Toxicological Sciences 54, 322-329 (2000)
Copyright © 2000 by the Society of Toxicology
Relationships between DNA Incorporation, Mutant Frequency, and Loss of Heterozygosity at the TK Locus in Human Lymphoblastoid Cells Exposed to 3'-Azido-3'-deoxythymidine
,1
* Wadsworth Center, New York State Department of Health, Albany, New York 12201–0509;
School of Public Health, State University of New York at Albany, Albany, New York 12203; and
Laboratory of Cellular Carcinogenesis and Tumor Promotion, Division of Basic Sciences, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892
3'-Azido-3'-deoxythymidine (AZT), a thymidine analogue widely used in the treatment of AIDS patients and for prevention of the onset of AIDS in HIV-seropositive individuals, causes tumors in mice exposed as adults or in utero. The purpose of this study was to investigate the potential mechanisms of AZT mutagenicity and carcinogenicity by quantifying the incorporation of AZT into cellular DNA, measuring AZT-induced thymidine kinase (TK) mutant frequencies (Mfs), and determining the percentage of loss of heterozygosity (LOH) in spontaneous or AZT-induced TK mutants in the human lymphoblastoid cell line, TK6. Cells were exposed to 300 µM AZT for 0, 1, 3, or 6 days, or to 0, 33, 100, 300, or 900 µM AZT for 3 days (n = 5 flasks/group). The effects of exposure concentration on incorporation of AZT into cellular DNA were evaluated by an AZT radioimmunoassay, and the effects of duration and concentration of AZT exposure on the TK Mfs were assessed by a cell-cloning assay. AZT was incorporated into DNA in a dose-related manner at concentrations up to 300 µM, above which no further increase was observed. TK Mf increased with the extended duration and with incremental concentrations of AZT exposure. There was a positive correlation (P = 0.036, coefficient = 0.903) between AZT-DNA incorporation and AZT-induced TK Mfs, suggesting that AZT incorporation into cellular DNA has a direct role in the genotoxicity of AZT. Southern blot analyses indicated that 84% (6.2 x 10–6/7.4 x 10 –6) of AZT-induced mutants were attributable to LOH, consistent with the known mechanism of AZT as a DNA chain terminator. Considering the importance of LOH in human carcinogenesis, AZT-induced LOH warrants further study.
Key Words: 3'-azido-3'-deoxythymidine; human lymphoblastoid cells; DNA incorporation; thymidine kinase; mutant frequency; loss of heterozygosity.
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