Dale M. Walker

Mechanistic Basis for Nonlinear Dose-Response Relationships for Low-Dose Radiation-Induced Stochastic Effects

The linear nonthreshold (LNT) model plays a central role in low-dose radiation risk assessment for humans. With the LNT model, any radiation exposure is assumed to increase ones risk of cancer. Based on the LNT model, others have predicted tens of thousands of deaths related to environmental exposure to radioactive material from nuclear accidents (e.g., Chernobyl) and fallout from nuclear weapons testing. Here, we introduce a mechanism-based model for low-dose, radiation-induced, stochastic effects (genomic instability, apoptosis, mutations, neoplastic transformation) that leads to a LNT relationship between the risk for neoplastic transformation and dose only in special cases. It is shown that nonlinear dose-response relationships for risk of stochastic effects (problematic nonlethal mutations, neoplastic transformation) should be expected based on known biological mechanisms. Further, for low-dose, low-dose rate, low-LET radiation, large thresholds may exist for cancer induction. We summarize previously published data demonstrating large thresholds for cancer induction. We also provide evidence for low-dose-radiation-induced, protection (assumed via apoptosis) from neoplastic transformation. We speculate based on work of others (Chung 2002) that such protection may also be induced to operate on existing cancer cells and may be amplified by apoptosis-inducing agents such as dietary isothiocyanates.

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 perinatal 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 γ and/or other mitochondrial elements) and leads over time to delayed, progressive cardiotoxicity.

Mutagenicity at the Hprt locus in T cells of female mice following inhalation exposures to low levels of 1,3-butadiene.

A study was conducted to test the hypothesis that repeated low level exposures to 1,3-butadiene (BD), approaching the OSHA occupational threshold for this chemical, produce a significant mutagenic response in mice. Female B6C3F1 mice (4-5 weeks of age) were exposed by inhalation for 2 weeks (6 h/day, 5 days/week) to 0 or 3 ppm BD, and then necropsied at 4 weeks after the cessation of exposures to measure the frequency of mutations (MF) at the Hprt locus using the T-lymphocyte clonal assay. At necropsy, T cells were isolated from spleen and cultured in the presence of mitogen, growth factors, and a selection agent. Cells were scored for growth on days 8-9 after plating to determine cloning efficiencies (CEs) and Hprt MFs. There was a marginal but significant reduction in the growth of splenic T cells from mice exposed to 3 ppm (n=27) compared with control mice (n=24) (P=0.004), suggesting the occurrence of BD-induced cytotoxicity at this low exposure concentration. In addition, the average Hprt MF in mice exposed to 3 ppm BD [1.54+/-0.82 (S.D.)x10(-6)] was significantly increased by 1.6-fold over the average control value of 0.96+/-0.51 (S.D.)x10(-6) (P=0.004). Comparisons of these data to earlier Hprt mutagenicity studies of mice exposed to high concentrations of BD (where significant mutagenic but not cytotoxic effects were observed) indicate that the ability to detect the cytotoxic and mutagenic responses of T cells to low levels of BD was enhanced by using a much larger sample size than usual for both the control and treatment groups. Additional analyses of the quantitative relationships between CE and MF demonstrated that CE had no significant effect upon MF values in sham-exposed control mice or mice exposed to low-level BD. Furthermore, the approaches for assessing the impact of CE and clonality on Hprt MFs in these control and BD-exposed mice were applied with the same rigor as in in vivo Hprt mutagenicity studies in human children. The overall study results support the conclusion that short-term low-level BD exposure is mutagenic in the mouse.

WR1065 mitigates AZT‐ddI‐induced mutagenesis and inhibits viral replication

The success of nucleoside reverse transcriptase inhibitors (NRTIs) in treating HIV‐1 infection and reducing mother‐to‐child transmission of the virus during pregnancy is accompanied by evidence that NRTIs cause long‐term health risks for cancer and mitochondrial disease. Thus, agents that mitigate toxicities of the current combination drug therapies are needed. Previous work had shown that the NRTI‐drug pair zidovudine (AZT)–didanosine (ddI) was highly cytotoxic and mutagenic; thus, we conducted preliminary studies to investigate the ability of the active moiety of amifostine, WR1065, to protect against the deleterious effects of this NRTI‐drug pair. In TK6 cells exposed to 100 μM AZT‐ddI (equimolar) for 3 days with or without 150 μM WR1065, WR1065 enhanced long‐term cell survival and significantly reduced AZT‐ddI‐induced mutations. Follow‐up studies were conducted to determine if coexposure to AZT and WR1065 abrogated the antiretroviral efficacy of AZT. In human T‐cell blasts infected with HIV‐1 in culture, inhibition of p24 protein production was observed in cells treated with 10 μM AZT in the absence or presence of 5–1,000 μM WR1065. Surprisingly, WR1065 alone exhibited dose‐related inhibition of HIV‐1 p24 protein production. WR1065 also had antiviral efficacy against three species of adenovirus and influenza A and B. Intracellular levels of unbound WR1065 were measured following in vitro/in vivo drug exposure. These pilot study results indicate that WR1065, at low intracellular levels, has cytoprotective and antimutagenic activities against the most mutagenic pair of NRTIs and has broad spectrum antiviral effects. These findings suggest that the activities have a possible common mode of action that merits further investigation. Environ. Mol. Mutagen. 2009.

Low-Dose Radiation and Genotoxic Chemicals Can Protect Against Stochastic Biological Effects

A protective apoptosis-mediated (PAM) process that is turned on in mammalian cells by low-dose photon (X and γ) radiation and appears to also be turned on by the genotoxic chemical ethylene oxide is discussed. Because of the PAM process, exposure to low-dose photon radiation (and possibly also some genotoxic chemicals) can lead to a reduction in the risk of stochastic effects such as problematic mutations, neoplastic transformation (an early step in cancer occurrence), and cancer. These findings indicate a need to revise the current low-dose risk assessment paradigm for which risk of cancer is presumed to increase linearly with dose (without a threshold) after exposure to any amount of a genotoxic agent such as ionizing radiation. These findings support a view seldom mentioned in the past, that cancer risk can actually decrease, rather than increase, after exposure to low doses of photon radiation and possibly some other genotoxic agents. The PAM process (a form of natural protection) may contribute substantially to cancer prevention in humans and other mammals. However, new research is needed to improve our understanding of the process. The new research could unlock novel strategies for optimizing cancer prevention and novel protocols for low-dose therapy for cancer. With low-dose cancer therapy, normal tissue could be spared from severe damage while possibly eliminating the cancer.

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 ∼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. Environ. Mol. Mutagen., 2009.

Peripheral Nerve Sheath Tumors, Rat

Peripheral nerve sheath tumors are round to oval, soft to firm, discrete, unencapsulated masses which range up to 10 cm along the longest axis. The cut surface is usually smooth, shiny, and translucent or white. Subcutaneous tumors may have an ulcerated surface.

The stress response resolution assay. I. Quantitative assessment of environmental agent/condition effects on cellular stress resolution outcomes in epithelium

The events or factors that lead from normal cell function to conditions and diseases such as aging or cancer reflect complex interactions between cells and their environment. Cellular stress responses, a group of processes involved in homeostasis and adaptation to environmental change, contribute to cell survival under stress and can be resolved with damage avoidance or damage tolerance outcomes. To investigate the impact of environmental agents/conditions upon cellular stress response outcomes in epithelium, a novel quantitative assay, the “stress response resolution” (SRR) assay, was developed. The SRR assay consists of pretreatment with a test agent or vehicle followed later by a calibrated stress conditions exposure step (here, using 6‐thioguanine). Pilot studies conducted with a spontaneously‐immortalized murine mammary epithelial cell line pretreated with vehicle or 20 µg N‐ethyl‐N‐nitrososurea/ml medium for 1 hr, or two hTERT‐immortalized human bronchial epithelial cell lines pretreated with vehicle or 100 µM zidovudine/lamivudine for 12 days, found minimal alterations in cell morphology, survival, or cell function through 2 weeks post‐exposure. However, when these pretreatments were followed 2 weeks later by exposure to calibrated stress conditions of limited duration (for 4 days), significant alterations in stress resolution were observed in pretreated cells compared with vehicle‐treated control cells, with decreased damage avoidance survival outcomes in all cell lines and increased damage tolerance outcomes in two of three cell lines. These pilot study results suggest that sub‐cytotoxic pretreatments with chemical mutagens have long‐term adverse impact upon the ability of cells to resolve subsequent exposure to environmental stressors. Environ. Mol. Mutagen. 54:268–280, 2013.

Frequencies of micronucleated reticulocytes, a dosimeter of DNA double-strand breaks, in infants receiving computed tomography or cardiac catheterization

The use of computed tomography (CT scans) has increased dramatically in recent decades, raising questions about the long-term safety of CT-emitted x-rays especially in infants who are more sensitive to radiation-induced effects. Cancer risk estimates for CT scans typically are extrapolated from models; therefore, new approaches measuring actual DNA damage are needed for improved estimations. Hence, changes in a dosimeter of DNA double-strand breaks, micronucleated reticulocytes (MN-RETs) measured by flow cytometry, were investigated in mice and infants exposed to CT scans. In male C57BL/6N mice (6-8 weeks-of-age), there was a dose-related increase in MN-RETs in blood samples collected 48h after CT scans delivering targeted exposures of 1-130 cGy x-rays (n=5-10/group, r=0.994, p=0.01), with significant increases occurring at exposure levels as low as 0.83 cGy x-rays compared to control mice (p=0.002). In paired blood specimens from infants with no history of a prior CT scan, there was no difference in MN-RET frequencies found 2h before (mean, 0.10±0.07%) versus 48h after (mean, 0.11±0.05%) a scheduled CT scan/cardiac catheterization. However, in infants having prior CT scan(s), MN-RET frequencies measured at 48h after a scheduled CT scan (mean=0.22±0.12%) were significantly higher than paired baseline values (mean, 0.17±0.07%; p=0.032). Increases in baseline (r=0.722, p<0.001) and 48-h post exposure (r=0.682, p<0.001) levels of MN-RETs in infants with a history of prior CT scans were significantly correlated with the number of previous CT scans. These preliminary findings suggest that prior CT scans increase the cellular responses to subsequent CT exposures. Thus, further investigation is needed to characterize the potential cancer risk from single versus repeated CT scans or cardiac catheterizations in infants.

Antiretroviral activity of aminothiols, WR2721 and WR1065

The aminothiol drug WR2721 (amifostine, Ethyol), and its free-thiol metabolite WR1065, are cytoprotective agents. Ethyol is licensed to attenuate toxicity in cancer patients receiving chemo- and radiation-therapy. We found that WR1065 mitigates Zidovudine (AZT)-induced mutagenesis, and designed studies to ascertain that AZT antiretroviral activity was not altered in the presence of this aminothiol. Fresh peripheral blood mononuclear cells were cultured with phytohemaglutinin (PHA) for 48 hr. The resulting PHA-stimulated blasts were infected for 2 hr with HIV-1BZ-167 before the addition of AZT, WR1065, or Ethyol dephosphorylated by preincubation with alkaline phosphatase. Cells were cultured for an additional 72 hr and assayed for cytotoxicity (trypan blue) and HIV-1 replication (p24 ELISA kit). AZT concentrations of 0.8, 3.0, and 5.0 μM inhibited virus replication by 85–96%, and the same AZT doses combined with 5–26 μM WR1065 inhibited virus replication by 91–97%. When tested alone, AZT at 7.75 μM inhibited virus replication by 96.7 ± 5.1% (mean ± SD, n = 4) with 54 ± 15% target cell survival. Ethyol at 50 μM inhibited virus replication by 75.4 ± 16.6% (mean ± SD, n = 4) and cell survival was 53 ± 19%. Similarly, WR1065 concentrations of 50 and 100 μM blocked virus replication by 83.2 ± 18.6% (n = 5) and 92.0 ± 11.0% (n = 5), respectively, with cell survival at 55 ± 22% and 42 ± 18%, respectively. The data show that Ethyol and WR1065 do not compromise the antiretroviral efficacy of AZT. Interestingly, when used alone, these aminothiols also substantially inhibit HIV-1 replication. (Supported in part by R01 CA95741 to VEW; patent pending).