Epigenetic studies in insects and the valproic acid perspective.

Valproic acid in association with sodium valproate (VPA) is an important anticonvulsant drug used for decades to treat neurological disorders. VPA also acts as an epigenetic modulator by inhibiting histone deacetylases, permitting histone acetylation, affecting the DNA and histone methylation status and gene expression, and inducing chromatin remodeling. Insects represent an important animal model for studies in several areas of science. Their high phenotypic plasticity makes them alternative models for epigenetic studies. This brief review emphasizes recent reports on insect epigenetics and the contribution of studies on the VPA action in insects, including effects on epigenetic markers, extending the pharmacological understanding of the potential of this drug, and demonstrating the usefulness of insects as an alternative animal model to drug studies.

Standardization of bulb and root sample sizes for the Allium cepa test.

Although the Allium cepa test has been widely used to identify potentially cytotoxic and genotoxic pollutants in aquatic environments, variable non-standardized choices have been made regarding the number of plant bulbs and roots analyzed. We propose numbers for bulbs and roots per bulb when comparing the frequencies of micronuclei, mitotic anomalies and mitotic index with this test. Roots that had been treated with aqueous solutions, such as water samples collected in August 2007 from the Paraíba do Sul River at the Brazilian cities of Tremembé and Aparecida; negative and positive controls were used for bioassays. The presence of pollutants in the river water had been presumed based on our previous cytological data and an official report by the São Paulo State Environmental Agency (Brazil) on presence of fecal contaminants (Tremembé and Aparecida) and elevated dissolved aluminium (Aparecida) in the water under study. The sampling of ten bulbs and five roots per bulb was found adequate for comparative studies to evaluate with the A. cepa test the potential damage inflicted by pollutants in aquatic environments. Furthermore, even one bulb and one root per bulb was sufficient in discerning this damage, thereby shortening the time required to attain a statistically confident comparative evaluation. However, to allow for the use of statistical programs based on the evaluation of average values, and to avoid criticism based on genetic variability, we propose that three bulbs and three roots per bulb be considered as standard sample sizes for the A. cepa test.

Eye pigments of the blood-sucking insect, Triatoma infestans Klug (Hemiptera, Reduviidae).

The pigmentation of black (wild) and red (mutant) eyes of Triatoma infestans was studied spectrophotometrically and compared with red-eyed (wild) and white-eyed (mutant) forms of Drosophila melanogaster. The spectral absorption profiles of the black and red eye pigments of T. infestans were similar to each other and to that of the wild-type eyes of D. melanogaster. The similarity to the wild form of D. melanogaster indicated that both eye forms of T. infestans contained ommochromes of the xanthommatin type, a finding confirmed by ascending paper chromatography. Pteridines, melanins, and ommins were not detected as eye pigments in T. infestans. The eye color difference in T. infestans was assumed to be a function of the xanthommatin concentration, with a smaller content of ommochrome in red eyes, although this probably did not affect the insect's visual acuity. These data support other findings regarding the similarities between black- and red-eyed specimens of T. infestans for other characteristics.

Feulgen-DNA absorption spectrum of euchromatin and constitutive heterochromatin.

When the heterochromatin and euchromatin of Triatoma infestans are maximally depurinated, their Feulgen-DNA spectral profiles exhibit a shoulder at about lambda = 530 nm. The shoulder is especially prominent in the curves of the heterochromatin. It is assumed that this pattern is related to richness in repetitive DNA in the heterochromatin, favoring appearance of Schiff reagent molecules di-substituted with adjacent apurinic acid aldehydes.

Experimentally induced heat-shock tolerance in Panstrongylus megistus (Hemiptera: Reduviidae).

The survival and molting incidence of fifth-instar nymphs of Panstrongylus megistus (Burmeister, 1835), a vector of Chagas' disease, were investigated following sequential heat shocks in which a mild shock (35 or 40 degrees C, 1 h) preceded a more drastic one (40 degrees C, 12 h). The shocks were separated by 8-, 18-, 24-, or 72-h periods at 28 degrees C. The heat-shock tolerance response was more effective when the first shock was given at 40 degrees C. When the period between shocks was 18 h, the tolerance to sequential shocks (in terms of specimen survival) weakened, which suggested a transient control of the process that enables the organism to circumvent the unfavorable effects of severe shock. In terms of molting incidence, the heat-shock tolerance was only demonstrated when the period between the first shock at 40 degrees C for 1 h and the second shock at 40 degrees C for 12 h was > or = 24 h. These results are the first to demonstrate the induction of heat-shock tolerance in a blood-sucking hemipteran.

Apoptosis and catastrophic cell death in benzo[a]pyrene-transformed human breast epithelial cells.

Apoptosis and mitotic death, bi- and multinucleation, giant cells and micronucleation were investigated in human breast epithelial cell lines transformed by benzo[a]pyrene (BP) (BP1, BP1-E and BP1-E1 cells) and in BP1 cells transfected with the c-Ha-ras oncogene (BP1-Tras cells). Since BP induces apoptosis and the abnormal expression of ras genes elicits catastrophic mitosis, both cell death phenomena were expected to occur in this system, especially in BP1-Tras cells. Regardless of the cell line considered, single-nucleate cells were found to be eliminated preferentially through apoptosis, while bi- and multinucleate cells were eliminated through catastrophic mitosis. Apoptosis and catastrophic mitosis were observed in all cell lines but were significantly more frequent in BP1-Tras cells. The abnormal expression of Ha-ras in the latter cells may enhance in this system the effects of the BP apoptosis path reported for BP-transformed Hepa 1c1c7 hepatoma cells. Transfection with the ras oncogene also enhanced the mitotic disturbances, which produced multi- and micronucleation and mitotic death, possibly because of the genomic instability promoted by this oncogene in the BP-transformed cell line.

Computer assessment of chromatin patterns on some germ line cells of the hemipteran, Triatoma infestans Klug.

The digitized nuclear images of some Feulgen-stained primary and secondary spermatocytes of Triatoma infestans were analyzed with computer programs. Different cell types could be mathematically discriminated in terms of distance of the condensed chromatin areas from the center of the nucleus, relative circumference of these areas, and staining intensity ratio of the condensed chromatin areas with respect to the whole nucleus. The method appears promising for further comparative studies involving the whole spermatogenetic process of T. infestans. It is probably advantageous for the analysis of other hemipteran (and even non-hemipteran) spermatogeneses.

Optical anisotropy of toluidine blue-stained transformed cell nuclei.

Changes in chromatin order with cell transformation were studied in terms of optical anisotropy (birefringence) in toluidine blue-stained NIH 3T3 and Balb/3T3 cells. The transformed NIH 3T3 cell lines used were obtained by transfection with the T24 cell H-ras oncogene and the whole genomic DNA of MCF-7 human breast carcinoma cells. C2PO and PAP2 cell lines were used as representatives of Ha-ras-transformed NIH 3T3 cells with poor and high metastatic ability, respectively. The chromatin of all cells examined exhibited metachromasy and a birefringence of greenish-yellow interference color. The highest birefringence intensity was found in the chromatin granules of the most frequent nuclear phenotype of ras- and MCF-7 DNA-transformed cells, with the exception of the cells with high metastatic ability, in which a faint birefringence was observed. The differences in chromatin birefringence intensity are assumed to indicate differences in chromatin stereoarrangement with cell transformation. In the case of the highly tumorigenic and/or highly metastatic transformants the faint birefringence is assumed to be associated with the heterogeneous and complex physiological processes that may require a relatively less ordered arrangement of the chromatin.

Interference image analysis of heat-shocked HeLa cells.

HeLa cells were studied with the interference microscope 1 h after heat shock at temperatures of 40 degrees C and 43 degrees C and also under conditions of recovery from the shock. The aim was to investigate changes in patterns of cellular dry mass distribution with the heat shock, based on variation of interference colours in interphase cells. A change in concentration and distribution of a partly high-salt-resistant material in the nuclear and perinuclear regions of the cells was found to be induced by the heat shock at 43 degrees C, a situation which reverted to control under recovery conditions. A similar interference image response was obtained for the heat shock assay at 40 degrees C, but it was detected only during the 4 h recovery period, suggesting that it could have been elicited later. The material induced by the heat shock and visualized by the analysis of interference images is assumed to be a part of the nuclear matrix-intermediate filament cell fraction.

Absorption spectrum of Feulgen-stained polytene chromosomes of Drosophila melanogaster.

Hetero- and euchromatic polytene chromosome regions of early and fully grown larvae of Drosophila melanogaster had their Feulgen absorption curves determined cytophotometrically. Hydrolysis conditions adequate to induce maximal DNA depurination were used. No differences in curve shapes specially concerning the absorption shoulder at the 520 less than or equal to lambda less than or equal to 550 nm spectral range were detected with advancing polytenization or when comparing the absorption patterns for the hetero- and euchromatic regions to each other. It is considered that prominence of the Feulgen absorption shoulder could be related to the amount of Schiff molecules di-substituted with apurinic acid aldehydes and to the relatively larger resistance of these apurinic acid fragments towards solubilization with acid hydrolysis due to their binding to nuclear proteins. It is therefore assumed that no significant differences owing to the above-cited factors exist for the chromosome regions analyzed at 2 phases of the polytenization process. Consequently, the replication of reiterated sequences from the main band DNA which occurs in polytene chromosome regions of Drosophila melanogaster appears not to affect the Feulgen absorption spectrum of this material.

Investigation of hycanthone binding to DNA in chromatin with different supra-organization, composition, and function.

Hycanthone is a drug used for the clinical treatment of schistosomiasis, although inducing chromosomes breaks and mutagenesis when it enters the DNA double helix. Based on visual observation of characteristics of basophilia and anisotropy after toluidine blue staining, a preferential binding of hycanthone to heterochromatin could be demonstrated for the nuclei of the Malpighian tubules of Triatoma infestans. In other cellular systems like cattle kidney cells in culture, plant cells, and mouse lymphocytes, the drug could be demonstrated to bind heterochromatin and euchromatin, irrespective of the packing state of the latter. When penetrating the various heterochromatin types (with the exception of T. infestans), the drug induced a chromatin loosening that could favor incidence of chromatin breaks. The variation of hycanthone binding to DNA in different cell types is possibly related to differences in composition, stereo-arrangement and stability of the DNA-protein complexes involved.

Lability to acid hydrolysis in some different DNA-protein complexes of spermatozoa.

The Feulgen hydrolysis kinetics was investigated in spermatozoa with different composition in DNA-protein complexes. The species used were: Bos taurus (arginine rich nuclear protein also containing cystine residues), Pichroplus bergi, Triatoma infestans (arginine-rich nuclear protein), Lytechinus variegatus and Apis mellifera (lysine-rich nuclear protein). The spermatozoa were subjected to Feulgen's reaction, after varying the fixative type and the hydrolysis times. Feulgen-DNA values were obtained with an automatic scanning cytophotometric procedure. Differences were demonstrated in the hydrolysis kinetics as a function of differences in composition of the DNA-protein complexes being present in the spermatozoon chromatin. Differences in the profiles of Feulgen hydrolysis curves, having for basis the fixation, were rather clear for bull, grasshopper, and blood-sucking insect spermatozoa than for the sea-urchin and bee spermatozoa. The different hydrolysis kinetics of chromatin of blood-sucking insect spermatozoa compared to that of grasshopper, sea-urchin, and bee sperm cells suggests that, although the first 2 materials contain an arginine-rich "germinative" protein and the latter 2 ones contain a lysine-rich protein, these differ to each other. The DNA depurination was obtained more quickly for T. infestans (20 min) and P. bergi (10 min) spermatozoa when they were fixed in the ethanol-acetic acid (EA) mixture. Morphologically anomalous bull spermatozoa fixed in the EA mixture presented a quicker depurination (30 min) as compared to the normal cells (1 h). The fast lability to acid hydrolysis in the abnormal cells is certainly due to anomalies in their basic nuclear "germinative" protein. In the formalin fixed materials the maximal depurination was obtained earlier in bull spermatozoa (30 min) followed by sperm cells of P. bergi, T. infestans, L. variegatus (all of them one-hour hydrolysis) and finally Apis mellifera (2 h hydrolysis). The presence of secondary peaks at the descending branch of the hydrolysis curves of grasshopper and sea-urchin spermatozoa, indicates for these, more than 1 kind of apurinic-acid protein complexes. The spermatozoa bearing arginine-and/or cystine-rich nuclear protein contain a more easily soluble apurinic acid protein complex. Due to the differences in hydrolysis kinetics of chromatin in spermatozoa containing different nuclear "germinative" proteins, this cellular type does not appear indicated as a haploid control for evaluation of Feulgen-DNA contents of diploid and polyploid somatic cells.

Critical electrolyte concentration and dispersion of birefringence in heterochromatin and euchromatin of copper sulfate-treated specimens of Triatoma infestans Klug.

Binding of toluidine blue molecules to DNA phosphates under Mg2+ competitive staining conditions and dispersion of birefringence were studied in heterochromatin and euchromatin of Malpighian tubule cells of the hemipteran, Triatoma infestans; 24 h after treatment with copper sulfate. The objective was to detect whether copper binds to chromatin DNA, affecting the critical electrolyte concentration (CEC) value and the chromatin anisotropy in these chromatin types. A decrease in CEC values as measured by metachromasy abolition, and in the concentration of Mg2+ leading to a change from anomalous dispersion of birefringence to normal dispersion of birefringence was found for the heterochromatin and euchromatin of two different nuclear types in the copper-treated material. Protein dissociation from DNA, leading to increased numbers of DNA phosphates available for dye binding, promoted by Mg2+ concentrations above that corresponding to the CEC point is suggested. Based on anisotropy findings, the binding of copper to DNA was assumed not to affect the usual molecular orientation of the DNA.

Basic fuchsins and the Schiff-aldehyde reaction. I. Spectral absorption characteristics in solution.

Spectral absorption profiles at the UV and visible ranges were detected spectrophotometrically for solutions of various basic fuchsins, for the SCHIFF reagent prepared from these stains and for the end product from SCHIFF-formaldehyde reaction. The aim was to detect whether absorption spectra could vary as a function of the basic fuchsin origin up to the point of being suspected of affecting potentially the spectrum of FEULGEN-stained chromatin. The results indicate a constancy in the spectral absorption characteristics for the absorption profiles at the visible range, when one considers SCHIFF reagents prepared from various basic fuchsins added with aldehyde. However, it is possible that size, stereoarrangement and electron distribution of the aldehyde- (eventually, ketone-) bearing molecule involved in SCHIFF-carbonyl reactions would function as auxochroms, the spectral characteristics of which could be affected by dye content and contaminants of the basic fuchsin.

Anisotropic properties of the myelin sheath.

Form birefringence curves were determined for fixed (and unfixed rat axons before and after lipid extraction. The total detected birefringence was assumed to be due to the macromolecular array of myelin sheath components (phospholipids, cholesterol, and proteins). Unfixed nerves displayed negative form birefringence. Their form birefringence curve exhibited a = refractive index match point positioned at n = 1.46 (intrinsic birefringence). Formalin fixation induced decrease in the optical retardation values but did not affect the profile of the form birefringence curves. After lipid removal, however, the anisotropic patterns of the fixed and unfixed nerves changed. A positive form birefringence was then exhibited, which is attributed to the macromolecular orientation of the protein framework of the myelin sheath. Changes in the shape of the form birefringence curve and in the localization (and number) of the refractive index match point were found. They varied as lipids had been removed from nerves subjected or not to fixation. Therefore, the form birefringence of the myelin sheath proteins plays a part in the total phenomen observed in the whole nerves interfering with that displayed by phospholipids and cholesterol of the mentioned structure.

[Survival and occurrence of molting in Triatoma infestans Klug (Hemiptera, Reduviidae) after temperature shock]. / Sobrevivência e ocorrência de muda em Triatoma infestans Klug (Hemiptera, Reduviidae) após choque de temperatura.

Survival and molting occurrence were studied in specimens of Triatoma infestans over 30 days after temperature shocks. Hyperthermal and hypothermal shocks could be found to affect both survival and molting incidence as a function of temperature and period of the development phase and sex of the specimens. Considering the various test conditions, the shock at 0 degree C for 12 h was found to elicit the most deleterious effect, whereas shocks at 40 degrees C and 0 degree C even for 1 h are interpreted as affecting the hormonal balance which controls molting. Cases of a rise in post-shock survival are suggested to have been favored by heat-shock protein action.

Experimentally induced heat- and cold-shock tolerance in adult Panstrongylus megistus (Burmeister) (Hemiptera, Reduviidae).

The survival rate of domestic male and female adult Panstrongylus megistus was studied after sequential heat and cold shocks in order to investigate shock tolerance compared to that previously reported for nymphs. Sequential shocks were such that a milder shock (0 degree C, 5 degrees C, 35 degrees C, or 40 degrees C for 1 h) preceded a severe one (0 degree C or 40 degrees C for 12 h), separated by intervals of 8, 18, 24, and 72 h at 28 degrees C (control temperature). The preliminary thermal shock induced tolerance to the more severe one, although tolerance intensity depended on the initial shock temperature and the interval between treatments. Despite the observed tolerance, the survival rate for insects subjected to both shocks decreased when compared to that of individuals subjected to a single mild shock. When tolerance differed with sex, females showed greater values than males. In contrast to the response detected in nymphs, for which higher heat tolerance values were sustained for intervals of up to 24 h (preliminary shock, 35 degrees C) or even longer (preliminary shock, 40 degrees C) between sequential shocks, significant values were verified in adults only for shock intervals of up to 8 h (preliminary shock, 40 degrees C). While findings for nymphs exhibited considerable cold-shock tolerance under conditions in which preliminary shocks were given at 5 degrees C or 0 degree C and the periods between shocks were up to 72 h long, the adults were shown to be capable of acquiring a substantial tolerance response to a more severe cold shock only when the preliminary shock was given at 0 degree C and shock interval surpassed 18 h. It is assumed that the mechanisms involved in the cellular protection of P. megistus under sequential temperature shocks (heat shock protein action?) may loose effectiveness with insect development.

Apoptotic ratios and mitotic abnormalities in 17-ß-estradiol-transformed human breast epithelial MCF-10F cells.

Treatment of human breast epithelial cells MCF-10F with 17-ß-estradiol has been reported to result in E2-transformed cells which have given rise to highly invasive C5 cells that in turn generate tumors in SCID mice. From these tumors, various cell lines, among which C5-A6-T6 and C5-A8-T8, were obtained. Although different phases of the tumorigenesis process in this model have been studied in molecular biology and image analysis assays, no cytological data on apoptotic ratios and mitotic abnormalities have been established to accompany the various steps leading to 17-ß-estradiol-treated MCF-10F cells to tumorigenesis. Here we detected that the apoptotic ratio decreases with the transformation and tumorigenesis progress, except for the tumor cell line C5-A8-T8, probably on account of its more intense proliferation rate and a more rapid culture medium consumption. Increased frequency of mitotic abnormalities contributed by triple- and tetrapolar metaphases, and by lagging chromosomes and chromosome bridges observed at the anaphase found by transformation and tumorigenesis progress. However, no difference was found under these terms when the C5-A6-T6 and C5-A8-T8 tumor cell lines were compared to each other. Present findings are in agreement with the nuclear instability and enrichment of dysregulated genes in the apoptotic process promoted by transformation and tumorigenesis in 17-ß-estradiol-treated MCF-10F cells.

Extended chromatin fibers and chromatin organization.

Studies of chromatin extensibility have revealed the flow of chromatin and DNA from cell nuclei and chromosomes in response to gravity or mechanical stretch following lysis by hypertonic saline and detergent solutions. Since this phenomenon was first reported, the technical methods by which extended chromatin fibers (ECFs) may be analyzed have been improved. These methods include topochemical assays, fluorescence in situ hybridization, immunofluorescence, electron microscopy, and polarization microscopy. Chromatin and DNA "halos" also have been studied in materials subjected to lysis, especially in a horizontal position or after cytocentrifugation. The analysis of ECF formation is useful not only as a tool for detecting the positioning of certain DNA signals on chromatin filaments, but also for describing diverse DNA-protein associations that may be related to varying transcriptional activities and chromatin supraorganization. A brief review of the methods and applications of ECF formation is presented here. We focus on light microscopy studies of ECF formation in mouse hepatocytes under different chromatin supraorganization and physiological conditions and in sperm cells with different DNA-protein complexes.

Prevalence and intensity of infection, metacyclogenesis and nuclear phenotypes in Panstrongylus megistus (Burmeister, 1835) after ingestion of Trypanosoma cruzi (Chagas, 1909) II and subjection to heat shock.

This study aimed to contribute to our knowledge of the parasite-vector interaction associated with Trypanosoma cruzi (Chagas, 1909) infection in Panstrongylus megistus (Burmeister, 1835), an important vector of Chagas' disease in Brazil. The prevalence and intensity of T. cruzi infection, the incidence of metacyclogenesis and the frequency of nuclear phenotypes in Malpighian tubules were investigated in nymphs of P. megistus, reared at 28 °C and subjected to heat shock (40 °C, 1 hour) two days after infection with T. cruzi II (Y strain). Following the 45-day post-infection period, the frequency of epimastigotes was much higher than that of trypomastigotes in both heat-shocked and non-shocked insects, and the prevalence of infection was not altered by heat shock. Fewer epimastigotes and trypomastigotes were found in the infected insects subjected to the heat shock, indicating that the multiplication and metacyclogenesis of the parasites were affected by the stress. In infected specimens heat shock promoted an increased frequency of cell nuclei with heterochromatin decondensation, a cell survival response to stress, and did not affect insect survival. The effects of infection and heat shock, especially on the multiplication and metacyclogenesis of T. cruzi, and the observed resistance to heat shock developed by P. megistus nymphs are suggestive that they should be considered when adequate conditions for rearing these infected insects in the laboratory are pursued.