Dose rate dependent reduction in chromatin accessibility at transcriptional start sites long time after exposure to gamma radiation.

Ionizing radiation (IR) impact cellular and molecular processes that require chromatin remodelling relevant for cellular integrity. However, the cellular implications of ionizing radiation (IR) delivered per time unit (dose rate) are still debated. This study investigates whether the dose rate is relevant for inflicting changes to the epigenome, represented by chromatin accessibility, or whether it is the total dose that is decisive. CBA/CaOlaHsd mice were whole-body exposed to either chronic low dose rate (2.5 mGy/h for 54 d) or the higher dose rates (10 mGy/h for 14 d and 100 mGy/h for 30 h) of gamma radiation (60Co, total dose: 3 Gy). Chromatin accessibility was analysed in liver tissue samples using Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-Seq), both one day after and over three months post-radiation (>100 d). The results show that the dose rate contributes to radiation-induced epigenomic changes in the liver at both sampling timepoints. Interestingly, chronic low dose rate exposure to a high total dose (3 Gy) did not inflict long-term changes to the epigenome. In contrast to the acute high dose rate given to the same total dose, reduced accessibility at transcriptional start sites (TSS) was identified in genes relevant for the DNA damage response and transcriptional activity. Our findings link dose rate to essential biological mechanisms that could be relevant for understanding long-term changes after ionizing radiation exposure. However, future studies are needed to comprehend the biological consequence of these findings.

Calibration of the comet assay using ionising radiation.

Several trials have attempted to identify sources of inter-laboratory variability in comet assay results, aiming at achieving more equal responses. Ionising radiation induces a defined level of DNA single-strand breaks (per dose/base pairs) and is used as a reference when comparing comet results but relies on accurately determined radiation doses. In this ring test we studied the significance of dose calibrations and comet assay protocol differences, with the object of identifying causes of variability and how to deal with them. Eight participating laboratories, using either x-ray or gamma radiation units, measured dose rates using alanine pellet dosimeters that were subsequently sent to a specialised laboratory for analysis. We found substantial deviations between calibrated and nominal (uncalibrated) dose rates, with up to 46% difference comparing highest and lowest values. Three additional dosimetry systems were employed in some laboratories: thermoluminescence detectors and two aqueous chemical dosimeters. Fricke's and Benzoic Acid dosimetry solutions gave reliable quantitative dose estimations using local equipment. Mononuclear cells from fresh human blood or mammalian cell lines were irradiated locally with calibrated (alanine) radiation doses and analysed for DNA damage using a standardised comet assay protocol and a lab-specific protocol. The dose response of eight laboratories, calculated against calibrated radiation doses, was linear with slope variance CV= 29% with the lab-specific protocol, reduced to CV= 16% with the standard protocol. Variation between laboratories indicate post-irradiation repair differences. Intra-laboratory variation was very low judging from the dose response of 8 donors (CV=4%). Electrophoresis conditions were different in the lab-specific protocols explaining some dose response variations which were reduced by systematic corrections for electrophoresis conditions. The study shows that comet assay data obtained in different laboratories can be compared quantitatively using calibrated radiation doses and that systematic corrections for electrophoresis conditions are useful.

Perturbed transcriptional profiles after chronic low dose rate radiation in mice.

Adverse health outcomes of ionizing radiation given chronically at low dose rates are highly debated, a controversy also relevant for other stressors. Increased knowledge is needed for a more comprehensive understanding of the damaging potential of ionizing radiation from all dose rates and doses. There is a lack of relevant low dose rate data that is partly ascribed to the rarity of exposure facilities allowing chronic low dose rate exposures. Using the FIGARO facility, we assessed early (one day post-radiation) and late (recovery time of 100-200 days) hepatic genome-wide transcriptional profiles in male mice of two strains (CBA/CaOlaHsd and C57BL/6NHsd) exposed chronically to a low dose rate (2.5 mGy/h; 1200h, LDR), a mid-dose rate (10 mGy/h; 300h, MDR) and acutely to a high dose rate (100 mGy/h; 30h, HDR) of gamma irradiation, given to an equivalent total dose of 3 Gy. Dose-rate and strain-specific transcriptional responses were identified. Differently modulated transcriptional responses across all dose rate exposure groups were evident by the representation of functional biological pathways. Evidence of changed epigenetic regulation (global DNA methylation) was not detected. A period of recovery markedly reduced the number of differentially expressed genes. Using enrichment analysis to identify the functional significance of the modulated genes, perturbed signaling pathways associated with both cancer and non-cancer effects were observed, such as lipid metabolism and inflammation. These pathways were seen after chronic low dose rate and were not restricted to the acute high dose rate exposure. The transcriptional response induced by chronic low dose rate ionizing radiation suggests contribution to conditions such as cardiovascular diseases. We contribute with novel genome wide transcriptional data highlighting dose-rate-specific radiation responses and emphasize the importance of considering both dose rate, duration of exposure, and variability in susceptibility when assessing risks from ionizing radiation.

Impact of multigenerational exposure to AgNO3 or NM300K Ag NPs on antioxidant defense and oxidative stress in Caenorhabditis elegans.

Adaptation of the nematode Caenorhabditis elegans towards NM300K silver nanoparticles (Ag NPs) has previously been demonstrated. In the current study, the sensitivity to a range of secondary stressors (CeO2 NP, Ce3+, Cu2+, Cd2+, and Paraquat) following the multigenerational exposure to silver nanoparticles (Ag NPs NM300K) or AgNO3 was investigated. This revealed improved tolerance to the ROS inducer Paraquat with higher fecundity after pre-exposure to Ag NP, indicating an involvement of reactive oxygen species (ROS) metabolism in the adaptive response to NM300K. The potential contribution of the antioxidant defenses related to adaptive responses was investigated across six generations of exposure using the sod-1::GFP reporter (GA508), and the Grx1-roGFP2 (GRX) biosensor strains. Results showed an increase in sod-1 expression by the F3 generation, accompanied by a reduction of GSSG/GSH ratios, from both AgNO3 and Ag NP exposures. Continuous exposure to AgNO3 and Ag NP until the F6 generation resulted in a decreased sod-1 expression, with a concomitant increase in GSSG/GSH ratios. The results thus show that despite an initial enhancement, the continuous exposure to Ag caused a severe impairment of the antioxidant defense capacity in C. elegans.

Ionizing radiation, genotoxic stress, and mitochondrial DNA copy-number variation in Caenorhabditis elegans: droplet digital PCR analysis.

Mitochondria are vulnerable to the effects of ionizing radiation; damage to mitochondrial DNA (mtDNA) may be more extensive and persistent than damage to nuclear DNA (nDNA). Variation in mtDNA copy number has been proposed as a marker for mitochondrial dysfunction in response to ionizing radiation. We have developed a precise and sensitive duplex droplet digital PCR (ddPCR) method for quantitation of the mtDNA/nDNA ratio in the model organism Caenorhabditis elegans. The effect on this ratio was investigated over a wide range of doses (0.03-72 Gy) of chronic gamma irradiation. Five mitochondrial targets and two nuclear reference genes were amplified pairwise in duplex PCR format (one mitochondrial and one nuclear target per PCR) by both ddPCR and quantitative PCR (qPCR). The results showed that ddPCR but not qPCR enabled detection of a significant increase in mtDNA copy number (1.6 ± 0.1-fold) for nematodes exposed to high doses (≥24 Gy). Thus, ddPCR provided higher precision and greater sensitivity than qPCR for detection of mtDNA copy number variation. The variation followed a Hill-type dose response with threshold 10.3 ± 1 Gy. This strongly suggests that chronic genotoxic stress affects mtDNA replication. The duplex ddPCR method is a novel, high-precision, sensitive tool for determination of mitochondrial DNA copy number variation and function in C. elegans.

Using prediction models to identify miRNA-based markers of low dose rate chronic stress.

Robust biomarkers of exposure to chronic low dose stressors such as ionizing radiation, particularly following chronic low doses and dose-rates, are urgently needed. MicroRNAs (miRNA) have emerged as promising markers of exposure to high dose and dose-rate. Here, we evaluated the feasibility of classifying γ-radiation exposure at different dose rates based on miRNA expression levels. Our objective was to identify miRNA-signatures discriminating between exposure to γ-radiation or not, including exposure to chronic low dose rates. We exposed male CBA/CaOlaHsd and C57BL/6NHsd wild-type mice to 0, 2.5, 10 and 100 mGy/h γ-irradiation (3 Gy total-dose). From an initial screening of 576 miRNAs, a set of 21 signature-miRNAs was identified based on differential expression (>± 2-fold or p < 0.05). This 21-signature miRNA panel was investigated in 39 samples from 4/5 livers/group/mouse strain. A set of significantly differentially expressed miRNAs was identified in all γ-irradiated samples. Most miRNAs were upregulated in all γ-irradiated groups compared to control, and functional analysis of these miRNAs revealed involvement in several cancer-related signaling pathways. To identify miRNAs that distinguished exposed mice from controls, nine prediction methods; i.e., six variants of generalized regression models, random-forest, boosted-tree and nearest-shrunken-centroid (PAM) were used. The generalized regression methods seem to outperform the other prediction methods for classification of irradiated and control samples. Using the 21-miRNA panel in the prediction models, we identified sets of candidate miRNA-markers that predict exposure to γ-radiation. Among the top10 miRNA predictors, contributing most in each of the three γ-irradiated groups, three miRNA predictors (miR-140-3p, miR-133a-5p and miR-145a-5p) were common. Three miRNAs, miR-188-3p/26a-5p/26b-5p, were specific for lower dose-rate γ-radiation. Similarly, exposure to the high dose-rates was also correctly predicted, including mice exposed to X-rays. Our approach identifying miRNA-based signature panels may be extended to classify exposure to environmental, nutritional and life-style-related stressors, including chronic low-stress scenarios.

Gamma radiation induces life stage-dependent reprotoxicity in Caenorhabditis elegans via impairment of spermatogenesis.

The current study investigated life stage, tissue and cell dependent sensitivity to ionizing radiation of the nematode Caenorhabditis elegans. Results showed that irradiation of post mitotic L4 stage larvae induced no significant effects with respect to mortality, morbidity or reproduction at either acute dose ≤6 Gy (1500 mGy·h-1) or chronic exposure ≤15 Gy (≤100 mGy·h-1). In contrast, chronic exposure from the embryo to the L4-young adult stage caused a dose and dose-rate dependent reprotoxicity with 43% reduction in total brood size at 6.7 Gy (108 mGy·h-1). Systematic irradiation of the different developmental stages showed that the most sensitive life stage was L1 to young L4. Exposure during these stages was associated with dose-rate dependent genotoxic effects, resulting in a 1.8 to 2 fold increase in germ cell apoptosis in larvae subjected to 40 or 100 mGy·h-1, respectively. This was accompanied by a dose-rate dependent reduction in the number of spermatids, which was positively correlated to the reprotoxic effect (0.99, PCC). RNAseq analysis of nematodes irradiated from L1 to L4 stage revealed a significant enrichment of differentially expressed genes related to both male and hermaphrodite reproductive processes. Gene network analysis revealed effects related to down-regulation of genes required for spindle formation and sperm meiosis/maturation, including smz-1, smz-2 and htas-1. Furthermore, the expression of a subset of 28 set-17 regulated Major Sperm Proteins (MSP) required for spermatid production was correlated (R2 0.80) to the reduction in reproduction and the number of spermatids. Collectively these observations corroborate the impairment of spermatogenesis as the major cause of gamma radiation induced life-stage dependent reprotoxic effect. Furthermore, the progeny of irradiated nematodes showed significant embryonal DNA damage that was associated with persistent effect on somatic growth. Unexpectedly, these nematodes maintained much of their reproductive capacity in spite of the reduced growth.

Adaptive tolerance to multigenerational silver nanoparticle (NM300K) exposure by the nematode Caenorhabditis elegans is associated with increased sensitivity to AgNO3.

Toxic effects of silver nanoparticles (Ag NPs) are, in most cases, measured within a single generation, while information regarding multigenerational exposure remains scarce. The current study assessed changes in toxic response (reproduction, fertility, and development) towards Ag NPs (NM300K; uncoated, 16.7 ± 6.5 nm) compared to AgNO3 over six generations, following chronic exposure of the model organism Caenorhabditis elegans. This revealed that AgNO3 exposure was associated with no changes in susceptibility to Ag. In contrast, multigenerational exposure to sub-lethal concentrations of Ag NPs resulted in persistent delayed development, but rendered increased tolerance to Ag NP with respect to fertility and fecundity. The results thus permit inference of a difference in toxic mode of action of the two forms of Ag, which instigate different response patterns. Results reveal a novel mechanism for the adaptation toward Ag NPs, where increased reproductive fitness occurs at the expense of somatic growth. This adaptive mechanism was, however associated with increased susceptibility to AgNO3 with respect to growth, fertility and reproduction. The current study thus demonstrates that a nano-specific resistance can be developed by C. elegans. Importantly, this adaptation renders increased vulnerability to another environmental stressor, and thus exposure to a second contaminant could be detrimental to such populations.

Lung effects of 7- and 28-day inhalation exposure of rats to emissions from 1st and 2nd generation biodiesel fuels with and without particle filter - The FuelHealth project.

Increased use of 1st and 2nd generation biofuels raises concerns about health effects of new emissions. We analyzed cellular and molecular lung effects in Fisher 344 rats exposed to diesel engine exhaust emissions (DEE) from a Euro 5-classified diesel engine running on B7: petrodiesel fuel containing 7% fatty acid methyl esters (FAME), or SHB20 (synthetic hydrocarbon biofuel): petrodiesel fuel containing 7% FAME and 13% hydrogenated vegetable oil. The Fisher 344 rats were exposed for 7 consecutive days (6 h/day) or 28 days (6 h/day, 5 days/week), both with and without diesel particle filter (DPF) treatment of the exhaust in whole body exposure chambers (n = 7/treatment). Histological analysis and analysis of cytokines and immune cell numbers in bronchoalveolar lavage fluid (BALF) did not reveal adverse pulmonary effects after exposure to DEE from B7 or SHB20 fuel. Significantly different gene expression levels for B7 compared to SHB20 indicate disturbed redox signaling (Cat, Hmox1), beta-adrenergic signaling (Adrb2) and xenobiotic metabolism (Cyp1a1). Exhaust filtration induced higher expression of redox genes (Cat, Gpx2) and the chemokine gene Cxcl7 compared to non-filtered exhaust. Exposure time (7 versus 28 days) also resulted in different patterns of lung gene expression. No genotoxic effects in the lungs were observed. Overall, exposure to B7 or SHB20 emissions suggests only minor effects in the lungs.

Ionizing radiation does not impair the mechanisms controlling genetic stability during T cell receptor gene rearrangement in mice.

PURPOSE: To determine whether low dose/low dose rate radiation-induced genetic instability may result from radiation-induced inactivation of mechanisms induced by the ATM-dependent DNA damage response checkpoint. To this end, we analysed the faithfulness of T cell receptor (TR) gene rearrangement by V(D)J recombination in DNA from mice exposed to a single dose of X-ray or chronically exposed to low dose rate γ radiation. MATERIALS AND METHODS: Genomic DNA obtained from the blood or the thymus of wild type or Ogg1-deficient mice exposed to low (0.1) or intermediate/high (0.2-1 Gy) doses of radiation either by acute X-rays exposure or protracted exposure to low dose-rate γ-radiation was used to analyse by PCR the presence of illegitimate TR gene rearrangements. RESULTS: Radiation exposure does not increase the onset of TR gene trans-rearrangements in irradiated mice. In mice where it happens, trans-rearrangements remain sporadic events in developing T lymphocytes. CONCLUSION: We concluded that low dose/low dose rate ionizing radiation (IR) exposure does not lead to widespread inactivation of ATM-dependent mechanisms, and therefore that the mechanisms enforcing genetic stability are not impaired by IR in developing lymphocytes and lymphocyte progenitors, including BM-derived hematopoietic stem cells, in low dose/low dose rate exposed mice.

Genotoxic effects of high dose rate X-ray and low dose rate gamma radiation in ApcMin/+ mice.

Risk estimates for radiation-induced cancer in humans are based on epidemiological data largely drawn from the Japanese atomic bomb survivor studies, which received an acute high dose rate (HDR) ionising radiation. Limited knowledge exists about the effects of chronic low dose rate (LDR) exposure, particularly with respect to the application of the dose and dose rate effectiveness factor. As part of a study to investigate the development of colon cancer following chronic LDR vs. acute HDR radiation, this study presents the results of genotoxic effects in blood of exposed mice. CBAB6 F1 Apc+/+ (wild type) and ApcMin/+ mice were chronically exposed to estimated whole body absorbed doses of 1.7 or 3.2 Gy 60 Co-γ-rays at a LDR (2.2 mGy h-1 ) or acutely exposed to 2.6 Gy HDR X-rays (1.3 Gy min-1 ). Genotoxic endpoints assessed in blood included chromosomal damage (flow cytometry based micronuclei (MN) assay), mutation analyses (Pig-a gene mutation assay), and levels of DNA lesions (Comet assay, single-strand breaks (ssb), alkali labile sites (als), oxidized DNA bases). Ionising radiation (ca. 3 Gy) induced genotoxic effects dependent on the dose rate. Chromosomal aberrations (MN assay) increased 3- and 10-fold after chronic LDR and acute HDR, respectively. Phenotypic mutation frequencies as well as DNA lesions (ssb/als) were modulated after acute HDR but not after chronic LDR. The ApcMin/+ genotype did not influence the outcome in any of the investigated endpoints. The results herein will add to the scant data available on genotoxic effects following chronic LDR of ionising radiation. Environ. Mol. Mutagen. 58:560-569, 2017. © 2017 The Authors Environmental and Molecular Mutagenesis published by Wiley Periodicals, Inc. on behalf of Environmental Mutagen Society.

The Pig-a Gene Mutation Assay in Mice and Human Cells: A Review.

This MiniReview describes the principle of mutation assays based on the endogenous Pig-a gene and summarizes results for two species of toxicological interest, mice and human beings. The work summarized here largely avoids rat-based studies, as are summarized elsewhere. The Pig-a gene mutation assay has emerged as a valuable tool for quantifying in vivo and in vitro mutational events. The Pig-a locus is located at the X-chromosome, giving the advantage that one inactivated allele can give rise to a mutated phenotype, detectable by multicolour flow cytometry. For in vivo studies, only minute blood volumes are required, making it easily incorporated into ongoing studies or experiments with limited biological materials. Low blood volumes also allow individuals to serve as their own controls, providing temporal information of the mutagenic process, and/or outcome of intervention. These characteristics make it a promising exposure marker. To date, the Pig-a gene mutation assay has been most commonly performed in rats, while reports regarding its usefulness in other species are accumulating. Besides its applicability to in vivo studies, it holds promise for genotoxicity testing using cultured cells, as shown in recent studies. In addition to safety assessment roles, it is becoming a valuable tool in basic research to identify mutagenic effects of different interventions or to understand implications of various gene defects by investigating modified mouse models or cell systems. Human blood-based assays are also being developed that may be able to identify genotoxic environmental exposures, treatment- and lifestyle-related factors or endogenous host factors that contribute to mutagenesis.

The Agaricus blazei-Based Mushroom Extract, Andosan™, Protects against Intestinal Tumorigenesis in the A/J Min/+ Mouse.

BACKGROUND: The novel A/J Min/+ mouse, which is a model for human Familial Adenomatous Polyposis (FAP), develops spontaneously multiple adenocarcinomas in the colon as well as in the small intestine. Agaricus blazei Murill (AbM) is an edible Basidiomycetes mushroom that has been used in traditional medicine against cancer and other diseases. The mushroom contains immunomodulating ß-glucans and is shown to have antitumor effects in murine cancer models. Andosan™ is a water extract based on AbM (82%), but it also contains the medicinal Basidiomycetes mushrooms Hericeum erinaceus and Grifola frondosa. METHODS AND FINDINGS: Tap water with 10% Andosan™ was provided as the only drinking water for 15 or 22 weeks to A/J Min/+ mice and A/J wild-type mice (one single-nucleotide polymorphism (SNP) difference), which then were exsanguinated and their intestines preserved in formaldehyde and the serum frozen. The intestines were examined blindly by microscopy and also stained for the tumor-associated protease, legumain. Serum cytokines (pro- and anti-inflammatory, Th1-, Th2 -and Th17 type) were measured by Luminex multiplex analysis. Andosan™ treated A/J Min/+ mice had a significantly lower number of adenocarcinomas in the intestines, as well as a 60% significantly reduced intestinal tumor load (number of tumors x size) compared to control. There was also reduced legumain expression in intestines from Andosan™ treated animals. Moreover, Andosan™ had a significant cytotoxic effect correlating with apoptosis on the human cancer colon cell line, Caco-2, in vitro. When examining serum from both A/J Min/+ and wild type mice, there was a significant increase in anti-tumor Th1 type and pro-inflammatory cytokines in the Andosan™ treated mice. CONCLUSIONS: The results from this mouse model for colorectal cancer shows significant protection of orally administered Andosan™ against development of intestinal cancer. This is supported by the finding of less legumain in intestines of Andosan™ treated mice and increased systemic Th1 cytokine response. The mechanism is probably both immuno-modulatory and growth inhibition of tumor cells by induction of apoptosis.

Gamma radiation at a human relevant low dose rate is genotoxic in mice.

Even today, 70 years after Hiroshima and accidents like in Chernobyl and Fukushima, we still have limited knowledge about the health effects of low dose rate (LDR) radiation. Despite their human relevance after occupational and accidental exposure, only few animal studies on the genotoxic effects of chronic LDR radiation have been performed. Selenium (Se) is involved in oxidative stress defence, protecting DNA and other biomolecules from reactive oxygen species (ROS). It is hypothesised that Se deficiency, as it occurs in several parts of the world, may aggravate harmful effects of ROS-inducing stressors such as ionising radiation. We performed a study in the newly established LDR-facility Figaro on the combined effects of Se deprivation and LDR γ exposure in DNA repair knockout mice (Ogg1(-/-)) and control animals (Ogg1(+/-)). Genotoxic effects were seen after continuous radiation (1.4 mGy/h) for 45 days. Chromosomal damage (micronucleus), phenotypic mutations (Pig-a gene mutation of RBC(CD24-)) and DNA lesions (single strand breaks/alkali labile sites) were significantly increased in blood cells of irradiated animals, covering three types of genotoxic activity. This study demonstrates that chronic LDR γ radiation is genotoxic in an exposure scenario realistic for humans, supporting the hypothesis that even LDR γ radiation may induce cancer.

Natural killer cells in free-living Mus musculus have a primed phenotype.

Recent reports have shown that natural killer (NK) cells may be long-lived, possess memory-like features and may need microbial priming to become fully reactive. Thus, the notion that these cells are typically innate, nonadaptive lymphocytes has been challenged. If microbial priming is essential for functional maturity, it is necessary to raise the question whether NK cells of laboratory mice, kept under strict hygienic conditions, represent these cells adequately. In their natural habitat, mice will encounter microbes to a greater extent, and we here investigated whether NK cells of feral mice showed signs of being primed. In comparison with C57BL/6 mice raised under specific pathogen-free conditions, NK cells from feral mice had high expression of CD69, KLRG1, granzyme B and NKp46 and a higher proportion of CD27+ cells, mostly CD11b-, as well as a higher presence in peripheral lymph nodes. Following cytokine stimulation, feral mouse NK cells had quickly inducible CD25 expression and a stronger interferon-gamma response. These findings indicate a high degree of pre-activation of NK cells of free-living mice, indicating a strong environmental impact on NK cells, which may be highly relevant for interpretation of studies in the mouse model.

Differentiation of naive CD4+ T cells into CD4+CD25+FOXP3+ regulatory T cells by continuous antigen stimulation.

Human CD4+CD25+ regulatory T (T(R)) cells express the transcription factor forkhead box p3 (FOXP3) and have potent immunosuppressive properties. While naturally occurring T(R) cells develop in the thymus, adaptive T(R) cells develop in the periphery from naive CD4+ T cells. Adaptive T(R) cells may express cyclooxygenase type 2 (COX-2) and suppress effector T cells by a PGE(2)-dependent mechanism, which is reversible with COX inhibitors. In this study we have characterized the differentiation of naive CD4+ T cells into adaptive T(R) cells in detail during 7 days of continuous antigen stimulation. After 2 days of stimulation of CD4+CD25- T cells, the cells expressed FOXP3 and COX-2 and displayed potent immunosuppressive properties. The suppressive phenotype was present at all observed time-points from Day 2, although suppression was merely present at Day 7. The adaptive T(R) cells expressed cell surface markers consistent with an activated phenotype and secreted high levels of TGF-beta, IL-10, and PGE(2). However, the suppressive phenotype was found exclusively in cells that proliferated upon activation. These data support the notion that activation of naive CD4+ T cells leads to concomitant acquisition of effector and suppressive properties.