Chemical carcinogenesis by DMBA (7, 12-dimethylbenzanthracene) in female BALB/c mice: new facts / Carcinogenese quimica por DMBA (7, 12-dimethylbenzanthracene) em camundongos femeas BALB/c: novos fatos

Polycyclic aromatic hydrocarbons are known carcinogens used in rodent experimental models. In this study, the carcinogen DMBA (7,12-dimethylbenzanthracene) was administered by gavage, diluted in corn oil, to female BALB / c mice at hebdomadary doses of 1 mg per animal for 1, 3, 6 or 9 weeks. Animals were weighed and monitored weekly until death. Remaining animals were euthanized at the age of 53 weeks. At necropsy, representative fragments of neoplasms were collected and routinely processed for histopathological analysis. Of all mice that received DMBA, 68.57% developed some type of tumor. Of the 70 mice treated with various doses of DMBA, 22 (31.43%) developed mammary tumors. The adenoacanthoma was the most commonly (18.75%) diagnosed histological type of breast cancer. Lung (15.71%), lymphoid tissue (11.43%), stomach (7.14%) and skin (2.86%) were also primary sites of tumor development. One third (33.33%) of the mice receiving 1 mg of DMBA developed lung cancer. Therefore, the administration of DMBA was shown to be an efficient model of carcinogenesis in mice, especially for the study of breast cancer, when using the highest dose, and lung, when using the lowest dose. Carcinogenesis models have been used for several purposes in cancer research. These results represent new facts for a classic carcinogenesis model.

Hidrocarbonetos policíclicos e aromáticos são carcinógenos usados em modelos experimentais em roedores. Neste estudo, o carcinógeno DMBA (7,12-dimetilbenzantraceno) foi administrado por gavagem, diluído em óleo de milho, para camundongos BALB/c em doses hebdomadárias de 1 mg por animal por 1, 3, 6 ou 9 semanas. Os animais foram pesados e monitorados semanalmente até a morte. Os animais remanescentes foram eutanasiados com a idade de 53 semanas. Na necroscopia, fragmentos representativos das neoplasias foram colhidos e rotineiramente processados para exame histopatológico. De todos os animais que receberam DMBA, 68,57% desenvolveram algum tipo de tumor. Entre os 70 camundongos tratados com diferentes doses de DMBA, 22 (31,43%) desenvolveram neoplasias mamárias. O adenoacantoma foi o tumor mamário mais comumente diagnosticado (18,75%). Pulmões (15,71%), tecido linfoide (11,43%), estômago (7,14%) e pele (2,86%) foram também locais primários de desenvolvimento de neoplasias. Um terço (33,33%) dos camundongos que receberam 1 mg de DMBA desenvolveram neoplasias pulmonares. Portanto, a administração de DMBA foi considerada um modelo eficiente de carcinogênese em camundongos, especialmente para o estudo de neoplasias mamárias, quando a maior dose é utilizada, e de neoplasias pulmonares, quando utilizada a menor dose. Os modelos de carcinogênese química têm sido usados para diversos estudos na pesquisa em câncer, os resultados aqui apresentados mostram novos fatos para um modelo clássico de carcinogênese.

ABCB1 and ABCC1 expression in peripheral mononuclear cells is influenced by gene polymorphisms and atorvastatin treatment.

This study investigated the effects of atorvastatin on ABCB1 and ABCC1 mRNA expression on peripheral blood mononuclear cells (PBMC) and their relationship with gene polymorphisms and lowering-cholesterol response. One hundred and thirty-six individuals with hypercholesterolemia were selected and treated with atorvastatin (10 mg/day/4 weeks). Blood samples were collected for serum lipids and apolipoproteins measurements and DNA and RNA extraction. ABCB1 (C3435T and G2677T/A) and ABCC1 (G2012T) gene polymorphisms were identified by polymerase chain reaction-restriction (PCR)-RFLP and mRNA expression was measured in peripheral blood mononuclear cells by singleplex real-time PCR. ABCB1 polymorphisms were associated with risk for coronary artery disease (CAD) (p<0.05). After atorvastatin treatment, both ABCB1 and ABCC1 genes showed 50% reduction of the mRNA expression (p<0.05). Reduction of ABCB1 expression was associated with ABCB1 G2677T/A polymorphism (p=0.039). Basal ABCB1 mRNA in the lower quartile (<0.024) was associated with lower reduction rate of serum low-density lipoprotein (LDL) cholesterol (33.4+/-12.4%) and apolipoprotein B (apoB) (17.0+/-31.3%) when compared with the higher quartile (>0.085: LDL-c=40.3+/-14.3%; apoB=32.5+/-10.7%; p<0.05). ABCB1 substrates or inhibitors did not affect the baseline expression, while ABCB1 inhibitors reversed the effects of atorvastatin on both ABCB1 and ABCC1 transporters. In conclusion, ABCB1 and ABCC1 mRNA levels in PBMC are modulated by atorvastatin and ABCB1 G2677T/A polymorphism and ABCB1 baseline expression is related to differences in serum LDL cholesterol and apoB in response to atorvastatin.

Hepatic granulomas induced by Schistosoma mansoni in mice deficient for connexin 43 present lower cell proliferation and higher collagen content.

Granuloma formation involves a coordinated interaction between monocytes and macrophages, epithelioid cells, lymphocytes, eosinophils, neutrophils and fibroblasts. It has been established that extracellular communication via cytokines is important for the assembly of granulomas. However, the importance of gap junctions and intercellular communication to granuloma formation and development had never been assessed. Connexins are proteins that form gap junctions, and connexin 43 (Cx43) is present in macrophages, lymphoid cells, myelogenous cells, fibroblasts and others. We analyzed the effect of heterologous deletion of Gja1 (Cx43 gene) on the formation and development of hepatic granulomas induced by Schistosoma mansoni eggs. Heterozygous (Cx43(+/-)) and wild-type (Cx43(+/+)) mice were infected subcutaneously with S. mansoni cercarie and evaluated after 6, 8 and 12 weeks. Granuloma cells express Cx43, as revealed by real-time PCR in isolated granulomas, and by immunohistochemistry. Cx43 expression was reduced in Cx43(+/-) mice, as expected. No differences in the average area of granulomas or number of cells per granuloma were observed between mice of different genotypes. However, granuloma cells from Cx43(+/-) mice displayed a reduced index of the proliferating cell nuclear antigen (PCNA) labeling at 8 and 12 weeks post-infection. Moreover, Cx43(+/-) granulomas unexpectedly presented a higher degree of fibrosis, quantified by morphometric analysis in Sirius Red-stained slides. Our results indicate that the deletion of one allele of the Cx43 gene, and possibly the reduced gap junction intercellular communication capacity (GJIC), may impair the interactions between granuloma cells, reducing their proliferation and increasing their collagen content, thereby modifying the characteristics of S. mansoni granuloma in mice.

Altered expression of connexins in urethane-induced mouse lung adenomas.

Lung carcinogenesis is a multistep process whose molecular alterations can be studied in mouse models. Urethane, a specific lung tumor carcinogen, can induce adenomas in mice. Mouse lung alveolar cells reportedly generate lung neoplasms, and express connexins 26, 32, 43 and 46. The aim of the present study was to evaluate the expression of connexins in urethane-induced lung adenomas. Fifteen-day-old CD1 male mice received 2 i.p. injections of urethane (1.5 g/kg bw). The mice were euthanized 25 weeks after urethane injection, and lung adenomas were quantified. Lung tissue and lung adenomas were harvested and the RNA was extracted. The expression of connexins 26, 32, 43 and 46 was evaluated by Real-Time PCR, and these proteins were identified by Western blot. Immunohistochemistry revealed the distribution pattern of these connexins in lung tissue and adenomas. The treatment with urethane was associated with the downregulation of Cx26, 32 and 46 expressions, and with the upregulation of Cx43 expression in lung tissue. Surprisingly, in lung adenomas Cx32 and Cx43 expressions were not detected, although the expression of connexins 26 and 46 was present. Western blot and immunohistochemistry corroborated the RT-PCR data. These results may indicate a role of Cx32 and Cx43 in urethane-induced lung carcinogenesis, since their absence may contribute to the development of urethane induced lung tumors. The role of Cx26 and Cx46 is yet to be determined.

Chemopreventive effects of Paullinia cupana Mart var. sorbilis, the guaraná, on mouse hepatocarcinogenesis.

Guaraná (Paullinia cupana) is originally from Amazon, Brazil. Its effects on mouse hepatocarcinogenesis have been investigated in this study. Mice were treated with N-nitrosodiethylamine (DEN), received three different doses of P. cupana added to commercial food, and euthanized after 25 weeks. Gross lesions were quantified, and preneoplastic lesions (PNL) were histologically measured. Cellular proliferation was evaluated by immunobloting for the proliferating cell nuclear antigen (PCNA). The incidence and multiplicity of macroscopic lesions were reduced. The PNL number and PCNA expression were reduced in the highest P. cupana dose. According to these results, guaraná presented inhibitory effects on DEN hepatocarcinogenesis in mice.

Defective gap junctional intercellular communication in the carcinogenic process.

Gap junctions are membrane structures made of intercellular channels which permit the diffusion from cytoplasm to cytoplasm of small hydrophilic molecules. Nearly 40 years ago, the loss of functional gap junctions has been described in cancer cells and led to the hypothesis that such type of intercellular communication is involved in the carcinogenesis process. From this time, a lot of data has been accumulated confirming that gap junctions are frequently decreased or absent in cancer cells whatever their tissue and species origins. Here, we review such data by insisting on the possible links existing between altered gap-junctional intercellular communication capacity (or the altered expression of their constitutive proteins, the connexins) and the stages of cancer progression in various cancer models. Then, we analyse particular aspects of the disturbance of connexin-mediated communication in cancer such as the cytoplasmic localization of connexins, the lack of heterologous communication between cancer cells and normal cells, the role of connexin gene mutations in cancer. In a separate part of the review, we also analyse the disturbance of gap-junctional intercellular communication during the late stages of cancer (invasion and metastasis processes).

Increased susceptibility to urethane-induced lung tumors in mice with decreased expression of connexin43.

Gap junction intercellular communication capacity and connexin expression are reportedly decreased in human lung cancer. The mechanisms by which connexins, the gap junction proteins, act as tumor suppressors are unclear. In order to understand the involvement of connexins in tumorigenesis, we analyzed the effect of the heterologous deletion of Gja1 [the connexin43 (Cx43) gene] on the development of lung adenomas in mice. Heterozygous (Cx43(+/-)) and wild-type mice (Cx43(+/+)) were treated or not with single doses of urethane at 15 and 17 days after birth. Twenty-five weeks later, both the number and size of nodules were increased in Cx43(+/-) mice as compared with Cx43(+/+) mice. Moreover, the lesions were histologically more aggressive in the heterozygous mice. However, no increase in spontaneous lesions was observed in the lungs of untreated Cx43(+/-) mice. Heterozygous mice effectively presented lower expression of Cx43 genes and decreased amounts of Cx43. In conclusion, our results indicate that deletion of one allele of the Cx43 gene clearly favors the carcinogenic effect of urethane administration and results in a higher susceptibility to lung adenoma formation in mice.

Role of antioxidant systems in induced nutritional pancreatic atrophy in chicken.

One-day-old chicks were reared using diets differing in their vitamin E and/or selenium content. The purpose of this research was to detect any possible imbalance in the antioxidant defense system, which could be related to development of nutritional pancreatic atrophy. Mitochondrial membranes from animals deficient in both nutrients, or just vitamin E, submitted to peroxidizability 'in vitro' had the production of TBARS greatly enhanced. Measurements of the 2-GSH/GSSG ratio suggested that selenium and vitamin E, the latter in higher magnitude, were responsible for maintenance of the reducing capacity of the cell. Enzymatic defense systems against oxidative stress were also studied. The results indicated that the total antioxidant enzymatic activity of pancreatic cells was not sufficient to scavenge all the ROS generated in the nutritionally deficient animals. The present study suggests that nutritional deficiency of selenium and/or vitamin E generates one imbalance between pro-oxidant and antioxidant systems in chicken pancreas, leading to oxidative stress and pancreatic atrophy.