Search

Cancer is referred to as the "Emperor of all maladies" accounting for the second-highest mortality rates worldwide. Major factors associated with cancer lethality are uncontrolled proliferation, metastasis, and frequent recurrence. The conventional therapeutic drugs used in cancer therapy have been associated with numerous damaging side-effects that call for the use of alternative therapeutic options. The natural plant compounds (NPCs) have been found to be effective against diverse groups of diseases including cancer. Among the different types, the polyphenolic phytochemicals like curcumin, (-)epigallocatechin-3-gallate, Resveratrol, and nimbolide which are predominant parts of daily dietary intake have proved their potency in reducing the aggressive properties of cancer. Here, we have highlighted the mechanisms through which these NPCs influence growth, metastatic potential, and the drug-resistant behavior of different cancer types. Moreover, we have also emphasized on their function as modulators of the immune system as well as the metabolic properties of the tumor. The role of these phytochemicals in reducing cancer progression has been highlighted when administered unaided or in combination with similar group of compounds. Moreover, their ability to enhance the drug-sensitivity of cancer cells which accounts for their use in combination with conventional chemotherapeutics has also been discussed in this article. Therefore, co-administration of these phytochemicals with chemically similar group members or with conventional chemotherapeutics may prove to be an effective treatment strategy for cancer.

Aims Protective efficacy of N‑acetylcysteine (NAC) was assessed against sub-acute diisopropyl phosphorofluoridate (DFP) poisoning in mice. Main methods Mice were allocated into nine groups of six each: vehicle control; DFP (0.125 LD50 ≈ 0.483 mg/kg bwt, s.c.); DFP + Atropine (ATR, 10 mg/kg bwt, i.p., 0 min); DFP + Pralidoxime (2-PAM, 30 mg/kg bwt, i.m., 0 min); DFP + NAC (150 mg/kg bwt, i.p., −60 min); DFP + ATR + NAC; DFP + 2-PAM + NAC; DFP + ATR + 2-PAM; and DFP + ATR + 2-PAM + NAC. Animals received various treatments for 21 d daily. Plasma butyrylcholinesterase (BChE) was measured after 7, 14 and 21 d of exposure. Brain acetylcholinesterase (AChE) and reduced glutathione (GSH), malondialdehyde (MDA), glutathione peroxidase (GPx), glutathione reductase (GR), catalase (CAT), and superoxide dismutase (SOD) were measured (brain, liver and kidney) after 21 d of exposure. Histopathology, immunohistochemistry, and Western blot for inducible nitric oxide synthase (iNOS) and c-fos were also performed. Key findings DFP significantly reduced BChE and AChE levels. Diminished GSH, CAT, SOD (brain and liver), GPx, GR, and elevated MDA (Brain) levels were also observed. DFP caused notable histopathology (brain, liver and kidney) and over expression of iNOS, and c-fos proteins (brain). NAC enhanced the protective efficacy of ATR and 2-PAM in most parameters, without any appreciable protection in iNOS and c-fos expression. Significance NAC as an adjunct with ATR and 2-PAM, exhibited marked beneficial effects against sub-acute DFP poisoning, indicating its possible implications in the management of OP poisoning.

In this study, we catalog structure activity relationships (SAR) of several short chain fatty acid (SCFA)-modified hexosamine analogs used in metabolic glycoengineering (MGE) by comparing in silico and experimental measurements of physiochemical properties important in drug design including of. We then describe the impact of these compounds on selected biological parameters that influence the pharmacological properties and safety of drug candidates by monitoring P-glycoprotein (Pgp) efflux, inhibition of cytochrome P450 3A4 (CYP3A4), hERG channel inhibition, and cardiomyocyte cytotoxicity. These parameters are influenced by length of the SCFA (e.g., acetate vs. n-butyrate), which are added to MGE analogs to increase the efficiency of cellular uptake, the regioisomeric arrangement of the SCFAs on the core sugar, the structure of the core sugar itself, and by the type of N-acyl modification (e.g, N-acetyl vs. N-azido). By cataloging the influence of these SAR on pharmacological properties of MGE analogs, this study outlines design considerations for tuning the pharmacological, physiochemical, and the toxicological parameters of this emerging class of small molecule drug candidates.

Aims Comparative sub-acute toxicity, including tolerance and dependence potential of fentanyl and its three novel and potent analogues was determined in mice. Main methods Comparative sub-acute (21 d, intraperitoneal; 1/10 LD50) toxicity of fentanyl and its three novel analogues viz., N-(1-(2-phenoxyethyl)-4-piperidinyl) propionanilide (2), N-isopropyl-3-(4-(N-phenylpropionamido)piperidin-1-yl)propanamide (5), and N-t-butyl-3-(4-(N-phenylpropionamido)piperidin-1-yl)propanamide (6) was determined in mice. Animals were observed for additional seven days to assess the recovery. The brain, liver and kidney toxicity was assessed on the basis of various biochemical, oxidative, histological, and neuroadaptive markers. The expression levels of key neuronal markers associated with drug tolerance and dependence were investigated by western blot and immunohistochemistry. Key findings Fentanyl and its analogues caused abnormal levels of liver and kidney specific biomarkers in plasma. Brain malondialdehyde (MDA) levels were raised by all the treatments and kidney MDA level by analogue 6 (21 d). Reduced glutathione levels in brain, liver, and kidney were diminished by all the treatments (21 & 28 d) and a significant change in the levels of antioxidant enzymes was also produced mainly after 21 d. The deleterious effects of fentanyl and its analogues were further substantiated by corresponding histopathological changes in brain, liver and kidney (21 & 28 d). These compounds were also found to produce neuroadaptive changes as evidenced by the increased expression levels of c-Fos, glucocorticoid receptor, N-methyl- d -aspartate receptor1 and μ-opioid receptor (21 & 28 d). Significance Three novel analogues of fentanyl were envisaged to have alternative therapeutic potentials. However, their comparative sub-acute toxicity revealed undesirable side effects, thereby masking their therapeutic ability.

Sodium butyrate (NaBu) is not only well-known for enhancing protein production, but also degrades glycan quality. In this study, butyrate supplied by the precursor molecule 1,3,4-O-Bu3 ManNAc is applied to overcome the negative effects of NaBu on glycan quality while simultaneously increasing the productivity of the model recombinant erythropoietin (EPO). The beneficial impact of 1,3,4-O-Bu3 ManNAc on EPO glycan quality, while evident in wild-type CHO cells, is particularly pronounced in glycoengineered CHO cells with stable overexpression of β-1,4- and β-1,6-N-acetylglucosaminyltransferases (GnTIV and GnTV) and α-2,6-sialyltransferase (ST6) enzymes responsible for N-glycan antennarity and sialylation. Supplementation of 1,3,4-O-Bu3 ManNAc achieves approximately 30% sialylation enhancement on EPO protein in wild-type CHO cells. Overexpression of GnTIV/GnTV/ST6 in CHO cells increases EPO sialylation about 40%. Combining 1,3,4-O-Bu3 ManNAc treatment in glyocengineered CHO cells promotes EPO sialylation about 75% relative to EPO from wild-type CHO cells. Moreover, a detailed mass spectrometric ESI-LC-MS/MS characterization of glycans at each of the three N-glycosylation sites of EPO showed that the 1st N-site is highly sialylated and either the negative impact of NaBu or the beneficial effect 1,3,4-O-Bu3 ManNAc treatments mainly affects the 2nd and 3rd N-glycan sites of EPO protein. In summary, these results demonstrate 1,3,4-O-Bu3 ManNAc can compensate for the negative effect of NaBu on EPO glycan quality while simultaneously enhancing recombinant protein yields. In this way, a platform that integrates glycoengineering with metabolic supplementation can result in synergistic improvements in both production and glycosylation in CHO cells.

Cyanide-induced chemical hypoxia is responsible for pronounced oxidative damage in the central nervous system. The disruption of mitochondrial oxidative metabolism has been associated with upregulation of uncoupling proteins (UCPs). The present study addresses the dose- and time-dependent effect of sub-acute cyanide exposure on various non-enzymatic and enzymatic oxidative stress markers and their correlation with inducible-nitric oxide synthase (iNOS) and uncoupling protein-2 (UCP-2) expression. Animals received (oral) triple distilled water (vehicle control), 0.25 LD50 potassium cyanide (KCN) or 0.50 LD50 KCN daily for 21 d. Animals were sacrificed on 7, 14 and 21 d post-exposure to measure serum cyanide and nitrite, and brain malondialdehyde (MDA), reduced glutathione (GSH), glutathione disulfide (GSSG), cytochrome c oxidase (CCO), superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione reductase (GR) and catalase (CA) levels, together with iNOS and UCP-2 expression, and DNA damage. The study revealed that a dose- and time-dependent increase in cyanide concentration was accompanied by corresponding CCO inhibition and elevated MDA levels. Decrease in GSH levels was not followed by reciprocal change in GSSG levels. Diminution of SOD, GPx, GR and CA activity was congruent with elevated nitrite levels and upregulation of iNOS and UCP-2 expression, without any DNA damage. It was concluded that long-term cyanide exposure caused oxidative stress, accompanied by upregulation of iNOS. The upregulation of UCP-2 further sensitized the cells to cyanide and accentuated the oxidative stress, which was independent of DNA damage.

Osteoarthritis (OA) is a degenerative joint disease characterized by articular cartilage degradation. Because OA has a multifactorial nature and complex interrelationship of the individual elements of a whole joint, there is a need for comprehensive therapeutic approaches for cartilage tissue engineering, which simultaneously address multiple aspects of disease etiology. In this work, we investigated a multifunctional carbohydrate-based drug candidate, tri-butanoylated N-acetyl-D-galactosamine analog (3,4,6-O-Bu(3)GalNAc) that induced cartilage tissue production by human mesenchymal stem cells (hMSCs) and human OA chondrocytes by modulating Wnt/β-catenin signaling activity. The dual effects promoted chondrogenesis of human MSC and reduced inflammation of human OA chondrocytes in in vitro cultures. Translating these findings in vivo, we evaluated therapeutic effect of 3,4,6-O-Bu(3)GalNAc on the rat model of posttraumatic OA when delivered via local intra-articular sustained-release delivery using microparticles and found this method to be efficacious in preventing OA progression. These results show that 3,4,6-O-Bu(3)GalNAc, a disease modifying OA drug candidate, has promising therapeutic potential for articular cartilage repair.

The chemical additive sodium butyrate (NaBu) has been applied in cell culture media as a direct and convenient method to increase the protein expression in Chinese hamster ovary (CHO) and other mammalian cells. In this study, we examined an alternative chemical additive, 1,3,4-O-Bu3 ManNAc, for its effect on recombinant protein production in CHO. Supplementation with 1,3,4-O-Bu3 ManNAc for two stable CHO cell lines, expressing human erythropoietin or IgG, enhanced protein expression for both products with negligible impact on cell growth, viability, glucose utilization, and lactate accumulation. In contrast, sodium butyrate treatment resulted in a ∼20% decrease in maximal viable cell density and ∼30% decrease in cell viability at the end of cell cultures compared to untreated or 1,3,4-O-Bu3 ManNAc treated CHO cell lines for both products. While NaBu treatment enhanced product yields more than the 1,3,4-O-Bu3 ManNAc treatment, the NaBu treated cells also exhibited higher levels of caspase 3 positive cells using microscopy analysis. Furthermore, the mRNA levels of four cell apoptosis genes (Cul2, BAK, BAX, and BCL2L11) were up-regulated more in sodium butyrate treated wild-type, erythropoietin, or IgG expressing CHO-K1 cell lines while most of the mRNA levels of apoptosis genes in 1,3,4-O-Bu3 ManNAc treated cell lines remained equal or increased only slightly compared to the levels in untreated CHO cell lines. Finally, lectin blot analysis revealed that the 1,3,4-O-Bu3 ManNAc-treated cells displayed higher relative sialylation levels on recombinant EPO, consistent with the effect of the ManNAc component of this additive, compared to control while NaBu treatment led to lower sialylation levels than control, or 1,3,4-O-Bu3 ManNAc-treatment. These findings demonstrate that 1,3,4-O-Bu3 ManNAc has fewer negative effects on cell cytotoxicity and apoptosis, perhaps as a result of a more deliberate uptake and release of the butyrate compounds, while simultaneously increasing the expression of multiple recombinant proteins, and improving the glycosylation characteristics when applied at comparable molarity levels to NaBu. Thus, 1,3,4-O-Bu3 ManNAc represents a highly promising media additive alternative in cell culture for improving protein yields without sacrificing cell mass and product quality in future bioproduction processes.

This report describes the metabolic glycoengineering (MGE) of intracellular esterase activity in human colon cancer (LS174T) and Chinese hamster ovary (CHO) cells. In silico analysis of the carboxylesterases CES1 and CES2 suggested that these enzymes are modified with sialylated N-glycans, which are proposed to stabilize the active multimeric forms of these enzymes. This premise was supported by treating cells with butanolylated ManNAc to increase sialylation, which in turn increased esterase activity. By contrast, hexosamine analogues not targeted to sialic acid biosynthesis (e.g., butanoylated GlcNAc or GalNAc) had minimal impact. Measurement of mRNA and protein confirmed that esterase activity was controlled through glycosylation and not through transcription or translation. Azide-modified ManNAc analogues widely used in MGE also enhanced esterase activity and provided a way to enrich targeted “glycoengineered” proteins (such as CES2), thereby providing unambiguous evidence that the compounds were converted to sialosides and installed into the glycan structures of esterases as intended. Overall, this study provides a pioneering example of the modulation of intracellular enzyme activity through MGE, which expands the value of this technology from its current status as a labeling strategy and modulator of cell surface biological events.

Postmortem stability of cyanide biomarkers is often disputed. We assessed the time and temperature-dependent changes in cytochrome c oxidase (CCO) activity and cyanide concentration in various organs of mice succumbing to cyanide. Immediately after death, excised mice organs and mice cadavers were stored at room temperature (35°C ± 5°C) or in frozen storage (-20°C ± 2°C). At various times after death, CCO activity and cyanide concentrations were measured in excised mice organs or organs removed from mice cadavers. The study revealed that (i) measuring both the biomarkers in mice cadavers was more reliable compared to excised mice organs, (ii) measuring temporal CCO activity and cyanide concentration in vital organs from mice cadavers (room temperature) was reliable up to 24 h, and (iii) CCO activity in the brain and lungs and cyanide concentration in organs from mice cadavers (frozen) were measurable beyond 21 days. This study will be helpful in postmortem determination of cyanide poisoning.

Due to several limitations of existing cyanide antidotes, α-ketoglutarate (α-KG) has been proposed as a promising treatment for cyanide.This study reports the accelerated stability and bioassay of a new oral α-KG formulation.Amber-colored PVDF bottles containing 100 ml of 10% α-KG in 70% sorbitol, preservative (sodium methyl paraben and sodium propyl paraben), sweetener (sodium saccharine), flavor (American ice-cream soda and peppermint) and color (tartrazine), at pH 7.0-8.0 were stored in stability chamber (40 ± 2 °C and 75 ± 5% humidity) for 6 months in a GMP compliant facility. Various physical (pH, color, evaporation, extractable volume and clarity), chemical (identification and quantification of active ingredient) and microbiological (total aerobic count) analyses, together with protection studies were carried periodically in mice. Acute toxicity of the formulation and bioavailability of α-KG were assessed in rats at the beginning of the experiment.No physical changes and microbiological growth were observed in the formulation. After 6 months, α-KG content in the formulation diminished by ∼24% but its protective efficacy against cyanide remained at 5.9-fold. Protection was further characterized spectrophotometrically by disappearance of α-KG spectrum in the presence of cyanide, confirming cyanohydrin formation. Oral LD50 of α-KG formulation in rats was7.0 g/kg body weight, and did not produce any acute toxicity of clinical significance. Also, an appreciable amount of α-KG was measured in blood.As per the guidelines of International Conference on Harmonization, the new α-KG formulation exhibited satisfactory stability, bioefficacy and safety as cyanide antidote.

Cyanogens include complex nitrile-containing compounds that can generate free cyanide of toxicological significance. Acute toxicity, time-dependent cyanide generation and cytochrome oxidase (CYTOX) inhibition in soft tissues, and urinary thiocyanate levels were measured after acute cyanogen intoxication in rats. Order of cyanogens in terms of LD₅₀ was: malononitrile (MCN)>propionitrile (PCN)≈sodium nitroprusside (SNP)>acrylonitrile (ACN)>succinonitrile (SCN)>acetonitrile (ATCN) for oral, and SNP>MCN>ACN>PCN>SCN>ATCN for intraperitoneal and subcutaneous routes. MCN was most toxic by oral (LD₅₀=66.4 mg/kg) and SNP by intraperitoneal (LD₅₀=16.7 mg/kg) and subcutaneous (LD₅₀=11.9 mg/kg) routes. Minimum survival time (25 min) was recorded after 4.0 LD₅₀ ATCN. Order of cyanogens (0.75 LD₅₀; oral) on the basis of maximum blood cyanide and time of peak cyanide generation were: ATCN>SNP>SCN>PCN>MCN>ACN, and MCN (30 min)

A desirable feature of many therapeutic glycoprotein production processes is to maximize the final sialic acid content. In this study, the effect of applying a novel chemical analog of the sialic acid precursor N-acetylmannosamine (ManNAc) on the sialic acid content of cellular proteins and a model recombinant glycoprotein, erythropoietin (EPO), was investigated in CHO-K1 cells. By introducing the 1,3,4-O-Bu3 ManNAc analog at 200-300 µM into cell culture media, the intracellular sialic acid content of EPO-expressing cells increased ∼8-fold over untreated controls while the level of cellular sialylated glycoconjugates increased significantly as well. For example, addition of 200-300 µM 1,3,4-O-Bu3 ManNAc resulted in >40% increase in final sialic acid content of recombinant EPO, while natural ManNAc at ∼100 times higher concentration of 20 mM produced a less profound change in EPO sialylation. Collectively, these results indicate that butyrate-derivatization of ManNAc improves the capacity of cells to incorporate exogenous ManNAc into the sialic acid biosynthetic pathway and thereby increase sialylation of recombinant EPO and other glycoproteins. This study establishes 1,3,4-O-Bu3 ManNAc as a novel chemical supplement to improve glycoprotein quality and sialylation levels at concentrations orders of magnitude lower than alternative approaches. Biotechnol. Bioeng. 2017;114: 1899-1902. © 2017 Wiley Periodicals, Inc.

Tissue-engineering strategies offer promising tools for repairing cartilage damage; however, these strategies suffer from limitations under pathological conditions. As a model disease for these types of nonideal systems, the inflammatory environment in an osteoarthritic (OA) joint limits the efficacy of engineered therapeutics by disrupting joint homeostasis and reducing its capacity for regeneration. In this work, we investigated a sugar-based drug candidate, a tributanoylated N-acetyl-d-glucosamine analogue, called 3,4,6-O-Bu3GlcNAc, that is known to reduce nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling in osteoarthritis. 3,4,6-O-Bu3GlcNAc not only inhibited NFκB signaling but also exerted chondrogenic and anti-inflammatory effects on chondrocytes isolated from patients with osteoarthritis. 3,4,6-O-Bu3GlcNAc also increased the expression of extracellular matrix proteins and induced cartilage tissue production in three-dimensional in vitro hydrogel culture systems. To translate these chondrogenic and anti-inflammatory properties to tissue regeneration in osteoarthritis, we implanted 3,4,6-O-Bu3GlcNAc-loaded poly(lactic-co-glycolic acid) microfiber scaffolds into rats. The drug-laden scaffolds were biocompatible, and when seeded with human OA chondrocytes, similarly promoted cartilage tissue formation. 3,4,6-O-Bu3GlcNAc combined with the appropriate structural environment could be a promising therapeutic approach for osteoarthritis.

Metabolic oligosaccharide engineering is a maturing technology capable of modifying cell surface sugars in living cells and animals through the biosynthetic installation of non-natural monosaccharides into the glycocalyx. A particularly robust area of investigation involves the incorporation of azide functional groups onto the cell surface, which can then be further derivatized using “click chemistry.” While considerable effort has gone into optimizing the reagents used for the azide ligation reactions, less optimization of the monosaccharide analogues used in the preceding metabolic incorporation steps has been done. This study fills this void by reporting novel butanoylated ManNAc analogues that are used by cells with greater efficiency and less cytotoxicity than the current “gold standard,” which are peracetylated compounds such as Ac4ManNAz. In particular, tributanoylated, N-acetyl, N-azido, and N-levulinoyl ManNAc analogues with the high flux 1,3,4-O-hydroxyl pattern of butanoylation were compared with their counterparts having the pro-apoptotic 3,4,6-O-butanoylation pattern. The results reveal that the ketone-bearing N-levulinoyl analogue 3,4,6-O-Bu3ManNLev is highly apoptotic, and thus is a promising anti-cancer drug candidate. By contrast, the azide-bearing analogue 1,3,4-O-Bu3ManNAz effectively labeled cellular sialoglycans at concentrations ∼3 to 5-fold lower (e.g., at 12.5 to 25 μM) than Ac4ManNAz (50 to 150 μM) and exhibited no indications of apoptosis even at concentrations up to 400 μM. In summary, this work extends emerging structure activity relationships that predict the effects of short chain fatty acid modified monosaccharides on mammalian cells and also provides a tangible advance in efforts to make metabolic oligosaccharide engineering a practical technology for the medical and biotechnology communities.

D-Threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP), a glycosphingolipid synthesis inhibitor, holds promise for the treatment of atherosclerosis and cardiac hypertrophy but rapid in vivo clearance has severely hindered translation to the clinic. To overcome this impediment, we used a materials-based delivery strategy wherein D-PDMP was encapsulated within a biodegradable polymer composed of poly ethylene glycol (PEG) and sebacic acid (SA). PEG-SA was formulated into nanoparticles that were doped with (125)I-labeled PEG to allow in vivo bio-distribution and release kinetics of D-PDMP to be determined by using γ-scintigraphy and subsequently, by mass spectrometry. Polymer-encapsulation increased the residence time of D-PDMP in the body of a treated mouse from less than one hour to at least four hours (and up to 48 h or longer). This substantially increased in vivo longevity provided by polymer encapsulation resulted in an order of magnitude gain in efficacy for interfering with atherosclerosis and cardiac hypertrophy in apoE-/- mice fed a high fat and high cholesterol (HFHC) diet. These results establish that D-PDMP encapsulated in a biodegradable polymer provides a superior mode of delivery compared to unconjugated D-PDMP by way of increased gastrointestinal absorption and increased residence time thus providing this otherwise rapidly cleared compound with therapeutic relevance in interfering with atherosclerosis, cardiac hypertrophy, and probably other diseases associated with the deleterious effects of abnormally high glycosphingolipid biosynthesis or deficient catabolism.

Time-dependent cyanide generation and acute toxicity of six different cyanogens were reported earlier, out of which malononitrile (MCN), propionitrile (PCN), and sodium nitroprusside (SNP) were found to be very toxic. We report here 14 d sub-acute toxicity of MCN, PCN, and SNP (oral; 1/10 LD 50 daily) in female rats, and its amelioration by α-ketoglutarate (α-KG; oral; 5.26 mmol/kg; +5 min), a potential cyanide antidote. Significant decrease in white blood cells (PCN, SNP), platelets count (PCN), and blood glucose levels (MCN, PCN, SNP) was accompanied by elevated levels of alanine aminotransferase, lactate dehydrogenase (MCN, PCN, SNP), and aspartate aminotransferase (PCN, SNP). Oxidative damage was evidenced by diminished total antioxidant status in plasma and enhanced malondialdehyde levels in liver and kidney. This was accompanied by diminished levels of reduced glutathione, glutathione peroxidase, glutathione reductase, superoxide dismutase, and catalase in the brain, liver and kidney. We also observed increased levels of blood cyanide and thiocyanate, together with inhibition of cytochrome c oxidase and thiosulfate-sulfur transferase activities in total brain and liver homogenate, respectively. Cyanogens also produced several histological changes in all the organs studied. Post-treatment with α-KG significantly abrogated the toxicity of cyanogens, indicating its utility as an antidote for long-term cyanogen exposure.

Based on accumulating evidence that the 3D topography and the chemical features of a growth surface influence neuronal differentiation, we combined these two features by evaluating the cytotoxicity, proliferation, and differentiation of the rat PC12 line and human neural stem cells (hNSCs) on chitosan (CS), cellulose acetate (CA), and polyethersulfone (PES)-derived electrospun nanofibers that had similar diameters, centered in the 200 to 500 nm range. None of the nanofibrous materials were cytotoxic compared to 2D (e.g., flat surface) controls; however, proliferation generally was inhibited on the nanofibrous scaffolds although to a lesser extent on the polysaccharide-derived materials compared to PES. In an exception to the trend towards slower growth on the 3D substrates, hNSCs differentiated on the CS nanofibers proliferated faster than the 2D controls and both cell types showed enhanced indication of neuronal differentiation on the CS scaffolds. Together, these results demonstrate beneficial attributes of CS for neural tissue engineering when this polysaccharide is used in the context of the defined 3D topography found in electrospun nanofibers.

Inflammation and tissue degeneration play key roles in numerous rheumatic diseases, including osteoarthritis (OA). Efforts to reduce and effectively repair articular cartilage damage in an osteoarthritic environment are limited in their success due to the diseased environment. Treatment strategies focused on both reducing inflammation and increasing tissue production are necessary to effectively treat OA from a tissue-engineering perspective. In this work, we investigated the anti-inflammatory and tissue production capacity of a small molecule 3,4,6-O-tributanoylated-N-acetylglucosamine (3,4,6-O-Bu3GlcNAc) previously shown to inhibit the nuclear factor κB (NFκB) activity, a key transcription factor regulating inflammation. To mimic an inflammatory environment, chondrocytes were stimulated with interleukin-1β (IL-1β), a potent inflammatory cytokine. 3,4,6-O-Bu3GlcNAc exposure decreased the expression of NFκB target genes relevant to OA by IL-1β-stimulated chondrocytes after 24 h of exposure. The capacity of 3,4,6-O-Bu3GlcNAc to stimulate extracellular matrix (ECM) accumulation by IL-1β-stimulated chondrocytes was evaluated in vitro utilizing a three-dimensional hydrogel culturing system. After 21 days, 3,4,6-O-Bu3GlcNAc exposure induced quantifiable increases in both sulfated glycosaminoglycan and total collagen. Histological staining for proteoglycans and type II collagen confirmed these findings. The increased ECM accumulation was not due to the hydrolysis products of the small molecule, n-butyrate and N-acetylglucosamine (GlcNAc), as the isomeric 1,3,4-O-tributanoylated N-acetylglucosamine (1,3,4-O-Bu3GlcNAc) did not elicit a similar response. These findings demonstrate that a novel butanoylated GlcNAc derivative, 3,4,6-O-Bu3GlcNAc, has the potential to stimulate new tissue production and reduce inflammation in IL-1β-induced chondrocytes with utility for OA and other forms of inflammatory arthritis.

We assessed the dose-dependent effect of potassium cyanide (KCN) on thiosulfate sulfurtransferase (TST), 3-mercaptopyruvate sulfurtransferase (3-MPST), and cystathionine λ-lyase (CST) activities in mice. The time-dependent effect of 0.5 LD50 KCN on cyanide level and cytochrome c oxidase (CCO), TST, 3-MPST, and CST activities was also examined. Furthermore, TST, 3-MPST, and CST activities were measured in stored mice cadavers. Hepatic and renal TST activity increased by 0.5 LD50 KCN but diminished by ≥2.0 LD50. After 0.5 LD50 KCN, the elevated hepatic cyanide level was accompanied by increased TST, 3-MPST, and CST activities, and CCO inhibition. Elevated renal cyanide level was only accompanied by increased 3-MPST activity. No appreciable change in enzyme activities was observed in mice cadavers. The study concludes that high doses of cyanide exert saturating effects on its detoxification enzymes, indicating their exogenous use during cyanide poisoning. Also, these enzymes are not reliable markers of cyanide poisoning in autopsied samples.

Ammonia is a widely used industrial chemical that is recognized as a potent neurotoxin and environmental pollutant. The present study addresses the oxidative stress and tissue pathology caused by 4 weeks of exposure to ammonium acetate (AMA; 100 mg/kg daily; orally) in rats, and their response to oral treatments with alpha-ketoglutarate (A-KG; 1.0 g/kg), a potential cyanide antidote, and/or N-acetyl cysteine (NAC; 10 mg/kg), an antioxidant. The organ–body weight index of brain and liver was significantly increased by AMA but kidney was unaffected. Also, plasma ammonia levels were significantly elevated without any concomitant change in blood gas status and hematology but levels of catalase, superoxide dismutase, glutathione peroxidase, glutathione reductase and reduced glutathione (GSH) in the brain and liver were diminished, accompanied by elevated levels of malondialdehyde. Levels of glutathione disulfide (GSSG) were unaffected, but the ratio of GSH:GSSG was reduced. Plasma alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase and total bilirubin were raised but urea, uric acid and creatinine levels were not altered. AMA also caused temporal, hepatic and renal pathology. However, the renal pathology was not supported by any biochemical alterations. A-KG or NAC alone afforded less protection against AMA as compared to both given together. The protective efficacy of A-KG can be ascribed to its ability to detoxify ammonia and additionally both A-KG and NAC have antioxidant properties as well. The study suggests a new therapeutic regimen for ammonia poisoning.

Cyanide is a potential suicidal, homicidal and chemical warfare agent. It produces histotoxic hypoxia following inhibition of cytochrome c oxidase, a terminal respiratory chain enzyme. The profound metabolic changes lead to neurotoxicity including alterations in the levels of neurotransmitters. The present study addressed the effect of acute exposure of lethal and sub-lethal doses of potassium cyanide (KCN; 0.75 or 2.0 LD50; po) on the levels of neurotransmitters in discrete brain regions of rats and its response to treatment with alpha-ketoglutarate (alpha-KG; 0.5 g/kg; po; -10 min) alone or with sodium thiosulphate (STS; 1.0 g/kg; ip; -15 min). KCN significantly decreased norepinephrine, dopamine and 5-hydroxytryptamine levels in different brain regions which were resolved by alpha-KG and/or STS. Corpus striatum and hippocampus were more sensitive as compared to cerebral cortex and hypothalamus. alpha-KG, a potential cyanide antidote alone or with STS showed neuroprotective effects against cyanide.

Oxidative stress plays a crucial role in mediating cyanide toxicity. The present study addresses the effect of cyanide on activity and gene-expression profile of certain antioxidant enzymes and the expression of heat shock protein (HSP-70) in different organs of rats. Rats were treated with 0.50 LD(50) (7.0 mg/kg) of potassium cyanide (KCN; oral) and/or alpha-ketoglutarate (A-KG; 1.0 g/kg; oral) daily for 14 days, and various biochemical variables were measured in brain, liver, and kidney after 7 and 14 days of treatments and a 7-day recovery period. Cyanide significantly reduced the activity of glutathione peroxidase (GPx), glutathione reductase (GR), superoxide dismutase (SOD), and catalase (CA) in all the organs after 7 days, while the activity of GPx in brain, liver, and kidney, GR in liver, and CA in brain remained diminished up to 14 days. The gene-expression profile of corresponding enzymes did not show any difference between the control and treatment groups. Elevated levels of malondialdehyde were observed in brain and kidney 7 and 14 days after cyanide. Cyanide also increased the expression of HSP-70 activity in brain after 7 days alone. Regression of toxicity was observed after the withdrawal of KCN. Treatment of A-KG was found to prevent all the biochemical alterations caused by cyanide. This study reveals that oxidative stress caused by cyanide was independent of the expression of antioxidant enzyme activity at the gene level, and all changes responded favorably to A-KG, indicating its therapeutic potential.

Oral toxicity of several cyanogens and their reversal by alpha-ketoglutarate (A-KG; oral) were studied in rats. LD(50) of acetonitrile (ATCN), acrylonitrile (ACN), malononitrile (MCN), propionitrile (PCN), sodium nitroprusside (SNP), and succinonitrile (SCN) was 4891, 143.3, 69.8, 122.9, 69.8 and 488.7 mg/kg, respectively while the protection index of A-KG (ratio of LD(50) of cyanogens in the presence or absence of A-KG) was>2.0 against MCN (7.6), PCN (2.7) and SNP (3.6) only. We further studied the efficacy of A-KG against acute toxicity of these three cyanogens (0.75 LD(50)) on various hematological and biochemical variables in blood and soft tissues 24h post-exposure. We observed increase in white blood cells (SNP), plasma alanine (PCN, SNP) and aspartate (PCN) aminotransferase, lactate dehydrogenase (MCN, PCN, SNP), Na(+) (MCN, PCN) and cyanide (PCN), and decrease in K(+) (MCN, SNP) accompanied by an increase in brain, kidney and liver malondialdehyde (PCN), decrease in brain glutathione peroxidase, glutathione reductase (PCN, SNP), reduced glutathione (MCN, PCN, SNP) and cytochrome oxidase (PCN), liver rhodanese (PCN, SNP), and kidney cytochrome oxidase (PCN). The study indicates that (i) PCN was most toxic among all the cyanogens and (ii) beside cyanide, A-KG could be considered as an effective antidote for cyanogens.

Cyanide is a rapidly acting neurotoxin that necessitates immediate, vigorous therapy. The commonly used treatment regimen for cyanide includes the intravenous administration of sodium nitrite (SN) and sodium thiosulphate (STS). Due to many limitations of these antidotes, a search for more effective, safer molecules continues. Cyanide is known to react with carbonyl compounds to form the cyanohydrin complex. The present study addresses the efficacy of several carbonyl compounds and their metabolites or nutrients with alpha-ketoglutaric acid (A-KG), citric acid, succinic acid, maleic acid, malic acid, fumaric and oxaloacetic acid, glucose, sucrose, fructose, mannitol, sorbitol, dihydroxyacetone, and glyoxal (5 or 10 mM; -10 min) against toxicity of potassium cyanide (KCN; 10 mM) in rat thymocytes in vitro. Six hours after KCN, cell viability measured by MTT assay and crystal violet dye exclusion revealed maximum cytoprotection by A-KG, followed by oxaloacetic acid. A-KG also resolved the leakage of intracellular lactate dehydrogenase, loss in nuclear integrity (propidium iodide staining), and altered mitochondrial membrane potential (rhodamine 123 assay) as a result of cyanide toxicity. Protection Index (ratio of LD(50) of KCN in protected and unprotected animals; PI) of all the compounds (oral; 1.0 g/kg; -10 min) determined in male mice, revealed that maximum protection was afforded by A-KG (7.6 PI), followed by oxaloacetic acid (6.4 PI). Comparative evaluation of various salts of A-KG alone or with STS (intraperitoneal; 1.0 g/kg; -15 min) showed that maximum protection was conferred by disodium anhydrous salt of A-KG, which also significantly prevented the inhibition of brain cytochrome oxidase caused by 0.75 LD(50) KCN. This study indicates the potential of A-KG as alternative cyanide antidote.

Cyanide is a rapidly acting neurotoxin that inhibits cellular respiration and energy metabolism leading to histotoxic hypoxia. This results in the dissipation of mitochondrial membrane potential (MMP) accompanied by decreased cellular ATP content which in turn is responsible for increased levels of intracellular calcium ions ([Ca 2+ ] i ) and total lactic acid content of the cells. Rat pheochromocytoma (PC12) cells possess much of the biochemical machinery associated with synaptic neurons. In the present study, we evaluated the cytoprotective effects of alpha-ketoglutarate (A-KG) and N -acetylcysteine (NAC) against cyanide-induced cytotoxicity and altered energy metabolism in PC12 cells. Cyanide-antagonism by A-KG is attributed to cyanohydrin formation whereas NAC is known for its antioxidant properties. Data on leakage of intracellular lactate dehydrogenase and mitochondrial function (MTT assay) revealed that simultaneous treatment of A-KG (0.5 mM) and NAC (0.25 mM) significantly prevented the cytotoxicity of cyanide. Also, cellular ATP content was found to improve, followed by restoration of MMP, intracellular calcium [Ca 2+ ] i and lactic acid levels. Treatment with A-KG and NAC also attenuated the levels of peroxides generated by cyanide. The study indicates that combined administration of A-KG and NAC protected the cyanide-challenged PC12 cells by resolving the altered energy metabolism. The results have implications in the development of new treatment regimen for cyanide poisoning.

Toxicological evaluation of two recently reported treatment protocols for cyanide—SN (sodium nitrite) + DMAP (4-dimethylaminophenol) and SN + HA (hydroxylamine)—was carried out in male rats. Both treatments produced transient hyperammonaemia and decreased mean arterial pressure. Heart rate decreased and respiratory rate increased, but these changes reached the level of significance only after SN + HA. Histopathological lesions in lung, liver (SN+HA) and kidney (SN+DMAP) were predominantly in the vicinity of blood vessels. The results indicate toxic effects in both treatment groups at a dose known to induce methaemoglobin concentration to the level of antidotal efficiency in cyanide intoxication.

Articular cartilage has a limited ability to self-repair because of its avascular nature and the low mitotic activity of the residing chondrocytes. There remains a significant need to develop therapeutic strategies to increase the regenerative capacity of cells that could repair cartilage. Multiple cell types, including chondrocytes and mesenchymal stem cells, have roles in articular cartilage regeneration. In this study, we evaluated a platform technology of multiple functionalized hexosamines, namely 3,4,6-O-tributanoylated-N-acetylgalactosamine (3,4,6-O-Bu3GalNAc), 3,4,6-O-tributanoylated-N-acetylmannosamine (3,4,6-O-Bu3ManNAc) and 3,4,6-O-Bu3GlcNAc, with the potential ability to reduce NFκB activity. Exposure of IL-1β-stimulated chondrocytes to the hexosamine analogs resulted in increased expression of ECM molecules and a corresponding improvement in cartilage-specific ECM accumulation. The greatest ECM accumulation was observed with 3,4,6-O-Bu3GalNAc. In contrast, mesenchymal stem cells (MSCs) exposed to 3,4,6-O-Bu3GalNAc exhibited a dose dependent decrease in chondrogenic differentation as indicated by decreased ECM accumulation. These studies established the disease modification potential of a hexosamine analog platform on IL-1β-stimulated chondrocytes. We determined that the modified hexosamine with the greatest potential for disease modification is 3,4,6-O-Bu3GalNAc. This effect was distinctly different with 3,4,6-O-Bu3GalNAc exposure to chondrogenic-induced MSCs, where a decrease in ECM accumulation and differentiation was observed. Furthermore, these studies suggest that NFκB pathway plays a complex role cartilage repair.

Combined Therapeutic Potential of meso -2,3-Dimercaptosuccinic Acid and Calcium Disodium Edetate on the Mobilization and Distribution of Lead in Experimental Lead Intoxication in Rats. Flora, S. J. S., Bhattacharya, R., and Vijayaraghavan, R. (1995). Fundam. Appl. Toxicol. 25, 233-240. Asymptomatic lead poisoning remains a serious public health problem in developed and developing countries. Chelation therapy particularly with calcium disodium ethelenediamine tetra-acetic acid (CaNa 2 EDTA) is often used therapeutically to reduce the body burden of lead. This chelating drug has serious side effects and drawbacks primarily related to redistribution of lead, nephrotoxicity, and essential metal depletion. The present study was planned to determine the effectiveness of CaNa 2 EDTA and meso -2,3-dimercaptosuccinic acid (DMSA) used in combination. Both drugs, when administered individually, resulted in significant urinary excretion of lead and lowered the tissue lead burden. Combined treatment with CaNa 2 EDTA and DMSA elicits an additive response in promoting urinary lead elimination, depleting body lead burden, and restoring altered lead-sensitive biochemical variables. Further, no redistribution of lead to brain or any other soft organ following combined DMSA-CaNa 2 EDTA treatment was observed indicating a definite advantage of combined therapy over the conventional treatment with CaNa 2 EDTA or DMSA alone. However, an elevation of serum transaminase activity, creatinine level, and depletion of blood zinc level may limit the usefulness of this combined treatment.