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Azoxymethane (AOM)-induced Aberrant Crypt Foci (ACF) in rats Abhishek Ajmani 1461060015

Abberant cryptic fociAberrant 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 ACFAccording to this classification, it consists of ACF of non-dysplastic, dysplasticand mixed types.

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 surroundingmucosa.

B. Dysplastic ACFThese 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.

C. Mixed ACFACF with mixed histology contain various proportions of pure adenomatous dysplasia and purehyperplastic features without dysplasiaMechanism 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 0Normal controlAzoxymethane-ACF control

ACF Rats + 5-FU 35 mg/kg for 5 days Blood collectionand sacrifice

Week 10ACF Rats + Gynura procumbens (250 mg/kg)ACF Rats + Gynura procumbens (500 mg/kg)Day 1Week 4Week 6

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 activityMalondialdehyde (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.Thank You