Fate of Pathologically Bound Oxygen Resulting from Inhalation of Labeled Ozone in Rats

Abstract

Inhaled ozone (O₃) reacts chemically with respiratory tract biomolecules where it forms covalently bound oxygen adducts. We investigated the fate of these adducts following inhalation exposure of rats to labeled ozone (¹⁸O₃, 2 ppm, 6 hr or 5 ppm, 2 hr). Increased ¹⁸O was detected in blood plasma at 7 hr post exposure and was continuously present in urine for 4 days. Total ¹⁸O excreted was -53% of the estimated amount of ¹⁸O₃ retained by the rats during ¹⁸O₃ exposure suggesting that only moderate recycling of the adduct material occurs. The time course of excretion, as well as properties of the excreted ¹⁸O were determined to provide guidance to future searches for urinary oxidative stress markers. These results lend plausibility to published findings that O₃ inhalation could exert influences outside the lung, such as enhancement of atherosclerotic plaques.

Keywords

ozone oxidative stress biomarkers adducts excretion

Introduction

Ozone (O₃) pollution of ambient air affects a worldwide population where exposure to O₃ has been shown to be associated with a variety of cardiopulmonary health impairments.^1–4 Due to its low water solubility and high chemical reactivity, O₃ is able to pass through the nose into the lung where it causes injury and forms stable adducts and reactive intermediates such as peroxides, aldehydes and carbonyls.^5,6 These intermediates are believed to be responsible for the reported oxidation of molecules in the blood^7,8 since O₃ itself is so reactive that it is not expected to penetrate beyond the respiratory tract surface.^9,10 Animal studies have shown that O₃ can affect extrapulmonary sites such as enhancement of atherosclerotic plaques and vascular injury in susceptible animals,^11,12 however, the mechanisms responsible for these effects are unknown. O₃ has been considered to be a good model oxidant for the elucidation of clinical markers of in vivo oxidative stress. A published series of studies in rats showed that some traditional measures of oxidative stress (isoprostanes and malondialdehyde in blood plasma and urine) that were effective markers of CCl₄–-induced oxidative stress to the liver, were not effective following inhalation of O₃.^13–15

We embarked on the present study with the goal of tracing the fate of O₃ reaction products that might enter the circulation and be excreted. Our previous studies showed that exposure to ¹⁸O-labeled ozone (¹⁸O₃) results in measurable ¹⁸O in nasal and bronchoalveolar lavage fluid (BALF), and that the concentration of ¹⁸O in BALF is related to the level of injury.^16–18 We hoped to elucidate methods for detection of oxidative stress clinically as well as shed light on the mechanism by which O₃ induces extrapulmonary effects. Relatively high exposure concentrations of ¹⁸O₃ were employed (4-12 fold higher than maximal ambient levels) because we were searching for chemicals in blood and urine after a large dilution from their pulmonary concentration. O₃ at these concentrations induces pulmonary edema in the rats which is detectable as increased total protein in BALF.¹⁶

Methods

Animals

Male 60 day old Fischer 344 rats, (Charles River Laboratories, Raleigh, NC) were housed in temperature and humidity controlled rooms (20 °C-25 °C, 35%-70% relative humidity) with a 12 hr light/dark cycle (light period = 06:00 to 18:00). Standard rat chow (ProLab, Brentwood, MO) and water were provided ad libitum. The rats had free access to deionized, reverse-osmosis-treated water and received autoclaved NIH 31 rodent chow (Zeigler Bros., Gardners, PA). All experiments were performed according to the United States Environmental Protection Agency Guidelines for the Care and Handling of Experimental Animals.

¹⁸O₃ and O₃ exposures

Rats were exposed to ¹⁸O₃ or O₃ in individual stainless steel wire mesh cages inside a 135 liter Rochester chamber at an airflow rate of 1.6 m³/hr. Control rats were exposed to filtered room air. ¹⁸O₃ was generated from ¹⁸O₂ using a corona discharge unit from a commercial NOx monitor (Bendix Corp., Louisburg, WV). Efficiency of conversion from ¹⁸O₂ was approximately 1.5%. This resulted in an excess ¹⁸O₂ concentration of 130 ppm over a natural abundance background of 400 ppm ¹⁸O₂ (ambient air contains 0.2% ¹⁸O₂). We have shown previously that this small increase in abundance of ¹⁸O₂ does not result in an appreciable increase in ¹⁸O in tissues.¹⁶ Chamber O₃ concentration was monitored with a Dasibi model 1003 AH O₃ monitor (Dasibi Environmental, Glendale, CA). Pre-exposures to unlabeled O₃ were performed similarly.

Experimental design

Table 1 shows a summary of the five experiments reported here. Experiment 1 employed a lower ¹⁸O₃ concentration for a longer time than subsequent experiments. Urine collection times were 07:00-08:00 and 17:00-18:00 for 4 days post ¹⁸O₃ exposure on all experiments.

Table 1

Summary of experiments performed in the present study.

Expt.	Purpose	Exposure	Groups	Comments
1	Demonstrate feasibility of detecting ¹⁸O₃ reaction products in urine and their molecular weight.	2 ppm ¹⁸O₃, 6 hr and air at 09:00-15:00.	Urine was collected twice daily for 4 days (n = 6 rats/group).	A sample of urine from each rat was dialyzed to retain the MW > 500 fraction which was analyzed separately for ¹⁸O content. Urease treatment was also done.
2	Demonstrate that ¹⁸O₃ reaction products are of respiratory origin rather than licked off the fur.	5 ppm ¹⁸O₃, 2 hr and air at 08:00-10:00.	Urine was collected twice daily for 4 days. A separate group of rats were bathed in detergent solution immediately post ¹⁸O₃ exposure (n = 4 rats/group).	Other measurements made included urine dry weight protein, albumin, urea, creatinine, and stability to heat.
3	Demonstrate the effect of pre-exposure to unlabeled O₃ one week before the ¹⁸O₃ exposure.	Pre-exposure to air, 2 or 5 ppm unlabeled O₃ for 2 hr was followed one week later by exposure of all rats to 5 ppm ¹⁸O₃ for 2 hr.	Urine was collected daily for 4 days from air, 2 and 5 ppm pre-exposed groups (n = 6 rats/group).	Other measurements made included urine volume, dry weight, urea and creatinine.
4	Quantify ¹⁸O label in bronchoalveolar lavage fluid (BALF) and in blood following ¹⁸O₃ exposure.	5 ppm ¹⁸O₃, 2 hr and air.	Blood was drawn and BALF cells and supernatants collected from separate groups of rats at 2, 7 and 16 hr post exposure (n = 6 rats/group).	BALF protein was also measured as an indicator of ¹⁸O₃ induced alveolar injury which resulted from loss of the blood-air barrier of the lung.
5	Examine the time course of appearance of ¹⁸O in urine following intratracheal instillation of BSA or PC pre-labeled in vitro with ¹⁸O₃.	Instilled 3O ug of ¹⁸O/rat in 14 mg/rat of BSA or PC.	Urine was collected for 4 days from saline, ¹⁸O-BSA and ¹⁸O-PC rats (n = 5 rats/group).	Other measurements made included urine volume, dry weight, urea and creatinine.

In experiment 2, the ¹⁸O₃ exposed rats were divided into two groups and half of the rats were bathed in detergent to remove ¹⁸O that could have been present as a reaction product with lipids or proteins on the fur and licked off during the urine collection period. Bathing was done immediately post ¹⁸O₃ exposure by immersion of rats briefly anesthetized with 5% halothane (Aldrich, Milwaukee, WI) in 0.4 liters of 0.1% sodium dodecyl sulfate. They were then rinsed in warm tap water and dried. A sample of the washing solution was lyophilized and the ¹⁸O content of the residue determined. In experiment 3, rats were pre-exposed a week prior to unlabeled O₃ to determine whether the pre-exposure would affect the subsequent elimination of ¹⁸O in the urine. The ¹⁸O₃ exposure involved three groups of rats with differing pre-exposure to unlabeled O₃.

Experiment 4 examined the quantities of ¹⁸O present in blood plasma and BALF. Rats were exposed similar to the urine studies and at 2, 7 and 16 hr post exposure they were anesthetized with pentobarbital (50 mg/kg body weight) and 5 mL of blood was removed from the dorsal aorta proximal to its bifurcation into the common iliac arteries. Immediately following exsanguination, lungs were lavaged with 37 °C saline (30 mL/kg body wt.) as previously described.¹⁹ BALF was centrifuged (400 × g, 15 min, 4 °C) and cell pellets and supernatants assayed for ¹⁸O and total protein.

Experiment 5 examined the time course of appearance of ¹⁸O in urine following intratracheal instillation of BSA or PC pre-labeled in vitro with ¹⁸O₃ (see details below).

Urine collection and preparation

Rats were housed individually in 4.4 liter volume (20 cm diam. × 14 cm high) plastic metabolism cages (Nalge Nunc, Rochester, NY) for seven days prior to exposure for acclimation to the cages. Following exposures to ¹⁸O₃, rats were returned to the metabolism cages and urine was collected for 4 days. The temperature of the urine collection tubes was maintained at 4 °C by enclosing them in copper tubing through which cooled ethylene glycol was circulated. Urine samples were centrifuged to remove extraneous debris (400 × g, 15 min, 4 °C), volumes were recorded and the supernatants were removed and stored at -80 °C. In the first two studies, the mg dry weight excreted per hour of urine collection was determined.

¹⁸O determination

The main purpose of the study was to examine ¹⁸O concentrations in urine and related tissues of the rats after exposure to ¹⁸O₃. All samples for ¹⁸O analysis were stored frozen (-80 °C) until lyophilization and then at 4 °C for a maximum of two months. Analysis for ¹⁸O content was performed on the dried material as previously described.¹⁶ Briefly, -0.5 mg of each sample was weighed into a silver cup and subjected to elemental analysis for oxygen content in a Carlo Erba elemental analyzer (model 1106, Fisions Inc., Danvers, MA and Elemental Microanalysis, Manchester, MA). This analyzer converted all oxygen in the sample to carbon monoxide which exited the analyzer in a helium stream. The effluent of the analyzer was directed by continuous flow through columns where the sample was further oxidized to CO₂ (140 °C, I₂O₅ granules) and a cold trap (-57 °C) to remove formed I₂. A small capillary stream of the resulting gas was pulled into the vacuum of an isotope-ratio mass spectrometer (model SIRA 10, VG Isogas, Cheshire, UK). The ¹⁸O/¹⁶O ratio of unknown samples was determined by reference to standards included in each sample run.

Preparation and intratracheal instillation of ¹⁸O-labeled lipid and protein solutions

Egg phosphatidylcholine (PC, Avanti Polar Lipids, Alabaster, AL) was dissolved in chloroform, dried under a stream of N₂ gas, then suspended by sonication in distilled water to achieve a concentration of 100 mg/mL. Bovine serum albumin (BSA) (Sigma, St. Louis, MO) was dissolved in distilled water to 100 mg/mL. Each solution was then exposed to 26 ppm ¹⁸O₃ for 1 hr in 125 mL flasks. ¹⁸O₃ was allowed to flow through a glass tube directly into each solution at a flow rate of 3.9 mL/min. After labeling, samples of each solution were lyophilized and stored at -20 °C. This dry material had stable concentrations of ¹⁸O over a -1 year period. BSA was labeled to achieve 2.33 mg ¹⁸O/g dry wt., and PC was labeled to a value of 2.14 mg ¹⁸O/g dry wt.

Intratracheal instillations were performed on rats anesthetized with 5% halothane. A 16 gauge cannula was inserted into the trachea after which an 18 gauge cannula attached to a syringe containing the solution to be instilled (0.3 mL of 45 mg/mL BSA or PC) was injected through the 18 gauge cannula into the lungs. This resulted in 31.4 μg ¹⁸O (1.7 umoles) per rat for ¹⁸O-BSA and 28.9 μg ¹⁸O (1.6 umoles) per rat for ¹⁸O-PC (see results of instillations in Table 2). The instillations had no noticeable toxic effect on the rats.

Table 2

The levels of retained or instilled ¹⁸O (per rat) following inhalation of ¹⁸O₃ or intratracheal instillation of ¹⁸O labeled protein or lipid: percentage of ¹⁸O retained in different tissue pools relative to exposure levels.

Treatment	¹⁸O retained by or instilled into the whole animal		Measured ¹⁸O in tissue pool
Treatment	Method of prediction of retained ¹⁸O	umoles ¹⁸O/rat	Tissue pool	umole/rat	Retained/measured, %
¹⁸O₃, 5 ppm 2 hr	Allometric equation and % uptake calculations	4.0	Urine	2.1	53
¹⁸O₃, 5 ppm 2 hr	Allometric equation and % uptake calculations	4.0	BAL fluid	0.8	20
¹⁸O labeled bovine serum albumin	Amount instilled intratracheally	1.8	Urine	1.0	54
¹⁸O labeled phosphatidyl choline	Amount instilled intratracheally	1.6	Urine	0.2	12

Dialysis and heat stability of ¹⁸O in urine

Dialysis of urine in Expt. 1 was performed by adding a 2.0 mL aliquot of each urine sample to 500 MW cutoff SpectroPor DispoDialyzer® dialysis tubes (Spectrum, Laguna Hills, CA). Urine was dialyzed against 8 liters of distilled water for 24 hr at 4 °C. To determine the heat lability of the ¹⁸O label, dried samples of the urine from ¹⁸O₃-exposed rats were heated for 30 min in a ceramic radiant heater (Omega Engineering, Inc., Stamford, CT) controlled by a rheostat (Superior Electric Co., Bristol, CT) and monitored by thermocouple (Omega type K). After cooling, the samples were again weighed and ¹⁸O contents determined.

Biochemical analyses

Urinary creatinine and urea were determined by coupled enzyme reaction (Sigma Diagnostics, St. Louis, MO). Total protein was analyzed by a Coomassie blue colorimetric method (Pierce Rockford IL). Albumin was analyzed using an immunoprecipitation based kit (DiaSorin, Stillwater, MN). These assays were adapted for use on a Cobas Fara II clinical analyzer (Roche Diagnostics, Branchburg, NJ). Some samples of urine were treated to convert the urea to CO₂ and NH₃ using urease (Sigma Type III from Jack Beans, final concentration of 1.7 U/mL). Samples were incubated 18 hr at 4 °C.

Data analysis

¹⁸O/¹⁶O ratios were derived from the mass spectrometer as ‘delta values’ relative to high and low standards containing known ¹⁸O/¹⁶O ratios included in each sample run. Delta values of ¹⁸O₃ exposed and air exposed samples were compared to determine whether the ¹⁸O₃ exposed samples were elevated (t-test, P value ≤ 0.05) above the air exposed samples. In all but experiment 5, ¹⁸O₃ treated samples were significantly elevated above natural abundance samples. Levels of ¹⁸O enrichment due to the ¹⁸O₃ exposures were determined by subtracting the mean natural abundance of ¹⁸O (~0.2 atom%) from all samples. The natural abundance ¹⁸O concentration was obtained from analysis of the same type of samples from air-exposed rats. Units of ¹⁸O enrichment were converted from umoles ¹⁸O/mole of total oxygen to ug ¹⁸O/gram dry weight by use of the mean percentage of oxygen in the dry samples (obtained as output from the elemental analyzer). The ‘excess ¹⁸O in samples resulting from ¹⁸O₃ exposure’ will hereafter be termed simply ‘¹⁸O incorporation’ or ‘¹⁸O.’

Elemental oxygen and nitrogen content of urine Since the elemental oxygen percentage (%O) of the samples was used in the calculations of ¹⁸O incorporation per gram dry weight (see Methods), we report that %O of the lyophilized urine pooled across collection times was 21.3% ± 0.3% (n = 42). The %N was also measured and averaged 25.4% ± 0.4% (n = 42). Exposure to ¹⁸O₃ did not significantly affect these percentages. Following dialysis, the urinary % N was reduced to 10.5% ± 0.9%, while the %O was not changed. Following urease treatment of urine %N was reduced by ~43%.

Results

The following results suggest that ¹⁸O₃ reaction products leave the respiratory tract, pass through the blood and are excreted in the urine. The time course of appearance of the ¹⁸O label in blood and urine, as well as the properties of the labeled material are reported.

Urinary ¹⁸O following ¹⁸O₃ exposure

Experiment 1 results showed that urinary ¹⁸O concentration (per gram of dry weight) was significantly elevated on all 4 days following ¹⁸O₃ exposure (Fig. 1), decreasing from 16 to 7 μg ¹⁸O/g dry during this period. Following dialysis to remove material smaller than 500 Daltons, the urine dry weight was reduced to about one fifth of its original value and the ¹⁸O concentration was increased ~60%. Urease treatment caused a ~50% higher ¹⁸O/gram dry weight in the first urine collection sample, however, all later samples showed insignificant changes in ¹⁸O content.

Figure 1

The time course of excretion of ¹⁸O in the urine of F344 rats following exposure to ¹⁸O³ (2 ppm, 6 hr), and the effect of removal molecules, 500 MW by dialysis. Excess ¹⁸O was easily detectable in all urine samples for 4 days following the ¹⁸O₃ exposure. Darkened bars represent periods of night time (18:00 to 06:00) in this and subsequent figures.

Effect of washing the fur

Washing the fur of the rats immediately after the ¹⁸O₃ exposure did not appear to alter the urinary ¹⁸O (Fig. 2). The dried washing solution contained ~230 ug ¹⁸O (13 umoles of ¹⁸O)/rat or about 5 times the amount recovered in urine.

Figure 2

Time course similar to that shown in Figure 1, but following a higher exposure level for a shorter time (5 ppm, 2 hr) and showing the effect of washing the fur of half of the rats to remove the possible influence of licking ¹⁸O from the fur. Note that the ¹⁸O appeared to be unaffected by the washing step, meaning that the ¹⁸O appears to be of respiratory origin. The amount of ¹⁸O recoverable in the dried wash fluid was -13 umole/rat or about 5 times the amount excreted into the urine.

Night versus day excretion

In the first three experiments there was a tendency for urine to be more concentrated at the morning collection time than at the evening collection. This created a sawtooth appearance of the time course of ¹⁸O disappearance. The possibility that the rate of ¹⁸O excreted per hour might also be higher during the night was investigated by obtaining the product of the ¹⁸O concentration (per gram dry) and the grams dry weight excreted per hour at the different collection periods. Figure 3 shows the rate of excretion of urine dry weight (mg dry weight per hour) during the times preceding each urine collection period versus the urine collection time. These data seemed to explain the sawtooth pattern of ¹⁸O excretion since the excretion rate of dry material was 1.6 to 3.9 fold higher during the night than during the day. There was a visual tendency for higher rates of dry weight excretion at the later times of urine collection in all exposures suggesting that at the early times there was a stress-induced reduction of excretion rates. This reduction in rate was observed in the air exposed and ¹⁸O₃ exposed, however, it was more prolonged after ¹⁸O₃ exposure (Fig. 3). Thus, both the rate of ¹⁸O excretion and urine dry weight excretion appeared to be higher during the night than during the day.

Figure 3

Rate of urine dry weight excreted (per hour per rat) following exposure to 5 ppm ¹⁸O₃ for 2 hr. Note that the excretion rate was higher during night time periods compared to daytime periods and that ¹⁸O₃ exposure appeared to lower the dry weight excretion at the early sampling times.

Effect of pre-exposure to O₃

The possibility that pre-exposure to O₃ might induce an adaptive response measurable by altered excretion of ¹⁸O₃ products was addressed in the third experiment which first exposed rats to air, 2 or 5 ppm O₃ (2 hr) then followed up with a second exposure a week later of all rats to 5 ppm ¹⁸O₃. We analyzed the ¹⁸O disappearance curve following ¹⁸O₃ exposure by selecting only the data for the urine collected in the morning. This led to smooth logarithmic washout curves with high R values (Fig. S1). Equations shown on the figures describe the fitted trend lines for the logarithmic decline in ¹⁸O over time. Rats pre-exposed to O₃ had a slightly steeper slope and higher Y intercept than the air pre-exposed group, however, these changes were small (<17%) and of questionable biological significance.

Excreted ¹⁸O compared to inhaled and retained ¹⁸O₃

The total amount of ¹⁸O found in urine of rats exposed to ¹⁸O₃ over the four day collection period was 2.1 μmols (Table 2), and the calculated amount of ¹⁸O that should have been retained per rat was 4.0 μmoles (see Supplement A for calculations). Thus, 53% of the amount of ¹⁸O retained by each rat following the ¹⁸O₃ exposure appeared to be excreted into the urine over the four post exposure days (Table 2).

¹⁸O in bronchoalveolar lavage fluid

We compared the quantities of ¹⁸O detected in the BALF supernatants of rats after ¹⁸O₃ exposure with the amounts excreted in urine by performing an experiment in which BALF was collected after exposure to ¹⁸O₃. Rats exposed to 5 ppm ¹⁸O₃ (2 hr) showed high levels of BALF extracellular protein (~3 mg/mL) compared to normal BALF protein levels (~0.1 mg/mL, Fig. 4). Protein levels decreased ~30% over 16 hr. ¹⁸O concentration in the BALF supernatant was ~150 ug ¹⁸O/g dry and decreased ~74% over 16 hours (Fig. 4). We estimate that the increase in BALF protein corresponds to about 0.3 mL of plasma leakage into the air spaces of the lung at the 2 hr post exposure time (Supplement E).

Figure 4

Disappearance of ¹⁸O and total protein from bronchoalveolar lavage fluid (BALF) low speed supernatants following exposure to ¹⁸O₃ (5 ppm, 2 hr). Normal protein concentrations in BALF are -0.1 mg/mL and are elevated by ¹⁸O₃ exposure. The amount of ¹⁸O present in the BALF at 2 hr post exposure was about 20% of the amount of ¹⁸O₃ calculated to be removed from respired air (see Table 2 and Supplement C).

¹⁸O in blood

The loss over time of ¹⁸O in BALF suggested that there should be a corresponding appearance of ¹⁸O in the blood. We estimated that if all of the ¹⁸O present in BALF at 2 hr post exposure (13.8 ug/rat–-see Supplement A) were immediately added to the blood plasma, the level of ¹⁸O would be ~24 ug/g dry which is much higher than our measured concentration of ~1.8 ug/g dry at 7 hr post exposure but not at other times (Fig. 5 and Supplement D). We did not find detectable ¹⁸O in red blood cells following ¹⁸O₃ exposure.

Figure 5

Concentration of excess ¹⁸O in blood plasma from rats breathing 5 ppm ¹⁸O₃ for 2 hours at 2, 7 and 16 hr post exposure. Significantly elevated plasma ¹⁸O was observed at 7 hr post exposure.

¹⁸O in urine of rats intratracheally instilled with ¹⁸O-PC and ¹⁸O-BSA

We investigated the possibility that simple transport of ozonized lipids or proteins from the pulmonary airways might account for the appearance of ¹⁸O in blood and urine following ¹⁸O₃ exposure by intra-tracheally instilling ¹⁸O PC or ¹⁸O-BSA generated by ¹⁸O₃ exposure in vitro. Amounts of ¹⁸O instilled were targeted to be similar to what was achieved following inhalation exposures to ¹⁸O₃ (see Table 2). A small increase in urinary ¹⁸O was observed in all ¹⁸O-BSA and ¹⁸O-PC instilled rats (Fig. S2). Due to problems encountered with the pyrolysis of urine samples, and also to relatively low levels of ¹⁸O detected in these experiments, it was not possible to obtain the desired statistical rigor or to define a clear washout behavior for ¹⁸O PC or ¹⁸O-BSA. We estimate that ~12.3% and ~54% of the instilled ¹⁸O-PC or ¹⁸O-BSA, respectively, was excreted into the urine over the four days of collection (Fig. S2 and Table 2).

Biochemical measurements made on urine

In addition to the dry weight of the urine samples, we measured urinary volume, creatinine, urea, total protein and albumin as potential denominators for expressing the ¹⁸O found in the urine. We found that urine volumes and dry weights (per day per rat) were correlated and that dry weights were always ~100 mg/mL of urine. Urinary albumin concentrations were always very low (< 6 ug/mL). Urinary urea showed values of 1000-2500 mg/dL (Fig. S3) and were elevated in both the air and ¹⁸O₃ exposed rat urine at the early collection times. Urinary creatinine ranged from ~30-150 mg/dL and showed a similar increase at the first collection times for both air and ¹⁸O₃ exposed rats. The ¹⁸O₃ exposed rats had a more prolonged elevation of creatinine and urea levels than the air exposed rats (Fig. S4). Intratracheal instillation of ¹⁸O-BSA, ¹⁸O-PC and sham saline increased urinary urea and creatinine for the first two days of collection similar to the inhalation exposures to air and ¹⁸O₃. There was no difference between the three treatments (data not shown).

Heat stability of ¹⁸O in urine

Samples of lyophilized urine from ¹⁸O₃ exposed rats were heated from 200 °C to 500 °C and remaining weights and ¹⁸O contents graphed (Fig. S5). Whereas the sample weights fell off rapidly to ~40% of the original dry weight as heat was increased to 200 °C, the ¹⁸O concentration was unaffected. As temperatures were further raised to 400 °C, ¹⁸O concentration fell to ~40% of unheated samples while sample weights did not show a further decrease. At 500 °C, both sample weight and ¹⁸O concentration were decreased to ~20% of unheated values.

Discussion

We report here that the use of ¹⁸O₃ enabled quantification of the generalized product of ¹⁸O₃ reactions originating in the respiratory tract in urine and blood. These findings appear to be the first proof that O₃ reaction products leave the respiratory tract, pass through the blood, and are excreted in the urine. They also appear to be the first application of ¹⁸O technology to measuring products of oxidative stress in urine. The time course of appearance of ¹⁸O in blood and urine, as well as properties of the labeled material provide insights that may be useful in explaining extrapul-monary effects of O₃. For example, atherosclerotic plaque formation has been shown to be enhanced by O₃ exposure. It is possible that oxidized proteins and lipids leaving the lung through the pulmonary veins could deposit in the walls of arteries leaving the heart. The detection of ¹⁸O in blood plasma and urine proves that the reaction products of ¹⁸O₃ pass through the blood; however, the lower-than-expected ¹⁸O levels in blood plasma may suggest significant binding of ¹⁸O-labeled products to vascular endothelium. Our previous attempts to measure excess ¹⁸O in red blood cells have not been successful. The percentage of ¹⁸O label excreted over 4 days relative to the amount deposited through inhalation was high (53%) suggesting that little recycling of ¹⁸O₃ reaction product occurs. It also implies that the oxygen addition reactions induced by ¹⁸O₃ are irreversible, damaging, and must be removed. Our finding that pre-exposure of the rats to O₃ one week prior to the ¹⁸O₃ exposure did not appear to alter the urinary disappearance curve of ¹⁸O suggests that adaptation to the oxidative stress of ¹⁸O₃ does not involve altering the rate of adduct removal.

Our observation that ¹⁸O was relatively heat stable and also enriched in the high molecular weight fraction of the urine might guide future efforts to focus on specific chemical biomarkers. Details about the rates and times of excretion of ¹⁸O might simplify and give direction to future urine collection for biomarker measurement. A published series of studies showed that some traditional measures of oxidative stress (isoprostanes and malondialdehyde in blood plasma and urine) that were effective following CCl₄–-induced oxidative stress to the liver, were not effective following inhalation of O₃ in the rat at the same level of exposure as that employed here.^13–15 Our quantitation of ¹⁸O in the blood plasma and urine suggests the possibility of finding other biomarkers that could be more effective in the future.

The ¹⁸O label in urine could have originated from injured cells in the lung or vasculature that were replaced by proliferative repair or from simple transport of extracellular ¹⁸O-containing adducts of proteins and lipids. If simple transport of the labeled proteins in BALF were to occur, we would have expected to recover in urine about the same amount of ¹⁸O that was present in the BALF supernatant fraction. We detected about 1/5th as much ¹⁸O in BALF as the estimated ¹⁸O₃ retained by the rat. This percentage was lower than the percentage of ¹⁸O that was excreted into urine (see Table 2 and Supplement C). Most of the ¹⁸O present in the BALF was associated with plasma proteins that had leaked into the injured airway lumen during the ¹⁸O₃ exposure because of damage to the air-blood barrier of the lung. Our experiments with intratracheally instilled serum albumin or phosphatidyl choline pre-labeled in vitro by exposure to ¹⁸O₃ showed that ozonated proteins and surfactant lipid can leave the lung and appear in the urine, however, concentrations detected were lower than expected. Only about 12% of the instilled ¹⁸O-PC and 54% of ¹⁸O-BSA appeared to be recoverable in the 4 days of urine collection post exposure (Table 2). A previous study instilled ¹²⁵Iodine labeled serum albumin into the alveoli and reported transport into the blood minutes following its instillation.²⁰ Previous studies of vascular injections of radiolabeled precursors of surfactant proteins and lipids showed that turnover of surfactant occurs rapidly–-on the order of hours–-rather than days as we observed here with ¹⁸O₃ reaction products. A high level of recycling of labeled surfactant lipids was also reported in normal rats²¹ in contradiction to the present study where ¹⁸O₃ reaction products appeared to be in large part excreted. It appears, therefore, that the injured lung may release ¹⁸O₃ reaction products slowly (over days) in comparison to the normal turnover of proteins and surfactant lipids (over hours). The slow transport of labeled material through the blood or possibly sequestration and slow release of label from the vascular endothelium may explain why it was difficult to detect ¹⁸O in blood plasma even though the quantities of ¹⁸O passing through the blood are significant (see Supplement D).

It appeared that the elevated levels of urea and creatinine we observed following exposure or intra-tracheal instillations could have been due to the reduced urinary volumes and dry weights at the early times after exposures. Reduced water and food consumption, along with a concomitant decrease in urine volume excretion often occurs due to stress. The wire mesh exposure cages appear to induce a stress response even in control rats that is manifest as hyper-thermia that lasts about 2 hr.²²

The present study is limited due its exploratory and descriptive nature. The calculations of percentages of recovered ¹⁸O in urine could be affected by estimates of ¹⁸O₃ inhaled and retained by the rats that are based on inexact allometric equations. ¹⁸O measurement of tissue samples suffer from four sources of error: (1) preparatory column conditioning, (2) instrument drift, (3) sample memory effects, and (4) dependence on accurate background ¹⁸O measurements included in each sample run.²³ It might be difficult to perform the present study at lower (and less injurious) exposure concentrations of ¹⁸O₃ because our sensitivity of detection of ¹⁸O in tissues was at the lower limit. Urine samples showed more variability and difficulties due to column conditioning (possibly related to the presence of inorganics) than plasma samples. Tracing the fate of oxygen using a stable isotope such as ¹⁸O, is necessary because radioactive forms of oxygen have extremely short half lives (< 134 sec).

In summary, we have shown that ¹⁸O₃ exposure of rats results in pathologically bound oxygen that is excreted into urine over a period of 4 or more days. Our findings suggest that new biomarker molecules specific to ozonized lung tissue could be identified in the future. The demonstrated transport of reaction products of O₃ formed in the lung or in the blood passing through the lung during exposure lends plausibility to published findings that O₃ inhalation could exert influences outside the lung. Future studies should search for O₃ reaction products in the vascular endothelium and investigate the chemical structures of oxidized biomolecules in urine.

Author Contributions

Conceived and designed the experiments: GEH. Analysed the data: GEH and RS. Wrote the first draft of the manuscript: GEH and RS. Contributed to the writing of the manuscript: RS. Agree with manuscript results and conclusions: GEH, RS and JM. Jointly developed the structure and arguments for the paper: GEH and RS. Made critical revisions and approved final version: GEH, RS and JM. All authors reviewed and approved of the final manuscript.

Funding

The study was performed in-house by the U.S. Environmental Protection Agency.

Disclaimer

The research described in this article has been reviewed by the National Health and Environmental Effects Research Laboratory, United States Environmental Protection Agency and approved for publication. Approval does not signify that the contents necessarily reflect the views and policies of the agency, nor does mention of trade names or commercial products constitute endorsement or recommendation for use.

Competing Interests

Author(s) disclose no potential conflicts of interest.

Disclosures and Ethics

As a requirement of publication the authors have provided signed confirmation of their compliance with ethical and legal obligations including but not limited to compliance with ICMJE authorship and competing interests guidelines, that the article is neither under consideration for publication nor published elsewhere, of their compliance with legal and ethical guidelines concerning human and animal research participants (if applicable), and that permission has been obtained for reproduction of any copyrighted material. This article was subject to blind, independent, expert peer review. The reviewers reported no competing interests.

Footnotes

Acknowledgements

The authors express appreciation to Drs. Christopher Gordon, Maria Kadiiska and Daniel Costa for review of the manuscript, Tony McDonald and Kay Crissman for assistance with urine collection, James Lehmann for intratracheal instillations, Shirley Henry and Bobby Crissman for assistance with ¹⁸O analyses, and Judy Richards for urinary biochemical analyses.

Supplementary Data

References

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Fate of Pathologically Bound Oxygen Resulting from Inhalation of Labeled Ozone in Rats

Abstract

Keywords

Introduction

Methods

Animals

18O3 and O3 exposures

Experimental design

Urine collection and preparation

18O determination

Preparation and intratracheal instillation of 18O-labeled lipid and protein solutions

Dialysis and heat stability of 18O in urine

Biochemical analyses

Data analysis

Results

Urinary 18O following 18O3 exposure

Effect of washing the fur

Night versus day excretion

Effect of pre-exposure to O3

Excreted 18O compared to inhaled and retained 18O3

18O in bronchoalveolar lavage fluid

18O in blood

18O in urine of rats intratracheally instilled with 18O-PC and 18O-BSA

Biochemical measurements made on urine

Heat stability of 18O in urine

Discussion

Author Contributions

Funding

Disclaimer

Competing Interests

Disclosures and Ethics

Footnotes

Acknowledgements

Supplementary Data

References

¹⁸O₃ and O₃ exposures

¹⁸O determination

Preparation and intratracheal instillation of ¹⁸O-labeled lipid and protein solutions

Dialysis and heat stability of ¹⁸O in urine

Urinary ¹⁸O following ¹⁸O₃ exposure

Effect of pre-exposure to O₃

Excreted ¹⁸O compared to inhaled and retained ¹⁸O₃

¹⁸O in bronchoalveolar lavage fluid

¹⁸O in blood

¹⁸O in urine of rats intratracheally instilled with ¹⁸O-PC and ¹⁸O-BSA

Heat stability of ¹⁸O in urine