Ecotoxicity evaluation of polymeric nanoparticles loaded with ascorbic acid for fish nutrition in aquaculture.

BACKGROUND: Ascorbic acid (AA) is a micronutrient essential for the mechanisms of reproduction, growth, and defense in fish. However, the biosynthesis of this micronutrient does not occur in fish, so it must be supplied with food. A difficulty is that plain AA is unstable, due to the effects of light, high temperature, and oxygen, among others. The use of nanoencapsulation may provide protection and preserve the physicochemical characteristics of AA for extended periods of time, decreasing losses due to environmental factors. METHOD: This study evaluated the protective effect of nanoencapsulation in polymeric nanoparticles (chitosan and polycaprolactone) against AA degradation. Evaluation was made of the physicochemical stability of the nanoformulations over time, as well as the toxicological effects in zebrafish (Danio rerio), considering behavior, development, and enzymatic activity. For the statistical tests, ANOVA (two-way, significance of p < 0.05) was used. RESULTS: Both nanoparticle formulations showed high encapsulation efficiency and good physicochemical stability during 90 days. Chitosan (CS) and polycaprolactone (PCL) nanoparticles loaded with AA had mean diameters of 314 and 303 nm and polydispersity indexes of 0.36 and 0.28, respectively. Both nanosystems provided protection against degradation of AA exposed to an oxidizing agent, compared to plain AA. Total degradation of AA was observed after 7, 20, and 480 min for plain AA, the CS nanoparticle formulation, and the PCL nanoparticle formulation, respectively. For zebrafish larvae, the LC50 values were 330.7, 57.4, and 179.6 mg/L for plain AA, the CS nanoparticle formulation, and the PCL nanoparticle formulation, respectively. In toxicity assays using AA at a concentration of 50 mg/L, both types of nanoparticles loaded with AA showed lower toxicity towards the development of the zebrafish, compared to plain AA at the same concentration. Although decreased activity of the enzyme acetylcholinesterase (AChE) did not affect the swimming behavior of zebrafish larvae in the groups evaluated, it may have been associated with the observed morphometric changes, such as curvature of the tail. CONCLUSIONS: This study showed that the use of nanosystems is promising for fish nutritional supplementation in aquaculture. In particular, PCL nanoparticles loaded with AA seemed to be most promising, due to higher protection against AA degradation, as well as lower toxicity to zebrafish, compared to the chitosan nanoparticles. The use of nanotechnology opens new perspectives for aquaculture, enabling the reduction of feed nutrient losses, leading to faster fish growth and improved sustainability of this activity.

Exploring the Toxicity, Lung Distribution, and Cellular Uptake of Rifampicin and Ascorbic Acid-Loaded Alginate Nanoparticles as Therapeutic Treatment of Lung Intracellular Infections.

Nanotechnology is a very promising technological tool to combat health problems associated with the loss of effectiveness of currently used antibiotics. Previously, we developed a formulation consisting of a chitosan and tween 80-decorated alginate nanocarrier that encapsulates rifampicin and the antioxidant ascorbic acid (RIF/ASC), intended for the treatment of respiratory intracellular infections. Here, we investigated the effects of RIF/ASC-loaded NPs on the respiratory mucus and the pulmonary surfactant. In addition, we evaluated their cytotoxicity for lung cells in vitro, and their biodistribution on rat lungs in vivo after their intratracheal administration. Findings herein demonstrated that RIF/ASC-loaded NPs display a favorable lung biocompatibility profile and a uniform distribution throughout lung lobules. RIF/ASC-loaded NPs were mainly uptaken by lung macrophages, their primary target. In summary, findings show that our novel designed RIF/ASC NPs could be a suitable system for antibiotic lung administration with promising perspectives for the treatment of pulmonary intracellular infections.

In Situ Synthesis of FeOCl in Hollow Dendritic Mesoporous Organosilicon for Ascorbic Acid-Enhanced and MR Imaging-Guided Chemodynamic Therapy in Neutral pH Conditions.

Chemodynamic therapy (CDT) based on the Fenton reaction is a promising strategy for nonlight cancer treatment. However, the traditional Fenton reaction is only efficient in strongly acidic conditions (pH = 2-4), resulting in the limited curative effect in a weakly acidic tumor microenvironment (TME). Herein, we first developed a simple in situ growth method to confine FeOCl nanosheets into hollow dendritic mesoporous organosilicon (H-DMOS) nanoparticles to obtain FeOCl@H-DMOS nanospheres. Ascorbic acid (AA) was then absorbed on the nanosystem as a H2O2 prodrug and, meanwhile, was used for the regeneration of Fentons reagent for Fe2+. Finally, poly(ethylene glycol) (PEG) was coated on FeOCl@H-DMOS-AA to enhance the permeability and retention (EPR) effect in tumor tissue. The as-fabricated FeOCl@H-DMOS-AA/PEG can generate a large amount of highly toxic hydroxyl radicals (•OH) by catalyzing H2O2 even in neutral pH conditions with the help of AA. As a result, the effect of CDT has been markedly enhanced by the increased amount of H2O2 and the efficient Fenton reaction in mild acidic TME, which can remove almost all of the tumors in mice. In addition, FeOCl also endows the nanosystem with T2-weighted MR imaging capability (r2 = 34.08 mM-1 s-1), thus realizing the imaging-guided cancer therapy. All in all, our study may contribute a new direction and may have a bright future for enhanced CDT with a neutral pH range.

A novel screening test to predict the developmental toxicity of drugs using human induced pluripotent stem cells.

In vitro human induced pluripotent stem (iPS) cells testing (iPST) to assess developmental toxicity, e.g., the induction of malformation or dysfunction, was developed by modifying a mouse embryonic stem cell test (EST), a promising animal-free approach. The iPST evaluates the potential risks and types of drugs-induced developmental toxicity in humans by assessing three endpoints: the inhibitory effects of the drug on the cardiac differentiation of iPS cells and on the proliferation/survival of iPS cells and human fibroblasts. In the present study, the potential developmental toxicity of drugs was divided into three classes (1: non-developmentally toxic, 2: weakly developmentally toxic and 3: strongly developmentally toxic) according to the EST criteria. In addition, the type of developmental toxicity of drugs was grouped into three types (1: non-effective, 2: embryotoxic [inducing growth retardation/dysfunction]/deadly or 3: teratogenic [inducing malformation]/deadly) by comparing the three endpoints. The present study was intended to validate the clinical predictability of the iPST. The traditionally developmentally toxic drugs of aminopterin, methotrexate, all-trans-retinoic acid, thalidomide, tetracycline, lithium, phenytoin, 5-fluorouracil, warfarin and valproate were designated as class 2 or 3 according to the EST criteria, and their developmental toxicity was type 3. The non-developmentally toxic drugs of ascorbic acid, saccharin, isoniazid and penicillin G were designated as class 1, and ascorbic acid, saccharin and isoniazid were grouped as type 1 while penicillin G was type 2 but not teratogenic. These results suggest that the iPST is useful for predicting the human developmental toxicity of drug candidates in a preclinical setting.

Labile iron affects pharmacological ascorbate-induced toxicity in osteosarcoma cell lines.

Vitamin C and iron are both important nutrients for humans and involved in several physiological processes. The biological activities of vitamin C and iron are based on their abilities to accept or donate electrons. Although vitamin C is well known as an excellent electron donor in physiological conditions, it also has pro-oxidant properties, especially with catalytic metal iron. Cancer cells have a higher iron requirement than normal cells, which allows pharmacological ascorbate to kill cancer cells selectively. In this study, we demonstrated that the levels of H2O2 in cells were significantly raised after treated with pharmacological ascorbate, and intracellular labile iron could increase pharmacological ascorbate-mediated oxidative stress by Fenton reaction. Catalytic metal iron plays opposite roles in and outside cells. Intracellular excess labile iron improved ascorbate-induced toxicity, while the excess labile iron in the medium abolished ascorbate-induced toxicity. Fe3+ and Fe2+ have the same effect on ascorbate-induced toxicity, but Fe3+ chelator deferoxamine (DFO) has a profound inhibition effect than Fe2+ chelator 2,2'-bipyridyl (BIP) on ascorbate-induced toxicity. The influence of intracellular labile iron and ascorbate on the ferritin expression may cause selective sensitivity in osteosarcoma cell lines on pharmacological ascorbate. High iron requirement of many cancer cells facilitates pharmacological ascorbate on cancer treatment. In addition, increasing iron content in tumour tissue may be effective strategies to improve the effects of pharmacological ascorbate.

The negative effect of magnetic nanoparticles with ascorbic acid on peritoneal macrophages.

Superparamagnetic iron oxide nanoparticles (SPIOn) are widely used as a contrast agent for cell labeling. Macrophages are the first line of defense of organisms in contact with nanoparticles after their administration. In this study we investigated the effect of silica-coated nanoparticles (γ-Fe2O3-SiO2) with or without modification by an ascorbic acid (γ-Fe2O3-SiO2-ASA), which is meant to act as an antioxidative agent on rat peritoneal macrophages. Both types of nanoparticles were phagocytosed by macrophages in large amounts as confirmed by transmission electron microscopy and Prusian blue staining, however they did not substantially affect the viability of exposed cells in monitored intervals. We further explored cytotoxic effects related to oxidative stress, which is frequently documented in cells exposed to nanoparticles. Our analysis of double strand breaks (DSBs) marker γH2AX showed an increased number of DSBs in cells treated with nanoparticles. Nanoparticle exposure further revealed only slight changes in the expression of genes involved in oxidative stress response. Lipid peroxidation, another marker of oxidative stress, was not significantly affirmed after nanoparticle exposure. Our data indicate that the effect of both types of nanoparticles on cell viability, or biomolecules such as DNA or lipids, was similar; however the presence of ascorbic acid, either bound to the nanoparticles or added to the cultivation medium, worsened the negative effect of nanoparticles in various tests performed. The attachment of ascorbic acid on the surface of nanoparticles did not have a protective effect against induced cytotoxicity, as expected.

The Interrelationship of Pharmacologic Ascorbate Induced Cell Death and Ferroptosis.

Pharmacologic ascorbate induced cell death and ferroptosis share common features such as iron dependency, production of ROS, lipid peroxidation, caspase independency and the possible involvement of autophagy. These observations lead us to hypothesize that ferroptosis may also be involved in cancer cell death due to pharmacologic ascorbate treatment. Thus cell death of HT-1080 cell line was induced by ferroptosis inducers and pharmacologic ascorbate then the mechanism of cell death was compared. The EC50 value of pharmacologic ascorbate on HT-1080 cell line was found to be 0.5 mM that is in the range of the most ascorbate sensitive cell lines. However either of the specific inhibitors of ferroptosis (ferrostatin-1 and liproxstatin-1) could not elevate the viability of pharmacologic ascorbate treated cells suggesting that ferroptosis was not involved in the pharmacologic ascorbate induced cell death. α-tocopherol that could effectively elevate the viability of erastin and RSL3 treated HT1080 cells failed to mitigate the cytotoxic effect of pharmacologic ascorbate further strengthened this assumption. Furthermore at lower concentrations (0.1-0.5 mM) ascorbate could avoid the effects of ferroptosis inducers. Our results indicate that pharmacologic ascorbate induced cytotoxicity and ferroptosis - albeit phenotypically they show similar traits - are governed by different mechanisms.

α-Tocopherol-ascorbic acid hybrid antioxidant based cationic amphiphile for gene delivery: Design, synthesis and transfection.

Natural antioxidants and vitamins have potential to protect biological systems from peroxidative damage induced by peroxyl radicals, α-tocopherol (Vitamin E, lipid soluble) and ascorbic acid (vitamin C, water soluble), well known natural antioxidant molecules. In the present study we described the synthesis and biological evaluation of hybrid of these two natural antioxidants with each other via ammonium di-ethylether linker, Toc-As in gene delivery. Two control cationic lipids N14-As and Toc-NOH are designed in such a way that one is with ascorbic acid moiety and no tocopherol moiety; another is with tocopherol moiety and no ascorbic acid moiety respectively. All the three cationic lipids can form self-assembled aggregates. The antioxidant efficiencies of the three lipids were compared with free ascorbic acid. The cationic lipids (Toc-As, N14-As and Toc-NOH) were formulated individually with a well-known fusogenic co-lipid DOPE and characterization studies such as DNA binding, heparin displacement, size, charge, circular dichroism were performed. The biological characterization studies such as cell viability assay and in vitro transfection studies were carried out with the above formulations in HepG2, Neuro-2a, CHO andHEK-293T cell lines. The three formulations showed their transfection efficiencies with highest in Toc-As, moderate inN14-As and least in Toc-NOH. Interestingly, the transfection efficiency observed with the antioxidant based conjugated lipid Toc-As is found to be approximately two and half fold higher than the commercially available lipofectamine 2000 at 4:1 charge ratio in Hep G2 cell lines. In the other cell lines studied the efficiency of Toc-As is found to be either higher or similarly active compared to lipofectamine 2000. The physicochemical characterization results show that Toc-As lipid is showing maximum antioxidant potency, strong binding with pDNA, least size and optimal zeta potential. It is also found to be least toxic in all the cell lines studied especially in Neuro-2a cell lines when compared to other two lipids. In summary, the designed antioxidant lipid can be exploited as a delivering system for treating ROS related diseases such as malignancy, brain stroke, etc.

Artefacts with ascorbate and other redox-active compounds in cell culture: epigenetic modifications, and cell killing due to hydrogen peroxide generation in cell culture media.

This letter discusses the various artefacts that can arise when ascorbate or other redox-active compounds are added to cell culture media.

Combination of urea-crosslinked hyaluronic acid and sodium ascorbyl phosphate for the treatment of inflammatory lung diseases: An in vitro study.

This in vitro study evaluated, for the first time, the safety and the biological activity of a novel urea-crosslinked hyaluronic acid component and sodium ascorbyl phosphate (HA-CL - SAP), singularly and/or in combination, intended for the treatment of inflammatory lung diseases. The aim was to understand if the combination HA-CL - SAP had an enhanced activity with respect to the combination native hyaluronic acid (HA) - SAP and the single SAP, HA and HA-CL components. Sample solutions displayed pH, osmolality and viscosity values suitable for lung delivery and showed to be not toxic on epithelial Calu-3 cells at the concentrations used in this study. The HA-CL - SAP displayed the most significant reduction in interleukin-6 (IL-6) and reactive oxygen species (ROS) levels, due to the combined action of HA-CL and SAP. Moreover, this combination showed improved cellular healing (wound closure) with respect to HA - SAP, SAP and HA, although at a lower rate than HA-CL alone. These preliminary results showed that the combination HA-CL - SAP could be suitable to reduce inflammation and oxidative stress in lung disorders like acute respiratory distress syndrome, asthma, emphysema and chronic obstructive pulmonary disease, where inflammation is prominent.

Evaluation of Uracil, Sodium Ascorbate, and Rosiglitazone as Promoters of Urinary Bladder Transitional Cell Carcinomas in Male Sprague-Dawley Rats.

The purpose of this study was to establish a 2-stage model of urinary bladder carcinogenesis in male Sprague-Dawley rats to identify tumor promoters. In phase 1 of the study, rats ( n = 170) were administered 100 mg/kg of the tumor initiator, N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN), twice weekly by oral gavage (po) for a period of 6 weeks. Phase 2 consisted of dividing rats into 4 groups ( n = 40 per group) and administering one of the following for 26 weeks to identify putative tumor promoters: (1) vehicle po, (2) 25 mg/kg/day rosiglitazone po, (3) 5% dietary sodium l-ascorbate, and (4) 3% dietary uracil. Rats were necropsied after 7.5 months, and urinary bladders were evaluated by histopathology. BBN/vehicle treatments induced the development of urothelial hyperplasia (83%) and papillomas (15%) but no transitional cell carcinomas (TCCs). Rosiglitazone increased the incidence and severity of papillomas (93%) and resulted in TCC in 10% of treated rats. Uracil was the most effective tumor promoter in our study and increased the incidence of papillomas (90%) and TCC (74%). Sodium ascorbate decreased the incidence of urothelial hyperplasia (63%) and did not increase the incidence of urothelial papillomas or TCC. These data confirm the capacity of our 2-stage model to identify urinary bladder tumor promoters.

The Effect of Ascorbic Acid over the Etoposide- and Temozolomide-Mediated Cytotoxicity in Glioblastoma Cell Culture: A Molecular Study.

AIM: Glioblastoma (GBM) is one of the lethal central nervous system tumors. One of the widely used chemical agents for the treatment of glioblastoma is temozolomide. It is an orally administered, deoxyribonucleic acid (DNA) alkylating agent. DNA alkylation triggers the death of tumor cells. However, some tumor cells are able to repair this type of DNA damage and thus lower the therapeutic effect of temozolomide. Laboratory and clinical studies indicate that temozolomide"s anticancer effects might be strengthened when combined with other chemotherapeutic agents like etoposide or antioxidant agents like ascorbic acid. In this study, we aimed to evaluate the cytotoxic and oxidative stress effects of ascorbic acid (1000 ?M), temozolomide (100 ?M) and etoposide (25 ?M) agents alone and in dual and triple combinations in a glioblastoma U87 MG cell culture. MATERIAL AND METHODS: The cytotoxic and oxidative stress effects were investigated by the 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) and liquid chromatography tandem-mass spectrometry (LC-MS/MS) analysis methods. RESULTS: Cytotoxicity tests showed that etoposide, temozolomide, "etoposide+ascorbic acid", "temozolomide+ascorbic acid", "temozolomide+etoposide" and "temozolomide+etoposide+ascorbic acid" combinations have anti-proliferative effects. The maximum anti-proliferation response was observed in the "temozolomide+etoposide+ascorbic acid"-added group. Similarly LCMS/ MS analyses showed that minimum oxidative DNA damage occurred in the "temozolomide+etoposide+ascorbic acid"-added group. CONCLUSION: Ascorbic acid decreases the cytotoxic and genotoxic effect of etoposide and etoposide-temozolomide combination but it has no meaningful effect on temozolomide"s toxicity.

Augmentation of intracellular iron using iron sucrose enhances the toxicity of pharmacological ascorbate in colon cancer cells.

Pharmacological doses (> 1mM) of ascorbate (a.k.a., vitamin C) have been shown to selectively kill cancer cells through a mechanism that is dependent on the generation of H2O2 at doses that are safely achievable in humans using intravenous administration. The process by which ascorbate oxidizes to form H2O2 is thought to be mediated catalytically by redox active metal ions such as iron (Fe). Because intravenous iron sucrose is often administered to colon cancer patients to help mitigate anemia, the current study assessed the ability of pharmacological ascorbate to kill colon cancer cells in the presence and absence of iron sucrose. In vitro survival assays showed that 10mM ascorbate exposure (2h) clonogenically inactivated 40-80% of exponentially growing colon cancer cell lines (HCT116 and HT29). When the H2O2 scavenging enzyme, catalase, was added to the media, or conditionally over-expressed using a doxycycline inducible vector, the toxicity of pharmacological ascorbate was significantly blunted. When colon cancer cells were treated in the presence or absence of 250µM iron sucrose, then rinsed, and treated with 10mM ascorbate, the cells demonstrated increased levels of labile iron that resulted in significantly increased clonogenic cell killing, compared to pharmacological ascorbate alone. Interestingly, when colon cancer cells were treated with iron sucrose for 1h and then 10mM ascorbate was added to the media in the continued presence of iron sucrose, there was no enhancement of toxicity despite similar increases in intracellular labile iron. The combination of iron chelators, deferoxamine and diethylenetriaminepentaacetic acid, significantly inhibited the toxicity of either ascorbate alone or ascorbate following iron sucrose. These observations support the hypothesis that increasing intracellular labile iron pools, using iron sucrose, can be used to increase the toxicity of pharmacological ascorbate in human colon cancer cells by a mechanism involving increased generation of H2O2.

Pharmacologic ascorbate induces neuroblastoma cell death by hydrogen peroxide mediated DNA damage and reduction in cancer cell glycolysis.

An ascorbate-mediated production of oxidative stress has been shown to retard tumor growth. Subsequent glycolysis inhibition has been suggested. Here, we further define the mechanisms relevant to this observation. Ascorbate was cytotoxic to human neuroblastoma cells through the production of H2O2, which led to ATP depletion, inhibited GAPDH, and non-apoptotic and non-autophagic cell death. The mechanism of cytotoxicity is different when PARP-dependent DNA repair machinery is active or inhibited. Ascorbate-generated H2O2 damaged DNA, activated PARP, depleted NAD+, and reduced glycolysis flux. NAD+ supplementation prevented ATP depletion and cell death, while treatment with a PARP inhibitor, olaparib, preserved NAD+ and ATP levels but led to increased DNA double-strand breakage and did not prevent ascorbate-induced cell death. These data indicate that in cells with an intact PARP-associated DNA repair system, ascorbate-induced cell death is caused by NAD+ and ATP depletion, while in the absence of PARP activation ascorbate-induced cell death still occurs but is a consequence of ROS-induced DNA damage. In a mouse xenograft model, intraperitoneal ascorbate inhibited neuroblastoma tumor growth and prolonged survival. Collectively, these data suggest that ascorbate could be effective in the treatment of glycolysis-dependent tumors. Also, in cancers that use alternative energy metabolism pathways, combining a PARP inhibitor with ascorbate treatment could be useful.

Baicalein prevents 6-OHDA/ascorbic acid-induced calcium-dependent dopaminergic neuronal cell death.

6-OHDA plus ascorbic acid (AA) has long been used to induce Parkinson's disease in rodents, while only 6-OHDA is commonly used to induce cell damage in cellular PD models. AA was believed to act as an anti-oxidant to prevent the degradation of 6-OHDA; however, some studies suggested that AA dramatically enhanced the selectivity and toxicity of 6-OHDA. To understand the mechanisms by which 6-OHDA/AA induces cell death, we established a 6-OHDA/AA cell toxicity model in human dopaminergic neuroblastoma SH-SY5Y cells. We confirmed that the toxicity of 6-OHDA was dramatically increased in the presence of AA, and the toxicity can be prevented by a flavonoid, baicalein. Mechanistically, our research reveals that 6-OHDA/AA induces cell death mainly through the interruption of intracellular calcium homeostasis, which leads to calpain activation and mitochondrial damage. Baicalein prevents 6-OHDA/AA-induced intracellular calcium elevation as well as consequent mitochondria damage. Taken together, our study confirms that 6-OHDA/AA is a more sensitive model for inducing neuronal lesion in vitro and reveals the central role of intracellular calcium in 6-OHDA/AA-induced cell death. Our studies further show that baicalein prevents 6-OHDA/AA-induced cell death by inhibiting intracellular calcium elevation.

Mechanism of melatonin protection against copper-ascorbate-induced oxidative damage in vitro through isothermal titration calorimetry.

AIMS: Involvement of oxidative stress in cardiovascular diseases is well established. Melatonin's role as an antioxidant and free radical scavenger via its receptor dependent and receptor independent pathways is well known. The aim of this study is to identify and elaborate upon a third mechanism by which melatonin is able to abrogate oxidative stress. MAIN METHODS: Oxidative stress was induced in vitro, by copper (0.2mM)-ascorbate (1mM) in isolated goat heart mitochondria, cytosol and peroxisomes and they were co-incubated with graded doses of melatonin. Similar experiments in a cell-free chemical system involving two pure antioxidant enzymes, Cu-Zn superoxide dismutase and catalase was also carried out. Biochemical changes in activity of these antioxidant enzymes were analysed. Isothermal titration calorimetric studies with pure Cu-Zn superoxide dismutase and catalase were also carried out. KEY FINDINGS: Incubation with copper-ascorbate led to alteration in activity of Cu-Zn superoxide dismutase and catalase which were found to be protected upon co-incubation with melatonin (80µM for catalase and 1µM for others). Results of isothermal titration calorimetric studies with pure Cu-Zn superoxide dismutase and catalase along with different combinations of copper chloride, ascorbic acid and melatonin suggest that when melatonin is present in the reaction medium along with copper-ascorbate, it restrains the copper-ascorbate molecules by binding with them physically along with scavenging the free radicals generated by them. SIGNIFICANCE: The present study suggests that possibly, binding of melatonin with antioxidant enzymes masks the vulnerable sites of these antioxidant enzymes, thus preventing oxidative damage by copper-ascorbate molecules.

Co-delivery of docetaxel and palmitoyl ascorbate by liposome for enhanced synergistic antitumor efficacy.

Palmitoyl ascorbate (PA) as an antioxidant has the potential for the treatment of cancer. In the present study, a nanocarrier system was developed for co-delivery of docetaxel (DOC) with palmitoyl ascorbate and the therapeutic efficacy of a combination drug regimen was investigated. For this purpose, different ratios of docetaxel and palmitoyl ascorbate were co-encapsulated in a liposome and they all showed high encapsulation efficiency. The average diameters of the liposomes ranged from 140 to 170 nm. Negative zeta potential values were observed for all systems, ranged from -40 mV to -56 mV. Studies on drug release and cellular uptake of the co-delivery system demonstrated that both drugs were effectively taken up by the cells and released slowly. Moreover, the liposome loading drugs with DOC/PA concentration ratio of 1:200 showed the highest anti-tumor activity to three different types of tumor cells. The higher in vivo therapeutic efficacy with lower systemic toxicity of the DOC-PA200-LPs was also verified by the H22 tumor bearing mice model. Our results showed that such co-loaded delivery systems could serve as a promising therapeutic approach to improve clinical outcomes against hepatic carcinoma.

Antioxidant defense system parameters in isolated fish hepatocytes exposed to bisphenol A - Effect of vitamin C.

In this study, isolated hepatocytes of pearl mullet (Alburnus tarichi) were exposed to bisphenol A (BPA) at concentrations of 25, 50, 100, and 200 µM for 24 h. Moreover, an in vitro antioxidant concentration of vitamin C (50 µM) was administrated to the culture medium along with the BPA exposures. Next, the antioxidant defense system parameters were analyzed. According to the results, the highest concentration of BPA (200 µM) proved to be severely toxic for the cells. The increased activities of superoxide dismutase (SOD) and glutathione-S-transferase (GST), the fluctuated activities of glutathione peroxidase (GPx), and the decreased content of reduced glutathione (GSH) were compared to the control group after the BPA exposures. Vitamin C co-administration was found to cause further increases in the SOD, GPx, and GST activities in some of the experimental groups and vitamin C could not restore the GSH content. Malondialdehyde (MDA) levels were observed to be unaffected in all exposure groups. These results show that BPA causes alterations in the antioxidant defenses of the isolated fish hepatocytes. In addition, vitamin C co-administration along with BPA was found to be non-protective against BPA-induced oxidative stress, consequently, aggravated a negative BPA impact.

Curcumin has protective and antioxidant properties on bull spermatozoa subjected to induced oxidative stress.

Over the past decades, there has been an emphasis on assessment of the use of natural compounds in the prevention or repair of oxidative injury to spermatozoa. Curcumin (CUR) is a natural phenol with powerful antioxidant properties. The aim of the present study was to examine if CUR could reverse reactive oxygen species (ROS)-mediated alterations to the motility, viability and intracellular antioxidant profile of bull spermatozoa subjected to a prooxidant (i.e., ferrous ascorbate - FeAA). Spermatozoa were washed from recently collected semen samples, suspended in 2.9% sodium citrate and subjected to CUR treatment (5, 10, 25 and 50µmol/L) in the presence or absence of FeAA (150µmol/L FeSO4 and 750µmol/L ascorbic acid) during a 6h in vitro culture. Spermatozoa motility characteristics were assessed using the SpermVision computer-aided spermatozoa analysis (CASA) system. Cell viability was examined with the metabolic activity (MTT) assay, ROS generation was quantified using luminometry and the nitroblue-tetrazolium (NBT) test was used to quantify the intracellular superoxide formation. Cell lysates were prepared at the end of the culture to assess the intracellular activity of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) as well as the concentrations of glutathione (GSH) and malondialdehyde (MDA). Treatment with FeAA led to a reduced spermatozoa motility (P<0.001), viability (P<0.001) and decreased the antioxidant characteristics of the samples (P<0.001) but increased the ROS generation (P<0.001), superoxide production (P<0.001) and lipid peroxidation (P<0.001). The CUR treatment led to a preservation of spermatozoa motion (P<0.001), mitochondrial activity (P<0.001) and antioxidant characteristics (P<0.05 with SOD and GSH; P<0.01 with CAT and GPx), revealing the concentration range of 25-50µmol/L CUR to be the most effective for sustaining spermatozoa viability. Data from the present study suggest that CUR exhibits significant protective and ROS-scavenging characteristics which may prevent oxidative insults to spermatozoa and thus preserve the functional activity of male gametes.