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Formation and Removal of Benzo(a)pyrene Adducts of DNA in Hamster Tracheal Epithelial Cells¹

Departments of Biochemistry [A. E., E. B.] and Pathology [B. T. M.] and The Vermont Regional Cancer Center, University of Vermont College of Medicine, Burlington, Vermont 05405 [A. E., B. T. M., E. B.]

A cloned cell line derived from normal hamster tracheal epithelium has been characterized with respect to its response to the environmental pollutant and carcinogen benzo(a)pyrene [B(a)P]. These cells metabolize B(a)P to ultimate reactive forms as assayed by alkylation of DNA. Alkylation with radiotracer amounts of B(a)P was maximum at 8 hr, at which time 70% of the applied hydrocarbon had been converted to water-soluble forms. At longer incubation times, the rate of removal of adducts exceeded the rate of formation. When B(a)P-containing medium was replaced with fresh medium at two or four hr, a subsequent biphasic removal of adducts occurred, a rapid removal for the first four hr postincubation and then a slower repair. About 50% of the DNA-bound hydrocarbon remained in DNA after 48 hr. Cells were able to divide in the presence of these lesions, undergoing five doublings (five days), while only 60% of the adducts were removed from the DNA.

Integrity of DNA during this period was monitored by the alkaline elution technique. A toxic dose of B(a)P was required to cause any increase in the rate of elution. Minimal single-strand breakage was observed from two to eight hr of B(a)P treatment, but at 15 hr DNA appeared normal. Comparison was made with a nontoxic dose of methyl methanesulfonate which caused very rapid elution of DNA after only one hr treatment.

At least 15 deoxyribonucleoside-bound B(a)P adducts were separated by high-pressure liquid chromatography. Four adducts, probably deoxyadenosine-B(a)P, were removed almost completely in 24 hr, while the others appeared to be poorly removed. The possible significance to neoplasia of persistent and repairable lesions is discussed.

¹ Supported by NIH Grant CA23514 and National Institute for Occupational Safety and Health Grant 00888.

Received 9/10/80. Accepted 3/19/81.