Human exposure to diesel exhaust induces CYP1A1 expression and AhR activation without a coordinated antioxidant response.

BACKGROUND: Diesel exhaust (DE) induces neutrophilia and lymphocytosis in experimentally exposed humans. These responses occur in parallel to nuclear migration of NF-κB and c-Jun, activation of mitogen activated protein kinases and increased production of inflammatory mediators. There remains uncertainty regarding the impact of DE on endogenous antioxidant and xenobiotic defences, mediated by nuclear factor erythroid 2-related factor 2 (Nrf2) and the aryl hydrocarbon receptor (AhR) respectively, and the extent to which cellular antioxidant adaptations protect against the adverse effects of DE. METHODS: Using immunohistochemistry we investigated the nuclear localization of Nrf2 and AhR in the epithelium of endobronchial mucosal biopsies from healthy subjects six-hours post exposure to DE (PM10, 300 µg/m3) versus post-filtered air in a randomized double blind study, as a marker of activation. Cytoplasmic expression of cytochrome P450s, family 1, subfamily A, polypeptide 1 (CYP1A1) and subfamily B, Polypeptide 1 (CYP1B1) were examined to confirm AhR activation; with the expression of aldo-keto reductases (AKR1A1, AKR1C1 and AKR1C3), epoxide hydrolase and NAD(P)H dehydrogenase quinone 1 (NQO1) also quantified. Inflammatory and oxidative stress markers were examined to contextualize the responses observed. RESULTS: DE exposure caused an influx of neutrophils to the bronchial airway surface (p = 0.013), as well as increased bronchial submucosal neutrophil (p < 0.001), lymphocyte (p = 0.007) and mast cell (p = 0.002) numbers. In addition, DE exposure enhanced the nuclear translocation of the AhR and increased the CYP1A1 expression in the bronchial epithelium (p = 0.001 and p = 0.028, respectively). Nuclear translocation of AhR was also increased in the submucosal leukocytes (p < 0.001). Epithelial nuclear AhR expression was negatively associated with bronchial submucosal CD3 numbers post DE (r = -0.706, p = 0.002). In contrast, DE did not increase nuclear translocation of Nrf2 and was associated with decreased NQO1 in bronchial epithelial cells (p = 0.02), without affecting CYP1B1, aldo-keto reductases, or epoxide hydrolase protein expression. CONCLUSION: These in vivo human data confirm earlier cell and animal-based observations of the induction of the AhR and CYP1A1 by diesel exhaust. The induction of phase I xenobiotic response occurred in the absence of the induction of antioxidant or phase II xenobiotic defences at the investigated time point 6 h post-exposures. This suggests DE-associated compounds, such as polycyclic aromatic hydrocarbons (PAHs), may induce acute inflammation and alter detoxification enzymes without concomitant protective cellular adaptations in human airways.

Heparin-binding protein in ventilator-induced lung injury.

BACKGROUND: Although mechanical ventilation is often lifesaving, it can also cause injury to the lungs. The lung injury is caused by not only high pressure and mechanical forces but also by inflammatory processes that are not fully understood. Heparin-binding protein (HBP), released by activated granulocytes, has been indicated as a possible mediator of increased vascular permeability in the lung injury associated with trauma and sepsis. We investigated if HBP levels were increased in the bronchoalveolar lavage fluid (BALF) or plasma in a pig model of ventilator-induced lung injury (VILI). We also investigated if HBP was present in BALF from healthy volunteers and in intubated patients in the intensive care unit (ICU). METHODS: Anaesthetized pigs were randomized to receive ventilation with either tidal volumes of 8 ml/kg (controls, n = 6) or 20 ml/kg (VILI group, n = 6). Plasma and BALF samples were taken at 0, 1, 2, 4, and 6 h. In humans, HBP levels in BALF were sampled from 16 healthy volunteers and from 10 intubated patients being cared for in the ICU. RESULTS: Plasma levels of HBP did not differ between pigs in the control and VILI groups. The median HBP levels in BALF were higher in the VILI group after 6 h of ventilation compared to those in the controls (1144 ng/ml (IQR 359-1636 ng/ml) versus 89 ng/ml (IQR 33-191 ng/ml) ng/ml, respectively, p = 0.02). The median HBP level in BALF from healthy volunteers was 0.90 ng/ml (IQR 0.79-1.01 ng/ml) as compared to 1959 ng/ml (IQR 612-3306 ng/ml) from intubated ICU patients (p < 0.001). CONCLUSIONS: In a model of VILI in pigs, levels of HBP in BALF increased over time compared to controls, while plasma levels did not differ between the two groups. HBP in BALF was high in intubated ICU patients in spite of the seemingly non-harmful ventilation, suggesting that inflammation from other causes might increase HBP levels.

Effects of 17ß-estradiol on proliferation, cell viability and intracellular redox status in native human lens epithelial cells.

PURPOSE: The purpose of this study was to examine the effects of 17ß-estradiol on proliferation, cell death and redox status in cultured human lens epithelial cells (HLECs). METHODS: HLECs were exposed to 17ß-estradiol after which cell viability was measured by 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) and the number of mitotic and apoptotic cell nuclei was determined after staining with Hoechst 33342. Apoptosis was also determined by measuring caspase-3 activity and propidium iodide was used to determine the proportion of non-viable cells. Pro- and antioxidative effects of 17ß-estradiol was investigated by measuring peroxides, superoxides and glutathione, using dichlorofluorescein diacetate (DCFH-DA), dihydroethidium (HET), and monochlorobimane (MCB), respectively. Effects on mitochondrial membrane potential were determined using 5,5',6,6'-tetrachloro-1,1',3,3'- tetraethylbenzimidazolylcarbocyanine iodide (JC-1). The ability of 17ß-estradiol to prevent reactive oxygen species (ROS)-production in HLECs after exposure to 25 µM H2O2 for 24h was also measured. RESULTS: This study demonstrates increased mitotic activity in HLECs exposed to physiologic concentrations of 17ß-estradiol (1 nM). Pharmacological concentrations of 17ß-estradiol caused increased number of apoptotic cell nuclei and caspase-3 activation. Physiologic concentrations of 17ß-estradiol (0.1-10 nM) stabilized the mitochondrial membrane potential. Similar or slightly higher concentrations of 17ß-estradiol (0.01-1 µM) protected against H2O2-induced oxidative stress as evident by decreased levels of peroxides and superoxides. CONCLUSIONS: The present study demonstrates mitogenic and anti-oxidative effects of 17ß-estradiol at physiologic concentrations, whereas pharmacological levels induced oxidative stress and acted pro-apoptotic in cultured lens cells.

Ozone exposure enhances mast-cell inflammation in asthmatic airways despite inhaled corticosteroid therapy.

Asthmatics are recognised to be more susceptible than healthy individuals to adverse health effects caused by exposure to the common air pollutant ozone. Ozone has been reported to induce airway neutrophilia in mild asthmatics, but little is known about how it affects the airways of asthmatic subjects on inhaled corticosteroids. We hypothesised that ozone exposure would exacerbate the pre-existent asthmatic airway inflammation despite regular inhaled corticosteroid treatment. Therefore, we exposed subjects with persistent asthma on inhaled corticosteroid therapy to 0.2 ppm ozone or filtered air for 2 h, on 2 separate occasions. Lung function was evaluated before and immediately after exposure, while bronchoscopy was performed 18 h post exposure. Compared to filtered air, ozone exposure increased airway resistance. Ozone significantly enhanced neutrophil numbers and myeloperoxidase levels in airway lavages, and induced a fourfold increase in bronchial mucosal mast cell numbers. The present findings indicate that ozone worsened asthmatic airway inflammation and offer a possible biological explanation for the epidemiological findings of increased need for rescue medication and hospitalisation in asthmatic people following exposure to ambient ozone.

Diesel exhaust exposure enhances the ozone-induced airway inflammation in healthy humans.

Exposure to particulate matter and ozone cause adverse airway reactions. Individual pollutant effects are often addressed separately, despite coexisting in ambient air. The present investigation was performed to study the effects of sequential exposures to diesel exhaust (DE) and ozone on airway inflammation in human subjects. Healthy subjects underwent bronchoscopy with bronchoalveolar lavage (BAL) and bronchial wash (BW) sampling on two occasions. Once following a DE exposure (with 300 mug.m(-3) particles with a 50% cut-off aerodynamic diameter of 10 mum) with subsequent exposure to O(3) (0.2 ppm) 5 h later. The other bronchoscopy was performed after a filtered air exposure followed by an ozone exposure, using an identical protocol. Bronchoscopy was performed 24 h after the start of the initial exposure. Significant increases in neutrophil and macrophage numbers were found in BW after DE followed by ozone exposure versus air followed by ozone exposure. DE pre-exposure also raised eosinophil protein X levels in BAL compared with air. The present study indicates additive effects of diesel exhaust on the ozone-induced airway inflammation. Together with similar results from a recent study with sequential diesel exhaust and ozone exposures, the present data stress a need to consider the interaction and cumulative effects of different air pollutants.

Lower airways inflammation in allergic rhinitics: a comparison with asthmatics and normal controls.

BACKGROUND: Allergic rhinitis (AR) and asthma represent a continuum of atopic disease. AR is believed to pre-dispose an individual to asthma. Compared with asthmatics and normal controls, the inflammatory response in the lower airways of rhinitics is not fully elucidated. To test the hypothesis that the inflammatory response in the airways of subjects with AR is at a level intermediate between that in normal controls and asthmatics, we have characterized bronchial inflammation and cytokine mRNA levels in non-asthmatic allergic rhinitics and compared it with subjects with allergic asthma and with normal controls. METHODS: Endobronchial mucosal biopsies were obtained at bronchoscopy from 14 allergic rhinitics, 16 asthmatics and 21 normal controls. Biopsies were embedded into glycol methacrylate resin for immunohistochemical analysis of cellular inflammation and snap frozen for semi-quantitative PCR analysis of cytokine mRNA levels. RESULTS: Airway inflammation in rhinitic subjects was characterized by an increase in submucosal eosinophils, mast cells and the mRNA expression of TNF-alpha, at an intermediate level between healthy and asthmatics. In addition, CD3(+) and CD8(+) lymphocytes in the epithelium, the endothelial expression of vascular adhesion molecule-1 and IL-1 beta mRNA were higher in the allergic rhinitics compared with both normal controls and asthmatics, whereas growth-related oncogene alpha-mRNA was decreased in AR compared with both healthy and asthmatics. Airway inflammation in the asthmatic group was characterized by higher numbers of eosinophils and mast cells, together with an increase in TNF-alpha-mRNA compared with both healthy and rhinitics. IFN-gamma mRNA was the highest in normal controls and lowest in the asthmatics. CONCLUSIONS: In individuals with AR the present data suggest an intermediate state of airway inflammation between that observed in normal individuals and subjects with clinical asthma. It is also indicated that IFN-gamma production by CD8(+) T lymphocytes could be protective against the development of airway hyperresponsiveness. Further work is needed to evaluate this hypothesis.

Airway antioxidant and inflammatory responses to diesel exhaust exposure in healthy humans.

Pulmonary cells exposed to diesel exhaust (DE) particles in vitro respond in a hierarchical fashion with protective antioxidant responses predominating at low doses and inflammation and injury only occurring at higher concentrations. In the present study, the authors examined whether similar responses occurred in vivo, specifically whether antioxidants were upregulated following a low-dose DE challenge and investigated how these responses related to the development of airway inflammation at different levels of the respiratory tract where particle dose varies markedly. A total of 15 volunteers were exposed to DE (100 microg x m(-3) airborne particulate matter with a diameter of <10 microm for 2 h) and air in a double-blinded, randomised fashion. At 18 h post-exposure, bronchoscopy was performed with lavage and mucosal biopsies taken to assess airway redox and inflammatory status. Following DE exposure, the current authors observed an increase in bronchial mucosa neutrophil and mast cell numbers, as well as increased neutrophil numbers, interleukin-8 and myeloperoxidase concentrations in bronchial lavage. No inflammatory responses were seen in the alveolar compartment, but both reduced glutathione and urate concentrations were increased following diesel exposure. In conclusion, the lung inflammatory response to diesel exhaust is compartmentalised, related to differing antioxidant responses in the conducting airway and alveolar regions.

Corneal endothelial integrity in mice lacking extracellular superoxide dismutase.

PURPOSE: To evaluate corneal endothelial morphology in mice without secreted extracellular superoxide dismutase (SOD) in normal ageing and in a lipopolysaccharide (LPS)-induced inflammation model and to measure the contents of SOD isoenzymes in the mouse cornea and the superoxide radical concentrations in corneas with and without extracellular SOD. METHODS: The central corneal endothelium of wild-type and extracellular SOD-null mice were studied in micrographs at eight different ages and after a unilateral intravitreal injection of LPS, with the contralateral eye serving as the control. The activities of the SOD isoenzymes in the mouse cornea were determined with a direct assay, the superoxide radical concentration was assessed by lucigenin-induced chemiluminescence, and the extracellular SOD distribution was mapped with immunohistochemistry. RESULTS: The activities of the cytosolic Cu- and Zn-containing SOD, the mitochondrial Mn-containing SOD and extracellular SOD were 4300, 15, and 340 U/g wet weight, respectively. Extracellular SOD was found in the epithelium, stroma, and endothelium. The concentration of extracellular superoxide radicals was doubled in extracellular SOD-null corneas, and the endothelial cell density decreased more with age in extracellular SOD-null than in wild-type control corneas. In the LPS-induced inflammation model, the cell density decreased more, and the cells became more irregular in extracellular SOD-null than in wild-type corneas. CONCLUSIONS: In the mouse cornea, absence of extracellular SOD leads to a higher concentration of extracellular superoxide radicals, an enhancement in the spontaneous age-related loss of endothelial cells, and an increased susceptibility to acute inflammatory endothelial damage. Extracellular SOD is likely to have a protective role in the corneal endothelium.

In vitro photochemical cataract in mice lacking copper-zinc superoxide dismutase.

We here evaluate cataract formation in mice lacking the cytosolic copper-zinc superoxide dismutase (CuZn-SOD) in an in vitro model using irradiation with visible light and riboflavin as a photosensitizing agent. Isolated, cultured lenses from wild-type and CuZn-SOD-null mice were irradiated for 1.5 h by a daylight fluorescent light after preincubation with 10 microM riboflavin for 24 h. Cataract formation was evaluated daily with digital image analysis and ocular staging, and after 5 d 86Rb uptake and water contents of the lenses were determined. Basal superoxide concentrations in freshly isolated lenses from wild-type and CuZn-SOD-null mice were determined with lucigenin-derived chemiluminescense, and enzymatic activities of all three SOD isoenzymes in the murine lens were determined with a direct spectrophotometric method. The cytosolic CuZn-SOD accounts for 90% of the total SOD activity of the murine lens. CuZn-SOD-null lenses showed a doubled basal superoxide concentration, and were more prone to develop photochemical cataract in the present model with more opacification, more hydration, and less 86Rb uptake than lenses from wild-type mice. We conclude that CuZn-SOD is an important superoxide scavenger in the lens, and that it may have a protective role against cataract formation.

Superoxide dismutase isoenzymes in the normal and diseased human cornea.

PURPOSE: The human cornea, a tissue much exposed to oxidative stress, is rich in extracellular superoxide dismutase (SOD). In this study, the contents and distributions of the SOD isoenzymes in the normal human cornea were compared with those in corneas affected by keratoconus and bullous keratopathy. METHODS: The central and peripheral parts of normal human corneas were analyzed separately. Central corneal buttons were obtained from patients with keratoconus and bullous keratopathy who were undergoing primary keratoplasty or retransplantation. SOD enzymatic activities were determined by a direct spectrophotometric method, and extracellular SOD and the cytosolic Cu- and Zn-containing SOD (CuZn-SOD) proteins were determined with ELISA and studied with immunohistochemistry. RESULTS: The total SOD content, and particularly the extracellular SOD content, was lower in the central than in the peripheral normal cornea. CuZn-SOD and extracellular SOD were demonstrated in all three corneal layers. CuZn-SOD was found in cells, whereas extracellular SOD appeared to be localized on cell surfaces, in basal membranes, and in the stroma. In keratoconus, corneal levels of extracellular SOD were half those in the control corneas, whereas CuZn-SOD and the mitochondrial Mn-containing SOD levels were normal. In bullous keratopathy, apart from edematous dilution, SOD isoenzyme levels were essentially normal. In a remarkable finding, the same pattern in SOD isoenzyme levels as in the original disease was also found at retransplantation. CONCLUSIONS: Extracellular SOD and CuZn-SOD show markedly different distribution patterns within the human cornea. Extracellular SOD activity in the central cornea is halved in keratoconus, compared with that in normal control corneas. The finding of a similar reduction at retransplantation in keratoconus suggests reduced corneal extracellular SOD synthesis in cells of the host as a cause of the low enzyme levels.

Aqueous humor lidocaine concentrations in topical and intracameral anesthesia.

PURPOSE: To assess aqueous humor lidocaine concentrations in 2 common regimens of topical anesthesia and after intracameral injection of the anesthetic agent. SETTING: University hospital eye clinic. METHODS: Twenty patients having routine cataract surgery were randomized into 3 groups: 1 given 3 drops of lidocaine 4% before surgery; 1 given 6 drops; 1 given 3 drops plus an intracameral injection of 0.1 mL lidocaine 1%. Lidocaine concentration was measured in aqueous humor samples taken before surgery. RESULTS: With 3 drops, aqueous lidocaine concentration was 1.4 micrograms/mL +/- 0.5 (SD) and with 6 drops, 4.3 +/- 1.5 micrograms/mL (P = .0015). With an intracameral injection, it was 341.8 +/- 152.6 micrograms/mL. CONCLUSION: Measurable amounts of lidocaine entered the anterior chamber in topical anesthesia, and more entered when more drops were given. It is likely that concentrations in this range could anesthetize the iris, but they are far lower than concentrations after an intracameral injection.

Small incision single-suture-loop pupilloplasty for postoperative atonic pupil.

In this small incision technique to correct postoperative atonic pupil in the aphakic or pseudophakic eye, the pupil is sutured and constricted with a 10-0 polypropylene loop through 3 1.0 mm stab incisions. The surgery is easier to perform than previous methods, appears safe and reproducible, and requires little special equipment. Although the pupil's shape may become slightly irregular, its size is fairly easy to regulate. This new method should be useful in correcting postoperative atonic pupil, and the small incision technique will likely minimize surgical trauma and induced astigmatism.

Sub-Tenon's anesthesia with a retained catheter in ocular surgery of longer duration.

A sub-Tenon's anesthesia technique was developed that is suitable for surgical procedures of longer duration. After application of a small amount of anesthetic agent in the standard manner, a thin polyethylene catheter is introduced under Tenon's capsule and fixated. The catheter is used to inject more anesthetic agent during the procedure. This anesthesia regimen provides unlimited time for surgery without the risks of losing anesthetic effect and without using a potentially toxic, long-term local anesthetic agent.

A simplified method for silicone intubation in lacrimal drainage obstruction.

PURPOSE: To simplify the surgical procedure in silicone tube implantation for lacrimal drainage obstruction. METHODS: To reduce the problem of retrieving the leader from under the inferior turbinate, I developed a method where I probe the canaliculi with a pigtail probe, allowing a leader to pass through both canaliculi simultaneously. The leader is then retrieved under direct visualization to minimize trauma. RESULTS: This new method necessitates only one retrieval of the leader in the nasal cavity instead of two. This single retrieval is also made easier. CONCLUSIONS: A surgically demanding and time-consuming procedure is made easier for the surgeon and the inconvenience for the patient is reduced.

Superoxide dismutase isoenzymes in the human eye.

PURPOSE: The authors determined the levels of the secreted, interstitially located extracellular superoxide dismutase (EC-SOD), the cytosolic copper- and zinc-containing SOD (CuZn-SOD), and the mitochondrial manganese-containing SOD (Mn-SOD) in tears, cornea, sclera, aqueous humor, lens, vitreous body, and retina of the human eye. METHODS: SOD enzymatic activity was determined by the direct spectrophotometric method using KO2. The amounts of EC-SOD and CuZn-SOD protein were also determined using enzyme-linked immunosorbent assay. RESULTS: Tears, aqueous humor, and vitreous body contained little SOD activity. Here ascorbate is the major scavenger of superoxide radicals. Of the ocular tissues studied, the lens contained the least SOD activity, 15-fold less than that of the average human tissue. The majority came from CuZn-SOD, but the lens also contained considerable amounts of inactive enzyme. There was some Mn-SOD, but the EC-SOD content was negligible. The cornea and sclera contained several times more SOD activity than the lens, and the EC-SOD contents were equal to or larger than those of CuZn-SOD. The retina contained much CuZn-SOD activity, but the EC-SOD and Mn-SOD activities were low. CONCLUSIONS: Ocular tissues and fluids, except for those in the retina, are less protected against superoxide radicals than extraocular tissues and fluids. The large amounts of EC-SOD in the sclera and cornea may be related to the risk for photochemical production of superoxide in these tissues. If inflammatory reactions occur, ocular tissues are at risk for damage induced by superoxide radicals and peroxynitrite, the reaction product with nitric oxide.

Scleral suturing of intraocular lenses.

This paper describes a simple, safe, and reproducible method for scleral suturing intraocular lenses. The method uses standard tools and equipment.