X-irradiated human lymphocytes with unstable aberrations and their preferential elimination by p53/survivin-dependent apoptosis.

PURPOSE: To investigate whether unstable types of chromosomal aberrations are more effective in priming apoptotic cell death in comparison with stable ones. Also, to highlight the phase of the cell cycle at which apoptosis occurs and the mechanism of its execution. MATERIALS AND METHODS: G0 human peripheral blood lymphocytes were X-irradiated in the presence or absence of the repair inhibitor cytosine arabinoside (Ara-C). After irradiation, the lymphocytes were analysed for induction of dicentrics, translocations, apoptosis, p53 and survivin expression at various recovery times. RESULTS: A preferential elimination of cells bearing dicentrics with respect to those with balanced translocations was observed. There was a time-dependent correlation between the decrease in the frequency of dicentrics and the increase in the per cent of apoptotic cells. Most of the apoptotic cells were labelled with bromodeoxyuridine and were mononucleated in cytochalasin B-treated cells cultures (blocked cytokinesis). However, after continuous colcemid treatment, the apoptotic pathway was not induced. Moreover, in the G2/M-phase, an increase in p53 and a decrease in survivin occurred that were X-ray and Ara-C dose dependent. CONCLUSIONS: The apoptotic process is primed when the dicentric-bearing human peripheral blood lymphocytes attempt to exit from metaphase. It is possible that unstable aberrations generate changes in the mitotic spindle causing mechanical tension at the kinetochore, activating the mitotic checkpoint and the execution of p53/survivin-dependent apoptosis.

Effects of gatifloxacin on phagocytosis, intracellular killing and oxidant radical production by human polymorphonuclear neutrophils.

The ingestion and killing of bacteria by phagocytic cells is an important step in the sequence of interactions between invading microorganisms and host defense systems and may be affected by antibiotics. We investigated the effects of gatifloxacin on the phagocytosis, killing and oxidative bursts of human polymorphonuclear neutrophils (PMNs). The percentage phagocytosis and the phagocytosis index were unaffected by exposure of Escherichia coli strains to sub-MICs of gatifloxacin to a 1/64 dilution. However a significant increase in percentage intraphagocytic killing and the killing index occurred in one E. coli strain at 1/32 MIC and in two strains at 1/16 MIC. The incubation of PMNs with sub-MICs and supra-MICs of gatifloxacin (to 32 MIC) did not affect the oxidative bursts.

Pifithrin-alpha, an inhibitor of p53, enhances the genetic instability induced by etoposide (VP16) in human lymphoblastoid cells treated in vitro.

Recent studies indicate that p53-dependent apoptosis induced in normal tissues during chemo- and radiotherapy can cause severe side effects of anti-cancer treatments that limit their efficiency. The aim of the present work was to further characterise the role of p53 in maintaining genomic stability and to verify whether the inhibition of p53 function in normal cells by pifithrin-alpha (PFT-alpha) may contribute in reducing the side effects of cancer therapy. Two human lymphoblastoid cell lines, derived from the same donor, TK6 (p53 wild type) and WTK1 (p53 mutated) have been treated with an anti-neoplastic drug, the etoposide (VP16), an inhibitor of DNA topoisomerase II in presence or in absence of the p53 inhibitor PFT-alpha. Following treatments with VP16 on TK6 and WTK1, we observed a higher induction of chromosome aberrations in WTK1 (p53 mutated) and of apoptosis in TK6 (p53 wild-type) cells. The p53 inhibition by PFT-alpha in VP16 treated TK6 cells produced an increase of chromosomal aberrations and a reduction of apoptosis. Therefore, the temporary suppression of the function of p53 by PFT-alpha, increasing the survival of the normal cells, could be a promising approach to reduce the side-effects of cancer therapy but it is important to consider that the surviving cells could be genetically modified and consequently the risk of secondary tumours could be increased.

The SH-metabolite I of erdosteine, a mucolytic drug, enhances the inhibitory effect of salbutamol on the respiratory burst of neutrophils.

Reactive oxygen species (ROS) are a common denominator of airway inflammation associated with chronic obstructive pulmonary disease (COPD) and asthma, as well as with less frequent lung diseases such as idiopathic pulmonary fibrosis (IPF), acute respiratory distress syndrome (ARDS) and cystic fibrosis (CF). The most frequently administered drugs used to treat these diseases are bronchodilators, antioxidant/antiphlogistic agents and mucoactive drugs. The metabolization of the mucoactive drug erdosteine produces an active metabolite (Met I) with a reducing SH group. In addition to its mucolytic action, Met I also has useful antioxidant activity. The various activities of beta 2-agonists include their ability to reduce the respiratory burst of neutrophils and the subsequent release of ROS. beta 2-Agonists and mucoactive drugs may be administered to the same patients during the treatment of lung diseases. The aim of this study was to investigate the ability of Met I to potentiate the activity of salbutamol in inhibiting the in vitro respiratory burst of neutrophils by means of chemiluminescence. The combination of Met I 5 and 10 micrograms/ml with salbutamol 10(-5), 10(-6) and 10(-7) M led to a significant reduction in respiratory bursts when the neutrophils were stimulated with the soluble stimulant N-formyl-methionylleucyl-phenylalanine (fMLP). The combinations of the two drugs that reduced the respiratory bursts when a particulate stimulus (Candida albicans) was used were those containing 10(-5) M of salbutamol. The reasons for this different behavior remain unclear and raise questions about the specific roles, sites and mechanisms of action of the different types of stimulation undergone by the respiratory airways.

Detection of DNA primary damage by premature chromosome condensation in human peripheral blood lymphocytes treated with methyl methanesulfonate.

Methyl methanesulfonate (MMS) is a direct acting methylating agent which produces apurinic sites that are transformed into DNA single-strand breaks by base excision repair. MMS-induced DNA lesions have to be transformed by DNA synthesis in order to give rise to chromosomal damage. In this study the premature chromosome condensation (PCC) technique was used in G(1) human lymphocytes treated with MMS to investigate whether, with this technique, chromosomal damage could be detected without the cell needing to undergo DNA synthesis. A dose-dependent increase in chromosomal fragmentation was indeed observed in G(1) lymphocytes. MMS treatment at 1.3, 2.5 and 5 mM was characterized by the appearance of highly fragmented chromosomes. This observation induced us to further investigate whether this effect was more connected with triggering of apoptotic cell death than a consequence of the PCC technique. Data obtained by nuclear morphology analysis, by Trypan blue exclusion assay and pulsed field gel electrophoresis seem to suggest that the observed chromosome fragmentation could be due to the onset of apoptosis. Consequently, one should bear in mind that the PCC technique can overestimate chromosomal damage when apoptosis is also induced.

The involvement of chromatin condensation in camptothecin-induced chromosome breaks in G0 human lymphocytes.

In the present study we evaluated campthotecin (CPT)-induced chromosomal damage in human lymphocytes in the G0 phase of the cell cycle as revealed by the premature chromosome condensation technique. The results obtained here indicate that CPT was able to induce chromosome fragments in the G0 phase of the cell cycle of human lymphocytes as detected in prematurely condensed chromosomes. This result appears to be rather surprising, since the DNA lesions produced by CPT (e.g. 'protein concealed' DNA single-strand breaks) should not produce any damage in G0. A possible explanation for this result could come from much evidence to suggest that chromatin condensation processes are significantly involved in the conversion of DNA lesions into chromosome breaks in prematurely condensed chromosomes. The unexpected clastogenic behaviour of CPT can be explained taking into account the chromosome condensation induced by mitosis promoting factors when human lymphocytes are fused in G0, thus converting the 'protein concealed' DNA single-strand breaks induced by CPT into chromosome breaks. The same perspective should be taken into consideration for breaks induced by CPT under normal physiological conditions in the G2 phase of the cell cycle.

Evaluation of bleomycin-induced chromosome aberrations under simulated microgravity conditions in human lymphocytes using "FISH" techniques.

In the present investigation we report the effects of simulated microgravity conditions (clinostat) on the induction of chromosomal aberrations in human lymphocytes in vitro by (R) Bleomycin. Chromosomal aberrations have been analysed by means of fluorescent in situ hybridisation (FISH) and chromosome-specific composite DNA probes (chromosome painting). The results obtained show that, under simulated microgravity conditions, the levels of both symmetrical and asymmetrical (dicentrics, rings), the number of cells bearing "complex" aberrations and hence the total numbers of aberrations were significantly elevated at any of the dose-levels assayed, compared to the parallel treatments performed as 1g control ("ground"). Furthermore, the ratio symmetrical:asymmetrical translocations was markedly elevated under simulated microgravity conditions, compared to the findings usually observed under "normal" 1g conditions. On these bases, we are much inclined to believe that simulated microgravity, rather than limiting the resealing of DNA double strand breaks (DSB's) induced by genotoxic agents is influencing in terms of enhancement the misrejoining of DSB's which is actually responsible for the fixation of the original lesions to DNA into chromosomal aberrations. In addition, the possible different misrepair processes leading to the formation of symmetrical and asymmetrical translocations might be differentially influenced by microgravity being the symmetrical translocations significantly more represented.

Effects of rokitamycin on phagocytosis and release of oxidant radicals of human polymorphonuclear leukocytes.

A rational choice of an antimicrobial agent must take into account not only the activity against the specific pathogen but also any possible negative or positive effects on the host defense system. Rokitamycin (RKM) is an orally active 16-membered-ring macrolide; there are no reports of specific investigations of these activities in the literature. Polymorphonuclear neutrophils (PMNs) from healthy adult donors were incubated in medium alone or in medium containing increasing concentrations (1, 10, 50, 100 micrograms/ml) of RKM. In unwashed PMNs phagocytosis was unaffected by RKM, while luminol-amplified chemiluminescence (LACL) was significantly reduced by 50 and 100 micrograms/ml. When PMNs were washed after incubation phagocytosis was not modified but LACL was significantly restored. These characteristics of RKM were similar to those of roxithromycin and can be put in correlation with the cellular/extracellular ratio (30.5 for PMNs and 120 for macrophages) that was similar to that of roxithromycin but higher than of other macrolides. The molecular mechanisms by which high concentrations of these two macrolides produce such an impairment of LACL are still unclear. RKM has no unwanted effects on PMNs because the serum concentrations that can be obtained with the highest doses administered to man are lower than the concentrations which did not affect PMN functions in our study.

Penetration of brodimoprim into human neutrophils and intracellular activity.

The entry of an antibiotic into phagocytes is a prerequisite for its intracellular bioactivity against susceptible facultative or obligatory intracellular microorganisms. Brodimoprim is a dimethoxybenzylpyrimidine that has recently entered into clinical use, and its uptake into and elimination from human polymorphonuclear neutrophils (PMNs), together with its effects on normal phagocytic and antimicrobial mechanisms, have been investigated. Brodimoprim uptake by PMNs was determined by a velocity-gradient centrifugation technique under various experimental conditions and was expressed as the ratio of the intracellular to the extracellular drug concentration (C/E) in comparison with the C/E of trimethoprim, which was used as a control drug. After incubation with 7.5 micrograms of brodimoprim per ml, PMNs accumulated brodimoprim (C/E, 74.43 +/- 12.35 at 30 min) more avidly than trimethoprim (C/E, 20.97 +/- 6.61 at 30 min). The cellular uptake of brodimoprim was not affected by temperature, 2,4-dinitrophenol, or potassium fluoride and was increased with an increase in the pH of the medium. It was reduced in formaldehyde-killed PMNs. The efflux of brodimoprim was very rapid (46% after 5 min). The liposolubility of brodimoprim was about three times that of trimethoprim, as was the uptake. Therefore, a possible passive transmembrane diffusion mechanism might be proposed. Brodimoprim did not decrease either phagocytosis or phagocyte-mediated bactericidal activity, nor did it affect oxidative burst activity, as investigated by luminol-amplified chemiluminescence. On the basis of the pharmacokinetic data for brodimoprim, the concentration of 7.5 micrograms/ml was chosen as the highest concentration attainable in serum by oral therapy, and at this concentration of brodimoprim, the amount of drug that penetrated into PMNs was able to maintain its antimicrobial activity without interfering with the functions of the PMNs.

Development and validation of alternative metabolic systems for mutagenicity testing in short-term assays.

We present here the results obtained within the framework of an EU funded project aimed to develop and validate alternative metabolic activating systems to be used in short-term mutagenicity assays, in order to reduce the use of laboratory animals for toxicology testing. The activating systems studied were established cell lines (Hep G2, CHEL), genetically engineered V79 cell lines expressing specific rat cytochromes P450, erythrocyte-derived systems, CYP-mimetic chemical systems and plant homogenates. The metabolically competent cell lines were used as indicator cells for genotoxic effects as well as for the preparation of external activating systems using other indicator cells. The following endpoints were used: micronuclei, chromosomal aberrations and sister chromatid exchanges, mutations at the hprt locus, gene mutations in bacteria (Ames test), unscheduled DNA synthesis and DNA breaks detected in the comet assay. All metabolic systems employed activated some promutagens. With some of them, promutagens belonging to many different classes of chemicals were activated to genotoxicants, including carcinogens negative in liver S9-mediated assays. In other cases, the use of the new activating systems allowed the detection of mutagens at much lower substrate concentrations than in liver S9-mediated assays. Therefore, the alternative metabolizing systems, which do not require the use of laboratory animals, have a substantial potential in in vitro toxicology, in the basic genotoxicity testing as well as in the elucidation of activation mechanisms. However, since the data basis is much smaller for the new systems than for the activating systems produced from subcellular liver preparations, the overlapping use of both systems is recommended for the present and near future. For example, liver S9 preparations may be used with some indicator systems (e.g., bacterial mutagenicity), and metabolically competent mammalian cell lines may be used with other indicator systems (e.g., a cytogenetic endpoint) in a battery of basic tests.

Influence of brodimoprim on polymorphonuclear leukocyte phagocytosis and oxidant radical production.

Antibiotics not only reach the site of infection, but also penetrate cyclically, during a treatment, into polymorphonuclear leukocytes (PMNs) and may influence their functions positively or negatively. With reference to these aspects, the influence of brodimoprim (BMP), a dimethoxybenzylpyrimidine recently entered into clinical use, on human PMN phagocytosis and oxidant radical production (chemiluminescence) was investigated. PMNs from healthy adult donors were incubated for 50 min in medium alone or in medium containing increasing concentrations (3.7, 7.5, 15, and 30 micrograms/ml) of BMP and trimethoprim (TMP). In unwashed PMNs, phagocytosis was not modified by BMP, but was significantly reduced by 30 micrograms/ml TMP; chemiluminescence was significantly reduced by 15 and 30 micrograms/ml BMP and by all concentrations of TMP. When PMNs were washed after incubation, phagocytosis was unaffected and chemiluminescence was significantly restored. BMP at therapeutic concentrations did not influence PMNs and was less toxic than TMP.