Measurements by filter elution of DNA single- and double-strand breaks in rat hepatocytes: effects of nitrosamines and gamma-irradiation

Abstract

This work presents a filter elution method for measuring DNA single- and double-strand breaks in primary rat hepatocytes without radioactive labeling of DNA. We have studied the effects of a series of nitrosamines and of gamma-irradiation on DNA single- and double-strand break induction. The repair of DNA single-strand breaks in the hepatocytes was measured after treatment with 60Co, 1-methyl-1-nitrosourea, and N-nitrosodimethylamine. The hepatocytes were isolated by ethylene glycol-bis(beta-aminoethyl ether)-N,N'-tetra acetic acid-collagenase perfusion and had a mean viability of 91 +/- 4% (S.D.). The isolated cells were treated for varying lengths of time with nitrosamines in suspension culture in L-15 medium containing 10% fetal bovine serum. After treatment, the cells were chilled, loaded onto 2 micrometers polycarbonate filters, and lysed in a 2% sodium dodecyl sulfate-proteinase K solution, pH 9.6. The DNA was eluted from the filter at either native or denaturing pH with fractions collected every 3 hr. The quantity of DNA in each fraction was determined by measuring the fluorescent product formed between it and diaminobenzoic acid after ethanol-sodium acetate precipitation and trapping of the DNA on 0.2-micrometer polycarbonate filters. The results show that the carcinogens, N-nitrosodimethylamine, N-nitrosodiethylamine, N-nitrosodipropylamine, N-nitrosodibutylamine, and 1-nitrosopiperidine all made dose- and time-related increases in the number of single-strand breaks in rat hepatocytes. N-Nitrosodiphenylamine produced small numbers of single-strand breaks. No double-strand breaks were formed by any of the nitrosamines. Single-strand breaks induced by N-nitrosodimethylamine were repaired very slowly relative to repair of either gamma-ray of 1-methyl-1-nitrosourea-induced single-strand breaks. This system has many advantages for studying carcinogen metabolism and DNA damage in hepatocytes, one of the major target cells for many carcinogens.-