Multiple mechanisms underlying troglitazone-induced mitochondrial permeability transition

Troglitazone, a thiazolidinedione class antidiabetic drug, was withdrawn from the market because of its severe idiosyncratic hepatotoxicity. It causes a mitochondrial permeability transition (MPT), which may in part contribute to its hepatotoxicity. In the present study, the mechanism of troglitazone mitochondrial toxicity was investigated in isolated rat liver mitochondria. Mitochondrial swelling induced by 10 μM troglitazone was attenuated by bromoenol lactone (BEL), an inhibitor of Ca²⁺-independent phospholipase A₂ (iPLA₂). In contrast, that induced by 50 μM troglitazone was exacerbated by BEL. This exacerbation was diminished by addition of 2 mM glutathione, an antioxidant. Oxygen consumption by state 3 respiration in isolated mitochondria was also decreased by troglitazone, but it was not affected by BEL. Mitochondrial swelling induced by 10 μM troglitazone was completely attenuated in the absence of Ca²⁺ while that induced by 50 μM troglitazone was not affected. Addition of 1 μM cyclosporin A (CsA), an inhibitor of MPT pores, completely attenuated swelling induced by 10 μM troglitazone while it only partly diminished that induced by 50 μM troglitazone. Thus, the MPT induced by 10 and 50 μM troglitazone are regulated by different mechanism; the MPT induced by 10 μM troglitazone is regulated by the activation of iPLA₂ and caused by the opening of CsA-regulating MPT pores followed by accumulation of Ca²⁺ in mitochondria, while that induced by 50 μM troglitazone is partly regulated by reactive oxygen species and mainly caused by the opening of CsA-insensitive MPT pores.     

Toxigenicity of enniatins from Western Australian Fusarium species to brine shrimp (Artemia franciscana)

The high prevalence (14 of 24 isolates) of enniatin-producing isolates from Western Australian Fusarium species isolated from pasture legumes associated with sheep feed refusal and rat deaths, and the high toxicity of their crude extracts to brine shrimp (Artemia franciscana) from a previous study warranted further investigation of this class of mycotoxin. Crude extracts from Fusarium acuminatum, Fusarium avenaceum, Fusarium tricinctum and Fusarium sambucinum, along with enniatins A, A1, B and B1 purified from a Western Australian strain of F. acuminatum using semi-preparative HPLC, were bioassayed using brine shrimp. All Fusarium isolates produced both enniatins B and B1, except for F. tricinctum WAC 8019, and 11 of the 17 isolates produced enniatin A1. Overall, all of the F. avenaceum isolates produced high amounts of enniatins, in particular enniatin B. One isolate of F. acuminatum (WAC 5715) and of F. tricinctum (WAC 11486) also produced high amounts of both enniatins B and B1. Only F. acuminatum WAC 5715 produced enniatin A among the tested isolates. All four purified enniatins A, A1, B, B1, individually and in combination, caused brine shrimp toxicity after 6 h of exposure, implicating that this emerging class of mycotoxin as a cause of the acute toxicity to brine shrimp observed. The mixture of all four enniatins was the most toxic to brine shrimp compared to purified individual enniatins, where the relative toxicity order was B > B1 > A1 > A. Enniatin B was the individual most toxic enniatin with some bioactivity at 5 μg/mL and almost 100% brine shrimp death at 50 μg/mL after 24 h of exposure. This study is the first report to confirm the acute toxicity of enniatins A, A1, B and B1 to brine shrimp, and also highlights the need for further investigation of the potential toxicity of these cyclic hexadepsipeptides to animals and humans.     

Titanium dioxide nanoparticles impair lung mitochondrial function

Titanium dioxide nanoparticles (TiO₂ NPs) are used in an increasing number of human products such as cosmetics, sunscreen, toothpaste and paints. However, there is clear evidence about effects associated to TiO₂ NPs exposure, which include lung inflammation and tumor formation and these effects are related to reactive oxygen species (ROS) formation. The ROS generation could be attributed to a mitochondrial dysfunction. Even though, it has been shown that TiO₂ NPs exposure can induce some alterations in mitochondria including cytochrome c release to cytosol, change in mitochondrial permeability and decrease of mitochondrial membrane potential (ΔΨ_m), there is no information about the changes in mitochondrial function induced by TiO₂ NPs. We hypothesized that TiO₂ NPs effects are associated with mitochondrial dysfunction and redox unbalance. To test our hypothesis we isolated mitochondria from lung tissue of rats and exposed them to 10 (g TiO₂ NPs (particle size < 25 nm)/mg protein for 1 h. Our results showed that TiO₂ NPs decreases NADH levels and impairs ΔΨ_m and mitochondrial function accompanied by ROS generation during mitochondrial respiration.     

Sequence Divergence of the Enniatin Synthase Gene in Relation to Production of Beauvericin and Enniatins in Fusarium Species

Beauvericin (BEA) and enniatins (ENNs) are cyclic peptide mycotoxins produced by a wide range of fungal species, including pathogenic Fusaria. Amounts of BEA and ENNs were quantified in individual rice cultures of 58 Fusarium strains belonging to 20 species, originating from different host plant species and different geographical localities. The species identification of all strains was done on the basis of the tef-1α gene sequence. The main aim of this study was to analyze the variability of the esyn1 gene encoding the enniatin synthase, the essential enzyme of this metabolic pathway, among the BEA- and ENNs-producing genotypes. The phylogenetic analysis based on the partial sequence of the esyn1 gene clearly discriminates species producing exclusively BEA from those synthesizing mainly enniatin analogues.     

Quercetin 3-O-methyl ether protects FL83B cells from copper induced oxidative stress through the PI3K/Akt and MAPK/Erk pathway

Quercetin is a bioflavonoid that exhibits several biological functions in vitro and in vivo. Quercetin 3-O-methyl ether (Q3) is a natural product reported to have pharmaceutical activities, including antioxidative and anticancer activities. However, little is known about the mechanism by which it protects cells from oxidative stress. This study was designed to investigate the mechanisms by which Q3 protects against Cu^2 +-induced cytotoxicity. Exposure to Cu^2 + resulted in the death of mouse liver FL83B cells, characterized by apparent apoptotic features, including DNA fragmentation and increased nuclear condensation. Q3 markedly suppressed Cu^2 +-induced apoptosis and mitochondrial dysfunction, characterized by reduced mitochondrial membrane potential, caspase-3 activation, and PARP cleavage, in Cu^2 +-exposed cells. The involvement of PI3K, Akt, Erk, FOXO3A, and Mn-superoxide dismutase (MnSOD) was shown to be critical to the survival of Q3-treated FL83B cells. The liver of both larval and adult zebrafish showed severe damage after exposure to Cu^2 + at a concentration of 5 μM. Hepatic damage induced by Cu^2 + was reduced by cotreatment with Q3. Survival of Cu^2 +-exposed larval zebrafish was significantly increased by cotreatment with 15 μM Q3. Our results indicated that Cu^2 +-induced apoptosis in FL83B cells occurred via the generation of ROS, upregulation and phosphorylation of Erk, overexpression of 14-3-3, inactivation of Akt, and the downregulation of FOXO3A and MnSOD. Hence, these results also demonstrated that Q3 plays a protective role against oxidative damage in zebrafish liver and remarked the potential of Q3 to be used as an antioxidant for hepatocytes.     

The marine toxin palytoxin induces necrotic death in HaCaT cells through a rapid mitochondrial damage

Palytoxin (PLTX) is one of the most toxic algal biotoxin known so far. It transforms the Na⁺/K⁺-ATPase into a cationic channel inducing a massive intracellular Na⁺ influx. However, from a mechanistic point of view, the features and the intracellular pathways leading to PLTX-induced cell death are still not completely characterized. This study on skin HaCaT keratinocytes demonstrates that PLTX induces necrosis since propidium iodide uptake was observed already after 1 h toxin exposure, an effect that was not lowered by toxin removal. Furthermore, necrotic-like morphological alterations were evidenced by confocal microscopy. Apoptosis occurrence was excluded since no caspases 3/7, caspase 8, and caspase 9 activation as well as no apoptotic bodies formation were recorded. Necrosis was preceded by a very early mitochondrial damage as indicated by JC-1 fluorescence shift, recorded already after 5 min toxin exposure. This shift was totally abolished when Na⁺ and Ca²⁺ ions were withdrawn from culture medium, whereas cyclosporine-A was ineffective, excluding the occurrence of a controlled biochemical response.     

Cadmium inhibits acid secretion in stimulated frog gastric mucosa

Cadmium, a toxic environmental pollutant, affects the function of different organs such as lungs, liver and kidney. Less is known about its toxic effects on the gastric mucosa. The aim of this study was to investigate the mechanisms by which cadmium impacts on the physiology of gastric mucosa. To this end, intact amphibian mucosae were mounted in Ussing chambers and the rate of acid secretion, short circuit current (I_sc), transepithelial potential (V_t) and resistance (R_t) were recorded in the continuous presence of cadmium. Addition of cadmium (20 µM to 1 mM) on the serosal but not luminal side of the mucosae resulted in inhibition of acid secretion and increase in NPPB-sensitive, chloride-dependent short circuit current. Remarkably, cadmium exerted its effects only on histamine-stimulated tissues. Experiments with TPEN, a cell-permeant chelator for heavy metals, showed that cadmium acts from the intracellular side of the acid secreting cells. Furthermore, cadmium-induced inhibition of acid secretion and increase in I_sc cannot be explained by an action on: 1) H₂ histamine receptor, 2) Ca²⁺ signalling 3) adenylyl cyclase or 4) carbonic anhydrase. Conversely, cadmium was ineffective in the presence of the H⁺/K⁺-ATPase blocker omeprazole suggesting that the two compounds likely act on the same target. Our findings suggest that cadmium affects the functionality of histamine-stimulated gastric mucosa by inhibiting the H⁺/K⁺-ATPase from the intracellular side. These data shed new light on the toxic effect of this dangerous environmental pollutant and may result in new avenues for therapeutic intervention in acute and chronic intoxication.     

Adenosine A3 receptor-mediated cardioprotection against doxorubicin-induced mitochondrial damage

Cardiotoxicity associated with doxorubicin (DOX) treatment limits the therapeutic efficiency of this drug against cancer. 2-Chloro-N(6)-(3-iodobenzyl)adenosine-5′-N-methyluronamide (Cl-IB-MECA), a selective agonist of A₃ adenosine receptor (A₃R), reduces DOX toxicity in newborn rat cultured cardiomyocytes. The study's aim was to determine whether the protection demonstrated by Cl-IB-MECA attenuates cardiac depression in vivo. In addition, we wished to examine whether this protective pathway affects the sarcoplasmic reticulum (SR) calcium uptake and release, as well as intramitochondrial Ca²⁺ accumulation induced by DOX.Rats were injected every alternate day (6 times) with (1) saline, (2) 2.5 mg/kg i.p. DOX, (3) 33 μg/kg i.v. Cl-IB-MECA, (4) DOX + Cl-IB-MECA. Left ventricular functions were assessed by invasive (pressure) and non-invasive (echocardiography) techniques at the end of the injection period and 4 weeks later. Cytosolic and intramitochondrial calcium levels were measured with indo-1 and rhod-2 probes. SR Ca²⁺ content was determined by exposing cultured rat cardiomyocytes to caffeine.Echocardiography data demonstrate left ventricular wall thinning (23%), an increase in the end systolic dimension (170%) and decreased fractional shortening (35 ± 5% vs. 54 ± 5%, p < 0.01) in DOX-treated animals, compared to the control group. DOX increased Ca²⁺ levels in the cytosol and in mitochondria by diminishing the SR Ca²⁺ uptake. Pretreatment with Cl-IB-MECA attenuated left ventricular dysfunction, improved SR calcium storage capacity and prevented mitochondrial Ca²⁺ overload.We conclude that the adenosine A₃ receptor agonist is effective in vivo against DOX cardiotoxicity via the restoration of Ca²⁺ homeostasis and prevention of mitochondrial damage that occurs as a result of Ca²⁺ overload.     

Activation of K+ channels and Na+/K+ ATPase prevents aortic endothelial dysfunction in 7-day lead-treated rats

Seven day exposure to a low concentration of lead acetate increases nitric oxide bioavailability suggesting a putative role of K⁺ channels affecting vascular reactivity. This could be an adaptive mechanism at the initial stages of toxicity from lead exposure due to oxidative stress. We evaluated whether lead alters the participation of K⁺ channels and Na⁺/K⁺-ATPase (NKA) on vascular function. Wistar rats were treated with lead (1st dose 4 μg/100 g, subsequent doses 0.05 μg/100 g, im, 7 days) or vehicle. Lead treatment reduced the contractile response of aortic rings to phenylephrine (PHE) without changing the vasodilator response to acetylcholine (ACh) or sodium nitroprusside (SNP). Furthermore, this treatment increased basal O₂⁻ production, and apocynin (0.3 μM), superoxide dismutase (150 U/mL) and catalase (1000 U/mL) reduced the response to PHE only in the treated group. Lead also increased aortic functional NKA activity evaluated by K⁺-induced relaxation curves. Ouabain (100 μM) plus L-NAME (100 μM), aminoguanidine (50 μM) or tetraethylammonium (TEA, 2 mM) reduced the K⁺-induced relaxation only in lead-treated rats. When aortic rings were precontracted with KCl (60 mM/L) or preincubated with TEA (2 mM), 4-aminopyridine (4-AP, 5 mM), iberiotoxin (IbTX, 30 nM), apamin (0.5 μM) or charybdotoxin (0.1 μM), the ACh-induced relaxation was more reduced in the lead-treated rats. Additionally, 4-AP and IbTX reduced the relaxation elicited by SNP more in the lead-treated rats. Results suggest that lead treatment promoted NKA and K⁺ channels activation and these effects might contribute to the preservation of aortic endothelial function against oxidative stress.     

The signalling role of action potential depolarization in insulin secretion: Metabolism-dependent dissociation between action potential increase and secretion increase by TEA

The K⁺ channel blocker, TEA is known to increase action potential amplitude and insulin secretion of mouse β-cells when added to a nutrient secretagogue. In the presence of a maximally effective sulfonylurea concentration (2.7 μM glipizide) the nutrient secretagogue α-ketoisocaproic acid (KIC, 10 mM) strongly increased insulin secretion (about elevenfold). Instead of enhancing the effect of KIC, TEA reduced the KIC-induced secretion by more than 50%. Also, the secretion rate produced by 2.7 μM glipizide alone was significantly reduced by TEA. In contrast, TEA enhanced the insulinotropic effect of glipizide when a basal glucose concentration (5 mM) was present. In the presence as well as in the absence of glucose glipizide produced a plateau depolarization with superimposed action potentials. Under both conditions, TEA increased the glipizide-induced action potential amplitude and further elevated the cytosolic free calcium concentration ([Ca²⁺]_i) with an oscillatory characteristic. These effects depended on the activity of L-type Ca²⁺ channels, even though the effect of TEA differed from that of 1 μM of the Ca²⁺ channel opener, Bay K8644, which primarily increased action potential duration. TEA did not negatively affect parameters of β-cell energy metabolism (NAD(P)H fluorescence and ATP/ADP ratio), rather, it slightly increased NAD(P)H fluorescence. Apparently, TEA inhibits insulin secretion in the absence of glucose in spite of a persistent ability to block K⁺ ion conductance. Thus, the signalling role of action potential depolarization in insulin secretion may require reconsideration and ion conductance-independent actions of K⁺ channels may be involved in this paradox effect of TEA.     

Effect of heparin treatment on the expression and activity of different ion-motive P-type ATPase isoforms from mouse extensor digitorum longus muscle during degeneration and regeneration after Bothrops jararacussu venom injection

Ca²⁺ ions are essential to myonecrosis, a serious complication of snake envenomation, and heparin seems to counteract this effect. We investigated the effect of local injection of Bothrops jararacussu venom in mouse fast-twitch extensor digitorum longus (EDL) muscle, without or with heparin, on functional/molecular alterations of two central proteins involved in intracellular Ca²⁺ homeostasis, sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA) and Na⁺/K⁺-ATPase. EDL-specific SERCA1 isoform expression dropped significantly just after venom administration (up to 60% compared to control EDL values at days 1 and 3; p < 0.05) while SERCA2 and Na⁺/K⁺-ATPase α₁ isoform expression increased at the same time (3-6- and 2-3-fold, respectively; p < 0.05). Although not significant, Na⁺/K⁺-ATPase α₂ isoform followed the same trend. Except for SERCA2, all proteins reached basal levels at the 7th day. Intravenous heparin treatment did not affect these profiles. Ca²⁺-ATPase activity was also decreased during the first days after venom injection, but here heparin was effective to reinstate activity to control levels within 3 days. We also showed that B. jararacussu venom directly inhibited Ca²⁺-ATPase activity in a concentration-dependent manner. Our results indicate that EDL SERCA and Na⁺/K⁺-ATPase are importantly affected by B. jararacussu venom and heparin has protective effect on activity but not on protein expression.     

MRP2 and the handling of mercuric ions in rats exposed acutely to inorganic and organic species of mercury

Mercuric ions accumulate preferentially in renal tubular epithelial cells and bond with intracellular thiols. Certain metal-complexing agents have been shown to promote extraction of mercuric ions via the multidrug resistance-associated protein 2 (MRP2). Following exposure to a non-toxic dose of inorganic mercury (Hg²⁺), in the absence of complexing agents, tubular cells are capable of exporting a small fraction of intracellular Hg²⁺ through one or more undetermined mechanisms. We hypothesize that MRP2 plays a role in this export. To test this hypothesis, Wistar (control) and TR⁻ rats were injected intravenously with a non-nephrotoxic dose of HgCl₂ (0.5 μmol/kg) or CH₃HgCl (5 mg/kg), containing [²⁰³Hg], in the presence or absence of cysteine (Cys; 1.25 μmol/kg or 12.5 mg/kg, respectively). Animals were sacrificed 24 h after exposure to mercury and the content of [²⁰³Hg] in blood, kidneys, liver, urine and feces was determined. In addition, uptake of Cys-S-conjugates of Hg²⁺ and methylmercury (CH₃Hg⁺) was measured in inside-out membrane vesicles prepared from either control Sf9 cells or Sf9 cells transfected with human MRP2. The amount of mercury in the total renal mass and liver was significantly greater in TR⁻ rats than in controls. In contrast, the amount of mercury in urine and feces was significantly lower in TR⁻ rats than in controls. Data from membrane vesicles indicate that Cys-S-conjugates of Hg²⁺ and CH₃Hg⁺ are transportable substrates of MRP2. Collectively, these data indicate that MRP2 plays a role in the physiological handling and elimination of mercuric ions from the kidney.     

Minocycline and doxycycline,but not other tetracycline-derived compounds,protect liver cells from chemical hypoxia and ischemia/reperfusion injury by inhibition of the mitochondrial calcium uniporter

Minocycline, a tetracycline-derived compound, mitigates damage caused by ischemia/reperfusion (I/R) injury. Here, 19 tetracycline-derived compounds were screened in comparison to minocycline for their ability to protect hepatocytes against damage from chemical hypoxia and I/R injury. Cultured rat hepatocytes were incubated with 50 μM of each tetracycline-derived compound 20 min prior to exposure to 500 μM iodoacetic acid plus 1 mM KCN (chemical hypoxia). In other experiments, hepatocytes were incubated in anoxic Krebs–Ringer–HEPES buffer at pH 6.2 for 4 h prior to reoxygenation at pH 7.4 (simulated I/R). Tetracycline-derived compounds were added 20 min prior to reperfusion. Ca^2 + uptake was measured in isolated rat liver mitochondria incubated with Fluo-5N. Cell killing after 120 min of chemical hypoxia measured by propidium iodide (PI) fluorometry was 87%, which decreased to 28% and 42% with minocycline and doxycycline, respectively. After I/R, cell killing at 120 min decreased from 79% with vehicle to 43% and 49% with minocycline and doxycycline. No other tested compound decreased killing. Minocycline and doxycycline also inhibited mitochondrial Ca^2 + uptake and suppressed the Ca^2 +-induced mitochondrial permeability transition (MPT), the penultimate cause of cell death in reperfusion injury. Ru360, a specific inhibitor of the mitochondrial calcium uniporter (MCU), also decreased cell killing after hypoxia and I/R and blocked mitochondrial Ca^2 + uptake and the MPT. Other proposed mechanisms, including mitochondrial depolarization and matrix metalloprotease inhibition, could not account for cytoprotection. Taken together, these results indicate that minocycline and doxycycline are cytoprotective by way of inhibition of MCU.     

Underlying mechanism of actions of tefluthrin,a pyrethroid insecticide,on voltage-gated ion currents and on action currents in pituitary tumor (GH3) cells and GnRH-secreting (GT1-7) neurons

Tefluthrin is a synthetic pyrethroid and involved in acute neurotoxic effects. How this compound affects ion currents in endocrine or neuroendocrine cells remains unclear. Its effects on membrane ion currents in pituitary tumor (GH₃) cells and in hypothalamic (GT1-7) neurons were investigated. Application of Tef (10 μM) increased the amplitude of voltage-gated Na⁺ current (I_Na), along with a slowing in current inactivation and deactivation in GH₃ cells. The current–voltage relationship of I_Na was shifted to more negative potentials in the presence of this compound. Tef increased I_Na with an EC₅₀ value of 3.2 ± 0.8 μM. It also increased the amplitude of persistent I_Na. Tef reduced the amplitude of L-type Ca²⁺ current. This agent slightly inhibited K⁺ outward current; however, it had no effect on the activity of large-conductance Ca²⁺-activated K⁺ channels. Under cell-attached voltage-clamp recordings, Tef (10 μM) increased amplitude and frequency of spontaneous action currents, along with appearance of oscillatory inward currents. Tef-induced inward currents were suppressed after further application of tetrodotoxin, riluzole or ranolazine. In GT1-7 cells, Tef also increased the amplitude and frequency of action currents. Taken together, the effects of Tef and its structural related pyrethroids on ion currents can contribute to the underlying mechanisms through which they affect endocrine or neuroendocrine function in vivo.     

The insulinotropic effect of fluoroquinolones

Antimicrobial fluoroquinolones induce, with strongly varying frequency, life-threatening hypoglycemias, which is explained by their ability to block K_ATP channels in pancreatic B-cells and thus to initiate insulin secretion. In apparent contradiction to this, we observed that none of the fluoroquinolones in this study (gatifloxacin, moxifloxacin, ciprofloxacin, and a number of fluorophenyl-substituted compounds) initiated insulin secretion of perifused mouse islets when the glucose concentration was basal (5 mM). Only when the glucose concentration was stimulatory by itself (10 mM), the fluoroquinolones enhanced secretion. The fluoroquinolones were ineffective on SUR1 Ko islets, which do not have functional K_ATP channels. All of these fluoroquinolones depolarized the membrane potential of mouse B-cells (patch-clamping in the whole-cell mode). Using metabolically intact B-cells (perforated-patch mode) however, 100 μM of gatifloxacin, ciprofloxacin or moxifloxacin were unable to depolarize when the glucose concentration was 5 mM, whereas other K_ATP channel blockers (tolbutamide and efaroxan) remained effective. Only at a very high concentration (500 μM) gatifloxacin and moxifloxacin, but not ciprofloxacin induced repetitive depolarizations which could be antagonized by diazoxide. In the presence of 10 mM glucose all fluoroquinolones which enhanced secretion markedly elevated cytosolic calcium concentration ([Ca²⁺]_i). In the presence of 5 mM glucose gatifloxacin and moxifloxacin at 500 μM but not at 100 μM elevated [Ca²⁺]_i. It is concluded that fluoroquinolones in the clinically relevant concentration range are not initiators, but rather enhancers of glucose-induced insulin secretion. The block of K_ATP channels appears necessary but not sufficient to explain the hypoglycemic effect of fluoroquinolones.     

Dual action of a dinoflagellate-derived precursor of Pacific ciguatoxins (P-CTX-4B) on voltage-dependent K and Na channels of single myelinated axons

The effects of Pacific ciguatoxin-4B (P-CTX-4B, also named gambiertoxin), extracted from toxic Gambierdiscus dinoflagellates, were assessed on nodal K⁺ and Na⁺ currents of frog myelinated axons, using a conventional voltage-clamp technique. P-CTX-4B decreased, within a few minutes, both K⁺ and Na⁺ currents in a dose-dependent manner, without inducing any marked change in current kinetics. The toxin was more effective in blocking K⁺ than Na⁺ channels. P-CTX-4B shifted the voltage-dependence of Na⁺ conductance by about 14 mV towards more negative membrane potentials. This effect was reversed by increasing Ca²⁺ in the external solution. A negative shift of about 16 mV in the steady-state Na⁺ inactivation-voltage curve was also observed in the presence of the toxin. Unmodified and P-CTX-4B-modified Na⁺ currents were similarly affected by the local anaesthetic lidocaine. The decrease of the two currents by lidocaine was dependent on both the concentration and the membrane potential during pre-pulses. In conclusion, P-CTX-4B appears about four times more effective than P-CTX-1B to affect K⁺ channels, whereas it is about 50 times less efficient to affect Na⁺ channels of axonal membranes. These actions may be related to subtle differences between the two chemical structures of molecules.     

Toxic effects of diazinon and its photodegradation products

The toxic effects of diazinon and its irradiated solutions were investigated using cultivated human blood cells (lymphocytes and erythrocytes) and skin fibroblasts. Ultra Performance Liquid Chromatography (UPLC)–UV/VIS system was used to monitor the disappearance of starting diazinon during 115-min photodegradation and formation of its by-products (diazoxon and 2-isopropyl-6-methyl-4-pyrimidinol (IMP)) as a function of time. Dose-dependent AChE and Na⁺/K⁺-ATPase inhibition by diazinon was obtained for all investigated cells. Calculated IC₅₀ (72 h) values, in M, were: 7.5 × 10⁻⁶/3.4 × 10⁻⁵, 8.7 × 10⁻⁵/6.6 × 10⁻⁵, and 3.0 × 10⁻⁵/4.6 × 10⁻⁵ for fibroblast, erythrocyte and lymphocyte AChE/Na⁺/K⁺-ATPase, respectively. Results obtained for reference commercially purified target enzymes indicate similar sensitivity of AChE towards diazinon (IC₅₀ (20 min)-7.8 × 10⁻⁵M), while diazinon concentrations below 10 mM did not noticeably affect Na⁺/K⁺-ATPase activity. Besides, diazinon and IMP induced increasing incidence of micronuclei (via clastogenic mode of action) in a dose-dependent manner up to 2 × 10⁻⁶ M and significant inhibition of cell proliferation and increased level of malondialdehyde at all investigated concentrations. Although after 15-min diazinon irradiation formed products do not affect purified commercial enzymes activities, inhibitory effect of irradiated solutions on cell enzymes increased as a function of time exposure to UV light and resulted in significant reduction of AChE (up to 28–45%) and Na⁺/K⁺-ATPase (up to 35–40%) at the end of irradiation period. Moreover, photodegradation treatment strengthened prooxidative properties of diazinon as well as its potency to induce cytogenetic damage.     

Regulation of NO-dependent acetylcholine relaxation by K channels and the Na-K ATPase pump in porcine internal mammary artery

This study was designed to determine whether K⁺ channels play a role in nitric oxide (NO)-dependent acetylcholine relaxation in porcine internal mammary artery (IMA). IMA segments were isolated and mounted in organ baths to record isometric tension. Acetylcholine-elicited vasodilation was abolished by muscarinic receptor blockade with atropine (10^-6 M). Incubation with indomethacin (3 × 10⁻⁶ M), superoxide dismutase (150 U/ml) and bosentan (10⁻⁵ M) did not modify the acetylcholine response ruling out the participation of cyclooxygenase-derivates, reactive oxygen species or endothelin. The relaxation response to acetylcholine was strongly diminished by NO synthase- or soluble guanylyl cyclase-inhibition using l-NOArg (10⁻⁴ M) or ODQ (3 × 10⁻⁶ M), respectively. The vasodilation induced by acetylcholine and a NO donor (NaNO₂) was reduced when rings were contracted with an enriched K⁺ solution (30 mM), by voltage-dependent K⁺ (K_v) channel blockade with 4-amynopiridine (4-AP; 10⁻⁴ M), by Ca²⁺-activated K⁺ (K_Ca) channel blockade with tetraethylammonium (TEA; 10⁻³ M), and by apamin (5 × 10⁻⁷ M) plus charybdotoxin (ChTx; 10⁻⁷ M) but not when these were added alone. In contrast, large conductance K_Ca (BK_Ca)_, ATP-sensitive K⁺ (K_ATP) and inwardly rectifying K⁺ (K_ir) channel blockade with iberiotoxin (IbTx; 10⁻⁷ M), glibenclamide (10⁻⁶ M) and BaCl₂ (3 × 10⁻⁵ M), respectively, did not alter the concentration-response curves to acetylcholine and NaNO₂. Na⁺−K⁺ ATPase pump inhibition with ouabain (10⁻⁵ M) practically abolished acetylcholine and NaNO₂ relaxations. Our findings suggest that acetylcholine-induced relaxation is largely mediated through the NO-cGMP pathway, involving apamin plus ChTx-sensitive K⁺ and K_v channels, and Na⁺−K⁺-ATPase pump activation.     

Dehydromonocrotaline induces cyclosporine A-insensitive mitochondrial permeability transition/cytochrome c release

Monocrotaline (MCT) is a pyrrolizidine alkaloid present in plants of the genus Crotalaria that causes cytotoxicity and genotoxicity in animals and humans. It is well established that the toxicity of MCT results from its hepatic bioactivation to dehydromonocrotaline (DHM), an alkylating agent, but the exact mechanism of action remains unknown. In a previous study, we demonstrated DHM's inhibition of mitochondrial NADH-dehydrogenase activity at micromolar concentrations, which is an effect associated with a significant reduction in ATP synthesis. As a follow-up study, we have evaluated the ability of DHM to induce mitochondrial permeability transition (MPT) and its associated processes in isolated rat liver mitochondria. In the presence of 10 μM Ca²⁺, DHM (50-250 μM) elicited MPT in a concentration-dependent, but cyclosporine A-independent manner, as assessed by mitochondrial swelling, which is associated with mitochondrial Ca²⁺ efflux and cytochrome c release. DHM (50-250 μM) did not cause hydrogen peroxide accumulation but did deplete endogenous glutathione and NAD(P)H, while oxidizing protein thiol groups. These results potentially indicate the involvement of mitochondria, via apoptosis, in the well-documented cytotoxicity of monocrotaline.     

Infection by mycotoxigenic fungal species and mycotoxin contamination of maize grain in Umbria, central Italy

Surveys were carried out in 2006 and 2007 in Umbria (central Italy) to evaluate the presence of mycotoxigenic fungi and mycotoxins in maize grain sampled at harvest. Fusarium spp., were the most abundant species detected in maize kernels, followed by Aspergillus species of sections Flavi and Nigri and by Penicillium spp. Among Fusarium species, F. verticillioides was the most prevalent species, as detected by PCR directly on the kernels and on the fungi isolated from the kernels, followed by F. proliferatum and F. subglutinans. Fumonisins were the predominant mycotoxins with values, on average, of 4.3 and 5.7 mg kg⁻¹, in 2006 and 2007, respectively, with a maximum of 76.3 mg kg⁻¹ in the second year. Deoxynivalenol ranged from 0.2 to 3.98 mg kg⁻¹ in 2006 (average 1.04 mg kg⁻¹) and from undetectable levels to 14 mg kg⁻¹ in 2007 (average 0.86 mg kg⁻¹). Aflatoxins, analyzed only in 2007, averaged 26.3 μg kg⁻¹, with a maximum of 820 μg kg⁻¹. Zearalenone content was always very low. Results indicate that EU legal limits for these mycotoxins were rarely exceeded with low levels across most of the examined area, suggesting that this region could be considered suitable for the production of healthy maize.