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Benzodiazepines, a significant group of newly recognised water contaminants, are psychotropic medications prescribed for common anxiety symptoms and sleep disorders. They resist efficient degradation during sewage treatment and endure in aquatic environments. Their presence in aquatic matrices is increasing, particularly after the recent pandemic period, which has led many people to systematically use benzodiazepines to manage anxiety. In previous studies, an important interference of this class of drugs on both the larval and adult stages of some aquatic species has been demonstrated, with effects on behaviour and embryonic development. This study examined the influence of delorazepam, a diazepam metabolite, on Artemia salina development to gain insight into responses in naupliar larvae. Results demonstrated that treatments (1, 5, and 10 µg/L) increase the hatching percentage and induce a desynchronisation in growth. Mortality was only slightly increased (close to 10% at six days post-hatching), but lipid reserve consumption was modified, with the persistence of lipid globules at the advanced naupliar stages. Locomotory activity significantly decreased only at 10 µg/L treatment. No teratogenic effects were observed, though modest damages were noticed in the posterior trunk and eyes, two targets of environmental toxicity. The negative impact of delorazepam on Artemia salina adds to those already reported in other species of invertebrates and vertebrates, which are not yet considered targets of these drugs. This study underscores the need for further research and immediate attention to this class of contaminants and the importance of monitoring their presence during environmental risk assessments.

In recent decades, cadmium has emerged as an environmental stressor in aquatic ecosystems due to its persistence and toxicity. It can enter water bodies from various natural and anthropogenic sources and, once introduced into aquatic systems, can accumulate in sediments and biota, leading to bioaccumulation and biomagnification in the food chain. For this reason, the effects of cadmium on aquatic life remain an area of ongoing research and concern. In this paper, a multidisciplinary approach was used to assess the effects of long-term exposure to an environmental concentration on the hepatopancreas of farmed juveniles of sea bream, Sparus aurata. After determining metal uptake, metallothionein production was assessed to gain insight into the organism's defence response. The effects were also assessed by histological and ultrastructural analyses. The results indicate that cadmium accumulates in the hepatopancreas at significant concentrations, inducing structural and functional damage. Despite the parallel increase in metallothioneins, fibrosis, alterations in carbohydrate distribution and endocrine disruption were also observed. These effects would decrease animal fitness although it did not translate into high mortality or reduced growth. This could depend on the fact that the animals were farmed, protected from the pressure deriving from having to search for food or escape from predators. Not to be underestimated is the return to humans, as this species is edible. Understanding the behaviour of cadmium in aquatic systems, its effects at different trophic levels and the potential risks to human health from the consumption of contaminated seafood would therefore be essential for informed environmental management and policy decisions., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Psychotropic drugs and benzodiazepines are nowadays among the primary substances of abuse. This results in a large and constant release into aquatic environments where they have potentially harmful effects on non-target organisms and, eventually, human health. In the last decades, evidence has been collected on the possible interference of benzodiazepines with reproductive processes, but data are few and incomplete. In this study, the possible negative influence of delorazepam on fertilization and embryo development has been tested in Paracentrotus lividus , a key model organism in studies of reproduction and embryonic development. Sperm, eggs, or fertilized eggs have been exposed to delorazepam at three concentrations: 1 μg/L (environmentally realistic), 5 μg/L, and 10 μg/L. Results indicate that delorazepam reduces the fertilizing capacity of male and female gametes and interferes with fertilization and embryo development. Exposure causes anatomical anomalies in plutei, accelerates/delays development, and alters the presence and distribution of glycoconjugates such as N-Acetyl-glucosamine, α-linked fucose, and α-linked mannose in both morulae and plutei. These results should attract attention to the reproductive fitness of aquatic species exposed to benzodiazepines and pave the way for further investigation of the effects they may exert on human fertility. The presence of benzodiazepines in the aquatic environment raises concerns about the reproductive well-being of aquatic species. Additionally, it prompts worries regarding potential impacts on human fertility due to the excessive use of anxiolytics.

Benzodiazepines, psychotropic drugs, are ubiquitous in the aquatic environment due to over-consumption and inefficient removal by sewage treatment plants. Bioaccumulation with consequent behavioral and physiological effects has been reported in many aquatic species. However, the responses are species-specific and still poorly understood. To improve the knowledge, we exposed the freshwater snail Planorbarius corneus to 1, 5, or 10 µg/L of delorazepam, the most widely consumed benzodiazepine in Italy. Conventional behavioral tests were used to assess the effects on locomotor and feeding behavior. Histological and biochemical analyses were also performed to detect possible changes in the structure and composition of the foot mucus and glands. The results show a paradoxical response with reduced feeding activity and locomotor hyperactivity. Pedal mucus was altered in texture but not in composition, becoming particularly rich in fibrous collagen-like material, and a significant change in the protein composition was highlighted in the foot. In conclusion, exposure to delorazepam induces disinhibited behavior in Planorbarius corneus , potentially increasing the risk of predation, and an increase in mucus protein production, which, together with reduced feeding activity, would severely compromise energy resources.

Delorazepam, a derivative of diazepam, is a psychotropic drug belonging to the benzodiazepine class. Used as a nervous-system inhibitor, it treats anxiety, insomnia, and epilepsy, but is also associated with misuse and abuse. Nowadays benzodiazepines are considered emerging pollutants: conventional wastewater treatment plants indeed are unable to eliminate these compounds. Consequently, they persist in the environment and bioaccumulate in non-target aquatic organisms with consequences still not fully clear. To collect more information, we investigated the possible epigenetic activity of delorazepam, at three concentrations (1, 5 and 10 μg/L) using Xenopus laevis embryos as a model. Analyses demonstrated a significant increase in genomic DNA methylation and differential methylation of the promoters of some early developmental genes (otx2, sox3, sox9, pax6, rax1, foxf1, and myod1). Moreover, studies on gene expression highlighted an unbalancing in apoptosis/proliferation pathways and an aberrant expression of DNA-repair genes. Results are alarming considering the growing trend of benzodiazepine concentrations in superficial waters, especially after the peak occurred as a consequence of the pandemic COVID-19, and the fact that benzodiazepine GABA-A receptors are highly conserved and present in all aquatic organisms., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Exposure to metal nanoparticles is potentially harmful, particularly when occurring during embryogenesis. In this study, we tested the effects of commercial AuNPs and AgNPs, widely used in many fields for their features, on the early development of Xenopus laevis , an anuran amphibian key model species in toxicity testing. Through the Frog Embryo Teratogenesis Assay- Xenopus test (FETAX), we ascertained that both nanoparticles did not influence the survival rate but induced morphological anomalies like modifications of head and branchial arch cartilages, depigmentation of the dorsal area, damage to the intestinal brush border, and heart rate alteration. The expression of genes involved in the early pathways of embryo development was also modified. This study suggests that both types of nanoparticles are toxic though nonlethal, thus indicating that their use requires attention and further study to better clarify their activity in animals and, more importantly, in humans.

Milk is an important source of nutrients and energy, but there are still many uncertainties regarding the health effects of milk and dairy products consumption. Milk from different species varies in physicochemical and nutritional properties. We previously showed that dietary supplements with different milks in rats trigger significant differences in metabolic and inflammatory states, modulating mitochondrial functions in metabolically active organs such as the liver and skeletal muscle. Here, we have deepened the effects of isoenergetic supplementation of milk (82 kJ) from cow (CM), donkey (DM) or human (HM) on hepatic metabolism to understand the interlink between mitochondrial metabolic flexibility, lipid storage and redox state and to highlight the possible role of two hepatocyte aquaporins (AQPs) of metabolic relevance, AQP8 and AQP9, in this crosstalk. Compared with rats with no milk supplementation, DM- and HM-fed rats had reduced hepatic lipid content with enhanced mitochondrial function and decreased oxidative stress. A marked reduction in AQP8, a hydrogen peroxide channel, was seen in the liver mitochondria of DM-fed rats compared with HM-fed, CM-fed and control animals. DM-fed or HM-fed rats also showed reduced hepatic inflammatory markers and less collagen and Kupffer cells. CM-fed rats showed higher hepatic fat content and increased AQP9 and glycerol permeability. A role of liver AQP8 and AQP9 is suggested in the different metabolic profiles resulting from milk supplementation.

In vitro systems have been mainly promoted by authorities to sustain research by following the 3Rs principle, but continuously increasing amounts of evidence point out that in vivo experimentation is also of extreme relevance. Xenopus laevis , an anuran amphibian, is a significant model organism in the study of evolutionary developmental biology, toxicology, ethology, neurobiology, endocrinology, immunology and tumor biology; thanks to the recent development of genome editing, it has also acquired a relevant position in the field of genetics. For these reasons, X. laevis appears to be a powerful and alternative model to the zebrafish for environmental and biomedical studies. Its life cycle, as well as the possibility to obtain gametes from adults during the whole year and embryos by in vitro fertilization, allows experimental studies of several biological endpoints, such as gametogenesis, embryogenesis, larval growth, metamorphosis and, of course, the young and adult stages. Moreover, with respect to alternative invertebrate and even vertebrate animal models, the X. laevis genome displays a higher degree of similarity with that of mammals. Here, we have reviewed the main available literature on the use of X. laevis in the biosciences and, inspired by Feymann's revised view, "Plenty of room for biology at the bottom", suggest that X. laevis is a very useful model for all possible studies., Competing Interests: The authors declare no conflicts of interest.

The International Parkinson's and Movement Disorder Society (MDS) criteria for progressive supranuclear palsy (PSP) have broadened the clinical spectrum of the disease and established phenotypic characterization according to the predominant manifestation at onset. The objective of this study is to describe clinical/cognitive and imaging features of a monocentric cohort of PSP patients, highlighting different patterns of functional disability according to the assigned phenotype. We retrospectively reviewed clinical/imaging data of 53 PSP patients diagnosed with probable PSP according to the MDS criteria and 40 age/sex-matched healthy controls (HCs). Neurological/neuropsychological assessments were performed using standardized scales, as well as comprehensive magnetic resonance imaging (MRI) morphometric measurements. In our cohort, there were 24/53 PSP-RS (Richardson's syndrome), 13/53 PSP-P (Parkinsonism), 7/53 PSP-PGF (Progressive gait freezing), and 9/53 PSP-Cog (Cognitive impairment). PSP-Cog presented the worst motor profiles, the highest percentages of dementia and impaired functional autonomy; 4/9 PSP-Cog and 2/7 PSP-PGF died. PSP-P had the lowest motor/cognitive burden. All MRI parameters had good discriminative efficacy vs. HCs, with P/M 2.0 discriminating PSP-PGF from PSP-RS and PSP-Cog. We highlighted discrete clinical and imaging patterns that best characterize different PSP phenotypes. PSP-Cog and PSP-PGF/RS manifest greater incidence of dementia and motor disability, respectively, while PSP-P has a more benign course. The identification of different phenotypes may be the expression of different progression patterns requiring tailored approaches in terms of follow-up and treatment. These findings support the concept of discrete patterns of Tau pathology within the PSP spectrum and encourage research for phenotype-specific outcome measures., (© 2023. The Author(s).)

Psychoactive compounds, and benzodiazepines (BZPs) in particular, represent an important class of emerging pollutants due to their large (ab)use and high resistance to degradation. Nowadays it is known that sewage treatment does not completely eliminate these substances and, therefore, BZPs and their metabolites reach concern levels in most aquatic environments all over Europe, ranging from µg/L to ng/L. In this study, we investigated the effects of delorazepam on Mytilus galloprovincialis, a model organism in toxicity testing and a key species in coastal marine ecosystems. Given its psychoactive activity, the study primarily addressed discovering the effects on behavior, by conventional valve opening and closure tests. Possible cytotoxic activity was also investigated by analyzing valve abductor muscles, gills histology, and correlated oxygen consumption. Results demonstrate negative effects on mussel behavior, interference with metabolism, and alteration of gill morphology and protein content. In conclusion, delorazepam confirms its toxicity to aquatic environments, highlighting the possibility that BZDs can ultimately affect the structure of the food web and the functions of the coastal ecosystems., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

It is known that ototoxicity is the main cause of toxicity induced by aminoglycoside antibiotics. Effects on cochlea and vestibule in vertebrates are variable, depending on the typology of the aminoglycoside and the animal model examined. Despite this, they are routinely used to prevent postoperative and urinary tract infections and in the treatment of tuberculosis and cystic fibrosis. Gentamicin causes hearing loss by damaging stereocilia and by causing degeneration of hair cells due to free radical formation and eventual activation of caspase-dependent pathways. Its toxicity increases with the frequency of administration, dose concentration, and duration of treatment. Turnover of new hair cells may occur spontaneously, throughout life, or may be triggered by an acoustic or ototoxic insult to replace dead cells. Turnover and repair of damage are common in fish and amphibians and in birds' vestibule. In contrast, in the papilla basilaris of birds, and in the vestibule of mammals, hair cell regeneration is activated only after damage. Sensory epithelium repair and hair cell regeneration also occur in the reptiles' vestibule, but no data is available on regeneration or repair in the basilar papilla, involved in sound perception. The purpose of this work is therefore to assess the damage induced by gentamicin on the papilla basilaris of a reptile model organism, the Lacertidae Podarcis siculus. Recovery was also evaluated 3, 8 and 18 days after the end of exposure, in absence of gentamicin and in presence of the otoprotective salicylate. Scanning electron microscopy (SEM) was carried out to check for morphological damage while the occurrence of cell proliferation events was evaluated by fluorescence microscopy, after administration of 5-Bromo-2'-deoxyuridine (BrdU). Results show that salicylate administration facilitates recovery and reduces damage to hair cells after gentamicin treatment. Following the incorporation of bromodeoxyuridine, we demonstrated that sensory epithelium repair and hair cell regeneration have occurred, and that the recovery is due to either proliferation of the supporting cells and/or self-repair of hair cell bundles in the weakly damaged sensory cells., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

During development, sexual differentiation results in physiological, anatomical and metabolic differences that implicate not only the gonads but also other body structures. Sex in Leopard geckos is determined by egg incubation temperature. Based on the premise that the developmental decision of gender does not depend on a single gene, we performed an analysis on E. macularius to gain insights into the genes that may be involved in gonads' sexual differentiation during the thermosensitive period. All the genes were identified as differentially expressed at stage 30 during the labile phase of sex differentiation. In this way, the expression of genes known to be involved in gonadal sexual differentiation, such as WNT4 , SOX9 , DMRT1 , Erα , Erβ , GnRH , P450 aromatase , PRL and PRL-R , was investigated. Other genes putatively involved in sex differentiation were sought by differential display. Our findings indicate that embryo exposure to a sex-determining temperature induces differential expression of several genes that are involved not only in gonadal differentiation, but also in several biological pathways ( ALDOC , FREM1 , BBIP1 , CA5A , NADH5 , L1 non-LTR retrotransposons , PKM ). Our data perfectly fit within the new studies conducted in developmental biology, which indicate that in the developing embryo, in addition to gonadal differentiation, sex-specific tissue and metabolic polarization take place in all organisms.

The role of the liver in autism spectrum disorders (ASD), developmental disabilities characterized by impairments in social interactions and repetitive behavioral patterns, has been poorly investigated. In ASD, it has been shown a dysregulation of gut-brain crosstalk, a communication system able to influence metabolic homeostasis, as well as brain development, mood and cognitive functions. The liver, with its key role in inflammatory and metabolic states, represents the crucial metabolic organ in this crosstalk. Indeed, through the portal vein, the liver receives not only nutrients but also numerous factors derived from the gut and visceral adipose tissue, which modulate metabolism and hepatic mitochondrial functions. Here, we investigated, in an animal model of ASD (BTBR mice), the involvement of hepatic mitochondria in the regulation of inflammatory state and liver damage. We observed increased inflammation and oxidative stress linked to hepatic mitochondrial dysfunction, steatotic hepatocytes, and marked mitochondrial fission in BTBR mice. Our preliminary study provides a better understanding of the pathophysiology of ASD and could open the way to identifying hepatic mitochondria as targets for innovative therapeutic strategies for the disease.

Benzodiazepines, psychotropics drugs used for treating sleep disorders, anxiety and epilepsy, represent a major class of emerging water pollutants. As occurs for other pharmaceutical residues, they are not efficiently degraded during sewage treatment and persist in effluent waters. Bioaccumulation is already reported in fish and small crustaceans, but the impact and consequences on other "non-target" aquatic species are still unclear and nowadays of great interest. In this study, we investigated the effects of a pharmaceutical preparation containing the benzodiazepine delorazepam on the embryogenesis of Xenopus laevis, amphibian model species, taxa at high risk of exposure to water contaminants. Environmental (1 μg/L) and two higher (5 and 10 μg/L) concentrations were tested on tadpoles up to stage 45/46. Results demonstrate that delorazepam interferes with embryo development and that the effects are prevalently dose-dependent. Delorazepam reduces vitality by decreasing heart rate and motility, induces marked cephalic and abdominal edema, as well as intestinal and retinal defects. At the molecular level, delorazepam increases ROS production, modifies the expression of some master developmental genes and pro-inflammatory cytokines. The resulting stress condition significantly affects embryos' development and threatens their survival. Similar effects should be expected as well in embryos belonging to other aquatic species that have not been yet considered targets for these pharmaceutical residues., (Copyright © 2022 Elsevier B.V. All rights reserved.)

In aquatic organisms, cadmium exposure occurs from ovum to death and the route of absorption is particularly wide, being represented by skin, gills and gastrointestinal tract, through which contaminated water and/or preys are ingested. It is known that cadmium interferes with the gut; however, less information is available on cadmium effects on an important component of the gut, namely goblet cells, specialized in mucus synthesis. In the present work, we studied the effects of two sublethal cadmium concentrations on the gut mucosa of Danio rerio . Particular attention was paid to changes in the distribution of glycan residues, and in metallothionein expression in intestinal cells. The results show that cadmium interferes with gut mucosa and goblet cells features. The effects are dose- and site-dependent, the anterior gut being more markedly affected than the midgut. Cadmium modifies the presence and/or distribution of glycans in the brush border and cytoplasm of enterocytes and in the goblet cells' cytoplasm and alters the metallothionein expression and localization. The results suggest a significant interference of cadmium with mucosal efficiency, representing a health risk for the organism in direct contact with contamination and indirectly for the trophic chain.

The exposure of organisms to the nanoparticulate is potentially hazardous, particularly when it occurs during embryogenesis. The effects of commercial SiO 2 NPs in early development were studied, using Xenopus laevis as a model to investigate their possible future employment by means of the Frog Embryo Teratogenesis Assay-Xenopus test (FETAX). The SiO 2 NPs did not change the survival but produced several abnormalities in developing embryos, in particular, the dorsal pigmentation, the cartilages of the head and branchial arches were modified; the encephalon, spinal cord and nerves are anomalous and the intestinal brush border show signs of suffering; these embryos are also bradycardic. In addition, the expression of genes involved in the early pathways of embryo development was modified. Treated embryos showed an increase of reactive oxygen species. This study suggests that SiO 2 NPs are toxic but non-lethal and showed potential teratogenic effects in Xenopus. The latter may be due to their cellular accumulation and/or to the effect caused by the interaction of SiO 2 NPs with cytoplasmic and/or nuclear components. ROS production could contribute to the observed effects. In conclusion, the data indicates that the use of SiO 2 NPs requires close attention and further studies to better clarify their activity in animals, including humans., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Tattooing is a technique that introduces colored substances under the skin in order to color it permanently. Decomposition products of tattoo pigments produce numerous damages for the skin and other organs. We studied the effects of a commercial red ink tattoo, PR170, on Xenopus laevis embryos and Daphnia magna nauplii using concentrations of 10, 20, and 40 mg/L. For Xenopus , we applied the FETAX protocol analyzing survival, malformations, growth, heart rate, and the expression of genes involved in the development. In D. magna , we evaluated the toxicity with an immobilization test. Moreover, we investigated the production of ROS, antioxidant enzymes, and the expression of the ATP-binding cassette in both models. Our results indicate that PR170 pigment has nanoparticle dimensions, modifies the survival and the ATP-binding cassette activity, and induces oxidative stress that probably produces the observed effects in both models. Deformed embryos were observed in Xenopus , probably due to the modification of expression of genes involved in development. The expression of pro-inflammatory cytokines was also modified in this amphibian. We think that these effects are due to the accumulation of PR170 and, in particular, to the presence of the azoic group in the chemical structure of this pigment. Further studies needed to better understand the effects of commercial tattoo inks.

In our previous studies, a ketorolac-galactose conjugate (ketogal) showed prolonged anti-inflammatory and analgesic activity, causing less gastric ulcerogenic effect and renal toxicity than its parent drug ketorolac. In order to demonstrate the safer profile of ketogal compared to ketorolac, histopathological changes in the small intestine and liver using three staining techniques before and after repeated oral administration in mice with ketorolac or an equimolecular dose of its galactosylated prodrug ketogal were assessed. Cytotoxicity and oxidative stress parameters were evaluated and compared in ketorolac- and ketogal-treated Human Primary Colonic Epithelial cells at different concentrations and incubation times. Evidence of mitochondrial oxidative stress was found after ketorolac treatment; this was attributable to altered mitochondrial membrane depolarization and oxidative stress parameters. No mitochondrial damage was observed after ketogal treatment. In ketorolac-treated mice, severe subepithelial vacuolation and erosion with inflammatory infiltrates and edematous area in the intestinal tissues were noted, as well as alterations in sinusoidal spaces and hepatocytes with foamy cytoplasm. In contrast, treatment with ketogal provided a significant improvement in the morphology of both organs. The prodrug clearly demonstrated a safer profile than its parent drug both in vitro and ex vivo, confirming that ketogal is a strategic alternative to ketorolac.

Obesity and associated metabolic disturbances, which have been increasing worldwide in recent years, are the consequences of unhealthy diets and physical inactivity and are the main factors underlying non-communicable diseases (NCD). These diseases are now responsible for about three out of five deaths worldwide, and it has been shown that they depend on mitochondrial dysfunction, systemic inflammation and oxidative stress. It was also demonstrated that several nutritional components modulating these processes are able to influence metabolic homeostasis and, consequently, to prevent or delay the onset of NCD. An interesting combination of nutraceutical substances, named DMG-gold, has been shown to promote metabolic and physical wellness. The aim of this research was to investigate the metabolic, inflammatory and oxidative pathways modulated by DMG-gold in an animal model with diet-induced obesity. Our data indicate that DMG-gold decreases the metabolic efficiency and inflammatory state and acts as an antioxidant and detoxifying agent, modulating mitochondrial functions. Therefore, DMG-gold is a promising candidate in the prevention/treatment of NCD.

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., (Copyright © 2019 Elsevier Inc. All rights reserved.)