Bioassay-directed chemical analysis and detection of mutagenicity in ambient air of the coke oven.

In the present study, we summarize the results of studies on the mutagenic potential of the main fractions and subfractions of extractable organic material (EOM) in the ambient air at the workplaces of the coke oven. The objective of our experiments was to apply the Bioassay-Directed Chemical Analysis (with the use of the Ames test) for the identification of the differences in the mutagenicity of these fractions, in relationship to the complex mixture of EOM in occupational air. From the evaluation of results, it is possible to deduce the following conclusions: (1) The comparison of the mutagenicity in the main fractions (basic, acidic, neutral) demonstrates the existence of differences in mutagenic potential. Of the total mutagenicity, 20.4% is in the basic fraction, 25.4% in the acidic fraction and 54.2% in the neutral fraction. (2) In general, 90.1% of the mutagenicity found in the basic, acidic and neutral fractions together was associated with the requirement of metabolic activation in vitro (+S9). In the case of the neutral fraction, it was 51.8%. (3) These results also suggest that frameshift mutations are the major component (53.8%) of the total mutagenicity of the main fractions. (4) With regards to the mutagenicity of organic compounds in the neutral fraction it appeared that genotoxicants of its subfractions (slightly and moderately polar and aromatic) play the main role. Carcinogenic aromatic hydrocarbons (PAH) and genotoxic nitrocompounds play an important role as determinants of the mutagenic potential of complex mixtures of harmful compounds in ambient air. This is confirmed first by the results of short-term bacterial tests.

DNA adduct formation in mammalian cell cultures by polycyclic aromatic hydrocarbons (PAH) and nitro-PAH in coke oven emission extract.

Mammalian cells in culture were used to study the genotoxic potential of coke oven emissions constituting a complex mixture of chemicals. For this purpose, particle extracts and some polycyclic aromatic and nitroaromatic hydrocarbons (PAH and nitro-PAH) occurring in these mixtures were assayed for DNA adduct formation using the -postlabeling technique. In primary cultures of rat hepatocytes, benzo[a]pyrene (B[a]P), benz[a]anthracene (B[a]A) and benzo[b]fluoranthene (B[k]F) caused DNA adduct levels in the range of 1 adduct/108 nucleotides. 4-Nitropyrene (4-NP), 6-nitrochrysene (6-NC), 3-nitrofluoranthene (3-NF) caused DNA adduct levels that were by one to two orders of magnitude higher. The crude particle extract and its fractions differing in acidity and polarity induced the formation of DNA reactive material within diagonal radioactive zones (DRZ) on the autoradiograms. On a weight base, the neutral aromatic fraction contributed by more than 80% to the total adduct level in hepatocytes. To examine whether the PAH- and nitro-PAH-DNA derived adducts can be further differentiated, hepatocyte cultures were preincubated with 2,3,7,8-tetrachloro-p-dioxin (TCDD) to induce the activity of cytochrome P450 1A1. TCDD pretreatment strongly increased the levels of PAH-DNA adducts, whereas, the levels of nitro-PAH adducts were markedly decreased. NCI-H322 cells, a human lung tumor cell line derived from Clara cells, exhibited PAH-DNA adduct levels between 10 and 100, and nitro-PAH-DNA adducts at levels between 0.2 to about 30 adducts per 108 nucleotides, respectively. In contrast to hepatocytes, incubations with extractable organic matter (EOM) and the neutral aromatic EOM fraction displayed several distinct spots in the chromatograms of NCI-H322 cells. The major spot was assigned by cochromatography to be identical with the major DNA adduct formed by incubation with B[a]P alone. In V79NH cells, a Chinese hamster lung cell line expressing nitro-PAH activating enzymes, but virtually no cytochrome P450 activity, PAH-derived DNA adducts were not detectable. Nitro-PAH-derived DNA adducts, however, were formed at levels between 10 and 300 adducts/108 nucleotides. The slightly and the moderately polar EOM fraction caused the formation of distinct adduct spots suggesting the occurrence of nitro-PAH in these fractions. GC/MS analyses revealed the presence of twelve PAH in the aromatic fraction, at a total amount of about 10% (w/w), and of four nitro-PAH in the slightly polar and the acidic fraction amounting to about 0.2% (w/w). In conclusion, our results indicate that PAH and nitro-PAH contribute to the genotoxicity of coke oven emissions. Using DNA adduct analysis in rat hepatocytes (+/-pretreatment with TCDD) and in NCI-H322 and in V79NH cells offers a promising approach to determine the genotoxic activity of PAH and nitro-PAH in any complex environmental samples.

Genotoxicity of coke-oven and urban air particulate matter in in vitro acellular assays coupled with 32P-postlabeling and HPLC analysis of DNA adducts.

This study is an in vitro part of the ongoing biomarker studies with population from a polluted region of Northern Bohemia and coke-oven workers from Czech and Slovak Republics. The aim of this study is to compare DNA adduct forming ability of chemical compound classes from both the urban and coke-oven extractable organic mass (EOM) of airborne particles. The crude extracts were fractionated into seven fractions by acid-base partitioning and silica gel column chromatography. In in vitro acellular assays we used calf thymus DNA (CT DNA) with oxidative (+S9) and reductive activation mediated by xanthine oxidase (+XO) under anaerobic conditions. Both the butanol and nuclease P1 versions of 32P-postlabeling for detection of bulky aromatic and/or hydrophobic adducts were used. The results showed that the spectra of major DNA adducts resulting from both the in vitro assays are within the fractions similar for both the urban and coke-oven samples. The highest DNA adduct levels with S9-activation were detected for the neutral aromatic fraction, followed by slightly polar and acidic fractions for both samples. With XO-mediated metabolism, the highest DNA adduct levels were detected for both the acidic fractions. Assuming additivity of compound activities, then the acidic fraction, which in the urban sample comprises a major portion of EOM mass (28%), may contain the greatest activity in both in vitro assays (39 and 69%, +S9 and +XO, respectively). In contrast, the aromatic fraction constituting only 8% of total urban EOM mass may account for comparable activity (34%) with organic acids. The highest DNA adduct forming activity of the coke-oven sample accounts for the aromatic fraction (82 and 63%, +S9 and +XO, respectively) that also contains the greatest portion of the total EOM (48%). To characterize some of the specific DNA adducts formed, we coupled TLC on 20x20 cm plates with HPLC analysis of 32P-postlabeled adducts. In both S9-treated samples of the aromatic fraction, we tentatively identified DNA adducts presumably diolepoxide-derived from: 9-hydroxy-benzo[a]pyrene (9-OH-B[a]P), benzo[a]pyrene-r-7,t-8-dihydrodiol-t-9,10-epoxide[+/-] (anti-BPDE), benzo[b,j,k]fluoranthenes (B[b]F, B[j]F, B[k]F), chrysene (CHRY), benz[a]-anthracene (B[a]A) and indeno[cd]pyrene (I[cd]P). These DNA adducts accounted for about 57% of total DNA adducts detected in both S9-treated samples of the aromatic fraction. DNA adducts of XO-treated samples were sensitive to nuclease P1 and HPLC profiles of the major adducts were markedly different from the major adducts of S9-treated samples. However, the combination of TLC and HPLC did not confirm the presence of DNA adducts derived from 1-nitropyrene (1 NP), 9-nitroanthracene (9 NA) and 3-nitrofluoranthene (3 NF) that were detected by GC-MS in the slightly polar fraction. We concluded that the chemical fractionation procedure facilitates the assessing of DNA adduct forming ability of different chemical compound classes. However, based on the results obtained with the whole extracts, it does not fulfil a task of the actual contribution of individual fractions within the activity of the whole extracts. Our results are the first in detecting of DNA adducts derived from urban air and coke-oven particulate matter.