DNA Damage in Bat Blood Leukocytes Using a Chromatin Dispersion Test (CDT): Biomarker of Environmental Genotoxicity.

Environmental pollutants produce adverse effects on organisms and ecosystems. Biomonitoring and biomarkers offer a reasonable approach to make these assessments. Induced genetic changes can be using as a biomarker in organisms that react to a given compound in the ecosystem. Monitoring environmental genotoxicity necessitates the choice of model animals known as "sentinels or biological monitors" and the suitability of validated tests for DNA damage evaluation. We aimed to estimate the DNA damage produced by thermal stress in the leukocytes of the Mexican free-tailed bat (Tadarida brasiliensis). The DNA damage in bat leukocytes exposed to different temperatures (35 °C, 45 °C, and 55 °C) was evaluated by the adapted chromatin dispersion test (CDT) and the results were confirmed by the alkaline comet test. The CDT permitted a clear representation of leukocytes with fragmented DNA and of nonfragmented DNA. In addition, we detected nuclear anomalies in relation to cell death cellular swelling, nuclear fragmentation, and chromatin lysis. The alkaline comet assay revealed that the halos of diffuse chromatin include fragmented DNA. The assay of the method employing the CDT is well established, precise, and cost-effective for the routine quantitative analysis of DNA damage on the effect of the leukocytes of bats exposed to thermal stress. This could also apply as a sensitive screening tool for the evaluation of genotoxicity in environmental protection programs.

Metal Contents in Feathers from Birds (Rhynchopsitta terrisi, and Columba livia) with Different Ecological Niches.

The metal contaminants can be utilized as an ecological tool to analyze niche partition in birds. As environmental contamination biological indicators, essential (Zn, Cu, and Cr) and non-essential (Pb and Cd) metals in the flight feathers of the Maroon-fronted Parrot and Pigeon with different ecological niches were assessed. The feathers of the parrot were gathered at a national park (Parque Nacional Cumbres de Monterrey) and the feathers of pigeons were collected at an urban site, that is, the city of Monterrey, Mexico. An atomic absorption spectrophotometer was used to establish the concentration of metals in the feathers. Zn, Cu, Cr, Pb, and Cd were detected in the two studied samples. The results obtained in this study exhibited an increase in metal concentrations in pigeon feathers with respect to parrot feathers. In conclusion, employing parrot and pigeon feathers comprises an important tool to track trace-metal occurrence in the environment and metal accumulation in birds. This information is crucial to possess in order to minimize exposure to essential metals in species of wild birds with different ecological niches.

Thermal stress induces pyknosis in pigeon erythrocytes: digital image analysis.

Pyknosis or hypercondensation of chromatin is informative in the understanding of nucleosomal packing in translationally inactive chromatin and in the compression of cell death. However, mechanisms that result in the formation of avian erythrocytes with variant nuclear morphology are poorly understood.Purpose: In this work, we evaluated pyknosis in pigeon erythrocytes treated with thermal stress using Digital Image Analysis (DIA).Materials and methods: Pigeon erythrocytes were treated at thermal stress (33 °C, 43 °C, and 53 °C), and nuclear modifications were analyzed by DIA.Results: Our results showed that thermal stress induced DNA condensation. Based on DNA fluorescent staining and compaction, four subclasses with progressively more pyknotic nuclei each could be distinguished. Alkaline comet assay showed that the presence of pyknotic nuclei was associated with the DNA fragmentation typical of apoptosis. DIA analysis showed a decrease of nuclear area and a significant increase of fluorescence intensity with respect to non-pyknotic nucleus. Additionally we observed nuclear dissolution events associated with swell and loose membrane integrity.Conclusion: These findings can contribute to the evaluation of health and metabolic status in diagnostic cytology, especially in neoplastic conditions and infection by microorganisms.

Quick assessment of DNA damage in cervical epithelial cells using a chromatin dispersion test.

PURPOSE: This study was aimed to quantify genomic DNA breakages in the cervical epithelium cells of patients diagnosed with different grades of cervical lesions using a quick test based on chromatin dispersion after controlled protein depletion. The association between the progressive stages of cervical dysplasia and the levels of DNA damage, taking into account the presence of papillomavirus human (HPV) infection, was investigated. METHODS: A hospital-based unmatched case-control study was conducted during 2018 with a sample of 78 women grouped according to histological diagnosis as follows: 23 women with low grade-squamous intraepithelial lesion (LG-SIL), 34 women with high grade- squamous intraepithelial lesion (HG-SIL), and three women with cervical carcinoma (CC). In parallel, 15 women without cervical lesions were included as a Control cohort. DNA damage levels in cervical epithelial cells were assessed using the chromatin dispersion test (CDT) and controlled in parallel with DNA breakage detection coupled with florescent in situ hybridization (DBD‒FISH) using whole genomic DNA probes. RESULTS: CDT produces different morphotypes in the cervical epithelium that can be associated with the level of DNA breakage revealed with DBD‒FISH. A significant increase of DNA damage was correlated with the histological progression of the patients and human papillomavirus (HPV) infection. CONCLUSION: The CDT is a simple, accurate and inexpensive morphological bioassay to identify different levels DNA damage that can be associated with the level of abnormal cells present in the cervical epithelium in patients who commonly present HPV infection.

DBD-FISH, an effective marker for detecting genotoxicity in buccal mucosa exfoliated cells of patients with oral cancer.

Oral squamous cell carcinoma (OSCC) is characterized by increased genetic instability as an essential variable of event of neoplastic transformation. The aim of this study was to evaluate genomic instability in exfoliated cells from the buccal mucosa of patients with OSCC vs. the control group, using DNA Breakage Detection/Fluorescence In Situ hybridization (DBD-FISH). Exfoliated cells from the buccal mucosa were obtained from 38 patients with oral cancer (case group) and from 10 individuals without oral lesions (control group). DNA damage was evaluated by DBD-FISH using the whole-genome DNA probe and digital imaging analysis. Collaterally, HPV infection was determined utilizing the INNO-LiPA HPV kit. Patients with OSCC showed an increase in the hybridization signal five times more intense than that of the baseline level of DNA damage detected in control individuals. The best cutoff value for predicting oral squamous cell carcinoma was 67.46, and an Odds Ratio (OR) value of 87. HPV detection analysis revealed than one patient with OSCC (2.6%) was positive for HPV. All controls were negative HPV. In conclusion, DBD-FISH permitted the clear visualization of level high of DNA damage in the buccal epithelial cells of patients with OSSC respect to control group. Chromosome instability in oral mucosa may be an individual marker of malignant transformation in OSCC.

Evaluation of the genotoxic effect of heavy metals in pigeons from urban and rural habitat in Monterrey, Mexico, using the chromatin dispersion assay.

PURPOSE: Evaluate genotoxic effect of heavy metals on Pigeon Erythrocytes (PE) from urban and rural habitat (outside of the city) in Monterrey, Mexico, using the chromatin dispersion assay. MATERIALS AND METHODS: We quantified metals concentrations (Cd, Hg, Cu and Pb) in tail feathers of 22 pigeons from an urban and a rural site in northeastern Mexico. DNA damage in peripheral blood erythrocytes was measured by chromatin dispersion assay in 13 pigeon living in urban habitat and in nine living in rural habitat as the control. MicroNucleus (MN) test was used to confirm levels of DNA damage. RESULTS: Birds in urban habitat had highest concentrations in feathers for all the metallic elements analysed with respect to birds in rural habitat. Concentrations of Cu and Hg showed a significant increase (p < 0.05). Our results showed a significant increase of DNA damage in urban-habitat pigeons compared with that of pigeons in rural area. These results were confirmed by a MN test. CONCLUSIONS: Our preliminary findings demonstrate that PE examination via chromatin dispersion assay is a reliable, precise and inexpensive morphological bioassay for evaluating environmental genotoxicity associated with heavy metals. Further studies for evaluating the individual participation of contaminants in DNA damage are needed.

Detection of DNA damage in pigeon erythrocytes using a chromatin dispersion assay.

The monitoring of environmental genotoxicity requires the selection of model organisms as 'sentinels' as well as the development of sensitive and reliable tests for the assessment of DNA damage. The aims of this study were to quantify genomic DNA strand breakage in the erythrocytes of Columba livia induced by thermal stress using the modified chromatin dispersion test and to validate the results by alkaline comet assay and DNA breakage detection-fluorescence in situ hybridization (DBD-FISH). The chromatin dispersion test allowed for clear visualization of erythrocyte cells with DNA damage and of cells with no DNA damage. DNA damage increased significantly with increase in temperature. Additionally, we observed nuclear abnormalities associated with apoptosis, such as karyorrhexis (nuclear disintegration) and karyolysis (nuclear dissolution). These results were validated by alkaline comet assay and DBD-FISH. In conclusion, this procedure is a reliable, precise, and inexpensive morphological bioassay for routine quantitative analysis of DNA breakage in pigeon erythrocytes induced by thermal stress. This method could also be useful as a practical screening tool for genotoxicity testing in environmental care.

Reduced sperm DNA longevity is associated with an increased incidence of still born; evidence from a multi-ovulating sequential artificial insemination animal model.

PURPOSE: Using a rabbit model, we assessed the influence of sperm DNA longevity on female reproductive outcomes. METHODS: Semen was collected from 40 bucks, incubated at 38 °C for 24 h, and the rate of sperm DNA fragmentation (rSDF) was determined using the sperm chromatin dispersion assay. Males were allocated into high rSDF (>0.5 units of increase per hour) or low rSDF (<0.5 units of increase per hour) groups. High and low rSDF semen samples were sequentially artificially inseminated into the same doe to reduce female factor variability, and pregnancy outcomes were recorded. RESULTS: While there was no difference in SDFs between rSDF groups immediately after collection (T0), differences were significant after 2 h of incubation; SDFs determined at collection and rSDF behaved as independent characters (Pearson correlation = 0.099; P = 0.542). Following artificial insemination, the rate of stillborn pups was significantly higher in does inseminated by males with a high rSDF (14/21) compared to those with low rSDF (15/6); (contingency χ(2) 5.19; p = 0.022). The risk of stillborn when low rSDF rabbits were used for insemination was 0.16, but increased to 0.36 when high rSDF animals were used (odds ratio = 2.85; 95 % confidence interval = 1.4-2.7). CONCLUSION(S): Dynamic assessment of SDF coupled with natural multiple ovulation, high fecundity of the rabbit and control over female factor influence, provided a useful experimental model to demonstrate the adverse effect of reduced sperm DNA longevity on reproductive outcome.

DNA damage in spermatozoa from infertile men with varicocele evaluated by sperm chromatin dispersion and DBD-FISH.

PURPOSE: Evaluation of DNA integrity is an important test, possessing greater diagnostic and prognostic significance for couples requiring assisted reproduction. In this study, we evaluate the levels of DNA damage in infertile patients with varicocele with respect to fertile males by the sperm chromatin dispersion (SCD) test. The presence of DNA breaks in spermatozoa was confirmed by DNA breakage detection-fluorescence in situ hybridization (DBD-FISH). METHODS: In this study, the frequency of sperm cells with fragmented DNA was studied in a group of 20 infertile patients with varicocele and compared with 20 fertile males. The spermatozoa were processed to classify different levels of DNA fragmentation using the Halosperm(®) kit, an improved SCD test, and DBD-FISH. RESULTS: Patients with varicocele showed 25.54 ± 28.17 % of spermatozoa with fragmented DNA, significantly higher than those of the group of fertile subjects (11.54 ± 3.88 %). The proportion of degraded cells in total sperm cells with fragmented DNA was sixfold higher in the case of patients with varicocele. The presence of DNA breaks in spermatozoa was confirmed by DBD-FISH. 5-bp Classical satellite-2 regions showed greater sensitivity to damage or "breakage" than alphoid satellite regions. CONCLUSIONS: Our finding preliminary demonstrated an increase of DNA fragmentation associated to severe sperm damage, in infertile patients with varicocele with respect to fertile males. 5-bp Classical satellite-2 regions showed greater sensitivity to damage or "breakage" than alphoid satellite regions.

Evaluation of environmental genotoxicity by comet assay in Columba livia.

The concentrations of recognized or suspected genotoxic and carcinogenic agents found in the air of large cities and, in particular, developing countries, have raised concerns about the potential for chronic health effects in the populations exposed to them. The biomonitoring of environmental genotoxicity requires the selection of representative organisms as "sentinels," as well as the development of suitable and sensitive assays, such as those aimed at assessing DNA damage. The aim of this study was to evaluate DNA damage levels in erythrocytes from Columba livia living in the metropolitan area of Monterrey, Mexico, compared with control animals via comet assay, and to confirm the results via Micronuclei test (MN) and DNA breakage detection-fluorescence in situ hybridization (DBD-FISH). Our results showed a significant increase in DNA migration in animals from the area assayed compared with that observed in control animals sampled in non-contaminated areas. These results were confirmed by MN test and DBD-FISH. In conclusion, these observations confirm that the examination of erythrocytes from Columba livia via alkaline comet assay provides a sensitive and reliable end point for the detection of environmental genotoxicants.

[Maternal perception of her child's weight and unrelated children less than 1 year old]. / Percepción materna del peso del hijo y de niños no emparentados menores de un año.

AIMS: To evaluate the maternal perception of their child's weight (MPCW) and perception of unrelated children's weight. DESIGN: Cross-sectional. LOCATION: Maternal and Child Nursing Health Department at 6 Units of Family Medicine. PARTICIPANTS: 486 dyads (mother and child under 1 year). MAIN MEASUREMENTS: The following question was applied: "I think my child is", and images were provided according the child's gender. Children's weight and height were measured. RESULTS: A total of 20.5% of the mothers of overweight (OW) children accurately perceived this situation, while none of the mothers of obese (OB) children did (κ=0.14±0.03, Z=5.36, p=.001). By images, 63.3% of mothers of OW children and 33.3% of mothers of OB children perceived this situation (κ=0.01±0.02, Z=0.73, p=.46). Most mothers selected the image of OW child as the image of a healthy child (κ=-0.04±0.01, Z=-2.65, p=.008), the image of a child under 1 year (κ=-0.01±0.02, Z=-0.86, p=.38) and the image that they would like their child to look like (κ=0.0004±0.01, Z=0.02, p=.98). CONCLUSION: The mothers do not perceive the OW-OB of their children.

Human papillomavirus infections in Mexican women with normal cytology, precancerous lesions, and cervical cancer: type-specific prevalence and HPV coinfections.

The prevalence and genotype distribution of human papillomavirus (HPV) provides the basis for designing HPV prevention programs. The prevalence rates of type-specific HPV and coinfections in samples of Mexican women were investigated in 822 women aged 18-87 years. HPV detection was performed using a Linear Array™ genotyping test. HPV infection was found in 12.4% of controls, 46.3% of those with cervical intraepithelial neoplasia 1, and 100% of those with cervical intraepithelial neoplasia 3 or cervical cancer. HPV 16 was the most prevalent type in all diagnosis groups. The HPV types most frequently found in cervical cancers were 16, 18, 45, 52, 58, and 39; HPV types 16, 62, 51, 84, 18, 53, and CP6108 were the most prevalent in control women. Considering HPV-positive samples only, coinfections occurred most often in controls (63%) and were less frequent in those with cervical cancer (26%). The most frequent viral types in coinfections with HPV 16 in control women were HPV 62, 51, and 84; in women with cervical cancers, HPV 18, 39, and 70 were most common. In conclusion, in addition to HPV types 16 and 18, types 45, 39, 58, 52, and 71 were found in cervical cancers in Mexican women (78%); among them, only 65% were attributable to HPV types 16 and 18. Therefore, it is necessary to consider these viral types in the design of new vaccines, and to determine whether certain HPV types coinfecting with HPV 16 in precursor lesions determine tumor progression or regression.

Use of the DBD-FISH technique for detecting DNA breakage in response to high doses of X-rays.

The aim of this study was to generate a dose-response curve using the DNA breakage detection-fluorescent in situ hybridization (DBD-FISH) test as a biomarker of initial genetic effects induced by high doses of X-rays. A dose-response curve was obtained by measuring the ex vivo responses to increasing doses (0-50 Gy) of X-rays in the peripheral blood lymphocytes of ten healthy donors. The overall dose-response curve was constructed using integrated density (ID; area × fluorescence intensity) as a measure of genetic damage induced by irradiation. The correlation coefficient was high (r = 0.934, b(0) = 10.408, and b(1) = 0.094). One-way ANOVA with the Student-Newman-Keuls test for multiple comparisons showed significant differences among the average ln ID values according to dose. Our results suggest the usefulness of the DBD-FISH technique for measuring intrinsic individual cellular radio sensitivity ex vivo.

DBD-FISH Using Specific Chromosomal Region Probes for the Study of Cervical Carcinoma Progression.

Genomic instability is an important biomarker in the progression of cervical carcinoma. DBD-FISH (DNA breakage detection-fluorescence in situ hybridization) is a sensitive method that detects strand breaks, alkali-labile sites, and incomplete DNA excision repair in cells of the cervical epithelium. This technique integrates the microgel immersion of cells from a vaginal lesion scraping and the DNA unwinding treatment with the capacity of FISH integrated into digital image analysis. Cells captured within an agarose matrix are lysed and submerged in an alkaline unwinding solution that generates single-stranded DNA motifs at the ends of internal DNA strand breaks. After neutralization, the microgel is dehydrated and the cells are incubated with DNA-labeled probes. The quantity of a hybridized probe at a target sequence corresponds to the measure of the single-stranded DNA produced during the unwinding step, which is equivalent to the degree of local DNA breakage. DNA damage does not show uniformly throughout the entire DNA of a cell; rather, it is confined to specific chromosomal sites. In this chapter, an overview of the technique is supplied, focusing on its ability for assessing the association between DNA damage in specific sequences and in the progressive stages of cervical carcinoma.

5-bp Classical Satellite DNA Loci from Chromosome-1 Instability in Cervical Neoplasia Detected by DNA Breakage Detection/Fluorescence in Situ Hybridization (DBD-FISH).

We aimed to evaluate the association between the progressive stages of cervical neoplasia and DNA damage in 5-bp classical satellite DNA sequences from chromosome-1 in cervical epithelium and in peripheral blood lymphocytes using DNA breakage detection/fluorescence in situ hybridization (DBD-FISH). A hospital-based unmatched case-control study was conducted in 2011 with a sample of 30 women grouped according to disease stage and selected according to histological diagnosis; 10 with low-grade squamous intraepithelial lesions (LG-SIL), 10 with high-grade SIL (HG-SIL), and 10 with no cervical lesions, from the Unidad Medica de Alta Especialidad of The Mexican Social Security Institute, IMSS, Mexico. Specific chromosome damage levels in 5-bp classical satellite DNA sequences from chromosome-1 were evaluated in cervical epithelium and peripheral blood lymphocytes using the DBD-FISH technique. Whole-genome DNA hybridization was used as a reference for the level of damage. Results of Kruskal-Wallis test showed a significant increase according to neoplastic development in both tissues. The instability of 5-bp classical satellite DNA sequences from chromosome-1 was evidenced using chromosome-orientation FISH. In conclusion, we suggest that the progression to malignant transformation involves an increase in the instability of 5-bp classical satellite DNA sequences from chromosome-1.

Chromatin Dispersion Test to Asses DNA Damage in Cervical Epithelial Cells.

The chromatin dispersion test (CDT) is based on the removal of nuclear proteins under the assumption that cells with fragmented DNA produce a typical halo of circular DNA loops, which is absent in cells with non-fragmented DNA. This method represents a simple, rapid, accurate, highly reproducible, and inexpensive technique to assess nuclear DNA damage in somatic cells. The visualization of DNA damage and the capacity of the test to provide a threshold value to discriminate between high and low levels of cervical lesions would aid in determining the malignant transformation. All of these advantages associated with the CDT protocol could promote this technique as a tool for the quick and reliable diagnosis of cervical epithelial disorders, even at primary-care centers.

Evaluation of DNA damage in obese children using the chromatin dispersion test.

Childhood obesity predicts adult obesity and may increase the lifetime risk of adverse health outcomes. Obesity is characterized by oxidative stress that can induce DNA damage; however, studies of childhood and adolescent obesity are scarce. We investigated DNA damage due to obesity in Mexican children using the chromatin dispersion test (CDT). We evaluated DNA damage to peripheral lymphocytes of 32 children grouped according to body mass index as normal weight (controls), overweight and obese groups using guidelines from the Centers for Disease Control (CDC). We found that the greatest DNA damage occurred in cells of obese children compared to normal weight and overweight children. Our findings support preventive action to obviate adverse health outcomes due to obesity.

Evaluation of DNA single and double strand breaks in women with cervical neoplasia based on alkaline and neutral comet assay techniques.

A hospital-based unmatched case-control study was performed in order to determine the relation of DNA single (ssb) and double (dsb) strand breaks in women with and without cervical neoplasia. Cervical epithelial cells of 30 women: 10 with low grade squamous intraepithelial lesions (LG-SIL), 10 with high-grade SIL (HG-SIL), and 10 without cervical lesions were evaluated using alkaline and neutral comet assays. A significant increase in global DNA damage (ssb + dsb) and dsb was observed in patients with HG-SIL (48.90 ± 12.87 and 23.50 ± 13.91), patients with LG-SIL (33.60 ± 14.96 and 11.20 ± 5.71), and controls (21.70 ± 11.87 and 5.30 ± 5.38; resp.). Pearson correlation coefficient reveled a strong relation between the levels ssb and dsb (r(2) = 0.99, P = 0.03, and r(2) = 0.94, P = 0.16, resp.) and progression of neoplasia. The increase of dsb damage in patients with HG-SIL was confirmed by DNA breakage detection-FISH (DBD-FISH) on neutral comets. Our results argue in favor of a real genomic instability in women with cervical neoplasia, which was strengthened by our finding of a higher proportion of DNA dsb.

Entamoeba histolytica: soluble and membrane-associated neutral sphingomyelinase-C and other unidentified esterase activity.

Sphingomyelinase (SMase) activity was measured in Entamoeba histolytica particulate and soluble subcellular fractions. The effects on SMase of incubation time, total protein concentration, pH, and several divalent cations were determined. SMase-C and other unidentified esterase activity were detected in soluble and particulate fractions. SMase-C was 94.5-96.0% higher than the unidentified esterase activity. Soluble and insoluble SMase-C specific activities increased with protein dose and incubation time. Soluble and insoluble SMase-C activities were maximum at pH 7.5 and were dependent on Mg(2+), Mn(2+), or Co(2+), and inhibited by Zn(2+), Hg(2+), Ca(2+), and EDTA. SMase-C was active in the pH range of 3-10 and its maximum activity was at pH 7.5. The soluble and insoluble SMases have remarkably similar physicochemical properties, strongly suggesting that E. histolytica has just one isoform of neutral SMase-C that had not been described before and might be essential for E. histolytica metabolism or virulence.

Chromosomal abnormalities and polymorphic variants in couples with repeated miscarriage in Mexico.

Cytogenetic studies have an important role in the evaluation of couples with repeated miscarriages and poor obstetric history. To estimate the prevalence of chromosomal abnormalities and polymorphic variants in 158 couples with repeated miscarriages, a cross-sectional study was conducted in Monterrey, Mexico from 1995 to 2003. Peripheral blood lymphocytes were cultured for chromosomal studies using standard methods. Twelve couples showed chromosomal abnormalities (7.60%), two Robertsonian translocations (1.27%), two balanced translocations (1.27%), one inversion (0.63%), and one a novel insertion (0.63%). This insertion [46, XX, ins (15;8) (q26;p11p23)] is unique, and is the third reported in association with repeated abortion. Mosaicism was observed in six couples (3.80%, three with structural abnormalities and three with numerical abnormalities). A female to male ratio of 1.4:1 was observed. In addition to these chromosomal abnormalities, polymorphic variants in constitutive heterochromatin of the 1qh+, 9qh+, and 16qh+ chromosomes were observed in 25 couples (15.82%), of the Yqh+ chromosome in 21 couples (13.29%), and of satellite in 35 couples (22.15%). In conclusion, chromosome analysis is necessary for appropriate clinical management of these patients.