Ozone

Endpoint summary

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Objective of study:

absorption

Qualifier:

no guideline followed

Principles of method if other than guideline:

Research study

GLP compliance:

no

Specific details on test material used for the study:

Ozone was generated in-situ using an uv ozone generator

Radiolabelling:

no

Species:

human

Sex:

male

Details on test animals or test system and environmental conditions:

Ten male subjects, 19-32 years of age, were recruited from the community in and around Chapel Hill, North Carolina. Excluded from participation in the study was anyone who smoked, had a history of asthma, allergic rhinitis, cardiac disease, acute respiratory disease within the previous 4 weeks. Screening procedures included a medical history, a physical examination, and a complete blood count plus differential white cell count. Accepted subjects were informed of the purpose of the study, the experimental methods, and the potential risks of participation before signing a statement of informed consent. This study was approved by the Committee on the Protection of the Rights of Human Subjects of the University of North Carolina School of Medicine.

Route of administration:

inhalation: gas

Vehicle:

other: air

Details on exposure:

The exposure apparatus was located in a 4 x 6 x 3.2-m stainless-steel walk-in environmental chamber. The chamber was maintained at 22°C and 40% relative humidity. Ozone was produced for the chamber by passing incoming air through a series of ultraviolet light O3 generator tubes. Ozone concentration was monitored by chemiluminescence detection using Bendix Model 8002 analyzers which were calibrated against an ultraviolet O3 photometer (Dasibi Model 1003AH). A detailed description of the environmental chamber operation has been published.
The subject exposure apparatus consisted of 4-in. stainless-steel pipe which opened to the environmental chamber at one end and attached to a fan/damper assembly at the other end. Chamber air was drawn down through the apparatus by the fan/damper assembly. Ports were cut into the apparatus for sampling of O3, O2, and CO2. A face port was also cut into the pipe for the subject to insert his nose and mouth and breathe from the airstream flowing through the apparatus. The exposure apparatus flow rate was approximately 40 L/min. At this rate, the downstream (relative to the face port) dilution of exhaled air with the airstream was similar to that used in the rat and guinea pig studies. Testing indicated that this rate was sufficient to prevent the rebreathing of downstream air by the subject and to supply an adequate volume of upstream air during maximum inspiratory flow. Flow measurements were determined using a Roots meter (Model 5M). Additionally, flows were checked for accuracy with a Gilibrator Primary Flow Calibrator (Gilian Instrument Corp., West Caldwell, NJ).

Duration and frequency of treatment / exposure:

Single 10 min exposure, followed by a 2nd 10 min exposure two weeks later

Dose / conc.:

0.3 ppm

No. of animals per sex per dose / concentration:

n=10 subjects

Control animals:

no

Positive control reference chemical:

not applicable

Details on study design:

Subjects were measured, using the same experimental protocol, on two visits. These visits were separated by at least 14 days.
On the day of an exposure, the subject completed a symptom questionnaire and performed forced expiratory maneuvers. These consisted of three forced vital capacity (FVC) maneuvers performed on a 12-1 dry seal spirometer (CPI model 220). Peak expiratory flow rate (PEF) and forced expiratory volume at 1 sec (FEV, 0) were calculated for each maneuver. The subject was then fitted with a telemetered ECG monitor and the Respitrace bands and exposed to clean air in the exposure chamber. The first 5 min for system equilibration followed by 10 min sampling to collect air exposure data. Similar runs were performed for nose and mouth breathing. Subsequently the whole procedure was repated, but with exposure to ozone. The percentage of O3 uptake was calculated using O3 upstream (µg/L); O3 downstream (µg/L); O3 background loss (µg/L); SAF is the system airflow (L/min); and VE (L/min).

Details on dosing and sampling:

Breathing parameters.
Subject chest wall movements caused by breathing efforts were measured using Respitrace inductance plethysmography chest bands (Respitrace, Ambulatory Monitoring, Inc., Ardsley, NY). The fitted Respitrace was calibrated by having the subject quietly breathe into a rolling seal spirometer that had been previously calibrated with a 1-liter syringe.
Tidal breathing measurements were obtained dunng the experiment by sampling the Respitrace signal at 12 msec intervals for 20 sec each minute. Breathing parameters reported represent averaged values from all breaths completed during this 20-sec sampling period (usually four to six breaths). These include tidal volume (VT), breathing frequency (f) expiratory minute volume (VE); maximum inspiratory and expiratory flows (Vimax. Vemax), inspiratory and expiratory times (Ti, Te), oxygen consumption and carbon dioxide production and the respiratory quotient (VO2, VCO2, RQ).

Statistics:

All breathing parameters and the percentage of O3 uptake were examined for time-related trends over the 10-min exposure period using linear regressions. These were calculated for each subject and breathing mode during each exposure. Because no trends were found, the individual minute by minute data were averaged for each subject dunng each exposure for each breathing mode. These means were used as the unit of observation for the subsequent analyses.
A two-way multivariate analysis of variance (MANOVA) was used to examine the effects of "route" of exposure (nasal or oral), "visit" (first or second), and the interaction between these two factors on the percentage of O3 uptake. Significance of an effect was determined using the Hotelling- Lawley trace. Because there were only two categories for each main effect companson (i.e., nasal versus oral or visit 1 versus visit 2), no further subtesting was required for significant main effects. Interactions between route and visit were tested using paired / tests. Three-way MANOVAs were used to examine the effects of route, visit, and exposure (air versus O3) and all possible interactions in the breathing data.
Pearson correlation coefficients were calculated to test for linear relationships between the percentage of O3 uptake and the breathing parameters, spirometric measurements of lung function, and subject weight and height.

Preliminary studies:

no

Details on absorption:

There was no significant effect of visit on the percentage of O3 uptake, demonstrating the reproducibility of the measurement. There was a slight but significant route effect such that the mean percentage of O3 uptake when breathing orally was 76.5% ± 3.3 SE compared to 73.1% ± 3.0 when breathing nasally. There was no interaction between visit and route.
The relative extent of intra- and intersubject variability was assessed. The variability between subjects was substantial. The lowest percentage of O3 uptake was observed in one subject, ranging from about 50 to 60% whether breathing orally or nasally. Among the nine other subjects, the percentage of O3 uptake ranged between about 65 and 95%, a difference of about 30%. In general, the variability in percentage of O3 uptake for an individual subject was less than this although in some cases (e.g., oral breathing), there was a difference of approximately 25% between the first and second visits. The average difference in percentage of uptake between the first and second visits for oral breathing was -3.1% ± 3.7 SE and for nasal breathing was -0.2% ± 3.9. Neither of these values are statistically significantly different from zero, suggesting that the percentage of uptake is generally not influenced between visits.
No statistically significant differences were found between spirometric measurements made on the first and second visits. The absence of statistically significance changes in breathing parameters among the exposure periods indicates that the subjects were breathing at steady state and that the O3 exposures were low enough not to affect ventilation.

Details on distribution in tissues:

no

Details on excretion:

no

Metabolites identified:

no

Details on metabolites:

no

Bioaccessibility (or Bioavailability) testing results:

no

Conclusions:

In conclusion, the study provides information on the absorption of ozone in healthy adult male subjects during normal quiet breathing by mouth and by nose under steady-state exposure (0.3 ppm for 10 min) and breathing conditions. Among the 10 subjects, the average percentage of O3 uptake was slightly, but statistically significantly greater, with oral breathing (76%) than with nasal breathing (73%). The percentage of O3 uptake ranged from approximately 50 to over 95% in 10 subject indicating the difference between subjects. This study also illustrated that the percentage of O3 uptake is reproducible within subjects, varying by about 25% or less.

Executive summary:

In this study, 10 healthy adult male subjects were exposed to 0.3 ppm O3 while seated and breathing naturally through the nose or mouth. Total respiratory tract O3 uptake, spontaneous breathing parameters, and respiratory gas exchange were measured for 10 min under steady-state conditions. The exposure protocol was replicated in each subject approximately 2 weeks after the first visit. On each visit, health exams were performed and spirometric lung measurements were obtained. The experimental design provided comparisons of total O3 uptake during nasal and oral breathing, differences in uptake in an individual at two time points, and an examination of between-subject variability in O3 uptake. Exposure to O3 had no effect on the breathing parameters or gas exchange. Oral and nasal breathing frequency averaged 16.2 ± 1.1 (SE) and 16.0 ± 1.2 breaths per minute with tidal volumes averaging 651 ± 46 and 669 ± 67 mL, respectively. A significant correlation (p < 0.01) was found for the minute volume during resting breathing with the percentage of uptake. The percentage of O3 uptake was consistently higher (p = 0.02) during oral breathing (76.5% ± 3.3) than during nasal breathing (73.1% ± 3.0) although this difference may not be biologically significant. The variability in percentage of uptake between subjects was substantial with calculated uptakes ranging from 51 to 96%, a difference of about 45%. Variability in percentage of uptake for an individual was less with the maximal difference between the first and second visits being about 20%; the average difference, however, was only about 3%. It is concluded that total percentage of O3 uptake is approximately 75% in adult males during resting breathing. It is slightly greater during oral than during nasal breathing, will vary considerably among subjects, and is moderately reproducible within a subject.

Reference 2

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Objective of study:

absorption

Qualifier:

no guideline followed

Principles of method if other than guideline:

Research study

GLP compliance:

no

Specific details on test material used for the study:

Ozone was generated in-situ using an UV ozone generator.

Radiolabelling:

no

Species:

rat

Strain:

other: Fischer 344, Long Evans, Sprague-Dawley

Sex:

male

Details on test animals or test system and environmental conditions:

Male rats of the Fischer 344 (F-344), Sprague-Dawley (SD), and Long-Evans (LE) strains were purchased from Charles River Breeding Laboratories. Kingston. New York. Animals were held in quarantine (SD, and LE rats for 1-2 weeks and F-344 rats for 4-6 weeks) in a facility maintained at 21-24°C with a 12-hr-on-12-hr-off light schedule. Food and water were available ad libitum until the time of the experiment (Purina Rodent Lab Chow No. 500. St. Louis, MO).

Route of administration:

inhalation: gas

Vehicle:

other: air

Details on exposure:

Each animal was restrained and sealed inside a body plethysmograph with its head extending into a glass coneshaped, nose-only, exposure chamber. The glass chamber facilitated precise placement of the nose within the airstream so that exposures between animals were consistent. Flow past the nose was adequate to prevent rebreathing of upstream air (1200 mL/min). The exposure atmosphere was produced using two tanks of Zero-grade air (Union Carbide Corp..Linde Division, Danbury, CT); one was metered through a uv ozone generator while the other supplied clean air for dilution. Uniform flow through each section of the system was maintained using mass flow meters and was monitored with pressure gauges. All pipes and fittings were made of stainless steel and were baffled to assure even distribution of 0s as it passed the nose. Static pressure in the airstream, at the nose and at analyzer inputs, were slightly greater than atmospheric.

Duration and frequency of treatment / exposure:

Single 1 hour exposure

Dose / conc.:

0.3 ppm

Remarks:

all strains

Dose / conc.:

0.6 ppm

Remarks:

one additional group of F-344 rats

No. of animals per sex per dose / concentration:

n=9 per group

Control animals:

no

Positive control reference chemical:

not applicable

Details on study design:

Rats were nose only exposed to 0.3 or 0.6 ppm ozone for one hour. Based on the ozone concentration in inspired and expired air the percentage of ozone retained was calculated. In addition lung function parameters were accessed in this study.
The testing protocol was as follows: With air flowing through the system an animal was secured in the plethysmograph and its head was sealed inside the exposure cone. A 30-min air exposure period commenced allowing time for the animal to acclimate to restraint and to the apparatus. Control readings were collected during the last 10 min of this period. Ozone was introduced into the airstream at a predetermined setting to achieve the desired concentration for exposure. After 60 min of O3 exposure the animal was injected intraperitoneally with a lethal dose of sodium pentobarbital without disturbing the position of the nose in the airstream.

Details on dosing and sampling:

The output from a pressure transducer, used to measure tidal volume, and the outputs from three gas analyzers (O3, O2, and CO2 were recorded silmutaneously on a polygraph and by computer. Data, which printed-out once a minute during each experiment, included tidal volume (VT), breathing frequency (f) expiratory minute volume (VE). maximum inspiratory and expiratory flows (Vimax. Vemax). inspiratory and expiratory times (Ti, Te), oxygen consumption and carbon dioxide production and the respiratory quotient (VO2, VCO2. RQ), quantities of O3 inhaled and O3 retained per minute (O3IN. O3RT), and the computed O3 percentage uptake by the animal (O3UP).

Statistics:

Control period data were analyzed using one-way analysis of variance (ANOVA) to test for preexposure differences among the treatment groups. Exposure-period data were analyzed in two stages. First, an autoregressive model was fit for each animal. Second, the parameters estimated in the first part (intercept, slope, and an autoregressive error term) were analyzed in a multivariate analysis of variance (MANOVA) to test for differences in response among the treatment groups. Significant multivariate effects were subtested using one-way ANOVAs to determine which estimated parameter was significant. Significant ANOVA effects were examined with Duncan’s multiple range test to test for statistical differences among the treatments.

Preliminary studies:

no

Details on absorption:

This study showed that O3 uptake (O3UP) was not significantly affected by O3 concentration or by time. Mean values for O3 uptake in rats are 45.0, 43.5, 45.7 and 47.6% for F-344 (0.6 ppm), F-344 (0.3 ppm), Sprague-Dawley (0.3 ppm) and Long Evans (0.3 ppm), respectively.

Review of the O3 exposure period data revealed that there were no differences between exposure groups for any of the functional variables. Ventilation was found to diminish over time and at the same rate, regardless of O3 exposure concentration, with statistically significant decreases seen in tidal volume (VT), expiratory minute ventilation (VE), carbon dioxide production (VCO2) and oxygen consumption (VO2).The actual quantity of O3 taken up per minute by the F-344 rat during exposure increased as O3 concentration increased and decreased over time similar to time trends seen in the ventilatory measurements.

Details on distribution in tissues:

no

Details on excretion:

no

Metabolites identified:

no

Details on metabolites:

no

Bioaccessibility (or Bioavailability) testing results:

no

Conclusions:

In conlusion, the study provides information on the absorption of ozone in three different straine of rats rats following a nose only exposure to 0.3 or 0.6 ppm for 1 hour. The study shows that more ozone is retained as ozone exposure concentration increased. The percentage uptake was constant over time and equal for each strain, averaging around 45% for all three strains.

Executive summary:

Ozone uptake was assessed in awake, spontaneously breathing Fischer-344 Sprague-Dawley, and Long-Evans rats to provide data on the dosimetry of O3 in small laboratory animals. Breathing measurements and O3 exposure data were obtained using a head-out body plethysmograph connected to a nose-only exposure system. The fractional uptake of O3 was computed by measuring flow and O3 concentration both upstream and downstream from the nose. The quantity of O3 removed by the animal O2 consumption, CO2 production, and tidal breathing measurements were automatically recorded once each minute. All animal types were exposed for 1 hr to 0.3 ppm O3. Other Fischer-344 rats were also exposed for 1 hr to 0.0 or to 0.6 ppm O3. Exposure concentrations of O3 had no significant effect on percentage O3 uptake in Fischer-344 rats. Results showed that percentage O3 uptake (47%) did not differ significantly among the three strains of rats. Similarly, percentage O3 uptake was independent of animal age, lung weight, and lung volume as well as normal variations encountered in the resting breathing measures. However, species-specific ventilation and O3 concentration were the primary determinants of the accumulated lung dose of O3 during the exposures.

Description of key information

Inhalation is the primary route of exposure. The substance is a corrosive gas, which reacts instantly with proteins and lipids on the surface of the respiratory tract. The calculated percentage of ozone uptake in the total respiratory tract in resting humans (namely adult men) is approximately 75% (Wiester et al., 1996). Ozone is not expected to be absorbed systemically via the respiratory route; it is instead quenched by endogenous substances on the surface of the respiratory tract without the involvement of any metabolizing processes.

Key value for chemical safety assessment

Bioaccumulation potential:

no bioaccumulation potential

Additional information

No studies, which were performed according to internationally accepted guidelines (e.g. OECD Test Guideline 417) for evaluation of the toxicokinetics [i.e. Absorption, Distribution, Metabolism and Elimination (ADME)] of pure ozone, were found in the public domain or in the archives of the applicants. However, a selection of the large amount of existing literature has been identified in the public domain and reviewed for the ADME properties of ozone. The summary below does give a good illustration of the ADME properties of ozone in human and animal species.

Detailed discussion of the studies

Absorption

Ozone uptake at inhalation was assessed in awake, spontaneously breathing Sprague-Dawley rats exposed to 0.0, 0.3 or 0.6 ppm ozone for 1 hour. Ozone uptake was calculated by measuring flow and ozone concentration both upstream and downstream from the nose. Total respiratory system uptake of ozone was found to be approximately 40% of that inspired and did not vary with ozone concentration nor with time during the 1-hour exposure. The quantity of ozone inhaled and retained by the rat increased proportionally with ozone concentration (Wiester et al., 1987).

In a second study, Wiester et al., 1988 confirmed these initial data, in which ozone uptake was assessed in awake, spontaneously breathing Fischer-344, Sprague-Dawley, and Long-Evans rats and Hartley guinea pigs. Animals were exposed for 1 hour to 0.0, 0.3 or 0.6 ppm ozone. The fractional uptake of ozone was computed by measuring flow and ozone concentration inhaled versus exhaled. In this study, exposure concentrations of ozone had no significant effect on percentage ozone uptake in Fischer-344 rats, and the percentage of ozone uptake (47%) did not differ significantly among three strains of rats nor between rats and guinea pigs. Similarly, percentage ozone uptake was independent of animal age, lung weight, and lung volume as well as normal variations encountered in the resting breathing measurements. However, species-specific ventilation and ozone concentration were the primary determinants of the accumulated lung dose of ozone during the exposures.

The percentage of ozone uptake in the total respiratory tract in humans has also been studied by Wiester et al., 1996. Ten healthy adult male subjects were exposed twice within a 2-week interval to 0.3 ppm ozone while seated and breathing naturally through the nose or mouth. Total respiratory tract ozone uptake was measured for 10 min under steady-state conditions. The percentage of ozone uptake was consistently higher during oral breathing (76.5% ± 3.3 SE) than during nasal breathing (73.1% ± 3.0) although the difference was not biologically significant. Between subjects the variability in percentage of uptake was substantial, ranging from 51 to 96%. Variability in percentage of uptake for an individual was less with the maximal difference between the first and second exposure being about 20%; the average difference, however, was only about 3%. The authors concluded that calculated total percentage of ozone uptake is approximately 75% in (non-exercising) adult men.

Rigas et al., 2000 studied the ozone uptake in healthy adults inhaling 0.2 or 0.4 ppm ozone through an oral mask while exercising continuously to elicit a minute volume of 20 L/min for 60 min or 40 l/min for 30 min. The ozone absorption was determined on a breath-by-breath basis as the ratio of ozone uptake to the inhaled ozone dose. The mean ozone uptake was 86%. Although concentration, minute volume, and exposure duration all had statistically significant effects on ozone uptake, the magnitudes of these effects were small compared with inter-subject variability. In a study on inter-subject variations in 28 female and 32 male nonsmokers being exposed continuously for 1 hour to 0.25 ppm ozone while exercising on a cycle ergometer at a constant ventilation rate of 30 L/min, the individual values of ozone uptake ranged from 70% to 98% among all the subjects with significant differences existing between men and women. Intersubject differences were found to be inversely correlated with breathing frequency and directly correlated with tidal volume (Reeser et al., 2005).

Bolus-response studies in human have also been performed to investigate ozone absorption as well as distribution following ozone inhalation. In a bolus-response study subjects receive an injection with the inspired air of a known volume and concentration of ozone at a predetermined point during inspiration. The fraction of ozone absorbed during a single breath was measured over a range of airway penetration volumes from 20 to 200 mL, with inspiratory and expiratory flows fixed at a nominal value of 250 mL/s (quiet breathing). The data indicated that 50% of the inhaled ozone was absorbed at a penetration of 70 mL which roughly corresponds to the upper airways, and essentially complete absorption occurred at a penetration of 180 mL, which roughly corresponds to the 16th airway generation (the beginning of the proximal alveolar region) (Hu et al., 1992).

In a study with increased respiratory flows between 150 and 1000 mL/s the relationship between ozone uptake and penetration volume was investigated. During quiet oral breathing at 250 mL/s, ozone uptake increased smoothly as penetration volume increased with 50% of the inhaled ozone absorbed in the upper airways and the remainder absorbed within the lower conducting airways such that no ozone reached the respiratory air space. The effect of increasing the respiratory flow was such that significantly less ozone was absorbed in the upper airways and lower conducting airways and some ozone reached the respiratory air space. As an example, at 1000 mL/s, only 10% of the inhaled ozone was absorbed in upper airways, and 65% was absorbed in the conducting airways such that 25% reached the respiratory air space (Hu et al., 1994).

Kabel et al., 1994 showed that during nasal breathing at a constant respiratory flow of 250 mL/s, ozone absorption increased smoothly as penetration volume increased with 80% of the inhaled ozone absorbed in the upper airways and 90% absorbed at the distal end of the trachea, respectively. Oral breathing caused a distal shift of the absorption-penetration volume distribution to the extent that absorption in the upper airways was reduced to 50% and inhaled ozone was 90% absorbed only after a bolus reached the 13th bronchial generation. Therefore, an exercise-induced change from nasal to oral breathing can render the distal lung more susceptible to ozone damage because of an elevation in ozone dose. It was also shown that changing the peak inhaled bolus concentration over a 10-fold range of 0.4 to 4 ppm ozone did not affect the absorption penetration volume distribution.

Similar results on ozone uptake were reported by Ultman et al., 1994, who found that with quiet mouth breathing, 50% of the ozone was absorbed in the mouth and oropharynx, and the remainder was absorbed within the conducting airways. When breathing nasally, about 80% of the ozone was absorbed in the upper airways, showing that the nose protects the lungs from ozone exposure. With increasing flow rates, more ozone reached and was absorbed by the lower airways and gas-exchange tissues in the lungs. During exercise, which entails both oral breathing and high flow rates, the dose rate of ozone to the lower airways and gas-exchange tissues would be more than three times the dose rate than when at rest.

Distribution

There has been limited use of isotopically-labeled ozone (¹⁸ozone) to investigate the distribution of ozone upon inhalation. The amount of ozone-derived-oxygen in the lungs of mice was determined by measuring the amount of¹⁸oxygen bound to organic constituents of lung tissue following exposure to 1 ppm¹⁸ozone for 60 min. Ozone-derived-oxygen accumulated in lung at a rate of 4.38 pmol/mg dry weight/min. Ozone-derived-oxygen had a half-life of approximately 6 h in lung. The authors estimated that a minimum of 44 pmol of ozone reacted with lung every minute of exposure to 1 ppm ozone (Santrock et al., 1989).

Studies with¹⁸ozone in rats and human to compare the incorporation of ozone-derived-oxygen in lung were done by Hatch et al., 1994. Eight human male volunteers were exposed to 0.4 ppm¹⁸ozone for 2 h with 15-min alternating periods of heavy treadmill exercise and rest. Rats were exposed identically but without exercise. Results showed that the exercising humans had four- to five-fold higher ozone-derived-oxygen concentrations in all of their bronchoalveolar lavage constituents than did the rats. The human volunteers also had significant increases in all of the effects markers after 0.4 ppm ozone, whereas the rats did not. Rats that were exposed to higher concentrations (2.0 ppm)¹⁸ozone had levels of ozone-derived-oxygen in bronchoalveolar lavage that were more comparable to, but still lower than, those of exercising humans. Changes in all other exposure effects markers in those rats were comparable or higher than in exercising humans. Therefore, it appears that ozone toxicity in resting rats underestimates effects in exercising humans because rats have a lower than expected dose of ozone to the distal lung.

Metabolism

A consensus opinion now exists that ozone does not penetrate deeply into the epithelial cell layer. Because of its high reactivity, ozone reacts directly with poly-unsaturated fatty acids (PUFA), antioxidants and proteins in the epithelial lining fluid (ELF) layer and the epithelial cellular membranes.

Reaction with fatty acids

The reactions of ozone with poly-unsaturated fatty acids have been extensively described by Pryor and co-workers (Pryor et al., 1991; 1992; 1996). The reactions involve an initial reaction of ozone with poly-unsaturated fatty acids across a carbon-carbon double bond, the yield being a 1,2,3-trioxalone which rearranges via carbonyl oxide and aldehyde intermediates to an ozonide via a Criegee reaction. This ozonide then decomposes to yield lipid peroxides or hydroperoxides. Work done by Pryor et al., 1996 provided evidence that, although ozonides are likely to break down to lipid peroxides in the lipophilic environment of biomembranes, in the aqueous environment of the airways the ozonides are likely to decompose via hydroxyhydroperoxy intermediates to yield further aldehyde products and hydrogen peroxide, which could then partake in metal-catalyzed Fenton reactions to generate hydroxyl radicals. Pryor et al. reported in 1996 for the first time the detection of hexanal, heptanal, and nonanal in the bronchoalveolar lavage (BAL) of rats exposed to 0.5 to 10 ppm ozone. These three aldehydes primarily result from the Criegee ozonation of specific mono- or polyunsaturated fatty acids that are present in significant amounts in the rat lung; e.g., palmitoleic acid gives heptanal, oleic gives nonanal, and linoleic and arachidonic can give hexanal. The detection and quantitation of aldehydes directly demonstrated that unsaturated fatty acids undergo Criegee ozonation in the lung when ozone is inhaled. The authors at that time suggested that these aldehydes, as well as other types of lipid oxidation products (such as hydroxyhydroperoxides and Criegee ozonides), may act as signal transduction molecules, activating lipases and causing the release of inflammatory molecules by a variety of pathways yet to be elucidated.

In a study by Uppu et al., 1995, ozone-induced oxidative damage to lipids caused changes in some of the unsaturated fatty acids in the lipid fraction of red blood cell (RBC) membranes. Significant amounts of hexanal, heptanal, and nonanal are formed from the ozonation of unsaturated fatty acids. Furthermore, ozonolysis of cholesterol and the formation of the reaction product 3β-hydroxy-5-oxo-5,6-secocholestan-6-al in lung tissue extracts from rats exposed to 1.3 ppm ozone for 12 h is described Pryor et al., 1991. More recent studies in mice detected several ozone-derived cholesterol derivatives, such as epoxycholesterol, that have been shown to be cytotoxic in human bronchial epithelial cells (Pulfer et al., 2005).

In healthy humans, exposure to 0.22 ppm ozone for 4 h increased levels of nonanal in BAL shortly after exposure, but not of hexanal. Following a second exposure, levels of both aldehydes returned to baseline by 18 h after the last exposure (Frampton et al., 1999).

In a study using proton transfer reaction-mass spectrometry for direct air analysis of volatile products resulting from the reactions of ozone with human skin lipids of humans exposed to 15 ppb ozone, Wisthaler and Weschler, 2009 reported reaction products that contain carbonyl, carboxyl, or α-hydroxy ketone groups. Among these, three previously unreported dicarbonyls have been identified, and two previously unreported α-hydroxy ketones have been tentatively identified. The authors stated that the results are fully consistent with the Criegee mechanism for ozone reacting with squalene, the single most abundant unsaturated constituent of skin lipids, and several unsaturated fatty acid moieties in their free or esterified forms.

Reaction with biological antioxidants

Postlethwait et al., 1998 studied the ozone absorption characteristics of ELF constituent mixtures and measured hexanal, heptanal, and nonanal concentrations as a function of ascorbic acid concentrations. Exposures of isolated rat lungs, BAL fluid and egg phosphatidylcholine liposomes were conducted. Ozone absorption by ascorbic acid , uric acid, and albumin exceeded that by egg phosphatidylcholine and glutathione (GSH), but ozone reaction with egg phosphatidylcholine occurred when ascorbic acid concentrations were reduced. Aldehydes were produced in low yield during lung and BAL fluid exposures in a time- and ozone concentration dependent manner. Reducing the ascorbic acid content of BAL fluid lowered the ozone uptake but increased aldehyde yields. Conversely, ascorbic acid addition to egg phosphatidylcholine increased ozone uptake but reduced aldehyde yields. It seems that ozone reacts simultaneously and competitively with its reductors.

Exposure of rat alveolar macrophages isolated by pulmonary lavage to 0.45 ppm ozone revealed a bi-phasic response in GSH, initially leakage of both reduced- and oxidized GSH increased through 30 min of exposure, then it declined (reduced GSH) or leveled off (oxidized GSH) (Banks et al., 1990).

Ballinger et al., 2005 utilized a red cell membrane (RCM) model to investigate the role of antioxidants in the interaction of ozone with the ELF. The authors concluded that although ozone diffusion through the RCM cannot be completely ruled out, reactive product(s) derived from ozone ascorbic acid and/ or GSH reactions, possibly O₃^*-or¹O₂, likely initiated RCM oxidation. This may suggest that in vivo, such secondary reactive species permeated through the ELF leading to cellular perturbations. Based on this in vitro work it is not unlikely that reactions in the ELF are followed by reactions with antioxidants (e.g., reduced ascorbate and reduced GSH), which can lead to secondary reactive oxygen species (ROS) production.

A study in fifteen healthy subjects exposed to 0.2 ppm ozone for 2 h, with bronchial wash, bronchoalveolar lavage, and biopsy sampling, demonstrated that ozone elicits a broad spectrum of airway antioxidant responses, with initial losses of vitamin C and urate followed by a phase of augmentation of low-molecular-weight antioxidant concentrations at the air–lung interface. During the ozone challenge significant losses of nasal lining fluid urate and vitamin C were observed, which resolved 6 h post exposure. At this time point, enhanced concentrations of total GSH, vitamin C, and urate were seen in bronchial airway lavages. In bronchoalveolar lavage, increased concentrations of total GSH, vitamin C, urate, α-tocopherol, and extracellular superoxide dismutase occurred 6 h post ozone. In alveolar leukocytes significant losses of GSH were observed, whereas ascorbate concentrations in endobronchial mucosal biopsies were elevated after ozone at 6 h post exposure this time (Behndig et al., 2009). This study clearly shows the involvement of the anti-oxidant machinery at low level ozone exposure.

Reactions with amino acids and proteins

Mudd et al., 1969 studied the reaction of ozone with amino acids and proteins and established the following order, from high to low, of susceptibility of amino acids in aqueous solutions to oxidation by ozone: cysteine, methionine, tryptophan, tyrosine, histidine, cystine, and phenylalanine. The other amino acids found in proteins were unaffected by ozone. The oxidations of tyrosine and histidine were dependent on pH, being greater under alkaline conditions. The oxidation of pancreatic ribonuclease by ozone resulted in a decrease in enzymic activity. The amino acids most affected by treatment with ozone were tyrosine and histidine. Reaction of avidin with ozone caused changes in the absorption spectrum characteristic of the oxidation of tryptophan. The biotin binding capacity of avidin was lost after treatment of avidin with ozone.Pryor and Uppu, 1993, revealed that oxidation of tryptophan in different proteins resembles the formation of aCriegee ozonide or a tautomer of the Criegee ozonide and the formation of hydrogen peroxide, similarly as in the oxidation of unsaturated fatty acids.

When human red blood cell (RBC) membranes were exposed to low levels of ozone in vitro, oxidative damage to proteins caused significant decreases in the content of thiol groups, the fluorescence of protein-tryptophan residues, and also a loss of activity of membrane-bound acetylcholinesterase (Uppu et al., 1995).

Elimination

A certain part of the inhaled ozone is removed from the body via the expired air. The ozone which is absorbed gets eliminated from the body by reaction with endogenous substrates, that after biochemical/biological conversion are available to the body as new substrates. It has been shown that ozone has a half-life of approximately 6 hours in mouse lungs (Santrock et al., 1989).