Acute illness and recovery in adult female rats following ingestion of a tooth whitener containing 6% hydrogen peroxide.

PURPOSE: To determine whether ingestion of a tooth whitener containing 6% hydrogen peroxide as bleaching agent affected the gastric mucosa of adult, female laboratory rats. MATERIALS AND METHODS: Thirty-six fasting rats were intubated with a single bolus (5 g/kg body weight) of the tooth whitener Natural White which contains 6% hydrogen peroxide. Thirty-two control rats received deionized water. Rats were necropsied 15 minutes, 2 hours, 1 and 2 weeks after whitener ingestion. The gastric mucosa was examined histologically and blood hematocrit, glucose, BUN, and bilirubin measured. RESULTS: Six of the 36 rats died within 2 hours of receiving whitener. Within 15 minutes of whitener ingestion, stomachs were grossly bloated with gas. Histological observation showed that gastric mucosal cells were vacuolated and gastric glands dilated. After 2 hours, gastric epithelial and glandular cells had sloughed off into the gastric lumen. Mean blood glucose and hematocrit were significantly (P < 0.05) elevated (233 +/- 41 mg/ml and 50.5 +/- 0.9%) over mean control values (129 +/- 8 mg/ml and 42.8 +/- 2.4%). Within 1 week, the stomachs of experimental rats were no longer bloated, the gastric mucosa appeared normal histologically, but mean blood hematocrit was significantly (P < 0.05) decreased (38.0 +/- 1.5%) from mean control value (43.3 +/- 0.5%). There were no significant differences in mean blood values 2 weeks after whitener ingestion.

Acute toxicological effects of ingested tooth whiteners in female rats.

Tooth whiteners are considered as cosmetic agents to be used for bleaching teeth. Since tooth whitener may be swallowed during the whitening procedure, studies were conducted to determine whether ingestion of tooth whitener containing carbamide peroxide resulted in toxic effects. Adult female rats were used, and vaginal smears were examined daily to determine whether the animals were ovulating. Following an overnight fast, a single bolus of a commercial tooth whitener (5 g of tooth whitener/kg fasting body weight) was administered by gavage. Control rats received de-ionized water. After 2 h, mean respirations per min of animals receiving the tooth whitener Quik Start (contains 35% carbamide peroxide) decreased from 169 to 55, and body temperature decreased from 38.4 to 34 degrees C. Other distress signs included: labored breathing, loss of righting reflex, partial eye closure, bloody urine, and incontinence. Three of 22 animals (3/22) died within 48 h, of gastric hemorrhaging. Eight/10 rats stopped ovulating. At necropsy 2 weeks post-dosing, 10/19 animals had grossly bloated stomachs, and mucosal necrosis was observed histologically in 3. Animals receiving White & Brite or Nu-Smile (containing 10 or 15% carbamide peroxide, respectively) exhibited similar but milder symptoms. The data indicate that ingestion of large doses of commercial preparations of tooth whiteners may be acutely toxic, sometimes fatal, to female laboratory rats.

Factors influencing synthesis and mineralization of bone matrix from fetal bovine bone cells grown in vitro.

This study of the in vitro synthesis and mineralization of bovine bone demonstrates that sheets of mineralized matrix can be produced consistently within 18-24 days of cell isolation. Mineralization surpasses that achieved by other systems with other species: The deposition of mineral extends beyond nodules to form branching trabeculae and then solid wafers of bone. Comparison of the fetal age of the bone source, enzyme digestion methods, seeding density, culture surface, nutritive media, and concentration of fetal calf serum and other additives, including insulin and ascorbic acid, has yielded a set of optimal culture conditions. In the presence of ascorbic acid and beta-glycerol phosphate, insulin has a dose-dependent effect on the morphology of the mineralized bone matrix produced. Quantitative analysis shows that in these cultures calcium accumulates most rapidly between days 6 and 10 after the introduction of mineralization medium but that mineral accretion continues throughout 14-16 days of culture. Alkaline phosphatase levels rise up to 200-fold, concomitant with a rapid increase in the number of cells per culture during the early mineralization phases; both fall as mineralization proceeds. This system has been used to study the induction of mRNA of type I collagen, alkaline phosphatase, and several noncollagenous bone proteins during the course of mineralization. Because of the degree of mineralization achieved with this system, it has many potential applications.

The isolation and primary culture of putative human root odontoblasts.

The use of procedures adapted from a routinely successful method of culturing bovine bone has led to the first system for the study of dentinogenesis in vitro. Two types of cells have been grown from pulp obtained from the growing root tips of impacted third molars extracted from 14- to 19-years olds: (1) epithelial-like cells that are probably derived from fragments of the epithelial root sheath and (2) odontoblast-like cells. The cultured epithelial-like cells grow out in distinctive rounded plaques while the odontoblast-like cells are tethered to and/or grow on top of the epithelial-like cells. The odontoblast-like cells produce mineralized matrix by 10 days when cultured on a defined mineralization formula containing conditioned medium obtained from fetal bovine bone cell cultures. Growth factors in this conditioned medium are important to cell proliferation and growth and to the synthesis of mineralized matrix. Sequential enzyme digestion in dispase and dispase/collagenase in serum-free Dulbecco's Modified Eagle's Medium is essential to obtaining adequate cell yields from the apical 3-5 mm of the developing root. Reduction of the number of fibroblasts by treating cultures with dispase in Tyrode's solution midway through the initial growth period enhances the purity of these cell cultures.

The toxin kainic acid: a study of avian nerve and glial cell response utilizing tritiated kainic acid and electron microscopic autoradiography.

Three questions are asked regarding the toxin kainic acid (KA). Does it destroy specific glial cells as well as neurons? Does KA gain access to the cytoplasm in intact cells and to which organelles does it bind? Intracerebral injections of tritiated KA into the pigeon (Columba livia) paleostriatal complex (basal ganglia) coupled with electron microscopic autoradiography revealed the following major points. Kainic acid destroyes oligodendrocytes, with pathophysiology apparent by 30 min after challenge with KA leading to cell destruction by 4 h. The response of astrocytes at the longest observation period (4 h) involves swelling of perivascular endfeet and processes in the neuropil. Reactive microglial-like cells show an accumulation of label in their cytoplasm, but no apparent morphological changes. The label appears in the cytoplasm of intact cells, both glia and neurons early after challenge with the toxin. Label is associated (bound) with mitochondria at an incidence significantly above chance at 30 min, 2 and 4 h after challenge with KA. Two hours after exposure to KA is the critical period where metabolic, physiological and morphological changes occur that lead to cell death. Cell destruction may be a consequence of KA-induced energy depletion. Kainate may interfere with adequate energy production by uncoupling glycolysis and the Krebs cycle in the mitochondria.

A simple method using alizarin red S for the detection of calcium in epoxy resin embedded tissue.

This report presents a simple procedure for staining 1-2 microns epoxy plastic sections of cells and mineralizing matrix present in fetal bovine bone tissue cultures. A 0.3% aqueous toluidine blue O solution was used as a cellular stain and was followed with 2% alizarin red S for the detection of calcium at sites of mineralization. Effects of concentration and pH of alizarin red S on the penetration of epon embedded thick sections were investigated. Optimal staining was achieved with a 2% aqueous alizarin red S solution adjusted to a pH of 5.5-6.5. This staining procedure provides unusually clear contrast between mineral and bone cells in plastic sections for light microscopy.

Fetal bovine bone cells synthesize bone-specific matrix proteins.

We isolated cells from both calvaria and the outer cortices of long bones from 3- to 5-mo bovine fetuses. The cells were identified as functional osteoblasts by indirect immunofluorescence using antibodies against three bone-specific, noncollagenous matrix proteins (osteonectin, the bone proteoglycan, and the bone sialoprotein) and against type 1 collagen. In separate experiments, confluent cultures of the cells were radiolabeled and shown to synthesize and secrete osteonectin, the bone proteoglycan and the bone sialoprotein by immunoprecipitation and fluorography of SDS polyacrylamide gels. Analysis of the radiolabeled collagens synthesized by the cultures showed that they produced predominantly (approximately 94%) type I collagen, with small amounts of types III and V collagens. In agreement with previous investigators who have employed the rodent bone cell system, we confirmed in bovine bone cells that (a) there was a typical cyclic AMP response to parathyroid hormone, (b) freshly isolated cells possessed high levels of alkaline phosphatase, which diminished during culture but returned to normal levels in mineralizing cultures, and (c) cells grown in the presence of ascorbic acid and beta-glycerophosphate rapidly produced and mineralized an extracellular matrix containing largely type I collagen. These results show that antibodies directed against bone-specific, noncollagenous proteins can be used to clearly identify bone cells in vitro.

Acute exposure to hymenoxon: electron microscopic study of the mouse liver.

Young, male mice (25 to 30 g) were given oral doses of hymenoxon, a sesquiterpene lactone, for 5, 10, or 20 days. Hymenoxon, isolated from Hymenoxys odorata DC, was dissolved in dimethyl sulfoxide (DMSO) and water (1:1, v/v) and was administered daily at a dosage level of 100 mg/kg of body weight for 5, 10, or 20 days. Two control groups were maintained; 1 group was given DMSO and water, and 1 group was given water only. Twenty-four hours after the last dosing, the mice were euthanatized and their livers were processed for transmission electron microscopy. Hepatocyte organelles of hymenoxon-treated mice appeared normal although the bile canaliculi contained a fine granular material and membranous structures, including myelin figures. The canaliculi of hymenoxon-treated mice were also markedly dilated, compared with bile canaliculi in the control groups. The mean area of bile canaliculi of the 5-, 10-, and 20-day mice was 14.08 microns2, 15.65 microns2, and 17.56 microns2, respectively, compared with 7.73 microns2 for the canaliculi of DMSO + water-treated mice. The P values were less than 0.001, 0.02, and 0.05 for the 5-, 10-, and 20-day hymenoxon-treated mice, respectively. Seemingly, the mouse was not a good model for studying hepatic ultrastructural changes produced by hymenoxon, using dosages less than or equal to 100 mg/kg/day for 20 days.

Calcium and parotid gland secretion: a cytochemical study.

Acinar cells of parotid glands from untreated, pilocarpine-treated, and atropine treated rats were studied, using a potassium pyroantimonate technique and X-ray microanalysis of calcium localization at the ultrastructural level. This was done in order to identify intracellular compartmentalisation of calcium and to elucidate any calcium translocation that might occur during the secretory process. Calcium pyroantimonate complexes were shown to be most prevalent on the plasma membrane of the non-secreting cells. These membranes gave up their calcium complexes during secretion. Conversely, the mitochondrial precipitates increased during secretion indicating an increase in intracellular calcium. The function of calcium in parotid gland secretion and the association of calcium with other cell structures are discussed.

Sporidesmin toxicity in rabbits: biochemical and morphological changes.

The earliest lesion in rabbits dosed orally with 2 mg of sporidesmin per kg of body weight was necrosis of occasional hepatocytes 1 day after dosing. The most consistent lesion was a severe necrotizing cholangitis of medium and large-sized intrahepatic and extrahepatic bile ducts, first seen 2 days after dosing. Similar lesions were also present in the gall bladder of some rabbits. Expansion of portal triads with fibrous tissue and proliferating bile ductules progressed to pseudo-lobulation by 21 days. Other hepatic changes observed irregularly included large infarcts at the periphery of some lobes, and multiple small foci of coagulation necrosis in midzonal and periportal regions. Vascular necrosis and thrombosis, invariably adjacent to necrotic bile ducts, was presumably responsible for the hepatic necrosis. Serum cholesterol and total bilirubin concentrations and GGT activity reached peaks 15 days after dosing and were useful indicators of the severity of biliary lesions. Serum ID activity was the most useful indicator of hepatic necrosis following oral dosing with sporidesmin. The similarity between hepatobiliary lesions observed in sheep and rabbits with experimental sporidesmin toxicity suggests that the rabbit would be a useful model for studying methods of treatment and prevention of "facial eczema" in ruminants.

Intracellular calcium localization in stimulated, non-stimulated and repressed eccrine sweat glands.

Acinar cells of eccrine sweat glands from control, pilocarpine-treated, and atropine-treated rats were studied using a potassium pyroantimonate technique for calcium localization at the ultrastructural level. This was done in order to identify intracellular compartmentalization of calcium and to elucidate any calcium translocation that might occur during the secretion process. Calcium-pyroantimonate complexes were identified in the mitochondria, plasma membrane and cytoplasmic vesicles of the untreated and the atropine-treated specimens. These complexes decreased drastically in the actively secreting cells. The function of calcium in sweat gland secretion and the action of the utilized pharmacological agents on membrane calcium are discussed.

Nonspecific esterase activity in plastic-embedded tissues using Meldola Blue.

A method for the demonstration of nonspecific esterase activity in plastic-embedded tissues using Meldola Blue is described. Although Meldola Blue does not function as an electron carrier in this method, it may account for the short incubation time and excellent localization of the reaction product. Use of Meldola Blue is an advancement in the demonstration of enzyme activity in plastic sections.

Ultrastructural changes in livers of young and aging rats exposed to methylated benzenes.

ULtrastructural examination was conducted on livers of young rats which were given (intraperitoneal injection) several methylated benzenes (73 mg/kg of body weight) for 3 consecutive days and on livers of aging rats (12 to 19 months old) which were fed methylated benzenes in the diet (200 mg/1,000 g of feed) for periods of 1, 2, 3, and 6 months. The young animals demonstrated white, raised, nodular lesions along the free margins of the liver. The lesions in young animals consisted of lipid droplets surrounded by macrophages and fibroblasts. Some fibroblasts in these lesions appeared to be active in collagen synthesis. Mast cells and eosinophils also occurred in the lesions. Hepatocytes in these young animals appeared morphologically normal. In contrast, hepatocytes in the aging rats developed 2 types of vacuoles. The 1st type of vacuole was bound by a double-unit membrane--the other unit membrane possibly derived from smooth endoplasmic reticulum; it contained a mottled, globular, electron-lucent material. The 2nd type of vacuole was peripherally located in hepatocytes, was single membrane-bound, and occasionally was observed opening into the space of Disse (spatium perisinusoideum). Our observations indicated that acute exposures (3 days) to at least some methylated benzenes does not cause ultrastructurally observable abnormalities in hepatocytes of young rats, but chronic oral ingestion of some can cause formation of vacuolar structures in aging rat hepatocytes.

Intracellular calcium localization in stimulated and non-stimulated extraorbital lacrimal glands of rats.

Acinar cells of extraorbital lacrimal glands from control, pilocarpine-treated, atropine-treated and atropine + pilocarpine-treated rats were studied using a potassium pyroantimonate technique and X-ray microanalysis for calcium localization at the ultrastructural level. This was done in order to identify intracellular compartmentalization of calcium and to elucidate any calcium translocation that might occur during the secretory process. Calcium-pyroantimonate complexes were identified in the mitochondria, plasma membrane and cytoplasmic vesicles of the untreated specimens and in the plasma membrane of atropine-treated specimens, these complexes decreased drastically in the actively-secreting cells. The function of calcium in lacrimal gland secretion and the action of pilocarpine and atropine on membrane calcium are discussed.

Vascular supply pattern to rat caudatoputamen and globus pallidus: scanning electronmicroscopic study of vascular endocasts of stroke-prone vessels.

The caudatoputamen (CP) and globus pallidus (GP) are supplied by vessels often involved with stroke in both rat and human. The pattern of vascular supply to the CP and GP in rat has, in contrast to humans, been only partially described. The vascular pattern to the rat CP and GP is described utilizing vascular endocasts and scanning electronmicroscopy in aging, normotensive rats. Endocasts were produced by intra-cardiac infusion of Batson's Corrosion Compound. The vascular pattern is complex, involving 1) recurrent vessels from the anterior cerebral artery, 2) branches from the arterial circle rostral or caudal to the origin of the middle cerebral artery (MCA), 3) up to 6 branches from the MCA, and 4) 2 major branches from the caudal part of the arterial circle. The vessels in groups 1--3 were serpentine, their luminal diameters abruptly reduced at branch points, and the angle of departure from the parent vessels approximated 90 degrees. These vessels supplied much of the CP and GP, while group 4 supplied the caudal CP with vessels arranged in a lattice-like fashion from the 2 penetrating parental arteries.

Necrotizing effects of kainic acid on neurons in the pigeon brain: histological observations.

The neurotoxin kainic acid (KA) has been shown to destroy neurons in the pigeon paleostriatal complex (PC), the avian analogue of the caudato-putamen and globus pallidus. In this earlier study the movement disorders in pigeons were strikingly similar to those reported by others in rats following intracerebral injection of KA into the corpus striatum. The toxic influences of KA on other parts of the pigeon brain have not been described. Therefore, KA was injected into areas of the telencephalon, diencephalon, mesencephalon and cerebellum. Areas sensitive to KA showed a marked cell loss and the neuropil exhibited spaces that contained fragments of necrotic neurons. The injection sites were invaded by glia and granulocytes. Kainic acid had a local necrotizing effect; for example, it destroys neurons in the PC, nucleus rotundus, nucleus spiriformis lateralis, nucleus ruber and neurons of the cerebellar cortex. An apparent long-distance effect of KA was also observed, since intracerebral injections of KA into the PC was followed by cell loss in the ipsilateral nucleus of the ansa lenticularis. Kainic acid has proved to be a potent neurotoxin with a pronounced necrotizing effect upon neurons in the pigeon brain.

A technique for improved fixation of the pigeon central nervous system for electron microscopy.

The central nervous system (CNS) of the pigeon has been difficult to fix with consistency, and consequently this problem has impeded ultrastructural studies of various parts of the pigeon brain. Here we describe a method for effective fixation of the pigeon CNS and discuss the three principal problems associated with good fixation of this animal's brain. The animal was deeply anesthetized and the thoracic cavity was opened without collapsing the pectoral girdle upon the brachiocephalic trunks and the common carotids. The perfusion pressure was raised to 140-150 mm Hg to overcome the high resistance of the small diameter, long common carotids. Heparin was added to the wash buffer to retard coagulation of blood in the vascular bed of the brain. The method is not foolproof, but with care excellent fixation can be achieved.

Irradiated salivary glands in the rhesus monkey. A light microscopic study.

Female rhesus monkeys (Macaca mulatta) received fractionated doses of orthovoltage irradiation to the submandibular and sublingual glands. Changes in the glands varied from serous cell degranulation and degeneration in the submandibular glands to acinar cell necrosis and fibrosis in the sublingual glands. Acute inflammation was absent in all irradiated glands. In all glands, the microvasculature appeared normal.