The Ca²âº-calmodulin-Ca²âº/calmodulin-dependent protein kinase II signaling pathway is involved in oxidative stress-induced mitochondrial permeability transition and apoptosis in isolated rat hepatocytes.

Oxidative stress (OS) is a common event in most hepatopathies, leading to mitochondrial permeability transition pore (MPTP) formation and further exacerbation of both OS from mitochondrial origin and cell death. Intracellular Ca²âº increase plays a permissive role in these events, but the underlying mechanisms are poorly known. We examined in primary cultured rat hepatocytes whether the Ca²âº/calmodulin (CaM)-dependent protein kinase II (CaMKII) signaling pathway is involved in this process, by using tert-butyl hydroperoxide (tBOOH) as a pro-oxidant, model compound. tBOOH (500 µM, 15 min) induced MPTP formation, as assessed by measuring mitochondrial membrane depolarization as a surrogate marker, and increased lipid peroxidation in a cyclosporin A (CsA)-sensitive manner, revealing the involvement of MPTPs in tBOOH-induced radical oxygen species (ROS) formation. Intracellular Ca²âº sequestration with BAPTA/AM, CaM blockage with W7 or trifluoperazine, and CaMKII inhibition with KN-62 all fully prevented tBOOH-induced MPTP opening and reduced tBOOH-induced lipid peroxidation to a similar extent to CsA, suggesting that Ca²âº/CaM/CaMKII signaling pathway fully mediates MPTP-mediated mitochondrial ROS generation. tBOOH-induced apoptosis, as shown by flow cytometry of annexin V/propidium iodide, mitochondrial release of cytochrome c, activation of caspase-3 and increase in the Bax-to-Bcl-xL ratio, and the Ca²âº/CaM/CaMKII signaling antagonists fully prevented these effects. Intramitochondrial CaM and CaMKII were partially involved in tBOOH-induced MPTP formation, since W7 and KN-62 both attenuated the tBOOH-induced, MPTP-mediated swelling of isolated mitochondria. We concluded that Ca²âº/CaM/CaMKII signaling pathway is a key mediator of OS-induced MPTP formation and the subsequent exacerbation of OS from mitochondrial origin and apoptotic cell death.

Poly(vinyl chloride) Films Incorporated with Antioxidant ZnO-Flavonoid Nanoparticles: A Strategy for Food Preservation.

Antioxidant films were prepared using poly(vinyl chloride) (PVC) incorporated with 0.5% or 1.0% zinc oxide (ZnO)-flavonoid (quercetin or morin) nanoparticles (NPZnO-Q% or NPZnO-M%) via the casting method. NP incorporation within the polymer matrix influenced the structural, morphological, optical, and thermal properties of the PVC-based films, as well as their antioxidant activity as assessed using the DPPH radical scavenging method. Our results indicated that increasing ZnO-flavonoid NP concentration increased films thickness, while reducing ultraviolet light (UV) transmittance but conserving transparency. The presence of NPZnO-Q% or NPZnO-M% improved the surface uniformity and thermal stability of the active films. In terms of antioxidant activity, there was an enhancement in the DPPH radical scavenging capacity (PVC/ZnO-Q1.0% > PVC/ZnO-Q0.5% > PVC/ZnO-M0.5% > PVC/ZnO-M1.0% > PVC), suggesting that the packaging can help protect food from oxidative processes. Therefore, these antioxidant films represent an innovative strategy for using as active food packaging material, especially intended for aiding in quality preservation and extending the shelf life of fatty foods.

Potential of Salvinia biloba Raddi for removing atrazine and carbendazim from aquatic environments.

In this exploratory study, naturally occurring Salvinia biloba Raddi specimens were assessed for atrazine and carbendazim polluted water remediation. Experiments were carried out over 21 days in glass vessels containing deionized water artificially contaminated with 0, 5, 10, and 20 mg L-1 of atrazine or carbendazim. Atrazine had a pronounced detrimental impact on S. biloba, as no biomass development was observed in all macrophytes exposed to this herbicide in the entire concentration range. However, carbendazim-treated plants were able to grow and survive in the polluted medium even when subjected to the highest concentration of this fungicide (i.e., 20 mg L-1). In addition, increased chlorosis and necrosis were also detected in plants subjected to carbendazim as a result of the high phytotoxicity caused by atrazine. A maximal removal efficiency of ~ 30% was observed for both pesticides at 5 mg L-1 and decreased with increasing concentrations of the pollutants. The spectrum of the FTIR-ATR analysis revealed the existence of various functional groups (e.g., amide, carboxyl, hydroxyl, phosphate, sulfate) on the plants, which could be related to pesticide biosorption. In addition, at the end of the 21-day assay, seven carbendazim-resistant bacteria could be isolated from the roots of fungicide-treated plants. Therefore, the use of autochthonous free-floating S. biloba macrophytes for phytoremediation of aquatic environments contaminated with carbendazim shows great promise. Still, additional research is required to further elucidate the plant-mediated carbendazim elimination process and the role of the herbicide-resistant bacteria, and seek alternative species capable of mitigating atrazine contamination.

Evaluation of Cadmium Bioaccumulation-Related Physiological Effects in Salvinia biloba: An Insight towards Its Use as Pollutant Bioindicator in Water Reservoirs.

Free-living macrophytes play an important role in the health of aquatic ecosystems. Therefore, the use of aquatic plants as metal biomonitors may be a suitable tool for the management of freshwater reservoirs. Hence, in this study, we assessed the effects of cadmium (Cd) in Salvinia biloba specimens collected from the Middle Paraná River during a 10-day experiment employing artificially contaminated water (100 µM Cd). S. biloba demonstrated a great ability for Cd bioaccumulation in both the root-like modified fronds (named "roots") and the aerial leaf-like fronds (named "leaves") of the plants. Additionally, Cd toxicity was determined by the quantification of photosynthetic pigments (chlorophylls a and b, and carotenoids), flavonoids, and soluble carbohydrate contents in S. biloba over time (1, 3, 5, 7, and 10 days). In general, deterioration was more pronounced in leaves than in roots, suggesting a greater implication of the former in long-term Cd sequestration in S. biloba. Deleterious effects in the appraised parameters were well correlated with the total amount of Cd accumulated in the leaves, and with the qualitative changes observed in the plants' phenotype during the 10-day metal exposure assay. The flavonoids and carotenoids in leaves were highly affected by low Cd levels followed by root carbohydrates. In contrast, chlorophylls and root flavonoids were the least impacted physiological parameters. Therefore, our results demonstrate that S. biloba displays dissimilar organ-linked physiological responses to counteract Cd phytotoxicity and that these responses are also time-dependent. Though further research is needed, our work suggests that easy-handled physiological data obtained from autochthonous free-floating S. biloba specimens may be used as a valuable tool for metal-polluted water biomonitoring.

Oxidative stress induces actin-cytoskeletal and tight-junctional alterations in hepatocytes by a Ca2+ -dependent, PKC-mediated mechanism: protective effect of PKA.

Oxidative stress elevates Ca2+ and, presumably, activates Ca2+ -dependent PKCs. We analyzed the participation of Ca2+ -dependent PKCs in actin disorganization and tight-junctional impairment induced by the pro-oxidant tert-butylhydroperoxide (tBOOH) in isolated rat hepatocyte couplets. tBOOH (100 microM) augmented radical oxygen species (ROS), as indicated by increased lipid peroxidation (+217%, p < 0.05) and intracellular production of 2',7'-dichlorofluorescein (+36%, p < 0.05). Cytosolic Ca2+ and PKCalpha translocation to membrane, an indicator of PKCalpha activation, were also elevated by tBOOH (+100 and +79%, respectively, p < 0.05). tBOOH increased the number of couplets displaying membrane blebs (+278%, p < 0.001) and caused redistribution of F-actin. tBOOH induced tight-junctional impairment, as indicated by a reduction in the percentage of couplets retaining presecreted cholyllysylfluorescein in their canalicular vacuoles (-54%, p < 0.001). tBOOH induced redistribution of the tight-junctional-associated protein ZO-1. All these events were prevented by the panspecific PKC inhibitors H7 and staurosporine, the Ca2+ -dependent PKC inhibitor Gö6976, the intracellular Ca2+ chelator BAPTA/AM, and the PKA activator dibutyryl-cyclic AMP. Furthermore, PKC inhibition and PKA activation not only prevented but also fully reversed tBOOH-induced blebbing. Conversely, tBOOH-induced ROS formation and Ca2+ elevation remained unchanged. We conclude that ROS induce hepatocellular actin-cytoskeleton rearrangement and tight-junctional impairment by a PKC-mediated, Ca2+ -dependent mechanism, which is counteracted by PKA.

Oxidative stress induces internalization of the bile salt export pump, Bsep, and bile salt secretory failure in isolated rat hepatocyte couplets: a role for protein kinase C and prevention by protein kinase A.

We have shown that Ca2+-mediated protein kinase C (PKC) activation induces impairment of bile salt secretory function and F-actin redistribution in hepatocyte couplets. Because oxidative stress induces Ca2+ elevation, we tested here whether PKC inhibition or protein kinase A (PKA) activation, which often counteracts PKC-dependent effects, can prevent and reverse these alterations. The pro-oxidant compounds tert-butylhydroperoxide (tBOOH, 100 microM) and 2,3-dimethoxy-1,4-naphthoquinone (30 microM), reduced by -41% and -29%, respectively, the percentage of couplets accumulating the fluorescent bile salt analog, cholyl-lysylfluorescein in their canalicular vacuoles (p < 0.01). tBOOH-induced bile salt secretory failure was accompanied by internalization of the canalicular bile salt export pump (Bsep), and disarrangement of cytoskeletal F-actin. All these deleterious effects were fully prevented by the intracellular Ca2+ chelator BAPTA/AM (20 microM), the pan-specific PKC inhibitors H7 (100 microM) and staurosporine (1 microM), the inhibitor of Ca2+-dependent PKCs, Gö6976 (2 microM), and the PKA activator dibutyryl-cAMP (500 microM). H7, Gö6976, and dibutyryl-cAMP not only prevented but also fully reversed the decrease in the cholyl-lysyl-fluorescein accumulation. In conclusion, these results suggest that low levels of oxidative stress impair bile salt secretion by internalizing Bsep through a Ca2+-dependent, PKC-mediated mechanism, and that inhibition of PKC, or activation of PKA, prevents and reverses these effects. Alterations in actin organization may be a causal factor.

Silibinin prevents cholestasis-associated retrieval of the bile salt export pump, Bsep, in isolated rat hepatocyte couplets: possible involvement of cAMP.

Estradiol-17beta-d-glucuronide (E(2)17G) and taurolithocholate (TLC) induce acute cholestasis-associated with retrieval of the bile salt export pump (Bsep), which parallels with alteration in transport activity. cAMP stimulates the apically directed vesicular trafficking of transporters, partially preventing these alterations. The hepatoprotector, silymarin, which inhibits cAMP-phosphodiesterase, prevents the cholestasis induced in vivo by both estrogens and TLC. We aimed to assess the ability of silibinin (Sil), the silymarin active component, to prevent the retrieval of Bsep induced by TLC and E(2)17G, and the associated alteration in its transport function. The possible involvement of cAMP as a second messenger and the intracellular signalling pathways implicated were also evaluated. Functional studies were performed analysing the proportion of isolated rat hepatocyte couplets (IRHC) accumulating the fluorescent bile salt analogue, cholyl-lysylfluorescein (CLF), into their sealed canalicular vacuoles. Cellular localisation of Bsep was assessed by immunofluorescent staining. Intracellular levels of cAMP were measured by ELISA. Sil (2.5microM) elevated by 40+/-3% intracellular cAMP, and mimicked the ability of dibutyryl-cAMP (10microM) to prevent internalisation of Bsep and the TLC (2.5microM)- and E(2)17G (50microM)-induced impairment in the capacity of IRHC to accumulate CLF apically. Preventive effects of Sil and dibutyryl-cAMP were not abolished by the specific protein kinase A inhibitors, KT5720 and H89. Contrarily, the intracellular Ca(2+) chelator, BAPTA/AM, significantly blocked the protective effect of both compounds. We conclude that Sil prevented TLC- and E(2)17G-induced bile salt secretory failure, at least in part, by avoiding redistribution of Bsep, by a mechanism probably involving cAMP-induced cytosolic Ca(2+) elevations.

Signaling modulation of bile salt-induced necrosis in isolated rat hepatocytes.

Hydrophobic bile salts induce either necrosis or apoptosis depending on the severity of the injury caused by them. Since bile salt-induced apoptosis is influenced by Ca2+- and protein kinase-signaling pathways, and both necrosis and apoptosis share common initiating mechanisms, we analyzed whether these signaling cascades also influence bile salt-induced necrosis in isolated rat hepatocytes. Taurochenodeoxycholate (TCDC, 0.25-1.50 mM, 2 h) reduced, in a dose-dependent manner, the percentage of viable hepatocytes, and increased the release of the cytosolic enzyme, lactate dehydrogenase (LDH) and alanine aminotransferase (ALAT), and that of the plasma membrane enzyme, alkaline phosphatase (AP). The PKC inhibitors, H7 (100 microM) and chelerythrine (2.5 microM), both prevented significantly TCDC-induced necrosis. On the contrary, the PKA activator, dibutyryl-cAMP, exacerbated TCDC-induced cell damage in a dose-dependent manner; this effect was more likely due to cAMP-mediated PKA activation, as the PKA inhibitor, KT5720 (1 microM), counteracted this effect. Instead, the intracellular Ca2+ chelator, BAPTA/AM (20 microM), was without effect. TCDC (1 mM) increased lipid peroxidation from 0.7 +/- 0.2 to 7.5 +/- 0.9 nmol of malondialdehyde per mg of protein, p < 0.001; the addition of the free radical scavenger, diphenyl-p-phenylendiamine, completely blocked this increase and prevented significantly TCDC-induced necrosis. PKC inhibition induced only a slight attenuation of TCDC-induced lipid peroxidation. Possible mechanisms accounting for the modulatory effect of signal transduction pathways on TCDC-induced necrosis, including signaling influence on TCDC transport events and TCDC-induced oxidative stress, are discussed.

Inhibition of rat liver UDP-glucuronosyltransferase by silymarin and the metabolite silibinin-glucuronide.

The inhibitory effects of silymarin, its main constituent silibinin and the metabolite silibinin-glucuronide on UDP-glucuronosiltransferase (UGT) were evaluated in rat hepatic microsomes. Three substrates were chosen to cover both UGT1A and UGT2B family isozymes: bilirubin (substrate of UGT1A1), p-nitrophenol (UGT1A6) and ethinylestradiol (UGT2B1 and 2B3 for position C17 and UGT1A1 for position C3). The study of p-nitrophenol and bilirubin glucuronidation indicated that silymarin (SM) and silibinin glucuronide (SB-G) were enzyme inhibitors. The kinetic analysis showed that the type of inhibition was competitive in all cases and the Ki obtained were: for p-nitrophenol glucuronidation, KiSB-Gapp: 14+/-1 microg/ml and KiSMapp: 51+/-10 microg/ml and for bilirubin glucuronidation, KiSB-Gapp: 16+/-3 microg/ml. In turn, ethinylestradiol glucuronidation was not affected by any of the compounds studied suggesting that the inhibitory effect was restricted to UGT1A isozymes. Similar studies performed using human hepatic microsomes showed that SM and SB-G were also inhibitors of human UGT1A isozymes. In conclusion, administration of silymarin or its main constituent silibinin could lead to the decrease in the glucuronidation of substrates whose conjugation depends on UGT1A isozymes in a process mediated by silibinin-glucuronide, though their effect in humans needs further investigation.

Characterization of the degradation performance of the sulfamethazine antibiotic by photo-Fenton process.

The present study provides results describing the degradation performance of the Sulfamethazine (SMT) antibiotic via photo-Fenton treatment. Experiments were carried out using 1 L solution samples of SMT (50 mg L(-1)) under different conditions. HPLC results reveal that both Fenton and photo-Fenton reactions were able to completely remove SMT antibiotic from the studied samples in less than 2 min treatment. Half-life times and kinetic parameters (assuming a pseudo-first-order kinetics at reaction initial stage, far from the equilibrium) for SMT degradation were determined and discussed. Hence, appropriate Fenton reagent loads are given to attain different targets proposed. TOC and HPLC data also revealed the presence of reaction intermediates; thus toxicity assays were performed regarding bacterial growth rate. The toxicity of an SMT solution was shown to increase during its degradation by means of photo-Fenton reactions.