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Summary: Environmental contaminants have previously been demonstrated to cause bone deformities mediated through the aryl hydrocarbon receptor (AhR) in fish and mammals. Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous in the environment, many of them capable of activating AhR. In the present study, fish scales were utilized as a model system to examine possible AhR‐mediated effects of PAHs on bone forming osteoblasts and bone resorptive osteoclasts, using the AhR‐ligand benzo(a)pyrene (BaP) as a model compound. Elasmoid scales from goldfish and zebrafish were exposed to 0.005–50 μM BaP for up to 48 hr, and the activity of osteoblastic and osteoclastic markers were measured, as well as mRNA levels of bone related genes and cyp1a and cyp3a. Using the sp7:luciferase zebrafish assay, a decrease in sp7 promoter activation was observed at the two highest concentrations (5 and 50 μM). Gelatin zymography revealed significantly reduced activity of the osteoclastic protease matrix metalloproteinase 9 (Mmp9) at the highest concentration. Furthermore, transcriptional analysis showed a dose‐dependent increase in cyp1a, however, no significant differential expression was observed for the bone related genes. The findings indicate that BaP might decrease differentiation and activation of osteoblasts, and reduce osteoclastic activity, and thus ultimately cause decreased bone formation. Further investigation is necessary in order to confirm the role of AhR in mediating these effects. [ABSTRACT FROM AUTHOR]

Valproic acid (VPA) is an anti-epileptic drug known to cause congenital craniofacial abnormalities, including orofacial clefts (OFC). The exact mechanisms by which VPA leads to craniofacial skeletal malformations are poorly understood. In this study, we investigated the effects of VPA on cartilage and bone formation in the zebrafish larval head during 1-13 hpf (early) and 25-37 hpf (late) development in which cranial neural crest cells (CNCCs) arise and then proliferate and differentiate, respectively. Double-staining for cartilage and bone at 5 dpf revealed that VPA reduced cartilage and bone formation in a dose-dependent manner after both early or late exposure. Several different CNCC-derived cartilage and bone elements were affected in both groups. In the early group (100 μM VPA), the posterior head length and the ethmoid plate were reduced in length (both p < 0.01), while mineralization of 4 out of 9 bone elements was often lacking (all p < 0.01). In the late group (100 μM VPA), also the posterior head length was reduced as well as the length of the ceratohyals (both p < 0.01). Similar to early exposure, mineralization of 3 out of 9 bone elements was often lacking (all p < 0.01). These results indicate that both CNCC formation (early) and differentiation (late) are hampered by VPA treatment, of which the consequences for bone and cartilage formation are persistent at 5 dpf. Indeed, we also found that the expression of several genes related to cartilage and bone was upregulated at 5 dpf. These data indicate a compensatory reaction to the lack of cartilage and bone. Altogether, VPA seems to induce craniofacial malformations via disturbed CNCC function leading to defects in cartilage and bone formation., Competing Interests: Declaration of competing interest None., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The regulation of skin darkness in vertebrates is mediated by alpha-melanophore-stimulating-hormone (alphaMSH). For this action, alphaMSH binds to the melanocortin (MC)-1 receptor, a 7-transmembrane receptor located in melanophore cell membranes. The Mozambique tilapia, Oreochromis mossambicus, can change the hue of its body in response to a change in background, a process that may involve alphaMSH and the MC1R. Scale melanophores were isolated from tilapia that were acclimatised for 25 days to a black, control grey or white background and then tested for their sensitivity to des-, mono-, and di-acetylated alphaMSH. On all backgrounds, mono-acetylated alphaMSH was the dominant isoform present in pituitary homogenates. Mono-acetylated alphaMSH also had the highest potency to disperse melanosomes. Black background adapted fish showed the highest dispersing response to alphaMSH, independent of the isoform applied. We elucidated the nucleotide and amino acid sequence of the tilapia MC1R. We show that its expression in skin does not change when tilapia are acclimatised for 25 days to a black, grey or white background, while a clear change in hue is visible. This finding, combined with the absence of differential MC1R gene expression following background acclimation indicates that the increased sensitivity to alphaMSH is most likely a result of changes in the intracellular signalling system in melanophores of black background adapted fish, rather than up-regulation of the MC1R.

Pituitary melanotropes release alpha-melanocyte-stimulating hormone (alpha-MSH) and acetylated beta-endorphin (NAc beta-end) during stress responses. However, effects of stressors on plasma concentrations of these hormones are highly inconsistent among fish species. Here, we show that also within a species, the common carp (Cyprinus carpio), fish sometimes respond with elevated alpha-MSH and NAc beta-end plasma levels, and at other times not. The origin of this variable response was investigated by (1) studying the effects of corticotropin-releasing hormone (CRH) on alpha-MSH and NAc beta-end release in vitro, (2) establishing where in the second messenger pathway coupled to CRH receptors melanotrope responsiveness is determined, and (3) testing modulatory actions of other hypothalamic factors (here opioid beta-endorphin). Melanotropes were in a high or low responsive state to CRH in vitro, which was especially evident when tissue was tested from fish kept at higher ambient water temperatures, and this correlates with the variability in alpha-MSH and NAc beta-end responses in vivo. Relative rates of alpha-MSH and NAc beta-end release following stimulation with CRH in vitro match plasma level changes in vivo, and this indicates that the CRH pathway does act in vivo. cAMP did not stimulate melanotropes in the low responsive state to release hormones in vitro. Thus, the mechanism that determines the cell status, occurs downstream of cAMP accumulation. Opioid beta-endorphin differentially modulated the actions of CRH, as NAc beta-end, but not alpha-MSH, release was inhibited. This response was not observed in the stress paradigms studied. We conclude that the variation in alpha-MSH and NAc beta-end stress responses in vivo correlates with many CRH responses in vitro; whether a cell is in a high or low responsive state to CRH is determined downstream of accumulation of the second messenger. We propose that melanotropes have to be in the high responsive state to be activated by CRH during stress in carp and other teleosts.

We elucidated the structure of the principle factors regulating the initiation of the acute stress response in common carp: corticotrophin-releasing hormone (CRH), CRH-receptor 1 (CRH-R1) and CRH-binding protein (CRH-BP). Phylogenetic analyses reveal that these proteins are evolutionarily well conserved in vertebrates. CRH and CRH-BP expression are not co-localised in the same hypothalamic perikarya. On the contrary, CRH-BP expression is limited to the perimeter of the nucleus preopticus (NPO), but is abundant in other regions, including an area directly rostral from, and in close proximity to, the NPO. Despite the lack of co-expression, the nerve fibres projecting onto both the rostral pars distalis (rPD) as well as the large fibre bundles projecting onto the pars intermedia (PI) contain CRH as well as CRH-BP, suggesting that both ACTH release from the rPD as well as the release of PI melanotrope content is regulated via CRH and CRH-BP. Finally, we show via real-time quantitative PCR that expression of hypothalamic CRH and CRH-BP following a 24 h restraint significantly increases, whereas PD CRH-R1 expression decreases; this reflects desensitisation of the PD for hypothalamic CRH output. We conclude that these factors are actively involved in the regulation of acute stress responses in the teleost fish.

MSH is a pituitary hormone derived by post-translational processing from POMC and involved in stress and background adaptation. N-terminal acetylation of MSH to monoacetyl alpha-MSH or diacetyl alpha-MSH increases the bioactivity of the peptide. The aim of this study was to characterize alpha-MSH acetylation in the sea bream (Sparus aurata L.) pituitary gland in response to the stressors air exposure and confinement, as well as in fish adapted for 15 days to a white, gray or black background. Pituitary homogenates were purified by reversed-phase HPLC (RP-HPLC). The alpha-MSH content of fractions was measured by RIA. Immunoreactive RP-HPLC fractions were further analyzed by electrospray mass spectrometry and the peptide sequence determined as SYSMEHFRWGKPV-NH2. In the pituitary gland of sea bream, des-, mono- and diacetyl alpha-MSH were identified. Then plasma alpha-MSH levels were measured in sea bream adapted to different backgrounds. Surprisingly, we found the highest plasma alpha-MSH levels in white-adapted as compared with black-adapted sea bream with intermediate values for gray-adapted fish. This observation is in contrast with results that have been obtained in eel, trout or terrestrial vertebrates. Next, des-, mono- and diacetyl alpha-MSH forms were measured in homogenates of the pituitary gland and in plasma of sea bream exposed to air, to confinement, or to different backgrounds. Monoacetyl alpha-MSH was the predominant form in all control and experimental groups. The lowest content of monoacetyl alpha-MSH relative to des- and diacetyl alpha-MSH was found in white-adapted fish. Levels of des- and diacetyl alpha-MSH forms were similar under all conditions. We observed that monoacetyl alpha-MSH is the most abundant isoform in the pituitary gland after background adaptation, confinement and air exposure, in sea bream. These data indicate that the physiologically most potent isoform of alpha-MSH may vary from species to species.

Ninety-nine adults with an enlarged retrorectal space on barium enema examination have been evaluated. Measurements were based on previously described methods using the lateral view of the barium filled rectum. In 38.4% of the cases there was no lesion (normal variations). The rest were classified as inflammatory conditions, tumours, and miscellaneous lesions. It is concluded that an increased width of the presacral space per se does not necessarily connote a lesion. Pathological widening of the space is usually associated with changes of the contour of the rectum, abnormalities of the sacrum, or other alterations of the presacral soft tissues.

In an effort to avert the cumulative effects of unresolved emotional problems on children's social and school adjustment, a psychosocial phase was added to a pediatric multiphasic examination. Based upon a cumulative stress concept, the screening procedures included child behavior and family stress questionnaires for parents, and abbreviated standard psychological tests for children, administered by specially trained aides. Computerized results were reported to the child's pediatrician. Follow-up by mental health counselors attached to the pediatric clinic was provided for patients identified as being at high risk of serious psychosocial problems. Evidence of validity of the screen, factors affecting the scores, and effectiveness of clinical follow-up of high-risk patients are discussed. Valid semicomputerized screening of school-age children for serious psychosocial problems can be carried out routinely and at relatively low cost by paraprofessional personnel in a pediatric setting. Impediments to effective use of the screening results on the part of both health care provider and patient are discussed.

The need for multiphasic screening tests in children lies in three major areas: detection of chronic complications of acute conditions, detection of abnormalities that might contribute to acute conditions, and detection of chronic invidious conditions in which early intervention might be beneficial.