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.

Hypoxanthine-guanine phosphoribosyltransferase reporter gene mutation for analysis of in vivo clonal amplification in patients with HTLV type 1-associated Myelopathy/Tropical spastic paraparesis.

We tested a surrogate selection approach utilizing mutation at a reporter gene [hypoxanthine-guanine phosphoribosyltransferase (hprt)] as a probe for in vivo cell division, for detection of clonal T cell expansion in human T lymphotropic (HTLV-1) carriers. Peripheral blood samples from HTLV-1-infected individuals with HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) were tested to determine the hprt mutant frequency (Mf). Wild-type and hprt mutant T cell clones were isolated, and clonal identity determined by multiplex PCR and DNA sequencing of T cell receptor (TCR) variable region beta-chain (TCR BV) and third complementarity determining regions (CDR3). Seven samples from HAM/TSP patients were tested, and Mfs were within the normal range for adults (mean 11.3 x 10(-6), max 22.4 x 10(-6), min 5.6 x 10(-6)). The frequency of HTLV-1 infection in wild-type and hprt mutant T cells from HAM/TSP patients was determined to identify enrichment in the mutant fraction of cells. This analysis was performed on 196 isolates from 6 individuals with HAM/TSP. In each case, there is enrichment for virally infected cells in the hprt mutant fraction of isolates. Ten mutant and eight wild-type isolates from sample LS42A (Mf 8.4 x 10(-6)) were tested for clonality by TCR BV PCR and sequencing. Of the 10 hprt mutants, there were two in vivo-expanded clones (four isolates with two identical TCRs, or 80% unique TCR sequences). These studies may provide new insights into the precise mechanism of HTLV-1 leukemogenesis, and aid in the study of mutator phenotypes generated by a combination of Tax-mediated in vivo expansion and mutagenesis.

Mitochondrial genome damage associated with cigarette smoking.

We have investigated the level of mitochondrial DNA (mtDNA) damage and deletions in bronchoalveolar lavage tissues from smokers and nonsmokers using quantitative, extra-long PCR and a "common" mtDNA deletion assay. Smokers had 5.6 times the level of mtDNA damage, 2.6 times the damage at a nuclear locus (beta-globin gene cluster), and almost 7 times the level of a 4.9-kb mtDNA deletion compared to nonsmokers, although the latter increase was not significant. Although both genomes (mitochondrial and nuclear) showed significantly increased levels of DNA damage in smokers (mtDNA P = 0.00072; beta-globin P = 0.0056), the relative differences were greatest in the mtDNA. Damage to the mtDNA may inhibit oxidative phosphorylation and, therefore, potentially cause or contribute to chronic lung disease and cancer. Consequently, the mtDNA may be a sensitive biomarker for environmentally induced genetic damage and mutation.