Genotoxicity of 3 '-azido-3 '-deoxythymidine in the human lymphoblastoid cell line, TK6: relationships between DNA incorporation, mutant frequency, and spectrum of deletion mutations in HPRT

Perinatal treatment with 3'-azido-3'-deoxythymidine (AZT) has been found to reduce the rate of maternal-infant transmission of HIV; however, AZT is ge notoxic in mammalian cells in vitro and induces tumors in the offspring of mice treated in utero. The purpose of the present study was to investigate the relationships between incorporation of AZT into DNA, and the frequency and spectrum of mutations at the HPRT locus of the human lymphoblastoid cel l line, TK6, following in vitro exposures to AZT. Cells were cultured in me dium containing 0 or 300 mu M AZT for 1, 3, or 6 day(s) (n = 5/group). The effects of exposure duration on incorporation of AZT into DNA and HPRT muta nt frequency were determined using an AZT radioimmunoassay and a cell cloni ng assay, respectively. AZT accumulated in DNA in a supralinear manner, app roaching a plateau at 6 days of treatment (101.9 +/- 14.7 molecules AZT/10( 6) nucleotides). After 3 days of AZT exposure, HPRT mutant frequency was si gnificantly increased (1.8-fold, p = 0.016) compared to background (mutant frequency = 3.78 X 10(-6)). Multiplex PCR amplification of genomic DNA was used to determine the frequency of exon deletions in HPRT mutant clones fro m untreated cells versus AZT-treated cells. Molecular analyses of AZT-induc ed mutations revealed a significant difference in the frequency of total ge ne deletions (44/120 vs. 18/114 in controls, p = 0.004 by the Mann-Whitney U-statistic). In fact, the Chi-square test of homogeneity demonstrate that the differences between the control and AZT-treatment groups is attributed mainly to this increase in total gene deletion mutations (p = 0.00001). The se data indicate that the primary mechanism of AZT mutagenicity in human TK 6 cells is through the production of large deletions which occur as a resul t of AZT incorporation into DNA and subsequent chain termination. The data imply that perinatal chemoprophylaxis with AZT may put children of HIV-infe cted women at potential risk for genetic damage. (C) 1999 Elsevier Science B.V. All rights reserved.