Histopathological effects of long-term exposure to realistic concentrations of cadmium in the hepatopancreas of Sparus aurata juveniles.

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.

Salicylate attenuates gentamicin-induced ototoxicity and facilitates the recovery in the basilar papilla of the lizard Podarcis siculus.

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.

Effects of Cadmium Exposure on Gut Villi in Danio rerio.

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.

Long term exposure to cadmium: Pathological effects on kidney tubules cells in Sparus aurata juveniles.

The effects of an exposure to cadmium chloride 0.47µM for 150days were studied in kidneys of juveniles Sparus aurata by a multidisciplinary approach so to correlate uptake and detoxification potential to changes in brush border and glycocalyx sugar composition. Results demonstrated that cadmium concentration in kidney significantly increased from day 30 reaching a plateau on day 120 while metallothioneins reached a peak on day 90 and by day 120 were already decreasing to control values. Cytological damage was extensive on day 90, clearly detectable at both structural and ultrastructural levels, in tubular cells and brush-border. Staining with a panel of four lectins revealed a significant increase in N-Ac-Gal and a decrease in mannose in the glycocalyx and the tubular basal membranes. From day 120, when cadmium concentration was high and metallothionein concentration decreasing, a clear recovery was observed in tubular cells morphology and sugar composition. Possible significance of these apparently contrasting data are discussed.

Novel Approach for Evaluation of Bacteroides fragilis Protective Role against Bartonella henselae Liver Damage in Immunocompromised Murine Model.

Bartonella henselae is a gram-negative facultative intracellular bacterium and is the causative agent of cat-scratch disease. Our previous data have established that Bacteroides fragilis colonization is able to prevent B. henselae damages through the polysaccharide A (PSA) in an experimental murine model. In order to determine whether the PSA is essential for the protection against pathogenic effects of B. henselae in immunocompromised hosts, SCID mice were co-infected with B. fragilis wild type or its mutant B. fragilis ΔPSA and the effects of infection on murine tissues have been observed by High-Frequency Ultrasound (HFUS), histopathological examination, and Transmission Electron Microscopy (TEM). For the first time, echostructure, hepatic lobes length, vascular alterations, and indirect signs of hepatic dysfunctions, routinely used as signs of disease in humans, have been analyzed in an immunocompromised murine model. Our findings showed echostructural alterations in all infected mice compared with the Phosphate Buffer Solution (PBS) control group; further, those infected with B. henselae and co-infected with B. henselae/B. fragilis ΔPSA presented the major echostructural alterations. Half of the mice infected with B. henselae and all those co-infected with B. henselae/B. fragilis ΔPSA have showed an altered hepatic echogenicity compared with the renal cortex. The echogenicity score of co-infected mice with B. henselae/B. fragilis ΔPSA differed significantly compared with the PBS control group (p < 0.05). Moreover the inflammation score of the histopathological evaluation was fairly concordant with ultrasound findings. Ultrastructural analysis performed by TEM revealed no significant alterations in liver samples of SCID mice infected with B. fragilis wild type while those infected with B. fragilis ΔPSA showed the presence of collagen around the main vessels compared with the PBS control group. The liver samples of mice infected with B. henselae showed macro-areas rich in collagen, stellate cells, and histiocytic cells. Interestingly, our data demonstrated that immunocompromised SCID mice infected with B. henselae and co-infected with B. henselae/B. fragilis ΔPSA showed the most severe morpho-structural liver damage. In addition, these results suggests that the HFUS together with histopathological evaluation could be considered good imaging approach to evaluate hepatic alterations.

Structural and functional changes in the zebrafish (Danio rerio) skeletal muscle after cadmium exposure.

This report describes the alterations induced by an environmentally realistic concentration of cadmium in skeletal muscle fibre organization, composition, and function in the teleost zebrafish. Results demonstrate that the ion induces a significant quantitative and qualitative deterioration, disrupting sarcomeric pattern and altering glycoprotein composition. These events, together with a mitochondrial damage, result in a significant reduction in swimming performance. In conclusion, the evidence here collected indicate that in presence of an environmental cadmium contamination, important economic (yields in fisheries/aquaculture), consumer health (fish is an important source of proteins), and ecological (reduced fitness due to reduced swimming performance) consequences can be expected.

Cadmium effects on the retina of adult Danio rerio.

The aim of this work is to describe the effects of cadmium pollution on the vision of adult zebrafish, Danio rerio. Retinal morpho-cytological alterations were investigated by light and electron microscopy, while the functionality of cadmium-exposed retinae was assessed by re-illumination behavioral tests with white or colored light. Our results demonstrate that cadmium toxicity causes significant degeneration and loss of organization at both macro and microscopic levels. These alterations impair functional responses particularly through an increase in light sensitivity. Metallothioneins were not seen to be up-regulated, while the recovery of visual acuity is due to a regenerative process by Müller cells.

Assembly of the auditory circuitry by a Hox genetic network in the mouse brainstem.

Rhombomeres (r) contribute to brainstem auditory nuclei during development. Hox genes are determinants of rhombomere-derived fate and neuronal connectivity. Little is known about the contribution of individual rhombomeres and their associated Hox codes to auditory sensorimotor circuitry. Here, we show that r4 contributes to functionally linked sensory and motor components, including the ventral nucleus of lateral lemniscus, posterior ventral cochlear nuclei (VCN), and motor olivocochlear neurons. Assembly of the r4-derived auditory components is involved in sound perception and depends on regulatory interactions between Hoxb1 and Hoxb2. Indeed, in Hoxb1 and Hoxb2 mutant mice the transmission of low-level auditory stimuli is lost, resulting in hearing impairments. On the other hand, Hoxa2 regulates the Rig1 axon guidance receptor and controls contralateral projections from the anterior VCN to the medial nucleus of the trapezoid body, a circuit involved in sound localization. Thus, individual rhombomeres and their associated Hox codes control the assembly of distinct functionally segregated sub-circuits in the developing auditory brainstem.

Evidence of Bacteroides fragilis protection from Bartonella henselae-induced damage.

Bartonella henselae is able to internalize endothelial progenitor cells (EPCs), which are resistant to the infection of other common pathogens. Bacteroides fragilis is a gram-negative anaerobe belonging to the gut microflora. It protects from experimental colitis induced by Helicobacter hepaticus through the polysaccharide A (PSA). The aim of our study was to establish: 1) whether B. fragilis colonization could protect from B. henselae infection; if this event may have beneficial effects on EPCs, vascular system and tissues. Our in vitro results establish for the first time that B. fragilis can internalize EPCs and competes with B. henselae during coinfection. We observed a marked activation of the inflammatory response by Real-time PCR and ELISA in coinfected cells compared to B. henselae-infected cells (63 vs 23 up-regulated genes), and after EPCs infection with mutant B. fragilis ΔPSA (≅90% up-regulated genes) compared to B. fragilis. Interestingly, in a mouse model of coinfection, morphological and ultrastructural analyses by hematoxylin-eosin staining and electron microscopy on murine tissues revealed that damages induced by B. henselae can be prevented in the coinfection with B. fragilis but not with its mutant B. fragilis ΔPSA. Moreover, immunohistochemistry analysis with anti-Bartonella showed that the number of positive cells per field decreased of at least 50% in the liver (20±4 vs 50±8), aorta (5±1 vs 10±2) and spleen (25±3 vs 40±6) sections of mice coinfected compared to mice infected only with B. henselae. This decrease was less evident in the coinfection with ΔPSA strain (35±6 in the liver, 5±1 in the aorta and 30±5 in the spleen). Finally, B. fragilis colonization was also able to restore the EPC decrease observed in mice infected with B. henselae (0.65 vs 0.06 media). Thus, our data establish that B. fragilis colonization is able to prevent B. henselae damages through PSA.

Impairment of circulating endothelial progenitors in Down syndrome.

BACKGROUND: Pathological angiogenesis represents a critical issue in the progression of many diseases. Down syndrome is postulated to be a systemic anti-angiogenesis disease model, possibly due to increased expression of anti-angiogenic regulators on chromosome 21. The aim of our study was to elucidate some features of circulating endothelial progenitor cells in the context of this syndrome. METHODS: Circulating endothelial progenitors of Down syndrome affected individuals were isolated, in vitro cultured and analyzed by confocal and transmission electron microscopy. ELISA was performed to measure SDF-1α plasma levels in Down syndrome and euploid individuals. Moreover, qRT-PCR was used to quantify expression levels of CXCL12 gene and of its receptor in progenitor cells. The functional impairment of Down progenitors was evaluated through their susceptibility to hydroperoxide-induced oxidative stress with BODIPY assay and the major vulnerability to the infection with human pathogens. The differential expression of crucial genes in Down progenitor cells was evaluated by microarray analysis. RESULTS: We detected a marked decrease of progenitors' number in young Down individuals compared to euploid, cell size increase and some major detrimental morphological changes. Moreover, Down syndrome patients also exhibited decreased SDF-1α plasma levels and their progenitors had a reduced expression of SDF-1α encoding gene and of its membrane receptor. We further demonstrated that their progenitor cells are more susceptible to hydroperoxide-induced oxidative stress and infection with Bartonella henselae. Further, we observed that most of the differentially expressed genes belong to angiogenesis, immune response and inflammation pathways, and that infected progenitors with trisomy 21 have a more pronounced perturbation of immune response genes than infected euploid cells. CONCLUSIONS: Our data provide evidences for a reduced number and altered morphology of endothelial progenitor cells in Down syndrome, also showing the higher susceptibility to oxidative stress and to pathogen infection compared to euploid cells, thereby confirming the angiogenesis and immune response deficit observed in Down syndrome individuals.

Bap170, a subunit of the Drosophila PBAP chromatin remodeling complex, negatively regulates the EGFR signaling.

BAP and PBAP constitute the two different forms of the Drosophila melanogaster Brahma chromatin remodelers. A common multisubunit core, containing the Brahma ATPase, can associate either with Osa to form the BAP complex or with Bap170, Bap180, and Sayp to constitute the PBAP complex. Although required for many biological processes, recent genetic analyses revealed that one role of the BAP complex during Drosophila wing development is the proper regulation of EGFR target genes. Here, we show that Bap170, a distinctive subunit of the PBAP complex, participates instead in the negative regulation of EGFR signaling. In adults, loss of Bap170 generates phenotypes similar to the defects induced by hyperactivation of the EGFR pathway, such as overrecruitment of cone and photoreceptor cells and formation extra veins. In genetic interactions, bap170 mutations suppress the loss of veins and photoreceptors caused by mutations affecting the activity of the EGFR pathway. Our results suggest a dual requirement of the PBAP complex: for transcriptional repression of rhomboid and for efficient expression of argos. Interestingly, genetic evidence also indicates that Bap170-mediated repression of rho is inhibited by EGFR signaling, suggesting a scenario of mutual antagonism between EGFR signaling and PBAP function.

Detrimental effects of Bartonella henselae are counteracted by L-arginine and nitric oxide in human endothelial progenitor cells.

The recruitment of circulating endothelial progenitor cells (EPCs) might have a beneficial effect on the clinical course of several diseases. Endothelial damage and detachment of endothelial cells are known to occur in infection, tissue ischemia, and sepsis. These detrimental effects in EPCs are unknown. Here we elucidated whether human EPCs internalize Bartonella henselae constituting a circulating niche of the pathogen. B. henselae invades EPCs as shown by gentamicin protection assays and transmission electron microscopy (TEM). Dil-Ac-LDL/lectin double immunostaining and fluorescence-activated cell sorting (FACS) analysis of EPCs revealed EPC bioactivity after infection with B. henselae. Nitric oxide (NO) and its precursor l-arginine (l-arg) exert a plethora of beneficial effects on vascular function and modulation of immune response. Therefore, we tested also the hypothesis that l-arg (1-30 mM) would affect the infection of B. henselae or tumor necrosis factor (TNF) in EPCs. Our data provide evidence that l-arg counteracts detrimental effects induced by TNF or Bartonella infections via NO (confirmed by DETA-NO and L-NMMA experiments) and by modulation of p38 kinase phosphorylation. Microarray analysis indicated several genes involved in immune response were differentially expressed in Bartonella-infected EPCs, whereas these genes returned in steady state when cells were exposed to sustained doses of l-arg. This mechanism may have broad therapeutic applications in tissue ischemia, angiogenesis, immune response, and sepsis.