Relative mutagenic potencies of several nucleoside analogs, alone or in drug pairs, at the HPRT and TK loci of human TK6 lymphoblastoid cells.

Experiments were performed to investigate the impact of didanosine (ddI), lamivudine (3TC), and stavudine (d4T) on cell survival and mutagenicity in two reporter genes, hypoxanthine-guanine phosphoribosyltransferase (HPRT) and thymidine kinase (TK), using a cell cloning assay for assessing the effects of individual nucleoside analogs (NRTIs)/drug combinations in human TK6 B-lymphoblastoid cells. Three-day treatments with 0, 33, 100, or 300 microM ddI, 3TC, or ddI-3TC produced positive trends for increased HPRT and TK mutant frequencies. While dose-related trends were too small to reach significance after treatments with d4T or d4T-3TC, pairwise comparisons with control cells indicated that exposure to 100 microM d4T or d4T-3TC caused significant elevations in HPRT mutants. Measurements of mutagenicity in cells exposed to d4T (or d4T-3TC) were complicated by the cytotoxicity of this NRTI. Enhanced increases in mutagenic responses to combined NRTI treatments, compared with single drug treatments, occurred as additive to synergistic effects in the HPRT gene of cells exposed to 100 microM ddI-3TC or 100 microM d4T-3TC, and in the TK gene of cells exposed to 100 or 300 microM ddI-3TC. Comparisons of these data to mutagenicity studies of other NRTIs in the same system (Meng Q et al. [2000c]: Proc Natl Acad Sci USA 97:12667-126671; Torres SM et al. [2007]: Environ Mol Mutagen) indicate that the relative mutagenic potencies for all drugs tested to date are: AZT-ddI > ddI-3TC > AZT-3TC congruent with AZT-3TC-ABC (abacavir) > AZT >/=ddI > d4T-3TC > 3TC > d4T >/= ABC. These collective data suggest that all NRTIs with antiviral activity against HIV-1 may cause host cell DNA damage and mutations, and impose a cancer risk.

Mutagenicity of zidovudine, lamivudine, and abacavir following in vitro exposure of human lymphoblastoid cells or in utero exposure of CD-1 mice to single agents or drug combinations.

Experiments were performed to investigate the impact of zidovudine (AZT), lamivudine (3TC), and abacavir (ABC) on cell survival and mutagenicity in two reporter genes, hypoxanthine-guanine phosphoribosyltransferase (HPRT) and thymidine kinase (TK), using cell cloning assays for assessing the effects of individual drugs/drug combinations in (1) TK6 human lymphoblastoid cells exposed in vitro and (2) splenic lymphocytes from male CD-1 mice exposed transplacentally on days 12-18 of gestation. In TK6 cells, dose-related increases in HPRT and TK mutant frequencies were found following 3 days of exposure to AZT or 3TC alone (33, 100, or 300 microM), or to equimolar amounts of AZT-3TC. Compared with single drug exposures, AZT-3TC coexposures generally yielded enhanced elevations in HPRT and TK mutant frequencies. Mutagenicity experiments with ABC alone, or in combination with AZT-3TC, were complicated by the extreme cytotoxicity of ABC. Exposure of cells either to relatively high levels of AZT-3TC short-term (100 microM, 3 days), or to peak plasma-equivalent levels of AZT-3TC for an extended period (10 microM, 30 days), resulted in similar drug-induced mutagenic responses. Among sets of mice necropsied on days 13, 15, or 21 postpartum, Hprt mutant frequencies in T-cells were significantly elevated in the AZT-only (200 mg/kg bw/day) and AZT-3TC (200 mg AZT + 100 mg 3TC/kg bw/day) groups at 13 days of age. These results suggest that the mutagenicity by these nucleoside analogs is driven by cumulative dose, and raises the question of whether AZT-3TC has greater mutagenic effects than AZT alone in perinatally exposed children.

Mitochondrial toxicity in hearts of CD-1 mice following perinatal exposure to AZT, 3TC, or AZT/3TC in combination.

Antiretroviral therapies based on nucleoside reverse transcriptase inhibitors (NRTIs), like zidovudine (3'-azido-3'-deoxythymidine; AZT) and lamivudine ((-)2',3'-dideoxy-3'-thiacytidine; 3TC), markedly reduce mother-to-child transmission of the human immunodeficiency virus (HIV). However, AZT induces damage in nuclear DNA of mice exposed in utero and postnatally, and mitochondrial DNA (mtDNA) damage has been observed in both human and mouse neonates following perinatal exposure to AZT and AZT/3TC in combination. To provide animal data modeling the NRTI-induced heart damage reported in human infants, we treated pregnant CD-1 mice throughout gestation and treated their pups by direct gavage from postnatal day (PND) 4 through PND 28 with daily doses of 150 mg/kg body weight (bw)/day AZT, 75 mg/kg bw/day 3TC, 125/62.5 mg/kg bw/day AZT/3TC, or the vehicle control. Half the pups were euthanized on PND 28; the remainder received no further dosing, and were euthanized at week 10. Heart tissue was collected, total DNA was extracted, and mtDNA copy number relative to nuclear DNA copy number, mtDNA damage, and mtDNA mutation assays were performed using PCR-based methods. Analyses revealed increases in mtDNA lesions in 4-week-old males and females treated with AZT or 3TC, but not in 10-week-old mice, suggesting that the damage resolved after treatment ceased. Interestingly, 10-week-old females treated with AZT/3TC had significant increases in mtDNA damage. Point mutations were elevated in 10-week-old females treated with AZT or AZT/3TC, but not 3TC; no increases in mutations were seen in either gender at 4 weeks of age. Our data suggest that AZT/3TC combination treatment produces greater mtDNA damage than either agent individually, and that female mice are more sensitive than males to AZT/3TC-induced mtDNA damage.

Age-, gender-, and species-dependent mutagenicity in T cells of mice and rats exposed by inhalation to 1,3-butadiene.

Experiments were performed: (i) to investigate potential age- and gender-dependent differences in mutagenic responses in T cells following exposures of B6C3F1 mice and F344 rats by inhalation for 2 weeks to 0 or 1250 ppm butadiene (BD), and (ii) to determine if exposures for 2 weeks to 62.5 ppm BD produce a mutagenic effect in female rats. To evaluate the effect of age on mutagenic response, mutant manifestation curves for splenic T cells of female mice exposed at 8-9 weeks of age were defined by measuring Hprt mutant frequencies (MFs) at multiple time points after BD exposure using a T cell cloning assay and comparing the resulting mutagenic potency estimate (calculated as the difference of areas under the mutant manifestation curves of treated versus control animals) to that reported for female mice exposed to BD in the same fashion beginning at 4-5 weeks of age. The shapes of the mutant T cell manifestation curves for spleens were different [e.g., the maximum BD-induced MFs in older mice (8.0+/-1.0 [S.D.]x10(-6)) and younger mice (17.8+/-6.1 x 10(-6)) were observed at 8 and 5 weeks post-exposure, respectively], but the mutagenic burden was the same for both age groups. To assess the effect of gender on mutagenic response, female and male rodents were exposed to BD at 4-5 weeks of age and Hprt MFs were measured when maximum MFs are expected to occur post-exposure. The resulting data demonstrated that the pattern for mutagenic susceptibility from high-level BD exposure is female mice>male mice>female rats>male rats. Exposures of female rats to 62.5 ppm BD caused a minor but significant mutagenic response compared with controls (n=16/group; P=0.03). These results help explain part of the differing outcomes/interpretations of data in earlier Hprt mutation studies in BD-exposed rodents.

In situ analysis of interleukin-1-induced transcription of cox-2 and il-8 in cultured human myometrial cells.

The specific binding of transcription factors to DNA has been shown to be inhibited by chromatin structure and increased by cooperative interactions with other proteins. Consequently, in situ analysis using chromatin immunoprecipitation offers the most accurate view of transcriptional control. Transient transfection studies and in vitro analyses of IL-1-induced cox-2 transcription in a number of cell types have indicated regulation by either nuclear factor kappa B (NF-kappa B) or CCAAT/enhancer binding protein (C/EBP beta), or both acting cooperatively. To determine the mechanisms of COX-2 (cyclooxygenase or prostaglandin endoperoxide synthase) induction in cultured human myometrial cells in situ, we examined the cross-linking of the RelA subunit of NF-kappa B and C/EBP beta to the cox-2 promoter and flanking sequences. As a control, we inspected the interaction of these transcription factors with the IL-8 gene, which has been shown in other cell types to be activated by the cooperative interaction of NF-kappa B and C/EBP beta. Indeed, both transcription factors were cross-linked to the il-8 promoter after IL-1 treatment, but only RelA was cross-linked to cox-2 DNA. The il-8 promoter was also found to physically interact with proteins cross-linked to sites further upstream. IL-1 treatment also increased polymerase II cross-linking to both promoters and increased histone H4 acetylation at specific sites. These results indicate that modification of chromatin structure is part of the response to IL-1 stimulation. Chromatin immunoprecipitation thus provides critical insight into the mechanisms of COX-2 and IL-8 expression in human myometrial cells.

Persistence of mitochondrial toxicity in hearts of female B6C3F1 mice exposed in utero to 3'-azido-3'-deoxythymidine.

Cardiac toxicity has been associated with HIV infection and exposure to nucleoside reverse transcriptase inhibitors (NRTIs), but the role of the latter in the development of cardiac disease of HIV-infected patients is uncertain. To investigate the cardiotoxicity of transplacentally administered zidovudine (AZT) or AZT plus lamivudine (3TC) in the absence of HIV infection, we evaluated several biomarkers of cardiac mitochondrial structure and cardiac structure and function in a B6C3F1 mouse model. In utero exposure to AZT-3TC resulted in ultrastructural pathology, loss of mitochondria, and altered echocardiographic measurements in newborn mice. Cardiac pathology and dysfunction persisted into the adult life of female mice exposed in utero to AZT, as evidenced by significant dose-dependent heart enlargement, clusters of atypical mitochondria and myofibril alterations, significantly increased cytochrome c oxidase activity, and significantly higher numbers of mutations in mitochondrial tRNA genes compared with unexposed controls at 18 to 24 mo of age. These data led to the hypothesis that the long-term pathology of peri-natal exposure to these NRTIs is related to persistent mitochondrial DNA mutations in cardiac tissue; that is, the primary damage during drug treatment is mutational (as opposed to affecting polymerase gamma and/or other mitochondrial elements) and leads over time to delayed, progressive cardiotoxicity.

Mutational analysis of the mitochondrial tRNA genes and flanking regions in umbilical cord tissue from uninfected infants receiving AZT-based therapies for prophylaxis of HIV-1.

A sensitive vertical denaturing gradient gel electrophoresis (DGGE) method, using 13 unipolar psoralen-clamped PCR primer pairs, was developed for detecting sequence variants in the 22 tRNA genes and flanking regions (together spanning approximately 21%) of the human mitochondrial genome. A study was conducted to determine (i) if mitochondrial DNA (mtDNA) polymorphisms and/or mutations were detectable in healthy newborns and (ii) if prepartum 3'-azido-2',3'-dideoxythymidine (AZT) based HIV-1 prophylaxis was associated with significant increases in mtDNA mutations and changes in the degree of heteroplasmy of sequence variants in uninfected infants born to HIV-1-infected mothers. DGGE analysis of umbilical cord tissue (where vascular endothelium and smooth muscle cells are the major source of mtDNA) showed that mtDNA sequence variants were significantly elevated by threefold in AZT-treated infants compared with unexposed controls (P < 0.001), with 24 changes observed in 19/52 (37%) treated newborns (averaging 0.46 changes/subject) versus only eight changes found in 7/55 (13%) unexposed newborns (averaging 0.15 changes/subject). Six distinct sequence variants occurring in unexposed controls were predominately synonymous and homoplasmic, representing previously reported polymorphisms. Uninfected infants exposed to a combination of AZT and 2',3'-dideoxy-3'-thiacytidine and "maternal HIV-1" had a significant shift in the spectrum of mutations (P = 0.04) driven by increases in nonsynonymous heteroplasmic sequence variants at polymorphic sites (10 distinct variants) and novel sites (four distinct variants). While the weight of evidence suggests that prepartum AZT-based prophylaxis produces mtDNA mutations, additional research is needed to determine the degree to which fetal responses to maternal HIV-1 infection, in the absence of antiretroviral treatment, contribute to prenatal mtDNA mutagenesis.

Interleukin-1-induced NF-kappaB recruitment to the oxytocin receptor gene inhibits RNA polymerase II-promoter interactions in cultured human myometrial cells.

The myometrial oxytocin receptor (OTR) is highly regulated during pregnancy, reaching maximal concentrations near term. These levels are then abruptly reduced in advanced labour and the post-partum period. Our goal was to examine the molecular basis for this reduction, using chromatin immunoprecipitation (ChIP). Interleukin-1alpha (IL1A) treatment of cultured human myometrial cells has previously been shown to reduce steady-state levels of OTR mRNA. We show further that IL1A reduced RNA polymerase II cross-linking to the otr promoter, as reflective of transcriptional inhibition. IL1A also increased the recruitment of nuclear factor kappaB (NF-kappaB) to a site 955 bp upstream from the transcriptional start site. Inhibition of NF-kappaB activation negated the effects of IL1A on polymerase II dissociation, indicating a causal relationship, at least in part, between recruitment of NF-kappaB and detachment of polymerase from the otherwise constitutively active otr promoter. IL1A treatment also resulted in increased histone H4 acetylation in the otr promoter region. Whereas NF-kappaB recruitment and histone acetylation are generally associated with activation of gene expression, our findings show that both processes can be involved in dissociation of RNA polymerase II from an active promoter. The results of these studies suggest that the elevation of IL1 in the myometrium occurring at the end of pregnancy initiates the process of down-regulation of OTRs in advanced labour, resulting in the desensitization of the myometrium to elevated levels of OT in the blood during lactation.