G. E. Suhasini, M. Nirmala, Rashmi Shiva Kumar and Archana Giri

JNTU, Hyderabad, India.

Abstract

The newly synthesised 2-(2-2 benzylidene hydrazinyl)-5,5 diphenyl-1,5dihydro -4-imidazol-4one was found to have anthelmintic, ,antibacterial and antifungal activity . Since the Compound has the potential for further development .Genotoxicity of the new Compound was studied for visible changes to chromosome structure and morphology .They have played a very important part as indicators of genetic damage in both clinical and cancer studies. The Chromosome Aberration Assay Invitro is a useful and sensitive test for detection of Genotoxins .There are four common types of structural Chromosome Aberrations are 1.Deletions   2.Duplications     3.Inversions     4.Translocations. Micronuclei are indicators for fixed genomic damage in cells .The Synthesised compound did not show any Chromosomal Aberrations or formation Micronuclei

Keywords

Genotoxicity; Mutagenecity; Chromosomal aberrations; Micro nuclei; Carcinogenecity

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Copy the following to cite this article: Suhasini G. E, Nirmala M, Kumar R. S, Giri A. Genotoxicity of Newly Synthesised Imidazole-2(2-2 benzylidene hydrazinyl)-5,5 diphenyl-1,5dihydro -4-imidazol-4one(4-chloro derivative). Biomed Pharmacol J 2012;5(2)

Copy the following to cite this URL: Suhasini G. E, Nirmala M, Kumar R. S, Giri A. Genotoxicity of Newly Synthesised Imidazole-2(2-2 benzylidene hydrazinyl)-5,5 diphenyl-1,5dihydro -4-imidazol-4one(4-chloro derivative). Biomed Pharmacol J 2012;5(2). Available from: http://biomedpharmajournal.org/?p=2495

Introduction

Mutations in DNA may lead to cancer that disrupts  these orderly process by disrupting the programming regulating process .in cultured cells certain phenotypic alterations that are characteristic of tumerogenic cells will be inducted and the cell transforms and acquire the characteristics of malignant cells.

Most  aberration inducing agents can produce lesions into chromatin at all stages of the cell cycle. (Cancer research 34, 2266-2273, sept1974)

Chromosome aberration assay in-vitro is a useful and sensitive test for detection of Geno toxins. The accumulation of genetic changes may lead to genetic instability which may result in cancer it has been demonstrated that Micronuclei frequencies besides chromosome aberration frequencies are a cytogenetic end

point providing strong evidence for the association between the extent of chromosomal changes and cancer risk.

Materials and Methods

Preparation of s9 mix

Mitomycin c stock solution                : 0.05mg in 100mlof sterile water

Mitomycin c working solution           : 10µl of stock solution to 50µl

Cyclophosphamide stock solution     : 0.05mg in 100ml of sterile water.

Cyclophosphamide working solution: 10µl of stock solution to 50ml using

Sterile water.

NADPH solution                               : 1.5mg in 5ml distilled water

0.4M Magnesium Chloride and 0.65M Potassium Chloride solution

Glucose -6-Phosphate solution                   : 700mg in 5ml distilled water.

9   0.1M Dipotassium Hydrogen Phosphate    : 175 g in 1000ml.

10.1M Potassium Dihydrogen Phosphate: 136g in 1000ml.

11.0.2M Phosphate Buffer solution: pH 7.4(80.2mlof K2HPO4 and KH2PO4 were mixed and the PH was adjusted to 7.4 and was made to 500ml.

0.15 M Potassium Chloride solution:13.5g in 1.2L of water.

12.Normal Saline :27g in 3000ml

13.Geisma stain

14.Trypsene –Versene solution

Preparation of culture media

Powdered Dulbecco’s modified medium with 10% foetal Calf Serum.

1X McCoy’s 5a medium with Hepes buffer plus 15% Foetal Calf serum

Methodology

procedure for cell culture

The CHO cells have been used to study the chromosomal aberration   induced by the new compound in comparison with the Mitomycin and Cyclophosphamide.The CHO cells are fibroblastic in nature with 12-14 hour cell cycle time and at Mitosis the cells   roundup and should be easy to dislodge by

shaking.The ‘Mitotic Shake Off Method’ was followed to remove the culture cells .Monolayer cultures of CHO cells approximately 60-80% confluent were for the study .in   the glass bottle of  25cm2

1,5×106 cells Produced, Control, Mitomycin, Cyclophosphamide and the Imidazole derivative. All  the  cultures were prepared in duplicate. The Monolayer were rinsed with about 10ml of pre-

warmed Phosphate Buffer Saline (PBS) and the PBS was discarded.The cells were rinsed with 3-5ml of pre-warmed Trypsin –Versene solution and excess removed immediately. The cells were observed for signs of rounding up and detaching from the glass bottle (5-10min) then resuspended the cells in 10ml of McCoy’s

5a medium with 15% Fetal Calf Serum.

Procedure for chromosomal aberration studies       

After the cells were exposed to the test materials the steps followed were

Colchicine solution of 0.2ml of a 10µg/ml was added two hours before harvesting.

At the end of the incubation period

(1.5CELL CYCLES),each glass bottle was shook gently to dislodge mitotic cells and transfer the cell suspension to labeled 15ml centrifuge tube and centrifuged at low speed (600rpm) for 5 min.

The supernatant was removed and the cells were gently resuspended in the residual medium and add freshly prepared  5ml of 0.075M Potassium Chloride.

The contents were left for three min at room temperature ,then were centrifuged for 5minutes and the supernatant was discarded.

5ml of freshly prepared Methanol : Acetic acid fixative (3:1) was added drop wise manner while shaking the tube gently to resuspend the cells.

The contents were centrifuged at a higher speed (1200 rpm) for 5 min and the supernatant was discarded.

The last two steps were repeated once again.Five ml of fresh fixative was added and the tubes allowed to stand for 30 minutes.

Then the contents were centrifuged ,supernatant was removed  and the cell button was re-suspended in 3-4 drops of fixative.

Using a Pasteur Pipette 3-4 drops of the suspension was transferred onto a microscope slide, previously soaked in alcohol  ,polished with lens tissue and cooled to 4°c from a few inches  above the slide and were checked for the degree of spreading of the metaphases.

The slides were dried and stained with Geimsa.

Atleast four slides were prepared from each culture.

The slides were rinsed off the stain with phosphate buffer and were shaken thoroughly to remove buffer and then air dried.

The slides were dipped in Sulphur free Xylene for a few seconds, and few drops of mounting medium.

Then the cover slip was placed without air bubbles and left overnight  Until mounting sets.

The slides were observed under 100X magnification with oil immersion using Carl  Zeiss Axioplan microscope.

The plates were observed for any kind of aberrations in the chromosomes.

Procedure for micronucleus test

After the cells are exposed to test materials using the previous procedure (used  in chromosomal aberration)

Cytochalasin-B solution of 0.2ml of a 24µg/ml was added sixhrs before harvesting.

At the end of the incubation period ,each glass bottle was  shook gently to dislodge mitotic cells and transfer the cell  suspension to labelled 15ml centrifuge tube and centrifuged at  low speed (600rpm) for 5 min.

The supernatant was discarded. 5ml of freshly prepared Methanol: Acetic acid fixative ((3:1) was

added drop wise manner while shaking the tube gently to  resuspend the cells.

The contents were centrifuged at a higher speed (1200rpm) for 5 min and the supernatant was discarded.

Five ml of freshly prepared Methanol : Acetic acid fixative(3:1) was added drop-wise manner while shaking the tube gently to re suspend the cells.

The contents were centrifuged at a higher speed(1200 rpm) for  five minutes and the supernatant was discarded.

Five ml of fresh fixative was added and the tubes were  allowed to stand for 30 minutes.

Then the were centrifuged, supernatant was removed and the cell button was resuspended in 3-4 drops of fixative.

Using a Pasteur Pipette 3 to 4 drops of the suspension was  transferred onto a microscope slide, previously soaked in alcohol, polished with lens tissue and cooled to 4  degree centigrade, from  a few inches above the slide and to check the degree of spreading of the cells.

The slides were Air dried and stained with Giemsa.

Atleast four slides were prepared for each culture. Each slide was rinsed with 5ml geimsa in horizontal staining  over a sink for 5 min.

The slides were rinsed off with Phosphate buffer, and were shaken thoroughly to remove buffer and then air dried.

The intensity of staining was checked  before mounting.The slides were dipped in sulphur- free xylene for a few seconds , add then in a few drops of mounting medium(eg DPX).

The cover slip was placed after expelling the air bubbles.

The slides were left for over night until the   mounting medium completely sets.

The slides were observed under 100X magnification with oil   immersion using Carl Zeiss Axioplan  microscope to observe the   presence of Micronuclei.

Results  and discussion 

Positive control Mitomycin C without s9 fraction, showed aberrations  like gaps , fragments, deletions and breakage showing that it is a mutagen.

Cyclophosphamide is mutagenic in the presence of s9   fraction. It produced gaps , fragments, deletions and breakages showing that it  is a mutagen.

Test material   did not produce any gaps, fragments ,deletions and breakages and no formation of

micronuclei, showing that it is not mutagenic and not carcinogenic.

Chromosomal aberrations                   

controls	Breaks in chromosomes	fragments	Trinucleate chromatids	deletions
Mitomycin c	present	present	present	present
Cyclophosphamide with s9	Present	Present	Present	present
Test sample	absent	Absent	Absent	absent

 

Micronuclei   formation 

Controls	micronuclei	Inference
Mitomycin c	present	mutagenic
cyclophosphamide	present	mutagenic
Test sample	absent	Non-mutagenic

 

The above results show that

Chromosomal Aberrations are present in the form of breaks in Chromosomes, Fragments , Trinucleate Chromatids and Deletions in the slides of Mitomycin C and  Cyclophosphamide.

But such Aberrations are not present in the slides of test sample in concentration of 100mg/ml.

Mirco Nuclei are seen in the slides of Mitomycin C and Cyclophosphamide .but MicroNuclei  are not present in the slides of test sample in concentration of 100mg/ml.

Conclusion

since the compound has Anthelmintic, Antibacterial, Antifungal activity after the study it is found that it has no Mutagenicity and Carcinogenicity .Further activity study can be done on this Compound.

Reference

1. Mayer ,a.l (1983).Invitro transformation assays for chemical carcinogens. mutation research 115,323-338.
2. Rebert Combes, Michael Balls , Rodger Curren, Michael Fishbach, Norbert Fuseing , David Kirkland, Claude Lasne, Joseph Landolph, Robert Leboeuf, Hans Arquqrdt, et al;  Cell transformation
3. Assays as Predictors of Human Carcinogenicity .thr report and recommendations of ECVAM workshop391-3 ,ATLA 27,745-767,1999.
4. Ayako Sakai, BALB/c3T3 cell tranfsformation assays for the assessment of chemical carcinogenecity, National Institute of Health Sciences, Hatano Research Institute, Food and Drug Safety  Center,729-5 ochiai , hadano kanagawa , 257-8523, Japan, AATEX 14, Special Issue , 36-373 proc 6^th world congress on Alternatives and Animal Use in the Life Sciences, August 21-25, 2007, Tokyo, Japan.
5. Albrecht Poth , Andreas hepperheimer and Susanne bohnenberger rcc cytotest cells research gmbh in den leppsteinweisen 19, d- 64380 rossodorf, Germany, AATEX 14, Special Issue , 519-521 proc 6^th world congress on Alternatives and Animal Use in the Life Sciences,August 21-25,2007, Tokyo, Japan
6. Fellows and o’donovan (2007) assessed the genotoxicity in mammalian cell  in Vitro in the various test systems, shown that the trypan blue exclusion, MI and binucleate incidence all grossly underestimate cytotoxicity in comparison with cell growth will enhance the acceptable level of toxicity.
7. Takeiri etal (2006) demonstrates that the newly established GDLI cell line is a useful tool to detect and analyse various mutations including large deletions in mammalian cells.
8. Steiblen et al.,(2005) evaluated mouse splenocyte to be an acceptable alternative to that of human lymphocytes during the evaluation of  aneugens.
9. Li et al.,(2007) in vitro reporter gene assays could provide an ideal system for quick assessment or screening of agents with genotoxic potential.
10. Mutagenicity : ejchart et al.,(1996) investigated in salmonella typhimurium TA98 and TA102 and suggested  that the involvement of active oxygen species in bleomycin induced mutagenesis and an absence of their participation in DOX induced muatagenesis.
11. Sayer et al.,(2006) brevetoxins(pbTxs) are potent  inducers of CHO-K1-BH4 chromosome damage.
12. Mutat res .1993 oct., 297(3): 281-92
13. Benigni R ,Andreoli C lab of  Com Toxicology and Ecotoxicology, Rome, Italy : Rodent carcinogenicity and toxicity, in vitro mutagenicity, and their physical chemical determinants
14. Andiron et al., (1995) activity of anthracycline, moflomycin was  studied  by  the  ames  test in four strain of S.typhimurium( TA97a, TA98, TA100 and TA102) and compared to the DOX, and observed , the low mutagenicity of moflomycin to have potential advantage over Dox.
15. Doppalapudi et al.,(2007) evaluated eleven candidate chemoprotective agents with the potential for inhibiting carcinogenesis in humans at increased risk of cancer and proved that none of the agents induced micronuclei in mouse bone marrow erythrocytes.
16. Estimation of carcinogenicity : in the preclinical safety evaluation conducted by Buckwold etal., (2006) on human omega-interferon (IFN-omega) induce reduction in the levels of hepatitis c virus RNA
17. Redbook 2000: iv. c. 1. b in vitro mammalian chromosomal aberration test  Brusick.,D.J, Principles of Genetic Toxicology,2^nd  edition, plenum, New York.