Oral malodorous compound causes caspase-8 and -9 mediated programmed cell death in osteoblasts.

BACKGROUND AND OBJECTIVE: Hydrogen sulfide (H(2) S) is one of two volatile sulfur compounds that are known to be the main cause of oral malodor; the other is methyl mercaptan. Other known volatiles existing in mouth air do not contribute significantly to oral malodor originating in the oral cavity. Hydrogen sulfide is also known to be an etiological factor in periodontal disease. However, the effects of H(2) S on alveolar bone remain unclear. The objectives of this study were to determine the apoptotic effects of H(2) S on osteoblasts and to verify the apoptotic molecular pathways. MATERIAL AND METHODS: A clonal murine calvaria cell line was incubated with 50 ng/mL of H(2) S. To detect apoptosis, the cells were analysed by flow cytometry and ELISA. Mitochondrial membrane depolarization was assessed using flow cytometry as well. ELISA was used to evaluate the release of cytochrome c into the cytosol and to assess Fas ligand, p53, tumor necrosis factor α, interleukin IL1-α IL-ß, IL-2, IL-4, IL-10, interferon-γ, granulocyte-colony stimulating factor and granulocyte-macrophage colony stimulating factor. Caspase-3, -8 and -9 activities were estimated. Expression of BAX and Bcl-2 was assessed by real-time quantitative RT-PCR. DNA fragmentation was detected by single-cell gel electrophoresis. Fas receptors were evaluated by western blotting. RESULTS: After H(2) S incubation, apoptotic levels increased significantly in a time-dependent manner. Mitochondrial membrane depolarization, the release of cytochrome c, p53 and caspase-3, -8 and -9 and DNA fragmentation were all significantly greater. BAX gene activity was upregulated, whereas Bcl-2 remained low. Fas ligand/Fas receptor, tumor necrosis factor α and other cytokines were not increased to a significant degree. CONCLUSION: At less-than-pathological concentrations in gingival crevicular fluid, H(2) S induces apoptosis in osteoblasts. The molecular mechanisms underlying the apoptotic process include p53, a mitochondrial pathway and caspase-8 activation.

Oral malodorous compound triggers mitochondrial-dependent apoptosis and causes genomic DNA damage in human gingival epithelial cells.

BACKGROUND AND OBJECTIVE: Volatile sulfur compounds are the main compounds causing halitosis. One of these compounds, hydrogen sulfide (H(2)S), which is responsible for physiological halitosis, is reported also to have periodontal pathogenic activities. Hydrogen sulfide has been shown to activate the apoptotic process in different tissues. Apoptosis plays an important role in the development of periodontitis. The aim of this study was to determine whether H(2)S causes apoptosis in human gingival epithelial cells and to examine the cellular signaling pathway initiating the process. MATERIAL AND METHODS: Human gingival epithelial cells were incubated with 50 ng/mL H(2)S in air contining 5% CO(2) for 24, 48 or 72 h. To detect apoptosis, the cells were stained with annexin V and 7-amino actinomycin D, and analyzed using flow cytometry. Reactive oxygen species, mitochondrial membrane depolarization and release of cytochrome C into the cytosol were assessed using flow cytometry and enzyme-linked immunosorbent assay. Activity levels for the key apoptotic enzymes caspase-9, -8 and -3 were also determined. Genomic DNA damage was detected using single-cell gel electrophoresis. RESULTS: Apoptosis was significantly increased to 24.5 +/- 5.7 at 24 h and 41.5 +/- 8.9% at 48 h (p < 0.01). Reactive oxygen species were enhanced and mitochondrial membrane depolarization was collapsed. Cytochrome C release was dramatically increased (0.12 +/- 0.02 vs. 0.02 +/- 0.01 at 24 h and 0.21 +/- 0.02 vs. 0.02 +/- 0.01 ng/mL at 48 h; p < 0.05). Caspase-9 and -3 were strongly activated, while caspase-8 activity remained low. The percentage of DNA strand breaks increased, especially at 48 h. CONCLUSION: Hydrogen sulfide induces apoptosis in human gingival epithelial cells by activating the mitochondrial pathway.

Ecotoxicological characterization of tannery wastewater in Dhaka, Bangladesh.

Tanning industries are one of the main economic activities in Bangladesh. It has been well documented that wastewater discharged from tanneries without appropriate treatment results in detrimental effects on the ecosystem. No ecotoxicity evaluation of any aquatic environment in Bangladesh has been conducted so far. In this study a battery of toxicity bioassays and chemical analysis were carried out from water samples obtained from three sampling points: upstream from discharging site on River Buriganga (S1), raw wastewater effluent (S2), and downstream the discharging sluice gate (S3), in the Hazaribagh tannery area of Dhaka City, Bangladesh. While S1 and S3 water samples did not show significant toxicity in the bioassays tested, S2 exhibited high acute toxicity to the bacterium Vibrio fischeri (15-min Microtox test, EC50 = 9.8%), the higher plant Lactuca sativa (5-day root elongation inhibition test, EC50 = 14.2%), and the microcrustacean Daphnia magna (24-hour mobility test, EC50 = 31.5%). The results suggested that the raw wastewater effluent had detrimental effects on broad spectrum of organisms in the aquatic ecosystem and bacterium was the most sensitive. The chemical analysis revealed that sample S2 contained an extremely high concentration of chromium (47 g l(-1)). Additionally microbiological analysis indicated that the sampling area is impacted by fecal pollution, increasing the environmental health risk for its inhabitants.

Toxicity of four antifouling biocides and their mixtures on the brine shrimp Artemia salina.

Zinc pyrithione (ZPT), Copper pyrihione (CPT), Chlorothalonil and Diuron are four of the most widely used as alternative to tributlytin (TBT) antifouling biocides in boat paints. As most previous laboratory bioassays for these biocides have been conducted solely based on acute tests with a single compound, information on the possible combined toxicity of these common biocides to marine organisms are limited. In this study, the toxicity of binary (in several proportions), ternary and quaternary mixtures were evaluated using the brine shrimp Artemia salina as test organism. Mixture toxicities were studied using the concentration addition model (isobolograms and toxic unit summation), and the mixture toxicity index (MTI). The ZPT-CPT combination had a strictly synergistic effect which requires attention because the coexistence of ZPT and CPT in the marine environment, due to transchelation of ZPT, may occur. The binary mixtures of Diuron with the metal pyrithiones exhibited various interactive effects (synergistic, antagonistic or additive) depending on concentration ratios, whereas all binary mixtures that contained Chlorothalonil exhibited antagonistic effects. The different types of combined effects subsequent to proportion variation of binary mixtures underline the importance of the combined toxicity characterization for various ratios of concentrations. The four ternary mixtures tested, also exhibited various interactive effects, and the quaternary mixture exhibited synergism. The models applied were in agreement in most cases. The observed synergistic interactions underline the requirement to review water quality guidelines, which are likely underestimating the adverse combined effects of these chemicals.

Acceleration of fracture healing in nonhuman primates by fibroblast growth factor-2.

One of the greatest needs in the clinical bone field is a bioactive agent to stimulate bone formation. We previously reported that fibroblast growth factor-2 (FGF-2) exhibited strong anabolic actions on bone formation in models of rodents and dogs. Aiming at a clinical application, this study was undertaken to clarify the effect of a single local application of recombinant human FGF-2 on fracture healing in nonhuman primates. After a fracture was created at the midshaft of the right ulna of animals and stabilized with an intramedullary nail, gelatin hydrogel alone (n = 10) or gelatin hydrogel containing 200 microg FGF-2 (n = 10) was injected into the fracture site. Although 4 of 10 animals treated with the vehicle alone remained in a nonunion state even after 10 weeks, bone union was complete at 6 weeks in all 10 animals treated with FGF-2. Significant differences in bone mineral content and density at the fracture site between the vehicle and FGF-2 groups were seen at 6 weeks and thereafter. FGF-2 also increased the mechanical property of the fracture site. We conclude that FGF-2 accelerates fracture healing and prevents nonunion in primates, and therefore propose that it is a potent bone anabolic agent for clinical use.

An oral adsorbent ameliorates renal overload of indoxyl sulfate and progression of renal failure in diabetic rats.

Otsuka Long-Evans Tokushima Fatty (OLETF) rats were established as a new model of non-insulin-dependent diabetes mellitus. An oral adsorbent, AST-120, is effective in removing such uremic toxins as indoxyl sulfate and delays the progression of chronic renal failure (CRF). This study was designed to determine the effects of AST-120 on the progression of CRF in uninephrectomized OLETF (1/2NxOLETF) rats and the localization of indoxyl sulfate in their kidneys. Four weeks after unilateral nephrectomy, 14 OLETF rats were divided into two groups; AST-120-administered and control 1/2NxOLETF rats. Long-Evans Tokushima Otsuka rats, which are genetically similar to the OLETF rats but not diabetic, were also included. After the administration of AST-120 for 36 weeks, we examined the effects of AST-120 on renal functional and pathological changes in the three groups. The control 1/2NxOLETF rats showed marked hyperglycemia, hyperlipidemia, renal failure, glomerular sclerosis, and tubulointerstitial injury. The administration of AST-120 to the 1/2NxOLETF rats retarded the progression of renal dysfunction and fibrosis, as well as hyperlipidemia, and reduced serum and urinary levels of indoxyl sulfate. Immunohistochemistry showed that AST-120 markedly reduced the overload of indoxyl sulfate in tubular epithelial cells, especially dilated tubules, of the 1/2NxOLETF rats. In conclusion, AST-120 delayed the progression of renal failure and fibrosis in 1/2NxOLETF rats and decreased the overload of indoxyl sulfate on renal tubular cells.

Bone regeneration by basic fibroblast growth factor complexed with biodegradable hydrogels.

The objective of this study is to enhance the bone induction activity of basic fibroblast growth factor (bFGF) for reconstruction of skull bone defects which has been clinically recognized as almost impossible. For this purpose, we prepared biodegradable hydrogels from gelatin with an isoelectric point of 4.9 which is capable of polyionic complexing with basic bFGF. When implanted in rabbit skull defects of 6 mm in diameter (6 defects per experimental group), the gelatin hydrogels incorporating 100 microg of bFGF promoted bone regeneration at the defect in marked contrast to free bFGF of the same dose, finally closing the bone defects after 12 weeks of implantation as is apparent from histological examination. In dual energy X-ray absorptometry analysis, the bone mineral density at the skull defects enhanced by the hydrogels was significantly higher than that by free bFGF at doses ranging from 2 to 200 microg/defect (P < 0.05). The extent of bone regeneration induced by gelatin hydrogels incorporating 100 microg of bFGF increased with a decrease in their water content. Histological examination indicated that more slowly degrading hydrogels of lower water content prolonged the retention period of osteoblasts in the bone defects. This led to enhanced bone regeneration compared with faster degrading hydrogels of higher water content. It was concluded that this biodegradable hydrogel system was a promising surgical tool to assist self-reconstruction of the skull bone.

Promoted bone healing at a rabbit skull gap between autologous bone fragment and the surrounding intact bone with biodegradable microspheres containing transforming growth factor-beta1.

This study is a trial to promote repairing of the rabbit skull bone gap between an autologous bone flap and the intact bone with biodegradable gelatin microspheres containing transforming growth factor-beta1 (TGF-beta1). A 10-mm diameter bone defect was prepared in rabbit skulls by drilling out a bone flap of 6 mm in diameter. After a surrounding gap defect of 2 mm was created and treated with 0.5 microg of free TGF-beta1 and gelatin microspheres containing 0.5 microg of free TGF-beta1, the circular autologous bone flap was placed in the center. Significant bone healing at the gap defect was observed 3 weeks after implantation of the TGF-beta1-containing gelatin microspheres. The bone mineral density (BMD) was significantly higher than that of other experimental groups. On the contrary, when applied with free TGF-beta1, a fibrous tissue initially infiltrated into the gap defect, resulting in impairing bone healing. The tissue response was similar to that at the defect implanted with empty gelatin microspheres and TGF-beta1-free phosphate-buffered saline solution alone. There was more space in the gap-filling bone in the 16-week view than the 3-week view. It is possible that this was an intermediate step along the way toward normal healing and formation of cancellous bone. We conclude that gelatin microspheres containing TGF-beta1 show promise as an agent to promote bone regeneration of subcritical size defects between surgically positioned autologous bone flaps and surrounding host bone.

Oral adsorbent ameliorates renal TGF-beta 1 expression in hypercholesterolemic rats.

BACKGROUND: A spontaneously hypercholesterolemic Imai rat has recently been reported as a model of focal glomerulosclerosis that causes nephrotic syndrome followed by renal failure. This study was designed to determine if an oral adsorbent, AST-120, ameliorates renal lesions and TGF-beta 1 expression in the rats. METHODS: AST-120 was given orally to the Imai rats for 32 weeks, and renal function and pathology were compared between the AST-120-administered and control Imai rats. RESULTS: AST-120-administered rats showed significantly lower level of blood urea nitrogen, serum creatinine, urinary protein, serum total-cholesterol, serum triglyceride, and serum and urinary indoxyl sulfate, and significantly higher levels of serum albumin and creatinine clearance than control rats. AST-120 reduced the glomerular sclerosis index, interstitial fibrosis area, and the extent of glomerular lipid deposition. Immunohistochemistry demonstrated that AST-120 reduced the expression of transforming growth factor (TGF)-beta 1 and tissue inhibitor of metalloproteinase (TIMP)-1 as well as interstitial infiltration of macrophages in the renal cortex of the Imai rats. CONCLUSIONS: AST-120 prevented the progression of nephrotic syndrome and renal failure in the Imai rats by ameliorating glomerular sclerosis and interstitial fibrosis, accompanied with reduced expression of TGF-beta 1 and TIMP-1, and reduced infiltration of macrophages in the kidneys.

Stimulation of bone formation by intraosseous application of recombinant basic fibroblast growth factor in normal and ovariectomized rabbits.

The effect on intraosseous bone formation of a single local injection of recombinant human basic fibroblast growth factor into the distal femur was examined in normal and ovariectomized rabbits. In normal rabbits, basic fibroblast growth factor increased bone mineral density around the injected site in a dose-dependent manner at 4 weeks, with significant effects at concentrations of 400 micrograms and greater. Doses of 400 and 1,600 micrograms of basic fibroblast growth factor increased bone mineral density by 8 and 9%, respectively, compared with the opposite control femur. Histological examination showed that basic fibroblast growth factor (400 micrograms) induced the proliferation or recruitment of undifferentiated mesenchymal cells around the existing trabeculae at 3 days after the injection. For the first 2 weeks, osteoid formation was strongly stimulated, and this was followed by mineral apposition for another 2 weeks, at which time the femurs were harvested. Consequently, basic fibroblast growth factor stimulated intraosseous bone formation at 4 weeks. We speculate that the direct action of basic fibroblast growth factor on bone formation may be to stimulate proliferation or recruitment of minimally differentiated mesenchymal cells and to initiate the cascade of events in later stages of bone formation. In ovariectomized rabbits, basic fibroblast growth factor (400 micrograms) also increased bone mineral density, histomorphometrical bone formation markers, and trabecular connectivity to levels similar to those in rabbits who had received sham operations.

Single local injection of recombinant fibroblast growth factor-2 stimulates healing of segmental bone defects in rabbits.

The effects of a single local injection of recombinant human fibroblast growth factor-2 on the healing of segmental bone defects were evaluated in rabbits. One month after the external fixator originally designed for this experiment was installed in the tibia of the rabbit, a 3-mm bone defect was created by an osteotomy in the middle of the tibia and 0, 50, 100, 200, or 400 microg of fibroblast growth factor-2 in 100 microl of saline solution was injected into the defect. Injection of the growth factor increased the volume and mineral content of newly made bone at the defect in a dose-dependent manner with significant effects at concentrations of 100 microg or greater. These significant effects were observed at 5 weeks and later. One hundred micrograms of the growth factor increased the volume and mineral content of newly made bone by 95 and 36%, respectively, at 5 weeks. These results indicate that a single local injection of fibroblast growth factor-2 stimulates the healing of segmental defects. We speculate that such an injection could be clinically useful for the healing of fractures even when the fracture gap is rather large.

Calcified metastatic brain tumor.

The case of a 57-year-old woman with a calcified metastatic brain tumor, histologically confirmed to be a squamous cell carcinoma, is reported. This patient is unusual because this metastatic squamous cell carcinoma contained an extraordinary huge conglomerated calcification, that was well-defined radiographically. This case is documented with a discussion of the pathogenesis of the calcification.

Subarachnoid hemorrhage possibly caused by a saccular carotid artery aneurysm within the cavernous sinus. Case report.

Aneurysms arising from the intracavernous portion of the internal carotid artery very rarely rupture. A patient is presented in whom rupture of an aneurysm wholly within the cavernous sinus caused a subarachnoid hemorrhage. The aneurysm was successfully clipped via a direct surgical approach. The possible mechanism by which subarachnoid hemorrhage occurred is briefly discussed.

Trigeminal neuralgia caused by compression from arteries transfixing the nerve. Report of three cases.

The authors present three patients with trigeminal neuralgia due to compression by an artery that transfixed the sensory root of the fifth cranial nerve. These cases represented 0.8% of 384 patients with trigeminal neuralgia treated by microvascular decompression at the authors' clinic during the past 12 years. In the remaining 381 cases, the compressing vessels were successfully removed from the trigeminal nerve without much difficulty, for an initial cure rate of 94.3%. In the three cases reported, however, the compressing artery penetrating the nerve could not easily be maneuvered away from the nerve. In the first two cases, partial rhizotomy perpendicular to the axis of the nerve at the site of arterial transfixion made it possible to separate the artery from the nerve. However, these two patients developed postoperative facial sensory impairment. In the third case, rhizotomy was performed longitudinal to the axis of the nerve at the site of arterial transfixion, making it possible to reposition the artery peripherally beyond the root entry zone of the nerve without causing any postoperative sensory deficits of the face. No recurrent pain has developed in more than 2 1/2 years since surgery in any of these three cases. When performing microvascular decompression surgery on patients in whom the compressing artery penetrates the nerve, the technique used in our third patient is the procedure of choice.

Germinoma in siblings: case reports.

We present the first reported cases of germinoma occurring in siblings of different genders. One tumor occurred in the left basal ganglia of a 7-year-old boy and the other in the suprasellar region of his 9-year-old sister. Both tumors were diagnosed as germinoma of two-cell pattern based on light and electron microscopic studies. Possible genetic factors responsible for this phenomenon are discussed.

Pneumocephalus associated with benign brain tumor: report of two cases.

Two cases of spontaneous pneumocephalus, which developed after ventriculoperitoneal shunt procedures for severe hydrocephalus caused by benign brain tumors, are reported. In both cases there was no previous history of cerebrospinal fluid leakage. Operation revealed that both patients had many small defects of the dura mater and the bone in the middle cranial fossa, which were plugged by necrotic brain tissue. These defects were remote from the sites of the original tumors but may have been produced by long-standing raised intracranial pressure, and they presumably allowed air to enter after intracranial pressure was reduced by shunting. Repair of the defects prevented further intracranial air retention.

Intracranial double teratomas.

A case of double teratomas located in the pineal region and the fourth ventricle is presented. A simultaneous occurrence from nonmetastatic, separate origins seems rare in cases of mature teratomas. Computerized tomographic scans led to detection of another asymptomatic teratoma. Surgical treatment produced good results.

Spinal epidural meningioma in childhood.

A spinal epidural meningioma in a 14-year-old boy is presented and the literature reviewed. He had several café-au-lait spots and had previously had a subcutaneous lipoma removed from the mid-lumbosacral region.

Extradural nasal and orbital extension of glioblastoma multiforme without previous surgical intervention.

A case of extradural nasal and orbital extension of a glioblastoma multiforme in the absence of previous surgical intervention is presented. Such penetration by a glioma is rare due to the natural resistance of the dura mater.

Calcification of a ventriculoperitoneal shunt tube. Case report.

A 16-year-old boy who had undergone a ventriculoperitoneal (VP) shunt because of hydrocephalus at 8 years of age complained of pain around the right neck and chest. He concomitantly had a slight fever of unknown etiology, which had been lasting for several years. Skull and chest roentgenograms revealed an unusual calcified shadow around the shunt tube. After removal of the shunt apparatus, his pain and fever disappeared. Silicone tubes used in a VP shunt apparatus may induce fibrous connective tissue proliferation around the tubes in both children and adults, but no reports of radiologically verified calcification of a VP shunt tube are found in the literature, to the best of our knowledge. The possible mechanism of calcification of the VP shunt tube is discussed.