Daniel B. Menzel

Ozone: An overview of its toxicity in man and animals

Ozone is one of the most toxic and ubiquitous air pollutants. This review focuses on the toxic effects of ozone in animals and on the similarities and disimilarities between the toxic effects in animals and humans. The molecular basis for the toxicity of ozone is discussed, based on the vigorous oxidizing properties of ozone. Despite the existence of anatomical differences between human, subhuman primate, and dog lungs versus common experimental rodent lungs, the anatomical lesion of ozone inhalation occurs at the functionally equivalent site of the junction between the conducting airway and the respiratory region. Ciliated cells of the upper airways and the type 1 cell of the centriacinar region are most affected. Type 2 cell proliferation is a hallmark of ozone toxicity. A wide variety of biochemical and physiological changes have been noted in several animal species and in humans. Considerable evidence for a free-radical-mediated or lipid peroxide-mediated toxicity is evident, especially in the induction of the glutathione peroxidase system and the protective effects of vitamins C and E. Ozone appears to be a weak mutagen and to produce chromosomal abnormalities. Defects in defense against airborne infection are present in animals, which are more susceptible to airborne infection after ozone exposure. Epidemiological studies, however, fail to detect increased respiratory infections in humans due to ozone. Despite the variety of toxic effects, few qualitative differences between species are apparent; rather, quantitative differences do occur. Ozone may thus be an ideal compound for quantitative extrapolation of toxicity from animals to humans.

Similarity between man and laboratory animals in regional pulmonary deposition of ozone.

Predicted pulmonary ozone (O3) dose curves obtained by model analysis of the transport and removal of O3 in the lungs of guinea pigs, rabbits, and man indicate that a general similarity exists among these species in the shapes of the dose curves. An overview of the major features of the lower airway mathematical model used is presented. This model predicts that the respiratory bronchioles receive the maximum O3 dose. For exposures corresponding to tracheal O3 concentrations greater than 100 micrograms/m3 (0.05 ppm), the predicted respiratory bronchiolar dose for rabbits was found to be twice that for guinea pigs and 80% of that for man. Sensitivity analyses are presented for model parameters relating to the treatment of the chemical reactions of O3 with the mucous layer. The role of tidal volume in the determination of pulmonary uptake of O3 in man is examined. The consistency and similarity of the dose curves for the three species lend strong support to the validity of extrapolating to man the results obtained on animals exposed to O3.

Stimulation of oxidant production in alveolar macrophages by pollutant and latex particles.

Abstract Air pollutant dusts as well as chemically defined particles were examined for their activating effect on oxidant production (O2− and H2O2) in guinea pig alveolar macrophages (AM). Oxidant production was measured as chemiluminescence of albumin-bound luminol. All particles examined stimulated the AM in a dose-dependent manner to different maximal levels of oxidant production. Amphibole asbestos samples were the most active of all agents studied. Various immune reactants as well as silica, metal-oxide-coated fly ash, polymethyl methacrylate beads, and chrysotile asbestos had intermediate activity, while fugitive dusts, polybead carboxylate microspheres, glass and latex beads, uncoated fly ash, and fiberglass had the lowest activity. In addition to direct stimulatory action on AM, particles also lowered the subsequent responsiveness of the cells to the bacterial peptide stimulant, N-formylmethionyl phenylalanine. This effect was only partially due to the cytotoxicity of the particles. While some relationship appeared to exist between stimulatory activity and cytotoxicity of the particles, the exact role of O2− in mediating cytotoxic effects is still open to question. Oxidant production in AM is a parameter which may be important in determining the pathological effects of particles as well as of materials adsorbed to their surfaces.

A model of the regional uptake of gaseous pollutants in the lung

An ozone (O3) dosimetry model is presented that takes into account convection and diffusion of O3 in the lumen and airspaces of the lower respiratory tract and transport and chemical reactions in the mucous and surfactant layers and in the underlying tissue and capillaries. The model was applied to human airway morphometric data. Values for the chemical and physical parameters that define the liquid tissue and blood compartments were based on reported experimental data. Simulation results illustrate the variability of results due to an uncertainty in the knowledge of transport parameters, liquid, tissue, and blood compartment thicknesses, and chemical reaction rates. Results were most sensitive to mucous compartment thickness and reaction rate constant and least sensitive to transport and blood parameters. Exercise was simulated, showing little effect on tracheobronchial uptake but a pronounced effect on pulmonary uptake.

Picomole analysis of glutathione, glutathione disulfide, glutathione S-sulfonate, and cysteine S-sulfonate by high-performance liquid chromatography

A method for simultaneous detection of picomole quantities of glutathione (GSH), glutathione disulfide (GSSG), glutathione S-sulfonate (GSSO3H), and cysteine S-sulfonate (CYSSO3H) by high-performance liquid chromatography has been developed. Compounds are separated by anion-exchange chromatography using a citric acid buffer system, and then derivatized postcolumn using o-phthalaldehyde with 2-mercaptoethanol, heated to 70 degrees C, and detected by fluorescence. The compounds elute with retention times of 12.5 min for GSH, 27.5 min for CYSSO3H, 29.8 min for GSSG, and 33.0 minutes for GSSO3H, with detection limits of 10, 200, 10, and 50 pmol, respectively. Recoveries are 103% for GSH, 102% for GSSG, 100% for CYSSO3H, and 96% for GSSO3H. Determination of target compounds in cells is described.

Effect of culture conditions on glutathione content in A549 cells

The effects of varying culture conditions on glutathione content in A549 (human type II lung tumor derived) cells were examined. Parameters studied were growth time, serum concentration, and the presence or absence of a mixture of insulin, transferrin, and selenous acid. Glutathione content increased with serum concentration. When cells were grown with serum, glutathione increased sharply 24 hours after passage and decreased thereafter. Insulin, transferrin, and selenous acid had little effect on cell growth or glutathione content. Replacement of media with fresh media containing 10% serum did not prevent the growth dependent decrease in glutathione. These results demonstrate that glutathione content in A549 cells is strongly affected by culture conditions.

Heinz bodies formed in erythrocytes by fatty acid ozonides and ozone.

Ozonides of the methyl esters of oleic, linoleic, linolenic and arachidonic acids were found to produce Heinz body inclusions in human and mouse erythrocytes. No simple relationships between structure and activity were noted. Concomitant with Heinz body formation, methemoglobin and loss of cellular thiols were observed. Methyl ozonides readily oxidized glutathione and 1 mole of oxidized glutathione was formed per mole of methyl oleate ozonide. Methyl ozonides catalyzed the formation of disulfide-linked interchain polymers between hemoglobin and ovalbumin. Heinz bodies were not produced with ozone in the absence of unsaturated lipids. Heinz bodies were observed in the blood of mice exposed to ozone (0.85 ppm) for 48 hours. These observations suggest that fatty acid ozonides could serve as a toxic chemical species formed on ozone inhalation and could explain the divergent protective effects of lipid antioxidants and thiol generating systems in vivo.

Nasopharyngeal removal of ozone in rabbits and guinea pigs.

In estimating pollutant concentrations responsible for observed pulmonary effects, nasopharyngeal removal of the pollutant plays an important role. The nasopharyngeal removal of ozone (O3) in anesthetized male guinea pigs and male and female rabbits was determined by drawing O3 through the isolated upper airways at a constant flow rate which approximated the animals respiratory minute volume. The tracheal O3 concentration in rabbits and guinea pigs was markedly similar and was linearly related to the chamber concentration of O3 over a range of 196--3920 micrograms/m3 (0.1--2.0 ppm O3). Regression analyses showed that O3 removal in the nasopharyngeal region is approximately 50% in both species. Both rabbit sexes responded similarly over the concentration range studied. Exposures of guinea pigs to O3 concentrations between 3920 and 5880 micrograms/m3 (2.0 and 3.0 ppm) showed that, at these higher concentrations, relatively more O3 is removed by the upper airways.

Influence of ozone on pentobarbital-induced sleeping time in mice, rats, and hamsters

Abstract Studies were conducted to investigate the effect of ozone (O 3 ) in prolonging pentobarbital (PEN)-induced sleeping time (S.T.). Since O 3 is a common air pollutant, an O 3 -induced alteration of mechanisms of drug action could have public health implications. It was shown that a 5-hr exposure to 1960 μg O 3 /m 3 (1 ppm) caused an increased PEN-induced S.T. in female mice (three strains), rats, and hamsters. This response was not observed in male rats or in male mice even when the latter were exposed for 5 hr/day for 3 days. Male hamsters were affected, but less so than females. Ozone concentration and time relationships were investigated in female mice. Concentrations from 196 to 9800 μg O 3 /m 3 (0.1 to 5 ppm) increased PEN-induced S.T. However, as the concentration was decreased, an increasing number of daily 3-hr exposures were needed to cause an effect. Once the maximal effect was observed, further daily exposures resulted in a dissipation or a disappearance of the effect. Also, recovery occurred within 24 hr after exposure ceased. Other experiments were performed in which mice received a continuous exposure to a C × T (concentration × time) of 5, ranging from 196 μg/m 3 (0.1 ppm) × 50 hr to 1960 μg/m 3 (1 ppm) × 5 hr. For most of the concentrations, the magnitudes of the O 3 effects were roughly equivalent. These data are interpreted as a systemic effect of O 3 on mechanisms of the termination of action of pentobarbital.

Glutathione S-sulfonate, a sulfur dioxide metabolite, as a competitive inhibitor of glutathione S-transferase, and its reduction by glutathione reductase

Glutathione S-sulfonate (GSSO3H) is a reaction product of glutathione disulfide (GSSG) and sulfite, the hydrated form of sulfur dioxide. In the present study, GSSO3H was found to be a potent competitive inhibitor of the glutathione S-transferases (GST) in the rat liver (Ki = 14 microM) and lung (Ki = 9 microM), and in human lung tumor-derived A549 cells (Ki = 4 microM). GSSO3H was also reduced by a cytosolic enzyme in the rat liver (Km = 313 microM) and lung (Km = 200 microM), and human lung A549 cells (Km = 400 microM). These results suggest that SO2 may affect the detoxification of xenobiotic compounds by inhibiting, via formation of GSSO3H, the enzymatic conjugation of glutathione (GSH) and reactive electrophiles. Although GSSO3H can be enzymatically degraded, the high substrate Km value suggests that this compound may not be readily reduced at low concentrations.