GENOTOXICITY OF COKE-OVEN AND URBAN AIR PARTICULATE MATTER IN IN-VITRO ACELLULAR ASSAYS COUPLED WITH P-32 POSTLABELING AND HPLC ANALYSIS OFDNA-ADDUCTS

This study is an in vitro part of the ongoing biomarker studies with p opulation from a polluted region of Northern Bohemia and coke-oven wor kers from Czech and Slovak Republics. The aim of this study is to comp are DNA adduct forming ability of chemical compound classes from both the urban and coke-oven extractable organic mass (EOM) of airborne par ticles. The crude extracts were fractionated into seven fractions by a cid-base partitioning and silica gel column chromatography. In in vitr o acellular assays we used calf thymus DNA (CT DNA) with oxidative (+S 9) and reductive activation mediated by xanthine oxidase (+XO) under a naerobic conditions. Both the butanol and nuclease P1 versions of P-32 -postlabeling for detection of bulky aromatic and/or hydrophobic adduc ts were used. The results showed that the spectra of major DNA adducts resulting from both the in vitro assays are within the fractions simi lar for both the urban and coke-oven samples. The highest DNA adduct l evels with S9-activation were detected for the neutral aromatic fracti on, followed by slightly polar and acidic fractions for both samples. With XO-mediated metabolism, the highest DNA adduct levels were detect ed for both the acidic fractions. Assuming additivity of compound acti vities, then the acidic fraction, which in the urban sample comprises a major portion of EOM mass (28%), may contain the greatest activity i n both in vitro assays (39 and 69%, +S9 and +XO, respectively). In con trast, the aromatic fraction constituting only 8% of total urban EOM m ass may account for comparable activity (34%) with organic acids. The highest DNA adduct forming activity of the coke-oven sample accounts f or the aromatic fraction (82 and 63%, +S9 and +XO, respectively) that also contains the greatest portion of the total EOM (48%). To characte rize some of the specific DNA adducts formed, we coupled TLC on 20 x 2 0 cm plates with HPLC analysis of P-32-postlabeled adducts. In both SE -treated samples of the aromatic fraction, we tentatively identified D NA adducts presumably diolepoxide-derived from: 9-hydroxy-benzo[a]pyre ne (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), chryse ne (CHRY), benz[a]-anthracene (B[a]A) and indeno[cd]pyrene (I[cd]P). T hese DNA adducts accounted for about 57% of total DNA adducts detected in both SE-treated samples of the aromatic fraction. DNA adducts of X O-treated samples were sensitive to nuclease P1 and HPLC profiles of t he major adducts were markedly different from the major adducts of SE- treated samples. However, the combination of TLC and HPLC did not conf irm the presence of DNA adducts derived from 1-nitropyrene (1 NP), 9-n itroanthracene (9 NA) and 3-nitrofluoranthene (3 NF) that were detecte d by GC-MS in the slightly polar fraction. We concluded that the chemi cal fractionation procedure facilitates the assessing of DNA adduct fo rming ability of different chemical compound classes. However, based o n the results obtained with the whole extracts, it does not fulfil a t ask of the actual contribution of individual fractions within the acti vity of the whale extracts. Our results are the first in detecting of DNA adducts derived from urban air and coke-oven particulate matter. ( C) 1998 Elsevier Science B.V. All rights reserved.