DNA methylation profiling of asbestos-treated MeT5A cell line reveals novel pathways implicated in asbestos response.

Occupational and environmental asbestos exposure is the main determinant of malignant pleural mesothelioma (MPM), however, the mechanisms by which its fibres contribute to cell toxicity and transformation are not completely clear. Aberrant DNA methylation is a common event in cancer but epigenetic modifications involved specifically in MPM carcinogenesis need to be better clarified. To investigate asbestos-induced DNA methylation and gene expression changes, we treated Met5A mesothelial cells with different concentrations of crocidolite and chrysotile asbestos (0.5 ÷ 5.0 µg/cm2, 72 h incubation). Overall, we observed 243 and 302 differentially methylated CpGs (≥ 10%) between the asbestos dose at 5 µg/cm2 and untreated control, in chrysotile and crocidolite treatment, respectively. To examine the dose-response effect, Spearman's correlation test was performed and significant CpGs located in genes involved in migration/cell adhesion processes were identified in both treatments. Moreover, we found that both crocidolite and chrysotile exposure induced a significant up-regulation of CA9 and SRGN (log2 fold change > 1.5), previously reported as associated with a more aggressive MPM phenotype. However, we found no correlation between methylation and gene expression changes, except for a moderate significant inverse correlation at the promoter region of DKK1 (Spearman rho = - 1, P value = 0.02) after chrysotile exposure. These results describe for the first time the relationship between DNA methylation modifications and asbestos exposure. Our findings provide a basis to further explore and validate asbestos-induced DNA methylation changes, that could influence MPM carcinogenesis and possibly identifying new chemopreventive target.

The role of iron impurities in the toxic effects exerted by short multiwalled carbon nanotubes (MWCNT) in murine alveolar macrophages.

Lung toxicity mediated by multiwalled carbon nanotubes (MWCNT) has been widely demonstrated and recently associated with induction of carcinogenic asbestos-like effects, but the chemical features that drive this toxic effect have still not been well elucidated. The presence of metals as trace contaminants during MWCNT preparation, in particular iron (Fe) impurities, plays an important role in determining a different cellular response to MWCNT. Our goal was to clarify the mechanisms underlying MWCNT-induced toxicity with correlation to the presence of Fe impurities by exposing murine alveolar macrophages to two different MWCNT samples, which differed only in the presence or absence of Fe. Data showed that only Fe-rich MWCNT were significantly cytotoxic and genotoxic and induced a potent cellular oxidative stress, while Fe-free MWCNT did not exert any of these adverse effects. These results confirm that Fe content represents an important key constituent in promoting MWCNT-induced toxicity, and this needs to be taken into consideration when planning new, safer preparation routes.

Pleiotropic effects of cardioactive glycosides.

Cardioactive glycosides, like digoxin, ouabain and related compounds, are drugs that inhibit Na(+)/K(+)-ATPase and have a strong inotropic effect on heart: they cause the Na(+)/Ca(2+) exchanger to extrude Na+ in exchange with Ca(2+) and therefore increase the [Ca(2+)](i) concentration. For this reason, some of these drugs are currently used in the treatment of congestive heart failure and cardiac arrhythmias. Recently it has been discovered that cardiac glycosides exert pleiotropic effects on many aspects of cell metabolism. Na(+)/K(+)-ATPase is not the exclusive target, as they affect the cell response to hypoxia, modulate several signaling pathways involved in cell death and proliferation, regulate the transcription of different genes and modify the pharmacokinetics of other drugs, by altering the expression and activity of drug-metabolizing enzymes. Some of these effects are related to the steroid structure of glycosides, a property which also makes them fine modulators of the synthesis of cholesterol and steroid hormones. Moreover, new endogenously synthesized glycosides have been discovered in the last years: these molecules are involved in the balance of salt and in the control of blood pressure. This review will focus on the recent studies which have demonstrated that exogenous and endogenous glycosides, besides playing a role as inotropic agents, are also important in the pathogenesis and therapy of different human diseases, such as stroke, diabetes, neurological diseases and cancer.

Fluoride effects: the two faces of janus.

The behavior of fluoride ions in the human organism is a classic example of double-edged sword. On the one hand the daily supplementation with fluoride is undoubtedly an important preventing factor in protecting teeth from caries, and, as an important mitogenic stimulus for osteoblasts, it may enhance mineral deposition in bone, but on the other hand fluoride, above a threshold concentration, has been demonstrated to be toxic. We present here a brief review of fluoride metabolism and exposure, its use in caries prevention and its effects on bone, followed by an updating about the main hypotheses concerning its mechanism of action and toxicity. The effects of fluoride have been related mainly to its ability to evoke the activation of G proteins and the inhibition of phosphotyrosine phosphatases, leading to an intracellular increase of tyrosine phosphorylation and activation of the mitogen-activated protein kinase pathway, and its capacity to cause generation of reactive oxygen species. We present also a unifying hypothesis accounting for these apparently different effects, although the available experimental models and conditions are highly variable in the literature. A lot of experiments still need to be performed to clarify the positive and negative effects of fluoride. Finding the mechanisms accounting for fluoride toxicity is an important point: indeed, the use of fluoride has been proposed in the preparation of new biomaterials to be inserted in the bone, in order to improve their stable and safe integration.

Asbestos induces doxorubicin resistance in MM98 mesothelioma cells via HIF-1alpha.

Human malignant mesothelioma (HMM), which is strongly related to asbestos exposure, exhibits high resistance to many anticancer drugs. Asbestos fibre deposition in the lung may cause hypoxia and iron chelation at the fibre surface. Hypoxia-inducible factor (HIF)-1alpha, which is upregulated by a decreased availability of oxygen and iron, controls the expression of membrane transporters, such as P-glycoprotein (Pgp), which actively extrude the anticancer drugs. The present study aimed to assess whether asbestos may play a role in the induction of doxorubicin resistance in HMM cells through the activation of HIF-1alpha and an increased expression of Pgp. After 24-h incubation with crocidolite asbestos or with the iron chelator dexrazoxane, or under hypoxia, HMM cells were tested for HIF-1alpha activation, Pgp expression, accumulation of doxorubicin and sensitivity to its toxic effect. Crocidolite, dexrazoxane and hypoxia caused HIF-1alpha activation, Pgp overexpression and increased resistance to doxorubicin accumulation and toxicity. These effects were prevented by the co-incubation with the cell-permeating iron salt ferric nitrilotriacetate, which caused an increase of intracellular iron bioavailability, measured as increased activity of the iron regulatory protein-1. Crocidolite, dexrazoxane and hypoxia induce doxorubicin resistance in human malignant mesothelioma cells by increasing hypoxia-inducible factor-1alpha activity, through an iron-sensitive mechanism.

Insulin stimulates glucose transport via nitric oxide/cyclic GMP pathway in human vascular smooth muscle cells.

OBJECTIVE: In cultured human vascular smooth muscle cells, insulin increases cyclic GMP production by inducing nitric oxide (NO) synthesis. The aim of the present study was to determine whether in these cells the insulin-stimulated NO/cyclic GMP pathway plays a role in the regulation of glucose uptake. METHODS AND RESULTS: Glucose transport in human vascular smooth muscle cells was measured as uptake of 2-deoxy-d-[3H]glucose, cyclic GMP synthesis was checked by radioimmunoassay, and GLUT4 recruitment into the plasma membrane was determined by immunofluorescence. Insulin-stimulated glucose transport and GLUT4 recruitment were blocked by an inhibitor of NO synthesis and mimicked by NO-releasing drugs. Insulin- and NO-elicited glucose uptake were blocked by inhibitors of soluble guanylate cyclase and cyclic GMP-dependent protein kinase; furthermore, glucose transport was stimulated by an analog of cyclic GMP. CONCLUSIONS: Our results suggest that insulin-elicited glucose transport (and the corresponding GLUT4 recruitment into the plasma membrane) in human vascular smooth muscle cells is mediated by an increased synthesis of NO, which stimulates the production of cyclic GMP and the subsequent activation of a cyclic GMP-dependent protein kinase.

Iron inhibits the nitric oxide synthesis elicited by asbestos in murine macrophages.

Crocidolite fibers stimulated nitric oxide synthase (NOS) activity and expression in glial and alveolar murine macrophages: this effect was inhibited by iron supplementation and enhanced by iron chelation. We suggest that in these cells crocidolite stimulates NOS expression by decreasing the iron bioavailability and activating an iron-sensitive transcription factor.

Signaling pathway of nitric oxide-induced acrosome reaction in human spermatozoa.

Nitric oxide (NO) has been recently shown to modulate in vitro motility, viability, the acrosome reaction (AR), and metabolism of spermatozoa in various mammalian species, but the mechanism or mechanisms through which it influences sperm functions has not been clarified. In human capacitated spermatozoa, both the intracellular cGMP level and the percentage of AR-positive cells were significantly increased after 4 h of incubation with the NO donor, sodium nitroprusside (SNP). SNP-induced AR was significantly reduced in the presence of the soluble guanylate cyclase (sGC) inhibitors, LY83583 and ODQ; this block was bypassed by adding 8-bromo-cGMP, a cell-permeating cGMP analogue, to the incubation medium. Finally, Rp-8-Br-cGMPS and Rp-8-pCPT-cGMPS, two inhibitors of the cGMP-dependent protein kinases (PKGs), inhibited the SNP-induced AR. Furthermore, SNP-induced AR did not occur in Ca2+ -free medium or in the presence of the protein kinase C (PKC) inhibitor, calphostin C. This study suggests that the AR-inducing effect of exogenous NO on capacitated human spermatozoa is accomplished via stimulation of an NO-sensitive sGC, cGMP synthesis, and PKG activation. In this effect the activation of PKC is also involved, and the presence of extracellular Ca2+ is required.

Human vascular smooth muscle cells express a constitutive nitric oxide synthase that insulin rapidly activates, thus increasing guanosine 3':5'-cyclic monophosphate and adenosine 3':5'-cyclic monophosphate concentrations.

AIMS/HYPOTHESIS: Insulin incubation of human vascular smooth muscle cells (hVSMC) for 120 min increases both guanosine 3':5'-cyclic monophosphate (cGMP) and adenosine 3':5'-cyclic monophosphate (cAMP) and these effects are blocked by inhibiting nitric oxide synthase (NOS). These data suggest that insulin activates a constitutive Ca2+-dependent NOS (cNOS), not described at yet in hVSMC. To test this hypothesis, we evaluated in hVSMC: i) the kinetics of the insulin-induced enhancement of the two cyclic nucleotides; ii) the ability of nitric oxide (NO) to increase both cyclic nucleotides; iii) NO involvement in the short-term influence of insulin on both cyclic nucleotides; iv) the ability of insulin to increase NO production in a few minutes; v) the presence of a cNOS activity; vi) the expression of mRNA for cNOS. METHODS: In hVSMC incubated with insulin, NO donors and the Ca2+ ionophore ionomycin, we measured cAMP and cGMP (RIA); in hVSMC incubated with insulin and ionomycin we measured NO, evaluated as L-(3H)-citrulline production from L-(3H)-arginine; by northern blot hybridization, we measured the expression of cNOS mRNA. RESULTS: i) By incubating hVSMC with 2 nmol/l insulin for 0-240 min, we observed an increase of both cGMP and cAMP (ANOVA: p = 0.0001). Cyclic GMP rose from 0.74 +/- 0.01 to 2.62 +/- 0.10 pmol/10(6) cells at 30 min (p = 0.0001); cAMP rose from 0.9 +/- 0.09 to 11.65 +/- 0.74 pmol/10(6) cells at 15 min (p=0.0001). ii) Sodium nitroprusside (100 mol/l) and glyceryltrinitrate (100 micromol/l) increased both cGMP and cAMP (p = 0.0001). iii) The effects of insulin on cyclic nucleotides were blocked by NOS inhibition. iv) An increase of NO was observed by incubating hVSMC for 5 min with 2 nmol/l insulin (p = 0.0001). v) Ionomycin (1 micromol/l) enhanced NO production (p = 0.0001) and increased both cyclic nucleotides (p = 0.0001). vi) hVSMC expressed mRNA of cNOS. CONCLUSION/INTERPRETATION: Human VSMC express cNOS, which is rapidly activated by insulin with a consequent increase of both cGMP and cAMP, suggesting that insulin-induced vasodilation in vivo is not entirely endothelium-mediated.

Follicular fluid proteins stimulate nitric oxide (NO) synthesis in human sperm: a possible role for NO in acrosomal reaction.

Nitric oxide (NO) is a free radical involved in the regulation of several functions of the male genitourinary system. It is produced by neurons and the endothelium and epithelia of reproductive system; it mediates penile erection and regulates sperm motility, viability, and metabolism. Here we show that human spermatozoa exhibit a detectable NO synthase (NOS) activity, measured both as ability of the intact sperm and cell lysate to convert L-[3H]arginine into L-[3H]citrulline and as 24 h accumulation of extracellular nitrite in intact sperm suspensions. NOS activity (identified as an endothelial isoform) was inhibited by L-canavanine and N(G)-monomethyl-L-arginine, and nitrite accumulation was inhibited by the NO scavenger hemoglobin; both enzyme activity and nitrite production were increased by a 24 h incubation of spermatozoa with protein-enriched extracts of human follicular fluid (PFF); a significant increase of citrulline synthesis was observed only after a 4 h incubation with 40% PFF, a time period during which acrosomal reactivity was significantly increased. PFF-induced acrosomal reaction was inhibited by L-canavanine and hemoglobin, and the NO donors sodium nitroprusside (SNP), S-nitroso-N-acetyl-penicillamine (SNAP), and DETA NONOate were able to increase the percentage of reacted spermatozoa. Our results suggest that NO synthesized by human sperm may play a role in follicular fluid-induced acrosomal reaction.

Chloroquine stimulates nitric oxide synthesis in murine, porcine, and human endothelial cells.

Nitric oxide (NO) is a free radical involved in the regulation of many cell functions and in the expression of several diseases. We have found that the antimalarial and antiinflammatory drug, chloroquine, is able to stimulate NO synthase (NOS) activity in murine, porcine, and human endothelial cells in vitro: the increase of enzyme activity is dependent on a de novo synthesis of some regulatory protein, as it is inhibited by cycloheximide but is not accompanied by an increased expression of inducible or constitutive NOS isoforms. Increased NO synthesis is, at least partly, responsible for chloroquine-induced inhibition of cell proliferation: indeed, NOS inhibitors revert the drug-evoked blockage of mitogenesis and ornithine decarboxylase activity in murine and porcine endothelial cells. The NOS-activating effect of chloroquine is dependent on its weak base properties, as it is exerted also by ammonium chloride, another lysosomotropic agent. Both compounds activate NOS by limiting the availability of iron: their stimulating effects on NO synthesis and inhibiting action on cell proliferation are reverted by iron supplementation with ferric nitrilotriacetate, and are mimicked by incubation with desferrioxamine. Our results suggest that NO synthesis can be stimulated in endothelial cells by chloroquine via an impairment of iron metabolism.