Vibration Rather than Microgravity Affects Bone Metabolism in Adult Zebrafish Scale Model.

Gravity and mechanical forces cause important alterations in the human skeletal system, as demonstrated by space flights. Innovative animal models like zebrafish embryos and medaka have been introduced to study bone response in ground-based microgravity simulators. We used, for the first time, adult zebrafish in simulated microgravity, with a random positioning machine (RPM) to study bone remodeling in the scales. To evaluate the effects of microgravity on bone remodeling in adult bone tissue, we exposed adult zebrafish to microgravity for 14 days using RPM and we evaluated bone remodeling on explanted scales. Our data highlight bone resorption in scales in simulated microgravity fish but also in the fish exposed, in normal gravity, to the vibrations produced by the RPM. The osteoclast activation in both rotating and non-rotating samples suggest that prolonged vibrations exposure leads to bone resorption in the scales tissue. Stress levels in these fish were normal, as demonstrated by blood cortisol quantification. In conclusion, vibrational mechanical stress induced bone resorption in adult fish scales. Moreover, adult fish as an animal model for microgravity studies remains controversial since fish usually live in weightless conditions because of the buoyant force from water and do not constantly need to support their bodies against gravity.

A Pilot Prospective Study Evaluating the Effect of Curcuma-Based Herbal Food Supplement on the Outcome of In Vitro Fertilization in Patients Testing Positive for Four Immunological Biomarkers.

Background and Objectives: Inflammation and oxidative stress have been described to reduce the chance for pregnancy instauration and maintenance. NOFLAMOX, a recently developed herbal preparation with recognized antioxidant and anti-inflammatory properties, can represent an interesting treatment to increase the chance of pregnancy, both physiological or after in vitro fertilization (IVF). The aim of this study was to assess NOFLAMOX's effect; a population with unexplained infertility was screened for the recently described IMMUNOX panel based on four immunological biomarkers with a prospective study approach. Materials and Methods: Patients with unexplained infertility and positive for at least one of the biomarkers of the IMMUNOX panel were included in this study and treated with NOFLAMOX for three months prior to an IVF cycle. Results: Eighty-six patients (n = 86) were screened with the IMMUNOX panel and the forty-seven (54.5%) found positive were included in this study. In more detail, 11 were positive for TNFα (23.4%), 18 (38.3%) for glycodelin (GLY), 29 (61.7%) for Total Oxidative Status (TOS), and 32 (68.1%) for Complement Activity Toxic Factor (CATF). After three months of treatment, a significant reduction in the number of IMMUNOX-positive patients was observable, with 26 patients who turned IMMUNOX-negative displaying a quantitative statistically significant variation of 100% (11/11), 38.9% (7/18), 65.5% (18/29), and 75% (24/32), for TNFα, glycodelin, TOS, and CATF, respectively. Followed in the subsequent IVF cycle, this NOFLAMOX-treated population showed a pregnancy rate of 42.3% compared to the 4.7% of the IMMUNOX-positive group of patients. Conclusions: Taken together, the results of this study suggest that NOFLAMOX could represent an interesting option for those patients with unexplained infertility of inflammatory/oxidative origin. Further studies are needed to confirm these results and explore possible strategies to restore fertility in women with immune-mediated sterility.

Molecular Hydrogen Prevents Osteoclast Activation in a Glucocorticoid-Induced Osteoporosis Zebrafish Scale Model.

Antioxidants represent a powerful tool for many human diseases and, in particular, molecular hydrogen has unique characteristics that make it a very promising therapeutic agent against osteoporosis. Zebrafish scales offer an innovative model in which new therapeutic approaches against secondary osteoporosis are tested. Scale bone loss obtained by prednisolone (PN) treatment is characterized by increased osteoclast activity and decreased osteoblast activity highlighted with bone enzymatic assays. We used this read-out system to test the therapeutic effects of hydrogen-rich water (HRW), an innovative antioxidant approach. HRW prevented osteoclast activation and bone loss in PN-treated fish scales, as verified by both biochemical and histochemical tartrate-resistant alkaline phosphatase assays. On the other hand, HRW treatment did not prevent PN-dependent osteoblast suppression, as measured by alkaline phosphatase activity. Moreover, HRW treatment did not facilitate the reparation of resorption lacunae induced in scales by PN. Our study highlighted a specific effect of HRW on adult osteoclast activity but not in osteoblasts, introducing an intriguing new antioxidant preventive approach against osteoporosis.

Soybean Meal-Dependent Acute Intestinal Inflammation Delays Osteogenesis in Zebrafish Larvae.

Foods are known to be modulators of inflammation and skeletal development. The intestine plays an essential role in the regulation of bone health mainly through the regulation of the absorption of vitamin D and calcium; in fact, inflammatory bowel diseases are often related to bone health issues such as low bone mineral density, high fracture risk, osteoporosis and osteopenia. Considering the complexity of the pathways involved, the use of a simple animal model can be highly useful to better elucidate the pathogenic mechanisms. Soybean flour with a high saponin content has been used in many studies to induce intestinal inflammation in zebrafish larvae. Using a 50% soybean meal (SBM), we analyzed the effects of this soy-induced inflammatory bowel disease on zebrafish larval osteogenesis. Soybean meal induces intestinal functional alterations and an inflammatory state, highlighted by neutral red staining, without altering the general development of the larvae. Our data show that the chondrogenesis as well as endochondral ossification of the head of zebrafish larvae are not affected by an SBM-diet, whereas intramembranous ossification was delayed both in the head, where the length of the ethmoid plate reduced by 17%, and in the trunk with a delayed vertebral mineralization of 47% of SBM larvae. These data highlight that diet-dependent bowel inflammation can differently modulate the different mechanisms of bone development in different zones of the skeleton of zebrafish larvae.

Aerophobin-1 from the Marine Sponge Aplysina aerophoba Modulates Osteogenesis in Zebrafish Larvae.

Longer life expectancy has led to an increase in efforts directed to the discovery of new healing agents for disorders related to aging, such as bone diseases. Harboring an incredible variety of bioactive metabolites, marine organisms are standing out as fruitful sources also in this therapeutic field. On the other hand, the in vivo zebrafish model has proven to be an excellent low-cost screening platform for the fast identification of molecules able to regulate bone development. By using zebrafish larvae as a mineralization model, we have thus evaluated the effects of the crude acetonic extract from the marine sponge Aplysina aerophoba and its bromotyrosine components on bone development. Obtained results led to the selection of aerophobin-1 (1) as a promising candidate for applications in regenerative medicine, paving the way for the development of a novel therapeutic option in osteoporosis treatment.

Soy diet induces intestinal inflammation in adult Zebrafish: Role of OTX and P53 family.

Inflammatory bowel diseases (IBDs) are a group of inflammatory conditions of the colon and small intestine, including Crohn's disease and ulcerative colitis. Since Danio rerio is a promising animal model to study gut function, we developed a soy-dependent model of intestinal inflammation in adult zebrafish. The soya bean meal diet was given for 4 weeks and induced an inflammatory process, as demonstrated by morphological changes together with an increased percentage of neutrophils infiltrating the intestinal wall, which developed between the second and fourth week of treatment. Pro-inflammatory genes such as interleukin-1beta, interleukin-8 and tumour necrosis factor alpha were upregulated in the second week and anti-inflammatory genes such as transforming growth factor beta and interleukin-10. Interestingly, an additional expression peak was found for interleukin-8 at the fourth week. Neuronal genes, OTX1 and OTX2, were significantly upregulated in the first two  weeks, compatible with the development of the changes in the gut wall. As for the genes of the p53 family such as p53, DNp63 and p73, a statistically significant increase was observed after two weeks of treatment compared with controls. Interestingly, DNp63 and p73 were shown an additional peak after four weeks. Our data demonstrate that soya bean meal diet negatively influences intestinal morphology and immunological function in adult zebrafish showing the features of acute inflammation. Data observed at the fourth week of treatment may suggest initiation of chronic inflammation. Adult zebrafish may represent a promising model to better understand the mechanisms of food-dependent intestinal inflammation.

Herbal Preparation (Bromelain, Papain, Curcuma, Black Pepper) Enhances Mineralization and Reduces Glucocorticoid-Induced Osteoporosis in Zebrafish.

Natural foods with antioxidant properties, such as curcuma, papain, bromelain and black pepper, have been indicated as a potential natural therapeutic approach against osteoporosis. Zebrafish are an excellent animal model to study the effects of herbal preparations on osteogenesis and bone metabolism, both in physiological and in pathological conditions. Our study was aimed at evaluating whether curcuma-bromelain-papain-pepper herbal preparation (CHP) administered in embryos and adult fish is capable of promoting bone wellness in physiological and osteoporotic conditions. The effect of CHP has been studied in embryonic osteogenesis and glucocorticoid-induced osteoporosis (GIOP) in an adult fish model in which drug treatment induces a bone-loss phenotype in adult scales very similar to that which characterizes the bones of human patients. CHP prevented the onset of the osteoporotic phenotype in the scales of GIOP in adult zebrafish, with the osteoblastic and osteoclastic metabolic activity maintaining unaltered. CHP is also able to attenuate an already established GIOP phenotype, even if the alteration is in an advanced phase, partially restoring the normal balance of the bone markers alkaline phosphatase (ALP) and tartrate-resistant acid phosphatase (TRAP) and stimulating anabolic reparative processes. The results obtained indicated CHP as a potential integrative antioxidant therapy in human bone-loss diseases.

Soybean Meal-Dependent Intestinal Inflammation Induces Different Patterns of Bone-Loss in Adult Zebrafish Scale.

Inflammatory bowel disease have been linked to several health issues, including high risk of low bone mineral density. Danio rerio (zebrafish) is a good model to verify the effects of intestinal inflammation, since its gastrointestinal and immune systems are closely related to that of mammalians. Zebrafish is also a powerful model to study bone metabolism using the scale as the read-out model. Food strongly impacts zebrafish gut physiology, and it is well known that soybean meal induces intestinal inflammation. Adult zebrafish fed with defatted soybean meal (SBM) exhibited an intestinal inflammation evidenced by morphological alterations, inflammatory infiltrate, and increased mRNA expression of inflammatory cytokines (IL-1ß, IL-6, IL-8, IL-10, TGFß, TNF-α). The peak of acute intestinal inflammation, spanning between week 2 and 3, correlates with a transitory osteoporosis-like phenotype in the scale border. Later, a chronic inflammatory condition, associated with persistent IL-8 expression, correlates with the progression of resorption lacunae in the scale center. Both types of resorption lacunae were associated with intense osteoclastic tartrate-resistant acid phosphatase (TRAP) activity. After 3 weeks of SBM treatment, osteoclast activity decreased in the scale border but not in the center. At the same time, alkaline phosphatase (ALP) is activated in the border to repair the bone matrix. This model can contribute to elucidate in vivo the molecular mechanisms that links intestinal inflammation and bone metabolism in IBD.

Molecular hydrogen enhances osteogenesis in Danio rerio embryos.

Molecular hydrogen (H2 ) has been reported to have important biological effects on bone tissue in several in vitro and in vivo models. Danio rerio (zebrafish) embryo is a good model to study osteogenesis because of its transparency, size and rapid development. In zebrafish embryo, hydrogen-rich water (HRW) does not affect vitality or growth rate up to 15%. In addition, 7% HRW treatment enhances zebrafish embryo osteogenesis, increasing the vertebral mineralization rate. In conclusion, we demonstrated the beneficial effects of hydrogen on anabolic bone functions in vivo.

Age-dependent modulation of bone metabolism in zebrafish scales as new model of male osteoporosis in lower vertebrates.

After middle age, in human bone, the resorption usually exceeds formation resulting in bone loss and increased risk of fractures in the aged population. Only few in vivo models in higher vertebrates are available for pathogenic and therapeutic studies about bone aging. Among these, male Danio rerio (zebrafish) can be successfully used as low vertebrate model to study degenerative alterations that affect the skeleton during aging, reducing the role of sex hormones.In this paper, we investigated the early bone aging mechanisms in male zebrafish (3, 6, 9 months old) scales evaluating the physiological changes and the effects of prednisolone, a pro-osteoporotic drug.The results evidentiated an age-dependent reduction of the mineralization rate in the fish scales, as highlighted by growing circle measurements. Indeed, the osteoblastic ALP activity at the matrix deposition site was found progressively downregulated.The higher TRAP activity was found in 63% of 9-month-old fish scales associated with resorption lacunae along the scale border. Gene expression analysis evidentiated that an increase of the tnfrsf1b (homolog of human rank) in aging scales may be responsible for resorption stimulation.Interestingly, prednisolone inhibited the physiological growth of the scale and induced in aged scales a more significant bone resorption compared with untreated fish (3.8% vs 1.02%). Bone markers analysis shown a significant reduction of ALP/TRAP ratio due to a prednisolone-dependent stimulation of tnfsf11 (homolog of human rankl) in scales of older fish.The results evidentiated for the first time the presence of a senile male osteoporosis in lower vertebrate. This new model could be helpful to identify the early mechanisms of bone aging and new therapeutic strategies to prevent age-related bone alterations in humans.

Liquiritigenin reduces osteoclast activity in zebrafish model of glucocorticoid-induced osteoporosis.

Drug and therapies currently used to treat human bone diseases have a lot of severe side effects. Liquiritigenin is a flavonoid extracted from Glycyrrhiza glabra roots which has been reported to have positive effects in vitro on osteoblasts activity and bone mineralization as well as inhibitory effect on osteoclasts differentiation and activity in vitro. The present study was aimed to evaluate the in vivo effects of liquiritigenin on bone structure and metabolism in physiological and pathological conditions using Danio rerio as experimental animal model. Treatments with liquiritigenin were performed on embryos to evaluate the osteogenesis during skeletal development. Other treatments were performed on adult fish affected by glucocorticoid-induced osteoporosis to assay the therapeutic potential of liquiritigenin in the reversion of bone-loss phenotype in scale model. Liquiritigenin treatment of zebrafish embryo significantly enhances the osteogenesis during development in a dose-dependent manner. In addition, liquiritigenin inhibits the formation of the osteoporotic phenotype in adult zebrafish model of glucocorticoid-induced osteoporosis preventing osteoclast activation in scales. Interestingly, liquiritigenin does not counteract the loss of osteoblastic activity in scales. The liquiritigenin exhibits in vivo anti-osteoporotic activity on adult fish scale model. It can be considered a good candidate to develop new drugs against osteoporosis.

Zebrafish Models of Human Skeletal Disorders: Embryo and Adult Swimming Together.

Danio rerio (zebrafish) is an elective model organism for the study of vertebrate development because of its high degree of homology with human genes and organs, including bone. Zebrafish embryos, because of the optical clarity, small size, and fast development, can be easily used in large-scale mutagenesis experiments to isolate mutants with developmental skeletal defects and in high-throughput screenings to find new chemical compounds for the ability to revert the pathological phenotype. On the other hand, the adult zebrafish represents another powerful resource for pathogenic and therapeutic studies about adult human bone diseases. In fish, some characteristics such as bone turnover, reparation, and remodeling of the adult bone tissue cannot be found at the embryonic stage. Several pathological models have been established in adult zebrafish such as bone injury models, osteoporosis, and genetic diseases such as osteogenesis imperfecta. Given the growing interest for metabolic diseases and their complications, adult zebrafish models of type 2 diabetes and obesity have been recently generated and analyzed for bone complications using scales as model system. Interestingly, an osteoporosis-like phenotype has been found to be associated with metabolic alterations suggesting that bone complications share the same mechanisms in humans and fish. Embryo and adult represent powerful resources in rapid development to study bone physiology and pathology from different points of view.

Liquiritigenin Reduces Blood Glucose Level and Bone Adverse Effects in Hyperglycemic Adult Zebrafish.

Diabetes mellitus is a metabolic disease characterized by chronic hyperglycemia that induces other pathologies including diabetic retinopathy and bone disease. Adult Danio rerio (zebrafish) represents a powerful model to study both glucose and bone metabolism. Then, the aim of this study was to evaluate the effects of liquiritigenin (LTG) on blood glucose level and diabetes complications in hyperglycemic adult zebrafish. LTG is a flavonoid extracted from Glycyrrhiza glabra roots which possess important antioxidant, anti-inflammatory, and anti-diabetic properties. During four weeks of glucose treatment, LTG significantly prevented the onset of the hyperglycemia in adult zebrafish. Moreover, hyperglycemic fish showed increased advanced glycation end-products (AGEs) and parathormone levels whereas LTG completely prevented both of these metabolic alterations. Large bone-loss areas were found in the scales of glucose-treated fish whereas only small resorption lacunae were detected after glucose/LTG treatment. Biochemical and histological tartrate resistant acid phosphatase (TRAP) assays performed on explanted scales confirmed that LTG prevented the increase of osteoclastic activity in hyperglycemic fish. The osteoblastic alkaline phosphatase (ALP) activity was clearly lost in scales of glucose-treated fish whereas the co-treatment with LTG completely prevented such alteration. Gene expression analysis showed that LTG prevents the alteration in crucial bone regulatory genes. Our study confirmed that LTG is a very promising natural therapeutic approach for blood glucose lowering and to contrast the development of bone complications correlated to chronic hyperglycemia.

Metabolic and bone effects of high-fat diet in adult zebrafish.

An increase of visceral fat affects human bone health causing fragility, mechanical strength reduction, and increased propensity to fractures because of impaired bone matrix microstructure and aberrant bone cell function. Adult Danio rerio (zebrafish) represents a powerful model to study both metabolic diseases and bone metabolism. The aim of this study was to generate an obese adult zebrafish by high-fat diet and evaluate metabolic and bone tissue effects. Fish blood glucose and insulin levels were found to be altered in high-fat diet fish revealing a failure in ß-cells insulin production. Blood analysis of adipokines revealed significant alterations in adiponectin and leptin levels that are common in human and other obesity animal models. Advanced glycation end products (AGEs), derived from hyperglycemia condition, were found to be altered too. All these alterations were associated with an impaired bone metabolism. The scales of high-fat diet fish shown bone resorption lacunae associated with an intense osteoclastic tartrate-resistant acid phosphatase (TRAP) activity, whereas alkaline phosphatase (ALP) decreased. These data suggest that an imbalance of fat metabolism alters energy metabolism generating an osteoporosis-like phenotype in adult zebrafish scales. The zebrafish obesity model can contribute to elucidate in vivo the molecular mechanisms of metabolic changes in human obese patients.

Rapid and cost-effective xenograft hepatocellular carcinoma model in Zebrafish for drug testing.

We developed a novel, rapid and cost-effective Zebrafish xenograft model of hepatocellular carcinoma (HCC) for drug screening in the disease. Following injection into the yolk sack of Zebrafish larvae of the human HCC cell line JHH6 stained by a vital dye, tumor mass growth was followed by fluorescence microscopy and by human Ki67 quantification. Tumor induced neo-angiogenesis was evaluated by alkaline phosphatase staining of the vessels, by using the Tg(fli1:EGFP)y1 strain of Zebrafish and by the quantification of the zebrafish vascular endothelial growth factor and of its receptor. We show that it is feasible to micro-inject JHH6 in Zebrafish larvae, that injected cells can grow for different days and that this induces a marked neo-angiogenesis. Finally, we show that our model allows testing the effects of anti-HCC drugs such as Bortezomib. Compared to more complex HCC mouse models, our model is far less expensive, faster to set up and does not need immunosuppressant treatment. Finally, the model makes use of JHH6, an aggressive form of HCC cell line never tested before in Zebrafish. In conclusion, the possibility to test anti HCC/neo-angiogenesis drugs makes our JHH6 model useful to select therapeutic molecules for a highly vascularized tumor such as HCC.

Chronic hyperglycemia affects bone metabolism in adult zebrafish scale model.

Type II diabetes mellitus is a metabolic disease characterized by chronic hyperglycemia that induce other pathologies including diabetic retinopathy and bone disease. The mechanisms implicated in bone alterations induced by type II diabetes mellitus have been debated for years and are not yet clear because there are other factors involved that hide bone mineral density alterations. Despite this, it is well known that chronic hyperglycemia affects bone health causing fragility, mechanical strength reduction and increased propensity of fractures because of impaired bone matrix microstructure and aberrant bone cells function. Adult Danio rerio (zebrafish) represents a powerful model to study glucose and bone metabolism. Then, the aim of this study was to evaluate bone effects of chronic hyperglycemia in a new type II diabetes mellitus zebrafish model created by glucose administration in the water. Fish blood glucose levels have been monitored in time course experiments and basal glycemia was found increased. After 1 month treatment, the morphology of the retinal blood vessels showed abnormalities resembling to the human diabetic retinopathy. The adult bone metabolism has been evaluated in fish using the scales as read-out system. The scales of glucose-treated fish didn't depose new mineralized matrix and shown bone resorption lacunae associated with an intense osteoclast activity. In addition, hyperglycemic fish scales have shown a significant decrease of alkaline phosphatase activity and increase of tartrate-resistant acid phosphatase activity, in association with alterations in other bone-specific markers. These data indicates an imbalance in bone metabolism, which leads to the osteoporotic-like phenotype visualized through scale mineral matrix staining. The zebrafish model of hyperglycemic damage can contribute to elucidate in vivo the molecular mechanisms of metabolic changes, which influence the bone tissues regulation in human diabetic patients.

Tenofovir and bone: age-dependent effects in a zebrafish animal model.

BACKGROUND: New drugs against HIV infection are associated with important side effects, including impaired bone health. An important reduction of bone mineral density has been repeatedly described in adult but not in young patients receiving tenofovir disoproxil fumarate (TDF) as part of their antiretroviral regimen. The present study aimed to verify the presence and the characteristics of the adverse effects of TDF on skeletal tissue by using the Danio rerio (zebrafish) model. METHODS: TDF administration via microinjection in the yolk has been used for embryo study. Water treatment with TDF and scale analysis by morphological, histological and biochemical experiments have been used for adult animals. RESULTS: TDF administration via microinjection in the yolk did not induce any significant effect on mineralization rate and did not produce deformity of the skeletal structure, or growth or vitality impairment of the embryos. TDF exposure in adult zebrafish induced an age-related reduction of the osteoblastic activity marker alkaline phosphatase (up to -44%) and an increase of the osteoclastic activity marker tartrate-resistant acid phosphatase (up to +57%) in the scales. Old fish treated with TDF exhibited an osteoporotic-like phenotype with resorption area along the edge of the scale. CONCLUSIONS: This study demonstrated that in zebrafish, as in humans, TDF shows a catabolic bone effect related to age and underlined the ultimate utility of using teleost fish as a model to assess the effects of drugs on bone tissue.

Danio rerio: the Janus of the bone from embryo to scale.

Danio rerio (zebrafish), like the Roman god Janus, is an old animal model which is recently emerged and looks to the future with an increasing scientific success. Unlike other traditional animal models, zebrafish represents a versatile way to approach the study of the skeleton. Transparency of the larval stage, genetic manipulability and unique anatomical structures (scales) makes zebrafish a powerful and versatile instrument to investigate the bone tissue in terms of structure and function. Like Janus, zebrafish offers two different faces, or better, two models in one animal: larval and adult stage. The embryo can be used to isolate new genes involved in osteogenesis by large-scale mutagenesis screenings. The behavior of bone cells and genes in osteogenesis can be investigate by using transgenic lines, vital dyes, mutants and traditional molecular biology techniques. The adult zebrafish represents an important resource to study the pathways related to the bone metabolism and turnover. In particular, the properties of the caudal fin allow to study mechanisms of bone regeneration and reparation whereas the elasmoid scale represents an unique tool to investigate the bone metabolism under physiological or pathological conditions.

Alendronate rescued osteoporotic phenotype in a model of glucocorticoid-induced osteoporosis in adult zebrafish scale.

Long-term effects of glucocorticoid treatment in humans induce bone loss and increase the risk of fracture in the skeleton. The pathogenic mechanisms of glucocorticoid-induced osteoporosis (GIOP) are still unclear. The GIOP and its effects have been reproduced in several animal models including Danio rerio (zebrafish) embryo. The treatment of adult fish with prednisolone (PN) has shown a dose-dependent decrease of mineralized matrix in the scales. Large resorption lacunae are characterized by single TRAP-positive cells which migrate to the margin of the scale merging into a multinucleated structures. The treatment with PN of cultured scales did not increase TRAP activity suggesting that the massive presence of osteoclasts in the resorption sites could be likely the result of a systemic recruitment of monocyte-macrophage precursors. We observed that treatment with PN induced a significant decrease of the alkaline phosphatase (ALP) activity in scale scleroblasts if compared with untreated controls. Then, we investigated the total mineral balance under prednisolone treatment using a time-dependent double live staining. The untreated fish fully repaired the resorption lacuna induced by prednisolone, whereas treated fish failed. The presence of osteoclast resorption fingerprints on new matrix suggested that the osteoclast activity counterbalances the osteodepositive activity exerted by scleroblasts. The treatment with PN in association with alendronate (AL) has surprisingly resulted in a significant decrease of TRAP activity and increase of ALP compared to PN-treated fish in biochemical and histological assays confirming the action of alendronate against GIOP in fish as well in humans.