52, 162–167 (1999)
Copyright © 1999 by the Society of Toxicology
TOXICOLOGICAL SCIENCES
Ozone Carcinogenesis Revisited
Hanspeter Witschi,* ,† ,1 Imelda Espiritu,* Kent E. Pinkerton,* ,‡ Kerry Murphy,* and Robert R. Maronpot§
*Institute of Toxicology and Environmental Health, †Department of Molecular Biosciences, ‡Department of Anatomy/Physiology and Cell Biology, School
of Veterinary Medicine, University of California, Davis, California 95616; and §National Institute of Environmental Health Sciences,
Research Triangle Park, North Carolina 27709
Received April 22, 1999; accepted July 19, 1999
The question was asked whether ozone would act as a lung
carcinogen in mice. To test the hypothesis, female strain A/J mice
were exposed for 6 h/day, 5 days/week to 0.12 ppm, 0.5 ppm, or 1.0
ppm of ozone; control animals were kept in filtered air. No ozonerelated deaths were observed at any time during the experiment.
After 5 months, one-third of the animals were killed. The remaining animals were split into two groups: exposure to ozone continued for one group, whereas the other group was transferred into
filtered air. Four months later, these animals were killed. No
significant increase in lung tumor multiplicity (average number of
tumors per lung) or lung tumor incidence (percentage of tumorbearing animals) was found in the animals exposed to ozone when
compared to animals kept in filtered air, regardless of ozone
concentration. Morphometric analysis of lungs of animals exposed
to the highest ozone concentration (1.0 ppm) showed a small,
statistically not significant increase in centriacinar lesions. It was
concluded that ozone is not a lung carcinogen in strain A/J mice at
those exposure levels. Moreover, this mouse strain appears to be
particularly resistant towards chronic ozone toxicity.
Key Words: ozone; carcinogen, lung tumor, strain A/J mice
Ozone is a major constituent of photochemical smog. It has
been suspected for some time that ambient air pollutants are a
risk factor for the development of lung cancer. The urban-rural
gradient in lung cancer incidence strongly suggests such a
possibility (Katsouyanni and Pershagen, 1997). However, it
has not yet been possible to unequivocally link ozone and lung
cancer in people. A significant positive association was recently found between lung cancer and the number of days
where particulate matter in the ambient air (PM 10) exceeded 40
mg/m 3. As far as ozone was concerned, an association between
excess lung cancer risk and exceedance of ozone thresholds of
80 ppb (current EPA standard: 120 ppb) was found only for
males, but not for females (Beeson et al. 1998).
Whether ozone has carcinogenic potential has been repeatedly examined in isolated cell systems or in experimental
animals. A review of the data on ozone carcinogenesis in 1988
1
To whom correspondence should be addressed at ITEH, University of
California, One Shields Avenue, Davis, CA 95616. Fax: (530) 752-5300.
E-mail: [email protected].
concluded that in vitro evidence for clastogenic and mutagenic
action was quite good (Witschi, 1988). Animal studies were
less conclusive. Early experiments usually used ozone concentrations higher than 1 ppm and gave equivocal responses. In
one study, ozone was labeled a tumor accelerator (Stokinger,
1962). In 1985, Hasset et al. concluded that exposure of strain
A/J mice to 0.3 or 0.5 ppm ozone alone was sufficient to
increase the number of lung tumors (Hassett et al., 1985). In a
paper published not much later, it was claimed that exposure to
0.8 ppm ozone significantly increased lung tumor incidence
(Last et al., 1987). Experiments using strain A/J mice thus
provided some evidence that ozone could produce lung tumors
in mice.
The United States National Toxicology Program and the
Health Effects Institute attempted to obtain a more definite
answer about ozone as an animal carcinogen (Catalano et al.,
1995). Male and female F344/N rats and B6C3F1 mice were
examined. The protocol called for ozone exposure 6 h/day, 5
days/week, plus corresponding control groups. Ozone concentrations were 0.12 ppm, 0.5 ppm, and 1.0 ppm. Exposures were
for 24 and 30 months. In female mice the incidence of alveolar/
bronchiolar adenomas was significantly increased in the highest dose group. In males, the increase was suggestive, but not
statistically significant (Herbert et al., 1996). No increase in
neoplasms at any site that could have been attributed to ozone
was found in rats (Boorman et al., 1994). Although the results
of this major study were conclusive, it was still of some interest
to reexamine the effects of chronic ozone exposure in the
reportedly more sensitive A/J mouse. We found no evidence
for ozone carcinogenesis in A/J mice.
MATERIALS AND METHODS
Animals. Female strain A/J mice were purchased from Jackson Laboratories, Bar Harbor, ME. Randomly chosen animals were sent to the Comparative Pathology Laboratory, UC Davis, for a standard rodent health surveillance screen. No evidence for infectious disease (pathogenic agents) or
presence of parasites or ova in pelage and cecum were reported. Histopathology was not processed, as no significant lesions were noted. Serology was
negative for mouse hepatitis virus, Sendai virus, Reovirus type 3, pneumonia
virus, parvo, ectromelia, and mycoplasma pulmonis.
Exposure system. The exposure system has been described in detail (Gelzleichter et al., 1992). Ozone was generated from medical grade 100% oxygen by
162
OZONE CARCINOGENESIS REVISITED
TABLE 1
Ozone Concentrations
Target
ppm
n
Mean
ppm
6 SD
Minimum
ppm
Maximum
ppm
0.12
0.50
1.00
8141
8152
8190
0.12
0.50
1.01
0.012
0.025
0.034
0.07
0.40
0.78
0.18
0.59
1.13
Note. n, sample size (number of measurements).
silent electric arc discharge. It was mixed into a stream of filtered air of 50%
humidity. Chamber concentrations of ozone were continuously monitored with a
calibrated UV photometer (Dasibi) and recorded every 2 min. The Dasibi ozone
analyzer was calibrated against a Dasibi UV photometer, which in turn was
calibrated against a National Bureau of Standards Reference photometer located at
the California Air Resources Board Assurance Standard Laboratory, Sacramento,
CA. Data on actual chamber concentrations are presented in Table 1.
Experimental protocol. After a suitable acclimatization period, the mice
were divided at random into four groups and exposed to the same ozone
concentrations as had been used in the National Toxicology Program carcinogenesis study with ozone: 0.12, 0.5, and 1.0 ppm (Herbert et al., 1996).
Exposures were 6 h/day, 5 days/week. At all times during the experiment,
including times of ozone exposure, conventional laboratory chow and water
were provided ad libitum. The animals were observed daily and weighed
weekly. After 5 months of exposure, half of the filtered air controls and
one-third in each group of the ozone exposed animals were killed (Group A).
The remaining ozone-exposed mice were divided into two groups: one group
was kept in ozone (Group B), whereas the other group was transferred into
filtered air (Group C). This protocol was adopted because it had been found in
experiments involving tobacco smoke that lung tumors in A mice became fully
expressed only when the animals were given a recovery period in fresh air
following smoke exposure (Witschi et al., 1997). The animals in Groups B and
C were killed 9 months after the beginning of the experiment.
Tissue preparation. Animals were killed by pentobarbital overdose. For
analysis of tumor incidence and multiplicity, the lungs were manually expanded to inspiratory volume by intratracheal instillation of Tellyesniczky’s
fluid and fixed for at least 24 h. The number of tumor nodules visible on the
lung surface was counted. The results were expressed as tumor incidence, i.e.,
percentage of animals with one or several lung tumors and as tumor multi-
163
plicity, the average number of tumors per lung. Tumor multiplicity was
calculated as average number of tumors per lung of all, including non–tumorbearing animals, as well as for tumor-bearing animals only. Histopathology of
lung tumors was done on H&E stained paraffin sections.
Morphometric analysis. To determine the extent of tissue changes by
repeated exposure to ozone up to 9 months in duration, the lungs of mice
exposed to filtered air or to 1.0 ppm ozone were examined morphometrically.
All lobes were embedded in immunobed. Each block was sectioned at 1.5 to
2.0 micron thickness. Sections for analysis were selected by random number
generation. A minimum of four bronchiole alveolar duct junction isolations in
longitudinal profile were examined. This anatomic region of the lung represents the abrupt transition from conducting airway to gas exchange region.
These anatomical sites were identified in longitudinal profile as a terminal
bronchiole opening onto an alveolar duct that immediately bifurcated into two
daughter alveolar ducts with an intervening alveolar bifurcation tissue ridge
(Fig. 1). Four anatomical sites within this region were analyzed: 1) bronchiolar
epithelium of the terminal bronchiole, 2) the first septal tip of the alveolar
outpocketing, 3) the first alveolar outpocket, and 4) the first alveolar duct
bifurcation or tissue ridge separating the two daughter alveolar duct generations of a second order. The bronchiolar epithelium was analyzed only within
the most distal portion of the terminal bronchiole just prior to the transition of
bronchiolar epithelium to alveolar epithelium. The first septal tip is represented
by the intervening alveolar septal tissue between the first and second alveolar
outpocketings immediately distal to the terminal bronchiole. First, alveolar
septal tips were analyzed on both sides of the terminal bronchiole profile. The
first alveolar outpocketing was represented by the first alveolus immediately
distal to the terminal bronchiole. Again both alveolar outpocketings immediately arising from the terminal bronchiole were analyzed. The first alveolar
duct bifurcation ridge, illustrated in Figure 2, typically consisted of the tissue
bifurcation ridge and down to the first alveolar outpocketing. Each of these
regions was captured at high magnification under oil (633 objective immersion lens). This level of resolution allowed us to examine the tissue volume
normalized to surface area of the bronchiole or alveolar duct. We were able to
further resolve epithelial tissue volume from septal wall tissue wall volume as
well as to determine the capillary volume fraction within each of these regions.
Each field was captured in random orientation, with the exception of the
terminal bronchiole in which the bronchiolar epithelium was oriented in a
horizontal fashion relative to the viewing field. Each region was examined
using Stereology Toolbox (Davis, CA) for analysis of volume and surface area
measurements. A cycloid grid consisting of 45 lines and 135 points were used
for all counts within the bronchiolar epithelium. For alveolar tissue analysis, a
21-line test lattice grid was used. For this analysis, a minimum of four
FIG. 1. Bronchiolar alveolar duct junction within the lungs of filtered air control (panel A) or ozone exposed (panel B) mouse. Each profile is in longitudinal
orientation with symmetrical bifurcation of the alveolar ducts and an intervening first alveolar bifurcation ridge (arrow). Scale bar is 500 mm.
164
WITSCHI ET AL.
FIG. 2. The first alveolar duct bifurcation is shown in panel A (arrow). In panel B, the first alveolar septal tip (arrow) and first alveolar outpocketing (asterisk)
are shown. Each of these anatomical regions is an enlargement from Figure 1A. Scale bar is 50 mm.
bronchiolar alveolar duct junctions were used with two images per terminal
bronchiole analyzed, two first alveolar septal tips analyzed, two first alveolar
outpockets analyzed, and one first alveolar duct bifurcation tissue ridge analyzed. All data counted as points or intercepts with the ends or along the length
of each test lattice line were tallied and organized within a spreadsheet.
Formulas used to stereologically derive tissue densities were used for analysis
(Pinkerton et al., 1982, 1992; Weibel, 1979).
Statistical analysis. Data on lung tumor multiplicity were analyzed by
ANOVA followed by the Tukey-Kramer Multiple Comparisons test. Lung
tumor incidences were compared using Fisher’s exact test. For analysis of the
morphometric data, two-way analysis of variance (ANOVA) was used to
determine potential differences between filtered air control and ozone exposed
animals. A p value of 0.05 or less was considered to be significant.
RESULTS
During the entire experimental period, no ozone-related
deaths were observed. The weight gains in the exposed animals, regardless of ozone concentration, were comparable to
the weight gains in animals kept in filtered air (Table 2). A first
group of animals was killed after a 5-month exposure to ozone
(Group A). The data are shown in Table 3. In the animals
exposed to 1.0 ppm ozone, lung tumor multiplicity, calculated
as average for all animals, was twice the control value. However, the difference was statistically not significant, nor was the
TABLE 2
Body Weights (g) of Animals Exposed to Ozone
Treatment
Initial
After 5 months
(Group A)
After 9 months
(Group B)
Filtered air
0.12 ppm
0.5 ppm
1.0 ppm
18.8 6 1.4 (96)
19.1 6 1.9 (96)
18.7 6 1.4 (96)
18.5 6 1.5 (100)
22.4 6 1.5 (35)
22.7 6 1.9 (35)
22.9 6 1.6 (35)
22.6 6 1.7 (35)
23.7 6 1.5 (29)
23.8 6 2.1 (30)
25.1 6 1.7 (32)
23.9 6 1.7 (35)
Note. All data are means 6 SD with number of animals in parentheses.
increase in lung tumor incidence. The number of lung tumors
per tumor-carrying animal was similar in all groups.
The second group of animals was exposed to ozone for a
total of 9 months (Group B). The data are shown in Table 4.
There were no statistically significant differences in lung tumor
multiplicity between controls and ozone-exposed mice,
whether calculated for all animals or for tumor-bearing animals
only. The highest lung tumor incidence (statistically different
from control and 1 ppm group) was seen in the mice exposed
to 0.5 ppm ozone.
A third group of animals was exposed first for 5 months to
ozone, then removed and kept in filtered air chambers for
another 4 months (Group C). The results are presented in Table
5. In this group, there was a statistically significant increase in
lung tumor multiplicity (calculated for all animals) in the mice
exposed to the lowest ozone concentration (0.12 ppm). Animals kept in the two higher concentrations had lung tumor
multiplicities only slightly above control values. There appeared to be a negative dose response. When lung tumor
multiplicities were calculated per tumor-bearing animals only,
similar numbers were found in all groups. The 0.12 ppm group
also had a significantly higher lung tumor incidence compared
to all the other groups.
Histologically most tumors were typical solid alveolar/bronchiolar adenomas comprising a monomorphic proliferation of
alveolar type II cells that filled alveolar spaces without compression of adjacent pulmonary parenchyma. Only occasional
papillary adenomas were seen. Alveolar/bronchiolar carcinomas generally arose within existing adenomas and were recognized by focal areas manifesting a different growth pattern
from the adenoma with minimal cellular pleomorphism and
crowding.
The lungs were also examined for overall histopathologic
alterations. Examination of the tissues under the light microscope revealed a striking absence of inflammatory changes in
165
OZONE CARCINOGENESIS REVISITED
TABLE 3
Lung Tumor Data in Strain A/J Mice Exposed for 5 Months to
Ozone (Group A)
TABLE 5
Lung Tumor Data in Strain A/J Mice Exposed for 5 Months
to Ozone, Followed by a 4-Month Recovery Period in
Air (Group C)
Lung tumor multiplicity a
Lung tumor multiplicity b
Exposure
Filtered air
0.12 ppm
0.50 ppm
1.00 ppm
All animals
0.11 6 0.05 (35)
0.09 6 0.05 (35)
0.14 6 0.07 (35)
0.23 6 0.07 (35)
Tumor-bearing
animals only
1.00 6 0.00 (3)
1.00 6 0.00 (3)
1.3 6 0.3 (4)
1.00 6 0.00 (8)
Lung tumor
incidence b
3/35 (9%)
3/35 (9%)
4/35 (11%)
8/35 (23%)
Note. Animals were exposured for 6 h/day, 5 days/week for 5 months.
a
Number of tumors per lung. All data given as mean 6 SEM, number of
animals in brackets.
b
Number of tumor-bearing animals per total number of animals at risk
(percentage in parentheses).
airways and lung parenchyma. Quantitative morphometry was
performed on the lungs of animals exposed to filtered air and
to 1.0 ppm ozone for 5 and 9 months. Table 6 provides all
tissue volumes normalized per surface area for each anatomical
region of the bronchiolar alveolar duct junction. It was anticipated that the most obvious changes produced by ozone would
be an increase in tissue volume in the first septal tip. Such an
increase was indeed seen after 5 and after 9 months’ ozone
exposure. However, the increase over control values was statistically not significant, because not all animals showed the
increased tissue volume. In the group exposed for 5 months,
only three out of five animals had values higher than the
highest control values; at 9 months, 4 out of 5 animals showed
such ozone-related changes. Because of the resulting large
standard deviations, statistical significance was not reached,
although individual animals clearly showed volume changes in
the septal tip tissues that were indicative of ozone exposure.
TABLE 4
Lung Tumor Data in Strain A/J Mice Exposed for 9 Months to
Ozone (Group B)
Lung tumor multiplicity b
Exposure a
All animals
Tumor-bearing
animals only
Lung tumor
incidence c
Filtered air
0.12 ppm
0.50 ppm
1.00 ppm
0.83 6 0.19 (29)
1.12 6 0.20 (31)
1.25 6 0.16 (32)
0.97 6 0.19 (35)
1.71 6 0.22 (14)
1.84 6 0.18 (19)
1.54 6 0.14 (26)
1.70 6 0.21 (20)
15/30 (48%)
19/31 (61%)
26/32 (81%)*
20/35 (57%)
a
Exposures for 6 h/day, 5 days/week for 5 months.
Number of tumors per lung. All data given as mean 6 SEM, number of
animals in brackets.
c
Number of tumor-bearing animals per total number of animals at risk
(percentage in parentheses).
* Significantly different (p , 0.05) from control and 1.0 ppm groups
(Fisher’s exact test).
b
Exposure a
All animals
Tumor-bearing
animals only
Lung tumor
incidence c
Filtered air
0.12 ppm
0.50 ppm
1.00 ppm
0.83 6 0.19 (29) d
1.93 6 0.25 (29)*
1.20 6 0.27 (30)
0.97 6 0.17 (34)
1.71 6 0.22 (14)
2.15 6 0.25 (26)
1.80 6 0.19 (20)
1.57 6 0.16 (21)
14/29 (48%)
26/29 (90%)*
20/30 (66%)
21/34 (62%)
a
Exposures for 6 h/day, 5 days/week for 5 months.
Number of tumors per lung. All data given as mean 6 SEM; number of
animals in parentheses.
c
Number of tumor-bearing animals per total number of animals at risk
(percentage in parentheses).
d
Same animals as in Table 4.
* Significantly different (p , 0.05) from all other groups (ANOVA and
Fisher’s exact test).
b
DISCUSSION
The strain A/J mouse lung tumor assay has often been used
as a convenient short-term assay for carcinogenesis (Stoner and
Shimkin, 1985), although it has some limitations and might not
be optimal for all classes of carcinogens (Maronpot et al.,
1986). Comparatively few assays have been performed in
which strain A/J mice have been exposed to an airborne carcinogen (summarized in Witschi et al., 1995). From the previous studies on ozone carcinogenicity in A/J mice, it could be
anticipated that ozone would give only a weak or no carcinogenic response (Hassett et al., 1985; Last et al., 1987). A study
with B6C3F1 mice led to similar conclusions (Herbert et al.,
1996). During the last few years, we have shown that environmental tobacco smoke, also a comparatively weak carcinogen,
is able to produce reproducibly a positive response in the lungs
of strain A/J mice (Witschi, 1998). One key element in these
studies was that the mice had to be given a recovery period in
air following tobacco smoke exposure in order to allow for full
tumor expression (Witschi et al., 1997). Our reevaluation of
ozone carcinogenicity was modeled after these previous experiences. For ozone exposure, we duplicated the NTP protocol
and exposed the most sensitive species, female mice, and from
the tobacco smoke studies we duplicated the recovery phase in
case this would be necessary for full expression of the tumorigenic potential of ozone.
The tumor response was negative. Although in animals
exposed for 5 months only to ozone there appeared to be a
doubling of lung tumor multiplicity in the highest dose group,
there was absolutely no sign of a significantly increased tumor
response after the full 9 months. Although the average number
of tumors per lung was somewhat higher in all mice exposed to
ozone than in the controls, there was no indication whatever of
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WITSCHI ET AL.
TABLE 6
Morphometric Characteristics of Anatomic Landmarks of the Centriacinus in the Mouse Lung
Tissue volumes per surface area (m 3/m 2) a
(5 mo exposure)
First alveolar septal tip
First alveolar outpocket
First alveolar duct bifurcation tissue ridge
Bronchiolar epithelium a
Tissue volumes per surface area (m 3/m 2) a
(9 mo exposure)
Filtered air
1.0 ppm ozone
Filtered air
1.0 ppm ozone
1.482 6 0.059
2.089 6 0.198
1.677 6 0.140
6.132 6 0.596
1.681 6 0.084
2.491 6 0.201
1.633 6 0.150
6.224 6 0.788
1,430 6 0.097
2,319 6 0.246
1.741 6 0.239
6.521 6 0.765
2.091 6 0.408
2.525 6 0.137
1.634 6 0.155
6.527 6 0.451
Note. All values are given as mean 6 SEM; n 5 5 mice throughout.
a
Volume/basal lamina surface area.
a dose response. A significantly increased tumor incidence was
found only in the group exposed to the middle ozone concentration of 0.5 ppm.
In animals allowed to recover for 4 months in air following
the initial 5-month exposure to ozone, there was only one
group where tumor multiplicity and tumor incidence were
significantly higher than in all other groups. This was in the
lowest ozone group (0.12 ppm). There is no explanation for
this observation. The absence of any positive response in all
other groups, regardless of ozone concentration and duration of
exposure, makes this singular finding spurious and of no significance for interpretation of the data. It should be added that
in the two previous studies that examined the effects of ozone
in the lungs of strain A mice, the results also could be interpreted to mean that ozone would not be carcinogenic. In the
study by Hassett et al. (1985), one experiment gave a positive
result, whereas the other one did not. In the study by Last et al.
(1987), the positive response was probably due more to abnormally low control values than to a true carcinogenic effect of
ozone.
The possibility has also been raised that ozone would act as a
tumor promoter. Early on, Stokinger had labeled ozone a tumor
accelerator (Stokinger, 1962). Later Hassett et al. did not find
evidence that ozone would promote the development of lung
tumors in A mice. In mice pretreated with carcinogens, ozone
actually reduced the number of developing lung tumors (Last et
al., 1987). In hamsters treated with NNK and exposed to 0.8 ppm
ozone, no enhanced tumor formation, but rather a slight decrease
in the incidence of peripheral lung tumors was observed (Witschi
et al., 1993a,b). A study with rats also came to the conclusion that
ozone would not act as an enhancer of carcinogen-induced lung
tumor development in rat lung (Boorman et al., 1994). The overall
balance of available evidence thus strongly suggests that ozone
has no tumor-promoting potential.
The most surprising aspect of the present study was the lack
of significant histopathologic effects. In the NTP carcinogenesis assay, dose-related non-neoplastic lesions in the airways
and lungs of the exposed animals were quite a prominent
feature (Herbert et al., 1996, Zhang et al. 1995). The complete
absence of inflammatory changes and, more strikingly, the
paucity of anatomical changes within the centriacinar region
(i.e., the bronchiolar alveolar duct junction) seen in some but
not all of the examined lungs, was quite surprising. This region
typically is considered a very important and critical site of
oxidant-induced tissue injury in the lungs of a wide variety of
species including rats and monkeys. The lack of more uniform
and more widely distributed tissue changes at a high concentration of ozone (i.e., 1 ppm) was unexpected.. It is possible
that in strain A/J mice the epithelial and interstitial cells of the
alveolar duct junction are unusually resistant to the effects of
oxidant gases, although our observation does not seem to fit the
recent finding that strain A/J mice are most sensitive to the
acute effects of ozone with a mean survival time of only 6 h in
10 ppm (Prows et al., 1997). These discrepancies need further
exploration.
In conclusion, there are several compelling mechanistic reasons, such as its radiomimetic and clastogenic effects or the
ability to produce increased cell proliferation in the respiratory
tract that could conceivably make ozone a pulmonary carcinogen. However, the preponderance of evidence from several
animal studies does not support or warrant such a conclusion.
ACKNOWLEDGMENTS
We wish to thank Brian Tarkington and Tim Duval for their excellent help
in performing the ozone exposures. This research was made possible by grants
ES00628 and ES05707 from the National Institute of Environmental Health
Sciences (NIEHS). The findings presented are solely the responsibility of the
authors and do not necessarily represent the official views of the NIEHS, NIH.
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