**6. In vitro studies**

A wide variety of in vitro systems was developed in order to study the genotoxicity of chemicals and their mixtures, including complex mixtures of environmental pollutants adsorbed onto respirable air particles (PM2.5). Complex mixtures of organic compounds to which humans are exposed through air pollution are only partially characterized with respect to their chemical composition due to difficulties with chemical analysis of the individual components. Therefore, assays based on biological effects of complex mixture components may be a suitable alternative to a circumstantial chemical analysis. Using rat liver microsomal fraction (S9), it has been repeatedly shown that PAHs formed DNA adducts after metabolic activation by P450 enzymes to diol epoxides. This activation system may be used in acellular assay coupled with 32P-postlabeling to assess genotoxic potential of complex environmental mixtures via the analysis of DNA forming activity of the mixtures in native DNA [106-109].

The first study comparing the biological activities of complex mixtures from urban air particles PM10 was published by Binkova et al. [110]. HiVol samples were collected during the winter (October-March) and summer (April-September) seasons in the years 1993-1994, extracts (EOM-extractable organic matter) were analyzed in several fractions using in vitro acellular assay (calf thymus DNA with/without rat liver microsomal S9 fraction) with DNA adduct analysis by 32P-postlabeling (Teplice district: winter: PM10 69.3 µg/m3 , B[a]P 7.4 ng/m3 , summer: PM10 36.5 µg/m3 , B[a]P 0.8 ng/m3 ; Prachatice district: winter: PM10 29.6 µg/m3 , B[a]P 5.4 ng/m3 ; summer: PM10 23.6 µg/m3 , B[a]P 0.7 ng/m3 ). The highest total DNA adduct levels were observed in the neutral fraction, especially in the aromatic subfraction with metabolic activation, which contained mainly PAHs and their methylderivatives. The major PAH-DNA adducts contributed about 50% of the total DNA adducts resulting from all of the crude extracts using S9-metabolic activation. These results indicate that PAHs are a major source of genotoxic activities of organic mixtures associated with urban air particles.

This approach was later repeatedly used in different localities with different level of PM10 and PM2.5 pollution. When EOM extracted from these particles was analyzed, total PAH-DNA adducts highly correlated with concentrations of B[a]P and c-PAHs [111-113]. All studies showed that a cell-free system in conjunction with the sensitive 32P-postlabeling is a suitable model to detect genotoxic potential of EOMs, particularly those containing c-PAHs, as well as to distinguish between direct and indirect genotoxicants in the complex environmental pollutants. Those results indicate that c-PAHs contribute predominantly to the total genotox‐ icity of various EOMs. The strong correlation between B[a]P and other c-PAH content in all EOMs tested in these studies (r = 0.98; p<0.001) suggests that B[a]P may be used as an indicator of other c-PAHs in mixtures.

PCR) analysis showed a significant decrease in expression of *APEX*, *ATM*, *FAS*, *GSTM1*, *IL1B* and *RAD21* in subjects from Ostrava, in a comparison of winter and summer seasons. In the control subjects, an increase in gene expression was observed for *GADD45A* and *PTGS2*. The Rossner et al. [105] concluded that high concentrations of pollutants in Ostrava do not increase the number of deregulated genes. This may be explained by adaptation of humans to chronic exposure to air pollution. To further explain this phenomenon analyses focused on regulation

For the first time, this study measures the levels of biomarkers in subjects exposed to air pollutants in this region. Simultaneous assessment of oxidative stress markers, DNA adducts, chromosomal aberrations and transcriptomics is a new approach that can bring more clarity

A wide variety of in vitro systems was developed in order to study the genotoxicity of chemicals and their mixtures, including complex mixtures of environmental pollutants adsorbed onto respirable air particles (PM2.5). Complex mixtures of organic compounds to which humans are exposed through air pollution are only partially characterized with respect to their chemical composition due to difficulties with chemical analysis of the individual components. Therefore, assays based on biological effects of complex mixture components may be a suitable alternative to a circumstantial chemical analysis. Using rat liver microsomal fraction (S9), it has been repeatedly shown that PAHs formed DNA adducts after metabolic activation by P450 enzymes to diol epoxides. This activation system may be used in acellular assay coupled with 32P-postlabeling to assess genotoxic potential of complex environmental mixtures via the analysis of DNA forming activity of the mixtures in native DNA [106-109]. The first study comparing the biological activities of complex mixtures from urban air particles PM10 was published by Binkova et al. [110]. HiVol samples were collected during the winter (October-March) and summer (April-September) seasons in the years 1993-1994, extracts (EOM-extractable organic matter) were analyzed in several fractions using in vitro acellular assay (calf thymus DNA with/without rat liver microsomal S9 fraction) with DNA adduct

analysis by 32P-postlabeling (Teplice district: winter: PM10 69.3 µg/m3

, B[a]P 0.7 ng/m3

were observed in the neutral fraction, especially in the aromatic subfraction with metabolic activation, which contained mainly PAHs and their methylderivatives. The major PAH-DNA adducts contributed about 50% of the total DNA adducts resulting from all of the crude extracts using S9-metabolic activation. These results indicate that PAHs are a major source of genotoxic

This approach was later repeatedly used in different localities with different level of PM10 and PM2.5 pollution. When EOM extracted from these particles was analyzed, total PAH-DNA adducts highly correlated with concentrations of B[a]P and c-PAHs [111-113]. All studies

, B[a]P 0.8 ng/m3

activities of organic mixtures associated with urban air particles.

, B[a]P 7.4 ng/m3

; Prachatice district: winter: PM10 29.6 µg/m3

). The highest total DNA adduct levels

,

, B[a]P

of mRNA expression are necessary.

to the mechanisms of pollution effects.

**6. In vitro studies**

626 Current Air Quality Issues

summer: PM10 36.5 µg/m3

; summer: PM10 23.6 µg/m3

5.4 ng/m3

Topinka et al. [114] used acellular assay for the DNA adduct analysis of EOM according to the size fraction of particulate matter: 1-10 µm, 0.5-1 µm, 0.17-0.5 µm and <0.17 µm and the concentration of c-PAHs. The fraction of 0.5-1µm, that formed 37-46% of total PM mass, was the major carrier of c-PAHs, and induce highest genotoxicity detected as DNA adducts by 32Ppostlabeling.

Numerous studies analyzing the effect of c-PAHs, particularly B[*a*]P, on oxidative damage in cell lines *in vitro* or in animals *in vivo* have been published [115-121], but the results are conflicting, probably because of differences in the experimental protocols. Gabelova et al. [115] did not find any significant increase of oxidative DNA damage measured by a single cell gel electrophoresis assay (the Comet assay) in HepG2 cells treated with 7.5 µM B[*a*]P for 2, 24 or 48 h. In the study by Park et al. [116], B[*a*]P at concentrations up to 10 µM induced nonspecific DNA damage measured by the Comet assay after 24 h treatment of HepG2 cells. Lipid peroxidation, analyzed as malondialdehyde (MDA) levels, was increased but showed no doseresponse. The authors concluded that oxidative DNA damage is probably related to B[*a*]P toxicity. In the A549 cell line, Garcon et al. [117] tested the effect of B[*a*]P treatment on lipid peroxidation measured as MDA levels. They treated the cells for 72 h with 0.05 µM B[*a*]P but found no increase. They hypothesized that the antioxidant defenses of the cells prevented the induction of lipid peroxidation by B[*a*]P [118, 119].

Rossner et al. [122] investigated the ability of organic extracts of size segregated aerosol particles (EOM; three fractions of PM, aerodynamic diameter 1–10 µm, 0.5–1µm and 0.17– 0.5µm) to induce oxidative damage to DNA in an in vitro acellular system of calf thymus (CT) DNA with and without S9 metabolic activation. PM was collected in the Czech Republic at four places with different levels of air pollution. Levels of 8-oxodG tended to increase with decreasing sizes of PM. S9 metabolic activation increased the oxidative capacity of PM.

These results indicate that smaller size fractions are more potent inducers of oxidative damage to DNA. This observation is in agreement with other studies [123-125]. In these studies, however, water-soluble PM extracts were used. Moreover, end-point parameters for measur‐ ing the potency of PM to induce oxidative damage differed from Rossner's approach. There are only two other reports that used organic PM extracts in the acellular CTDNA system [124, 126] and only one that tested S9 metabolic activation of extracts [126], but none of these analyzed the oxidative capacity of individual size fractions. Thus, Rossner's results [122]) are probably the first showing that the ability of organic PM extracts to induce oxidative damage to DNA also increases with decreasing sizes of particles. This trend seems to be less pro‐ nounced after S9 metabolic activation of EOM. Thus, the presence of PAHs in EOM is probably not the only factor responsible for oxidative damage induction by PM organic extracts.

On the other hand, results of Rossner et al. [122] showed that metabolic activation of PAHs plays at least a partial role in the induction of oxidative damage to DNA because 8-oxodG levels in CT-DNA incubated with S9 fraction were significantly higher than in samples without S9 metabolic activation. Also, they observed a positive correlation between c-PAHs concen‐ trations and 8-oxodG levels induced by PM. This correlation was stronger and statistically significant when PM extracts were incubated with S9 metabolic fraction.

The oxidative capacity of PM extracts increases with increasing levels of air pollution. Smaller size fractions of PM induce higher oxidative damage, which is caused partly by higher content of c-PAHs and partly by other unidentified factors.
