Preparation of Test Compounds

The oil-based preparation used in the present study was supplied by Global Life Technologies Corp (GLT) (Chevy Chase, MD) and is a member of their Nozin® brand product line. The study formulation contains the following components: soy oil - 69.18%; coconut oil - 20.00%; orange oil - 4.90%; aloe vera oil - 4.90%; peppermint oil - 0.75%; vitamin E - 0.27%. All components of the test formulation are USP-grade and have been individually evaluated and identified by the FDA to fall under the Generally Recognized as Safe classification. This formulation has been demonstrated to be without irritating or inflammatory effects in an in vivo mammalian mucosal test system in studies carried out on behalf of GLT by North American Science Associates, Inc., an independent FDA-approved safety testing agency. The oil-based preparation was administered as supplied in both in vivo and in vitro experiments, as described below.

For the human nasal studies, sterile water without additives, containing 0.75% peppermint oil as a scented masking agent, was selected for use as the sham test agent. Because saline, itself, has been reported to reduce inflammatory cell number in the nose [37], water was considered to represent an appropriate vehicle against which to compare the oil-based preparation.

Subjects

This study was conducted as prescribed by the research protocol reviewed and approved by the Institutional Review Board of the Johns Hopkins Bloomberg School of Public Health. The study employed a single blind cross-over design, as described in the treatment and exposure protocol, below. Nine healthy adult men and women (22 to 40 years of age) were recruited into the ozone exposure study after obtaining informed consent (Table 1). Subjects were excluded if they had a history of chronic respiratory disease, cardiovascular disease or upper respiratory infection during the previous four weeks, if they were "smokers" or if they indicated an inability to sustain light exercise for at least 30 min. "Non-smokers" were defined as those individuals with a lifetime total of fewer than 3 pack-years plus abstinence from smoking of at least one year prior to the study. Subjects were required to refrain from taking prescription and non-prescription anti-inflammatory medications for the week prior to, and for the duration of, the 3-week study period. One subject was removed from the study after the initial nasal lavage indicated the presence of very high numbers of leukocytes in the nose (>100,000/ml), suggesting the presence of a latent upper respiratory infection. A second subject withdrew himself for reasons unrelated to the study.

Table 1 Exposure of Healthy Subjects to Ozone Full size table

Consistent with Institutional Review Board approval and following the same exclusion and informed consenting procedures described above, a second cohort of 12 different healthy adult subjects (9 men and 3 women) 22 to 62 years of age were recruited to assess the effects of the oil preparation on baseline antioxidant gene expression in the nasal epithelium (Table 2).

Table 2 Activation of Nrf2 by Components of the Natural Oil Preparation Full size table

Cells

BEAS-2B Cells

Cells of the BEAS-2B human bronchial epithelial cell line were obtained from the American Type Culture Collection (ATCC, Bethesda, MD). Cultures were expanded by growth on T-75 plastic flasks in DMEM/F-12 (1:1) medium (Invitrogen, Grand Island, NY) and seeded on 6- or 12-well Falcon filter inserts ( 0.4 μm pore size; Becton Dickinson, Franklin Lakes, NJ) and grown to confluence with the same medium above and below prior to treatment.

H23 Cells

Cells of the NIH-H23 human lung cell line were purchased from the American Type Culture Collection. H23 cells were transfected with a plasmid vector (pGL3 vector with a minimal promoter) purchased from Promega Corporation, Madison, WI, expressing the firefly luciferase gene driven by a minimal TATA-like promoter. Upstream to the promoter, a short DNA fragment containing the Nrf2 binding site found in the NQO1 gene promoter was cloned, as previously described in BEAS-2B cells [38]. H23 cells expressing the reporter plasmid were selected using blasticidin as the antibiotic. Several clones were screened using the luciferase assay and one clone exhibiting maximum luciferase activity was selected for detailed characterization of its Nrf2 activation profile. Sulforaphane, a naturally-occurring isothiocyanate known to activate Nrf2 [39] was used to demonstrate that Nrf2 dependent luciferase reporter activity in the H23-ARE-luciferase cells was dose-dependent and linked to downstream antioxidant enzyme gene activation [38]. These cells were cultured and seeded on filter inserts, as described above, and used in all assessments of Nrf2 activation in the present study.

Treatment and Exposure of Subjects to Ozone

The human subjects component of the study was carried out in the Health Effects Assessment Laboratory (HEAL) in the Department of Environmental Health Sciences of the Bloomberg School of Public Health. The purpose of this study was to test the hypothesis that oil treatment would mitigate ozone-induced upper respiratory system neutrophil inflammation. A single blind, non-randomized design was chosen to enable the identification and elimination from further unnecessary participation any individuals who were unresponsive to this level of ozone exposure. The masking scent in both sham and test preparations kept subjects blinded to the treatments in each arm. The study design is depicted in Figure 1. On the first day of Arm 1 of the protocol, the absence of baseline inflammation was confirmed in each subject by determining that inflammatory cell concentrations fell within normal limits (<20,000 cells/ml nasal lavage) (Figure 2). On the second day of Arm 1, the aqueous control preparation containing 0.75% peppermint oil as a masking agent was administered in a single-blinded manner as a single 50 μl application in each nostril using a metered spray applicator (model VP7/50 18/415 + poussoir 232 NA/B) manufactured by Aptar (Le Vaudreuil, France). Immediately following nasal treatment, subjects were exposed to 0.25 ppm O 3 for 120 min. with alternating 30 min periods of rest and light exercise consisting of slowly walking on a treadmill. Exposures took place in a temperature- and humidity-controlled chamber as previously described [40]. To optimize upper respiratory targeting, subjects were visually monitored after being instructed to chew gum with a closed mouth for the duration of the exposure period. Eighteen hours following exposure, subjects underwent nasal lavage to assess post-exposure. After a 7-10 day washout period, a second 3-day study period was repeated in Arm 2 following the same procedures, but associated with the nasal spray application of the oil-based test agent.

Figure 1 Depiction of the ozone exposure intervention protocol. Full size image

Figure 2 Intervention in oxidant pollutant exposure-induced inflammation at 18 hrs by topical application of the oil preparation. Individual data showing the upper respiratory inflammatory responses of subjects exposed to ozone (0.25 ppm, 2 hr) when pretreated with 50 μl of scented sterile water (sham) or a mixture of natural oils administered by aerosol spray to each nostril. Each subject is represented by the same symbol in both arms of the study; numbers correspond to subject numbers in Table 1. Points connected by dashed lines represent means of each group. * indicates significant difference from baseline (P < 0.05) by paired t analysis. Full size image

Assessment of Nasal Inflammation

Nasal lavage was carried out according to a standardized procedure. With the subject seated in a chair and the head tilted backwards, 5 ml of 37°C Ringer's lactate was instilled by pipette into each nostril. After 5-10 seconds, the head was brought forward and the fluid expelled into a basin by gentle blowing. This procedure was repeated 4 times. Following centrifugation, the cells from all 4 tubes were pooled by re-suspension in phosphate buffered saline for cellular analysis.

Counts of inflammatory cells were made using a hemocytometer and calculated as total inflammatory cells per ml of nasal lavage return. Return volumes, which averaged 84% of the 40 ml instilled volume, were very consistent within each subject and were used to normalize the inflammatory cell return. In healthy adults without respiratory disease or allergic symptoms, ozone exposure elicits a predominantly polymorphonuclear neutrophilic (PMN) inflammatory response [41]. In the present study, PMNs comprised greater than 95% of the inflammatory cells recovered in nasal lavage fluid. Thus, the total number of leukocytes retrieved by lavage was used as an index of nasal inflammation.

Cell-free supernatant from nasal lavage samples was stored at -80°C prior to assay for the presence of the inflammatory mediators IL-6 and IL-8 by ELISA (R & D Systems, Minneapolis, MN).

Nasal symptoms prior to and at eighteen hours post exposure in each of the two arms were scored following a standard procedure [42, 43] by having the subjects make a mark on a horizontal 100 mm line indicating the level of the symptom described, with the least sensation at the far left and the most at the far right. Scores were determined by measuring the distance in mm from the left end of the line and the change in numerical values between the two arms were compared.

Assessment of Nasal Epithelial Gene Expression

Collection of nasal mucosal epithelial cells was made from the upper and lower aspects of the inferior medial turbinates of the right and left nostrils using a nasal mucosal curette (Rhino-probe®). Epithelial biopsy samples were taken prior to and 8 hours following administration of the oil-based test agent or the scented control preparation utilized in the ozone study. Using a metered spray applicator, 50 microliters of each of the two agents was administered in a single-blinded and random manner to one or the other of the two nostrils. Using this design, each turbinate provided its own baseline value for gene expression and the two agents were tested simultaneously in the same individual. Preliminary experiments in several subjects demonstrated that prior sampling on the turbinate at a site distant from the second sample site had no effect on baseline expression of the heme oxygenase-1 (HO-1) target gene in the second sample in the absence of treatment (first to second expression ratio = 0.96 ± 0.06; mean ± SEM, n = 8 sample pairs from 9 subjects). Biopsy samples were frozen in liquid nitrogen and stored for RNA extraction and PCR analysis as described below.

Treatment of cells in culture

After ensuring that the surfaces of BEAS-2B epithelial cell cultures were free of liquid, 200 μl of control agent (HBSS or soy oil, as indicated) or test oil preparation were added to the apical surfaces and evenly distributed by rotation. It was found that the soy oil component of the test preparation was indistinguishable from HBSS as a negative control and, thus, soy oil was used in the majority of cell culture studies as the control; ratio of threshold cycle for HO-1 gene expression HBSS:soy = 1:1.01 ± 0.09 (±SD; n = 6). After treatment, the cultures were returned to the incubator for 15 min. prior to removal of the treatment fluids by suction. The surfaces were then gently washed twice with 500 μl of warmed (37°C) HBSS, and the cultures were returned to the incubator for the designated periods of time prior to extraction of RNA or protein. In two series of experiments, control and oil-treated cells underwent further challenge at 12 hours with lipopolysaccharide (LPS, 3 μg/ml medium, Escherichia coli, serotype 055.B5 - Sigma.) for 4 hours prior to RNA extraction. The removal of treatment oil by suction and the repeated aqueous wash and removal of wash fluid and floating oil by suction ensured the complete removal of oil from the cultures prior to subsequent treatment. In the four sets of experiments in which activation of Nrf2 by individual treatment oil constituents and four sets in which the dose-dependency of Nrf2 activation by orange oil was assessed, duplicate cultures were extracted for measurement of luciferase activity at the times indicated, as described below.

Determination of Gene and Protein Expression

Real Time RT-PCR

Total RNA was extracted from cultured cells and from nasal mucosal epithelial cells obtained by biopsy using the RNeasy kit (Qiagen) and was quantified by UV absorbance spectrophotometry. The reverse transcription reaction was performed by using the high capacity cDNA synthesis kit (Applied Biosytems) in a final volume of 20 μl containing 1 μg of total RNA, 100 ng of random hexamers, 1X reverse transcription buffer, 2.5 mM MgCl 2 , 1 mM dNTP, 20 units of multiscribe reverse transcriptase, and nuclease free water. Quantitative real time RT-PCR analyses of Human heme oxygenase-1 (HO-1), NAD(P)H:quinone oxidoreductase 1 (NQO1), glutamate cysteine ligase-modulatory subunit (GCLm), glutamate cysteine ligase-catalytic subunit (GCLc), and tumor necrosis factor alpha (TNFα) were performed on cell and nasal biopsy extracts using primers and probe sets from Applied Biosystems. Assays were performed by using the ABI 7000 Taqman system (Applied Biosystems). β-actin was used for normalization.

Western Blot Analysis

To obtain total protein lysates, cells were lysed in RIPA buffer containing Halt Protease Inhibitor cocktail (Pierce, Rockford, Illinois, United States) and centrifuged at 12,000 g for 15 min at 4°C. Protein concentrations of the supernatant were measured using Bio-Rad protein assay (Bio-Rad, CA). To detect the translocation of Nrf2 protein to the nucleus, nuclear protein was isolated using the NE-PER protein isolation kit (Pierce, Rockford, IL). For immunoblot analysis, 20 μg of total protein lysate or 20 μg of nuclear protein lysate was resolved on 12% SDS-PAGE gels. Proteins were transferred onto PVDF membranes and blocked with PBS- Tween (0.1% Tween-20 in PBS, pH 7.2) supplemented with 5% low fat milk powder (w/v) for 2 hr at room temperature. All primary antibodies were diluted in PBS-Tween (0.1%) with 5% nonfat dry milk and incubated overnight at 4°C. Following antibodies were used for immunoblotting: anti-HO1 (Abcam), anti-NQO1 (Novus Biologicals), anti-GCLm, and anti-GAPDH (Imgenex, Sorrento Valley, CA), anti-Nrf2 and anti-lamin B (Santa Cruze Biotechnology (Santa Cruze, CA). After washing the primary antibody, the membranes were incubated with horseradish peroxidase conjugated anti-rabbit, anti-mouse or anti-goat antibody (~1:2500 in 0.1% Tween-20, with 5% low fat milk powder (w/v) for 1 hr at room temperature. Membranes were again washed with PBS-Tween (0.1%) and secondary antibodies were visualized by enhanced chemiluminescence detection system (Amersham Biosciences, NJ). Densitometric measurement of individual target protein lots were normalized to GAPDH or lamin B and quantified using the Image J (NIH) software package for graphic display.

Determination of Nrf2 Activation

Changes in activation of the Nrf2 transcription factor in cultured H23 ARE cells in response to treatment were determined as previously described [44]. In brief, cells grown to 70% confluence on 12-well inserts were treated as indicated for 15 min and incubated for 12 hr at 37°C. Rinsed cells were fully lysed and luciferase luminescence was generated using the E4030 Luciferase Assay System (Promega) and detected with a TD-20/20 Luminometer (Turner Designs). Luminescence was normalized to the protein content of each lysate as determined using the Bio-Rad Protein Assay System and Microplate Reader. Nrf2 activation levels were expressed as Relative Luminescence Units/ug protein.

Statistics

Nasal lavage and biopsy data were tested for differences between control and oil-treatments using paired-t analyses. Comparisons of cell culture data were made using Student's t analyses. In instances of a lack of normality, the Wilcoxon Signed Rank Test was used. In all cases, P values < 0.05 were considered significant. Statistical analyses were carried out with SigmaStat Statistical software (Jandel Scientific, San Rafael, CA).