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Risk comparisons between limit values for ionizing radiation, PAH, and benzene in Sweden.

The background of regulatory limit values for carcinogenic agents in Sweden is discussed and exemplified with the ambient and occupational air pollutants benzene and PAH (especially benzo[a]pyrene, BaP), ionizing radiation, and radon. The estimated cancer risks at different limit values are compared, as is the estimated number of cancer cases annually due to existing pollutant levels. Although the individual lifetime cancer risks are much higher at the occupational limit values for benzene and BaP than what is recommended for the general public, the estimated number of cancer cases annually is lower at existing pollutant levels. The individual cancer risk at the occupational dose limit for ionizing radiation is comparable to the occupational cancer risk with BaP, but higher than the one for benzene. At the dose limit for the general public, the radiation risk would be higher than what is recommended for individual air pollutants. However, doses from man-made radiation are usually much lower than the dose limit due to optimization. Thus, the estimated number of cancer cases annually due to radiation is low and comparable to the estimated number due to the chemical air pollutants discussed. In contrast, the lung cancer risk with radon in dwellings both at limit values and existing levels is high and comparable to occupational limits for the chemical carcinogens and for radiation. The estimated number of cancer cases annually at existing radon concentrations (1100 cases) is much higher than for the other discussed pollutants (0.03-7 cases each) and also higher than the estimated number of lung cancer cases due to total air pollution (approximately 100 cases).

Benzene↗

Quantitative analysis of triglycerides using atmospheric pressure chemical ionization-mass spectrometry.

Atmospheric pressure chemical ionization-mass spectrometry (APCI-MS) was used for quantitative analysis of triglycerides (TG) separated by reverse-phase high-performance liquid chromatography. APCI-MS was used for analysis of mono-acid TG standards containing deuterated internal standard, of a synthetic mixture of heterogeneous TG, of randomized and normal soybean oils and of randomized and normal lard samples. Quantitation of the TG by four approaches based on APCI-MS were compared, and these were compared to quantitation obtained using liquid chromatography (LC) with flame-ionization detection (FID). The APCI-MS methods were based on (i) calibration curves from data for mono-acid TG standards, (ii) response factors obtained from a synthetic mixture of TG, (iii) response factors calculated from comparison of randomized samples to their statistically expected compositions, and (iv) response factors calculated from comparison of fatty acid (FA) compositions calculated from TG compositions to FA compositions obtained by calibrated gas chromatography (GC) with FID. Response factors derived from a synthetic mixture were not widely applicable to samples of disparate composition. The TG compositions obtained using APCI-MS data without application of response factors had average relative errors very similar to those obtained using LC-FID. Numerous TG species were identified using LC/APCI-MS which were undetected using LC-FID. Two quantitation methods, based on response factors calculated from randomized samples or on response factors calculated from FA compositions, both gave similar results for all samples. The TG compositions obtained using response factors calculated from FA compositions showed less average relative error than was obtained from LC-FID data, and were in good agreement with predicted compositions for the synthetic mixture and for randomized soybean oil and lard samples.

Chromatography, High Pressure Liquid↗

[Death inducing ligands in combination with ionizing radiation: objective and current knowledge].

BACKGROUND: Apart from optimization of the radiation technology, future new strategies in radiation oncology will focus on the biological optimisation leading to improved adaptation of the tumor therapy on each tumor entity. In this regard, different areas of biological research may be distinguished: prediction, development of new cytotoxic agents, improvement of the tolerance of normal tissue and the optimisation of radiochemotherapy. MATERIAL AND METHOD: For the development of new strategies the knowledge of molecular mechanisms of radiation induced cell death is essential. In the present article a novel biological strategy for optimisation of radiotherapy is introduced. RESULTS: Distinct pathways mediate apoptosis in response to death receptor stimulation and ionizing radiation although an overlapping set of molecules is involved. TRAIL (TNF alpha-related apoptosis inducing ligand) is a type-II membrane protein belonging to the TNF family, which preferentially induces apoptotic cell death in a wide variety of tumor cells but not in normal cells. Based on the assumption of distinct signalling pathways, combination of TRAIL and ionizing radiation seems ideal to increase the therapeutic efficacy. Our study presents the rationale, own data, data of other groups as well as the current status of the latest findings regarding the function and physiological role of the TRAIL death ligand. CONCLUSIONS: Apoptosis resistance towards ionising radiation is not associated with a general apoptosis resistance. Death ligands, for example TRAIL trigger apoptosis even in cells not undergoing apoptosis in response to radiation. A combination of both modalities induces additive or synergistic effects in regard to apoptosis induction and eradication of clonogenic tumor cells. Thus, the combination of TRAIL with radiation proofs that novel treatment strategies may be developed on the basis of molecular or cell biological research.

Animals↗

[Health risks of ionizing radiation].

Ionizing radiation from natural or man-made sources may affect human health. The main consequence of low and moderate radiation doses is the induction of cancer. To quantify these effects both epidemiological studies and experimental radiobiological research are needed. The ensuing results of the studies are used to establish radiation protection principles and dose limits to protect the public from the effects of ionizing radiation.

Environmental Exposure↗

Development of a simplified method for the simultaneous determination of retinol, alpha-tocopherol, and beta-carotene in serum by liquid chromatography-tandem mass spectrometry with atmospheric pressure chemical ionization.

A new and simple method for the determination of fat-soluble vitamins (retinol, alpha-tocopherol, and beta-carotene) in human serum was developed and validated by using liquid chromatography-tandem mass spectrometry with atmospheric pressure chemical ionization (LC-APCI-MS-MS). Different solvent mixtures were tested to obtain deproteinization and extraction of the analytes from the matrix. As a result, a volume of 240 microL of a 1:1 (v/v) ethanol/ethyl acetate mixture added to 60 microL of serum was found to be suitable for both protein precipitation and antioxidants solubilization, giving the best recovery for all three analytes. Deproteinized samples (20 microL) were injected after dilution, without the need for concentration or evaporation to dryness and reconstruction of the sample. Vitamins were separated on a C-8 column using a 95:5 (v/v) methanol/dichloromethane mixture and ionized in the positive-ion mode; detection was performed in the selected-reaction monitoring mode. Linearity of the LC-APCI-MS-MS method was established over 5 orders of magnitude for retinol and alpha-tocopherol, whereas in the case of beta-carotene it was limited to 4 orders. Lower limits of quantitation were 1.7, 2.3, and 4.1 nM for retinol, alpha-tocopherol, and beta-carotene, respectively. Serum concentrations of retinol, alpha-tocopherol, and alpha+beta-carotene determined in a group of healthy volunteers were 2.48, 38.07, and 0.50 microM, respectively, in samples collected in winter ( n=122) and 2.69, 45.88, and 0.90 microM during summer ( n=66).

Adult↗

Accurate determination of allelic frequencies in mitochondrial DNA mixtures by electrospray ionization time-of-flight mass spectrometry.

The mitochondrial locus 16519T/C was used as a model for the evaluation of the benefits of ion-pair reversed-phase high-performance liquid chromatography on-line hyphenated to electrospray ionization time-of-flight mass spectrometry (ICEMS assay) for the determination of allelic frequencies of single nucleotide polymorphisms. This marker has gained interest in forensic science owing to its ability to increase the discrimination power of mitochondrial DNA testing as a consequence of its high variability across various populations. In a first set of experiments, artificial mitochondrial DNA mixtures prepared from all four theoretically possible 16519 alleles served as samples. Any allele occurring at a frequency of as low as 1-5% was unequivocally detectable irrespective of the kind of allelic mixture. Measured and expected allelic frequencies correlated well following correction of observed experimental bias, which was most probably attributable to differential PCR amplification and/or preferential ionization. For thirteen different T/C mixtures with C contents in the range 1.0-99.0%, an average error of 1.2% and a maximum error of 2.2% were observed. Furthermore, ICEMS was applied to the quantitative genotyping of eight selected individuals of which four were heteroplasmic with C contents in the range 1.9-34.1%. To check the reliability of these results, allelic proportions were additionally determined by a cloning assay. The results of the two assays correlated well (R (2)=0.9971). In all cases, deviations were obtained that were smaller than 5.4%. The overall observed assay performance suggests that the described mass spectrometric technique represents one of the most powerful assays for the determination of allelic frequencies available today.

DNA, Mitochondrial↗

Prognostic relevance of previous exposure to ionizing radiation in well-differentiated thyroid cancer.

BACKGROUND AND AIMS: Although exposure to ionizing radiation is a well-known risk factor for well-differentiated thyroid cancer, its effects on tumor behavior remain unclear. This study was undertaken to address this question. MATERIALS AND METHODS: We randomly selected 426 patients who underwent thyroidectomy for cancer between 1964 and 2000 and divided them into two groups: previously exposed (ExR); not exposed to radiation (nExR). Data were retrospectively collected. RESULTS: There were 340 female patients and 86 male, age 9-89 years with mean follow-up of 56 months. The ExR group (n=68) was smaller than the nExR group (n=358). Most patients in the ExR group (64.5%) had previously received therapeutic radiation, whereas 25% were occupationally exposed and 10.5% were at risk from environmental radiation. Both groups were similar with regard to extent of thyroidectomy and adjuvant treatment, but neck dissections were more frequent in the nExR group. There were no significant differences between both groups for age, gender, tumor multicentricity, frequency of microcarcinoma, histology, lymph node disease, distant metastasis, and local recurrence. Incidental microcarcinomas were more frequent in the ExR (35.3%) group than in the nExR group (22.1%). Mean tumor size in the ExR group (18.7 mm) was smaller than in the nExR group (22.3 mm). CONCLUSION: Ionizing radiation increases risk for well-differentiated thyroid cancer (WDTC) but not adverse cancer behavior. Surgeons should be aware of the high incidence of microcancer among patients with previous exposure to radiation.

Adenocarcinoma, Follicular↗

Probing structural changes in the alpha and beta domains of copper- and silver-substituted metallothionein by emission spectroscopy and electrospray ionization mass spectrometry.

Steady-state emission spectra, excited-state lifetimes, kinetic data, and mass spectroscopic properties are reported for Ag(I)- and mixed Ag(I)/Cu(I)-substituted alpha and beta domains of recombinant human metallothionein (MT1a). Kinetic analysis of the changes in the Cu(I) emission spectra during the stepwise displacement of Cu(I) ions by Ag(I) at room temperature shows that the rate of displacement of Cu(I) is unexpectedly slow. Although the first Ag(I) added results in major changes in the Cu(I)-MT binding site, Cu(I) displacement by Ag(I) does not take place until the addition of the third Ag(I), and is completed by the addition of the seventh Ag(I). The emission from Ag(I) and mixed Cu(I)/Ag(I)-MT species at 77 K shows that the band maxima shift as a function of Ag(I) loading, which can be correlated with shifts in coordination geometry from trigonal to digonal. Two phosphorescence lifetimes were detected for the Ag(I)-substituted alpha and beta domains of MT, which are attributed to the presence of Ag(I) ions in two different environments. The lifetime of Ag(I)-substituted MT was found to be shorter when the Ag(I)-MT species were formed by Ag(I) additions to the Cu(I)-substituted alpha and beta fragments than when the Ag(I)-MT species were formed from the apo-alpha and apo-beta fragments, suggesting the formation of structurally different Ag(I)-MT clusters. Electrospray ionization mass spectrometric studies suggest the metallation reactions of Ag(I) with MT take place in a series of steps to form a series of Ag(I)-substituted MT species. Ag(I)-substituted MT species are not detected until past the addition of 3 mol equiv of Ag(I), suggesting that cluster formation begins only at this point, stabilizing the metallated species sufficiently to survive ionization.

Binding Sites↗

Effects of ionizing radiation on brain tissue surrounding arteriovenous malformations: an experimental study in a rat caroticojugular fistula model.

Arteriovenous malformation (AVM) may change the cerebral hemodynamics. The purpose of this study was to detect the effects of ionizing radiation (IR) on tissues surrounding AVM in a rat caroticojugular fistula model. Forty rats were divided into four groups. Eight weeks after caroticojugular fistulas and chronic hypoperfusion were created in groups 1 and 2, IR was administered to groups 1 and 3. Group 4 was the control. Brain tissue samples were taken 72 h after irradiation. Comet assay to detect DNA strand breaks (DSB), terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) assay for apoptosis, and free radical measurement were performed. Although the difference between fistula plus irradiation (group 1) and fistula (group 2) was statistically insignificant in terms of DSB and free radical measurement, apoptotic cell count was significantly higher in group 1. Nonetheless, apoptotic cell count corresponded well with both free radicals and DSB in the irradiated group (group 3). Ionizing radiation resulted in significant apoptosis in both groups with or without fistulas. Chronic hypoperfusion might not prevent cerebral damage after IR. Optimal care should be taken with brain tissue around AVM during radiotherapy, regardless of presence or absence of the "steal" phenomenon.

Animals↗

Ionizing radiation induces a p53-dependent apoptotic mechanism in ARPE-19 cells.

PURPOSE: To investigate the molecular mechanisms for cell growth inhibition or apoptosis in human retinal pigment epithelium (RPE) cells after ionizing radiation. METHODS: Cell survival studies, a TdT-mediated dUTP-biotin nick-end labeling (TUNEL) assay, and a caspase-3 immunocytochemical analysis were performed on irradiated ARPE-19 cell cultures at different time periods. Transcriptional levels of p53, p21, Bax, Fas/Fas-L, vascular endothelial growth factor ( VEGF), and pigment epithelium-derived growth factor ( PEDF) were evaluated by semiquantitative reverse transcriptional polymerase chain reaction. Mutations in the p53 gene were analyzed by DNA sequencing. Protein levels of p53, VEGF, and PEDF were evaluated by Western blot. RESULTS: Cell viability was inversely related to radiation dose. TUNEL-positive cells were detected 6 h after radiation exposure. Caspase-3 immunocytochemical analysis revealed increased immunoreactivity in the TUNEL-positive cells. Levels of p53, p21, and Bax mRNA were greatest at the 2-h postradiation period. VEGF and PEDF mRNA and protein levels were constant. Protein levels of p53 were increased at the 4- and 6-h postradiation period. CONCLUSIONS: Ionizing radiation induces apoptosis in normal proliferating RPE cells through p53 activation, without affecting expression of VEGF or PEDF. We documented a molecular basis for explaining the decrease in effectiveness of radiation therapy, particularly for age-related macular degeneration. In the clinical setting, selection of appropriate radiation therapy methods and the doses for specific diseases need careful evaluation.

Apoptosis↗

Characterization of mixtures of alkyl polyglycosides (Plantacare) by liquid chromatography-electrospray ionization quadrupole time-of-flight mass spectrometry.

PURPOSE: Industrial products of alkyl polyglycosides (APGs) are widely used as well tolerated surfactants in washing and cleaning agents. APGs belong to the class of nonionic surfactants and are mixtures of molecules consisting of a hydrocarbon chain with Y carbon atoms linked to X sugar residues. Physicochemical properties of APG products strongly depend on the molecular composition. Therefore, a detailed analytical investigation of technical-grade APGs is presented. METHODS: Complex mixtures of alkyl/alkenyl polyglycosides (APGs, Plantacare) and mixtures of APGs with other surfactants were analyzed by reversed-phase liquid chromatography-electrospray ionization quadrupole time-of-flight mass spectrometry (LC/ESI-QTOF-MS) using methanol-water as mobile phase and gradient elution. RESULTS: Analytes were separated according to the chain length of the alkyl homologs. Under the soft ionization of the ESI technique, mainly [M+Na]+ ions were observed, which proved to be very stable. Additionally [M+H]+ ions were detected. The QTOF mass spectrometer allows to identify even high molecular mass components in the mixtures, and APGs up to eight sugar residues were found. From these data, the alkyl/alkenyl chain lengths and the degree of oligomerization of the sugar moiety for different technical-grade APGs were calculated. Using MS/MS experiments, additional structural and chemical information was obtained. CONCLUSIONS: The presented LC/ESI-QTOF-MS approach allows to analyze and characterize various APG products, such as Plantacare. The ability of this LC/ESI-QTOF-MS approach to analyze mixtures of APGs with other surfactants is demonstrated.

Chemistry, Pharmaceutical↗

Cohort Studies and Multi-omics Approaches to Low-Dose Ionizing Radiation-Induced Cardiovascular Disease: A Comprehensive Review.

The effect of low-dose ionizing radiation exposure on the risk of cardiovascular disease (CVD) represents a significant concern in the field of radiation protection. The prevailing approach to mitigating the adverse effects of low-dose or low-dose-rate radiation does not currently incorporate the potential risk of CVD, despite the possibility that such risk may be a substantial contributor to overall health hazards. Current evidence suggests a potential association between radiation exposure and CVD; however, the overall findings remain inconclusive. This is particularly due to the uncertainty surrounding the influence of significant non-radiation risk factors on the associations reported in epidemiological studies. It is difficult to discern the underlying connection in observational epidemiology when there is substantial variation in baseline risk factors. The paucity of epidemiological research in this domain is being partially offset by the advancement of multi-omics approaches. These methods assist in identifying radiosensitive targets, comprehending underlying biological processes, and pinpointing biomarkers. This, in turn, fortifies the evidence gleaned from epidemiological studies. In this review, we delve into the body of epidemiological research pertaining to CVD induced by low-dose ionizing radiation and the application of multi-omics techniques. The integration of these two methodologies holds the promise of identifying specific molecules or biological pathways that can be employed to validate endpoints related to radiation risk assessment.

Humans↗

The induction of reciprocal translocations in mouse germ cells by chemicals and ionizing radiations. III. Differential effect of two doses of adriamycin combined with 5 or 9 Gy of gamma-rays.

The induction of reciprocal translocations in mouse germ cells by combined treatments with chemicals and ionizing radiations has been studied. Male mice were intraperitoneally injected with doses of 5 or 10 mg/kg of adriamycin (ADR) and irradiated with doses of 5 or 9 Gy of gamma-rays 24 h later. Three types of response were found after analyzing diakinesis-metaphase I multivalent configurations: potentiation, with the dose of 5 mg/kg of ADR plus 9 Gy; subadditivity, with the dose of 5 mg/kg of ADR plus 5 Gy; and additivity, with the dose of 10 mg/kg of ADR plus 5 or 9 Gy. According to these results, the subadditive effect observed with the lower dose of ADR plus 5 Gy cannot be explained under the assumption that depletion of any kind of spermatogonia is sufficient for modifying the chromosomal response of stem cells to ionizing radiations. The role of DNA repair mechanisms modulating the response of spermatogonial cells to combined treatments is discussed under the assumption that some repair mechanisms can be triggered by treatment with a low dose of a chemical and these repair mechanisms can reduce cell mortality. Consequently, a higher frequency of more radioresistant cells can survive.

Animals↗

Attenuation and cross-attenuation in taste aversion learning in the rat: studies with ionizing radiation, lithium chloride and ethanol.

The preexposure paradigm was utilized to evaluate the similarity of ionizing radiation, lithium chloride and ethanol as unconditioned stimuli for the acquisition of a conditioned taste aversion. Three unpaired preexposures to lithium chloride (3.0 mEq/kg, IP) blocked the acquisition of a taste aversion when a novel sucrose solution was paired with either the injection of the same dose of lithium chloride or exposure to ionizing radiation (100 rad). Similar pretreatment with radiation blocked the acquisition of a radiation-induced aversion, but had no effect on taste aversions produced by lithium chloride (3.0 or 1.5 mEq/kg). Preexposure to ethanol (4 g/kg, PO) disrupted the acquisition of an ethanol-induced taste aversion, but not radiation- or lithium chloride-induced aversions. In contrast, preexposure to either radiation or lithium chloride attenuated an ethanol-induced taste aversion in intact rats, but not in rats with lesions of the area postrema. The results are discussed in terms of relationships between these three unconditioned stimuli and in terms of implications of these results for understanding the nature of the proximal unconditioned stimulus in taste aversion learning.

Animals↗

Ionizing radiation and genetic risks. IV. Current methods, estimates of risk of Mendelian disease, human data and lessons from biochemical and molecular studies of mutations.

This paper is aimed at a synthesis of conclusions and concepts from the first three papers of this series and an inquiry of their relevance to the estimation of the risk of autosomal dominant and X-linked diseases in man, due to exposure to ionizing radiation. For a population under conditions of continuous irradiation, the doubling-dose method (DD method) enables the prediction of the excess risk of dominant and X-linked diseases at equilibrium. Per unit dose, this quantity is the product of the natural prevalence of these diseases (assumed to be 10,000/10(6) livebirths) and the reciprocal of the DD. The DD currently used is 1 Gy and is based primarily on data on the induction of recessive specific-locus mutations in male mice. The estimate of risk to the first generation is derived from that at equilibrium; the figure is about 15% of the equilibrium value (i.e., 15 cases/10(6) livebirths/cGy). With the direct method, the first-generation risk of dominant disease is estimated using data on the induction of dominant skeletal and cataract mutations in male mice and a number of correction factors. The estimates are about 10-20 cases and 0-9 cases, respectively, for irradiation of males and females, per 10(6) livebirths/cGy. In the Japanese studies, no significant adverse genetic effects, attributable to exposure of the parents to the atomic bombs, could be demonstrated with respect to any of the endpoints used. Most of the latter are clinically and socially relevant but mutationally insensitive. On the basis of these data, Neel and colleagues have estimated that the gametic DD for genetic effects of radiation in man is at least about 4-5 times the 1 Gy value thus far used. The concepts, assumptions, and the data-base used with the DD method have been re-examined. Arguments are advanced to support the thesis that ionizing radiation is probably not very efficient in inducing the very specific molecular changes that are known to underlie spontaneous mutations which cause naturally occurring dominant genetic diseases. It is suggested that (i) the DD estimate of 1 Gy that is used to estimate risk for autosomal dominant and X-linked diseases is conservative and (ii) the 1% prevalence figure for these diseases that is used for this purpose may be too high. If these suggestions are correct, then the estimate of risk for the dominant and X-linked diseases may need to be revised downwards.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

Selective c-Jun overexpression is associated with ionizing radiation-induced apoptosis in the developing cerebellum of the rat.

Immunohistochemistry to Bcl-2, Bax, c-Myc, c-Fos, Fos-related, c-Jun, Jun B and Jun D was used to study the involvement of these factors in ionizing radiation-induced apoptosis in the cerebellum of the developing rat. Selective c-Jun overexpression was observed during the whole process of radiation-induced cell death. Furthermore, c-Jun overexpression was restricted to apoptotic cells, as shown by double labeling with the method of in situ labeling of nuclear DNA fragmentation and c-Jun immunohistochemistry. This is the first in vivo evidence that selective c-Jun overexpression is associated with apoptotic cell death in the developing nervous system following ionizing radiation.

Animals↗

Ionizing radiation exposure of LDEF (pre-recovery estimates).

The long duration exposure facility (LDEF), launched into a 258 nautical mile orbit with an inclination of 28.5 degrees, remained in space for nearly 6 yr. The 21,500 lb NASA satellite was one of the largest payloads ever deployed by the Space Shuttle. LDEF completed 32,422 orbits and carried 57 major experiments representing more than 200 investigators from 33 private companies, 21 universities and nine countries. The experiments covered a wide range of disciplines including basic science, electronics, optics, materials, structures and power and propulsion. A number of the experiments were specifically designed to measure the radiation environment. These experiments are of specific interest, since the LDEF orbit is essentially the same as that of the Space Station Freedom. Consequently, the radiation measurements on LDEF will play a significant role in the design of radiation shielding of the space station. The contributions of the various authors presented here attempt to predict the major aspects of the radiation exposure received by the various LDEF experiments and therefore should be helpful to investigators who are in the process of analyzing experiments which may have been affected by exposure to ionizing radiation. The paper discusses the various types and sources of ionizing radiation including cosmic rays, trapped particles (both protons and electrons) and secondary particles (including neutrons, spallation products and high-LET recoils), as well as doses and LET spectra as a function of shielding. Projections of the induced radioactivity of LDEF are also discussed.

Computer Simulation↗

Toward fingerprinting cellular lipidomes directly from biological samples by two-dimensional electrospray ionization mass spectrometry.

Cellular lipids are composed of linear combinations of aliphatic chains covalently attached to backbones and/or head groups that collectively represent the cellular lipidome. Herein we present a novel two-dimensional electrospray ionization mass spectrometric approach to fingerprint most of the major and many of the minor lipid classes in the hepatic cellular lipidome, which collectively represent >80% of the total lipid mass, directly from their chloroform extracts. Through lipid class-selective intrasource ionization and subsequent analysis of two-dimensional cross-peak intensities, the chemical identity and mass composition of individual molecular species of most mouse hepatic lipid classes were determined from its chloroform extract. This new integrated platform provides a robust foundation for the automated analysis of multidimensional mass spectra to advance the level of analytical scripting in lipidomics to a new plateau.

Animals↗