Implications of recent epidemiologic studies for the linear nonthreshold model and radiation protection.

The recently published NCRP Commentary No. 27 evaluated the new information from epidemiologic studies as to their degree of support for applying the linear nonthreshold (LNT) model of carcinogenic effects for radiation protection purposes (NCRP 2018 Implications of Recent Epidemiologic Studies for the Linear Nonthreshold Model and Radiation Protection, Commentary No. 27 (Bethesda, MD: National Council on Radiation Protection and Measurements)). The aim was to determine whether recent epidemiologic studies of low-LET radiation, particularly those at low doses and/or low dose rates (LD/LDR), broadly support the LNT model of carcinogenic risk or, on the contrary, demonstrate sufficient evidence that the LNT model is inappropriate for the purposes of radiation protection. An updated review was needed because a considerable number of reports of radiation epidemiologic studies based on new or updated data have been published since other major reviews were conducted by national and international scientific committees. The Commentary provides a critical review of the LD/LDR studies that are most directly applicable to current occupational, environmental and medical radiation exposure circumstances. This Memorandum summarises several of the more important LD/LDR studies that incorporate radiation dose responses for solid cancer and leukemia that were reviewed in Commentary No. 27. In addition, an overview is provided of radiation studies of breast and thyroid cancers, and cancer after childhood exposures. Non-cancers are briefly touched upon such as ischemic heart disease, cataracts, and heritable genetic effects. To assess the applicability and utility of the LNT model for radiation protection, the Commentary evaluated 29 epidemiologic studies or groups of studies, primarily of total solid cancer, in terms of strengths and weaknesses in their epidemiologic methods, dosimetry approaches, and statistical modelling, and the degree to which they supported a LNT model for continued use in radiation protection. Recommendations for how to make epidemiologic radiation studies more informative are outlined. The NCRP Committee recognises that the risks from LD/LDR exposures are small and uncertain. The Committee judged that the available epidemiologic data were broadly supportive of the LNT model and that at this time no alternative dose-response relationship appears more pragmatic or prudent for radiation protection purposes.

Scope of radiological protection control measures.

In this report, the Commission recommends approaches to national authorities for their definition of the scope of radiological protection control measures through regulations, by using its principles of justification and optimisation. The report provides advice for deciding the radiation exposure situations that should be covered by the relevant regulations because their regulatory control can be justified, and, conversely, those that may be considered for exclusion from the regulations because their regulatory control is deemed to be unamenable and unjustified. It also provides advice on the situations resulting from regulated circumstances but which may be considered by regulators for exemption from complying with specific requirements because the application of these requirements is unwarranted and exemption is the optimum option. Thus, the report describes exclusion criteria for defining the scope of radiological protection regulations, exemption criteria for planned exposure situations, and the application of these concepts in emergency exposure situations and in existing exposure situations. The report also addresses specific exposure situations such as exposure to low-energy or low-intensity adventitious radiation, cosmic radiation, naturally occurring radioactive materials, radon, commodities, and low-level radioactive waste. The quantitative criteria in the report are intended only as generic suggestions to regulators for defining the regulatory scope, in the understanding that the definitive boundaries for establishing the situations that can be or need to be regulated will depend on national approaches.

A novel protein C-factor VII chimera provides new insights into the structural requirements for cytoprotective protease-activated receptor 1 signaling.

Essentials The basis of cytoprotective protease-activated receptor 1 (PAR1) signaling is not fully understood. Activated protein C chimera (APCFVII-82 ) was used to identify requirements for PAR1 signaling. APCFVII-82 did not initiate PAR1 signaling, but conferred monocyte anti-inflammatory activity. APC-specific light chain residues are required for cytoprotective PAR1 signaling. SUMMARY: Background Activated protein C (APC) cell signaling is largely reliant upon its ability to mediate protease-activated receptor (PAR) 1 proteolysis when bound to the endothelial cell (EC) protein C (PC) receptor (EPCR). Furthermore, EPCR-bound PC modulates PAR1 signaling by thrombin to induce APC-like EC cytoprotection. Objective The molecular determinants of EPCR-dependent cytoprotective PAR1 signaling remain poorly defined. To address this, a PC-factor VII chimera (PCFVII-82 ) possessing FVII N-terminal domains and conserved EPCR binding was characterized. Methods Activated PC-FVII chimera (APCFVII-82 ) anticoagulant activity was measured with calibrated automated thrombography and activated FV degradation assays. APCFVII-82 signaling activity was characterized by the use of reporter assays of PAR1 proteolysis and EC barrier integrity. APCFVII-82 anti-inflammatory activity was assessed according to its inhibition of nuclear factor-κB (NF-κB) activation and cytokine secretion from monocytes. Results PCFVII-82 was activated normally by thrombin on ECs, but was unable to inhibit plasma thrombin generation. Surprisingly, APCFVII-82 did not mediate EPCR-dependent PAR1 proteolysis, confer PAR1-dependent protection of thrombin-induced EC barrier disruption, or limit PAR1-dependent attenuation of interleukin-6 release from lipopolysaccharide (LPS)-stimulated macrophages. Interestingly, EPCR occupation by active site-blocked APCFVII-82 was, like FVII, unable to mimic EC barrier stabilization induced by PC upon PAR1 proteolysis by thrombin. APCFVII-82 did, however, diminish LPS-induced NF-κB activation and tumor necrosis factor-α release from monocytes in an apolipoprotein E receptor 2-dependent manner, with similar efficacy as wild-type APC. Conclusions These findings identify a novel role for APC light chain amino acid residues outside the EPCR-binding site in enabling cytoprotective PAR1 signaling.

Isolation and characterization of a 1-beta-D-arabinofuranosylcytosine-resistant Chinese hamster ovary cell mutant that is also X-ray sensitive and is noncomplementary with ataxia telangiectasia cells.

In order to study the mechanism of induction of mutations and chromosome aberrations by ionizing radiations, it is particularly useful to have available radiation-sensitive mutants. While several X-ray-sensitive rodent cell lines are available, they have been selected rather nonspecifically. It was determined that selection for resistance to the DNA replication/repair inhibitor, 1-beta-D-arabinofuranosylcytosine (ara-C), would permit production of a set of X-ray-sensitive mutant cell lines that would be defective in the resynthesis step of excision or recombination repair. Such mutant cells could also be used for the isolation and characterization of human DNA repair genes. In particular, it was predicted that the repair gene defective in individuals with ataxia telangiectasia (AT) might be amenable to study with ara-C-resistant (X-ray-sensitive) mutants, since additional studies, presented here, have shown that AT cells are resistant to ara-C. In the long term, it is hoped that determining the specific defect in AT might lead to an understanding of the possible role of defective repair in tumor induction and/or progression. The general approach used to isolate ara-C-resistant Chinese hamster ovary cell mutants was to treat cells with ethyl methanesulfonate and select in increasing concentrations of ara-C. Although several mutants were isolated, one in particular, Ara-CR213, has been studied most extensively. It was selected largely because it shows the greatest sensitivity to X-rays. Ara-CR213 cells were hypersensitive to the killing effect of X-rays with an LD10 of 2.5 Gy as compared to the wild-type cells that had an LD10 of 6 Gy. The mutant showed an increased frequency of X-ray-induced chromosomal aberrations in the G1 and G2 stages of the cell cycle compared to wild-type frequencies. There was no increase in sister chromatid exchange levels. All of these observations in Ara-CR213 are very similar to those made with AT cells in our and other laboratories. Even more important, complementation analysis of Ara-CR213 x AT hybrid cells indicated that the gene responsible for X-ray sensitivity of AT is also mutated in Ara-CR213 cells. Thus, Ara-CR213 appears to have a mutant phenotype and probably genotype that is very similar to, if not exactly the same as, those of AT. This makes it quite different from other X-ray-sensitive cells that have been isolated in other laboratories.

Differential sensitivity of a mouse myeloid leukemia cell line and normal mouse bone marrow cells to X-ray-induced chromosome aberrations.

Cell line ML-1 was established from a myelogenous leukemia of an RFM mouse. The ML-1 cells and in vitro normal mouse bone marrow cells were analyzed to determine if there was a differential sensitivity to X-ray-induced chromosome aberrations in G1 cells and/or differences in postirradiation cell cycle progression. Cells identified as being in G1 at the time of irradiation by their staining pattern after replication in 5-bromodeoxyuridine were analyzed for all types of chromosomal aberrations following X-ray doses of 0.5, 1.0, 1.5, and 2.0 Gy. ML-1 cells showed a greater sensitivity to the induction of both chromosome-type aberrations (dicentrics and terminal deletions) and chromatid-type aberrations (exchanges and deletions) compared to normal mouse bone marrow cells, which only contained chromosome-type aberrations. The presence of chromatid-type aberrations in the ML-1 cells and not normal bone marrow cells suggested a differential progression through the cell cycle for the two cell types after irradiation. Mitotic index and flow cytometric analyses were performed and showed that both cell types have a delay in progression from G2 into mitosis, but only the normal mouse bone marrow cells have a delay in progression from G1 into S, as well as delayed progression through the S phase following X-irradiation. These results indicate that the ML-1 leukemia cells have an increased radiosensitivity. This may be due to a defect in their ability to respond to DNA damage as evidenced by their lack of a G1- and S-phase delay which allows normal cells an increased time to repair DNA damage before replication. These same characteristics have been observed in ataxia telangiectasia cells and may well represent a general feature of cells with increased radiosensitivity.

Thrombomodulin enhances complement regulation through strong affinity interactions with factor H and C3b-Factor H complex.

INTRODUCTION: Coagulation and complement systems are simultaneously activated at sites of tissue injury, leading to thrombin generation and opsonisation with C3b. Thrombomodulin (TM) is a cell-bound regulator of thrombin activation, but can also enhance the regulatory activity of complement factor H (FH), thus accelerating the degradation of C3b into inactive iC3b. OBJECTIVES: This study sought to determine the biophysical interaction affinities of two recombinant TM analogs with thrombin, FH and C3b in order to analyze their ability to regulate serum complement activity. METHODS: Surface plasmon resonance (SPR) analysis was used to determine binding affinities of TM analogs with FH and C3b, and compared to thrombin as positive control. The capacity of the two recombinant TM analogs to regulate complement in serum was tested in standard complement hemolytic activity assays. RESULTS: SPR analysis showed that both TM analogs bind FH and C3b-Factor H with nanomolar and C3b with micromolar affinity; binding affinity for its natural ligand thrombin was several fold higher than for FH. At a physiological relevant concentration, TM inhibits complement hemolytic activity in serum via FH dependent and independent mechanisms. CONCLUSIONS: TM exhibits significant binding affinity for complement protein FH and C3b-FH complex and its soluble form is capable at physiologically relevant concentrations of inhibiting complement activation in serum.

N-linked glycan truncation causes enhanced clearance of plasma-derived von Willebrand factor.

Essentials von Willebrands factor (VWF) glycosylation plays a key role in modulating in vivo clearance. VWF glycoforms were used to examine the role of specific glycan moieties in regulating clearance. Reduction in sialylation resulted in enhanced VWF clearance through asialoglycoprotein receptor. Progressive VWF N-linked glycan trimming resulted in increased macrophage-mediated clearance. Click to hear Dr Denis discuss clearance of von Willebrand factor in a free presentation from the ISTH Academy SUMMARY: Background Enhanced von Willebrand factor (VWF) clearance is important in the etiology of both type 1 and type 2 von Willebrand disease (VWD). In addition, previous studies have demonstrated that VWF glycans play a key role in regulating in vivo clearance. However, the molecular mechanisms underlying VWF clearance remain poorly understood. Objective To define the molecular mechanisms through which VWF N-linked glycan structures influence in vivo clearance. Methods By use of a series of exoglycosidases, different plasma-derived VWF (pd-VWF) glycoforms were generated. In vivo clearance of these glycoforms was then assessed in VWF-/- mice in the presence or absence of inhibitors of asialoglycoprotein receptor (ASGPR), or following clodronate-induced macrophage depletion. Results Reduced amounts of N-linked and O-linked sialylation resulted in enhanced pd-VWF clearance modulated via ASGPR. In addition to this role of terminal sialylation, we further observed that progressive N-linked glycan trimming also resulted in markedly enhanced VWF clearance. Furthermore, these additional N-linked glycan effects on clearance were ASGPR-independent, and instead involved enhanced macrophage clearance that was mediated, at least in part, through LDL receptor-related protein 1. Conclusion The carbohydrate determinants expressed on VWF regulate susceptibility to proteolysis by ADAMTS-13. In addition, our findings now further demonstrate that non-sialic acid carbohydrate determinants expressed on VWF also play an unexpectedly important role in modulating in vivo clearance through both hepatic ASGPR-dependent and macrophage-dependent pathways. In addition, these data further support the hypothesis that variation in VWF glycosylation may be important in the pathophysiology underlying type 1C VWD.

The effect of cytosine arabinoside on the frequency of single-strand breaks in DNA of mammalian cells following irradiation or chemical treatment.

1-beta-D-Arabinofuranosyl cytosine (araC), a pyrimidine nucleoside analog used in the treatment of malignant tumors [1, 2], inhibits ultraviolet repair of DNA in a reversible manner. The inhibition occurs during the resynthesis-ligation step and is apparent at all sites undergoing repair. By use of araC it was possible to substantiate the reported observation that the initial velocities of ultraviolet repair are dose dependent and that hamster and human cells are more efficient that mouse cells in excising DNA damage after fluences of less than 50 J/m2. araC does not strongly inhibit gamma-ray-induced repair, although alkali-labile sites are removed more slowly in araC-treated cells. Repair of damage to DNA by N-methyl-N-nitrosoguanidine, mitomycin C, 4-nitroquinoline oxide and 8-hydroxyquinoline is strongly inhibited by araC.

Extrapolations are the Achilles heel of risk assessment.

This Reflections article considers the problems associated with the various extrapolations that are required for the estimation of human cancer risks from exposure to environmental carcinogens at low doses. These include extrapolation between species (particularly rodent to human), from responses at high doses to those at low doses, and among different stages of life. Reductions in uncertainty in risk estimates are closely coupled to the ability to conduct reliable extrapolations. The best way forward appears to be the use of data on mechanisms of carcinogenesis to develop bioindicators of responses related to the pathway to tumor formation. Such an approach is proposed based on the phenotypes represented by the six acquired characteristics forming the Hanahan-Weinberg model for carcinogenesis (The Hallmarks of Cancer). In addition, approaches can be established that use the Hanahan-Weinberg model as the basis for the collection and/or analysis of microarray or similar data. The reduction in reliance on default options and safety factors in the risk assessment process is a real possibility.

von Willebrand factor arginine 1205 substitution results in accelerated macrophage-dependent clearance in vivo.

BACKGROUND: Enhanced von Willebrand factor (VWF) clearance is important in the etiology of type 1 and type 2 von Willebrand disease (VWD). More than 20 different VWF point mutations have already been reported in patients with enhanced clearance. These include the VWD-Vicenza variant, which is characterized by an Arg1205His substitution in the VWF D3 domain. Critically, however, the molecular mechanisms through which single amino acid substitutions in VWF result in enhanced clearance of this complex multimeric glycoprotein have not been defined. OBJECTIVES: In this study, we have investigated the biological basis underlying the enhanced clearance of the VWF-R1205H variant. METHODS: Using VWF(-/-) mice, in vivo clearance rates were determined for a series of full-length and truncated recombinant VWF variants. In addition, the role of macrophages in modulating enhanced VWD-Vicenza clearance was investigated using clodronate liposome administration. RESULTS: Our findings demonstrate that substitutions of R1205 with histidine, cysteine or serine all result in markedly reduced survival of full-length recombinant VWF. Importantly, D'A3 fragments containing these same R1205 substitutions also demonstrated significantly enhanced clearance. In contrast to the reduced in vivo survival observed with R1205H, clearance of R1204H was not enhanced. Recent studies have demonstrated that hepatic and splenic macrophages play key roles in regulating VWF clearance. Importantly, macrophage-depletion also served to markedly attenuate the enhanced clearance phenotypes associated with VWF-R1205H, VWF-R1205S and VWF-R1205C. CONCLUSIONS: Collectively, these novel findings demonstrate a specific and critical role for the R1205 residue in modulating macrophage-mediated clearance of VWF in vivo.

Telomeres, telomerase and chromosome stability.

Telomeres in most species consist of repeat units of a small number of nucleotides that together with secondary structures and associated proteins stabilize the linear chromosomal DNA molecule. Chromosomes lose a small amount of telomeric DNA after each cell replication. It has been proposed that when telomeres shorten below a critical length, a DNA damage response pathway is activated and induces cell cycle arrest. In cells such as stem cells that maintain a proliferative capacity, telomere length is maintained by the reverse transcriptase, telomerase. In addition, telomerase activity is present in 90% of primary human tumors, suggesting a role for telomerase in providing a proliferative capacity to cells, which is a requirement in progression to malignancy. Telomerase activity can be involved in chromosome healing, although telomerase-independent processes also appear to be capable of capping broken chromosome ends. This review describes the structure and maintenance of telomeres, the importance of a critical telomere length to cell proliferation and the telomeric status of broken chromosome ends produced during development or by spontaneous or induced DNA damages.

Myeloid leukemia in male RFM mice following irradiation with fission spectrum neutrons or gamma rays.

The induction of myeloid leukemia following fission neutron irradiation was examined over the 0-80 rad dose range. Over this dose range the dose response could be described by the linear regression equation: y = 0.94 + 0.18X. A comparison of these data with data obtained following gamma irradiation from this study and a previous study indicated that the relative biological effectiveness for myeloid leukemia induction was 2.8. These results appear to be compatible with those reported by other investigators.

Modulation of restriction enzyme-induced damage by chemicals that interfere with cellular responses to DNA damage: a cytogenetic and pulsed-field gel analysis.

The electroporation of restriction enzymes into mammalian cells results in DNA double-strand breaks that can lead to chromosome aberrations. Four chemicals known to interfere with cellular responses to DNA damage were investigated for their effects on chromosome aberrations induced by AluI and Sau3AI; in addition, the number of DNA double-strand breaks at various times after enzyme treatment was determined by pulsed-field gel electrophoresis (PFGE). The poly(ADP-ribose) polymerase inhibitor 3-aminobenzamide (3AB) dramatically increased the yield of exchanges and deletions and caused a small but transitory increase in the yield of double-strand breaks induced by the enzymes. 1-beta-D-Arabinofuranosylcytosine, which can inhibit DNA repair either by direct action on DNA polymerases alpha and delta or by incorporation into DNA, potentiated aberration induction but to a lesser extent than 3AB and did not affect the amount of DNA double-strand breakage. Aphidicolin, which inhibits polymerases alpha and delta, had no effect on AluI-induced aberrations but did increase the aberration yield induced by Sau3AI. The postreplication repair inhibitor caffeine had no effect on aberration yields induced by either enzyme. Neither aphidicolin nor caffeine modulated the amount of DNA double-strand breakage as measured by PFGE. These data implicate poly(ADP-ribosyl)ation and polymerases alpha and delta as important components of the cellular processes required for the normal repair of DNA double-strand breaks with blunt or cohesive ends. Comparison of these data with the effect of inhibitors on the frequency of X-ray-induced aberrations leads us to the conclusion that X-ray-induced aberrations can result from the misjoining or nonrejoining of double-strand breaks, particularly breaks with cohesive ends, but that this process accounts for only a portion of the induced aberrations.

Mutations induced by ionizing radiation in a plasmid replicated in human cells. I. Similar, nonrandom distribution of mutations in unirradiated and X-irradiated DNA.

The Escherichia coli supF gene encoding the suppressor tyrosine tRNA in a human shuttle plasmid, pZ189, was used as a target for molecular analysis of X-ray-induced mutations in human lymphoblastoid cells. Following replication of the in vitro-irradiated plasmid in human cells, the mutant supF-containing molecules were cloned by phenotypic screening in E. coli and the nature of the mutations was determined by direct sequencing of the tRNA gene. At 160 Gy the mutant frequency was 13 times (0.39%) that observed in unirradiated controls (0.031%). When control plasmid was replicated directly in E. coli, the mutant frequency was 16 times less than that of the plasmid passaged through the human cells. The distribution of mutations was highly nonrandom and remarkably similar in both irradiated and control DNAs. The majority of the mutations were transitions involving G.C pairs and occurred selectively at most 5'-TC (3'-AG) sequences. These mutations at C's were preferentially distributed in the nontranscribed strand. We propose that mutations in the control plasmid result from oxidative damages that occur during and/or after its incorporation into human cells and that these damages are similar to those induced by ionizing radiation. The hot spots for mutations suggest that the proximate nucleotide sequence and the overall conformation of the target DNA are important in the production and/or processing of these damages during repair and replication.

Mutations induced by ionizing radiation in a plasmid replicated in human cells. II. Sequence analysis of alpha-particle-induced point mutations.

The human shuttle plasmid pZ189, containing the Escherichia coli supF gene as the mutational target, was irradiated in vitro with 210Po alpha particles and transfected into human lymphoblastoid cells. Plasmids which were replicated in human cells were recovered and those containing mutant supF genes were isolated by phenotypic screening in E. coli. The mutations were characterized by sequencing the tRNA gene. The mutant frequency increased linearly with the alpha-particle dose and, at 259 Gy, it was 16 times (0.29%) that observed in unirradiated controls (0.018%). The distribution of alpha-particle-induced point mutations was highly nonrandom and similar to that observed in the unirradiated or X-irradiated plasmid DNAs. The majority of the mutations were G.C----A.T transitions and occurred selectively at most 5'-TC (3'-AG) and 5'-CC (3'-GG) sequences. For the unirradiated control DNA, these mutations at C's (G's) were preferentially located in the nontranscribed strand, similar to the observation previously made for mutations in X-irradiated DNA. Such a strand bias was not observed for mutations in the alpha-particle-irradiated DNA. The data suggest that, although similar types of point mutations are induced in unirradiated, X-irradiated, and alpha-particle-irradiated DNAs, the mechanisms of their induction and the exact nature of the lesions involved may be quite different.

Medium spiny neuron projection from the rat striatum: an intracellular horseradish peroxidase study.

The morphological features of striatal projection neurons and the responses of these neurons to electrical stimulation of the substantia nigra were studied in rats through the methods of intracellular recording and intracellular labeling with the enzyme horseradish peroxidase. Under urethane anesthesia, single striatal neurons were first analyzed for responsiveness to nigral stimuli and then filled electrophoretically with the enzyme in order to permit subsequent serial reconstruction. The axon of the medium spiny neuron was found to form an extensive collateral plexus within the striatum before entering the globus pallidus or the internal capsule. These medium spiny projection neurons responded to nigral stimuli with monosynaptic excitation.

Electrophysiological and horseradish peroxidase studies of precerebellar afferents to the nucleus interpositus anterior. I. Climbing fiber system.

The input to the nucleus interpositus anterior (NIA) of the cat from the inferior olive (IO) was studied by stimulating the IO while recording intracellularly from the NIA, and by the retrograde transport of horseradish peroxidase (HRP). Stimulation of the IO evoked monosynaptic EPSPs in NIA neurons. The cells labeled in the IO following electrophoretic and pressure injection of HRP into NIA were located in the rostral parts of the dorsal and medial accessory olive. Stimulation of the IO also polysynaptically evoked an IPSP and a late disinhibitory depolarization. Data were presented which indicated that these potentials were mediated by the Purkinje cells of the cerebellar cortex.

Future of germ cell cytogenetics.

The celebration of the 25th Anniversary of the Environmental Mutagen Society provides an excellent opportunity to assess the status of research in a broad range of areas, with an emphasis on the directions in which they are going. This chapter concentrates on the analysis of chromosomal alterations in mammalian germ cells. The future developments in germ cell cytogenetics research will build heavily upon techniques developed over the past 25 years. With these it is possible to assess numerical and structural alterations in the male in differentiating spermatogonia, spermatocytes, and post-meiotic cells (at the first cleavage division) and for the female in oocytes and the zygote. The most predictable advances will be in the identification of specific alterations through FISH of interphase spermatozoa in humans and further improvements with the human sperm/hamster egg in vitro fertilization technique. Of particular importance is the fact that this will allow for the study of effects in human germ cells. From a more speculative viewpoint it might be possible to assess the role of particular genomic organization on genetic outcomes by direct observation; these might include genomic imprinting and the visual separation of male and female genomes. The overall aim of germ cell cytogenetic studies will remain as improving our ability to identify and estimate the true genetic risk in humans.