Characterization of the effects of antiepileptic drugs on bone metabolism: in vitro studies with human osteoblastic and osteoclastic cells

Bone is constantly being molded and shaped by the action of osteoclasts and osteoblasts. A proper equilibrium between both cell types metabolic activities is required to ensure an adequate skeletal tissue structure, and it involves resorption of old bone and formation of new bone tissue. It is reported that treatment with antiepileptic drugs (AEDs) can elicit alterations in skeletal structure, in particular in bone mineral density. Nevertheless, the knowledge regarding the effects of AEDs on bone cells are still scarce. In this context, the aim of this study was to investigate the effects of five different AEDs on human osteoclastic, osteoblastic and co-cultured cells. Osteoclastic cell cultures were established from precursor cells isolated from human peripheral blood and were characterized for tartrate-resistant acid phosphatase (TRAP) activity, number of TRAP+ multinucleated cells, presence of cells with actin rings and expressing vitronectin and calcitonin receptors and apoptosis rate. Also, the involvement of several signaling pathways on the cellular response was addressed. Osteoblastic cell cultures were obtained from femur heads of patients (25-45 years old) undergoing orthopaedic surgery procedures and were then studied for cellular proliferation/viability, ALP activity, histochemical staining of ALP and apoptosis rate. Also the expression of osteoblast-related genes and the involvement of some osteoblastogenesis-related signalling pathways on cellular response were addressed. For co-cultured cells, osteoblastic cells were firstly seeded and cultured. After that, PBMC were added to the osteoblastic cells and co-cultures were evaluated using the same osteoclast and osteoblast parameters mentioned above for the corresponding isolated cell. Cell-cultures were maintained in the absence (control) or in the presence of different AEDs (carbamazepine, gabapentin, lamotrigine, topiramate and valproic acid). All the tested drugs were able to affect osteoclastic and osteoblastic cells development, although with different profiles on their osteoclastogenic and osteoblastogenic modulation properties. Globally, the tendency was to inhibit the process. Furthermore, the signaling pathways involved in the process also seemed to be differently affected by the AEDs, suggesting that the different drugs may affect osteoclastogenesis and/or osteoblastogenesis through different mechanisms. In conclusion, the present study showed that the different AEDs had the ability to directly and indirectly modulate bone cells differentiation, shedding new light towards a better understanding of how these drugs can affect bone tissue.

Modulation of osteoclastogenesis by antihypertensive drugs

Despite its rigid structure, bone is a dynamic tissue that is in constant remodeling. This process requires the action of the bone-resorbing osteoclasts and the bone-synthesing osteoblasts. One of the adverse effects attributed to some antihypertensive agents is the ability to alter normal bone metabolism. However, their effective actions on human bone cells remain to be clarified. In this work, the effects of five calcium channel blockers, a class of antihypertensive drugs (AHDs), were investigated on osteoclastic differentiation. Osteoclastic cell cultures were established from precursor cells isolated from human peripheral blood, and were maintained in the absence (control) or in the presence of 10-8-10-4 M of different AHDs (amlodipine, felodipine, diltiazem, lercanidipine and nifedipine). Cell cultures were characterized throughout a 21 day period for tartrate-resistant acid phosphatase (TRAP) activity, number of TRAP+ multinucleated cells, presence of cells with actin rings and expressing vitronectin and calcitonin receptors, and apoptosis rate. Also, the involvement of several signaling pathways on the cellular response was addressed. It was observed that the tested AHDs had the ability to differentially affect osteoclastogenesis. At low doses, amlodipine and felodipine caused an increase on osteoclastic differentiation, while the other drugs inhibited it. At higher doses, all the molecules caused a decrease on the process. The tested AHDs also showed different effects on the analysed signaling pathways. In conclusion, AHDs appeared to have a direct effect on human osteoclast precursor cells, affecting their differentiation. Interestingly, some of them increased while others inhibited the process. Unraveling the mechanisms beneath these observations might help to explain the adverse effects on bone tissue described for this drug class.

Ciprofloxacin-imprinted polymeric receptors as ionophores for potentiometric transduction

A 3D-mirror synthetic receptor for ciprofloxacin host–guest interactions and potentiometric transduction is presented. The host cavity was shaped on a polymeric surface assembled with methacrylic acid or 2-vinyl pyridine monomers by radical polymerization. Molecularly imprinted particles were dispersed in 2-nitrophenyl octyl ether and entrapped in a poly(vinyl chloride) matrix. The sensors exhibited a near-Nernstian response in steady state evaluations. Slopes and detection limits ranged from 26.8 to 50.0mVdecade−1 and 1.0×10−5 to 2.7×10−5 mol L−1, respectively. Good selectivity was observed for trimethoprim, enrofloxacin, tetracycline, cysteine, galactose, hydroxylamine, creatinine, ammonium chloride, sucrose, glucose, sulphamerazine and sulfadiazine. The sensors were successfully applied to the determination of ciprofloxacin concentrations in fish and in pharmaceuticals. The method presented offered the advantages of simplicity, accuracy, applicability to colored and turbid samples and automation feasibility, as well as confirming the use of molecularly imprinted polymers as ionophores for organic ion recognition in potentiometric transduction.

Histamine induces ATP release from human subcutaneous fibroblasts via pannexin-1 hemichannels leading to Ca2+ mobilization and cell proliferation

Changes in the regulation of connective tissue ATP-mediated mechano-transduction and remodeling may be an important link to the pathogenesis of chronic pain. It has been demonstrated that mast cell-derived histamine plays an important role in painful fibrotic diseases. Here we analyzed the involvement of ATP in the response of human subcutaneous fibroblasts to histamine. Acute histamine application caused a rise in intracellular Ca2+ ([Ca2+]i) and ATP release from human subcutaneous fibroblasts via H1 receptor activation. Histamine-induced [Ca2+]i rise was partially attenuated by apyrase, an enzyme that inactivates extracellular ATP, and by blocking P2 purinoceptors with pyridoxal phosphate-6-azo(benzene-2,4-disulfonic acid) tetrasodium salt and reactive blue 2. [Ca2+]i accumulation caused by histamine was also reduced upon blocking pannexin-1 hemichannels with 10Panx, probenecid, or carbenoxolone but not when connexin hemichannels were inhibited with mefloquine or 2-octanol. Brefeldin A, an inhibitor of vesicular exocytosis, also did not block histamine-induced [Ca2+]i mobilization. Prolonged exposure of human subcutaneous fibroblast cultures to histamine favored cell growth and type I collagen synthesis via the activation of H1 receptor. This effect was mimicked by ATP and its metabolite, ADP, whereas the selective P2Y1 receptor antagonist, MRS2179, partially attenuated histamine-induced cell growth and type I collagen production. Expression of pannexin-1 and ADPsensitive P2Y1 receptor on human subcutaneous fibroblasts was confirmed by immunofluorescence confocal microscopy and Western blot analysis. In conclusion, histamine induces ATP release from human subcutaneous fibroblasts, via pannexin-1 hemichannels, leading to [Ca2+]i mobilization and cell growth through the cooperation of H1 and P2 (probably P2Y1) receptors.

Bradykinin-induced Ca2+ signaling in human subcutaneous fibroblasts involves ATP release via hemichannels leading to P2Y12 receptors activation

Background: Chronic musculoskeletal pain involves connective tissue remodeling triggered by inflammatory mediators, such as bradykinin. Fibroblast cells signaling involve changes in intracellular Ca2+ ([Ca2+]i). ATP has been related to connective tissue mechanotransduction, remodeling and chronic inflammatory pain, via P2 purinoceptors activation. Here, we investigated the involvement of ATP in bradykinin-induced Ca2+ signals in human subcutaneous fibroblasts. Results: Bradykinin, via B2 receptors, caused an abrupt rise in [Ca2+]i to a peak that declined to a plateau, which concentration remained constant until washout. The plateau phase was absent in Ca2+-free medium; [Ca2+]i signal was substantially reduced after depleting intracellular Ca2+ stores with thapsigargin. Extracellular ATP inactivation with apyrase decreased the [Ca2+]i plateau. Human subcutaneous fibroblasts respond to bradykinin by releasing ATP via connexin and pannexin hemichannels, since blockade of connexins, with 2- octanol or carbenoxolone, and pannexin-1, with 10Panx, attenuated bradykinin-induced [Ca2+]i plateau, whereas inhibitors of vesicular exocytosis, such as brefeldin A and bafilomycin A1, were inactive. The kinetics of extracellular ATP catabolism favors ADP accumulation in human fibroblast cultures. Inhibition of ectonucleotidase activity and, thus, ADP formation from released ATP with POM-1 or by Mg2+ removal from media reduced bradykinin-induced [Ca2+]i plateau. Selective blockade of the ADP-sensitive P2Y12 receptor with AR-C66096 attenuated bradykinin [Ca2+]i plateau, whereas the P2Y1 and P2Y13 receptor antagonists, respectively MRS 2179 and MRS 2211, were inactive. Human fibroblasts exhibited immunoreactivity against connexin-43, pannexin-1 and P2Y12 receptor. Conclusions: Bradykinin induces ATP release from human subcutaneous fibroblasts via connexin and pannexin-1-containing hemichannels leading to [Ca2+]i mobilization through the cooperation of B2 and P2Y12 receptors.

Iron Metabolism: From Health to Disease

Background Iron is vital for almost all living organisms by participating in a wide range of metabolic processes. However, iron concentration in body tissues must be tightly regulated since excessive iron may lead to microbial infections or cause tissue damage. Disorders of iron metabolism are among the most common human diseases and cover several conditions with varied clinical manifestations. Methods An extensive literature review on the basic aspects of iron metabolism was performed, and the most recent findings on this field were highlighted as well. Results New insights on iron metabolism have shed light into its real complexity, and its role in both healthy and pathological states has been recognized. Important discoveries about the iron regulatory machine and imbalances in its regulation have been made, which may lead in a near future to the development of new therapeutic strategies against iron disorders. Besides, the toxicity of free iron and its association with several pathologies has been addressed, although it requires further investigations. Conclusion This review will provide students in the fields of biochemistry and health sciences a brief and clear overview of iron physiology and toxicity, as well as imbalances in the iron homeostasis and associated pathological conditions.

Ciprofloxacin-imprinted polymeric receptors as ionophores for potentiometric transduction

A 3D-mirror synthetic receptor for ciprofloxacin host–guest interactions and potentiometric transduction is presented. The host cavity was shaped on a polymeric surface assembled with methacrylic acid or 2-vinyl pyridine monomers by radical polymerization. Molecularly imprinted particles were dispersed in 2-nitrophenyl octyl ether and entrapped in a poly(vinyl chloride) matrix. The sensors exhibited a near-Nernstian response in steady state evaluations. Slopes and detection limits ranged from 26.8 to 50.0 mV decade−1 and 1.0 × 10−5 to 2.7 × 10−5 mol L−1, respectively. Good selectivity was observed for trimethoprim, enrofloxacin, tetracycline, cysteine, galactose, hydroxylamine, creatinine, ammonium chloride, sucrose, glucose, sulphamerazine and sulfadiazine. The sensors were successfully applied to the determination of ciprofloxacin concentrations in fish and in pharmaceuticals. The method presented offered the advantages of simplicity, accuracy, applicability to colored and turbid samples and automation feasibility, as well as confirming the use of molecularly imprinted polymers as ionophores for organic ion recognition in potentiometric transduction.

Sertraline accumulation and effects in the estuarine decapod Carcinusmaenas: Importance of the history of exposure to chemical stress

Sertraline is widely prescribed worldwide and frequently detected in aquatic systems. There is, however, a remarkable gap of information on its potential impact on estuarine and coastal invertebrates. This study investigated sertraline accumulation and effects in Carcinus maenas. Crabs from a moderately contaminated (Lima) and a low-impacted (Minho) estuary were exposed to environmental and high levels of sertraline (0.05, 5, 500 μg L−1). A battery of biomarkers related to sertraline mode of action was employed to assess neurotransmission, energy metabolism, biotransformation and oxidative stress pathways. After a seven-day exposure, sertraline accumulation in crabs’ soft tissues was found in Lima (5 μg L−1: 15.3 ng L−1 ww; 500 μg L−1: 1010 ng L−1 ww) and Minho (500 μg L−1: 605 ng L−1 ww) animals. Lima crabs were also more sensitive to sertraline than those from Minho, exhibiting decreased acetylcholinesterase activity, indicative of ventilatory and locomotory dysfunction, inhibition of anti-oxidant enzymes and increased oxidative damage at ≥0.05 μg L−1. The Integrated Biomarker Response (IBR) index indicated their low health status. In addition, Minho crabs showed non-monotonic responses of acetylcholinesterase suggestive of hormesis. The results pointed an influence of the exposure history on differential sensitivity to sertraline and the need to perform evaluations with site-specific ecological receptors to increase relevance of risk estimations when extrapolating from laboratory to field conditions.

Transgenerational Inheritance of Paternal Neurobehavioral Phenotypes: Stress, Addiction, Ageing and Metabolism

Epigenetic modulation is found to get involved in multiple neurobehavioral processes. It is believed that different types of environmental stimuli could alter the epigenome of the whole brain or related neural circuits, subsequently contributing to the long-lasting neural plasticity of certain behavioral phenotypes. While the maternal influence on the health of offsprings has been long recognized, recent findings highlight an alternative way for neurobehavioral phenotypes to be passed on to the next generation, i.e., through the male germ line. In this review, we focus specifically on the transgenerational modulation induced by environmental stress, drugs of abuse, and other physical or mental changes (e.g., ageing, metabolism, fear) in fathers, and recapitulate the underlying mechanisms potentially mediating the alterations in epigenome or gene expression of offsprings. Together, these findings suggest that the inheritance of phenotypic traits through male germ-line epigenome may represent the unique manner of adaptation during evolution. Hence, more attention should be paid to the paternal health, given its equivalently important role in affecting neurobehaviors of descendants.