Interactions between miRNAs and Double-Strand Breaks DNA Repair Genes, Pursuing a Fine-Tuning of Repair.

The repair of DNA damage is a crucial process for the correct maintenance of genetic information, thus, allowing the proper functioning of cells. Among the different types of lesions occurring in DNA, double-strand breaks (DSBs) are considered the most harmful type of lesion, which can result in significant loss of genetic information, leading to diseases, such as cancer. DSB repair occurs through two main mechanisms, called non-homologous end joining (NHEJ) and homologous recombination repair (HRR). There is evidence showing that miRNAs play an important role in the regulation of genes acting in NHEJ and HRR mechanisms, either through direct complementary binding to mRNA targets, thus, repressing translation, or by targeting other genes involved in the transcription and activity of DSB repair genes. Therefore, alteration of miRNA expression has an impact on the ability of cells to repair DSBs, which, in turn, affects cancer therapy sensitivity. This latter gives account of the importance of miRNAs as regulators of NHEJ and HRR and places them as a promising target to improve cancer therapy. Here, we review recent reports demonstrating an association between miRNAs and genes involved in NHEJ and HRR. We employed the Web of Science search query TS ("gene official symbol/gene aliases*" AND "miRNA/microRNA/miR-") and focused on articles published in the last decade, between 2010 and 2021. We also performed a data analysis to represent miRNA-mRNA validated interactions from TarBase v.8, in order to offer an updated overview about the role of miRNAs as regulators of DSB repair.

Sustained Activation of TNFα-Induced DNA Damage Response in Newly Differentiated Adipocytes.

The response to DNA damage is the mechanism that allows the interaction between stress signals, inflammatory secretions, DNA repair, and maintenance of cell and tissue homeostasis. Adipocyte dysfunction is the cellular trigger for various disease states such as insulin resistance, diabetes, and obesity, among many others. Previously, our group demonstrated that adipogenesis per se, from mesenchymal/stromal stem cells derived from human adipose tissue (hASCs), involves an accumulation of DNA damage and a gradual loss of the repair capacity of oxidative DNA damage. Therefore, our objective was to identify whether healthy adipocytes differentiated for the first time from hASCs, when receiving inflammatory signals induced with TNFα, were able to persistently activate the DNA Damage Response and thus trigger adipocyte dysfunction. We found that TNFα at similar levels circulating in obese humans induce a sustained response to DNA damage response as part of the Senescence-Associated Secretory Phenotype. This mechanism shows the impact of inflammatory environment early affect adipocyte function, independently of aging.

As-Cd-Pb Mixture Induces Cellular Transformation via Post-Transcriptional Regulation of Rad51c by miR-222.

BACKGROUND/AIMS: Exposure to heavy metals is today a threat to society. The understanding of the molecular processes related to diseases related to exposure to metals mixture involve changes in the expression of microRNAs. Changes on microRNAs expression may alter several cellular processes, among them, DNA repair inhibition has been described as an essential event leading to the initiation of metal-induced carcinogenesis. METHODS: We evaluate the miR-222 expression in the two-stage transformation Balb/c 3T3 cell assay treated with As-Cd-Pb mixture. RESULTS: We could appreciate that up-regulation of miR-222 reduces the expression both gene and as a protein expression of Rad51c by RT-PCR and immunoblot, respectively. CONCLUSION: Here, we demonstrate that the mixture of As-Cd-Pb at epidemiologically relevant concentrations induces miR-222 up-regulation, which directly negatively regulates Rad51c expression and impairs homologous recombination of DNA during the initiation stage of cell transformation. This inhibition triggers morphological transformation in a murine two-stage Balb/c 3T3 cell assay, suggesting that this small RNA acts as an initiator of the carcinogenesis process.

Glutathione depletion triggers actin cytoskeleton changes via actin-binding proteins.

The importance of glutathione (GSH) in alternative cellular roles to the canonically proposed, were analyzed in a model unable to synthesize GSH. Gene expression analysis shows that the regulation of the actin cytoskeleton pathway is strongly impacted by the absence of GSH. To test this hypothesis, we evaluate the effect of GSH depletion via buthionine sulfoximine (5 and 12.5 mM) in human neuroblastoma MSN cells. In the present study, 70% of GSH reduction did not induce reactive oxygen species, lipoperoxidation, or cytotoxicity, which enabled us to evaluate the effect of glutathione in the absence of oxidative stress. The cells with decreasing GSH levels acquired morphology changes that depended on the actin cytoskeleton and not on tubulin. We evaluated the expression of three actin-binding proteins: thymosin ß4, profilin and gelsolin, showing a reduced expression, both at gene and protein levels at 24 hours of treatment; however, this suppression disappears after 48 hours of treatment. These changes were sufficient to trigger the co-localization of the three proteins towards cytoplasmic projections. Our data confirm that a decrease in GSH in the absence of oxidative stress can transiently inhibit the actin binding proteins and that this stimulus is sufficient to induce changes in cellular morphology via the actin cytoskeleton.

Assessing genotoxicity of diuron on Drosophila melanogaster by the wing-spot test and the wing imaginal disk comet assay.

The aim of this study was to evaluate the genotoxicity of the herbicide diuron in the wing-spot test and a novel wing imaginal disk comet assay in Drosophila melanogaster. The wing-spot test was performed with standard (ST) and high-bioactivation (HB) crosses after providing chronic 48 h treatment to third instar larvae. A positive dose-response effect was observed in both crosses, but statistically reduced spot frequencies were registered for the HB cross compared with the ST. This latter finding suggests that metabolism differences play an important role in the genotoxic effect of diuron. To verify diuron's ability to produce DNA damage, a wing imaginal disk comet assay was performed after providing 24 h diuron treatment to ST and HB third instar larvae. DNA damage induced by the herbicide had a significantly positive dose-response effect even at very low concentrations in both strains. However, as noted for the wing-spot test, a significant difference between strains was not observed that could be related to the duration of exposure between both assays. A positive correlation between the comet assay and the wing-spot test was found with regard to diuron genotoxicity.

Metal mixture (As-Cd-Pb)-induced cell transformation is modulated by OLA1.

Environmental pollutants are complex mixtures in which metals are ubiquitous. Metal mixtures of arsenic, cadmium and lead are present in the occupational environment and generate health effects such as cardiovascular, renal and cancer diseases. Cell transformation induced by metal mixtures that depend on reactive oxygen species (ROS) generation, cell viability maintenance and avoidance of senescence was previously reported by our group. The aim of the present study was to explore the role of a Obg-like ATPase1 (OLA1) in the cell transformation of BALB/c 3T3 A31-1-1 clonal cells induced by a metal mixture (2 µM NaAsO2, 2 µM CdCl2 and 5 µM Pb(C2H3O2)2 3H2O) through ROS generation. The interest in OLA1 is justified because this protein has been proposed to be a negative regulator of the cellular antioxidant response. Small interfering RNA (siRNA) was used to knockdown OLA1 before the initiation stage of the transformation assay. We evaluated (ROS) and OLA1 protein expression throughout the initiation and promotion stages of transformation. OLA1 knockdown modulated metal mixture-induced cell transformation more strongly when the metal mixture was an initiator stimulus than when it was a promoter. The ability of the metal mixture to initiate cell transformation was diminished by OLA1 knockdown, an effect that depended on intracellular ROS levels. The effect of OLA1 was synergistic with N-Acetyl-l-cysteine (NAC) co-treatment. Oxidative stress-associated transcription factors Egr1 and Smad were also down-regulated by the OLA1 knockdown, contributing to the rescue of metal mixture cell transformation.

Oxidative stress during courtship affects male and female reproductive effort differentially in a wild bird with biparental care.

Oxidative stress has been suggested as one of the physiological mechanisms modulating reproductive effort, including investment in mate choice. Here, we evaluated whether oxidative stress influences breeding decisions by acting as a cost of or constraint on reproduction in the brown booby (Sula leucogaster), a long-lived seabird with prolonged biparental care. We found that during courtship, levels of lipid peroxidation (LP) of males and females were positively associated with gular skin color, a trait presumably used in mate choice, while levels of reactive oxygen species (ROS) were higher as laying approached and in early breeding pairs. Evidence of a constraining effect of oxidative stress for females was suggested by the fact that females with higher ROS during courtship laid smaller first eggs and had chicks with lower rates of body mass gain, and higher female LP was associated with lower offspring attendance time. No evidence of an oxidative cost of parental effort was found; from courtship to parental care, levels of ROS in males and females decreased, and changes in LP levels were non-significant. Finally, using a cross-fostering experiment we found that offspring ROS was unrelated to rearing and genetic parents' ROS. Interestingly, offspring LP was positively associated with the LP during courtship of both the rearing parents and the genetic father, suggesting that offspring LP might have both a genetic and an environmental component. Hence, in the brown booby, oxidative stress may be a cost of investment in reproductive traits before egg laying and constrain females' investment in eggs and parental care.

Causes of genome instability: the effect of low dose chemical exposures in modern society.

Genome instability is a prerequisite for the development of cancer. It occurs when genome maintenance systems fail to safeguard the genome's integrity, whether as a consequence of inherited defects or induced via exposure to environmental agents (chemicals, biological agents and radiation). Thus, genome instability can be defined as an enhanced tendency for the genome to acquire mutations; ranging from changes to the nucleotide sequence to chromosomal gain, rearrangements or loss. This review raises the hypothesis that in addition to known human carcinogens, exposure to low dose of other chemicals present in our modern society could contribute to carcinogenesis by indirectly affecting genome stability. The selected chemicals with their mechanisms of action proposed to indirectly contribute to genome instability are: heavy metals (DNA repair, epigenetic modification, DNA damage signaling, telomere length), acrylamide (DNA repair, chromosome segregation), bisphenol A (epigenetic modification, DNA damage signaling, mitochondrial function, chromosome segregation), benomyl (chromosome segregation), quinones (epigenetic modification) and nano-sized particles (epigenetic pathways, mitochondrial function, chromosome segregation, telomere length). The purpose of this review is to describe the crucial aspects of genome instability, to outline the ways in which environmental chemicals can affect this cancer hallmark and to identify candidate chemicals for further study. The overall aim is to make scientists aware of the increasing need to unravel the underlying mechanisms via which chemicals at low doses can induce genome instability and thus promote carcinogenesis.

Genetic structure and diversity of animal populations exposed to metal pollution.

Studying the genetic diversity of wild populations that are affected by pollution provides a basis for estimating the risks of environmental contamination to both wildlife, and indirectly to humans. Such research strives to produce both a better understanding of the underlying mechanisms by which genetic diversity is affected,and the long-term effects of the pollutants involved.In this review, we summarize key aspects of the field of genetic ecotoxicology that encompasses using genetic patterns to examine metal pollutants as environmental stressors of natural animal populations. We address genetic changes that result from xenobiotic exposure versus genetic alterations that result from natural ecological processes. We also describe the relationship between metal exposure and changes in the genetic diversity of chronically exposed populations, and how the affected populations respond to environmental stress. Further, we assess the genetic diversity of animal populations that were exposed to metals, focusing on the literature that has been published since the year 2000.Our review disclosed that the most common metals found in aquatic and terrestrial ecosystems were Cd, Zn, Cu and Pb; however, differences in the occurrence between aquatic (Cd=Zn>Cu>Pb>Hg) and terrestrial (Cu>Cd>Pb>Zn>Ni)environments were observed. Several molecular markers were used to assess genetic diversity in impacted populations, the order of the most common ones of which were SSR's > allozyme > RAPD's > mtDNA sequencing> other molecular markers.Genetic diversity was reduced for nearly all animal populations that were exposed to a single metal, or a mixture of metals in aquatic ecosystems (except in Hyalella azteca, Littorina littorea, Salmo trutta, and Gobio gobio); however, the pattern was less clear when terrestrial ecosystems were analyzed.We propose that future research in the topic area of this paper emphasizes seven key areas of activity that pertain to the methodological design of genetic ecotoxicological studies. Collectively, these points are designed to provide more accurate data and a deeper understanding of the relationship between alterations in genetic diversity of impacted populations and metal exposures. In particular, we believe that the exact nature of all tested chemical pollutants be clearly described, biomarkers be included, sentinel organisms be used, testing be performed at multiple experimental sites, reference populations be sampled in close geographical proximity to where pollution occurs, and genetic structure parameters and high-throughput technology be more actively employed. Furthermore, we propose a new class of biomarkers,termed "biomarkers of permanent effect," which may include measures of genetic variability in impacted populations.

A metal mixture induces transformation upon antioxidant depletion in a hepatic cell line.

INTRODUCTION: Metals are ubiquitous soil, air, and water pollutants. A mixture of arsenic cadmium and lead, in particular, has commonly been found in the vicinity of smelter areas. The mixture of As-Cd-Pb has been shown to be carcinogenic, and transforming potential and oxidative stress have been proposed as principal mechanisms involved in this process. The aim of this work was to explore the role of the antioxidant barrier in the establishment of cell transformation upon chronic exposure to a metal mixture containing 2 µM NaAsO(2), 2 µM. CdCl(2), and 5 µM Pb(C(2)H(3)O(2))(2)∙3H(2)O in WRL-68 cells-a non-transformed human hepatic cell line. MATERIAL AND METHODS: In this study, we used a WRL-68 cell model of human embryonic hepatic origin treated with antioxidant inhibitors (L-Buthionine-sulfoxamine and aminotriazole) to test the role of the antioxidant barrier in the establishment of cell transformation upon chronic exposure to a metal mixture of As-Cd-Pb (2 µM NaAsO(2), 2 µM CdCl(2) and 5 µM Pb(C(2)H(3)O(2))(2)∙3H(2)O). We evaluated oxidative damage markers, including reactive oxygen species, lipid peroxidation, and genotoxicity, as well as antioxidant response markers, including glutathione concentration, catalase activity, and superoxide dismutase activity, which promote morphological transformation, which can be quantified by foci formation. RESULTS: As expected, we found an increase in the intracellular concentration of the metals after treatment with the metal mixture. In addition, treatment with the metal mixture in addition to inhibitors resulted in a large increase in the intracellular concentration of cadmium and lead. Our results describe the generation of reactive oxygen species, cytotoxicity, genotoxicity, and oxidative damage to macromolecules that occurred exclusively in cells that were morphologically transformed upon exposure to a metal mixture and antioxidant barrier inhibition. CONCLUSION: Our results show the importance of the antioxidant barrier role in the protection of cellular integrity and the transformation potential of this metal mixture via free radicals.

Effect of Air Pollution on the Basal DNA Damage of Mother-Newborn Couples of México City.

Environmental pollution of megacities can cause early biological damage such as DNA strand breaks and micronuclei formation. Comet assay tail length (TL) reflects exposure in the uterus to high levels of air pollution, primarily ozone and air particles (PM10), including mothers' smoking habits during pregnancy, conditions which can lead to low birth weight. In this biomonitoring study, we evaluated basal DNA damage in the cord blood cells of newborn children from Mexico City. We found a correlation between DNA damage in mothers and their newborns, including various parameters of environmental exposure and complications during pregnancy, particularly respiratory difficulties, malformations, obstetric trauma, neuropathies, and nutritional deficiencies. Mothers living in the southern part of the city showed double DNA damage compared to those living in the northern part (TL 8.64 µm vs. 4.18 µm, p < 0.05). Additionally, mothers' DNA damage correlates with exposure to NOx (range 0.77-1.52 ppm) and PM10 (range 58.32-75.89 µg/m3), as well maternal age >29. These results highlight the sensitivity of the comet assay in identifying differential in utero exposure for newborns whose mothers were exposed during pregnancy. They also suggest the importance of antioxidants during pregnancy and the role of the placental barrier in protecting the newborn from the DNA-damaging effects of oxidative pollution.

Role of Ape1 in Impaired DNA Repair Capacity in Battery Recycling Plant Workers Exposed to Lead.

Exposure to lead in environmental and occupational settings continues to be a serious public health problem. At environmentally relevant doses, two mechanisms may underlie lead exposition-induced genotoxicity, disruption of the redox balance and an interference with DNA repair systems. The aim of the study was to evaluate the ability of lead exposition to induce impaired function of Ape1 and its impact on DNA repair capacity of workers chronically exposed to lead in a battery recycling plant. Our study included 53 participants, 37 lead exposed workers and 16 non-lead exposed workers. Lead intoxication was characterized by high blood lead concentration, high lipid peroxidation and low activity of delta-aminolevulinic acid dehydratase (δ-ALAD). Relevantly, we found a loss of DNA repair capacity related with down-regulation of a set of specific DNA repair genes, showing specifically, for the first time, the role of Ape1 down regulation at transcriptional and protein levels in workers exposed to lead. Additionally, using a functional assay we found an impaired function of Ape1 that correlates with high blood lead concentration and lipid peroxidation. Taken together, these data suggest that occupational exposure to lead could decrease DNA repair capacity, inhibiting the function of Ape1, as well other repair genes through the regulation of the ZF-transcription factor, promoting the genomic instability.

Induction of oxidative stress by low doses of lead in human hepatic cell line WRL-68.

Even though the molecular mechanisms by which lead induces toxicity and cancer have been intensely studied for many years, its carcinogenic mechanisms are not well understood yet. Several possible mechanisms have been examined to gain understanding on the carcinogenic properties of lead, which include mitogenesis, alteration of gene expression, and oxidative damage, among others. The aim of the present study was to explore the induction of oxidative damage at low lead concentrations using human embryonic hepatic cells WRL-68. Our results showed induction of reactive oxygen species, changes in the superoxide dismutase and catalase activity, as well as an induction of lipidperoxidation and DNA damage. However, after 5 weeks of exposure, these alterations returned to their basal levels. These results taking together indicate that at low concentrations, lead is able to establish an oxidative stress scenario; however under optimal antioxidant defense the oxidative scenario could be abolished through an adaptative process.

miR-27b-3p a Negative Regulator of DSB-DNA Repair.

Understanding the regulation of DNA repair mechanisms is of utmost importance to identify altered cellular processes that lead to diseases such as cancer through genomic instability. In this sense, miRNAs have shown a crucial role. Specifically, miR-27b-3 biogenesis has been shown to be induced in response to DNA damage, suggesting that this microRNA has a role in DNA repair. In this work, we show that the overexpression of miR-27b-3p reduces the ability of cells to repair DNA lesions, mainly double-stranded breaks (DSB), and causes the deregulation of genes involved in homologous recombination repair (HRR), base excision repair (BER), and the cell cycle. DNA damage was induced in BALB/c-3T3 cells, which overexpress miR-27b-3p, using xenobiotic agents with specific mechanisms of action that challenge different repair mechanisms to determine their reparative capacity. In addition, we evaluated the expression of 84 DNA damage signaling and repair genes and performed pathway enrichment analysis to identify altered cellular processes. Taken together, our results indicate that miR-27b-3p acts as a negative regulator of DNA repair when overexpressed.

DNA Integrity Estimated via the Comet Assay Reflects Oxidative Stress and Competitive Disadvantage in Developing Birds.

AbstractIncreases in DNA degradation have been detected in numerous situations in which organisms are exposed to pollutants. However, outside of the ecotoxicological literature, few studies have investigated whether there exists important variation in DNA integrity in free-living, healthy animals. Using the alkaline version of the comet assay to estimate DNA integrity in blood samples, we aimed to evaluate whether DNA integrity during early life is associated with nestlings' age, body mass, within-brood status, and oxidative stress using nestlings from a wild population of spotless starlings (Sturnus unicolor) as a model. We found important levels of variation in DNA integrity, suggesting the possibility that DNA integrity may have implications for offspring fitness. DNA integrity was dependent on the developmental stage, being lower at hatching than at the end of the nestling period. DNA integrity was also negatively related to the levels of oxidative damage at hatching and positively associated with wing length at fledging. In addition, position within the size hierarchy of the brood at fledging explained differences in DNA integrity, with higher levels in core than in marginal nestlings. Finally, despite extensive within-individual variation along nestling's age, we found DNA integrity during early life to be moderately repeatable within broods. Hence, DNA integrity in early life appears to be mainly affected by environmental factors, such as natural stressors. Our results suggest that measuring the variation in DNA integrity may be a fruitful approach for the assessment of individual fitness in natural populations and can be applied to studies in developmental biology and ecology.

Post-transcriptional regulation of Rad51c by miR-222 contributes cellular transformation.

DNA repair inhibition has been described as an essential event leading to the initiation of carcinogenesis. In a previous study, we observed that the exposure to metal mixture induces changes in the miR-nome of the cells that was correlated with the sub-expression of mRNA involved in processes and diseases associated with metal exposure. From this analysis, one of the miRNAs that shows changes in its expression is miR-222, which is overexpressed in various cancers associated with exposure to metals. In silico studies showed that a possible target for the microRNA-222 could be Rad 51c, a gene involved in the double-stranded DNA repair. We could appreciate that up-regulation of miR-222 reduces the expression both gene and as a protein expression of Rad51c by RT-PCR and immunoblot, respectively. A luciferase assay was performed to validate Rad51c as miR-222 target. Neutral comet assay was performed in order to evaluate DNA double-strand breaks under experimental conditions. Here, we demonstrate that miR-222 up-regulation, directly regulates Rad51c expression negatively, and impairs homologous recombination of double-strand break DNA repair during the initiation stage of cell transformation. This inhibition triggers morphological transformation in a two-stage Balb/c 3T3 cell assay, suggesting that this small RNA acts as an initiator of the carcinogenesis process.

MicroRNAs Regulate Metabolic Phenotypes During Multicellular Tumor Spheroids Progression.

During tumor progression, cancer cells rewire their metabolism to face their bioenergetic demands. In recent years, microRNAs (miRNAs) have emerged as regulatory elements that inhibit the translation and stability of crucial mRNAs, some of them causing direct metabolic alterations in cancer. In this study, we investigated the relationship between miRNAs and their targets mRNAs that control metabolism, and how this fine-tuned regulation is diversified depending on the tumor stage. To do so, we implemented a paired analysis of RNA-seq and small RNA-seq in a breast cancer cell line (MCF7). The cell line was cultured in multicellular tumor spheroid (MCTS) and monoculture conditions. For MCTS, we selected two-time points during their development to recapitulate a proliferative and quiescent stage and contrast their miRNA and mRNA expression patterns associated with metabolism. As a result, we identified a set of new direct putative regulatory interactions between miRNAs and metabolic mRNAs representative for proliferative and quiescent stages. Notably, our study allows us to suggest that miR-3143 regulates the carbon metabolism by targeting hexokinase-2. Also, we found that the overexpression of several miRNAs could directly overturn the expression of mRNAs that control glycerophospholipid and N-Glycan metabolism. While this set of miRNAs downregulates their expression in the quiescent stage, the same set is upregulated in proliferative stages. This last finding suggests an additional metabolic switch of the above mentioned metabolic pathways between the quiescent and proliferative stages. Our results contribute to a better understanding of how miRNAs modulate the metabolic landscape in breast cancer MCTS, which eventually will help to design new strategies to mitigate cancer phenotype.

DNA-AP sites generation by etoposide in whole blood cells.

BACKGROUND: Etoposide is currently one of the most commonly used antitumor drugs. The mechanisms of action proposed for its antitumor activity are based mainly on its interaction with topoisomerase II. Etoposide effects in transformed cells have been described previously. The aim of the present study was to evaluate the genotoxic effects of this drug in non-transformed whole blood cells, such as occurs as collateral damage induced by some chemotherapies. METHODS: To determine etoposide genotoxicity, we employed Comet assay in two alkaline versions. To evaluate single strand breaks and delay repair sites we use pH 12.3 conditions and pH >13 to evidence alkali labile sites. With the purpose to quantified apurinic or apyrimidine (AP) sites we employed a specific restriction enzyme. Etoposide effects were determined on whole blood cells cultured in absence or presence of phytohemagglutinin (PHA) treated during 2 and 24 hours of cultured. RESULTS: Alkaline (pH > 13) single cell gel electrophoresis (SCGE) assay experiments revealed etoposide-induced increases in DNA damage in phytohemaglutinine (PHA)-stimulated blood and non-stimulated blood cells. When the assay was performed at a less alkaline pH, 12.3, we observed DNA damage in PHA-stimulated blood cells consistent with the existence of alkali labile sites (ALSs). In an effort to elucidate the molecular events underlying this result, we applied exonuclease III (Exo III) in conjunction with a SCGE assay, enabling detection of DNA-AP sites along the genome. More DNA AP-sites were revealed by Exo III and ALSs were recognized by the SCGE assay only in the non-stimulated blood cells treated with etoposide. CONCLUSION: Our results indicate that etoposide induces DNA damage specifically at DNA-AP sites in quiescent blood cells. This effect could be involved in the development of secondary malignancies associated with etoposide chemotherapy.

Environmental and occupational biomonitoring using the Comet assay.

Biomonitoring of human populations exposed to potential mutagens or carcinogens can provide an early detection system for the initiation of cell disregulation in the development of cancer. In recent years, the Comet assay, also known as a "single cell gel" (SCG) electrophoresis assay, has become an important tool for assessing DNA damage in exposed populations. This is the method of choice for population-based studies of environmental and occupational exposure to air pollutants, metals, pesticides, radiation, and other xenobiotics as we show in this review. To appreciate the role of the Comet assay in the field of biomonitoring, we review data from 122 studies that employed the assay. These studies evaluated environmental versus occupational exposures and the levels of DNA damage in cells of individuals exposed in each case. Our review of the literature reveals the importance of the need to establish standard methodological conditions that affect unwinding and electrophoresis times and tail values (tail length, tail DNA, tail moment), with the goal of being able to compare data collected in different laboratories throughout the world. The Comet assay is susceptible to subtle artifacts of manipulation depending on the type and timing of sampling performed. Therefore, in the reporting of DNA damage detected by the Comet assay, the context of how the DNA damage was created also needs to be reported and considered in the interpretation of Comet assay results. The success of the Comet assay is reflected by its use over the past 20 years in the field of biomonitoring, and by the increasing number of studies that continue to report its use. As the shortcomings of the assay are identified and considered in the interpretation of DNA damage detection, the Comet assay will continue to provide improved reliability as a biomarker in human biomonitoring studies.

Cholesterol potentiates beta-amyloid-induced toxicity in human neuroblastoma cells: involvement of oxidative stress.

Alterations in brain cholesterol concentration and metabolism seem to be involved in Alzheimer's disease (AD). In fact, several experimental studies have reported that modification of cholesterol content can influence the expression of the amyloid precursor protein (APP) and amyloid beta peptide (Abeta) production. However, it remains to be determined if changes in neuronal cholesterol content may influence the toxicity of Abeta peptides and the mechanism involved. Aged mice, AD patients and neurons exposed to Abeta, show a significant increase in membrane-associated oxidative stress. Since Abeta is able to promote oxidative stress directly by catalytically producing H(2)O(2) from cholesterol, the present work analyzed the effect of high cholesterol incorporated into human neuroblastoma cells in Abeta-mediated neurotoxicity and the role of reactive oxygen species (ROS) generation. Neuronal viability was studied also in the presence of 24S-hydroxycholesterol, the main cholesterol metabolite in brain, as well as the potential protective role of the lipophilic statin, lovastatin.