Study of the antineoplastic action of Tabebuia avellanedae in carcinogenesis induced by azoxymethane in mice 1 Estudo da ação antineoplásica da Tabebuia avellanedae ( Ipê-Roxo ) na carcinogênese induzida pelo azoximetano em camundongos

Purpose: To study the antitumor action of Tabebuia avellanedae in experimentally induced colon carcinogenesis by azoxymethane in mice. Methods: Fifty (n=50) mice were divided into five groups: in group I azoxymethane (AOM) was administered, in Group II β-lapachone, in group III vehicle (diluent) and in group IV vehicle + AOM and finally in group V β-lapachone + AOM. Results: It was observed the presence of aberrant crypt foci in all animals of groups I and IV, 50% in group II and 90% in group V. Conclusion: The β-lapachone extracted from the Tabebuia avellanedae showed no protective effect of lesions induced by azoxymethane in colon of mice.


Introduction
The Tabebuia avellanedae a typical tree of the Brazilian savannah, has been used for years in folk medicine as treatment for various diseases 1 .In the '60s and '70s, there was significant investment in studies of substances extracted from this plant, especially lapachol and β-lapachone, which started to be commercialized for use in adjuvant chemotherapy for treatment of leukemia 1,2 .
Lapachol and its derivatives have very important properties in the induction of apoptosis, by acting on topoisomerases enzymes I and II, and any change in the balance between these enzymes is sufficient to induce apoptosis -which is the programmed cell death.The biochemistry of induction is not totally clear, however, the information available in the literature can point to an action of inducing apoptosis through inhibition of topoisomerase-DNA 1 complex.
The inhibitory action on repairing systems seems to be the only mode of action of β-lapachone, as fungi, which do not express the topoisomerases, are also inhibited by this quinone 1 .Apparently, the β-lapachone has a critical role as xenobiotic on more than one intracellular target, and not only topoisomerase I. Another hypothesis was the attack by β-lapachone to specific points in the catalytic cycle that expresses the action of topoisomerase I, for example, the checkpoints G1 and S, by action other than a simple merge.It was recently reported that β-lapachone induces apoptosis of malignant epithelial cells, human glioma, and human retinal pigments 1 .
There are few scientific studies that used lapachol and its derivatives as antineoplastic agents, especially in the study of colon cancer 6 .The main objective of this study was to evaluate the antitumor action of lapachol in experimentally induced colonic carcinogenesis by azoxymethane in mice.

Methods
The procedures were approved by the Ethics Committee in the use of animals, protocol No. 71/2007.
Fifty 8-week-old male mice were use, average weight 20 grams, from UFMS Central Animal Colony.They were held at the Laboratory of Carcinogenesis, Department of Surgical Clinic of UFMS in boxes with polypropylene with galvanized cap, standard size for six animals and adjustment period of 7 days.
The 50 animals were randomly divided into five groups according to protocol: Group I: 10 (n) mice were submitted to two doses of azoxymethane, one in the first week and another in the second; Group II: 10 (n) mice were submitted to β-lapachone in daily doses for 6 weeks; Group III: 10 (n) mice were submitted to vehicle (diluent) of β-lapachone in daily doses for 6 weeks; Group IV: 10 (n) mice were submitted to vehicle (diluent) of β-lapachone in daily doses for 6 weeks and two doses of azoxymethane, one in the first week and another in the second week; Group V: 10 (n) mice were submitted to β-lapachone in daily doses for six weeks and two doses of azoxymethane, one in the first week and another on Monday.

Administration of substances
The beta-lapachone was diluted in solution (vehicle) of 20% ethanol, and was administered daily by oral gavage at 25mg/kg for 6 weeks and in the control group it was administered vehicle with the same volume and frequency.Azoxymethane at a dose of 15mg/Kg weight / dose diluted in 0.9% saline solution subcutaneously in two doses during the experiment -1 st and 2 nd weeks.

Euthanasia
Mice were identified, weighed and euthanasia was performed with thiopental infusion at a dose of 150mg/kg, intraperitoneally.Then, in a supine position, it was made a median incision of the skin, extending from the xiphoid process to pubis; dieresis of abdominal wall plans, identification of the ileocaecal region and repair with forceps; total colectomy after section of the entire length of mesocolon, excluding the cecum and rectum, which were not used in the experiment.

Histopathology
The preparation of the piece to histopathology was made by opening the colon in the anti-mesenteric border, washing the mucosa with a ringer solution; colon fixation on cardboard and placed in formol lampooned in 10%, for 24 hours, then, inclusion in paraffin, diereses with microtome in proximal edge, at 3 cm level using the entire length of the width of the rectum in longitudinal section, and placed on glass slides for histological processing with hematoxylin and eosin (HE).
We performed histological and morphological study, categorizing them into: hyperplastic aberrant crypt focus: presents a moderate hypercellularity, with slight enlargement of light and abnormal form, dysplastic aberrant crypt focus: besides the enlargement of light, there is enough tortuosity of the crypt that is markedly hypercellular and pseudostratification; microadenoma: severely hypercellular, presence of cell differentiation, hyperchromasia and loss of cellular polarity 7 .

Statistical analysis
Statistical analysis was performed using the chi square test and the z test, considering a significance level of 5%.

Results
After histopathological analysis, we found the result shown in Table 1.It was considered positive the presence of hyperplastic or dysplastic aberrant crypt focus.It was not found the presence of microadenomas.According to the presence or absence of histopathological criteria, we observed a significant difference (p<0.05) between groups I and II, and III I, II and III, II and IV, III and IV and III and V.Among groups I and IV, and V I, II and V and IV and V no significant differences were observed.

Discussion
The focus of aberrant crypt has been used for early detection of factors influencing colorectal carcinogenesis 8,9 , found in rodents treated with carcinogens, and in humans 10,11,12 .Experimental evidence supports the hypothesis that this type of lesion is pre-neoplastic [11][12][13][14][15][16] , consisting of crypts that have expanded lumen and abnormal form 10,16 probably evolved in time with proliferative, hyperplastic, dysplastic phenomena, and finally, small adenomas 9,10 .There was need to hold two control groups of β-lapachone vehicle, since the diluent is alcohol (ethanol 20%) 2 , from which group III, treated only with vehicle, showed no histopathological changes, as seen in literature 17 .Group IV showed similar changes to group I due to the use of tumor promoter (AOM).

Groups
Lapachol and its derivative β-lapachone are naphthoquinones with therapeutic potential against some types of cancer 2 .The β-lapachone exhibits in vitro activity against many types of different lineages of cells, especially malignant cancers of lung, breast, colorectal, prostate, melanoma and leukemia.Despite the broad spectrum of bioactivities, the mechanisms of action of β-lapachone in experimental models are not well delineated.And yet, there is a strong interest focused on the commercial use of the substance, which can be demonstrated by several patents granted in recent years involving this quinone, probably as a guarantee for commercial use in the future 1 .
In vitro study it is known that β-lapachone has activity against approximately 60 human tumor cell lines, among them colon cancer 18 .It also induces apoptosis of breast cancer cells; it has considerable anticancer activity in multiple myeloma cells, inhibition of NO synthase, which may lead to new findings on the development of anti-inflammatory; at low concentrations it can induce cell death of prostate cancer; it is cytotoxic against various tumor cells, including activity against cells resistant to other chemotherapics 1 .
In group II we found hyperplastic aberrant crypt foci in 50% of cuts.Studies show that Lapachol require increased levels to achieve a cytotoxic effect, but in larger doses the major toxic effects of Lapachol arise, such as nausea, vomiting, reversible increase of prothrombin time.The clinical study of Lapachol has not been determined yet as it hasn't achieved a high plasma level which apparently produce a toxic effect.In the study by Dinnen et al. 21, it is demonstrated that the concentration of Lapachol, required to induce cell differentiation is lower than necessary to promote a cytotoxic effect [17][18][19][20][21] .
This study only found aberrant crypt foci of hyperplastic and dysplastic types; the first type was found in all animals with histopathological changes, and the second was uncommon in all groups.Most of the work makes only descriptive evaluation of histopathology slides stained by using hematoxylin and eosin, and present evaluation results with immunohistochemistry, genetics (DNA) or biochemicals 1,10,11,13,15,16,22 .
Only one animal in group V showed no pathological changes, which brought no significant changes in reducing aberrant crypt foci in mice that were induced with azoxymethane colon tumor and treated with β-lapachone.Thus, there was evidence that β-lapachone extracted from the Tabebuia avellanedae inhibits the appearance of lesions in the colon of mice when administered in AOM-induced model.

Conclusions
β-lapachone extracted from Tabebuia avellanedae, at the concentration used, showed no protective effect of lesions induced by azoxymethane in the colon of mice.The β-lapachone was considered capable of inducing lesions in the colon of mice, as compared to control.And the azoxymethane is an excellent model to induce aberrant crypt foci in hyperplastic and dysplastic colon of mice.

TABLE 1 -
Animals per group according to the presence or not of histological changes (n=10) Histopathological changes characterized by the presence of hyperplastic or dysplastic aberrant crypt focus in slides stained with HE.

TABLE 2 -
Paired comparison between groups according to the presence of pathological changes in animals