Aluminum exposure alters the pedal mucous secretions of the chocolate-band snail, Eobania vermiculata (Gastropoda: Helicidae).

Aluminum (Al) is used in everyday life and present in food drugs, packaging, industry, and agriculture. Although it is the most common metal in the Earth crust, a correlation has been demonstrated between its presence and various pathologies, even serious ones, especially of a neurological type. However, there is a histological gap regarding the role Al can have in contact with the covering and secreting epithelia. The alterations of the ventral and dorsal foot mucocytes and their secretions of the snail Eobania vermiculata caused by Al were investigated in situ by histochemical and lectin-histochemical techniques. Administration to different experimental groups took place for 3 and 9 days with 50 and 200 µM of AlCl3. Several types of mucocytes were detected with a prevalent secretion of acid glycans in the foot of E. vermiculata. Sulfated glycans prevail in the dorsal region, with one type showing only fucosylated residues and another also having galactosaminylated and glycosaminylated residues. Carboxylated glycans prevail in the ventral region, with presence of galactosaminylated, glycosaminylated, and fucosylated residuals in both cells. Snails treated presented a general decrease of mucin amount in the secreting cells and affected the mucus composition. These changes could alter the rheological and functional properties of the mucus with possible implications for the health of the treated animals. RESEARCH HIGHLIGHTS: Snails were fed with Al-contaminated lettuce at different concentrations. In the foot mucocytes produced mucus with prevailing acidic glycans. In the treated resulted a reduction in the amount of mucus and an alteration of glycan composition.

Synthesis and Degradation of Poly(ADP-ribose) in Zebrafish Brain Exposed to Aluminum.

Poly(ADPribosyl)ation is a post-translational protein modification, catalyzed by poly(ADP-ribose) polymerase (PARPs) enzymes, responsible for ADP-ribose polymer synthesis (PAR) from NAD+. PAR turnover is assured by poly(ADPR) glycohydrolase (PARGs) enzymes. In our previous study, the altered histology of zebrafish brain tissue, resulting in demyelination and neurodegeneration also with poly(ADPribosyl)ation hyperactivation, was demonstrated after aluminum (Al) exposure for 10 and 15 days. On the basis of this evidence, the aim of the present research was to study the synthesis and degradation of poly(ADP-ribose) in the brain of adult zebrafish exposed to 11 mg/L of Al for 10, 15, and 20 days. For this reason, PARP and PARG expression analyses were carried out, and ADPR polymers were synthesized and digested. The data showed the presence of different PARP isoforms, among which a human PARP1 counterpart was also expressed. Moreover, the highest PARP and PARG activity levels, responsible for the PAR production and its degradation, respectively, were measured after 10 and 15 days of exposure. We suppose that PARP activation is related to DNA damage induced by Al, while PARG activation is needed to avoid PAR accumulation, which is known to inhibit PARP and promote parthanatos. On the contrary, PARP activity decrease at longer exposure times suggests that neuronal cells could adopt the stratagem of reducing polymer synthesis to avoid energy expenditure and allow cell survival.

Aluminum induces a stress response in zebrafish gills by influencing metabolic parameters, morphology, and redox homeostasis.

Environmental air pollution and resulting acid rain have the effect of increasing aluminum levels in water bodies. We studied the effects of aluminum on fish gills, the tissue most exposed to aluminum, using zebrafish as an experimental model. Adult zebrafish were exposed to an aluminum concentration found in polluted environments (11 mg/L) for 10, 15 and 20 days and the effects on gill morphology, redox homeostasis (ROS content, NADPH oxidase, NOX, activity, oxidative damage, antioxidant enzymes, total antioxidant capacity, in vitro susceptibility to oxidants) and on behavioural and metabolic parameters (routine respiratory oxygen consumption rMO2, tail-beating frequency, cytochrome oxidase activity and muscle lactate content) were evaluated. Exposure to aluminum affects branchial histology, inducing alterations in primary and secondary lamellae and redox homeostasis, modifying ROS levels, NOX activity, lipid and protein oxidative damage, antioxidant enzymes, and total antioxidant capacities, and increases rMO2. The effects exhibited a time-dependent behaviour, suggesting the activation of an adaptive response. These changes are associated with a transition of muscle metabolism from aerobic to anaerobic, as suggested by the increase in muscle lactate content, which is probably functional to preserve locomotor performance. Overall, the results here reported provide new insights into the toxicity mechanisms of Al exposure on gill tissue and the subsequent adaptive response of aquatic species.

Histological alterations and oxidative stress in adult zebrafish muscle after aluminium exposure.

Aluminium (Al) is among the most abundant metals in nature, and its presence in the environment is further increasing by anthropogenic activities. In water bodies, the Al concentrations ranged between 0.001 and 50 mg/L, raising concerns about the health of aquatic organisms. For this reason, zebrafish was chosen as the model, since it is well suited for ecotoxicological studies. Adult specimens were exposed to 11 mg/L of Al for 10, 15 and 20 days to assess both the morphology and the oxidative state of muscle tissue. Considering the involvement of ROS, the activity of the main antioxidant enzymes, metallothioneins contents, but also oxidative damage and enzymes involved in energy consumption and neuromuscular transmission were assessed. Collected data showed an increase in the thickness of the endomysium and resorbed myofibrils in the organisms exposed to Al for 10 days, and an increase of myotomes' size in the organisms exposed to Al for 15 days. Moreover, the organisms exposed for less time to Al, it was evident an activation of anaerobic metabolism and the increased activity of antioxidant enzymes such as superoxide dismutase, glutathione peroxidase and glutathione S-transferases. However, these effects stabilized with increasing exposure time. In addition, only after 20 days of treatment did the oxidative damage to the proteins and the activity of acetylcholinesterase increase while the levels of metallothioneins and the lipid peroxidation were lower for all treated animals when compared to the control group. Overall, the biochemical and histological changes induced by aluminium exposure in the muscular tissue represent a relevant contribution to understanding the environmental risk due to the diffusion of this metal within the aquatic compartment.

Aluminium exposure leads to neurodegeneration and alters the expression of marker genes involved to parkinsonism in zebrafish brain.

Aluminium, despite being extremely widespread in the world, is a non-essential metal to human metabolism. This metal is known to have toxic effects on a variety of organs including the brain and is considered an etiological factor in neurodegenerative diseases. However, the molecular mechanisms by which aluminium exerts neurotoxic effects are not yet completely understood. Zebrafish is an animal model also used to study neurodegenerative diseases since the overall anatomical organization of the central nervous system is relatively conserved and similar to mammals. Adult zebrafish were exposed to 11 mg/L of Al for 10, 15, and 20 days and the neurotoxic effects of aluminium were analysed by histological, biochemical, and molecular evaluations. Histological stainings allowed to evaluation of the morphology of the brain parenchyma, the alteration of myelin and the activation of neurodegenerative processes. The expression of the Glial Fibrillary Acidic Protein, a marker of glial cells, was evaluated to observe the quantitative alteration of this important protein for the nervous system. In addition, the poly(ADP-ribose) polymerase activity was measured to verify a possible oxidative DNA damage caused by exposure to this metal. Finally, the evaluation of the markers involved in Parkinsonism was assessed by Real-Time PCR to better understand the role of aluminium in the regulation of genes related to Parkinson's neurodegenerative disease. Data showed that aluminium significantly affected the histology of cerebral tissue especially in the first periods of exposure, 10 and 15 days. This trend was also followed by the expression of GFAP. At longer exposure times, there was an improvement/stabilization of the overall neurological conditions and decrease in PARP activity. In addition, aluminium is involved in the deregulation of the expression of genes closely related to Parkinsonism. Overall, the data confirm the neurotoxicity induced by aluminium and shed a light on its involvement in neurodegenerative processes.

Apoptosis, oxidative stress and genotoxicity in developing zebrafish after aluminium exposure.

Aluminium is a non-essential metal and potentially toxic to organisms whose environmental concentration increases due to pollution. In our previous studies, the behavioral changes induced by aluminium were already shown on zebrafish, a model organism widely used for ecotoxicology screening. To examine in depth the knowledge about the toxicity mechanism induced by this metal, zebrafish embryos, at 6 hpf, have been exposed to 50, 100 and 200 µM of AlCl3 for 72 h. Phenotypic alterations, apoptosis and oxidative stress responses have been assessed by evaluations of antioxidant defence and changes in metabolism at the end of treatment. The mRNA expression level of c-fos, appa and appb as marker genes of neural development and function were analyzed by qPCR for the highest used concentration. The data showed that aluminium significantly affected the development of zebrafish inducing morphological alterations and cell death. The oxidative state of larvae was altered, although the formation of reactive oxygen species and the levels of metallothioneins, and the activity of some antioxidant enzymes, decreased at the maximum concentration tested. In addition, at this concentration, the expression of the evaluated genes increased. The comprehensive information obtained gives a realistic snapshot of the aluminium toxicity and provides new information on the mechanism of action of this metal.

The Interplay between Light Quality and Biostimulant Application Affects the Antioxidant Capacity and Photosynthetic Traits of Soybean (Glycine max L. Merrill).

This paper evaluates the combined effect of biostimulant and light quality on bioactive compound production and seedling growth of soybean (Glycine max L. Merrill) plants. Germinated seeds pre-treated with different concentrations (0.01%, 0.05%, 0.5%) of an amino acid-based biostimulant were grown for 4 days at the dark (D), white fluorescent light (FL), full-spectrum LED (FS), and red-blue (RB) light. Potential changes in the antioxidant content of sprouts were evaluated. Part of the sprouts was left to grow at FL, FS, and RB light regimes for 24 days to assess modifications in plants' anatomical and physiological traits during the early developmental plant stage. The seed pre-treatment with all biostimulant concentrations significantly increased sprout antioxidant compounds, sugar, and protein content compared to the control (seeds treated with H2O). The positive effect on bioactive compounds was improved under FS and RB compared to D and FL light regimes. At the seedling stage, 0.05% was the only concentration of biostimulant effective in increasing the specific leaf area (SLA) and photosynthetic efficiency. Compared to FL, the growth under FS and RB light regimes significantly enhanced the beneficial effect of 0.05% on SLA and photosynthesis. This concentration led to leaf thickness increase and shoot/root ratio reduction. Our findings demonstrated that seed pre-treatment with proper biostimulant concentration in combination with specific light regimes during plant development may represent a useful means to modify the bioactive compound amount and leaf structural and photosynthetic traits.

Exposure to aluminium causes behavioural alterations and oxidative stress in the brain of adult zebrafish.

Aluminium (Al) water pollution is an increasing environmental problem. Accordingly, this study aimed to find out more about its toxic effects on aquatic organisms. Adult zebrafish were exposed to 11 mg/L of Al and the behavioural responses and its correlation with brain oxidative stress, antioxidant-defences, changes in metabolism and neurotransmission were assessed at 10, 15 and 20 days of exposure. The behavioural and locomotory responses, suggest an increase in the anxiety state, especially observed in animals exposed to Al for 15 days. The reactive oxygen species increased in a time-dependent trend, while the oxidative damage varied over exposure time. The activity of antioxidant enzymes, as superoxide dismutase, glutathione peroxidase and glutathione S-transferases, and the metallothioneins levels increased after short-term exposures and tended to decrease or stabilize at longer times. The results contribute to understand the toxic mechanisms activated by Al highlighting correlations like behavioural disorders and oxidative state.

Adverse effects of E150d on zebrafish development.

E150d is a food additive used to increase palatability and impart colour to foods and drinks. Known as 'caramel dye', it contains 4-methylimidazole, a cytotoxic molecule for animal models and human. Recently, the growing consumption of E150d causes an increasing release of this additive into the environment, particularly in water bodies. For this reason, in this study it was assessed the toxic effect of E150d on zebrafish embryos, a conventional aquatic model organism. Six hours post fertilization embryos were treated with two different concentrations of E150d (0.3 g/L and 0.6 g/L) for 72 h and their embryonic development was studied. It emerged that this food additive induced toxic effects on hatching, survival, embryos phenotype and cardiac beat with a dose-dependent trend. Furthermore, it impaired swimming performance and induced damages in skeletal muscles and pericardial cavity. Data obtained showed the risk associated with the dispersion of E150d in water bodies suggesting that a greater attention should be paid in avoiding an unnecessary use so to preserve human and environmental health.

Eobania vermiculata as a potential indicator of nitrate contamination in soil.

The effects of nitrates were analysed on the land snail Eobania vermiculata, a good bioindicator to assess the effects of certain pollutants in soil. It is known that the molluscs are very sensitive to contamination substances and can be used as sentinel organism for environmental pollution assessment. The nitrates are present in fertilizers and in food additives and their excess can not only be harmful to the environment but also dangerous for the humans. Indeed, in the mammals the nitrates are converted into nitrites and can cause a series of complications as the formation of methaemoglobin and cancers. In this study, adult organisms of E. vermiculata were exposed to soil containing 2000 mg/L of nitrates for 30 days to evaluate the stool microbiome and the histological changes at the level of the foot. Eggs of these snails were similarly treated to observe their hatching, survival and development. Histological changes were observed at level of the foot of adult snails exposed to nitrate and in their stools was evident an increase of bacteria, especially those that have a high ability to exploit nitrates and nitrogen as nutrients. Instead, the treated eggs showed changes in hatching, hypopigmentation of newborn snails and a decrease of their survival in time. The overall information obtained from these endpoints can provide important information regarding the quality of the environment. In addition, they also showed that the invertebrate organism E. vermiculata despite being a simple organism is very useful and efficient for ecotoxicological studies.

Impact of copper in Xenopus laevis liver: Histological damages and atp7b downregulation.

Copper is an essential micronutrient but its excess in the dietary can be toxic. Both copper deficiency and abundance can occur in natural conditions and can lead to pathological dysfunctions. Many of the toxic effects of copper, such as increased lipid peroxidation in cell membranes and DNA damage, are due to its role in the generation of oxygen free radicals. Copper is released into the environment by both natural sources and human activities and it can damage organisms and ecosystems. In the present work the effects of copper has been studied on Xenopus laevis, an interesting model organism, after three weeks of exposure at 1 mg/L of CuCl, concentration allowed in the water for human use. The effects of this metal were analysed on the liver at light microscope by Hematoxylin-Eosin, Mallory, Pas and Perls stainings to evaluate the general histology, the glycogen metabolism and presence of hemosiderin. Moreover the number and area of melanomoacrophages, known as inflammation parameters, were assessment. Finally, we investigated the expression of atp7b gene and localization of respective ATP7B protein, the membrane protein involved in Cu detoxication. The achieved results showed that copper, even at a low concentration, causes serious histological alterations of liver. It induces an increase in the size and number of melanomacrophages and higher amount of hemosiderin in the treated than controls. Moreover, it alters the gene expression and localization of ATP7B protein. The data are indicative that an exposition at low and chronic concentration of copper in Xenopus laevis damages seriously the liver. For this reason it's important to consider this metal one of the pollutants involved in the decline of the amphibians and for its possible effects in other vertebrates including humans.

Dietary Supplementation with Fish Oil or Conjugated Linoleic Acid Relieves Depression Markers in Mice by Modulation of the Nrf2 Pathway.

Inflammation and oxidative stress play an important role in the pathogenesis of depressive disorders and nuclear erythroid related factor 2 (Nrf2), a regulator of RedOx homeostasis and inflammation, is a promising target for depression prevention/treatment. As fish oil (FO) and conjugated linoleic acid (CLA) are known Nrf2 inducers, their protective ability is comparatively evaluated in a murine model of depression (MRL/MpJ-Faslpr ). Oxidative stress, fatty acids content, and critical factors reflecting brain functioning-namely brain-derived neurotrophic factor (BDNF), synaptic markers, and cholinergic signaling-are preliminarily evaluated in the frontal cortex of 8-week (Young) and in 22-week old animals (Old), which are used as model of depression. These markers are measured in Old mice at the end of a 5-week pretreatment with FO or CLA (728 or 650 mg kg-1 , respectively). Old mice exhibit disrupted Redox homeostasis, compensatory Nrf2 hyperactivation, lower docosaheaxaenoic acid (DHA), and lower BDNF and synaptic function proteins compared to Young mice. FO and CLA treatment relieves almost all the pathophysiological hallmarks at a level comparable to Young mice. Presented data provide the first evidence for the comparable efficacy of FO or CLA supplementation in preventing depression signs in Old MRL/lpr mice, likely through their ability of improving Nrf2-mediated antioxidant defenses.

Effects of four food dyes on development of three model species, Cucumis sativus, Artemia salina and Danio rerio: Assessment of potential risk for the environment.

Food dyes, or color additives, are chemicals added to industrial food products and in domestic cooking to improve the perceived flavor and attractiveness. Of natural and synthetic origin, their safety has been long discussed, and concern for human safety is now clearly manifested by warnings added on products labels. Limited attention, however, has been dedicated to the effects of these compounds on aquatic flora and fauna. For this reason, the toxicity of four different commercially available food dyes (cochineal red E120, Ponceau red E124, tartrazine yellow E102 and blue Patent E131) was assessed on three different model organisms, namely Cucumis sativus, Artemia salina and Danio rerio that occupy diverse positions in the trophic pyramid. The evidence collected indicates that food dyes may target several organs and functions, depending on the species. C. sativus rate of germination was increased by E102, while root/shoot ratio was ∼20% reduced by E102, E120 and E124, seed total chlorophylls and carotenoids were 15-20% increased by E120 and 131, and total antioxidant activity was ∼25% reduced by all dyes. Mortality and low mobility of A. salina nauplii were increased by up to 50% in presence of E124, E102 and E131, while the nauplii phototactic response was significantly altered by E102, E120 and E124. Two to four-fold increases in the hatching percentages at 48 h were induced by E124, E102 and E131 on D. rerio, associated with the occurrence of 20% of embryos showing developmental defects. These results demonstrated that the food dyes examined are far from being safe for the aquatic organisms as well as land organisms exposed during watering with contaminated water. The overall information obtained gives a realistic snapshot of the potential pollution risk exerted by food dyes and of the different organism' ability to overcome the stress induced by contamination.

Effects of aluminium and cadmium on hatching and swimming ability in developing zebrafish.

Aluminium and cadmium are biologically non-essential metals with a role in neurodegenerative and neuromuscular diseases. As an attractive model for neurobehavioural studies, zebrafish at 6 h post fertilization were exposed to 9, 18, 36 and 72 µM CdCl2 and 50, 100 and 200 µM AlCl3, respectively, for 72 h, and motility such as distance moved, mean velocity, cumulative movement, meander and heading were measured by DanioVision equipment. The hatching time was also analysed. A delay in the exit from the chorion was observed in all treated larvae with respect to the controls. CdCl2 acted on the exit from the chorion of larvae with a dose-dependent delay. By contrast, the delay caused by AlCl3 was greater at low concentrations. A dose-dependent reduction in swimming performance was observed in the larvae exposed to CdCl2. Instead, for those exposed to AlCl3, swimming performance improved at higher concentrations although values were in general lower than those of control. All the parameters had a similar trend except the meander parameter which showed a dose-dependent reduction. These data show that cadmium and aluminium can delay hatching and alter swimming ability in the early developmental stages of zebrafish, albeit with different effects, suggesting that exposure to sublethal concentrations of both metals can change behavioural parameters.

Neurodegeneration in zebrafish embryos and adults after cadmium exposure.

Cadmium is a biologically non-essential metal. It is also toxic to many organs including the brain. The aim of this study was to analyse the neurodegenerative effects of this metal in embryos and adults of zebrafish exposed to sub-lethal concentrations of cadmium. The study was performed by cytochemical stainings. Six hours after fertilisation (hpf) zebrafish embryos were treated for 24 hours with 9 λM of cadmium and subsequently stained with Acridine orange in whole mount to detect apoptosis in the brain. Adult zebrafish were treated for 16 days with the same concentration of cadmium, and cell death in the brain was detected by Fluoro-Jade B staining at 2, 7 and 16 days of treatment. An increase in cell death was observed only at 16 days of treatment in adults, while an increase in apoptotic events was revealed in the brain of embryos after 24 h of treatment. This evidence is indicative that cadmium, even at a sub-lethal concentration, induces cell death in the brain of embryos but also in adults of zebrafish in which the phenomenon appears time-dependent.Â.