Ozone

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Acceptable, well-documented publication which meets basic scientific principles

Data source

Reference

Reference Type:

publication

Title:

Inhalation of ozone induces DNA strand breaks and inflammation in mice

Author:

Bornholdt J. et al.

Year:

2002

Bibliographic source:

Mutation Research 520, 63-72

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

Principles of method if other than guideline:

The genotoxic and inflammatory effects of ozone were investigated in female mice exposed to 1 and 2 ppm ozone once for 90 min. The tail moment in bronchoalveolar lavage (BAL) cells from mice was determined by the comet assay as a measure of DNA strand breaks at different intervals after termination of the exposure. To determine whether the exposures were mutagenic, MutaTMMice were exposed to 2 ppm ozone, 90 min per day for 5 days and examined for induction of mutations.

GLP compliance:

not specified

Type of assay:

mammalian comet assay

Test material

Test material information

Constituent 1

Reference substance name:

Ozone

EC Number:

233-069-2

EC Name:

Ozone

Cas Number:

10028-15-6

Molecular formula:

O3

IUPAC Name:

trioxygen

Test material form:

gas

Specific details on test material used for the study:

Ozone was generated photochemically by a thermostated mercury lamp in a flow of 0.15 L 99.999% oxygen.

Test animals

Species:

mouse

Strain:

other: female BALB/c mice and Muta TM mice

Sex:

female

Details on test animals or test system and environmental conditions:

The mice were allowed to acclimatise for minimally 7 days, were housed in polypropylene cages with sawdust bedding, were given a standard diet and water ad libitum. They were kept at controlled temperature (20 ± 2 °C), humidity (50 ± 10%) and a 12 h light/dark cycle. The mean weight at the time of exposure for the BALB/c mice was 20.6 g with S.D. of 1.6 g. The mean weight for the Muta TM mice was 26.0 g with S.D. of 3.4 g.

Administration / exposure

Route of administration:

inhalation: gas

Vehicle:

air

Details on exposure:

Both the BALB/c mice and the Muta TM mice were exposed to ozone for 90 min in an 18 L glass/stainless steel chamber. Ozone was generated photochemically by a thermostated mercury lamp in a flow of 0.15 L 99.999% oxygen. The ozone was led through 2 m of Teflon tube and mixed into the flow of 24.5 L/min ambient air just before the chamber. The ozone concentration in the breathing zone was measured throughout the entire exposure by an API photometric O3 analyser model 400 (API, San Diego, CA). Control animals (both BALB/c and Muta TM Mice) were exposed to ambient air or ambient air plus 0.15 L/min 99.999% oxygen.

Duration of treatment / exposure:

90 min

Frequency of treatment:

BALB/c mice received a single exposure. Muta TM Mice were exposed on five consecutive days.

Post exposure period:

After exposure the BALB/c mice were allowed to recover between 20 and 1400 min before they were sacrificed to study the time course of the effects after the exposure. Muta Tm mice were allowed to recover for 14 days before sacrifice.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

1 ppm (nominal)

Remarks:

BALB C mice

Doses / concentrations 2

Dose / conc.:

2 ppm (nominal)

Remarks:

BALB/c mice and Muta M Mice

No. of animals per sex per dose:

single exposure: 3 to 8 females/ exposure group
repeated exposure: 5 females / exposure group

Control animals:

yes, concurrent vehicle

other: air + 0.6% oxygen

Positive control(s):

not applicable

Examinations

Tissues and cell types examined:

Bronchoalveolar lavage (BAL) and lung tissue cells, blood

Details of tissue and slide preparation:

- BALB/C mice (single exposure): The animals were anaesthetised and a 0.8 mL blood sample was withdrawn from the heart, for analysis of oxidised protein in the serum. The blood was stabilised in 72 microliter 0.17 M K2EDTA and kept on ice until plasma was isolated by centrifugation at 4 °C and 12000 × g for 10 min. Immediately after withdrawing the heart blood, a bronchoalveolar lavage (BAL) was performed three times with 1 mL of 0.9% sterile saline through the trachea. The BAL was immediately put on ice until recovering the cells at 1250 rpm at 4 °C for 10 min after maximum 90 min. The cells were re-suspended in Merchant’s medium (4 °C) and used directly in the comet assay and for cell identification. The lungs were frozen immediately in cryotubes (NUNC) in liquid nitrogen and stored at −80 °C.

- Muta Mice (repeated exposure): Mice were allowed to recover for 14 days before sacrifice. The mice were anaesthetised and 0.8 mL blood was withdrawn from the heart. The blood was stabilised in 72 microliter 0.17 M K2EDTA and stored at −80 °C. The lungs were isolated, quickly frozen in liquid nitrogen and stored at −80 °C. DNA was isolated from the right lung of the mice using. The vectors were packaged into phages. Phages with mutations in the cII gene were screened by use of the positive selection model Select-cII mutation detection system for Big Blue rodents.

- Cell viability: The total number and viability of the cells were determined by staining with 0.25% trypan blue exclusion.

- Cell identification: Cells were collected on microscope slides by centrifugation at 1000 rpm for 4 min. The slides were fixed with 96% ethanol and stained with May–Grünwald–Giemsa stain. Two hundred cells were evaluated for each preparation.

- Cytokine (interleukin-6, TNF alpha, interleukin-1) and ERCC1 mRNAs: Lung tissue was homogenised with an ultrathorax and RNA and subsequently cDNA was prepared and stored at -80 °C. (RT-)PCR was used to evaluated the levels on cytokines and ERCC1.

- 8-oxo-deoxyguanosine (8-oxo-dG) content in lung tissue from BALB/c mice: 8-oxo-dG relative to dG was measured in lung tissue by HPLC with electrochemical detection.

- Comet assay: To investigate the extent of DNA strand breaks caused by ozone, the tail moment in the comet assay was determined in the lung tissue and in the freshly prepared BAL cells. A frozen lung was immediately placed in a stainless steel cylindrical sieve in 2mL Merchant’s medium. After disruption of the tissue the extract was filtered through a 53 µm nylon mesh. The cell concentration was adjusted to 1 × 10^6 cells/mL with Merchant’s medium. The BAL cells were used without further preparation. One percent (w/v) normal melting point and 1% (w/v) low melting point agarose solutions were freshly prepared with PBS (0.14M NaCl, 1.8mM KH2PO4, 8.1mM Na2HPO4, 2.7mM KCl, pH 7.4). First a layer of agarose was prepared by transfer of 95 µl normal melting point agarose at 65 °C onto a fully frosted slide. A second layer of agarose was prepared by mixing 95 µl low melting point agarose with 10 µl cell suspension (1×10^6 cells/mL) at 37 °C. The mixture was applied onto the first layer of agarose and covered with a cover slip. The embedded cells were lysed in ice-cold lysis buffer (2.5M NaCl, 0.1M Na2EDTA, 10mM Tris, 1% Triton X-100, pH 10) overnight. After lysis the slides were washed briefly in freshly made ice-cold alkaline electrophoresis solution (0.3M NaOH, 1mM Na2EDTA, pH >13). The slides were then placed in a Maxicell EC360M horizontal electrophoresis tank containing ice-cooled alkaline electrophoresis solution for 40 min. Electrophoresis was carried out at 25V and 292–296mA for 20 min. The slides were washed two times for 5 min with cold (4 °C) neutralising buffer (0.4M Tris, pH 7.5). The DNA was stained by applying 25 µl of 0.6 µM TOTO TM-1 iodide to each gel. The samples were analysed on a Leica DM BL fluorescence microscope with 400× magnification, a 450–490 nm excitation filter and a LP520 suppression filter. Using a Kinetics® image analysing system (version 3.0) the DNA damage was measured as tail moment. For each sample, three gels were analysed and 25 cells on each gel were measured

Evaluation criteria:

DNA strand breaks in BAL cells and lung tissue were evaluated by a comet assay. The tail was defined as the area where the intensity was lower than 10% of intensity in the head. Due to day-to-day variation in the comet assay, each tail moment was normalised by dividing the tail moment by the mean of tail moments of the untreated control mice for that day.

Statistics:

To define the time period when an effect on DNA strand breaks and IL-6 mRNA expression could be detected a 95% reference interval was determined for the control group. The interval was given by x¯ ± (sk), where s is the standard derivation for (n − 1) and k is defined as, k = (√(1/n) + 1)t (f )1−(α/2). The effect time window border lines were defined by the first two ubsequent data that were outside the reference interval. To test for dose effect in the time interval defined where the effect occurred, we used a Student’s t-test and ANOVA. The mutation frequencies, 8-oxo-dG/dG results, protein oxidation and cell identification data were all compared by the Student’s t-test or the modified Student’s t-test. Homogeneity was tested with a variance ratio test.

Results and discussion

Test results

Test results 1

Sex:

female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

not examined

Remarks on result:

other: lung tissue were used for the comet assay

Test results 2

Sex:

female

Genotoxicity:

negative

Remarks:

after 200 min recovery

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

not examined

Remarks on result:

other: BAL cells used for the comet assay

Test results 3

Sex:

female

Genotoxicity:

positive

Remarks:

within 0-200 min recovery

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

not examined

Remarks on result:

other: BAL cells used for the comet assay

Test results 4

Sex:

female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

not examined

Remarks on result:

other:

Remarks:

No mutations observed in the cII gene in lung homogenates from Muta TM mice

Additional information on results:

BALB/c mice (single exposure): see Table1 and Figure 1.
The recovery period when the tail moments were increased above the reference interval was in the time interval 0–200 min after the exposure. During this time, the tail moments were dose-dependently increased (P< 0.001, ANOVA) with a 1.6- and 2.6-fold increase over the untreated controls at 1 and 2 ppm, respectively. No changes were observed after 200 min. Cells prepared from the lung tissue of mice exposed to 2 ppm ozone had no increase in tail moment compared to the air-exposed mice. Staining and microscope analysis of the cell composition in the BAL fluid revealed that most cells were macrophages, and a small but significant infiltration of polymorphic neutrophils or lymphocytes occurred at 2 ppm ozone. The viability of BAL cells was 90–97% and showed no difference between cells from exposed and unexposed mice. No difference in the 8-oxo-dG/dG ratio between mice exposed to 1 or 2 ppm ozone and the unexposed mice could be detected at any time after exposure. mRNA level of ERCC1 was not changed due. IL-6 was induced up to 150-fold in the ozone-exposed animals (2 ppm) sacrificed after a recovery period of 150–200 min but had returned to control values at 16 hours. Other cytokines, IL-1 and TNF alpha were not affected.

Muta TM mice (multiple treatment):
The Muta TM mice did not show a difference in mutation frequency between exposed and control animals. In contrast, the ozone-treated mice tended to have fewer mutations that controls.

Any other information on results incl. tables

Table 1: DNA single strand breaks in BALB/c mice after 90 min exposure to air, 1 or 2 ppm ozone measured in a comet assay

	BAL cells	Lung cells
Air Ozone(1ppm) Ozone(2ppm)	1.01 ± 0.35   (n= 21) 1.56 ± 0.68∗   (n= 11) 2.57 ± 0.66∗∗ (n= 12)	1.0 ± 0.6 (n= 6) n.d. 1.1 ± 0.9 (n= 7)

The values are the mean ± S.D. of the relative tail moments for all mice in the recovery period between 0 and 200 min.

∗ P <0.05 for difference to air (Student’s t-test).

∗∗ P <0.01 for difference to air (Student’s t-test).

Applicant's summary and conclusion

Conclusions:

The data show supportive information that an acute 90 min exposure to 1-2 ppm ozone concentrations caused DNA strand-breaks in BAL cells in the lungs of mice shortly after exposure and reversal there-off. No mutagenic effect were detected at the transgenic cII gene in the lungs of MutaTM mouse model.

Executive summary:

The genotoxic and inflammatory effects of ozone were investigated in female mice exposed to ozone for 90 min. The tail moment in bronchoalveolar lavage (BAL) cells from BALB/c mice was determined by the comet assay as a measure of DNA strand breaks. Lung tissue and BAL was isolated from animals with a different recovery period after exposure. Within the first 200 min after exposure, the BAL cells from the mice exposed to 1 or 2 ppm ozone had 1.6- and 2.6-fold greater tail moments than unexposed mice. 200 min after exposure there was no effect observed anymore. It could be ruled out that the effect during the first 200 min was due to major infiltration of lymphocytes or neutrophils. No increase of tail moments in comparison to the negative control was observed in lung tissue cells from BALB/c mice. Unexpectedly, ozone had no effect on the content of 8-oxo-deoxyguanosine (8-oxo-dG) in nuclear DNA or on oxidized amino acids in the lung tissue. The mRNA level of the repair enzyme ERCC1 was not increased in the lung tissue. Inflammation was measured by the cytokine mRNA level in lung homogenates. An up to 150-fold induction of interleukin-6 (IL-6) mRNA was detected in the animals exposed to 2 ppm ozone compared to the air-exposed control mice. Also at 1 ppm ozone, the IL-6 mRNA was induced. The large induction of IL-6 mRNA in the lung took place after DNA strand breaks were induced in BAL. This does not support the notion that inflammatory reactions are the cause of DNA damage. To determine whether these exposures were mutagenic, Muta Mice were exposed to 2 ppm ozone, 90 min per day for 5 days. No treatment-related mutations could be detected in the cII transgene. These results indicate that a short episode of ozone exposure at five times the threshold limit value (TLV) in US induces lung inflammatory mediators and DNA damage in the cells in the lumen of the lung. This was not reflected by an induction of mutations in the lung of Muta TM Mice. This data support the idea that acute exposure to ozone and related damage can be handled by the cell through activation of repair mechanisms or increase of antioxidant levels and subsequently to avoid the introduction of cellular DNA mutations.