Organic osmolytes increase expression of specific tight junction proteins in skin and alter barrier function in keratinocytes.

BACKGROUND: The epidermal barrier is important for water conservation, failure of which is evident in dry-skin conditions. Barrier function is fulfilled by the stratum corneum, tight junctions (TJs, which control extracellular water) and keratinocyte mechanisms, such as organic osmolyte transport, which regulate intracellular water homeostasis. Organic osmolyte transport by keratinocytes is largely unexplored and nothing is known regarding how cellular and extracellular mechanisms of water conservation may interact. OBJECTIVES: We aimed to characterize osmolyte transporters in skin and keratinocytes, and, using transporter inhibitors, to investigate whether osmolytes can modify TJs. Such modification would suggest a possible link between intracellular and extracellular mechanisms of water regulation in skin. METHODS: Immunostaining and quantitative polymerase chain reaction of organic osmolyte-treated organ-cultured skin were used to identify changes to organic osmolyte transporters, and TJ protein and gene expression. TJ functional assays were performed on organic osmolyte-treated primary human keratinocytes in culture. RESULTS: Immunostaining demonstrated the expression of transporters for betaine, taurine and myo-inositol in transporter-specific patterns. Treatment of human skin with either betaine or taurine increased the expression of claudin-1, claudin-4 and occludin. Osmolyte transporter inhibition abolished this response. Betaine and taurine increased TJ function in primary human keratinocytes in vitro. CONCLUSIONS: Treatment of skin with organic osmolytes modulates TJ structure and function, which could contribute to the epidermal barrier. This emphasizes a role for organic osmolytes beyond the maintenance of intracellular osmolarity. This could be harnessed to enhance topical therapies for diseases characterized by skin barrier dysfunction.

CRISPR/Cas9 mediated generation of an ovine model for infantile neuronal ceroid lipofuscinosis (CLN1 disease).

The neuronal ceroid lipofuscinoses (NCLs) are a group of devastating monogenetic lysosomal disorders that affect children and young adults with no cure or effective treatment currently available. One of the more severe infantile forms of the disease (INCL or CLN1 disease) is due to mutations in the palmitoyl-protein thioesterase 1 (PPT1) gene and severely reduces the child's lifespan to approximately 9 years of age. In order to better translate the human condition than is possible in mice, we sought to produce a large animal model employing CRISPR/Cas9 gene editing technology. Three PPT1 homozygote sheep were generated by insertion of a disease-causing PPT1 (R151X) human mutation into the orthologous sheep locus. This resulted in a morphological, anatomical and biochemical disease phenotype that closely resembles the human condition. The homozygous sheep were found to have significantly reduced PPT1 enzyme activity and accumulate autofluorescent storage material, as is observed in CLN1 patients. Clinical signs included pronounced behavioral deficits as well as motor deficits and complete loss of vision, with a reduced lifespan of 17 ± 1 months at a humanely defined terminal endpoint. Magnetic resonance imaging (MRI) confirmed a significant decrease in motor cortical volume as well as increased ventricular volume corresponding with observed brain atrophy and a profound reduction in brain mass of 30% at necropsy, similar to alterations observed in human patients. In summary, we have generated the first CRISPR/Cas9 gene edited NCL model. This novel sheep model of CLN1 disease develops biochemical, gross morphological and in vivo brain alterations confirming the efficacy of the targeted modification and potential relevance to the human condition.

Differential expression and functionality of ATP-binding cassette transporters in the human hair follicle.

BACKGROUND: ATP-binding cassette (ABC) transporters are involved in the active transport of an extremely diverse range of substrates across biological membranes. These transporters are commonly implicated in the development of multidrug resistance and are also involved in numerous physiological and homeostatic processes, including lipid transport, cell migration and differentiation. OBJECTIVES: To close the knowledge gap in the expression of ABC transporters in the human hair follicle (HF). METHODS: Quantitative polymerase chain reaction (qPCR) of ABC genes and immunofluorescence microscopy analysis of cryosections of human HFs. RESULTS: By qPCR analysis, numerous members of the ABC transporter superfamily, such as ABCB1, ABCG2 and ABCA12, were found to be transcribed in full-length human scalp HFs. Immunofluorescence microscopy demonstrated that the intrafollicular protein expression of different xenobiotic ABC transporters (ABCB1, ABCC1, ABCC4, ABCG2) varies greatly, with ABCG2 expression restricted primarily to the epithelial stem cell region of the outer root sheath (bulge), whereas expression of ABCB1, ABCC1 and ABCC4 was more widespread. Lipid transporters ABCA1, ABCA12 and ABCA4 were almost uniformly expressed throughout the HF epithelium. Functional ABCB1/G2 activity was demonstrated by exclusion of the substrate dye, Hoechst 33342. In the bulge, this was reversed by ABCB1 and ABCG2 inhibition. CONCLUSIONS: These data encourage further investigation of ABC transporters as potentially important regulators of HF epithelial biology. Clinically, pharmacological modulation of the activity of selected intrafollicular ABC transporters may permit novel therapeutic interventions, such as protecting HF stem cells from chemotherapy-induced damage, counteracting cholesterol-associated hypertrichosis, and manipulating the intrafollicular prostaglandin balance in androgenetic alopecia.

Rab3Gap1 mediates exocytosis of Claudin-1 and tight junction formation during epidermal barrier acquisition.

Epidermal barrier acquisition during late murine gestation is accompanied by an increase in Akt kinase activity and cJun dephosphorlyation. The latter is directed by the Ppp2r2a regulatory subunit of the Pp2a phosphatase. This was accompanied by a change of Claudin-1 localisation to the cell surface and interaction between Occludin and Claudin-1 which are thought to be required for tight junction formation. The aim of this study was to determine the nature of the barrier defect caused by the loss of AKT/Ppp2r2a function. There was a paracellular barrier defect in rat epidermal keratinocytes expressing a Ppp2r2a siRNA. In Ppp2r2a knockdown cells, Claudin-1 was located to the cytoplasm and its expression was increased. Inhibiting cJun phosphorylation restored barrier function and plasma membrane localisation of Claudin-1. Expression of the Rab3 GTPase activating protein, Rab3Gap1, was restored in Ppp2r2a siRNA cells when cJun phosphorylation was inhibited. During normal mouse epidermal development, Claudin-1 plasma membrane localisation and Rab3Gap1 cell surface expression were co-incident with Akt activation in mouse epidermis, strongly suggesting a role of Rab3Gap1 in epidermal barrier acquisition. Supporting this hypothesis, siRNA knockdown of Rab3Gap1 prevented plasma membrane Claudin-1 expression and the formation of a barrier competent epithelium. Replacing Rab3Gap1 in Ppp2r2a knockdown cells was sufficient to rescue Claudin-1 transport to the cell surface. Therefore these data suggest Rab3Gap1 mediated exocytosis of Claudin-1 is an important component of epidermal barrier acquisition during epidermal development.

Altered claudin expression is a feature of chronic plaque psoriasis.

Epithelial tight junctions play a central role in cell-cell adhesion and are necessary for the selective paracellular movement of ions. Claudins are key components of tight junctions and their expression is altered in gut epithelia in a variety of inflammatory enteropathies, including ulcerative colitis and Crohn's disease. Psoriasis is a chronic inflammatory skin disease affecting approximately 2% of the western population, with significantly increased occurrence in individuals with Crohn's disease. Initial studies investigated the expression of claudins in skin of healthy volunteers and patients with chronic plaque psoriasis. We report here that claudins-1 and -3 are the major protein species present in the epidermis of healthy skin; they are expressed on the surface of epidermal keratinocytes, consistent with their localization to tight junctions. In plaques of psoriasis, claudin-1 was not identifiable in the epidermis, although typical staining patterns were observed in clinically normal, uninvolved skin of patients with psoriasis. Claudin-3 was present in the epidermal granular cell layer in normal skin, but was only identified within the cytosol of epidermal keratinocytes in both involved and uninvolved skin of psoriasis patients. We examined further whether exposure of keratinocytes in vitro to pro-inflammatory cytokines mimicked the observed changes in claudin expression seen in chronic plaque psoriasis; lipopolysaccharide, interferon-gamma and tumour necrosis factor-alpha had no effect on claudin protein expression or distribution. Addition of interleukin-1beta, however, resulted in down-regulation of claudins-1 and -3. Tumour necrosis factor-alpha and interleukin-1beta were further used in an in vivo model of skin inflammation; interleukin-1beta alone modulated claudin protein expression in this system. These data demonstrate that epidermal claudin expression is altered in chronic plaque psoriasis and that expression is in part modulated by interleukin-1beta.

Resolution of P-glycoprotein and non-P-glycoprotein effects on drug permeability using intestinal tissues from mdr1a (-/-) mice.

1. Intestinal xenobiotic transporters are a significant barrier to the absorption of many orally administered drugs. P-glycoprotein (PGP) is the best known, but several others, including members of the multidrug resistance-associated protein (MRP) family, are also expressed. Definitive information on their precise effect on intestinal drug permeability is scarce due to a lack of specific inhibitors and the difficulty of studying non-PGP activity in the presence of high PGP expression. 2. We have investigated the in vitro use of intestinal tissues from PGP knockout (mdr1a (-/-)) mice as a tool for dissecting the mechanisms of intestinal drug efflux. The permeability characteristics of digoxin (DIG), paclitaxel (TAX) and etoposide (ETOP) were measured in ileum from mdr1a (-/-) and wild-type (FVB) mice mounted in Ussing chambers. 3. DIG and TAX exhibited marked efflux across FVB tissues (B-A : A-B apparent permeability (P(app)) ratio 10 and 17 respectively) which was absent in mdr1a (-/-) tissues, confirming that PGP is the sole route of intestinal efflux for these compounds. The A-B P(app) of both compounds was 3 - 5 fold higher in mdr1a (-/-) than in FVB. 4. Polarized transport of ETOP in FVB tissues was reduced but not abolished in mdr1a (-/-) tissues. Residual ETOP efflux in mdr1a (-/-) tissues was abolished by the MRP inhibitor MK571, indicating involvement of both PGP and MRP. 5. MK571 abolished calcein efflux in mdr1a (-/-) tissues, while quinidine had no parallel effect in FVB tissues, suggesting involvement of MRP but not PGP. 6. Tissues from mdr1a (-/-) mice provide a novel approach for investigating the influence of PGP ablation on intestinal permeability and for resolving PGP and non-PGP mechanisms that modulate drug permeability.

Kinetic profiling of P-glycoprotein-mediated drug efflux in rat and human intestinal epithelia.

Intestinal drug efflux mediated by P-glycoprotein and other ABC transporters is widely accepted as a reason for low or variable oral absorption. However, little is known about species and regional differences in P-glycoprotein so the functional and predictive relevance of observations made in cell models such as Caco-2 is uncertain. The aim of this study was to define the kinetics of drug efflux in rat and human intestinal tissues in vitro using the "reference" substrates digoxin and vinblastine. The expression and functional role of other ABC transporters in the transport of these compounds was also investigated. Saturable, verapamil-sensitive efflux of digoxin was observed in all intestinal regions. Apparent affinity of the efflux process varied within a relatively narrow range (50-92 microM), increasing in rat from small to large intestine. In contrast, maximal transporter activity varied over a 4- to 5-fold range with ileum > jejunum > colon. Similar regional differences in efflux were also observed with vinblastine. Maximal efflux levels were similar in Caco-2 and ileum for both substrates, suggesting that Caco-2 may quantitatively predict small intestinal drug efflux. Digoxin efflux kinetics was virtually identical in rat and human colon. Inhibitor studies showed that digoxin and vinblastine efflux in intestinal tissues was mediated by P-glycoprotein, although a minor component could be attributed to multidrug resistance-related protein (MRP)-like transporters in Caco-2. This study has analyzed the differential functional expression of drug efflux along the gastrointestinal tract. Such data will be critical in developing predictive models of P-glycoprotein-mediated efflux using information gathered from in vitro systems.

Calcium mobilization is required for spreading in human osteoblasts.

Adhesion-induced changes in intracellular calcium concentration ([Ca2+]i) were measured in populations of human osteoblasts spreading on bone matrix proteins. In cells spreading on collagen type I, fibronectin, or laminin, average values for [Ca2+]i were found to increase approximately 2x over baseline and then decline. The speed with which [Ca2+]i increased and declined was dependent upon the matrix protein on which the cells were plated but was generally complete within 1 hour from the time of plating. Calcium mobilization was found to be due to influx of calcium across the osteoblast plasma membrane and was integrin dependent. Carboxyamido triazole (CAI), a specific inhibitor of nonvoltage-dependent calcium channels, or BAPTA-AM, a chelator of intracellular calcium, inhibited osteoblast adhesion and spreading on collagen type I, fibronectin and laminin in a dose-dependent manner. In conclusion, these results demonstrate that calcium mobilization is induced upon integrin-ligand contact and that calcium influx is required for cell adhesion and spreading.

Preclinical safety evaluation of rhuMAbVEGF, an antiangiogenic humanized monoclonal antibody.

Recombinant humanized antivascular endothelial growth factor (rhuMAbVEGF) is a monoclonal IgG1 antibody that is being developed as an antiangiogenic agent for use in treating a variety of solid tumors. Preclinical safety studies included an immunohistochemical tissue cross-reactivity study, in vitro hemolytic potential and blood compatibility studies, and multiple dose toxicity studies. Toxicity studies were conducted in cynomolgus monkey because rhuMAbVEGF is pharmacologically active in this species and does not bind rat or mouse vascular endothelial growth factor (VEGF). Following twice weekly administration of rhuMAbVEGF for 4 or 13 wk, young adult cynomolgus monkeys exhibited physeal dysplasia characterized by a dose-related increase in hypertrophied chondrocytes, subchondral bony plate formation, and inhibition of vascular invasion of the growth plate. In addition, decreased ovarian and uterine weights and an absence of corpora lutea were observed in females receiving 10 and 50 mg/kg/dose in the 13-wk study. Both the physeal and ovarian changes were reversible with cessation of treatment. No other treatment-related effects were observed following rhuMAbVEGF administration at doses up to 50 mg/kg. These findings indicate that VEGF is required for longitudinal bone growth and corpora lutea formation and that rhuMAbVEGF can reversibly inhibit physiologic neovascularization at these sites.

Determinants of potassium channel assembly localised within the cytoplasmic C-terminal domain of Kv2.1.

The C-terminal domain of the voltage-gated potassium channel Kv2.1 is shown to have a role in channel assembly using dominant negative experiments in Xenopus oocytes. Kv2.1 channel polypeptides were co-expressed with a number of polypeptide fragments of the cytosolic C-terminus and the assembly of functional channel homotetramers quantified electrophysiologically using the two electrode voltage clamp technique. Co-expression of C-terminal polypeptides corresponding to the final 440, 318, 220 and 150 amino acid residues of Kv2.1 all resulted in a significant reduction in the functional expression of the full-length channel. A truncated version of Kv2.1 lacking the final 318 amino acids of the C-terminal domain (Kv2. 11-535) exhibited similar electrophysiological properties to the full-length channel. Co-expression with either the 440 or 318 residue polypeptides resulted in a reduction in the activity of the truncated channel. In contrast, the 220 and 150 residue C-terminal fragments had no effect on Kv2.11-535 activity. These data demonstrate that C-terminal interactions are important for driving Kv2.1 channel assembly and that distinct regions of the C-terminal domain may have differential effects on the formation of functional tetramers.

Determination of low-molecular-mass antioxidant concentrations in human respiratory tract lining fluids.

Antioxidants present within lung epithelial lining fluids (ELFs) constitute an initial line of defense against inhaled environmental oxidants such as ozone, nitrogen oxides, and tobacco smoke, but the antioxidant composition of human ELFs is still incompletely characterized. We analyzed ELF concentrations of the low-molecular-mass antioxidants ascorbate, urate, glutathione (GSH), and alpha-tocopherol by obtaining bronchoalveolar lavage (BAL) and nasal lavage fluids from healthy nonsmoking volunteers and compared two different BAL procedures. ELF dilution by the lavage procedures was estimated by measurement of urea in recovered BAL fluids in comparison with those in blood plasma from the same subjects. The results indicated that a recently developed single-cycle BAL procedure minimizes influx of non-ELF urea into the instilled fluid and thus allows for a more accurate determination of ELF antioxidant concentrations. Using this procedure, we determined that bronchoalveolar ELF contains 40 +/- 18 (SD) microM ascorbate, 207 +/- 167 microM urate, 109 +/- 64 microM GSH, and 0.7 +/- 0.3 microM alpha-tocopherol (n = 12 subjects). Similar analysis of nasal lavage fluid yielded nasal ELF levels of 28 +/- 19 microM ascorbate and 225 +/- 105 microM urate (n = 12 subjects), whereas GSH was undetectable (<0.5 microM). Our results demonstrate that ascorbate and urate are major low-molecular-mass ELF antioxidants in both the upper and lower respiratory tract, whereas GSH is present at significant concentrations only in bronchoalveolar ELF.

Increased histamine and 5-HT in portal vein plasma and mesenteric lymph during brief ischemia and reperfusion.

Brief mesenteric ischemia (10 min) can stimulate both visceral A delta- and C-fiber afferents and evoke reflex excitation of the cardiovascular system. We have shown that exogenous histamine causes reflex cardiovascular responses and that intra-arterial injection of 5-hydroxytryptamine (5- HT) into a mesenteric artery stimulates visceral A delta- and C-fiber afferents. We therefore hypothesized that brief abdominal ischemia is associated with release of histamine and 5-HT into the interstitium, where these mediators could stimulate or sensitize ischemically sensitive visceral afferent nerve endings. Accordingly, we measured concentrations of histamine and 5-HT in portal venous blood plasma and intestinal lymph fluid in cats. Cannulas were placed in a portal vein and in an intestinal lymphatic duct distal to the lymph node. Lymph and plasma histamine and 5-HT concentrations were measured by high-performance liquid chromatography before, during, and immediately after 10-min occlusion of the descending thoracic aorta. Histamine concentration increased significantly (P < 0.01) in portal venous blood plasma from a preocclusion level of 2.2 +/- 0.6 to 4.6 +/- 1.0 and 6.4 +/- 1.3 nmol/ml and in lymph fluid from a preocclusion level of 3.4 +/- 1.0 to 6.3 +/- 1.3 and 6.4 +/- 1.3 nmol/ml (n = 18) during brief ischemia and reperfusion, respectively. Also, the 5-HT concentration was significantly (P < 0.01) elevated in portal venous blood plasma from a preocclusion concentration of 1.1 +/- 0.5 to 2.7 +/- 0.8 and 2.5 +/- 0.8 nmol/ml and in lymph from a preocclusion level of 1.8 +/- 0.7 to 4.0 +/- 1.4 and 4.6 +/- 1.3 nmol/ml (n = 13) during brief ischemia and reperfusion, respectively. Because visceral afferent nerve endings are located in the interstitium, elevation of the interstitial concentration of histamine and 5-HT may contribute to the stimulation or sensitization of these nerve terminals during the brief ischemia and reperfusion period.

Production of hydroxyl radicals in contracting skeletal muscle of cats.

Reactive oxygen species increase during exhaustive contraction of skeletal muscle, but characterization of the specific species involved and their rates of production during nonexhaustive muscle contraction have not been investigated. We hypothesized that the production rate of hydroxyl radical (.OH) increases in contracting muscle and that this rate is attenuated by pretreatment with deferoxamine (Def) or dimethylthiourea (DMTU). We measured the rate of production of .OH before, during, and after 5 min of intermittent static contraction of the triceps surae muscles in cats (n = 6) using the formation of p-, m-, and o-tyrosines by hydroxylation of phenylalanine. L-Phenylalanine (30 mg/kg i.v.) was administered to each animal 3 min before contraction. Blood samples were collected from the popliteal vein 1 min before contraction; 1, 3, and 4.5 min during contraction; and 1 min after contraction. During and after contraction, the cumulative production rates of p-, m-, and o-tyrosines were elevated by 42.84 +/- 5.41, 0.25 +/- 0.04, and 0.21 +/- 0.03 nmol.min-1.g-1, respectively, compared with noncontracting triceps surae muscles. Pretreatment with Def (10 mg/kg i.v.; n = 5) or DMTU (10 mg/kg i.v.; n = 4) decreased the cumulative rates of production of p-, m-, and o-tyrosines during and after contraction. Additionally, the rate of tyrosine production increased in proportion to the percentage of maximal tension developed by the triceps surae muscles. These results directly demonstrate that .OH is produced in vivo in the skeletal muscle of cats during intermittent static contraction and that production can occur before the onset of fatigue.

Hydroxyl radical production during myocardial ischemia and reperfusion in cats.

We previously showed that generation of reactive oxygen species during myocardial ischemia and reperfusion stimulates cardiac sympathetic afferent nerve endings. We hypothesized that, in this feline model of brief ischemia and reperfusion, HO. is produced during ischemia and the rate and concentration of production of HO.during reperfusion is dependent on the duration of myocardial ischemia. Therefore, we evaluated the time dependency of production of HO. during reperfusion after 2, 5, and 10 min of reversible occlusion of the left anterior descending (LAD) coronary artery to induce ischemia in cats (n = 10). Blood samples collected from the coronary vein at 0.25, 1, 2, and 4 min after 2 min of ischemia revealed net cumulative rate of production of p-, m-, and o-tyrosine of 99 +/- 31, 10 +/- 5.1, and 0.8 +/- 0.2 nmol.min-1.g-1, respectively. After 5 min of ischemia, net cumulative rates of production of p-, m-, and o-tyrosine during reperfusion were 177 +/- 63, 74 +/- 26, and 1.6 +/- 0.8 nmol.min-1.g-1, respectively, whereas after 10 min of ischemia production rates were 153 +/- 42, 78 +/- 29, and 2.1 +/- 0.5 nmol.min-1.g-1, respectively. The highest rate of production of tyrosines was observed immediately after ischemia, perhaps indicating a washout of HO.-derived products that had accumulated in the myocardium during ischemia. To evaluate production of HO. during ischemia, deoxygenated saline (PO2 10 +/- 0.9 mmHg) containing phenylalanine was perfused into the ischemic coronary vascular bed through a cannula placed in the LAD (n = 16). Perfusate was collected from the coronary vein during the 10 min of ischemia. Net production of HO. during ischemia, measured by the production of p-, m-, and o-tyrosine, was 82 +/- 11, 6.6 +/- 0.4, and 1.7 +/- 0.3 nmol.min-1.g-1, respectively. Pretreatment with deferoxamine (10 mg/kg, n = 7) or dimethylthiourea (10 mg/kg, n = 6) decreased net production of HO. during ischemia and reperfusion. These results demonstrate that HO. is produced during brief ischemia and reperfusion, with the greatest amount being produced immediately after ischemia. Additionally, we show that the duration of brief ischemia determines the rate of production of HO. during reperfusion.

An assessment of the hidden and total antibiotic costs of four parenteral cephalosporins.

The aim of this study was to compare the hidden costs, and their impact on total antibiotic costs, of ceftriaxone therapy with those of cefotaxime, ceftazidime and cefuroxime in nosocomial infection. The total antibiotic costs of 7-day standard courses of the 4 cephalosporins were compared. The costs were divided into 3 parts: (i) the cost of the drug itself; (ii) the preparation and administration (labour) costs; and (iii) the consumables and waste costs. The latter 2 costs together comprised the hidden cost of an antibiotic course. Hidden costs were higher for cefotaxime, ceftazidime and cefuroxime, which are normally administered 3 times a day, than for ceftriaxone, which is administered once daily. The percentage contribution of hidden costs to total antibiotic costs increased with decreasing antibiotic cost, and were lower with higher dosages of all antibiotics. With cefotaxime, ceftazidime and cefuroxime, and with ceftriaxone at the lower dosage given by bolus intravenous (IV) injection, the labour component of hidden costs exceeded the consumables/waste component. However, when costs were calculated for ceftriaxone administered at the higher dosage by IV infusion, the costs of consumables and waste were greater than the labour costs. Ceftriaxone had the lowest hidden costs of all the antibiotics studied. The total antibiotic cost of low dosage ceftriaxone (1 g per dose) was comparable with that of cefuroxime, and was substantially less than the costs of cefotaxime and ceftazidime. At the high ceftriaxone dosage (2g per dose), the total antibiotic cost of cefuroxime was less than that of ceftriaxone; however, the total antibiotic cost of ceftriaxone remained substantially less than that of cefotaxime or ceftazidime.

Tyrosine modification by reactive nitrogen species: a closer look.

Peroxynitrite (ONOO-) is a powerful oxidant and cytotoxic species formed by the rapid reaction between nitrogen monoxide (nitric oxide, .NO) and superoxide (O2.-). At neutral pH ONOO- is partly protonated and this protonated form, peroxynitrous acid (ONOOH), decomposes rapidly to nitrate, forming (an) intermediate(s) with reactivity similar to .OH and .NO2. Peroxynitrite can hydroxylate and nitrate aromatic rings, and aromatic nitration of phenols such as tyrosine by ONOOH is proposed to proceed via a radical mechanism, with intermediate formation of .NO2. Modification of tyrosine by .NO2 also involves nitration via a radical mechanism. Aromatic nitration of phenols by ONOO- has been shown to be enhanced by superoxide dismutase or Fe(3+)-EDTA, which were proposed to catalyze heterolytic cleavage of ONOOH to form a nitrating species similar to the nitronium ion (NO2+). We investigated possible mechanisms of tyrosine modification by various reactive nitrogen species, including ONOO-, 3-morpholinosydnonimine (SIN-1), and .NO2. Reaction of tyrosine with ONOO- leads to formation of 3-nitrotyrosine and dityrosine, indicating intermediate formation of tyrosyl radicals. The pH dependence of formation of both 3-nitrotyrosine and dityrosine by ONOO- suggests that intermediate formation of ONOOH is required. Qualitatively similar results were obtained when ONOOH was generated continuously by H2O2 and NaNO2 at mildly acidic pH or with SIN-1, a compound which at neutral pH releases both .NO and O2.-, presumably producing ONOO-. However, relatively low yields of nitrotyrosine were obtained with SIN-1, possibly because of competing reactions of tyrosyl radicals with .NO or O2.-. Possible involvement of .NO2 in tyrosine modification by ONOO- was studied using hydroxyl radical scavengers, which can increase the radical yield during decomposition of ONOOH and thereby enhance generation of .NO2. Hydroxyl radical scavengers did not affect tyrosine modification by .NO2 directly and slightly inhibited tyrosine modification by authentic ONOO-. However, when ONOO- was produced at a slower rate, either by SIN-1 or by H2O2/NaNO2 at acidic pH, hydroxyl radical scavengers were found to significantly enhance tyrosine nitration. Our results suggest that ONOO- or ONOO(-)-generating systems induce nitration of tyrosine (or tyrosine residues in proteins) via intermediate formation of tyrosyl radicals and .NO2.

Oxidative damage by ozone and nitrogen dioxide: synergistic toxicity in vivo but no evidence of synergistic oxidative damage in an extracellular fluid.

Inhalation of ozone (O3) and/or nitrogen dioxide (.NO2) is associated with the development of inflammation in the respiratory tract and various alterations in pulmonary functions. Respiratory tract lining fluids (RTLFs) represent the first biological fluids coming into contact with these inhaled toxicants. Using plasma as a surrogate for RTLFs, we have previously shown that O3 [Cross, Motchnik, Bruener, Jones, Kaur, Ames and Halliwell (1992) FEBS Lett. 298, 269-272] and .NO2 [Halliwell, Hu, Louie, Duvall, Tarkington, Motchnik and Cross (1992) FEBS Lett. 313, 62-66] are both capable of depleting antioxidants and damaging proteins and lipids. O3 particularly damages proteins, whereas .NO2 induces the peroxidation of lipids and nitrates aromatic amino acids. It has been reported that O3 and .NO2 cause synergistic toxicity in rodents [Gielzleichter, Witschi and Last (1992) Tox. Appl. Pharmacol. 116, 1-9]. In the present chapter, we review evidence showing that combined exposure of these two oxidant gases to human plasma fails to exert synergistic oxidative damage to plasma constituents, and in fact, O3 and .NO2 antagonize each other's actions. We conclude that the potentiating effect of these two gases on morbidity and mortality in rodents represents a complex interactive biological effect rather than a simple synergistic oxidative effect in extracellular fluids.

Oxidants, antioxidants, and respiratory tract lining fluids.

Respiratory tract lining fluids (RTLFs) are a heterogeneous group of substances covering the respiratory tract epithelial cells (RTECs) from nasal mucosa to alveoli. Antioxidant contained in the RTLFs can be expected to provide an initial defense against inhaled environmental toxins. The major antioxidants in RTLF include mucin, uric acid, protein (largely albumin), ascorbic acid, and reduced glutathione (GSH). RTLF antioxidants can be augmented by such processes as transudation/exudation of plasma constituents; RTEC secretory processes, including glandular mucus secretion; and cellular antioxidants derived from lysis of RTECs and of inflammatory cells. The antioxidant composition of RTLFs and their role in modulating normal and pathophysiologic RTEC functions under conditions of oxidative stress are yet to be fully characterized.