At termination they were examined grossly for pathological changes, and the heart, liver, spleen, kidneys, adrenal glands, testes or ovaries, brain, thymus and lung were weighed. In addition to these organs, lymph nodes (cervical, mesenteric), the aorta, salivary gland, bone and bone marrow (sternum, femur), trachea, thyroid, tongue, esophagus, stomach, duodenum, small intestine, large intestine, pancreas, urinary bladder, seminal vesicle, prostate gland, epididymis, oviduct, uterus, vagina, pituitary gland, sciatic nerve, skeletal muscle, spinal cord, eyes and their accessory organs were excised and fixed in 10% buffered formalin. Testes of five males each of the control and high dose groups were fixed in Bouin’s solution. Paraffin-embedded tissue sections of all organs/tissues were routinely prepared and stained with hematoxylin and eosin for histopathological examination. All organs and tissues in the control and high dose group animals were compared. Histopathology was also extended to all tissues of low and middle dose groups in which lesions were found in the high dose group.
2.3. Statistics The Duncan’s new multiple range test was employed for comparison of body weights, organ weights (both absolute and relative), and hematology and serum biochemistry data between control and treated groups. For all comparisons, p-values less than 5% (p < 0.05) were considered to be statistically significant (StatView, SAS Institute Inc., NC, USA).
3. Results 3.1. In-life parameters No mortality or obvious clinical signs were evident in any of the animals throughout the experimental period. During the experiment the body weights and cumulative body weight gains were significantly reduced from week 2 to the end of the experiment in the high dose males (group 4) and at week 8 and 13 in the high dose females (group 8), compared to the respective controls (p < 0.05 or p < 0.01) (Fig. 1). The food consumption and water intake values showed no difference among the groups during the experiment (data not shown). Total intake of mastic gum for 13 weeks was 0 (group 1), 2.7 (group 2), 8.4 (group 3) and 26.5 g (group 4) in male, and 0 (group 5), 1.8 (group 6), 5.5 (group 7) and 17.1 g (group 8) in female rats. 3.2. Organ weights Body weights and absolute organ weights are given in Table 1. Absolute liver weights were significantly increased in group 2 (p < 0.05) and groups 3, 4 and 8 (p < 0.01), with dose-dependence. Absolute thymus weights were significantly decreased in groups 2–4 (p < 0.01), albeit without dose-dependence, and absolute weights of spleen (group 2), left testes (groups 2 and 4) and thymus (group 6) showed significant differences from control values (p < 0.05), but again there was no correlation with dose. Relative organ weights are shown in Table 2. Relative liver weights were also significantly increased in group 2
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J.S. Kang et al. / Food and Chemical Toxicology 45 (2007) 494–501 350 300 250 **
**
**
**
**
**
200
**
**
**
gram
* **
150
Group 1 (0%)
**
Group 2 (0.22%)
100
Group 3 (0.67%) Group 4 (2%)
50
4 and 8 (p < 0.01, p < 0.05, respectively), albumin in groups 3, 4 and 8 (p < 0.05, p < 0.01, p < 0.05, respectively), c-GTP in groups 7 and 8 (p < 0.01), total cholesterol in groups 4, 7, and 8 (p < 0.05, p < 0.01, p < 0.01, respectively), BUN in group 8 (p < 0.05), and calcium in group 4 (p < 0.01). There were significant decreases of creatinine in groups 2–4 (p < 0.05, p < 0.01, p < 0.01, respectively), triglycerides in groups 3, 4 and 7 (p < 0.05, p < 0.01, p < 0.05, respectively), sodium in group 7 (p < 0.01), and inorganic phosphorus in groups 6 and 7 (p < 0.05). There were significant increases of ALP in groups 4 and 8 (p < 0.01, p < 0.05, respectively), and decreases in groups 6 and 7 (p < 0.01).
0 0
1
2
3
4
5
6
7 8 Weeks
9
10
11
12
13
200 180 160 **
**
140
gram
120 100
3.4. Macroscopic and microscopic findings Macroscopic examination at necropsy revealed no gross lesions, and microscopic examination also revealed no treatment-related findings in any organs examined. Extended microscopic examination of liver, thymus, lymph nodes (cervical, mesenteric) and spleen in low and middle doses of male and female also showed no histopathological findings.
Group 5 (0%)
80
4. Discussion
Group 6 (0.22%)
60
Group 7 (0.67%)
40
Group 8 (2%)
20 0 0
1
2
3
4
5
6 7 Weeks
8
9
10
11
12
13
Fig. 1. Change of body weights during the experiment: (a) males and (b) females. *,**Significantly different from the control group at the level of p < 0.05, p < 0.01, respectively.
(p < 0.05) and groups 3, 4, 7 and 8 (p < 0.01) with dosedependence. There were increased relative weights of the heart (p < 0.01) and spleen (p < 0.05) in group 4, right kidney in groups 4 and 8 (p < 0.05), left kidney in group 8 (p < 0.05), right and left testes in group 4 (p < 0.01), and brain in groups 4 and 8 (p < 0.01, p < 0.05, respectively) compared with controls. Relative thymus weights were significantly increased in groups 2 and 6 (p < 0.05, p < 0.01, respectively), but there was no correlation with dose. 3.3. Hematology and serum biochemistry Several alterations were observed in hematological parameters (Table 3), including increased WBC in group 4 and platelets in groups 3 and 4, and decreased MCV in group 4 (p < 0.05). There were significant decreases of MCH in groups 7 and 8 (p < 0.05, p < 0.01, respectively) and of MCHC in group 8 (p < 0.01), along with significant increases of segmented neutrophils and lymphocytes in group 3 (p < 0.05), but with no dose dependence. Data for serum biochemistry are shown in Table 4. There were significant increases of total proteins in groups
Dietary treatment of mastic gum for 13 weeks in the present study caused decreased body weights at the high dose, especially in males, and increased liver weights in a dose-related manner in both genders. However, macroscopic and microscopic examinations revealed no treatment-related findings in any organs, including in the liver. Generally, non-adverse effects can be defined as biological effects that do not cause biochemical, morphological, or physiological changes that affect the general well-being, growth, development or life span of an animal. As minor elevations in liver weight that are not accompanied by any morphological change in the organ or any change in liver function may be considered as not adverse and an increase of liver weight by induced hypertrophy and hyperplasia, at least up to 20% in the absence of other liver pathology, is often regarded as a non-adverse effect in rodents (Karbe et al., 2001; Lewis et al., 2002; Williams and Iatropoulos, 2002), it seems that liver weight increase without any morphological changes is adaptive response in this study. On the while, it should be concerned that the increase of liver weight with mastic gum showed dose-dependence in this study, and the maximal percentage increase in liver weight was about 22% in the highest dose male rats in this study. So, it is reasonable that 2% administration of mastic gum in the diet for 13 weeks induce adverse effect. However, in all doses, we could not identify any histopathological findings in the liver, in line with the absence of clinical chemistry changes relating to liver. It has a possibility that there are a limited number of morphologic changes that can be discerned using conventional light microscopy in the liver (Hardisty and Brix, 2005). As to heterogeneity
Table 1 Body weights and absolute organ weights of male and female F344 rats treated with mastic gum Male
Group Mastic gum (%)
1 0
2 0.22
3 0.67
4 2
Female 5 0
6 0.22
7 0.67
8 2
No. of rates examined Initial body weight (g) Final body weight (g)
10 114 + 3 307 ± 10
10 115 + 5 311 ± 13
10 113 + 4 304 ± 20
10 115 + 3 283 ± 14**
10 101 + 2 170 ± 6
10 100 + 2 168 ± 4
10 99 + 3 165 ± 3
10 100 + 3 164 ± 5**
Organ Heart (g) Liver (g) Spleen (g) Rt.Kidney (g) Lt.Kidney (g) Rt.Adrenal gl. (mg) Lt.Adrenal gl. (mg) Rt.Testis (g) Lt.Testis (g) Rt.Ovary (mg) Lt.Ovary (mg) Brain (g) Thymus (g) Lung (g)
0.89 ± 0.07 6.76 + 0.38 0.59 + 0.05 0.91 ± 0.07 0.95 ± 0.10 0.02 ± 0.01 0.02 ± 0.01 1.49 ± 0.08 1.50 ± 0.06 – – 1.94 + 0.07 0.21 ± 0.03 0.93 ± 0.09
0.91 ± 0.08 7.17 + 0.40* 0.64 + 0.04* 0.94 ± 0.06 0.94 ± 0.06 0.02 ± 0.01 0.02 ± 0.00 1.52 ± 0.05 1.56 ± 0.07* – – 1.96 + 0.03 0.16 ± 0.06** 0.94 ± 0.05
0.88 ± 0.07 7.49 + 0.60** 0.62 + 0.04 0.91 ± 0.05 0.94 ± 0.07 0.02 ± 0.01 0.02 ± 0.01 1.51 ± 0.11 1.54 ± 0.08 – – 1.94 + 0.04 0.17 ± 0.03** 0.94 ± 0.05
0.93 ± 0.08 8.26 + 0.52** 0.60 + 0.07 0.90 ± 0.08 0.92 ± 0.04 0.02 ± 0.00 0.02 ± 0.01 1.55 ± 0.10 1.57 ± 0.05* – – 1.93 + 0.06 0.16 ± 0.03** 0.89 ± 0.05
0.58 ± 0.04 3.67 + 0.23 0.40 + 0.02 0.53 ± 0.03 0.53 ± 0.05 0.02 ± 0.00 0.02 ± 0.01 – – 0.03 + 0.01 0.03 + 0.01 1.82 + 0.04 0.15 ± 0.02 0.73 ± 0.06
0.57 ± 0.03 3.68 + 0.16 0.40 + 0.04 0.53 ± 0.03 0.53 ± 0.03 0.02 ± 0.01 0.02 ± 0.01 – – 0.03 + 0.01 0.04 + 0.01 1.81 + 0.07 0.12 ± 0.04* 0.72 ± 0.08
0.60 ± 0.08 3.98 + 0.17 0.38 + 0.02 0.53 ± 0.04 0.53 ± 0.02 0.02 ± 0.01 0.02 ± 0.01 – – 0.03 + 0.01 0.03 + 0.01 1.80 + 0.02 0.14 ± 0.02 0.70 ± 0.03
0.60 ± 0.04 4.95 + 0.32** 0.41 + 0.01 0.54 ± 0.04 0.55 ± 0.03 0.02 ± 0.00 0.02 ± 0.01 – – 0.03 + 0.01 0.03 + 0.01 1.81 + 0.05 0.16 ± 0.02 0.69 ± 0.06
Data represent mean ± SD. *, **Significantly different from control group at the level of p < 0.05, p < 0.01, respectively.
J.S. Kang et al. / Food and Chemical Toxicology 45 (2007) 494–501
Sex
497
498
Sex
Male
Group Mastic gum (%)
1 0
2 0.22
3 0.67
4 2
5 0
Female 6 0.22
7 0.67
8 2
No. of rats examined
10
10
10
10
10
10
10
10
Organ Heart (%) Liver (%) Spleen (%) Rt.Kidney (%) Lt.Kidney (%) Rt.Adrenal gl. (%) Lt.Adrenal gl. (%) Rt.Testis (%) Lt.Testis (%) Rt.Ovary (%) Lt.Ovary (%) Brain (%) Thymus (%) Lung (%)
0.29 ± 0.02 2.20 ± 0.08 0.19 ± 0.02 0.30 ± 0.02 0.31 ± 0.03 0.01 ± 0.00 0.01 ± 0.00 0.49 ± 0.02 0.49 ± 0.02 – – 0.63 + 0.03 0.07 ± 0.01 0.30 ± 0.02
0.29 ± 0.02 2.31 ± 0.06* 0.21 ± 0.01 0.30 ± 0.02 0.30 ± 0.01 0.01 ± 0.00 0.01 ± 0.00 0.49 ± 0.02 0.50 ± 0.03 – – 0.63 + 0.03 0.05 ± 0.02* 0.30 ± 0.01
0.29 ± 0.02 2.46 ± 0.10** 0.20 ± 0.01 0.30 ± 0.01 0.31 ± 0.01 0.01 ± 0.00 0.01 ± 0.00 0.50 ± 0.03 0.51 ± 0.02 – – 0.64 + 0.03 0.05 ± 0.01 0.31 ± 0.01
0.33 ± 0.04** 2.92 ± 0.08** 0.21 ± 0.02* 0.32 ± 0.02* 0.33 ± 0.02 0.01 ± 0.00 0.01 ± 0.00 0.55 ± 0.05** 0.56 ± 0.03** – – 0.68 + 0.03** 0.06 ± 0.01 0.32 ± 0.01
0.34 ± 0.02 2.15 ± 0.16 0.24 ± 0.01 0.31 ± 0.02 0.31 ± 0.03 0.01 ± 0.00 0.01 ± 0.00 – – 0.02 + 0.01 0.02 + 0.01 1.07 + 0.04 0.09 + 0.01 0.43 ± 0.03
0.34 ± 0.02 2.20 ± 0.09 0.24 ± 0.03 0.31 ± 0.02 0.32 ± 0.02 0.01 ± 0.00 0.01 ± 0.00 – – 0.02 + 0.00 0.02 + 0.01 1.08 + 0.03 0.07 ± 0.02** 0.43 ± 0.04
0.37 ± 0.05 2.41 ± 0.10** 0.23 ± 0.01 0.32 ± 0.02 0.32 ± 0.01 0.01 ± 0.00 0.01 ± 0.00 – – 0.02 + 0.00 0.02 + 0.01 1.09 + 0.02 0.09 ± 0.01 0.43 ± 0.01
0.36 ± 0.02 3.02 ± 0.17** 0.25 ± 0.01 0.33 ± 0.01* 0.33 ± 0.01* 0.01 ± 0.00 0.01 ± 0.00 – – 0.02 + 0.00 0.02 + 0.01 1.10 + 0.03* 0.09 ± 0.01 0.42 ± 0.03
Data represent mean ± SD. *, **Significantly different from control group at the level of p < 0.05, p < 0.01, respectively.
J.S. Kang et al. / Food and Chemical Toxicology 45 (2007) 494–501
Table 2 Relative organ weights of male and female F344 rats treated with mastic gum
Item
WBC RBC Hb Hct Platelet MCV MCH MCHC Unseg. Neutrophila Seg. neutrophila Lymphocyte Monocyte Eosinophil Basophil Abn. Lymphocytea
Sex
Male
Female
Group Mastic gum (%)
1 0
2 0.22
3 0.67
4 2
5 0
6 0.22
7 0.67
8 2
/MCLa e4/ MCL g/dl % e4/ MCL FLa PGa % % % % % % % %
2820.0 ± 418.5 873.9 ± 23.7 14.7 + 0.3 47.2 + 1.5 57.8 ± 8.0 53.9 ± 1.1 16.9 ± 0.2 31.3 + 0.5 1.0 ± 0.0 17.4 ± 9.3 79.1 ± 9.7 2.1 ± 1.2 0.4 ± 0.5 0.0 ± 0.0 0.1 ± 0.3
2850.0 ± 552.3 877.8 ± 16.1 14.8 + 0.3 47.0 + 1.0 60.5 ± 6.4 53.4 ± 0.5 16.9 ± 0.3 31.5 + 0.5 0.9 ± 0.2 23.1 ± 4.1 72.7 ± 6.3 2.5 ± 2.4 0.8 ± 1.3 0.0 ± 0.0 0.0 ± 0.0
2850.0 ± 779.2 873.8 ± 18.4 14.7 + 0.3 46.7 + 1.3 66.2 ± 5.6* 53.4 ± 0.7 16.9 ± 0.2 31.5 + 0.5 1.2 ± 0.4 27.2 ± 7.4* 69.0 ± 7.6* 2.3 ± 1.4 0.3 ± 0.5 0.0 ± 0.0 0.0 ± 0.0
3730.0 ± 561.8* 872.7 ± 22.5 14.5 + 0.3 46.0 + 1.2 66.4 ± 5.5* 52.7 ± 0.8* 16.6 ± 0.3 31.5 + 0.7 1.0 ± 0.0 19.3 ± 6.5 76.8 ± 7.4 2.4 ± 2.1 0.5 ± 0.7 0.0 ± 0.0 0.0 ± 0.0
2877.8 ± 774.2 849.6 ± 38.3 15.5 + 0.7 49.5 + 2.7 62.6 ± 8.8 58.1 ± 1.4 18.3 ± 0.2 31.3 + 0.9 1.2 ± 0.4 19.6 ± 9.2 76.4 ± 9.3 2.2 ± 1.2 0.6 ± 0.9 0.0 ± 0.0 0.0 ± 0.0
3140.0 ± 1079.3 843.1 ± 30.6 15.4 + 0.6 48.9 + 2.1 61.8 ± 6.9 58.1 ± 1.0 18.3 ± 0.3 31.5 + 0.7 1.1 ± 0.4 15.8 ± 6.7 81.0 ± 6.8 1.9 ± 1.2 0.2 ± 0.4 0.0 ± 0.0 0.0 ± 0.0
3140.0 ± 658.6 846.9 ± 20.4 15.3 + 0.5 48.9 + 1.3 63.6 ± 7.6 57.8 ± 0.9 18.0 ± 0.1* 31.2 + 0.4 1.1 ± 0.3 13.4 ± 5.7 82.7 ± 4.7 2.4 ± 1.2 0.4 ± 0.8 0.0 ± 0.0 0.0 ± 0.0
3400.0 ± 1047.6 844.3 ± 27.7 15.0 + 0.6 49.2 + 2.0 61.1 ± 5.3 58.2 ± 1.2 17.8 ± 0.2** 30.5 + 0.4** 1.0 ± 0.0 13.8 ± 12.7 83.6 ± 12.4 1.4 ± 0.5 0.2 ± 0.4 0.0 ± 0.0 0.0 ± 0.0
Data represent mean ± SD. *, **Significantly different from control group at the level of p < 0.05, p < 0.01, respectively. a Unseg. Neutrophil: unsegmented neutrophil; Seg. Neutrophil: segmented neutrophil; Abn. Lymphocyte: abnormal lymphocyte; MCL: microliter; FL: femtoliter; PG: picogram.
J.S. Kang et al. / Food and Chemical Toxicology 45 (2007) 494–501
Table 3 Complete blood count of male and female F344 rats treated with mastic gum
499
500
Item
Total proteins Albumin A/G ratio AST ALT ALP c-GTP Creatinine Triglycerides Total cholesterol Total bilirubin BUN Sodium Potassium Chloride Calcium Inoranic phosphorus
Sex
Male
Group Mastic gum (%)
1 0
2 0.22
3 0.67
4 2
5 0
6 0.22
7 0.67
8 2
g/dl g/dl
6.4 + 0.1 3.4 ± 0.1 1.2 ± 0.1 120.2 ± 17.1 54.3 ± 2.9 454.7 ± 31.2 1.0 ± 0.0 0.42 ± 0.03 65.8 ± 13.4 62.7 ± 3.7 0.1 ± 0.0 24.0 ± 3.3 142.1 ± 1.1 4.4 ± 0.4 100.5 ± 1.1 10.2 ± 0.3 8.4 ± 2.6
6.5 ± 0.2 3.5 ± 0.1 1.2 ± 0.1 129.3 ± 38.7 61.9 ± 15.2 437.2 ± 27.3 1.0 ± 0.0 0.39 ± 0.03* 54.2 ± 18.7 63.0 ± 4.4 0.1 ± 0.0 22.0 ± 3.2 142.0 ± 1.1 4.5 ± 0.4 100.5 ± 1.4 10.2 ± 0.2 8.5 ± 2.4
6.6 ± 0.2 3.6 ± 0.1* 1.2 ± 0.1 116.7 ± 20.2 60.0 ± 7.7 454.4 ± 19.5 1.0 ± 0.0 0.37 ± 0.02** 50.0 ± 12.0* 66.0 ± 4.4 0.1 ± 0.0 23.7 ± 3.1 142.0 ± 1.3 4.6 ± 0.5 100.1 ± 1.3 10.4 ± 0.2 8.6 ± 2.6
6.9 ± 0.3** 3.7 ± 0.1** 1.2 ± 0.1 105.3 ± 15.7 58.6 ± 4.8 499.0 ± 31.7** 1.3 ± 0.7 0.34 ± 0.04** 33.2 ± 8.2** 70.5 ± 6.2* 0.1 ± 0.0 26.3 ± 2.3 141.6 ± 2.0 4.6 ± 0.5 99.5 ± 1.6 10.8 ± 0.2** 8.5 ± 1.9
6.4 ± 0.3 3.5 ± 0.2 1.2 ± 0.1 129.5 ± 25.0 40.8 ± 5.9 328.8 ± 32.1 1.2 ± 0.4 0.31 ± 0.02 40.5 ± 12.3 88.3 ± 6.3 0.1 ± 0.0 19.4 ± 2.2 142.6 ± 1.2 4.6 ± 0.6 100.4 ± 1.3 10.4 ± 0.4 9.3 ± 1.6
6.3 ± 0.3 3.5 ± 0.1 1.3 ± 0.0 126.4 ± 32.7 39.4 ± 5.8 273.3 ± 24.1** 1.3 ± 0.5 0.32 ± 0.04 41.2 ± 17.5 91.2 ± 8.7 0.1 ± 0.0 18.7 ± 2.9 141.4 ± 2.0 4.4 ± 0.4 100.9 ± 2.0 10.4 ± 0.4 7.2 ± 1.8*
6.5 ± 0.2 3.6 ± 0.1 1.2 ± 0.1 144.0 ± 50.0 41.1 ± 8.1 268.2 ± 27.5** 2.7 ± 1.7** 0.32 ± 0.03 25.4 ± 13.2* 98.7 ± 8.0** 0.1 ± 0.0 19.4 ± 2.0 140.1 ± 1.4** 4.3 ± 0.5 101.5 ± 1.0 10.3 ± 0.3 7.0 ± 1.6*
6.7 ± 0.3* 3.7 ± 0.1* 1.2 ± 0.1 122.8 ± 34.5 43.6 ± 3.2 361.3 ± 43.8* 10.4 ± 0.7** 0.29 ± 0.03 34.0 ± 11.4 118.9 ± 10.5** 0.1 ± 0.0 22.6 ± 1.9* 141.2 ± 1.8 4.6 ± 0.5 100.3 ± 2.7 10.5 ± 0.2 7.3 ± 1.6
IU/l IU/l IU/l IU/l mg/dl mg/dl mg/dl mg/dl mg/dl mEq/l mEq/l mEq/l mg/dl mg/dl
Female
Data represent mean ± SD. *, **Significantly different from control group at the level of p < 0.05, p < 0.01, respectively.
J.S. Kang et al. / Food and Chemical Toxicology 45 (2007) 494–501
Table 4 Blood clinical chemistry of male and female F344 rats treated with mastic gum
J.S. Kang et al. / Food and Chemical Toxicology 45 (2007) 494–501
and complexity in the liver, new technologies and approaches, such as microarray analysis and proteomics, may help understanding of toxicity (Malarkey et al., 2005). Although there was a decrease in absolute thymus weight in male rats, the difference of relative thymus weight did not show dose-dependence. Furthermore, histopathological examination of lymphoid organs from all groups did not show any abnormal findings. Therefore, we conclude that mastic gum did not exert a direct immunotoxic effect or a generalized stress. However, as mastic gum is associated with allergenicity, including contact dermatitis (Keynan et al., 1987, 1997; Wakelin, 2001), it may induce immunological response under some conditions. As the conventional examination of lymphoid system tissues is generally not sufficient to identify subtle lymphoid system changes (Haley et al., 2005), enhanced histopathology including immunohistochemical techniques is warranted in further studies. Some alterations of hematological parameters were also evident, including increases of WBCs and PLT in males. As recent report suggested pharmacological effects on peripheral blood mononuclear cells (Dedoussis et al., 2004), there is a possibility that mastic gum may influence hematological parameters in some unknown way. By serum biochemistry, increased total cholesterol was seen in high dose males and females, along with increased c-GTP in females. However, increases of total protein, albumin, ALP, calcium and BUN in males and/or females were minimal, and decreases of creatinine and triglyceride had little toxicological significance. No observed adverse effect level (NOAEL) is the highest exposure level that there are no statistically significant increase in the frequency or severity of adverse effect between the exposed population and its control (Lewis et al., 2002). In this study, low and middle dose treatment of mastic gum induced liver weight increase that were not accompanied by any morphological change or any change in liver function, so it may be considered as not adverse. As dietary treatment of mastic gum for 13 weeks caused decreased body weights at the high dose, especially in males, and increased liver weights in a dose-related manner in both genders without any morphological findings, it is concluded that the administration of it has a NOAEL of 0.67% in the diet. Further studies are warranted to ascertain mechanisms that are responsible for the decrease of body weight and increase of liver weight and alteration of specific hematological and serum chemical parameters. Acknowledgements We would like to thank Ms. Kaori Touma, Masayo Imanaka, and Shoko Araki for their technical assistance, and Ms. Mari Dokoh, Yuko Onishi, and Yoko Shimada for their help during preparation of this manuscript. This research was supported by Grants-in-Aid from the Ministry of Health, Labour and Welfare of Japan.
501
References Adams, T.B., Smith, R.L., 2004. Issues and challenges in the safety evaluation of food flavors. Toxicol. Lett. 149, 209–213. Al-Habbal, M.J., Al-Habbal, Z., Huwez, F.U., 1984. A double-blind controlled clinical trial of mastic and placebo in the treatment of duodenal ulcer. Clin. Exp. Pharmacol. Physiol. 11, 541–544. Al-Said, M.S., Ageel, A.M., Parmar, N.S., Tariq, M., 1986. Evaluation of mastic, a crude drug obtained from Pistacia lentiscus for gastric and duodenal anti-ulcer activity. J. Ethnopharmacol. 15, 271–278. Bebb, J.R., Bailey-Flitter, N., Ala’Aldeen, D., Atherton, J.C., 2003. Mastic gum has no effect on Helicobacter pylori load in vivo. J. Antimicrob. Chemother. 52, 522–523. Dedoussis, G.V., Kaliora, A.C., Psarras, S., Chiou, A., Mylona, A., Papadopoulos, N.G., Andrikopoulos, N.K., 2004. Antiatherogenic effect of Pistacia lentiscus via GSH restoration and downregulation of CD36 mRNA expression. Atherosclerosis 174, 293–303. Ford, R.A., Api, A.M., Letizia, C.S., 1992. Monographs on fragrance raw materials. Food Chem. Toxicol. 30 (Suppl), 1S–138S. Haley, P., Perry, R., Ennulat, D., Frame, S., Johnson, C., Lapointe, J.M., Nyska, A., Snyder, P., Walker, D., Walter, G., 2005. STP position paper: best practice guideline for the routine pathology evaluation of the immune system. Toxicol. Pathol. 33, 404–407, discussion 408. Hardisty, J.F., Brix, A.E., 2005. Comparative hepatic toxicity: prechronic/ chronic liver toxicity in rodents. Toxicol. Pathol. 33, 35–40. Huwez, F.U., Thirlwell, D., Cockayne, A., Ala’Aldeen, D.A., 1998. Mastic gum kills Helicobacter pylori. N. Eng. J. Med. 339, 1946. Johnson, F.M., 2002. How many food additives are rodent carcinogens? Environ. Mol. Mutagen 39, 69–80. Karbe, E., Williams, G.M., Lewis, R.W., Kimber, I., Foster, P.M., 2001. Distinguishing between adverse and non-adverse effects. Session Summary. J. Toxicol. Pathol. 14, 321–325. Keynan, N., Geller-Bernstein, C., Waisel, Y., Bejerano, A., Shomer-Ilan, A., Tamir, R., 1987. Positive skin tests to pollen extracts of four species of Pistacia in Israel. Clin. Allergy 17, 243–249. Keynan, N., Tamir, R., Waisel, Y., Reshef, A., Spitz, E., Shomer-Ilan, A., Geller-Bernstein, C., 1997. Allergenicity of the pollen of Pistacia. Allergy 52, 323–330. Lewis, R.W., Billington, R., Debryune, E., Gamer, A., Lang, B., Carpanini, F., 2002. Recognition of adverse and nonadverse effects in toxicity studies. Toxicol. Pathol. 30, 66–74. Loughlin, M.F., Ala’Aldeen, D.A., Jenks, P.J., 2003. Monotherapy with mastic does not eradicate Helicobacter pylori infection from mice. J. Antimicrob. Chemother. 51, 367–371. Lucas, C.D., Hallagan, J.B., Taylor, S.L., 2001. The role of natural color additives in food allergy. Adv. Food Nutr. Res. 43, 195–216. Malarkey, D.E., Johnson, K., Ryan, L., Boorman, G., Maronpot, R.R., 2005. New insights into functional aspects of liver morphology. Toxicol. Pathol. 33, 27–34. Parke, D.V., Lewis, D.F., 1992. Safety aspects of food preservatives. Food Addit. Contam. 9, 561–577. Poulsen, E., 1991. Safety evaluation of substances consumed as technical ingredients (food additives). Food Addit. Contam. 8, 125–133. Simon, R.A., 2003. Adverse reactions to food additives. Curr. Allergy Asthma Rep. 3, 62–66. Spott, D.A., Shelley, W.B., 1970. Exanthem due to contact allergen (benzoin) absorbed through skin. JAMA 214, 1881–1882. Takahashi, K., Fukazawa, M., Motohira, H., Ochiai, K., Nishikawa, H., Miyata, T., 2003. A pilot study on antiplaque effects of mastic chewing gum in the oral cavity. J. Periodontol. 74, 501–505. Wakelin, S.H., 2001. Allergic contact dermatitis from mastic in compound mastic paint. Contact Dermatitis 45, 118. Williams, G.M., Iatropoulos, M.J., 2002. Alteration of liver cell function and proliferation: differentiation between adaptation and toxicity. Toxicol. Pathol. 30, 41–53.
Evaluation of the toxicity of mastic gum with 13 weeks dietary administration to F344 rats Jin Seok Kang, Hideki Wanibuchi, Elsayed I. Salim, Anna Kinoshita, Shoji Fukushima
*
Department of Pathology, Osaka City University Medical School, 1-4-3 Asahi-machi, Abeno-ku, Osaka 545-8585, Japan Received 28 November 2005; accepted 24 September 2006
Abstract Dietary toxicity of mastic gum, a natural food additive, was studied in male and female F344 rats fed 0%, 0.22%, 0.67% and 2% levels mixed into powdered basal diet for 13 weeks. No mortality or obvious clinical signs were observed in any of the animals throughout the experimental period. Body weights were significantly reduced in the high dose-treated group from week 2 to the end of the experiment in males, and at weeks 8 and 13 in females. There were increased absolute and relative liver weights in a dose-related manner or limited to the high dose group males or females, along with changes in hematological parameters, including increased WBC and platelet in high dose males. Altered serum biochemistry parameters included increases of total proteins, albumin, and total cholesterol in both sexes, and c-GTP in females only. However, macroscopic examination at necropsy revealed no gross lesions, and microscopic examination also revealed no treatment-related findings in any organs examined. As dietary treatment of mastic gum for 13 weeks in the present study caused decreased body weights at the high dose, especially in males, and increased liver weights in a dose-related manner in both genders without any morphological findings, it is concluded that the administration of it has a no observed adverse effect level (NOAEL) of 0.67% in the diet. Ó 2006 Elsevier Ltd. All rights reserved. Keywords: Mastic gum; Toxicity; F344 rats
1. Introduction Food additives are chemicals which are added to food during processing, either as coloring agents, preservatives, antioxidants, sweeteners or flavoring materials. In the modern society, with the advent of processed foods, there has been an inevitable use of food additives. Most commonly employed food additives are safe, but some have demonstrated unexpected toxicity or carcinogenicity Abbreviations: (A/G), albumin/globulin ratio; (ALT), alanine aminotransferase; (ALP), alkaline phosphatase; (AST), aspartate aminotransferase; (BUN), blood urea nitrogen; (c-GTP), c-glutamyl transpeptidase; (Hb), hemoglobin concentration; (Hct), hematocrit; (MCV), mean corpuscular volume; (MCH), mean corpuscular hemoglobin; (MCHC), mean corpuscular hemoglobin concentration; (NOAEL), no observed adverse effect level; (RBC), red blood cell count; (WBC), white blood cell count. * Corresponding author. Tel.: +81 6 6645 3735; fax: +81 6 6646 3093. E-mail address: [email protected] (S. Fukushima). 0278-6915/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2006.09.013
(Parke and Lewis, 1992; Johnson, 2002), and the usage of some is associated with allergies and asthma (Lucas et al., 2001; Simon, 2003). Therefore, considerable controversy has arisen regarding the benefits and potential hazards of food additives, and achievement of some degree of consensus is necessary for safety evaluation (Poulsen, 1991; Adams and Smith, 2004). Due to a general lack of toxicological data for some natural food additives, risk assessment and safety evaluation often cannot be undertaken. Therefore, a need exists to increase the toxicological database for natural food additives. Mastic gum is a hard, transparent resin collected from the mastic tree (Pistacia lentiscus) found chiefly in Mediterranean countries. The resin may be chewed as a gum or it may be added to bakery goods or alcoholic beverages (Ford et al., 1992). Mastic gum is known to have some pharmacological activities. It has been used for the
J.S. Kang et al. / Food and Chemical Toxicology 45 (2007) 494–501
treatment of stomach disorders, and recent research suggests it may have potential benefit for the treatment of oral, gastric and duodenal ailments (Al-Habbal et al., 1984; AlSaid et al., 1986; Huwez et al., 1998; Takahashi et al., 2003), although there have been contrary reports (Bebb et al., 2003; Loughlin et al., 2003). Toxicological data of mastic gum have been reported concerning acute toxicity, skin irritation and phototoxicity in animals and humans (Spott and Shelley, 1970; Keynan et al., 1987, 1997; Ford et al., 1992). In the present experiment, the general toxicity of mastic gum was evaluated with 13 weeks dietary administration to male and female F344 rats. 2. Material and methods 2.1. Animals and the test chemical Five-week-old F344 rats (40 males and 40 females) were obtained from Charles River Japan, Inc. (Atsugi, Japan), and housed in a room maintained on a 12 h light/dark cycle, at constant temperature (23 ± 1 °C) and humidity (50 ± 5%). They were allowed free access to powdered CRF-1 basal diet (Oriental Yeast, Tokyo, Japan) and tap water throughout the experiment. Mastic gum was obtained from Nakamura Chiro Association (Tokyo, Japan). For setting of the high dose, we carried out a preliminary test by adding mastic gum at concentrations of 0%, 0.56%, 1.67% and 5% into powdered CRF-1 basal diet (Oriental Yeast, Tokyo, Japan), which was then fed to F344 rats for seven days. Since decreased body weight and increased liver weight were evident at the highest-dose in males, along with increased liver weight in females, 2% was selected as the highest dose for the 13 weeks toxicity test. For dietary administration, mastic gum was mixed at concentrations of 0%, 0.22%, 0.67% and 2% into a CRF-1 powdered basal diet. The stability in the powder diet was evaluated and no decomposition was confirmed after storage for 56 days at room temperature. Diets were therefore prepared twice during this experiment.
2.2. Experimental design Male and female, six-week-old animals were randomly allocated to eight groups. Groups 1–4 (males) and groups 5–8 (females), ten rats per group, received diets containing mastic gum at doses of 0%, 0.22%, 0.67% and 2%, respectively, for 13 weeks. All procedures were approved by the Institutional Animal Care and Use Committee of Osaka City University Medical School. The animals were observed for clinical signs and mortality daily. Body weights, food consumption and water intake were measured every week, and the mean food consumption or water intake/animal/day was calculated per group in each sex. At the end of the experiment, all animals were fasted overnight and euthanized by exsanguination under ether anesthesia. Blood was taken from the abdominal aorta for hematology and blood chemistry analyses. Hematological examinations were performed for the following parameters: white blood cell count (WBC), red blood cell count (RBC), hemoglobin concentration (Hb), hematocrit (Hct), platelet count, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC) and differential WBC counts. Serum biochemistry was performed for the following parameters: total proteins, albumin, albumin/globulin ratio (A/G), aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), c-glutamyl transpeptidase (c-GTP), creatinine, triglycerides, total cholesterol, total bilirubin, blood urea nitrogen (BUN), sodium, potassium, chloride, calcium and inorganic phosphorus.
495
Analyses for hematological examination and serum chemistry were conducted at Mitsubishi Kagaku Bio-Clinical Laboratories, Inc., using automatic analyzing machines, SE-9000/Sysmex and AU5200/Olympus, for blood and serum analyses, respectively. All animals were subjected to a complete necropsy. At termination they were examined grossly for pathological changes, and the heart, liver, spleen, kidneys, adrenal glands, testes or ovaries, brain, thymus and lung were weighed. In addition to these organs, lymph nodes (cervical, mesenteric), the aorta, salivary gland, bone and bone marrow (sternum, femur), trachea, thyroid, tongue, esophagus, stomach, duodenum, small intestine, large intestine, pancreas, urinary bladder, seminal vesicle, prostate gland, epididymis, oviduct, uterus, vagina, pituitary gland, sciatic nerve, skeletal muscle, spinal cord, eyes and their accessory organs were excised and fixed in 10% buffered formalin. Testes of five males each of the control and high dose groups were fixed in Bouin’s solution. Paraffin-embedded tissue sections of all organs/tissues were routinely prepared and stained with hematoxylin and eosin for histopathological examination. All organs and tissues in the control and high dose group animals were compared. Histopathology was also extended to all tissues of low and middle dose groups in which lesions were found in the high dose group.
2.3. Statistics The Duncan’s new multiple range test was employed for comparison of body weights, organ weights (both absolute and relative), and hematology and serum biochemistry data between control and treated groups. For all comparisons, p-values less than 5% (p < 0.05) were considered to be statistically significant (StatView, SAS Institute Inc., NC, USA).
3. Results 3.1. In-life parameters No mortality or obvious clinical signs were evident in any of the animals throughout the experimental period. During the experiment the body weights and cumulative body weight gains were significantly reduced from week 2 to the end of the experiment in the high dose males (group 4) and at week 8 and 13 in the high dose females (group 8), compared to the respective controls (p < 0.05 or p < 0.01) (Fig. 1). The food consumption and water intake values showed no difference among the groups during the experiment (data not shown). Total intake of mastic gum for 13 weeks was 0 (group 1), 2.7 (group 2), 8.4 (group 3) and 26.5 g (group 4) in male, and 0 (group 5), 1.8 (group 6), 5.5 (group 7) and 17.1 g (group 8) in female rats. 3.2. Organ weights Body weights and absolute organ weights are given in Table 1. Absolute liver weights were significantly increased in group 2 (p < 0.05) and groups 3, 4 and 8 (p < 0.01), with dose-dependence. Absolute thymus weights were significantly decreased in groups 2–4 (p < 0.01), albeit without dose-dependence, and absolute weights of spleen (group 2), left testes (groups 2 and 4) and thymus (group 6) showed significant differences from control values (p < 0.05), but again there was no correlation with dose. Relative organ weights are shown in Table 2. Relative liver weights were also significantly increased in group 2
496
J.S. Kang et al. / Food and Chemical Toxicology 45 (2007) 494–501 350 300 250 **
**
**
**
**
**
200
**
**
**
gram
* **
150
Group 1 (0%)
**
Group 2 (0.22%)
100
Group 3 (0.67%) Group 4 (2%)
50
4 and 8 (p < 0.01, p < 0.05, respectively), albumin in groups 3, 4 and 8 (p < 0.05, p < 0.01, p < 0.05, respectively), c-GTP in groups 7 and 8 (p < 0.01), total cholesterol in groups 4, 7, and 8 (p < 0.05, p < 0.01, p < 0.01, respectively), BUN in group 8 (p < 0.05), and calcium in group 4 (p < 0.01). There were significant decreases of creatinine in groups 2–4 (p < 0.05, p < 0.01, p < 0.01, respectively), triglycerides in groups 3, 4 and 7 (p < 0.05, p < 0.01, p < 0.05, respectively), sodium in group 7 (p < 0.01), and inorganic phosphorus in groups 6 and 7 (p < 0.05). There were significant increases of ALP in groups 4 and 8 (p < 0.01, p < 0.05, respectively), and decreases in groups 6 and 7 (p < 0.01).
0 0
1
2
3
4
5
6
7 8 Weeks
9
10
11
12
13
200 180 160 **
**
140
gram
120 100
3.4. Macroscopic and microscopic findings Macroscopic examination at necropsy revealed no gross lesions, and microscopic examination also revealed no treatment-related findings in any organs examined. Extended microscopic examination of liver, thymus, lymph nodes (cervical, mesenteric) and spleen in low and middle doses of male and female also showed no histopathological findings.
Group 5 (0%)
80
4. Discussion
Group 6 (0.22%)
60
Group 7 (0.67%)
40
Group 8 (2%)
20 0 0
1
2
3
4
5
6 7 Weeks
8
9
10
11
12
13
Fig. 1. Change of body weights during the experiment: (a) males and (b) females. *,**Significantly different from the control group at the level of p < 0.05, p < 0.01, respectively.
(p < 0.05) and groups 3, 4, 7 and 8 (p < 0.01) with dosedependence. There were increased relative weights of the heart (p < 0.01) and spleen (p < 0.05) in group 4, right kidney in groups 4 and 8 (p < 0.05), left kidney in group 8 (p < 0.05), right and left testes in group 4 (p < 0.01), and brain in groups 4 and 8 (p < 0.01, p < 0.05, respectively) compared with controls. Relative thymus weights were significantly increased in groups 2 and 6 (p < 0.05, p < 0.01, respectively), but there was no correlation with dose. 3.3. Hematology and serum biochemistry Several alterations were observed in hematological parameters (Table 3), including increased WBC in group 4 and platelets in groups 3 and 4, and decreased MCV in group 4 (p < 0.05). There were significant decreases of MCH in groups 7 and 8 (p < 0.05, p < 0.01, respectively) and of MCHC in group 8 (p < 0.01), along with significant increases of segmented neutrophils and lymphocytes in group 3 (p < 0.05), but with no dose dependence. Data for serum biochemistry are shown in Table 4. There were significant increases of total proteins in groups
Dietary treatment of mastic gum for 13 weeks in the present study caused decreased body weights at the high dose, especially in males, and increased liver weights in a dose-related manner in both genders. However, macroscopic and microscopic examinations revealed no treatment-related findings in any organs, including in the liver. Generally, non-adverse effects can be defined as biological effects that do not cause biochemical, morphological, or physiological changes that affect the general well-being, growth, development or life span of an animal. As minor elevations in liver weight that are not accompanied by any morphological change in the organ or any change in liver function may be considered as not adverse and an increase of liver weight by induced hypertrophy and hyperplasia, at least up to 20% in the absence of other liver pathology, is often regarded as a non-adverse effect in rodents (Karbe et al., 2001; Lewis et al., 2002; Williams and Iatropoulos, 2002), it seems that liver weight increase without any morphological changes is adaptive response in this study. On the while, it should be concerned that the increase of liver weight with mastic gum showed dose-dependence in this study, and the maximal percentage increase in liver weight was about 22% in the highest dose male rats in this study. So, it is reasonable that 2% administration of mastic gum in the diet for 13 weeks induce adverse effect. However, in all doses, we could not identify any histopathological findings in the liver, in line with the absence of clinical chemistry changes relating to liver. It has a possibility that there are a limited number of morphologic changes that can be discerned using conventional light microscopy in the liver (Hardisty and Brix, 2005). As to heterogeneity
Table 1 Body weights and absolute organ weights of male and female F344 rats treated with mastic gum Male
Group Mastic gum (%)
1 0
2 0.22
3 0.67
4 2
Female 5 0
6 0.22
7 0.67
8 2
No. of rates examined Initial body weight (g) Final body weight (g)
10 114 + 3 307 ± 10
10 115 + 5 311 ± 13
10 113 + 4 304 ± 20
10 115 + 3 283 ± 14**
10 101 + 2 170 ± 6
10 100 + 2 168 ± 4
10 99 + 3 165 ± 3
10 100 + 3 164 ± 5**
Organ Heart (g) Liver (g) Spleen (g) Rt.Kidney (g) Lt.Kidney (g) Rt.Adrenal gl. (mg) Lt.Adrenal gl. (mg) Rt.Testis (g) Lt.Testis (g) Rt.Ovary (mg) Lt.Ovary (mg) Brain (g) Thymus (g) Lung (g)
0.89 ± 0.07 6.76 + 0.38 0.59 + 0.05 0.91 ± 0.07 0.95 ± 0.10 0.02 ± 0.01 0.02 ± 0.01 1.49 ± 0.08 1.50 ± 0.06 – – 1.94 + 0.07 0.21 ± 0.03 0.93 ± 0.09
0.91 ± 0.08 7.17 + 0.40* 0.64 + 0.04* 0.94 ± 0.06 0.94 ± 0.06 0.02 ± 0.01 0.02 ± 0.00 1.52 ± 0.05 1.56 ± 0.07* – – 1.96 + 0.03 0.16 ± 0.06** 0.94 ± 0.05
0.88 ± 0.07 7.49 + 0.60** 0.62 + 0.04 0.91 ± 0.05 0.94 ± 0.07 0.02 ± 0.01 0.02 ± 0.01 1.51 ± 0.11 1.54 ± 0.08 – – 1.94 + 0.04 0.17 ± 0.03** 0.94 ± 0.05
0.93 ± 0.08 8.26 + 0.52** 0.60 + 0.07 0.90 ± 0.08 0.92 ± 0.04 0.02 ± 0.00 0.02 ± 0.01 1.55 ± 0.10 1.57 ± 0.05* – – 1.93 + 0.06 0.16 ± 0.03** 0.89 ± 0.05
0.58 ± 0.04 3.67 + 0.23 0.40 + 0.02 0.53 ± 0.03 0.53 ± 0.05 0.02 ± 0.00 0.02 ± 0.01 – – 0.03 + 0.01 0.03 + 0.01 1.82 + 0.04 0.15 ± 0.02 0.73 ± 0.06
0.57 ± 0.03 3.68 + 0.16 0.40 + 0.04 0.53 ± 0.03 0.53 ± 0.03 0.02 ± 0.01 0.02 ± 0.01 – – 0.03 + 0.01 0.04 + 0.01 1.81 + 0.07 0.12 ± 0.04* 0.72 ± 0.08
0.60 ± 0.08 3.98 + 0.17 0.38 + 0.02 0.53 ± 0.04 0.53 ± 0.02 0.02 ± 0.01 0.02 ± 0.01 – – 0.03 + 0.01 0.03 + 0.01 1.80 + 0.02 0.14 ± 0.02 0.70 ± 0.03
0.60 ± 0.04 4.95 + 0.32** 0.41 + 0.01 0.54 ± 0.04 0.55 ± 0.03 0.02 ± 0.00 0.02 ± 0.01 – – 0.03 + 0.01 0.03 + 0.01 1.81 + 0.05 0.16 ± 0.02 0.69 ± 0.06
Data represent mean ± SD. *, **Significantly different from control group at the level of p < 0.05, p < 0.01, respectively.
J.S. Kang et al. / Food and Chemical Toxicology 45 (2007) 494–501
Sex
497
498
Sex
Male
Group Mastic gum (%)
1 0
2 0.22
3 0.67
4 2
5 0
Female 6 0.22
7 0.67
8 2
No. of rats examined
10
10
10
10
10
10
10
10
Organ Heart (%) Liver (%) Spleen (%) Rt.Kidney (%) Lt.Kidney (%) Rt.Adrenal gl. (%) Lt.Adrenal gl. (%) Rt.Testis (%) Lt.Testis (%) Rt.Ovary (%) Lt.Ovary (%) Brain (%) Thymus (%) Lung (%)
0.29 ± 0.02 2.20 ± 0.08 0.19 ± 0.02 0.30 ± 0.02 0.31 ± 0.03 0.01 ± 0.00 0.01 ± 0.00 0.49 ± 0.02 0.49 ± 0.02 – – 0.63 + 0.03 0.07 ± 0.01 0.30 ± 0.02
0.29 ± 0.02 2.31 ± 0.06* 0.21 ± 0.01 0.30 ± 0.02 0.30 ± 0.01 0.01 ± 0.00 0.01 ± 0.00 0.49 ± 0.02 0.50 ± 0.03 – – 0.63 + 0.03 0.05 ± 0.02* 0.30 ± 0.01
0.29 ± 0.02 2.46 ± 0.10** 0.20 ± 0.01 0.30 ± 0.01 0.31 ± 0.01 0.01 ± 0.00 0.01 ± 0.00 0.50 ± 0.03 0.51 ± 0.02 – – 0.64 + 0.03 0.05 ± 0.01 0.31 ± 0.01
0.33 ± 0.04** 2.92 ± 0.08** 0.21 ± 0.02* 0.32 ± 0.02* 0.33 ± 0.02 0.01 ± 0.00 0.01 ± 0.00 0.55 ± 0.05** 0.56 ± 0.03** – – 0.68 + 0.03** 0.06 ± 0.01 0.32 ± 0.01
0.34 ± 0.02 2.15 ± 0.16 0.24 ± 0.01 0.31 ± 0.02 0.31 ± 0.03 0.01 ± 0.00 0.01 ± 0.00 – – 0.02 + 0.01 0.02 + 0.01 1.07 + 0.04 0.09 + 0.01 0.43 ± 0.03
0.34 ± 0.02 2.20 ± 0.09 0.24 ± 0.03 0.31 ± 0.02 0.32 ± 0.02 0.01 ± 0.00 0.01 ± 0.00 – – 0.02 + 0.00 0.02 + 0.01 1.08 + 0.03 0.07 ± 0.02** 0.43 ± 0.04
0.37 ± 0.05 2.41 ± 0.10** 0.23 ± 0.01 0.32 ± 0.02 0.32 ± 0.01 0.01 ± 0.00 0.01 ± 0.00 – – 0.02 + 0.00 0.02 + 0.01 1.09 + 0.02 0.09 ± 0.01 0.43 ± 0.01
0.36 ± 0.02 3.02 ± 0.17** 0.25 ± 0.01 0.33 ± 0.01* 0.33 ± 0.01* 0.01 ± 0.00 0.01 ± 0.00 – – 0.02 + 0.00 0.02 + 0.01 1.10 + 0.03* 0.09 ± 0.01 0.42 ± 0.03
Data represent mean ± SD. *, **Significantly different from control group at the level of p < 0.05, p < 0.01, respectively.
J.S. Kang et al. / Food and Chemical Toxicology 45 (2007) 494–501
Table 2 Relative organ weights of male and female F344 rats treated with mastic gum
Item
WBC RBC Hb Hct Platelet MCV MCH MCHC Unseg. Neutrophila Seg. neutrophila Lymphocyte Monocyte Eosinophil Basophil Abn. Lymphocytea
Sex
Male
Female
Group Mastic gum (%)
1 0
2 0.22
3 0.67
4 2
5 0
6 0.22
7 0.67
8 2
/MCLa e4/ MCL g/dl % e4/ MCL FLa PGa % % % % % % % %
2820.0 ± 418.5 873.9 ± 23.7 14.7 + 0.3 47.2 + 1.5 57.8 ± 8.0 53.9 ± 1.1 16.9 ± 0.2 31.3 + 0.5 1.0 ± 0.0 17.4 ± 9.3 79.1 ± 9.7 2.1 ± 1.2 0.4 ± 0.5 0.0 ± 0.0 0.1 ± 0.3
2850.0 ± 552.3 877.8 ± 16.1 14.8 + 0.3 47.0 + 1.0 60.5 ± 6.4 53.4 ± 0.5 16.9 ± 0.3 31.5 + 0.5 0.9 ± 0.2 23.1 ± 4.1 72.7 ± 6.3 2.5 ± 2.4 0.8 ± 1.3 0.0 ± 0.0 0.0 ± 0.0
2850.0 ± 779.2 873.8 ± 18.4 14.7 + 0.3 46.7 + 1.3 66.2 ± 5.6* 53.4 ± 0.7 16.9 ± 0.2 31.5 + 0.5 1.2 ± 0.4 27.2 ± 7.4* 69.0 ± 7.6* 2.3 ± 1.4 0.3 ± 0.5 0.0 ± 0.0 0.0 ± 0.0
3730.0 ± 561.8* 872.7 ± 22.5 14.5 + 0.3 46.0 + 1.2 66.4 ± 5.5* 52.7 ± 0.8* 16.6 ± 0.3 31.5 + 0.7 1.0 ± 0.0 19.3 ± 6.5 76.8 ± 7.4 2.4 ± 2.1 0.5 ± 0.7 0.0 ± 0.0 0.0 ± 0.0
2877.8 ± 774.2 849.6 ± 38.3 15.5 + 0.7 49.5 + 2.7 62.6 ± 8.8 58.1 ± 1.4 18.3 ± 0.2 31.3 + 0.9 1.2 ± 0.4 19.6 ± 9.2 76.4 ± 9.3 2.2 ± 1.2 0.6 ± 0.9 0.0 ± 0.0 0.0 ± 0.0
3140.0 ± 1079.3 843.1 ± 30.6 15.4 + 0.6 48.9 + 2.1 61.8 ± 6.9 58.1 ± 1.0 18.3 ± 0.3 31.5 + 0.7 1.1 ± 0.4 15.8 ± 6.7 81.0 ± 6.8 1.9 ± 1.2 0.2 ± 0.4 0.0 ± 0.0 0.0 ± 0.0
3140.0 ± 658.6 846.9 ± 20.4 15.3 + 0.5 48.9 + 1.3 63.6 ± 7.6 57.8 ± 0.9 18.0 ± 0.1* 31.2 + 0.4 1.1 ± 0.3 13.4 ± 5.7 82.7 ± 4.7 2.4 ± 1.2 0.4 ± 0.8 0.0 ± 0.0 0.0 ± 0.0
3400.0 ± 1047.6 844.3 ± 27.7 15.0 + 0.6 49.2 + 2.0 61.1 ± 5.3 58.2 ± 1.2 17.8 ± 0.2** 30.5 + 0.4** 1.0 ± 0.0 13.8 ± 12.7 83.6 ± 12.4 1.4 ± 0.5 0.2 ± 0.4 0.0 ± 0.0 0.0 ± 0.0
Data represent mean ± SD. *, **Significantly different from control group at the level of p < 0.05, p < 0.01, respectively. a Unseg. Neutrophil: unsegmented neutrophil; Seg. Neutrophil: segmented neutrophil; Abn. Lymphocyte: abnormal lymphocyte; MCL: microliter; FL: femtoliter; PG: picogram.
J.S. Kang et al. / Food and Chemical Toxicology 45 (2007) 494–501
Table 3 Complete blood count of male and female F344 rats treated with mastic gum
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500
Item
Total proteins Albumin A/G ratio AST ALT ALP c-GTP Creatinine Triglycerides Total cholesterol Total bilirubin BUN Sodium Potassium Chloride Calcium Inoranic phosphorus
Sex
Male
Group Mastic gum (%)
1 0
2 0.22
3 0.67
4 2
5 0
6 0.22
7 0.67
8 2
g/dl g/dl
6.4 + 0.1 3.4 ± 0.1 1.2 ± 0.1 120.2 ± 17.1 54.3 ± 2.9 454.7 ± 31.2 1.0 ± 0.0 0.42 ± 0.03 65.8 ± 13.4 62.7 ± 3.7 0.1 ± 0.0 24.0 ± 3.3 142.1 ± 1.1 4.4 ± 0.4 100.5 ± 1.1 10.2 ± 0.3 8.4 ± 2.6
6.5 ± 0.2 3.5 ± 0.1 1.2 ± 0.1 129.3 ± 38.7 61.9 ± 15.2 437.2 ± 27.3 1.0 ± 0.0 0.39 ± 0.03* 54.2 ± 18.7 63.0 ± 4.4 0.1 ± 0.0 22.0 ± 3.2 142.0 ± 1.1 4.5 ± 0.4 100.5 ± 1.4 10.2 ± 0.2 8.5 ± 2.4
6.6 ± 0.2 3.6 ± 0.1* 1.2 ± 0.1 116.7 ± 20.2 60.0 ± 7.7 454.4 ± 19.5 1.0 ± 0.0 0.37 ± 0.02** 50.0 ± 12.0* 66.0 ± 4.4 0.1 ± 0.0 23.7 ± 3.1 142.0 ± 1.3 4.6 ± 0.5 100.1 ± 1.3 10.4 ± 0.2 8.6 ± 2.6
6.9 ± 0.3** 3.7 ± 0.1** 1.2 ± 0.1 105.3 ± 15.7 58.6 ± 4.8 499.0 ± 31.7** 1.3 ± 0.7 0.34 ± 0.04** 33.2 ± 8.2** 70.5 ± 6.2* 0.1 ± 0.0 26.3 ± 2.3 141.6 ± 2.0 4.6 ± 0.5 99.5 ± 1.6 10.8 ± 0.2** 8.5 ± 1.9
6.4 ± 0.3 3.5 ± 0.2 1.2 ± 0.1 129.5 ± 25.0 40.8 ± 5.9 328.8 ± 32.1 1.2 ± 0.4 0.31 ± 0.02 40.5 ± 12.3 88.3 ± 6.3 0.1 ± 0.0 19.4 ± 2.2 142.6 ± 1.2 4.6 ± 0.6 100.4 ± 1.3 10.4 ± 0.4 9.3 ± 1.6
6.3 ± 0.3 3.5 ± 0.1 1.3 ± 0.0 126.4 ± 32.7 39.4 ± 5.8 273.3 ± 24.1** 1.3 ± 0.5 0.32 ± 0.04 41.2 ± 17.5 91.2 ± 8.7 0.1 ± 0.0 18.7 ± 2.9 141.4 ± 2.0 4.4 ± 0.4 100.9 ± 2.0 10.4 ± 0.4 7.2 ± 1.8*
6.5 ± 0.2 3.6 ± 0.1 1.2 ± 0.1 144.0 ± 50.0 41.1 ± 8.1 268.2 ± 27.5** 2.7 ± 1.7** 0.32 ± 0.03 25.4 ± 13.2* 98.7 ± 8.0** 0.1 ± 0.0 19.4 ± 2.0 140.1 ± 1.4** 4.3 ± 0.5 101.5 ± 1.0 10.3 ± 0.3 7.0 ± 1.6*
6.7 ± 0.3* 3.7 ± 0.1* 1.2 ± 0.1 122.8 ± 34.5 43.6 ± 3.2 361.3 ± 43.8* 10.4 ± 0.7** 0.29 ± 0.03 34.0 ± 11.4 118.9 ± 10.5** 0.1 ± 0.0 22.6 ± 1.9* 141.2 ± 1.8 4.6 ± 0.5 100.3 ± 2.7 10.5 ± 0.2 7.3 ± 1.6
IU/l IU/l IU/l IU/l mg/dl mg/dl mg/dl mg/dl mg/dl mEq/l mEq/l mEq/l mg/dl mg/dl
Female
Data represent mean ± SD. *, **Significantly different from control group at the level of p < 0.05, p < 0.01, respectively.
J.S. Kang et al. / Food and Chemical Toxicology 45 (2007) 494–501
Table 4 Blood clinical chemistry of male and female F344 rats treated with mastic gum
J.S. Kang et al. / Food and Chemical Toxicology 45 (2007) 494–501
and complexity in the liver, new technologies and approaches, such as microarray analysis and proteomics, may help understanding of toxicity (Malarkey et al., 2005). Although there was a decrease in absolute thymus weight in male rats, the difference of relative thymus weight did not show dose-dependence. Furthermore, histopathological examination of lymphoid organs from all groups did not show any abnormal findings. Therefore, we conclude that mastic gum did not exert a direct immunotoxic effect or a generalized stress. However, as mastic gum is associated with allergenicity, including contact dermatitis (Keynan et al., 1987, 1997; Wakelin, 2001), it may induce immunological response under some conditions. As the conventional examination of lymphoid system tissues is generally not sufficient to identify subtle lymphoid system changes (Haley et al., 2005), enhanced histopathology including immunohistochemical techniques is warranted in further studies. Some alterations of hematological parameters were also evident, including increases of WBCs and PLT in males. As recent report suggested pharmacological effects on peripheral blood mononuclear cells (Dedoussis et al., 2004), there is a possibility that mastic gum may influence hematological parameters in some unknown way. By serum biochemistry, increased total cholesterol was seen in high dose males and females, along with increased c-GTP in females. However, increases of total protein, albumin, ALP, calcium and BUN in males and/or females were minimal, and decreases of creatinine and triglyceride had little toxicological significance. No observed adverse effect level (NOAEL) is the highest exposure level that there are no statistically significant increase in the frequency or severity of adverse effect between the exposed population and its control (Lewis et al., 2002). In this study, low and middle dose treatment of mastic gum induced liver weight increase that were not accompanied by any morphological change or any change in liver function, so it may be considered as not adverse. As dietary treatment of mastic gum for 13 weeks caused decreased body weights at the high dose, especially in males, and increased liver weights in a dose-related manner in both genders without any morphological findings, it is concluded that the administration of it has a NOAEL of 0.67% in the diet. Further studies are warranted to ascertain mechanisms that are responsible for the decrease of body weight and increase of liver weight and alteration of specific hematological and serum chemical parameters. Acknowledgements We would like to thank Ms. Kaori Touma, Masayo Imanaka, and Shoko Araki for their technical assistance, and Ms. Mari Dokoh, Yuko Onishi, and Yoko Shimada for their help during preparation of this manuscript. This research was supported by Grants-in-Aid from the Ministry of Health, Labour and Welfare of Japan.
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