Miguel Betancourt

Assessment of DNA damage in leukocytes from infected and malnourished children by single cell gel electrophoresis/comet assay

The alkaline single cell gel (SCG) assay is a sensitive electrophoretic technique for detecting the presence of DNA single strand breaks and alkali-labile damage in individual cells. This technique was used to assess and compare the level of DNA damage in peripheral blood leukocytes/lymphocytes from well-nourished children with infection, well-nourished children with infection and under drug treatment, and from malnourished infected children with and without previous drug treatment. The present study shows that severe infection is associated with a significant increase in DNA damage. The average migration length was five times greater in severely infected well-nourished children compared to that found in healthy, well-nourished children. The results obtained in this study indicate that malnutrition is another factor associated with an increase in DNA migration. The average tail length for malnourished, severely infected children was twice as great as that obtained for cells from well-nourished, severely infected children. We also detected a variable increase in DNA migration associated with treatment for severe infection. Nevertheless, the excessive heterogeneity, the concurrent number of drugs used and the limited size of the treated population precludes an accurate assessment of which drugs were involved in the increase in DNA damage. Further studies will be necessary involving a large number of patients to address the relation between levels of DNA damage and specific kinds of infection, various drug treatments, and the type and severity of malnutrition. The increased level of DNA damage in severely infected and malnourished children could be related to negative effects such as a deficient immune response resulting in an increased susceptibility to malignant transformation.

Changes in the distribution of lectin receptors during capacitation and acrosome reaction in boar spermatozoa.

Sperm glycocalyx modifications are known to occur during capacitation and the acrosome reaction (AR). These changes are very important for gamete recognition and fertilization in mammals but are not fully understood. The purpose of this study was to determine the distribution of surface carbohydrates in boar spermatozoa during capacitation and the AR. These processes may be associated with specific changes in the content and distribution of surface carbohydrates. Thirty-nine ejaculates from fertile boars of various breeds were analyzed. N-Acetylglucosamine and sialic acid, mannose and fucose residues were detected by fluorescence microscopy and flow cytometry using FITC-conjugated lectins. Triticum vulgaris agglutinin (WGA) bound on the head and tail of fresh sperm, and fluorescence intensity (FI) decreased in capacitated sperm (6751 to 5621 fluorescence units (FU), P<0.05), and decreased further in acrosome-reacted sperm (5240 FU, P<0.05). Concanavalia ensiformis agglutinin (Con-A) bound homogeneously on the head and the midpiece of fresh sperm with a FI of 5335 FU, and increased in capacitated sperm (5957 FU, P<0.05) mainly on the acrosomal region. In acrosome-reacted sperm, fluorescence was concentrated on the border of the acrosomal region (5608 FU, P<0.05). It was not possible to detect Ulex europaeus agglutinin (UEA) by fluorescence microscopy. However, flow cytometry revealed UEA receptors (187 FU), with a nonsignificant decreased number in capacitated (142 FU) and AR sperm (142 FU). Labeling patterns were similar in all breeds. Sperm glycocalyx modifications observed in this study provide insights to the molecular modifications accompanying capacitation and the AR. This kind of study could improve the diagnosis of reproductive problems of subfertile boars and males of other species.

Uptake, cellular distribution and DNA damage produced by mercuric chloride in a human fetal hepatic cell line

A human hepatic cell line (WRL-68 cells) was employed to investigate the uptake of the toxic heavy metal mercury. Hg accumulation in WRL-68 cells is a time and concentration dependent process. A rapid initial phase of uptake was followed by a second slower phase. The transport does not require energy and at low HgCl2 concentrations (<50 microM) Hg transport occurs by temperature-insensitive processes. Subcellular distribution of Hg was: 48% in mitochondria, 38% in nucleus and only 8% in cytosolic fraction and 7% in microsomes. Little is known at the molecular level concerning the genotoxic effects following the acute exposure of eucaryotic cells to low concentrations of Hg. Our results showed that Hg induced DNA single-strand breaks or alkali labile sites using the single-cell gel electrophoresis assay (Comet assay). The percentage of damaged nucleus and the average length of DNA migration increased as metal concentration and time exposure increased. Lipid peroxidation, determined as malondialdehyde production in the presence of thiobarbituric acid, followed the same tendency, increased as HgCl2 concentration and time of exposure increased. DNA damage recovery took 8 h after partial metal removed with PBS-EGTA.

Effects of the Insecticides Malathion and Diazinon on the Early Oogenesis in Mice In Vitro

Malathion and diazinon are two of the most commonly used organophosphorous (OP) agrochemicals. Several studies show that these pesticides exert several effects on mammalian spermatogenesis. Nevertheless, there are no studies concerning their effects on oogenesis. The objective of this study was to evaluate the effects of these insecticides on the viability of in vitro cultured mouse oocytes during the early oogenesis and to get a further understanding of the molecular mechanisms by which OP insecticides act and affect germinal cells. Oocytes were cultured from fetal ovaries for 10 days, when most oocytes had reached the diplotene stage (germinal vesicle stage). Cultures were exposed to different concentrations of malathion or diazinon for 24 h, and the effect on oocyte viability was assessed. Gene expression in oocytes exposed to the insecticides was analyzed by generating cDNA libraries and performing differential screenings. Results show a significant decrease in oocytes survival after 24‐h exposure to 250 μM malathion or 900 nM diazinon, and the effect of these insecticides on the regulation of genes encoding proteins involved in transcription (BP75), translation (ribosomal protein S5), and mitochondrial function (cytochrome oxidase subunits I and III), providing evidence for OP insecticides as toxicants for mammals oocytes during the early oogenesis.

Differential effects of herbicides atrazine and fenoxaprop-ethyl, and insecticides diazinon and malathion, on viability and maturation of porcine oocytes in vitro.

Exposure to pesticides may be a major cause of reproductive dysfunction in humans and animals. Atrazine and fenoxaprop-ethyl, widely used herbicides, and malathion and diazinon, organophosphate insecticides, are considered only slightly toxic to vertebrates; however, there is evidence of greater effects on reproductive function. The aim of this study was to evaluate the effect of these pesticides on oocyte viability and in vitro maturation. Gametes were matured in increasing concentrations of the pesticides and then stained with MTT to evaluate viability and bisbenzimide to assess the maturation stage, in the same oocyte. Atrazine had no effect on viability but maturation was significantly reduced, while fenoxaprop-ethyl affected both parameters. The insecticides affected viability and maturation but to a different degree. The four pesticides showed a more pronounced effect on maturation than on viability, due to a blockage at germinal vesicle stage.

Sister-chromatid exchange (SCE) and cell proliferation in lymphocytes from infected and non-infected children with severe protein calorie malnutrition (PCM)

The frequency of sister-chromatid exchanges (SCE) and the rate of cell proliferation were evaluated through differential staining of sister chromatids in mitogen-stimulated cultured lymphocytes sampled from five well-nourished children, from seven severely malnourished children infected with bacterium, and from 10 severely malnourished children following treatment for infection with antimicrobial drugs 2 weeks before blood sampling. The replication indices at 48 h of culture were higher in both groups of malnourished children than in the well-nourished children, indicating either a faster response to PHA and/or a shorter cell cycle in lymphocytes of these patients. The average frequency of SCE per mitosis was also significantly higher than in the control group. The mitotic index was similar in the three groups of children. The lack of significant difference in response between the two groups of malnourished children suggests that the effects observed at the cytogenetic level are caused by severe malnutrition per se, and not by any associated infection.

Viability, maturation and embryo development in vitro of immature porcine and ovine oocytes vitrified in different devices.

This study was designed to evaluate the effects of vitrification on immature porcine and ovine oocytes, collected at a slaughterhouse, by performing vitrification in devices with different volumes. Viability was evaluated both before and after vitrification and maturation. Immediately after warming, the percentage of viable pig oocytes was 81% regardless the type of device, while in the control (after oocyte selection) was 95%. The viability of matured pig oocytes after warming, vitrified in beveled edge open straws (BES) was 6%, in small-open-pulled-straw (SOPS) was 17% and in cryotop was 4%, while the viability of the control group was 86%. The viability and maturation results were similar with all devices. Embryo development (ED) was observed in fresh porcine oocytes with 15% 2-8 cell embryos, 7% morulae and 3% blastocysts, and non-embryo cleavage was observed in warmed oocytes. The viability of sheep oocytes immediately after warming averaged 90% in all devices, while that of the control (after oocyte selection) averaged 95%. The viability of warmed oocytes after maturation was: BES 21%, SOPS 30%, cryotop 21% and control group 86%; while maturation values were 11, 21, 34 and 70%, respectively. After vitrification, the highest ED was achieved with ovine oocytes vitrified in SOPS, with 17% morulae development and it was the only device in which blastocysts developed. A direct relationship was observed between viability and actin filament integrity in both species.

Expression of lectin receptors on the membrane surface of sperm of fertile and subfertile boars by flow cytometry.

Studies suggest that carbohydrates are important in different stages of fertilization. Plasma membrane changes accompanying in vitro capacitation and acrosome reaction (AR), such as removal or appearance of specific glycoproteins, have been studied using lectins that bind specifically to carbohydrate residues. In specialized artificial insemination farms and semen production centers, identification of boars with decreased fertilization ability (subfertility) is a newborn necessity. This investigation is a sequential study to determine the kinetics of surface carbohydrates turnover during in vitro capacitation and AR in fertile and subfertile boar sperm. Flow cytometry determinations of the binding of three FITC-labeled lectins were assessed. WGA binding was significantly lower in fresh, capacitated, and acrosome-reacted sperm of subfertile boars than in fertile boars. Con-A binding was not significantly different in fresh sperm of fertile and subfertile boars. However, Con-A labeling in capacitated, and acrosome-reacted sperm differed significantly in both groups. UEA binding increased only in capacitated sperm of subfertile boars. These findings could be used as indicators of capacitation and AR and may also be a good indicator of sperm fertilizing ability in boars.

Analysis of Mitomycin C-induced micronuclei in lymphocytes from malnourished infected children

The purpose of this study was to determine if peripheral blood lymphocytes from malnourished children with gastrointestinal or respiratory bacterial infection show increased frequencies of Mitomycin C (MMC)‐induced micronuclei as compared to well‐nourished, infected children. The results indicate that cells from malnourished, infected children had greater chromosome damage. This may indicate that such children would be more susceptible to environmental damage and malignant transformation.

Effect of porcine follicular fluid proteins and peptides on oocyte maturation and their subsequent effect on in vitro fertilization.

The follicular fluid (FF) is a microenvironment that contains molecules involved in oocyte maturation, ovulation, and fertilization. Characterizing the proteins and peptides present in the FF could be useful for determining which proteins and peptides to use as a supplement for culture media. Biologically active peptides produced during the maturation or degradation of functional proteins are called cryptides. The aim of this study was to identify the proteins and cryptides in porcine FF that could stimulate porcine oocyte in vitro maturation (IVM) and in vitro fertilization (IVF) when added to culture maturation medium. Five FF protein fractions (F1-F5) were obtained by ionic exchange chromatography, resolved by SDS-PAGE, and identified by tandem mass spectrometry. These fractions had effects on IVM and/or IVF. The F1 fraction, which was composed of immunoglobulin fragments, cytokeratin, transferrin, and plasminogen precursor increased IVM and IVF. The F2, F3, and F4 fractions reduced the percentage of oocytes in first metaphase. Additionally, the F3 fraction, which was composed of immunoglobulins and transthyretin, interfered with germinal vesicle breakdown. The F5 fraction, which was mainly composed of serum albumin and keratin, favored germinal vesicle breakdown and promoted IVM. Most of the 31 proteins which were associated with the immune response and inflammatory processes could be related to oocyte maturation and fertilization. Some of the identified proteins were present in more than one fraction; this could be explained by a change in their isoelectric points, because of the loss of part of the amino acid sequence or a change in the glycosylation status of the protein. Improved oocyte IVM and IVF will increase embryo production, which in turn will contribute to the efficiency of assisted reproduction in various mammalian species.