Apoptotic volume decrease (AVD) in A549 cells exposed to water-soluble fraction of particulate matter (PM10).

Exposure to atmospheric particulate matter (PM) is recognized as a human health risk factor of great concern. The present work aimed to study the cellular mechanisms underlying cytotoxic effects of airborne particulate matter <10 µm in size (PM10), sampled in an urban background site from January to May 2020, on A549 cells. In particular, the study addressed if PM10 exposure can be a main factor in the induction of the Apoptotic Volume Decrease (AVD), which is one of the first events of apoptosis, and if the generation of intracellular oxidative stress can be involved in the PM10 induction of apoptosis in A549 cells. The cytotoxicity of PM10 samples was measured by MTT test on cells exposed for 24 h to the PM10 aqueous extracts, cell volume changes were monitored by morphometric analysis of the cells, apoptosis appearance was detected by annexin V and the induction of intracellular oxidative stress was evaluated by the ROS sensitive CM-H2DCFDA fluorescent probe. The results showed cytotoxic effects ascribable to apoptotic death in A549 cells exposed for 24 h to aqueous extracts of airborne winter PM10 samples characterized by high PM10 value and organic carbon content. The detected reduced cell viability in winter samples ranged from 55% to 100%. Normotonic cell volume reduction (ranging from about 60% to 30% cell volume decrease) after PM10 exposure was already detectable after the first 30 min clearly indicating the ability of PM10, mainly arising from biomass burning, to induce Apoptotic Volume Decrease (AVD) in A549 cells. AVD was prevented by the pre-treatment with 0.5 mM SITS indicating the activation of Cl- efflux presumably through the activation of VRAC channels. The exposure of A549 cells to PM10 aqueous extracts was able to induce intracellular oxidative stress detected by using the ROS-sensitive probe CM-H2DCFDA. The PM10-induced oxidative stress was statistically significantly correlated with cell viability inhibition and with apoptotic cell shrinkage. It was already evident after 15 min exposure representing one of the first cellular effects caused by PM exposure. This result suggests the role of oxidative stress in the PM10 induction of AVD as one of the first steps in cytotoxicity.

Relationship among parameters of lake polluted sediments evaluated by multivariate statistical analysis.

In the attempt to assess the relationship and interdependency among sediment toxic pollutants, in particular heavy metals, polycyclic aromatic hydrocarbons (PAH), and linear alkyl sulfonates (LAS) and some of the sediment typical components: inorganic carbon (IC), organic material (OM) and acid volatile sulphides (AVS), multivariate techniques of statistical analysis have been applied to a set of chemical data obtained by the analysis of the sediments of the Trasimeno Lake, a central Italy lake characterized by a large surface (128 km(2)) and a low mean depth (about 4.5 m). The results of principal component analysis (PCA) show interrelationships between: OM content and PAH, Pb, and Cu concentrations of the sediments, LAS and AVS, and AVS and IC. The effect of the different sampling periods on sediment composition and contamination level, and the clustering of the sampling sites as a consequence of pollutant load are also shown. The principal component bi-plot of the variables and samples indicates that PAH have the greatest influence on the separation of samples in the different sampling periods.

Cancer chemoprevention by hydroxytyrosol isolated from virgin olive oil through G1 cell cycle arrest and apoptosis.

Recent epidemiological evidence and animal studies suggest a relationship between the intake of olive oil and a reduced risk of several malignancies. The present study assesses the effect of hydroxytyrosol, a major antioxidant compound of virgin olive oil, on proliferation, apoptosis and cell cycle of tumour cells. Hydroxytyrosol inhibited proliferation of both human promyelocytic leukaemia cells HL60 and colon adenocarcinoma cells HT29 and HT29 clone 19A. The con-centrations of hydroxytyrosol which inhibited 50% of cell proliferation were approximately 50 and approximately 750 micromol/l for HL60 and both HT29 and HT29 clone 19A cells, respectively. At concentrations ranging from 50 to 100 micromol/l, hydroxytyrosol induced an appreciable apoptosis in HL60 cells after 24 h of incubation as evidenced by flow cytometry, fluorescence microscopy and internucleosomal DNA fragmentation. Interestingly, no effect on apoptosis was observed after similar treatment of freshly isolated human lymphocytes and polymorphonuclear cells. The DNA cell cycle analysis, quantified by flow cytometry, showed that the treatment of HL60 cells with hydroxytyrosol 50-100 micromol/l arrested the cells in the G0/G1 phase with a concomitant decrease in the cell percentage in the S and G2/M phases. These results support the hypothesis that hydroxytyrosol may exert a protective activity against cancer by arresting the cell cycle and inducing apoptosis in tumour cells, and suggest that hydroxytyrosol, an important component of virgin olive oil, may be responsible for its anticancer activity.

Protective activity of butyrate on hydrogen peroxide-induced DNA damage in isolated human colonocytes and HT29 tumour cells.

Epidemiological studies support the involvement of short-chain fatty acids (SCFA) in colon physiology and the protective role of butyrate on colon carcinogenesis. Among the possible mechanisms by which butyrate may exert its anti-carcinogenicity an antioxidant activity has been recently suggested. We investigated the effects of butyrate and mixtures of SCFA (butyrate, propionate and acetate) on DNA damage induced by H(2)O(2) in isolated human colonocytes and in two human colon tumour cell lines (HT29 and HT29 19A). Human colonocytes were isolated from endoscopically obtained samples and the DNA damage was assessed by the comet assay. H(2)O(2) induced DNA damage in normal colonocytes in a dose-dependent manner which was statistically significant at concentrations over 10 microM. At 15 microM H(2)O(2) DNA damage in HT29 and HT29 19A cells was significantly lower than that observed in normal colonocytes (P < 0.01). Pre-incubation of the cells with physiological concentrations of butyrate (6.25 and 12.5 mM) reduced H(2)O(2) (15 microM) induced damage by 33 and 51% in human colonocytes, 45 and 75% in HT29 and 30 and 80% in HT29 19A, respectively. Treatment of cells with a mixture of 25 mM acetate + 10.4 mM propionate + 6.25 mM butyrate did not induce DNA damage, while a mixture of 50 mM acetate + 20.8 mM propionate + 12.5 mM butyrate was weakly genotoxic only towards normal colonocytes. However, both mixtures were able to reduce the H(2)O(2)-induced DNA damage by about 50% in all cell types. The reported protective effect of butyrate might be important in pathogenetic mechanisms mediated by reactive oxygen species, and aids understanding of the apparent protection toward colorectal cancer exerted by dietary fibres, which enhance the butyrate bioavailability in the colonic mucosa.

Influence of culture conditions on the DNA-damaging effect of benzene and its metabolites in human peripheral blood mononuclear cells.

The DNA-damaging ability of benzene and its metabolites on peripheral blood mononuclear cells (PBMC) has been investigated by using the alkaline comet assay. The PBMC were incubated with different compounds in two different media for 2 and 24 hr at concentrations that did not affect cell viability and the DNA damage was quantified by a computerized image analysis system. Benzene and phenol (5 mM) did not show any genotoxic activity after 2 hr of incubation in the two media tested, phosphate-buffered saline (PBS) and RPMI containing 5% of heat-inactivated fetal calf serum (RPMI + 5% FCS), whereas phenol was genotoxic and cytotoxic at 10 mM after 24 hr of incubation in RPMI + 5% FCS. All other benzene metabolites were genotoxic at micromolar concentrations when incubated in PBS with the following decreasing order of potency: benzenetriol, catechol, hydroquinone, and benzoquinone. When the PBMC were incubated in RPMI + 5% FCS, the effect of catechol (200-600 microM) and benzenetriol (10 microM) was reduced, whereas the genotoxicity of benzenetriol at high concentrations (50-100 microM) and hydroquinone (150-2500 microM) was not affected. In contrast, the effect of benzoquinone at 5 and 10 microM was greatly enhanced when the cells were incubated in RPMI + 5% FCS. This effect resulted mainly from the presence of serum in the medium and it was almost completely inhibited by boiling the serum (100 degrees C, 5 min) and was partially reduced by extensive dialysis. Benzoquinone was the most damaging compound when tested under more physiological conditions, thereby supporting the general observation that it is the most myelotoxic benzene metabolite.

Antioxidants prevent the lymphocyte DNA damage induced by PMA-stimulated monocytes.

Oxidative stress has been related to several degenerative diseases such as cancer and coronary heart disease. Reactive oxygen species can damage different cellular macromolecules, including DNA, which is directly responsible for mutation and carcinogenesis. In this study, monocytes that were activated by phorbol 12-myristate 13-acetate (PMA) were coincubated with lymphocytes, and the DNA damage was measured by single-cell gel electrophoresis (comet) assay. Stimulation of monocytes with PMA activates the "respiratory burst," which evokes DNA damage in lymphocytes. The extent of the damage is related to the concentration of monocytes and the exposure time. Exogenous addition of superoxide dismutase did not prevent the DNA damage, which suggests that superoxide ions are not directly responsible for the damage. Partial protection was observed when catalase was included (60% protection), which indicates that other reactive species, in addition to H2O2, are responsible for the damage. In this system, the protective activity of natural antioxidants at different concentrations was also investigated. After coincubation of PMA-activated monocytes with lymphocytes in the presence of each antioxidant for one hour at 37 degrees C, the lymphocyte DNA damage was determined. All the compounds protected the lymphocytes to a certain degree, with a maximum effect at different concentrations: 41% protection with 1 microM ascorbic acid, 55% protection with 40 microM alpha-tocopherol, 50% protection with 3 microM beta-carotene, and 56% protection with 5 microM quercetin. On the basis of these results, we maintain that this "ex vivo model," more closely related to physiological conditions, could be used to test the antioxidant activity of different compounds.

Priming effect of benzo[a]pyrene on monocyte oxidative metabolism: possible mechanisms.

Monocytes, separated from human peripheral blood, were preincubated with different polycyclic aromatic hydrocarbons (PAHs) for 24 h and the production of superoxide ions (O*2-) was then measured using as a stimulating agent phorbol 12-myristate 13-acetate. A significantly enhanced O*2- production is only observed when the cells are treated with benzo[a]pyrene (B[a]P); benzo[e]pyrene, benzo[a]anthracene and 3-methylcholanthrene induce a small but not significant increase of O*2-. Anthracene has no effect, while phenanthrene slightly inhibits. The priming activity of B[a]P is unrelated to variations in intracellular Ca2+ ([Ca2+]i), as demonstrated by the inability of B[a]P to increase [Ca2+]i concentration in both monocytes and the promonocytic cell line U937. Furthermore, in monocytes the sarcoplasmic/endoplasmic reticulum Ca2+ -ATPase inhibitor, thapsigargin, which can increase [Ca2+]i evokes a differentiation-like event associated with a decrease in the production of superoxide ions. These results further support that the enhancing activity of B[a]P on monocytes superoxide production is not mediated by an increase of [Ca2+]i. In contrast, the role of the aryl hydrocarbon receptor (AhR) in B[a]P-induced superoxide ion enhancement is suggested by the inhibitory effect of the specific antagonist alpha-naphthoflavone (alphaNF), while the tumor necrosis factor (TNF-alpha) is not involved in the phenomenon. Thus, the interaction of B[a]P with its cytosolic receptor and either the metabolism of the compound into reactive intermediates or the over-expression of some unknown genes seem to be involved in an essential step in this process.

Comparison of fluorogenic and conventional membrane filter media for enumerating coliform bacteria.

4-Methylumbelliferyl-beta-D-galactoside and 4-methylumbelliferyl-beta-D-glucuronide were added to MacConkey broth and their diagnostic powers for total coliforms (TC) and Escherichia coli, respectively, were tested by membrane filtration at primary isolation. Examining water samples from different sources proved the usefulness of fluorogenic rather than reference media both as regards recovery efficiency and rapidity (possible within 12 h) of analyses. The recoveries obtained by fluorogenic and conventional tests for both TC and E. coli were correlated. Values were comparable in surface water samples, while a higher sensitivity of fluorogenic media was observed in samples of shallow contaminated ground water. Results seem to indicate that the use of fluorogenic membrane filtration analysis for colimetric indicators could be favourably considered especially for sanitary surveying of drinking water.

Numerical analysis and DNA base compositions of some thermophilic Bacillus species.

Morphological, physiological, and biochemical characteristics and the DNA base compositions of 133 thermophilic Bacillus strains were determined. A total of 54 of these strains were received as identified species (mainly Bacillus stearothermophilus, Bacillus coagulans, Bacillus brevis, and Bacillus licheniformis) from international culture collections, and 79 newly isolated strains, which were isolated mainly from sugar diffusion juices of Italian plants, were also examined. Numerical taxonomy techniques (simple matching coefficient and unweight pair grouping using the mathematical average) and DNA G + C values showed that the strains aggregated into nine clusters. Both B. licheniformis and B. brevis were well separated from the other organisms. B. stearothermophilus and B. coagulans were confirmed as separate clusters and exhibited greater heterogeneity than previously shown. The B. stearothermophilus strains clustered into four groups, three of which have been recognized previously by other authors; the members of the fourth group had distinctive characteristics, including considerable biochemical inertness, an inability to grow at temperatures greater than 60 degrees C, and a high G + C content. Within the B. coagulans cluster the strains with characteristics very similar to those of the new species Bacillus smithii clustered together. However, the remaining strains were still clearly separated into two groups; one of these groups was considered B. coagulans sensu stricto, and the other was distinguished by morphological and biochemical criteria, such as spores which do not swell the sporangia, utilization of citrate, a higher proteolytic activity, and acidification of some carbohydrates. Our results were confirmed by comparing them with distinctive characteristics of recently described thermophilic Bacillus species.

[Tuberculin survey and its meaning for a correct prevention in a high risk population. Epidemiological data relative to students of the faculty of medicine and soldiers residing in Ferrara (author's transl)]. / Indagine tubercolinica e suo significato ai fini di una correta prevenzione in popolazione ad alto rischio. Dati epidemiologici in studenti della Facoltà di Medicina ed in militari di Ferrara.

The Authors list the results obtained from a tuberculin survey carried out on students of the Faculty of Medicine of the University of Ferrara (school year 1977/1978) and on enlisting soldiers. A different skin positivity is here confirmed: a higher percentage in males (27.41%) than in females (17.91%). A higher frequency of positive responses is noted in students originating from the centre-south of Italy. Such tendency is principally due to the greater differences in skin positivity found in females. Students over 26 years of age show a skin positivity of more than 40%, whilst for 17 to 20 years old skin positivity ranges between 22% to 25%. The frequency of skin positivity in soldiers confirms the data found in male students of the same age group. Post vaccine control, although limited to a smaller group of students, shows a definite turn towards positivity with an average figure in both sexes of 63%. The Authors, although admit having carried out their survey on a small population, believe to have contributed with useful information on the epidemiological situation of tuberculosis in age groups with scarce statistical data.

Sensitivity to lytic agents and DNA base composition of several aerobic spore-bearing bacilli.

The authors studied the possible relationship between a genetic characteristic, like DNA base composition, and certain phenotypic characteristics, i.e., sensitivity to lytic agents, morphology of colonies, and biochemical reactions in 34 strains of spore-bearing bacilli. From the results obtained two groups of bacilli have been identified. The first group includes the species B. subtilis, B. pumilus, B. licheniformis, and B. firmus and one strain of B. megaterium. The mean value of the GC% of the DNA is 44.22 +/- 1.76. All the strains examined are highly sensitive to lysozyme and resistant to sodium lauryl sulphate (S.L.S.); the surface colonies have a "rhizoid" appearance and the microcolonies on slide microculture are star-shaped. The second group includes the species B. cereus, B. cereus var. mycoides, B. anthracis, and B. thuringiensis. The mean value of the GC% of the DNA is 33.65 +/- 0.59. All the strains belonging to this group are resistant to both lysozyme and S.L.S., and the surface macro-colonies and the microcolonies have a "medusae head" appearance. The two groups also have certain different biochemical reactions; e.g., anaerobic growth and the egg yolk reaction, with few exception, are negative for the first group and positive for the second; furthermore, the strains in the first group (with rare exceptions) cause fermentation in the three carbohydrates, glucose, arabinose, and xylose, while glucose only is fermented by all strains with one exception in the second group. The position of B. megaterium is not yet clear, although one strain may certainly be included in the first group. Lysis by lipase is extremely variable and does not correlate with any of the other characteristics studied. The other species studied in relation to the characteristics, considered in our research (B. coagulans, B. macerans, B. polymyxa, B. laterosporus, B. alvei, B. circulans, B. stearothermophilus, and B. brevis), are not susceptible to grouping, either in the first, or in the second or even in a separate group.

[Cellular lysis and dissociative phase of the genus Bacillus]. / Lisi cellulare e fasi dissociative del genere Bacillus

The sensitivity of the dissociative variants "R", "RS" and "S" of the spore-forming bacilli to some lytic agents (lysozyme, sodium lauryl sulphate, lipase, trypsin and some of their associations) has been studied. The research has been carried out on 32 strains: 14 "R", 14 "RS" and 4 "S" of different species of the genus Bacillus. The results have shown that the sensitivity to the studied lytic agents is strictly correlated, not to the species, but to the dissociative phases: these results are in accordance with those obtained in previous researches, carried out by the same Authors, on other characters, both morphological and biological, of the same variants. Thus, also through this way, it is possible to reach the conclusion that within this genus, there is more similarity among the different species of the same dissociative phase than among different dissociative variants of the same species. The results obtained in the present study allow to advance hypothesis, based also on the data of the literature, about the composition of the cell walls of the three above mentioned dissociative variants of the strains belonging to the genus Bacillus.

[Dissociative phases and pathogenicity of different species of the Bacillus genus]. / Fasi dissociative e patogenicitá di specie diverse del genere "Bacillus"

The previously postulated hypothesis, according to which different species of the genus Bacillus show strictly similar morphological and biological properties when the same variants are considered, has been confirmed by the present research. The "S" (smooth) variants of the five studied species (B. anthracis, B. subtilis, B. cereus, B. megaterium, B. mesentericus) are all lethal, at the experimented dose, for mice, whereas the "R" (rough, "star-shaped" colonies) variant of the same strains of the same species are all not pathogenic for the same animals. Likewise the "S" variants of three species tested in guinea pigs showed to be pathogenic; particularly B. anthracis and B. subtilis were lethal, whereas B. cereus caused a black eschar like that one described in the cutaneous anthrax. The "RS" variant ("medusae head" surface colonies) is not pathogenic for mice and guinea pigs (even B. anthracis) if the tested strains are cultivated for years in ordinary solid nutrient media; the same morphological variants are strongly pathogenic (also B. subtilis), when the strains are recently isolated from infected animals. The similarity between the same "S" variants of different species is proved also by the protection given by anti-anthrax serum to animals infected by B. subtilis.