1p36 is a chromosomal site of genomic instability in cervical intraepithelial neoplasia.

We investigated the association between progressive stages of cervical neoplasia and DNA damage in 1p36 DNA sequences of chromosome 1 in cervical epithelium using DNA breakage detection/fluorescence in situ hybridization (DBD-FISH). We used a hospital based unmatched case control study of 29 women that were grouped according to disease stage and selected according to histological diagnosis: 10 with low grade squamous intraepithelial lesions (LG-SILs), 10 with high grade SILs (HG-SILs) and nine with no cervical lesions; the 1pter sequence was used as internal control. We found a significant increase in the number of patients with HG-SIL compared to patients with LG-SILs or with no cervical lesions. 1p36 Genomic instability was validated by DBD-FISH using neutral comets. Genetic instability at specific gene loci, such as 1p36, might be characteristic of cervical cancer progression. DBD-FISH appears to be a useful approach for detecting and comparing damage to specific chromosomal regions related to the progression of cervical cancer.

Evaluation of oxidative DNA damage in pigeon erythrocytes using DNA breakage detection-fluorescence in situ hybridization (DBD-FISH).

DNA breakage detection-fluorescence in situ hybridization (DBD-FISH) enables detection and quantification of DNA breakage in the entire genome or within specific DNA sequences in single cells. We used this method to visualize and evaluate DNA damage in pigeon erythrocytes that were induced by elevated temperature and hydrogen peroxide. We also examined morphological changes in the cell nuclei. DBD-FISH demonstrated a significant increase of DNA damage in a temperature dependent manner, which resulted in nuclear abnormalities associated with apoptotic cells. These cells gave strong nuclear fluorescent signals that indicated cell death.

The presence of human papillomavirus in semen does not affect the integrity of sperm DNA.

It remains unknown whether human papillomaviruses (HPVs) in semen affect sperm DNA integrity. We investigated whether the presence of these viruses in semen was associated with an elevated sperm DNA fragmentation index. Semen samples of 22 normozoospermic patients undergoing infertility treatment, nine fertile donors and seven fertile men with a risk of HPV infection (genital warts or condylomas) were included in the study. The samples were examined by an INNO-LiPA test PCR-based reverse hybridisation array that identifies 28 types of HPVs as simple or multiple infections. Sperm DNA integrity was determined by sperm chromatin dispersion assay (SCD). Our preliminary findings demonstrate an increase in HPV infection in infertile men with respect to fertile men. However, the sperm DNA fragmentation index was not increased in semen containing these viruses.

Absence of toxicity and genotoxicity in an extract of Rubus coriifolius.

Rubus coriifolius Focke is a wild plant from the Rosaceae family. It grows in both Guatemala and Mexico. The polar extract of the aerial parts of this plant has antibacterial, anti-inflammatory, and anti-protozoal activities. These properties may explain the traditional use of this plant. In vivo and in vitro assays were used to assess the genotoxic and toxic effects of an ethanol extract of the aerial parts of R. coriifolius. Three groups of rats were orally administered the R. coriifolius extract diluted in ethanol (5%) at doses of 1.89 mg/kg body weight (low dose), 4.72 mg/kg body weight (medium dose), and 9.44 mg/kg body weight (high dose) for 3 weeks. Genotoxic/cytotoxic effects induced by the R. coriifolius ethanol extract were evaluated in vivo by a micronuclei (MN) test in rat's bone marrow cells and in vitro by MN and sister chromatid exchange (SCE) in human lymphocyte cultures. In vivo genotoxicity analyses revealed that the average number of micronucleated polychromatic erythrocytes and the polychromatic erythrocyte/red blood cell ratio at all doses were not significantly different from those of the negative control. In vitro genotoxicity analyses showed that MN, SCE, and proliferative index frequencies in a human lymphocyte cell culture were not significantly different from those of the negative control. These results demonstrate that the ethanol extract of R. coriifolius aerial parts is not toxic or mutagenic (in vitro and in vivo) and does not affect cell proliferation at the concentrations analyzed.

Digital image analysis of AgNORs in cervical smears of women with premalignant and malignant lesions of the uterine cervix.

We performed a hospital-based, unmatched case-control study to investigate the association between progressive stages of cervical neoplasia and digital analysis of cell proliferation by silver stained nucleolus organizer region associated proteins (AgNORs). We measured cell proliferation levels in the cervical epithelial cells of 10 women with low grade squamous intraepithelial lesions (LG-SIL), eight with high grade squamous intraepithelial lesions (HG-SIL), 11 with cervical cancer (CC) and eight with no cervical lesions (controls) using the AgNORs technique. Cell proliferation was measured by digital image analysis (DIA). DIA revealed increased total areas of AgNORs in HG-SIL and CC compared to LG-SIL and control patients. AgNORs with a kidney or cluster shape exhibited greater areas than those with a spherical or long shape. We propose a cut-off of 118 pixels to differentiate benign (control and LG-SIL) from malignant (HG-SIL and CC) lesions. DIA of AgNORs is a simple and inexpensive method for studying proliferation. The increased total area of AgNORs in malignant lesions provides information regarding cell behavior and may be related to cervical carcinogenesis; however, further validation studies are required to establish its usefulness in cytological analysis.

Localisation and quantification of alkali-labile sites in human spermatozoa by DNA breakage detection-fluorescence in situ hybridisation.

The localisation and quantification of constitutive alkali-labile sites (ALSs) were investigated using a protocol of DNA breakage detection plus fluorescence in situ hybridisation (DBD-FISH) and alkaline single-cell gel electrophoresis (SCGE or comet assay), in spermatozoa of infertile and fertile men. Semen samples from 10 normozoospermic patients undergoing infertility treatment and 10 fertile men were included in this study. ALSs were localised and quantified by DBD-FISH. The region most sensitive to alkali treatment in human spermatozoa was located in the basal region of the head. ALSs were more frequent in spermatozoa of infertile men than in those of fertile men. These results were confirmed by SCGE comet assays. In conclusion, the most intense localisation of hybridisation signals in human spermatozoa, representing the highest density of constitutive ALSs, was not randomly distributed and was predominantly located in the base of the head. Moreover, infertile men presented with an increase in ALS frequency. Further studies are necessary to determine the association between ALS, sperm chromatin organisation and infertility.

Chromosomal damage as prognosis marker in cervical carcinogenesis.

Cancer of the uterine cervix is the third most common cancer in women worldwide and the most common cancer among Mexican and Latin American women. Risk factors that have been associated with the development of cervical intraepithelial neoplasia suggest that Human Papillomavirus (HPV) types 16, 18, 31, and 33 entail a high risk of developing a malignancy of this type. The accumulation of genetic alterations allows the growth of neoplastic cells; chromosomal instability is an event that occurs in the precancerous stages. The candidate cancer risk biomarkers include cytogenetic endpoints, such as chromosomal aberrations, sister chromatid exchange, micronuclei, and the outcomes of comet assay and DNA breakage detection-fluorescence in situ hybridization. The patterns identified in these cytogenetic studies indicate that chromosomal instability is a transient and chromosomally unstable intermediate in the development of cervical lesions. In this context, the mechanisms that may underlie the progressive increase in genetic instability in these patients seem to be related directly to HPV infection. The studies discussed in this paper show that chromosomal instability may serve as a biomarker by predicting the progression of cervical intraepithelial neoplasia. Nevertheless, these results should be validated in larger, prospective studies.

DNA breakage detection-fluorescence in situ hybridization (DBD-FISH) in buccal cells.

DNA breakage detection-fluorescence in situ hybridization (DBD-FISH) is a recently developed technique that allows cell-by-cell detection and quantification of DNA breakage in the whole genome or within specific DNA sequences. The present investigation was conducted to adapt the methodology of DBD-FISH to the visualization and evaluation of DNA damage in buccal epithelial cells. DBD-FISH revealed that DNA damage increased significantly according to H2O2 concentration (r2=0.91). In conclusion, the DBD-FISH technique is easy to apply in buccal cells and provides prompt results that are easy to interpret. Future studies are needed to investigate the potential applicability of a buccal cell DBD-FISH model to human biomonitoring and nutritional work.

Constitutive heterochromatin polymorphisms in human chromosomes identified by whole comparative genomic hybridization.

Whole comparative genomic hybridization (W-CGH) is a new technique that reveals cryptic differences in highly repetitive DNA sequences, when different genomes are compared using metaphase or interphase chromosomes. W-CGH provides a quick approach to identify differential expansion of these DNA sequences at the single-chromosome level in the whole genome. In this study, we have determined the frequency of constitutive chromatin polymorphisms in the centromeric regions of human chromosomes using a whole-genome in situ cross-hybridization method to compare the whole genome of five different unrelated individuals. Results showed that the pericentromeric constitutive heterochromatin of chromosome 6 exhibited a high incidence of polymorphisms in repetitive DNA families located in pericentromeric regions. The constitutive heterochromatin of chromosomes 5 and 9 was also identified as highly polymorphic. Although further studies are necessary to corroborate and assess the overall incidence of these polymorphisms in human populations, the use of W-CGH could be pertinent and of clinical relevance to assess rapidly, from a chromosomal viewpoint, genome similarities and differences in closely related genomes such as those of relatives, or in more specific situations such as bone marrow transplantation where chimerism is produced in the recipient.

Total homocysteine levels in healthy children from the Monterrey metropolitan area, Mexico.

Currently, there are indications for determining hyperhomocysteinemia in adulthood as risk factors for cardiovascular diseases, psychiatric disorders, pregnancy complications, birth defects, cognitive impairment in the elderly, in addition to cancer. If hyperhomocysteinemia is determined from childhood, it may be modulated with the provision of an opportunity for public health intervention. The objective of this descriptive study was to determine total homocysteine (tHcy) levels in healthy children from the Monterrey metropolitan area in Mexico. In a peripheral-blood sample collected from 56 healthy children aged 2-10 years, we determined tHcy concentration by high performance liquid chromatography (HPLC) with fluorescence detection. The geometric mean +/- SD was 9.78 +/- 1.73 micromol/l. tHcys of the children studied were homogeneous by age cohort and gender. Nutritional state was classified by body mass index (BMI). Sixty five percent of children who participated in the study had normal BMI, and 96% of the children belong to the low socioeconomic status. In conclusion, to our knowledge this is the first-ever information on homocysteine (Hcy) prevalence in a population of healthy Mexican children. tHcy concentration was higher than that reported in other populations studies. This preliminary study could constitute the baseline for future public health studies.

Association between human papilloma virus-type infections with micronuclei frequencies.

To determine the association between Human papillomavirus (HPV)-type infections with the frequency of Micronucleus (MN), a hospital-based, unmatched case-control study was carried out. We evaluated and compared the average number of MN/1,000 cells among three groups of Mexican females. Twenty one women ranging in age from 31-56 years and divided into three groups were studied. Group I comprised seven control women without cervical lesions and with HPV-negative, Group II was composed of seven women with Squamous intraepithelial lesions (SIL) infected with low-risk HPV low-risk, and Group III was made up of seven women with SIL infected with high-risk HPV infection. Analysis of variance (ANOVA) test revealed differences among Groups I (5.14+/-3.02), II (13.43+/-3.41), and III (25.43+/-3.41) (F=67.46; P=0.0001). We demonstrated an association between HPV type infection and higher MN frequencies. However, a larger controlled study with sufficient follow-up will be required to further evaluate the usefulness of this test in the clinical management of women with HPV infection.

Shared Y chromosome repetitive DNA sequences in stallion and donkey as visualized using whole-genomic comparative hybridization.

The genome of stallion (Spanish breed) and donkey (Spanish endemic Zamorano-Leonés) were compared using whole comparative genomic in situ hybridization (W-CGH) technique, with special reference to the variability observed in the Y chromosome. Results show that these diverging genomes still share some highly repetitive DNA families localized in pericentromeric regions and, in the particular case of the Y chromosome, a sub-family of highly repeated DNA sequences, greatly expanded in the donkey genome, accounts for a large part of the chromatin in the stallion Y chromosome.

Koilocytes are enriched for alkaline-labile sites.

This study investigated possible variations in the chromatin structure of koilocytes resulting from human papillomavirus (HPV) infection. Alkaline-labile sites (ALS) were detected with the DNA breakage detection­fluorescence in situ hybridization (DBD-FISH) technique using a whole human genome DNA probe obtained from individuals without koilocytosis. The variable levels of ALS present were measured quantitatively using image analysis after whole-genome DNA hybridization. A significant increase in the number of ALS was observed in koilocytes compared with normal cells. We demonstrated that the presence of ALS could be an indicator of chromatin change in koilocytes caused by HPV infection.

Whole-comparative genomic hybridization in domestic sheep (Ovis aries) breeds.

Whole-comparative genomic hybridization (W-CGH) allows identification of chromosomal polymorphisms related to highly repetitive DNA sequences localized in constitutive heterochromatin. Such polymorphisms are detected establishing competition between genomic DNAs in an in situ hybridization environment without subtraction of highly repetitive DNA sequences, when comparing two species from closely related taxa (same species, sub-species, or breeds) or somewhat related taxa. This experimental approach was applied to investigating differences in highly repetitive sequences of three sheep breeds (Castellana, Ojalada, and Assaf). To this end, W-CGH was carried out using mouflon (sheep ancestor) chromosomes as a common target to co-hybridize equimolar quantities of two genomic DNAs obtained from either Castellana, Ojalada or Assaf sheep breeds. The results showed that the amount of constitutive heterochromatin is greater in all pericentromeric heterochromatin regions of acrocentric chromosomes than in metacentric or sex chromosomes. Additionally, when W-CGH was performed using DNAs from the Iberian breeds Castellana and Ojalada, chromosomal pericentromeric regions revealed quantitatively and qualitatively a presence of DNA families similar to that obtained from any of the above-cited breeds. On the contrary, when the DNA used in W-CGH experiments was obtained from Assaf, as compared to either Castellana or Ojalada, two different pericentromeric DNA families of highly repetitive sequences could be detected. Lastly, sex chromosomes were shown to be homogeneous among all breeds and thus revealed no detectable constitutive heterochromatin. W-CGH results were confirmed using DNA breakage detection-FISH experiments (DBD-FISH) carried out on lymphocytes. As a whole, the results showed that two different repetitive DNA families are present in the pericentromeric heterochromatin of the sheep breeds studied here. Additionally, they suggest a differential presence of these distinct repetitive DNA families in Castellana and Ojalada breeds as compared to the Assaf breed. Finally, the results of W-CGH after using mouflon as the targeted chromosomes also show that the two DNA families are present in the ancestor.

Assessment of sperm DNA fragmentation in stallion (Equus caballus) and donkey (Equus asinus) using the sperm chromatin dispersion test.

Sperm DNA fragmentation (sDF) is an important parameter to assessing sperm quality. Information about sperm quality is not available for donkeys, especially in some breeds at risk of extinction. The objectives of this research were to test the four commercial variants of sperm chromatin dispersion test (SCD; sperm Halomax test), originally developed to assess sDF in boars, bulls, rams and stallions, in order to scrutinize their applicability in the study of sDF in a donkey breed at risk of extinction (Zamorano-Leonesa), for which there is no specific test available to analyze sperm at present. Only the SCD test, originally developed for stallions, produced stable and consistent results, and was deemed suitable to assess DNA fragmentation in sperm samples from donkeys. Image analysis was used to compare differences between the SCD methodology applied to stallion and donkey semen samples processed under the same experimental conditions. The extent of SCD in the SCD test was approximately 20% lower in donkey sperm than in stallion sperm. Yet, the ratio of chromatin sperm dispersion achieved in fragmented and unfragmented nuclei did not differ significantly between species. These data suggest that a similar protein depletion treatment can cause differences in protein removal in equivalent cells from different species and that sperm chromatin may be organized differently in stallions and donkeys.

DNA fragmentation in frozen sperm of Equus asinus: Zamorano-Leonés, a breed at risk of extinction.

The dynamics of sperm DNA fragmentation (sDF) and sperm viability were analyzed in frozen-thawed sperm samples of Equus asinus (Zamorano-Leonés), a breed at risk of extinction. Sperm DNA fragmentation was assessed using an adaptation of the sperm chromatin dispersion test developed for stallions in five different frozen samples. Sperm were thawed and incubated at different temperatures (37 degrees C, 25 degrees C, and 4 degrees C) and sDF was assessed at different times and compared. The mean sDF after thawing at the beginning of the experiment was 18.20+/-14.77% and did not differ significantly from the results of a neutral comet assay (22.0+/-19.34%). The tendency in the sDF of all donkeys indicated that sperm DNA is more sensitive to breakage when incubated at 37 degrees C than when incubated at 25 degrees C or 4 degrees C. Interestingly, the tendency was not the same when different animals were compared, and differences in sDF dynamics were established among individuals. sDF correlated negatively with sperm viability in some individuals but not in others. From a conservation perspective, sDF analysis may offer a new way to assess sperm quality in endangered breeds in order to identify and select the best semen samples for artificial reproduction purposes. In particular, we recommend for artificial insemination the use of semen samples with a slow increase in sDF with time after thawing.

[Assessing sperm DNA damage]. / Evaluación del daño en el DNA espermático.

Infertility affects almost 20% of couples in reproductive age and the male factor being responsible of 50% of this infertility. Among the classic parameters that determine a good seminal quality such as sperm motility, sperm morphology or the quality of the of acrosomes and/or sperm membranes, the integrity of the DNA molecule is crucial to carry out a successful fertilization. Nevertheless, the study of this parameter has not been straightforward approached. This fact has shunned its incorporation, as a routine technique, within a standard seminogram. The aim of the present review is to summarize and update those technologies that are considered more successful to study sperm DNA fragmentation with special emphasis to: 1) the levels of technological complexity and the possibility of its use in laboratories of andrology, according with the equipment and the resources available, and 2) the effects and possible implications of high level of sperm DNA fragmentation for fecundation, embryo development and fertility.

Frequency of micronuclei in Mexicans with type 2 diabetes mellitus.

A case-control study was carried out on a sample of 15 Mexican patients (40-56 years old) with type 2 diabetes mellitus (DM2) that had developed five years and been treated with oral hypoglycemic drugs (sulfonylurea and/or metformin), with no microvascular or macrovascular complications. The aim of this study was to assess whether Mexican patients with DM2 differed from a control group in the frequency of micronuclei (MN). A control group of 10 individuals without DM2 (38-54 years old) was included. The frequency of MN in binucleated lymphocytes was analyzed according to the Fenech criteria. At time being this investigation should be considered as a preliminary study in which the influence of potential confounders cannot be adequately assessed. However, our result showed a MN frequency significant increase in DM2 patients (6.53 +/- 2.03 per 1000 cells) relative to that of the control group (3.10 +/- 1.79 per 1000 cells). MN may constitute a possible component of a panel of biomarkers for the risk of DM2. This cytogenetic damage also indicates an enhanced risk of cancer, as has been found in previous studies. These results should be validated by other researchers.

Chromosomal abnormalities and polymorphisms in Mexican infertile men.

A cross-sectional study was conducted to estimate the prevalence of chromosome abnormalities and normal variable chromosome features (polymorphisms) in infertile men from northeastern Mexico. Karyotyping was carried out in 326 men with diagnosis of infertility. The sperm counts showed 204 patients with oligozoospermia, 87 with azoospermia and 35 normozoospermia. Five patients with oligozoospemia and two with azoospermia presented chromosome abnormalities. Nonzoospermic men did not show chromosomal abnormalities. Polymorphisms of heterochromatin and satellite length showed a significant increased in oligozoospermic and azoospermic men with respect to normozoospermic men, respectively. This study reports the prevalence of chromosome abnormalities, polymorphisms of heterochromatin length, and polymorphisms in satellites in Mexican infertile men. The prevalence in this study was similar to other studies in world literature.

Polymorphism of Ag(+)-NORs in cervical smears from women with cervical cancer.

OBJECTIVE: To evaluate Ag(+)-stained (Ag(+)-NOR) polymorphism in four groups of patients with various grades of cervical lesions and in a control group. STUDY DESIGN: Forty-five women were selected, diagnosed and classified on the bases of the Pap smear and colposcopy/biopsy at Hospital de Ginecologia y Obstetricia del Instituto Mexicano del Seguro Social in Monterrey, Mexico. Five categories were considered: (1) inflammatory, (2) low grade squamous intraepithelial lesions (LSILs), (3) high grade squamous intraepithelial lesions (HSILs), (4) invasive cervical cancer, and (5) normal. The cervical smears were stained by the Ag(+)-NOR method. One hundred cells per slide were counted and classified according to the polymorphism of Ag(+)-NOR dots: typical (spherical) and atypical (large, kidney shaped and clustered). The four shapes of Ag(+)-NORs were quantified by percentage and transformed using the arcsine root procedure. RESULTS: Statistical analysis showed a significant decrease in spherical shape according to neoplastic development. The three atypical shapes showed a significant increase in patients with HSIL and invasive carcinoma in respect to LSIL. Principal components analysis grouped the data at five locations in the plane formed by the first two principal components according to the diagnosis. CONCLUSION: These findings suggest the potential diagnostic and prognostic value of the determination of Ag(+)-NOR polymorphism in cervical cytology studies.