Sodium fusidate loaded apatitic calcium phosphates: Adsorption behavior, release kinetics, antibacterial efficacy, and cytotoxicity assessment.

The present work reports the adsorption, release, antibacterial properties, and in vitro cytotoxicity of sodium fusidate (SF) associated with a carbonated calcium phosphate bone cement. The adsorption study of SF on cement powder compared to stoichiometric hydroxyapatite and nanocrystalline carbonated apatite was investigated to understand the interaction between this antibiotic and the calcium phosphate phases involved in the cement formulation and setting reaction. The adsorption data revealed a fast kinetic process. However, the evolution of the amount of adsorbed SF was well described by a Freundlich-type isotherm characterized by a low adsorption capacity of the materials toward the SF molecule. The in vitro release results indicated a prolonged and controlled SF release for up to 34 days. The SF amounts eluted daily were at a therapeutic level (0.5-2 mg/L) and close to the antibiotic minimum inhibitory concentration (0.1-0.9 mg/L). Furthermore, the release data fitting and modeling suggested that the drug release occurred mainly by a diffusion mechanism. The antibacterial activity showed the effectiveness of SF released from the formulated cements against Staphylococcus aureus. Furthermore, the biological in vitro study demonstrated that the tested cements didn't show any cytotoxicity towards human peripheral blood mononuclear cells and did not significantly induce inflammation markers like IL-8.

Immunogenicity Risk Assessment of Process-Related Impurities in An Engineered T Cell Receptor Cellular Product.

Cell therapies such as genetically modified T cells have emerged as a promising and viable treatment for hematologic cancers and are being aggressively pursued for a wide range of diseases and conditions that were previously difficult to treat or had no cure. The process development requires genetic modifications to T cells to express a receptor (engineered T cell receptor (eTCR)) of specific binding qualities to the desired target. Protein reagents utilized during the cell therapy manufacturing process, to facilitate these genetic modifications, are often present as process-related impurities at residual levels in the final drug product and can represent a potential immunogenicity risk upon infusion. This manuscript presents a framework for the qualification of an assay for assessing the immunogenicity risk of AA6 and Cas9 residuals. The same framework applies for other residuals; however, AAV6 and Cas9 were selected as they were residuals from the manufacturing of an engineered T cell receptor cellular product in development. The manuscript: 1) elucidates theoretical risks, 2) summarizes analytical data collected during process development, 3) describes the qualification of an in vitro human PBMC cytokine release assay to assess immunogenicity risk from cellular product associated process residuals; 4) identifies a multiplexed inflammatory innate and adaptive cytokine panel with pre-defined criteria using relevant positive controls; and 5) discusses qualification challenges and potential solutions for establishing meaningful thresholds. The assessment is not only relevant to establishing safe exposure levels of these residuals but also in guiding risk assessment and CMC strategy during the conduct of clinical trials.

Assessment of reproductive toxicity and genotoxicity of Aconiti Lateralis Radix Praeparata and its processed products in male mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Aconiti Lateralis Radix Praeparata (Chinese name: Fuzi), the root of Aconitum carmichaelii Debx., is a representative medicine for restoring yang and rescuing patient from collapse. However, less studies had been reported on the reproductive toxicity and genotoxicity of Fuzi. According to the principle of reducing toxicity and preserving efficiency, only processed products of Fuzi are commonly applied in clinic, including Baifupian, Heishunpian and Danfupian. However, whether processing could alleviate the reproductive toxicity and genotoxicity of Fuzi had not been revealed. AIM OF THE STUDY: To assess the effect and possible mechanism of Fuzi and its processed products on reproductive toxicity and genotoxicity in male mice. MATERIALS AND METHODS: Aqueous extracts of Fuzi and its processed products (Baifupian, Heishunpian and Danfupian, 5.85 g/kg) were administrated by gavage once daily for fourteen consecutive days. The reproductive toxicity was evaluated by testis weight, testis ratio, testis histopathology, sperm count, sperm viability rate and sperm deformity rate. The genotoxicity was evaluated by comet assay and micronucleus test in sperm, peripheral blood cell and bone marrow cell. Possible mechanisms of attenuating toxicity by processing were analyzed by detecting the level of testosterone, superoxide dismutase (SOD), glutathione (GSH), malondialdehyde (MDA) and catalase (CAT). RESULTS: Fuzi significantly caused different degrees of reproductive toxicity and genotoxicity, specifically reducing the weight and testicular coefficient of testis, causing obvious pathological changes in testicular tissue, reducing sperm count and sperm viability rate, increasing sperm deformity rate and DNA damage in sperm/peripheral blood cells/bone marrow cells. Moreover, Fuzi decreased the level of testosterone, SOD, GSH and CAT, while increased the level of MDA in serum. Notably, the reproductive toxicity and genotoxicity induced by the processed products, especially Heishunpian and Danfupian, were significantly lowered compared to Fuzi. Processing could increase the level of testosterone, SOD, GSH, CAT and decrease the level of MDA compared to Fuzi. CONCLUSION: Fuzi and its processed products had reproductive toxicity and genotoxicity, but the toxicity of processed products was significantly weakened compared to Fuzi. The protective mechanism of processing to reduce the toxicity of Fuzi might be related to increasing the level of testosterone and decreasing oxidative stress.

Genotoxic risk assessment and mechanism of DNA damage induced by phthalates and their metabolites in human peripheral blood mononuclear cells.

The human genome is persistently exposed to damage caused by xenobiotics, therefore the assessment of genotoxicity of substances having a direct contact with humans is of importance. Phthalates are commonly used in industrial applications. Widespread exposure to phthalates has been evidenced by their presence in human body fluids. We have assessed the genotoxic potential of selected phthalates and mechanism of their action in human peripheral blood mononuclear cells (PBMCs). Studied cells were incubated with di-n-butyl phthalate (DBP), butylbenzyl phthalate (BBP) and their metabolites: mono-n-butylphthalate (MBP), mono-benzylphthalate (MBzP) in the concentrations range of 0.1-10 µg/mL for 24 h. Analyzed compounds induced DNA single and double strand-breaks (DBP and BBP ≥ 0.5 µg/mL, MBP and MBzP ≥ 1 µg/mL) and more strongly oxidized purines than pyrimidines. None of the compounds examined was capable of creating adducts with DNA. All studied phthalates caused an increase of total ROS level, while hydroxyl radical was generated mostly by DBP and BBP. PBMCs exposed to DBP and BBP could not completely repair DNA strand-breaks during 120 min of postincubation, in opposite to damage caused by their metabolites, MBP and MBzP. We have concluded that parent phthalates: DBP and BBP caused more pronounced DNA damage compared to their metabolites.

Population Pharmacokinetics of Intracellular 5-Fluorouridine 5'-Triphosphate and its Relationship with Hand-and-Foot Syndrome in Patients Treated with Capecitabine.

Capecitabine is an oral pro-drug of 5-fluorouracil. Patients with solid tumours who are treated with capecitabine may develop hand-and-foot syndrome (HFS) as side effect. This might be a result of accumulation of intracellular metabolites. We characterised the pharmacokinetics (PK) of 5-fluorouridine 5'-triphosphate (FUTP) in peripheral blood mononuclear cells (PBMCs) and assessed the relationship between exposure to capecitabine or its metabolites and the development of HFS. Plasma and intracellular capecitabine PK data and ordered categorical HFS data was available. A previously developed model describing the PK of capecitabine and metabolites was extended to describe the intracellular FUTP concentrations. Subsequently, a continuous-time Markov model was developed to describe the development of HFS during treatment with capecitabine. The influences of capecitabine and metabolite concentrations on the development of HFS were evaluated. The PK of intracellular FUTP was described by an one-compartment model with first-order elimination (ke,FUTP was 0.028 h-1 (95% confidence interval 0.022-0.039)) where the FUTP influx rate was proportional to the 5-FU plasma concentrations. The predicted individual intracellular FUTP concentration was identified as a significant predictor for the development and severity of HFS. Simulations demonstrated a clear exposure-response relationship. The intracellular FUTP concentrations were successfully described and a significant relationship between these intracellular concentrations and the development and severity of HFS was identified. This model can be used to simulate future dosing regimens and thereby optimise treatment with capecitabine.

Safety and bioactivity assessment of aqueous extract of Thai Henna (Lawsonia inermis Linn.) Leaf.

The worldwide demand for a natural dye by the cosmetic and food industry has recently gained interest. To provide scientific data supporting the usage of Thai henna leaf as a natural colorant, the phytochemical constituents, safety, and bioactivity of aqueous extract of the henna leaf by autoclave (HAE) and hot water (HHE) were determined. HAE contained a higher amount of total phenolic and flavonoid contents than HHE. The major constituents in both extracts were ferulic acid, gallic acid, and luteolin. The extracts displayed no marked mutagenic activity both in vitro and in vivo mammalian-like biotransformation. HAE and HHE also exhibited non-cytotoxicity to human immortalized keratinocyte cells (HaCaT), peripheral blood mononuclear cells (PBMCs), and murine macrophage RAW 264.7 cell line with IC20 and IC50 > 200 µg/ml. The extracts exhibited antioxidant and anti-inflammatory activity as evidenced by significant scavenging of ABTS and DPPH radicals and decreasing NO levels in LPS-induced RAW 264.7 cells. The antioxidant and anti-inflammatory properties of the extracts might be attributed to their phenolic and flavonoid contents. In conclusion, the traditional use of henna as a natural dye appears not to exert toxic effects and seems biosecure. Regarding safety, antioxidant, and anti-inflammatory properties, the aqueous extract of Thai henna leaf might thus serve as a readily available source for utilization in commercial health industries.

Comparative Assessment of Leukocyte Infiltrate Composition in the Uterine and Vaginal Tissues in Rats with Experimental Endomyometritis Treated with Interferon-γ at Different Times of the Day.

The cell composition of leukocyte infiltrates in the endometrium, myometrium, and vaginal walls was studied in Wistar rats with modeled chronic endomyometritis after administration of IFNγ (0.1 µg/100 g body weight) in different daily regimens (10.00 or 20.00). Morning injections of this cytokine ameliorated inflammatory infiltration of the uterine wall and vagina, but increased the content of neutrophils in the endometrium. Evening cytokine injections reduced neutrophilic infiltration, enhanced mononuclear infiltration, and had no effect on plasmacytic infiltration of the uterine and vaginal walls. In the vaginal wall, both IFNγ administration schedules decreased neutrophil content. The data indicate the necessity to take into account the circadian rhythms in IFN therapy.

Assessment of Structural and Functional Comparability of Biosimilar Products: Trastuzumab as a Case Study.

BACKGROUND: Biotherapeutics are protein products generated using recombinant DNA technology and manufactured in prokaryotic or eukaryotic cells. It is often said that "the process is the product" and thereby the effect of the manufacturing process is etched on the final product in the form of its heterogeneity. For any biotherapeutic, the acceptable range of the critical quality attributes is defined based on the expected impact of a specific variation on the product stability, safety, and efficacy. For a biosimilar to receive regulatory approval, the manufacturer must demonstrate analytical and clinical comparability with the originator product. As this is mandatory, every biosimilar manufacturer performs this exercise for each biosimilar product under development. However, few reports of thorough evaluation of the quality of biosimilar products are available in the literature. OBJECTIVE: We examined the structural and functional comparability of biosimilars of trastuzumab, a humanized monoclonal antibody biotherapeutic. The originator product, Herclon (Roche), was compared with four marketed biosimilars: Trasturel from Reliance Life Sciences, Canmab from Biocon, Vivitra from Zydus Ingenia, Hertraz from Mylan. METHODS: Structural comparability was established using mass spectrometry and spectroscopic techniques such as Fourier transform infrared spectroscopy, differential light scattering, circular dichroism, and fluorescence spectroscopy. Stability was compared by performing accelerated thermal stress studies. Functional comparability was established via surface plasmon resonance and biological assays such as antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity. RESULTS: With respect to comparability, one biosimilar exhibited significant differences in multiple attributes, such as lower percentage of monomer content and main charge variant species, lower percentage of aglycosylated glycoform G0, and lower estimated potency values. CONCLUSIONS: Overall, the results indicated general similarity with respect to structure and function, but we found variations with respect to size heterogeneity, charge heterogeneity, and glycosylation pattern in each of the biosimilars.

Pharmacodynamic assessment of cell-bound natalizumab on PBMC samples stored in liquid nitrogen.

Natalizumab is a monoclonal IgG4 antibody used for treatment of relapsing remitting MS. Natalizumab interferes with lymphocyte migration by blocking alpha-4 integrin (CD49d). Saturation levels of alpha-4 integrin on circulating T cells by natalizumab have been associated with clinical effectiveness of therapy. However, in most cases, measurements have been carried out using freshly isolated PBMCs. The aim of this study was to set up and evaluate a method to measure relative levels of cell-bound natalizumab using frozen PBMC samples. A new method was set up to measure cell-bound natalizumab by flow cytometry on T cell subsets using fully saturated cells as a 100% reference. A comparison was made between spike samples and samples of natalizumab-treated MS patients freshly isolated and stored in liquid nitrogen. Cell-bound natalizumab could be measured (using an anti-IgG4 antibody) on cells stored in liquid nitrogen. Natalizumab was found to slowly dissociate from the cells during isolation and subsequent sample work-up. This dissociation was more pronounced for monovalent natalizumab resulting from Fab arm exchange (the predominant isoform in patients) than bivalent natalizumab straight from the vial. We established a correction factor to account for this phenomenon. The resulting method has good accuracy compared to assessing fresh cells. The inter-assay precision (%CV) is ca. 12% using frozen cells. In conclusion, we established a method to assess relative levels of cell-bound natalizumab on cells obtained from frozen PBMC samples.

In vitro and in vivo assessment of the proresolutive and antiresorptive actions of resolvin D1: relevance to arthritis.

BACKGROUND: Resolvin D1 (RvD1), an important member of resolvins, exerts a wide spectrum of biological effects, including resolution of inflammation, tissue repair, and preservation of cell viability. The aim of the present study is to investigate the anti-arthritic potential and clarify the bone protective actions of RvD1 in vitro and in vivo. METHODS: RAW264.7 cells were treated with 50 ng/ml LPS for 72 h in the presence or absence of RvD1 (0-500 nM). Primary human monocytes were treated with M-CSF + RANKL for 14 days ± RvD1 (0-500 nM) with or without siRNA against RvD1 receptor FPR2. Expressions of inflammatory mediators, degrading enzymes, osteoclasts (OC) formation, and bone resorption were analyzed. The therapeutic effect of RvD1 (0-1000 ng) was carried out in murine collagen antibody-induced arthritis. Arthritis scoring, joint histology, and inflammatory and bone turnover markers were measured. RESULTS: RvD1 is not toxic and inhibits OC differentiation and activation. It decreases bone resorption, as assessed by the inhibition of TRAP and cathepsin K expression, hydroxyapatite matrix resorption, and bone loss. In addition, RvD1 reduces TNF-α, IL-1ß, IFN-γ, PGE2, and RANK and concurrently enhances IL-10 in OC. Moreover, in arthritic mice, RvD1 alleviates clinical score, paw inflammation, and bone and joint destructions. Besides, RvD1 reduces inflammatory mediators and markedly decreases serum markers of bone and cartilage turnover. CONCLUSION: Our results provide additional evidence that RvD1 plays a key role in preventing bone resorption and other pathophysiological changes associated with arthritis. The study highlights the clinical relevance of RvD1 as a potential compound for the treatment of inflammatory arthritis and related bone disorders.

An Assessment of the Ability of Submicron- and Micron-Size Silicone Oil Droplets in Dropped Prefillable Syringes to Invoke Early- and Late-Stage Immune Responses.

A number of biopharmaceuticals are available as lyophilized formulations along with a prefilled syringe (PFS) containing water for injection (WFI). Submicron- and micron-size droplets of lubricating silicone oil (SO) applied to the inner surface of the PFS barrel might migrate into the WFI, to which protein pharmaceuticals can adsorb, potentially inducing an immune response. In the present study, we subjected siliconized cyclo-olefin polymer PFSs filled with WFI to dropping stress to simulate actual shipping conditions as well as evaluated the risk associated with the released SO droplets. The results confirmed the undesirable effects of SO on therapeutic proteins, including adsorption to SO droplets and increased secretion of several innate cytokines from human peripheral blood mononuclear cells of a small donor panel. Assessment of immunogenicity in vivo using BALB/c mice revealed a slight increase in the plasma concentrations of antidrug antibodies over 21 days in response to SO-containing antibody samples compared to the absence of SO. These results indicate that SO droplets form complexes with pharmaceutical proteins that can potentially invoke early- and late-stage immune responses. Therefore, the use of SO-free cyclo-olefin polymer PFSs as primary containers for WFI could contribute to the enhanced safety of reconstituted biopharmaceuticals.

In vitro toxicity assessment of zinc and nickel ferrite nanoparticles in human erythrocytes and peripheral blood mononuclear cell.

Ferrite nanoparticles (NPs) have gained attention in biomedicine due to their many potential applications, such as targeted drug delivery, their use as contrast agents for magnetic resonance imaging and oncological treatments. The information about the risk effects of ferrite NPs in human blood cells is, however, scarce. To assess their potential toxicity, in vitro studies were carried out with magnetite and zinc, nickel and nickel­zinc ferrites NPs at different concentrations (50, 100 and 200 µg·ml-1). The toxicity of the ferrite NPs was evaluated in humans by determining red blood hemolysis, by measuring the content of total proteins, and by assaying catalase and glutathione-S-transferase activities. Our results show that nickel­zinc ferrite lead to hemolysis, and that magnetite, zinc and nickel­zinc ferrites increase glutathione-S-transferase activity. No significant changes in human peripheral blood mononuclear cells viability were observed after the treatment with the four different ferrite NPs in vitro.

An in vitro assessment for evaluating the efficiency of ß-d-mannuronic acid (M2000) in myelodysplastic syndrome.

ß-d-Mannuronic acid (M2000), a novel non-steroidal anti-inflammatory drug (NSAID) with immunosuppressive properties, has been previously shown to exhibit potential therapeutic effects on autoimmune diseases. Immunosuppression therapy has been a standard approach for myelodysplastic syndrome (MDS) for many years. We evaluated the effect of M2000 on isolated peripheral blood mononuclear cells (PBMCs) from patients with MDS. The PBMCs were isolated from 13 patients with MDS and 13 normal donors. The cells were then treated with low, moderate, and high doses of M2000 and diclofenac as a control group. The level of interleukin (IL)-6, tumor necrosis factor alpha (TNF-α), IL-3, granulocyte colony-stimulating factor (G-CSF) gene expression and the serum level of IL-6 and TNF-α production were evaluated by real-time polymerase chain reaction and enzyme-linked immunosorbent assay methods, respectively. Our findings indicated a significant reduction in the production of IL-6 and TNF-α as inflammatory cytokines. Furthermore, the level of G-CSF gene expression was significantly increased. In conclusion, M2000, a newly designed NSAID, has a remarkable effect on isolated PBMC in patients with MDS, which might bring a potential hope for its oral administrations in these patients.

Homogeneous BTK Occupancy Assay for Pharmacodynamic Assessment of Tirabrutinib (GS-4059/ONO-4059) Target Engagement.

Bruton's tyrosine kinase (BTK) is a clinically validated target for B-cell leukemias and lymphomas with FDA-approved small-molecule inhibitors ibrutinib and acalabrutinib. Tirabrutinib (GS-4059/ONO-4059, Gilead Sciences, Inc., Foster City, CA) is a second-generation, potent, selective, irreversible BTK inhibitor in clinical development for lymphoid malignancies, including chronic lymphocytic leukemia (CLL) and diffuse large B-cell lymphoma (DLBCL). An accurate pharmacodynamic assay to assess tirabrutinib target coverage in phase 1/2 clinical studies will inform dose and schedule selection for advanced clinical evaluation. We developed a novel duplex homogeneous BTK occupancy assay based on time-resolved fluorescence resonance energy transfer (TR-FRET) to measure free and total BTK levels in a multiplexed format. The dual-wavelength emission property of terbium-conjugated anti-BTK antibody served as the energy donor for two fluorescent energy acceptors with distinct excitation and emission spectra. The assay was characterized and qualified using full-length purified recombinant human BTK protein and peripheral blood mononuclear cells derived from healthy volunteers and patients with CLL. We demonstrated assay utility using cells derived from lymph node and bone marrow samples from patients with CLL and DLBCL. Our TR-FRET-based BTK occupancy assay provides accurate, quantitative assessment of BTK occupancy in the clinical trial program for tirabrutinib and is in use in ongoing clinical studies.

Critical issues in genotoxicity assessment of TiO2 nanoparticles by human peripheral blood mononuclear cells.

In the last years, a number of in vitro studies have been performed to assess the genotoxic activity of titanium dioxide (TiO2 ). To resolve the contradictory results, in this study, we investigated the genotoxic activity of commercial TiO2 nanoparticles (NPs) and microparticles of different forms (anatase, rutile and mix of both). We evaluated micronucleus formation in stimulated lymphocytes, as well as DNA strand breaks and 8-oxo-7,8-dihydro-2'-deoxyguanosine in peripheral blood mononuclear cells (PBMCs), a mixed population of lymphocytes and monocytes. Different responses to TiO2 exposure were obtained depending on the assay. Both TiO2 NPs and microparticles and all the crystalline forms elicited a significant increase in 8-oxo-7,8-dihydro-2'-deoxyguanosine and DNA strand breaks in the whole PBMC population, without a concurrent increase of micronuclei in proliferating lymphocytes. The distribution of DNA damage in PBMCs, detected by the comet assay, that measures DNA damage at level of single cells, indicated the presence of a more susceptible cell subpopulation. The measurement of side scatter signals by flow cytometry highlighted the preferential physical interaction of TiO2 particles with monocytes that also displayed higher reactive oxygen species generation, providing a mechanistic explanation for the different responses observed in genotoxicity assays with PBMCs and lymphocytes. This study confirmed the suitability of human PBMCs as multi-cell model to investigate NP-induced DNA damage, but suggested some caution in the use of stimulated lymphocytes for the assessment of NP clastogenicity.

Assessment of polycyclic aromatic hydrocarbon exposure, lung function, systemic inflammation, and genotoxicity in peripheral blood mononuclear cells from firefighters before and after a work shift.

Firefighting is regarded as possibly carcinogenic, although there are few mechanistic studies on genotoxicity in humans. We investigated exposure to polycyclic aromatic hydrocarbons (PAH), lung function, systemic inflammation and genotoxicity in peripheral blood mononuclear cells (PBMC) of 22 professional firefighters before and after a 24-h work shift. Exposure was assessed by measurements of particulate matter (PM), PAH levels on skin, urinary 1-hydroxypyrene (1-OHP) and self-reported participation in fire extinguishing activities. PM measurements indicated that use of personal protective equipment (PPE) effectively prevented inhalation exposure, but exposure to PM occurred when the environment was perceived as safe and the self-contained breathing apparatuses were removed. The level of PAH on skin and urinary 1-OHP concentration were similar before and after the work shift, irrespective of self-reported participation in fire extinction activities. Post-shift, the subjects had reduced levels of oxidatively damaged DNA in PBMC, and increased plasma concentration of vascular cell adhesion molecule 1 (VCAM-1). The subjects reporting participation in fire extinction activities during the work shift had a slightly decreased lung function, increased plasma concentration of VCAM-1, and reduced levels of oxidatively damaged DNA in PBMC. Our results suggest that the firefighters were not exposed to PM while using PPE, but exposure occurred when PPE was not used. The work shift was not associated with increased levels of genotoxicity. Increased levels of VCAM-1 in plasma were observed. Environ. Mol. Mutagen. 59:539-548, 2018. © 2018 Wiley Periodicals, Inc.

Toxicity assessment of mycotoxins extracted from contaminated commercial dog pelleted feed on canine blood mononuclear cells.

Raw ingredients of pet food are often contaminated with mycotoxins. This is a serious health problem to pets and causes emotional and economical stress to the pet owners. The aim of this study was to determine the immunotoxicity of the most common mycotoxins (aflatoxin, fumonisin, ochratoxin A and zearalenone) by examining 20 samples of extruded dry dog food found on the South African market [10 samples from standard grocery store lines (SB), 10 from premium veterinarian lines (PB)]. Pelleted dog food was subjected to extraction protocols optimized for the above mentioned mycotoxins. Dog lymphocytes were treated with the extracts (24 h incubation and final concentration 40 µg/ml) to determine cell viability, mitochondrial function, oxidative stress, and markers of cell death using spectrophotometry, luminometry and flow cytometry. Malondialdehyde, a marker of oxidative stress showed no significant difference between SB and PB, however, GSH was significantly depleted in SB extract treatments. Markers of apoptosis (phosphatidylserine externalization) and necrosis (propidium iodide incorporation) were elevated in both food lines when compared to untreated control cells, interestingly SB extracts were significantly higher than PB. We also observed decreased ATP levels and increased mitochondrial depolarization in cells treated with both lines of feed with SB showing the greatest differences when compared to the control. This study provides evidence that irrespective of price, quality or marketing channels, pet foods present a high risk of mycotoxin contamination. Though in this study PB fared better than SB in regards to cell toxicity, there is a multitude of other factors that need to be studied which may have an influence on other negative outcomes.

Biogenic synthesis of gold nanoparticles from Terminalia arjuna bark extract: assessment of safety aspects and neuroprotective potential via antioxidant, anticholinesterase, and antiamyloidogenic effects.

The development of neuroprotective drugs through eco-friendly production routes is a major challenge for current pharmacology. The present study was carried out to synthesize gold nanoparticles (AuNPs) through biogenic route using ethanolic bark extract of Terminalia arjuna, a plant of high interest in Asian traditional medicine, and to evaluate its neuroprotective effects. The synthesized AuNPs were characterized by UV-Vis spectroscopy, FTIR spectroscopy, XRD, FESEM, EDX, HRTEM, DLS, and zeta potential analyses. UV-Vis spectroscopy showed a characteristics SPR absorption band at 536 nm specific for AuNPs. XRD, TEM, and FESEM analyses revealed the formation of face-centered cubic crystalline, spherical and triangular shaped AuNPs, with size ranging between 20 and 50 nm. DLS and ZP analysis illustrated that the average size of AuNPs was 30 nm, which was found to be stable at 45 mv. The neuroprotective potential of AuNPs was evaluated by assessing its antioxidant, cholinesterase inhibitory, and antiamyloidogenic activities. AuNPs showed dose-dependant inhibition of acetylcholinesterase and butyrylcholinesterase with IC50 value of 4.25 ± 0.02 and 5.05 ± 0.02 µg/ml, respectively. In vitro antioxidant assays illustrated that AuNPs exhibited the highest reducing power and DPPH radical scavenging activity. In addition, AuNPs also efficiently suppressed the fibrillation of Aß and destabilized the preformed mature fibrils. Results of toxicity studies in PBMC and adult zebra fish illustrated that AuNPs are non-toxic and biocompatible. Overall, our results highlighted the AuNPs promising potential in terms of antioxidant, anticholinesterase, antiamyloidogenic effects, and non-lethality allowing us to propose these nanomaterials as a suitable candidate for the development of drugs helpful in the treatment of neurodegenerative disorders like Alzheimer's disease. Graphical abstract ᅟ.

Assessment of DNA damage in blood lymphocytes of bakery workers by comet assay.

The comet assay is widely used in screening and identification of genotoxic effects of different substances on people in either their working or living environment. Exposure to fuel smoke leads to DNA damage and ultimately different types of cancer. Using a comet assay, the present study aimed to assess peripheral blood lymphocyte DNA damage in people working in bakeries using natural gas, kerosene, diesel, or firewood for fuel compared to those in the control group. The subjects of this study were 55 people in total who were divided into four experimental groups, each of which comprised of 11 members (based on the type of fuel used), and one control group comprised of 11 members. Using CometScore, the subjects' peripheral blood lymphocytes were examined for DNA damage. All bakers, that is, experimental subjects, showed significantly greater peripheral blood lymphocyte DNA damage compared to the individuals in the control group. There was greater peripheral blood lymphocyte DNA damage in bakers who had been using firewood for fuel compared to those using other types of fuel to such an extent that tail moments (µm) for firewood-burning bakers was 4.40 ± 1.98 versus 1.35 ± 0.84 for natural gas, 1.85 ± 1.33 for diesel, and 2.19 ± 2.20 for kerosene. The results indicated that burning firewood is the greatest inducer of peripheral blood lymphocytes DNA damage in bakers. Nonetheless, there was no significant difference in peripheral blood lymphocyte DNA damage among diesel and kerosene burning bakers.

Assessment of gold nanoparticles on human peripheral blood cells by metabolic profiling with 1H-NMR spectroscopy, a novel translational approach on a patient-specific basis.

Human peripheral blood cells are relevant ex vivo models for characterizing diseases and evaluating the pharmacological effects of therapeutic interventions, as they provide a close reflection of an individual pathophysiological state. In this work, a new approach to evaluate the impact of nanoparticles on the three main fractions of human peripheral blood cells by nuclear magnetic resonance spectroscopy is shown. Thus, a comprehensive protocol has been set-up including the separation of blood cells, their in vitro treatment with nanoparticles and the extraction and characterization of metabolites by nuclear magnetic resonance. This method was applied to assess the effect of gold nanoparticles, either coated with chitosan or supported on ceria, on peripheral blood cells from healthy individuals. A clear antioxidant effect was observed for chitosan-coated gold nanoparticles by a significant increase in reduced glutathione, that was much less pronounced for gold-cerium nanoparticles. In addition, the analysis revealed significant alterations of several other pathways, which were stronger for gold-cerium nanoparticles. These results are in accordance with the toxicological data previously reported for these materials, confirming the value of the current methodology.