Lack of effect of long-term fluoride ingestion on blood chemistry and frequency of sister chromatid exchange in human lymphocytes.

Two studies were conducted to assess the potential for adverse physiologic and genotoxic effects of long-term fluoride ingestion in adults residing in three communities with varying water fluoride levels (0.2 ppm, 1.0 ppm, and 4.0 ppm). All were long-time (> or = 30 years) residents of their respective communities. Plasma and urine samples were collected for fluoride analyses. Additional plasma was collected to determine blood chemistry, and plasma lymphocytes were examined to determine the frequency of sister chromatid exchange. Significant differences in urine (P = 0.001) and plasma (P = 0.0001) fluoride levels were found in the three communities. Seven of the blood parameters were statistically different among the communities, although all were within the defined normal range of the pathology laboratory. Sister chromatid exchange frequency was statistically higher in the 4.0 ppm fluoride community as compared to the other communities. Because of the higher SCE frequency in the 4.0 ppm fluoride community, a second study was performed to determine if the increased frequency was potentially related to the fluoride level of the communal water supply. Of the 58 adults recruited; 30 had used city water and 28 had used well water (< or = 0.3 ppm fluoride) as their principal water source for 30 years. Data analyses showed that the sister chromatid exchange frequency did not differ between the groups, indicating that the increased sister chromatid exchange frequency was not related to the fluoride level of the communal water. The investigation provided evidence that the long-term ingestion of water containing 4.0 ppm fluoride did not have any clinically important physiologic or genotoxic effects in healthy adults.

Evaluation of mutagenicity of restorative dental materials using the Ames Salmonella/microsome test.

Compounds of five commercially available dental material kits were examined for mutagenic potential by use of the Ames Salmonella/microsome test. Dimethyl sulfoxide (DMSO) was used as the solvent for all materials, except MONO-LOK2 Primer and Right-On Activator, which were dissolved in 95% ethanol. The Tenure, Fuji, and Gluma materials were initially screened at 2 mg (solids) or 2 microL (liquids) per plate, without S9 (Aroclor 1254-induced rat liver microsomes), and dose-response studies were conducted for those materials showing potential mutagenicity. The components of three dental kits, MONO-LOK2, Right-On, and Gluma, were further examined, both with and without S9 activation, at doses of 0.2, 4, 20, 100, 500, and 2500 micrograms (nL for liquid components) per plate. The data showed no mutagenic potential for the components of Fuji Glass Ionomer (Type III), Tenure Dentin Bonding System, and MONO-LOK2 kits, or Right-On Paste. However, Gluma 3, a component of the Gluma/Lumifor Bonding System, exhibited mutagenic activity in a dose-response manner. The mutagenicity of Gluma 3 was demonstrated in repeated experiments. Gluma 4 and the resin of the Gluma dental kit, as well as Right-On Activator, caused a slight increase in the number of revertants, and judgment regarding their mutagenicity could not be made conclusively. The results of this study indicate that some commercial dental materials may not have been adequately tested for mutagenicity, and that a reliable test program for evaluation of mutagenicity of dental materials is needed so that their safe use in dental practice can be ensured.

Genotoxic evaluation of chronic fluoride exposure: micronucleus and sperm morphology studies.

The purpose of this study was to investigate the genotoxic effects of chronic fluoride exposure on mammalian cells in vivo by use of the mouse bone-marrow micronucleus test and the sperm morphology methodology. Mice of genotype B6C3F1 were obtained at weaning and maintained on a low-fluoride diet (less than 0.2 ppm F) ad libitum throughout the experiment. The animals were randomly assigned to seven groups and given fluoride (as sodium fluoride) in concentrations ranging from 1.0 to 75 ppm in the drinking water. Negative (distilled water) and positive (cyclophosphamide) controls were included. After a 21-week treatment period, the animals were killed by cervical dislocation, and blood was obtained by cardiac puncture. Slides of femur marrow cells were prepared and blindly examined for the frequency of micronucleated polychromatic erythrocytes (MN-PCE). Slides of sperm from the cauda epididymides of the male mice were also prepared and similarly examined for morphological abnormalities. Weight of the testes was recorded, and the plasma, humeri, testes, and carcasses were saved for fluoride analyses. Analyses of bone and plasma fluoride confirmed the effective absorption of fluoride following ingestion. The frequency of MN-PCE, the count of abnormal sperm, and the weight of the testes for mice chronically exposed to fluoride, in doses ranging from approximately 0.3 to 23 mg/kg/day, were not significantly different from those of the negative control animals. The results of this study support the view that fluoride has no genotoxic effects.

Genotoxic evaluation of chronic fluoride exposure: sister-chromatid exchange study.

As part of a continuing investigation, this study was conducted to examine the genotoxic effects of chronic exposure to sodium fluoride (NaF) in drinking water on the frequency of sister-chromatid exchange (SCE) in the bone-marrow cells of male Chinese hamsters. Animals at about 3 weeks of age were randomly assigned to 6 groups, each with at least 3 hamsters, and were maintained on a low fluoride diet (less than 0.2 ppm F) throughout the experiment. At 4 weeks of age, the animals in groups I-V began to receive drinking water containing fluoride at concentrations of 0, 1, 10, 50 and 75 ppm, respectively. Group VI was treated with cyclophosphamide and served as the positive control. The animals were sacrificed at 24 weeks of age by cervical dislocation. The humeri and plasma were analyzed for fluoride content, which was found to increase with the increase in fluoride concentration in drinking water. Slides of chromosomes from bone-marrow cells were prepared and blindly examined for the frequency of SCE. The mean scores of SCE for the hamsters receiving drinking water containing F concentrations up to 75 ppm for 21 weeks ranged from 4.28 to 6.28 per cell, and were not significantly different from those of the negative controls (4.60-5.44/cell). The results indicated that chronic fluoride exposure had no effect on the frequency of SCE in Chinese hamster bone-marrow cells under the conditions of the present investigation.

Measurement of enamel demineralization using microradiography and confocal microscopy. A correlation study.

Substantial amounts of tooth minerals are lost during dental caries formation. Transversal microradiography, a well-accepted method used to quantify mineral loss, is a time-consuming technique which requires a thin enamel section (100 microns) and involves the use of x-rays. In an attempt to solve these difficulties, a procedure has been developed in which a human tooth specimen with demineralized enamel is cut in half (HT), stained with a fluorescent dye (rhodamine B) and analyzed using a laser scanning confocal microscope. A series of three studies was conducted to correlate measurements of enamel demineralization obtained from enamel thin (100 microns) sections (TS) using transversal microradiography with three parameters (area of the lesion; total and average dye fluorescence intensities) measured on the same TS or on a thicker section (HT) of the same specimen by laser scanning confocal microscopy. Results showed that a 0.1 mM rhodamine B solution provided the most adequate imaging conditions for confocal microscopy. Pearson's correlation coefficients, calculated between microradiography and confocal microscopy data obtained using a 0.1 mM rhodamine B solution, were: delta Z vs. HT lesion area = 0.95; delta Z vs. HT total fluorescence = 0.80; delta Z vs. HT average fluorescence = 0.74; delta Z vs. TS lesion area = 0.95; delta Z vs. TS total fluorescence = 0.74; delta Z vs. TS average fluorescence = 0.55. All these correlations coefficients were statistically significant (p < 0.01). It is concluded that in enamel demineralization studies statistically significant correlations exist between parameters measured using transversal microradiography and parameters quantified using confocal microscopy.

Changes in neutrophil actin and shape during sequestration induced by complement fragments in rabbits.

Complement fragment-induced sequestration of neutrophils within the lungs may be mediated by stimulus-induced decreases in the deformability of neutrophils, prolonging their lung capillary transit times. As changes in deformability often occur through changes in cytoskeletal proteins, this study determined whether the distribution of actin within intracapillary neutrophils was altered by intravascular complement fragments and whether sequestered neutrophils were less deformed. Ultrathin cryosections of lung tissue from rabbits given an infusion of complement fragments or saline were immunolabeled with anti-actin antibodies. The number of gold particles/microvillus and the density of gold particles/microgram 2 cytoplasm in the submembrane and the central region of intracapillary neutrophils was quantitated. Neutrophil shape was evaluated using laser confocal microscopy. In control rabbits, the ratio of submembrane/central gold was always greater than one and most neutrophils were elongated, 97% having shape factors > 1.10. The ratio of submembrane/central gold was greater in complement-treated rabbits (5.1 +/- 0.9) than controls (2.6 +/- 0.4; P < 0.026). The number of gold particles/microvillus was also increased in complement-treated rabbits (3.9 +/- 0.5) compared with controls (2.3 +/- 0.5; P < 0.045). Neutrophils were more often spherical when rabbits received complement fragments for 1.5 minutes than in control lungs or after 15-minute infusions. These data suggest that complement fragments induce a rapid redistribution of actin from the central to the submembrane region and the microvilli and result in more round neutrophils. This redistribution may decrease the deformability of neutrophils by altering the stiffness of the submembrane region and/or by preventing the microvilli from flattening.

Detection of mutans streptococci in secondary carious lesions using immunofluorescent techniques and confocal laser scanning microscopy.

Secondary caries is one of the major reasons causing restoration failure; however, little is known of its microbial etiology, mainly because of the difficulties in eliminating bacterial contamination during collection and sample preparation. Therefore, the purpose of this study was to investigate the feasibility of immunofluorescent techniques and confocal laser scanning microscopy for identification and quantification of bacteria in secondary carious lesions. Thirty-six extracted human teeth, clinically diagnosed as having secondary caries, were used in the study. The teeth were sectioned in half across the secondary carious lesion. One half of each tooth was processed using the Brown and Hopps histologic staining method for bacterial detection. Sections (100 microns thick) were obtained from the other half of each tooth for immunofluorescence labeling to detect and identify mutans streptococci in the subsurface of the lesion using confocal imaging techniques. Mutans streptococci were detected in 88.9% of the samples analyzed with the immunofluorescent technique. The Brown and Hopps histopathologic examination demonstrated evidence of bacteria in 94.4% of the samples. In addition, quantification of bacteria was conducted by digitalization of confocal images. The results indicated that the immunofluorescence confocal laser scanning microscopy technique was sensitive and specific for detection and quantification of mutans streptococci in secondary carious lesions.

High-efficiency gene transfer into normal and adenosine deaminase-deficient T lymphocytes is mediated by transduction on recombinant fibronectin fragments.

Primary human T lymphocytes are powerful targets for genetic modification, although the use of these targets in human gene therapy protocols has been hampered by low levels of transduction. We have shown previously that significant increases in the transduction of hematopoietic stem and progenitor cells with retroviral vectors can be obtained by the colocalization of the retrovirus and target cells on specific fibronectin (FN) adhesion domains (H. Hanenberg, X. L. Xiao, D. Dilloo, K. Hashino, I. Kato, and D. A. Williams, Nat. Med. 2:876-882, 1996). We studied the transfer of genes into primary T lymphocytes by using FN-assisted retroviral gene transfer. Activated T lymphocytes were infected for three consecutive days on the recombinant FN fragment CH-296 with a retroviral vector encoding the murine B7-1 protein. Transduced lymphocytes were analyzed for murine B7-1 expression, and it was found that under optimal conditions, 80 to 89% of the CD3+ lymphocytes were transduced. Gene transfer was predominantly augmented by the interaction between VLA-4 on the T lymphocytes and the FN adhesion site CS-1. Adenosine deaminase (ADA)-deficient primary T lymphocytes transduced on CH-296 with a retrovirus encoding murine ADA (mADA) exhibited levels of mADA activity severalfold higher than the levels of the endogenous human ADA protein observed in normal human T lymphocytes. Strikingly, the long-term expression of the transgene was dependent on the activation status of the lymphocytes. This approach will have important applications in human gene therapy protocols targeting primary T lymphocytes.

An in vitro microbial model for studying secondary caries formation.

Secondary caries is a major reason for the replacement of restorations. Because it is hypothesized that the development of secondary caries is closely associated with pathogenic oral bacteria, an in vitro microbial model has been developed to produce secondary carious lesions. A mixture of overnight cultures of Streptococcus mutans and Lactobacillus casei in dextrose-free trypticase soy broth, supplemented with 5% sucrose (TSBS), at 37 degrees C was used in this model as the inoculum for the experimental groups. Uninoculated control groups were incubated with medium only. Groups of human tooth specimens restored using composite, together with their respective controls, were exposed for 7 or 12 days to circulating cycles of TSBS (30 min each, 3 times per day) and a mineral wash solution (for a total of 22.5 h per day), at 37 degrees C. The pH of the experimental groups dropped to 4.l-4.5 during the test periods. The pH of the control groups remained at 6.8-7.0. The inoculated bacteria remained viable throughout the study. No contamination of experimental or control samples occurred. Laser scanning confocal microscopy demonstrated the development of incipient surface and wall lesions in all the specimens of experimental groups in as few as 7 days. Reproducibility of the model was confirmed in a second investigation. Therefore, it was concluded that this model can be used for studying the microbial etiology and prevention of secondary caries.