Azoxymethane (AOM)-induced Aberrant

Crypt Foci (ACF) in rats

Abhishek Ajmani
1461060015

Abberant cryptic foci

Aberrant crypt foci (ACF) are clusters of abnormal tube-like glands in the lining of the colon
and rectum. Aberrant crypt foci form before colorectal polyps and are one of the earliest changes
seen in the colon that may lead to cancer. ACF are, as opposed to normal epithelial cells,
apoptosis resistant. When looking for aberrant crypt foci with microscopy, methylene blue is
used as a staining agent. The resulting figure is fairly easy to detect under the microscope at low
magnification (x40).
1. Pit pattern and the Kudo classification.
Kudo et al. developed the current classification system for pit pattern
description.

2. Histological Classification of ACF

According to this classification, it consists of ACF of
dysplastic
and mixed types.

non-dysplastic,

A. ACF with Normal Mucosa (Non-Dysplastic)

1. Non-Hyperplastic ACF. This subtype of ACF possess enlarged crypts at least
1.5 times larger than normal, but lack significant abnormalities in the
epithelial cells lining the crypts.
2. Hyperplastic ACF.
This class embodies the overall pathology of the hyperplastic polyp. They
maintain larger crypts with an elongated appearance, having both side and
apical branching. The crypt profile is serrated and may exhibit cellular tufting
slightly above the surrounding
mucosa.
B. Dysplastic ACF
These ACF demonstrate significant variability from the normal crypt pit
pattern. Goblet cells are decreased and mucin production is depleted. The
epithelial cells are enlarged, stretched and show stratification with
depolarized nuclei.

Mechanism of Azoxymethane induced Aberrant

Cryptic Foci
Azoxymethane is a colon carcinogen and used commonly to induce aberrant
crypt foci in the rat colon.
After administration, AOM is metabolised into methylazoxymethanol by
CYP2E1, which causes DNA mutations. Mutation of K-ras activates this
pathway and its downstream PI3K/Akt pathway and MAPK pathway. Mutation
of -catenin also prevents it from being degraded by GSK-3 and accumulation
of -catenin leads to cell proliferation. TGF-, a pro-apoptotic protein, is
inhibited. All of these changes form the basis of AOM carcinogenesis.

Experimental
Protocols
Injection of Azoxymethane solution in
normal saline (15 mg/kg)
Day 0

Day 1

Week 4

Week 6

Blood
collection
and sacrifice
Week 10

Normal control
Azoxymethane-ACF control
ACF Rats + 5-FU 35 mg/kg
for 5 days
ACF Rats + Gynura procumbens
(250 mg/kg)
ACF Rats + Gynura procumbens
(500 mg/kg)

Parameters checked

Gross evaluation of clone mucosa

The number of ACF per colon and the number of aberrant crypts in each focus were
determined with the aid of light microscope. Scoring was based on number of ACF
identified in colon.

Histopathological examination
Tumor histology was looked upon under this step.

Immunohistochemical staining
Detection of proliferating cell nuclear antigen (PCNA), and Bcl-2 protein was done.

Antioxidant activity
Malondialdehyde (MDA) for lipid peroxidation levels. Glutathione-S-transfer(GST) and
superoxide dismutase (SOD) were also assessed in colon tissue homogenate

Biochemical analysis
Determination of glucose, albumin, alanine aminotransferase (ALT), alkaline phosphatase
(ALP), aspartate aminotransferase (AST), creatinine and urea levels was done.

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