In vivo ozone exposure does not increase DNA single-strand breaks in human peripheral lymphocytes

Abstract

In this randomized parallel study, we examined whether an acute ozone (O₃) exposure leads to increased DNA strand breaks in human lymphocytes. The groups were exposed to 0.21 ppm O₃ or filtered air for two hours. 30min and 4.5 h after exposure, DNA damage was determined in isolated lymphocytes using the Fast Micromethod. There was no detectable effect after O₃ exposure. We conclude that an acute O₃ exposure at the tested concentration does not lead to persistent DNA damage.

Introduction

Ozone (O₃) is one of the major urban air pollutants known for its negative effects on different health issues. ¹ Atmospheric O₃ concentration is subjected to substantial temporal and spatial variation. Since major influencing factors are solar radiation and air pollution (e.g., traffic exhausts) with nitric oxides (NOx) and volatile organic compounds (VOCs), higher O₃ concentrations are usually found in urban areas during the summer months. ² In addition to this, seasonal variation is also considered, which varies over the day at sunny days in urban areas. NOx are involved not only in the formation of O₃ in the presence of sunlight but also in its breakdown during the night. This daily variation is less distinct in rural areas due to lower NOx concentrations. Average O₃ concentrations have declined over the past years, ^3,4 but still peak values can reach concentrations up to 0.2 ppm in urban areas. ⁵ Inhalative exposure to O₃ may increase with respiratory minute volume as a result of physical workload due to outdoor work or sports. O₃ is also produced during welding and plays a role as an indoor workplace contaminant. ⁶ Various epidemiological studies have shown an increase in mortality, morbidity, and bronchial carcinoma associated with O₃ exposure. ^{7 –9}

O₃ is a reactive oxygen species, which is capable of reacting with a variety of biological target structures such as olefin structures or electron donors. The chemical reaction between O₃ and the olefin structures follows the mechanism of ozonolysis and leads to the production of hydrogen peroxide (H₂O₂) and aldehydes. ^10,11 Similar reactions can occur with proteins and amino acids. ¹² The products formed with the latter class of compounds (electron donors) are mainly radicals such as hydroxy radicals that can trigger chain reactions with other structures leading to substantial cellular damage. ¹¹

According to Pryor, ¹¹ 90% of the reactions take place in the epithelial lining fluid (ELF) and the remaining 10% at cell membranes consisting of polyunsaturated fatty acids, proteins, amino acids or electron donors such as vitamins or glutathione. Due to the lipophilic nature of the cell membrane, mostly Criegee ozonides are produced. In total, 10% of the products are radicals starting a chain reaction accountable for 50% of the damage. When O₃ reacts with the constituents of ELF aldehydes, H₂O₂ and Criegee ozonides are produced. Diaz-Llera et al., ¹³ in their study, supposed that O₃-induced DNA damage is mediated by H₂O₂, which is considered as a reaction product of O₃ in the organism. They were able to prevent O₃-induced DNA damage in human peripheral blood leukocytes in vitro by the addition of H₂O₂-degrading enzyme catalase. In contrast, Gabrielson et al. ¹⁴ postulated that different reactive species such as aldehydes play a more important role in cytotoxicity in epithelial cells of the bronchial system.

Because of its quick absorption and the high reactivity of O₃, products of secondary and tertiary reaction may be accountable for the damage in peripheral blood cells. To investigate O₃-related DNA damage in respiratory tissue many in vivo studies have been conducted, which are presented in the following: The ability of O₃ to induce DNA damage in lymphocytes has been demonstrated in animal experiments. ¹⁵ Haney et al. ¹⁶ also found a significant increase in DNA single-strand breaks in bronchoalveolar lavage cells of mice after exposure to 0.25 or 0.5 ppm O₃ for 3 h.

This study simulates the impact of O₃ concentrations that are found in the atmosphere. Our question was whether an experimental acute exposure of human subjects to 0.21 ppm O₃ causes DNA damage in the peripheral lymphocytes.

Methods

Study design and outside O₃ concentration

We designed a randomized parallel study with 40 healthy, nonsmoking male subjects exposed to 0.21 ppm O₃ or filtered ambient air for 2 h in an exposure chamber at the University Medical Center, Johannes Gutenberg University, Mainz, Germany. O₃ concentrations are the highest during summer time. To minimize previous O₃ contact before the experiment and to reduce O₃ exposure for the control group, the study was conducted during the winter season. To reduce the possible influence of other irritants, the measurements were performed on days without smog, fog, or atmospheric inversion. Subjects were advised to avoid excessive outdoor activities prior to their exposure. They did not take any medication. We measured the atmospheric O₃ concentration on the exposure days to exclude relevant environmental exposure using an ultraviolet photometric ambient O₃ analyzer/calibrator (Thermo Environmental Instruments, Franklin, Massachusetts, USA). Three subjects were subsequently excluded because urine samples indicated inhalative smoking. ¹⁷ Therefore, 19 subjects (median age: 25 years) were included in the placebo and 18 in the O₃ group (median age: 23.5 years). During exposure, subjects had to cycle on an ergometer with a power of 100 W two times for 15 min to provide sufficient inhalation. At three different time points (directly before exposure as well as 30 min and 4.5 h afterward) 18 ml of ethylenediaminetetraacetic acid (EDTA)–blood were taken to investigate DNA single-strand breaks in the lymphocytes. The time span up to 30 min after exposure was used to collect exhaled breath condensate for other tests, which was the primary focus of this study and made an earlier blood sampling impossible. The collected condensate was used in a pilot study to evaluate an experimental method. It was not used to investigate DNA damage and no concern of this investigation.

The study has been approved by the local ethics committee.

Lymphocyte isolation

Lymphocytes were separated from other blood fractions by Ficoll gradient centrifugation as described by Schröder et al. ¹⁸ The cells were cryopreserved for 24 h at −80°C and finally stored in liquid nitrogen. For the analysis of single-strand breaks, the cell concentration was adjusted to 1 × 10⁵ cells/ml.

DNA damage analysis using Fast Micromethod

In order to analyze possible DNA damage, the Fast Micromethod was used, which is a very sensitive method to detect DNA single-strand breaks. ¹⁹ This method uses a fluorescent dye, PicoGreen (Molecular Probes, Leiden, the Netherlands), which preferentially binds to double-stranded DNA but not to single-stranded DNA. After adding an alkaline solution the cellular double-stranded DNA starts to unwind and the fluorescence decreases. The rate of DNA unwinding and hence the decrease in fluorescence intensity correlates with the number of single-strand breaks. The determination of the DNA damage was performed as described by Schröder et al. ¹⁸ Aliquots of the cell suspensions were added into the wells of a 96-well microplate and lysed with PicoGreen-containing lysing solution with urea, sodium dodecyl sulfate, and EDTA. DNA unwinding was started after an incubation period of 40 min by adding the alkaline solution and fluorescence was measured with a Fluoroskan II spectrometer (Labsystems, Helsinki, Finland; excitation at 485 nm and emission at 538 nm). The strand scission factor (SSF) was calculated for the time points of 30 min and 4.5 h after exposure, using the preexposure samples as intraindividual baseline values.

Statistics

SSF was calculated for the samples collected 30 min and 4.5 h after exposure. Values of both the groups were compared using Mann–Whitney test for nonparametrical, independent samples. Significance level was set at p < 0.05.

Results

The mean outdoor atmospheric O₃ concentration during the experiments was 0.03 ppm (SD = 0.01). There were no statistical differences between the SSF values of the O₃ group and the control group, determined 30 min after exposure (median SSF values O₃ vs. ambient air (control): −0.00 vs. 0.01; p = 0.80; Figure 1). The SSF values calculated 4.5 h after exposure (−0.02 vs. −0.00) were also not statistically different (p = 0.82; Figure 2). These results show that there was no increase in DNA damage at both time points after exposure.

Figure 1.

Strand scission factors of the control group versus the ozone-exposed group 30 min after exposure.

Figure 2.

Strand scission factors of the control group versus the ozone-exposed group 4.5 h after exposure.

Discussion

Our study did not show any difference in the extent of DNA single-strand breaks in the human lymphocytes after an acute O₃ exposure as measured by Fast Micromethod. In comparison, the animal studies as mentioned in the introduction described O₃-induced effects. However, these effects were related to higher O₃ concentrations than 0.21 ppm. Exposure of humans to comparable concentrations was not possible due to ethical concerns. Therefore, the lack of any visible effect could be due to an exposure at a comparably low O₃ concentration.

Other in vivo studies investigating systemic DNA damage in peripheral blood cells of animals showed conflicting results. Chromosome aberrations have been reported in the peripheral lymphocytes of Chinese hamsters after an exposure of the animals to 0.24 or 0.33 ppm O₃ for 5 h; no decrease in DNA damage could be detected 2 weeks after exposure. ¹⁵ In contrast, Tice et al. ²⁰ who studied Chinese hamsters at different concentrations (up to 2.0 ppm for 6 h) did not find any increase in chromosome aberrations.

There are only a few studies on DNA damage in vivo in human subjects, also with inconsistent results. Merz et al. ²¹ exposed 6 subjects to 0.5 ppm for 6 and 10 h, leading to chromosomal aberrations in peripheral lymphocytes, which were interpreted as single-strand breaks. McKenzie et al. ²² investigated chromosomal damage in male subjects at concentrations of 0.35 and 0.52 ppm for 4 h. After exposure, no damage could be detected in the lymphocytes. In a further study, Guerrero et al. ²³ exposed volunteers to 0.5 ppm O₃ for 2 h and compared DNA damage in lymphocytes taken before and directly after exposure. No difference in sister chromatid exchange rates could be detected. Our study did not reveal an induction of DNA damage by exposure to O₃. The Fast Micromethod used in this study is an established procedure to assess DNA damage and repair in human peripheral blood cells. ^{24 –26} The Fast Micromethod and their modifications are among the most sensitive methods for the assessment of DNA strand breaks and have several advantages compared to other methods. ²⁷ A previous study showed that the sensitivity and specificity of our method is comparable to other established assays for the detection of DNA damage (e.g., comet assay). ²⁸

In this study, a 30-min delay between the end of exposure and blood sampling might be a limitation. Other studies showed that single-strand breaks can be repaired quickly. ²⁹ If in our study a small number of systemic DNA single-strand breaks in lymphocytes had occurred, this damage would have probably been repaired after 30 min. Therefore, it cannot be excluded that our experimental design was not capable to detect early low levels of induced DNA damage. However, it can be concluded that an acute exposure to 0.21 ppm O₃ does not lead to a critical damage in peripheral blood cells in human subjects.

Different studies showed an association between ambient O₃ concentrations and DNA damage in the peripheral blood cells after chronic exposure in residents of highly polluted cities. There were positive correlations between O₃ exposure and detectable lesions in leukocytes and lymphocytes. ^30,31 These long-term results cannot be directly compared with our findings that reflect an acute exposure, but they show that there might be a damaging potency of repetitive contact with high O₃ levels. Also, the influence of other pollutants cannot be ruled out in those environmental studies. It might be an interesting subject to investigate DNA damage in the peripheral blood cells after a sudden increase in environmental O₃ concentrations in terms of an acute exposure during one or more days.

Conclusions

In our study, an acute exposure of male subjects to 0.21 ppm O₃ did not lead to increased DNA damage in the peripheral blood lymphocytes when compared with a control group. Possible reasons for this observation might be that the applied concentration of 0.21 ppm is too low to induce relevant damage in peripheral blood cells or a fast repair after the exposure.