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Revista do Colégio Brasileiro de Cirurgiões

Print version ISSN 0100-6991

Rev. Col. Bras. Cir. vol.40 no.2 Rio de Janeiro Mar./Apr. 2013 



Hepatic repercussions of azoxymethane-induced colorectal carcinogenesis



Idália Maria Brasil Burlamaqui, UFC-CEI; Conceição Aparecida DornelasII; Paulo Roberto Carvalho AlmeidaII; Francisco Vagnaldo Fechine JamacaruIII; Daniel Magalhães Coutinho MotaIV; Francisco José Cabral MesquitaIV; Lara Albuquerque de BritoV; Lara Burlamaqui VerasVI; Lusmar Veras RodriguesVII

IPhD Graduate, Post-Graduation Program, UFCE
IIAssociate Professor, Department of Pathology and Forensic Medicine, UFCE
IIIVisiting Professor, Department of Pharmacology, UFCE
IVResident, Faculty of Medicine, UFCE
VMedical School Graduate, Faculty of Medicine, UFCE
VIMedical School Graduate, Faculty of Medicine of Juazeiro, CE
VIIAssistant Professor, Department of Surgery, UFCE

Address correspondence to




OBJECTIVE: To evaluate the hepatic effects of colonic carcinogenesis induced by azoxymethane at different doses and times of exposure in rats.
METHODS: Forty-four Wistar rats were divided into four groups. The animals were eight weeks at the beginning of the experiment. group 1 received 1.0ml of saline intraperitoneally once a week for two weeks. Group 2 received 15 mg/kg of azoxymethane intraperitoneally once a week for two weeks. These animals were killed at the 15th week of the experiment. The animals of group 3 received saline intraperitoneally once a week for two weeks. Group 4 animals received 20mg/kg of azoxymethane intraperitoneally once a week for two weeks. These animals were killed at the 26th week of the experiment. The fragments of liver tissue were stained with hematoxylin and eosin and evaluated microscopically.
RESULTS: Groups 1 and 2 differed significantly in relation to steatosis, no difference having been found between group 3 and group 4. However, in group 4 we observed pre-neoplastic lesions (foci of altered, clear, vacuolated, basophilic, amphophilic tigroid, oncocytic, small or acidophilus cells, spongiosis and peliosis) and neoplastic lesions (adenomas and colangiomas) containing atypical hepatocytes in between, not identified in group 3.
CONCLUSION: In the model of colorectal carcinogenesis, preneoplastic and neoplastic hepatic lesions appear and evolve in proportion to the time of exposure and dose of azoxymethane.

Key words: Colonic neoplasms. Colorrectal neoplasms. Tumor makers, biological. Azoxymethane. Fatty liver.




Colorectal cancer (CRC) is among the most frequent types of malignancies. It is the third most common cancer in the world in both genders, the fourth most common type in men and third in women 1. Is the fourth most commonly diagnosed cancer in the United States, with 178,000 new cases per year, and a mortality of about 47 per 100,000 inhabitants 2.

The cause of CRC is the result of a complex interaction of external variables, such as environmental and dietary agents, and internal factors, of somatic or hereditary nature 3. When detected at early stages, it has a greater chance of cure and survival 4.

Considering the importance of neoplastic disease and the need to understand the pathophysiology of the emergence of early lesions, several colorectal carcinogenesis models are udes 5,6. The model Bird promotes carcinogenesis by 1,2 dimethylhydrazine (DMH) or azoxymethane (AOM) and assesses the formation of aberrant crypts in colonic mucosa of rodents, being widely used in experimental research. The lesions induced by AOM - K-ras, APC, and p53 mutations - are similar to CRC in humans and can also be found in other organs such as liver, small intestine and peritoneum 7-9.

There are few studies on liver injury caused by AOM 10,11 during colorectal carcinogenesis induced according to the Bird model 12-16.

The aim of this study is to evaluate the relationship between dose and exposure time of azoxymethane on the liver repercussions during colonic carcinogenesis in rats.



The survey was conducted after approval by the Ethics Committee on Animal Research of the Federal University of Ceará (CEPA / UFC), protocol number 09 on February, 3rd 2009, and according to the International Standards for Biomedical Research on Animals.

We used 44 rats (Rattus norvegicus albinus, mammalia Rodentia, Muridae) of Wistar strain, obtained from the Central Animal Facility of the Federal University of Ceará, eight-weeks old and weighing 180g. The animals were kept in individual cages with polypropylene lid with zinc galvanized wire, lined with wood shavings, in the laboratory of Experimental Surgery, Department of Surgery, Faculty of Medicine, Federal University of Ceará. They remained housed in appropriate conditions, relative humidity around 50%, and average temperature of 25º C, light-dark cycle of 12/12 hours and ventilation. They received water and food ad libitum.

The animals were identified, weighed and randomly divided into four groups. After weaning, they were fed standard Biobase Biotec Rats and Mice chow composed of 59% carbohydrate (CHO), 29% protein (NSP) and 12% lipids (LIP).

Groups were composed as follows: Group 1 - (G1) control group with mice not exposed to AOM (n = 9) - animals received an injection of 1.0 ml of sterile 0.9% saline, intraperitoneally, once a week for two weeks and were killed at 15 weeks; Group 2 - (G2) group study with rats exposed to AOM 15 mg / kg (n = 9) - animals injected with AOM 15 mg / kg, intraperitoneally once a week for two weeks and were killed at 15 weeks; Group 3 - (G3) control group with mice not exposed to AOM (n = 14): animals received an injection of 1.0 ml of 0.9% sterile saline solution, intraperitoneally once a week for two weeks and were killed at 26 weeks; Group 4 - (G4) group study with rats exposed to AOM 20 mg / kg (n = 12): animals injected with AOM 20 mg / kg, intraperitoneally once a week for two weeks and were killed at 26 weeks.

At the 15th week (groups 1 and 2) and at week 26 (groups 3 and 4) after the first injection, the animals were anesthetized with 80 mg/kg ketamine and 8 mg/kg xylazine intraperitoneally and set in supine position for surgery. They then underwent laparotomy through a xypho-pubic midline incision exposing the peritoneal cavity to total proctocolectomy and hepatectomy. These organs were weighed, measured and evaluated for the presence of macroscopic lesions. Samples of liver lobes were cleaved and sent for routine histopathology. The colon was opened at the anti-mesocolic edge, washed with saline and extended in kraft paper, folded on its axis concentrically and immersed in 10% formalin solution for fixation and subsequent studies. Then the animals were killed by hypovolemic shock after section of the abdominal aorta.

All organs were cleaved and two fragments of liver (right and left lateral lobe) were removed, as well as the lesions macroscopically observed. The tissues were fixed in 10% buffered formalin for 24 hours and then taken to histotechnical processing. After embedment in paraffin, 5µm thick cuts were made and stained with hematoxylin and eosin (H/E).

The study variables are nominal. We used the chi-square test to compare non-paired samples in contingency tables 2 x 2, comparing groups 1 and 2 and groups 3 and 4 for the presence of hepatic steatosis. The significance level was 5%, which was statistically significant when Á <0,05.



Histopathological examination of the liver showed mild steatosis in all groups. It was less frequent in group 1, in which animals were younger and did not receive AOM.

Groups 1 and 2 differed significantly (x2 = 7.54, Á = 0.011) in relation to hepatic steatosis at euthanasia (15 weeks after injection of saline and 15 mg/kg AOM, respectively) (Tables 1 and 2). Groups 3 and 4 did not differ (x2 = 2.08, Á = 0.216) in relation to hepatic steatosis at euthanasia (26 weeks after injection of saline and 20 mg/kg AOM, respectively) (Tables 1 and 3).



Premalignant and malignant lesions, however, have only been observed in the rats in from group 4, which received higher dose AOM (20 mg/kg). Of the premalignant lesions, the most common were amphophilic cells, seen in six animals in Group 4 (Figures 1 and 2). The most frequent neoplastic lesions were colangiomas and adenomas. We observed one case of carcinoma in situ and one hepatocellular carcinoma. Spongiosis and peliosis were also common in this group.






The focus of aberrant crypts, pre-neoplastic lesions of the colon mucosa, was initially described by Bird 7. It has the characteristic of being induced by specific carcinogen dose-dependent manner and is seen in the colonic mucosa early in two to four weeks after initiation of dosing. The size and multiplicity of crypts increase with time, and characteristics, such as proliferation and tumor dysplasia, are predictors of outcome 6-9.

The AOM is a metabolite of DMH, whose mechanism of induction of preneoplastic lesions is attributed to increased expression of c-fos gene and reduced c-myc gene, as well as the mutated K-ras gene alterations similar to those observed in spontaneous carcinogenesis in humans 17,18. AOM is usually preferred over DMH due to being more powerful and requiring few reactions to its activation. It is activated in the liver by N-oxidation, generating reactive compounds essential for chemical carcinogenesis (metilazoximetanol ion and methyl diazoxide) being brought to the colon into the bloodstream or via bile as a glucuronide conjugate. After activation, the methylated DNA is mainly at positions N7-guanine and O6-guanine 19.

In the present study, preneoplastic lesions in the liver appeared and evolved proportionally to the dose and duration of exposure to the carcinogen.

AOM causes hepatocyte proliferation. This condition is still considered preneoplastic, as hepatocytes do not seem to have any degree of autonomous growth, and called foci and nodules of altered hepatocytes. Three hepatocytic lineages can be identified during the development of tests for liver tumors in rodents: glycogenolyitic-basophilic, anphophilic-basophilic and xenomorphic-basophilic 20,21.

Preneoplastic (foci of altered, clear, vacuolated, basophilic, amphophilic, tigroid, oncocytic, small and acidophilus cells, with spongiosis and peliosis) and neoplastic (adenoma and cholangioma) lesions can be distinguished from the non-transformed tissue surrounding them by changes in the expression of various enzymes, including adenosine triphosphatase (ATPase), glucose-6-phosphatase, g-glutamyl transpeptidase (gGT) and placental glutathione S-transferase (GST-P); these are commonly used as malignant disease markers 22 -24.

Steatosis was seen more frequently in group 2 than in group 1. However, the difference between groups 3 and 4 was significant. On the other hand, group 4 animals with displayed preneoplastic and neoplastic lesions, possibly due to longer AOM exposure (11 weeks longer). Morphometric studies showed that foci of vacuolated cells (fat cells) can develop cell glycogen and gradually evolve into mixed cells and hepatic malignancy 22.

Comparing the groups 1 (15 weeks) and 3 (26 weeks), steatosis was more evident in the latter, where the only variable was time (11 weeks). The liver of senile animals can present with degeneration, with areas of fatty, lighter, vacuolated and ballooned cells, with bulkier and irregular nuclei 25.

In this study, AOM caused preneoplastic and neoplastic liver injury, proportional to the dose and duration of exposure, similar to the findings of Bird in colorectal carcinogenesis. However, more studies are necessary to strengthen these findings.

In conclusion, in the model of colorectal carcinogenesis involving rats, preneoplastic and neoplastic liver injuries appear and evolve in proportion to the duration of exposure and the dose of AOM.



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Address correspondence to:
Idália Maria Brazil Burlamaqui

Received on 15/06/2012
Accepted for publication 12/08/2012
Conflict of interest: none
Source of funding: National Council for Scientific and Technological Development (CNPq); Post Graduation Program in Surgery of the Federal University of Ceará and the Laboratory of Experimental Surgery (LABCEX), Federal University of Ceará, Faculty of Medicine.



Work conducted at the Laboratory of Experimental Surgery (LABCEX), Post-Graduation Program in Surgery, Faculty of Medicine, Federal University of Ceará - UFCE, Fortaleza, Ceará State - CE, Brazil.

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