Chronic Experiments Research Articles

Mimicking the Tendon Microenvironment to Enhance Skeletal Muscle Adhesion and Longevity in a Functional Microcantilever Platform.

Microcantilever platforms are functional models for studying skeletal muscle force dynamics in vitro. However, the contractile force generated by the myotubes can cause them to detach from the cantilevers, especially during long-term experiments, thus impeding the chronic investigations of skeletal muscles for drug efficacy and toxicity. To improve the integration of myotubes with microcantilevers, we drew inspiration from the elastomeric proteins, elastin and resilin, that are present in the animal and insect worlds, respectively. The spring action of these proteins plays a critical role in force dampening in vivo. In animals, elastin is present in the collagenous matrix of the tendon which is the attachment point of muscles to bones. The tendon microenvironment consists of elastin, collagen, and an aqueous jelly-like mass of proteoglycans. In an attempt to mimic this tendon microenvironment, elastin, collagen, heparan sulfate proteoglycan, and hyaluronic acid were deposited on a positively charged silane substrate. This enabled the long-term survival of mechanically active myotubes on glass and silicon microcantilevers for over 28 days. The skeletal muscle cultures were derived from both primary and induced pluripotent stem cell (iPSC)-derived human skeletal muscles. Both types of myoblasts formed myotubes which survived for five weeks. Primary skeletal muscles and iPSC-derived skeletal muscles also showed a similar trend in fatigue index values. Upon integration with the microcantilever system, the primary muscle and iPSC-derived myotubes were tested successively over a one month period, thus paving the way for long-term chronic experiments on these systems for both drug efficacy and toxicity studies.

A Novel 3D‐Printed/Porous Conduit with Tunable Properties to Enhance Nerve Regeneration Over the Limiting Gap Length

AbstractEngineered grafts constitute an alternative to autologous transplant for repairing severe peripheral nerve injuries. However, current clinically available solutions have substantial limitations and are not suited for the repair of long nerve defects. A novel design of nerve conduit is presented here, which consists of a chitosan porous matrix embedding a 3D‐printed poly‐ε‐caprolactone mesh. These materials are selected due to their high biocompatibility, safe degradability, and ability to support the nerve regeneration process. The proposed design allows high control over geometrical features, pores morphology, compression resistance, and bending stiffness, yielding tunable and easy‐to‐manipulate grafts. The conduits are tested in chronic animal experiments, aiming to repair a 15‐mm long gap in the sciatic nerve of rats, and the results are compared with an autograft. Electrophysiological and nociception tests performed monthly during a 4‐month follow‐up show that these conduits allow a good degree of muscle functional recovery. Histological analyses show abundant cellularization in the wall and in the lumen of the conduits and regenerated axons within all rats treated with these grafts. It is suggested that the proposed conduits have the potential to repair nerves over the limiting gap length and can be proposed as strategy to overcome the limitations of autograft.

Acute and chronic toxicity of manganese to tropical adult coral (Acropora millepora) to support the derivation of marine manganese water quality guideline values

Adult corals are among the most sensitive marine organisms to dissolved manganese and experience tissue sloughing without bleaching (i.e., no loss of Symbiodinium spp.) but there are no chronic toxicity data for this sensitive endpoint. We exposed adult Acropora millepora to manganese in 2-d acute and 14-d chronic experiments using tissue sloughing as the toxicity endpoint. The acute tissue sloughing median effect concentration (EC50) was 2560 μg Mn/L. There was no chronic toxicity to A. millepora at concentrations up to and including the highest concentration of 1090 μg Mn/L i.e., the chronic no observed effect concentration (NOEC). A coral-specific acute-to-chronic ratio (ACR) (EC50/NOEC) of 2.3 was derived. These data were combined with chronic toxicity data for other marine organisms in a species sensitivity distribution (SSD). Marine manganese guidelines were 190, 300, 390 and 570 μg Mn/L to provide long-term protection of 99, 95, 90, and 80 % of marine species, respectively.

EVALUATION OF TOXICITY OF BENZIMIDAZOLE DERIVATIVES AND THEIR EFFECT ON FUNCTIONAL PARAMETERS OF CERIODAPHNIA DUBIA RICHARD, 1894

Recently, due to active development of the pharmaceutical industry, an acute problem of environmental pollution with synthetic biologically active compounds has arisen. Benzimidazole cycle is one of the most frequently encountered pharmacophores in drugs. Substances containing even microquantities of benzimidazole have a toxic effect on living systems. Besides, being alien to nature, benzimidazoles are not included in metabolic processes and do not decompose for a long time. Evaluation of benzimidazole toxicity, its stability in the aquatic environment, benzimidazole impact on biological parameters will make it possible to predict possible changes in the populations of ceriodaphnia and other aquatic organisms.
 The purpose of the work is to evaluate the toxicity of benzimidazole derivatives and their effect on the survival and fertility of Ceriodaphnia dubia Richard, 1894.
 Materials and Methods. In acute and chronic experiments, the authors determoined parameters standard for toxicological studies: median lethal concentrations (LC50), life expectancy, total number of offspring, number of litters per female, and average hatching time. The stability of the toxicity of benzimidazole derivatives was established in terms of Ceriodaphnia dubia lethality during a 30-day exposure.
 Results. The authors evaluated the toxicity of benzimidazole and its seven derivatives for ceriodaphnia in terms of the 48-hour median lethal concentration, depending on their structure. The highest toxicity was noted for 5-bromo-2-(trifluoromethyl)-1H-benzimidazole (LC50 2.4 mg/l), the lowest one for 2-methyl-1H-benzimidazole (LC50 109.7 mg/l). It has been shown that long-term exposure (38 days) to potassium salt of pyrido[1,2-a]benzimidazole-7-carboxylic acid (LC50 69.18 mg/l) at a concentration of 20 mg/l in a chronic experiment does not lead to a statistically significant change in survival and fertility parameters of ceriodaphnia. Stability evaluation of toxicity of the potassium salt of pyrido[1,2-a]benzimidazole-7-carboxylic acid and residual toxicity of its degradation products revealed a relatively low stability of the compound toxicity.
 Conclusion. The toxicological evaluation of benzimidazole derivatives for hydrobionts showed the correlation between the degree of their toxicity and structural features.

Effects of diesel exhaust particles on the health and survival of the buff-tailed bumblebee Bombus terrestris after acute and chronic oral exposure

The diesel-powered transportation sector is a major producer of environmental pollution in the form of micro- and nanoscale diesel exhaust particles (DEP). Pollinators, such as wild bees, may inhale DEP or ingest it orally through plant nectar. However, if these insects are adversely affected by DEP is largely unknown. To investigate potential health threats of DEP to pollinators, we exposed individuals of Bombus terrestris to different concentrations of DEP. We analysed the polycyclic aromatic hydrocarbons (PAH) content of DEP since these are known to elicit adverse effects on invertebrates. We investigated the dose-dependent effects of those well-characterized DEP on survival and fat body content, as a proxy for the insects’ health condition, in acute and chronic oral exposure experiments. Acute oral exposure to DEP showed no dose-dependent effects on survival or fat body content of B. terrestris. However, we could show dose-dependent effects after chronic oral exposure with high doses of DEP where significantly increased mortality was observed. Further, there was no dose-dependent effect of DEP on the fat body content after exposure. Our results give insights into how the accumulation of high concentrations of DEP e.g., near heavily trafficked sites, can influence insect pollinators’ health and survival.

Environmental exposure and ecotoxicological properties of a new-generation fluorosurfactant (cC6 O4 ): A comparison with selected legacy perfluoroalkyl acids.

Cyclic C6 O4 (cC6 O4 , CAS number 1190931-27-1) is a new-generation polyfluorinated alkyl substance (PFAS) used as a polymerization aid in the synthesis of fluoropolymers, which has been produced in Italy since 2011. A review of the properties of cC6 O4 , focused on environmental distribution and ecotoxicology, was conducted. The EQuilibrium Criterion model was applied, using default environmental scenarios, to estimate environmental distribution and fate. In a situation of static thermodynamic equilibrium in a closed system (Level I), cC6 O4 distributes mainly to water (97.6%) and in a minimal amount to soil (2.3%). In a more realistic scenario (Level III), with dynamic conditions in an open system, with advection in air and water and with equal emissions in air and water, the major amount of the compound is transported through water advection. Monitoring data, mainly referring to surface and groundwater, are available for water bodies close to the production sites (maximum measured concentration 52 μg/L) as well as for a wider area in the river Po watershed with concentrations generally lower than 1 μg/L. Few values are also available for concentration in biota. Effect data indicate low toxicity on all tested organisms with no observed effect concentration (NOEC) values always higher than the maximum concentrations tested (100 mg/L for acute tests). Bioaccumulation potential is also very low. A comparison with selected widely used PFAS with five to eight C atoms indicates that cC6 O4 is substantially less dangerous to aquatic organisms. For the time being, an ecological risk for the aquatic ecosystem may be excluded even in directly exposed ecosystems. However, for a complete assessment of the suitability of cC6 O4 as a substitute forother PFAS (namely, perfluorooctanoic acid), more comprehensive chronic experiments are necessary, to produce realistic NOEC, as well as higher tier experiments (e.g., mesocosms) capable of providingecologically relevant endpoints. Moreover, a more accurate evaluation of the environmental persistence would be necessary. Integr Environ Assess Manag 2023;19:1636-1648. © 2023 SETAC.

Clarification of hypertension mechanisms provided by the research of central circulatory regulation.

Sympathoexcitation, under the regulatory control of the brain, plays a pivotal role in the etiology of hypertension. Within the brainstem, significant structures involved in the modulation of sympathetic nerve activity include the rostral ventrolateral medulla (RVLM), caudal ventrolateral medulla (CVLM), nucleus tractus solitarius (NTS), and paraventricular nucleus (paraventricular). The RVLM, in particular, is recognized as the vasomotor center. Over the past five decades, fundamental investigations on central circulatory regulation have underscored the involvement of nitric oxide (NO), oxidative stress, the renin-angiotensin system, and brain inflammation in regulating the sympathetic nervous system. Notably, numerous significant findings have come to light through chronic experiments conducted in conscious subjects employing radio-telemetry systems, gene transfer techniques, and knockout methodologies. Our research has centered on elucidating the role of NO and angiotensin II type 1 (AT1) receptor-induced oxidative stress within the RVLM and NTS in regulating the sympathetic nervous system. Additionally, we have observed that various orally administered AT1 receptor blockers effectively induce sympathoinhibition by reducing oxidative stress via blockade of the AT1 receptor in the RVLM of hypertensive rats. Recent advances have witnessed the development of several clinical interventions targeting brain mechanisms. Nonetheless, Future and further basic and clinical research are needed.

Co-exposure to environmentally relevant concentrations of cadmium and polystyrene nanoplastics induced oxidative stress, ferroptosis and excessive mitophagy in mice kidney

Nanoplastics (NPs) are defined as a group of emerging pollutants. However, the adverse effect of NPs and/or heavy metals on mammals is still largely unclear. Therefore, we performed a 35-day chronic toxicity experiment with mice to observe the impacts of exposure to Cadmium (Cd) and/or polystyrene nanoplastics (PSNPs). This study revealed that combined exposure to Cd and PSNPs added to the mice's growth toxicity and kidney damage. Moreover, Cd and PSNPs co-exposure obviously increased the MDA level and expressions of 4-HNE and 8-OHDG while decreasing the activity of antioxidase in kidneys via inhibiting the Nrf2 pathway and its downstream genes and proteins expression. More importantly, the results suggested for the first time that Cd and PSNPs co-exposure synergistically increased iron concentration in kidneys, and induced ferroptosis through regulating expression levels of SLC7A11, GPX4, PTGS2, HMGB1, FTH1 and FTL. Simultaneously, Cd and PSNPs co-exposure further increased the expression levels of Pink, Parkin, ATG5, Beclin1, and LC3 while significantly reducing the P62 expression level. In brief, this study found that combined exposure to Cd and PSNPs synergistically caused oxidative stress, ferroptosis and excessive mitophagy ultimately aggravating kidney damage in mice, which provided new insight into the combined toxic effect between heavy metals and PSNPs on mammals.

Combined effects of two abiotic stressors (salinity and temperature) on a laboratory-simulated population of Daphnia longispina

Sea level rise is expected to continue apace, with a concomitant rise in temperature on the globe's surface. Natural populations genetic pool compromised by increased salinity would contribute to decrease resilience under future temperature changes. Therefore, this work aimed to understand the long-term effects of salinization on the genetic diversity of a cladoceran-simulated laboratory population under different temperature regimes. For that, six clonal lineages of the cladoceran Daphnia longispina were chosen based on their reported differential lethal sensitivity (LCx) to salinity. The chronic experiment was initiated when each individual clonal lineage reached the population’s carrying capacity, and then were exposed to the LC70,48 h for the most tolerant clonal lineage of D. longispina (corresponding to 5.91 mS/cm) to 17°C, 20°C and 23°C for at least 30 days. Salinity affected D. longispina survival and reproduction, with the disappearance of salt-tolerant earlier than salt-sensitive lineages after chronic exposures. Different sensitivity ranks were observed for clonal lineages when comparing short-term and chronic survival, most probably due to acclimation-driven population recovery. Non-optimal tested temperatures (17°C and 23°C) enhanced negative effects of salinity through loss of the most sensitive clonal lineages, suggesting a potential future synergistic effect between both abiotic factors.

Multigenerational effects of the insecticide Pyriproxyfen and recovery in Daphnia magna

In the present study, an ecotoxicological approach to the evaluation of the insecticide Pyriproxifen in the crustacean Daphnia magna was done. Acute toxicity tests (48 h), feeding behavior test (5 h) and chronic toxicity test (21 days) were carried out on a parental daphnid generation (F0). Pyriproxifen D. magna EC50 value in our experimental conditions was 336.47 μg/L. Based on this result, sublethal test concentrations were selected for the feeding study and the F0 chronic experiment. Filtration and ingestion rates of D. magna exposed animals did not show any significant difference respect to control daphnids. However, daphnids from the parental F0 generation when exposed to the insecticide during 21 days showed a decreased in all the reproductive parameters tested (mean total neonates per female, mean brood size, time to first brood, and mean number of broods per female) as well as in the population statistic growth rate (r), although survival was not affected. On the other hand, offspring from F0 females exposed to the highest Pyriproxifen concentration (14.02 μg/L) were separated in two F1 generation experiments. One group was transferred during 21 days to a medium free of toxicant (F1 generation-TC group) while the other group was exposed during 21 days to the same insecticide concentration as their mothers (14.02 μg/L) (F1 generation-TT group). Results from both experiments determined a decreased in most of the reproductive parameters which was higher in the F1-TT group, although some of them were recovered in the F1-TC group. On the other hand, the morphological analysis of the daphnids showed that the coloration pattern was altered in the daphnids exposed to the insecticide, together with a significant size decreased, and neonates from F0 progeny with the same morphological abnormality. Finally, we determined that the insecticide caused the appearance of males among the offspring generated by the F0.

Soil Respiration Responses to Throughfall Exclusion Are Decoupled From Changes in Soil Moisture for Four Tropical Forests, Suggesting Processes for Ecosystem Models

AbstractClimatic drying is predicted for many tropical forests yet models remain poorly parameterized for these ecosystems, hampering predictions of forest‐climate interactions. We applied an integrated model–experiment approach, parameterizing an ecosystem model with tropical forest observational data and comparing model predictions to a field drying manipulation. We hypothesized that drying suppresses soil CO2 fluxes (i.e., respiration) in relatively dry tropical forests but increases CO2 fluxes in wetter tropical forests by alleviating anaerobiosis. We measured soil CO2 fluxes during wet‐dry cycles from 2015 to 2022 in four Panamanian forests that vary in rainfall and soil fertility. Measured soil CO2 fluxes declined in the dry season and peaked in the early wet season ahead of peak soil moisture, resulting in lower soil moisture optima for respiration than previously modeled. We then parameterized the model using field data and the new moisture‐respiration response functions. The updated model predicted increased soil CO2 fluxes with drying in wetter and fertile forests and suppressed fluxes in drier, infertile forests. In contrast to model predictions, a chronic throughfall exclusion experiment initially suppressed soil respiration across forests, with sustained suppression for four years in the wettest forest only (−28% ± 4% during the dry season). In the fertile forest, drying eventually elevated CO2 fluxes over this period (+75% ± 28% during the late wet season). The unexpected negative drying effect in the wettest, infertile forest could have resulted from reduced vertical flushing of nutrients into soils. Including hydro‐nutrient interactions in ecosystem models could improve predictions of tropical forest‐climate feedbacks.

Neuroprotective Activity of Ethanolic Extract of Red Ginger Containing 6-Shogaol on Scopolamine-Induced Memory Impairment in Alzheimer's Mice

Red ginger contains 6-shogaol, which has a neuroprotective effect and is crucial in several brain diseases, including Alzheimer’s. This study investigated the neuroprotective effect of red ginger extract (RGE) containing 6-shogaol on scopolamine-induced memory loss in mice. Male Balb/C mice (n = 30; 6–7 weeks old) were divided into six groups: normal, Alzheimer, drug control (donepezil), and RGE (100, 200, and 400 mg/kg BW). In the acute experiment, the mice were treated 60 minutes before the test, followed by 1 mg/kg of scopolamine 30 minutes later. Thirty minutes later, the mice were placed individually in a Y-maze to observe spontaneous alteration activity (SAA). In the chronic experiment, the mice were treated once daily for seven days. On days 8–14, the mice were administered scopolamine and treatment. SAA was observed every 3–4 days. On day 15, malondialdehyde (MDA) and acetylcholinesterase (AChE) levels in the serum and brain were determined. RGE treatment 400 mg/kg BW, containing 1.664 µg of 6-shogaol per 100 mg of ethanol RGE, reduced memory loss better than the other two doses. RGE successfully decreased MDA and increased AChE in the serum and brain. RGE also showed effectively to improve memory in Alzheimer’s disease.

Two genes related to apoptosis in the hepatopancreas of juvenile prawn, Macrobrachium nipponense: Molecular characterization and transcriptional response to nanoplastic exposure

Nanoplastics have been widely found in the global water environment, causing plastic pollution and affecting human beings and numerous organisms. Studies involving freshwater crustacean exposure to nanoplastics, however, are limited. In this study, juvenile prawns (Macrobrachium nipponense) were exposed to 75 nm polystyrene nanoplastics at different concentrations (0, 5, 10, 20, or 40 mg/L) for a 28-d chronic exposure experiment. To study the effects of exposure to nanoplastics on hepatopancreas cell apoptosis, C-Jun N-terminal kinase (JNK) and phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha (PIK3CA) genes were selected, and hepatotoxic enzyme activities and Toll pathway- and apoptosis-related gene expression were determined. For the first time, full-length Mn-JNK and Mn-PIK3CA cDNAs were cloned from M. nipponense. Homologous comparisons showed that JNK and PIK3CA had conserved functional sequences. The apoptosis rate in the high-concentration nanoplastic group (40 mg/L) was significantly higher than in the low-concentration nanoplastic (5 mg/L) and control groups (0 mg/L). The alanine aminotransferase (ALT), aspartate aminotransferase (AST), glutamyl transpeptidase (GGT) and xanthine oxidase (XOD) enzyme activities in the hepatopancreas increased with exposure to higher concentrations of nanoplastics. In addition, the levels of apoptosis- and Toll pathway-related gene expression and JNK and PIK3CA gene expression were initially increased, then decreased with exposure to higher concentrations of nanoplastics. This study showed that polystyrene nanoplastics activate toll-related pathways leading to apoptosis and hepatopancreas damage, which provides theoretical support for future aquatic toxicological research.

Effects of salinity on growth, physiology, biochemistry and gut microbiota of juvenile grass carp (Ctenopharyngodon idella)

Grass carp (Ctenopharyngodon idella) is among the most important freshwater fish species in China. However, it remained unclear how salinity could affect grass carp. Two experiments were performed. The first experiment was a 4-day acute salt tolerance experiment with six salinities (0, 4, 8, 12, 16, and 20 ppt). The second experiment was an 8-week chronic salt stress experiment with three salinities (0, 2 and 6 ppt). To investigate the intestinal bacterial community of grass carp from three salinities (0, 2, and 6 ppt), the 16S rDNA sequencing was performed. The results showed that grass carp exhibited great adaptability to low salinity (2 ppt), with no significant difference in growth and maintained stable physiological and immune status. However, exposed to high salinity (6 ppt) caused significant deleterious effects on grass carp, including growth inhibition as well as physiological and immune-related changes. The gut microbiota in grass carp changed with salinity. With the increase of salinity, the proportion of beneficial bacteria in the gut of grass carp gradually decreased, while some harmful bacteria gradually occupied the dominant position. Changes in gut microbial composition ultimately affected the growth of grass carp. This study helps further clarify the effects of salinity on grass carp.

Characterization of a chloroacetanilide derivative in a chronic oral toxicology study in rats

Introduction. For the treatment of agricultural crops, an original herbicide of selective action was created - propisochlor, a derivative of chloroacetanilide. Its characteristics in an acute toxicological experiment didn’t reveal the features of the pathogenesis of intoxication. The identification of regularities in the manifestation of the toxic properties of propisochlor in various regimens with the oral route of exposure is necessary at the stage of production design. Moreover, it is necessary to substantiate a number of hygienic regulations for safe production and its further application in agriculture, which lies in the plane of solving the leading state tasks aimed at protecting human health and its environment, and meets the requirements of international legislation. A special role in the development of such methods and methods belongs to the sanitary-toxicological study of new xenobiotics and generic molecules, as a result of which threshold and subthreshold doses of pesticide preparations and their active substances are determined. The aim of this study was to characterize propisochlor in a chronic oral toxicology study in rats. To achieve the goal, it was necessary to solve the following tasks: to study the chronic oral effect of the chloroacetanilide derivative on the rat organism; establish a dose that does not have a visible undesirable effect (NOELch); set the lowest dose at which no adverse effects are observed (LOAELch); calculate the allowable daily intake, or allowable daily exposure (ADD, or ADI, or PDE) for a person. Material and methods. The object of testing in this work was a herbicide from the class of chloroacetanilide, propisochlor. The test system in the chronic experiment was white male rats, on which the influence of the test compound was evaluated in dynamics after 1, 3, 6 and 12 months according to the state of integral indicators. Results. The parameters of the oral toxicity of the test compound were determined and the changes in the studied integral indicators were evaluated at various stages of the experiment in a chronic experiment. Conclusion. Based on the comparative characterization of a new herbicide propisochlor in a chronic oral toxicology study in rats, a non-acting dose (NOEL), the lowest effective dose (LOAEL), and an acceptable daily dose for humans (ADI, or PDE) are justified.

Acute Exposure of Zebrafish (Danio rerio) to the Next-Generation Perfluoroalkyl Substance, Perfluoroethylcyclohexanesulfonate, Shows Similar Effects as Legacy Substances.

Perfluoroethylcyclohexanesulfonate (PFECHS) is an emerging perfluoroalkyl substance (PFAS) that has been considered a potential replacement for perfluorooctanesulfonic acid (PFOS). However, there is little information characterizing the toxic potency of PFECHS to zebrafish embryos and its potential for effects in aquatic environments. This study assessed toxic potency of PFECHS in vivo during both acute (96-hour postfertilization) and chronic (21-day posthatch) exposures and tested concentrations of PFECHS from 500 ng/L to 2 mg/L. PFECHS was less likely to cause mortalities than PFOS for both the acute and chronic experiments based on previously published values for PFOS exposure, but exposure resulted in a similar incidence of deformities. Exposure to PFECHS also resulted in significantly increased abundance of transcripts of peroxisome proliferator activated receptor alpha (pparα), cytochrome p450 1a1 (cyp1a1), and apolipoprotein IV (apoaIV) at concentrations nearing those of environmental relevance. Overall, these results provide further insight into the safety of an emerging PFAS alternative in the aquatic environment and raise awareness that previously considered "safer" alternatives may show similar effects as legacy PFASs.

Ecotoxicological Differences of Antimony (III) and Antimony (V) on Earthworms Eisenia fetida (Savingy).

In this study, we assessed the acute and chronic toxic effects of Sb (III) and Sb (V) on Eisenia fetida (Savingy) (E. fetida) by applying the filter paper contact method, aged soil treatment, and avoidance test experiment. In the acute filter paper contact test, the LC50 values for Sb (III) were 2581 mg/L (24 h), 1427 mg/L (48 h), and 666 mg/L (72 h), which were lower than Sb (V). In the chronic aged soil exposure experiment, when the Sb (III)-contaminated soil was aged 10 d, 30 d, and 60 d after exposure for 7 d, the LC50 value of E. fetida was 370, 613, and >4800 mg/kg, respectively. Compared to Sb (V) spiked soils aged only for 10 d, the concentrations causing 50% mortality significantly increased by 7.17-fold after 14 days of exposure in soil aged for 60 d. The results show that Sb (III) and Sb (V) could cause death and directly affect the avoidance behavior of E. fetida; yet, the toxicity of Sb (III) was higher than that of Sb (V). Consistent with the decrease in water-soluble Sb, the toxicity of Sb to E. fetida was greatly reduced with time. Therefore, in order to avoid overestimating the ecological risk of Sb with varying oxidative states, it is important to consider the forms and bioavailability of Sb. This study accumulated and supplemented the toxicity data, and provided a more comprehensive basis for the ecological risk assessment of Sb.

Responses of oxidative stress biomarkers of freshwater fish (Oreochromis niloticus) exposed to Cr6+, Hg2+, Ni2+ and Zn2+ in differing calcium levels

Freshwaters from different geographical locations show different hardness, affecting metal uptake and toxicity in fish. The most important ion that determines water hardness is calcium. In this study, acute and chronic effects of metals on the oxidative stress biomarkers in the liver of freshwater fish (Oreochromis niloticus) were investigated in differing Ca2+ (30, 60 and 120 mg Ca2+/L) levels. Fish were exposed to Cr6+, Ni2+ and Zn2+ (30 μM) and Hg2+ (0.3 μM) for 3 days in acute experiments, while they were exposed to Cr6+, Ni2+ and Zn+2 (10 μM) and Hg2+ 0.03 μM) for 30 days in chronic experiments. Data showed that the oxidative stress biomarkers significantly (p < 0.05) altered after metal exposures at all calcium levels, though there was no significant change (p > 0.05) among calcium controls. In both acute and chronic exposures, catalase CAT) and superoxide dismutase (SOD) activities increased significantly, while glutathione peroxidase (GPX), glutathione reductase (GR) and glutathione S-transferase (GST) activities decreased. There were significant decreases in total glutathione (GSH) levels in acute exposures, though GSH levels increased in chronic exposures. Malondialdehyde (MDA) levels significantly increased in both durations. The highest significant alterations in the biomarkers occurred at the lowest Ca2+ levels. GPX and GST were found to be the most sensitive enzymes in all exposures and the least alterations in biomarker response occurred in fish exposed to Ni2+. This study demonstrated that calcium levels (hardness) were important factors in the evaluation of metal toxicity for freshwater fish.

A Model Framework for the Estimation of Animal ‘Suffering’: Its Use in Predicting and Retrospectively Assessing the Impact of Experiments on Animals

This paper presents and illustrates, with a working example, a hypothesis for the assessment of ongoing severity before and during an experiment that will enable humane endpoints and intervention points to be applied accurately and reproducibly, as well as helping to implement any national legal severity limits in subacute and chronic animal experiments, e.g., as specified by the competent authority. The underlying assumption of the model framework is that the degree of deviation from normality of specified measurable biological criteria will reflect the level of pain, suffering, distress and lasting harm incurred by or during an experiment. The choice of criteria will normally reflect the impact on an animal and have to be chosen by scientists and those caring for the animals. They will usually include measurements of good health such as temperature, body weight, body condition and behaviour, which vary according to the species, husbandry and experimental protocols and, in some species, unusual parameters such as time of the year (e.g., migrating birds). In animal research legislation, endpoints or severity limits may be specified so that individual animals do not suffer unnecessarily or endure severe pain and distress that is long-lasting (Directive 2010/63/EU, Art.15.2). In addition, the overall severity is estimated and classified as part of the harm: benefit licence assessment. I present a mathematical model to analyse the measurement data to determine the degree of harm (or severity) incurred. The results can be used to initiate alleviative treatment if required or if permitted during the course of an experiment. In addition, any animal determined to have breached the severity classification of a procedure can be humanely killed, treated or withdrawn from the experiment. The system incorporates the flexibility to be used in most animal research work by being tailored to the research, the procedures carried out and the species under investigation. The criteria used to score severity can also be used as additional scientific outcome criteria and for an analysis of the scientific integrity of the project.

Zinc-Boron Complex-Based Dietary Supplements for Longevity and Healthy Life.

The nutritional role of zinc (Zn) and boron (B) in the plant-animal-human food chain is highly topical worldwide research. Our data indicated that Zn-B complex (ZBC)-based dietary supplements can be used as stable non-toxic formulations, with high intestinal absorption rate, inducing alpha2-macroglobulin (A2M) expression for longevity and healthy life. ZBC is metabolized by hydrolysis, mainly at the absorption site (intestinal level), and most of it is excreted in the urine. Within seven hours from the administration in mice, almost the entire amount of orally absorbed ZBC is eliminated in a metabolized form. The highest amount of A2M protein in mouse liver was determined by immunoabsorbance assay in the chronic experiment (1000mg/kg of ZBC), followed by the subchronic experiment (at the same dose of ZBC), and by the acute experiment (5000mg/kg of ZBC).