José Luis Avanzo

Effect of vitamin E and selenium on resistance to oxidative stress in chicken superficial pectoralis muscle.

One-day-old chicks were reared using diets that differed in their vitamin E and/or selenium content. In chicks depleted of both selenium and vitamin E, signs of exudative diathesis on the superficial pectoralis muscle were observed. The purpose of this research was to determine the defective points of the antioxidant defense system, which made this tissue highly susceptible to nutritionally-induced oxidative stress. Vitamin E, and selenium in lower magnitude, were the factors that strikingly affected the course of mitochondrial lipid peroxidation. Animals fed diets deficient in vitamin E and selenium displayed the lowest reduced glutathione level and glutathione peroxidase activity. The decreased levels of reduced glutathione were not due to a defective activity of glutathione reductase, which was increased in both mitochondria and cytosol. The absence of vitamin E was linked to lowering of mitochondrial thiol levels. The Glutathione peroxidase/Cu,Zn-superoxide dismutase ratio was 2.8 in animals fed selenium and vitamin E, and decreased to 0.13 in animals deficient in both nutrients. This change was indicative of oxidant-induced damage mediated by hydrogen peroxide. Catalase activity increased in an attempt to counteract the decrease in glutathione peroxidase activity. The results obtained showed that alpha-tocopherol and Se deficiencies caused multiple alterations in the antioxidant system and adversely affected the redox state of chicken superficial pectoralis muscle.

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.

Deletion of a single allele of Cx43 is associated with a reduction in the gap junctional intercellular communication and increased cell proliferation of mouse lung pneumocytes type II

Abstract.  Objectives: Connexins (Cx) are proteins that form the gap junctional channels at neighbouring plasma membranes between adjacent cells. Cxs are involved in cell communication, which is reportedly correlated with cell proliferation and differentiation. Alterations in connexin expression and/or gap junctional intercellular communication (GJIC) capacity have long been postulated to be important in a number of pathological conditions including cancer. This study was performed to determine the consequences of the deletion of a single allele of Gja1 (Cx43 gene) in Alveolar Type II cells (APTIIs), and its impact on GJIC and cell proliferation. Material and methods: In order to do so, APTIIs from wild type (Cx43+/+) and heterozygous (Cx43+/–) mice were harvested and cultured for 4 days. The GJIC capacity was evaluated by scrape‐loading method, with the transfer of lucifer yellow dye. The expression of Cx43 was evaluated by immunofluorescence method and Western blotting. Cell proliferation was evaluated by 3‐(4,5‐dimethylthazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide assay. Results: It was observed that GJIC capacity was significantly reduced and cell proliferation index was significantly higher in Cx43+/– cells compared to Cx43+/+ cells. Conclusions: These results show that knocking out one allele of Cx43 leads to a lower cell to cell communication capacity, and consequently induces a higher cell proliferation. Because chemically induced lung adenomas in mice are known to originate from APTIIs, these alterations may play a critical role in their susceptibility to lung carcinogenesis.

Higher susceptibility of spontaneous and NNK-induced lung neoplasms in connexin 43 deficient CD1 × AJ F1 mice: Paradoxical expression of connexin 43 during lung carcinogenesis

Connexins (Cxs) are proteins that form the communicating gap junctions, and reportedly have a role in carcinogenesis. Here, we evaluated the importance of Connexin43 (Cx43) in spontaneous and 4‐(methylnitrosamino)‐1‐(3‐pyridyl)‐1‐butanone (NNK)‐induced lung carcinogenesis. Male wild‐type (Cx43+/+) and hemizygote (Cx43+/−) CD1 × AJ F1 mice were injected with NNK or saline. After 60 weeks mice were euthanized; lung nodules were counted, measured, and fixed in formalin or snap frozen. Immunohistochemistry for Cx43 and Beta‐catenin (β‐catenin) was performed and Cx43 mRNA expression was evaluated by real‐time PCR. Cx43 deletion significantly increased the incidence and number of spontaneous nodules in the CD1 × AJ F1 mice and the number of gross lesions and the aggressiveness of lesions in NNK‐treated mice. Cx43 mRNA increased significantly and was correlated with the aggressiveness of tumors, although lesions from Cx43+/− mice expressed less Cx43 RNAm than their counterparts. Lung parenchyma presented a Cx43 immunostaining pattern with points or plaques between cells. In hyperplasias and adenomas, Cx43 was found in the membrane and in cytoplasm. Malignant lesions presented increased Cx43 in cytoplasm and a few membrane spots of immunostaining. β‐catenin was weakly expressed in lung parenchyma. Though hyperplasias presented some cells with nuclear β‐catenin, NNK‐induced tumors contained a higher number of this staining pattern. Also, no difference in β‐catenin occurred between both genotypes independently of the histological grade. In summary, our results indicate that Cx43 acts as a tumor suppressor gene in early lung tumorigenesis and loses this property in advanced carcinogenesis. Therefore, Cxs are better classified as conditional tumor suppressors.

Chemical carcinogenesis by 7,12-dimethylbenzanthracene in balb/c mice

Patients and methods The carcinogen DMBA was administered by gavage to 70 Balb/c female mice, diluted in corn oil, at hebdomadary doses of 1 mg per animal for 1, 3, 6 or 9 weeks. Others 20 animals were used in the control group, who received only corn oil administration. Animals were weighed and monitored weekly until death, remaining animals were euthanized at age of 53 weeks. At necropsy, representative fragments of tumors were harvested, routinely processed for microscopy and classified agreeing with International Agency for Research on Cancer (IARC).

Expression and distribution of connexin 32 in rat liver with experimentally induced fibrosis

The connexin 32 (Cx32) is a protein that forms the channels that promote the gap junction intercellular communication (GJIC) in the liver, allowing the diffusion of small molecules through cytosol from cell-to-cell. Hepatic fibrosis is characterized by a disruption of normal tissue architeture by cellular lesions, and may alter the GJIC. This work aimed to study the expression and distribution of Cx32 in liver fibrosis induced by the oral administration of dimethylnitrosamine in female Wistar rats. The necropsy of the rats was carried out after five weeks of drug administration. They presented a hepatic fibrosis state. Sections from livers with fibrosis and from control livers were submitted to immunohistochemical, Real Time-PCR and Western-Blot analysis to Cx32. In fibrotic livers the Cxs were diffusely scattered in the cytoplasm, contrasting with the control livers, where the Cx32 formed junction plaques at the cell membrane. Also it was found a decrease in the gene expression of Cx32 without reduction in the protein quantity when compared with controls. These results suggest that there the mechanism of intercellular communication between hepatocytes was reduced by the fibrotic process, which may predispose to the occurrence of a neoplastic process, taken in account that connexins are considered tumor suppressing genes.