IN VITRO CHROMOSOMAL ABERRATION TESTS.pptx

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1. IN VITRO CHROMOSOMAL ABERRATION TESTS Presented By: Dinam Gyatso Lepcha 20HMPL03 15-07-2021 1

2. INTRODUCTION The adverse health effects caused by the Geno toxins in vivo are of concern. The effects of exposure to relatively low concentrations of Geno toxic substances may not be immediately apparent, this means that there are no warning signs so that exposure can be avoided The short term in vitro mammalian cell chromosome aberrations test is used to assess the potential Genotoxic hazard of test substances Abnormality in Chromosome can be of following Types Deletion Duplication Inversion translocation 15-07-2021 2

3. METHODS White Blood Cell Culture Dosing Formulation: Cyclophosphamide(treat cancer), anhydrous dimethyl sulphoxide as a vehicle, S9 fraction Sampling, Slide preparation and Staining: KCl in water(0.56%) Methanol : Glacial acetic Acid(3:1 v/v) Microscopic Slides Conduct of Test: High quality metaphase preparation is preferred, use of a trained and experienced Observer 15-07-2021 3

4. METHODS Study Design: Culture initiation Add WBC sample to culture medium including PHA and incubate(37±1°C) for 48 Hrs, cells mostly in G0 of cell cycle Start of Exposure: 0hrs Add dosing formulations dimethyl sulphoxide & incubate for 3Hrs, cells at all stages of cell cycle End of Exposure: 3 hrs Remove dosing Formulation. Incubate for 15Hrs. Cells should have undergone S phase since 0 Hr 15-07-2021 4

5. Study Design Metaphase Arrest: 18 hrs Add Colcemid. Incubate for 2 hrs. Cells should be at first M phase since 0 hrs Harvest: 20 hrs Hypotonic to swell cells, fixative cell suspension on slides, stain and cover slip 15-07-2021 5

6. White Blood Culture Blood is to be taken by addition of Heparin tubes to avoid clotting for Blood collection The sample is been then incubated at 37°C±1°C throughout with gentle mixing of the cultures. A humidified atmosphere of 5% CO2 in air may be introduced at the culture Lymphocytes are grown as suspension cultures in sterile, disposable centrifuge tubes A healthy donor who do not smoke and willing to donate blood on a regular basis is selected , absence of viral diseases such as hepatitis and HIV are important both for protection of operators and for the success of the assay The Lymphocytes are tested for a good response to PHA giving an acceptable mitotic index(MI) and AGT. 15-07-2021 6

7. WHITE BLOOD CULTURE The Lymphocytes should have a normal karyotype and exhibit levels of chromosome aberrations that falls within a normal range with references to the historical negative control database Prior to vein puncture, it should be ascertained that the potential donor has not knowingly been exposed to ionizing radiation, hazardous chemicals or has suffered from viral infections in the 2 weeks period before giving blood Heparinised blood, 0.4 ml is added to the warmed supplemented RPMI1640 medium so that the final volume following addition of S-9 mix/KCl and the dosing formulation is 10ml These suspension cultures are incubated for approximately 48 hrs Even small but prolonged reductions in incubator temperature can affect the AGT and this may not be obvious as it is likely to be measured peroidically 15-07-2021 7

8. PREPARATION, ADDITION & REMOVAL OF DOSING FORMULATION Add approximately 10 mg CPA and add sufficient amount of DMSO to prepare the stock solution that is 100x strong Prepare a alternative stock solution that is 10x strong and dissolve in water for injection then sterilize to avoid microbial contamination The stock solution is diluted to provide six concentration of CPA , in the presence of S-9 mix it The S-9 fraction is thawed just before required and the S-9 mixture is prepared The CPA dosing formulation(0.1 ml) is added to each culture to achieve the required final concentration of CPA in a total of 10ml, culture exposed to CPA in absence of S-9 receives 0.5 ml of KCl Incubation is continued for 3 hrs 15-07-2021 8

9. PREPARATION, ADDITION & REMOVAL OF DOSING FORMULATION Culture tubes are centrifuged at approximately 300x g for 10 mins, the supernatant is carefully removed and cells resuspended in 10 ml RPMI medium without supplements, prewarmed to 37°C. This process is repeated so that there have been two changes of medium, after the third centrifugation, the cells are resuspended in 10ml complete RPMI medium, prewarmed at 37°C and incubation is continued 15-07-2021 9

10. SAMPLING, SLIDE PREPARATION AND STAINING Colcemid are added 2-3 hrs before sampling to arrest dividing cells at metaphase At the sampling time, cultures are centrifuged at 300x g for 10 mins, the supernatant carefully removed and cells resuspended in 4ml warmed KCl at 37°C for 15 mins to allow cells to grow Cells are fixed by gently mixing suspension of cells with 6 ml fixative . It is very important to avoid clumping of the lymphocytes, the tubes containing cells suspension in fixative and hypotonic solution are centrifuged at approximately 300 x g for 10mins The supernatant is removed and discarded into a labelled container for disposal The cells are resuspended by slow addition of fresh, cold fixative, mixing using a vortex mixer to avoid cell clumping The tubes containing the suspension are centrifuged, this process is repeated until the cells pallets no longer contain traces of red blood cells 15-07-2021 10

11. SAMPLING, SLIDE PREPARATION AND STAINING Lymphocytes are usually kept at 1-10°C at least overnight to ensure adequate fixation Cells suspension are centrifuged and resuspended in a minimal amount of freshly prepared fixative , several drops of cell suspension are placed on cold microscope After the slides have dried the cells are stained for 5 mins in filtered 4% Giemsa in pH 6.8 Buffer The slides are rinsed, dried and mounted with coverslips 15-07-2021 11

12. STAINING Stain the slides in a solution of Hoechst 33258 for 25 mins protected from sunlight Rinse slides thoroughly in MacIlvaine's buffer pH 8.0 twice, the second time being in the buffer prewarmed to 40°C , place the slides flat in a tray and add sufficient prewarmed buffer to cover the slides causing the chromatid arm to swell Expose the slides to the UV light at approximately 366 nm for 25-40 mins Remove the slides and rinse thoroughly in PBS at pH 6.8 Stain for 10 mins in filtered 4% Giemsa stain in PBS at pH 6.8, rinse the slides thoroughly, first in PBS at pH6.8 and then in water, shake off excess moisture, air dry and mount with coverslip 15-07-2021 12

13. ANALYSIS OF CELLS IN FIRST, SECOND AND THIRD DIVISION The AGT is calculated as follows: proliferative index(PI)= %cells M1 2(%cellsM2) 3(%cells M3) 100 Average Generation time= Hours in BrdU PI The AGT should be in the region of 12-14hrs for human lymphocytes, longer than 16hrs indicates that culture conditions are suboptimal and the source of the problem should be identified and rectified 15-07-2021 13

14. ASSESSMENT OF MITOTIC INHIBITION Mitotic index = number of cells in mitosis X 100 Total no of cells observed Mitotic inhibition%=[1-{mean Mlt/Mean Mlc}] X 100 Where Mlt= MI in cell exposed to test substance Mlc= MI in solvent control cells 15-07-2021 14

15. ANALYSIS AND INTERPRETATION OF RESULTS After completion of analysis and decoding, the proportions of aberrant cells per culture (often expressed as a percentage, since 100 cells are routinely analyzed) are tabulated as: 1.Cells with structural aberrations including gaps 2.Cells with structural aberrations excluding gaps (this information is used to draw a conclusion as to the clastogenic potential of a test substance as gaps may occur by non-genotoxic modes of action) 3.Polyploid, endoreduplicated, or hyper diploid cells (An increase in polyploidy may indicate that a chemical has the potential to induce numerical aberrations, when use of the in vitro micronucleus test should be considered.) 15-07-2021 15

16. The acceptance and evaluation criteria to determine the outcome of the test should be specified in advance. An acceptable test, assuming good cell growth, would be likely to have: 1.Homogeneity between replicate cultures 2.The proportion of cells with structural aberrations (excluding gaps) in vehicle control cultures should fall within the historical negative control range. In the absence of an historical negative control range, an approximate guide would be that the frequency of cells with aberrations should be <5%. This low frequency means that there may not be any aberrations seen in 50 or even 100 cells. 3.At least 160 cells out of an intended 200 should be suitable for analysis at each concentration of CPA, unless 10 or more cells per slide showing structural aberrations other than gaps only have been observed during analysis . 15-07-2021 16

17. The following evaluation criteria may be used for ascribing the potential of a test article to induce chromosome aberrations in a valid assay. A proportion of cells with structural aberrations at one or more concentrations exceeds the concurrent vehicle control values and the historical negative control (normal) range (if available) in both replicate cultures. A statistically significant increase in the proportion of cells with structural aberrations (excluding gaps) is observed (p≤0.05). Evidence of a concentration-related trend in the proportion of cells with structural aberrations (excluding gaps) will be judged to support the conclusion Results that only partially satisfy the above criteria need to be dealt with on a case-by-case basis .. 15-07-2021 17

18. A test article that satisfies none of the above criteria may be considered not to have the potential to induce chromosome aberrations. The criteria to establish a negative response are more stringent, and two independent experiments are required currently. The distinction between an indirect-acting and a direct-acting genotoxin is important in predicting in vivo risk arising from a positive in vitro assay 15-07-2021 18

19. THANK YOU 15-07-2021 19