Ana María Salazar

Induction of p53 protein expression by sodium arsenite

Arsenic is carcinogen for humans and has been shown to act as an enhancer in initiated animal models. In a previous work we found impairment of lymphocyte proliferation in arsenic-exposed individuals and in vitro we obtained dose-related inhibition of mitotic response and lymphocyte proliferation. Intrigued by these effects and based on the role of p53 on cell proliferation, we tested different concentrations of sodium arsenite for their ability to induce the expression of tumor suppressor gene p53 in different cell lines (HeLa, C-33A. Jurkat) and a lymphoblast cell line transformed with Epstein-Barr virus (LCL-EBV). We also evaluated changes in their viability after 24 h arsenic treatment; C-33A cells showed the higher sensitivity to arsenic treatment while HeLa, Jurkat and LCL-EBV cells showed similar cytotoxicity curves. Immunoblots showed an increased expression of p53 gene with 1 microM sodium arsenite in Jurkat cells and 10 microM sodium arsenite in HeLa and LCL-EBV cells. In addition, we transfected Jurkat cells and human lymphocytes with wild-type and mutated p53 genes; lymphocytes and Jurkat cells that received the mutated p53 showed increased sensitivity to arsenic cytotoxicity. Data obtained indicate that arsenic induces p53 expression and that cells with a functional p53 contend better with damage induced by this metalloid.

Progesterone receptor isoforms expression in the prepuberal and adult male rat brain

Progesterone receptor (PR) isoforms expression was determined in several regions of the prepuberal and adult male rat brain by using reverse transcription coupled to polymerase chain reaction. Rats under a 14:10-h light-dark cycle, with lights on at 0600 h were used. We found that in the hypothalamus of prepuberal animals the expression of both PR isoforms was similar, whereas PR-A expression was higher than that of PR-B in adults. In the cerebellum PR-B expression was predominant in both prepuberal and adult rats. In both ages PR-A and PR-B exhibited a non-significant tendency to be predominant in the hippocampus and the preoptic area respectively. In the frontal cortex and the olfactory bulb PR isoforms were expressed at a similar level. These results indicate a differential expression pattern of PR isoforms in the male rat brain and suggest that the tissue-specific expression of PR-A and PR-B is important for the appropriate response of each cerebral region to progesterone.

Genotoxic effects of metronidazole

Metronidazole (MTZ) is an effective agent used in the treatment of parasitic infections. Its genotoxic effects have been shown in a variety of prokaryotic systems; however, negative results have been reported in human in vivo studies. Due to its wide spread use, a study was performed to evaluate the chromosomal aberration frequencies in peripheral blood lymphocyte cultures from 10 individuals, before and after metronidazole treatment. A significant increase in the percentage of cells with chromatid and isochromatid breaks was observed after metronidazole treatment (1500 mg per day for 10 days). The percentages of cells with aberrations did not correlate with the levels of MTZ found in plasma. Individual variability was observed with respect to both the induction of aberrations and the concentration of MTZ in plasma. They could represent differences at the metabolic level, since metronidazole is known to be biotransformed by a polymorphic P450 cytochrome, and its metabolites have shown mutagenic activity.

Arsenite reduces insulin secretion in rat pancreatic β-cells by decreasing the calcium-dependent calpain-10 proteolysis of SNAP-25

An increase in the prevalence of type 2 diabetes has been consistently observed among residents of high arsenic exposure areas. We have previously shown that in rat pancreatic beta-cells, low arsenite doses impair the secretion of insulin without altering its synthesis. To further study the mechanism by which arsenite reduces insulin secretion, we evaluated the effects of arsenite on the calcium-calpain pathway that triggers insulin exocytosis in RINm5F cells. Cell cycle and proliferation analysis were also performed to complement the characterization. Free [Ca2+]i oscillations needed for glucose-stimulated insulin secretion were abated in the presence of subchronic low arsenite doses (0.5-2 microM). The global activity of calpains increased with 2 microM arsenite. However, during the secretion of insulin stimulated with glucose (15.6 mM), 1 microM arsenite decreased the activity of calpain-10, measured as SNAP-25 proteolysis. Both proteins are needed to fuse insulin granules with the membrane to produce insulin exocytosis. Arsenite also induced a slowdown in the beta cell line proliferation in a dose-dependent manner, reflected by a reduction of dividing cells and in their arrest in G2/M. Data obtained showed that one of the mechanisms by which arsenite impairs insulin secretion is by decreasing the oscillations of free [Ca(2+)]i, thus reducing calcium-dependent calpain-10 partial proteolysis of SNAP-25. The effects in cell division and proliferation observed with arsenite exposure can be an indirect consequence of the decrease in insulin secretion.

Relationship between micronuclei formation and p53 induction.

Human exposure to multiple chemicals compromises the integrity of genetic material. Hence, it is essential to determine the extent of DNA damage induced by xenobiotics. In cell lines, the induction of p53 expression in response to treatments with DNA-damaging agents has been proposed as a tool for the detection of genotoxic damage, although a direct correlation between a marker of chromosomal damage and p53 expression has not previously been studied. The micronucleus assay is a widely used genotoxicity test that has been shown to detect structural and numerical chromosomal damage. The present study was designed to characterize the relationship between micronuclei and p53 induction. RKO cells were cultured and treated with non-cytotoxic concentrations of colchicine, vinblastine, bleomycin or arsenic. Mannitol and clofibrate, which are non-genotoxic chemicals, were also included. The frequency of micronuclei was evaluated using the cytokinesis-block assay, and p53 induction was measured by Western blot assay. Our data showed that a significant induction of micronuclei and of p53 protein occurred only with the genotoxic chemicals. No differences in p53 induction were associated with the clastogenic or aneuplodogenic potential of the chemical exposure. The linear regression analysis revealed a direct relationship between p53 levels and the induction of micronuclei (p=0.0001, r(2)=0.9372), indicating that the level of p53 is associated with chromosomal damage.

p53 Expression in circulating lymphocytes of non-melanoma skin cancer patients from an arsenic contaminated region in Mexico. A pilot study

Arsenic is a common environmental toxicant and epidemiological studies associate arsenic exposure with various pathologic disorders and several types of cancer. Skin cancers are the most common arsenic-induced neoplasias and the prevalence of skin lesions has been reported to be significantly elevated in individuals exposed to arsenic via drinking water in México. Being lymphocytes the main cells used for human monitoring, we evaluated the expression of p53 protein in the lymphocytes from 44 healthy individuals and 19 samples from individuals living in a chronic arsenicism endemic region. Of the latter group, 12 individuals had non-melanoma skin cancer and 9 of them expressed p53 in the circulating lymphocytes, whereas only one of the 7 non-cancer arsenic exposed individuals expressed it. In the healthy non-arsenic exposed group only one from 44 individuals expressed the protein. These results suggest a clear relationship between non-melanoma skin cancer and p53 expression in circulating lymphocytes. p53 expression in circulating lymphocytes should be evaluated as a potential biomarker of effect or susceptibility.

Role of Calpain-10 in the Development of Diabetes Mellitus and Its Complications

Calpain activity has been implicated in several cellular processes such as cell signaling, apoptosis, exocytosis, mitochondrial metabolism and cytoskeletal remodeling. Evidence has indicated that the impairment of calpain expression and the activity of different calpain family members are involved in diverse pathologies. Calpain-10 has been implicated in the development of type 2 diabetes, and polymorphisms in the CAPN10 gene have been associated with an increased risk of developing this disease. The present work focused on the molecular biology of calpain-10, supporting its key participation in glucose metabolism. Current knowledge regarding the role of calpain-10 in the development of type 2 diabetes mellitus and diabetes-related diseases is additionally reviewed.

Immunological mechanisms involved in the protection against intestinal taeniosis elicited by oral immunization with Taenia solium calreticulin

Oral immunization with functional recombinant Taenia solium calreticulin (rTsCRT) induces 37% reduction in tapeworm burden in the experimental model of intestinal taeniosis in hamsters. Furthermore, tapeworms recovered from vaccinated animals exhibit diminished length, being frequently found in more posterior parts of the small intestine. The aim of this study was to analyze the immunological mechanisms involved in protection in response to rTsCRT oral immunization. Hamsters were orally immunized with rTsCRT using cholera toxin (CT) as adjuvant, weekly for 4 weeks. Fifteen days after the last boost animals were challenged with four T. solium cysticerci. Reduction in the adult worm recovery and increased transcription of mRNA for IL-4 and IFN-γ in the mucosa of rTsCRT+CT immunized animals were observed. Immunization also induced goblet cell hyperplasia in the mucosa surrounding the implantation site of the parasite. Specific IgG and IgA antibodies in serum and fecal supernatants were detected after the second immunization, being more pronounced after challenge. Our data suggest that oral vaccination with rTsCRT+CT regulates a local expression of IL-4 and IFN-γ, stimulating secretion of IgA that, together with the increase of goblet cells and mucin production, could result in an unfavorable environment for T. solium promoting an impaired tapeworm development.

Fanconi anemia cells with unrepaired DNA damage activate components of the checkpoint recovery process

BackgroundThe FA/BRCA pathway repairs DNA interstrand crosslinks. Mutations in this pathway cause Fanconi anemia (FA), a chromosome instability syndrome with bone marrow failure and cancer predisposition. Upon DNA damage, normal and FA cells inhibit the cell cycle progression, until the G2/M checkpoint is turned off by the checkpoint recovery, which becomes activated when the DNA damage has been repaired. Interestingly, highly damaged FA cells seem to override the G2/M checkpoint. In this study we explored with a Boolean network model and key experiments whether checkpoint recovery activation occurs in FA cells with extensive unrepaired DNA damage.MethodsWe performed synchronous/asynchronous simulations of the FA/BRCA pathway Boolean network model. FA-A and normal lymphoblastoid cell lines were used to study checkpoint and checkpoint recovery activation after DNA damage induction. The experimental approach included flow cytometry cell cycle analysis, cell division tracking, chromosome aberration analysis and gene expression analysis through qRT-PCR and western blot.ResultsComputational simulations suggested that in FA mutants checkpoint recovery activity inhibits the checkpoint components despite unrepaired DNA damage, a behavior that we did not observed in wild-type simulations. This result implies that FA cells would eventually reenter the cell cycle after a DNA damage induced G2/M checkpoint arrest, but before the damage has been fixed. We observed that FA-A cells activate the G2/M checkpoint and arrest in G2 phase, but eventually reach mitosis and divide with unrepaired DNA damage, thus resolving the initial checkpoint arrest. Based on our model result we look for ectopic activity of checkpoint recovery components. We found that checkpoint recovery components, such as PLK1, are expressed to a similar extent as normal undamaged cells do, even though FA-A cells harbor highly damaged DNA.ConclusionsOur results show that FA cells, despite extensive DNA damage, do not loss the capacity to express the transcriptional and protein components of checkpoint recovery that might eventually allow their division with unrepaired DNA damage. This might allow cell survival but increases the genomic instability inherent to FA individuals and promotes cancer.

Genotoxicity induced by Taenia solium and its reduction by immunization with calreticulin in a hamster model of taeniosis.

Genotoxicity induced by neurocysticercosis has been demonstrated in vitro and in vivo in humans. The adult stage of Taenia solium lodges in the small intestine and is the main risk factor to acquire neurocysticercosis, nevertheless its carcinogenic potential has not been evaluated. In this study, we determined the genotoxic effect of T. solium infection in the hamster model of taeniosis. In addition, we assessed the effect of oral immunization with recombinant T. solium calreticulin (rTsCRT) plus cholera toxin as adjuvant on micronuclei induction, as this protein has been shown to induce 33–44% protection in the hamster model of taeniosis. Blood samples were collected from the orbital venous plexus of noninfected and infected hamsters at different days postinfection, as well as from orally immunized animals, to evaluate the frequency of micronucleated reticulocytes as a measure of genotoxicity induced by parasite exposure and rTsCRT vaccination. Our results indicate that infection with T. solium caused time‐dependent DNA damage in vivo and that rTsCRT immunization reduced the genotoxic damage induced by the presence of the tapeworms. Environ. Mol. Mutagen. 54:347–353, 2013.