Role of Ocimum sanctum as a Genoprotective Agent on Chlorpyrifos-Induced Genotoxicity

Protective effect of Ocimum sanctum was evaluated on chlorpyrifos-induced genotoxicity in in vivo and in vitro models. Two different concentrations of pesticide were taken, i.e., 1/5 and 1/15 of LD50 of chlorpyrifos for the in vivo study. Rats were pre-treated orally with O. sanctum extract (OE) at 50 mg/kg b.wt. For the in vitro studies, human lymphocyte cultures were exposed to 75 μg/ml chlorpyrifos with and without OE. Structural and numerical (both aneuploidy and euploidy types) chromosomal aberrations (CAs) were scored for the assessment of induced genotoxic effects, while the variation in mitotic index (MI) was considered as a monitor for induced cellular toxicity. The same concentration of the pesticide (75 μg/ml) was taken to study the DNA damage by comet assay. Results showed that lymphocytes treated with the pesticide exhibited increased DNA damage but the increase was statistically insignificant (P>0.05). In rats pretreated with OE, a significant (P<0.01) increase in MI was observed and there was a significant decrease in the frequency of aberrant cells as compared to the rats treated with chlorpyrifos alone. A significant (P<0.05) increase in CA was observed in cultures treated with 75 μg/ml chlorpyrifos as compared to controls, which decreased significantly (P<0.05) with OE pretreatment.


INTRODUCTION
C h l o r p y r i f o s , a n o n -s y s t e m i c b ro a d -s p e c t r u m organophosphate insecticide, is used for the control of a large number of insect pests of various crops. It is a cholinesterase inhibitor. [1] Since Ocimum sanctum leaf extract has time tested healing value in the traditional Indian medicinal system, it was thought that it would be interesting to know if it has a genoprotective effect against aberrations induced by chlorpyrifos in in vivo mouse system.

Original Article
There is a continued interest and need to identify and develop non-toxic genoprotective compounds. An efficient genoprotectant could prove useful in occupational and therapeutic settings where genotoxic chemicals are used or where exposure occurs. O. sanctum, commonly called "Tulsi" (Family Labiateae), is easily available in the whole of tropical and subtropical India. It is held sacred by Hindus, and various parts of the plant have been traditionally used in Ayurveda and Siddha systems of medicine for the treatment of diverse hepatic disorders, cold, cough and as an antidote for snakebite. [2] It has also been reported to have anticarcinogenic activity, [3] as well as radioprotective effects. [4] Flavonoids isolated from O. sanctum scavenged the free radicals in vitro and showed antilipoperoxidant activity in vivo at a very low concentration. [5] It is well known that pesticides are genotoxic to experimental subjects (rat/mice) and have been shown to cause the same effects in human subjects also. [6] The extensive application of pesticides in modern agriculture requires an intensive investigation of the impact of these chemicals on the environment and public health. With the dispersal of hundreds of millions of kilograms each year, these agents must be analyzed for their mutagenic properties.
Therefore, looking at the extensive application of chlorpyrifos and the possible genoprotective role of O. sanctum, it was considered worthwhile to undertake this study. Thus, the present study was undertaken to investigate the genoprotective effect of O. sanctum extract (OE) on mitotic index (MI) and chromosomal aberration (CA) percentage in bone marrow cells of rats induced with 1/15 and 1/5 LD 50 of chlorpyrifos. Our goal was also to evaluate the cytogenetic effects of single exposure to chlorpyrifos although pesticide sprayers receive a chronic exposure to this commonly used pesticide.

Comet assay to assess DNA damage
Comet assay or single cell gel electrophoresis assay was used for the evaluation of DNA damage in individual cells. [7] The experimental sample consisted of blood subjected to chlorpyrifos at 75 µg/ml (the concentration is one-fourth of what is sprayed by farmers in the field) for 2 hours at 37ºC. Blood sample treated with 40 mM H 2 O 2 for 10 min at room temperature formed the positive control. The negative control was untreated blood. A small number of cells are immersed in agarose gel, lysed, subjected to an electrophoretic field and then stained with silver stain. [8] The assay was run in triplicate for experimental and control samples. Fifty cells for each sample were scored for DNA damage visually under the light microscope and were classified into six categories [9] as shown below: Category A: Undamaged cells Categories B-E: Cells with progressively greater DNA damage Category F: Apoptotic cells

In vitro lymphocyte culture and in vivo studies
Preparation of OE Fresh leaves of O. sanctum, collected locally, were air dried, powdered and extracted with 50% ethyl alcohol and 50% distilled water in a soxhlet apparatus by refluxing for 68 hours (at 4 hours/day for 17 days) at 60°C. The extract was evaporated to obtain it in a powder form. For oral administration, the extract was constituted in 0.5 ml distilled water and administered at 50 mg of extract/kg b.wt. to rats, since this dose of OE gave protection against radiation injury. [10] The dose of the insecticide was calculated as 1/15 and 1/5 of the recommended LD 50 (135 mg/kg b.wt.) for rats. The rats were sacrificed 24 hours later and bone marrow preparations were made in the usual manner. The same experimental schedule was followed for 1/15 LD 50 of chlorpyrifos. For the controls, the rats were given 0.5 ml distilled water orally for 21 days.

Human lymphocyte culture
The assessment of the genoprotective role of OE was also carried out in vitro in cultured human lymphocytes. The chromosome preparations were made from peripheral blood cultures following the method of Moorhead et al. [11] As a first step, the CA percentage was assessed using 75 µg/ml. In the other samples, O. sanctum extract was added at zero hour at 12 µg/ml and chlorpyrifos was added after 48 hours to the culture at 75 µg/ml.

Statistical analysis
The data were analyzed using student's t-test.

RESULTS AND DISCUSSION
Comet assay to assess DNA damage The assay was run in triplicate for experimental and control samples. Fifty cells for each sample were scored for DNA damage visually under the light microscope and were classified into six categories as shown in Table 1.  In the second treatment (1/15 LD 50 ), the mean value of CA for controls was 2.0±0.9 and it was 3.5±0.30 for animals treated with only chlorpyrifos. However, there was an increase in the frequency of aberrant cells in bone marrow of rats treated with 1/15 LD 50 of chlorpyrifos but the increase was not significant. In the animals pretreated with OE, there was a decrease in the frequency of aberrant cells (mean CA% 2.5±0.8) as compared to the chlorpyrifos treated rats [ Table 3].

In vitro study
A significant increase (P<0.05) in CAs was observed in lymphocytes treated with chlorpyrifos and a statistically significant (P<0.05) decrease was found in cultures pretreated with OE. MI decreased significantly (P<0.05) in cultures treated with chlorpyrifos and a slight increase in MI was found in OE pretreated lymphocytes but the increase was not statistically significant [ Table 4]. Thus, the study showed that 75 µg/ml of chlorpyrifos caused statistically nonsignificant damage to DNA as determined by comet assay. There seems to be no other reference available on such assessment of genotoxicity of chlorpyrifos. However, similar work has been done on some other pesticides and herbicides which are outlined below.  Assessment of genotoxic effects of chlorpyrifos and acephate by comet assay in mice leukocytes was done by Rahman et al. [12] Evaluation of herbicide-induced DNA damage in human lymphocytes by comet assay was done by Ribas et al, [13] They found that alachlor, atrazine, maleic hydrazide, paraquat and trifluralin gave positive results for genotoxicity by increasing the comet tail length. Chlorpyrifos-induced DNA damage in rat liver and brain cells was assessed through comet assay by Mehta et al., [14] who classified the DNA damage in various classes from zero to four.
DNA damaging effects of pesticides were measured by comet assay and CAs in Chinese hamster ovary cells by Vigreux et al, [15] and they found that chlorothalonil was toxic to CHOK1 cells but carbendazim did not induce DNA strand breaks in comet assay. Occupational exposure of workers employed in pesticide production was found to cause an increase in mean tail length of comet in a study by Paramjit et al. [16] In the classification of pesticides, chlorpyrifos is classified as a "moderately hazardous pesticide" by World Health Organization (WHO). [1] Our results indicate a moderate toxicity of chlorpyrifos at a concentration of 75 µg/ml of blood, which agrees with the WHO data.
This study was also aimed to evaluate the genoprotective effects of O. sanctum on chlorpyrifos-induced genotoxicity. The use of pesticides has become a routine mainly in underdeveloped countries, but the genotoxic potential of these substances is not yet well established. [17] Most of the farmers responsible for the application are at risk for cytotoxicity and genotoxicity.
Awa et al, [18] detected a positive correlation between the risk of genetic diseases in populations and the level of cytogenetic damage, whereas Au et al, [19] hypothesized that CAs were in the background of carcinogenesis and that the determination of their incidence was an important parameter for the effect of various agents on the health status of mammals and man. Thus, the increased frequency of CAs is related to higher risk of development of malignancies.
Maximum number of aneuploidy cells was observed in mice treated with 1/5 of LD 50 of chlorpyrifos and the number significantly decreased in mice pretreated with OE. There was a total absence of metaphase plates showing precocious centromeric separation in rats pretreated with OE; perhaps the flavonoids and other active components help in polymerization of spindle fibers so that all the cell divisions are in phase.
Radiation and chemical toxins produce biological damage by forming reactive oxygen species like singlet oxygen and superoxides, hydroxyl and hydroperoxy radicals, hydrogen peroxide and organic peroxides. [20] The genoprotective effect of O. sanctum is associated with the presence of its flavonoids, such as orientin and vicenin, which take part in scavenging reactive intermediates that are capable of binding to proteins and DNA. [5] Chlorpyrifos was found to increase the activities of superoxide dismutase, glutathione peroxidase and catalase. Melatonin causes decrease in the above enzymes and an increase in thiobarbituric acid reactive substances. Production of reactive oxygen species could be a cause of DNA damage.
In vitro and in vivo generation of reactive oxygen species, DNA damage and lactate dehydrogenase (LDH) leakage by selected pesticides was studied by Bagchi et al. [21] According to them, brain lipid peroxidation and DNA single strand breaks are two indices of oxidative stress and oxidative tissue damage.
Thus, the most likely mechanism of DNA damage and chromosome breakage by chlorpyrifos seems to be through the production of reactive oxygen species, and the present investigation indicates that the pre-treatment of rats with OE at 50 mg/kg per day for 21 days has a significant (P<0.001) positive effect in the MI depression caused by