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Mode of action of mutagenic and oncogenic synthetic quinoline N-oxides Laishes, Brian Anthony

Abstract

The molecular events in cells exposed to mutagenic and oncogenic quinoline N-oxides were examined by analyzing single-strand breakage of DNA. Secondary cultures of embryonic Syrian-hamster cells and a line of BHK-21 cells were used. The choice of nitroquinoIine N-oxides afforded a series of water-soluble chemicals with three relative degrees of oncogenicity: highly oncogenic 4-nitroquinoIine 1-oxide (4NQO), weakly oncogenic 3-methyI-4-nitroquinoIine 1-oxide (3-methyI-4NQO), and the non-oncogenic 3-nitroquinoIine 1-oxide (3NQO) and 8-nitroquinoIine 1-oxide (8NQO). The detection of subtle alterations in DNA sedimentation velocity was greatly improved by a double-label procedure. Cells treated for 2 hours with the various nitroquinoIine N-oxide compounds were prelabelled with 0.05 uCi/ml ¹⁴C-thymidine for 24 hours. Untreated control cells were labelled with 0.25 uCi/ml ³H-thymidine for 24 hours. Aliquots of quinoline N-oxide treated and untreated cells were mixed, layered on the alkaline sucrose gradient, centrifuged at 25,000 rpm for 300 minutes, and the amount of ¹⁴C (treated) and ³H(untreated) radioactivity in each fraction of the gradient was determined. The alkaline sucrose gradient technique was modified in the following ways: (1) a PBS cell suspension was layered directly onto a 2% sucrose solution overlay, on the gradient, to reduce DNA-shearing forces believed previously encountered with a 0.5M NaOH overlay, (2) cell lysis and centrifugation were carried out at 4° rather than room temperature, and (3) the number of cells layered per gradient was reduced to 12,000. A correlation between the degree of oncogenicity of nitroquinoline N-oxides and their capacity to induce DNA single-strand breakage was indicated, although limited because of the low sensitivity of the sucrose gradient technique. Single-strand breaks of DNA occurred when celIs were treated for 2 hours with 5x10⁻⁶M 4NQO but were not detectable when exposed to 4NQO at a 1x10⁻⁶M concentration or less. However, when cells were treated with the weakly oncogenic 3-methyI-4NQO (5x10⁻⁶M) or the non-oncogenic 3NQO and 8NQO (5x10⁻⁶M) there were either no singIe-strand breaks produced or the frequency was too small to be recognized. The repair of single-strand breaks was measured by sampling 4NQO-exposed cells after various periods post-treatment and estimating the amount of ¹⁴C(treated)-DNA (in percent of total ¹⁴C counts) over and above the amount of ³H(untreated)-DNA (in percent of total ³H counts) occurring in the top half of each gradient. Substantial repair at 24 hours was indicated by less than 4% ¹⁴C-DNA above ³H-DNA as opposed to 29% ¹⁴C-DNA above ³H-DNA at 0 hours post-treatment incubation. Caffeine (1,3,7-trimethyIxanthine), added at a concentration of 4x10⁻³M to cell cultures, greatly reduced DNA single-strand breaks induced by 4NQO (4x10⁻⁶M). Attempts were made to correlate the capacity of synthetic quinoline N-oxides to induce DNA single-strand breaks with their capacity to invoke charge-transfer complexes with one or more DNA nucleotides, and so this study suggests approaches by which biological phenomena can be interpreted, ultimately, in terms of relative electron charge densities of molecules.

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