Free radical biology and pathology

A free radical is defined as any species capable of independent existence (hence the term “free”) that contains one or more unpaired electrons. The following are examples of free radicals²: superoxide radical O₂^.-, hydroxy radical OH, hydrogen peroxide H₂O₂ (if two hydroxy radicals ever meet, they can join their unpaired electrons and make oxygen-oxygen covalent bonds giving H₂O₂. However, if one extra electron is added to H₂O₂, it makes OH⁻ and with the addition of an extra e⁻ we have an hydroxy radical). Ultraviolet radiation produces hydroxy radicals.Other molecules of concern are oxides of nitrogen, ozone and singlet oxygen (rearrangement of electrons that produces very rapid oxidation). So we are stuck with the conundrum of having essential life giving chemicals in themselves causing toxicity-oxidation. Hemoglobin makes superoxide radicals — oxyhemoglobin slowly releases superoxide radical (O₂^.-) and forms ferric hemoglobin (methemoglobin) which is unable to bind and transport O₂. This release of O₂^.- may happen only one in a thousand cycles of O₂ binding and release, but there is a large mass of hemoglobin in the body and so the total body O₂^.- produced by this mechanism is significant — in the human body it has been estimated that up to 3% of hemoglobin releases O₂^.-.Unpaired electrons make for very unstable, highly reactive atoms and/or molecules. Paired electrons, by the way of contrast, are the characteristic of a far more stable state.This is a very hazardous, unnatural and unstable state, because electrons normally come in pairs. This odd, unpaired electron in a free radical causes it to collide with other molecules so it can steal an electron from them, which changes the structure of these other molecules and causes them to also become free radicals. This can create a self perpetuating chain reaction in which the structure of millions of molecules are altered in a matter of nano-seconds wreaking havoc with genetic material (DNA), protein molecules, enzymes and cells.Free radicals are “capable of reversibly or irreversibly damaging compounds of all biochemical classes, including nucleic acids, protein and free amino acids, lipids and lipoproteins, carbohydrates, and connective tissue macromolecules. These species may (also) have an impact on such cell activities as membrane function, metabolism, and gene expression.” In the human it is now recognized that free radicals are contributing causes to a large number of disease entities.     

The Control of Reactive Oxygen Species Influences Porcine Oocyte In Vitro Maturation

The aim of this study was to examine the effect of varying intracellular reactive oxygen species (ROS) levels during oocyte in vitro maturation with enzymatic ROS production systems (xanthine + xanthine oxidase or xanthine + xanthine oxidase + catalase), scavenger systems (catalase or superoxide dismutase + catalase) or cysteine on porcine oocyte maturation. Oocyte ROS levels showed an increase when H₂O₂ or O₂?^‐ production systems were added to the culture medium (p < 0.05). On the other hand, the presence of ROS scavengers in the maturation medium did not modify oocyte ROS levels compared with the control after 48 h of maturation, but the addition of cysteine induced a decrease in oocyte ROS levels (p < 0.05). The ROS production systems used in this work did not modified the percentage of oocyte nuclear maturation, but increased the decondensation of sperm head (p < 0.05) and decreased the pronuclear formation (p < 0.05). In turn, the addition of O₂?^‐ and H₂O₂ scavenging systems during in vitro maturation did not modify the percentage of oocytes reaching metaphase II nor the oocytes with decondensed sperm head or pronuclei after fertilization. However, both parameters increased in the presence of cysteine (p < 0.05). The exogenous generation of O₂?^‐ and H₂O₂ during oocyte in vitro maturation would not affect nuclear maturation or later sperm penetration, but most of the spermatozoa cannot progress to form the pronuclei after fusion with the oocyte. The decrease in endogenous ROS levels by the addition of cysteine would improve pronuclear formation after sperm penetration.     

Calcium homeostasis and its relationship to superoxide production in blood and milk neutrophils of lactating goats

Polymorphonuclear neutrophils (PMN), which comprise over 70% of the somatic cells in goat milk, are a major cellular component of innate immunity in the goat mammary gland. However, the function of milk PMNs is modified after diapedesis compared to PMNs in blood. As many aspects of PMN activity depend directly on intracellular Ca²⁺ concentration ((Ca²⁺)_i), the present study aimed to determine the changes in Ca²⁺ homeostasis of milk PMNs from lactating goats compared to autologous blood PMNs, and to examine the significance of these variations to the immuno-competency of milk PMNs. The intracellular Ca²⁺ store of freshly prepared milk cells was estimated from the elevation of (Ca²⁺)_i after ionomycin treatment, which was found to be significantly less than blood PMNs. Replenishment of the intracellular Ca²⁺ store in milk cells after intracellular Ca²⁺ depletion by Bapta-AM followed by spiking with 2.5 mM Ca²⁺ for 20 min was also compared to that of blood PMNs, showing that after depletion/spiking the intracellular Ca²⁺ store in milk cells was much less than blood PMNs. The production of superoxide anion (O₂^?) in vitro in response to (Ca²⁺)_i-dependent or (Ca²⁺)_i-independent modulators was used to evaluate the relevance of altered Ca²⁺ homeostasis on the immuno-competency of milk cells compared to blood PMNs. The results indicated that milk cells produced similarly low levels of O₂^? as blood PMNs when treated with ionomycin. However, the amount of O₂^? produced by milk cells in response to phorbol 12-myristate 13-acetate (PMA) stimulation, although greater than ionomycin treatment, was significantly less than that of blood PMNs. The capacity for O₂^? production by both cell types in response to PMA reverted to the resting state with use of the protein kinase C (PKC) inhibitor, staurosporine. In conclusion, the current study demonstrated an irreversible shortage of intracellular Ca²⁺ in the milk PMNs of lactating goats compared to blood PMNs. It also showed that preliminary O₂^–production, primed by ionomycin treatment, remained unchanged in milk PMNs, despite the shortage in intracellular Ca²⁺, but decreased O₂^? production capacity, mediated via the PKC pathway, in milk PMN. It is suggested that the defects in Ca²⁺ homeostasis in milk PMNs of lactating goats is partially attributable for the post-diapedesis functionality modifications.     

The chemiluminescent response of bovine polymorphonuclear leucocytes isolated from milk and blood

Polymorphonuclear leucocytes (PMN) were isolated from milk and blood of healthy cows, and the generation of reactive oxygen by the two cell populations was compared by measuring chemiluminescence (CL) after stimulation with zymosan. The ratio of milk to blood PMN CL was relatively constant in a given animal, but varied widely between different cows, ranging from 0.3 to 1.3.The relative contributions of various oxygen species to CL was studied by measuring quenching using different oxygen scavengers. While the relative contributions of H₂O₂, ^?O₂ and ′O₂ seemed to be similar in both milk and blood PMN, the OH· radical was clearly more prominent in PMN isolated from milk than from blood. In addition, blood PMN CL was more dependent on the presence of glucose in the reaction medium than milk PMN CL. Furthermore, the CL response to phorbol myristate acetate, to the Ca ionophore A23187 and to Sendai virus was different in the two cell types. The results suggest that CL generation in milk PMN differs from that in blood PMN in quantitative as well as qualitative aspects.     

Response of Thawed Epidi dymal Red Deer Spermatozoa to Increasing Concentrations of Hydrogen Peroxide,and Importance of Individual Male Variability

Oxidative stress represents a challenge during sperm manipulation. We have tested the effect of increasing hydrogen peroxide (H₂O₂) levels on red deer spermatozoa after cryopreservation, and the role of male‐to‐male variation in that response. In a first experiment, eight thawed samples were submitted to 0, 25, 50, 100 and 200 μm H₂O₂ for 2 h at 37°C. Intracellular reactive oxygen species (H₂DCFDA‐CM) increased with H₂O₂ concentration, but we only detected a decrease in sperm function (motility by CASA and chromatin damage by sperm chromatin structure assay) with 200 μm . Lipoperoxidation assessed by the thiobarbituric acid reactive substance (TBARS) method increased slightly with 50 μm H₂O₂ and above. In a second experiment, samples from seven males were submitted to 0 and 200 μm H₂O₂ for 2 h, triplicating the experiment within each male. Males differed at thawing and regarding their response to incubation and H₂O₂ presence. We found that the kinematic parameters reflected male‐to‐male variability, whereas the response of the different males was similar for lipid peroxidation and viability. A multiparametric analysis showed that males grouped differently if samples were assessed after thawing, after incubation without H₂O₂ or after incubation with H₂O₂. Red deer spermatozoa are relatively resilient to H₂O₂ after thawing, but it seems to be a great male‐to‐male variability regarding the response to oxidative stress. The acknowledgement of this individual variability might improve the development of optimized sperm work protocols.     

The effect of conglutinin on production of reactive oxygen species in bovine granulocytes

Conglutinin is a high molecular-weight lectin originally detected in bovine serum. It belongs to the family of collectins that bind sugar residues in a Ca²⁺-dependent manner and are effector molecules in innate immunity. Conglutinin appears to play an important role in immune defense mechanisms, showing antiviral and antibacterial activities when tested in vivo and in vitro. The present study evaluated the effect of conglutinin on the respiratory bursts in bovine peripheral phagocytes. Using nitroblue tetrazolium and hydrogen peroxide assays, we showed that sugar ligand-bound conglutinin stimulated the production of superoxide and H₂O₂ in granulocytes whereas the non-sugar-bound form of conglutinin inhibited these processes. These results indicate that both forms of conglutinin are able to interact with surface leukocyte receptors but have opposite effects on phagocytic activity. Our findings suggest that conglutinin bound to sugar residues on microbial surfaces can induce oxygen burst in phagocytes, and thereby mediates the elimination of pathogens and prevents the spread of infection.     

The Effect of Activation of Granulocytes on Enzyme Release and Hydrogen Peroxide and Superoxide Production in Buffaloes

Singh, V.K., More, T. and Singh, S., 1997. The effect of activation of granulocytes on enzyme release and hydrogen peroxide and superoxide production in buffaloes. Veterinary Research Communications, 21 (4), 241-247Polymorphonuclear cells kill microorganisms by the stock of antibiotic proteins and peptides stored in their lysosomal granules and have the ability to produce reactive oxygen intermediates (ROI) such as H₂O₂, O ₂ ^– , and HOCl. Since the components involved in the microbicidal functions of buffalo (Bos bubalis) polymorphonuclear cells (PMN) have not been characterized, an assessment was made of the levels of various enzymes, the extent of extracellular release of these enzymes, and also their ability to produce H₂O₂/O ₂ ^– upon activation with opsonized zymosan (OZ) or lipopolysaccharide (LPS). Using GPC-HPLC, OZ was shown to be a more potent secretagogue than LPS, causing a significantly greater release of low-molecular-weight components. Varying levels of the enzymes (myeloperoxidase, lactate dehydrogenase, acid and alkaline phosphatases, -galactosidase, -D-glucuronidase, elastase and lysozyme) were recorded in the buffalo PMN and both the activators (OZ and LPS) caused significant release of all the enzymes except alkaline phosphatase. Both the activators also caused a significant increase in H₂O₂/O ₂ ^– production by the PMN. However, OZ caused a more pronounced activation than LPS. The studies revealed the presence of oxygen-dependent and oxygen-independent microbicidal systems with buffalo PMN, which responded more effectively to zymosan activation.     

Ouabain-sensitive respiration and protein synthesis in duodenal mucosa and liver in rats fed increasing levels of pea fibre or protein and housed in 18 or 28°C environments

Seventy‐two Wistar rats were used in two studies to investigate the effect of environmental temperature (18 or 28°C), and increasing levels of dietary fibre (low, 68 g/kg dry matter (DM); medium 110 g/kg DM; high, 157 g/kg DM) and protein (low, 91 g/kg DM; medium, 171 g/kg DM; high, 262 g/kg DM) on respiration attributable to Na⁺,K⁺‐ATPase activity and protein synthesis in duodenal mucosa and liver of rats. In vitro O₂ consumption in tissues was measured polarographically using a Clark‐style YSI biological O₂ monitor. Whole‐body O₂ consumption was measured with two open‐circuit respiration chambers. Whole‐body O₂ consumption was higher (p < 0.05) at 18°C than at 28°C. Rats fed the low protein diet had significantly higher (p < 0.05) whole‐body O₂ consumption than those fed the medium or high protein diet. Compared with 28°C, the environmental temperature of 18°C caused an increase (p < 0.05) in total O₂ consumption and O₂ consumption attributable to Na⁺,K⁺‐ATPase activity in duodenal mucosa. There was no effect (p > 0.05) of environmental temperature on total O₂ consumption, Na⁺,K⁺‐ATPase activity attributable to protein synthesis dependent on O₂ consumption in the liver. Total O₂ consumption and O₂ consumption attributable to Na⁺,K⁺‐ATPase activity increased (p < 0.05) in duodenal mucosa in rats fed the low level of dietary fibre compared with rats fed the medium level of dietary fibre. In vitro O₂ consumption determined in duodenal mucosa and in liver did not always correspond to whole‐body O₂ consumption. This may indicate that respiration in the duodenum and liver adapts differently and may not reflect changes in whole‐body respiration in response to dietary modification and changes in thermal environment.     

Effect of dihydrotestosterone on mouse embryonic stem cells exposed to H2O2-induced oxidative stress

Oxidative stresses induced by reactive oxygen species (ROS) have been shown to be involved in several physiological and pathophysiological processes, such as cell proliferation and differentiation. Steroid hormones can protect cells against apoptosis or induce cell proliferation by several mechanisms. Among androgenic hormones, dihydrotestosterone (DHT) is generated by a 5α-reduction of testosterone. Unlike testosterone, DHT cannot be aromatized to estradiol, therefore DHT is considered a pure androgenic steroid. This study was conducted to examine the effect of DHT (10^-7 M) on H₂O₂ (10^-3 M) -induced injuries in mouse embryonic stem (ES) cells. H₂O₂ induced ROS generation and increased lipid peroxide formation and DNA fragmentation. These effects of H₂O₂ were inhibited by pretreatment with DHT. H₂O₂ also increased the phosphorylation of p38 MAPK, SAPK/JNK and nuclear factor kappa B (NF-κB), but DHT blocked these effects. Moreover, H₂O₂ decreased DNA synthesis and the levels of cell cycle regulatory proteins [cyclin D1, cyclin E, cyclin-dependent kinase (CDK) 2, and CDK 4]. These effects of H₂O₂ were inhibited by pretreatment with DHT. In conclusion, DHT may partially prevent H₂O₂-induced cell injury through inhibition of ROS and ROS-induced activation of p38 MAPK, SAPK/JNK and NF-κB in mouse ES cells.     

Quantitative morphology in canine cutaneous soft tissue sarcomas

Stained cytological specimens from 24 dogs with spontaneous soft tissue sarcomas [fibrosarcoma (n = 8), liposarcoma (n = 8) and haemangiopericytoma (n = 8)], and 24 dogs with reactive connective tissue lesions [granulation tissue (n = 12) and dermal fibrosis (n = 12)] were analysed by computer‐assisted nuclear morphometry. The studied morphometric parameters were: mean nuclear area (MNA; µm²), mean nuclear perimeter (MNP; µm), mean nuclear diameter (MND mean; µm), minimum nuclear diameter (D_min; µm) and maximum nuclear diameter (D_max; µm). The study aimed to evaluate (1) possibility for quantitative differentiation of soft tissue sarcomas from reactive connective tissue lesions and (2) by using cytomorphometry, to differentiate the various histopathological soft tissue sarcomas subtypes in dogs. The mean values of all nuclear cytomorphometric parameters (except for D_max) were statistically significantly higher in reactive connective tissue processes than in soft tissue sarcomas. At the same time, however, there were no considerable differences among the different sarcoma subtypes. The results demonstrated that the quantitative differentiation of reactive connective tissue processes from soft tissue sarcomas in dogs is possible, but the same was not true for the different canine soft tissue sarcoma subtypes. Further investigations on this topic are necessary for thorough explication of the role of quantitative morphology in the diagnostics of mesenchymal neoplasms and tumour‐like fibrous lesions in dogs     

The effects of physostigmine on the electroretinogram in the beagle dog

The purpose of the study was to correlate electroretinogram (ERG) parameters with increasing levels of plasma, erythrocyte and ocular tissue cholinesterase inhibition using the beagle dog as a model for human neurovisual toxicity. The anticholinesterase compound physostigmine was administered at various steady-state intravenous infusion rates based on pharmacokinetic estimates of plasma and red blood cell cholinesterase inhibition. The most sensitive parameter was the b-wave amplitude of the rod response, which was significantly depressed compared to pretreatment at all levels of acute cholinesterase depression. The overall maximal ERG response demonstrated a trend of declining a-and b-wave amplitudes, which corresponded with the increased levels of cholinesterase depression, but these differences were not significant. The depression of the electroretinogram rod and cone amplitudes appeared to parallel plasma cholinesterase inhibition more closely than erythrocyte cholinesterase activity. Ocular tissue cholinesterase activity was significantly depressed in the retina (70%), cornea (60%) and dorsal rectus extraocular muscle (46%). Electroretinography may be a useful physiological tool for evaluating the ocular toxicity of certain chemicals or pharmaceuticals associated with cholinesterase biomarker activity.Abbreviations AChE acetylcholinesterase - Amp amplitude - BuChE butyrylcholinesterase - C _p plasma level - C _ss steady-state plasma concentration - D _L loading dose - e ^–kt the rate of elimination over time (t) - ERG electroretinogram - k elimination rate constant - Lat latency - MBW metabolic body weight - O₁–O₅ consecutive oscillatory potential wavelets - PreTx pretreatment - R rate of infusion - R _Inf rate of infusion - V _d volume of distribution     

Pharmacokinetics of hemoglobin-based oxygen carrier hemoglobin-glutamer-200 bovine (oxyglobin) in the horse

Oxyglobin (OXY) is a hemoglobin‐based oxygen carrier (HBOC) made of glutaraldehyde‐polymerized bovine hemoglobin (bHb). Products similar to OXY are under development for use as temporary blood substitutes in trauma, shock and anemia. Since they all may increase blood O₂‐carrying capacity and thus, possibly tissue oxygenation, they may also be used to enhance performance of both equine and human athletes. That is why HBOCs are banned from use in athletic competition. Our goal was to determine the pharmacokinetics of OXY after intravenous (IV) infusion to horses. Blood and urine samples were collected from adult horses that received an IV dose of 32.5 g of OXY. Concentrations of OXY in plasma and urine were quantified using a newly developed LC/Q‐TOF‐MS/MS detection technique. Level of quantification (LOQ) was 50 μg mL^–1. The decline of the plasma concentration‐time curve of the HBOC was described by a 2‐compartment model (C1 and C2). The median distribution alpha (t1/2k1,0) and elimination beta (t1/2k2,0) half‐lives were 1.3 and 12.0 hours, respectively. The bHb molecules in OXY are not of uniform size and vary substantially in molecular weight (MW). Of the OXY molecules 53% were eliminated in C1, which represented the smaller MW molecules and 47% in C2, which represented the larger MW bHb. The maximal 0‐time plasma concentration was 662.0 μg/mL and declined to 97.1 μg mL^–1 at 24 h. The area below the plasma concentration‐time curve was 5143 μg h^–1 mL^–1. The volumes of C₁ and C₂ were 86.9 and 63.9 mL kg^–1, respectively. Oxyglobin was not detected in urine. This study shows the detection and quantification in equine plasma of a HBOC following IV infusion and demonstrates the short half‐life of about 50% of infused bHb molecules.     

Innate immunity and host defense peptides in veterinary medicine

Recent years have witnessed a surge in interest directed at innate immune mechanisms. Proper conceptualization of the key elements of innate immunity, however, is still a work in progress, because most research in immunology traditionally has been focused on components of the acquired immune response. The question of why an animal stays healthy in a world filled with many dangers is perhaps as interesting as why it sometimes surrenders to disease. Consequently, studies with an increased focus on inborn mechanisms of animal host defense may help further the development of appropriate preventative and therapeutic measures in veterinary medicine. Host defense peptides (HDPs) are central effector molecules of innate immunity, and are produced by virtually all living species throughout the plant and animal kingdoms. These gene-encoded peptides play a central role in multiple, clinically relevant disease processes. Imbalances in the expression of HDPs can lead to overt pathology in different organ systems and cell types in all species studied. In addition, HDPs are an ancient group of innate chemical protectors, which are now evaluated as model molecules for the development of novel natural antibiotics and immunoregulatory compounds. This review provides an overview of HDPs and is aimed at veterinary practitioners as well as basic researchers with an interest in comparative immunology involving small and large animal species.     

Phenotypic and functional analysis of bovine peripheral blood dendritic cells before parturition by a novel purification method

Dendritic cells (DCs) are specialized antigen presenting cells specializing in antigen uptake and processing, and play an important role in the innate and adaptive immune response. A subset of bovine peripheral blood DCs was identified as CD172a⁺/CD11c⁺/MHC (major histocompatibility complex) class II⁺ cells. Although DCs are identified at 0.1%–0.7% of peripheral blood mononuclear cells (PBMC), the phenotype and function of DCs remain poorly understood with regard to maintaining tolerance during the pregnancy. All cattle used in this study were 1 month before parturition. We have established a novel method for the purification of DCs from PBMC using magnetic‐activated cell sorting, and purified the CD172a⁺/CD11c⁺ DCs, with high expression of MHC class II and CD40, at 84.8% purity. There were individual differences in the expressions of CD205 and co‐stimulatory molecules CD80 and CD86 on DCs. There were positive correlations between expression of cytokine and co‐stimulatory molecules in DCs, and the DCs maintained their immune tolerance, evidenced by their low expressions of the co‐stimulatory molecules and cytokine production. These results suggest that before parturition a half of DCs may be immature and tend to maintain tolerance based on the low cytokine production, and the other DCs with high co‐stimulatory molecules may already have the ability of modulating the T‐cell linage.     

The pathology of brucellosis reflects the outcome of the battle between the host genome and the Brucella genome

The successful co-existence of each Brucella spp. with its preferred host is the outcome of ancient co-evolutionary relationships and selection pressures that often result in a stalemate where the pathogen has evolved to survive within the biological systems of the host, and the host has evolved innate and acquired immune systems which allow controlled survival of infection by the pathogen, ultimately supporting the survival of the host-pathogen system. In general, Brucella spp. have evolved a similar fundamental pathogenesis of facultative intracellular parasitism though the predominant route of natural exposure varies from oropharynx to genital tract, as does the preferred tissue and cellular tropism, e.g. non-professional placental trophoblasts, fetal lung, professional macrophages of reticulendothelial system, and the male and female reproductive tracts. The morphogenesis of the pyogranulomatous lesions stimulated by Brucella reflects the nature of the persistent parasitism, i.e. genome versus genome. The question is, how can this perplexing array of survival mechanisms be unraveled? Fortunately, the integration of real-time image analysis, cell biology, genome-wide analysis, proteomics and bioinformatics holds the most promise ever for the global analysis of the Brucella infectious process and the host:pathogen interface leading to a clearer understanding of the interactions of these biological systems. These discoveries will be expected to provide a frameshift in rationales for interrupting and/or controlling brucellosis at host and/or pathogen levels.     

Nitric Oxide and Superoxide Anion Production During Heparin‐Induced Capacitation in Cryopreserved Bovine Spermatozoa

The aim of this work was to quantify NO, O₂^? and ONOO^? production during heparin‐induced capacitation of cryopreserved bovine spermatozoa. A time dependent hyperbolic increase was observed for heparin‐dependent capacitation, O₂ uptake, and NO production. Conversely, O₂^? production was increased during the first 15 min of incubation, showing a decrease from this time until 45 min. At 15 min of heparin incubation, a threefold increase in O₂ consumption (5.9 ± 0.6 nmol/min × 10⁷ cells), an enhancement in NO release (1.1 ± 0.2 nmol/min × 10⁷ cells), and a five‐fold increase in O₂^? production (1.3 ± 0.07 nmol/min × 10⁷ cells), were observed. Peroxynitrite production rate was estimated taking into account NO and O₂^? generation and the second‐order rate constant of the reaction between these species. To conclude, heparin‐induced capacitation of cryopreserved bovine spermatozoa activates (i) mitochondrial O₂ uptake by high ADP levels due to increased energy requirements, (ii) NO production by a constitutive NOS and (iii) O₂^? production by a membrane‐bound NAD(P)H oxidase. The products of both enzymes are released to the extracellular space and could be involved in the process of sperm capacitation.     

Pituitary function in the acute phase response in domestic farm animals: cytokines,prostaglandins, and secretion of ACTH

Contained in this report is a review of available data on pituitary cytokines in domestic species of agricultural importance. The concept is advanced that the pituitary gland is essential to appropriate generation of host defense mechanisms and thus should be considered among other tissues contributing to innate immunity. The functions of these intrapituitary cytokines, principally IL-6, are discussed in the context of potential regulation of the pituitary-adrenal axis (ACTH secretion) via intrapituitary PGE₂ generation during the acute-phase response to infectious/inflammatory stimuli. Data from other species are cited as appropriate for comparative purposes and elaboration of proposed mechanisms. However, the scope of the review is not intended to comprehensively cover the vast literature on proinflammatory cytokines and prostaglandins generated peripherally and centrally during host responses to inflammatory stimuli.     

1,25‐Dihydroxyvitamin D3 and its analogues increase catalase at the mRNA,protein and activity level in a canine transitional carcinoma cell line

Antioxidant enzymes, such as catalase, superoxide dismutases (SOD), MnSOD and Cu/ZnSOD, protect cells by scavenging reactive oxygen species (ROS). Numerous studies have reported the anti‐cancer effects of 1,25‐dihydroxyvitamin D₃ (calcitriol) and its related analogues, seocalcitol and analogue V. In this study, canine bladder transitional cell carcinoma (cbTCC) cells were used to determine effects of calcitriol and its related analogues on antioxidant enzyme gene expression, protein expression and activity. Catalase mRNA was increased in response to calcitriol (10^?7 M), and seocalcitol (10^?7 and 10^?9 M). MnSOD mRNA was decreased in response to calcitriol at 10^?7 M. Catalase was significantly increased in response to calcitriol (10^?7 and 10^?9 M), and seocalcitol (10^?9 M). Catalase enzymatic activity increased in response to calcitriol, seocalcitol and analogue V (10^?9 M). In addition, global gene expression analysis identified the involvement of mitogen‐activated protein kinase (MAPK) signalling in cbTCC's response to calcitriol and seocalcitol treatment.     

The effects of hyperoxia on the biosynthesis of cyclooxygenase products and haemodynamic response to nitric oxide synthase inhibition with L-NAME in endotoxaemic pigs

The interaction between constitutive nitric oxide and oxygen may depend on the degree of tissue oxygenation and may play a critical role in the pathophysiological response to endotoxaemia. We investigated if hyperoxia (100% O₂) attenuated the systemic and pulmonary vasoconstriction and increased biosynthesis of thromboxane B₂ (TXB₂) and 6-keto-prostaglandin (PG) F_1α induced by inhibition of nitric oxide synthase with N^G-nitro-l -arginine-methyl-ester (L-NAME) in a porcine model of endotoxaemia. Twenty-two domestic, random source pigs, weighing 15.4 ± 2.7 kg (mean ± standard deviation) were the subjects of this study. Pigs were anaesthetized with isoflurane in 100% O₂, orotracheally intubated and ventilated to maintain normocapnia, and then instrumented for haemodynamic monitoring. Following instrumentation, pigs were maintained at an end-tidal isoflurane concentration of 2%. Pigs were randomly assigned to treatment groups: saline + 30% O₂ (Control, n = 6); Escherichia coli lipopolysaccharide (5 μg/kg/h from 1 to 2 h followed by 2 μg/kg/h from 2 to 5 h) + 30% O₂ (LPS, n = 4); L-NAME (0.5 mg/kg/h, from 0 to 5 h) + LPS + 100% O₂ (n = 6); and L-NAME + LPS + 30% O₂ (n = 6). L-NAME and endotoxin significantly (P < 0.05) increased mean arterial pressure, mean pulmonary arterial pressure, and systemic and pulmonary vascular resistance index beginning at 90 min. When results were pooled across all time periods, mean arterial pressure and mean pulmonary arterial pressure were significantly higher in the L-NAME + LPS + 30% O₂ group than all other groups, reflecting pulmonary and systemic vasoconstriction. Hyperoxia attenuated the L-NAME + LPS-induced increases in TXB₂ and 6-keto-PGF_1α concentrations at 90 and 120 min and 120 min, respectively, although the differences were not statistically significant. These results support the observation that nitric oxide synthase inhibition with L-NAME has deleterious haemodynamic effects in this model of endotoxaemia. The temporal attenuation of L-NAME-induced pulmonary and systemic vasoconstriction by hyperoxia suggested that the haemodynamic effects of acute endotoxaemia were in part influenced by the relative amounts of nitric oxide and oxygen present.