Mutation Research/Genetic Toxicology and Environmental Mutagenesis
Inhalation of ozone induces DNA strand breaks and inflammation in 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].
Section snippets
Animals
Female BALB/c mice were purchased from M & B A/S, Ry, Denmark and female Muta™Mice (CD2-lacZ/HazfBR) were purchased from Covance Research Products, Denver, US. The animals were allowed to acclimatise for minimally 7 days, were housed in polypropylene cages with sawdust bedding (Lignolcel S8, Brogaarden, Denmark), were given a standard diet (Altromin Standard Diet no. 1324, Brogaarden, Denmark) and water ad libitum. They were kept at controlled temperature (20±2 °C), humidity (50±10%) and a 12 h
Results
The aim of this study was to examine the acute genotoxic and inflammatory responses after a short-term exposure to ozone. We used the tissue from the same mouse in as many assays as possible. However, for some assays there was not enough tissue. Therefore, the number of data varies for different end points. In order to generate the ozone a flow of 0.150 l/min O2 was used. This slightly elevated the oxygen concentration in the air by 0.6%. To exclude that the added oxygen itself had a DNA
Discussion
In Nordic surveys, the lung cancer incidence is consistently higher in welders and electrical workers: two occupations that also experience a higher risk of ozone exposures [6]. Ozone has been shown to induce cancer in mice in a long-term study [23]. Despite this fact, the knowledge of the mechanisms behind the action of ozone is limited. Ozone is also known as an airway irritant that causes inflammation [24]. The intention of this study was to investigate a potential genotoxic and inflammatory
Acknowledgements
This work was supported by the Danish Research Council (Grant no. 9801314) and the Danish Environmental Research Program.
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