Transplacental mutagenicity of N-ethyl-N-nitrosourea at the hprt locus in T-lymphocytes of exposed B6C3F1 mice.

Previous studies have compared age-related differences in total mutagenic burden in mice of differing age (preweanling, weanling, or young adult) after single intraperitoneal (i.p.) injections of ethylnitrosourea (ENU). The purpose of the present investigation was to determine the effects of time elapsed since treatment on the frequency of hprt mutant T-cells (Mf) from mice treated transplacentally with single acute vs. multiple split doses of ENU. To this end, pregnant C57BL/6 mice (n = 13-16/group), which had been bred to C3H males, were given i.p. injections of 40 mg ENU/kg bw in a single dose on day 18 of gestation, in a split dose of 6 mg ENU/kg bw on days 12 through 18 of gestation, or DMSO vehicle alone. Groups of pups were necropsied on days 10, 13, 15 (single dose only), 17, 20, 40, and 70 postpartum for T-cell isolations and hprt Mf measurements using the T-cell cloning assay. The time required to reach maximum Mfs in T-cells isolated from thymus of transplacentally treated animals was 2 weeks, the same time span as previously observed after ENU treatment of adult, weanling, and preweanling mice. Mfs in T-cells isolated from spleens of control animals averaged 2.1 +/- 0.3 (SE) x 10(-6). In spleens of mice treated transplacentally with ENU in a single dose, Mfs reached a maximum at 15 days postpartum [84.7 +/- 15.8 (SE) x 10(-6)] and decreased to lower but still elevated levels at 40 days postpartum. In spleens of mice treated transplacentally with ENU in a split dose, Mfs reached a maximum at 13 days postpartum [74.0 +/- 16.3 (SE) x 10(-6)] and decreased to background levels at 40 days postpartum. The areas under the curves describing the change in hprt Mfs over time for ENU-treated vs. control mice estimate the mutagenic potency for transplacental single- and split-dose exposures to be 1.9 and 0.8 x 10(3), respectively. Comparison of the mutagenic potency estimates for mice exposed to ENU in utero to 4-week-old mice given a similar dose of the same lot number of ENU indicates that the mouse is more susceptible to ENU-induced mutagenesis during fetal life.

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 genotoxic 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 cell line, TK6, following in vitro exposures to AZT. Cells were cultured in medium containing 0 or 300 microM AZT for 1, 3, or 6 day(s) (n = 5/group). The effects of exposure duration on incorporation of AZT into DNA and HPRT mutant frequency were determined using an AZT radioimmunoassay and a cell cloning assay, respectively. AZT accumulated in DNA in a supralinear manner, approaching 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 significantly 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 from untreated cells versus AZT-treated cells. Molecular analyses of AZT-induced mutations revealed a significant difference in the frequency of total gene 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). These data indicate that the primary mechanism of AZT mutagenicity in human TK6 cells is through the production of large deletions which occur as a result of AZT incorporation into DNA and subsequent chain termination. The data imply that perinatal chemoprophylaxis with AZT may put children of HIV-infected women at potential risk for genetic damage.

Detection of cyclophosphamide-induced mutations at the Hprt but not the lacI locus in splenic lymphocytes of exposed mice.

The relative sensitivities and specificities of the endogenous Hprt gene and the lacI transgene as mutational targets were evaluated in splenic lymphocytes from male standard B6C3F1 mice (only Hprt assayed) and from lacI transgenic B6C3F1 mice treated at 6-7 weeks- of-age with the indirect-acting agent, cyclophosphamide (CP). To define the effects of the time elapsed since CP treatment on Hprt mutant frequencies (Mfs), nontransgenic mice were given single i.p. injections of 25 mg CP/kg or vehicle (PBS) alone and then necropsied 2, 4, 6, 8, or 10 weeks after treatment. Peak Mfs were found at 6 weeks postexposure, with mean Mf values ranging from 2.27 to 3.27 x 10(-5) using two different lots of CP in standard packaging (compared with mean control Mf values of 0.14 to 0.26 x 10(-5) in various experiments). To determine the dose response for Hprt Mfs, nontransgenic mice were given single doses of 0, 12.5, 25, 50, or 100 mg CP/kg and necropsied 4 weeks postexposure. These treatments produced a supralinear dose response curve for CP-induced Hprt Mfs. Based on these experiments, CP mutagenicities at Hprt and lacI were compared in transgenic mice treated with 0, 25, or 100 mg CP/kg (using another lot of CP in ISOPAC((R)) bottles; Sigma) and necropsied 6 weeks later. There was a significant increase in Hprt Mfs in treated transgenic mice (100 mg CP/kg: 0.75 +/- 0.09 x 10(-5); 25 mg CP/kg: 0.39 +/- 0.05 x 10(-5)) versus controls (0.10 +/- 0.01 x 10(-5)); however, the Mfs in lacI of lymphocytes from the same CP-treated animals were not significantly different from controls (100 mg CP/kg: 9.4 +/- 1.1 x 10(-5); 25 mg CP/kg: 6.7 +/- 0. 8 x 10(-5); control: 7.7 +/- 0.7 x 10(-5)). Hprt mutational spectra data in CP-treated transgenic and nontransgenic mice were different from those of control mice, whereas the spectra of mutations in lacI of lymphocytes from Big Blue((R)) transgenic mice were not significantly changed after CP treatment. These data indicate that, under these treatment conditions, CP-induced mutations in splenic lymphocytes were detectable in the Hprt gene but not the lacI transgene of this nontarget tissue for CP-induced cancer.