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³²P-Postlabeling Analysis of Aromatic DNA Adducts in Fish from Polluted Areas¹

Environmental Carcinogenesis Unit, British Columbia Cancer Research Centre, Vancouver, British Columbia, Canada V5Z1L3 [B. P. D.], and Roswell Park Memorial Institute, Buffalo, New York 14203 [J. J. B., A. M.]

Brown bullheads (Ictalurus nebulosus) were sampled from sites in the Buffalo and Detroit Rivers where fish are exposed to high levels of sediment bound polycyclic aromatic hydrocarbons, and suffer from an elevated frequency of liver cancer. DNA was isolated from the livers of these wild fish and from control specimens which were raised in clean aquariums. DNA was enzymatically digested to normal and adducted nucleotides, and hydrophobic/bulky adducts were enriched in the digests either by preparative reverse-phase high-pressure liquid chromatography, or selective nuclease P₁ dephosphorylation of normal nucleotides. Aromatic DNA-carcinogen adducts were then quantitated using ³²P-postlabeling analysis. Using both adduct enrichment procedures, chromatograms derived from DNA of fish from polluted areas showed a diffuse diagonal radioactive zone not present in DNA from aquarium raised fish. The diagonal zone appeared to consist at least in part of multiple overlapping discrete adduct spots which could be partially separated by gradient high-pressure liquid chromatography prior to ³²P-postlabeling analysis, and most of which were more strongly retained on a reverse-phase column than the major benzo(a)pyrene-DNA adduct. The behavior of the adducts in the diagonal radioactive zone and of their unlabeled precursors is consistent with their identification as nucleotide adducts of a variety of bulky hydrophobic aromatic environmental compounds. Total pollution-related adduct levels as analyzed by HPLC adduct enrichment and ³²P-postlabeling were 70.1 ± 29 (SD) nmol/mol normal nucleotide in fish from the Buffalo River, and 52 and 56 nmol/mol for two specimens from the Detroit River.

¹ This research was supported in part by Grant A3403 from the Natural Sciences and Engineering Research Council, Canada and by cooperative agreement CR 812575-02-0 with the U. S. Environmental Protection Agency.

Received 6/ 9/87. Revised 9/21/87. Accepted 9/22/87.