Different effects of two cyclic chalcone analogues on redox status of Jurkat T cells

Chalcones are intermediary compounds of the biosynthetic pathway of the naturally flavonoids. Previous studies have demonstrated that chalcones and their conformationally rigid cyclic analogues have tumour cell cytotoxic and chemopreventive effects. It has been shown that equitoxic doses of the two cyclic chalcone analogues (E)-2-(4′-methoxybenzylidene)-(2) and (E)-2-(4′-methylbenzylidene)-1-benzosuberone (3) have different effect on cell cycle progress of the investigated Jurkat cells. It was also found that the compounds affect the cellular thiol status of the treated cells and show intrinsic (non-enzyme-catalyzed) reactivity towards GSH under cell-free conditions. In order to gain new insights into the cytotoxic mechanism of the compounds, effects on the redox status and glutathione level of Jurkat cells were investigated. Detection of intracellular ROS level in Jurkat cells exposed to 2 and 3 was performed using the dichlorofluorescein-assay. Compound 2 did not influence ROS activity either on 1 or 4 h exposure; in contrast, chalcone 3 showed to reduce ROS level at both timepoints. The two compounds had different effects on cellular glutathione status as well. Compound 2 significantly increased the oxidized glutathione (GSSG) level showing an interference with the cellular antioxidant defence. On the contrary, chalcone 3 enhanced the reduced glutathione level, indicating enhanced cellular antioxidant activity. To investigate the chalcone–GSH conjugation reactions under cellular conditions, a combination of a RP-HPLC method with electrospray ionization mass spectrometry (ESI-MS) was performed. Chalcone–GSH adducts could not be observed either in the cell supernatant or the cell sediment after deproteinization. The investigations provide further details of dual – cytotoxic and chemopreventive – effects of the cyclic chalcone analogues.     

Different effects of two cyclic chalcone analogues on redox status of Jurkat T cells

Chalcones are intermediary compounds of the biosynthetic pathway of the naturally flavonoids. Previous studies have demonstrated that chalcones and their conformationally rigid cyclic analogues have tumour cell cytotoxic and chemopreventive effects. It has been shown that equitoxic doses of the two cyclic chalcone analogues (E)-2-(4′-methoxybenzylidene)-(2) and (E)-2-(4′-methylbenzylidene)-1-benzosuberone (3) have different effect on cell cycle progress of the investigated Jurkat cells. It was also found that the compounds affect the cellular thiol status of the treated cells and show intrinsic (non-enzyme-catalyzed) reactivity towards GSH under cell-free conditions. In order to gain new insights into the cytotoxic mechanism of the compounds, effects on the redox status and glutathione level of Jurkat cells were investigated. Detection of intracellular ROS level in Jurkat cells exposed to 2 and 3 was performed using the dichlorofluorescein-assay. Compound 2 did not influence ROS activity either on 1 or 4 h exposure; in contrast, chalcone 3 showed to reduce ROS level at both timepoints. The two compounds had different effects on cellular glutathione status as well. Compound 2 significantly increased the oxidized glutathione (GSSG) level showing an interference with the cellular antioxidant defence. On the contrary, chalcone 3 enhanced the reduced glutathione level, indicating enhanced cellular antioxidant activity. To investigate the chalcone–GSH conjugation reactions under cellular conditions, a combination of a RP-HPLC method with electrospray ionization mass spectrometry (ESI-MS) was performed. Chalcone–GSH adducts could not be observed either in the cell supernatant or the cell sediment after deproteinization. The investigations provide further details of dual – cytotoxic and chemopreventive – effects of the cyclic chalcone analogues.     

Toxicity of the veterinary sulfonamide antibiotic sulfamonomethoxine to five aquatic organisms

The purpose of this study was to investigate the acute and chronic toxicity of sulfamonomethoxine (SMM) to aquatic organisms to evaluate its impact at different trophic levels in the ecosystem. Regarding the growth inhibition of microalgae, SMM exhibited 72-h median effective concentration (EC₅₀) values of 5.9 mg L⁻¹ for freshwater Chlorella vulgaris and 9.7 mg L⁻¹ for marine Isochrysis galbana. In a study on the cladocerans, SMM exhibited acute toxicity and 48-h median lethal concentrations of 48 mg L⁻¹ for Daphnia magna and 283 mg L⁻¹ for D. similis. An examination of chronic toxicity revealed that SMM inhibited the brook production of the cladocerans and exhibited 21-day EC₅₀ values of 14.9 mg L⁻¹ for D. magna and 41.9 mg L⁻¹ for D. similis. This study investigated the potentially adverse effects of SMM on aquatic organisms and revealed that microalgae exhibited higher sensitivity to SMM than cladocerans did. The residue of SMM in water is recommended to be carefully evaluated to reduce ecological impacts after applied to cultured animals.     

Effect of melamine on potassium currents in rat hippocampal CA1 neurons

As an industrially synthesized chemical, melamine has been applied in a wide range of areas. However, many questions on the adverse effect and toxicity of melamine have been emerged, recently. In this investigation, the cytotoxicity of melamine on PC12 cells was evaluated. Furthermore, the effect of melamine on the transient outward potassium current (I_A) and the delayed rectifier potassium current (I_K) in hippocampal CA1 pyramidal neurons of rat was studied using whole-cell patch-clamp technique. The results showed that melamine-induced cell death in a concentration and time-dependent manner, and produced a concentration-dependent inhibition in amplitudes of I_A and I_K at any concentrations (5 × 10⁻⁴, 5 × 10⁻⁵, and 5 × 10⁻⁶ g/ml). Moreover, at higher concentration (5 × 10⁻⁴ g/ml), melamine had observable effects of the steady-state inactivation of I_A, that is melamine shifted inactivation curve of I_A towards hyperpolarization. The spontaneous firing frequency was increased as well. These results suggest that the regulation of I_A and I_K induced by melamine would make neurons display aberrant firing properties and abnormal neuronal discharge, which could be a possible underlying mechanism for the melamine-induced neurotoxicity.     

Comparative evaluation of two dye probes in the rat everted gut sac model for unambiguous classification of P-gp substrate and inhibitor

IntroductionP-glycoprotein (P-gp) plays a crucial role in beta-amyloid efflux from the blood–brain barrier thus becoming a promising pharmacological target in the treatment of Alzheimer's disease (AD). The increase of P-glycoprotein expression and activity by a P-gp inducer could be an effective pharmacological strategy in slowing or halting the progression of AD. Commonly used in vitro methods to classify a P-gp interacting molecule as substrate, inhibitor, modulator or inducer are not always confirmed by in vivo experiments. Here we validate the new dye-probe beta-amyloid (1–40) HiLyte Fluor? TR-labeled (Ab-HiLyte) (Anaspec) P-gp mediated transport in the ex vivo rat everted gut sac assay by using MC18 or MC266, a fully characterized P-gp inhibitor and substrate, respectively, and compare it with the commonly used dye rhodamine.MethodsMale Wistar rats' everted intestines were divided into sacs, each sac was filled with 10 μM Ab-HiLyte with or without 50 μM of MC18 or MC266. Ab-HiLyte concentrations in mucosal fluid were measured spectrophotometrically at 594 nm at each appropriate time.ResultsThe Ab-HiLyte P-gp mediated efflux had a K = 1.00 × 10^? 2 min^? 1 and t_1/2 = 68.74 min, while in the presence of MC18, the Ab-HiLyte efflux turned out to be reduced by an order of magnitude (K = 1.65 × 10^? 3 min^? 1) and the half life is extremely increased (t_1/2 = 419 min). A P-gp substrate, like MC266, determines no change in the efflux of Ab: the kinetic constant and the half life turned out to be unmodified (K = 1.81 × 10^? 2 min^? 1 and t_1/2 = 38.28 min).DiscussionThe results demonstrate that the new dye probe, Ab-HiLyte, could be a probe of choice to unequivocally distinguish between a P-gp substrate and an inhibitor. This is particularly important as different groups obtain a controversial classification of the same compound.     

Effects of Zizyphus lotus L. (Desf.) polyphenols on Jurkat cell signaling and proliferation

We assessed the effects of Zizyphus lotus L. (Desf.) polyphenols (ZLP) on T-cell signaling and proliferation. Our results showed that ZLP exerted no effect on the increases in intracellular free calcium concentrations, [Ca^2 +]_i, in human Jurkat T-cells. However, ZLP modulated the thapsigargin-induced increases in [Ca^2 +]_i in these cells. ZLP treatment was found to decrease the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2). In addition, ZLP induced a rapid (t_1/2 = 33 s) and dose-dependent decrease in intracellular pH (pH_i) in human Jurkat T-cells. Furthermore, ZLP significantly curtailed T-cell proliferation by diminishing their progression from S to G2/M phase of cell cycle, and the expression of interleukin-2 (IL-2) mRNA. Taken together, the results of the present study demonstrate that ZLP modulate cell signaling and exert immunosuppressive effects in human T-cells.     

Down-regulation of carboxylesterases 1 and 2 plays an important role in prodrug metabolism in immunological liver injury rats

Liver plays a central role in xenobiotics metabolism, thus affecting the in vivo disposition and therapeutic effects of drugs. Carboxylesterases (CESs), with the main isoforms CES1 and CES2, are important in the metabolism of ester-type prodrugs. However, influences of immunological liver injury on the activity of CES remain undefined. In the present study, we demonstrated treatment with lipopolysaccharide (LPS) suppressed the activities of CES1 and CES2. The decreased activities of CES1 and CES2 were preliminarily assessed by the hydrolysis assay for their common substrate p-nitrophenyl acetate (PNPA) with rat hepatic microsomal enzyme. Subsequently, RT-PCR results showed that the levels of CES1 mRNA and mRNA of CES2 (AB010635) and CES2 (AY034877) in the model group were significantly lower than those of the normal control group (P < 0.05). Western blot results showed that the expressions of CES1 and CES2 proteins were decreased (P < 0.05). To further clarify the effects of LPS on the metabolic activities of CESs, pharmacokinetic studies were performed in rats by utilizing imidapril and irinotecan (CPT-11) as the specific substrates for CES1 and CES2, respectively. After treatment with LPS, AUC_0 -∞ and C_max of imidaprilat were decreased from 2084.86 ± 340.66 ng · h^− 1 · mL^− 1 and 234.66 ± 68.85 ng · mL^− 1 to 983.87 ± 315.34 ng · h^− 1 · mL^− 1 and 113.1 ± 19.69 ng · mL^− 1 (P < 0.05), respectively. Moreover, AUC_0 -∞ and C_max of SN-38 were decreased from 8100 ± 918.6 ng · h^− 1 · mL^− 1 and 144.67 ± 20.28 ng · mL^− 1 to 3270 ± 500.5 ng · h^− 1 · mL^− 1 and 56.19 ± 10.38 ng · mL^− 1 (P < 0.05), respectively. In summary, immunological liver injury remarkably attenuated the expressions and metabolic activities of CES1 and CES2, which may be associated with the regulatory effects of cytokines under inflammation.     

Inhibitory effects and mechanism of 25-OH-PPD on glomerular mesangial cell proliferation induced by high glucose

ObjectiveTo investigate the protective effects and potential mechanism of the compound 25-OH-PPD (PPD) on the glomerular mesangial cells (GMC) under high glucose condition.MethodsThe hypertrophic GMC cells were established by DMEM containing glucose and randomly divided into five groups, including the normal control group (Control), the high glucose model group (HG, 25 mmol L⁻¹), the PPD low dose group (1 μmol L⁻¹, PPD-L), the PPD middle dose group (5 μmol L⁻¹, PPD −M) and the PPD high dose group (10 μmol L⁻¹, UCN-H). The GMC were incubated for 48 h under different treatment factors. Total protein content was determined by Lowry method. The diameter of the single GMC and volume were measured by computer photograph analysis system. The GMC cell viability was analyzed by MTT assay. The level of malondialdehyde (MDA), the content of glutathione (GSH) and superoxide dismutase (SOD) activity were measured by ELISA. [Ca²⁺]_і transient was measured by Till image system and by cell-loading Fura-2/AM. The expression of COX-1 and COX-2 were also determined using ELISA method.ResultsThe viability of GMC and the total protein content were decreased in HG group, different dosage PPD group could increase these indexes (P < 0.05). The level of MDA was increased, the content of GSH and SOD was decreased in HG group, while PPD could reduce the MDA and enhance GSH and SOD (P < 0.05). Following treatment with different dosage (PPD-L, PPD-M or PPD-H), the [Ca²⁺]_і transient was reduced (P < 0.05 or P < 0.01). Moreover, the expression of COX-1 was decreased while COX-2 expression was increased in different dosage PPD groups.ConclusionThe protective effects of PPD on GMC from HG-induced hypertrophy may be associated with the inhibition of [Ca²⁺]_і transient and decreasing expression of COX-1 via the oxidative-stress injure pathway.     

Mild caloric restriction reduces blood pressure and activates endothelial AMPK-PI3K-Akt-eNOS pathway in obese Zucker rats

Genetic obesity models exhibit endothelial dysfunction associated to adenosine monophosphate-activated protein kinase (AMPK) dysregulation. This study aims to assess if mild short-term caloric restriction (CR) restores endothelial AMPK activity leading to an improvement in endothelial function. Twelve-week old Zucker lean and obese (fa/fa) male rats had access to standard chow either ad libitum (AL, n = 8) or 80% of AL (CR, n = 8) for two weeks. Systolic blood pressure was significantly higher in fa/fa AL rats versus lean AL animals, but was normalized by CR. Endothelium-dependent relaxation to acetylcholine (ACh, 10^− 9 to 10^− 4 M) was reduced in fa/fa AL compared to control lean AL rats (p < 0.001), and restored by CR. The AMPK activator AICAR (10^− 5 to 8·10^− 3 M) elicited a lower relaxation in fa/fa AL rings that was normalized by CR (p < 0.001). Inhibition of PI3K (wortmannin, 10^− 7 M), Akt (triciribine, 10^− 5 M), or eNOS (L-NAME, 10^− 4 M) markedly reduced AICAR-induced relaxation in lean AL, but not in fa/fa AL rats. These inhibitions were restored by CR in Zucker fa/fa rings. These data show that mild short-term CR improves endothelial function and lowers blood pressure in obesity due to the activation of the AMPK–PI3K–Akt–eNOS pathway.     

Activity of the enzymes of the antioxidative system in cadmium-treated Oxya chinensis (Orthoptera Acridoidae)

One purpose in this research was to determine the toxic effects of Cd on antioxidant enzymes of Oxya chinensis (Orthoptera: Acridoidae). Changes in the activities of the antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), and guaiacol peroxidase (GPx) were measured in O. chinensis insects injected with Cd²⁺. Fifth-nymphs of O. chinensis insects were injected with Cd²⁺ at different concentrations (0, 0.55 × 10⁻⁴, 1.10 × 10⁻⁴, 1.65 × 10⁻⁴, 2.20 × 10⁻⁴, and 2.75 × 10⁻⁴ g g⁻¹). An increase in SOD activity in O. chinensis was observed at 1.10 × 10⁻⁴ to 2.75 × 10⁻⁴ g g⁻¹ Cd²⁺. The SOD activity was lower at 2.20 × 10⁻⁴and 2.75 × 10⁻⁴ g g⁻¹ than that at 1.10 × 10⁻⁴ and 1.65 × 10⁻⁴ g g⁻¹. It appears that SOD had a positive protective effect at low Cd²⁺ concentrations, and that this effect disappeared at high Cd²⁺ concentrations. CAT activity was accelerated to varying degrees at 1.10 × 10⁻⁴ to 2.75 × 10⁻⁴ g g⁻¹ for males and at 1.10 × 10⁻⁴, 2.20 × 10⁻⁴, and 2.75 × 10⁻⁴g g⁻¹ for females. CAT showed a strong detoxification effect with all treatments. GPx activity decreased with increasing Cd²⁺ concentration with all treatments for males and at 2.20 × 10⁻⁴ and 2.65 × 10⁻⁴g g⁻¹ for females. We showed that GPx activity had a weak detoxification function with all treatments for males and at high Cd²⁺ for females. Thus, CAT had a strong detoxification effect, whereas SOD had a medium and GPx had a weak detoxification effect. Among the three enzymes, CAT played an important role in the damaging mechanisms of reactive oxygen species in O. chinensis insects. Alterations of the antioxidant enzyme level under environmental stresses are suggested as indicators of biotic and abiotic stress.     

Oxidative stress,genotoxicity and cytotoxicity of 1-methyl-3-octylimidazolium chloride on Paramisgurnus dabryanus

This study evaluated the toxicity of 1-methyl-3-octylimidazolium chloride ([C₈mim]Cl) on Paramisgurnus dabryanus by enzyme analysis, comet assay, and apoptosis analysis. The study showed that [C₈mim]Cl had an obvious toxic effect inducing oxidative stress, genotoxicity, and cytotoxicity to fish liver cells. [C₈mim]Cl also induced changes in the activities of superoxide dismutase and catalase, and the glutathione content and malondialdehyde level in fish exposed at 20–80 mg L⁻¹. With increased exposure concentration and time, the four antioxidant enzyme activities, three different comet parameters and apoptosis rates of tested cells were significantly increased, with significant differences (P < 0.05 or P < 0.01) observed between control group and each treatment group. This study shows that [C₈mim]Cl could be a threat to aquatic organism health when accidentally released into aquatic ecosystems.     

Isothiazole dioxide derivative 6n inhibits vascular smooth muscle cell proliferation and protein farnesylation

Isothiazole dioxides have been shown to inhibit Trypanosoma brucei protein farnesyltransferase (PFTase) in isolated enzyme, but elicited only a minor effect on mammalian PFTase. In the present study we have evaluated the effect of 3-diethylamino-4-(4-methoxyphenyl)-isothiazole 1,1-dioxides with different substituents at C5, on rat PFTase and protein geranylgeranyltransferase-I (PGGTase-I) with the final aims to improve the potency against mammalian PFTase and to identify new compounds with antiproliferative properties. For these purposes, in vitro and cell culture models have been utilized. The results showed that isothiazole dioxides with C4–C5 double bond and sulfaryl substituted at the C5 position but none of the dihydro-derivatives, were able to inhibit in vitro PFTase in a concentration dependent manner (IC₅₀ ranging from 8.56 to 1015 μM). Among those, compound 6n (C5; methyl-S) displayed 500-fold higher inhibitory potency on PFTase than PGGTase-I. Compound 6n was shown to affect rat smooth muscle cell (SMC) proliferation at concentrations similar (IC₅₀ = 61.4 μM) to those required to inhibit [³H]-farnesol incorporation into cellular proteins (−44.1% at 100 μM). Finally, compound 6n interferes with rat SMC proliferation by blocking the progression of G0/G1 phase without inducing apoptosis, as assessed by [³H]-thymidine incorporation assay and flow cytometry analysis. Taken together, we described a new PFTase inhibitor containing the isothiazole dioxide moiety that affects mammalian protein farnesylation and SMC proliferation by inhibiting G0/G1 phase of the cell cycle.     

Chemical composition,antiproliferative and antioxidant properties of lipid classes in ordinary and dark muscles from chub mackerel (Scomber japonicus)

This study investigated to compare lipid profiles in ordinary and dark muscles from chub mackerel and to examine antiproliferative and antioxidative properties of lipid classes. The average levels of neutral lipids (NL), glycolipids (GL), and phospholipids (PL) in ordinary muscle were 92.32 ± 0.19%, 5.10 ± 0.48%, and 2.58 ± 0.46%; in dark muscle were 96.88 ± 0.15%, 2.59 ± 0.36%, and 0.54 ± 0.29%, respectively. The fatty acid composition indicated that PL had a higher percentage of PUFA (especially 22:6n?3) with lower percentages of SFA and MUFA compared to NL and GL (p < 0.05). The main ion peaks of GL in ordinary and dark muscles showed that monocharged and bischarged molecular ion were presented at m/z 876.9 and 438.8, respectively. In MTT assay, inhibition of AGS and HT-29 cell proliferation was greatest with the 0.5 and 1.0 mg mL^?1 GL treatments. The GL of ordinary muscle with 0.05 mg mL^?1 concentrations markedly decreased the levels of reactive oxygen species (ROS) induced by H₂O₂ compared to the control (p < 0.05). From our results, GL might have antiproliferative and antioxidant properties based on protective effect against the production of intracellular ROS.     

Cytotoxic effect of inositol hexaphosphate and its Ni(II) complex on human acute leukemia Jurkat T cells

Inositol hexaphosphate (InsP₆) is present in cereals, legumes, nuts and seed oils and is biologically active against some tumor and cancer cells. Herein, this study aimed at evaluating the cellular toxicity, antiproliferative activity and effects on cell cycle progression of free InsP₆ and InsP₆–Ni(II) of leukemic T (Jurkat) and normal human cells. Treatments with InsP₆ at concentrations between 1.0 and 4.0 mM significantly decreased the viability of Jurkat cells, but showed no cytotoxic effect on normal human lymphocytes. Treatment with InsP₆–Ni(II) complex at concentrations between 0.05 and 0.30 mM showed an anti-proliferative dose and a time-dependent effect, with significantly reduced cell viability of Jurkat cells but showed no cytotoxic effect on normal human lymphocytes as compared to the control. Ni(II) free ion was toxic to normal cells while InsP₆–Ni(II) had no cytotoxic effect. The InsP₆–Ni(II) complex potentiated (up to 10 ×) the antiproliferative effect of free InsP₆ on Jurkat cells. The cytometric flow assay showed that InsP₆ led to an accumulation of cells in the G0/G1 phase of the cell cycle, accompanied by a decrease in the number of cells in S and G2/M phases, whereas InsP₆–Ni(II) has led to an accumulation of cells in the S and G2/M phases. Our findings showed that InsP₆–Ni(II) potentiates cytotoxic effects of InsP₆ on Jurkat cells and may be a potential adjuvant in the treatment of cancer.     

Synthetic polysulfane derivatives induce cell cycle arrest and apoptotic cell death in human hematopoietic cancer cells

Natural polysulfanes including diallyltrisulfide (DATS) and diallyltetrasulfide (DATTS) from garlic possess antimicrobial, chemopreventive and anticancer properties. However these compounds exhibit chemical instability and reduced solubility, which prevents their potential clinical applicability. We synthesized six DATS and DATTS derivatives, based on the polysulfane motif, expected to exhibit improved physical and chemical properties and verified their biological activity on human leukemia cells.We identified four novel cytotoxic compounds (IC₅₀ values: compound 1, 24.96 ± 12.37 μM; compound 2, 22.82 ± 4.20 μM; compound 3, 3.86 ± 1.64 μM and compound 5, 40.62 ± 10.07 μM, compared to DATTS: IC₅₀: 9.33 ± 3.86 μM). These polysulfanes possess excellent differential toxicity, as they did not affect proliferating mononuclear blood cells from healthy donors.We further demonstrated ability of active compounds to induce apoptosis in leukemia cells by analysis of nuclear fragmentation and of cleavage of effector and executioner caspases. Apoptosis was preceded by accumulation of cells in G2/M phase with a pro-metaphase-like nuclear pattern as well as microtubular alterations. Prolonged and persistent arrest of cancer cells in early mitosis by the benzyl derivative identifies this compound as the most stable and effective one for further mechanistic and in vivo studies.     

CYP-450 isoenzymes catalyze the generation of hazardous aromatic amines after reaction with the azo dye Sudan III

This work describes the mutagenic response of Sudan III, an adulterant food dye, using Salmonella typhimurium assay and the generation of hazardous aromatic amines after different oxidation methods of this azo dye. For that, we used metabolic activation by S9, catalytic oxidation by ironporphyrin and electrochemistry oxidation in order to simulate endogenous oxidation conditions. The oxidation reactions promoted discoloration from 65% to 95% of Sudan III at 1 × 10⁻⁴ mol L⁻¹ and generation of 7.6 × 10⁻⁷ mol L⁻¹ to 0.31 × 10⁻⁴ mol L⁻¹ of aniline, o-anisidine, 2-methoxi-5-methylaniline, 4-aminobiphenyl, 4,4′-oxydianiline; 4,4′-diaminodiphenylmethane and 2,6-dimethylaniline. The results were confirmed by LC–MS–MS experiments. We also correlate the mutagenic effects of Sudan III using S. typhimurium with the strain TA1535 in the presence of exogenous metabolic activation (S9) with the metabolization products of this compound. Our findings clearly indicate that aromatic amines are formed due to oxidative reactions that can be promoted by hepatic cells, after the ingestion of Sudan III. Considering that, the use of azo compounds as food dyestuffs should be carefully controlled.     

Chicken egg yolk antibodies (IgY) modulate the intestinal mucosal immune response in a mouse model of Salmonella typhimurium infection

This study determined the effects of chicken egg yolk antibodies (IgY) on immune responses in the intestinal mucosal of mice infected with Salmonella typhimurium. Sixty, 28-day-old mice were divided into 4 groups and treated with streptomycin or sterile water for 2 days followed by 1 day without treatment. The control group was unchallenged whereas the mice in the other three groups were treated twice with 10⁹ CFU mL^− 1 S. typhimurium. For the next 3 days, control mice continued to receive no treatment whereas the mice in the remaining three groups were orally administered with 20 mg mL^− 1 of specific IgY, 20 mg mL^− 1 of nonspecific IgY or PBS. S. typhimurium activated gut-associated lymphoid tissue, increasing the release of IFN-γ and TNF-α in the mucosa and increased the number of activated T-lymphocytes and cytotoxic T-γδ. Specific IgY attenuated the increase in IFN-γ and TNF-α and the decrease in IL-10. S. typhimurium induced mobilization of CD8⁺ and CD8⁺ TCRγδ T cells in the epithelium and CD4⁺ and CD8⁺ T cells in the lamina propria reflecting an inflammatory process that was attenuated by IgY. These results suggest that specific IgY modulates intestinal mucosal immune responses during a S. typhimurium infection.     

In vivo sustained dermal delivery and pharmacokinetics of interferon alpha in biphasic vesicles after topical application

Biphasic vesicles, a novel nanostructured lipid-based delivery system show potential for topical application of interferon alpha (IFN α) for the treatment of human papillomavirus (HPV) infections (anogenital warts). Dermal delivery of IFN α encapsulated in biphasic vesicles (BPV-IFN α), applied topically to the skin, was characterized in a guinea pig model.BPV-IFN α (1 g, 2 MIU/g) was topically applied either as a single or multiple treatments on the skin of guinea pigs. As a comparison with currently used regimens, IFN α solution was administered intravenously or intradermally. Skin and serum samples were collected over 96 h, IFN α levels were determined by an antiviral assay, and half-life (t_1/2) and elimination (k) rates were calculated.Topical BPV-IFN α treatment resulted in maximum skin levels (about 100,000 U/100 cm²) of IFN α within 6 h and maintained for 72–96 h. Clearance from the skin after intradermal injections was initially fast (t_1/2 0.62 h, k 1.1179 h⁻¹), followed by a slower steady decrease after 6 h. After intravenous and intradermal administration, IFN α was rapidly cleared from the serum, t_1/2 0.75 h, k 0.9271 h⁻¹ and t_1/2 1.28 h, k 0.5421 h⁻¹, respectively, whereas after topical application, IFN α levels remained below 100 U/mL. Topical application of BPV- IFN α resulted in sustained delivery of biologically active IFN α locally into skin with minimal systemic exposure.     

A study of genotoxicity and oxidative stress induced by mercuric chloride in the marine polychaete Perinereis aibuhitensis

The marine polychaete worm Perinereis aibuhitensis was used to study the genotoxic effects of mercuric chloride by means of the comet assay and micronucleus (MN) test. P. aibuhitensis was subjected in vivo to two different concentrations of mercuric chloride (0.05 mg L⁻¹ and 0.5 mg L⁻¹) for 96 h. The comet assay of coelomocytes demonstrated that TailDNA% values increased with extended exposure to or increased concentrations of HgCl₂ (p < 0.01). The frequency of MNs was the highest in the treatment with 96 h of exposure at all concentrations (p < 0.01). The genotoxic effect of HgCl₂ was both dose- and time-dependent in exposed P. aibuhitensis. The activities of the antioxidant enzymes, superoxide dismutase (SOD) and glutathione peroxidases (GPx) were also estimated. Significant variations in antioxidant enzyme activities depended on the sampling time and the concentrations of mercuric chloride. Compared with the control, the activities of the antioxidant enzymes (SOD and GPx) were elevated at the lower concentration of mercuric chloride (0.05 mg L⁻¹) (p < 0.05) for shorter exposure periods (24 h and 72 h). At the higher concentration of mercury (0.5 mg L⁻¹), the activities of GPx and SOD were inhibited; no variation was observed. These results proved that the use of the comet assay and MN test in coelomocytes of P. aibuhitensis is appropriate for determining the levels of DNA damage and that P. aibuhitensis is a species that is sensitive to mercury pollutants. This species may be considered a suitable candidate for monitoring marine heavy metal pollution.     

Cytotoxicity and genotoxicity of phenazine in two human cell lines

Phenazine was recently identified as a drinking water disinfection byproduct (DBP), but little is known of its toxic effects. We examined in vitro cytotoxicity and genotoxicity of phenazine (1.9–123 μM) in HepG2 and T24 cell lines. Cytotoxicity was determined by an impedance-based real-time cell analysis instrument. The BrdU (5-bromo-2′-deoxyuridine) proliferation and MTT ((3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) viability assays were used to examine mechanisms of cytotoxicity. Genotoxicity was determined using the alkaline comet assay. Concentration-dependent cytotoxicity was observed in HepG2 cells, primarily due to an antiproliferative effect (BrdU 24 h IC₅₀: 11 μM; 48 h IC₅₀: 7.8 μM) observed as low as 1.9 μM. T24 cells experienced a minor antiproliferative effect (BrdU 24 h IC₅₀: 47 μM; 48 h IC₅₀: 17 μM). IC₅₀ values for HepG2 proliferation and viability were 54–77% lower compared to T24 cells. In both cell lines, IC₅₀ values for proliferation were 66–90% lower than those for viability. At phenazine concentrations producing equivalent cytotoxicity, HepG2 cells (1.9–30.8 μM) experienced no significant genotoxic effects, while T24 cells (7.7–123 μM) experienced significant genotoxicity at ⩾61.5 μM. While these effects were seen at phenazine concentrations above those found in disinfected water, the persistence of the antiproliferative effect and the differential toxicity in each cell line deserves further study.