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. Fo r this purpose, particle extracts and some polycyclic aromatic and nit roaromatic hydrocarbons (PAH and nitro-PAH) occurring in these mixture s were assayed for DNA adduct formation using the P-32-postlabeling te chnique. 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 D NA adduct levels in the range of 1 adduct/10(8) nucleotides. 4-Nitropy rene (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 diagon al radioactive zones (DRZ) on the autoradiograms. On a weight base, th e 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 culture s were preincubated with 2,3,7,8-tetrachloro-p-dioxin (TCDD) to induce the activity of cytochrome P450 1Al. TCDD pretreatment strongly incre ased the levels of PAH-DNA adducts, whereas, the levels of nitro-PAH a dducts were markedly decreased. NCI-H322 cells, a human lung tumor cel l line derived from Clara cells, exhibited PAH-DNA adduct levels betwe en 10 and 100, and nitro-PAH-DNA adducts at levels between 0.2 to abou t 30 adducts per 10(8) nucleotides, respectively. In contrast to hepat ocytes, incubations with extractable organic matter (EOM) and the neut ral aromatic EOM fraction displayed several distinct spots in the chro matograms of NCI-M322 cells. The major spot was assigned by cochromato graphy 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 exp ressing nitro-PAH activating enzymes, but virtually no cytochrome P450 activity, PAM-derived DNA adducts were not detectable. Nitro-PAH-deri ved DNA adducts, however, were formed at levels between 10 and 300 add ucts/10(8) nucleotides. The slightly and the moderately polar EOM frac tion caused the formation of distinct adduct spots suggesting the occu rrence of nitro-PAH in these fractions. GC/MS analyses revealed the pr esence of twelve PAH in the aromatic fraction, at a total amount of ab out 10% (w/w), and of four nitro-PAH in the slightly polar and the aci dic fraction amounting to about 0.2% (w/w). In conclusion, our results indicate that PAH and nitro-PAH contribute to the genotoxicity of cok e oven emissions. Using DNA adduct analysis in rat hepatocytes (+/- pr etreatment with TCDD) and in NCI-H322 and in V79NH cells offers a prom ising approach to determine the genotoxic activity of PAH and nitro-PA H in any complex environmental samples. (C) 1998 Elsevier Science B.V. All rights reserved.