Introduction

Many synthetic  and natural compounds have been reported to act as mutagens and/or carcinogens (Ames, 1983; Vargas et al., 1990).The hazards associated with exposure to carcinogenic and mutagenic    chemicals are of  prime concern as they are related to public health and as   well as being of research interest.

Identification of these relies on    experimental  in-vivo and in-vitro  genotoxic  methods including Ames test(bacterial mutation assay) and cultured mammalian cell systems such as human Peripheral Blood Lymphocytes (PBL) or Chinese Hamster Ovary (CHO) cells.

Capacity to damage the genome is an indicator of potential mutagenicity and mutagenicity is considered as key event in carcinogenicity. Genetic damage at the chromosomal level causes an alteration in either chromosome structure or number, and such alterations can be measured as Chromosomal aberration or Micronucleus frequency.

Chromosomal aberration in-vitro assay  is  to induce chromosomal damage as well as  gene  mutations  and the micronucleus (MN) test is a multi-target genotoxic endpoint, assessing not only clastogenic and aneugenic events but also some epigenetic effects, and  it is predictive for cancer.

For this purpose, a Chinese Hamster Ovary cell line (CHO), commonly used  in genetic toxicology studies , was selected as an in vitro biological model to assess  the in- vitro  studies.

Materials and Methods

CHO Cell line

Preparation of S9 mix : Wistar rats, Sodium Phenobarbital (30mg and 60mg/body weight)

Solutions:

Mitomycin C stock solution (Sigma Aldrich)     : 0.05mg in 100mlof sterile water

Mitomycin C working solution (Sigma Aldrich)  : 10µl of stock solution to 50µl

Cyclophosphamide stock solution(Sigma Aldrich) : 0.05mg in 100ml of sterile water

Cyclophosphamide working solution : 10µl of stock solution to 50µl .

NADPH solution (4mM)                                : 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.

1M Di potassium Hydrogen Phosphate    : 175 g in 1000ml.

1M Potassium Di hydrogen Phosphate : 136g in 1000ml.

0.2M Phosphate Buffer solution( pH 7.4) : 80.2mlof K2HPO4 and 19.8 ml

of 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.

Normal Saline :27g in 3000ml

Geisma stain

Trypsene –Versene solution(Invitrogen)

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 used and about 1.5 x 10⁶  cells were introduced into the glass bottle of  25cm²  containing culture medium , Control, Mitomycin, Cyclophosphamide and the test compound. 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 was 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 3 min. at room temperature ,then were centrifuged for 5 min. 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. 5 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.

At least 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 add few drops of mounting medium.

Then the cover slip was placed without air bubbles and left overnight  until  completely  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. 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  5 min. and the supernatant was discarded.

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

Then the contents 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 ^o C, 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.

At least 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 substance did not produce any gaps, fragments ,deletions and breakages and no formation of   micronuclei. It is likely that a chemical that results in a mutagenic  response in testing will possess the potential to manifest this mutagenic activity as adverse health effect like cancer and  so test substance  is neither  mutagenic nor carcinogenic.           

Chromosomal aberrations

Micronuclei   formation

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 good anti-inflammatory activity and  after the study it is found that it has no Mutagenicity and Carcinogenicity .Further activity study can be done on this Compound.

References

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