Mutagenicity monitoring of airborne particulate matter (PM10) in the Czech Republic.

The mutagenic activities associated with inhalable airborne particulate matter (PM10) collected over a year in four towns (Czech Republic) have been determined. The dichloromethane extracts were tested for mutagenicity using the Ames plate incorporation test and the Kado microsuspension test both with Salmonella typhimurium TA98 and its derivative YG1041 tester strains in the presence and absence of S9 mixture. The aim of this study was to assess the suitability of both bacterial mutagenicity tests and to choose the appropriate indicator strain for monitoring purposes. To elucidate the correlation between mutagenicity and polycyclic aromatic hydrocarbons (PAHs), the concentration of PAHs in the air samples were determined by GC/MS. In general, the significant mutagenicity was obtained in organic extracts of all samples, but differences according to the method and tester strain used were observed. In both mutagenicity tests, the extractable organic mass (EOM) exhibited higher mutagenicity in the YG1041 strain (up to 97 rev/microg in the plate incorporation and 568 rev/microg in the microsuspension tests) than those in TA98 (up to 2.2 rev/microg in the plate incorporation and 14.5 rev/microg in the microsuspension tests). In the plate incorporation test, the direct mutagenic activity in YG1041 was on average 60-fold higher and in microsuspension assay 45-fold higher with respect to strain TA98. In the presence of S9 mix, the mutagenic potency in YG1041 declined (P<0.001) in summer, but increased in TA98 (P<0.05) in samples collected during the winter season. The microsuspension assay provided higher mutagenic responses in both tester strains, but in both strains a significant decrease of mutagenic potency was observed in the presence of S9 mix (P<0.001 for YG1041, P<0.05 for TA98 in winter). The mutagenic potencies detected with both indicator strains correlated well (r=0.54 to 0.87) within each mutagenicity test used but not (for TA98) or moderately (r=0.44 to 0. 66 for YG1041) between both of the tests. The mutagenic activity (in rev/m(3)) likewise the concentration of benzo[a]pyrene and sum of carcinogenic PAHs showed seasonal variation with distinctly higher values during winter season. A correlation between the PAH concentrations and the mutagenicity results for the plate incorporation, but not for the microsuspension tests was found. In samples from higher industrial areas, the higher mutagenicity values were obtained in plate incorporation test with TA98 and in both tests with YG1041 in summer season (P<0.05). According to our results, plate incorporation test seems to be more informative than microsuspension assay. For routine ambient air mutagenicity monitoring, the use of YG1041 tester strain without metabolic activation and the plate incorporation test are to be recommended.     

Genotoxicity of environmental air pollution in three European cities: Prague, Kosice and Sofia

The genotoxic potential of extractable organic matter (EOM) associated with the respirable particulate matter (PM <10 microm) of atmospheric pollution has been determined in three European cities--Prague (Czech Republic, two monitoring sites, Libus and Smíchov), Kosice (Slovak Republic) and Sofia (Bulgaria) using the alkaline single-cell gel electrophoresis (the comet assay). The EOM samples were extracted by dichloromethane from ambient airborne particles collected daily (24 h intervals) during 3-month sampling periods in winter and summer seasons. The human metabolically competent cell line Hep G2 was used as a test system and benzo[a]pyrene (BaP), a known carcinogen, was applied as a positive control (internal standard) in each electrophoretic run. Two-hour exposure of Hep G2 cells to equivalent EOM concentrations ranging from 5 to 150 microg EOM/ml resulted in a linear dose-dependent increase of DNA migration (r > 0.9, P < 0.01). A less significant dose-response (r = 0.61) was only induced by the EOM sample from the locality Prague-Libus (PRG-LB) in the winter. Generally, a 1.5 to four-fold increase of DNA strand breaks over the background control level was determined in EOM-exposed cells. In order to compare the genotoxic potential of individual EOMs, a mathematical model was used to correct the 'real' data. No substantial location- or season-related differences were found in EOM genotoxicity (EOM microg/ml), except for the EOM sample from Sofia, collected in the summer. This EOM sample induced a nearly two-fold lower level of DNA damage in comparison with other EOMs. On the other hand, clear statistically significant location- and season-related differences (P < 0.001) in ambient air genotoxicity were determined when the EOM quantity per cubic meter of air (microg/m3) was taken into account. In that case, the genotoxicity of winter air pollution was six- to 10-fold higher in comparison with summer air. The air pollution genotoxicity in individual localities rose during the winter season in the order: PRG-LB < Kosice < Prague-Smíchov (PRG-SM) < Sofia, while during the summer season the highest ambient air genotoxicity was revealed in the locality Prague-Smíchov and approximately equal air pollution genotoxicity was determined among localities Prague-Libus, Kosice and Sofia (PRG-LB approximately Kosice approximately Sofia < PRG-SM). The greatest overall air pollution genotoxicity was determined in the locality Sofia during the winter season. In a time course study to evaluate the kinetics of DNA strand break rejoining it was shown that the level of DNA strand breaks in EOM-exposed cells has returned to near the background level within 24 h after the treatment.     

Genotoxicity of urban air pollutants in the Czech Republic. Part I. Bacterial mutagenic potencies of organic compounds adsorbed on PM10 particulates

As part of a long-term program to investigate the impact of air pollution on the health of a population in a polluted region in Northern Bohemia, mutagenicity of extractable organic matter (EOM) from air particles PM10 was investigated by the means of Salmonella typhimurium indicator strains TA98 and YG1041 using the Ames plate incorporation assay. The air samples were collected in both the polluted and the control districts during the summers and winters of 1993-1994. In the polluted district, the collection was repeated during the winter of 1996-1997. The crude extracts from filters pooled according to the locality and the season were fractionated by acid-base partitioning into acid, base, and neutral fractions. The neutral fractions were further fractionated by silica gel column chromatography into five subfractions. The induction of revertants with the crude extracts was higher in winter samples than in summer samples. Both indirect-acting and direct-acting mutagenicity were observed. The indirect mutagenic potency of aromatic subfractions containing polycyclic aromatic hydrocarbons (PAHs) was generally low. The mutagenic potency detected with TA98 was more distinct only in the winter sample 1993-1994 from the polluted area, where the aromatic subfraction accounted for 23% of total mutagenicity. In both strains, the highest direct-acting mutagenicity was found in slightly polar fractions containing nitro-PAHs. The mutagenic potency detected with YG1041 was about two orders of magnitude higher than that detected with TA98. No substantial locational- or time-related variances in the mutagenic potencies of EOM, or in the spectrum of chemical components identified in individual fractions were found. The polluted district, in comparison to the control district, was found to have higher amounts of EOM, carcinogenic PAHs and mutagenicity of air particles (rev/m(3)). The fractionating process, combined with the bacterial mutagenicity test, confirmed that nitro-derivatives are the most important contributors to the bacterial mutagenicity of air particles. However, this study did not fulfill the expectancy to bring substantially new, clear-cut information on the composition and the biological activity of air pollution in both districts.     

Sensitivity of different endpoints for in vitro measurement of genotoxicity of extractable organic matter associated with ambient airborne particles (PM10)

Sensitivity and correlations among three endpoints were evaluated to assess the genotoxic potential of organic complex mixtures in vitro. This study was focused on DNA adduct formation, DNA single strand break induction and tumour suppressor p53 protein up-regulation produced by extractable organic matter (EOM) absorbed on respirable particulate matter PM₁₀ (particulate matter < 10 μm) collected in three European cities (Prague, Sofia, Košice) during winter and summer period. To compare the sensitivity of particular endpoints for in vitro measurement of complex mixture genotoxicity, the metabolically competent human hepatoma cell line Hep G2 was treated with equivalent EOM concentration of 50 μg/ml. Cell exposure to EOMs resulted in significant DNA adduct formation and DNA strand break induction, however, a lack of protein p53 up-regulation over the steady-state level was found. While the maximum of DNA strand breaks was determined after 2 h cell exposure to EOMs, 24 h treatment interval was optimal for DNA adduct determination.

No substantial location- and season-related differences in EOM genotoxicity were detected using DNA strand break assessment. In agreement with these results no significant variation in DNA adduct levels were found in relation to the locality and season except for the monitoring site in Prague. The Prague EOM sample collected during summer period produced nearly three-fold lower DNA adduct level in comparison to the winter EOM sample.

Comparable results were obtained when the ambient air genotoxicity, based on the concentration of carcinogenic PAHs in cubic meter of air (ng c-PAHs/m³), was elicited using either DNA adduct or strand break determination. In general, at least six-fold higher genotoxicity of the winter air in comparison to the summer air was estimated by each particular endpoint. Moreover, the genotoxic potential of winter air revealed by DNA adduct assessment and DNA strand break measurement increased in the same order: Košice  Prague < Sofia.

Based on these data we suppose that two endpoints DNA breakage and DNA adduction are sensitive in vitro biomarkers for estimation of genotoxic activity of organic complex mixture associated with airborne particles. On the other hand, the measurement of protein p53 up-regulation manifested some limitations; therefore it cannot be used as a reliable endpoint for in vitro genotoxicity assessment.     

Temporal variation in the genotoxic potential of urban air particulate matter

The main aim of this study was to compare the genotoxic potential of organic extracts from urban air particles collected in three different sampling periods in the center of Prague (Czech Republic). For this purpose, we analyzed the DNA adduct forming activity of extractable organic matter (EOM) from urban air particles <10 microm (PM10) in the human hepatoma cell line HepG2. DNA adducts were analyzed by (32)P-postlabelling with nuclease P1 enrichment. PM10 concentrations were 36.9 microg/m(3), 62.6mug/m(3) and 39.0 microg/m(3), in summer 2000, winter 2001 and winter 2005, respectively. The corresponding EOM contents were 5.0 microg/m(3) (13.9% of PM10), 14.9 microg/m(3) (23.8%) and 6.7 microg/m(3) (17.2%). The total DNA adduct levels induced by 10 microg EOM/ml were 4.7, 19.5 and 37.2 adducts/10(8) nucleotides in summer 2000, winter 2001 and winter 2005, respectively. However, when the EOM quantities per cubic meter of air were taken into consideration, the summer sample exhibited a 10-fold lower genotoxicity than did those of winter, while the difference between the winter samples was not significant: 23.4 in summer 2000, 291 in winter 2001 and 249 in winter 2005 (in relative units). Although the PM10 concentration in air and the EOM content in particles in winter 2005 were significantly lower than in winter 2001, the genotoxic potential of the ambient air in these samples was almost equal. There were significant positive correlations between the B[a]P and c-PAH content in EOM from various sampling periods and the total DNA adduct levels detected in the EOM-treated samples. These findings support the hypothesis that the B[a]P and c-PAH content in EOM is the most important factor that determines its genotoxic potential. Thus, estimating the genotoxic potential of the ambient air and predicting health risk should be based mainly on the c-PAH concentration and the biological activity of the extracts, while the mass of particles and the EOM content do not seem to be crucial determinants of ambient air genotoxicity.     

Sensitivity of different endpoints for in vitro measurement of genotoxicity of extractable organic matter associated with ambient airborne particles (PM10)

Sensitivity and correlations among three endpoints were evaluated to assess the genotoxic potential of organic complex mixtures in vitro. This study was focused on DNA adduct formation, DNA single strand break induction and tumour suppressor p53 protein up-regulation produced by extractable organic matter (EOM) absorbed on respirable particulate matter PM₁₀ (particulate matter < 10 μm) collected in three European cities (Prague, Sofia, Košice) during winter and summer period. To compare the sensitivity of particular endpoints for in vitro measurement of complex mixture genotoxicity, the metabolically competent human hepatoma cell line Hep G2 was treated with equivalent EOM concentration of 50 μg/ml. Cell exposure to EOMs resulted in significant DNA adduct formation and DNA strand break induction, however, a lack of protein p53 up-regulation over the steady-state level was found. While the maximum of DNA strand breaks was determined after 2 h cell exposure to EOMs, 24 h treatment interval was optimal for DNA adduct determination.No substantial location- and season-related differences in EOM genotoxicity were detected using DNA strand break assessment. In agreement with these results no significant variation in DNA adduct levels were found in relation to the locality and season except for the monitoring site in Prague. The Prague EOM sample collected during summer period produced nearly three-fold lower DNA adduct level in comparison to the winter EOM sample.Comparable results were obtained when the ambient air genotoxicity, based on the concentration of carcinogenic PAHs in cubic meter of air (ng c-PAHs/m³), was elicited using either DNA adduct or strand break determination. In general, at least six-fold higher genotoxicity of the winter air in comparison to the summer air was estimated by each particular endpoint. Moreover, the genotoxic potential of winter air revealed by DNA adduct assessment and DNA strand break measurement increased in the same order: Košice  Prague < Sofia.Based on these data we suppose that two endpoints DNA breakage and DNA adduction are sensitive in vitro biomarkers for estimation of genotoxic activity of organic complex mixture associated with airborne particles. On the other hand, the measurement of protein p53 up-regulation manifested some limitations; therefore it cannot be used as a reliable endpoint for in vitro genotoxicity assessment.     

Genotoxicity of urban air pollutants in the Czech Republic. Part II. DNA adduct formation in mammalian cells by extractable organic matter

The study was aimed at determining the genotoxic potential of extractable organic matter (EOM) from ambient air particles PM10 (<10 micrometer) using mammalian cells in culture as test system. Air samples were collected in the course of summer and winter periods in two regions of the Czech Republic representing low and high levels of air pollution, the districts of industrial Teplice and rural Prachatice, respectively. EOM was fractionated by acid-base partitioning and silica gel column chromatography. Aliquots of fractions were incubated with cultured hepatocytes derived from male rats or Chinese hamster lung V79NH cells expressing nitroreductase activity but virtually no cytochrome P450 activity. DNA adduct levels were analyzed by 32P-postlabeling using butanol extraction for adduct enrichment. In hepatocytes, crude extracts caused the formation of substantial amounts of DNA reactive material being detectable in a broad diagonal radioactive zone (DRZ) in the chromatograms. Highest DNA adduct levels were found in the aromatic fractions and slightly polar fractions which contain most of the polycyclic aromatic hydrocarbons (PAH) and nitro-substituted PAH (nitro-PAH), respectively, comprising 75-90% of total adducts. This partitioning was independent of the sampling period and locality. In agreement with the higher average ambient air concentrations of PAH in the winter than the summer, 3-4-fold higher DNA adduct levels were detected in extracts sampled in the winter. Calculated on the basis of EOM/m(3), DNA adduct levels of samples collected in winter period were 10-fold higher than those collected in the summer period and 2-fold higher in Teplice than in Prachatice. Pretreatment of hepatocytes with 2,3,7,8-tetrachlorodibenzo-p-dioxin decreased DNA binding by 50-75%. In contrast to the findings in hepatocytes, in V79NH cells about 80% of the DNA adducts were caused by material in the slightly polar fractions appearing as distinct spots in the radiochromatograms. Seasonal variation of DNA adducts in V79NH cells was greater than variation between localities. Our results suggest that PAH as well as nitro-PAH are the main contributors to the genotoxicity of EOM derived from both industrial and rural areas. The results, furthermore, indicate that analysis of DNA adducts in mammalian cells in culture offers a suitable method for monitoring the genotoxicity of complex mixtures of environmental chemicals.     

Genotoxicity and embryotoxicity of urban air particulate matter collected during winter and summer period in two different districts of the Czech Republic

This study is the in vitro part of a long-term program to investigate the impact of air pollution on the health of a population in a polluted region of Northern Bohemia. In order to assess the possible health risks associated with a complex mixture of hundreds of organic compounds adsorbed to air particles, we used a biomarker-directed fractionation procedure to evaluate biological activities of different chemical compound classes. The extractable organic compounds from the air particles collected in both the polluted and the control districts during the summers and winters of 1993-1994 were investigated. The principal aim of this study was to compare the DNA binding activities of those compound classes using an in vitro acellular assay coupled with 32P-postlabeling and an embryotoxicity assay using Chick Embryotoxicity Screening Test (CHEST). In both assays, the highest activity was due to the neutral fractions from which the aromatic subfractions containing mainly polycyclic aromatic hydrocarbons (PAHs) and their methyl-derivates were the most active for both localities and seasons. A good correlation between the levels of DNA adduct formation using S9 metabolic activation and the ED50 for all different complex mixtures of organic compounds was observed (r=0.773, p<0.001). DNA adduct maps and high performance liquid chromatography (HPLC) profiles were similar for samples from both districts and seasons. The major DNA adducts resulting from the crude extracts were identical to those derived from aromatic fractions. The DNA adducts tentatively identified constituted about 50% of the total adducts formed by the crude extracts following S9-metabolic activation. Our results confirmed the similarities of the major ubiquitous emission sources of organic compounds in both districts. This is the first report in which the biological activities of complex mixtures in short-term assays with remarkably different endpoints such as DNA adduct formation and embryotoxicity have been compared.     

Assessment of oxidative DNA damage formation by organic complex mixtures from airborne particles PM(10)

The free radical generating activity of airborne particulate matter (PM₁₀) has been proposed as a primary mechanism in biological activity of ambient air pollution. In an effort to determine the impact of the complex mixtures of extractable organic matter (EOM) from airborne particles on oxidative damage to DNA, the level of 8-oxo-2′-deoxyguanosine (8-oxodG), the most prevalent and stable oxidative lesion, was measured in the human metabolically competent cell line Hep G2. Cultured cells were exposed to equivalent EOM concentrations (5–150 μg/ml) and oxidative DNA damage was analyzed using a modified single cell gel electrophoresis (SCGE), which involves the incubation of whole cell DNA with repair specific DNA endonuclease, which cleaves oxidized DNA at the sites of 8-oxodG. EOMs were extracted from PM₁₀ collected daily (24 h intervals) in three European cities: Prague (Czech Republic, two monitoring sites, Libuš and Smíchov), Košice (Slovak Republic) and Sofia (Bulgaria) during 3-month sampling periods in the winter and summer seasons. No substantial time- and dose-dependent increase of oxidative DNA lesions was detected in EOM-treated cells with the exception of the EOM collected at the monitoring site Košice, summer sampling. In this case, 2 h cell exposure to EOM resulted in a slight but significant increase of oxidative DNA damage at three from total of six concentrations. The mean 8-oxodG values at these concentrations ranged from 15.3 to 26.1 per 10⁶ nucleotides with a value 3.5 per 10⁶ nucleotides in untreated cells. B[a]P, the positive control, induced a variable but insignificant increase of oxidative DNA damage in Hep G2 cell (approximately 1.6-fold increase over control value).

Based on these data we believe that EOM samples extracted from airborne particle PM₁₀ play probably only a marginal role in oxidative stress generation and oxidative lesion formation to DNA. However, adsorbed organic compounds can undergo various interactions (additive or synergistic) with other PM components or physical factors (UV-A radiation) and in this way they might enhance/multiply the adverse health effects of air pollution.     

Evaluation of a battery of Salmonella typhimurium tester strains for biomonitoring of mutagenic polycyclic aromatic hydrocarbons, nitroarenes and aromatic amines

Various combinations of Salmonella typhimurium tester strains and S9 mix for bioactivation (TA98+S9 mix, TA98S; YG1041+S9 mix, YG1041S) and strain YG1041 in the absence of S9 mix (YG1041) were used to evaluate the mutagenic activity of eight polycyclic aromatic hydrocarbons (PAHs), seven nitroarenes (NAs) and seven aromatic amines (AAs). Three cigarette smoke extracts and two extracts of smokers' urine (SUE) were also included. Urinary mutagenicity was then determined on 31 individuals, potentially exposed to PAHs, for 0 h, 7 h, 12 h and 24 h. Concentrations of urinary 1-hydroxypyrene (1OHP) and 3-hydroxybenzo[a]pyrene (3OHBaP), the levels of atmospheric pyrene (Py) and benzo[a]pyrene (BaP), and particulate concentrations in air (AP) were also measured. PAHs could be detected by TA98S and YG1041S, with TA98S being more sensitive than YG1041S. While NAs could be detected by all combinations, YG1041 and YG1041S were more sensitive than TA98S. Although both YG1041S and TA98S could detect AAs, YG1041S was more sensitive than TA98S. Cigarette smoke extract contained mutagenic AAs and NAs, but AAs were the only mutagenic compounds detected in the extracts of smokers' urine. The concentrations of 1OHP (7 h and 12 h) were significantly higher than those at 0 h, but no difference could be detected with 3OHBaP. Correlations were found between Py and 1OHP (7 h and 24 h) and between BaP and 3OHBaP concentrations (7 h, 12 h and 24 h). A significantly elevated urinary mutagenicity was detected with YG1041S at 7h in the group of smokers. A good correlation was determined between AP and the test results with TA98S (7 h) and with YG1041 (0 h and 7 h). Urinary 1OHP correlated with the test results with YG1041S (0 h, 7 h and 12 h) while 3OHBaP correlated with those obtained with YG1041S (7 h). Overall, 21/31 individuals were occupationally exposed to AAs, 15/31 individuals were exposed to NAs, and 2/31 were exposed to PAHs as indicated by the Salmonella mutagenicity assay. The urine mutagenicity test was not effective at monitoring occupational exposure to PAHs. However, the correlation with AP implied the presence of unknown mutagenic atmospheric substances that could modulate the urinary mutagenicity.     

Assessment of oxidative DNA damage formation by organic complex mixtures from airborne particles PM10

The free radical generating activity of airborne particulate matter (PM₁₀) has been proposed as a primary mechanism in biological activity of ambient air pollution. In an effort to determine the impact of the complex mixtures of extractable organic matter (EOM) from airborne particles on oxidative damage to DNA, the level of 8-oxo-2′-deoxyguanosine (8-oxodG), the most prevalent and stable oxidative lesion, was measured in the human metabolically competent cell line Hep G2. Cultured cells were exposed to equivalent EOM concentrations (5–150 μg/ml) and oxidative DNA damage was analyzed using a modified single cell gel electrophoresis (SCGE), which involves the incubation of whole cell DNA with repair specific DNA endonuclease, which cleaves oxidized DNA at the sites of 8-oxodG. EOMs were extracted from PM₁₀ collected daily (24 h intervals) in three European cities: Prague (Czech Republic, two monitoring sites, Libuš and Smíchov), Košice (Slovak Republic) and Sofia (Bulgaria) during 3-month sampling periods in the winter and summer seasons. No substantial time- and dose-dependent increase of oxidative DNA lesions was detected in EOM-treated cells with the exception of the EOM collected at the monitoring site Košice, summer sampling. In this case, 2 h cell exposure to EOM resulted in a slight but significant increase of oxidative DNA damage at three from total of six concentrations. The mean 8-oxodG values at these concentrations ranged from 15.3 to 26.1 per 10⁶ nucleotides with a value 3.5 per 10⁶ nucleotides in untreated cells. B[a]P, the positive control, induced a variable but insignificant increase of oxidative DNA damage in Hep G2 cell (approximately 1.6-fold increase over control value).Based on these data we believe that EOM samples extracted from airborne particle PM₁₀ play probably only a marginal role in oxidative stress generation and oxidative lesion formation to DNA. However, adsorbed organic compounds can undergo various interactions (additive or synergistic) with other PM components or physical factors (UV-A radiation) and in this way they might enhance/multiply the adverse health effects of air pollution.     

In vitro genotoxicity of PAH mixtures and organic extract from urban air particles part II: human cell lines

Principal aims of this study were at first, to find a relevant human derived cell line to investigate the genotoxic potential of PAH-containing complex mixtures and second, to use this cell system for the analysis of DNA adduct forming activity of organic compounds bound onto PM10 particles. Particles were collected by high volume air samplers during summer and winter periods in three European cities (Prague, Kosice, and Sofia), representing different levels of air pollution. The genotoxic potential of extractable organic matter (EOM) was compared with the genotoxic potential of individual carcinogenic polycyclic aromatic hydrocarbons (c-PAHs) as well as their artificial mixtures. Metabolically competent human hepatoma HepG2 cells, confluent cultures of human diploid lung fibroblasts (HEL), and the human monocytic leukemia cell line THP-1 were used as models. DNA adducts were analyzed by ³²P-postlabeling. The total DNA adduct levels induced in HepG2 cells after exposure to EOMs were higher than in HEL cells treated under the same conditions (15–190 versus 2–15 adducts/10⁸ nucleotides, in HepG2 and HEL cells, respectively). THP-1 cells exhibited the lowest DNA adduct forming activity induced by EOMs (1.5–3.7 adducts/10⁸ nucleotides). A direct correlation between total DNA adduct levels and c-PAH content in EOM was found for all EOMs in HepG2 cells incubated with 50 μg EOM/ml (R = 0.88; p = 0.0192). This correlation was even slightly stronger when B[a]P content in EOMs and B[a]P-like adduct spots were analyzed (R = 0.90; p = 0.016). As THP-1 cells possess a limited metabolic capacity for most c-PAHs to form DNA reactive intermediates and are also more susceptible to toxic effects of PAHs and various EOM components, this cell line seemed to be an inappropriate system for genotoxicity studies of PAH-containing complex mixtures. The seasonal variability of genotoxic potential of extracts was stronger than variability among the three localities studied. In HepG2 cells, the highest DNA adduct levels were induced by EOM collected in Prague in the winter period, followed by Sofia and Kosice. However, in the summer sampling period, the order was quite opposite: Kosice > Sofia > Prague. When the EOM content per m³ of air was taken into consideration in order to compare real exposures of humans to genotoxic compounds in all three localities, extracts from respirable dust particles collected in Sofia exhibited the highest genotoxicity regardless of the sampling period. The results indicate that most of DNA adducts detected in cells incubated with EOMs have their origin in low concentrations of c-PAHs representing 0.03–0.17% of EOM total mass. Finally, our results suggest that HepG2 cells have a metabolic capacity for PAHs similar to human hepatocytes and represent therefore the best in vitro model for investigating the genotoxic potential of complex mixtures containing PAHs among the three cell lines tested in this study.     

Nucleotide Excision Repair Is Not Induced in Human Embryonic Lung Fibroblasts Treated with Environmental Pollutants

The cellular response to genotoxic treatment depends on the cell line used. Although tumor cell lines are widely used for genotoxicity tests, the interpretation of the results may be potentially hampered by changes in cellular processes caused by malignant transformation. In our study we used normal human embryonic lung fibroblasts (HEL12469 cells) and tested their response to treatment with benzo[a]pyrene (B[a]P) and extractable organic matter (EOM) from ambient air particles <2.5 µm (PM2.5) collected in two Czech cities differing in levels and sources of air pollution. We analyzed multiple endpoints associated with exposure to polycyclic aromatic hydrocarbons (PAHs) including the levels of bulky DNA adducts and the nucleotide excision repair (NER) response [expression of XPE, XPC and XPA genes on the level of mRNA and proteins, unscheduled DNA synthesis (UDS)]. EOMs were collected in the winter and summer of 2011 in two Czech cities with different levels and sources of air pollution. The effects of the studied compounds were analyzed in the presence (+S9) and absence (–S9) of the rat liver microsomal S9 fraction. The levels of bulky DNA adducts were highest after treatment with B[a]P, followed by winter EOMs; their induction by summer EOMs was weak. The induction of both mRNA and protein expression was observed, with the most pronounced effects after treatment with B[a]P (–S9); the response induced by EOMs from both cities and seasons was substantially weaker. The expression of DNA repair genes was not accompanied by the induction of UDS activity. In summary, our results indicate that the tested compounds induced low levels of DNA damage and affected the expression of NER genes; however, nucleotide excision repair was not induced.     

In vitro genotoxicity of PAH mixtures and organic extract from urban air particles part I: acellular assay

Acellular assay of calf thymus DNA ± rat liver microsomal S9 fraction coupled with ³²P-postlabelling was used to study the genotoxic potential of organic compounds bound onto PM10 particles collected in three European cities—Prague (CZ), Kosice (SK) and Sofia (BG) during summer and winter periods. B[a]P alone induced DNA adduct levels ranging from 4.8 to 768 adducts/10⁸ nucleotides in the concentration dependent manner. However, a mixture of 8 c-PAHs with equimolar doses of B[a]P induced 3.7–757 adducts/10⁸ nucleotides, thus suggesting the inhibition of DNA adduct forming activity by interaction among various PAHs. Comparison of DNA adduct levels induced by various EOMs indicates higher variability among seasons than among localities. DNA adduct levels for Prague collection site varied from 19 to 166 adducts/10⁸ nucleotides, for Kosice from 22 to 85 and for Sofia from 6 to 144 adducts/10⁸ nucleotides. Bioactivation with S9 microsomal fraction caused 2- to 7-fold increase in DNA adduct levels compared to −S9 samples, suggesting a crucial role of indirectly acting genotoxic EOM components, such as PAHs. We have demonstrated for the first time a significant positive correlation between B[a]P content in EOMs and total DNA adduct levels detected in the EOM treated samples (R = 0.83; p = 0.04). These results suggest that B[a]P content in EOM is an important factor for the total genotoxic potential of EOM and/or B[a]P is a good indicator of the presence of other genotoxic compounds causing DNA adducts. Even stronger correlation between the content of genotoxic compounds in EOMs and total DNA adduct levels detected (R = 0.94; p = 0.005) was found when eight c-PAHs were taken into the consideration. Our findings support a hypothesis that a relatively limited number of EOM components is responsible for a major part of its genotoxicity detectable as DNA adducts by ³²P-postlabelling.     

Seasonal variations of metal pollution and distribution,sources, and ecological risk of polycyclic aromatic hydrocarbons (PAHs) in sediment of the Al Hawizah wetland,Iran

The pollution of surface sediments of Al Hawizah wetland by metals and polycyclic aromatic hydrocarbons (PAHs) has been fully investigated. For determination of PAHs and metals concentration in sediments, eight sampling stations were selected in the study area. The results showed that the concentration of Mn is the highest, while the content of Cr is the lowest in both the seasons. The concentration of Cr and V is lower than mean crust content, while Cu concentration is more than mean crust content. The results obtained from Muller's geochemical index are indicative of range from uncontaminated to moderately contaminated. Based on potential ecological risk (RI), the Al Hawizah wetland had low ecological risk. The total PAHs concentrations ranged from 1071 to 15540 ng/g dry weight, with a mean of 9417.50 ng/g in the summer, while total amounts of PAHs in the winter ranged from 1542 to 17283 with a mean 10321.25 ng/g dry weight. The area of study was affected by pyrogenic and petrogenic sources (51.74 and 48.26%, respectively), in the winter. The concentrations of polycyclic aromatic hydrocarbons (PAHs) compounds were lower than effects range median (ERM) standard while were higher than effects range low (ERL) standard, except station 1, in both seasons.     

Mutagenic profile of rubber dust and fume exposure in two rubber tire companies

The aim of this study was to evaluate current mutagenic activity of ambient rubber dust and fume exposure in the mixing and curing departments of two rubber tire companies situated in The Netherlands and Sweden. Salmonella typhimurium strains YG1021, YG1024 and YG1041 were used to study the possible presence of mutagenic nitroarenes and aromatic amines. A large difference in mutagenic activity was found between the two companies. While the rubber tire company situated in The Netherlands revealed overall high mutagenic activity of rubber dust and fumes in the mixing and curing departments, respectively, 430 and 279 rev/m(3) (YG1041), the Swedish company showed almost no mutagenic activity, respectively, 18 and 54 rev/m(3) (YG1041). Further identification of the mutagenic profile showed that mutagenic activity was exclusively observed in S. typhimurium strains with elevated levels of O-acetyltransferase activity (YG1041 and YG1024) in the presence of a metabolic active liver S9 fraction, possibly indicating the presence of indirect mutagenic aromatic amines. These results show that although production processes and lay-out within rubber tire companies are comparable, differences in rubber chemicals used and overall level of control measures (e.g., good housekeeping, cleanliness) are likely to result in substantial differences in mutagenic exposure levels between companies.     

Genotoxic activity of extractable organic matter from urban airborne particles in Shanghai, China

The aim of this research is to investigate the impact of air pollution on the population in Shanghai. The genotoxicity of extractable organic matter (EOM) from the air particles was investigated by the means of the Salmonella plate incorporation assay, rat hepatocyte unscheduled DNA repair assay, and mice micronuclei test. The airborne particles were collected in 13 locations during the summer of 1992 and winter of 1993. The crude extracts were fractionated by acid-base partitioning into acid, base and neutral fractions. The neutral fractions were further fractionated by resin-silica gel column chromatography into three subfractions. The induction of revertants with the crude extracts was higher in winter samples than in summer samples. Both indirect-acting and direct-acting mutagenicity were observed. The mutagenicity was detected with TA98, but was not detected with TA100. The mutagenic activity was the greatest in the acid, aromatic and polar fractions from summer samples. The fractions from the winter samples did not show clear differences. There was no substantial location-related variance in the mutagenic potencies of EOM, but substantial location- or time-related variances in the mutagenic potencies of the airborne particles per cubic meter air were found. While rat hepatocyte unscheduled DNA synthesis (UDS) assay revealed genotoxicity for all the samples, there was no big variance in the genotoxicity of the fractions. The mouse micronuclei test showed results similar to the UDS assay. The difference of locality did not have statistical significance.     

Molecular epidemiology studies of carcinogenic environmental pollutants. Effects of polycyclic aromatic hydrocarbons (PAHs) in environmental pollution on exogenous and oxidative DNA damage

Exposure to high levels of environmental air pollution is known to be associated with an increased carcinogenic risk. The individual contribution to this risk derived from specific carcinogenic chemicals within the complex mixture of air pollution is less certain, but may be explored by the use of molecular epidemiological techniques. Measurements of biomarkers of exposure, of effect and of susceptibility provide information of potential benefit for epidemiological and cancer risk assessment. The application of such techniques has been mostly concerned in the past with the carcinogenic polycyclic aromatic hydrocarbons (c-PAHs) that are associated with particulate matter in air pollution, and has showed clear evidence of genotoxic effects, such as DNA adducts, chromosome aberrations (CA) and ras oncogene overexpression, in environmentally exposed Czech and Polish populations. We are currently extending these studies by an investigation of populations exposed to environmental pollution in three European countries, Czech Republic, Slovak Republic and Bulgaria. This pays particular attention to PAHs, but also investigates the extent of radically induced (oxidative) DNA damage in the exposed populations. Policemen, bus drivers and controls, who carried personal monitors to determine their exposures to PAHs have been studied, and blood and urine were collected. Antioxidant and dietary status were assessed in these populations. Stationary monitors were also used for ambient air monitoring. Amongst the parameters studied in the biological samples were: (a) exposure biomarkers, such as PAH adducts with DNA, p53 and p21(WAF1) protein levels, (b) oxidative DNA damage, (c) the biological effect of the exposure by measurement of chromosome damage by fluorescence in situ hybridisation (FISH) or conventional methods, and (d) polymorphisms in carcinogen metabolising and DNA repair enzymes. Repair ability was also measured by the Comet assay. In vitro systems are being evaluated to characterise the genotoxicity of the organic compounds adsorbed to air particles.     

Seasonal variations in the rates of aquatic and aerial respiration and ammonium excretion of the ribbed mussel, Geukensia demissa (Dillwyn)

Aquatic and aerial rates of oxygen consumption and ammonium excretion of ribbed mussels, Geukensia demissa (Dillwyn), collected from the mid-intertidal zone of a mid-Atlantic salt marsh, were measured under ambient conditions of food, temperature, and salinity over five seasons. Rates of aquatic respiration covaried with body size and season, as the rates were high and strongly related to mussel tissue weight in spring and summer but low and weight independent in winter. There was a significant interannual difference between summer of 1995 and 1996. Rates of aerial respiration also varied seasonally, with high rates of oxygen consumption in summer and low rates in winter. The magnitude of these seasonal variations were greater than those for aquatic respiration, and as a result, the ratio of aerial to aquatic respiration was higher in summer (0.61 and 0.52) than in winter (0.11). This indicates that G. demissa was able to actively regulate aerial respiration, thereby permitting high aerobic metabolism during prolonged periods of air exposure in summer. We hypothesize that such high rates of aerial respiration, during the seasons of high metabolic activity, are required to provide sufficient energy for mussels to facilitate food digestion during air exposure at low tide. Rates of ammonium excretion varied seasonally and increased with mussel weight in all seasons. The atomic ratio of oxygen to nitrogen (O:N), calculated from aquatic respiration vs. ammonium excretion, was significantly lower in autumn (26) than in other seasons (46–60) among which the O:N did not vary significantly.     

Application of Salmonella strains with altered nitroreductase and O-acetyltransferase activities to the evaluation of the mutagenicity of airborne particles

The Ames test was applied to evaluation of the mutagenicity of month's samples of airborne particles from the center of Wroc?aw (SW Poland) collected in August and December 1997. The strains used for the study were TA 98, TA 100 and their derivatives: TA 98 NR, YG 1021, YG 1024, YG 1026, YG 1029, YG 1041, YG 1042. Both studied samples were mutagenic for almost all tested strains, with the exception of the August sample which did not influence the strain TA 100 without the metabolic activation with the S9 fraction. The December sample exhibited higher genotoxic activity than the August sample. Mutagenicity ratios of the strains with reduced nitroreductase and O-acetyltransferase activities were higher, and of the strain without the nitroreductase--lower than those of the parent strains. This indicates that nitro and amino derivatives of PAHs are responsible for the significant proportion of total mutagenicity of the studied samples of particulates. Metabolic activation with the S9 fraction caused the increase of the mutagenic activity of the samples, which indicates the presence of promutagens. The GC-MS analysis revealed the presence of known indirect mutagens from the PAHs group.