NEEM LEAF EXTRACT PREVENTS METHOTREXATE INDUCED GENOTOXICITY IN THE SOMATIC CELLS OF DROSOPHILA MELANOGASTER”

Received on 11 February, 2014; received in revised form, 11 April, 2014; accepted, 13 June, 2014; published 01 August, 2014

NEEM LEAF EXTRACT PREVENTS METHOTREXATE INDUCED GENOTOXICITY IN THE SOMATIC CELLS OF DROSOPHILA MELANOGASTER”

T.R. Swain, M.R. Singha and R. Samal

Department of Pharmacology, S.C.B. Medical College, Cuttack-753007, Odisha, India

ABSTRACT:

Background &Objective: To explore possible protective effect of neem leaf extract against methotrexate induced genotoxicity in the somatic cells (wing primordial) of Drosophila melanogaster.

Methods: In the first phase of the experiment, neem leaf extract (NLE) at two doses was tested in the somatic cells (wing primordia) of Drosophila melanogaster (3^rd instar trans heterozygous larvae resulting from cross of two bioactive strains) to probe effect on mutagenicity. In the second phase, effect of NLE at two doses (2.5% & 5%) was studied to probe its role in prevention of methotrexate induced genotoxicity. Concurrent negative control was run simultaneously in each experiment, where the larvae were exposed to the solvent (distilled water). The effect was observed and quantified under microscope as the number of spots (Clones) per wing.

Results & interpretation: Only higher concentration of NLE (5%) showed significantly higher spontaneous mutation (2.7%) compared to untreated control group. Methotrexate induced 12.01 spots per wing (positive control). NLE at both doses  (2.5 % & 5%) when administered concurrently with methotrexate, average wing spot were reduced to 7.5 and 8.2 respectively which was significantly less compared to only methotrexate treated group.

Conclusion:  NLE at both doses shows promising beneficial effect in preventing methotrexate induced mutation in Drosophila melanogaster.

Neem leaf, Methotrexate, Genotoxicity, Drosophila Melanogaster

INTRODUCTION: People of India have been using different medicinal plants to combat diseases from time immemorial. One such extensively used medicinal plant in India is Neem (Azadirachta indica A. Juss) which has established a very important place in Ayurveda - the ancient Indian treatise on human health.

There are more than 135 active principles so far isolated from different parts of Neem tree including bark, seed, oil, leaves, flower, fruit, twig, root and gum ¹. These active principles are known to have anti-inflammatory, antipyretic, analgesic, immunostimulant, hypoglycemic, anti-ulcer, anti-fertility, anti-malarial, antifungal, and antiviral activities.

There is rapid expansion in the field of cancer and cancer chemotherapy is extensively prescribed to curb it. Most of anticancer agents posses serous side effect including genetic abnormality. Awasthy KS ² has studied Genotoxicity of a crude leaf extract of neem in male germ cells of mice.

On the other hand , anti mutagenic effect of neem leaves extract has been reported by many workers. ^3,4 Because of these inconclusive reports, present study was planned to explore the possible anti genotoxic potential of Neem leaf extract in a non-mammalian test - system (Drosophila melanogaster).

MATERIALS AND METHODS: Somatic Mutation and Recombination Tests (SMART) have mainly been a standardized test for genotoxicity studies ⁵. Two high bioactive strains of Drosophila melanogaster, viz., ORR; mwh/mwh and ORR;flr3/TM3, Ser were used. These stocks have high constitutive level of cytochrome P-450. As a result, they have high metabolic activity. The marker flr3 (flare) is a recessive autosomal marker on the left arm of chromosome 3 (3-38.8) and is expressed as a short, thick and misshaped wing hair. Since the marker is zygotic lethal in homozygous condition, it is kept on a balancer chromosome TM3. The allele mwh (multiple wing hairs) is also a recessive autosomal mutation located on the tip of chromosome 3 (3-0.3). In homozygous condition it is expressed as multiple wing hairs in contrast to a single trichome per wing cells of the wild type fly.⁶Anti-neoplastic drug methotrexate in pure form was used from laboratory and the Neem leaf extract (NLE) was obtained from Indian Institute of Herbs, Saharanpur (Batch No. RD-01).

For all treatment, methotrexate conc. was 0.01 micro unit. All the experiments were conducted at a controlled temperature of 25 ± 1⁰C and 60% relative humidity. NLE at two doses (2.5% & 5%) was prepared by proper dilution of stock, for the treatment of 3rd instar larvae. Concurrent control experiments were run simultaneously, where same age larvae were exposed to distilled water (solvent) alone.

The larvae, in batches, were transferred to glass vials containing 1.5gm of instant food rehydrated with desired concentrations of test compounds like methotrexate or NLE or combination of methotrexate and Neem extract, for the rest of their life, i.e., for 48 hrs and 72 hours. On eclosion after treatment, the adult flies were stored in 70% ethanol. The wings of transheterozygous flies were detached from adult body in Faure’s solution.

On eclosion (hatching), 40 wings (20 from male and 20 from female) of flies developing from the control and chemically treated larvae were mounted and screened for the induction of wing spots (clones). The frequencies of the spontaneous and induced spots were noted for further statistical analyses. On a clean slide the wings were spread out in a drop of Faure's solution, allowed to be firmly glued for 24 hrs, mounted finally with another drop of Faure's solution under a clean cover slip and charged with metal cubes to harden the preparations. The mutant mosaic spots (clone) i.e.; multiple wing hair (Mwh) or flare (Flr) hair in place of a single trichome or wing hair were scored in the treated wings and statistically analyzed. Thus the genetic changes induced by any mutagen are expressed phenotypic ally in the form of mutant mosaic spots on the wings of eclosing adults.

Microscopic Examination of Wings: The wings of adult flies, on eclosion, were stored in 70% ethanol and subsequently the wings of the flies were detached and mounted in Faure’s solution (Gum Arabic, glycerol, chloral hydrate and water). The mounted slides were charged with metal cubes which allowed the wings to dry up and harden without crumpling them. Both dorsal and ventral surfaces of the mounted wings were screened under a compound microscope at 400X magnification.  Only the distal wing compartments were screened ⁷.

The size and type of each spot (i.e., whether small or large and whether mwh and flr3 single or mwh/flr3 twin) was recorded for statistical analyses. The mutant clones were classified into three types i) small singles: Clones with one or two mutant cells ii) large singles: containing 3 or more cells and iii) twin: twin spots, containing adjacent mwh and flr3 cells ⁵.

Statistical Analysis: The frequencies of different types of spots were statistically evaluated on the basis of a multiple decision procedure ⁵ to determine whether the result was positive, weakly positive, inconclusive or negative. Since small single spots (s = 1-2) and total spots (T) have high spontaneous frequencies; m (multiplication factor) was fixed at a value of 2. On the other hand, since the large single spots(S>2) and twin spots (t) have low spontaneous frequencies, m was fixed at 5.

The data were evaluated at 5% level of significance. Conditional Binomial Test was applied to know the significance of the result.

RESULTS: Drosophila melanogaster possess spontaneous mutagenic potential in the form of development of wing spots that was observed microscopically in the present study. Eighty wings of adults developed from 3rd instar larvae grown on instant food supplemented & NLE in two strength (2.5% & 5%) alone or along with methotrexate were screened. On an average of 0.81 spots per wing was recorded following exposure of larvae to distilled water, which served as negative control. Eclosing adults were screened with 0.25 % conc. of NLE either at 48 or 72 hrs of exposure. NLE at 2.5% did not produce any significant change in number of spontaneous wing spots compared to control group at both at 48 and 72 hours (p > 0.05). On the contrary, 2.9 & 2.7 total spots per wing were recorded at the end of 72 hrs in 5% NLE treated group. This value was statistically significant compared to control group. All the spots were singles either with multiple wing hair (mwh)or Flr3 phenotype. In comparison to control group, the outcomes for total spots were statistically significant for 5% NLE only (Table 1).

TABLE 1: DATA ON THE WING MOSAIC ASSAY AFTER LARVAL EXPOSURE

n= 80 wings, NLE 2.5% n= 80 wings, MTX conc. 0.01 mm, NLE 2.5%, SS, smallsingle spots, LS, Large single spots, T S, twin spots,  * =  p < 0.05 compared to control

In order to explore antimutagenic potential of NLE, against methotrexate induced genotoxicity, the mwh t/t flr3 larvae were concurrently treated with 2.5% & 5% NLE. The experiment was repeated and when wing spot data obtained in the two different experiments did not differ significantly, the data were pooled.

After treatment with 0.01 mμ of methotrexate, on an average of 12.01 total spots per wing could be observed. When the induced spot frequencies were statistically compared with the spontaneous frequencies in the corresponding control, the outcome was highly significant (P < 0.001) (Table 2, Picture 1).

TABLE 2: EFFECT OF PRE, CONCURRENT AND POST TREATMENT OF NLE (2.5%) ON METHOTREXATE INDUCED MUTATION NLE 2.5% n= 80, MTX conc. 0.01 µm, NLE 2.5% , S S, SMALL SINGLE SPOTS ,LS, LARGE SINGLE SPOTS,  TS, TWIN SPOTS

* = p < 0.05, ** = p < 0.01 compared to control compared to methotrexate treated group

When the larvae were treated concurrently with 2.5% & 5% NLE, there was a drastic reduction in the spot frequencies compared to the corresponding frequency in the positive control group (methotrexate treatment). The data are summarized in Table 2. Following treatment of larvae with 2.5% NLE and methotrexate, on an average 7.5 spots per wing was observed. When the frequencies of the induced spots were compared with the frequency of the spots in the positive control wings and subjected for statistical evaluation the results was statistically significant.

PICTURE 1:

Similar number of wings of eclosing adults was screened following exposures to a mixture of methotrexate and 5ml of 5% NLE, an average of 8.2 spots per wing was observed at the end of 72 hours. Statistical evaluation of the data involving comparison of frequencies of different types of spots with positive control data (Methotrexate) revealed the outcomes to be significant. However, there was no significant difference with spots between two doses of NLE treated group. (P > 0.05)

DISCUSSION: In this study, both methotrexate and 5% NLE induced small and large single spots containing either mwh or flr3 cells in large frequencies. Although still it is difficult to comprehend the exact mechanism involved in the process of induction of wing spots, methotrexate is a standard agent used to induce genotoxicity ^{8, 9}. Hence, it is concluded that methotrexate is genotoxic in the wing disc cells following chronic larval exposures. Single spots in the adult wings are a result of either gene mutation or gene conversion in their corresponding wild type loci ⁵, whereas, the origin of the single spots with mwh phenotype could be due to segmental aneuploidy ¹⁰, nondisjunction ⁵ and mitotic recombinations ⁷ in the chromosome.

Studies on the area of antimutagenesis and anticarcinogenesis are primarily devoted to detect anti risk factors in human through several in vitroand in vivoshort term tests. 2.5% NLE (low dose) did not produce any significant wing spots spontaneously compared to distilled water treated control group. This suggests safety profile of NLE when used at low dose.

On the contrary, 5 % NLE (high dose) demonstrated genotoxicity in the form of somatic wing spots compared to untreated control. In all treatment groups number of small single and large single spots were more marked compared to twin spots. Both the doses of NLE could prevent development of wing spots to a significant extent when treated along with methotrexate. However this beneficial effect was not significantly enhanced with high dose NLE (p> 0.05). At higher dose rather NLE can favor mutation as observed in this system.

Awasthy et al ¹¹ have found that oral administration of crude ethanol extract of leafs of Neem significantly increased the incidence of structural mitosis disruptive changes in metaphase chromosomes of bone marrow of Swiss albino mice. The same investigators in 2001 had reported the genotoxicity of crude Neem leaf extract in male mice germ cells in the form of structural changes in meiotic chromosomes along with chromosome strand breakage or spindle disturbance and abnormal regulation of genes controlling sperm shape.

In our study, the average spot counts of flies eclosing from larvae treated with 2.5% NLE along with methotrexate were found to be significantly lower than the corresponding spots of methotrexate treatment alone group. The data clearly reveals that NLE has the potential to reduce the mutagenicity induced by other xenobiotic substances like Methotrexate. Drosophila treated with 2.5% NLE exhibited less number of total spots compared to 5% NLE, it may be presumed that NLE might have shielded the genetic material from mutagenic effect of Methotrexate.

The reduction of different spot frequencies in files concurrently treated with NLE led us to believe that NLE might have prevented the conversion of promutagens into mutagens by trapping free oxy-radicals. The gradual increase of spot count (Table 2) from 2.5% to 5% NLE treated group suggests that this beneficial effect of NLE is best appreciated when given concomitantly as a preventive agent. It appears that some of the active principles, if not all, of NLE are capable of inducing genotoxic effects.

We agree with Awasthy et al ⁹ in proposing that some constituents of NLE may induce such genotoxicity at higher dose. In addition to the possible genotoxicity of NLE stated above, there were reports of various other effects of extracts like chemo preventive effect ¹², effect on other xenobiotic detoxification mechanism ¹³, inhibition of aflatoxin production in fungai ¹⁴.

On the contrary, high dose NLE (5%) was found to increase the rate of mutation in comparison to spontaneous ones in our study. Thus, it can be presumed that while some of the principles of Neem can induce mutations, some others have the potency to prevent them.

Again, it can be derived from present study that the mild genotoxic effect of NLE can be prevented by further reducing the concentration of NLE to a safe level to be used as an antimutagenic agent. Active principles of neem leaf have not been separated in this study because of lack of proper facility which is a limitation of our study. The effects of different active principles of Azadirachta (neem), salannin, nimbin, and 6-desacetylnimbin on ecdysone 20-monooxygenase (E-20-M) has previously been examined in three insect species. Homogenates of wandering stage third instar larvae of Drosophila melanogaster, or abdomens from adult female

Aedes aegypti, or fat body or midgut from fifth instar larvae of Manduca. All these evaluated compounds derived from neem seeds, were found to inhibit, the E-20-M activity in three insect species in a dose-dependent fashion.  Anti-carcinogenic potential of Azadirachta indica leaf extract has been studied adopting protocol of benzo(a)pyrene-induced fore-stomach and 7,12-dimethyl benz(a) anthracene (DMBA)-induced skin papilloma genesis, which  revealed its potential to induce only the Phase-II enzyme activity associated mainly with carcinogen detoxification in liver of mice ⁸.

The antineoplastic drug methotrexate is found to be highly genotoxic in the somatic cells of drosophila melanogaster. The genotoxic effects of methotrexate in this test system have previously been reported. Also this Anti neoplastic drug was reported to produce genotoxic effects in other test systems ⁹.

CONCLUSIONS: Based on present experimental condition and result, methotrexate clearly proved genotoxicity in wing mosaic assay of Drosophila melanogaster. Neem leaf extract at 2.5 % dose, exerts a protective effect against the genotoxic action of anti-metabolite anticancer drug methotrexate.

The result of this study further stimulates to investigate the underlying mechanisms involved in the antimutagenic effect of NLE.

ACKNOWLEDGEMENTS: Authors thankfully acknowledge the help and support of Dr Nishigandha Panda of Ravenshaw University for carrying out this project work.

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   How to cite this article:

   Swain TR, Singha MR and Samal R: Neem leaf extract prevents Methotrexate induced genotoxicity in the somatic cells of Drosophila melanogaster. Int J Pharm Sci Res2014; 5(6): 3312-17.doi: 10.13040/IJPSR.0975-8232.5(8).3312-17

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