Mutation Research, 277 (1992) 221-238 © 1992 Elsevier Science Publishers B.V. All rights reserved 0165-1110/92/$15.00

221

MUTREV 07324

Review of the genotoxicity of ozone Katarina Victorin Institute of Environmental Medicine, Karolinska Instituter, S-104 01 Stockholm, Sweden (Received 16 December 1991) (Accepted 27 March 1992)

Keywords: Ozone; Genotoxicity; Mutagenicity

Summary Ozone is a powerful oxidant, reactive to biomolecules. In aqueous solution it decomposes to give hydrogen peroxide, superoxide and hydroxy radicals which can take part in secondary reactions. Ozone is a disinfectant that inactivates both viruses and bacteria. Although other reactions are primarily responsible for the inactivation, cellular D N A is also damaged. Ozone is genotoxic to microorganisms, plants and cell cultures in vitro. The results from in vivo cytogenetic studies with laboratory animals after inhalation exposure are contradictory. Chromosome aberrations in lymphocytes, but not SCEs, have been demonstrated in Chinese hamsters but not in mice. Chromatid deletions were induced in pulmonary macrophages in rats. No cytogenetic effects have been reported for bone marrow cells or spermatocytes. The few experimental and epidemiological studies with human subjects do not allow a conclusion on the cytogenetic effects of ozone in lymphocytes in humans. No life-long cancer studies have been performed with ozone. However, after 4 and 6 months of inhalation exposure, lung adenomas were induced in strain A / J mice, but not in Swiss-Webster mice.

Introduction, general toxicity Ozone is a ubiquitous air pollutant due to its formation in photochemical smog reactions between volatile hydrocarbons and nitrogen oxides. The environmental concentrations are usually highest outside city centers during the daytime and in the 6 months of summer. Other sources for local exposure to ozone are disinfection and bleaching processes, ultraviolet lamps and high-

Correspondence: Katarina Victorin, Institute of Environmental Medicine, Karolinska Institutet, Box 60208, S-104 01 Stockholm, Sweden.

voltage electric equipment. Some of the stratospheric ozone will contribute to the background concentration of ozone. The W H O European air quality 1-h guideline is 150-200 /~g/m 3 (0.076-0.1 ppm) and the 8-h guideline is 0.05-0.06 ppm (WHO, 1987). The US Federal ambient air quality standard is 0.12 ppm (1-h average not to be exceeded more than once a year) (EPA, 1991). Background levels of ozone range from 40 to 60 / x g / m 3 (0.02-0.03 ppm) as 24-h mean values, although maximal 24-h mean values of 1 2 0 / x g / m 3 (0.06 ppm) have been measured at sea level on the Atlantic Ocean and other remote areas.

222 Long-range transport of ozone may give maximum hourly concentrations of 0.10-0.15 ppm in rural areas. In certain parts of Europe and the USA urban 1-h mean concentrations may exceed 0.18 and 0.2 ppm, respectively (WHO, 1987). The American Environmental Protection Agency has summarized concentrations and trends of air pollutants in the United States (EPA, 1991). The data on ozone are based on 471 monitoring sites througout the country, most of them situated in urban areas. The annual second highest daily maximum 1-h concentration is on the average 0.12 ppm for all sites, with a slight (10%) decreasing trend during the years 1981-1990. Several reviews on the general toxicity and biochemical mechanisms of ozone have been published, and are referred to in the following paragraphs (Borek and Mehlman, 1983; Mentzel, 1984; Mehlman and Borek, 1987; WHO, 1987; Mustafa, 1990). Ozone is a powerful oxidant, and exerts its biological action by oxidative destruction of biomolecules. In general, two main mechanisms are important, namely the oxidation of polyunsaturated fatty acids to acid peroxides and the oxidation of sulfhydryl groups and amino acids of enzymes, proteins and peptides. This makes cellular membranes sensitive targets. Inhalation of ozone primarily affects the terminal bronchioles and the alveoli. The bronchiolar ciliated cells and the epithelial alveolar type I cells are damaged, and are later replaced with proliferating Clara cells in the airways and epithelial type II cells in the alveoli, respectively. When exposing laboratory animals for a few hours or days effects such as alterations in lung biochemistry, morphological alterations in the lung and a potentiation of bacterial lung infections have occurred with lowest observed effect levels in the range of 0.08-0.2 ppm. Controlled human studies (exposure 1-3 h) have reported impairment of pulmonary function, accompanied by respiratory and other symptoms at levels down to 0.12 ppm. Field studies and epidemiological studies have indicated a number of acute effects of ozone and other photochemical oxidants such as eye, nose and throat irritation, chest discomfort, cough and headache, and pulmonary function decrements in children and asthmatics. These

effects have occurred at levels down to approximately 0.1 ppm. A very small fraction of inhaled ozone is taken up by the blood. However, a wide range of extrapulmonary effects have been identified after ozone exposure, such as biochemical and morphological changes in red blood cells and changes in enzyme activities. In aqueous solution, ozone decomposes to give hydrogen peroxide, superoxide and hydroxyl radicals. It is believed that some of the ozone-induced free radicals in biological systems are derived from the oxidative decomposition of polyunsaturated fatty acids. In ozone reactions with the fatty acids, also epoxides and dialdehydes such as malonaldehyde are formed. It is possible that these secondary reaction products are causing damage to various extrapulmonary sites (Mustafa, 1990; Steinberg, 1990). In the following literature survey, no attempt is made to evaluate the genotoxicity of decomposition products or secondary reaction products from ozone. Studies on complex mixtures of photochemical oxidants are not included either. Only such studies are cited that have been published in the open scientific literature. Reactions with DNA and RNA

As early as 1954 it was shown that bubbling of ozone through a solution of DNA causes a rapid change in the UV spectra, probably resulting from effects on the constituent purines and pyrimidines (Christensen and Giese, 1954). The nucleotide bases thymine and guanine have been found to be the most sensitive to ozonation (Prat et al., 1968; Omura et al., 1972; Ishizaki et al., 1981; Shinriki et al., 1981, 1984). According to Van der Zee et al. (1987a), DNA damage proceeds both directly via ozone molecules and indirectly via hydroxyl radicals, when solutions of nucleotides or DNA are treated with ozone. Ozonation of supercoiled plasmid DNA converted the closed circular DNA to open circular DNA when treated in vitro (Sawadaishi et al., 1984). Ozone reacted preferentially with thymine and guanine residues, and the strand cleavage sites have been investigated (Sawadaishi et aI., 1985, 1986). The relaxation, linearization and

223 degradation of supercoiled plasmid DNA, indicating single- and double-strand breaks, was described by Hamelin (1985). A significant reduction of transforming ability of the plasmid also was observed in this study. In studies on the mechanism of virus inactivation by ozone, several authors have suggested that damage to the viral coat protein is primarily responsible for inactivation. The released nucleic acid is also inactivated (De Mik and De Groot, 1977; Kim et al., 1980; Sproul et al., 1982). However, in a study by Roy et al. (1981) it was concluded that, although alteration in the coat protein was observed, damage to the viral RNA (as demonstrated by sucrose gradient centrifugation) was the major cause of inactivation of poliovirus I in aqueous solution. Mura and Chung (1990) investigated the effect of ozone on in vitro transcription activity. Isolated T7 bacteriophage D N A solution was bubbled with ozone and the priming activity was studied by the kinetics of incorporation of 3Huridine into RNA. The rate of incorporation decreased with increasing treatment time with ozone. The results indicated that lesions produced in the structure of the DNA molecule interfere with its transcriptional ability, in part due to a decrease in chain initiation.

Genotoxic effects in microorganisms, plants and insects Ozone is used as a water disinfectant. The inactivation mechanisms for bacteria are probably more complex than in virus inactivation, since ozone attacks proteins and unsaturated lipids in the cell membrane, as well as enzymes within the cell (Ishizaki et al., 1987). In early studies by Scott and Lesher (1963), it was postulated that the primary attack of ozone is on the double bonds of unsaturated lipids in the cell membrane. Several studies have also revealed mutations and other genotoxic effects to cellular DNA, as reviewed below. Hamelin and Chung (1974) investigated the mutagenicity of ozone in an Escherichia coli K12 m e t h i o n i n e - and m a l t o s e - r e q u i r i n g strain, MQ259, scoring for Met + and Mal + revertants. Ozone exposure was by bubbling 0.05-50 ppm

ozone for 1-60 min through 5 ml of bacterial suspension at pH 5.0. Statistically significant increases in mutant frequencies per 106 cells occurred with 0.05-1.0 ppm ozone for >__5 min exposure with an optimum at 30-60 min. Concentrations higher than 1 ppm were highly toxic to the bacteria. The highest mutation rates were obtained with logarithmic growth phase cells, at low pH (pH 5.0) and in buffer. Ozone-induced Met + mutants were isolated after bubbling of 0.1 ppm ozone through the bacteria suspension for 60 min, and tested for the presence of other mutations. It was found that ozone also induces forward mutations in a wide range of genes responsible for the nutritional properties of E. coli (auxotroph mutants), as well as sensitivity to UV light and formation of capsular polysaccharide (mucoid mutants) (Hamelin and Chung, 1975) DNA is rapidly degraded, and the sensitivity of the polA strain of E. coli to the toxicity of ozone indicates that DNA polymerase I is involved in the repair of ozone-induced lesions to DNA (Hamelin et al., 1977; Hamelin and Chung, 1978; L'H6rault and Chung, 1984). Comparative ozone toxicity studies with different poIA mutants of E. coli K12 showed that, in addition to the polymerase activity of pol I, the 5 ' - 3 ' exonuclease activity is important in the repair of ozone,induced D N A damage (Parduez and Chung, 1988). After testing for streptomycin-resistant mutants (50 ppm ozone bubbled through 5 ml bacterial suspension for 1-20 min) in different strains of E. coli defective in DNA repair, it was suggested that ozone may be able to induce mutations both directly and indirectly via the rec-lex error-prone repair system (L'H6rault and Chung, 1984). Bubbling of 50 ppm ozone for 15 min through 3H-thymidine labeled E. coli bacterial suspension has also been shown to induce DNA single- and double-strand breaks, as indicated by the sedimentation rate in alkaline sucrose gradient centrifugation (Hamelin and Chung, 1989). DNA strand breaks in plasmid DNA in E. coli cells was also demonstrated in a study by Ishizaki et al. (1987). In experiments by Nover and Botzenhart (1985), E. coli bacterial suspensions were gassed

224 with 0.5 p p m ozone for 4.5 h, which induced forward mutations (base-pair substitutions and deletions). Comparative toxicity studies with different D N A repair-deficient mutant strains of another bacterial species, Bacillus subtilis, have also been performed (Song and Chung, 1983). As in the experiments by Chung and co-workers with E. coli mentioned above, treatment was by bubbling ozone through 5 ml of bacteria suspension. The results indicated that the recA and recC loci seem to be most important and perhaps the polA and uvrA loci may also be required for the repair of ozone-induced D N A damage. It should be noted that in all the experiments where exposure is by bubbling of ozone through a bacterial suspension the observed effects may be due not only to ozone, but also to degradation products of ozone in water, such as hydroxy radicals and hydrogen peroxide. The Ames Salmonella assay has been applied in three studies. Ozone in air was passed over agar plates with Salmonella TA100, without lids, in an exposure chamber for 20 h in one study, and 6 h in the other study. No mutagenic effect could be demonstrated in a single experiment with 0.5 p p m ozone (Shepson et al., 1985), or with several doses in the range of 0.1-2.0 p p m (Victorin and St,~hlberg, 1988). Concentrations of around 2 p p m and higher were toxic to the bacteria in the latter study, whereas no toxicity was observed in the experiment with 0.5 p p m ozone for 20 h by Shepson et al. (1985). In a recent study, Dillon et al. (1992) exposed several Salmonella strains to ozone in air. Strain TA102, but not strains TA100, TA104 or TA98, gave a slight mutagenic response after 35 min exposure to 0.02-0.04 and 0.2-0.5 ppm ozone but not at higher doses, probably due to toxicity. Control experiments with ozone were performed in a study dealing with the germicidal action of ozonated olefins in air. 3H-Thymidinelabeled E. coli bacteria were aerosolized in air containing 0.04 p p m ozone for 30-45 min. D N A was extracted from the bacteria and the induction of D N A strand breaks studied after fractionation by centrifugation in alkaline sucrose gradients. The results from ozone-induced breaks were inconclusive since aerosolization in the absence of

ozone gave comparable results (De Mik and De Groot, 1978). Dubeau and Chung (1982) studied different genetic effects of ozone in the yeast Saccharomyces cerevisiae. Exposure was by bubbling 50 p p m ozone through 5 ml of cellular suspension. An exposure time of 90 min was highly toxic to the cells. At shorter exposure times, 20-60 min, ozone induced a variety of genetic events, such as forward and reverse mutations as well as gene conversion and mitotic crossing-over. However, compared to other known mutagens, ozone appeared to be a weak mutagen. In an early study by Fetner (1958), root meristem cells of the plant Vicia faba were exposed to 4000 p p m ozone in air for 15, 30 and 60 min. The cells were exposed in interphase and analyzed at the first anaphase. The treatment induced chromosome-type, but not chromatid-type aberrations. The effect of ozone on Vicia faba was also studied by Janakiraman and Harney (1976), but in this case meiotic chromosomes of the buds of exposed plants were investigated for chromosomal effects. Exposure to ozone at a concentration of 2 p p m for 4 or 8 h, but not 1 ppm, caused chromosome-type aberrations 24 h after exposure. The early stages of meiosis seemed to be more susceptible to chromosome damage than later stages. The Tradescantia plant test system has been used to study genotoxic effects of volatile air pollutants both in the laboratory and for field monitoring. Schairer et al. (1979) used somatic mutations in stamen hairs in clone 4430 as the endpoint. Ozone exposure for 6 h at 5 p p m was positive in this system. Ma et al. (1982), however, reported ozone (5 p p m for 5.5 h) to be negative when chromosomal damage, as reflected by micronucleus formation in the meiotic pollen mother cells, was the endpoint. Erdman and Hernandez (1982) exposed male Drosophila virilis flies to 30 ppm ozone for 3 h. Longer exposure times were not tolerated by the flies. Ozone induced dominant lethals in the offspring, as calculated by the proportion of eggs that failed to develop into pupae. In general, postmeiotic cell stages of spermatogenesis were more sensitive to ozone-induced dominant lethals

225

than meiotic and premeiotic stages. For most mating periods, the control group had higher total number of eggs than those treated with ozone. In another study, this effect was also seen

along with a life-span shortening of male offspring from exposed females and a decrease in hatchability after repeated exposure to 27 ppm ozone during 1-2 h (Chigusa and Nakada, 1972).

TABLE 1 GENOTOXIC EFFECTS IN MICROORGANISMS, PLANTS AND INSECTS Test organism

Endpoint

Ozone exposure

Result

Reference Hamelin and Chung, 1974

E. coli K12

Reverse mutations

Bubbling of 0.05-1.0 ppm, 30-60 min, through cell suspension

+

E. coli K12

Forward mutations

Bubbling of 0.1 ppm, 60 min, through cell suspension

+

Hamelin and Chung, 1975

E. coli, different strains

Forward mutations, effect on rec-lex error-prone repair system

Bubbling of 50 ppm, 1-20 min, through cell suspension

+

L'H6rault and Chung, 1984

E. coli K12

Forward mutations

Bubbling of 0.5 ppm, 4.5 h, through cell suspension

Nover and Botzenhart, 1985

E. coli

DNA strand breaks

Bubbling of 50 ppm, 15 min, through cell suspension

Hamelin and Chung, 1989

E. coli MRE 162

DNA strand breaks

0.04 ppm, 30-45 min, in aerosolized bacteria

De Mik and DeGroot, 1978

Bacillus subtilis

DNA repair

Bubbling of 5-50 ppm, 30 min, through cell suspension

Salmonella TA100

Reverse mutations

0.5 ppm in air, 20 h

Shepson et al., 1985

Salmonella TA100

Reverse mutations

0.1-2.0 ppm, 6 h

Victorin and St~hlberg, 1988

Salmonella TA102

Reverse mutations

0.02-0.5 ppm, 35 min

Dillon et al., 1992

Salmonella TA1535, Reverse mutations TA98, TA100, TA104

0.02-0.5 ppm, 35 min

Dillon et al., 1992

Saccharomyces cerevisiae

Forward and reverse point mutations, gene conversion, mitotic crossing-over

Bubbling of 50 ppm, 60 or 90 min, through cell suspension

Dubeau and Chung, 1982

14cia faba

Chromosome-type aberrations in root meristem cells Chromatid-type aberrations

4000 ppm, 15, 30 or 60 min

Vicia faba

Chromosome aberrations in buds

2 ppm, 4 or 8 h

Tradescantia

Micronuclei in pollen

5 ppm, 5.5 h

Tradescantia

Somatic mutations in stamen hairs

5 ppm, 6 h

+

Shairer et al., 1979

Drosophila

Dominant lethals

30 ppm, 3 h

+

Erdman and Hernandez, 1982

+

+

Song and Chung, 1983

Fetner, 1958

Fetner, 1958

4000 ppm, 15, 30 or 60 min +

Janakiraman and Harney, 1976 Ma et al., 1982

226

Data evaluation Bubbling of ozone through a suspension of bacteria or yeast cells induces forward and reverse mutations, as well as D N A strand breaks. This effect is probably caused by both ozone and hydroxy radicals and hydrogen peroxide formed in aqueous solution. Mutations in bacteria after airborne exposure have been more difficult to show, probably partly because the ozone gas has to diffuse into the soft agar medium, and partly because ozone is bacteriotoxic at low doses. The negative result in the study by De Mik and De Groot (1978) can be easily explained by the low concentration of ozone tested. Chromosome aberrations have been induced in the plant Vicia faba, and in one out of two studies with Tradescantia. The induction of dominant lethals in Drosophila is not necessarily due to genotoxic effects, as other toxic effects were also seen. Overall, the data discussed indicate that ozone is genotoxic to microorganisms, plants and possibly insects (Table 1). This should be expected, considering the damaging effect of ozone to naked D N A and to viruses. Cell cultures

Cytogenetic effects Fetner (1962) was the first to demonstrate chromosomal effects of ozone in cell cultures, using the human cell line KB. The medium was removed from the culture bottles, and the cells were rinsed before exposure by inflow of 8 p p m ozone in oxygen for 5 or 10 min into the rotating bottles. Higher concentrations than this resulted in the cells dislodging from the glass surface. The cells were then incubated for 24 h in medium containing colchicine and metaphase cells analyzed for chromatid breaks. An increase in deletions was observed compared to control cells exposed to oxygen for 10 min. In a study of the toxicity of ozone in vitro, Sachsenmaier et al. (1965) exposed cultures of embryonic chick fibroblasts to 700-7000 ppm ozone for 30 min. The cells were grown on cover glasses in roller bottles, and exposed to ozone with a small amount of medium left in the bottle. After exposure, fresh medium without colchicine was added, and cytologic preparations were fixed

after 2-44 h. The high concentration of ozone caused strong alterations of both interphase and mitotic cells (rounded or lysed cells, shrunken nuclei, clumped metaphase chromosomes), but only at the monolayer part and top layer part of the cultures, which indicates little penetration effect of ozone. Gooch et al. (1976) exposed human peripheral leukocytes to ozone by two methods. One way was by bubbling known levels of ozone through a suspension of cells in Hanks' balanced salt solution and removing aliquots at various time intervals. This exposure was expressed in p p m / h and the actual concentration was not mentioned. The second method consisted of putting leukocytes into ozone-saturated phosphate-buffered saline (approx. 2 ppm) either 12 or 36 h after phytohemagglutinin (PHA) stimulation. The cells were reincubated after exposure, colchicine added after 56 h of total incubation time, and fixation of cells performed at 60 h. No chromosome or chromatid aberrations occurred with the second method (2 ppm ozone for up to 90 min). However, with the first method cells that were treated 36 h after P H A stimulation (presumably in Sphase) demonstrated an increase in the frequency of chromatid deletions at two of the three highest doses tested (7.23 p p m / h and 7.95 p p m / h ) . Treatment of the 12-h G 1 stage cells did not result in an appreciable level of chromatid damage. No mention was made of cytotoxic effects. Guerrero et al. (1979) treated 24-h cultures of human fetal lung cells (WI-38) in 100 m m petri dishes containing 12 ml serum-free Eagle's diploid medium, placed on a rocking table inside an exposure chamber. Cells were exposed to 0.25, 0.5, 0.75 or 1.0 p p m ozone for 1 h. Reincubation was performed with BrdU in the medium for 60 h and then the cultures were treated with colcemid for a additional 4 h. A dose-related increase in sister-chromatid exchanges (SCE) resulted from the ozone exposure. An increase in chromatid deletions also occurred at 1 ppm, but no increase in chromosome-type aberrations. Another mode of exposure was described by Rasmussen and Crocker (1982). V79 Chinese hamster lung fibroblasts were allowed to attach to cellulose m e m b r a n e filters (Millipore 33HAWP) for several hours, which were then

227 perfused with growth medium and placed inside an exposure chamber. Exposure by this method to 0.05 p p m ozone caused a substantial loss in the fraction of cells recovered from the filters. Ozone at 0.035 p p m did not a p p e a r to induce SCE in one experiment when cells that had been grown for 18 h in medium containing BrdU were exposed for 1 h and then further incubated for 3 - 4 h in medium containing BrdU and colcemid. Shiraishi and Bandow (1985) exposed Chinese V79 hamster cells by letting ozone into culture bottles containing 10 ml Hanks' balanced salt solution and placed on roller drums. After exposure for 2 h, the cells were incubated with BrdU for 1 day and then treated with colcemid for an additional 3 h. Ozone in 5 different concentrations from 0.13 to 1.0 p p m induced SCEs along with a dose-related cell growth inhibition (71% inhibition at 1 p p m ozone). In a review p a p e r on research at Fudan University in China, Hsueh and Ziang (1984) reported ozone induction of SCEs in human lymphocytes at concentrations of 0.15-0.75 ppm. Cell growth inhibition occurred with 0.75 p p m ozone when the exposure time was longer than 60 min. No details of the experimental conditions were given in the paper. The original data are presented in a p a p e r by Zhou et al. (1981), not available to the author of this review. Effects on D N A In a later study (Rasmussen, 1986), 14Cthymidine prelabeled V79 cells were exposed to ozone for 1 h by adding 2 ml serum-free basal medium to 60-mm tissue culture dishes and placing the dishes on a rocker platform in the exposure chamber. D N A replication, as measured by 3H-thymidine incorporation, was depressed in a dose-dependent manner over an ozone concentration range of 1-10 ppm. Concentrations higher than 2 p p m were toxic. The results were interpreted to indicate that ozone interacts with D N A in a way that inhibits replication. The low concentration of 0.035 p p m ozone gave no such effect (Rasmussen and Crocker, 1982). Induction of D N A single-strand breaks was the endpoint in three studies. In the first, Van der Z e e et al. (1987b) exposed washed lacthymidine labeled L929 murine fibroblast cell cul-

tures for 10 s every 30 s to a stream of ozone in oxygen at a very high concentration (25 /zmole O 3 / m l = 1200 g / m 3 =600,000 ppm). Using a D N A unwinding technique with fluorimetric analysis of DNA, ozone exposure for 2 h was shown to generate single-strand breaks. Using the alkaline elution technique there was no increased elution rate. Control experiments showed that the increased elution rate induced by X-rays was reduced when cells were pre-treated with ozone. This indicates that ozone had induced the generation of DNA-interstrand cross-links. According to the authors this might have obscured the resuits when measuring strand breaks with the alkaline elution technique. Ozone-induced DNA-protein cross-links were also formed. No mention was made in the p a p e r on cytotoxic effects. However, at the ozone concentrations used, the cells must have been killed by the treatment. Borek et al. (1988) exposed laC-thymidine D N A labeled cell cultures of the human epidermal cell line R H E K to 5 p p m ozone for 10 min. D N A single-strand breaks were measured with the alkaline elution technique and alkaline sucrose gradient centrifugation. No significant induction of D N A strand breaks was detected. However, the significance of this result might have been h a m p e r e d by the fact that the cells were frozen and thawed before analysis, and this treatment caused some D N A breaks in control cells compared to unfrozen controls, as detected with the more sensitive method, the alkaline elution technique. This D N A damage was detectable in control ceils at a level corresponding to that produced by 5 - 7 Gy of X-rays. The resolution of the analysis was sufficiently sensitive to identify ozone-induced D N A breaks, which would correspond to breaks produced by 2 - 3 Gy. Control experiments with ozonated buffer were performed in a study on formation of genotoxic reaction products by ozonation of arylamines (Kozumbo and Agarwal, 1990). CCD-18 Lu human lung fibroblasts and transformed type II epithelial A549 lung ceils were used as tester organisms. In control experiments, washed cell cultures were treated for 2 h with 1 ml of buffer that had been pre-exposed in a 35-mm dish to 1 p p m ozone in a humified ozone exposure chamber. The pre-labeled cells were analyzed for D N A

228

single-strand breaks by the alkaline elution technique. The ozone-exposed buffer caused no increase in single-strand breaks, compared to unexposed buffer.

Cell transformation in uitro In a study by Borek et al. (1986) it was shown that ozone induced neoplastic transformation in

primary hamster embryo cells and in mouse fibroblast cultures ( C 3 H / 1 0 T 1 / 2 ) . 2 ml of buffered saline was added to the cell cultures in 100-mm petri dishes, which were then exposed to 5 p p m ozone for 5 min in an exposure chamber. After incubation, transformation was scored using morphological criteria. Transformed cells were able to colonize in 0.3% agar. Ozone acted,synergisti-

TABLE 2 STUDIES U S I N G C E L L C U L T U R E S Cell line

Endpoint

Ozone exposure

Result

Reference

KB h u m a n cell line

Chromatid-type aberrations

8 ppm, 5 or 10 min

+

Fetner, 1962

Embryonic chick flbroblasts

Gross chromosomal effects

700-7000 ppm, 30 min

+

Sachsenmaier, 1965

H u m a n lymphocytes

Chromatid-type aberrations

Bubbling through cell suspension Bubbling through cell suspension Ozone-saturated buffer

+

Gooch et al., 1976

-

Gooch et al., 1976

-

Gooch et al., 1976

Chromosome-type aberrations Chromatid- and chromosome-type aberrations Wl-38 h u m a n cell line

SCE Chromatid-type aberrations Chromosome-type aberrations

0.25-1 ppm, 1 h 0.25-1 ppm, 1 h 0.25-1 ppm, 1 h

+ + (1 ppm)

Guerrero et al., 1979 Guerrero et al., 1979 Guerrero et al., 1979

V79 Chinese hamster cells V79 Chinese hamster cells V79 Chinese hamster cells

SCE

0.035 ppm, 1 h

-

Inhibition of D N A replication Inhibition of D N A replication

0.035 ppm, 1 h 1-10 ppm, 1 h

+

R a s m u s s e n and Crocker, 1982 R a s m u s s e n and Crocker, 1982 Rasmussen, 1986

V79 Chinese hamster cells

SCE

0.13-1.0 ppm, 2 h

+

Shiraishi and Bandow, 1985

H u m a n lymphocytes

SCE

0.15-0.75 ppm

+

Hsueh and Xiang, 1984 (Zhou et al., 1981)

L929 murine fibroblasts

D N A single-strand breaks D N A inter-strand cross-links

600,000 ppm, 2 h

+ +

Van tier Zee et al., 1987 Van der Zee et al., 1987

CCD-18Lv h u m a n lung fibroblasts

D N A single-strand breaks

Kozumbo and Agarwal, 1990

A549 h u m a n lung type II epithelial cells

D N A single-strand breaks

Ozonated buffer (buffer exposed to 1 ppm, 2 h) Ozonated buffer

H u m a n epidermal cell line R H E K

D N A single-strand breaks

5 ppm, 10 min

Borek et al., 1988

Primary hamster embryo cells C 3 H / 1 0 T 1 / 2 mouse fibroblasts

Neoplastic transformation

5 ppm, 5 min

+

Borek et al., 1986

Neoplastic transformation

5 ppm, 5 min

+

Borek et al., 1986

Neoplastic transformation Neoplastic transformation

1 or 10 ppm, 40 min 0.7 ppm, 40 min biweekly for 9 exposures

+

T h o m a s s e n et al., 1991 T h o m a s s e n et al., 1991

Primary rat tracheal epithelial cells

Kozumbo and Agarwal, 1990

229 cally with ionizing radiation, but only when delivered after radiation (Borek et al., 1989a). In a similar study (Borek et al., 1989b) ozone and UV light induced cell transformation in an additive fashion. Preneoplastic transformation was also shown to be induced by ozone in a study by Thomassen et al. (1991), however only after multiple exposures. Primary rat tracheal epithelial cells in culture dishes without lids, containing 5 ml of buffered saline, were exposed to ozone in a rocking exposure chamber. Single exposures were to 1 ppm or 10 ppm for 40 min. The multiple exposure was to 0.7 ppm twice weekly for 5 weeks. Data evaluation Ozone has been shown to induce chromatidtype aberrations, SCE and neoplastic transformation in cell cultures (Table 2). The negative SCE result by Rasmussen and Crocker (1982) is easily explained by the low ozone concentration that was used. Induction of single-strand breaks in D N A cannot be evaluated. The positive finding of Van der Zee et al. (1987) cannot be considered relevant, due to the extremely high concentration used. On the other hand, the negative result of Kozumbo and Agarwal (1990) can be explained by the low concentration of ozone tested. In vivo genotoxic effects - a n i m a l experiments

The first of a number of inhalation experiments with laboratory animals was performed by Zelac et al. (1971). They used female Chinese hamsters; four animals each were exposed to either 0.24 or 0.3 ppm ozone for 5 h, and four other animals served as controls. For the exposed animals, blood samples were taken from the eye orbital directly after exposure, and 6 and 15 days post exposure. Lymphocyte cultures were incubated for 3 days and chromosome spreads were scored for chromosome-type aberrations. The break frequency was significantly higher in the exposed groups compared to the control group immediately after exposure and 15 days after exposure, but not 6 days after exposure. In an attempt to duplicate the positive findings of Zelac et al., Tice et al. (1978) exposed Chinese

hamsters, both male and female, to 0.43 ppm ozone for 5 h. Blood samples were obtained by heart puncture from 3, 7 and 5 animals directly after, 1 week and 2 weeks after exposure, respectively. PHA-stimulated lymphocyte cultures were incubated for 52 h with colcemid during the last 4 h. An increase in chromatid-type aberrations (deletions and gaps) was noted 1 and 2 weeks after the treatment, but not immediately after treatment. No chromosome-type aberrations were seen. Direct bone marrow preparations from some animals (4, 3 and 4 animals directly after exposure, 1 week and 2 weeks after exposure, respectively) showed no induced aberrations. SCE frequencies in the cultures of peripheral blood (incubated with BrdU in the medium) from the Chinese hamsters was not increased (6, 7 and 5 animals); nor were SCEs in bone marrow cells from groups of 3 - 4 C 5 7 B L / 6 female mice, exposed to 2 ppm ozone for 6 h and examined 0, 1, 2 and 3 weeks after exposure. The animals were infused with BrdU and injected with colcemid before killing. Ozone exposure induced no changes in the cell replication rates. Gooch et al. (1976) exposed mice and hamsters to ozone. No mention was made of the number of animals used in the experiments. Young male mice, strain C3H, were exposed to 0.15 ppm or 0.21 ppm ozone for 5 h and 0.99 ppm for 2 h. Blood samples were obtained by cardiac puncture immediately after, and 2 weeks after exposure. Multiple leukocyte cultures from several animals were pooled in order to obtain sufficient numbers of mitoses for analysis. Colchicine was added after 48 h incubation time, and the cells were fixed at 52 h. No significant induction of chromosome damage, either chromatid or chromosome aberrations, resulted from the ozone exposure. Mouse spermatocyte preparations were made 8 weeks following exposure, and analyzed for the presence of reciprocal translocations. No such effect was seen. Nor was there any induction of chromatid or chromosome aberrations in bone marrow cells from Chinese hamsters exposed to 0.23 ppm ozone for 5 h or 5.2 ppm ozone for 6 h. Samples from the femurs were obtained 2, 6 and 12 h after the exposure, and the animals were injected i.p. with colchicine 2 h prior to being killed.

230

Another negative study on chromosome aberrations (including chromatid breaks) in bone marrow cells was reported by Zhurkov et al. (1979). They exposed male white rats, 5 in each dose group, to either 0.15 m g / m 3 (0.075 ppm) 8 h daily for 7 days or 5.6 m g / m 3 (2.8 ppm) continuously for 5 days. At the ozone level of 2.8 ppm, the bone marrow mitotic activity was substantially depressed. Pulmonary alveolar macrophages were used by Rithidech et al. (1990) to indicate genotoxic effects in lung ceils. Female F 3 4 4 / N rats, 5 in each dose group, were exposed to 0.12, 0.27 and 0.8 p p m ozone for 6 h. The animals were injected with colchicine 24 h after exposure and killed 4 h later. The exposure to ozone significantly in-

creased the proportion of cells with chromatid deletions and gaps. The proportion of cells with chromatid deletions was highest in the 0.27 ppm exposure group. Chromatid gaps were most frequent in the highest dose group, 0.8 ppm. This level of ozone also induced a significant increase in the mitotic index of the macrophages. No chromosome-type aberrations were seen. Thomassen et al. (1991) studied the preneoplastic transformation potency of ozone to rat tracheal epithelial cells after inhalation of 0.14, 0.6 or 1.2 ppm, 6 h / d a y , 5 d a y s / w e e k for 1, 2 or 4 weeks. Following exposure of male F 3 4 4 / N rats, the tracheal epithelium cells were isolated, plated and scored for preneoplastic variants after 5 weeks of incubation. There was no statistically

TABLE 3 G E N O T O X I C EFFECTS IN L A B O R A T O R Y ANIMALS Species

Endpoint

Ozone exposure

Result

Reference

Chinese hamster, females

Chromosome-type aberrations in lymphocytes

0.24 and 0.3 ppm, 5 h

+

Zelac et al., 1971

Mouse strain C3H, males

Chromosome- and chromatidtype aberrations in lymphocytes Reciprocal translocations in spermatocytes Chromosome- and chromatidtype aberrations in bone marrow cells

0.15 and 0.21 ppm for 5 h, 0.99 ppm for 2 h

Mouse strain C3H, males Chinese hamster

Chinese hamster males + females

Mouse strain C47BL/6, females

Chromosome-type aberrations in lymphocytes Chromatid-type aberrations in lymphocytes Chromosome- and chromatid-type aberrations in bone marrow cells SCE in lymphocytes SCE in bone marrow cells

Gooch et al., 1976

0.15 and 0.21 ppm for 5 h, 0.99 ppm for 2 h 0.23 ppm for 5 h, 5.2 ppm for 6 h

-

Gooch et al., 1976

-

Gooch et al., 1976

0.43 ppm, 5 h

-

Tice et al., 1978

0.43 ppm, 5 h

+

Tice et al., 1978

0.43 ppm, 5 h

-

Tice et al., 1978

0.43 ppm, 5 h 2.0 ppm, 6 h

-

Tice et al., 1978 Tice et al., 1978

Rat, males

Chromosome- and chromatidtype aberrations in bone marrow cells

0.075 ppm, 8 h daily for 7 days or 2.8 ppm continously for 5 days

Zhurkov et al., 1979

Rat strain F334/N, females

Chromosome-type aberrations in pulmonary macrophages Chromatid-type aberrations

0.12, 0.27 and 0.8 ppm, 6 h 0.12, 0.27 and 0.8 ppm, 6 h +

Rithidech et al., 1990

Preneoplastic transformation in tracheal epithelial cells

0.14, 0.6 and 1.2 ppm, 6 h/day, 5 days/week, for 1, 2 or 4 weeks

Rat strain F334/N, males

Rithidech et al., 1990 Thomassen et al., 1991

231

significant increase in transformation frequency compared to control rats. Hussein et al. (1985) m e a s u r e d an increased activity of the enzyme poly-ADPR synthetase in mouse lungs (strain A / I ) after exposure to 0.45 p p m for 5 - 7 days. The increased enzyme activity was interpreted by the authors to potentially reflect a response to lung cellular D N A repair, thus indicating that ozone might have caused D N A damage. However, as this enzyme is also stimulated by provoked cell proliferation this conclusion is quite uncertain. Data evaluation The results from the in vivo cytogenetic studies with laboratory animals are contradictory (Table 3). Concerning chromosome aberrations in lymphocytes, Zelac et al. (1971) noted chromosometype aberrations, but Tice et al. (1978) only saw chromatid-type aberrations at similar doses in Chinese hamsters. SCEs were not induced by the same treatment. In mice, no aberrations of either type were observed (Gooch et al., 1976). No cytogenetic effects have been seen in bone marrow cells or spermatocytes (Gooch et al., 1976; Tice et al., 1978; Zhurkov et al., 1979). As ozone is a directly acting, oxidative and reactive compound, it is plausible that it will be inactivated before it reaches target cells in the bone marrow. Its toxicity also prevents the testing of high concentrations. From this point of view it is not surprising that no effects were seen in bone marrow or spermatocytes. The lung would be the relevant target organ for the study of genotoxic effects of ozone after inhalation. However, only one relevant study with lung cells has been published (Rithidech et al., 1990), in which chromatid-type aberrations in lung macrophages were induced. The presented data are not easy to interpret. However, with regard to the positive findings of Zelac et al. (1971), Tice et al. (1978) and Rithidech et al. (1990), genotoxic effects in vivo cannot be ruled out, at least considering lung cells and lymphocytes.

Cytogenetic effects in humans In a study of the effect of ozone on pulmonary function in humans, chromosome aberrations in peripheral lymphocytes were also analyzed (Merz

et al., 1975). Two individuals were exposed to 0.5 p p m for 6 h and 4 individuals to 0.5 p p m for 10 h. The individuals served as their own control, blood samples being withdrawn both pre and post exposure. In addition, the group of 4 individuals was also tested 2 weeks and 6 weeks after exposure. The lymphocytes were cultured with P H A stimulation, treated with colchicine and fixed at 72 h. No chromosome-type aberrations were seen in any individuals. However, for most individuals an increase in achromatic lesions (gaps) and chromatid deletions was found after exposure. The number of chromatid deletions seemed to be highest 2 weeks after exposure. The statistical significance of the results was not evaluated. No mention was made of smoking habits, etc. G u e r r e r o et al. (1979) analyzed the SCE frequency in peripheral lymphocytes in 31 male and female college students 1 day before and immediately after exposure to 0.5 p p m ozone for 2 h. The lymphocyte cultures were grown with P H A for 24 h and then with BrdU for an additional 48 h. Colcemid was added during the last 3 h. There was no significant difference in the mean SCE frequency per chromosome for all subjects before compared to after exposure. The individual SCEs were not reported, nor was there any mention of smoking habits, etc. McKenzie et al. (1977) also have performed cytogenetic studies with ozone-exposed individuals. In their first study 26 healthy non-smoking male college students were exposed to 0.4 p p m for 4 h. Blood samples were withdrawn immediately before and after exposure, as well as 3 days, 2 weeks and 4 weeks after exposure. The pooled results from all individuals showed no statistically significant increase in the number of cells with chromosome- or chromatid-type aberrations (including gaps) after exposure. Their later studies were summarized in McKenzie (1982). Neither 0.6 p p m ozone for 2 h, 0.6 p p m ozone + histamine for 2 h nor 0.4 p p m ozone for 4 h repeatedly for 4 days gave statistically significant increases in structural aberrations or SCEs immediately after, 3 days after or 2 or 4 weeks after exposure ( > 10 subjects). No experimental details were given in this paper. Sarto and Viola (1980) noticed a statistically significantly increased frequency of chromatid

232

gaps, but no other chromosome aberrations, in lymphocytes from 10 workers exposed to nearly 0.3 p p m ozone for 1-3 years, compared to 10 unexposed controls. No mention was made of smoking habits or possible confounding air pollutants in the workplace (production of plastic bags).

Data eL'aluation As the inter-individual variation in chromosome aberration and SCE frequencies can be expected to be fairly high, either the investigated individuals should serve as their own control, or when comparing group means there should be a good control of confounding variables. In the study by Merz et al. (1975), most individuals experienced an increase in chromatid-type aberrations after exposure to ozone. However, only 6 individuals participated, and there were some data missing, which gives lesser weight to this study. In the other studies, group means were compared. In the two negative inhalation studies, McKenzie et al. (1977, 1982) included only nonsmokers, but in the study by Guerrero et al. (1979) smoking or other confounding factors were not mentioned. No mention of confounding factors was made in the positive study on ozone-exposed workers either (Sarto and Viola, 1980). Thus, both the negative study by Guerrero et al. and the positive study by Sarto and Viola must be regarded as inconclusive. In summary, the experimental and epidemiological studies with human

subjects do not allow a conclusion on the cytogenetic effects of ozone in lymphocytes (Table 4). Tumor induction

The carcinogenicity of ozone has been investigated to a limited extent. Stokinger (1965) reported that ozone induced lung tumors (adenomas) in a susceptible strain of mice (strain not mentioned), exposed to 1 ppm ozone daily for 15 months. However, no experimental details were presented in this article. W e r t h a m e r et al. (1970) exposed Swiss-Webster mice to 4.5 p p m ozone during 2 h every third day for a period of up to 75 days. A statistically significant increase in cellular alterations in the lung between ozone-exposed mice and control animals was reported, although the control data were not shown. The cellular alterations included hyperplasia, metaplasia and adenomas. There were also inflammatory reactions. Hassett et al. (1985) exposed female strain A / J mice to either 0.31 p p m intermittently for 103 h / w e e k every other week for 6 months, or to 0.5 p p m for 102 h / w e e k during the first week of each month for 6 months. In the first study, animals were killed 5 months and in the second study 3 months after the final ozone exposure. The presence of tumor nodules (adenomas) on the pleural surface of the lung was reported to be significantly higher in the exposed animals c o m -

TABLE 4 IN V I V O C Y T O G E N E T I C Endpoint Chromosome-type aberrations Chromatid-type aberrations

EFFECTS IN HUMANS (LYMPHOCYTES) N u m b e r of individuals

O z o n e exposure

Result

Reference

6

0.5 ppm, 6 h or 10 h

-

M e r z et al., 1975

6

0.5 ppm, 6 h or 10 h

+

M e r z et al., 1975

SCE

31

0.5 ppm, 2 h

-

G u e r r e r o et al., 1979

C h r o m o s o m e - and chromatid-type a b e r r a t i o n s or S C E

10-30

0.4 ppm, 4 h; once or r e p e a t e d l y for 4 days; 0.6 ppm, 2 h

-

M c K e n z i e et al., 1977; McKenzie, 1982

C h r o m a t i d gaps Other chromosomeand c h r o m a t i d - t y p e aberrations

10

0.3 ppm, 1-3 years 0.3 ppm, 1 - 3 years

+ -

Sarto and Viola, 1980 Sarto and Viola, 1980

233

In a similar study, Last et al. (1987) exposed both strain A / J and strain Swiss-Webster male mice to 0.4 or 0.8 p p m ozone for 8 h per night, 7 nights per week for 18 weeks. The animals were then killed. U p o n examination, 0.8, but not 0.4 ppm, induced a significant increase in lung adenomas in A / J mice, but not in the more resistant Swiss-Webster strain. However, later Witschi (1991) commented that the significant increase might have been due to abnormal low tumor incidences in the control groups. In one part of the experiment, the animals were pretreated with

pared to control animals in both studies. However, in a later p a p e r from the same research group (Mustafa et al., 1988) only the increase in the second study was referred to as being statistically significant. The tumor inducing activities of urethan, as a single i.p. injection, was not significantly enhanced by ozone pretreatment. However, when injection of the carcinogen urethan followed immediately after each ozone exposure, a higher than expected tumor incidence was produced. This indicates that ozone acted as a cocarcinogen.

DOSE

OZONE

(ug/ml

SA2

i

o.ooo,

0.01

0

ECB BSDI

ECK

ECFI

VFC BML

I

I

SIH

I ,

TSM

I ISCO SCFSCR •

I

I

100

10000

SlC

t

CHL CIH

I

CLA CVA

I1i

.......................................................................

Iiil i

o oo

SAO

iii;ii.iii

o,, .

.

.

.

.

.

.

-I

DIH

!"

iil i_

SLH

'i

EUK.I PLANTS I Iis, MA IN . ~ M A N S .............................................................. 'V°u~m=l

Fig. 1. Genetic activity profile of ozone. The mean value of the lowest effective dose (LED) for each test reporting positive results is shown as a vertical line drawn in the upward direction. Conversely, the mean value of the highest ineffective dose (HID) is drawn in the downward direction. A solid line represents the majority call for each test and a dashed line displays conflicting data. The dose units are / z g / m l for in vitro tests and m g / k g for in vivo tests. Note: The window for the H I D values has been lowered, showing a broader portion of "the zone of uncertainty" (area between the horizontal dashed lines), to display the H I D s for these test results.

234 TABLE 5 GENETIC ACTIVITY PROFILE DATA FOR OZONE End point

Test code

Test system

Results NM M

Dose (LED or H ID )

Reference

D D G

ECB BSD SA0

E. coli, strand b r e a k s / X - l i n k s / r e p a i r B. subtilis REC, differential toxicity S. typhimurium TA100, reverse mutation

+ + -

0 0 -

0.1000 0.1000 0.0040

Hamelin and Chung, 1989 Song and Chung, 1983 Victorin and St~thlberg, 1988

G G

SA0 SA0

G

SA2

S. typhimurium TA100, reverse mutation S. typhimurium TA100, reverse mutation S. typhimurium TA102, reverse mutation

+

+

0.0010 0.0170 0.0001

Shepson et al., 1985 Dillon et al., 1992 Dillon et al., 1992

G G G

SA4 SA5 SA9

S. typhimurium TA104, reverse mutation S. typhimurium TA1535, reverse mutation S. typhimurium TA98, reverse mutation

-

-

0.017(/ 0.0170 0.0170

Dillon et al., 1992 Dillon et al., 1992 Dillon et al., 1992

G G G

ECF ECF ECF

E. coli (excluding K12), forward mutation E. coli (excluding KI2), forward mutation E. coli (excluding K12), forward mutation

+ + +

0 0 0

0.0002 0.0010 0.1000

Hamelin and Chung, 1975 Nover and Botzenhart, 1985 L'H~rault and Chung, 1984

G R G

ECK SCG SCF

E. coli KI2, forward or reverse mutation S. cerecisiae, gene conversion S. ceret:isiae, forward mutation

+ (+ ) (+ )

0 0 0

0.0002 0.1000 0.1000

Hamelin and Chung, 1974 Dubeau and Chung, 1982 Dubeau and Chung, 1982

G G M

SCR TSM TSI

S. cerecisiae, reverse mutation Tradescantia species, mutation Tradescantia species, micronuclei

(+) + -

0 0 0

0.1000 0.0100 0.0100

Dubeau and Chung, 1982 Schairer and Sautkulis, 1982 Ma et al., 1982

C C C

VFC VFC DML

Vicia faba, chromosomal aberrations Vicia faba, chromosomal aberrations D. melanogaster, dominant lethals

+ + +

0 0 0

0.0040 8.0000 0.0600

Janakiraman and Harney, 1976 Fetner, 1958 Erdman and Hernandez, 1982

S S D

SIC SIC DIH

SCE, Chinese hamster cells in vitro SCE, Chinese hamster cells in vitro Strand b r e a k s / X - l i n k s , human cells in vitro

+ -

0 0 0

0.0002 0.0001 0.0002

Shiraishi and Bandow, 1985 Rasmussen and Crocker, 1982 Kozumbo and Agarwal, 1990

D S S

DIH SIH SIH

Strand b r e a k s / X - l i n k s , human cells in vitro SCE, other human cells in vitro SCE, other human cells in vitro

+ +

0 0 0

0.0100 0.0005 0.0003

Borek et al., 1988 Guerrero et al., 1979 Hsueh and Xiang, 1984

C C C

CHL CIH CIH

Chrom aberr., human lymphocytes in vitro Chrom aberr., other human cells in vitro Chrom aberr., other human cells in vitro

+ + +

0 0 0

0.0300 0.0005 0.0160

Gooch et al., 1976 Guerrero et al., 1979 Fetner, 1962

S C C

SVA CBA CBA

SCE, animals in vivo Chrom aberr., animal bone marrow in vivo Chrom aberr., animal bone marrow in vivo

-

0 0 0

2.0000 2.0000 0.1500

Tice et al., 1978 Gooch et al., 1976 Tice et al., 1978

C C C

CBA CLA CLA

Chrom aberr., animal bone marrow in vivo Chrom aberr., animal leucocytes in vivo Chrom aberr., animal leucocytes in vivo

+ -

0 0 0

2.0000 0.2500 0.3000

Zhurkov et al., 1979 Zelac et al., 1971 Gooch et al., 1976

C C S

CL A CVA SLH

Chrom aberr., animal leucocytes in vivo Chrom aberr., other animal cells in vivo SCE, human lymphocytes in vivo

+ + -

0 0 0

0.1500 0.3000 0.0500

Tice et al., 1978 Rithidech et al., 1990 McKenzie, 1982

S C C

SLH CLH CLH

SCE, human lymphocytes in vivo Chrom aberr., human lymphocytes in vivo Chrom aberr., human lymphocytes in vivo

-

0 0 0

0.0010 0.0700 0.0500

Guerrero et al., 1979 Sarto and Viola, 1980 McKenzie, 1982

C C P

CLH CLH SPM

Chrom aberr., human lymphocytes in vivo Chrom aberr., human lymphocytes in vivo Sperm morphology, mice

+ -

0 0 0

0.0500 0.1600 0.3000

McKenzie et al., 1977 Merz et al., 1975 Gooch et al., 1976

Endpoint indications: C, chromosome aberration; D, DNA damage; G, gene mutation; M, micronucleus; P, sperm morphology; R, recombination; S, SCE.

235 a single i.p. injection of urethan one day before ozone exposure started. In these animals, ozone caused a decrease in the n u m b e r of tumors per lung. This may be explained in terms of a cytotoxic action of ozone upon initiated cells that will grow into tumors (Witschi et al., 1991; Sweet et al., 1980). In contrast, the co-carcinogenic action of ozone observed by Hassett et al. (1985) might be explained by the cell proliferative activity of ozone, expanding the cell population at risk to undergo transformation. Thus, the sequence of exposure determines whether the effects of ozone on lung tumor development are beneficial or detrimental (Witschi, 1991). The effect of ozone on cancer cell metastasis in the lungs of mice has been studied in two papers. Kobayashi et al. (1987) exposed male C 3 H / H e mice to 0.1, 0.2, 0.4 and 0.8 p p m continuously for 1, 3, 7 and 14 days. Then a suspension of fibrosarcoma NR-FS cells was infused via the tail vein. The animals were killed after 14 days. A significantly increased rate of pulmonary metastasis was observed in the ozone-exposed mice. The maximal enhancement occurred in mice exposed to 0.8 p p m for 1 day. The prolonged exposure (14 days) gradually suppressed the enhancement. In a similar study by Richters (1988), no significantly enhanced cancer cell colonization of lungs was observed when male C 5 7 B L / 6 mice were exposed to 0.15 or 0.3 p p m ozone for 7 h / d a y , 5 d a y s / w e e k , for a total period of 12 weeks and then infused with m e l a n o m a cells (B16F10R1). O t h e r effects seen in animal experiments may be of relevance when discussing the tumor inducing activity of ozone. Cell proliferation (increased D N A replication) and m e t a p l a s i a / h y p e r p l a s i a have been induced in parts of the nasal epithelium in rats and monkeys (Reutzel et al., 1990; Johnson et al., 1990; H a r k e m a et al., 1987). Ozone also induces cell proliferation (increase in labeling index and mitotic index) of type II epithelial lung cells, alveolar macrophages, and lymphocytes of the lung (Hiroshima et al., 1987; Wright et al., 1987; El Sayed et al., 1990; Hotchkiss et al., 1989; Dziedzic et al., 1990). Data evaluation The purpose of the present review was to evaluate the genotoxicity, not the carcinogenicity

of ozone. A proper evaluation of the carcinogenic potential should await results from lifetime studies at maximally tolerated doses. The reactivity and genotoxic potential of ozone in vitro indicate that inhalation of ozone may involve a cancer risk to lung tissue. This is supported by the animal studies referred to in this chapter.

Genetic activity profile A graphic display of the data on the genetic and related effects of ozone is shown in Fig. 1 as a genetic activity profile (GAP). A complete listing of these data is given in Table 5. The profile was p r e p a r e d with the methodology previously described by Waters et al. (1988). The window showing the highest ineffective doses for the negative test results has been lowered in order to display the tow dose values for ozone. Consequently, the actual doses in units of / z g / m l or m g / k g are shown on a log scale.

Acknowledgements Dr. Michael D. Waters of the Genetic Toxicology Division, US Environmental Protection Agency, and H. Frank Stack and Marcus A. Jackson of Environmental Health Research and Testing (EHRT), Inc., Research Triangle Park, NC, USA, prepared the Genetic Activity Profile. Their work is highly appreciated. Also thanks to Dr. Siv Ljungquist for critically reviewing the manuscript.

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