[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
Cytology and Genetics 05/2014; 48(3):180-188. · 0.29 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: The presence of constitutive alkali-labile sites (ALS) has been investigated using a protocol of DNA breakage detection-fluorescence in situ hybridization and comet assay in spermatozoa of donkey (Equus asinus) and stallion (Equus caballus). These results were compared with those obtained using a similar experimental approach using somatic cells. The relative abundance of ALS was of the order of four times more in spermatozoa than in somatic cells. Alkali-labile sites showed a tendency to cluster localized at the equatorial-distal regions of the sperm. The amount of hybridized signal in the ALS in the sperm of donkey (Equus asinus) was 1.3 times greater than in stallion (Equus caballus), and the length of the comet tail obtained in donkey sperm was 1.6 times longer than that observed in stallion (P < 0.05); however, these differences were not appreciated in somatic cells. In conclusion, ALS localization in sperm is not a randomized event and a different pattern of ALS distribution occurs for each species. These results suggest that ALS represents a species-specific issue related to chromatin organization in sperm and somatic cells in mammalian species, and they might diverge even with very short phylogenetic distances.
[Show abstract][Hide abstract] ABSTRACT: 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.
International Journal of Molecular Sciences 01/2013; 14(2):4135-47. · 2.46 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Mammalian serum is essential for the growth of Giardia duodenalis cultivated under axenic conditions. Unfortunately, some factors present in bovine serum used as supplement in the culture medium may inhibit protozoal growth and activity. TYI-33-PACSR is a TYI medium supplemented with a serum replacement (PACSR) made up of Earle's amino acid solution, Diamond's vitamin-tween 80 mixtures and LCR (a lipid-cholesterol - rich mixture). PACSR was previously used in the culture media for axenic cultivation of Entamoeba histolytica and Trichomonas vaginalis. The main objective of this work was to demonstrate that TYI-33-PACSR is useful for axenic cultivation of G. duodenalis. Additionally, the activity of phospholipase A(2) (PLA A(2)) in the sub-cellular vesicular fraction (P30) of G. duodenalis grown in TYI-S-33 and TYI-33-PACSR was compared. All strains of Giardia grown in TYI-33-PACSR reached relative cellular densities of 91 to 95% compared to controls growing in serum-supplemented TYI-S-33 medium. Additionally, PLA A(2) activity was similar in the P30 sub-cellular fraction obtained from trophozoites growing in TYI-S-33 and TYI-33-PACSR. Thus, TYI-33-PACSR could be useful in analyzing the biological properties of G. duodenalis in the absence of serum.
[Show abstract][Hide abstract] ABSTRACT: A systematic search for a hidden Y-chromosome mosaicism, in Turner syndrome (TS) patients is justified by the evaluation of the risk of development of germ cell tumors. In this study, we analyzed cryptic Y-chromosome derivatives by polymerase chain reaction (PCR) coupled with fluorescence in situ hybridization (FISH) using Y-specific sequences in patients with TS, and validated this methodology. Unrelated patients with TS (n=32) of Mexican mestizo ethnic origin were diagnosed using cytogenetic analysis. Clinical assessment, endocrine evaluation, karyotyping, FISH and PCR analysis of the Y-chromosomal loci were performed. We found that 9.4% (3 out of 32) patients with TS had Y-chromosome material. Two patients showed Y-chromosome by conventional cytogenetics. One patient had no Y-chromosome by initial karyotyping (45, X) but was positive by lymphocyte PCR DNA analysis of the Y-sequence-specific sex-determining region Y (SRY) gene. Our results suggest that the detection of the Y-chromosome material using sensitive methods, such as PCR coupled with FISH, should be carried out in all patients with TS and should not be limited to TS patients with cytogenetically identifiable Y-chromosome and/or virilization.
[Show abstract][Hide abstract] ABSTRACT: 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.
European journal of histochemistry: EJH 01/2012; 56(4):e49. · 2.41 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
BioMed Research International 01/2012; 2012:385245. · 2.88 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To evaluate the association between the progressive stages of cervical dysplasia and DNA damage.
A hospital-based, unmatched case-control study was performed. DNA damage levels in the cervical epithelial cells of 30 women (10 with low-grade squamous intraepithelial lesions [LSIL], 10 with high-grade SIL [HSILI, and 10 with no cervical lesions) were evaluated using the DNA breakage detection-fluorescence in situ hybridization (DBD-FISH) technique. DNA damage levels were measured quantitatively using image analysis after whole-genomic DNA hybridization.
LSIL patients presented a hybridization signal 20 times greater than the signal in control individuals, which reflected the basal level of DNA damage detected, and HSIL patients showed 100 times the basal control signal. The Kruskal-Wallis test showed differences between both patient groups and the control and between the patients with LSIL and HSIL.
The DBD-FISH technique is easy to apply to cervical scrapings and provides prompt results. Our findings confirm that the grade of a cervical lesion correlates with the degree of genomic instability.
Analytical and quantitative cytology and histology / the International Academy of Cytology [and] American Society of Cytology 06/2011; 33(3):175-81. · 0.60 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The comet assay is a well-established, simple, versatile, visual, rapid, and sensitive tool used extensively to assess DNA damage and DNA repair quantitatively and qualitatively in single cells. The comet assay is most frequently used to analyze white blood cells or lymphocytes in human biomonitoring studies, although other cell types have been examined, including buccal, nasal, epithelial, and placental cells and even spermatozoa. This study was conducted to design a protocol that can be used to generate comets in subnuclear units, such as chromosomes. The new technique is based on the chromosome isolation protocols currently used for whole chromosome mounting in electron microscopy, coupled to the alkaline variant of the comet assay, to detect DNA damage. The results show that migrant DNA fragments can be visualized in whole nuclei and isolated chromosomes and that they exhibit patterns of DNA migration that depend on the level of DNA damage produced. This protocol has great potential for the highly reproducible study of DNA damage and repair in specific chromosomal domains.
Journal of Histochemistry and Cytochemistry 05/2011; 59(7):655-60. · 2.26 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: This pilot study analyzed and compared the presence of chromosome 8 aneusomy in Mexican women with breast cancer and adjacent, intraductal, proliferative lesions. To determine the chromosome 8 copy number, we performed fluorescence in situ hybridization in nine patients (1800 cells) who underwent mastectomy. We selected two tissue samples from each patient, one corresponding to the invasive ductal carcinoma (IDC) and the other adjacent to the intraductal proliferative lesion (IPL). Breast tissue from 17 autopsy samples (1700 cells) was used as a control. The number of cells with monosomy, disomy and polysomy per subject and type of tissue were compared among the three groups of tissue with the RxC statistical software package using 50,000 total replicates. Chromosome 8 aneusomy was found in 66 and 67% of cells from the IDC and IPL samples, respectively. Monosomy was detected significantly more frequently in IPL compared with IDC samples (49.11 vs. 27.11%; p=0.0000), whereas polysomy was significantly more frequent in IDC compared with IPL samples (40.11 vs. 16.99%; p=0.00000). Control cells showed 92.3% disomy. These findings suggest that polysomy of chromosome 8 is more frequently observed in IDC and that monosomy is more frequent in tissue of IPL. Therefore, monosomy may be considered as a primary preneoplastic event. Future studies should be performed to increase the amount of breast tissue with ductal proliferative changes and with cancer, in order to support the results of this pilot study.
[Show abstract][Hide abstract] ABSTRACT: To investigate the association between the progressive stage of cervical dysplasia and DNA damage by comet assay.
A hospital-based, unmatched, case-control study was performed. DNA damage levels (none, low, medium and high) in the cervical epithelial cells of 31 women (10 with low grade squamous intraepithelial lesions [LSIL], 10 with high grade [HSIL] and 11 with no cervical lesion) were evaluated using the comet assay.
A significant increase in medium DNA damage was observed in women with HSIL (17 +/- 8.9) relative to that in the control women (9 +/- 6.1). A significant increase in high DNA damage was also observed in women with LSIL (23 +/- 15.4) or HSIL (32 +/- 13.1) relative to that in the control women (12 +/- 7.9).
These findings confirm that the grade of a cervical lesion correlates with the degree of genomic instability.
Analytical and quantitative cytology and histology / the International Academy of Cytology [and] American Society of Cytology 08/2010; 32(4):207-13. · 0.60 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We undertook this study to compare the expression level of prostate apoptosis response-4 (Par-4) among patient outcome in two groups of women with breast cancer (short and long survival) and two groups without breast cancer (benign lesion and control).
We included breast specimens with nonhistological abnormalities (eight samples) as a control group. Semiquantitative and quantitative analysis of immunohistochemical staining by image analysis software were used to study the intensity of Par-4 expression. Both methods produced similar results (p>0.05).
No significant expression of Par-4 was observed in normal breast tissue. Benign lesions and breast cancer tissue showed strong nuclear expression of Par-4, predominantly on epithelial cells and specifically in ductal cells. Par-4 expression was lower in myoepithelial cells and there was no appreciable stromal staining. Significantly less Par-4 reactivity was detected in tissue from patients with a short survival compared with patients with benign lesions and those with a long survival.
Our findings suggest that a lower expression level of Par-4 is related to an unfavorable prognosis. A larger prospective study of samples of all patient groups with a longer follow-up is needed to validate this finding.
Archives of medical research 05/2010; 41(4):261-8. · 1.88 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
European journal of histochemistry: EJH 01/2010; 54(1):e2. · 2.41 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.