Tetrabromobisphenol A Promotes the Osteoclastogenesis of RAW264.7 Cells Induced by Receptor Activator of NF-kappa B Ligand In Vitro

BACKGROUND: Tetrabromobisphenol A (TBBPA), one of the most widely used brominated flame-retardants, is a representative persistent organic pollutants group. Studies on TBBPA toxicity have been conducted using various target cells; however, few studies have investigated TBBPA toxicity in bone cells. Therefore, this study investigated the in vitro effects of TBBPA on osteoclasts, a cell type involved in bone metabolism. METHODS: RAW264.7 cells were cultured in medium containing 50 ng/mL receptor activator of nuclear factor kappa B ligand (RANKL) and varying concentrations of TBBPA. To evaluate the effects of TBBPA on the differentiation and function of osteoclasts, osteoclast-specific gene expression, tartrate-resistant acid phosphatase (TRAP) activity, bone resorbing activity, mitochondrial membrane potential (MMP) and mitochondrial superoxide were measured. RESULTS: The presence of 20 μM TBBPA significantly increased TRAP activity in RANKL-stimulated RAW264.7 cells, the bone resorbing activity of osteoclasts, and the gene expression of Akt2, nuclear factor of activated T-cells cytoplasmic 1, and chloride channel voltage-sensitive 7. However, TBBPA treatment caused no change in the expression of carbonic anhydrase II, cathepsin K, osteopetrosis-associated transmembrane protein 1, Src, extracellular signal-related kinase, GAB2, c-Fos, or matrix metalloproteinase 9. Furthermore, 20 μM TBBPA caused a significant decrease in MMP and a significant increase in mitochondrial superoxide production. CONCLUSION: This study suggests that TBBPA promotes osteoclast differentiation and activity. The mechanism of TBBPA-stimulated osteoclastogenesis might include increased expression of several genes involved in osteoclast differentiation and reactive oxygen species production.

Ultrastructural changes of erythrocytes in whole blood after exposure to prospective in silico-designed anticancer agents: a qualitative case study

BACKGROUND: Novel, in silico-designed anticancer compounds were synthesized in our laboratory namely, 2-ethyl-3-O-sulphamoyl-estra-1,3,5(10),15-tetraen-17-ol (ESE-15-ol) and 2-ethyl-3-O-sulphamoyl-estra-1,3,5(10)16-tetraene (ESE-16). These compounds were designed to have improved bioavailability when compared to their source compound, 2-methoxyestradiol. This theoretically would be due to their increased binding affinity to carbonic anhydrase II, present in erythrocytes. Since the novel compounds under investigation are proposed to be transported within erythrocytes bound to carbonic anhydrase II, the morphological effect which they may exert on whole blood and erythrocytes is of great significance. A secondary outcome included revision of previously reported procedures for the handling of the whole blood sample. The purpose of this study was twofold. Firstly, the ultrastructural morphology of a healthy female's erythrocytes was examined via scanning electron microscopy (SEM) after exposure to the newly in silico-designed compounds. Morphology of erythrocytes following exposure to ESE-15-ol and ESE-16 for 3 minutes and 24 hours at 22°C were described with the use of SEM. The haemolytic activity of the compounds after 24 hours exposure were also determined with the ex vivo haemolysis assay. Secondly, storage conditions of the whole blood sample were investigated by determining morphological changes after a 24 hour storage period at 22°C and 37°C. RESULTS: No significant morphological changes were observed in the erythrocyte morphology after exposure to the novel anticancer compounds. Storage of the whole blood samples at 37°C for 24 hours resulted in visible morphological stress in the erythrocytes. Erythrocytes incubated at 22°C for 24 hours showed no structural deformity or distress. CONCLUSIONS: From this research the optimal temperature for ex vivo exposure of whole blood samples to ESE-15-ol and ESE-16 for 24 hours was determined to be 22°C. Data from this study revealed the potential of these compounds to be applied to ex vivo study techniques, since no damage occurred to erythrocytes ultrastructure under these conditions. As no structural changes were observed in erythrocytes exposed to ESE-15-ol and ESE-16, further ex vivo experiments will be conducted into the potential effects of these compounds on whole blood. Optimal incubation conditions up to 24 hours for whole blood were established as a secondary outcome.

The changes of carbonic anhydrase activity and its mRNA expression in rats cochlea after noise exposure / 临床耳鼻咽喉头颈外科杂志

OBJECTIVE@#To investigate the changes of carbonic anhydrase (CA) activity and its mRNA expression in rats cochlea after noise exposure.@*METHOD@#Twenty-four healthy Sprague Dawley rats were randomly allocated into 4 groups including 1 control group without contact of noise and 3 experimental groups exposed to a narrowband white noise of 4 kHz, 110 dB SPL 4h/d for 1 day, 1 week and 3 weeks, respectively. ABR thresholds were tested for each group at the time points described above. An immunohistochemical method was used to detect the expression of CA in the cochlea tissue. Differences in CAII mRNA expressions with and without noise exposure were examined using RT-PCR.@*RESULT@#The thresholds of ABR in noise exposed cochlea were increased compared with the control cochlea (P < 0.01). CA activity and the CAII mRNA expression in noise exposed cochlea were decreased compared with the control cochlea (P < 0.01). The increase of ABR thresholds and decreases of CA activity and the CAII mRNA expression showed a time dependent pattern as the extension of duration exposed to noise.@*CONCLUSION@#Noise exposure could downregulate the CA activity and CAII mRNA expression in the cochlea. CA was proably involved in the pathogenesis of noise-induced hearing loss.

A Case of Cavernous Sinus Thrombophlebitis and Meningitis as a Complication in Osteopetrosis

Osteopetrosis is a rare genetic bone disease characterized by increased bone density but prone to breakage due to defective osteoclastic function. Among two primary types of autosomal dominant osteopetrosis (ADO), osteopetrosis type II is characterized by sclerosis of bones, predominantly involving the spine, the pelvis, and the skull base. Fragility of bones and dental abscess are leading complications. This report presents a case of osteopetrosis in a 52-years-old female, which was complicated by the development of cavernous sinus thrombophlebitis and meningitis. She was suffered from multiple fractures since one year ago. Laboratory data revealed elevated serum levels of tartrate resistant acid phosphatase (TRAP) without carbonic anhydrase II DNA mutation. A thoracolumbar spine X-ray showed, typical findings of ADO type II (ADO II; Albers-Schonberg disease), prominent vertebral endplates so called the 'rugger jersey spine'. Her older sister also showed same typical spine appearance. We report a case of ADO II with cavernous sinus thrombophlebitis and meningitis that was successfully treated with long-term antibiotics with right sphenoidotomy.

Expression of carbonic anhydrase II in cultured rat osteoclasts after stress stimulation / 华西口腔医学杂志

<p><b>OBJECTIVE</b>To test expression of carbonic anhydrase II (Ca II) mRNA in osteoclasts which were applied with fluid shear stress.</p><p><b>METHODS</b>The bone marrow cells of Sprague-Dawley rats were cultured with the presence of 1,25-(OH)2D3 and dexamethasone. The osteoclast-like cells were identified by tartrate-resistant acid phosphatase(TRAP) staining and scanning electron microscope (SEM) observation, then purified with trypsin/ethylenediamine tetraacetic acid (EDTA). Different values and lasting time of steady fluid shear stress were exerted on the osteoclasts with parallel plate flow system. The Ca II expression of osteoclasts were detected by real time reverse transcription-polymerase chain reaction(RT-PCR) and nested polymerase chain reaction(PCR).</p><p><b>RESULTS</b>The levels of Ca II mRNA were down-regulated correspondingly with the increase of stress and time (P < 0.05).</p><p><b>CONCLUSION</b>It's indicated that steady fluid shear stress within a certain range may down-regulate the expression of Ca II in osteoclasts.</p>

The Molecular Mechanism of Baicalin on RANKL-induced Osteoclastogenesis in RAW264.7 Cells

This study examined the anti-osteoclastogenic effects of baicalin on receptor activator of NF-kB ligand (RANKL)-induced RAW264.7 cells. Baicalin is a flavonoid that is produced by Scutellaria baicalensis and is known to have multiple biological properties, including antibacterial, anti-inflammatory and analgesic effects. The effects of baicalin on osteoclasts were examined by measuring 1) cell viability; 2) the formation of tartrate-resistant acid phosphatase (TRAP) (+) multinucleated cells; 3) RANK/RANKL signaling pathways and 4) mRNA levels of osteoclast-associated genes. Baicalin inhibited the formation of RANKL-stimulated TRAP (+) multinucleated cells and also suppressed the RANKL-stimulated activation of p-38, ERK, cSrc and AKT signaling. Baicalin also inhibited the RANKL-stimulated degradation of IkappaB in RAW264.7 cells. In addition, the RANKL-stimulated induction of NFATc1 transcription factors was found to be abrogated by this flavonoid. Baicalin was further found to decrease the mRNA expression of osteoclast-associated genes, including carbonic anhydrase II, TRAP and cathepsin K in the RAW264.7 cells. Our data thus demonstrate that baicalin inhibits osteoclastogenesis by inhibiting the RANKL-induced activation of signaling molecules and transcription factors in osteoclast precursors.

Topiramato, efectos en el estado ácido base y consecuencias clínicas / Topiramate: Effects on the Acid-Base Status and Clinical Consequences

En la presente revisión se analiza cómo el topiramato afecta a corto y largo plazo el estado ácido base mediante la inhibición de la anhidrasa carbónica tipo II, generando una acidosis tubular renal mixta que provoca consecuencias clínicas de importancia como nefrolitiasis, osteoporosis y retraso en el crecimiento. Dado que los tratamientos con este fármaco son crónicos, pueden prevenirse estos sucesos de diversas maneras. Se ofrecen pautas para el manejo de las distintas situaciones clínicas

This review provides an analysis of the short-and long-term impact of Topiramate on the acid-base status through the inhibition of carbonic anhydrase II, which leads to a mixed renal tubular acidosis that causes significant clinical consequences such as nephrolithiasis, osteoporosis and growth delay. Because treatments with this drug are chronic, these events may be prevented in different ways. The author offers guidelines for the management of different clinical situations

Clinicopathological significance of the expression of carbonic anhydrase II in human pancreatic invasive ductal cancer / 中华外科杂志

<p><b>OBJECTIVE</b>To study the clinicopathological significance of the expression of carbonic anhydrase (CA)II protein and mRNA in primary invasive ductal cancer (IDC) of human pancreas.</p><p><b>METHODS</b>The expression of CAII protein in 33 paired paraffin embedded IDC specimens of the pancreas and paired adjacent non-cancerous pancreatic tissues was detected by immunohistochemistry. Western blot and reverse transcription polymerase chain reaction (RT-PCR) were used to examine the expression of CAII protein and mRNA level in 12 paired fresh IDC specimens of the pancreas and adjuvant non-cancerous pancreatic tissues. The relationship between the protein expression and clinicopathological features was analyzed.</p><p><b>RESULTS</b>Overexpression of CAII protein was shown in 11 cases of pancreatic IDC tissues (33.3%, 11/33), which was much lower than that in paired non-cancerous pancreatic tissues (72.7%, t = 6.275, P = 0.000). The expression of CAII protein had no correlation with tumor position (χ² = 0.992, P = 0.319), differentiation (χ² = 0.866, P = 0.352), TNM stage (χ² = 1.210, P = 0.271) and Lymph node metastasis (χ² = 0.798, P = 0.372), but had bordering statistic sig with the prognosis of the patients (χ² = 3.233, P = 0.072). The median survival time in the patients with high expression of CAII protein was 540 days, while that in the patients with low expression was 320 days. The expression of CAII protein and mRNA was lower in IDC than that in paired non-cancerous pancreatic tissues detected by Western blot and RT-PCR respectively (t = 3.399, P = 0.006; t = 2.281, P = 0.043).</p><p><b>CONCLUSION</b>CAII is down regulated in pancreatic IDC and might be relative with the prognosis.</p>

Enhanced Expression of Carbonic anhydrase II in Hypokalemic Rat Kidney / 체질인류학회지

A number of acid-base or electrolyte disorders are associated with decreased or increased HCO3- reabsorption in the renal tubules. The present study was to examine the alterations of expression and distribution of Carbonic anhydrase II in the kidneys of normal and potassium-depleted rats using Western blot analysis and immuno-histochemistry. Western blot analysis demonstrated that CA II protein, ~30 kDa at molecular mass, was abundantly expressed in normal group. All potassium-depleted groups showed slightly increased CA II protein compared to normal group. In control group, immunoreactivity of CA II protein was detected in the entire collecting duct. Signal intensity was prominent in the intercalated cells and weak in the principal cells of the cortical collecting ducts. In potassium-depleted groups, the pattern of cellular labeling of CA II protein was identical to that of normal group, but the signal intensity was decreased in cortical collecting duct, markedly increased in the inner stripe of outer medullary and inner medullary collecting ducts, and unchanged in the outer stripe of outer medullary collecting duct. These results suggest that chronic hypokalemia impact the expression pattern of CA II protein depending the portion of the collecting duct.

Expression of carbonic anhydrase II in human testes and spermatozoa and its clinical significance / 中华男科学杂志

<p><b>OBJECTIVE</b>To investigate the expression of carbonic anhydrase II (CA2) in human testes and spermatozoa, and to compare the expressions of CA2 in ejaculated spermatozoa between normozoospermic and asthenozoospermic men.</p><p><b>METHODS</b>The localization of CA2 in human testes was observed by immunohistochemistry, and that in human sperm by immunofluorescence. Western blot was used to detect the expression of CA2 in the semen samples obtained from 16 normozoospermic and 16 asthenozoospermic volunteers.</p><p><b>RESULTS</b>The CA2 protein was shown to be localized in the tail of elongating spermatids by immunohistochemistry and in the flagellum of human sperm by immunofluorescence. Western blot revealed an obviously increased expression of CA2 in the spermatozoa of asthenozoospermic patients, with statistically significant difference from the normozoospermic group (1.84 +/- 0.32 vs 1.41 +/- 0.26, P < 0.05).</p><p><b>CONCLUSION</b>The CA2 protein is expressed in the spermatogenic stage of elongating spermatids in human testes and localized in the sperm tail. The expression of CA2 is significantly increased in the spermatozoa of asthenozoospermic men, which might be responsible for low sperm motility.</p>

[Osteopetrosis with carbonic anhydrase II deficiency: about 24 cases]

Twenty four patients suffering from osteopetrosis caused by carbonic anhydrase II deficiency are colliged. This pathology seems to be frequent in Tunisia. Mental retardation is present in 52%, 85% of patients have short stature and 25% have optic atrophy. All affected subjects show craniofacial disproportion and dental anomalies. Twenty patients have at least one bone fracture. Metabolic acidosis is constant: it is profound during the first life decade. A severe selective reduction of carbonic anhydrase II in erythrocyte is confirmed in 18 cases. Osteosclerosis and defective skeletal modelling are constant, cerebral calcification can be seen at the scanner approximately at the age of two years and six months. All patients are homozygous for a splice junction mutation in intron 2 of the carbonic anhydrase II gene, this mutation doesn't seem to protect patients from bone fractures nor to induce a severe metabolic acidosis

Carbonic anhydrase II immunostaining in the cerebellum of postnatal mice / 대한해부학회지

The carbonic anhydrase II (CA-II) is specifically expressed in oligodendrocytes, the cells responsible for myelination in the central nervous system. However no direct evidence on relationship between myelin formation and CA-II immunoreactivity has been described. The aims of these studies are to investigate the relationship between CA-II and myelination during cerebellar development of mouse. Myelin staining was found on postnatal (P) 14, and its intensity increased in proportion to developmental age. CA-II positive oligodendrocytes were observed in the white matter of cerebellum on P 14 day. CA-II positive oligoden-drocytes also occured in the granular layer and Purkinje cell layers in the later stage of dvelopment. The parallel development in the CA-II expression and myelination during development suggests that CA-II in oligoendrocyte play a role to myelination.

Chromophobe Renal Cell Carcinoma

We report 13 chromophobe renal cell carcinomas (10.8%) observed among 120 renal cell carcinomas in adults. The average age was 53 (range: 34-72) years old, and 6 were males and 7 females. The mean tumor size was 10 (range: 5-17) cm, mean nuclear grade 2.4, and mean Robson's stage was 1.9. There were two distinct histologic variants; typical variant (n=9) and eosinophilic variant (n=4). Both of them showed typical light microscopic features and positive reaction with Hale's colloidal iron and carbonic anhydrase II, a marker protein of intercalated cells of renal collecting ducts. A strong positive immunoreactivity for epithelial membrane antigen was noted in the cytoplasm in 12 of 13 tumors. Numerous microvesicles, 180~440 nm in diameter, were identified ultrastructurally. DNA aneuploidy was found in 3 out of 10 cases. Neither local recurrence nor metastasis have been identified during the following period of 4~144 (mean 48) months.

Histopathology and Mainz Classification of Renal Cell Tumors: A Histogenetic Study and DNA Content Analysis

The Mainz classification for renal cell tumors was introduced in 1986 and it's utility has been reported in several histogenetic and genetic studies of renal cell tumors. We present a study of 127 cases of renal cell tumors with clinicopathologic correlation, DNA content analysis, and histogenesis studied by histochemical and immunohistochemical staining. The 127 renal cell tumors classified by the Mainz classification were 87 clear cell, 17 chromophilic, 13 chromophobe and 3 sarcomatoid renal cell carcinomas, 5 oncocytomas and 2 adenomas. These subtypes showed significant correlation not with age, sex, Robson's stage, DNA ploidy or tumor recurrence but with nuclear grade (p=0.001) and tumor size (p=0.001). Hall's colloidal iron (p=0.002) and carbonic anhydrase II (p=0.013) stains, representing the origin of distal nephron especially of collecting duct, were significantly correlated with specific subtypes of renal cell tumors, especially chromophobe cell renal carcinoma. This study demonstrates that the Mainz classification suggests several morphologically different subtypes and variants of renal cell tumors and that some of them may have originated from the distal nephron, particularly from the collecting duct.