Evaluation of Camphor Mutagenicity in Somatic Cells of Pregnant Rats

MATERIALS AND METHODS

Animals And Husbandry
Adult male (body weight, 150-200 g) and virgin female (body weight, 120-150 g) rat obtained from Laboratory Animal house of king Fahd Research Center at King Abulaziz University and maintained in constant temperature control rooms (22±2°C). The research project was conducted from 1/3/2008 to 1/3/2009. Animals received food and water then maintained on a 12 h light/12 h dark photoperiod. After one week of acclimatization, untreated females and males mated by overnight cohabitation (one male to three females). Females examined for the presence of a vaginal plug the following morning. The day a dropped copulatory plug found designated as day zero of gestation. The pregnant females divided into four groups according to the recommendations of Jamshidzadeh and Sajedianfard (2006) with slight modifications, each group consists of 30 pregnant females. On the 1st day of gestation, females of the first group given 1/2 mL corn oil daily by oral gavages and considered as control. The pregnant females of the other three groups given 1/2 mL of the different doses of camphor at the levels of 5, 10 and 20 mg kg^-1 b.wt. by using corn oil as vehicle. Group of ten females were given a single intraperitoneal injection of 25 mg kg^-1 b.wt. cyclophosphamide and considered as positive control. Pregnant females of the experimental and control groups sacrificed by cervical dislocation at 7, 14 and 20 day of gestation and their abdomens were opened to determine their gravid status.

Bone Marrow Chromosome Preparation
Cytogenetic analysis performed on bone-marrow cells according to the recommendations of Preston et al. (1987), with slight modifications. Five pregnant rats of each group at different intervals (7, 14 and 20 day) were injected with 1.0 mL of 0.5% colchicines intraperitoneally, 2 h prior to sacrificing the animals to arrest the mitotic division at metaphase stage. Bone marrow cells collected in saline from the femurs, then pipetting by using Pasteur pipette. The cells sediment by centrifugation at 1200 rpm for 10 min. Potassium chloride solution 0.56% added to sediment as hypotonic solution and incubated at 37°C for 30 min. Cells fixed in freshly prepared cold fixative (3-1 methanol-glacial acetic acid) for 15 min in refrigerator and then the mixture centrifuged for 10 min. The fixative was changed and left overnight in refrigerator and then centrifugation and fixation repeated three times at an interval of 20 min. The material resuspended in a small volume of the fixative, dropped on a slides which was dipped in 70% ethanol and flame-dried. Slides were stained in 10% phosphate buffered Giemsa (pH 6.8) for 35 min then washed in phosphate buffer solution. One hundred good metaphases for each animal examined microscopically and chromosome aberrations recorded.

Micronucleus Test
A micronucleus (MN) formed during the metaphase/anaphase transition of mitosis (cell division). It may arise from a whole lagging chromosome (aneugenic event leading to chromosome loss) or an acentric chromosome fragment detaching from a chromosome after breakage (clastogenic event) which do not integrate in the daughter nuclei. The bone marrow of five pregnant females of control and treated groups of different intervals were extracted, smear preparations made by using fetal calf serum and stained in 10% phosphate buffered Giemsa (pH 6.8) for 5 min according to Salamone et al. (1980). Polychromatic erythrocytes scored for micronuclei under the microscope. All slides, including those of positive and negative controls, should be independently coded before microscopic analysis. At least 2,000 immature erythrocytes per animal scored for the incidence of micronucleated immature erythrocytes.

Statistical Analysis
To evaluate the mutalpgenic effects of camphor the data analyzed using the ANOVA and Duncan’s comparison tests. The significance level of (p≤0.05) adopted to compare data within the same experiment.

RESULTS

In the present study, the oral administration of camphor to pregnant female rats at the first day of gestation induced an increase in the percentage of embryo failure (abortion) with increasing the dose, thus, the percentage of abortion in the dose of 5 mg kg^-1 b.wt. camphor was 73.3%, in the dose of 10 mg kg^-1 b.wt. was 80% and in the dose of 20 mg kg^-1 b.wt. was 93.3%.

The present study conducted in order to identify the possible mutagenicity, which may be caused by camphor in bone marrow of pregnant rats and to demonstrate that there is a relation between the abortion caused by camphor and the incidence of chromosomal aberrations.

The evaluation of mutagenicity was conducted using two methodologies: chromosome aberration and micronucleus test in bone marrow cells of pregnant rats at days 7, 14 and 20 of gestation. The structural chromosomal aberrations that were observed for were chromatid gap, chromatid break, deletions and centromeric attenuations. While, the numerical aberrations that were observed for were hypoploidy and polyploidy.

Chromosomal Analysis
The effects of different doses treatment of camphor on pregnant rat bone marrow at 7, 14 and 20 day of gestation were shown in (Table 1-3). The data represented by Mean±SD. Results showed that camphor causes a moderate increase in the frequencies of individuals and total chromosomal aberrations as compared to control at the three different doses and intervals, but these increase were non significant. In addition, the analysis of the frequencies of total chromosomal aberrations showed that there were non-significant increases in the mean values of total chromosomal aberrations of camphor doses at day seven and fourteen of gestation than control.

Table 1:	Mean value and standers deviations of different types of chromosomal aberrations induced by camphor in bone marrow cells of pregnant female rats at day seven of gestation

Table 2:	Mean value and standers deviations of different types of chromosomal aberrations induced by camphor in bone marrow cells of pregnant female rats at day fourteen of gestation
Each value represents the mean and standard deviations of five animals. Cht. means chromatid Means with different letters within each column are significant at 5% level

Table 3:	Mean value and standers deviations of different types of chromosomal aberrations induced by camphor in bone marrow cells of pregnant female rats at day twenty of gestation
Each value represents the mean and standard deviations of five animals. Cht. means chromatid Means with different letters within each column are significant at 5% level

Table 4:	Frequencies of micronuclei in pregnant female rat bone marrow of all experimental groups
Means with different letters within each column are significant at 5% level

While, at gestation day 20, there were no significant differences between camphor doses and control (Table 1-3).

Micronucleus Analysis
Results of micronucleus test in bone marrow cells of pregnant female rats of all experimental groups are summarized in Table 4. Data indicated that the dose of 5 mg kg^-1 b.wt. camphor induced non significant decrease in the frequencies of micro nucleated polychromatic erythrocytes (MNPCEs) at three intervals compared to control. However, doses of 10 and 20 mg kg^-1 b.wt. exhibited a non-significant increase in the frequency of MNPCEs at seven day of gestation when compared to negative control. While, there was no significant differences at fourteen and twenty days of gestation (Table 4).

Camphor against aflatoxin B1 in S. typhimurium TA100. From the previous finding, we can conclude that camphor at the concentrations tested do not has mutagenic activity and the abortion of treated pregnant rats are not due to the mutagenicity of camphor.

DISCUSSION

The essential oils and their monoterpenoid constituents have been widely used as fragrances in cosmetics, as flavouring food additives, as scenting agents in a variety of household products, as active ingredients in some old drugs and as intermediates in the synthesis of perfume chemicals (Gomes-Carneiro et al., 1998). Camphor is a pleasant- smelling cyclic ketone of the hydroaromatic terpene group. Its history dates to ancient Chinese medicine. Camphor was highly regarded as a circulatory and cardiac stimulant in the late 19th and early 20th centuries. Traditional uses have been as an abortifacient, contraceptive (Vasey and Karayannopoulos, 1972), cold remedy, aphrodisiac, antiaphrodisiac, suppressor of lactation and antiseptic (Compadre et al., 1986; Goldfrank et al., 2002).

In the present study, the oral administration of camphor to pregnant female rats induced an increase of embryo loss and the rate of embryo loses increases with an increasing dose of Camphor. Such result is in accordance with previous reports (Riggs et al., 1965; Weiss and Catalano, 1973) which showed that camphor could easily pass placental barrier and affect fetal development. Camphor ingestion might lead to abortion because camphor crosses the placenta and foetuses lack the enzymes to hydroxylate and conjugate with glucuronic acid (Rabl et al., 1997). In addition, 4-methyl-benzylidene camphor (4-MBC) exhibits estrogenic activity, is a preferential Estrogen Receptor (ER)-β ligand and interferes with development of female reproductive organs and brain of both sexes in rats (Seidlova-Wuttke et al., 2006; Durrer et al., 2007). 4-methylbenzylidene Camphor exert their effects on endocrine function disrupting including uterotrophic that is a preferential Estrogen Receptor (ER)-β ligand and interferes with development of female reproductive organs and brain of both sexes in rats.

In the present study, results show that camphor does not increase the frequencies of total chromosomal aberrations and number of micronuclei in bone marrow cells of pregnant rats. These results indicate that there was no evidence of mutagenicity of camphor at any of the concentrations tested. This is in agreement with earlier studies (Gomes-Carneiro et al., 1998; Knezevic-Vukcevic et al., 2006) in which Camphor did not exhibit mutagenic activity in the Ames test, by using the Salmonella/microsome assay (TA97a, TA98, TA100 and TA102 tester strains), without and with addition of an extrinsic metabolic activation system (lyophilized rat liver S9 fraction induced by Aroclor 1254). In addition, Chang et al. (1998, 2000) found that the camphorated phenolic compounds used in clinical dentistry as sedatives for the dental pulp did not cause genotoxic effects on cultured human pulp fibroblasts in vitro.

Micronucleus test result indicated that camphor 5 mg kg^-1 b.wt. dose showed non-significant decrease in the frequencies of micronuclei compare to those of control. This result may interprets by (Knezevic-Vukcevic et al., 2006) they showed that essential oil of sage and its fractions can be considered to contain substances with antimutagenic potential against UV-induced and possibly spontaneous mutations. In view of the composition of sage oil and fractions, obvious candidates are α-thujone, 1, 8-cineole, camphor and α-humulene. Meanwhile, Goel et al. (1989) demonstrated that camphor antagonized γ-radiation-induced increase in SCE frequency in mice bone marrow cells. In addition, Kim et al. (1992) reported an antimutagenic effect of cineole and disturbance exhibited by camphor oils. This latest depending on the test organism used, the effect could also be attributed to certain molecules that are involved in those oils. Therefore, further antimutagenesis and anticarcinogenesis studies are recommend.

ACKNOWLEDGMENTS

This research was supported by the National Basic Research Program of King Abdul Aziz University, Saudi Arabia (project No. 428/515). The skilful assistance of all staff members of the Biology Department, King Abdul Aziz University is gratefully acknowledged.