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5-Fluorouracil Incorporation into DNA of CF-1 Mouse Bone Marrow Cells as a Possible Mechanism of Toxicity¹

Departments of Pharmacology [J. D. S., R. B. D.] and Medicine [H. J. W., R. B. D.], Medical College of Virginia, Richmond, Virginia 23298

Isolated CF-1 mouse bone marrow cells were exposed for 1 hr to 5-fluorouracil (FUra) at concentrations from 1.8 to 50 µM and then washed and suspended in a soft agar growth medium to assess the effect on toxicity (measured as reduction in colony growth compared to control). These data were used to determine specific toxic concentrations ranging from 25 to 90% lethal doses. Subsequent studies examined in parallel the effect of these toxic concentrations of FUra on the possible sites of toxicity including: (a) inhibition of thymidylate synthetase activity using a modified ³H release assay; (b) incorporation of FUra into RNA (FUra-RNA); and (c) incorporation of FUra into DNA (FUra-DNA).

Thymidylate synthetase activity was slightly decreased (75% of control) after 1-hr exposure to a 50% lethal dose and was not significantly further reduced as the FUra concentration was increased to an 85% lethal dose. Furthermore, subsequent exposure of FUra-treated cells to a nontoxic thymidine dose (5 µM) failed to reverse toxicity. FUra-RNA increased during 1-hr exposure to increasing concentrations of FUra (25 to 90% lethal doses). Although initially suggesting a relationship between the level of FUra-RNA and toxicity, subsequent studies in cells exposed to FUra in the presence of uridine demonstrated a significantly decreased toxicity while, at the same time, a minimal decrease of FUra in RNA. In contrast, FUra-DNA was significantly decreased in the presence of uridine and correlated with decreased toxicity. In additional subsequent studies, an apparent decrease in subsequent DNA synthesis was observed (measured by ³²P or [³H]thymidine incorporation into DNA) as the level of FUra-DNA increased.

In conclusion, FUra is demonstrated to be incorporated into DNA of isolated CF-1 mouse bone marrow cells, and the level of FUra-DNA appears to be closely associated with toxicity and inhibition of further DNA synthesis. The parallel studies of thymidylate synthetase activity and FUra-RNA suggest that FUra-DNA may be an unrecognized mechanism of FUra toxicity in these cells.

¹ Supported by USPHS Grant CA 23412 and American Cancer Society Grant IN-105H.

² To whom requests for reprints should be addressed, at Box 540, Medical College of Virginia Station, Richmond, VA 23298.

Received 3/24/83. Accepted 12/20/83.

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