Comprehensive germline mutation analysis and clinical profile in a large cohort of Brazilian xeroderma pigmentosum patients.

BACKGROUND: Xeroderma pigmentosum (XP) patients present a high risk of developing skin cancer and other complications at an early age. This disease is characterized by mutations in the genes related to the DNA repair system. OBJECTIVES: To describe the clinical and molecular findings in a cohort of 32 Brazilian individuals who received a clinical diagnosis of XP. METHODS: Twenty-seven families were screened for germline variants in eight XP-related genes. RESULTS: All patients (N = 32) were diagnosed with bi-allelic germline pathogenic or potentially pathogenic variants, including nine variants previously undescribed. The c.2251-1G>C XPC pathogenic variant, reported as the founder mutation in Comorian and Pakistani patients, was observed in 15 cases in homozygous or compound heterozygous. Seven homozygous patients for POLH/XPV variants developed their symptoms by an average age of 7.7 years. ERCC2/XPD, DDB2/XPE and ERCC5/XPG variants were found in a few patients. Aside from melanoma and non-melanoma skin tumours, a set of patients developed skin sebaceous carcinoma, leiomyosarcoma, angiosarcoma, mucoepidermoid carcinoma, gastric adenocarcinoma and serous ovarian carcinoma. CONCLUSIONS: We reported a high frequency of XPC variants in 32 XP Brazilian patients. Nine new variants in XP-related genes, unexpected non-skin cancer lesions and an anticipation of the clinical manifestation in POLH/XPV cases were also described.

A genetic cluster of patients with variant xeroderma pigmentosum with two different founder mutations.

BACKGROUND: Xeroderma pigmentosum (XP) is a rare human syndrome associated with hypersensitivity to sunlight and a high frequency of skin tumours at an early age. We identified a community in the state of Goias (central Brazil), a sunny and tropical region, with a high incidence of XP (17 patients among approximately 1000 inhabitants). OBJECTIVES: To identify gene mutations in the affected community and map the distribution of the affected alleles, correlating the mutations with clinical phenotypes. METHODS: Functional analyses of DNA repair capacity and cell-cycle responses after ultraviolet exposure were investigated in cells from local patients with XP, allowing the identification of the mutated gene, which was then sequenced to locate the mutations. A specific assay was designed for mapping the distribution of these mutations in the community. RESULTS: Skin primary fibroblasts showed normal DNA damage removal but abnormal DNA synthesis after ultraviolet irradiation and deficient expression of the Polη protein, which is encoded by POLH. We detected two different POLH mutations: one at the splice donor site of intron 6 (c.764 +1 G>A), and the other in exon 8 (c.907 C>T, p.Arg303X). The mutation at intron 6 is novel, whereas the mutation at exon 8 has been previously described in Europe. Thus, these mutations were likely brought to the community long ago, suggesting two founder effects for this rare disease. CONCLUSIONS: This work describes a genetic cluster involving POLH, and, particularly unexpected, with two independent founder mutations, including one that likely originated in Europe.

A Mouse Model of Targeted Musashi1 Expression in Whole Intestinal Epithelium Suggests Regulatory Roles in Cell Cycle and Stemness.

The intestinal epithelium is very peculiar for its continuous cell renewal, fuelled by multipotent stem cells localized within the crypts of Lieberkühn. Several lines of evidence have established the evolutionary conserved RNA-binding protein Musashi1 as a marker of adult stem cells, including those of the intestinal epithelium, and revealed its roles in stem cell self-renewal and cell fate determination. Previous studies from our laboratories have shown that Musashi1 controls stem cell-like features in medulloblastoma, glioblastoma, and breast cancer cells, and has pro-proliferative and pro-tumorigenic properties in intestinal epithelial progenitor cells in vitro. To undertake a detailed study of Musashi1's function in the intestinal epithelium in vivo, we have generated a mouse model, referred to as v-Msi, overexpressing Musashi1 specifically in the entire intestinal epithelium. Compared with wild type litters, v-Msi1 mice exhibited increased intestinal crypt size accompanied by enhanced proliferation. Comparative transcriptomics by RNA-seq revealed Musashi1's association with gut stem cell signature, cell cycle, DNA replication, and drug metabolism. Finally, we identified and validated three novel mRNA targets that are stabilized by Musashi1, Ccnd1 (Cyclin D1), Cdk6, and Sox4. In conclusion, the targeted expression of Musashi1 in the intestinal epithelium in vivo increases the cell proliferation rate and strongly suggests its action on stem cells activity. This is due to the modulation of a complex network of gene functions and pathways including drug metabolism, cell cycle, and DNA synthesis and repair.

Control of endodermal endocrine development by Hes-1.

Development of endocrine cells in the endoderm involves Atonal and Achaete/Scute-related basic helix-loop-helix (bHLH) proteins. These proteins also serve as neuronal determination and differentiation factors, and are antagonized by the Notch pathway partly acting through Hairy and Enhancer-of-split (HES)-type proteins. Here we show that mice deficient in Hes1 (encoding Hes-1) display severe pancreatic hypoplasia caused by depletion of pancreatic epithelial precursors due to accelerated differentiation of post-mitotic endocrine cells expressing glucagon. Moreover, upregulation of several bHLH components is associated with precocious and excessive differentiation of multiple endocrine cell types in the developing stomach and gut, showing that Hes-1 operates as a general negative regulator of endodermal endocrine differentiation.

Bioinformatics construction of the human cell surfaceome.

Cell surface proteins are excellent targets for diagnostic and therapeutic interventions. By using bioinformatics tools, we generated a catalog of 3,702 transmembrane proteins located at the surface of human cells (human cell surfaceome). We explored the genetic diversity of the human cell surfaceome at different levels, including the distribution of polymorphisms, conservation among eukaryotic species, and patterns of gene expression. By integrating expression information from a variety of sources, we were able to identify surfaceome genes with a restricted expression in normal tissues and/or differential expression in tumors, important characteristics for putative tumor targets. A high-throughput and efficient quantitative real-time PCR approach was used to validate 593 surfaceome genes selected on the basis of their expression pattern in normal and tumor samples. A number of candidates were identified as potential diagnostic and therapeutic targets for colorectal tumors and glioblastoma. Several candidate genes were also identified as coding for cell surface cancer/testis antigens. The human cell surfaceome will serve as a reference for further studies aimed at characterizing tumor targets at the surface of human cells.

Differential gene expression analysis of iodide-treated rat thyroid follicular cell line PCCl3.

The inhibitory effect of supraphysiological iodide concentrations on thyroid hormone synthesis (Wolff-Chaikoff effect) and on thyrocyte proliferation is largely known as iodine autoregulation. However, the molecular mechanisms by which iodide modulates thyroid function remain unclear. In this paper, we analyze the transcriptome profile of the rat follicular cell lineage PCCl3 under untreated and treated conditions with 10(-3) M sodium iodide (NaI). Serial analysis of gene expression (SAGE) revealed 84 transcripts differentially expressed in response to iodide (p

Species distribution modelling and cpSSR reveal population history of the Neotropical annual herb Tithonia rotundifolia (Asteraceae).

The impacts of the historical geologic and climatic events on the diversity and genetic structure of Neotropical taxa have recently become a subject of study. However, annual plants associated with tropical dry forests remain under-studied. The exploration of additional taxa in contrasting environments will improve the current understanding of responses of the Neotropical biota to these events. Here, we explore the species distribution and geographic structure of the annual herb Tithonia rotundifolia. We sampled 175 individuals from 19 populations of T. rotundifolia. Species distribution modelling and six microsatellite chloroplast loci were used to infer its population history. We identified areas of historical climate suitability and then tested if there is genetic structuring among these areas. Haplotypes showed strong phylogeographic structure. Historical climatic suitability areas were found along the Pacific coast; however, a gap was found at the Isthmus of Tehuantepec (IT). Although Bayesian analysis showed population structuring, amova revealed that the IT is not its main driver. Instead, a subdivision into a higher number of regions had higher FCT values. Also, populations to the east of the IT showed evidence of recent population expansion and migration in a south-north direction. Pleistocene climate fluctuations partially explain the geographic structure of T. rotundifolia. However, life-history characteristics such as limited seed dispersal and the patchy distribution of suitable habitats explain the high haplotype diversity and population sub-structuring and diversity. Lastly, the absence of geographic structure of some haplotypes may indicate long-distance dispersal, or hybridisation with the closely related T. tubaeformis.

Peptides isolated from animal venom as a platform for new therapeutics for the treatment of Alzheimer's disease.

Alzheimer's disease (AD) is a progressive neurodegenerative disease that deeply affects patients, their family and society. Although scientists have made intense efforts in seeking the cure for AD, no drug available today is able to stop AD progression. In this context, compounds isolated from animal venom are potentially successful drugs for neuroprotection, since they selectively bind to nervous system targets. In this review, we presented different studies using peptides isolated from animal venom for the treatment of AD. This is a growing field that will be very helpful in understanding and even curing neurodegenerative diseases, especially AD.

MRPL53, a New Candidate Gene for Orofacial Clefting, Identified Using an eQTL Approach.

A valuable approach to understand how individual and population genetic differences can predispose to disease is to assess the impact of genetic variants on cellular functions (e.g., gene expression) of cell and tissue types related to pathological states. To understand the genetic basis of nonsyndromic cleft lip with or without cleft palate (NSCL/P) susceptibility, a complex and highly prevalent congenital malformation, we searched for genetic variants with a regulatory role in a disease-related tissue, the lip muscle (orbicularis oris muscle [OOM]), of affected individuals. From 46 OOM samples, which are frequently discarded during routine corrective surgeries on patients with orofacial clefts, we derived mesenchymal stem cells and correlated the individual genetic variants with gene expression from these cultured cells. Through this strategy, we detected significant cis-eQTLs (i.e., DNA variants affecting gene expression) and selected a few candidates to conduct an association study in a large Brazilian cohort (624 patients and 668 controls). This resulted in the discovery of a novel susceptibility locus for NSCL/P, rs1063588, the best eQTL for the MRPL53 gene, where evidence for association was mostly driven by the Native American ancestry component of our Brazilian sample. MRPL53 (2p13.1) encodes a 39S protein subunit of mitochondrial ribosomes and interacts with MYC, a transcription factor required for normal facial morphogenesis. Our study illustrates not only the importance of sampling admixed populations but also the relevance of measuring the functional effects of genetic variants over gene expression to dissect the complexity of disease phenotypes.

Mechanism of insulin receptor kinase inhibition in non-insulin-dependent diabetes mellitus patients. Phosphorylation of serine 1327 or threonine 1348 is unaltered.

The tyrosine kinase activity of insulin receptor isolated from the skeletal muscle of NIDDM patients has previously been found to be decreased compared with the activity of receptor from nondiabetic subjects but the mechanism underlying this defect is unknown. Phosphorylation of receptor serine/threonine residues has been proposed to exert an inhibitory influence on receptor tyrosine kinase activity and Ser 1327 and Thr 1348 have been identified as specific sites of phosphorylation in the insulin receptor COOH terminal domain. To address the potential negative regulatory role of phosphorylation of these residues in vivo, we assessed the extent of phosphorylation of each site in insulin receptor isolated from the skeletal muscle of 12 NIDDM patients and 13 nondiabetic, control subjects. Phosphorylation of Ser 1327 and Thr 1348 was determined using antibodies that specifically recognize insulin receptor phosphorylated at these sites. In addition, a phosphotyrosine-specific antibody was used to monitor receptor tyrosine phosphorylation. The extent of insulin-induced tyrosine autophosphorylation was decreased in receptor isolated from diabetic versus nondiabetic muscle, thus confirming earlier reports. In contrast, there was no significant difference in the extent of phosphorylation of either Ser 1327 or Thr 1348 in receptor isolated from diabetic or nondiabetic muscle as assessed by immunoprecipitation (Ser 1327: 5.6 +/- 1.6% diabetics vs. 4.7 +/- 2.0% control; Thr 1348: 3.8 +/- 1.0% diabetics vs. 3.2 +/- 1.2% control). Moreover, within each group there was no correlation between the level of tyrosine kinase activity and the extent of serine/threonine phosphorylation. It is concluded that the stoichiometry of serine/threonine phosphorylation of insulin receptor in vivo is low, and that increased phosphorylation of Ser 1327 or Thr 1348 is not responsible for the decreased insulin receptor tyrosine kinase activity observed in the skeletal muscle of NIDDM patients.

Acute hyperglycemia provides an insulin-independent inducer for GLUT4 translocation in C2C12 myotubes and rat skeletal muscle.

GLUT4 translocation and activation of glucose uptake in skeletal muscle can be induced by both physiological (i.e., insulin, nerve stimulation, or exercise) and pharmacological (i.e., phorbol ester) means. Recently, we demonstrated that high glucose levels may mimic the effects of phorbol esters on protein kinase C (PKC) and insulin receptor function (J Biol Chem 269:3381-3386, 1994). In this study, we tested whether the previously described effects of phorbol esters on translocation of GLUT4 in myotubes in culture and also in rat skeletal muscle might be mimicked by glucose. We found that stimulation of C2C12 myotubes with both insulin (10(-7) mol/l, 5 min) and glucose (25 mmol/l, 10 min) induces a comparable increase of the GLUT4 content in the plasma membrane. To test whether this effect occurs in intact rat skeletal muscle as well, two different model systems were used. As an in vitro model, isolated rat hindlimbs were perfused for 80 min with medium containing 6 mmol/l glucose +/- insulin (1.6 x 10(-9) mmol/l, 40 min) or 25 mmol/l glucose. As an in vivo model, acute hyperglycemia (> 11 mmol/l glucose, 20 min) was induced in Wistar rats by intraperitoneal injection of glucose under simultaneous suppression of the endogenous insulin release by injection of somatostatin. In both models, subcellular fractions were prepared from hindlimb skeletal muscle, and plasma membranes were characterized by the enrichment of the marker enzyme alpha 1 Na(+)-K(+)-ATPase.(ABSTRACT TRUNCATED AT 250 WORDS)

Growth hormone and prolactin stimulate the expression of rat preadipocyte factor-1/delta-like protein in pancreatic islets: molecular cloning and expression pattern during development and growth of the endocrine pancreas.

GH and PRL have been shown to stimulate proliferation and insulin production in islets of Langerhans. To identify genes regulated by GH/PRL in islets, we performed differential screening of a complementary DNA library from neonatal rat islets cultured for 24 h with human GH (hGH). One hGH-induced clone had 96% identity with mouse preadipocyte factor-1 (Pref-1, or delta-like protein (Dlk)]. The size of Pref-1 messenger RNA (mRNA) in islets was 1.6 kilobases, with two less abundant mRNAs of 3.7 and 6.2 kilobases. The Pref-1 mRNA content of islets from adult rats was only 1% of that in neonatal islets. Pref-1 mRNA was markedly up-regulated in islets from pregnant rats from day 12 to term compared with those from age-matched female rats. Two peaks in mRNA expression were observed during gestation, one on day 14 and the other at term, whereafter it decreased to nonpregnant levels. Pref-1 mRNA was up-regulated 3- to 4-fold in neonatal rat islets of Langerhans after 48-h culture with hGH, as found also with bovine GH or ovine PRL. During the development of pancreas from embryonic day 12 (E12) to postnatal day 4, we observed a 2-fold increase in Pref-1 mRNA on E17 and a 5-fold increase at birth, followed by a rapid decline on postnatal day 4. Pref-1 immunoreactivity was found in a subpopulation of insulin cells of neonatal islets of Langerhans. At an early embryonal stage (E13), most cells of the pancreatic anlage were Pref-1 positive, becoming predominantly restricted to the insulin-producing cells during development. In conclusion, these findings suggest that Pref-1 is involved in both differentiation and growth of beta-cells.

Rat endocrine pancreatic development in relation to two homeobox gene products (Pdx-1 and Nkx 6.1).

We studied the distribution of the homeodomain proteins Pdx-1 and Nkx 6.1 in the developing rat pancreas. During early development, nuclear staining for both Pdx-1 and Nkx 6.1 occurred in most epithelial cells of the pancreatic anlage. Subsequently, Nkx 6.1 became more beta-cell-restricted, and Pdx-1 also occurred in other islet cell types and in the duodenal epithelium. During early pancreatic development, cells co-storing insulin and glucagon were regularly detected. The vast majority of these did not possess nuclear staining for either Pdx-1 or Nkx 6.1. Subsequently, cells storing insulin only appeared. Such cells displayed strongly Pdx-1- and Nkx 6.1-positive nuclei. Therefore, Nkx 6.1, like Pdx-1, may be an important factor in pancreatic development and in mature insulin cell function.

Developmental expression of proprotein convertase 1/3 in the rat.

We have isolated a clone that has 3' end sequence identity with prohormone convertase 1/3 (PC1/3) from a rat islet cDNA library. Northern blot analysis and immunocytochemical studies have confirmed its presence in the endocrine pancreas. Analysis of poly A mRNA from various adult tissues demonstrated that it was relatively abundant in whole brain, lung and spleen, but not detectable in kidney, testis and heart. Using probes consisting of either the coding region or the 3' end sequences, the mRNA transcripts identified were 5.0, 3.0 and 8.5 kb. The 8.5 kb transcript detected has not been described previously. RT-PCR of RNA isolated from rat embryonic tissues using a primer set corresponding to the 3' end of the PC1/3 sequence showed a steady increase of expression in fetal pancreas and intestine during the course of development. In contrast, comparatively high and constant levels of PC1/3 expression were detected in fetal lung, whereas low and constant expression was detected in fetal liver. Double immuno-staining showed that PC1/3 was co-localised with insulin throughout development, and at mid-gestation, PC1/3 immunoreactivity could also be detected within glucagon-producing cells in the developing pancreas. Thus, we have identified a novel PC1/3 mRNA transcript in the rat by using sequence-specific probes and have demonstrated that the developmental expression of prohormone convertase PC1/3 is confined primarily to pancreas and intestine, suggesting that it may play a possible role in regulating growth and differentiation of these tissues.

Pulmonary lymphoid lesions in an experimental model of collapsing glomerulopathy in rats.

UNLABELLED: The characterization of lung damage in an experimental model of collapsing glomerulopathy (CG) in rats is described. METHODS: 12 rats were divided into two groups and injected intravenously (iv) with 1 mg/saline in a final volume of 1 ml/ day in the tail vein for 5 days, with fractionated serum from control and CG subjects. Proteinuria was quantified, and the Glomerular filtration rate was calculated based on creatinine clearance (CC). Rats were sacrificed by perfusion fixation at day 5. RESULTS: Rats injected with serum from CG patients developed proteinuria (p<0.001). A decrease in CC (0.68+/-0.19) in these rats was also observed. Glomerular tuft retraction and mesangial proliferation was observed in all rats receiving serum from the CG patients. Peribronchiolar infiltrate integrated mainly by lymphocytes, was identified in all CG rats. In some areas this infiltration disrupted the basement membrane and damaged the epithelium. No histopathological abnormalities in the kidney or lungs were found in rats receiving control serum. CONCLUSION: Patchy pulmonary lymphoid infiltrates were found in the CG model. Up to now there was no information about pulmonary lymphoid infiltration in CG patients. Besides fluid overload due to renal insufficiency or a nephrotic syndrome, other causes of pulmonary involvement in CG patients should be explored.

Comparison of two wild rodent species as sentinels of environmental contamination by mine tailings.

BACKGROUND: Contamination with heavy metals is among the most hazardous environmental concerns caused by mining activity. A valuable tool for monitoring these effects is the use of sentinel organisms. Particularly, small mammals living inside mine tailings are an excellent study system because their analysis represents a realistic approach of mixtures and concentrations of metal exposure. PURPOSE: We analyzed metal tissue concentrations and DNA damage levels for comparison between genders of a sentinel (Peromyscus melanophrys) and a nonsentinel (Baiomys musculus) species. Also, the relationship between DNA damage and the distance from the contamination source was evaluated. METHODS: This study was conducted in an abandoned mine tailing at Morelos, Mexico. Thirty-six individuals from both species at the exposed and reference sites were sampled. Metal concentrations in bone and liver of both species were analyzed by atomic absorption spectrophotometry, and DNA damage levels were assayed using the alkaline comet assay. RESULTS: In general, concentrations of zinc, nickel, iron, and manganese were statistically higher in exposed individuals. A significant effect of the organ and the site on all metal tissue concentrations was detected. Significant DNA damage levels were registered in the exposed group, being higher in B. musculus. Females registered higher DNA damage levels than males. A negative relationship between distance from the mine tailing and DNA damage in B. musculus was observed. CONCLUSIONS: We consider that B. musculus is a suitable species to assess environmental quality, especially for bioaccumulable pollutants--such as metals--and recommend that it may be considered as a sentinel species.

Temporal blastemal cell gene expression analysis in the kidney reveals new Wnt and related signaling pathway genes to be essential for Wilms' tumor onset.

Wilms' tumors (WTs) originate from metanephric blastema cells that are unable to complete differentiation, resulting in triphasic tumors composed of epithelial, stromal and blastemal cells, with the latter harboring molecular characteristics similar to those of the earliest kidney development stages. Precise regulation of Wnt and related signaling pathways has been shown to be crucial for correct kidney differentiation. In this study, the gene expression profile of Wnt and related pathways was assessed in laser-microdissected blastemal cells in WTs and differentiated kidneys, in human and in four temporal kidney differentiation stages (i.e. E15.5, E17.5, P1.5 and P7.5) in mice, using an orthologous cDNA microarray platform. A signaling pathway-based gene signature was shared between cells of WT and of earliest kidney differentiation stages, revealing genes involved in the interruption of blastemal cell differentiation in WT. Reverse transcription-quantitative PCR showed high robustness of the microarray data demonstrating 75 and 56% agreement in the initial and independent sample sets, respectively. The protein expression of CRABP2, IGF2, GRK7, TESK1, HDGF, WNT5B, FZD2 and TIMP3 was characterized in WTs and in a panel of human fetal kidneys displaying remarkable aspects of differentiation, which was recapitulated in the tumor. Taken together, this study reveals new genes candidate for triggering WT onset and for therapeutic treatment targets.

Vanadium pentoxide inhalation provokes germinal center hyperplasia and suppressed humoral immune responses.

Vanadium, an important air pollutant derived from fuel product combustion, aggravates respiratory diseases and impairs cardiovascular function. In contrast, its effects on immune response are conflicting. The aim of our work was to determine if spleens of vanadium-exposed CD1 mice showed histological lesions that might result in immune response malfunction. One hundred and twelve CD-1 male mice were placed in an acrylic box and inhaled 0.02 M vanadium pentoxide (V2O5); actual concentration in chamber approximately 1.4 mg V2O5/m(3)) for 1 hr/d, twice a week, for 12 wk. Control mice inhaled only vehicle. Eight mice were sacrificed prior to the exposures. Eight control and eight V2O5-exposed mice were sacrificed 24 hr after the second exposure of each week until the 12-wk study was over. Another 8 mice that completed the 12-wk regimen were immunized with recombinant Hepatitis B surface antigen (HBsAg; three times over an 8-wk period) before sacrifice and analyses of their levels of anti-HBsAg antibody (HBSAb) using ELISA. In all studies, at sacrifice, blood samples were obtained by direct heart puncture and the spleen was removed, weighed and processed for H-E staining and quantitation of CD19 cells. The results indicated that the spleen weight of V2O5-exposed animals peaked at 9 wk (546 +/- 45 vs. 274 +/- 27 mg, p < 0.0001) and thereafter progressively decreased (321 +/- 39 mg at 12 wk, p < 0.001; control spleen = 298 +/- 35 mg). Spleens of V2O5-exposed animals showed an increased number of very large and non-clearly delimited germinal centers (that contained more lymphocytes and megakaryocytes) compared to those of control mice. In addition, their red pulp was poorly delimited and had an increase in CD19+ cells within hyperplasic germinal nodes. The mean HBsAb levels in immunized control mice were greater than that in the exposed hosts (i.e., OD = 0.39 +/- 0.03 vs. 0.11 +/- 0.05, p < 0.01). HBsAb avidity dropped to a value of 40 in V2O5-exposed animals vs. 86 in controls (p < 0.0001). We conclude that the chronic inhalation of V2O5, a frequent particle (PM(2.5)) component, induces histological changes and functional damage to the spleen, each of which appear to result in severe effects on the humoral immune response.

Matrix metalloproteinases 2 and 9 in central nervous system and their modification after vanadium inhalation.

Vanadium (V) derivatives are well-known environmental pollutants and its toxicity has been related with oxidative stress. Toxicity after vanadium inhalation on the substantia nigra, corpus striatum, hippocampus and ependymal epithelium was reported previously. The purpose of this study was to analyse the role of matrix metalloproteinases 2 (MMP-2) and 9 (MMP-9) in the changes observed in brain tissue after chronic V inhalation. Mice were exposed to vaporized, vanadium pentoxide 0.02 m in deionized water for 1 h twice a week, and killed at 1 h, 1, 2 and 4 weeks after exposure. The brain was removed and the olfactory bulb, prefrontal cortex, striatum and hippocampus were dissected and the MMP content was obtained by zymography. The results showed that MMP-9 increased in all the structures at the end of the exposure, although in the hippocampus this increment was evident after 1 week of exposure. When MMP-2 was analysed in the olfactory bulb and prefrontal cortex it remained unchanged throughout the whole exposure, while in the hippocampus it increased at week 4, while in the striatum MMP-2 increased from the second week only, through the whole experiment. These results demonstrate that V increased MMPs in different structures of the CNS and this change might be associated with the previously reported modifications, such as dendritic spine loss and neuronal cell death. The modifications in MMPs could be related with blood-brain barrier (BBB) disruption which was reported previously. Oxidative stress might also be involved in the activation of these gelatinases as part of the different mechanisms which take place in V toxicity in the CNS.

DNA damage as an early biomarker of effect in human health.

In the last few decades the need for new approaches to assess DNA damage has been increasing due to the implications that different insults on genetic material may have on human health. In this context, the identification of how chemical agents with different mechanisms of action (i.e., antineoplastic drugs) damage DNA provides a good model to investigate some cellular and molecular mechanisms underlying the basis of genetic toxicology. The nasal epithelium is the first barrier with which environmental pollutants interact, and for this reason this epithelium can be useful as a sentinel in order to assess the interactions between the environment and the living organisms. Taking these phenomena into account and using a simple, sensitive and rapid method such as the single cell gel electrophoresis, we could obtain information and an initial approach on the DNA status. This assay in combination with other techniques that provide more information about other molecular parameters could give us a better view of the biological status of the living cell.