Indiscriminate use of pesticides appears to alter immune response in non-target organisms such as humans and other animals. Thus, immune modulation is considered as one of the potential risks and consequences following exposure to these chemicals. Because of the widespread usage, exposure to mixtures of pesticides during the lifetime of individuals is unavoidable and can result in potentiation of the toxic effects. Because immune cells are more susceptible to toxic insults at a lower dose than most other cell types, the effects of pesticides and their mixtures on murine splenocytes were evaluated. C57BL/6 male mouse splenocytes, in vitro, were exposed to permethrin and endosulfan, individually and in-combination (25-200 µM). The immunotoxic potential of these pesticides was monitored using a flow cytometric technique in combination with 7-Amino Actinomycin D (7-AAD) staining. Endosulfan exposures (25-150 µM) resulted in time- and dose-dependent increase in apoptotic and necrotic cell death in murine splenocytes, in vitro. Permethrin exposure (50-200 µM) resulted in neither a time-dependent/dose-dependent loss of splenocyte viability nor induction of apoptosis in splenocytes. With mixtures of permethrin and endosulfan, depressed viability and enhanced early apoptosis and late apoptosis/necrosis were observed. Exposure to mixtures of 50 µM endosulfan with 50 or 100 µM permethrin increased late apoptosis/necrosis compared to exposure to either chemical alone. DNA fragmentation, a hall mark of apoptosis was observed by DNA ladder technique, confirming the occurrence of apoptosis. Morphological observation using cytospun slides was also carried out to further confirm the presence of apoptosis and necrosis. These findings suggest that the immunotoxicity of endosulfan both individually and in mixtures with permethrin is associated with the occurrence of apoptotic and necrotic processes. Further, the ability of these pesticides to alter the oxidative status of the cells, via reactive oxygen species (ROS) generation and modulation of intracellular antioxidant enzymes levels, was investigated. We monitored the generation of ROS such as hydrogen peroxide (H₂O₂) with 2´, 7´- dichlorofluorescin diacetate (DCFH-DA) assay and superoxide anion (O₂⁻) with hydroethidine (HE) assay in combination with flow cytometry. Spectrophotometric techniques were used to measure antioxidant enzymes such as catalase (CAT), superoxide dismutase (SOD), glutathione reductase (GR) and glutathione peroxidase (GPX). Results of the analyses revealed that individual pesticides increased the production of H₂O₂ in a time and dose-dependent manner. Both time and dose-dependent increases in O₂⁻ production were caused by permethrin; whereas endosulfan exposure resulted in only a dose-dependent increase. However, exposure to mixtures of these pesticides had little or no effect on the generation of H₂O₂ and O₂⁻ radicals as compared to individual pesticides. The levels of SOD and GPX in pesticide-treated splenocytes were found to be not different from solvent control. An increase in GR and CAT levels in cells was noticed with permethrin (100 µM) exposure. These findings suggest that permethrin and endosulfan have the ability to affect the cellular oxidative status and can cause toxicity in immune cells, in vitro. / Master of Science

Yes / Platinum complexes are the most widely used anticancer drugs; however, new generations of agents are needed. The organoiridium(III) complex [(η5-Cpxbiph)Ir(phpy)(Cl)] (1-Cl), which contains π-bonded biphenyltetramethylcyclopentadienyl (Cpxbiph) and C^N-chelated phenylpyridine (phpy) ligands, undergoes rapid hydrolysis of the chlorido ligand. In contrast, the pyridine complex [(η5-Cpxbiph)Ir(phpy)(py)]+ (1-py) aquates slowly, and is more potent (in nanomolar amounts) than both 1-Cl and cisplatin towards a wide range of cancer cells. The pyridine ligand protects 1-py from rapid reaction with intracellular glutathione. The high potency of 1-py correlates with its ability to increase substantially the level of reactive oxygen species (ROS) in cancer cells. The unprecedented ability of these iridium complexes to generate H2O2 by catalytic hydride transfer from the coenzyme NADH to oxygen is demonstrated. Such organoiridium complexes are promising as a new generation of anticancer drugs for effective oxidant therapy. / We thank the ERC (247450), SNSF (PA00P2_145308 for N.P.E.B.), IAS (for I.R.C.), BBSRC (for J.M.H.), Science City (AWM and ERDF), and the EPSRC for support, and Prof. Timothy Bugg and members of EC COST Action CM1105 for stimulating discussions. We also thank Professor Pat Unwin, Mike Snowden, and Rob Lazenby for their help with the electrochemical experiments and the National Cancer Institute for NCI-60 human tumor cell panel screening.

Yes / Boron dipyrromethene derivates (BODIPYs) are promising photosensitisers (PSs) for cancer treatment using photodynamic therapy (PDT). This study investigates the functionalisation of graphene oxide (GO) with a BODIPY derivate for glutathione (GSH) depletion and PDT. The functionalisation of GO with a 3,5-dichloro-8-(4-boronophenyl) BODIPY via a diol derivatisation with the phenyl boronic acid moiety at the meso position of the BODIPY core, allowed to preserve the intrinsic properties of GO. We demonstrated that both chlorine atoms were substituted by GSH in the presence of glutathione transferase (GST), inducing a relevant bathochromic shift in the absorption/emission features and thus generating the active PS. Ex vitro assessment using cell lysates containing cytoplasmatic GST revealed the intracellular catalytic mechanism for the nucleophilic substitution of the GO-BODIPY adduct with GSH. Confocal microscopy studies showed important differences in the cellular uptake of free BODIPY and GO-BODIPY and revealed the coexistence of GO-BODIPY, GO-BODIPY-GS, and GO-BODIPY-GS2 species inside vesicles and in the cytoplasm of the cells after 24 h of incubation. In vitro biocompatibility and safety of GO and GO-BODIPY were evaluated in 2D and 3D models of prostate adenocarcinoma cells (PC-3), where no toxicity was observed up to 100 µg ml−1 of GO/GO-BODIPY in all treated groups 24 h post-treatment (cell viability > 90%). Only a slight decrease to 80% at 100 µg ml−1 was observed after 48 h of incubation. We demonstrated the efficacy of a GO adduct containing an α-chlorine-substituted BODIPY for the simultaneous depletion of intracellular GSH and the photogeneration of reactive oxygen species using a halogen white light source (5.4 mW cm−2) with a maximum in the range of 500–800 nm, which significantly reduced cell viability (<50%) after irradiation. Our study provides a new vision on how to apply BODIPY derivates and potentiate the toxicity of PDT in prostate and other types of cancer.

Master of Science / Department of Kinesiology / Timothy I. Musch / During exercise aged individuals exhibit endothelial dysfunction and decreased levels of whole-limb blood flow (BF), both of which may be linked mechanistically to age-related increases in reactive oxygen species (ROS). Ascorbic acid (AA) reduces levels of ROS and has been shown to alleviate vascular and hyperemic dysfunction at rest (Jablonski et al., 2007) and during small muscle mass exercise in humans (Kirby et al., 2009). However, the effect of AA on vascular function and BF to individual muscles during whole-body exercise is not known. PURPOSE: To test the hypothesis that a single high-dose infusion of AA would increase BF to the hindlimb musculature of old rats at rest and during treadmill running. METHODS: 18 old (~28 months) Fischer 344 x Brown Norway rats were randomized into rest (n=9) and exercise (n=9) groups. BF to the total hindlimb and individual muscles (28 individual muscles and muscle parts) was evaluated via radiolabeled microspheres before and after intra-arterial AA administration (76 mg/kg in 3 ml heparinized saline, 30 minute infusion) at rest and during submaximal treadmill running (20m/min, 5% grade). Total antioxidant capacity (TAC) and thiobarbituric acid reactive species (TBARS) were measured before and after AA to determine the ability of this specific dose of AA to increase levels of plasma antioxidants and decrease levels of ROS, respectively. RESULTS: At rest: AA increased TAC (~37%, P<0.05) but did not change TBARS (Pre: 6.8±0.7 vs Post: 7.0±1.0 µM, P>0.05). AA decreased total hindlimb BF (Pre: 25±3 vs Post: 16±2 ml/min/100g, P<0.05) and BF to 8 of the 28 muscles that were evaluated. During exercise: TAC was increased (~35%, P<0.05) and TBARS were decreased (Pre: 9.8±2.0 vs Post: 7.0±1.0 µM, P<0.05). However, there was no effect on either total hindlimb BF (Pre: 154±14 vs Post: 162±13, P>0.05) or BF to any of the individual muscles evaluated. CONCLUSIONS: Increased TAC via AA infusion reduces hindlimb muscle BF at rest but had no effect on BF during whole-body dynamic exercise. Thus, even though TBARS decreased, there was no evidence that AA supplementation increases blood flow to the locomotor muscles of old rats during whole-body exercise.

Blood cells or hemocytes play important roles in immunity. They are a major source of many immune-related molecules such as antibodies in adaptive immunity of vertebrates and prophenoloxidase (proPO) in invertebrates. In the crayfish Pacifastacus leniusculus, the proPO-system has been reported to be an important component of immune responses against microorganisms. In this study, several mutant strains of Aeromonas hydrophila were used to reveal that LPS (lipopolysaccharide) is an important factor for the pathogenicity of A. hydrophila, strongly inducing the proPO system and melanization. This proPO activating system is a multistep process, which has to be tightly controlled to avoid the harmful side effects of toxic intermediates. Many regulating factors have been reported to fine-tune the proPO-system. In this study, the cleavage of caspase-1-like activity was shown to be a novel negative regulator of PO activity in crayfish. Moreover, the fragments obtained by cleavage of proPO by the proPO-activating enzyme and caspase-1-like protein increased bacterial clearance. Thus, the peptides generated also have important biological functions. In addition to being a source of immune proteins, hemocytes also participate in phagocytosis, encapsulation, and nodulation. An infection normally causes a reduction of hemocyte numbers. Consequently, hemocyte homeostasis is important for maintaining appropriate hemocyte numbers in the circulation of the animal. This study shows that the reactive oxygen species level in the anterior proliferation center of crayfish hematopoietic tissue (HPT), together with cell proliferation, was increased during infection. Pl-β-thymosins were proposed to be involved in hemocyte homeostasis by increasing stem cell migration and thus increasing the circulating hemocyte number. Crayfish hemocyte numbers, as well astakine (Ast1 and Ast2) expression in hemocytes and HPT, were previously shown to be under circadian regulation. Here, we show that Ast1, Ast2, and proPO exhibit rhythmic expression in the crayfish brain similarly to their orthologs, prokineticin 1, prokineticin 2 and tyrosinase, respectively, in the zebrafish brain. Tyrosinase expression was detected in zebrafish brain cells while PO-positive cells were identified as hemocytes that had infiltrated into the crayfish brain. Therefore, this information suggests a close relationship between crayfish hemocytes and the crayfish brain as well as vertebrate neurons.

High circulating glucose and non-esterified free fatty acid (NEFA) levels can cause pancreatic β-cell failure. The molecular mechanisms of this β-cell glucolipotoxicity are yet to be established conclusively. In this thesis by exploring mitochondrial energy metabolism in INS-1E insulinoma cells and isolated pancreatic islets, a role of mitochondria in pancreatic β-cell glucolipotoxicity is uncovered. It is reported that prolonged palmitate exposure at high glucose attenuates glucose-stimulated mitochondrial respiration which is coupled to ADP phosphorylation. These mitochondrial defects coincide with an increased level of mitochondrial reactive oxygen species (ROS), impaired glucose-stimulated insulin secretion (GSIS) and decreased cell viability. Palmitoleate, on the other hand, does not affect mitochondrial ROS levels or cell viability and protects against the adverse effects of palmitate on these phenotypes. Interestingly, palmitoleate does not significantly protect against mitochondrial respiratory or insulin secretion defects and in pancreatic islets tends to limit these functions on its own. Furthermore, strong evidence suggests that glucolipotoxic-induced ROS are of a mitochondrial origin and these ROS are somehow linked with NEFA-induced loss in cell viability. To explore the mechanism of glucolipotxic-induced mitochondrial ROS and associated cell loss, uncoupling protein-2 (UCP2) protein levels and activity were probed in NEFA exposed INS-1E cells. It is concluded that UCP2 neither mediates palmitate-induced mitochondrial ROS production and the related cell loss, nor protects against these deleterious effects. Instead, UCP2 dampens palmitoleate protection against palmitate toxicity. Collectively, these data shed important new light on the area of glucolipotoxicity in pancreatic β-cells and provide novel insights into the pathogenesis of Type 2 diabetes.

Clostridium difficile (CD) is the leading cause of bacterial hospital-associated infection in North America. How CD colonizes the human host, including its response to the innate immune system and other stresses, is poorly understood. This work considers CD's defenses against two stresses found in the host - the antimicrobial peptide LL-37 and reactive oxygen species (ROS). LL-37 had bactericidal activity against CD. CD strains varied in their sensitivity to the peptide, and epidemic-associated strains were more resistant to LL-37 than others. CD became more resistant to LL-37 following exposure to sub-lethal concentrations of the peptide, suggesting the presence of inducible resistance mechanisms. A quantitative proteomics analysis revealed definite alterations in CD protein expression caused by LL-37. Specific changes included increased expression of DltB, a protein previously reported to confer resistance against other antimicrobial peptides. Notably, disruption of individual LL-37-induced genes did not sensitize CD to the peptide. This suggests functional redundancy, and that LL-37 may cause global changes in protein expression, not limited to antimicrobial peptide resistance determinants. One of the proteins most strongly induced by LL-37 was a predicted superoxide reductase (SOR). As CD is considered a strict anaerobe, expression of a predicted antioxidant protein was an interesting finding. Heterologous expression of CD SOR in a superoxide dismutase-deficient E. coli strain confirmed its action as a superoxide scavenger. Insertional inactivation of SOR rendered CD more sensitive to oxygen and ROS-generating compounds, indicating that SOR contributes to antioxidant defense in CD. SOR mutants were impaired in their ability to cause disease in hamsters, indicating a role for this protein in infection.

Infection is one of the factors that may contribute to non-healing of chronic wounds; the presence of antibiotic resistant bacteria serves to exacerbate the problem due to limited treatment options. Bacteria utilise several mechanisms to survive exposure to antibiotics, including synthesis of deactivating enzymes, target modification or substitution, changes to membrane permeability, upregulation of efflux pumps and the formation of a biofilm. Quorum sensing is a density-dependent mechanism of bacterial cell to cell communication that can be instrumental in co-ordinating biofilm initiation. Hyperbaric oxygen therapy (HBOT) is an option offered to some patients with chronic wounds, including diabetic foot ulcers. Evidence suggests that HBOT can reduce the incidence of major amputation in these patients. As well as the direct toxicity of increased tissue oxygenation on anaerobic bacteria HBOT may also increase levels of reactive oxygen and nitrogen species in the wound environment. This study aimed to investigate the effects of hyperoxia and oxidative damage on three specific mechanisms of antibiotic resistance: the activity of penicillinase, an antibiotic deactivating enzyme synthesised by bacteria; the activity of quorum sensing signalling molecules (AHLs); and biofilms and their associated bacteria. It also analysed the population dynamics of, primarily, bacteria in diabetic foot ulcers during HBOT, by the use of molecular analysis tools such as PCRDGGE. The presence of fungal species was investigated in wounds prior to HBOT and in two wounds at two points during HBOT. This study found that hydrogen peroxide, hypochlorous acid and peroxynitrite reduced the activity of penicillinase in vitro. Hypochlorous acid reduced the activity of a range of AHLs in vitro but not in vivo. Oxygen concentration did not have any impact on biofilm mass, nor did it significantly affect the ability of an oxidant-generating enzyme to reduce live bacterial cells within a biofilm. The population dynamics of bacterial species identified in all the wounds were complex and did not undergo identifiable changes during HBOT. Fungal species were identified in all wounds prior to HBOT, though different profiles were observed in the two wounds investigated during HBOT. These results suggest that oxidants could play a role in the attenuation of antibiotic resistance in chronic wound bacteria. It is unclear whether HBOT alters the population dynamics of non-healing wound microflora

In recent years, the causal relationship between inflammation and cancer has gained wider acknowledgement and acceptance. While various types of immune cells are involved in the process of inflammation, macrophages represent the major inflammatory component of many tumors. Derived from circulating monocytes, these cells migrate to tumor sites in response to molecular cues present within the tumor microenvironment. Once there, interactions with neoplastic cells shape the differentiation and functional orientation of macrophages into two phenotypically distinct subsets: the “classically” activated M1 macrophages and the “alternatively” activated M2 macrophages. The preeminent paradigm in macrophage-related cancer research is that within the tumor stoma, macrophages acquire an M2 phenotype characterized by production of pro-angiogenic factors, ECM degrading enzymes and up-regulation of anti-inflammatory responses, thereby promoting tumor progression. M1 macrophages, on the other hand are thought to exert anti-tumorigenic effects due to their production of pro-inflammatory cytokines, and reactive oxygen species (ROS). While the generation of ROS during immune responses is an important aspect of immune regulation and host defense, excessive ROS production has been implicated in the pathogenesis of various degenerative diseases, including cancer. Yet, despite the well-established role of M1 macrophages in generating high levels of ROS via NADPH oxidase (NOX), M1 macrophages are still largely viewed as anti-tumorigenic. Hence, this study reevaluates the complex interaction between prostate cancer (PCa) cells and tumor-associated macrophages (TAMs), and operates on the premise that PCa cells promote a pro-tumor microenvironment, denoted by increased inflammation and oxidative stress, in part, through M1 macrophage-mediated, NOX-derived ROS production. Accordingly, immunofluorescent analysis of prostate tissue microarrays demonstrated an influx of M1 macrophages in prostate carcinoma. Immature monocytes co-cultured with the poorly tumorigenic prostate cell line, LNCaP, demonstrated changes in morphology and protein expression consistent with M1 macrophage polarization. PCa cells co-cultured with M1 macrophages displayed significantly higher intracellular ROS levels. Furthermore, M1-mediated ROS generation through NOXs increased prostate cell invasiveness and anchorage-independent growth. Taken together, results from this study suggest a potentially novel pro-tumorigenic function of M1 macrophages in early PCa progression, and aid in understanding the complex role of inflammation in cancer.

Peroxynitrite (PN, ONOO-), formed by nitric oxide radical (•NO) and superoxide radical (O2•-), plays an important role in post-traumatic oxidative damage. In the early work, we determined the temporal characteristics of PN-derived oxidative damage in a rat spinal cord injury (SCI) model. Our results showed 3-nitrotyrosine (3-NT), a specific marker for PN, rapidly accumulated at early time points (1 hr, 3 hrs), after when it plateaued and the high level was sustained to 1 week post injury. The co-localization of 3-NT and lipid peroxidation derived-4-HNE observed in immunohistochemistry indicates PN is involved in lipid peroxidative as well as protein nitrative damage. PN-oxidative damage exacerbates intracellular Ca2+ overload, which activates Ca2+ dependent calpain-mediated cytoskeletal protein (α-spectrin) degradation. The 145 kD fragments of α-spectrin (SBDP 145), which are specifically generated by calpain, increased dramatically as early as 1 hr after injury although the peak increase did not occur until 72 hrs post injury. The high level waned back toward sham level at one week post injury. We then carried out experiments to evaluate the beneficial effects of tempol, a scavenger of PN-derived radicals, following SCI. Three pathological events including PN-induced oxidative damage, mitochondrial dysfunction and cytoskeletal degradation were investigated. Immunoblotting and immunohistochemical studies indicated PN-mediated oxidative damage including protein nitration, protein oxidation and lipid peroxidation, were all reduced by a single dose of tempol (300mg/kg, i.p) after SCI. Spinal cord (SC) mitochondrial dysfunction in terms of the respiratory control ratio (RCR) significantly improved by both 150 mg/kg and 300 mg/kg tempol treatments. Moreover, calpain-mediated proteolysis was significantly decreased by tempol, with greater effects on calpain-specific SBDP 145 observed. Direct PN-scavenging effect of tempol was confirmed in vitro. Exposure of healthy SC mitochondria to SIN-1, a PN donor in vitro, impaired mitochondrial respiration in a dose-dependent manner. Tempol was able to protect mitochondria against SIN-1-induced damage by improving mitochondrial function and decreasing mitochondrial 3-NT formation. These findings strongly support the concept that PN is a crucial player in the secondary damage following SCI. And tempol, by scavenging PN-induced free radicals, provides a promising pharmocotherapeutic strategy for treating acute SCI.