ASSIGNMENT NO.

Genotoxicity Study (In-vitro and In-vivo Micronucleus)

INTRODUCTION

 A micronucleus assay is a test used for toxicological testing of potential genotoxic

compounds.
 It is commonly used because of its simplicity, reliability and reproducibility.
 This assay is one of the most successful assays for genotoxic carcinogens, i.e.,
carcinogens that act by causing genetic damage.
 Micronuclei are morphologically identical to the main nuclei, but are smaller than it
and it is not connected to the main nuclei. It is non-retractile and they can therefore be
readily distinguished from staining particles

HISTORY

 The micronucleus was recognized in the end of the 19 th century when Howell and Jolly
found small inclusions in the blood taken from cats and rats.
 The small inclusions, called Howell-Jolly body, are also observed in the erythrocytes of
peripheral blood from severe anemia patients. These are the first description of the
micronucleus itself.
 In 1970, Boller and Schmid developed a test method to evaluate the frequency of
micronucleated erythrocytes among normal erythrocytes, which lack their own nuclei
during haematopoiesis, using bone marrow and peripheral blood cells of Chinese
Hamster treated with a strong alkylating agent, trenimon. They named this method as
"Mikrokern-Test (micronucleus test)".

MODELS OF MICRONUCLEUS ASSAY

1. In-vitro: OECD guideline No. 487

2. In-vivo: OECD guideline No. 474

In-vitro Micronucleus test

 The in vitro micronucleus assay is a genotoxicity test system used for the detection of
chemicals that induce the formation of small membrane-bound DNA fragments
such as micronuclei in the cytoplasm of interphase cells.

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 The assay thus has the potential to detect the activity of both clastogenic and aneugenic
chemicals.

Principle:

In-vitro cell cultures are exposed to the test substances both with and without
metabolic activation. After exposure to a test substance, and addition of cytochalasin B for
blocking cytokinesis cell cultures are grown for a sufficient period to allow chromosomal
damage to lead to the formation of micronuclei in bi- or multinucleated interphase cells.
Harvested and stained interphase cells are then analysed microscopically for the presence of
micronuclei. Micronuclei are scored in those cells that complete nuclear division following
exposure to the test item.

General Considerations:

a) Cell lines:
 Most commonly used cell lines are CHL/IU, CHO, SHE and V79
 Mouse lymphoma L5178Y cells are also used though they have interactions with the
cytochalasinB
 Cell types with low and stable frequency of micronuclei are mostly used because
frequency of micronuclei formation may influence the studies
b) Media:
 Cells are cultured in Phytohaemaglutinin (PHA) medium at 37°C.
 Cultures should not be contaminated with mycoplasma.
c) Metabolic activation:
 Cells should be treated with the test substance both in the presence and absence of an
appropriate metabolic activation system.
 1-10% v/v conc. of post mitochondrial fraction (S9) is the most commonly used
metabolic activating system prepared from liver of rodents.
 Initially it is treated with Aroclor1254 or in combination with phenobarbitone and
naphthoflavone.
d) Use of CyclochalasinB:
 CyclochalasinB helps in formation of binucleated cells by inhibiting the formation of
micro filament and cytokinesis.

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  It is used in the concentration of 3-6µg/ml to get 50% binucleated cells; along with
the test substance and it should be given at least 6hours before the first mitosis.
e) Controls:
 Positive controls: used to identify clastogens, aneugens and effectiveness of
exogenous metabolic activation system.
Examples: MitomycinC; Cytosine arabinoside
 Negative controls: consists of solvent alone doesn’t requires any metabolic activators

Procedure:

Step 1:

 Incubate cell lines and appropriate culture media according to test at 37°C.
 Cells are taken from the stock to prepare suspension and harvested to obtain the
monolayer of cells.
 Lithium Heparin is added in the blood sample resulting in separation of lymphocytes.
Step 2:
 Phytohaemaglutinin (induces cell division) is added to it and after 44 to 48 hrs this
culture is added to cell lines along with test chemical.
Step 3:
 Initial experiment is performed in the absence of S9 fraction. Cells should be exposed
to test compound and after 20 hrs remove the treatment medium by washing. Then
add the fresh media (CytochalasinB) and harvest for 1.5 to 2 cell cycle length.
 Then in presence of S9 fraction - Aroclor 1254 (enzyme for metabolic activation)
Cells should be exposed to test compound for 3-6hrs. Remove the S9 and treatment
medium by washing. Add fresh media (CytochalasinB) and harvest for 1.5- 2 cell
cycle length.

Analysis:

 All slides, including those of positive and negative controls, should be independently
coded before the microscopic analysis.
 In cytochalasin B-arrested cultures, micronucleus frequencies should be analysed in
2000 binucleated cells per concentration (1000 binucleated cells per culture, two cultures
per concentration).

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 With cytochalasin B a parallel scoring of micronuclei in mononucleated cells is optional
(1000 cells/culture).
 Evaluation of micronuclei formation can be known by reduction of cell proliferation.
 Cytokinesis block proliferation index: CBPI indicates the number of cell cycles per
cell during the period of exposure to cytochalasinB.

No. of Mononucleate cells + 2 x No. of binucleate cells + 3 x No. of multinucleate

CBPI =
cells Total no. of cells
CBPI = 1; indicates 100% cytotoxicity

Diag: In-vitro Micronucleus Assay

In-vivo Micronucleus test

 The in vivo micronucleus assay is a genotoxicity test system used for the detection of
damage induced by test substance to the chromosome or mitotic apparatus of
erythroblast.

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 They are done using – Peripheral Blood and Bone Marrow

Principle:

Rodents are treated with the test agent by appropriate route, bone marrow extracted
at appropriate times after treatment, smear slides are prepared either with whole bone
marrow or cellulose column-fractionated cell suspension, stained, coded, and analysed for
the toxicity (PCE to NCE ratio) and micro nucleated cell frequency.

Procedure:

5 animals should be treated with the test chemical.

Dosing done at 3 levels (low, medium, high doses)

After 24-48 hrs animals are sacrificed by carbon dioxide euthanasia

cut the ends of bone; flush the bone marrow with isotonic solution

Centrifuge the marrow suspension

Preparation of smear of sample collected

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 Diag: In-vivo Micronucleus Assay in Mouse Bone Marrow

Staining: Slides are stained using Giemsa or Fluorescent DNA specific dyes.
Example of fluorescent stains - Acridine orange
Hoechst + pyronin Y
May - Grunwald
May - Grunwald + Giemsa
Analysis:

 Flow Cytometry
 Laser scanning cytometry
 Image analysis

Test report:

 It should include the information about test substance, solvent, cells, test condition and
result.

Application:

 Distinguish aneugens from clastogens

 Perform long-term toxicological studies
 Examining the radioactive properties of H2 receptor antagonists
 Evaluation of radio sensitivity of human glioma cells

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