Inhalation of ozone induces DNA strand breaks and inflammation in mice

Abstract

Ozone (O₃) is a well-known oxidant pollutant present in photochemical smog. Although ozone is suspected to be a respiratory carcinogen it is not regulated as a carcinogen in most countries.

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. 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. After 200 min there was no effect. It could be ruled out that the effect during the first 200 min was due to major infiltration of lymphocytes or neutrophils. Unexpectedly, ozone had no effect on the content of 8-oxo-deoxyguanosine (8-oxo-dG) in nuclear DNA or on oxidised 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™Mice.

Introduction

Ozone is the major reactive oxygen species present in outdoor air and in the photochemical smog. Ozone in outdoor air is mainly due to photochemical production and transport from the stratosphere. In some cities the outdoor concentration locally peaks at 0.3 ppm [1], but it is usually about an order of magnitude lower. Epidemiological evidence suggests that episodes of ozone air pollution correlate with increased mortality rates [2] and respiratory illnesses [3]. There are geographic associations of ozone air pollution with lung cancer incidence [4]. There are also sources of ozone exposure in the occupational environment, for example, during welding. Recently developed welding techniques, which produce less particulate welding fumes, paradoxically produce more ozone [5].

In Denmark, there is a substantial variation in the lung cancer incidence between different occupations [6]. Smoking habits and lifestyle explain most of this variation, but not all. There are also strong indications of important exposures in the working environment. Standardised incidence rates in Denmark for lung cancer in trades that may be exposed to elevated concentrations of ozone are: drivers (135% for men), electrical workers (128% for men and 168% for women) and welders (122% for men) [6]. For ozone, the exposure limit in the occupational environment is 0.1 ppm during an 8 h averaging period in Denmark [7]. In US, the threshold limit value (TLV) is ranging from 0.05 ppm during heavy work, up to 0.1 ppm during light work. Up to 0.2 ppm is allowed in a period of <2 h, as long as the time-weighted average is not exceeded [8]. IARC has not evaluated whether ozone is carcinogenic to man. The American Conference of Governmental Industrial Hygienists considers ozone as “not classifiable as a human carcinogen” but as a “cause concern that (it) could be carcinogenic for humans” (Class A4) [8]. The German MAK commission also considers that there is “cause of concern that (it) could be carcinogenic to man” (Category 3) [9].

Because of its structure, ozone is so highly reactive that it is consumed in the lung lining fluid. Therefore, the effects on the lung cells may be due to the reaction products formed [10], [11], such as hydroxyl radicals, hydrogen peroxide, Crigee ozonides and aldehyde derivatives [11], [12], [13]. One or more of these products are presumed to interact with DNA and generate strand breaks or mutations. Ozone is also known as a potent inflammatory agent in the lung. It is, therefore, possible that the DNA damage is secondary to inflammation by the release of reactive oxygen species from inflammatory cells.

In this paper, we have investigated the induction of DNA damage and mutations in mice after acute exposures to ozone. We have also investigated the time course of DNA strand break formation after ozone exposure and the sequence of DNA damage and inflammation. We chose acute exposures because we wanted to investigate the direct effects of ozone. In contrast, long-term exposure is associated with severe chronic inflammatory conditions and histological changes in the lung. In an earlier study, we have shown that a single painting of coal tar on the skin of Muta™Mice is highly mutagenic to the epidermal cells [14].