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.

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.

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.

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.

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.

Radiation effects.

International Commission on Radiological Protection (ICRP) Committee 1 (C1) considers the risk of induction of cancer and heritable disease; the underlying mechanisms of radiation action; and the risks, severity, and mechanisms of induction of tissue reactions (formerly 'deterministic effects'). C1 relies upon the interpretation of current knowledge of radio-epidemiological studies; current information on the underlying mechanisms of diseases and radiation-induced disease; and current radiobiological studies at the whole animal, tissue, cell, and molecular levels. This overview will describe the activities of C1 in the context of the 2007 Recommendations of ICRP. In particular, the conclusions from the most recent C1 Task Group deliberations on radon and lung cancer, and tissue reactions will be discussed. Other activities are described in summary fashion to illustrate those areas that C1 judge to be likely to influence the development of the risk estimates and nominal risk coefficients used 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.

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.

Physicochemical analysis of phasic menstrual cycle effects on acid-base balance.

In accordance with Stewart's physicochemical approach, the three independent determinants of plasma hydrogen ion concentration ([H(+)]) were measured at rest and during exercise in the follicular (FP) and luteal phase (LP) of the human menstrual cycle. Healthy, physically active women with similar physical characteristics were tested during either the FP (n = 14) or LP (n = 14). Arterialized blood samples were obtained at rest and after 5 min of upright cycling at both 70 and 110% of the ventilatory threshold (T(Vent)). Measurements included plasma [H(+)], arterial carbon dioxide tension (Pa(CO(2))), total weak acid ([A(Tot)]) as reflected by total protein, and the strong-ion difference ([SID]). The transition from rest to exercise in both groups resulted in a significant increase in [H(+)] at 70% T(Vent) versus rest and at 110% T(Vent) versus both rest and 70% T(Vent). No significant between-group differences were observed for [H(+)] at rest or in response to exercise. At rest in the LP, [A(Tot)] and Pa(CO(2)) were significantly lower (acts to decrease [H(+)]) compared with the FP. This effect was offset by a reduction in [SID] (acts to increase [H(+)]). After the transition from rest to exercise, significantly lower [A(Tot)] during the LP was again observed. Although the [SID] and Pa(CO(2)) were not significantly different between groups, trends for changes in these two variables were similar to changes in the resting state. In conclusion, mechanisms regulating [H(+)] exhibit phase-related differences to ensure [H(+)] is relatively constant regardless of progesterone-mediated ventilatory changes during the LP.

Increased sensitivity to chromatid aberration induction by bleomycin and neocarzinostatin results from alterations in a DNA damage response pathway.

DNA damage response pathways coordinate the cellular response to DNA damage. To investigate the roles of tumor suppressor genes in these pathways, human lymphoblastoid cells (wild-type, p53-/-, ATM-/-) were treated for 1 h with 0-3 microg/ml of the radiomimetic compound bleomycin (BLM), and cells treated in G(2) were analyzed for chromatid aberrations. BLM-induced aberration frequencies were significantly increased, to the greatest extent in the ATM-/- cells and, to a lesser extent, in the p53-/- cells compared to wild-type cells. These observations are consistent with p53 and ATM acting in a damage response pathway activated by DNA strand breaks. The consequences of disrupting this pathway were further investigated by studies using wortmannin, a PI-3 kinase and DNA repair inhibitor. Wortmannin significantly increased the BLM-induced aberration frequencies in all but the ATM-/- cells, elevating the sensitivity of p53-/- cells to ATM-/- levels and that of wild-type cells to intermediate levels. These differential sensitivities suggest that the ATM phenotype is the result of dual cellular defects, one involving p53 and the other a wortmannin-sensitive component. Similar studies in Brca1+/- and Brca2+/- human lymphoblasts showed no increased sensitization to BLM in the absence of inhibitor, and differential sensitization by wortmannin. To determine if there was any substrate specificity for p53- and ATM-mediated DNA damage responses, chromatid aberrations were assessed in wild-type, p53-/-, and ATM-/- cells exposed to 0-0.4 microg/ml neocarzinostatin (NCS) for 1 h. In contrast to results with BLM, the p53-/- cells exhibited a low sensitivity to NCS-induced aberrations, similar to wild-type, while ATM-/- cells remained highly sensitive. This suggests that the response to BLM- and NCS-induced lesions involves different mechanisms.

A comparison of the roles of p53 mutation and AraC inhibition in the enhancement of bleomycin-induced chromatid aberrations in mouse and human cells.

Previous studies have shown that p53 is involved in the repair of bleomycin-induced DNA damage, and that the frequency of bleomycin-induced chromatid aberrations is elevated in G(2)-treated p53 null transgenic mouse embryo fibroblasts (MEF) as compared to isogenic controls. To further characterize p53-mediated DNA repair, we studied the effect of p53 status on the ability of the DNA repair inhibitor 1-ss-D-arabinofuranosylcytosine (AraC) to sensitize MEF to bleomycin-induced chromatid aberrations. Both p53+/+ and p53-/- MEF were treated in G(2) with 0 to 7.5 microg/ml bleomycin in the presence or absence of AraC (5x10(-5) M). The frequency of bleomycin-induced chromatid aberrations was significantly higher in p53-/- cells than wild-type cells in the absence of AraC. AraC treatment significantly increased the frequency of bleomycin-induced chromatid aberrations in p53+/+ MEF to the levels in p53-/- (no AraC) but had no effect in p53-/- MEF. These results suggest that an AraC-sensitive DNA repair component is altered or absent in p53-/- cells. Similar results were observed in p53-mutant WTK1 and wild-type TK6 human lymphoblast cells exposed to 0 to 3 microg/ml bleomycin in G(2). However, AraC did cause a small increase in bleomycin sensitivity in WTK1 cells. This difference from the p53-/- MEF response may be due to differences in p53-mutant phenotype. To determine whether mutation of p53 alters DNA replication fidelity, p53+/+ and p53-/- MEF were exposed to 0 to 1 microg/ml mitomycin C (MMC). MMC did not induce chromosome aberrations in either cell line treated in G(2) but did with the same effectiveness in both cell lines treated in S-phase. Thus, p53 deficiency does not affect DNA replication fidelity or the repair of MMC-induced DNA damage.

Effects of pregnancy and chronic exercise on maternal cardiac structure and function.

This study examined the interactive effects of pregnancy and aerobic conditioning on maternal cardiac structure and function. Effects of closely monitored cycle ergometer conditioning were studied during the second (TM2) and third trimesters (TM3) in 22 previously sedentary pregnant women (exercised group, EG) and a nonexercising pregnant control group with similar characteristics (CG, n = 19). Subjects were studied in the resting state by two-dimensional echocardiography and during cycle ergometer exercise at three steady-state power outputs at the start of TM2 (ENTRY), at the end of TM2 and TM3 (postconditioning), and 3-4 months postpartum (NPR, nonpregnant reference, CG only). Aerobic conditioning did not increase left ventricular dimensions beyond those attributable to pregnancy itself. In addition, in contrast with previous studies of nonpregnant women, physical conditioning during pregnancy did not reduce heart rate (HR) in the resting state. During exercise, the slope of the HR versus oxygen uptake (VO2) regression decreased significantly between preconditioning and the end of TM3 in the EG, suggesting that training-induced reductions in HR become more evident with increasing exercise intensity. Also, significant reductions in oxygen pulse (VO2/HR) were observed at all three work rates in the CG, but not in the EG. These findings support the hypothesis that the cardiovascular effects of aerobic conditioning are obscured by more powerful effects of pregnancy in the resting state but become "unmasked" during strenuous exercise.

Cytogenetic effects of ethylene oxide, with an emphasis on population monitoring.

Cytogenetic assays are an integral component of the battery of short-term assays that are used for the hazard identification component of a cancer risk assessment. The protocol for the conduct of such assays for maximal sensitivity for detecting clastogenicity has to be attendant to the mechanism of induction of the endpoint being assessed and the fact that several aberration types are cell lethal necessitates that analysis be for cells at their first posttreatment metaphase. Cytogenetic assays for human populating monitoring have been used for predicting potential for carcinogenicity in humans. However, the assays as typically conducted are not appropriate for chronic exposures because nontransmissible alterations are assessed. The use of fluorescent in situ hybridization (FISH) techniques for the assessment of transmissible changes such as reciprocal translocations are required to make population monitoring studies interpretable, and for removing some of the concern over the influence of confounders on outcome. The database for the cytogenetic effects of ethylene oxide in vitro and in vivo, with an emphasis on human population monitoring, has been critically reviewed. Based on the endpoints studied, the size of the study groups, the information on exposure, the nature of any exposure response data, and the possible influence of confounders (i.e., control matching), it is concluded that acute, high exposures to ethylene oxide with sampling shortly (a few days) after exposure can be detected by increases in chromosome aberrations or SCE in peripheral lymphocytes. Such increases are indicators of exposure to a genotoxic chemical and not predictors of subsequent adverse health effects to individuals. The effect of chronic and/or low level (less than about 25 ppm) exposures cannot be reliably evaluated using current methods. The use of FISH, for example, for assessing reciprocal translocation frequencies (as a measure of transmissible events) will greatly improve the ability to detect chronic exposures to clastogenic chemicals.

Attenuation of G1 checkpoint function by the non-genotoxic carcinogen phenobarbital.

Non-genotoxic chemical carcinogens are capable of inducing tumors in rodents without interacting with or directly altering the genetic material. Since a preponderance of evidence suggests that cancer results from the accumulation of genetic alterations, the mechanisms by which many non-genotoxic carcinogens induce genotoxic events remain unclear. The present study investigated whether the mitogenic, non-genotoxic carcinogen phenobarbital (PB) could alter cell-cycle checkpoint controls, thereby indirectly leading to the accumulation of genetic damage. Initial studies involved characterizing cell-cycle checkpoint responses to DNA damage in freshly isolated B6C3F1 mouse hepatocytes. These cells responded to bleomycin-induced DNA damage by arresting in G1 and G2. Cell-cycle arrest was coupled with p53 protein induction; however, p21WAF1 protein levels remained unchanged. Studies that utilized hepatocytes isolated from C57BL p53-/- mice showed that the DNA damage-induced G1 cell-cycle arrest was dependent on p53 function, but cell-cycle arrest in G2 was not affected by loss of p53. PB was able to delay and attenuate the G1 checkpoint response without altering G2 checkpoint function. A reduction in p53 protein, but not transcript levels, was observed in hepatocytes exposed to PB. Additionally, PB delayed and attenuated p53 protein induction during DNA damage, which suggests that changes in the p53 protein may be contributing to the attenuated G1 checkpoint response caused by PB. Altered G1 checkpoint function represents an epigenetic mechanism by which phenobarbital may prevent the detection and repair of DNA damage and indirectly increase the frequency of genotoxic events above that occurring spontaneously. Abrogation of checkpoint controls may, thus, play an important mechanistic role in mitogenic, non-genotoxic chemical carcinogenesis.

Acid-base regulation and control of ventilation in human pregnancy.

The purposes of this review were twofold: to apply modern physicochemical principles to explain changes in acid-base regulation and the control of ventilation in human pregnancy; and to demonstrate the value of pregnancy as a model for the study of endocrine effects on physiological control systems. Application of P.A. Stewart's approach (P.A. Stewart. Can. J. Physiol. Pharmacol. 61: 1444-1461, 1983) shows that lower values of plasma hydrogen ion concentration ([H+]) observed at rest and in association with exercise in pregnancy are the result of lower values for carbon dioxide tension (Pco2) and total weak acid ([A(tot)]). This effect is partly offset by a lower strong ion difference ([SID]). The ability to predict plasma [H+] at rest and following strenuous exercise in pregnancy (J.G. Kemp, F.A. Greer, and L.A. Wolfe. J. Appl. Physiol. 83: 644-651, 1997) supports the validity of Stewart's approach. Jennings and associates (D.B. Jennings. Can. J. Physiol. Pharmacol. 72: 1499-1512, 1994) have further demonstrated in animal models the involvement of plasma osmolality and circulating levels of angiotensin II (ANG II) and arginine vasopressin (AVP) in the chemical control of ventilation. We hypothesize that pregnancy-induced increases in respiratory sensitivity to carbon dioxide are the combined result of reduced plasma osmolality, reduced cerebrospinal fluid [SID], and augmented circulating levels of progesterone, ANG II, and AVP.