Formalin induced genotoxicity in rats and its amelioration with Eugenol and curcumin

Introduction

Some substancs caus genotoxicity by cellular damage. To assess genotoxicity, researchers research look for cellular and DNA damage. The major genotoxicity are mutations of genes, aberrations in chromosome and deffects in DNA. No single method is perfect to assess the cellular and DNA damage, different methods are employed. They are 1. One of the best test to observe Genotoxicity is to measure and test DNA damage in single cell which show both qualitative and quantitative measurement (Comet Assay), 2. Bone marrow micronucleus test (MNT) is the most accurate test for Genotoxicity with simultaneous assessment of aberration in chromosome, 3. Gene mutation and chromosomal aberration tests are for identification of actual lesions in the DNA molecule.

Results

Average number of micronuclei (MN) per 2000 PCE was significantly highest in Gr-II (Table 1) as compared to other groups. The micronuclei seen in Gr-I, Gr-VIII, and Gr-IX were on the lower side, and no significant difference was observed. In the formalin-treated group, this value was 3.93±0.36, which was significantly higher than both of the control groups, whereas the Gr-VI and Gr-VII values showed a decreasing trend from Gr-III, which was not significant. Similar trends were also seen for Gr-II in comparison with Gr-IV and Gr-values PCE (mean) per 200 total erythrocytes (TE) in bone marrow cells of rats was significantly lowest (59.33±3.32) in Gr-II, i.e., Cyclophosphamide-treated rats. The administration of Eugenol (Gr-IV) and Curcumin (Gr-V) resulted in improving the condition. The mean PCE values of Gr-I, Gr-VIII, and Gr-IX were significantly higher than those of other groups. In Gr-III rats, this value was 92.33±3.51, which was significantly different from both positive and negative controls. The values of Gr-VI and Gr-VII showed an improving tendency compared to Gr-III, which was nonsignificant (Table 2).

Chromosomal aberration assay

The mean structural chromosomal aberration in different groups from Gr-I to Gr-IX was observed as 1.16±0.12, 10.65±1.09, 4.45±0.23, 10.34±1.04, 10.11±0.92, 4.22±0.23, 4.10±0.32, 1.13±0.15, and 1.10±0.11, respectively. The highest pulverization was seen in Gr-II rats, while the least was observed in Gr-I, Gr-VIII, and Gr-IX. A significantly higher value was seen in the formalin-treated group (Gr-III) compared to the negative control group. Administration of Eugenol and Curcumin reduced the chromosomal aberration (CA) value, though the reduction was non-significant (Table 3).

Comet assay

The value of OTM per cell in Gr-II (2.37±0.017) rats was significantly highest from all other groups. The lowest values of OTM were seen in Gr-1, Gr-VIII & Gr-IX. In Formalin treated rats (Gr-III), thevalue0.63±0.052 was observed which differ significantly from both positive and negative control groups i.e. Gr I and Gr II. In Gr VI and VII this OTM value were observed 0.60±.009 and 0.59±0.022 respectively which was slightly lower than the Gr-III data. The data of OTM of Gr-IV and Gr-V showed slight decrease from Gr-II data which was non-significant (Table.4)

Discussion

Genotoxicity by substances and their amelioration by eugenol and surcumin were studied. Bone marrow micronucleus test (MNT) is a reliable genotoxic test that can predict 100% genotoxic potential (Rao et al., 2005). In the present study, the micronucleus assay revealed that the average number of micronuclei per 2000 PCE in formalin-treated rats (Gr-III) was significantly higher than that of the negative control group (Gr-I). However, this value was significantly lower than that of the positive control group. The increase in the number of micronuclei following formalin exposure is supported by the findings of Mert et al. (2014) [15] in Nile tilapia, Heddle et al. (1981) [7] , and Ma et al. (1985) [14] in mouse peripheral erythrocytes, as well as Norppa et al. (1985) [17] in human lymphocytes and Chinese hamster ovary cells. In contrast, Speit et al. (2006) [18] observed no significant increase in micronuclei in the nasal mucosa of rats at different doses of formalin. The administration of Eugenol (Gr-VI) and Curcumin (Gr-VII) in rats previously exposed to formalin caused a decrease in the MNT value, although this reduction was not statistically significant. The slight decrease observed may be attributed to the ameliorative effect of these herbal treatments. Similar protective effects were reported with citrus and ellagic acid by Hosseinimehr and Karami (2005) [8] , respectively, in studies on Cyclophosphamide-induced genotoxicity.

In the positive control group (Gr-II), a significantly highest MNT value per 2000 PCE was observed, which aligns with the findings of Samarth et al. (2018) [19] . The mean number of PCE per 200 total erythrocytes in bone marrow cells of rats was significantly lowest in Gr-II, i.e., Cyclophosphamide-treated rats. This significant reduction may be due to the known genotoxic effects of Cyclophosphamide. The administration of Eugenol (Gr-IV) and Curcumin (Gr-V) led to an improvement in this value, likely due to the ameliorative effects of these traditional therapeutic agents. The mean PCE values of Gr-I, Gr-VIII, and Gr-IX were significantly higher compared to other groups. In Gr-III rats, this value was 92.33±3.51, which was significantly different from both the positive and negative control groups. The values in Gr-VI and Gr-VII showed improvement compared to Gr-III, although the differences were not statistically significant. Different structural chromosomal aberrations such as breaks, caps, deletions, exchanges, and pulverization are detected in bone marrow metaphase analysis. These genetic damages are directly or indirectly used to assess the genotoxic effects of certain toxic chemicals (Jena et al., 2002) [9] . In the present study, the highest level of damage (pulverization) was observed in Gr-II rats, whereas the least damage was seen in Gr-I, Gr-VIII, and Gr-IX. A significantly higher value was observed in the formalinadministered group (Gr-III) compared to the negative control group. This elevated chromosomal aberration was also reported by Cortes et al. (1986) [5] in the root meristem of Allium cepa, Rieger and Michaelis (1960) [20] in the primary roots of Vicia faba, Ma et al. (1985) [14] in mouse peripheral erythrocytes, Norppa et al. (1985) [17] in Chinese hamster ovary cells, and Nocentini et al. (1980) [21] in monkey kidney cells. Bezerra et al. (2017) [2] observed the antioxidant, anti-carcinogenic, cytotoxic, and antitumor properties of Eugenol. The administration of Eugenol and Curcumin in both formalin-and cyclophosphamide-administered groups caused a reduction in chromosomal aberration (CA) values, although this reduction was not statistically significant. The decrease in CA values may be attributed to the ayurvedic and therapeutic properties of these compounds. The findings of Fanqiun and Zhijia (2017) [10] , Bezerra et al. (2017) [2] , and Binu et al. (2018) [3] on Eugenol, and Panda (2017) [22] on the anticancer properties of Curcumin, support these observations. The mean value of OTM (Olive Tail Moment) per cell in Gr-II animals was significantly higher than in all other groups. The lowest OTM values were observed in Gr-I, Gr-VIII, and Gr-IX. In formalin-administered rats (Gr-III), a value of 0.63±0.052 was recorded, which was significantly different from the negative control group. This increase in tail moment and tail DNA percentage is attributed to the damaging properties of formalin. Similar findings were reported by Lu et al. (2005) [12] , Kumarave et al. (2009) [11] , and Borkotoky et al. (2014) [1] for Nimesulide. In Gr-VI and Gr-VII, the OTM values were 0.60±0.009 and 0.59±0.022, respectively, which were slightly lower than the Gr-III value. The OTM data for Gr-IV and Gr-V showed a slight decrease compared to the Gr-II group, though the differences were non-significant. The reports of Fanqiun and Zhijia (2017) [10] , Bezerra et al. (2017) [2] , and Binu et al. (2018) [3] on Eugenol, and Panda (2017) [22] on the anticancer properties of Curcumin, are in agreement with the present findings.

Declaration of Interest

This was part masters programme of Dr. Sitesh Kumar Mohapatra, an M.V.Sc scholar. The authors declare no conflict of interest.

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